Abstracts of the Plenary and Symposia Talks; and Poster Presentations

Exp Clin Cardiol. 2011 Oct;16(Suppl A):1A.

1 Genome Wide Characterization of RNA Polymerase II-Dependant Transcription During Cardiac Hypertrophy

Abstract

Differential regulation of gene expression is the underlying mechanism involved in development and disease. During cardiac hypertrophy there are two distinct gene expression changes that occur in the myocyte. One is the total increase in cellular mRNA and protein that is commensurate with, and responsible for, the increase in cell mass and volume (∼30%). The second is selective, more dramatic, increases or decreases in specific genes that play a more specialized role in cell growth. Until recently it was assumed that transcriptional activation of genes is achieved by de novo recruitment of RNA polymerase II to promoters. However, this idea has recently been challenged by studies in Drosophila, which suggest that RNA polymerase II (pol II) is already bound to the promoter-proximal region of a gene. To understand the full nature of these transcriptional activities, we examined the RNA pol II bound to the genome before and after development of hypertrophy in a genome-wide approach. This was accomplished by pol II chromatin immunoprecipitation followed by extensive sequencing (ChIP-Seq). The results of this were aligned to the genome and viewed in both a quantitative and qualitative graphical format with Affymetrix’ Integrated Genome Browser software. Our results with poll ChIP-Seq not only identify widespread promoter-proximal RNA pol II pausing in the adult heart, but also reveal its synchronized release during cardiac hypertrophy, which recapitulates the neonatal heart pattern. We have also uncovered a unique aspect of RNA pol II transcription that has never been seen before, in the form of its stalling as it exits the gene’s 3’ boundary. The data provide unique mechanistic insights into the regulation of gene transcription during hypertrophy.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):1A.

2 Evaluation of Load-Independent Biventricular Contractility During Machine Perfusion of the Donor Heart

Ayyaz Ali ¹, B Xiang ¹, S Colah ¹, Paul Mundt ¹, Rakesh C Arora ¹, Steven R Large ¹, G Tian ¹, Darren H Freed ¹

Abstract

Donor hearts are commonly rejected for transplantation on the basis of apparent poor function. Ex-vivo machine perfusion may provide an opportunity to objectively evaluate donor heart function outside the milieu of the brain dead donor. This may also be useful for assessment of hearts from donors following circulatory death. We describe a method that enables assessment of right and left ventricular contractility during ex-vivo cardiac perfusion.

Hearts were retrieved from 60 kg pigs. Initial resuscitation was achieved via perfusion of the aortic root and coronary circulation. After recovery of cardiac function and rhythm, the left and right atrium were perfused to establish biventricular working mode. Pressure-volume loops were used to measure load-independent ventricular contractility, end-systolic pressure-volume relationship (ESPVR) and pre-load recruitable stroke work (PRSW). Dobutamine was administered to assess contractile reserve.

During working mode, the LV ESPVR was 23±11 and the LV PRSW was 67±7. Both measures increased significantly after infusion of dobutamine (LV ESPVR: 45±12, p=0.01; LV PRSW 102±18, p=0.03). The RV ESPVR (pre 4.1±2.3 vs. post 11.9±4.1) and RV PRSW (pre: 6.7±2.4 vs post. 8.7±4.6) were also measured in 2 animals and increased followed dobutamine infusion. Hearts were perfused for 219±64 minutes. Over the perfusion period, there was a trend towards increased cardiac weight (282±33 g to 332±63 g, p=0.16).

Load-independent biventricular contractility and contractile reserve can be measured during machine perfusion. Such methods may allow for a more discerning examination of the human donor heart, including organs from borderline and extended criteria donors.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):1A.

3 Protective Role of Oleic Acid Against TNF-alpha Induced Effects in Adult Rat Cardiomyocytes

A Al-Shudiefat ¹, A Sharma ¹, A Bagchi ¹, Anju Bajaj ¹, P Singal ¹

Abstract

BACKGROUND:

One of the important cytokines, tumor necrosis factor alpha (TNF-alpha), is produced by many cell types including macrophages, lymphocytes, and cardiomyocytes. Some of the adverse effects of TNF-alpha are suggested to be mediated by an increase in the oxidative stress. Oleic acid (OA) has been reported to decrease: low density lipoproteins (LDL); reactive oxygen species; blood pressure; and increase the expression of peroxisome proliferator-activated receptor (PPAR gamma). The latter is known to depress the expression of TNF-alpha. The purpose of this study was to explore the protective role of OA against TNF-alpha induced effects in rat cardiomyocytes.

Cardiomyocytes were isolated from rat hearts and grouped as follows: A) control; B) OA (50 μM); C) TNF-alpha (10 ng/ml); and D) TNF-alpha (10 ng/ml) + OA (50 μM). Following the exposure time of four hours, cells were analyzed for oxidative stress, apoptosis, cellular damage and viability. Oxidative stress analysis using the dye DCFDA showed that TNF-alpha increased production of reactive oxygen species significantly, while OA ameliorated this increase (P<0.05). Western blot analysis showed that TNF-alpha significantly (P<0.05) enhanced the expression of apoptotic proteins Bax, cleaved caspase3, cleaved PARP, Bnip3, and TGF beta, while OA prevented these changes. Apoptosis and cell damage by TNF- alpha was increased and viability was decreased significantly (P<0.05), while OA offered protection.

TNF- alpha induced cell injury and cell loss is significantly reduced by OA by blunting oxidative stress and apoptosis. (Supported by CIHR).

Exp Clin Cardiol. 2011 Oct;16(Suppl A):1A.

4 Delirium in the Postoperative Cardiac Surgery Patient

RC Arora ¹

Abstract

Delirium is a common and serious problem that occurs after cardiac surgery. Delirium is an acute, fluctuating disorder of attention and cognition and is the most common neurological complication after cardiac surgery. Analogous to cardiac surgery associated acute kidney injury or lung injury, delirium is a form of acute brain dysfunction. Not only is experiencing delirium a distressing condition for the patient and their families, postoperative delirium has been associated with increased 30-day and 5-year mortality, higher hospital costs, loss of functional independence and persistent, long-term adverse cognitive outcomes. Despite more than 40 years since the first report of delirium after heart surgery, we still only have a rudimentary understanding of the causes of delirium in this group of patients. Over the past two decades, cardiac surgical operations are being performed on older patients with recent coronary syndromes, higher New York Heart Association classifications, lower left ventricular ejection fractions and cardiogenic shock preoperatively. All of these features are associated with increased rates of postoperative delirium. Other important risk factors associated with postoperative delirium include advanced age, impaired cognitive and functional status, sensory impairment, concomitant medical illnesses, and multi-drug treatments. Therefore while it is unclear what the actual incidence of delirium is in the current era of cardiac surgery, rates will likely continue to increase due to the aging demographic of the “typical” cardiac surgery patient. Novel and effective treatment strategies are needed to reduce the incidence and severity of this life-threatening complication.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):2A.

5 Enhanced Expression of Gαq and PLCβ1 Contribute to Vascular Hypertrophy in Spontaneously Hypertensive Rats

ME Atef ¹, MB Anand-Srivastva ¹

Abstract

Gαq plays an important role in the regulation of vascular tone. Although the implication of Gαq protein and the associated signaling in the cardiac hypertrophy has been clearly shown, the role of Gαq in vascular hypertrophy is not yet fully understood. The present study was undertaken to investigate if the expression of Gαq and PLCβ1 proteins are upregulated in aortic vascular smooth muscle cells (VSMC) from 16 week-old spontaneously hypertensive rats (SHR), which has been shown to exhibit cardiac hypertrophy and to further explore the implication of endogenous angiotensin II (Ang II) and endothelin-1 (ET-1) and signaling mechanisms for this upregulation. The expression of Gαq and PLCβ1 proteins, as determined by Western blotting, was significantly increased in VSMCs from SHR compared to WKY rats. In addition, the protein and DNA synthesis, as determined by 3H-leucine and 3H-thymidine incorporation respectively, were significantly augmented in VSMCs from SHR. However, the antisense of Gαq attenuated the hypertrophy but not the proliferation. The increased expression of Gαq and PLCβ1 proteins was attenuated by AT1 receptor antagonist, losartan, and ET-A receptor antagonist, BQ123, but not by ET-B receptor antagonist, BQ788. The increased protein expression of Gαq and PLCβ1 as well as protein synthesis were also attenuated by N-acetylcysteine (NAC), a scavenger of superoxide anion, NAD(P)H oxidase inhibitor DPI, AG1024 (IGF-R inhibitor) and AG1478 (EGF-R inhibitor). These results suggest that VSMCs from 16-week-old SHR exhibit enhanced expression of Gαq and PLCβ1 as well as protein synthesis, which may be attributed to enhanced levels of Ang II, ET-1, growth factor receptor as well as oxidative stress and may be responsible for the hypertrophic response in VSMCs from SHR.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):2A.

6 Regulation of BNIP3 by Casein Kinase II Promotes Cell Survival of Ventricular Myocytes

Delphine Baetz ¹, Joe Gordon ^2,³, Hongying Gang ^2,³, Lorrie A Kirshenbaum ^2,³

Abstract

Oxygen deprivation for prolonged periods leads to cardiac cell death and ventricular dysfunction. Casein kinase 2 (CK2) is a multifunctional serine/threonine kinase essential for regulating a variety of cellular processes including cell survival. However, its function in cardiac muscle is not well established. In this report, we demonstrate signaling pathways mediated by CK2 are necessary and sufficient to suppress cell death of ventricular myocytes during hypoxia. Post-natal ventricular myocytes displayed a 4.0 fold increase (p<0.05) in myocyte death during hypoxia compared to normoxic control cells while hypoxia-induced cell death was significantly decreased in cells expressing wild type CK2. Treatment of cardiac myocytes with the CK2 inhibitor, DRB (5,6-Dichloro-1-β-D-Ribofuranosyl-Benzimidazole) significantly increased myocytes death that that can be rescued by expressing CK2. In parallel, western blot analysis revealed a marked reduction in CK2 expression levels, which coincided with a 13 fold reduction in CK2 kinase activity in hypoxic myocytes. Interestingly, we identified the hypoxia-inducible death protein BNIP3 to be a phosphoprotein and critical CK2 target. In vitro kinase assay verified phosphorylation of BNIP3 by CK2 on critical serine residues located in N-terminal PEST domain of BNIP3. To ascertain whether the CK2 site within the BNIP3 gene is necessary for the observed increased hypoxia induced cell death through BNIP3, we generated BNIP3 constructs in which the CK2 site had been mutated. We generated a constitutively phosphorylated form of BNIP3 named full BNIP S62D (FN S62D), and an unphosphorylable form of BNIP3 named Full BNIP S62A (FN S62A). Expression of BNIP3 defective for CK2 phosphorylation increased cell death, and is responsible for mitochondrial defect. Whereas expression of phosphorylated BNIP3 doesn’t cause mitochondrial defects and cell death of ventricular myocytes as compared to control cells.

To our knowledge, our data provides the first direct evidence for a post-transcriptional regulation of BNIP3 activity by CK2. Further, we show that BNIP3 ability to induce apoptosis is dependent on its phosphorylation status. In this study, we provide a novel insight into the cytoprotective properties of CK2 and suggest that CK2 can phosphorylate BNIP3.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):2A.

7 L-10-Induced Cardiomyocyte Survival Requires TLR 4 Regulated MyD88 Molecule

Ashim K Bagchi ¹, Anita K Sharma ¹, Ana Ludke ¹, Abd Al-Rahaman Al-Shudiefat ¹, Pawan K Singal ¹

Abstract

Activation of TLRs have been associated with the maintenance of cardiomyocyte contractile function via Interferon regulatory factor 3 (IRF3) and NF-kB signaling pathway. Protective effect of anti-inflammatory cytokine interleukin 10 (IL-10) against TNF-α-induced cardiomyocyte apoptosis have already been reported but its innate signaling mechanism is not yet understood. We have investigated the downstream innate signaling mechanism subsequent to IL-10 stimulation. Isolated rat cardiomyocytes were exposed to IL-10 for different time durations and were examined for different signaling molecule. IL-10 caused a significant increase in the expression of CD14, TLR4 and MyD88. Lipopolysaccharide (LPS) was used as a positive control. Activation of TLR4 led to phosphorylation of IRF3 and nuclear translocation of NF-kBp65. Phosphorylation of IRF3 possibly enhanced the synthesis of IL-1β instead of TNF-α as we found profound suppression of TNF-α compared to IL-1β mRNA activity. More interestingly, translational activity of IL-1β was elevated even after removal of IL-10 stimulation. There was an increased transcriptional activity in pro-survival, Bcl-xL expression that inhibited the caspase-3 activity for proteolytic cleavage of PARP, which suggests that IL-10 treatment could switch apoptosis signals to cell survival through IL-1β-mediated TLR4 activation. Inhibition of MyD88 modulated the above listed responses to IL-10 and there was an activation of PARP cleavage independent of caspase-3 activation. These data suggest that IL-10-induced cardiomyocyte survival upregulates TLR4 innate signaling through IL-1β-regulated MyD88 adaptor molecule, which is a key molecule in the IL-10-mediated TLR4 innate signaling pathway.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):2A.

8 Interdependancy of Calpain-Calpastatin and Ubiquitin-Proteasome Systems with Cardiac Ischemia/Reperfusion

Angelo Belcastro ¹

Abstract

The integrative role(s) of the proteolytic pathways to cardiac muscle proteolysis accompanying ischemia/reperfusion (I/R) is not well understood. Increasing evidence points to a complex and complicated relationship among the three primary pathways for regulating protein loss/degradation – i) ubiquitin-proteasome system (UPS); ii) calpain-calpastatin system; and iii) autophagy. The UPS is the central pathway for degradation of proteins in the heart, which is important for regulating protein quality. With I/R the activity of the UPS is increased and several myofibrillar- and membrane-associated proteins (such as myosin light chain-2, troponin-I, troponin-T and ryanodine receptors) are reported to be targeted for enhanced degradation by the UPS. Notwithstanding this targeted degradation the UPS does not initiate and participate directly in the disassembly of intact organelles, In this regard calpain and calpastatin have been implicated in the prerequisite dismantling of cytoskeleton-organelle structures and releasing proteins for subsequent targeting for the UPS. Indeed desmin, vimentin, and β-crystalin (all calpain substrates) have been modified and removed during ischemia/reperfusion. An increase in the activity ratio of calpain-calpastatin has been reported during I/R implicating a greater loss/modification of calpain-substrate proteins in the heart. For C-C to be considered an important regulatory – perhaps rate limiting step – feature of proteolysis, the C-C system must be able to modify/release calpain-substrate proteins within organelles at varying and selective rates. This presentation details the upstream importance of C-C in providing selected myofibrillar associated protein substrates for proteolysis.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):3A.

9 Redox-Sensitive Modulation of Signaling Pathways Involved in Thyroid Hormone-Induced Cardiac Hypertrophy and Failure

A Belló-Klein ¹, AS Araujo ¹

Abstract

Thyroid hormone-induced cardiac hypertrophy has been attributed to its classic action, but there are several data suggesting a nongenomic action, involving reactive oxygen species as molecular mediators. Thus, hyperthyroidism was induced by thyroxine (T4) (12 mg/L) in drinking water to male Wistar rats. After 14 days, cardiac hypertrophy was observed and, after 28 days, typical signals of heart failure were also seen. Lipid and protein oxidation were increased and the reduced to oxidized glutathione ratio was decreased in heart homogenates of hyperthyroid rats. Myocardial antioxidant enzyme activities increased, while vitamin C and total radical-trapping antioxidant potential levels were decreased in hyperthyroid animals. These alterations were attenuated by vitamin E (20 mg/kg/day s.c. during 28 days). Protein expression of the nuclear transcription factor Nrf2 and of the antioxidants thioredoxin-1 and peroxiredoxin-6 was found to be increased in the hyperthyroid and attenuated by vitamin E, which has also promoted a reduction in cardiac hypertrophy and left ventricle dysfunction induced by T4. The expression of some prosurvival (Akt, ERK1/2) and proapoptotic proteins (GSK3-beta, JNK) was quantified and showed to be elevated in the T4 group, except for JNK that remained unchanged. Vitamin E administration promoted a significant reduction in their expression. The influence of the balance between reactive oxygen and nitrogen species in the myocardial failure was also determined in this model. These results indicate that redox imbalance due to hyperthyroidism induces adaptation of antioxidant systems and suggest a redox activation of some signaling pathways, with NO balance acting as a mediator of this adaptive response in experimental hyperthyroidism.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):3A.

10 Regulation of Rac1 Activation by Calmodulin in Platelet and CHRF-288-11 Cells

RP Bhullar ^1,², B Xu ²

Abstract

Rac1, a small GTP-binding protein, is implicated in lamellipodia formation. Calmodulin (CaM), a ubiquitous Ca²⁺-binding protein, is able to interact with and regulate the activity of several small GTP-binding proteins. In this study, we have investigated if CaM interacts with Rac1 and regulates its activation. Pull down assay demonstrated that CaM from platelet lysate interacts in a calcium-dependent manner with GST-Rac1. In the reverse experiment, CaM coupled to Sepharose beads pulled down Rac1 from platelet lysate. Pure CaM was also able to bind Rac1 demonstrating direct Rac1-CaM. A stretch of 14 amino acids in Rac1 was identified as the CaM binding region. The scrambled form of the peptide did not bind CaM. The role of CaM in Rac1 activation was studied using freshly isolated human platelets. Maximal Rac1 activation in response to thrombin occurred at ∼60 sec. Incubation with the CaM specific inhibitor, W7, prior to thrombin challenge, abolished Rac1 activation. We further extended these studies using the megakaryocytic cell line, CHRF-288-11. Maximal Rac1 activation by thrombin or collagen was observed at 3 and 1 min respectively and this was inhibited by W7. The less potent CaM inhibitor, W5, had no effect on Rac1 activation by thrombin or collagen. Transient over-expression of CaM in CHRF cells increased the basal and thrombin-mediated Rac1 activation when compared to control. Results demonstrate that in platelets and CHRF cells, binding of CaM to Rac1 increases its activation and that CaM may play an important role in the regulation of cytoskeleton remodelling. In addition, the CHRF-288-11 cells may serve as a useful model system in place of platelets to understand the regulation of Rac1 by CaM.

Footnotes

Supported by the Heart & Stroke Foundation of Manitoba

Exp Clin Cardiol. 2011 Oct;16(Suppl A):3A.

11 Nuclear Membranes G-Proteins Coupled Receptors and Nuclear T-Tubules Contribute to Normal and Pathological Cardiovascular Cell Function

Ghassan Bkaily ¹, Danielle Jacques ¹

Abstract

Growing evidence in the literature including our work suggests that the nucleus is a cell within a cell. Recent work from our laboratory showed the presence of G-protein coupled receptors (GPCRs) in the nuclear envelope membranes of all the cell types tested, including human cardiac and vascular endothelial and smooth muscle cells (hVSMCs). In the present work, we report that direct activation of nuclear membrane GPCRs induces nuclear changes (nucleoplasmic ionic homeostasis) and promotes protein synthesis. In addition, some types of receptors are limited to the nuclear membranes. Furthermore, we demonstrate the presence of nuclear membranes’ invaginations in the nucleus, which are extensions of the nuclear envelope into the nucleoplasm. These new nuclear structures were called nuclear T-tubules. Similar to the nuclear envelope, the nuclear T-tubules possess GPCRs, such as Endothelin-1 and Angiotensin II receptors as well as nuclear pores and ionic transporters. These nuclear T-tubules are dynamic and appear to reflect the metabolic and physiopathologic states of the hVSMCs. Hence, nuclear T-tubules seem to be markers of hVSMCs pathology. In addition, the presence of nuclear T-tubules as well as nuclear membranes GPCRs were found to regulate basal cytosolic and nuclear free calcium levels. In conclusion, nuclear T-tubules and nuclear membranes GPCRs contribute to hVSMCs function.

Footnotes

This work is supported by the National Sciences and Engineering Research Council of Canada (NSERC) and the Heart and Stroke Foundation of Quebec (HSFQ)

Exp Clin Cardiol. 2011 Oct;16(Suppl A):3A.

12 Ezetimibe Inhibits the Absorption of Omega-3 Fatty Acids

DP Blackwood ¹, A Al-Busaidi ¹, RK La Vallee ¹, DS Jassal ¹, GN Pierce ¹

Abstract

INTRODUCTION:

Atherosclerosis is a leading cause of cardiovascular disease. The omega-3 fatty-acid, alpha linolenic acid (ALA), has been shown to be anti-atherogenic. The presence of elevated ALA in circulation may be an important benefit to cardiovascular health. Animal studies have demonstrated that the bioavailability of essential fatty acids is enhanced by a cholesterol supplemented diet. It follows, therefore, that certain cholesterol-lowering drugs may inhibit fatty acid absorption. The purpose of the study was to determine if a cholesterol absorption inhibitor (ezetimibe) will decrease circulating levels of ALA.

METHODS:

Patients between 44–80 years old, requiring statin therapy to regulate blood cholesterol levels, were randomly assigned to one of four groups for a 6 week trial: 1) placebo; 2) ezetimibe therapy; 3) a supplement of flaxseed oil (containing 1.0g ALA); or 4) ezetimibe and flaxseed oil supplementation.

RESULTS:

As expected, ingestion of flaxseed oil resulted in a significant increase in circulating ALA levels in patients who were not given ezetimibe. The presence of ezetimibe, however, attenuated circulating ALA levels even in the presence of flax oil supplementation. There were no significant differences amongst the groups in terms of circulating total cholesterol, LDL, HDL, or triglyceride levels.

CONCLUSION:

Our data demonstrates that ezetimibe therapy inhibits the absorption of omega-3 fatty acids. In view of the cardiovascular benefits that these patients may receive from high circulating levels of ALA, this represents a potentially serious negative drug-diet interaction.

Footnotes

This research was generously supported by the CIHR

Exp Clin Cardiol. 2011 Oct;16(Suppl A):4A.

13 FAT/CD36 and Fatty Acid Transport are Key Components of Muscle and Cardiac Energy Provision and Insulin Resistance

Arend Bonen ¹

Abstract

Fatty acids are important substrates involved in a myriad of metabolic and molecular functions. In skeletal muscle, fatty acids are key energy substrates that are readily oxidized or esterified into triacylglycerols, particularly when this tissue is exposed to metabolic stimuli. For many years it was thought that fatty acids entry into muscle and heart occurred via simple diffusion across the plasma membrane. Over the past decade this view has been altered substantially, as it is now accepted that fatty acid uptake by muscle occurs via a highly regulatable, protein-mediated mechanism involving the fatty acid transporter FAT/CD36. Recent work has shown that this protein is largely responsible for i) the acute and chronic regulation of fatty acid transport into muscle, ii) muscle fuel selection and iii) the classic adaptive responses to exercise training. In addition, FAT/CD36 is also a key component involved in the development of skeletal muscle and cardiac insulin resistance.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):4A.

14 Postconditioning Induced by BK_Ca Channel Opening in Isolated Ventricular Myocytes is Mediated by Reactive Oxygen Species

GH Borchert ¹, F Kolar ¹

Abstract

The aim was to find out whether activation of large conductance Ca²⁺-activated K⁺ channels (BK_Ca) can induce postconditioning-like effect and whether its protective mechanism involves signalling via reactive oxygen species (ROS). In order to target mitochondrial BK_Ca channels, we used isolated ventricular myocytes which do not contain functional BK_Ca channels in the sarcolemma. Cells were isolated from the left ventricles of adult male Wistar rats and subjected to a simulated ”ischemia/reperfusion” (25-min metabolic inhibition with NaCN and 2-deoxyglucose followed by 30-min reenergization). NS11021 (0.1 μM), a novel BK_Ca channel opener (1), or hydrogen peroxide (2 μM) added at reenergization increased cell survival (the number of rod-shaped cells) by about 30% and markedly reduced the release of lactate dehydrogenase (LDH). These cytoprotective effects of NS11021 were completely abolished by paxilline, a BK_Ca blocker, or tempol, a superoxide dismutase mimetic, but not by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K). NS11021 slightly but significantly increased fluorescence in DCF-DA-loaded myocytes indicating an increased ROS formation. The NS11021-induced ROS signal was abolished by paxilline or tempol. NS13558, an inactive analogue of NS11021, affected neither cell survival/LDH release nor DCF-DA fluorescence. These results suggest that pharmacological activation of mitochondrial BK_Ca channels effectively protects isolated cardiomyocytes against simulated ”reperfusion” injury and the mechanism of this form of postconditioning requires ROS signalling but not the activation of PI3-K/Akt pathway.

Reference

1.Nardi A, Olesen SP. Curr Med Chem. 2008;15:1126–46. doi: 10.2174/092986708784221412. [DOI] [PubMed] [Google Scholar]

Exp Clin Cardiol. 2011 Oct;16(Suppl A):4A.

15 Role of WNT/β-Catenin Signaling in Rejuvenating Myogenic Differentiation of Aged Mesenchymal Stem Cells from Cardiac Patients

KR Brunt ¹, Y Zhang ¹, A Mihic ¹, SH Li ¹, S Basmaji ¹, W Zhang ¹, M Li ¹, K Tsang ¹, TM Yau ¹, RK Li ¹

Abstract

INTRODUCTION:

Autologous stem cell therapy has not been as effective as forecast in preclinical studies. Patient age could be an important contributing factor to stem cell function. Our goal was to uncover age dependent mechanisms of stem cell function.

METHODS & RESULTS:

We counted bone marrow mesenchymal stem cells (MSC) from different patients to determine their growth kinetics and established an age dependent decline in proliferation. Myogenic differentiation following high-density culture of MSC and azacytidine treatment was also impaired relative to patient age. To understand why MSC have an altered state, we performed real-time PCR array analysis and determined that alterations in the basal WNT/β-catenin signaling network in MSC was highly correlated with patient age. Transcript levels of CTNNB1, LEF1, FZD8, WNT3A, and SFRP4 were negatively correlated with age, whereas FOSL1, LRP6, and FZD6 were positively correlated with age. At the protein level, translocation of β-catenin to the nucleus was impaired in older patients. However, MSC from aged patients that were repeatedly treated with lithium chloride – to maximize nuclear translocation of β-catenin during myogenic induction – recovered the capacity for myogenic differentiation; but failed to do so with knockdown of β-catenin using siRNA, thus establishing a central β-catenin role in age dependent decline of MSC function in cardiovascular patients.

CONCLUSIONS:

These findings show that the WNT signaling network is altered in human MSC resulting in reduced nuclear β-catenin bioavailability in aged patients. However, this is conditionally recoverable, providing a logical target for improving the function of bone marrow stem cells and their clinical utility in aged patients.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):4A.

16 Cystathionine Gamma-Lyase Deficient Smooth Muscle Cells Exhibit Redox Imbalance and Apoptosis Under Hypoxic Stress Conditions

Sean Bryan ¹, Guangdong Yang ¹, Rui Wang ¹, Neelam Khaper ¹

Abstract

Hydrogen sulfide (H₂S) has recently emerged as a novel and important gasotransmitter in the cardiovascular system, where it is generated mainly by cystathionine gamma-lyase (CSE). Abnormal metabolism and functions of the CSE/H₂S pathway have been linked to various cardiovascular diseases including atherosclerosis and hypertension. An important role for H₂S in regulating the balance between cellular growth and death has been demonstrated in vascular smooth muscle cells (SMCs). H₂S is increasingly recognized as a critical regulator of vascular integrity, but its role in SMCs during hypoxia has not been explored in a model of CSE-deficiency. In the present study, we analyzed cell viability, apoptosis, redox status, and mitochondrial activity in hypoxia-exposed (12h at 1%O₂) SMCs derived from mesenteric artery of CSE knockout (CSE-KO) mice and compared it to those from wild-type (CSE-WT) mice. CSE-KO cells exhibited redox imbalance and aberrant mitochondrial activity versus CSE-WT cells, indicating an essential regulatory role for endogenous CSE/H₂S pathway on SMC function. CSE-KO cells were also more susceptible to hypoxia-induced cell death, indicating a critical contribution of endogenous CSE/H₂S pathway to the hypoxia stress response. These findings support the concept that H₂S is a crucial regulator of vascular homeostasis, and that the CSE/H₂S pathway is essential for SMC survival under hypoxic conditions.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):4A–5A.

17 Incidence of Device Upgrades to Cardiac Resynchronization Therapy in a Heart Failure Population

Anjala Chelvanathan ¹, Mehrdad Golian ¹, Shelley Zieroth ¹, Colette Seifer ¹

Abstract

INTRODUCTION:

The use of implantable cardiac defibrillators (ICDs) has be shown to improve survival among patients who have New York Heart Association (NYHA) class II to IV heart failure with left ventricular systolic dysfunction (ejection fraction [EF] ≤35%) and has become the standard of care. Over the last decade, cardiac resynchronization therapy (CRT) has emerged as an important treatment modality in patients with heart failure. Upgrading from pacemakers (PPMs) or implantable cardiac defibrillators to cardiac resynchronization devices (CRTs) is an increasing occurrence. Our objectives were to determine the incidence of upgrades and to identify the characteristics of this patient population.

METHODS:

We identified the number of patients upgraded from pacemakers or ICDs to CRT at our institution over a 3 year period from January 2008 to December 2010.

RESULTS:

There were 125 CRT devices implanted during that period, 31 of which were upgrades. The median duration between original device implant and upgrade was 40 months (range 1 – 206). Eighty-four percent of patients were male. Mean age was 61 ± 13 years. Sixteen of 31 were upgrades from ICDs and 71% of the ICDs were implanted for primary prevention. Ninety percent were NYHA functional class III or IV and 52% had ischemic cardiomyopathy. Twenty-nine of 31 patients had a left ventricular ejection fraction less than 30%. Mean QRS duration was 166 ± 31 ms. Twenty-five leads were inserted via the coronary sinus.

CONCLUSION:

Upgrading cardiac rhythm devices is feasible and a common occurrence accounting for 25% of CRT insertions over a 3 year period.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):5A.

18 Clinical Predictors of Upgrade from ICD to CRT-D in Heart Failure Population

Anjala Chelvanathan ¹, Mehrdad Golian ¹, Shelley Zieroth ¹, James Tam ¹, Colette Seifer ¹

Abstract

BACKGROUND:

Implantable cardiac defibrillators (ICD’s), with or without cardiac resynchronization therapy (CRT), improves survival in the heart failure population. Patients who have received ICD’s, may subsequently meet criteria suggesting benefit from upgrading to a CRT device. Upgrading can be technically difficult; have associated morbidity and cost implications. The purpose of this study was to determine if there are any baseline clinical variables in patients who have ICD’s, which may predict subsequent upgrading to CRT.

METHODS:

We identified patients at a single centre, tertiary facility who were upgraded from ICD therapy to CRT between January 2008 and December 2010. We then compared the clinical characteristic of this group (CRT-upgrade group) to patients who received ICD’s (ICD group) during the same time period.

RESULTS:

Sixteen patients received upgrades from ICD to CRT. The mean age was 55.7 ± SD 8.9 years; 93.7% male. Two hundred and seventy four patients received an ICD during the same period, mean age 60.4 ± 13.3 years; 79.3% male.

CONCLUSION:

QRS width and left ventricular systolic dysfunction, may predict which ICD eligible patients may subsequently meet criteria for CRT.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):5A.

19 c-Ski Acts via Zeb2 to Increase Meox2 Expression and Modulate Cardiac Myofibroblast Phenotype

Ryan H Cunnington ¹, Josette M Douville ², Krista L Bathe ¹, Sunil G Rattan ¹, Jeffrey Wigle ², Ian MC Dixon ¹

Abstract

TGF-β₁/R-Smad signaling is commonly associated with activation of normally quiescent cardiac fibroblasts to secretory and contractile myofibroblasts. Cardiac myofibroblasts are responsible for progressive cardiac fibrosis in various aetiologies of heart failure. We have previously shown that c-Ski (Ski) treatment of primary cardiac myofibroblasts is associated with the diminution of the expression of myofibroblast phenotype markers as well as decreased type I collagen synthesis and contraction of these cells. Ski is a known repressor of TGF-β₁/R-Smad signaling which may act by blocking actions of R-Smads at the cellular nucleus and at the level of the TβR1 receptor. Zeb2 is a novel Smad interacting protein and transcription factor that is highly expressed in cardiac myofibroblasts. Recently we have shown that cardiac fibroblast to myofibroblast phenoconversion is associated with reduced Meox1 and Meox2 (a homeobox protein) mRNA expression in data gathered from P0, P1 and P2 cells. Further, Ad-Ski infection of P1 cells was associated with a rescue of Meox2 (but not Meox1) mRNA expression, and that Meox2 infection itself causes a decrease in myofibroblast marker expression and contractility. Nonetheless the mechanism of how Ski might influence Meox2 is unknown. We now show that Ski causes a dramatic reduction of Zeb2 protein expression and conclude that the mechanism of action of Ski in reducing the myofibroblast phenotype is due to the robust suppression of Zeb2 expression, followed by the derepression of Meox2 gene in these cells.

Footnotes

Supported by operating funds from the CIHR and HSFM

Exp Clin Cardiol. 2011 Oct;16(Suppl A):5A.

20 Sex as a Determinant of Cardiac Dysfunction: Potential Role of AKAP121

MP Czubryt ¹, A Cheung ¹

Abstract

The presence, prevalence and incidence of cardiovascular disease (CVD) is dependent on sex. Women are typically protected from CVD relative to men, an effect largely ascribed to estrogen. However, this generalized picture belies hidden complexity, since women have clearly worse outcomes and higher incidence with respect to certain CVDs such as diastolic failure. Furthermore, sex differences remain even after estrogen has been accounted for. There may thus be fundamental differences in the nature and function of cardiac tissue between men and women, including differences in how individual cells function in health and disease. In particular, the development and progression of cardiac hypertrophy may be quite different between the sexes. Our data indicates that physiologic cardiac hypertrophy induced by voluntary wheel running in mice occurs earlier in females than males. Conversely, pathologic hypertrophy due to pressure overload occurs more rapidly in males than females. The mechanisms underpinning these differences remain elusive, but our recent data suggests that the A-kinase anchor protein AKAP121 may play a role. Knock-down of AKAP121 resulted in spontaneous hypertrophy of isolated cardiomyocytes, while over-expression was protective. This effect was due to activation of the calcineurin-NFAT pathologic hypertrophic signaling pathway following knockdown. Interestingly, immunoprecipitated calcineurin consistently bound a ∼50 kDa protein that immunoreacted with anti-AKAP121 antibodies. We have observed that a similar ∼50 kDa band is expressed at much higher levels in cardiac tissue from females compared to males. Our results suggest that an AKAP121 derivative may bind to and repress calcineurin preferentially in females, which may contribute to the observed sex differences in development of hypertrophy.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):5A.

21 HMG-CoA Reductase Inhibitor Ameliorates Ischemia/Reperfusion Injury in Hypercholesterolemic Rabbits

Verónica D’Annunzio ¹, Martín Donato ¹, Bruno Buchholz ¹, Ana Quiroga ¹, Ricardo J Gelpi ¹

Abstract

We have previously shown that rosuvastatin reduced infarct size in the normo and hypercholesteremic rabbit’s heart, but we did not assess the ventricular function in a detailed form. Goal: to determine whether rosuvastatin administered during reperfusion improves postischemic ventricular dysfunction in hypercholesterolemic rabbit hearts. In group 1, isolated hearts were subjected to 30 min of ischemia and 30 min of reperfusion. In group 2 we added rosuvastatin after 30 min of ischemia and from the beginning of reperfusion. We performed additional experiments to evaluate ischemic vasodilatation response using adenosine during reperfusion (group 3), and rosuvastatin + adenosine (group 4).

LV developed pressure was lower (26.7±3.9 mmHg at 30 min R) compared with the preischemic values. Rosuvastatin attenuated postischemic systolic ventricular dysfunction, reaching 43.6±7.1 mmHg after 30 minutes of reperfusion (p<0.05 vs. control group). The LV end diastolic pressure (myocardial stiffness), increased during the reperfusion. However, this effect was abolished by rosuvastatin (p<0.05). +dP/dt_max and –dP/dt_max improved after rosuvastatin treatment. Ischemia induced coronary perfusion pressure (CPP) increase. Adenosine did not decrease CPP during reperfusion showing that vasodilatation is impaired after ischemia/reperfusion in hypercholesterolemic rabbit hearts, however, the administration of rosuvastatin with adenosine fully recovers coronary vasodilatation. Thus, rosuvastatin attenuated systolic and diastolic postischemic dysfunction after 30 min of global ischemia in hypercholesterolemic rabbit hearts improving myocardial perfusion.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):6A.

22 Resveratrol Modified Stem Cells in Cardiac Regeneration: Role of Micro RNA

Dipak K Das ¹, Narasimman Gurusamy ¹, Partha Mukhopadhyay ¹

Abstract

To study the efficiency of maintaining the reduced tissue environment via pretreatment with natural antioxidant resveratrol in stem cell therapy, we pre-treated male Sprague-Dawley rats with resveratrol (2.5 mg/kg/day gav-aged for 2 weeks). After occlusion of the left anterior descending coronary artery (LAD), adult cardiac stem cells stably expressing EGFP were injected into the border zone of the myocardium. One week after the LAD occlusion, the cardiac reduced environment was confirmed in resveratrol-treated rat hearts by the enhanced expression of nuclear factor-E2-related factor-2 (Nrf2) and redox effector factor-1 (Ref-1). In concert, cardiac functional parameters (left ventricular ejection fraction and fractional shortening) were significantly improved. The improvement of cardiac function was accompanied by the enhanced stem cell survival and proliferation as demonstrated by the expression of cell proliferation marker Ki67 and differentiation of stem cells towards the regeneration of the myocardium as demonstrated by the expression of EGFP 28 days after LAD occlusion in the resveratrol-treated hearts. Our results demonstrate that resveratrol maintained a reduced tissue environment by overexpressing Nrf2 and Ref-1 in rats resulting in an enhancement of the cardiac regeneration of the adult cardiac stem cells as demonstrated by increased cell survival and differentiation leading to cardiac function. The results further suggested a role of micro RNA in the resveratrol mediated stem cell survival.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):6A.

23 Pre-Clinical Strategies to Prevent Rejection of Allogeneic Mesenchymal Stem Cells by Injured Myocardium for Cardiac Regeneration

Sanjiv Dhingra ¹, Peng Li ¹, Xi-Ping Huang ¹, Jian Guo ¹, Jun Wu ¹, Faquan Zeng ¹, Pawan K Singal ¹, Richard D Weisel ¹, Ren-Ke Li ¹

Abstract

Mesenchymal stem cells (MSCs) are immuneprivileged, and clinical application of these cells for cardiac repair is under investigation. Recently we reported that differentiation of transplanted allogeneic MSCs in infarcted heart leads to loss of immuneprivilege characteristics and rejection of these cells by the host. Present study examines precise mechanisms of this immune switch in MSCs. The immuneprivilege in MSCs is maintained by immunosuppressive soluble factors. We treated rat MSCs with 5-azacytidine to induce myogenic differentiation and screened several soluble factors before and after differentiation. The prostaglandin E2 (PGE2) level decreased in differentiated MSCs that was associated with loss of immune-privilege as we observed increased cytotoxicity in MSCs caused by allogeneic T cells in the co-culture. Un-differentiated not the differentiated MSCs decreased the rate of T cell proliferation, changed the polarity of CD4+ T cells towards T regulatory cells. PGE2 treatment in differentiated cells prevented T cell mediated cytotoxicity, decreased the T cell proliferation. In the in vivo experiments, we maintained PGE2 levels by using biodegradable hydrogel. After 5 weeks of transplantation, allogeneic cells were rejected by the myocardium in the control group. In the PGE2 treated group 35% cells survived, and we observed a significant improvement in heart function. Thus differentiation of transplanted MSCs in myocardium leads to a decrease in PGE2 levels and loss of immuneprivilege. Maintaining the optimum level of PGE2 in MSCs preserves the immuneprivilege environment and ability of these cells for cardiac regeneration.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):6A.

24 NF-kB Activation Down Stream of mTOR Signaling Pathway is Suppressed in Hypoxia Induced Cardiac Cell Death

R Dhingra ¹, JW Gordon ¹, H Gang ¹, LA Kirshenbaum ^1,²

Abstract

The mammalian target of rapamycin (mTOR) is a highly conserved serine threonine kinase centrally involved in vital processes including growth, proliferation, and gene transcription. However, mTOR’s role in regulating cell survival under normal or disease conditions has not been determined. We have previously demonstrated that NF-kB activation was necessary and sufficient for basal cell survival and suppressing mitochondrial perturbations during hypoxic injury. Herein, we provide new compelling evidence that activation of IKKB-NF-kB signaling pathway down-stream of mTOR suppresses mitochondrial perturbations and cell death during metabolic stress imposed by hypoxia. Constitutive activation of mTOR in tuber sclerosis complex (TSC) 2−/− cells displayed marked increased mTOR targets p-P70S6, p-4EBP-1 and S6 protein. A significant increase in nuclear NF-kB activity and NF-kB gene transcription was also observed in TSC 2−/− cells. Interestingly, in in vitro models of hypoxia, we observed a marked reduction in mTOR and NF-kB activity. In addition, basal Bnip3 gene transcription and cell death were significantly increased in cells exposed to hypoxia and defective for mTOR activity. IKKB-mediated activation of NF-kB restored mTOR activity and suppressed cell death induced by Bnip3 in hypoxic cells. To our knowledge, our data provided the first direct evidence that operationally links mTOR and cell survival via IKKB-NF-kB mediated repression of Bnip3.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):6A.

25 Engineered Tissues for Heart Mending

Paolo Di Nardo ^1,²

Abstract

The poor knowledge of the progenitor cell biology and several technical drawbacks have determined low viability of the transplanted cells (<1% of the cells are detectable in the target area after 1 week) and insufficient mechanical support to the heart function, hampering the efficacy of cell therapies applied to the injured myocardium. Moreover, the strategy to administer stem cells by injection has demonstrated to be inadequate to allow a sufficient number of cells that could integrate in the myocardial architecture. Recent evidences have suggested that adult progenitor cells can be used to fabricate ex vivo engineered cardiac tissue to be implanted into the injured myocardium. Engineered tissues can be fabricated in the presence or in the absence of biocompatible polymeric scaffolds. Indeed, specifically manipulated biomaterials can be used to deliver signals sensed as “biologically relevant” by cells without embedding growth factors into the inert material, but this technology requires further efforts and investments to be clinically exploited. Conversely, it is possible to fabricate scaffoldless sheets made of human progenitor cells, possibly isolated from the heart of the same patient candidate to receive the cell treatment. The results so far obtained demonstrated that, when leant on the heart surface, the progenitor cells embedded into the scaffoldless sheets easily migrated into the myocardium differentiating in cardiomyocytes and integrating in the tissue architecture, as demonstrated by the proper connections established between the graft and host cells. Further exploitation of this technology can allow to fabricate safe and cost-effective human myocardium bio-substitutes to be used in the clinical setting.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):6A–7A.

26 Changes in Athlete’s Antioxidant State Following Sport Specific and Sport Nonspecific Bout of Exercise

Dusica Djordjevic ¹, Dragan M Djuric ², Dejan Cubrilo ¹, Vladimir Zivkovic ¹, Milena Vuletic ¹, Nevena Barudzic ¹, Dusko Kornjaca ¹, Vladimir LJ Jakovljevic ¹

Abstract

It is thought that the extent of redox homeostasis disturbance induced by an acute bout of exercise depends on many factors, inter alia, exercise mode, intensity and duration. The aim of our study was to compare the effects of a sport nonspecific exercise bout with sport specific bout of exercise on antioxidant state of athletes. 58 handball players were subjected to a maximal progressive exercise test on the bicycle ergometer and to a specific handball training. Intensity of exercise was evaluated by Polar Team² System for heart rate monitoring. Subjects were taken a blood sample immediately before and after exercise bouts. Blood analysis included measurement of superoxide dismutase activity, catalase activity and glutathione levels. Laboratory exercise test that lasted 10.57±0.24 minutes and in which levels of athlete’s heart rate corresponded to a submaximal and maximal zone of intensity for 5.58±1.27 minutes induced significant decrease of catalase activity (from 4.8±3.2 to 3.2±3.3 U/g of haemoglobin x 10³), while one and a half hour handball training during which players spent 44.71±10.52 minutes in submaximal and maximal zone of intensity induced significant decrease of superoxide dismutase activity (from 3441.7±3106.7 to 2442.9±2927.6 U/g of haemoglobin x 10³) and glutathione levels (from 47165.7±12123.7 to 36642.6±999.9 nmol/ml of red blood cells). Both exercise sessions induced depletion of antioxidants, which suggests that athletes experienced exercise-induced oxidative stress regardless of mode, intensity and duration of exercise.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):7A.

27 The Effects of Vibroacoustic Induced Microvibrations on Cardiovascular System in Rats

D Djurić ¹, V Jakovljević ¹, DJ Jovović ¹

Abstract

Vibroacoustic devices that transmit sound as vibration to the body have developed over the last 30 years; vibroacoustic therapy is used in clinical treatment (sedative music and pulsed, sinusoidal low frequency tones 20Hz–70Hz, played through a bed or chair). There has been a substantial amount of research investigating vibratory sensitivity in subjects including infrasound, ultrasound, noise and music. Rohracher (1946) has claimed firstly that the entire surface of the human and homothermic animal body exhibits continuous microvibrations with frequency 7–13 Hz due to muscular and cardiac activity (which should be changed during relaxation, exercise, disease or microgravity). In this research we have used vibroacoustic acute induced microvibrations (up to 60 min, frequency range 30Hz–18000 kHz, amplitude range 2.8 μm – 12.3 μm) to evaluate their influence on cardiovascular parameters in rats, and following stimulation with different applied level of vibroacoustic induced microvibrations (device Vitafon, St. Petersburg, Russian Federation). Certain ECG characteristics (frequency, rhythm, R wave amplitude), parameters of contractility in isolated non-paced heart, histological examination of cardiac tissue, haemodynamic characteristics (arterial blood pressure, cardiac output, carotid arterial flow, carotid vascular resistance), and oxidative stress markers in blood have been evaluated in rats. The obtained results clearly show beneficial effects of applied low-frequency vibroacoustic acute induced microvibrations on rat cardiovascular system (increased R wave, decreased arterial blood pressure, decreased carotid vascular resistance). Further investigations will be performed to make clear physiological mechanisms of such obtained results.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):7A.

28 MEOX Transcription Factors Activate p21 and p16 Expression and Induce Endothelial Cell Senescence

JM Douville ¹, D Cheung ¹, JT Wigle ¹

Abstract

Human atherosclerotic tissue contains a high proportion of senescent cells. MEOX1 and MEOX2 are homeodomain transcription factors that are expressed in the cells of the vasculature. Meox1/2 knockout mice studies have shown that these proteins have partially redundant functions during development, suggesting that they regulate transcription ofcommon genes. There are only two known MEOX2 target genes – p21 and p16 – both of which block cell cycle progression and are involved in the establishment of cellular senescence.

The objectives of our study were to determine whether MEOX1 is able to activate MEOX2 target genes; elucidate the mechanism of MEOX2 activation of p21 and p16 and identify novel target genes of the MEOX proteins in endothelial cells (ECs).

MEOX1/2 activated p21 and p16 gene transcription in ECs, however, MEOX1 was a much stronger activator of p16 expression. In addition, a DNA binding deficient version of MEOX2 was able to activate p21, but not p16, gene transcription. Furthermore, MEOX protein expression in ECs decreased cellular proliferation and increased senescence. To identify the site required for MEOX activation of the p16 promoter, we made DNA probes containing the homeodomain binding sites. MEOX1/2 bound a probe containing the proximal binding site. Microarray analysis revealed that MEOX1/2 decrease in the expression of genes involved in pseudouridination, blood coagulation and cell cycle regulation.

Taken together, we conclude that MEOX1 and MEOX2 bind to a key homeodomain binding site within the p16 promoter, while the regulation of the p21 gene is independent of DNA binding. Furthermore, both MEOX proteins can induce cellular senescence in ECs.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):7A.

29 Fish Oil Mediated Cardiovascular Complications in ApoE^KO Mice

Xiaozhou Du ¹, Sheena Bohonis ¹, Matthew Zeglinski ¹, Yan Le ², Zongjun Zhao ², Hope Anderson ², Roman Nepomuceno ¹, Erin Karlstedt ¹, Khuong Le ³, Mohammed Moghadasian ³, Davinder S Jassal ¹

Abstract

INTRODUCTION:

Atherosclerosis is the principle cause of cardiovascular disease worldwide. Due to their spontaneous development of atherosclerosis, apolipoprotein E knockout mice (ApoE^KO) are one of the best studied animal models of atherosclerosis. Although previous studies have evaluated the effects of fish oil in cardiovascular disease, little is known about its cardiac effects in the setting of ApoE deficiency.

METHODOLOGY:

A total of 20 six-week-old male ApoE^KO mice were randomized into safflower oil and fish oil treatment groups (n=10 per group). All animals were maintained on a high fat diet reconstituted with treatment oils (20% wt/wt) for 16 weeks. Serum lipid levels and blood pressure were serially measured. In vivo cardiac function was assessed weekly using murine echocardiography. Ventricular brain natriuretic peptide (BNP) expression was assessed at the end of the experiment as a biomarker of cardiac remodeling.

RESULTS:

Compared to the control group, plasma triglycerides were significantly increased in the fish oil treated group at week 8, while plasma total cholesterol decreased throughout the experiment. Fish oil treated mice also showed increase in systolic, diastolic and mean arterial blood pressure over the treatment course. The hemodynamic changes were accompanied by left ventricular hypertrophy (LVH), as observed on echocardiography. The development of LVH is supported by the increased myocardial BNP expression in fish oil treated mice.

CONCLUSION:

Fish oil supplemented high fat diet causes adverse cardiovascular effects in ApoE deficient mice.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):7A–8A.

30 Involvement of AMPK Signaling in the Regulation of Myocardial Calcium

Todd A Duhamel ¹, Marc P Morissette ¹, Riley A Epp ¹, Shanel E Susser ¹, Yan-Jun Xu ¹

Abstract

The 5′adenosine-mono-phosphate-activated protein kinase (AMPK) is a key protein involved in the regulation of energy metabolism. Intracellular calcium has a direct role in regulating cellular energy homeostasis. Therefore, we tested the hypothesis that AMPK regulates SERCA2a-dependent calcium transport mechanisms. Intracellular calcium levels were measured using fura2 under several conditions. Notably, the AMPK activator AICAR did not alter basal intracellular calcium levels, but did increase the thapsigargin-induced intracellular calcium changes (AICAR,196 ± 11 nM versus Control, 157 ± 8 nM) in H9C2 cells. An opposite effect was observed when myocytes were pre-treated with the AMPK inhibitor Compound C. In a different experiment, we determined that SERCA2a function is impaired in cardiac tissue samples isolated from AMPKalpha2 kinase-dead transgenic mice, which is a genetic manipulation that reduces AMPK activity by ∼70%, as compared to wild-type mice. Given that AMPK signaling is impaired in diabetic tissue and can be normalized by exercise training, we conducted a final experiment to demonstrate that exercise training enhances SERCA2a protein content and activity in a dose-dependent manner in the diabetic heart. Based on these data, we suggest that AMPK signaling has an important role for regulating myocardial calcium cycling.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):8A.

31 Impact of Age on Circulating Levels of Mammalian Enterolignans Following Flaxseed Consumption in Healthy Adults

AL Edel ¹, E Dibrov ¹, M Aliani ², GN Pierce ¹

Abstract

Lignans are polyphenolic compounds found in plants that are gaining significant interest due to their antioxidative and cardioprotective properties. Flaxseed is one of the richest sources of the lignan secoisolariciresinol diglucoside (SDG). SDG is converted by microflora in the gut to secoisolariciresinol and then further metabolized into the mammalian enterolignans, enterodiol (END) and/or enterolactone (ENL). It was hypothesized that age may influence the capacity of the gut to process SDG. Healthy adults were grouped into either an 18–29 or 45–69 year old group (n=10/group). Subjects were asked to consume one muffin containing 30g of ground flax-seed (0.3g of SDG) daily for one month. Enterolignans were isolated from plasma at baseline and one month using liquid-liquid extraction and analyzed by gas chromatography-mass spectroscopy. Baseline plasma samples contained either trace or undetectable amounts of enterolignans, however, following flaxseed consumption, almost all subjects reflected an increase. This increase was significant for the 45–69 year old group. It is well established that advancing age is associated with greater incidences of cardiovascular disease, which in addition to the aging process is also linked with oxidative damage. If SDG metabolites are more readily bioavailable in older individuals, the antioxidative capabilities of END and ENL will benefit this population greatly. Therefore, daily flaxseed consumption may be an important therapeutic pathway to address this problem.

Footnotes

Supported by CIHR, Flax2015, MMSF and ARDI

Exp Clin Cardiol. 2011 Oct;16(Suppl A):8A.

32 Cardiac Interstitial Fibrosis – Role of Immunoinflammatory Dysregulation

ML Entman ¹

Abstract

Our laboratory has demonstrated that interstitial fibrosis in the mouse heart is dependent upon the development of a class of fibroblasts arising directly from monocyte precursors. These CD45+ fibroblasts are dependent upon elevation of MCP-1, a classic chemokine for mononuclear cells. Deletion of MCP-1 inhibits interstitial fibrosis arising from intermittent ischemia or angiotensin II treatment. In the latter, hypertrophy is unaffected by MCP-1 deletion while fibrosis is virtually eliminated. The mechanism of fibrosis dictating this pathophysiologic observation relates to the MCP-1-dependent uptake of natural killer T lymphocytes and monocytes, which undergo Th1Th2 and M1M2 transition respectively. Transition to Th2 and M2 phenotypes is a classic defense mechanism against proinflammatory signaling which, when occurring on a chronic basis, promotes transition of M2 macrophages into myeloid-dependent (CD45⁺) fibroblasts. This pathophysiologic mechanism is also pertinent to cardiac interstitial fibrosis arising in the aging animal and after transaortic constriction. In contrast, fibroblasts arising by this mechanism are not critical to myocardial scar formation. Thus, the mechanisms of these cellular events and the factors which control them are potential targets for pharmacologic intervention without alteration of cardiac repair.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):8A.

33 Time Profile of the Expression of Redox-Sensitive Proteins Involved in Cardiac Remodeling Post-Miocardial Infarction

RO Fernandes ¹, PC Schenkel ^1,², AMV Tavares ¹, GP Diniz ³, MFM Ribeiro ¹, ASR Araujo ¹, ML Barreto-Chaves ³, A Belló-Klein ¹

Abstract

AIM:

To investigate the profile of renin angiotensin system and redox status, as well as its associations with some redox-sensitive prosurvival and proapoptotic proteins involved in the transition from myocardial infarction (MI) to heart failure in rats.

METHODS AND RESULTS:

Male Wistar rats were divided into 6 groups: 3 Sham-operated and 3 MI 2 days, 7 days and 28 days post surgical procedure. Cardiac function was analyzed by echocardiography, H₂O₂ concentration was measured spectrophotometrically, and thioredoxin-1, angiotensin II, AT1 and AT2 receptors, p-JNK/JNK, p-ERK/ERK, p-Akt/Akt, p-mTOR/mTOR and p-GSK3β/GSK3β myocardial immunocontent was evaluated by Western blot. Our results show a favorable balance for the Trx-1 over the concentration of H₂O₂ in the 2d-MI group. In this early period, redox-sensitive proteins involved in cell death were less active. In 7 days post-MI, there was a reversion of this balance, since H₂O₂ concentration increased, possibly through a higher RAS activation, surpassing Trx-1 counter-regulatory capacity. We also found, in this scenario, a prominent activation of JNK and GSK3β associated with the worsening of ventricular function. A similar scenario was observed in 28 days post-MI animals, except for a significant drop in Trx-1 concentration.

CONCLUSION:

Our findings present insight on the mechanisms involved in the endogenous adaptation to the ischemic injury, which can be useful for the design of therapeutical strategies targeting oxidative stress post-MI.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):8A.

34 Calcium-Activated Chloride Channels and TMEM16A – A New Therapeutic Target in Pulmonary Arterial Hypertension?

Abigail S Forrest ¹, Marissa Huebner ¹, Talia Joyce ¹, Ramon J Ayon ¹, Michael Wiwchar ¹, Cynthia Yenter ¹, Maria Valencik ², Linda Ye ¹, Cherie Singer ¹, Dayue Duan ¹, Iain Greenwood ³, Normand Leblanc ¹

Abstract

Pulmonary arterial hypertension (PAH) is a rare human disease with poor prognosis for survival and a high mortality rate. PAH is defined by a pulmonary artery pressure (PAP) exceeding 25 mm Hg at rest, and 30 mm Hg during physical activity. The cellular and molecular mechanisms triggering this disease are largely unknown. Ca²⁺-activated Cl^– channels (Cl_Ca) are excitatory anion channels activated by a rise in intracellular Ca²⁺ that could play a role in the enhanced vasoconstriction and remodeling of the pulmonary arterial (PA) vasculature in PAH. The purpose of the present study was to determine if Ca²⁺-activated Cl^– currents (I_Cl(Ca)) and the newly discovered gene postulated to encode for these channels, TMEM16A, are altered in PA smooth muscle cells (PASMCs) from monocrotaline (MCT)-treated rats. Rats were either injected IP with saline or a single dose of MCT (50 mg/kg) to induce PAH, and the animals sacrificed after 3 weeks. Consistent with PAH, rats treated with MCT displayed an increase in right ventricular weight with no change in left ventricular and septum weights. Patch clamp experiments in PASMCs from proximal conduit and small intralobar PA revealed that time- and voltage-dependent I_Cl(Ca) elicited by 500 nM internal free Ca²⁺ were more than ∼ 2-fold larger in the MCT vs. saline groups. Such findings were correlated with increased expression of TMEM16A mRNA transcript and protein levels by qRT-PCR and Western blot in the MCT vs. saline groups. In conclusion, the Cl_Ca/TMEM16A axis may represent a novel therapeutic target in the treatment of PAH.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):9A.

35 Age Difference Explains Sex Differences in Cardiac Treatment Rates after AMI

RR Fransoo ¹, PJ Martens ¹, HJ Prior ¹, E Burland ¹, D Chateau ¹, A Katz ¹

Abstract

Many investigators have reported higher rates of cardiac procedures for males than females after acute myocardial infarction (AMI), suggesting that men are treated more aggressively than women. However, others have reported no significant differences after controlling for age, resulting in uncertainty about the existence of a true gender bias in cardiac care. All studies show a higher rate of AMIs among males, and an older age profile among females.

In this study, a population-based cohort approach was used to calculate age-specific procedure rates by sex from comprehensive administrative data. Chi-square tests and generalized linear modeling were used to assess gender differences and interactions. For all four procedures studied (catheterization, angioplasty, stent insertion and bypass surgery), rates were significantly higher for males than females (p<0.01). However, age-specific rates revealed no significant differences by sex and a sharp decrease in intervention rates with age for both males and females. Generalized linear modeling confirmed that patient age was a significant predictor of intervention rates, whereas sex was not.

The significant sex difference in overall rates was completely confounded by the older age profile of female AMI patients compared to their male counterparts. In other words, it’s not that males get treated more aggressively than females, but rather that younger AMI patients are treated more aggressively older ones, and female AMI patients are older than males.

Preliminary results of ongoing research suggest that males are more likely than females to die of AMI without being hospitalized (ie sudden cardiac death).

Exp Clin Cardiol. 2011 Oct;16(Suppl A):9A.

36 Primary Human Mesenchymal Stem Cell Biology In Vitro

Darren H Freed ¹

Abstract

The ability of the heart to regenerate is limited. Cell transplantation in an effort to repair the wounded hearts of patients with ischemic heart disease has been undertaken with variable success. Various studies have shown an increase in heart function with mesenchymal stem cell (MSC) transplantation, but limited differentiation of transplanted cells to mature, functional cardiomyocytes. One explanation for this observation is that MSC spontaneously differentiate in culture, which limits their capacity for further differentiation in vivo. Furthermore, MSC viability may be impaired by medications that are commonly given to patients with ischemic heart disease. We examined MSC in vitro and compared their phenotypic and physiological characteristics to cultured cardiac fibroblasts. The specific role of non-muscle myosin-II (NMMII) was assessed, as was the response to HMG-CoA reductase inhbitors (“statins”).

In cell culture, primary human bone marrow cells increasingly expressed the myofibroblastic markers alpha smooth muscle actin (α-SMA), the embryonic isoform of smooth muscle myosin (SMemb), procollagen 1A2 and vimentin over serial passage. Collagen gel contraction assays revealed that MSCs contracted to a similar degree as cardiac myofibroblasts. MSC treated with TGFβ, showed increased contractile function compared to non-treated cells. Contraction of collagen gels could be inhibited with NMMII inhibitor, blebbistatin. HMG-CoA reductase inhibition caused a dose and time-dependent decrease in cell viability, an effect which could be reversed with mevalonate or adenoviral overexpression of IKKbeta.

Human MSC transform to myofibroblasts during cell expansion in vitro. These cells demonstrate similar contractile ability to cardiac myofibroblasts. These data suggest that MSCs differentiate readily in vitro to a myofibroblast phenotype that is unlikely to support further differentiation to cardiomyocytes that will integrate into the host myocardium to improve systolic function. In addition, cholesterol lowering medications that are commonly given to patients with ischemic heart disease may affect the viability of MSC following transplantation. Additional studies are warranted to address this spontaneous differentiation and improve cardiomyocyte differentiation potential. Furthermore, the contribution of MSCs to cardiac fibrosis needs to be investigated.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):9A.

37 Science Through Heart Education

Pallab (Paul) Ganguly ¹

Abstract

As the breadth and complexity of research in cardiovascular sciences grows, so does the need for expanded, up-to-the-minute educational opportunities for cardiovascular scientists, physicians, nurses, and technologists. Although these health professionals have responded by developing a range of educational initiatives that meet those needs, there is a real lack of a concerted effort that addresses science through cardiovascular education.

The time has come when the general public and global medical communities are eager to know the benefit from a holistic approach of science that cuts across bench research. Thus, worldwide reputation for excellence in training basic and clinical scientists in advanced techniques and innovations needs new resources of cardiovascular education.

It is proposed that twenty first century cardiovascular science can be focussed in a more effective and comprehensive manner if health professionals can work together to achieve a common goal, the Heart of Education. Such an effort should be directed to: 1) developing a better curriculum at the medical school 2) producing more clinician scientists 3) useful communication and interaction within scientists, physicians, nurses and technologists 5) translational research 4) public awareness and 5) participating in governmental health policies.

The success to this endeavour lies in introducing a program of Cardiovascular Education in Institutes actively engaged in basic and clinical research.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):9A.

38 Autophagy Regulates Transfat-Mediated Apoptosis in Primary Cardiac Myofibroblasts

S Ghavami ¹, RH Cunnington ¹, J Davies ¹, SG Rattan ¹, K Bathe ¹, B Yeganeh ¹, R Arora ¹, MJ Los ¹, DH Freed ¹, AJ Halayko ¹, T Klonisch ¹, GN Pierce ¹, IMC Dixon ¹

Abstract

Vaccenic acid (VA) and elaidic acid (EA) are the predominant transmonoene in ruminant fats and vegetable oil. Here, we investigated the mechanism of cell death induced by VA and EA on primary rat ventricular myofiboroblasts (rVF). MTT assay demonstrated that both VA and EA (200 M, 0–72 hrs) induced cell death in rVF (P < 0.001). FACS assay confirmed that both VA and EA induce apoptosis in rVA. rVF cells treated with VA and EA exhibited elevated cleaved caspase-9 and, -3 without activation of caspase-8. Both VA and EA decreased Bcl2/Bax expression ratio, as well as Bax translocation to mitochondria with subsequent decrease of mitochondria membrane potential () in rVA. Bax and Bax/Bak knockdown in mouse embryonic fibroblasts (MEF) inhibited VA and EA-induced cell death. Transmission electron microscopy revealed VA and EA (200 μM in 72 hours) induced autophagosomes formation in rVF. Immunobloting also confirmed the induction of autophagy markers: LC3-β lipidation, increased Atg5–12 formation, and increased beclin1 in rVF treated with VA and EA. Finally, delition of ATG3 and ATG5 significantly inhibit VA and EA-induced apoptosis (P < 0.001). In conclusion, we have shown for the first time that TFA induces simultaneous apoptosis (intrinsic pathway) and autophagy in rVF. Furthermore, TFA-induced autophagy is required for this proapoptotic effect. Further studies to address the effect of TFA on the heart may reveal significant translational value for prevention of TFA-linked heart disease.

Footnotes

Supported by CIHR and HSF of Manitoba

Exp Clin Cardiol. 2011 Oct;16(Suppl A):10A.

39 Apoptosis, Autophagy, and ER-Stress are Involved in Mevalonate Cascade Inihibiton Induced Cell Death in Human Atrial Fibroblasts

S Ghavami ^1,², B Yeganeh ^1,², G Stelmack ¹, HH Kashani ^1,², D Schaafsma ^1,², P Sharma ^1,², RH Cunnington ³, SG Ratan ³, K Bathe ³, MJ Los ⁴, T Klonisch ⁵, IMC Dixon ³, DH Freed ³, AJ Halayko ^1,^2,⁶

Abstract

HMG-CoA reductase inhibitors (statins) are cholesterol-lowering drugs and may underlie their beneficial effects on myocardial remodeling. Here, we identify the mechanisms of statins induced cell death in primary human atrial fibroblast (hATF) cells. Mevalonate cascade inhibition simultaneously induces activation of intrinsic apoptosis pathway, autophagy and unfolded protein response (UPR) in these cells indicating cooperation of mitochondria, endoplasmic reticulum (ER) and lysosomes in simvastatin-induced cell death. Inhibition of ER-dependent caspases (caspase-4, and-12), and lysosomal cathepsins (cathepsin-B and -L) significantly decrease simvastatin-induced cell death in hATF cells. On the other hand, simvastatin treatment altered the total and mitochondrial fraction balance of pro-apoptotic (Bax, Bad, PUMA, NOXA, and BNIP3) and anti-apoptotic (Bcl2, Bcl-XL and Mcl-1) bcl2 family members. Chemical inhibition of autophagy augments simvastatin-induced caspase activation, UPR and cell death. Likewise, Inhibition of simvastatin-induced autophagy using mouse embryo fibroblast ATG5 KO cells, augmented pro-apoptotic proteins and triggered the UPR as compared to the correspondence scramble cells. The data generated in this study demonstrates that mevalonate cascade inhibition-induced cell death is a complex mechanism, tightly regulated by apoptosis, autophagy, and UPR in which autophagy plays crucial regulatory function in this process.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):10A.

40 Preconditioning and Myocardium Functional Recovery Protection by Pantoprazole and Omeprazole in the Ischemia-Reperfusion in the Isolated Heart of Rats

Otoni Moreira Gomes ¹

Abstract

Results are presented of the myocardium functional recovery protection by pantoprazole and omeprazole in isolated hearts of rats submitted to ischemia and reperfusion with and without ischemic preconditioning. This study was approved by the Ethics Committee for Animal Experimentation of our institutions and was conducted according to the guidelines for animal experimentation of the Brazilian College on Animal Experimentation (COBEA). We studied the isolated hearts of 48 Wistar rats with 280 g mean body weight. After analgesia induced by sulfuric ether P.A inhalation and anaesthesia by intraperitoneal injection of ketamine 10 mg and xilazine 2 mg, their hearts were removed and perfused with Krebs-Henseleit solution (95% of O₂ and 5% of CO₂, 37 °C, 110–120 mmHg perfusion pressure, 8 mmHg ventricular diastolic pressure) in the São Francisco de Assis Truth is Jesus Cardiovascular Foundation disposable Langendorff modified system. The isolated hearts of the control groups were submitted to 20 min ischemia and 30 min reperfusion or submitted to 5 min ischemia and 5 min reperfusion precondictioning before the 20 min ischemia induction. Treated hearts were submitted to intracoronary injection of pantoprazole 100 μg or omeprazole 200 μg immediately before the 20 min ischemia induction period. The following parameters were registered after the 15 min stabilization period (t₀), and after the reperfusion period (t₃₀): heart rate (HR), coronary flow (CoF), systolic pressure (SP), +dP/dt and −dP/dt. The statiscal analysis method of Kruskal-Wallis (p<0.05) was employed. It was concluded that pantoprazole and omeprazole administration before ischemia induction significantly protected the myocardium function recovery with same protective results (p<0.05) obtained with the ischemic preconditioning technique.

Additional studies may precisely determine the interaction of proton pump blockers with the phenomena of ischemic myocardial pre- and post-conditioning, with a potential favorable resolution for both clinical therapeutic and preventive use in coronary heart diseases as well as in the myocardial protection for cardiac surgeries. In this respect, the preconditioning by H+/K+ pump blockers preoperatively can optimize the cardiac protection, replacing intermittent occlusions of the coronary arteries or the aorta itself with a parietal harmful potential whose efficacy has already been refuted and differing from the conclusion of the present study, which establishes a significant protection of myocardial contractility.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):10A.

41 Alternatively Spliced Bnip3 Isoform Promotes Cell Survival by Recruiting Bcl-2 to the IP3-Receptor

J W Gordon ¹, Y Hai ¹, H Gang ¹, LA Kirshenbaum ^1,²

Abstract

Alternative splicing provides a versatile mechanism by which cells generate proteins with different or even antagonistic properties. We previously identified a novel splice variant of Bnip3 (Bnip3ΔEx3) generated in cardiac myocytes subjected to hypoxia. Like full-length Bnip3 (Bnip3FL), Bnip3ΔEx3 forms a complex with the antiapoptotic proteins Bcl-2 and FKBP38; whereas, functional analysis of a Bnip3 mutant that lacks a Bcl-2 interacting domain acts as a more potent inducer of programmed cell death, that can not be inhibited by Bnip3ΔEx3. In silico analysis of Bnip3ΔEx3 reveals an endoplasmic reticulum (ER) targeting domain within its C-terminus, that is not present in Bnip3FL. We validated that Bnip3ΔEx3 is indeed localized to the ER and demonstrated that Bnip3ΔEx3 mutants that lack this ER targeting domain fail to antagonize cell death induced by Bnip3FL or hypoxia. Furthermore, we demonstrate that Bnip3ΔEx3 physically interacts with the IP3 receptor and serves to recruit Bcl-2 to the ER; whereas, Bnip3ΔEx3 fails to protect against programmed cell death in the presence of a Bcl-2 mutant that lacks its IP3 receptor interacting domain. Given the otherwise lethal consequences of de-regulated Bnip3FL expression in post-mitotic cells, our findings reveal a novel intrinsic defense mechanism that opposes the lethal consequences mitochondrial-ER cross-talk associated with programmed cell death in ventricular myocytes.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):10A.

42 Cardioprotectant and Islet Neogenic Potential of Novel Phytochemicals – Therapy for Diabetes

Sarita Gupta ¹

Abstract

Diabetes is not only a complex disorder but also a metabolic syndrome, which affects multiple systems. Type I diabetes is due to islet destruction where as type II is because of insulin resistance. Presentation will deal with the work of our lab where we systematically evaluated one of the herb Enicostemma littorale Blume (EL) for its antidiabetic effect. Hyperglycemia, hyperlipidemia and oxidative stress are major factors in the pathogenesis of diabetic complications. Protective effects of EL and its compounds were demonstrated on various models of diabetic complications. Presentation will emphasize on its cardio protective effect by demonstrating amelioration in oxidative stress, decrease in hypertension and hypercoagulibility of blood and platelets along with hypolipidemic effect.

One of the exciting areas is role of stem cell in differentiation and pathogenesis. Our lab has also reported novel activity of these compounds by demonstrating in vitro islet neogenic potential from adult progenitor (stem cell) of pancreatic and non pancreatic origin. Transplantation of these newly generated islets into alloxan-induced diabetic rats showed reversal of hyperglycemia. Thus it is an attractive strategy for treatment of diabetes. Experiments were also undertaken to understand the role of Poly (ADP Ribose). Polymerase enzyme in islet-neogenesis from progenitors and mechanism of action of these compounds will also be discussed.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):11A.

43 Prevention and Treatment of Heart Diseases by Ayurvedic Drugs

SK Gupta ¹

Abstract

Myocardial infarction is the leading cause of morbidity and mortality in industrialized societies and remains an important health problem worldwide. Studies conducted by WHO, estimate that by 2020, 50% of the world’s heart patients would be living in India. Therefore, in the present study therapeutic potential of a Polyherbal herbal combination (containing Withania somnifera [50 mg/kg] + Curcuma longa [100 mg/kg] + Ocimum sanctum [75 mg/kg]) in the treatment of acute MI was evaluated. The study was carried out in male albino wistar rats for a duration of 30 days. Efficacy of the polyherbal combination was evaluated in two internationally acclaimed and reproducible models: (1) Isoproterenol induced Model and (2) Ischaemia-Reperfusion Model; these are akin to human heart diseases. Chronic oral treatment with the Polyherbal combination resulted in significant enhancement of the myocardial endogenous antioxidants SOD, CAT, GSHPx (p<0.01) and reduction in myocardial TBARS level (p<0.05) as compared to the sham control group. Furthermore, the Polyherbal combination was effective enough to beneficially modify the ischemia-reperfusion-induced homodynamic alterations ([+]LVdP/dt, [−] LVdP/dt & LVED). Most importantly, the Polyherbal combination demonstrated significant anti-apoptotic effects i.e. decreased Bax (p<0.01), upregulated Bcl-2 (p<0.001) expression and attenuated TUNEL positivity (p<0.001). Also, preserved myocardial CPK activity (p<0.01) and histopathologic evaluation further confirm its myocardial salvaging effects. Results of the present study clearly indicate that there is strong scientific basis for the use of ayurvedic drugs in the prevention and treatment of cardiovascular diseases.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):11A.

44 Effect of Antioxidant and Anti-Inflammatory Agents in Experimental Ischemic Stroke Models

YK Gupta ¹

Abstract

Treatment of acute ischemic stroke remains a major challenge. Recombinant tissue plasminogen activator (rtPA), the only approved drug for the treatment, has a narrow time window and adverse effects like intracranial hemorrhage and progression of ischemia/reperfusion injury. Excitotoxicity, inflammatory cascade and release of free radicals during ischemia/reperfusion leads to progression of brain injury after stroke. Therefore, searching for drugs that can affect the progression of ischemia/reperfusion injury becomes essential. Approaches using anti- oxidants, anti-inflammatory, anti-apoptotic, anti-excitotoxic agents have been tried in experimental models of stroke. We investigated drugs of herbal origin and other neuroprotectants in middle cerebral artery occlusion model (MCAo) of transient focal cerebral ischemia in rats. The diffusion weighted magnetic resonance imaging was done for assessment of infarct area, behavioral parameters, brain oxidative stress, and inflammatory markers were studied to evaluate the neuroprotection potential of these investigational agents. Withania somnifera (1 g/kg), resveratrol (20 mg/kg), vineatrol (20 & 40 mg/kg), alpha-tocopherol (250 mg/kg), adenosine (500 mg/kg), melatonin (20 mg/kg), and TAK-044 (5 mg/kg), rapamycin (150 & 250 μg/kg) and mycophenolate mofetil (100 & 200 mg/kg) demonstrated neuroprotective effects in the MCAo model of stroke. These agents are to be further evaluated for their mechanism at the molecular level and efficacy when given in combination.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):11A.

45 Antioxidant Potential and Reactivity of Soy Derived Products and Applicability to Biological Systems

MK Hagen ^1,², AR Lehenbauer-Lüdke ³, AS Araujo ¹, RH Mendes ¹, TG Fernandes ¹, JMG Mandarino ⁴, SF Llesuy ⁵, EV de Jong ⁶, A Belló-Klein ¹

Abstract

The aim of this study was to analyze and compare the content of isoflavones in two soy products and its effectiveness as antioxidants in vitro. Isoflavones content was analyzed in soybean hypocotyl (SH) and isolated soy protein (ISP) through HPLC. The quality (TAR) and quantity (TRAP) of antioxidants present in samples were quantified by chemiluminescence. A free radical generating system was induced in rat liver homogenates to measure the inhibition of lipid peroxidation (LPO) by both products. The results showed that the amount of daidzin was nine times higher in SH, whereas the genistein content was five times higher in ISP. SH presented three fold increase in TAR, while both products exhibited same TRAP levels and inhibition of LPO. Although SH and ISP presented differences in the isoflavone content and antioxidant potential, both soy derived products could be used as alternative preventive and therapy for oxidative stress-related diseases.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):11A.

46 Free Radicals and Antioxidants – Quo Vadis?

B Halliwell ¹

Abstract

Reactive (oxygen) species are generated continually in the human body, both for useful purposes and by “accidents of chemistry”. They are significant contributors to age-related diseases and perhaps to the ageing process itself, e.g. they contribute to cancer, neurodegeneration, and probably to cardiovascular disease, although the evidence that they play major pathogenic roles in cardiovascular disease is less compelling. Iron in a “redox active” form plays a key role in facilitating damage by reactive species. Nevertheless, reactive species have beneficial effects in killing invading pathogens and facilitating signal transduction, especially in coordinating the inflammatory response and, in some cases (an example being myocardial stunning), the response of tissues to stress. Indeed, several epidemiological studies have found little protective effect of antioxidants against the development of cancer, cardiovascular or neurodegenerative disease (although some studies have been positive) whereas dietary fruits and vegetables appear more beneficial in health maintenance and disease prevention in at least some studies. Many constituents could contribute to these effects, including antioxidants and (seemingly paradoxically) pro-oxidants. The care needed to establish the true effects of antioxidants and pro-oxidants in cell culture and in vivo will be illustrated by recent data using “biomarkers” of oxidative damage and studies of ageing in C.elegans.

47 Alterations in Cardiolipin Metabolism in Heart Failure: Can these be Reversed?

Grant M Hatch ¹

Abstract

Heart failure (HF) occurs as a consequence of hypertension, ischemic heart disease, valvular disease or inherited idiopathic cardiomyopathy. According to the American Heart Association Statistical Committee, over 5,000,000 people suffer from this condition with over 600,000 new cases diagnosed each year. Therefore, to understand the underlying cause of this serious condition and to prevent its development, it is important to identify the pathophysiologic basis of the disease. Cardiolipin (CL) is a major mitochondrial membrane phospholipid, and tetralinoleoyl-CL (L4-CL) plays a key role in maintaining mitochondrial respiratory and cardiac function via modulation of the activities of enzymes involved in oxidative phosphorylation. In the mammalian heart, L4-CL comprises the majority of CL fatty acyl molecular species. CL is synthesized de novo in four unique steps and, subsequent to its synthesis, it is remodeled in order to obtain this unique L4-CL fatty acyl molecular species composition. Elevation in CL synthesis enhances formation of metabolically active mitochondrial membranes whereas loss of L4-CL results in disassembly of mitochondrial respiratory complexes, loss of mitochondrial complex proteins and reduced oxidative phosphorylation. We have observed in both left and right HF animal models that the level of L4-CL is dramatically reduced, as a result of alteration in both the de novo biosynthesis and remodeling of CL, and this reduction in L4-CL results in reduced mitochondrial function. In addition, we have observed reduced L4-CL in explanted hearts from human HF patients. We summarize our observations of reduced L4-CL in experimental and human HF, present a new model for the development of HF associated with reduced L4-CL, and discuss mechanisms for reversing the loss of L4-CL in HF.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):12A.

48 Increase of Endogenous Hydrogen Sulfide in Early Reperfusion Phase Plays a Crucial Role for Cardioprotection Induced by Ischemic Postconditioning

Yi-E Huang ^1,², Wei Xie ¹, Zhi-Han Tang ¹, Xin-Tian Shen ^1,², Xiang-Ping Peng ¹, Zhan-Zhi Zhao ¹, De-Bo Nie ¹, Lu-Shan Liu ¹, Zhi-Sheng Jiang ¹

Abstract

AIMS:

Hydrogen sulfide (H₂S), produced by cystanthionine-γ-lysase (CSE) in the cardiovascular system, has been positioned as the third gasotransmitter besides nitric oxide (NO) and carbon monoxide (CO). The present study aimed to investigate the role of H₂S in ischemic postconditioning (IPO) during the early period of reperfusion.

MAIN METHODS:

IPO with 6 episodes of 10 s reperfusion followed by 10 s ischemia (IPO 2’) was manipulated when reperfusion was started. Cardiodynamics and concentration of H₂S were measured at 1, 2, 3, 4, 5, 10, 20, 30, 60, 90 and 120 minutes of reperfusion. LDH and infarct size were determined at the end of the reperfusion.

KEY FINDINGS:

The concentration of H₂S was stable during whole experiment in CON group, while reached peak at the first minute of reperfusion in IR group, while the concentration of H₂S at the first minute of reperfusion in IPO 2’ group was higher than IR group, which correlated with cardioprotection including improved heart contractile function and reduced infarct size and LDH. However, the above effects of IPO 2’ was attenuated by pre-treatment with blockade of endogenous H2S production with DL-propargylglycine (PAG) for 20 minutes before global ischemia. Furthermore, we found other forms of IPO, IPO commencing at 1 minute after reperfusion (Delayed IPO) or lasting only for 1 minute (IPO 1’), failed to increase the concentration of H₂S and protect myocardium.

SIGNIFICANCE:

We conclude that increase of endogenous H₂S in early reperfusion phase plays a crucial role for cardioprotection induced by ischemic postconditioning.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):12A.

49 Cardiovascular Actions of Incretins: From Bench to Bedside

Mansoor Husain ¹

Abstract

Preclinical studies in isolated cells, tissues and animal models show that glucagon-like peptide 1 (GLP-1), as well as GLP-1 receptor agonists used in the treatment of type-2 diabetes, have the potential to exert beneficial cardiovascular effects. We will present some of these data, which enhance our understanding of GLP-1 and its potential cardioprotective effects in humans.

GLP-1 and its metabolite, GLP-1 (9–36), have been shown to exert cardioprotective effects, which only partly depend on the GLP-1 receptor. Our data show that both GLP-1 and GLP-1 (9–36) protect the heart from ischaemia-reperfusion injury, vasodilate mesenteric arteries and increase coronary flow. These effects are not entirely mediated through GLP-1 receptors (1). We show that these effects are also manifested in isolated cardiomyocytes and human endothelial cells (2).

We have also investigated the cardioprotective actions of dipeptidyl peptidase-4 (DPP-4) inhibitors and GLP-1 receptor agonists in mice, using experimental models of myocardial infarction (MI). In particular, we have looked at whether pre-treatment with a GLP-1 receptor agonist protects against myocardial injury in vivo following coronary occlusion. Liraglutide, a once-daily GLP-1 receptor agonist, improves survival following MI, reduces infarct size and activates pro-survival pathways in the mouse heart (3). DPP-4-knockout mice also exhibit a survival advantage over wild-type mice following experimental MI and increased expression and activity of cardioprotective genes (ANP, P-Akt and GSK3-b). Furthermore, pharmacological inhibition of DPP-4 with sitagliptin conferred a survival advantage over untreated controls (4).

In humans, too, GLP-1, DPP-4 inhibitors and the GLP-1 analogues currently in use for the treatment of type-2 diabetes have been shown to affect the cardiovascular system. Together, these findings increase our understanding of the cardioprotective actions of GLP-1-based therapies and provide mechanistic rationale for the emerging evidence of the cardiovascular effects of these agents in clinical studies.

References

1.Ban, et al. Circulation. 2008;117:2340–50. doi: 10.1161/CIRCULATIONAHA.107.739938. [DOI] [PubMed] [Google Scholar]
2.Ban, et al. Endocrinology. 2010;151:1520–31. doi: 10.1210/en.2009-1197. [DOI] [PubMed] [Google Scholar]
3.Noyan-Ashraf, et al. Diabetes. 2009;58:975–83. doi: 10.2337/db08-1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Sauvé, et al. Diabetes. 2010;59:1063–73. doi: 10.2337/db09-0955. [DOI] [PMC free article] [PubMed] [Google Scholar]

Exp Clin Cardiol. 2011 Oct;16(Suppl A):12A.

50 L-Cysteine Supplementation as an Adjuvant Therapy for Type 2 Diabetes

Sushil K Jain ¹

Abstract

Diabetes has become an epidemic and remains a major public health issue world wide. Intensive blood glucose control dramatically reduces the devastating complications that result from poorly controlled diabetes. Recent studies report lower blood levels of H₂S in type 2 diabetic patients. H₂S is emerging as an important signaling molecule. H₂S can be formed from L-cysteine (LC). Animal studies have demonstrated that supplementation with LC or protein rich in LC have improved glucose metabolism and that H₂S or H₂S donor supplementation ameliorated atherogenic processes. Our recent studies demonstrate that exogenous LC or H₂S supplementation increases glucose utilization, and inhibits IL-8 and MCP-1 secretion in high-glucose-treated cultured U937 monocytes. Further studies in ZDF rats supplemented with LC (1 mg, orally, daily) or vehicle for 8 weeks showed that LC supplementation significantly (p<0.05) increased blood levels of H₂S (37%) and NO₂ (30%) and lowered levels of glycated hemoglobin (9%), MCP-1 (31%), TNF-α (31%) and HOMA insulin resistance (25%) compared with levels seen in saline-supplemented D. The blood levels of glycated hemoglobin and IR showed a significant negative correlation (p<0.05) with concentrations of H₂S and nitrite in LC supplemented ZDF rats. Thus, the loss of H₂S in diabetes could represent a novel mechanism responsible for the impaired glucose homeostasis and cardiovascular complications in diabetes. This presentation will discuss cell culture and animal studies, and the molecular mechanisms that suggest a potential use for LC or H₂S containing compounds in controlling glucose metabolism in diabetes.

Footnotes

Supported by grants from NIDDK and the Office of Dietary Supplements (RO1 DK072433) and Malcolm-Feist Endowed Chair in Diabetes

Exp Clin Cardiol. 2011 Oct;16(Suppl A):13A.

51 Oxidative Stress in Health and Disease: From Basic Science to Applied Clinical Investigations

Vladimir Lj Jakovljevic ¹, Dusica Djordjevic ¹, Dejan Cubrilo ¹, Vladimir Zivkovic ¹, Milena Vuletic ¹, Nevena Barudzic ¹, Dusko Kornjaca ¹, Dragan M Djuric ²

Abstract

Cellular damage by reactive oxygen species (ROS) such as O₂- or hydroxyl radicals (OH-) is a significant causal factor involved in heart diseases, especially during myocardial ischemia-reperfusion. L-arginine/NO system and its main metabolic product, nitric oxide (NO), is bioregulatory system that plays an important role in cardiovascular homeostasis. The bioactivity of endothelium-derived NO is impaired in cardiovascular diseases, caused in part by the increased vascular production of O₂- and its reactive intermediates – hydrogen peroxide. Two crucial problems with NO are short half-life and rapid bioinactivation by reactive oxygens species (ROS) and forming reactive nitrogen species (RNS). Regarding this data, we tried to connect our basic exeperiments in isolated heart with some clinical events where oxidative stress is involved. One part of our basic investigation was to assess the effects of 1) hydrosoluble vitamins (Vitamin C and Folic acid) 2) the effects of specific phosphodisterase 5 (PDE5) inhibitors and 3) the effects of vibroacustic microvibrations on coronary flow (CF), nitrite outflow (NO), and dynamic of oxidative stress parameters in isolated rat heart. The results showed that applied vitamins and PDE5 inhibitors have different effects on isolated rat heart and opposite interaction with NO-system, while vibroacustic microvibrations do not affects heart function. On the other hand, the aim of our clinical studies were to examine parameters of oxidative stress in 1) sternous exercise, 2) during hyperbaric oxygenation and 3) in fetal distress. The results of our studies in top athletes suggested that the type of sport and training activity (anaerobic compared to aerobic) affects oxidative stress, as well as its dynamic in sternous exercise. Furthermore, programmed physical activity increases SOD activity as a first line in antioxidant defense, while excess H₂O₂ production depletes CAT activity. Finally, correlation of measured parameters were age specific. In the second part, influences of breathing oxygen under high pressure (HBOt) on potential proxidative activity in the diabetic patients with serious vascular complications were recorded. All examinated patients were treated with the same therapy protocol, and obtained results show that there was no statistically significant differences in the values of oxidative stress parameters during the HBOt compared to the values before the HBOt. And finally, in order to examine role of oxidative stress in fetal distress development, we tested 46 mothers and newborn children whose oxidative status was evaluated immediately after birth by tracking the concentration of superoxide anion radicals (O₂-), hydrogen peroxide (H₂O₂) and TBARS in blood plasma, as well as the activity of enzymes of the first line of anti-oxidative protection – superoxide dismutase (SOD) and catalase (CAT) in erythrocytes. The results of our research show that there is statistically significant difference in values of all parameters in children with and without distress and between mothers of newborn children with or without distress, while statistically significant difference in O₂- values was not noticed between mothers from the control group and from the examined group.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):13A.

52 High Molecular Weight (HI) FGF-2: An Old ‘New’ Player in Heart Disease

E Kardami ¹, J-J Santiago ¹, W Srisakuldee ¹, X Ma ¹, L McNaughton ¹, B Bestvater ¹, RR Fandrich ¹, B Nickel ¹, R Arora ¹, D Freed ¹, D Jassal ¹, P Singal ¹, P Cattini ¹

Abstract

Fibroblast growth factor 2 (FGF-2) is a multifunctional heparin-binding protein synthesized and accumulated predominantly by fibroblasts and myofibroblasts as high molecular weight (>20 kD, hi) and low molecular weight (lo) isoforms. Over the years, our laboratory has conducted several comparative studies regarding the biological activities of hi and lo FGF-2 in the normal and diseased hearts. We have thus shown that hi and lo FGF-2 exert distinct intracrine, autocrine and paracrine effects. Intracellularly, hi FGF-2, but not lo-FGF-2 promotes chromatin compaction leading to cell death; in addition hi FGF-2 exerts deleterious effects on cardiac mitochondria, while lo FGF-2 is beneficial. Both hi- and lo FGF-2 isoforms are secreted by cardiac fibroblasts in response to angiotensin II stimulation; hi FGF-2, but not lo-FGF-2, can then exert paracrine induction of cardiomyocyte hypertrophy, in vitro and in vivo. Hi FGF-2 can also exert autocrine upregulation of pro-fibrotic matricellular proteins, and proteins associated with the innate inflammatory response and the inflammasome. We have found hi FGF-2 to become upregulated in several experimental models with heart disease, such as diabetes or adriamycin associated cardiomyopathy, and pressure overload hypertrophy, suggesting that it plays a role in the development and/or maintenance of these pathologies. Finally, we have found that human heart-derived fibroblasts behave in a manner very similar to their rodent counterparts, expressing and secreting hi FGF-2, which is biologically active in that it can induce cardiomyocyte hypertrophy and promote fibroblast to myofibroblast conversion. In conclusion, our work has pointed to the hi FGF-2 isoform as a major ‘player’ in the promotion of maladaptive cardiac phenotype in chronic heart diseases.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):13A.

53 The Impact of Repeated Marathon Running on Cardiovascular Function in the Aging Population

E Karlstedt ¹, A Chelvanathan ¹, K Kumar ¹, N Bhullar ¹, M Lytwyn ¹, S Bohonis ¹, S Oomah ¹, R Nepomuceno ¹, J Du ¹, M Da Silva ¹, S Melnyk ¹, M Zeglinski ¹, S Sharma ¹, ID Kirkpatrick ¹, DS Jassal ¹

Abstract

BACKGROUND:

Several studies in younger individuals (18–40 years) have correlated elevations in cardiac biomarkers with injury following marathon running with transient and reversible right ventricular (RV) systolic dysfunction. Whether or not permanent cardiac injury occurs due to repeated marathon running in the aging population remains controversial.

OBJECTIVE:

The aims of this study were to 1) Assess the extent of cardiac dysfunction after completion of full marathon running in the elderly population, using cardiac biomarkers, TTE, cardiac computed tomography (CCT) and CMR; 2) Assess presence/distribution of myocardial injury using DE-CMR.

METHODS:

25 healthy volunteers (>50 yrs) participating in the 2010 and 2011 Full Manitoba Marathons were included. Cardiac biomarkers, TTE, and CMR were performed 1 week prior, immediately after, and 1 week after the race. Cardiac CT was done within 3 months of the marathon.

RESULTS:

All participants demonstrated elevated cTnT immediately post marathon; median value of 0.41 ug/L. On CMR, RV end-diastolic volume increased after the full marathon, and the RV ejection fraction was reduced, 49±3% compared with 65±2% at baseline (p<0.05). One participant showed delayed enhancement on CMR, involving the sub-endocardium of the anterior wall, with concomitant stenosis of the left anterior descending artery on CCT.

CONCLUSIONS:

Marathon running in the elderly is associated with a transient, yet reversible, increase in cardiac biomarkers and RV systolic dysfunction. Repeated marathon running in the aging population does not result in permanent myocardial injury.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):13A–14A.

54 GSK-3 and Pluripotency: The Central Role of Catenins in Mediating The Effects of GSK-3 Inhibitions on Embryotic Stem Cell Properties

K Kelly ¹, S Mahendram ¹, B Doble ¹

Abstract

Pluripotent stem cells possess the properties of self-renewal and pluripotency, which are controlled by a multitude of integrated cellular signaling pathways, including the beta-catenin/TCF pathway. Stimulation of the beta-catenin/TCF pathway through inhibition/ablation of GSK-3 results in elevated cytosolic and nuclear beta-catenin levels, increased expression of beta-catenin/TCF target genes, and reinforced pluripotency in mouse embryonic stem cells (ESCs). Surprisingly, we recently demonstrated that the effects of beta-catenin on pluripotency do not require the activation of well-established beta-catenin/TCF target genes. Indeed, the transcriptional transactivation domain of beta-catenin was dispensable for the effects of stabilized beta-catenin on pluripotency. In addition to beta-catenin, other members of the catenin superfamily (i.e. p120- and gamma-catenin/plakoglobin) are GSK-3 substrates. We find that p120 and plakoglobin are stabilized upon treatment of mouse ESCs with GSK-3 inhibitors, or in mouse ESCs lacking both isoforms of GSK-3. By using wild-type and beta-catenin knockout ESCs, we are currently examining whether these catenins, like beta-catenin, positively regulate the maintenance of pluripotency. Collectively, these studies will reveal mechanistic insight into how different members of the catenin superfamily regulate the pluripotent state. Our findings have implications not only for stem cell biology, but also for cancers in which catenins play an oncogenic role.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):14A.

55 Role of microRNAs in Cardiovascular Complications of Diabetes

Madhu Khullar ¹

Abstract

Cardiovascular complications account for significant morbidity and mortality in the diabetic population. Epidemiological and clinical trial data have also confirmed the greater incidence and prevalence of heart failure in diabetes. Heart failure in diabetes without known cardiac complications supports the existence of diabetic cardiomyopathy. Cardiomyocyte hypertrophy and myocardial fibrosis are the established pathological features of the diabetic heart and are associated with dysregulation of gene expression of key pathway genes.

The search for the basic mechanisms that are responsible for the development and progression of diabetic cardiomyopathy has been exhaustive; nonetheless, no single unifying mechanism has been uncovered that explains the development and progression of heart failure in diabetic patients. Central dogma of molecular biology states that expression of protein coding genes can be regulated at various levels from transcription to translation. In the past, most studies have focused on protein coding genes and their regulation at the transcriptional level. Recent studies have uncovered a potentially important role for a family of tiny noncoding regulatory RNAs, known as microRNAs (miRNAs or miRs), in the transcriptional and post transcriptional regulation of gene expression. Further, microRNAs have been reported to regulate diverse aspects of cardiac function and also play an important role in the pathogenesis of heart failure through their ability to regulate the expression levels of genes that govern the process of adaptive and maladaptive cardiac remodeling. However, our understanding of the role that microRNAs play in heart failure is limited. An overview of the putative role of micro RNAs targeting various pathways involved in the pathogenesis of cardiovascular complications in diabetes milieu will be elucidated.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):14A.

56 Role of Iroquois Homedomain Transcription Factors in Electrical and Contractile Properties of the Heart

Han Kim ¹, Anna Rosen ¹, Mark Davis ¹, Shan-Shan Zhang ¹, Benoit Bruneau ¹, Chi-Chung Hui ¹, Peter Backx ¹

Abstract

The Iroquois homeodomain transcription factors (Irx) are critical for global patterning in development. We have previously shown that Irx5 is a major determinant of spatial repolarization pattern of the heart by negatively regulating the transmural gradient of the cardiac transient outward K⁺ currents (I_to,f). Since I_to,f modulates both cardiac contractility and hypertrophy, and is invariably reduced in heart disease, contractility properties and responses to biomechanical stress were studied in mice lacking Irx5. As expected, the normal transmural gradient of excitation-contraction was eliminated in Irx5-deficient hearts in association with reduced cardiac contractility and Ca²⁺ transients reductions in the endocardium, not epicardium. Although structurally normal, Irx5-deficient hearts showed impaired cardiac hypertrophy, along with severe interstitial fibrosis and contractile deterioration, in response to biomechanical stress. Hypertrophy induced by norepineprine was also markedly reduced in cultured Irx5-deficient myocytes. Elimination I_to,f (via K_V4.2-ablation) abolished the effects of Irx5-deletion on contractility while having no effect on hypertrophic responses to pressure-overload. On the other hand, the activity of calcineurin (Cn)-Nfat (a major determinant of hypertrophic signaling) was reduced in Irx5-deficient hearts while the hypertrophy induced by over-expression of Cn in heart was also reduced in mice. Our results establish that Irx5 regulates cardiac contractility in I_to,f-dependent manner while modulating hypertrophic responses in an I_to,f-independent, Cn-Nfat-dependent manner.

We have also found that another Irx gene, Irx3, controls the electrical properties of the ventricular conduction system. Specifically, the loss of Irx3 leads to conduction block as well as deterioration of the conducting system in post-natal development. These changes are linked to the ability of Irx3 to antithetically regulate the two gap junction-forming connexins (Cx) of the ventricular conduction system: enhancing Cx40 and suppressing Cx43 expression. Irx3 directly represses Cx43 transcription and indirectly activates Cx40 transcription. Thus, Irx3 plays a critical role in the precise regulation of intercellular gap junction coupling and impulse propagation in the heart.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):14A.

57 Potassium and Sudden Cardiac Death

Keld Kjeldsen ¹

Abstract

A magnitude of 3 million people worldwide annually suffer sudden cardiac death, often emerging from a complex interplay of substrate and triggers. Disturbed potassium homeostasis among heart cells is such a trigger. Thus, hypokalemia and also more transient reductions in plasma potassium concentration are of importance. Hypokalemia is present in 7–17% of patients with cardiovascular disease, up to 20% of hospitalized patients and up to 40% of patients on diuretics. Importantly, inadequate management of hypokalemia was found in up to 24% of hospitalized patients. Hypokalemia is associated with increased risk of arrhythmia in patients with cardiovascular disease as well as increased all-course mortality, cardiovascular mortality and heart failure mortality by up to 10-fold. Long-term potassium homeostasis depends on renal potassium excretion. However, skeletal muscles play an important role in short-term potassium homeostasis primarily because skeletal muscles contain the largest single pool of potassium in the body. Moreover, due to the large content of sodium, potassium-pumps and potassium channels, the skeletal muscles possess a huge capacity for potassium exchange. Hypokalemia is in cardiovascular patients often caused by non-sparring diuretics, insufficient potassium intake, and shift of potassium into stores by increased potassium-uptake stimulated by catecholamines, beta-adrenoceptor agonists and insulin. Importantly, there is protection against further reduction in plasma potassium in severe hypokalemia. Interestingly, drugs with proven significant positive effect on mortality and morbidity in heart failure patients all increase plasma K concentration. Importantly, there is protection against further increase in plasma potassium in severe hyperkalemia. Thus, it may prove beneficial to pay more attention to hypokalemia and to maintain plasma potassium in the upper normal range. The more at risk of fatal arrhythmia and sudden cardiac death, the more attention should be given to the potassium homeostasis.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):14A–15A.

58 miR-1 and miR-301A Overexpression Impairs Collagen Gel Contraction in Human Mesenchymal Stem Cells

J Klassen ¹, S Neumann ¹, M Guirgis ¹, MA Ngo ¹, Y Li ¹, DH Freed ¹

Abstract

Previous data in our lab suggests that mesenchymal stem cells (MSCs) of bone marrow origin display a myofibroblast phenotype in vitro. Coincident with the onset of a myofibroblast phenotype, we observed a reduction in miR-301a expression. We hypothesize that microRNA signalling governs the differentiation of bone marrow derived MSCs to myofibroblasts, specifically miR-301a and miR-1.

The protocol received IRB approval. MSCs were isolated from patients undergoing open heart surgery and were cultured in standard conditions. Cells were plated on collagen gel substrates and were transfected with a 50nM concentration of pre-miR-1 and pre-miR-301a for 3 hours. The reduction in collagen gel area was analyzed 48 hours post transfection to assess MSC-myofibroblast function. qRT-PCR was used to analyze RNA profile and expression of myosin isoforms.

Human MSCs were found to express both NMM-IIA and IIB, and readily contracted collagen gels in the basal state, which was increased with TGF-β1. Overexpression of miR-301a or miR-1 resulted in reduced collagen gel contraction when compared with a negative scramble control. Cells transfected with miR-1 contracted less than transfection with miR-301a. Analysis with qRT-PCR revealed a decrease in non-muscle myosin IIA expression in both miR-1 and miR-301a transfected cells when compared with a negative scramble control. Smooth muscle myosin was also decreased in miR-301a transfected cells.

MSCs adopt a myofibroblastic phenotype when cultured in standard conditions, coincident with an altered microRNA signature. Overexpression of miR-301a and miR-1 reduced MSC contractility through reduced myosin expression. It is likely that miR-1 and miR-301a target additional proteins in these cells.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):15A.

59 Long-Acting Phosphodiesterase-5 Inhibitor, Tadalafil, Protects Against Myocardial Ischemia, Reperfusion Injury in Type 2 Diabetic Mice via Suppression of Oxidative Stress and Akt-AMPK Signaling

S Koka ¹, A Das ¹, FN Salloum ¹, R Kukreja ¹

Abstract

Diabetes is associated with increased risk for cardiovascular complications. Since phosphodiesterase-5 inhibitors (PDE51), restore nitric oxide signaling, we hypothesized that chronic treatment with long acting PDE5I, tadalafil, might improve cardiac complications associated with type 2 diabetes in db/db mice.

Diabetic db/db mice (n=24/group) were treated with vehicle or tadalafil (TAD. I mg/kg, i.p) for 28 days. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (Δψm) were measured following 40 min simulated ischemia (I) followed by I h of reoxygenation (R) in ventricular myocytes. A subset of mice hearts were subjected to I (30 min) and R (60 min) in Langendorff mode, and infarct size was measured. Another subset of mice hearts were used for the estimation of lipid peroxidation, GSSG/GSH, expression of pAKT and pAMPK. TAD treatment improved the metabolic status of db/db mice. TAD-treated mice showed reduced infarct size compared to the control db/db mice (21.2±1.8% vs 45.8±2.8%; p<O.O I). db/db mice demonstrated significantly increased ROS production, lipid peroxidation and GSSG/GSH ratio, which were attenuated in TAD-treated mice. TAD treatment also preserved the loss of Δψm and enhanced the expression of myocardial pAkt and pAMPK compared to the control (p<0.05). Furthermore, proteomic analysis by 2D-DIG electrophoresis revealed distinct favorable modulations in redox regulating proteins and chaperones following TAD treatment in diabetic hearts. We conclude that tadalafil treatment attenuates oxidative stress and improves mitochondrial integrity while providing powerful cardioprotective effects potentially through Akt-AMPK signaling in type 2 diabetes.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):15A.

60 Physiological Role of Slow Sodium Channels in Excitable Membranes

Krylov BV Pavlov ¹

Abstract

Slow sodium channels were discovered in 1981 (Kostyuk et al., 1981). The channels (TTX-resistant, TTX_r, Na_v1.8), attracted rapt attention of neuropharmacologists. It was shown that these channels play a crucial role in coding of nociceptive signals (Gold et al., 1996; Borovikova et al., 1997; Jarvis et al., 2007; Karymova et al., 2008).

Using patch-clamp method, quantum-chemical calculations and mathematical modeling, we investigated effects of gamma-pyrone and low-power CO₂-laser radiation on the membrane of nociceptive neurons. Short-term dissociated cell culture of dorsal root ganglion cells from new-born rats was prepared for experiments. These small dark cells are characterized by high density of the slow sodium channels Na_v1.8.

Two distinct molecular mechanisms of modulation of functional activity of slow sodium channels were observed. The first mechanism is based on receptor-coupled interaction. In this case, the attacking molecule interacts with the membrane receptor which is coupled to ionic channel under investigation. Earlier we have shown (Krylov et al., 1999) that morphine decreases voltage sensitivity of activation gating system of TTX-resistant sodium channels. The important role of signal transducer in this case is played by Na, K-ATPase. G-proteins do not take part in this mechanism. It is well-known that G-proteins are coupled to classical opioid receptors. This new non-pumping function of Na, K-ATPase indicates that the new family of opioid-like receptors is coupled to the slow sodium channels. These new receptors are also blocked by naltrexone or naloxone. But their physiological function principally differs from the function of classical opioid receptors. Our preclinical and clinical (first phase) investigations of the new analgesic “Anoceptin” that specifically activates new-found opioid-like receptors show its high efficiency and absence of negative side effects.

The second (transducer-coupled) mechanism of modulation of functional activity of slow sodium channels was investigated applying infrared (IR) radiation generated by a low-power CO₂-laser on the membrane of nociceptive neurons using the whole-cell patch-clamp method. Low-power IR radiation diminished the voltage sensitivity of activation gating machinery of slow sodium channels (Na_v1.8). Ouabain known to block both the transducing (Xie, 2001) and pumping functions of Na⁺,K⁺-ATPase eliminated IR irradiation effects. The molecular mechanism of interaction of CO₂-laser radiation with sensory membrane was suggested. The primary event of this interaction is energy absorption by ATP molecules. The transfer of vibrational energy from Na⁺,K⁺-ATPase-bound and excited ATP molecules to Na⁺,K⁺-ATPase activates this enzyme converting it to a signal transducer. This effect leads to a decrease in the voltage sensitivity of Na_v1.8 channels. The mechanism is elucidated due to the combined application of a very sensitive patch-clamp method and an optical facility with a controlled CO₂-laser. As a result, the mechanism of interaction between the non-thermal low-power IR radiation and nociceptive neuron membrane is suggested. Medicinal device for pain relief “Camertone” that consists of the low-power CO₂-laser is now under clinical tests.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):15A.

61 Phosphodiesterase-5 Inhibitors in Attenuation of Doxorubicin-Induced Cardiomyopathy

Rakesh C Kukreja ¹

Abstract

Doxorubicin (DOX) is one of the most effective and commonly used chemotherapeutic agents for treating cancer in both children and adults. However, its clinical utility is limited due to cumulative cardiotoxicity that can lead to heart failure. Most of the cellular events triggered by DOX contribute to cell death, which is the primary mechanism by which DOX induces cardiomyopathy. Several studies have shown that phosphoid-esterase-5 (PDE-5) inhibitors including sildenafil, vardenafil and tadalafil induce protective effect against ischemia/reperfusion injury in the heart. We also demonstrated that treatment with sildenafil prior to DOX treatment inhibited cardiomyocyte apoptosis, preserved mitochondrial membrane potential (Δψm), myofibrillar integrity and prevented left ventricular (LV) dysfunction as well as ST segment prolongation. These effects were duplicated by the use of long acting PDE-5 inhibitor, tadalafil, which also prevented cardiomyocyte apoptosis in DOX-induced cardiomyopathy through mechanisms involving up-regulation of cGMP, increase in protein kinase G activity and MnSOD level. Our most recent work showed that sildenafil is a powerful sensitizer of DOX-induced killing of prostate tumors in nude mice while providing concurrent cardioprotective benefit. These studies suggest that prophylactic treatment with PDE-5 inhibitors might become a promising therapeutic intervention for managing the clinical concern of DOX-induced cardiotoxicity in patients.

Footnotes

Supported in part by grants HL51045, HL59469, and HL79424 from the National Institutes of Health

Exp Clin Cardiol. 2011 Oct;16(Suppl A):15A–16A.

62 microRNAS May be Responsible for Angiogenic Defects Observed in Patients Suffering from Hereditary Hemorrhagic Telangiectasia (HHT)

Michael Kutryk ¹

Abstract

BACKGROUND:

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant genetic disorder characterized by cutaneous telangiectasia, recurrent epistaxis and visceral arteriovenous malformations. HHT is associated with mutations in transforming growth factor-β superfamily receptors, which include bone morphogenetic protein receptor-2, endoglin and activin receptor-like kinase 1 (ALK-1).

OBJECTIVE:

As circulating endothelial progenitor cells (EPCs) have been shown to play an important role in vascular homeostasis and repair, our objective was to determine if EPCs derived from patients with HHT display functional abnormalities, and if changes in the levels of microR-NAs might be involved.

METHODS AND RESULTS:

Mononuclear cells were isolated from venous blood of controls and patients with HHT. Early-outgrowth EPCs were obtained by 7 day culture on fibronectin-coated slides. The percentage of EPCs that stained positive for endothelial lectin or vascular endothelial growth factor receptor-2 was significantly reduced in patients with HHT. Cell migration was also impaired in HHT patients compared to controls. Significant reductions in endoglin, ALK-1 and endothelial nitric oxide synthase mRNA expression, as determined by real-time-PCR, were detected in EPCs from patients with HHT compared to controls. EPC microRNA profiling showed altered levels of key angiogenesis-related micro-RNAs in patients with HHT.

CONCLUSIONS:

Early outgrowth EPCs derived from patients with HHT demonstrated reduced survival and migration, impaired differentiation to an endothelial cell-like phenotype, and reduced expression of genes involved in growth and repair. Altered levels of microRNAs, that target genes involved in the proliferation and maturation of neo-vessels, suggests a possible mechanism for the disrupted angiogenic capacity of EPCs from patients with HHT. These findings might have relevance for understanding the development of the vascular lesions seen in patients with HHT.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):16A.

63 Expression of Endothelin vs No System Components During Initiation and Development of Isoproterenol-Induced Cardiac Hypertrophy

J Kyselovic ¹, L Pivackova ¹, B Gajdacova ¹, P Musil ¹, A Gazova ¹, P Ochodnicky ¹, J Klimas ¹, P Krenek ¹

Abstract

The effect of catecholamines on expression of endothelin and nitric oxide (NO) systems in heart failure (HF) is complex and remains controversial. We tested the correlation of heart pathophysiology with changes of genes expression of endothelin/NO system in isoproterenol (ISO) induced HF.

Male Wistar rats were administered ISO (5 mg/kg, i.p.) in a single dose and sarcrificed by CO₂ asphyxiation after 0.5, 1, 2, 4, 8 or 24 h and 1, 2, 4 or 8 days of treatment. Increased heart weight was observed already after 24 h after ISO (p<0,05), and progressively increased until the 8th day. Decreased indices of left ventricular systolic and diastolic function dp/dt_min and dp/dt_max (both P<0.05). Isolated atria from HF rats had a lower spontaneous beating rate (p<0.05). Ventricular myocytes from failing hearts had impaired cell shortening. We detected a transient decrease of ET-1 expression 30 min after ISO (p<0.05), followed by increase at 4 h (p<0.05), and remained increased until the end of the experiment. ETA expression transiently decreased between 0.5 and 8 h, returned to baseline and decreased again at days 4 and 8 (p<0.05). ETB receptor were increased at 2 and 4 (p<0.05), ECE-1 expression was down regulated on day 2 and 4 (p<0.05). Additionally, expressions of eNOS, iNOS and their allosteric modulators hsp90 were increased but not nNOS or caveolins.

Despite the increased capacity for NOS, NO does not sustain contractility of failing myocytes and may contribute to the decreased basal heart rate and it may accelerate beta-adrenergic stimulation of chronotropy. Changes in endothelin system components also participate on the pathogenesis of ISO-induced cardiac hypertrophy.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):16A.

64 CNS Effects of Aldosterone: Critical Role in Salt-Induced Hypertension and in Heart Failure

Frans HH Leenen ¹

Abstract

Sympatho-excitatory and pressor responses to aldosterone mediated by MR in the CNS have been well documented. MR activation by aldosterone appears to increase activity in angiotensinergic pathways involving the PVN by enhancing ENaC activity followed by “ouabain” release. This aldosterone-“ouabain” pathway can be activated by increases in CSF [Na⁺] or in circulating and central angiotensin II. Central infusion of an aldosterone synthase inhibitor, MR blocker or Digibind to bind “ouabain” prevent the sympatho-excitatory and pressor responses to both CSF [Na⁺] and Ang II, suggesting that the MR activation may be due to an increase in aldosterone locally produced in the CNS.

High salt diet increases CSF [Na⁺] in models of salt-sensitive hypertension such as Dahl S and SHR. The high salt-induced sympatho-excitation and hypertension in these models can be prevented by specific CNS blockade of any of the steps in the cascade from aldosterone synthase to AT1-receptors and appear therefore to be mediated by the Na⁺-induced activation of the hypothalamic aldosterone-“ouabain” pathway.

Post MI, sympathetic hyperactivity and activation of the circulatory/cardiac RAAS contribute to progressive remodeling of the heart and heart failure. Chronic activation of the hypothalamic aldosterone-“ouabain” pathway has emerged as the main mechanism contributing to persistent AT1-receptor stimulation in the CNS and thereby to the sympathetic hyperactivity as well as the chronic activation of the circulatory and cardiac RAAS. Considering the above described CNS effects of circulating Ang II, an initial increase in plasma Ang II post MI may activate neurons in circumventricular organs such as the SFO followed by a local increase in aldosterone biosynthesis. Not surprisingly, chronic central blockade of any step in the cascade also prevents a major part of the structural remodeling of the heart post MI and the decrease in LV function.

In conclusion, beyond its classical role as a hormone regulating renal Na⁺-transport, aldosterone exerts profound effects on arteries and the heart. Its central actions are recently emerging, and appear to play a critical role in cardiovascular homeostasis when challenged by high salt intake or decreased cardiac function.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):16A.

65 Bone Marrow Origins of Resident Cardiac Stem-Cell Niche Govern Repair after Infarction in Aged Mice

S-H Li ¹, Z Sun ¹, KR Brunt ¹, RD Weisel ¹, R-K Li ¹

Abstract

Heart failure may result from a loss of cardiomyocytes after myocardial infarction. The heart achieves limited repair through tissue resident and circulating stem cells. Resident cardiac stem cells are believed to be a legacy of development, residing in myocardial stasis but retaining a capacity for regeneration in times of injury. Here we uncover inceptive resident cardiac stem cells of hematopoietic origin that independently direct endogenous repair. Age negatively influences the cardiac recipient environment and the functional capacity of stem cell transplant. Whereas a young environment recuperates aged stem-cells, young stem cells perform inadequately in aged recipients. We address this paradox, revealing a third compartment: resident cardiac stem cells of hematopoietic origin that govern cardiac repair. Rejuvenation of aged bone marrow increases the regenerative capacity by restoring this resident cardiac stem cell niche. Cardiac resident stem cells play an important role in restoration of cardiac function and repair.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):16A–17A.

66 HMG-CoA Reductase Inhibitors Attenuate Human Mesenchymal Stem Cell Survival Through Down-Regulation of NF-kB

Y Li ¹, MA Ngo ¹, KK Sran ¹, RC Arora ¹, LA Kirshenbaum ¹, DH Freed ¹

Abstract

Circulating progenitor cells of bone marrow origin have been implicated in transplant cardiac allograft vasculopathy (CAV) and cardiac fibrosis. HMG-CoA reductase inhibitors (“statins”) have been shown to slow the progression of CAV and improve patient survival, presumably as a result of altered lipid metabolism. We examined the in vitro effects of three HMG-CoA reductase inhibitors atorvastatin, simvastatin and pravastatin on the viability of primary human bone marrow derived mesenchymal stem cells (MSC) and expression of NF-κB.

The protocol received IRB approval. Human MSC were isolated and cultured in standard conditions. Cells were treated with each drug at 0.1, 1.0 or 10 μM ± mevalonate. Cell viability was assessed using an optical MTT assay. NF-κB p65 expression was assessed by Western blot. Activation of the NF-κB pathway was achieved through overexpression of Ikk-κ.

Treatment of MSC with 1 and 10 μM simvastatin or atorvastatin resulted in progressively reduced cell viability to about 50% which was associated with a corresponding NF-κB p65 decline to 70 and 50% respectively. Viability could be rescued to over 90% by co-incubation with mevalonate or 65 – 90% by pretreatment with Ikk-κ expressing vector. Pravastatin did not affect MSC viability or NF-κB expression.

Mevalonate depletion through HMG-CoA reductase inhibition impairs the viability of primary human MSC in culture through down-regulating NF-κB p65. This represents an additional plieotropic effect of statins and may explain the beneficial effect of this therapy independent of its effect on lipid metabolism. The lack of effect with pravastatin treatment may be related to the hydrophilic characteristic of the molecule

Exp Clin Cardiol. 2011 Oct;16(Suppl A):17A.

67 Transplanted Embryonic Stem Cells Overexpressing Thymosin β4 Enhance Cardiomyogenesis and Attenuate Remodeling in the Infarcted Heart

Xilin Long ¹, Pawan K Singal ², Dinender K Singla ¹

Abstract

Whether genetically transformed embryonic stem (ES) cells with thymosin β4 (Tβ4) can enhance cardiomyogenesis and attenuate remodeling following transplantation in the infarcted heart remains unknown. Mouse stable ES cell lines, RFP-ESCs and Tβ4-ESCs, were established expressing RFP and RFP-Tβ4 fusion protein, respectively. Spontaneously beating embryoid bodies (EBs) were generated in the cell culture system. Enhanced expression of cardiac transcriptional factor GATA-4, Mef2c and Txb6 in Tβ4-EBs were confirmed with RT-PCR analysis. An increased number of EB area stained positive for sarcomeric α-actin and cardiac α-actinin in Tβ4-EBs compared with the RFP control, suggesting significantly increased functional cardiac myocytes. Furthermore, we transplanted Tβ4-ESCs in the infarcted heart, and animals were sacrificed at 2 weeks post-MI. There was a significant increase in cardiac myocytes compared with the RFP-ESCs control. Reduced apoptosis was evidenced by a decrease in TUNEL staining and caspase 3 activity. There was an increase in pAkt and a decrease in levels of PTEN, a negative regulator of Akt pathway in hearts injected with Tβ4-ESCs. Furthermore, cardiac fibrosis was significantly reduced. Echocardiography data showed an increase in fractional shortening and increase in ejection fraction, suggesting improved cardiac function in Tβ4-ESCs compared with MI and MI+RFP-ESCs groups. It is concluded that genetically modified Tβ4-ESCs enhance cardiac myocyte differentiation both in the cell culture system and in vivo. This was associated with a decrease in both cardiac apoptosis and fibrosis as well as an improved heart function.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):17A.

68 Garlic Extracts Prevents Oxidative Stress and Hypertrophy in Adult Cardiomyocytes

XL Louis ^1,^2,³, R Murphy ², SJ Thandapilly ^1,^2,³, L Yu ^1,³, T Netticadan ^1,^2,³

Abstract

In ancient times, plants were recognized for their medicinal properties. Later, the arrival of synthetic drugs pushed it to the backstage. However, from being merely used for food, plants are now been widely explored for their therapeutic values. In the current study, we used skin and flesh extracts from a variety of purple garlic to test its efficacy in preventing cardiomyocyte hypertrophy.

Hypertrophy was induced using norepinephrine (NE) in adult rat cardiomyocytes pretreated with garlic skin and flesh extracts. Fluorescent probe was used to measure oxidative stress in cardiomyocytes treated with and without extracts and NE. Pharmacological blockade of nitric oxide (NO), hydrogen sulfide (H₂S) and 5′ adenosine monophosphate-activated protein kinase (AMPK) were used to elucidate the mechanism of action of garlic extracts. Treatment with norepinephrine induced an increase in cell size; this increase was significantly prevented in cells treated with garlic skin and flesh extracts. Incubation with NE increased oxidative stress, however, incubation with extracts prevented this increase. NO, H₂S and AMPK blockers partially inhibited the beneficial effect of extracts on NE treated cardiomyocytes.

These results suggest that the purple variety of garlic extracts is effective in preventing NE induced cardiomyocyte hypertrophy by reducing oxidative stress. The beneficial effects may in part be mediated through AMPK, which in turn, may be activated by NO or H₂S.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):17A.

69 Vitamin C Modulates the Activation of Map Kinases and p53 in Doxorubicin-Induced Cardiomyocyte Apoptosis

A Ludke ¹, A K Sharma ¹, A K Bagchi ¹, A Bajaj ¹, Pawan K Singal ¹

Abstract

INTRODUCTION:

Improving the antioxidant defenses of cardiomyocytes is one strategy for cardiac protection against the cardiomyopathy caused by the chemotherapic anthracycline drug doxorubicin (Dox). However, there is a lack of scientific evidence to support a vitamin supplementation recommendation for cancer patients. We examined the effects of Vitamin C (Vit C), a potent antioxidant, on Dox-induced reactive oxygen species (ROS) and downstream steps leading to apoptosis in cardiomyocytes.

METHODS AND RESULTS:

Adult rat cardiomyocytes were incubated for 1, 3, 6, 12 and 24 h with Dox (10 μM), with and without Vit C (25 μM). Exposure to Dox resulted in an increase in JNK and p38 activity at 3 h, which was sustained up to 24 h; a transient increase in ERK 1/2 at 6 h, but a decrease at 12 h and 24 h. Activity of p53 began to rise at 3 h and continued to increase up to 24 h. These changes correlated with a decrease in the viability of cells and an increase in oxidative stress as well as markers of apoptosis in a time-dependent manner. Vit C (25 μM) was able to mitigate Dox-induced changes by decreasing ROS as well as there was a delayed and smaller increase in p38, JNK and p53. With Vit C, the increase in apoptosis markers was reduced while cardiomyocyte viability was improved. It appears that Dox-induced apoptosis is mediated by sustained activation of p38, JNK and p53, and Vit C is able to modulate these Dox-induced changes.

CONCLUSION:

The present findings suggest a nutritional approach as adjunct therapy to prevent Dox-induced cardiotoxicity and a better management of cancer patients.

Footnotes

Supported by Manitoba Heart and Stroke Foundation.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):17A.

70 Calrorie Restriction Increases Myocardial Telomerase Activity via Enhanced Autophagy Flux in the Diabetic Rat Heart

Naoki Makino ¹

Abstract

Caloric restriction (CR) is expected to retard cellular senescence and to attenuate the physiological decline in organ function. Therefore, the aim of the present study was to investigate the impact of long-term CR on cardiac senescence, in particular the effect of CR on the telomere biology associated with diabetes. Male 2-month-old OLETF diabetic rats were divided into ad libitum fed (AD) and CR (30% energy reduction) groups. At 10-month-old those experimental rats, the telomere biology, markers of cell senescence, the autophagy flux and LV function were examined. CR group revealed that the telomerase activity was not downregulated without telomere length shortening, and that protein expressions for Sirt 1 and FOX01 were increased, while, phospho-Akt and p53 were decreased, compared to AD group. The immunofluorescent labeling of CLIII protein was observed to be elevated in CR group. Echocardiography showed similar LV systolic function in both groups, but better LV diastolic function in the CR group. The present study indicates that CR increases telomerase activity and affects cardiac senescence which is may be associated with functional improvement in aged heart with diabetes. Those observations may be in part due to enhanced autophagy flux.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):18A.

71 Regulation of Cardiac Gene Expression by Beta-Blockers

John C McDermott ¹

Abstract

In contrast to the well characterized effects of β-blockers in modulating cardiac inotropic, chronotropic and lusitropic physiological parameters, there is a surprising lack of information concerning how β-blocker treatment affects cardiac gene expression. Recently, we have observed potent effects of β-adrenergic blockade on a key transcriptional regulator in the mammalian heart named Myocyte Enhancer Factor 2 (MEF2). MEF2 transcriptional regulatory proteins play a crucial role in orchestrating gene expression during ontogeny of the heart and also during pathologic and physiological adaptation of the heart. Studies to date indicate that MEF2 is repressed by activation of PKA signalling. We have observed that MEF2D is a direct target of PKA and this repressive phosphorylation is responsible for the inhibition of MEF2 function by β-adrenergic signaling. The potency of this repressive PKA effect on MEF2 led us to consider what happens in the heart where β-adrenergic signaling is of profound importance. In a transgenic MEF2 reporter mouse model, blockade of the β-adrenergic response using a β-1 selective anatagonist (Atenolol) results in a biphasic response. In the acute term (within 2 days) MEF2 activity is indeed de-repressed as one would predict. However, chronic β-blocker treatment (10 weeks in mice) resulted in an extensive down-regulation of MEF2 activity in the heart that we would not have predicted based on acute cell culture experiments. Importantly, we have recently observed that downregulation of MEF2C protein level occurs under pressure overload conditions when the mice are treated with β-blockers suggesting protection from cardiac hypertrophy. Since MEF2 regulates a large battery of cardiac genes we propose that this may have important clinical relevance for understanding the effects of chronic β-blocker treatment in heart disease patients.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):18A.

72 The Role of Fructose Ingestion on Blood Pressure Elevation and the Metabolic Syndrome

JH McNeill ¹, L Tran ¹, V Yuen ¹

Abstract

It has been known since the 1980s that feeding rats sucrose or fructose can increase blood pressure, insulin resistance, and lipids as well as blood levels of vasoactive substances such as norepinephrine and endothelin. While rats do not become obese, there is an increase in visceral fat content. The Metabolic Syndrome (MS) exhibits similar symptoms in humans along with the development of obesity. Each metabolic disturbance is a risk factor for cardiovascular disease and for the development of type 2 diabetes. As a result of these similarities the fructose-fed rat (FFR) has been used as a model to study the MS. As insulin resistance is a major risk factor in the development of MS a decrease is clinically important and should prevent the occurrence of other symptoms. Treatment of FFR with appropriate blocking agents decreases the change in blood pressure but does not always affect the metabolic changes. Decreasing the metabolic changes with type 2 diabetic drugs prevents both metabolic and vascular changes. Antioxidants have also reduced symptoms in FFR. Recent interest in fructose-induced toxicity has centered on the formation of uric acid, which is increased in both humans and rodents following high fructose intake. Drugs that block the synthesis of uric acid can reduce blood pressure and decrease other symptoms of the MS or of fructose feeding. Recent work in our lab has found that etanercept, metformin and prazosin blocked the increase in uric acid levels and blood pressure produced by fructose. Gonadectomized rats fed fructose did not show an increase in either blood pressure or uric acid levels. In summary, FFR exhibit many of the symptoms of the MS, in particular, an increase in blood pressure. Drugs that lower uric acid may prove to be of clinical importance in treating MS.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):18A.

73 Temporal Dystrophic Remodelling within the Intrinsic Cardiac Nervous System of the Streptozotocin Diabetic Rat Model

C Menard ¹, P Fernyhough ¹, M Durston ¹, E Zherebitskaya ¹, G Glazner ¹

Abstract

There is significant evidence to support the existence of “diabetic cardiomyopathy,” described as heart failure (HF) in diabetic individuals in the absence of obstructive coronary disease and hypertension. The underlying pathogenesis is only partially understood, but alterations in the autonomic nervous system’s (ANS) control of cardiac function have been implicated. An important component of the cardiac ANS is the intrinsic cardiac nervous system (ICNS). The ICNS behaves as a neuronal modulator of cardiac function, and has been called the “little brain on the heart”. While there have been several investigations into the effects of diabetes on extracardiac neurons, little is known about the alterations that occur in the ICNS. It is proposed that high glucose concentrations induce toxicity via oxidative stress, resulting in neuronal dystrophy and dysfunction. Our first aim was therefore to confirm that a process of dystrophic remodeling occurs within the ICNS of diabetic hearts. Our second aim was to examine the role of oxidative stress in the pathogenesis of neuronal dystrophy. Our preliminary data indicated that neuronal dystrophy occurs in the ICNS neurons of streptozotocin (STZ)-diabetic rats, and accumulates temporally within the disease process. It was also determined that an increase in reactive oxygen species (ROS) occurs in the neuronal processes of diabetic rats, indicating an association between oxidative stress and the development of a dystrophy. While our preliminary work provides novel insight for diabetes and cardiac research, more investigations are needed to further examine neuronal dysfunction and cell death, and to prove a causative role for oxidative stress in the development of dystrophy.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):18A.

74 Calreticulin and Protein Quality Control in Congential Heart Diseases

Marek Michalak ¹

Abstract

Calreticulin is an endoplasmic reticulum luminal Ca²⁺-buffering chaperone involved in protein quality control. It is generally accepted that the protein is involved in two major functions of the endoplasmic reticulum: regulation of intracellular Ca²⁺ homeostasis and folding of newly synthesized proteins and glycoproteins. Extensive physiological, cell biological, biochemical and molecular biological studies on the function of calreticulin indicate that the protein is a multi-process molecule affecting many cellular functions inside and outside of the ER environment. Calreticulin deficiency in mice is lethal in utero due to defects in heart development and impaired myofibrillogenesis. Calreticulin-deficient cells have disrupted translocation of MEF2C and NF-AT transcription factors due to decreased endoplasmic reticulum Ca²⁺ release, necessary for activation of calcineurin and subsequent translocation of transcription factors. The expression of calreticulin is elevated in the embryonic heart but it is down-regulated after birth. Increased expression of the protein in the developing heart results in AV block reminiscent of the complete heart block in children. Cardiac-specific over-expression of calreticulin in adult mice resulted in rapid progression of dilated cardiomyopathy and heart failure. Transgenic mice exhibited decreased expression level of sarcoplasmic reticulum Ca²⁺ cycling proteins. There was significantly reduced ejection fraction and cardiac output of left ventricle, and decreased systolic and diastolic function of left and right ventricle in calreticulin over-expressers. These findings underscore the importance of calreticulin and endoplasmic reticulum in cardiac physiology and indicate that up-regulation of calreticulin gene leads to dilated cardimyopathy and heart failure.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):19A.

75 Molecular Dynamics Underlying Conjugated Linoleic Acid-Induced Skeletal Muscle Glucose Transport

S Mohankumar ¹, C Taylor ¹, L Siemens ¹, P Zahradka ¹

Abstract

Conjugated linoleic acid (CLA), a dietary lipid, has been proposed as an anti-diabetic/obesity agent. However, studies addressing the mechanisms of CLA on skeletal muscle glucose transport are limited. Our study investigated the cellular dynamics of cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12) CLA isomers using L6 myotubes. Cells were treated without or with CLA isomers for 15 minutes and subsequently monitored for glucose uptake using isotope/fluorescently-labelled 2-deoxyglucose, intracellular Ca²⁺ (Cai²⁺) release using Fluo-4 AM and GLUT4 translocation using immunofluorescence as well as protein phosphorylation events using Western blotting. Acute exposure of myotubes to CLA stimulated the GLUT4 trafficking and glucose uptake by activating insulin-dependent signals, including phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit and Akt substrate-160 kDa (AS160). Intriguingly, t10,c12-CLA stimulated Cai²⁺ release and phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and AMP-activated protein kinase (AMPK) in a concentration-dependent manner, whereas c9,t11-CLA showed modest or no effects. Blocking PI3-kinase, Cai²⁺ release, CaMKII and AMPK abrogated CLA isomer-mediated AS160 phosphorylation and glucose uptake. Genetic knock down of CaMKII in myotubes using siRNA completely abolished CLA isomer-mediated glucose uptake. Furthermore, the evidence for a positive correlation between CaMKII and AMPK, in conjunction with inhibition of t10,c12-CLA-mediated AMPK activation by CaMKII blockers, indicates that CaMKII acts upstream of AMPK. These data establish that t10,c12-CLA acts via Cai²⁺-CaMKII-AMPK-AS160 to stimulate skeletal muscle glucose transport, whereas the mechanism of c9,t11-CLA remains unclear.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):19A.

76 Discovery of the Mechanism that Enables the Provision of Nutrients to Prolferating Cells

Salvador Moncada ¹

Abstract

Cell proliferation is accompanied by an increase in the utilization of glucose and glutamine. The proliferative response is dependent on a decrease in the activity of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdh1 which controls G1- to S-phase transition by targeting degradation motifs, including the KEN box. This occurs not only in cell cycle proteins but also in the glycolysis-promoting enzyme 6-phospho-fructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), as we have recently demonstrated in cells in culture as well as in proliferating human T lymphocytes. Moreover, we have found that glutaminase 1 is a substrate for this ubiquitin ligase and appears at the same time as PFKFB3 in proliferating cells. Glutaminase 1 is the first enzyme in glutaminolysis, which converts glutamine to lactate, yielding intermediates for cell proliferation. Thus APC/C-Cdh1 is responsible for the provision not only of glucose but also of glutamine and, as such, accounts for the critical step that links the cell cycle with the metabolic substrates essential for its progression. The relevance of these observations to understanding of the proliferative and metabolic changes that occur in cancer will be discussed.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):19A.

77 Resveratrol Normalizes SERCA2a Protein Content and Function in the Diabetic Heart

Marc P Morissette ^1,², Shanel E Susser ^1,², Riley A Epp ^1,², Darrell R Smith ^3,⁴, Paul Fernyhough ^3,⁴, Todd A Duhamel ^1,²

Abstract

Abnormal calcium transport and the down-regulation of sarcoplasmic reticulum calcium ATPase (SERCA2a) expression in the heart is known to directly contribute to diabetic cardiomyopathy. Resveratrol (RES) has previously been shown to improve cardiovascular function in diabetic rats. Therefore, we hypothesized that resveratrol supplementation would normalize SERCA2a protein expression and function in the type 1 diabetic heart. Twenty-four Sprague Dawley rats were split into one of three groups. The first group were age matched control animals (CON). The second group were made diabetic by injecting them with streptozotocin (STZ). The third group of rats were also injected with STZ, maintained for 9 weeks and then received resveratrol (5 mg/kg body wt) by gavage daily for the final 9 weeks of the study (STZ+RES). Left ventricle samples were collected and prepared for Western blotting and biochemical analyses. As expected, diabetes reduced SERCA2a protein content by 44% enhanced the ratio of phospholamban monomers by 5-fold, and inhibited SERCA2a Ca²⁺-sensitivity (Ca₅₀, 243±27 nM for STZ, as compared to 382±56 nM for CON). Resveratrol-treatment for the final 9 weeks of the study rescued each parameter. In conclusion, resveratrol supplementation counteracts the down regulation of SERCA2a protein expression and function in the diabetic heart.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):19A.

78 TNF-Regulated Cell Survival via HDAC-1 Regulated NF-kB Signaling Pathway in Ventricular Myocytes.

W Mughal ¹, H Gang ¹, R Dhingra ¹, Y Wang ¹, LA Kirshenbaum ¹

Abstract

Epigenetic changes by histone modifying proteins influence chromatin structure and gene transcription. The impact of histone deacetylases (HDACs) on the regulation of gene expression essential for cell survival in cardiac myocytes remains cryptic. In this report we provide new compelling evidence that targeted inactivation of the transcriptional activation domain of NF-kB by HDAC1 provokes autophagy and cell death of post-natal ventricular myocytes induced by TNF. Cells treated with TNF displayed an increase in NF-kB gene transcription with no apparent change in cell viability. NF-kB activity and basal cell survival was markedly decreased in cells in presence of HDAC1 compared to vector control cells or cells treated with TNF alone. Impairment of NF-kB dependent gene transcription by HDAC1 increased basal apoptosis and reduced cell viability. Interestingly, HDAC1 mediated repression of NF-kB signaling was accompanied by mitochondrial perturbations including loss in mitochondrial membrane potential, and permeability transition pore. Inhibition of HDAC1 activity with trichostatin A or mutations of HDAC1 (H141A) defective for histone deacetylation abrogated the inhibitory effects of HDAC1 on NF-kB and autophagy gene expression. IKKB-mediated activation of NF-kB signaling pathway suppressed mitochondrial perturbations and autophagy down-stream of TNF signaling. To our knowledge, our data provide the first direct evidence that TNF-regulated NF-kB signaling via HDAC-1 promotes cell survival of ventricular myocytes.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):19A–20A.

79 Impact of Thermal Therapy on Chronic Heart Failure

T Mussivand ¹, U Mustafa ¹

Abstract

BACKGROUND:

Thermal therapy utilizing heat energy to increase core body temperature up to 1°C has shown benefit in chronic heart failure patients. This increase in temperature sets off several physiological pathways by inducing nitric oxide, heat shock proteins, antioxidants and others. This has the potential to positively impact pathological issues associated with heart failure leading to improved clinical outcome. Far infrared radiation sauna and warm water immersion are two common approaches for thermal therapy. We are working on a method to increase core body temperature at will.

OBJECTIVE:

The aim of this research is to identify, analyze and report improvements in the clinical outcome of patients treated with thermal therapy.

METHODS:

51 published articles from 1980 to 2011 are reviewed, which focus on the use of thermal therapy in chronic heart failure patients from all NYHA classes.

RESULTS:

Thermal therapy has a positive impact and improves clinical parameters including; 1) ejection fraction, 2) stroke volume, 3) systolic/diastolic blood pressure, 4) cardiac dimensions, 5) cardiac volumes, 6) intracardiac pressures, 7) vascular endothelial functions, 8) ventricular arrhythmias, 9) neuroendocrine functions, 10) other disease related signs and symptoms and 11) prognosis of chronic heart failure. Upregulation of nitric oxide synthesis, heat shock proteins and antioxidants enzymes are examples of imp rovements on the molecular level after thermal therapy treatment. No negative side effects were reported.

CONCLUSION:

Thermal therapy may be an effective, low cost and non-invasive modality for heart failure patients. It has a positive impact on disease process and clinical outcomes of chronic heart failure. More clinical trials are needed to further illuminate the impact of thermal therapy on heart failure.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):20A.

80 The Role of Tissue Doppler Imaging Using Transesophageal Echocardiography in the Non-Invasive Assessment of Left Ventricular Filling Pressures During Cardiac Surgery

RG Nepomuceno ¹, K Kumar ¹, M Golian ¹, A Chalvanathan ¹, M Zeglinski ¹, S Bohonis ¹, S Mackenzie ¹, DS Jassal ¹

Abstract

BACKGROUND:

Cardiac surgical patients often require invasive pulmonary artery catheters (PAC) to determine left ventricular filling pressures (LVFP) in order to guide fluid management. Tissue Doppler imaging (TDI) as derived by transthoracic echocardiography (TTE), non-invasively assesses pulmonary capillary wedge pressure (PCWP), which is a surrogate for LVFP. With the use of TDI as derived by TTE, the ratio of transmitral inflow to mitral annular velocity during early diastole (E/E’) correlates with PCWP. However, little is known on the role of TDI using intra-operative transesophageal echocardiography (TEE) in the assessment of LVFP during cardiac surgery. Our objective was to determine if TDI indices obtained by intra-operative TEE during cardiac surgery accurately estimates PCWP using PAC as the gold standard.

METHODS:

Patients undergoing cardiac surgery were prospectively enrolled at a single tertiary care centre. Conventional and novel echocardiographic parameters were evaluated using intra-operative TEE with concurrent invasive PAC monitoring before and after cardiopulmonary bypass (CPB).

RESULTS:

The study population included 34 patients (26 males, mean age 64±9 years). The correlation between E/E’ using TEE and PCWP was poor to modest at best. Pre-CPB, the correlation ratio between mean E/E’ and PCWP was r=0.23. Post-CPB, the correlation ratio between mean E/E’ and PCWP was r=0.42.

CONCLUSION:

Intra-operative TEE was unable to accurately assess LVFP in patients undergoing cardiac surgery. The PAC continues to be the gold standard in the assessment of LVFP for this patient population.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):20A.

81 Resveratrol Prevents the Development of Hypertension-Induced Cardiac Impairment

Thomas Netticadan ¹

Abstract

OBJECTIVES:

Current treatments for hypertension and resultant heart failure have proven inadequate. Accordingly, it is of paramount importance to explore new strategies to prevent its development. Food-derived compounds, and in particular polyphenols, are gaining attention due to their cardioprotective properties. We examined the effect of the polyphenol resveratrol, present predominantly in grapes, in preventing the development of pathological cardiac hypertrophy and its deleterious effects on heart function.

METHODS:

In vivo studies: 10-week old spontaneously hypertensive rat (SHR), an established animal model of hypertension and heart failure, and its appropriate controls, were treated with and without resveratrol (2.5 mg/kg/day) for a period of 10 weeks. Echocardiography was done to assess cardiac structure and function in all animals. In vitro studies: normal adult rat heart cardiomyocytes were exposed to a high dose of norepinephrine (0.25 μM) a potent inducer of pathological hypertrophy. Norepinephrine exposed cardiomyocytes and its appropriate controls were treated with and without resveratrol (30 μM). To examine the mechanisms underlying resveratrol action, the effects of pharmacologic blockers of established anti-hypertrophic molecules nitric oxide (NO), AMPK and sirtuins were assessed in resveratrol treated norepinephrine exposed cardiomyocytes Morphometric analysis and protein synthesis measurements were conducted to characterize cardiomyocyte structure in all groups.

RESULTS AND CONCLUSIONS:

Pathological cardiac hypertrophy and impairment of heart function was observed in 20 week SHR; pre-treatment with resveratrol prevented these abnormalities. Exposure to norepinephrine resulted in cardiomyocyte hypertrophy; pre-treatment with resveratrol prevented this defect. Inhibitors of NO and AMPK, but not sirtuins, abolished the beneficial effects of resveratrol. On the basis of these results, we conclude that resveratrol may have potential in preventing the development of cardiac impairment in patients with hypertension. The beneficial effects of resveratrol appear to be mediated by the NO-AMPK signalling.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):20A.

82 Non-Muscle Myosin II Inhibition Attenuates Collagen Gel Contractility and Reduces Cell Viability in Human Bone Marrow Derived Mesenchymal Stem Cells

MA Ngo ¹, Y Li ¹, S Neumann ¹, RC Arora ¹, DH Freed ¹

Abstract

Matrix elasticity is important in mesenchymal stem cell (MSC) differentiation. We have shown that human MSCs adopt a myofibroblastic phenotype in culture, which may limit their capacity for further differentiation in vivo. Blebbistatin (BB), a specific inhibitor of non-muscle myosin II (NMM-II), may play a key role in modifying differentiation and proliferation in vitro as it abrogates the response of MSCs to matrix elasticity. We studied the effects of BB, NMM-IIA and IIB knockdown on human MSCs in vitro with respect to their contractility, viability and phenotype.

The protocol received IRB approval. MSCs were isolated from patients undergoing open heart surgery and cultured in standard culture conditions. Cells were plated onto collagen gel substrates, transfected, serum starved and treated with either TGFβ1 (10 ng/mL), BB (10 or 50 μM/mL), or both. The reduction in gel surface area and cell viability was measured. Phenotypic changes were assessed using immunofluorescent staining.

Human MSCs were found to express both NMM-IIA and IIB, but not IIC. BB treatment (50 μM/mL) completely inhibited basal and TGFβ1 induced contractility. Cell viability was also reduced, but was restored when co-treated with TGFβ1. Individual NMM-IIA or IIB siRNA knockdown caused no significant change in contractility or viability.

Human MSC adopt a myofibroblast phenotype in culture and display similar physiology to human cardiac fibroblasts with respect to collagen gel contraction. Inhibition of NMM-II reduces cell survival and limits myofibroblast function. Inhibition of individual NMM-II isoforms alone does not alter gel contraction and viability, indicating redundancy in the function of both isoforms.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):20A–21A.

83 Blebbistatin Attenuates Collagen Gel Contractility and Reduces Cell Viability in Human Bone Marrow Derived Mesenchymal Stem Cells

Melanie A Ngo ¹, Yun Li ¹, Shannon Neumann ¹, Rakesh C Arora ¹, Darren H Freed ¹

Abstract

BACKGROUND:

Previous data from our lab suggest that human bone marrow derived mesenchymal stem cells (MSCs) adopt a myofibroblastic phenotype in culture, which may limit their capacity for further differentiation in vivo and subsequently, their potential therapeutic use. This may be due to the effect of matrix elasticity in vitro. Blebbistatin (BB), a specific inhibitor of non-muscle myosin II (NMM-II), may play a key role in modifying differentiation and proliferation in vitro as it abrogates the response of MSCs to matrix elasticity. We examined the effects of BB and non-muscle myosin IIB knockdown on human bone marrow derived MSCs in vitro with respect to their contractility, viability and phenotype.

METHODS:

The protocol received IRB approval. Bone marrow derived MSCs were isolated from patients undergoing open heart surgery. Cells were cultured in standard DMEM F12 with 20% FBS conditions and plated onto collagen gel substrates. Cells were transfected with NMM-IIB siRNA, serum starved for 24 hours and treated with either TGFβ1 (10 ng/mL), BB (10 or 50 μM/mL), or both for an additional 24 hours. The reduction in gel surface area was then analyzed and cell viability was assessed using the live dead assay. Phenotypic changes following BB treatment was assessed using immunofluorescent staining.

RESULTS:

Compared to non-treated cells, low dose BB treatment (10 μM/mL) significantly reduced contractility while preserving its TGFβ1 responsiveness. However, high dose BB treatment (50 μM/mL) completely inhibited contractility and the cell’s TGFβ1 responsiveness. Further, high dose BB treatment significantly reduced cell viability, but was restored when treated in conjunction with TGFβ1. No significant changes in both contractility and viability were observed with NMM-IIB knockdown.

CONCLUSION:

Human MSC adopt a myofibroblast phenotype in culture and display similar physiology to human cardiac fibroblasts with respect to collagen gel contraction. Inhibition of NMM-II reduces cell survival and limits myofibroblast function. Specifically, inhibition of NMM-IIB alone does not play a major role in gel contraction and viability, thus suggesting that other NMM-II isoforms may be responsible.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):21A.

84 Carvedilol Suppresses Fatty Acid Oxidation and Stimulates Glycolysis in C2C12 Cells

A Onay-Besikci ¹, E Suzmecelik ¹, AT Ozcelikay ¹

Abstract

Beta adrenergic receptor blocking drugs (β-blockers) are used chronically in many cardiovascular diseases such as hypertension, ischemic heart disease, arrhythmia, and heart failure. Beneficial effects arise from the inhibition of sympathetic nervous system hyperactivity, reduction of heart rate, and remodeling by blocking the mitogenic effect of catecholamines. A possible effect of β-blockers on cardiac substrate metabolism has also been suggested. The direct effects of β-blockers on mouse C2C12 cells were investigated in this study. C2C12 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and differentiated into myotubes in the same medium that contained 1% FBS. Palmitic acid oxidation and glycolysis were measured by using [9,10-3H]palmitate and [5-3H] glucose, respectively. The amount of ³H₂O as an indicator of substrate usage was measured. Carvedilol inhibited palmitate oxidation and increased glycolysis by nearly 50%. Prazosin altered substrate metabolism in a similar fashion as carvedilol whereas propranolol or bisoprolol were devoid of metabolic effects. When added to mimic sympathetic activation, epinephrine stimulated glycolysis but did not alter fatty acid oxidation. Based on these results, carvedilol appears to have direct effects on substrate metabolism that are related to the blockade of α1 adrenergic receptors.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):21A.

85 Hormones and the Heart: Promises, Problems and Unresolved Issues

Suzanne Oparil ¹

Abstract

The relationship between sex hormones and cardiovascular function and disease has long been recognized. As early as the 1950s, researchers concluded that although levels of cholesterol played a major role in the development of cardiovascular disease, other factors, including gender and hormones, played a role as well. Since that time, despite extensive research focusing on the effects of estrogen on vascular function, the relationship remains poorly understood. Furthermore, clinical treatment of postmenopausal women with hormone replacement therapy continues to be controversial due to conflicting findings in clinical trials.

Until the 1990s, extensive observational data suggested that hormone replacement therapy was cardioprotective. However, results from the Heart and Estrogen/Progestin Replacement Study (HERS-I and II) did not confirm a protective effect of hormone replacement therapy on the heart. Later, data from the Women’s Health Initiative reported an increase in coronary heart disease risk in women treated with combined estrogen-progestin compared to placebo, while the Women’s Health Initiative unopposed-estrogen arm showed no increase in coronary heart disease events. Since release of the Women’s Health Initiative, follow-up analyses have shown that the timing of initiation of hormone replacement therapy makes a difference in outcomes. These analyses showed that younger postmenopausal women who initiate therapy at the time of menopause are not at increased risk of coronary heart disease events compared to women who initiate therapy at a later age.

This presentation will discuss the pathophysiologic effects of sex hormones on the vasculature, describe both clinical and basic research that has led us to our current understanding, and conclude with future perspectives on avenues of investigation that may lead to innovative treatments for postmenopausal women.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):21A.

86 Gender Differences in Cardiac Ischemia and Protection

B Ostadal ^1,², I Netuka ^1,³, J Besik ^1,³, I Ostadalova ^1,²

Abstract

It has been observed that significant gender differences exist already in normal heart. They involve among others cardiac growth, contractile function, calcium metabolism and function of mitochondria. Differences, characteristic of the normal myocardium, generate the logical presumption of the different reaction of the male and female heart to various pathogenic factors. Most of the experimental studies confirm the clinical observations: increased resistance of the female heart to ischemia/reperfusion injury was shown in dogs, rats, mice and rabbits. Furthermore, sex differences in the ischemic tolerance of the adult myocardium can be influenced by interventions (eg, hypoxia) imposed during the early phases of ontogenetic development. The already high tolerance of the adult female heart can be increased by adaptation to chronic hypoxia and ischemic preconditioning. It seems, however, that the protective effect depends on age: it was absent in young, highly tolerant heart but it appeared with the decrease of natural resistance during aging. Both experimental and clinical studies have indicated that female gender influences favorably also the adaptive response to myocardial infarction. Detailed molecular and cellular mechanisms of sex differences are still unknown; they involve genomic and non-genomic effects of sex steroid hormones, particularly estrogens. The cardiovascular system is, however, influenced not only by estrogens but also by other sex hormones, e.g. androgens. Moreover, steroid hormone receptors do not act alone but interact with a broad array of co-regulatory proteins to alter transcription. The differences deserve serious consideration in clinical practice in search for proper diagnostic and therapeutic procedures.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):21A–22A.

87 Women and the Management of Acute Coronary Syndrome

P Ostadal ¹

Abstract

In contrast to general perception, coronary heart disease (CHD) continues to be the leading cause of death among women in the developed countries. Moreover, mortality from CHD has almost not declined in women in the last years as it has in men. Despite these facts, females are still under-represented in the majority of clinical trials. It is not rare that women respond to cardiovascular therapeutic intervention differently from men; current literary evidence for the female-specific management in acute coronary syndrome (ACS) is still insufficient. At the time of presentation of ACS, women are typically older and more likely to have hypertension, diabetes and metabolic syndrome, but are less likely to smoke tobacco than men. Women with ACS are less frequently than men to undergo cardiac catheterization and subsequent revascularization procedure or to be discharged on evidence-based therapies. It has been shown, however, that women benefit from an invasive approach in ACS as much as men, despite their advanced age and slightly higher rate of bleeding complications. Also, administration of beta-blockers and statins is associated with comparable reduction of cardiovascular event rates in women and men. On the other hand, women may profit less than men from ACE-inhibitor therapy and experience side-effects more often. Regarding the antiplatelet agents, whereas both sexes benefit more or less equally from aspirin therapy, contradictory data are available on the efficacy of clopidogrel in women. It became evident that current practice in the management of CHD in women has clearly insufficient efficacy. There is a strong urgency for intensive gender differences research, and for development of a female-specific therapeutic strategy in ACS.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):22A.

88 Pulmonary and Systemic Vasodilator Responses to the Soluble Guanylyl Cyclase Activator, BAY 60-2770, Are Not Dependent on Endogenous Nitric Oxide or Reduced Heme

EA Pankey ¹, SN Murthy ¹, PJ Kadowitz ¹

Abstract

BAY 60-2770 is a nitric oxide (NO)-independent activator of soluble guany-lyl cyclase (sGC) that increases the catalytic activity of the heme-oxidized or heme-free form of the enzyme. In this study, responses to intravenous injections of the sGC activator BAY 60-2770 were investigated under baseline and elevated tone conditions induced by the thromboxane mimic U-46619 when NO synthesis was inhibited by N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME), when sGC activity was inhibited by ODQ, an agent that oxidizes sGC, and in animals with monocrotaline-induced pulmonary hypertension. The intravenous injections of BAY 60-2770 under baseline conditions caused small decreases in pulmonary arterial pressure, larger decreases in systemic arterial pressure, and no change or small increases in cardiac output. Under elevated tone conditions during infusion of U-46619, intravenous injections of BAY 60-2770 caused larger decreases in pulmonary arterial pressure, smaller decreases in systemic arterial pressure, and increases in cardiac output. Pulmonary vasodilator responses to BAY 60-2770 were enhanced by L-NAME or by ODQ in a dose that attenuated responses to the NO donor sodium nitroprusside. ODQ had no significant effect on baseline pressures and attenuated pulmonary and systemic vasodilator responses to the sGC stimulator BAY 41-8543. BAY 60-2770 and sodium nitroprusside decreased pulmonary and systemic arterial pressures in monocrotaline-treated rats in a nonselective manner. The present data show that BAY 60-2770 has vasodilator activity in the pulmonary and systemic vascular beds that is enhanced by ODQ and NOS inhibition, suggesting that the heme-oxidized form of sGC can be activated in vivo in an NO-independent manner to promote vasodilation.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):22A.

89 Thyroid Hormone and Cardiac Regeneration: From Prometheus Myth to Reality?

Constantinos Pantos ¹

Abstract

Nature’s models of regeneration provide substantial evidence that a natural healing process may exist in the heart. The potential for regeneration has been evolutionarily conserved in mammals and seems to be restricted to early developmental stages. This window of regeneration is reactivated during the disease state in which fetal gene reprogramming occurs in response to stress. Analogies exist between damaged and developing heart, indicating that a regulatory network which drives embryonic heart development may control aspects of heart regeneration. In this context, TH which is a critical regulator of the maturation of the myocardium appears to have a regenerative role later in adult life. Changes in thyroid hormone (TH)-thyroid hormone receptor (TR) homeostasis govern the return of the injured myocardium to the fetal phenotype. Accordingly, TH can induce cardiac regeneration by reactivating developmental gene programming. As a proof of concept, TH is found to be an independent determinant of functional recovery and mortality in patients with myocardial infarction. The potential of TH to regenerate/repair the myocardium is now awaited to be tested in large clinical trials.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):22A.

90 Cardiomyocyte Cell Cycle Regulation for Myocardial Repair

Kishore BS Pasumarthi ¹

Abstract

In adults, damaged myocardium is replaced by scar tissue because surviving cardiomyocytes cannot proliferate. In light of this issue, several groups have examined the effects of various interventions on cell cycle activity in embryonic and postnatal cardiomyocytes. To date, only a few factors were shown to stimulate the cardiomyocyte cell cycle in experimental models of myocardial injury. We showed that targeted expression of cyclin D2 (cycD2) can induce cardiomyocyte cell cycle activity in the infarct border zone and partially reduce infarct size, as well as improve myocardial function 5–6 months post-myocardial infarction (MI). However, additional studies are required to increase the magnitude of cardiac regeneration and functional restoration after myocardial injury. Thus, the goal of our research is to examine whether the combinatorial expression of multiple cell cycle proteins can increase regenerative capacity of adult cardiomyocytes. Further, we have found a novel cell cycle regulatory pathway in embryonic cardiomyocytes. This pathway is coupled to cell cycle exit via protein aggregation and endoplasmic reticulum (ER) stress associated protein degradation (ERAD) and is induced by overexpression of an alternative splice variant form of cyclin D2 (D2SV). We have also found that D2SV is upregulated during hypertrophy and myocardial infarction. Our preliminary data suggest that the cell cycle regulation via ER stress may be a major block for inducing a regenerative response in the mammalian heart. We are currently studying the mechanisms of D2SV cell cycle exit and determining the effects of blocking D2SV expression on cardiomyocyte cell cycle activity. These studies are useful to identify new targets for therapeutic manipulation of myocardial repair.

Footnotes

Supported by the Heart and Stroke Foundation of Nova Scotia

Exp Clin Cardiol. 2011 Oct;16(Suppl A):22A.

91 Effect of Nuclear Factor Kappa B Inhibition on Cardiovascular Remodelling In Experimental Hypertension

Olga Pechanova ¹, Stanislava Vrankova ¹, Jana Parohova ¹, Andrej Barta ¹, Pavol Janega ¹

Abstract

We aimed to analyze effects of nuclear factor-kappa B (NF-κB) inhibition on blood pressure (BP) regulation and cardiovascular remodelling. Adult 12-week-old male Wistar Kyoto rats (WKY) were treated with the nitric oxide synthase (NOS) inhibitor N^G-nitro-L-arginine-methyl ester (L-NAME, 40 mg/kg/day) for seven weeks. From the fourth week of L-NAME treatment, the NF-κB inhibitor lactacystin (1 mg/kg) was applied once a week. Furthermore, age-matched WKY received L-NAME or lactacystin alone for 7 or 3 weeks, respectively. Total NOS activity was determined in the left ventricle (LV) and aorta. The concentration of conjugated dienes, fibrosis, and collagen I and III levels were determined in the LV. The cross-sectional area (CSA) and wall thickness to internal diameter ratio (WT/ID) were measured in the aorta. L-NAME treatment increased BP significantly (145±2 mmHg vs. 110±3 mmHg in controls). The addition of lactacystin resulted in further significant increase in BP (161±3 mmHg). Similarly, lactacystin potentiated the increased conjugated dienes concentration induced by LNAME. Whereas L-NAME alone did not affect NOS activity, the addition of lactacystin decreased it in both tissues investigated. The addition of lactacystin did not affect LV hypertrophy, fibrosis, and collagen I and III, already increased by L-NAME; however, it further amplified CSA in the aorta increased by L-NAME alone. WT/ID increased significantly only after the addition of lactacystin. In conclusion, decreased NOS activity along with increased oxidative load may be responsible for decreased NO bioavailability and further BP increase after NF-κB inhibition in L-NAME-induced hypertension. Increased CSA and WT/ID could contribute to this hypertensive process.

Footnotes

Supported by grants APVV-0538-07, APVV-0742-10 and VEGA: 2/0190/11, 2/0178/09

Exp Clin Cardiol. 2011 Oct;16(Suppl A):22A–23A.

92 Cardiac Calsequestrin Plays a Pivotal Role in SR Calcium Transport and RyR Mediated Ca²⁺ Release

Muthu Periasamy ¹, Anuradha Kalyanasundaram ¹, Sandor Gyórke ¹

Abstract

Calsequestrin (CASQ2) was discovered nearly three decades ago but its role in cardiac calcium transport is just beginning to be understood. CASQ2 is the primary Ca²⁺ buffering protein in the cardiac SR lumen and maintains a fine balance between free vs. bound calcium ion concentration. CASQ2, along with Triadin (TRD1) and Junctin forms a lumenal regulatory complex and controls Ryanodine receptor (RyR2) activity including channel opening, closing and refractoriness. Recent studies have clearly demonstrated that mutations in CASQ2 can result in altered RYR2 function/Ca²⁺ leak and triggered arrhythmias, especially CPVT (Catecholamine induced Polymorphic Ventricular Tachycardia). In this context we have been studying the importance of CASQ2 using a CASQ2 KO mouse model. Although, initial studies from CASQ2 KO mice suggested that despite loss of CASQ2, the mice maintained cardiac function and Ca²⁺ handling is kept near normal, but were quite susceptible to triggered arrhythmias by catecholamines. However new data from our lab show that loss of CASQ2 predisposes mice to develop cardiac hypertrophy upon aging; we additionally obtained new exciting data to show that when Ca²⁺ load was increased by either SERCA overexpression or PLB ablation, these mice exhibit severe RYR2 mediated Ca²⁺ leak, develop cardiac hypertrophy and die of sudden cardiac death. These data provide novel information to suggest that CASQ2 plays in an important role in SR Ca²⁺ buffering function (Load) and stabilization of the Ryanodine receptor, thereby maintaining a fine balance between SR-load and release.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):23A.

93 Infection, Oxidized LDL and Atherosclerotic Heart Disease

GN Pierce ¹, JF Deniset ¹, E Dibrov ¹, MN Chahine ¹

Abstract

The direct atherogenic action of C pneumonia infection has now been identified. Circulating cholesterol can strongly modulate these atherogenic effects but the molecular mechanisms responsible for this are not clear. We have explored the mechanism whereby C pneumonia induces atherosclerosis and what factor within the high circulating cholesterol may be responsible for the effects. C pneumonia infection is associated with an increase in cell proliferation in the vasculature and this was strongly influenced by oxidized LDL. An induction of HSP60 expression was also involved in the mitogenic effects. The mitogenic effects of C pneumonia result in a thickening of the vascular wall under tissue culture conditions. This does not involve a host immune response but appears to be induced by a direct action on the vasculature. These effects are also accompanied by changes in vascular contractile function. Our data strongly support a role for infectious disease, and C pneumonia specifically, in atherogenic cardiovascular disease.

Footnotes

Supported by CIHR

Exp Clin Cardiol. 2011 Oct;16(Suppl A):23A.

94 Drugs in Pregnancy: Implications for a Cardiologist

K Rakusan ¹

Abstract

Therapeutic drugs are and will continue to be used during pregnancy and, in most cases, their use is justified. There are several clinical situations in which drug therapy may be necessary throughout pregnancy. Nevertheless, such use may result in various adverse events as presented. The safe use of drugs (including cardiovascular) during pregnancy depends on several factors including the time of application, the type of drug and its dose. Thus, the anticipated risks and benefits need to be carefully evaluated and described to the patients, before the administration of the drug, so that they can make an informed decision.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):23A.

95 Can Circulating Monocytes be used to Predict Risk of Macrovascular Disease in Type 2 Diabetes?

Surya Ramachandran ¹, Anila Venugopal ¹, K Satisha ¹, NS Prathapachandran ¹, Ajit Mullasari ¹, MR Pillai ¹, CC Kartha ¹

Abstract

Hyperglycemia induced proteome changes in monocytes is unknown despite a variety of studies on the mechanisms of endothelial and monocyte activation in diabetes.

Hyperglycemia is widely recognized as a potent stimulator of monocyte activity. To investigate whether monocytes under hyperglycemia secrete proteins significant to monocyte – endothelial interactions, we did a proteomic analysis of monocytes, primed with high glucose (HG). Monocytic cells (THP-1) were cultured in HG medium for 72 hours and their protein profile was analyzed using 2D gel electrophoresis. Fifteen spots were differentially expressed, which were identified using MALDI TOF/MS. One protein that revealed a marked decrease in expression as glucose concentration increased was identified as Cyclophilin A (CypA), which was validated by immunoblotting experiments. qRT-PCR assays revealed that the cellular mRNA level of this protein decreased in a dose-dependent manner. We have also found a relatively decreased expression of CyPA in monocytes of patients with type 2 diabetes mellitus. We conclude that CyPA is a secretory factor released by monocytes in response to oxidative stress induced by HG. CyPA has both paracrine and autocrine stimulatory effect on endothelial cells and vascular smooth muscle cells. CyPA secreted by monocytes could enhance monocyte adhesion to endothelial cells and, thus, have a role in the pathogenesis of atherosclerosis in type 2 diabetes. Whether CyPA could be a marker for monocyte activation in type 2 diabetes warrants scrutiny.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):23A.

96 Role of Histone Deacetylation and AMPK in Cardiac PGC-1-Alpha Gene Expression in Hypoxia and Ischemia

A Ramjiawan ¹, RA Bagchi ¹, L Albak ¹, A Blant ¹, MA Cavasin ², TR Horn ², TA McKinsey ², MP Czubryt ¹

Abstract

The transcriptional regulator PPARg coactivator-1a (PGC-1a) responds to signals indicating a deficient energy state or increased cellular workload, and in turn induces genes to augment mitochondrial biogenesis and fatty acid oxidation. Loss of PGC-1a expression results in mitochondrial dysfunction, and is associated with cardiac diseases including heart failure. We examined PGC-1a expression in oxygen (hypoxia) or fuel (ischemia) deficiency in isolated neonatal rat cardiomyocytes. Cells readily tolerated hypoxia up to 12 hours, but showed reduced PGC-1a mRNA expression concomitant with promoter histone deacetylation. Histone deacetylase inhibition during hypoxia restored PGC-1a expression, as did over-expression of estrogen-related receptor a (ERRa), another regulator of cardiac metabolic genes. We identified a conserved ERRa consensus binding site in the PGC-1a promoter and demonstrated via luciferase and chromatin immunoprecipitation assays that ERRa directly regulates PGC-1a expression. Extension of hypoxia to 24 hours, inducing ischemia as glucose in the culture medium is exhausted, resulted in an induction of PGC-1a expression that was augmented in glucose-free medium. Inhibition of AMP kinase (AMPK) blocked this effect, and ischemia resulted in phosphorylation of AMPK. A mouse model of hypobaric hypoxia demonstrated a significant decrease in PGC-1a mRNA and protein expression, confirming our in vitro data that oxygen deprivation is a negative regulator of PGC-1a in cardiac muscle. Our results indicate that PGC-1a expression responds to independent signals due to oxygen or fuel deprivation, and may contribute to the understanding of how PGC-1a expression is lost in the failing heart.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):23A–24A.

97 Prevention of Cardiovascular Disease and Diabetes Mellitus in Low and Middle Income Countries

SS Rastogi ¹, RB Singh ¹

Abstract

Hyperglycemia, as a component of metabolic syndrome, appears to be an important risk marker of vascular disease in most developing countries that are under transition from poverty to affluence. Despite a moderate increase in fat intake and low rates of obesity, the risk of coronary artery disease (CAD) and diabetes is rapidly increasing in most of the developing economies. It is a paradox that in some of these countries the increased risk of people to diabetes and CAD, especially at a younger age, is difficult to explain by conventional risk factors. It is possible that the presence of new risk factors, especially higher lipoprotein(a) (Lpa), hyperhomocysteinemia, insulin resistance, low high density lipoprotein cholesterol and poor nutrition during fetal life, infancy and childhood, may explain at least in part, the cause of this paradox. The prevalence of obesity, central obesity, smoking, physical inactivity and stress are rapidly increasing in low and middle income populations, due to economic development. In high income populations, there is a decrease in tobacco consumption, increase in physical activity and dietary restrictions, due to learning of the message of prevention, resulting into reduction in coronary and sroke mortality. Hypertension (5–10%), diabetes (3–5%) and CAD (3–4%) are very low in the adult, rural populations of India, China and in the African sub-continent, which has less economic development. However, in urban and immigrant populations of India and China, the prevalence of hypertension (>140/90, 25–30%), diabetes (6–18%) and CAD (7–14%) are significantly higher than they are in some of the high income populations. Mean serum cholesterol (180–200 mg/dl), obesity (5–8%) and dietary fat intake (25–30% en/day) are paradoxically not very high and do not explain the cause of increased susceptibility to CAD and diabetes in some South Asian countries. The force of lipid-related risk factors and refined starches and sugar appears to be greater in these populations due to the presence of the above factors and results into CVD and diabetes at a younger age in these countries. These findings may require modification of the existing American and European guidelines, proposed for prevention of CAD, in high income populations. Wild foods or designer foods (400–500 g/day) substitution (www.columbus-concept.com) for proatherogenic foods; in conjunction with moderate physical activity and cessation of tobacco, may be protective against deaths and disability due to CVD and diabetes in most of these countries.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):24A.

98 Lifestyle-Related Risk Factors and their Impact on Cardiac Response to Acute Ischaemia: Possibilities to Restore Impaired Ischaemic Tolerance of the Heart

T Ravingerova ¹, A Adameova ¹, S Carnicka ¹, V Ledvenyiova ¹, M Nemcekova ¹, E Galatou ², E Barlaka ², A Lazou ²

Abstract

Myocardial response to ischaemia may be altered by lifestyle-related risk factors (RF) including, besides metabolic disorders and stress, chronically elevated blood pressure leading to left ventricular hypertrophy. The risk is increasing with age, in particular, in females. On the other hand, some stressful stimuli including reactive oxyden species, hypoxia and high glucose, play a dual role in the pathogenesis of ischaemia/reperfusion injury (IRI) and trigger adaptive processes leading to enhanced ischaemic tolerance. Previously, suppression of endogenous protection against myocardial IRI by RF has not been considered. We aimed to verify the hypothesis that RF of lifestyle, such as hyperlipidemia and/or hypertension, may modify cardiac response to acute ischaemia not only by interference with pathophysiological mechanisms of IRI per se, but by via suppression of intrinsic adaptive mechanisms known as preconditioning (PC). While hypertrophied hearts of hypertensive (SHR) rats, male counterparts of age-matched females were more sensitive to IR manifested by a larger extent of irreversible injury (infarct size), acute STZ-induced diabetes (recently termed as metabolic preconditioning) or high fat-cholesterol diet alone did not impair cardiac susceptibility to ischaemia. However, combination of RF markedly exacerbated myocardial IRI. On the other hand, PC still conferred an effective protection against IRI, although its extent was lower in SHR and aged rats. Research shows that protective effects of adaptation may be attenuated in heart affected by RF, although the potential of intrinsic cardioprotection is still retained even in the pathologically altered myocardium that requires a higher intensity of the preconditioning stimulus. Pleiotropic effects of hypolipidemics, PPAR agonists, beyond their lipid-lowering properties indicate a promising approach to restore reduced myocardial ischaemic tolerance in these hearts.

Footnotes

Grants VEGA SR 1/0620/10, 2/0054/11, APVV-LPP-0393-09

Exp Clin Cardiol. 2011 Oct;16(Suppl A):24A.

99 Metabolic Syndrome in the Canadian Adult Population: The Canadian Health Measures Survey

ND Riediger ¹, I Clara ¹

Abstract

BACKGROUND:

Metabolic syndrome has been identified as a combination of co-occurring risk factors for cardiovascular disease. Data regarding the prevalence and description of metabolic syndrome in the Canadian population is not currently available.

PURPOSE:

To describe the prevalence and distribution of components of metabolic syndrome according to sociodemographic factors in a Canadian sample.

METHODS:

Data from the Canadian Health Measures Survey Cycle 1 conducted by Statistics Canada was used. This is a cross-sectional study of a representative sample of the Canadian population. Non-pregnant adults, 18 years and older were included. The prevalence of metabolic syndrome and its components according to age, sex, education, and income adequacy were estimated.

RESULTS:

The prevalence of metabolic syndrome in the Canadian population was estimated to be 19%. Age was the strongest predictor of metabolic syndrome, such that 17 % of those 18–39 years old had metabolic syndrome compared to 39% of those aged 60 years or older. Rates of metabolic syndrome were higher among those with lower levels of household education and income adequacy. Abdominal obesity was the most common component of the metabolic syndrome observed. However, low HDL cholesterol was the most common component found alone. The most common combination of components among those with metabolic syndrome meeting three criteria was abdominal obesity, low HDL cholesterol and hypertriglyceridemia. Females were more likely to be abdominally obese and males were more likely to have hypertriglyceridemia.

CONCLUSIONS:

Approximately one in five Canadian adults has the metabolic syndrome. Those with low income adequacy and education status have a higher prevalence.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):24A.

100 Peritoneal Dialysis for the Treatment of Refractory Congestive Heart Failure

Jacques Rizkallah ¹, Manish Sood ¹, Martina Reslerova ¹, Francisco Cordova ¹, Amrit Malik ¹, Shelley Zieroth ¹

Abstract

BACKGROUND:

The use of peritoneal dialysis (PD) for long-term management of diuretic resistant volume overload in heart failure (HF) is an under-utilized option. Literature on such use is scarce and its impact on mortality remains to be established in this high-risk cohort. We present our single center experience with palliative PD in end-stage HF and perform an adjunctive analysis of patient survival derived by the Seattle Heart Failure Model (SHFM).

METHODS:

A chart review of HF patients (transplant ineligible) receiving PD solely for HF volume management between 2007–11 (N=10). Clinical data was reviewed pre- and post-PD initiation. The SHFM score was applied based on clinical data at the time of PD initiation.

RESULTS:

Mean age was 58.3±12.7 years. Mean EF pre-PD was 24.5±6.0% with the majority NYHA class IIIB with moderate-severe right ventricular (RV) dysfunction. Weight decreased from an average 90.7±12.3 to 83.2±11.2 kg (p.016) and stabilization or improvement in diuretic response, and peripheral edema was noted. NYHA class improved in 7/10 patients. There was a significant decrease in re-hospitalizations from 3.2±2.5 to 0.1±0.3 (p.007) and average LOS from 37±36.7 to 0.78±2.3 days (p.019). The mean life expectancy for all patients was 2.2±2.2 years based on the SHFM. The calculated mean life expectancy for the 6 deceased patients was 1.5±1.2 years with actual survival at less than 50% of predicted at 0.7±0.4 years. There were no mortalities secondary to PD complications.

CONCLUSION:

The institution of PD for HF volume management was well tolerated and demonstrated favorable outcomes in the management of refractory HF. Survival was worse than predicted, perhaps as the SHFM is not applicable to this higher risk population with concomitant renal disease and RV dysfunction.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):25A.

101 The Association of Arterial Stiffness with the High Incidence of Cardiac Arrhythmias in Patients with Peripheral Arterial Disease

Delfin Rodriguez-Leyva ¹, Randy Guzman ², Grant N Pierce ³

Abstract

A susceptibility to arrhythmias may explain the increased risk for cardiovascular events in patients with peripheral artery disease (PAD). The purpose of the study was to identify the incidence and type of arrhythmias in patients with PAD and their relationship to the vascular abnormalities. This cross sectional study examined 110 PAD patients (73% male, 67 years old) with an ankle/brachial index (ABI) of <0.77. Cardiac arrhythmias were found in 37% of the subjects. Frequent premature ventricular and supraventricular contractions were the most common arrhythmias (78%). Most (65%) were observed at rest before exercise but were also detected during and after exercise. PR segment duration,QRS complex duration and QTc interval were significantly longer in patients with arrhythmias. QTc differences were significant for males but not for females. Both initial and absolute claudication distances were greater in patients with arrhythmias but ABI values were not. Age (>60 years), pre-existing coronary artery disease (CAD) and greater arterial stiffness (higher augmentation index) were identified as predictors of arrhythmias. Patients with PAD have a high incidence of cardiac arrhythmias that are concerning because they are most frequently observed at rest.Older age, concomitant CAD and greater arterial stiffness may pre-dispose PAD patients to arrhythmias. Better physician awareness of the potential presence of this condition in PAD patients and screening these patients with the use of a simple, non-invasive technique like PWA may allow for risk stratification.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):25A.

102 Effect of the Maleimide Derivative MI-1, a Novel Antitumor Agent, on the Rat Myocardium and Cardiomyocytes

V Rybalchenko ¹, I Kharchuk ¹, V Kyrychenko ¹, O Lynchak ¹

Abstract

The low toxicity is one of the advantages of a new generation of drugs compared with conventional anticancer preparations. The maleimide derivative 1-(4-chlorobenzyl)-3-chloro-4-(CF3-phenylamino)-1H-pyrrol-2,5-dione (MI-1) is an ATP-competitive low molecular inhibitor of the tyrosine kinases EGF-R, FGF-R1, IGF1-R, INS-R, SRK, YES, VEGF-R1-3, ZAP70 (inhibitor type I) and exhibits antiproliferative activity in cultures of transformed and cancerous human cells in vitro.

Intragastric administration of potent, targeted drug MI-1 during 20 weeks causes diffusional changes in rat myocardium, such as loosening and thinning of some muscle fibers, and increase of the size of cardiomyocytes nuclei. In the vasculature, MI-1 causes dilation and hyperemia of capillaries, appearance of the histolymphocyte infiltrates around them, thickening of the arteries and veins walls, and endothelial and perivascular edema.

MI-1 does not manifest the pronounced preconditioning effect, inhibits the processes of apoptosis and necrosis in the culture of neonatal rat cardiomyocytes and is able to modulate the activity of 20S- and 26S-proteasome. Thus, the histological changes in the myocardium and inhibition of the apoptosis and necrosis processes in the cultures of the rat cardiomyocytes indicate low cardiotoxicity of MI-1 compared to classical anticancer agents, and warrant the further studies of this new compound.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):25A.

103 Enhanced Levels of Angiotensin II Contribute to the Overexpression of Gi Proteins in Vascular Smooth Muscle Cells from SHR: Role of Epidermal Growth Factor Receptor Transactivation

Y-H Gomez Sandoval ¹, MB Anand-Srivastava ¹

Abstract

We earlier showed that the increased expression of Gi proteins exhibited by vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) was attributed to the enhanced levels of endogenous endothelin. Since the levels of angiotensin II (Ang II) are also enhanced in VSMC from SHR, the present study was undertaken to examine the role of enhanced levels of endogenous Ang II in the overexpression of Giα proteins in VSMC from SHR and to further explore the underlying mechanisms responsible for this increase. The enhanced expression of Giα-2 and Giα-3 proteins in VSMC from SHR compared to WKY was attenuated by the captopril, losartan and AG1478, inhibitors of angiotensin converting enzyme, AT1 receptor and epidermal growth factor receptor (EGFR) respectively as well as by the siRNAs of AT1, c-Src and EGFR. The enhanced inhibition of forskolin-stimulated adenylyl cyclase activity by low concentrations of GTPγS and of inhibitory responses of hormones on adenylyl cyclase activity in VSMC from SHR were also attenuated by losartan.. Furthermore, the enhanced phosphorylation of EGFR in VSMC from SHR was also restored to control levels by captopril, losartan, PP2, a c-Src inhibitor and N-acetyl-L-cysteine (NAC), superoxide anion (O₂⁻) scavenger, whereas enhanced ERK1/2 phosphorylation was attenuated by captopril and losartan. Furthermore, NAC also restored the enhanced phosphorylation of c-Src in SHR to control levels. These results suggest that the enhanced levels of endogenous Ang II in VSMC from SHR, transactivate EGFR, which through MAP kinase signaling, enhances the expression of Giα proteins and associated adenylyl cyclase signalling

Footnotes

Supported by CIHR

Exp Clin Cardiol. 2011 Oct;16(Suppl A):25A.

104 Impact of Diabetes-Associated LDL on Mitochondrial Respiration in Vascular Endothelial Cells

Ganesh Sangle ¹, Xueping Xie ¹, Subir Roy Chowdhury ¹, Garry Shen ¹

Abstract

Cardiovascular disease is the predominant cause of death in diabetic patients. Oxidative stress and endothelial dysfunction have been detected in vasculature of diabetic patients or animal models. Hyperglycemia and dyslipoproteinemia are biochemical markers for diabetes. Elevated levels of glycated LDL (gLDL) and oxidized LDL (oLDL) were frequently detected in diabetic patients. Our group reported that gLDL and oLDL increased the generation of reactive oxygen species from vascular endothelial cells (EC). The present study demonstrated that gLDL and oLDL reduced oxygen consumption in mitochondrial electron transfer chain (mETC) Complex I and IV in porcine aortic EC. Treatment with gLDL or oLDL reduced mitochondrial membrane potential in EC, and inhibited the activities of NADH dehydrogenase (ND, Complex I), succinate cytochrome c reductase (Complex II), ubiquinol cytochrome c reductase (Complex III) and cytochrome c oxidase (Complex IV) in EC. Abundanced of ND1 and cytochrome b (a subunit of Complex III enzyme) in EC were reduced following incubation with gLDL or oLDL. Treatment with gLDL or oLDL increased the abundance of ROS-associated with mitochondria in EC detected using immunohistochemistry and confocal microscopy. The results suggest that diabetes-associated LDL may inhibit activities of mETC enzymes through reducing abundances of multiple subunits in mETC enzymes, which was associated with increase of ROS in mitochondria of EC. Impairment in mitochondrial respiration in EC induced by diabetes-associated LDL may contribute to oxidative stress and endothelial dysfunction in vasculature of diabetes.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):26A.

105 Autocrine Effects of High Molecular Weight FGF-2 on Cardiac Myobroblasts Suggest Link to Fibrosis and Innate Inflammation

Jon-Jon Santiago ¹, Brian P Bestvater ¹, Vincent Chen ², Jie Wang ¹, Robert Fandrich ¹, Peter A Cattini ¹, Elissavet Kardami ¹

Abstract

BACKGROUND:

Previously we documented that high molecular weight (Hi)-FGF-2, rather than the commonly studied 18 kDa low molecular weight isoform Lo-FGF-2, is a potent inducer of cardiac hypertrophy in vitro and in vivo. We also showed that cardiac myofibroblasts are major producers of Hi-FGF-2 in the heart, and that angiotensin II promotes up-regulation of total and secreted myofibroblast-derived Hi-FGF-2 which induced myocytes hypertrophy in a paracrine mode. However, the potential autocrine FGF-2 isoform-specific actions on cardiac myofibroblasts have not been studied. Here, we examined the effect of extracellular-acting Hi- and Lo-FGF-2 on: (i) the secretome profile of myofibroblasts; (ii) expression of inflammation-associated cytokines; and (iii) the phenotypic transition of fibroblasts into activated/hypersecretory myofibroblasts.

METHODS/RESULTS:

Cardiac myofibroblast cultures, derived from neonatal rat pups (RCMFs) were treated with recombinant Hi-FGF-2 or Lo-FGF-2 (10 ng/mL). Conditioned medium from RCMFs was collected and analyzed by mass spec (LC-MS/MS). Hi-FGF-2 was found to be more potent compared to Lo-FGF-2 in stimulating secretion of several matricellular fibrosis-associated proteins including periostin, tenascin, plasminogen activator inhibitor-1, and follistatin-like protein 1. Although, qRT-PCR did not show differences in corresponding mRNA levels, Western blotting of conditioned media with specific antibodies validated the MS/MS data. We also examined how endogenous Hi-FGF-2 secretion may affect human atrial tissue-derived myofibroblasts (HCMFs), by incubating these cells with an anti-human-Hi-FGF-2-specific antibody preparation. Neutralizing the autocrine action of human Hi-FGF-2 resulted in down-regulation of α-smooth muscle actin, pro-collagen, periostin, and EDA-fibronectin, indicating a reversal from myofibroblast to fibroblast phenotype. In addition, stimulation of HCMFs with Hi-FGF-2 was more potent than Lo-FGF-2 in up-regulating expression of interleukin IL-1β, a major marker of the innate inflammatory response; and that blocking the autocrine action of endogenous Hi-FGF-2 with neutralizing anti-human-Hi-FGF-2 antibodies reduced endogenous interleukin IL-1β expression.

CONCLUSIONS:

Taken together our data indicate, for the first time, that Hi-FGF-2 rather that Lo-FGF-2, exerts potent autocrine effects on cardiac myofibroblasts. These include promoting the fibroblast-to-myofibroblast transition, secretion of fibrosis associated matricellular proteins, and stimulating expression of pro-inflammatory cytokines such IL-1β. Thus, inhibition of the autocrine effects of Hi-FGF-2 on cardiac fibroblasts and myofibroblasts is a potential new therapeutic target for preventing maladaptive cardiac remodelling.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):26A.

106 Diabetes Induces a State of Hyper-Phosphorylation of Connexin 43 in a Murine Model

A Sepehri ¹, JJ Santiago ¹, M Durston ¹, DH Freed ¹, P Fernyhough ¹, E Kardami ¹, RC Arora ¹

Abstract

INTRODUCTION:

There is growing evidence of the existence of diabetic cardiomyopathy, or heart failure in the absence of obstructive coronary heart disease associated with diabetes. We have observed that Connexin 43 (Cx43), the main gap junction in the heart, is upregulated in a diabetic rodent model. It is unclear if this upregulation represented the phosphorylated or unphosphorylated state. We therefore examined the phosphorylation state of the Cx43 and the signaling pathways of Neuregulin-1 (NRG-1) and FGF-2 with respect to their target, Cx43, in a murine model.

METHODS/RESULTS:

Diabetes was induced in Swiss Webster Mice by Streptozocin (2 injections of 90 mg/kg, 24 hrs apart) and carried until endpoints of reduced body weight and elevated blood glucose levels were achieved (n=12). Age matched controls (n=13) were used for comparison. Western blot analysis showed that the left ventricular tissue demonstrated an increase in total Cx43 levels. Analysis of the phosphorylation of key phosphorylation sites (serine 262 and 368) demonstrated a hyperphosphorylation state only at the serine 262 site. In addition, NRG-1 expression was decreased while its myocardial cell-surface receptor, ErbB2 was increased. We also found that the pro-hypertrophic high molecular weight FGF-2 was elevated in diabetics.

CONCLUSIONS:

The down-regulated NRG-1 expression is consistent with other heart failure models. Diabetes seems to cause a dichotomous cardiovascular response: a cytoprotective response, shown through the elevated P-S262 levels, and a pro-hypertrophic response, shown through elevated Hi-FGF-2. Determination of the mechanism through which diabetes acts to cause these changes can provide direction for future intervention.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):26A.

107 Simvastatin Inhibits the Profibrotic Response of Human Atrial Fibroblasts

D Schaafsma ¹, S Ghavami ¹, IMC Dixon ¹, AJ Halayko ¹

Abstract

BACKGROUND:

Atrial fibroblasts contribute to cardiac remodelling, including expansion of the cardiac interstitium and eventual fibrosis. Transforming growth factor (TGF)-β1 has emerged as a key mediator of this process. We investigated mevalonate cascade-associated signaling in TGFβ1-induced extracellular matrix (ECM) protein expression by primary human atrial fibroblasts.

METHODS:

We used simvastatin (10 μM) to inhibit 3-hydroxy-3-methly-glutaryl-coenzyme A (HMG-CoA) reductase which converts HMG-CoA to mevalonate. Selective inhibitors of geranylgeranyl transferase-1 (GGT1; GGTI-286, 10 μM) and farnesyl transferase (FT; FTI-277, 10 μM) were used to determine whether GGT1 and FT contribute to TGFβ1-induced ECM expression. In addition, we studied the effects of co-incubation with simvastatin and mevalonate (1mM), geranylgeranylpyrophosphate (GGPP, 30 μM) or farnesylpyrophosphate (FPP, 30 μM).

RESULTS:

Immunoblotting showed simvastatin significantly inhibited TGFβ1 (2.5 ng/ml, 48h)-induced collagen I and fibronectin expression. This was prevented by exogenous mevalonate or isoprenoids (GGPP or FPP). The effects of simvastatin were mimicked by GGTI-286, but not FTI-277, suggesting primary involvement of GGT1 in TGFβ1-induced signaling. In addition, regulatory Smad signaling, which is classically associated with TGFβ1 mediated intracellular events, was affected in a similar fashion. Thus, phosphorylation of Smad2/3 was inhibited by simvastatin, an effect that was mimicked by GGTI-286 and prevented by GGPP and mevalonate.

CONCLUSIONS:

We conclude that TGFβ1-induced ECM expression in atrial fibroblasts largely relies on activity of GGT1 and availability of isoprenoids. Our data indicate for the first time that TGFβ1-induced regulatory Smad signaling might be subject to isoprenoid-dependent signaling as well.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):26A–27A.

108 Role of Phosphorylation of Akt^ser473/Ask1^ser83 in the Modulation of Doxorubicin-Induced Cardiotoxicity Phosphorylation

Anita K Sharma ¹, Ana Ludke ¹, Ashim K Bagchi ¹, Abd-Al AR-Rahman Shudiefat ¹, Pawan K Singal ¹

Abstract

The clinical use of doxorubicin (DOX) against human malignancies is limited by its side effects of cardiomyopathy and heart failure. The underlying cell signaling mechanism of DOX-induced cardiotoxicity is still unclear. This study is investigating the role of Akt/Ask1 and the effect of trolox in the modulation of DOX-induced cardiotoxicity. Rat cardiomyocytes were exposed to DOX (5μM) for 24h with or without trolox (20μM) pretreatment for 4h. In a time course study, Akt activity decreased after 10m of exposure to DOX until 24h. p38 showed a biphasic change with an up-regulation up to 30min followed by another peak at 24h. JNK activity showed a steady increase up to 6h that remained consistent up to 24h. p53 activity showed a steady rise until 24h. Activation of Ask1 was not observed up to 6h, however, it was up-regulated at 24h. Trolox pretreatment down-regulated the DOX -induced increase in oxidative stress (OS) and promoted activity of Akt^ser473 and Ask1^ser83. Trolox attenuated above mentioned DOX-induced cell signaling changes as well as apoptosis. Inhibition of Akt using Wortmannin up- regulated phospho levels of AskThr845 and Bax/Bcl-xl and down regulated Ask1^ser83 suggesting the role of Akt^ser473/Ask1^ser83 in trolox- modulation of DOX- induced OS and apoptosis. This study shows that trolox could be used as a pharmacological modulator of DOX-induced OS and apoptosis by up-regulating phosphorylation of Akt^ser473/Ask1^ser83.

Footnotes

Supported by the Heart and Stroke foundation of Manitoba

Exp Clin Cardiol. 2011 Oct;16(Suppl A):27A.

109 New Insights on Actin-Myosin Regulation in Cardiac Muscle and Disease

F Sheikh ¹

Abstract

Actin-myosin interactions provide the driving force for each heartbeat. However, the molecular mechanisms underlying the regulation of these interactions in cardiac muscle, which are fundamental to heart function and are of central importance in heart disease and failure in humans, are still not well understood. For the last five decades, the obligatory step of Ca²⁺ binding to actin bound regulatory proteins (eg., troponin-tropomyosin complex) has contributed to the prevailing textbook view that the control of calcium cycling and actin-bound regulatory proteins dominate regulation of muscle contraction and underlie the dysregulation of contractile dynamics in heart failure. In contrast, myosin regulatory proteins such as the ventricular form of myosin regulatory light chain-2 (MLC2v) are viewed to play lesser roles, and thus, their regulation and function remain poorly understood in this context and especially in vivo. Using novel integrative gene-targeted animal and multi-scale computational models, we provide compelling mechanistic evidence that provides evidence of an indispensable role for MLC2v via its phosphorylation in regulating cardiac myosin cycling kinetics, which control actin-myosin interactions as well as surprisingly, feedback cooperatively to influence calcium-dependent activation of the thin filament. Loss of these mechanisms uncovers previously unrecognized early defects in the rates of twitch relaxation and ventricular torsion, which strikingly precede heart disease and failure in a novel non-phosphorylatable MLC2v mouse model. We show that in contrast to conventional views, there is a direct and early role for MLC2 in regulating actin-myosin interactions in striated muscle contraction and that this mechanism plays a critical role in heart failure.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):27A.

110 CLP-1/P-TEFb Pathway Regulates Cardiac Remodeling via Transcriptional Modulation of Hypertrophic Growth Response and Cell Regeneration

MAQ Siddiqui ¹, E Mascareno ¹, P Hong ¹, J Galatioto ¹, I Rozenberg ¹

Abstract

Cardiac hypertrophy, manifested by an increase in size of cardiomyocytes and proliferation of cardiac fibroblasts, is initially a compensatory response to cardiac insults. When the stress is chronic, the compensatory remodeling of the heart becomes decompensatory and is accompanied with loss of compliance. Our objective is to understand how the cardiac gene regulatory program controls the genetic response of the heart to stress. Our experimental design was based on our finding that cardiac lineage protein-1 (CLP-1) controls gene expression at the level of transcriptional elongation via its dynamic association/dissociation with/from the positive transcriptional elongation complex P-TEFb. We have previously shown that the CLP-1-null mice die during late embryonic stages E-17 to E-18 due to massive growth of the heart. The heterozygous CLP-1^+/− mice have no phenotypic abnormality, yet they exhibit enhanced susceptibility to hypertrophic agonists, such as, angiotensinII (Ang II). We investigated the role of CLP-1 in AngII induced ventricular remodeling in hypertrophy and fibrotic growth by crossing the CLP-1^+/− mouse with the transgenic mouse that carries overexpression of angiotensinogen (ANG) gene. We also examined the expression of atrial natriuretic peptide (ANP) and TGFb as they play counter regulatory roles cardiac hypertrophy and fibrosis. We observed that the haplo-deficiency of CLP-1 in the biogenic MHC-ANG/CLP^+/− mice caused pronounced changes in ventricular remodeling. There was an increase in LV mass, enhanced deposition of collagen, attenuated expression of ANP and an increase in the TGFb/Smad3 signaling accompanied with transdifferentiation of fibroblast into myofibroblast. Concomitantly, the deficiency of CLP-1 level also caused activation of Jak/STAT signaling. In a parallel study, we used an experimental muscle injury model to investigate the growth regulatory role of CLP-1. We observed that haplodeficiency of CLP-1 promotes post-injury expansion of the muscle progenitor cell pool and enhanced muscle cell regeneration. The reduced level of CLP-1 enhances proliferation to relieve CLP-1-dependent inhibition of PTEFb activity. These results demonstrate the role of CLP-1 in genetic control of hypertrophic growth and cell regeneration and provide the basis for modulation of this pathway for cell based therapy for muscle disorders.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):27A.

111 Can High w-6/w-3 Ratio Diets Predispose Cardiovascular Diseases: The Tsium Tsoum Concept

RB Singh ¹, Fabien De Meester ¹, Agniesca Wilczynska ¹

Abstract

Chronic diseases of affluence are also called noncommunicable diseases (NCD) by the World Health Organization. These diseases are a major impediment to human development, imposing large health and economic burden worldwide. These burdens are reciprocal because poverty is a potential cause of NCDs in developed countries and NCDs contribute to poverty. The world has been in a position to learn, the mechanism of transition from poverty to economic development and emergence of NCDs. The last decade of the last century offered us an opportunity to initiate action to counter growing epidemics of CVD including metabolic syndrome on both sides of the atlantic. When people learned the methods of prevention, there was a decrease in CVD in the western world but obesity continued to increase, resulting into an increase in the metabolic syndrome in both developed and developing economies. Western diet is characterized with energy dense, refined, ready prepared foods with a high glycemic index (e.g., refined starches; bread, biscuits, candies, cornflakes, peeza, potato chips, cola drinks and sugar) and unhealthy lipids (e.g., trans fats and saturated fats) poor in w-3 fatty acids, phytochemicals and fiber. Such diets have been adopted by increased number of people and populations in the Western world and in the urban populations of middle income countries in the last few decades. These diets are known to predispose inflammation and the epidemic of NCDs. Cardiovascular disease (CVD), diabetes mellitus, obesity, cancer, autoimmune diseases, rheumatoid arthritis, asthma and depression are associated with increased production of thromboxane A2(TXA2), leucotrienes, prostacyclin, interleukins-1 and 6, tumor necrosis factor-alpha and C-reactive proteins. Increased dietary intake of w-6 fatty acids is known to enhance all these biomarkers as well as atherogenicity of cholesterol which have adverse proinflammatry effects resulting into NCDs. Recent studies indicate that Tsim Tsoum approaches including a Mediterranean diet rich in fruits, vegetables, nuts, canola oil, olive oil characterized with low w-6/w-3 ratio in the diet, as well as physical activity, and meditation can modulate inflammation as well as body-mind interactions and may be protective against risk of CVD and all cause mortality. Inflammation appears to be an important unifying hypothesis, because one recent study has demonstrated that decline in inflammation can cause significant reduction in all cause mortality and cardiovascular events.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):27A–28A.

112 Metabolic Syndrome: A Disease of the Brain

RB Singh ¹, Fabien De Meester ², Agnieska Wilczynska ²

Abstract

INTRODUCTION:

Association between brain dysfunction and pathogenesis of metabolic syndrome leading to cardiovascular diseases, type 2 diabetes and insulin resistance are reviewed.

METHODS:

Medline search till Dec, 2009 and articles published in various national and international journals were reviewed. Experts working in the field were also consulted.

RESULTS:

Increased intake of refined carbohydrates, linoleic acid, saturated and total fat and low dietary n-3 fatty acids and other long chain polyunsarurated fatty acids (PUFA) in conjunction with sedentary behaviour and mental stress, and various personality traits can predispose inflammation and central obesity. There may be increased sympathetic activity with increased secretion of catecholamine, cortisol and serotonin and proinflammatory cytokines that appear to be underlying mechanisms of metabolic syndrome. Excess secretion of these neurotransmitters in conjunction of underlying long chain PUFA deficiency, may damage the neurons via proinflammatory cytokines, in the ventromedial hypothalamus and insulin receptors in the brain, especially during fetal life, infancy and childhood, resulting into their dysfunction. Since 30-50% of the fatty acids in the brain are long chain PUFA, especially omega-3 fatty acids, which are incorporated in the cell membrane phospholipids, it is possible that their supplementation may be protective. Omega-3 fatty acids are also known to enhance parasympathetic activity and increase the secretion of anti-inflammatory cytokines IL-4 and IL-10, as well as acetylecholine in the hippocampus. It is possible that marginal deficiency of long chain PUFA, especially n-3 fatty acids, due to poor dietary intake during the critical period of brain growth and development in the fetus and infant, and also possibly in the child, adolescents and adults, may enhance the release of tumor necrosis factor-alpha, interleukin-1, 2 and 6 and cause neuronal dysfunction. Experimental studies indicate that ventromedial hypothalamic lesion in rats induces hyperphagia, resulting into glucose intolerance and insulin resistance. Treatment with neuropeptide Y abolished the hyperphagia and ob mRNA(leptin mRNA) in these rats. Longterm infusion of norepinephrine and serotonin into the ventromedial hypothalamus, impaired pancreatic islet function in as much as, ventromedial hypothalamic norepinephrine and serotonin levels are elevated in hyperinsulinemic and insulin resistant animals. Treatment with insulin was associated with restoration of these hypothalamic neurotransmitter abnormalities indicating that a dysfunction of ventromedial hypothalamus can impair pancreatic beta cells resulting into metabolic abnormalities consistent with metabolic syndrome. Treatment with omega-3 fatty acids, meditation, beta blockers, ACE inhibitors, and oestrogen may have a beneficial influence on insulin receptors and ventromedial hypothalamic dysfunction. However, no definite and precise insight into the patho-physiological link between metabolic syndrome and brain and nutrition is available. Despite this weakness, epidemiological studies and intervention trials indicate that treatment with n-3 fatty acids may be applied to clinical practice and used to direct therapy for prevention of type 2 diabetes, hypertension, coronary artery disease, and atherosclerosis, indicating that metabolic syndrome may also respond to this treatment.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):28A.

113 Embryonic Stem Cells Improve Cardiac Function in Doxorubicin-Induced Cardiomyopathy Mediated Trhough Multiple Mechanisms

Dinender K Singla ¹, Aisha Ahmed ¹, Reetu Singla ¹, Binbin Yan ¹

Abstract

Whether transplanted embryonic stem (ES) cells or their conditioned medium (CM) in DOX induced cardiomyopathy (DIC) can repair and regenerate myocardium remains elusive. Therefore, we transplanted ES cells or CM in DIC to examine apoptosis, fibrosis, cytoplasmic vacuolization and myofibrillar loss and their associated Akt and ERK pathway. Moreover, we also determined activation of endogenous c-kit+ve cardiac stem cells (CSCs), levels of HGF and IGF-1, growth factors required for c-kit cell activation, and their differentiation into cardiac myocytes, which also contributes in cardiac regeneration and improved heart function. We generated DIC in C57Bl/6 mice (cumulative dose of DOX 12mg/kg body weight, i.p), and animals were treated with ES cells, CM or cell culture medium in controls. Two weeks post-DIC, ES cells or CM transplanted hearts showed a significant (p<0.05) decrease in cardiac apoptotic nuclei and their regulation with Akt and ERK pathway. Cardiac fibrosis observed in the ES cell or CM groups was significantly less compared with DOX and cell culture medium groups (p<0.05). Next, cytoplasmic vacuolization and myofibrillar loss was reduced (p<0.05) following treatment with ES cells or CM. Moreover, our data also demonstrated increased levels of c-kit+ve CSCs in ES cells or CM hearts and differentiated cardiac myocytes from these CSCs, suggesting endogenous cardiac regeneration. Importantly, the levels of HFG and IGF-1 were significantly increased in ES cells or CM transplanted hearts. In conclusion, we reported that transplanted ES cells or CM in DIC hearts significantly decreases various adverse pathological mechanisms as well as enhances cardiac regeneration that contributes to improved heart function.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):28A.

114 Proton Radiation-Induced Cardiovascular Toxicity: Pathology and Prevention

J Slezak ¹, N Tribulova ¹, M Ivanova ¹, J Styk ¹, K Frimmel ¹, T Ravingerova ¹, J Ruzicka ¹, L Okruhlicova ¹

Abstract

Proton beam radiotherapy provides an focused dose distribution and absence of exit dose, compared to conventional radiation therapy, whereby reduces irradiation to adjacent normal healthy tissue. However, some exposure of the heart and vessels is often unavoidable. Cardivascular adverse effect of radiation is especially pronounced in patients with tumors localized in the chest region.

The study concentrates on pathology of proton radiation-induced cardiovascular toxicity and on prevention of injury of healthy tissues in areas at risk. Proton-irradiation of the heart with a single dose of 20 Gy delivered to the heart region was performed on Wistar rats.

Adverse effect of ionizing radiation is mostly mediated by reactive oxygen and nitrogen species, which deplete antioxidant stores. Radiation damage of cardiovascular system shows that endothelial cells are the most radio Ionizing radiation Ionizing radiation sensitive part of vasculature. Cardiovascular injury is represented by inflammatory changes in myocardium and microvasculature, decrease in capillary density, intimal proliferation, mast cells decrease, lymphocyte adhesion, monocytes infiltration, microthrombi, nuclear chromatin changes, cardiomyocyte apoptosis, focal ischemia-like and fibrotic changes, as well as early endothelial substructural changes, starting one month after proton-irradiation.

Protection of normal tissue against radiation-induced damage may increase the therapeutic benefit of radiotherapy. Different more or less effective measures to prevent radiation-induced injury and to increase cardiovascular tolerance to irradiation of healthy tissue have been evaluated including treatment with antiinflammatory and fibrinolytic drugs, statins, NO donors, compounds with antioxidant properties and other more sophisticated agents. Further investigation is needed to determine the most effective prevention of radiation induced injury to healthy tissues accidentally targeted by radiation.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):28A–29A.

115 FGF-2 Stimulates Translocation of Connexin43/PKC-Epsilon to Cardiac Mitochondria, Increases Mitochondrial Cx43 Phosphorylation, and Prevents Calcium-Induced Permeability Transition

W Srisakuldee ¹, Z Makazan ¹, BE Nickel ¹, F Zhang ², KB Pasumarthi ², E Kardami ¹

Abstract

Fibroblast growth factor-2 (FGF-2) is protective against ischemia-, and/or reperfusion-induced cardiomyocyte cell death, by a mechanism dependent on connexin43 (Cx43) phosphorylation at protein kinase C (PKC)-ɛ target sites such as serine (S) 262. Cx43 localizes predominantly to intercalated disks but is also present at sub-sarcolemmal mitochondria, SSM, where it is considered important for a pro-survival response. We examined the effect of FGF-2 administration to the heart on, specifically, SSM-located Cx43 levels and phosphorylation in relation to SSM resistance to calcium-induced permeability transition (mPT).

Mitochondria (SSM) isolated from FGF-2-perfused hearts displayed 1.4-, 1.6-, and 2.0- fold increases in Cx43, PKCɛ, and TOM-20 translocase, compared to those from untreated hearts (controls), indicative of FGF-2-induced translocation. While control SSM had minimal levels of phospho-S262- and phospho-S368-Cx43, those from FGF-2-treated heart showed a 30- and 6-fold increase in, respectively, phospho-S262-Cx43, and phos-pho-S368-Cx43. Sensitivity to calcium-induced mPT (swelling), measured: spectrophotometrically as a drop in absorbance at 545 nm; by western blotting as cytochrome C release (both inhibitable by cyclosporine A) was significally reduced in SSM from FGF-2-treated hearts compared to controls. PMA elicited a similarly reduced sensitivity to calcium-induced mPTP in control SSM, in vitro; this was accompanied by a 14-fold increase in mitochondrial phospho-S262-Cx43 which was mediated by mitochondrial PKCɛ as it was blocked by a selective PKCɛ (ɛV1-2) inhibiting peptide.

Taken together, our data suggest that FGF-2 cardioprotection is mediated, at least in part, by decreased mitochondrial sensitivity to calcium-induced mPT, caused by translocation of Cx43/PKCɛ to mitochondria, and/or a concomitant, PKCɛ-mediated potent increase in mitochondrial phospho-S262-Cx43.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):29A.

116 Immortalized Stem Cell Lines as Standardized Research Tools for Cardiac Repair

Stefania Pagliari ^1,², Emilie Nouguerede ^1,³, Francesca Pagliari ^1,³, Giancarlo Forte ^1,², Paolo Di Nardo ^1,³

Abstract

Procedures to properly differentiate and efficiently implant stem cells into injured organs remain to be defined. Among the major causes is that each laboratory follows its own “recipe” often using inconsistent nomenclature and non-comparable experimental protocols. In this context, the absence of any standardization has negatively affected all efforts to implement cell treatments for heart repair. The availability of immortalized stem cell lines could allow to generate comparable results in different laboratories, above all to unravel basic mechanisms governing stem cell behavior. To this end, immortalized murine mesenchymal (MSC) and cardiac stem cell (CSC) lines (mTERT-MSC and mTERT-CSC) enriched for Linneg/Sca-1pos fraction have been obtained through the transfection of MSC and CSC with murine TERT and single-cell isolation. Such cell lines maintained the typical MSC and CSC self-renewal capacity and continuously expressed the specific phenotype. Moreover, mTERT-MSC and mTERT-CSC retained the functional features of freshly isolated cells in culture without evidence of senescence or spontaneous differentiation events. Then, mTERT-MSC and mTERT-CSC have been cultured onto various polymeric supports with different results. Nevertheless, such cell lines displayed the ability to preserve Sca-1 expression and to retain multilineage potential when appropriately stimulated on all of the scaffolds tested.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):29A.

117 SIRT1-Mediated Deacetylation Plays an Essential Role in Regulating Akt-Signaling

Nagalingam R Sundaresan ¹, Madhu Gupta ², Mahesh Gupta ¹

Abstract

INTRODUCTION:

Akt is considered to be the cardinal node in almost all cellular events, which help cells to live in peril and peace. Akt activation is three step process which includes (i) binding of Akt to PIP3, (ii) translocation of Akt from the cytosol to the membrane, and (c) phosphorylation of Akt by the upstream kinases, PDK1 and mTORC2. Recent studies have shown that SIRT1 influences Akt activation and promotes cell growth. This study was designed to test the mechanism through which SIRT1-regulates Akt activity.

METHODS AND RESULTS:

By biochemical analysis of Akt, we found that Akt is an acetylated protein under basal conditions. Growth factor stimulation of cells activates and deacetylates Akt. By time course analysis we found that deacetylation of Akt occurs prior its phosphorylation. To identify the HDAC which could deacetylate Akt, we analyzed SIRT1-KO and SIRT1 transgenic mice having cardiac-specific over expression of SIRT1. SIRT1-KO mice had smaller body size, reduced HW/BW ratio and reduced phosphorylation of Akt and its downstream targets, compared to control mice. On the other hand, SIRT1-Tg mice showed robust cardiac hypertrophy at adulthood, which was associated with deacetylation and phosphorylation of Akt, suggesting that SIRT1 may be the HDAC deacetylating Akt and promoting cardiac hypertrophy. In vitro studies showed that SIRT1 deacetylates Akt and that promotes Akt membrane localization and activation. Chemical agents inhibiting Akt blocked the pro-hypertrophic activity of SIRT1.

CONCLUSION:

These results uncovered a new step in Akt signaling and demonstrated that the pro-hypertrophic activity of SIRT1 is mediated through its ability to deacetylate and activate Akt.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):29A.

118 PCSK9 May Be A Player in Rat Myocardial Ischemia-Reperfusion Injury

Tang Zhi-Han ¹, Zeng Hai-Yan ¹, QU Shun-Lin ¹, LIU Lu-Shan ¹, Jiang Zhi-Sheng ¹

Abstract

OBJECTIVE:

To investigate the expression of PCSK9 and Caspase3 in the impaired myocardium by ischemia-reperfusion, and explore the role of PCSK9 in myocardial ischemia-reperfusion.

METHODS:

The left descending coronary artery (LAD) of rats was occluded for 45 min, followed by reperfusion for 6 h, 12 h or 24 h, respectively. The levels of lactate creatine kinase (CK), creatine kinase MB (CK-MB) and lactate dehydrogenase (LDH) in serum were measured by automatic biochemical analyzer and the pathological changes in myocardium of rat were examined by HE staining. DNA ladder detection was used for evaluation of myocardial cell apoptosis, and the expression of PCSK9 and Caspase3 in myocardium was detected by RT-PCR and Western blot.

RESULTS:

The levels of CK, CK-MB and LDH in ischemia-reperfusion group were increased by 3.22-fold, 1.54-fold and 3.02-fold after reperfusion of 6 h, respectively, reached the peak at 12h, and restored to normal level at 24 h. HE staining and DNA ladder detection indicated that the reperfusion-induced myocardial damage was increased in a time-dependent manner. The expression of PCSK9 mRNA in I/R 6 h, 12 h, 24 h group were 1.55-fold, 2.18-fold and 2.73-fold higher than control group, and a similar change was seen at the expression of PCSK9 protein. The levels of Caspase3 mRNA and protein were also significantly increased by ischemia-reperfusion injury in a time-dependent mode .

CONCLUSION:

PCSK9 and Caspase3 mRNA and protein levels were significantly increased in ischemia-reperfused myocardium in a time-dependent manner, which are consistent with the change of myocardial damage. These data indicate that PCSK9 may be invovled in myocardial ischemia-reperfusion injury.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):29A.

119 Law and Order Among Astronomical Numbers of Cardiac Sarcomeres

Henk EDJ ter Keurs ¹

Abstract

The macroscopic hallmarks of the normal heart beat are rapid onset of contraction and rapid relaxation as an inotropic response of both end diastolic volume and increased heart rate. At the microscopic level, the calcium ion (Ca²⁺) plays a crucial role in normal cardiac contraction. This paper reviews the cycle of Ca²⁺ fluxes during the normal heart beat, which underlie the coupling between excitation and contraction (ECC) and permit a highly synchronized action of about hundred million billion cardiac sarcomeres in the left ventricle. Length dependence of the response of the regulatory sarcomeric proteins mediates the Frank-Starling Law of the heart. We will discuss recent evidence regarding mechanisms underlying the length dependence of activation and present a simple model to account for the current observations and the Frank-Starling Law.

Ca²⁺ transport may go astray in heart disease, and both jeopardize the exquisite mechanism of systole and diastole and triggering arrhythmias. We will show that the interplay between weakened and strong segments in nonuniform cardiac muscle may further lead to mechano-electric feedback – or reverse excitation contraction coupling (RECC) mediating an early diastolic Ca²⁺ transient caused by the rapid force decrease during the relaxation phase. These rapid force changes in nonuniform muscle may cause arrhythmogenic Ca²⁺ waves to propagate by activation of neighbouring SR by diffusing Ca²⁺ ions.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):30A.

120 Effect of Resveratrol in Hypertension Induced Cardiac Dysfunction in SHR

SJ Thandapilly ¹, XL Louis ¹, T Yang ¹, DM Stringer ¹, L Yu ¹, S Zhang ¹, J Wigle ¹, E Kardami ¹, P Zahradka ¹, C Taylor ¹, HD Anderson ¹, T Netticadan ¹

Abstract

Cardiac hypertrophy is the compensatory enlargement of the heart aimed at reducing the stress caused by various diseases. Sustained hypertrophy, however, is detrimental and leads to heart failure. In this study, we examined the effect of resveratrol, a polyphenol, in preventing cardiac hypertrophy and impairment due to essential hypertension. Spontaneously hypertensive rats (SHRs) and control Wistar-Kyoto (WKY) rats were treated with resveratrol (2.5 mg/kg/day) for a period of 10 weeks. Blood pressure, cardiac structure and function were measured in all groups. In order to investigate the sub-cellular mechanism of resveratrol action, adult cardiomyocytes were pre-treated with resveratrol (30μm) and then exposed to norepinephrine (1 μm). Cardiomyocyte hypertrophy was determined by morphometry and ³H-phenylalanine incorporation assay. Resveratrol treatment significantly prevented the development of concentric hypertrophy, and contractile dysfunction in SHR without lowering blood pressure. The in vitro studies also showed that resveratrol prevented the adult cardiomyocyte hypertrophy induced by norepinephrine. Pharmacological inhibition of NO-AMPK signaling abolished the anti-hypertrophic action of resveratrol in cardiomyocytes. Consistent with the in vitro findings, the anti-hypertrophic effect of resveratrol in the SHR model was associated with activation of NO-AMPK signaling. Resveratrol treatment was beneficial in preventing the development of hypertrophy and cardiac dysfunction in SHR. Furthermore, NO-AMPK signaling may mediate the cardioprotective effects of resveratrol.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):30A.

121 Inhibition of Prolyl Hydroxylases as a Therapeutic Strategy for Ischemic Heart Disease

M Thirunavukkarasu ¹, B Oriowo ¹, L Zhan ¹, J Sanchez ¹, N Maulik ¹

Abstract

Oxygen supply declines under ischemic/hypoxic conditions in many human vascular diseases including ischemic heart disease. The resulting hypoxia causes functional impairement of cells as well as structural tissue damage and triggers a broad spectrum of cellular defenses such as angiogenesis, apoptosis. During hypoxia, reduced prolyl hydroxylase activity initiates a signaling cascade that includes the accumulation and activation of HIF-1 and hence they can be used as therapeutic targets to treat ischemic diseases. Intense research on PHDs and HIFs are required to identify and validate therapeutic targets for ischemic diseases by altering the glycolysis, apoptosis and angiogenesis pathways. Under normoxic conditions, hydroxylation of specific prolyl residues by the PHD oxygen sensors activates the interaction of HIF-1 with the von Hippel-Lindau protein targets HIF-1 for proteosomal degradation. In hypoxia, this interaction is actually suppressed due to lower hydroxyproline levels, and HIF-1 is stabilized and accumulates. Again, in the streptozotocin–induced diabetic animal model, myocardial ischemia/reperfusion-induced HIF-1 expression is found to be impaired, there was significant proteosomal degradation of HIF-1 and the impairment of angiogenesis. While PHDs were shown to hydroxylate HIF-1 in an oxygen concentration-dependent manner in vitro, but PHD2 has been found to be physiologically relevant regulator of HIF-1 stabilization. However the relevant physiological targets of PHD-1 and PHD-3 remain uncharacterized. If we understand the molecular mechanism and possibly reinforce targets we can bring these PHDs (inhibition) to the clinical scenerio.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):30A.

122 Adipose Tissue is a Potential Source of Multipotent Stem Cells for Treatment of Heart Failure

Ganghong Tian ¹

Abstract

Heart failure is the leading cause of death worldwide. Unfortunately, current therapies are unable to stop the pathological cascade of heart failure. Recent studies have shown that stem cells may be an effective therapeutic strategy for curing of the devastating disease. However, ideal cell types for treatment of heart failure and other degenerative diseases have not been defined.

Adipose tissue contains a type of adult stem cells, named adipose-derived stem cells (ASCs). In our preclinical studies, we have found that ASC can differentiate into several cell lineages and express various growth factors that are essential for tissue regeneration. The results indicate that ASC has a great potential for use in clinic for treatment of heart failure and other degenerative disease. In my presentation, I will summarize our preclinical work on characterization and evaluation of ASC, particularly on their ability in repairing injured heart muscle.

In addition, effective cell therapy requires more than injection of right type of cells. Its outcome profoundly depends on whether stem cells are viable and functional after transplantation. Reliable monitoring of implanted stem cells in vivo is very important to understand the mechanisms of cell therapy and to maximize its therapeutic benefits. Magnetic resonance imaging offers a great spatial resolution while positron emission tomography (PET) offers a great sensitivity, allowing detection of chemicals in a picomolar level. Dual-model probes that be visualized by MRI and PET would therefore have a wide research and clinical application in stem cell therapy, cardiology, oncology, and neurology. In my presentation I will briefly talk about our studies on monitoring of stem cells using both PET and MRI. I will also mention our work on development of dual-imaging nanoparticles for tracking stem cells.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):30A.

123 Genes Linking Several Complications of Type 2 Diabetes

J Tremblay ¹

Abstract

T2D and its complications are multifactorial traits known to have a strong genetic component with contributing environmental determinants. Micro- and macrovascular disorders are affecting many organs leading to severe complications including nephropathy, neuropathy, retinopathy and major adverse cardiovascular events (MACE) in about half of the patients suffering from T2D. We used ADVANCE cohort that is a randomized trial of blood pressure lowering and intensive glucose control on outcomes in patients with T2D to identify the genetic determinants of T2D complications. Total genomic DNA was extracted from ADVANCE patients of Caucasian origin (n=2313). Cases having several complications of T2D were compared to T2D patients without such complications. DNA was genotyped using Affymetrix GeneChip® SNP arrays 5.0 and 6.0 and genome wide association studies (GWAS) were performed with PLINK v1.07 to generate several lists of SNPs associated to either hypertension, atrial fibrillation, micro-macrovascular complication, myocardial infarction and angina, MACE, MACE and albuminuria or neuropathy. The cAMP-specific phosphodiesterase 4D (PDE4D) is a gene common to 7 outcomes including macrovascular disorders, hypertension, myocardial infarction and angina, MACE, MACE + albuminuria, atrial fibrillation and neuropathy. 27 SNPs located in introns of PDE4D represent three groups of SNPs that are associated to different phenotypes and that reside in different haploblocks in CEU. We also identified four genes that are associated to 6 complications, the CUB and sushi domain-containing protein 1 precursor (CSMD1), the ataxin2 binding protein 1 (A2BP1), the membrane associated guanylate kinase2 (MAGI2) and the ribosomal protein S6, 90kd, polypeptide 2 (RBS6KA2). Our results suggest a pleiotropic role of PDE4D and other genes in several complications of T2D.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):31A.

124 Targeting CaMKIIβ Isoforms in Heart Failure

Balwant S Tuana ¹

Abstract

The Ca²⁺/CaM-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is ubiquitously expressed. Distinct genes (α, β, δ, γ) encode different isoforms of CaMKII, which are further subjected to alternative splicing giving rise to tissue specific variants. Cardiac tissue is shown to have a predominance of CaMKII δB and δC isoforms which are unregulated in heart failure. We reported that cardiac and skeletal muscle express a 73 kDa CaMKII β4 isoform that was generated by alternative splicing of the CaMKIIβ gene. The CaMKIIβ4 isoform differs from the ubiquitous (brain) CaMKIIβ isoform by the presence of three exons encoding three proline rich repeats in the C-terminal association domain. A direct interaction of a kinase anchoring protein (αKAP) with CaMKIIβ4 can target the enzyme to the sarcoplasmic reticulum (SR). Furthermore, αKAP directly binds Serca2a and thus can recruit CaMKIIβ4 to phosphorylate phospholamban. CaMKIIβ4 can also assemble the glycolytic enzyme complex to potentially regulate the local production of ATP at the SR membrane. A role for CaMKIIβ4/αKAP in modulating cardiac function in health and disease will be discussed.

Footnotes

Supported by Canadian Institute of Health Research

Exp Clin Cardiol. 2011 Oct;16(Suppl A):31A.

125 Decreased Fatty Acid Oxidation in Malonyl CoA Decarboxylase Deficient Mice Does Not Exacerbate Age-Induced Insulin Resistance

John R Ussher ¹, Wendy Keung ¹, Vaninder K Sidhu ¹, David G Lopaschuk ¹, Liyan Zhang ¹, Jagdip S Jaswal ¹, Cory S Wagg ¹, Jason RB Dyck ¹, Gary D Lopaschuk ¹

Abstract

Aging is associated with the development of chronic diseases such as obesity, insulin resistance, and heart failure. A reduction in mitochondrial fatty acid oxidation is believed to be a key factor contributing to the progression of these diseases due to the accumulation of intracellular lipid intermediates. In order to address the contribution of impaired mitochondrial fatty acid oxidation to these age-related diseases, we utilized mice deficient for malonyl CoA decarboxylase (MCD^−/−), a model of reduced fatty acid oxidation. Mice were separated into 2 cohorts, one was allowed to age until death for survival analysis, while the other was subjected to a number of physiological parameters as they aged. As expected, decreased fatty acid oxidation in MCD^−/− mice was associated with the accumulation of lipid intermediates in both the muscle and liver. Despite this accumulation of lipid, MCD^−/− mice do not develop age-associated insulin resistance nor display any signs of hepatic disease, and on the contrary, have a significantly enhanced lifespan. Although fatty acid oxidation rates are decreased in hearts from aged MCD^−/− mice, these mice have improved cardiac efficiency and show no signs of cardiac dysfunction. Our data suggest that a life-long reduction in fatty acid utilization is compensated for by an enhancement in carbohydrate utilization, explaining why these animals do not develop premature disease in the presence of elevated lipid intermediates. Our data suggest that MCD inhibition may be beneficial in the battle against chronic disease in our elderly population.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):31A.

126 Implication of c-Src in Endothelin-1-Induced MAPK Activation and egr-1 Expression in Vascular Smooth Muscle Cells

G Vardatsikos ¹, AK Srivastava ¹

Abstract

We have previously shown that c-Src, a non-receptor protein tyrosine kinases (NR-PTK), is an upstream regulator of endothelin-1 (ET-1)-induced activation of protein kinase B (PKB) in vascular smooth muscle cells (VSMC). However, the role of c-Src in ET-1-induced mitogen activated protein kinase (MAPK) signaling remains controversial in this cell type. Furthermore, the signaling events downstream of these growth promoting pathways have not been clearly defined. Therefore, in the present studies, we have investigated the involvement of c-Src in MAPK activation and the regulation of early growth response factor-1 (egr-1), an atherogenic transcription factor, by ET-1. Treatment of VSMC with ET-1 induced the phosphorylation of MAPKs in A10 VSMC and in aortic VSMC isolated from 8–10 week old Sprague Dawley rats (SD VSMC). PP2, a Src family inhibitor, decreased phosphorylation of MAPKs induced by ET-1, in a dose-dependent fashion. ET-1 also up-regulated the expression of egr-1, which was inhibited by PP2. Further proof for the role of c-Src in this process was obtained by using mouse embryonic fibroblasts deficient in c-Src (Src^−/−MEF). ET-1-induced egr-1 expression, as well as MAPK activation, was found to be downregulated in Src^−/− MEF, as compared to MEF expressing normal Src levels. In summary, these data demonstrate that ET-1 signals the activation of MAPKs and up-regulates egr-1 expression through a c-Src dependent mechanism in VSMC.

Footnotes

Supported by grants from Canadian Institutes of Health Research

Exp Clin Cardiol. 2011 Oct;16(Suppl A):31A.

127 Implication of IGF-1R, ERK1/2 and c-Src in Endothelin-1-Induced Early Growth Response Factor-1 Expression in Vascular Smooth Muscle Cells (VSMC)

G Vardatsikos ¹, AK Srivastava ¹

Abstract

Aberrations in endothelin-1 (ET-1) signaling are thought to contribute to the development of vascular pathologies, such as atherosclerosis. This is believed to occur through the hyperactivation of growth promoting signal transduction pathways, including PI3K/PKB and the MAPK pathways, as well as regulation of transcription factors, such as early growth response factor-1 (egr-1), which was recently shown to be over-expressed in atherosclerotic plaque, and to play a role in multiple cardiovascular pathological processes. We have previously shown that IGF-1R and c-Src are upstream regulators of ET-1 activation of PKB in VSMC. However, the role of c-Src in ET-1-induced MAPK signaling remains controversial in VSMC, and the upstream mechanisms of ET-1-induced egr-1 expression remain unclear. Therefore, in the present studies, we have investigated the involvement of IGF-1R, c-Src and MAPKs in ET-1-induced egr-1 expression. ET-1 enhanced egr-1 protein levels in a time-dependent fashion in isolated aortic VSMC. AG1024 and PP2, selective pharmacological inhibitors of IGF-1R and c-Src, respectively, inhibited ET-1-induced egr-1 expression. PD98059, a selective inhibitor of MEK and the ERK1/2 cascade, was also able to inhibit ET-1-induced egr-1 expression. However, SB202190, a highly selective inhibitor of p38mapk, failed to inhibit ET-1-induced egr-1 expression in VSMC. In summary, these data demonstrate that ET-1 potently induces the expression of egr-1 in through a pathway implicating IGF-1R and c-Src, as well as ERK1/2, but not p38mapk, in VSMC.

Footnotes

Supported by grants from the CIHR

Exp Clin Cardiol. 2011 Oct;16(Suppl A):31A.

128 Protection Afforded by Inhibition of Adenylyl Cyclase Type 5

Stephen F Vatner ¹, Lin Yan ¹, Dorothy E Vatner ¹

Abstract

There are nine mammalian isoforms of adenylyl cyclase (AC). Type 5 AC (AC5) is one of the two major isoforms in the heart but is also present in most other organs. Overexpressing AC5 in the heart exacerbates chronic catecholamine-induced cardiomyopathy through increasing oxidative stress. Conversely, disrupting AC5 (AC5 knockout (KO)) prolongs longevity and protects against cardiac stress, either from chronic pressure overload or chronic beta adrenergic receptor stimulation. Similar results were found using a pharmacological inhibitor of AC5. The beneficial effects of inhibiting AC5 involved not only protection against development of heart failure, but also improved exercise capacity, increased insulin sensitivity and concomitant protection against obesity and glucose intolerance. Thus, inhibiting AC5 is a novel pathway with the potential of multi-target therapy for heart failure, obesity and diabetes.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):32A.

129 RNA Interference-Mediated Gene Silencing of the Negative SERCA2a Modulator Phospholamban

Roland Vetter ¹, Tobias Grössl ², Anja Geisler ², Sandra Pinkert ², Jens Kurreck ², Henry Fechner ^2,3

Abstract

Central players of cardiomyocyte Ca²⁺ regulation are the sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA2a) and its negative modulator phospholamban (PLN). The function of the PLN/SERCA2 system is critically dysregulated in failing hearts. Targeting the expression of PLN by RNA interference (RNAi) is a potential novel therapeutic approach to rescue SR Ca²⁺ handling in heart failure (HF). We have shown previously that AAV9 mediated delivery of small hairpin (sh) RNAs directed against PLN restores the hemodynamic parameters of failing heart in vivo. However, AAV9 vectors are also known to transduce the liver in addition to the heart. Moreover, the promoters typically used for expression of shR-NAs are known to be active in both organs. In contrast to conventional shRNAs, artificial microRNAs (amiR) represent a new class of shRNAs that can be expressed using tissue specific promoters. In this context, a known PLB silencing siRNA sequence was embedded into the environment of the miR155 and expressed under the control of a cardiac CMV-MLC0.26 hybrid promoter in cultured neonatal rat cardiomyocytes (CM). Treatment of the latter with a self complementary AAV6 vector expressing amiR155-PLN resulted in a time-dependent PLN mRNA and protein silencing that started at day 4 and reached its maximum (approx. −80%) at day 14 post transduction. By contrast, expressing the conventional shRNA resulted in an earlier and stronger PLN silencing. Both vectors did not alter the expression of SERCA2a and some other Ca²⁺-cycling cardiac proteins. PLN knockdown by these vectors caused a marked increase in the rate of cell homogenate SR Ca²⁺ uptake when measured at submicromolar Ca²⁺ concentration. Thus, amiR155-PLN appears to be an efficient and novel potential heart-specific therapeutic tool for PLN-silencing that could be of interest for the use in RNAi based gene therapeutic approaches to rescue SR Ca²⁺ handling in HF.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):32A.

130 FGF in the Heart: A Matter of Life and Death

J Wang ¹, ME Bock ¹, D Chan ¹, PA Cattini ¹

Abstract

Fibroblast growth factor (FGF)-16 plays an essential role during embryonic heart development and is expressed preferentially in the heart after birth, but its function here is unclear. A role in heart development was suggested by expression in the endocardium and epicardium at mid-gestation. We generated FGF-16 null mice that resulted in cardiac defects and death at embryonic day 11.5. In contrast to pre-birth, FGF-16 production switches to the myocardium in the perinatal rat and mouse heart, and is also detectable in the human heart. Direct effects of FGF-16 on postnatal cardiomyocytes are not well described. We showed, however, that FGF-16 interferes with protein kinase C (PKC) activation by FGF-2 in neonatal rat cardiomyocytes. FGF-2 is expressed in the myocardium but unlike FGF-16 is not cardiac-specific. FGF-2 is presumed to help maintain a healthy heart through its many effects, including on cardiac protection and cell growth, which are both linked to PKC signaling. This raises the possibility that FGF-16 contributes to control of the local “FGF” signaling environment in health and disease. Heart damage and cardiomyocyte loss can lead to heart dysfunction and failure. The demise of cardiomyocytes can result from vascular or ischemic heart disease, myocardial infarction and genetic cardiomyopathies, but also as a side effect of therapeutic agents, like the anti-cancer drug doxorubicin. Thus, an understanding of the endogenous cardioprotective environment that may include FGF-2 and its regulation by FGF-16, could contribute to the development of protective strategies that are heart-specific. We have started to characterize a doxorubicin model of neonatal rat cardiomyocyte injury, with a view to investigate if FGF-2 affects cell survival and whether FGF-16 modulates this effect.

Footnotes

Supported by the HSF of Manitoba

Exp Clin Cardiol. 2011 Oct;16(Suppl A):32A.

131 Alternatively Spliced Bnip3 Isoform Inhibits Hypoxia and Nutrient Deprivation-Induced Autophagy in Ventricular Myoctyes

Y Wang ¹, J W Gordon ¹, R Dhingra ¹, H Gang ¹, W Mughal ¹, LA Kirshenbaum ^1,²

Abstract

Autophagy is an evolutionary conserved process that allows cells to recycle their macromolecular constituents during times of cellular stress. Autophagy has been detected in several cardiac pathologies including ischemia-reperfusion injury, the infarct border zone, and in the progression to heart failure. In this report we show that metabolic stress imposed by hypoxia and/or nutrient deprivation, induces the expression of both full-length Bnip3 (Bnip3FL) and the spliced variant (Bnip3ΔEx3). In addition, we demonstrate that forced expression of Bnip3ΔEx3 could block autophagy induced by Bnip3FL during hypoxia or starvation. Furthermore, the autophagic response was markedly enhanced by genetic inhibition of Bnip3ΔEx3 by RNAi. Finally, we demonstrate that a shift from glucose to lipid metabolism to cardiac myocytes reduced autophagosome formation that was lost by inhibition of Bnip3ΔEx3. Our findings demonstrate a novel defense mechanism, where nutrient availability alters Bnip3 isoforms during hypoxia, which regulates autophagy.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):32A.

132 Hypomagnesemia-Induced Neurogenic Inflammation of the Heart

WB Weglicki ¹, IT Mak ¹, JJ Chmielinska ¹, JH Kramer ¹

Abstract

In animal models of hypomagnesemia due to magnesium-deficient diets early indicators of oxidative and nitrosative stress result from neuronal release of neuropeptides. With progressive hypomagnesemia significant cardiac inflammation and apoptosis occur; these changes can be partially reversed by the N-methyl D-aspartate (NMDA) receptor blockade that prevents loss of substance P (SP). In addition, treatment with SP receptor blockers reduces oxidative/nitrosative stress. Using echocardiography to assess cardiac systolic and diastolic dysfunction during hypomagnesemia we found significantly reduced ventricular fractional shortening and enhanced diastolic dysfunction, which were partially restored by SP receptor blockade.

In other studies with normomagnesemic rats, epidermal growth factor receptor (EGFR) blockade by a tyrosine kinase inhibitor, tyrphostin AG1478, decreased magnesium levels for more than a month when significant systolic dysfunction became evident. In recent years, cancer chemotherapy agents that block both EGF receptors as well as the EGFR tyrosine kinase have had increased clinical applications; antibodies to EGFR (e.g. cetuximab) have been reported to cause hypomagnesemia in many patients. Our findings of hypomagnesemia and cardiac dysfunction due to EGFR tyrosine kinase inhibition may have clinical relevance, particularly in patients who require combination therapy that includes other magnesium wasting drugs like cisplatin, which may result in even more severe hypomagnesemia and potentiation of cardiac side effects.

Footnotes

Supported by NIH RO1-HL-62282

Exp Clin Cardiol. 2011 Oct;16(Suppl A):32A–33A.

133 Can Long-Term Ventricular Assist Devices be Safely Implanted in Low-Volume Non-Heart Transplant Centers?

CW White ¹, A Chelvanathan ¹, S Zieroth ¹, DH Freed ¹

Abstract

Mechanical circulatory support (MCS) using an implantable ventricular assist device (VAD) has been established as an appropriate therapy for bridge to transplant (BTT) or transplant candidacy (BTC) in patients with advanced heart failure. Previous studies have suggested outcomes following VAD implantation may be dependent on institutional procedural volume. We report our non-heart transplant center’s experience with long-term VADs.

We conducted a retrospective review of all patients who received an implantable VAD over a two-year period (2008–2010) where there is access to a variety of MCS therapies including ECMO and short-term support devices.

During the study period, 41 patients received MCS of which 11 were supported with an implantable VAD (mean age 52 years, 82% male). Indication for device placement was post-cardiotomy in 36 %, ischemic cardiomyopathy in 36%, and non-ischemic cardiomyopathy in 28%. The majority of patients (64%) were INTERMACS category I. Prior to VAD placement, all patients required at least 1 inotropic medication, 64% had suffered a cardiac arrest, 64% required an intra-aortic balloon pump, and 72% required mechanical ventilation. ICU and 180-day survival were 100%. Survival on continued support or to transplantation was 82%, mean duration of support of 221 days with all surviving patients in NYHA class I or II.

Successful MCS programs can be safely and effectively established at low-volume non-heart transplant centers with survival rates comparable to INTERMACS registry data and high volume single centers, when managed by a specialty trained multidisciplinary MCS team. Specialty training among members of a multidisciplinary team and surgeon experience with a variety of MCS solutions may be more important determinants of outcome following implantation of long-term VADs.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):33A.

134 Regulation of Endothelial Proliferation Senescence by the Meox Family of Homeobox Genes

JT Wigle ¹, DC Cheung ¹, J Douville ¹

Abstract

The mesenchyme homeobox genes, MEOX1 and MEOX2, encode home-odomain transcription factor proteins. The homeodomain is an evolutionarily conserved DNA binding motif, however it has also been shown to mediate protein-protein interactions. MEOX2 is a key regulator of cardiovascular cell function and controls cell proliferation via induction of the cyclin-dependent kinase inhibitor, p21. Recently MEOX2 has been shown to induce senescence via induction of p16 transcription. Meox1/Meox2 gene knockout studies in mice have shown that the Meox proteins can partially compensate for one another during development; however MEOX1 function in the cardiovasculature has not yet been studied. The objectives of our study were to: 1) Establish the mechanism of MEOX2/MEOX1 regulation of p21 and p16 gene transcription 2) Determine the effects of increased MEOX1/MEOX2 expression on proliferation and senescence of endothelial cells. In order to fulfill these aims, MEOX proteins were expressed in primary human umbilical vein endothelial cells (HUVEC) via adenoviral transduction. The ability of MEOX proteins to regulate p21/p16 gene expression was determined by luciferase reporter gene assays, western blot analysis and quantitative real-time PCR. Cellular proliferation was assessed by bromodeoxyuridine (BrdU) labeling and senescence by staining for Beta-galactosidase. MEOX1 and MEOX2 were able to activate transcription of p21 and p16. This induction correlated with increased endothelial senescence and decreased endothelial proliferation. We also determined that a DNA binding deficient version of MEOX2 was able to activate p21 expression but was unable to activate p16 expression.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):33A.

135 New Concepts in Vascular Dysfunction

P Zahradka ¹

Abstract

Restenosis remains a significant problem even after the introduction of drug-eluting stents. Based on the evidence to date, a significant factor in the failure of these stents to block restenosis stems from the specific drug targets, which appear to have deleterious effects on normal vascular function. Consequently, identification of new targets may provide alternative options for therapeutic intervention. The release of Angiotensin II (AngII) in response to vascular injury promotes development of restenotic lesions. We have previously reported that the actions of AngII are transduced through both AT1 and AT2 receptor subtypes, but the signal transduction pathway(s) leading from the AT2 receptor were undefined. The importance of the AT2 receptor was established with the non-peptide antagonist PD123319, which prevented both proliferation and migration, and similar results were obtained after depletion of the AT2 receptor with lentivirus-directed shRNA. Additionally, we have found that ligand binding to the AT2 receptor results in phosphorylation of focal adhesion kinase (FAK), and inhibition of FAK activation also blocks cell proliferation and migration. Since the AT2 receptor mediates FAK phosphorylation at a site distinct from that obtained via the AT1 receptor, we propose that FAK has an essential integrative role as a conduit for the cellular actions of AngII. Furthermore, the AT2 receptor and FAK may represent novel targets for therapeutic intervention.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):33A.

136 Calcium Activates SERCA2 Gene Transcription via the Calcineurin-NFAT Pathway in Cardiomyocytes

A Zarain-Herzberg ¹, JA Fragoso-Medina ¹, R Estrada-Avilés ¹, G Rodríguez ¹

Abstract

The cardiac sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2) is vital for the proper contractile function in the heart. Decreased levels of SERCA2 mRNA and protein have been extensively demonstrated in animal models of cardiac hypertrophy and in patients with heart failure. Recent studies have shown that SERCA2 mRNA and protein levels are increased in response to increased cytoplasmic Ca²⁺ concentration ([Ca²⁺]c), and it has been suggested that the Calcineurin-NFAT pathway is probably one of the mechanisms by which SERCA2 gene expression is increased in response to high [Ca²⁺]c. The SERCA2 gene 5′-regulatory region contains 6 putative NFAT transcription factor binding elements that might be responsible for transcriptional activation, therefore, the goal of our work is to investigate whether activation of SERCA2 expression in response to increased [Ca²⁺]c is due to a transcriptional mechanism involving NFAT.

To increase [Ca²⁺]c primary cultures of neonatal rat cardiomyocytes were treated with thapsigargin (Tg) (0.2 μM) and the calcium ionophore A23187 (0.5 μM) for 12 hours. SERCA2 mRNA expression was increased 48% by Tg, and 56% by A23187. To understand if the mRNA increase was due to a transcriptional mechanism, transient transfections of the human SERCA2 promoter constructs in pGL3-basic were performed. The transcriptional activity was increased 2 to 3-fold when [Ca2+]c was increased and the calcineurin blocker Cyclosporine A abolished such increase. These results demonstrate that the rise of [Ca2+]c increases SERCA2 gene transcription mainly via the Calcineurin-NFAT pathway.

Acknowledgments

Supported by SEP-CONACYT 78750 and PAPIIT-UNAM IN204410 grants to A. Z.-H

Exp Clin Cardiol. 2011 Oct;16(Suppl A):33A–34A.

137 Chemotherapy Induced Cardiac Dysfunction is Potentiated by Congenital Absence of Nitric Oxide Synthase 3 (NOS3)

M Zeglinski ¹, S Bohonis ¹, J Lerner ¹, M da Silva ¹, A Sharma ¹, J Thliveris ¹, P Singal ¹, DS Jassal ¹

Abstract

BACKGROUND:

The recent addition of Trastuzumab (Trz) to Doxorubicin (Dox) in the adjuvant setting increases remission and reduces mortality in breast cancer patients. However, 25% of women develop Dox+Trz induced cardiac dysfunction. Despite the growing risk of heart failure in these breast cancer patients, little is known about the mechanisms associated with Dox+Trz induced cardiac dysfunction. The relationship between nitric oxide synthase 3 (NOS3) and oxidative stress has become of interest in the development of heart failure in breast cancer patients.

OBJECTIVE:

To determine whether congenital absence of NOS3 (NOS3^−/−) potentiates cardiac dysfunction in an acute female murine model of Dox+Trz induced cardiomyopathy.

METHODS:

90 C57Bl/6 female mice were treated with ip injections of Dox, Trz, or Dox+Trz. Echocardiography was preformed daily for 10 days. On day 10, mice were sacrificed and histological analysis was performed.

RESULTS:

WT female mice that received Dox+Trz demonstrated a decrease in LVEF from 75±3% at baseline to 56±2% (p<0.05). In contrast, NOS3^−/− mice that received Dox+Trz demonstrated a decrease in LVEF from 74±2% at baseline to 45±3% at day 10 (p<0.05). Furthermore, NOS3^−/− mice demonstrated decreased survival following the administration of Dox+Trz. Histology determined increased loss of cell integrity and dilation of the smooth endoplasmic reticulum in NOS3^−/− mice.

CONCLUSION:

NOS3^−/− mice treated with Dox+Trz demonstrated increased cardiac dysfunction as compared to WT. Further study is required to determine whether overexpression of NOS3 will be cardioprotective in this female model of Dox+Trz mediated cardiac dysfunction.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):34A.

138 Physical Interaction Between the Cardiac Rapidly (IKr) and Slowly (IKs) Activating Delayed Rectifier Potassium Channels

Shetuan Zhang ¹

Abstract

Cardiac repolarization is controlled by the rapidly (IKr) and slowly (IKs) activating delayed rectifier potassium channels. The human ether-a-go-go-related gene (hERG) encodes IKr, whereas KCNQ1 and KCNE1 together encode IKs. Decreases in IKr or IKs cause long QT syndrome (LQTS), a cardiac disorder with high risk of sudden death. A reduction in extracellular K⁺ concentration ([K⁺]o) induces LQTS and selectively causes endocytic degradation of mature hERG channels from the plasma membrane. In the present study, we investigated whether IKs compensates for the reduced IKr under low K⁺ conditions. Our data show that when hERG and KCNQ1 were expressed separately in human embryonic kidney (HEK) cells, exposure to 0 mM K⁺ for 6 h completely eliminated the mature hERG channel expression, but had no effect on KCNQ1. When hERG and KCNQ1 were coexpressed, KCNQ1 significantly delayed 0 mM K⁺-induced hERG reduction. As well, hERG degradation led to a significant reduction in KCNQ1 in 0 mM K⁺ conditions. An interaction between hERG and KCNQ1 was identified in hERG+KCNQ1 expressing HEK cells. Furthermore, KCNQ1 preferentially co-immunoprecipitated with mature hERG channels which are localized in the plasma membrane. Biophysical and pharmacological analyses indicate that although hERG and KCNQ1 closely interact with each other, they form distinct hERG and KCNQ1 channels. These data extend our understanding of delayed rectifier potassium channel trafficking and regulation as well as the pathology of LQTS.

Exp Clin Cardiol. 2011 Oct;16(Suppl A):34A.

139 Different Influence of Spontaneous Hypertension on Membrane Properties and ATP Production of Heart and Kidney Mitochondria in Rat: Effect of Captopril and Nifedipine, Adaptation and Dysadaptation

A Ziegelhöffer ¹, J Mujkošová ¹, O Uličná ², I Waczulíková ³, M Ferko ¹, N Vrbjar ¹, B Ziegelhöffer ⁴, T Ravingerová ¹

Abstract

In spite of numerous papers on hypertension (HYP), its influence on mitochondrial (MIT) function is still little understood. Present study deals with HYP-induced changes in function and properties of heart and kidney MIT of 16 week old male spontaneously hypertensive rats treated for the next 4 weeks daily with nifedipine (NIF 10 mg.kg-1) and/or captopril (CAP 80 mg.kg-1). Systolic blood pressure (SBP) and heart rate (HR) were monitored during- and the heart/body ratio (HW/BW) at termination of experiment. In isolated MIT we estimated the parameters of oxidative energy production (OEP): QO2, RCI index, ADP/O ratio, the rate of oxidative phosphorylation (ROP), further the fluidity (MF) and content of conjugated dienes (CD) in MIT membranes. Heart: The elevated SBP, HR and HW/BW characterising HYP were accompanied with elevated MIT OEP indicating an adaptive response to HYP-caused increase in energy demands of the myocardium. CAP and CAP+NIF almost similarly prevented the increase in SBP, HR and HW/BW removing also the reason for adaptive rise in ROP (all changes p<0.05–0.01). Kidney: HYP induced a dramatic drop in MIT OEP. Antihypertensive therapy even aggravated this energy insufficiency whereby CAP+NIF treatment was more detrimental than that with CAP alone. Heart and kidney MIT both exhibited moderately increased MF that was little influenced by CD content but was potentiated more with CAP than with CAP+NIF treatment.

Footnotes

Grants: VEGA 2/7126/27, 1/0755/09, 2/0054

Exp Clin Cardiol. 2011 Oct;16(Suppl A):34A.

140 Effects of Palladium Compounds on the Isolated Rat Heart: Focus on Oxidative Stress

Vladimir Zivkovic ¹, Dragan M Djuric ², Dusica Djordjevic ¹, Tanja Peric ¹, Milena Vuletic ¹, Nevena Barudzic ¹, Vladimir Jakovljevic ¹

Abstract

Taken into consideration limited data about effects of palladium on cardiovascular system and oxidative stress in rats, the aim of our study was to compare toxicity of inorganic and organic palladium compounds on the isolated rat heart and their effects on oxidative stress. The hearts (total number n=30, 6 for each experimental group) excised from Wistar albino rats, male sex, age 8 weeks, body mass 180–200 g, were retrogradely perfused according to the Langendorff technique at constant perfusion pressure (70 cmH₂O). After the insertion of sensor in the left ventricle, the parameters of heart function: (maximum rate of left ventricular pressure development (dP/dt max), systolic left ventricular pressure (SLVP), diastolic left ventricular pressure (DLVP), mean blood pressure (MBP) and heart rate (HR)), were continuously registered. Oxidative stress parameters (NO, TBARS, O₂⁻ and H₂O₂) were determined spectrophotometrically in coronary venous effluent. The experiments were performed during control conditions, and in presence of perfusion with incresing concentration of following: (triethanolamine (TEA), triethanolamine acetate (TEAA), palladium(II)chloride (PdCl₂), and trans-dichlorobis(triethanolamine-N)palladium(II) complex (trans-[PdCl₂(TEA)₂])) started every 30 minutes (30, 60, 90, 120 minutes). In our study, inorganic palladium compound (PdCl₂) induced clear depression of the isolated rat heart contractility, manifested as drop in diastolic and mean blood pressure, and as decrease of the heart rate. On the other hand, it seems that palladium, when bound in an organic compound (linked to TEA in Pd complex), does not contribute significantly to cardio-toxicity. Our results show that PdCl₂ may also have highest pro-oxidant potential (increase of O₂ and H₂O₂ levels), comparing with organic palladium compound.

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PERMALINK

Abstracts of the Plenary and Symposia Talks; and Poster Presentations

1 Genome Wide Characterization of RNA Polymerase II-Dependant Transcription During Cardiac Hypertrophy

Maha Abdellatif

Danish Sayed

Abstract

2 Evaluation of Load-Independent Biventricular Contractility During Machine Perfusion of the Donor Heart

Ayyaz Ali

B Xiang

S Colah

Paul Mundt

Rakesh C Arora

Steven R Large

G Tian

Darren H Freed

Abstract

3 Protective Role of Oleic Acid Against TNF-alpha Induced Effects in Adult Rat Cardiomyocytes

A Al-Shudiefat

A Sharma

A Bagchi

Anju Bajaj

P Singal

Abstract

BACKGROUND:

4 Delirium in the Postoperative Cardiac Surgery Patient

RC Arora

Abstract

5 Enhanced Expression of Gαq and PLCβ1 Contribute to Vascular Hypertrophy in Spontaneously Hypertensive Rats

ME Atef

MB Anand-Srivastva

Abstract

6 Regulation of BNIP3 by Casein Kinase II Promotes Cell Survival of Ventricular Myocytes

Delphine Baetz

Joe Gordon

Hongying Gang

Lorrie A Kirshenbaum

Abstract

7 L-10-Induced Cardiomyocyte Survival Requires TLR 4 Regulated MyD88 Molecule

Ashim K Bagchi

Anita K Sharma

Ana Ludke

Abd Al-Rahaman Al-Shudiefat

Pawan K Singal

Abstract

8 Interdependancy of Calpain-Calpastatin and Ubiquitin-Proteasome Systems with Cardiac Ischemia/Reperfusion

Angelo Belcastro

Abstract

9 Redox-Sensitive Modulation of Signaling Pathways Involved in Thyroid Hormone-Induced Cardiac Hypertrophy and Failure

A Belló-Klein

AS Araujo

Abstract

10 Regulation of Rac1 Activation by Calmodulin in Platelet and CHRF-288-11 Cells

RP Bhullar

B Xu

Abstract

Footnotes

11 Nuclear Membranes G-Proteins Coupled Receptors and Nuclear T-Tubules Contribute to Normal and Pathological Cardiovascular Cell Function

Ghassan Bkaily

Danielle Jacques

Abstract

Footnotes

12 Ezetimibe Inhibits the Absorption of Omega-3 Fatty Acids

DP Blackwood

A Al-Busaidi

RK La Vallee

DS Jassal

GN Pierce

Abstract

INTRODUCTION:

METHODS:

RESULTS:

CONCLUSION:

Footnotes

13 FAT/CD36 and Fatty Acid Transport are Key Components of Muscle and Cardiac Energy Provision and Insulin Resistance

Arend Bonen

Abstract

14 Postconditioning Induced by BKCa Channel Opening in Isolated Ventricular Myocytes is Mediated by Reactive Oxygen Species

GH Borchert

F Kolar

Abstract

14 Postconditioning Induced by BK_Ca Channel Opening in Isolated Ventricular Myocytes is Mediated by Reactive Oxygen Species