The inclusion of HIV-1 protease inhibitors (PIs) in the highly active antiretroviral therapy (HAART) regimen has improved the prognosis of HIV-1 infected patients. Long-term use of PIs is associated with metabolic disorders, insulin resistance syndrome (IRS) and cardiovascular diseases. We have hypothesized that PIs induce oxidative stress and antioxidant may ameliorate the side effects. We determined the levels of ROS and of anti-oxidant enzymes in the PI treated rat pancreatic beta-cells (INS-1). The ER stress molecules, levels of uncoupling protein 2 (UCP2) and ATP levels were also determined. Exposure of INS-1 cells to the nelfinavir significantly decreased pre-pro-insulin gene expression and insulin secretion within 24 hr. There was a significant increase in ROS levels and ER stress molecules (Grp78 and phosphoeIF2α) and a decrease in superoxide dismutase (Cu/Zn SOD) and glutathione levels. Mitochondrial ROS generation and mitochondrial SOD (MnSOD) in PI treated INS-1 cells were unaffected suggesting that mitochondrial ETC is not the source for the oxidative radicals. Simultaneous treatment with TQ (2.5 μM), an ingredient of black seed oil (BSO), significantly inhibited ROS and ER stress in cells exposed to nelfinavir and increased glucose mediated insulin secretion to normal levels. The in vivo study in rats (8 per group) included control (normal diet), diet mixed with nelfinavir, zidovudine and efavirenz, diet mixed with drugs and BSO and diet mixed with BSO. There was no significant change in body weight, post-prandial blood glucose levels and lipid profile of treated groups as compared to controls up to 7 months. Serum analysis at the end of the study in the HAART-group showed 2- and 5-fold increase in serum insulin and C-peptide levels, respectively, which was reduced to normal levels in rats that received HAART drugs in combination with BSO. These data demonstrate TQ/BSO as a potential therapeutic strategy for ameliorating IRS in HAART treated patients.
Platelet dysfunction plays an important role in the development of several cardiovascular diseases. Platelet activation and aggravation is considered to be central to arterial thrombus production. As a result, pharmacological intervention that influence platelet function have applications for the prevention and treatment of ACS, heart failure, hypertension, ischemia-reperfusion injury, stroke, atherosclerosis, atrial fibrillation as well as coronary and peripheral arterial disease. Although classical antiplatelet agents such as Aspirin, Thienopyridines and Glycoprotein IIb-IIIa inhibitors have proven beneficial effects for the treatment of some specific cardiovascular diseases, there are limitations for their use as these drugs target platelet functions directly. In contrast, newly developed antiplatelet agents have brought applications for the treatment of cardiovascular disease as they not only influence platelet dysfunctions, but also considered to affect cardiac and vascular smooth muscle cell functions. Natural food products and nutriceutical agents also appear to modify cardiovascular abnormalities by affecting various platelet functions.
Left ventricular assist devices (LVAD) are currently used to either “bridge” patients with terminal congestive heart failure (CHF) until cardiac transplantation is possible or optionally for patients with contraindications for transplantation (“destination therapy”). Mechanical support is associated with a marked decrease of cardiac dilation and hypertrophy as well as numerous cellular and molecular changes (“reverse cardiac remodeling”), which can be accompanied by improved cardiac function (“bridge to recovery”) in a relatively small subset of patients with heart transplantation no longer necessary even after removal of the device (“weaning”). In the recent past, novel pharmacological strategies have been developed and are combined with mechanical support, which has increased the percentage of patients with improved clinical status and cardiac performance. Gene expression profiles have demonstrated that individuals who recover after LVAD show different gene expression compared to individuals who do not respond to unloading. This methodology holds promise for the future to develop read out frames to identify individuals who can recover after support. Aside from describing the morphological changes associated with “reverse cardiac remodeling”, this talk will focus on signal transduction, transcriptional regulation, apoptosis, cell stress proteins, matrix remodeling, inflammatory mediators and aspects of neurohormonal activation in the failing human heart before and after ventricular unloading.
The vascular form of the Ehlers-Danlos syndrome, EDS type IV, is a result of mutations in the COL3A1 gene that encodes the chains of type III and alters the sequence in the triple-helical domain. Complications in afflicted patients include spontaneous ruptures of vessels and hollow organs, particularly the colon. The haploinsufficiency for one COL3A1 allele is one of the genotypes resulting in the EDS type IV. We studied 9 and 14 months old mice, +/− for Col3a1 “null” alleles. In the aortic wall of several +/− mice (but in none of the +/+) we found lesions accompanied with focal inflammation, fibrosis, and disruption of elastin. The biomechanical property of the colon was assessed by measurement of the pressure induced by increment injections of 0.9% sodium chloride into isolated segment. The slope of pressure-volume relationship was significantly less steep in +/− mice than in the +/+ (p<0.05). The average blowup pressure in the colon of the +/+ mice (234±10.1 mmHg) was 30% higher than in the colons of +/− mice (163±9.3 mmHg), The biomechanical weakness of the colon of +/− mice was associated with reduced mRNA expression and total protein concentration of collagen III detected by real time PCR and by LC/MS respectively. The biomechanical weakness of colon from +/− mice could serve as a measurable endpoint for assessment of treatment strategies in this mouse model.
Recent evidence indicates that nitrite can be reduced to nitric oxide (NO) and act as a selective hypoxia sensitive pulmonary vasodilator. However questions about species, route of administration and mechanism of nitrite activation remain unanswered. In the present study pulmonary vasodilator responses to sodium nitrite in the rat were investigated under baseline and elevated tone conditions and the effects of inhibitors of xanthine oxidoreductase, NO synthase and of hypoxia on responses to nitrite were investigated. These studies show that iv injections of sodium nitrite in dose of 10 to 100 umol/kg decrease pulmonary and systemic arterial pressure and increase cardiac output without changing left ventricular end diastolic pressure showing that nitrite anion decreases pulmonary and systemic vascular resistance. The decreases in pulmonary arterial pressure were greatly enhanced when baseline tone was increased with U46619 or hypoxia and responses to sodium nitrite were slower in onset than responses to nitroprusside. Vasodilator responses to sodium nitrite were enhanced by L-NAME and inhibited by allopurinol in a dose that did not alter responses to sodium nitroprusside or DEA/NO. Pulmonary vasodilator responses to sodium nitrite under elevated tone conditions were not altered by hypoxia and responses to hypoxia were enhanced by L-NAME but were not altered by allopurinol. These data indicate that nitrite is rapidly converted into a substance that has pulmonary vasodilator activity and that vasodilator responses are enhanced when tone is increased and NO synthesis is inhibited. These data indicate that nitrite is reduced to NO by an allopurinol sensitive mechanism. The response to sodium nitrite is not enhanced by hypoxia when tone is increased by U46619 or L-NAME. These data suggest that NO derived from NOS but not from the reduction of endogenous nitrite modulate the response to hypoxia and that nitrite is rapidly reduced to NO by xanthine oxidoreductase.
Cardiac fibroblasts synthesize collagens as part of the extracellular matrix, which contributes to the mechanical strength of the myocardium. In response to stresses such as myocardial infarction or hypertension, the heart undergoes remodeling marked by cardiomyocyte hypertrophy, alterations in chamber dimensions and phenotype conversion of fibroblasts into myofibroblasts that synthesize large amounts of fibrillar collagens. Excessive collagen synthesis impairs contraction and relaxation, eventually contributing to cardiac failure. The development of improved anti-fibrotic therapies requires a greater understanding of the control of collagen synthesis. Of note, the transcriptional regulators of collagen synthesis are not fully known. Stimulation of cardiac fibroblasts with the pro-fibrotic growth factor TGF-β induces a four-fold increase in scleraxis expression. Overexpression of scleraxis in NIH 3T3 fibroblasts is sufficient to up-regulate the expression of collagen Iα2. By in silico analysis, we have identified several putative scleraxis binding sites in the collagen Iα2 promoter which are conserved in mice, rats and humans. In in vitro luciferase assays, scleraxis strongly transactivated the collagen Iα2 promoter. Deletion experiments reveal that transactivation is critically dependent on the DNA-binding motif of scleraxis, and is attenuated by deletion of a protein-interaction moiety. Taken together, our results strongly support the hypothesis that scleraxis directly regulates cardiac collagen gene expression, and may play a key role in cardiac fibrosis. We report here that the basic helix-loop-helix transcription factor scleraxis regulates the expression of collagen Iα2, the primary collagen expressed in cardiac fibrosis. We show that scleraxis is expressed in cardiac fibroblasts, and its expression level increases two-fold as fibroblasts phenoconvert to myofibroblasts.
Acknowledgements
Heart & Stroke Foundation of Canada and Manitoba Medical Service Foundation.
Since atrial fibrillation (AF) which is more prevalent in hypertension constitutes a major risk for embolic stroke, the aim of the present study was to identify clinical phenotypes associated to AF in hypertensive patients (HT) in order to improve its prevention. 160 consecutive HT patients in sinus rhythm (80 with documented history of 1 or more episodes of AF and 80 with no known history of AF) and 30 normotensive subjects without history of AF underwent clinical, metabolic, ECG and a Doppler echocardiography evaluation. The activity of the autonomic nervous system was studied at rest in supine position and during active standing by measurement of blood pressure (BP), heart rate (HR), spectral analysis of short-term heart rate variability (HRV) in terms of low (LF) and high (HF) frequency power, and the ratio LF/HF, as well as plasma noradrenaline (NA) and adrenaline (A) levels by high-performance liquid chromatography technique. Plasma renin, aldosterone, atrial natriuretic peptide (ANP) and insulin levels were measured by radioimmunoassay kits. The mean age was not significantly different between the two groups of HT patients but the duration from the onset of hypertension was shorter in patients with a history of AF. This study enabled us to describe by simple, noninvasive techniques several distinctive characteristics in HT patients with a history of AF: 1) an increased systolic and pulse BP, 2) a prolonged duration of the P wave, 3) an increased LA size and a reduction in LA ejection fraction, 4) an impairment in autonomic modulation with a reduced cardiac sympathetic reactivity and an increased parasympathetic reactivity to standing and 5) lower ANF, renin, aldosterone and insulin levels. Our study shows that it is possible to identify several phenotypes associated to an increased risk for AF in HT patients thus allowing for a better prediction and prevention of this serious complication of hypertension in man.
It is now well known that the behaviour of subcellular organelles such as sarcolemma (SL), sarcoplasmic reticulum (SR) and myofibrils (MF) is dramatically altered in the ischemic heart disease. Although extensive research from our laboratory has revealed that the attenuated cardiac function in the ischemic heart disease is closely associated with remodeling of subcellular organelles (changes in biochemical and molecular composition) as a consequence of ischemia-reperfusion injury, the mechanisms of these alterations are not fully understood. By employing an isolated rat heart as a model of ischemia-reperfusion injury, we have demonstrated that the SL, SR and MF gene expression was depressed in ischemic-reperfused hearts and these alterations were attenuated by different antioxidant interventions including ischemic preconditioning. Furthermore, the changes in subcellular gene expression due to ischemia-reperfusion were simulated by interventions, which generate oxidative stress in the myocardium. These results suggest that the delayed recovery of cardiac function in the ischemic hearts upon reperfusion may be a consequence of the development of oxidative stress, and subsequent depression in SL, SR and MF gene expression and subcellular remodeling.
Acknowledgements
Supported by a grant from the Canadian Institutes of Health Research.
Despite the major effort to reduce symptoms and to improve prognosis of patients with arterial occlusive syndromes and secondary heart failure and amputations, a significant number of patients do not benefit from currently available therapies. We tested the use of autologous venous endothelial (EC) and smooth muscle cells (SMC) modified to express Ang-1 and VEGF for induction of therapeutic angiogenesis. We initially tested the combination of EC and SMC in vitro in a spheroid assay of angiogenesis. The combination of EC expressing Ang-1 and SMC expressing VEGF produced a coordinated sprouting of EC and SMC. Gene transfer to the cells was accomplished using pseudo-typed retroviral vectors. The same cell and gene combination produced arteriogenesis in vivo two weeks after injection in a miniature pig ischemic hind limb model. Arteriogenesis was evident by two-fold increase in hind limb arterial flow and 40% increase in number of arterial vessels in hind limb musculature (n=6). Bioavailability and toxicity studies in rabbits demonstrated no adverse effects and no systemic spread of injected cells beyond the ischemic hind limb after local, intra-arterial injection. Improved hind limb muscle perfusion, arterial blood flow, and increased number of collateral arteries were demonstrated at 3 weeks and persisted for up to 6 months. Based on the in vitro and animal data we designed a dose escalating, open label, non-randomized study of 12 patients with limiting claudication secondary to leg arterial occlusion. Patient enrolment was started in 2007. http://clinicaltrial.gov/ct2/show/NCT00390767
Autophagy is an intracellular process in which cell digests its own constituents via lysosomal degradative pathway. Though autophagy has been shown in several cardiac diseases like heart failure, hypertrophy and ischemia reperfusion injury, the role and the regulation of autophagy is still largely unknown. Studies have indicated that autophagy is induced during hypoxia-reoxygenation in cardiac cells. However, the exact role of autophagy induced in hypoxia-reoxygenation is not known. Hypoxic adaptation is known to induce survival signaling in cardiac cells, and further it is shown to protect them. BAG-1 is a multifunctional pro-survival molecule binds protect cardiac cells from apoptosis induced by several stimuli. In the present study, H9c2 cardiac myoblast cells were subjected to either 30 min of hypoxia followed by reoxygenation for 1 hr (HR) or hypoxic preconditioning (HPC), where the cells were subjected to 3 cyclic episodes of 5 min hypoxia and 5 min reoxygenation, followed by HR. Hypoxia was given by placing the cultured plates into an air-tight hypoxic chamber placed in a humidified 37°C CO2 incubator, and passing the mixture of 95% Nitrogen and 5% CO2. Reoxygenation was given by placing back the cultured plates in a humidified 37°C CO2 incubator saturated with air. We found that hypoxia-reoxygenation injury in H9c2 cells slightly induced the protein expression of LC3-II, an autophagic marker protein present in the autophagosomal membrane. Also, the protein expression of Bag-1 is also slightly induced during hypoxia-reoxygenation. However, hypoxic adaptation prior to hypoxia reoxygenation further induced the protein expression of LC3-II and BAG-1. Our immunofluorescence analysis further show that LC3-II level has been enhanced during hypoxic adaptation. Furthermore, treatment with BAG-1 siRNA attenuated hypoxic adaptation induced LC3-II and BAG-1 proteins in cardiac myoblast cells. Moreover, hypoxic adaptation-induced cardiac cell viability studied using 3,(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) was reduced by Bag-1 siRNA treatment. The release of cardiac cell death marker lactate dehydrogenase (LDH) studied using the culture supernatant was reduced by hypoxic adaptation, but Bag-1 siRNA treatment abolished the hypoxic adaptation-induced reduction of LDH release. These results indicate that cardiac protection elicited during hypoxic adaptation is mediated at least in part via upregulation of autophagy in association with BAG-1 protein.
Genomics is currently on a long journey from Mendelian traits to complex diseases. Over last two years, genomic determinants of several complex diseases were identified by whole genome association studies (WGAS). Such achievement was made possible by the availability of analytical tools, which progressed from 500 polymorphic markers in 2003, to the current million single nucleotide polymorphism (SNPs) and copy number variants (CNVs) on a single chip, in 2008. Integration of information obtained from DNA also includes epigenetics and structure of telomers. Progressively, this wealth of information will be subjected to systems medicine and even to diseasome integrative analysis. With genetically designed rats we have undertaken the resolution of the composite trait of hypertension in the search of its dynamic genetic architecture interacting with environmental stimuli such as diet, stress and therapeutic agents. We have determined, using systematic genome wide search, the age- and sex-specific determinants of cardiovascular traits. Among 540 traits, 12.5% were either age- or sex-specific and 20 were determined by specific SNPs/haplotypes. Routinely used sex and age adjustment does not overcome the need for specific evaluation. The power of WGAS and the dense coverage of genome with SNPs permit the analysis of shared and distinct allelic impact among discrete geoethnic groups and their specific target organ susceptibilities. Our ancestral genes shaped in their original environment were submitted to environmental selection through a long process of adaptation to different cycles of stress, infection, starvation and dietary habits resulting in our current allelic make-up. Functionality of our genome is submitted to rapid adaptation over the lifetime by processes such as imprinting. Together with industrialisation and transition economies, the final result is the current cardiovascular epidemics with obesity, type 2 diabetes and their outcomes.
Numerous pathological conditions are associated with genomic damage, such as cardiovascular disease and cancer. There is growing evidence that DNA is one of the most targets of oxidative attack, which may be induced for instance by advanced glycation end products (AGEs), angiotensin II (AngII), aldosterone, homocysteine and other uremic toxins (indoxyl sulphate). We analysed the induction and modulation of genomic damage in in vitro studies (kidney cells, colon and liver cells, in human promyelocytic (HL-60) cells), as well as in vivo in peripheral blood lymphocytes of patients with end-stage renal disease. For evaluation of genomic damage we assessed two biomarkers: the single cell gel electrophoresis (comet assay) and the micronucleus frequency (MF) test. In vitro we could show that various advanced glycation end products (AGE-BSA, carboxymethyllysine, methylglyoxal) induce DNA damage in the mentioned cell lines, which was suppressed both by antioxidants (N-acetylcysteine) and AngII type 1 receptor blockers (Stopper et al., 2003; Schupp et al., 2005). Incubation of renal tubular and HL-60 cells with AngII in concentrations between 85–350 nM led to a 6- to 15-fold increase in DNA damage in the comet assay and a 4-fold increase in the MF test. AngII-induced DNA damage could be prevented by coincubation with AngII type 1 receptor blocker, candesartan, but not by an AngII type 2 receptor antagonist. Measurement of reactive oxygen species (ROS) by flow cytometry showed an enhanced formation after exposure to AngII and a reduction of ROS after candesartan (Schupp et al., 2007). Chronic administration of candesartan to 15 maintenance hemodialysis patients resulted in a significant improvement of the enhanced DNA damage, as compared to untreated control patients. Also homocysteine induced in vitro genomic damage. Administration of folic acid in dialysis patients resulted in a significant decline of micronucleus frequency.
Impairment of NO-dependent vasodilation has recently gained diagnostic, prognostic and therapeutic significance in cardiovascular diseases. We analyzed activities of NO, EDHF and PGI2 in aorta in animal models of atherosclerosis (ApoE/LDLR−/− mice) and heart failure (Tgαq*44 mice) that represent models of primary and secondary type of endothelial dysfunction, respectively. In 2-month-old apoE/LDLR−/− mice atherosclerotic lesions (Oil Red staining, CD68 immunostaining) were virtually absent while they were detected in 4–8-month-old mice. Acetylcholine (Ach)-induced endothelium-dependent relaxation in thoracic aorta was already impaired in 2-month-old apoE/LDLR−/− mice and remained diminished in older apoE/LDLR−/− mice. Importantly, EDHF-mediated relaxation (inhibited by miconazol and TEA) as well as COX2-derived PGI2 were gradually up-regulated in apoE/LDLR−/− mice parallel to the progression of atherosclerosis. In 6-month-old Tgαq*44 mice cardiac function as measured by MRI in vivo was nearly normal, while it was profoundly impaired in 14-month-old Tgαq*44 mice. Ach-induced endothelium-dependent relaxation in thoracic aorta was preserved in 6-month-old Tgαq*44 mice while it was impaired in 14-month-old Tgαq*44 mice. Basal NO activity and SNAP-induced endothelium-independent relaxation remained unchanged in Tgαq*44 mice irrespectively to the age of animals. EDHF and PGI2 pathways were not upregulated in Tgαq*44 mice. In summary, in apoE/LDLR−/− mice impairment of NO-dependent vasodilation in aorta precedes the development of atherosclerotic plaques, while in Tgαq*44 mice it occurs subsequent to heart injury. Compensatory upregulation of EDHF (most likely involving cytP450-derived EETs) and COX-2 derived PGI2 pathways, are present in atherosclerosis but not in cardiomyopathy in Tgαq*44 mice. Consequently, pharmacology of endothelium targeted to EETs or COX-2/PGI2 may be more effective in atherosclerosis than in dilated cardiomyopathy.
Oxytocin (OT), a neurohypophysial hormone, is known to be also produced and released in the heart (1). It acts on its cardiac receptors to decrease the heart rate and force of contraction. During stress several cardiovascular parameters are altered and OT may play a direct as well as indirect role in these alterations. Here, we investigated relative expression of OT receptor in each heart chamber. Additionally, we studied the involvement of this system in the events underlying response of the heart to acute stress. We used two types of stress: restraint/immobilization (IMO) and this stressor combined with immersion of rats into water (IMO+C), which were applied for 1 hour to Wistar rats. Heart atria and ventricles were analyzed by real-time RT-PCR and immunohistochemistry. The quantitative RT-PCR reactions were carried out in an iCycler (BioRad). Relative expression of OT receptor mRNA was expressed as a ratio of target gene concentration to housekeeping gene. The basal expression of OT receptor mRNA was about 30 times higher in the atria than in the ventricles. IMO+C caused increase of the mRNA expression in both ventricles, however, there were no significant changes in the atria. After IMO, the relative expression of OT receptor mRNA in both atria and ventricles did not significantly differ to controls. Indirect immunofluorescence showed OT receptor-immunoreactivity on the surface of cardiomyocytes. In summary, exposure to the two types of restraint stressors caused differential changes in OT receptor mRNA production in the rat heart.
Acknowledgements
Supported by MSM 0021620806 and MSM 0021620819.
Reference
- 1.Jankowski M, et al. Proc Natl Acad Sci USA. 1998;95:14558–14563. doi: 10.1073/pnas.95.24.14558. [DOI] [PMC free article] [PubMed] [Google Scholar]
Histone acetylation status plays a role in the epigenetic modulation of gene expression in health and in disease and is maintained by a dynamic balance between the activities of histone acetyl transferases and histone deacetylases (HDACs). The anticancer prodrugs butyroyloxymethyl diethylphosphate (AN-7) and butyroyloxymethyl butyrate (AN-1) release, upon metabolic degradation, the HDAC inhibitor butyric acid and formaldehyde, and induce histone hyperacetylation. We tested the hypothesis that HDAC inhibition by these prodrugs confers protection against ischemia and chemotherapy induced cardiac injury. The oral administration of AN-7 and AN-1 to mice and rats for 90 min induced histone hyperacetylation in the hearts and preconditioned them for improved tolerance to ischemia and reperfusion (I/R) ex vivo (Langendorff setup). The recovery of the left ventricular developed pressure (LVP) was significantly higher, and the degree of irreversible ischemic damage (infarct size, TTC staining) was significantly lower in the treated compared to the untreated hearts. In neonatal rat cardiomyocytes, co-administration of the prodrugs with doxorubicin reduced LDH leakage and preserved the mitochondrial membrane potential compared to cardiomyocytes treated with doxorubicin alone. In the two experimental models, molecular markers of damage and protection denoted prodrug associated cardioprotection. Our findings indicate that the HDAC inhibitory prodrugs impart cardioprotection against I/R injury and chemotherapy carditoxicity and warrant further investigation towards implementation for clinical application.
Acknowledgements
Supported by grants from the Israeli Ministry of Science, Arts and Sports, and Israel Science Foundation.
Na+ transport-regulating mechanisms classically considered to reflect renal control of the blood pressure, i.e. aldosterone–mineralocorticoid receptors–epithelial sodium channels – Na+/K+-ATPase, have now been demonstrated to be present in the central nervous system, and contribute to regulation of cerebrospinal fluid [Na+] by the choroid plexus and to neuronal responsiveness to cerebrospinal fluid/brain [Na+]. Dysfunction of either or both can activate central nervous system pathways involving “ouabain” and angiotensin type 1 receptor stimulation. The latter causes sympathetic hyperactivity and adrenal release of marinobufagenin – a digitalis-like inhibitor of the α1 Na+/K+-ATPase isoform – both contributing to hypertension on high salt intake. Specific central nervous system blockade of mineralocorticoid receptors or epithelial sodium channels prevents the development of hypertension on high salt intake, irrespective of the presence of a “salt-sensitive kidney”. Variants in the coding regions of some of the genes involved in Na+ transport have been identified, but sodium sensitivity may be mainly determined by abnormal regulation of expression, pointing to primary abnormalities in regulation of transcription. Looking beyond the kidney is providing new insights into mechanisms contributing to salt-sensitive hypertension, which will help to dissect the genetic factors involved and to discover novel strategies to prevent and treat salt-sensitive hypertension.
To explore the relationship between variations, i.e., Val16Ala in manganese superoxide dismutase (Mn-SOD), Prol98Leu in glutathione peroxidase-1 (GPx-1) and promoter –262C/T in catalase (CAT) gene and type 2 diabetes mellitus (T2DM) with or without coronary heart disease (CHD) in Chinese. 292 unrelated Chinese were divided into T2DM (n=198) and non-DM and non-CHD control (n=94) groups. The T2DM group was subdivided into non-CHD (n=95) and CHD (n=103) group, respectively. The individual genotypes of Mn-SOD-Val16Ala, GPx-1-Prol98Leu and CAT –262C/T were determined by PCR-direct sequencing and PCR-RFLP. Genotypic and allelic frequencies and clinical characteristics were compared among the groups. 1. Genotypic distributions in Val16Ala of Mn-SOD were not different between control and T2DM group, even between non-CHD and CHD subgroups. The decreased Ala16 allelic frequency in Chinese were observed when compared with Caucasian (P<0.001). 2. Prol98Leu genotypic frequency in GPx-1 in T2DM group was obviously elevated when compared with control group (P=0.012, OR=2.254), no significant differences of them were shown between CHD and non-CHD subgroups. Decreased Leu198 allelic frequency in Chinese were observed when compared with Swedish population (P=0.001).3. Genotypic and allelic frequencies of CAT –262C/T were not shown differences between control and T2DM group and between CHD and non-CHD subgroups. There were significant differences in CAT –262C/T distribution between Chinese and other ethnic populations, such as Caucasian, Russian and British (P<0.001). Prol98Leu in GPx-1 increases the risk of T2DM, while Leu198 allele is not a risk factor of diabetic CHD. Val16Ala in MnSOD and –262C/T in CAT may not contribute to the development of T2DM with or without CHD in Chinese. In addition, our results suggested that genetic heterogeneities on these three variations existed between Chinese and other ethnic populations.
Increased insulin resistance is associated with shorter telomere (TM) length in leucocytes. It remains unknown that improving insulin sensitivity affects the TM length and telomerase activity in diabetic heart. Male OLETF rats as type 2 diabetic models were used in the study. PPAR-r agonist, pioglitazone (PIO;10 mg/kg/day) mixed with rat chow was administered to OLETF (n=6) from early to advanced diabetic stage (20 weeks to 40 weeks of ages). At 40 weeks of age, HOMA-IR determined from serum glucose and insulin levels was higher in PIO-treated OLETF (n=6) than the untreated OLETF (n=6). The length of TM DNA estimated as the TM/centromeric DNA contents ratio was decreased in OLETF (54±3.4% of control LETO rats) (mean±SE). These observations were inhibited in PIO-treated OLETF (86±4.2% of LETO). Cardiac telomerase activity by TRAP assay and telomerase reverse transcriptase mRNA expression showed both depressed to 36±1.4% and 26±1.8% of LETO, respectively. These reductions were also inhibited in PIO-treated OLETF to 146±5.7% and 91±3.7% of LETO, respectively. PIO administration to OLETF induced to decrease cardiac mass estimated by left ventricle to body weight ratio (OLETF+PIO vs. OLETF; 2.47±0.10 vs. 2.75±0.14 mg/g), but not in systolic function determined by echocardiography (fractional shortening), compared to untreated OLETF. All effects are significant with p<0.05. The present study suggests that improving insulin sensitivity may induce to cardiac remodeling through the inhibition of the shorten TM length and the depressed telomerase activity in diabetic heart, but not change in cardiac function.
Over the past few years the better understanding of the biology of angiogenesis has significantly increased the use of growth factors in the treatment and/or prevention of ischemic heart disease by inducing blood vessel formation. The GSK-3β/β-catenin axis is one of the most interesting pathways involved in the molecular mechanism of angiogenesis. Recently we have shown the critical role of GSK-3β/β-catenin in ischemic preconditioning (IP) mediated angiogenesis. We observed for the first time that IP significantly induces the β-catenin driven expression of VEGF, Bcl-2, & survivin in established chronic rat MI model. β-catenin is found to be a critical mediator during development of angiogenesis & it is phosphorylated by active GSK-3β in a cytosolic multiprotein degradation complex containing adenomatous polyposis coli protein, axin, & GSK-3β, thereby marking it for ubiquitination & subsequent degradation by the proteasomal pathway. During IP induction of the PI-3 kinase pathway leads to phosphorylation of GSK-3β (inactive), thereby leading to the release, stabilization, accumulation of β-catenin in the cytosol & subsequent translocation of β-catenin into the nucleus drives the expression of its target genes (VEGF, Bcl-2, survivin) through activation of the T-cell transcription factors/lymphoid-enhancer binding factor (TCF/LEF) transcription factors. These astounding results paved the way for the induction of angiogenesis in the ischemic heart by intramyocardial administration of adenoviral associated β-catenin (Ad-βcat) immediately after an MI which depicted similar patterns of cardioprotection as was observed with IP. The efficacy of β-catenin overexpression in the induction of collateral vessel formation and reducing ventricular remodeling was further ascertained by using the Ad-shRNA-β-catenin (Ad-shβcat) to silence the gene expression of β-catenin thereby abolishing the IP mediated cardioprotection.
Volatile anaesthetics emerged as important cardioprotective agents in both animal models of ischaemia/reperfusion injury and humans with coronary artery disease. Their administration before a prolonged ischemic episode is known as anaesthetic preconditioning whereas when given at the very onset of reperfusion the strategy is termed anesthetic postconditioning. Both types of anaesthetic conditioning reduce, albeit not to the same degree, the extent of myocardial injury and share similar, albeit not identical, intracellular signal transduction pathways with their widely investigated counterparts, ischaemic pre- and postconditioning. Moreover, compelling evidence points towards the inhibition of mitochondrial permeability transition pore (PTP) as the putative end-effector mechanism of cardioprotection. In addition, important interactions between cyclosporine A, the classic PTP desensitizer, and anaesthetic postconditioning have been lately reported. However, there are several inconsistencies in response to both investigated anaesthetics (isoflurane and sevoflurane) and protocols (pre- and postconditioning) which are far from being elucidated. The presentation will discuss some discrepancies in protective response relative to the: (i) experimental models and animal species, (ii) presence or absence of an additive benefit by combining anaesthetic pre-and postconditioning and the (iii) effect of cyclosporine A in the presence of volatile anaesthetics.
Acknowledgements
Supported by National University Research Council Grant ID 1254/2007 and Romanian Academy Grant 266-148/2008.
The incidence and prevalence of obesity and the metabolic syndrome have risen markedly in the past decade, representing a serious cardiovascular health hazard with significant morbidity and mortality. The etiology of the metabolic syndrome and its various pathogenic mechanisms are incompletely defined and under intense investigation. Contemporary research suggests that the adipocyte-derived hormone leptin may be an important factor linking obesity, the metabolic syndrome, and cardiovascular disorders. Although recent evidence indicates that under normal conditions leptin may be an important factor in regulating pressure and volume, during situations of chronic hyperleptinemia and leptin resistance, this hormone may function pathophysiologically for the development of hypertension and cardiac and renal diseases. Future research will determine if reduction of circulating leptin and/or blockade of its peripheral actions can confer cardiovascular and renal protection in hyperleptinemic patients with obesity and the metabolic syndrome.
Epidemiological and histological evidence were the first indications that infectious disease may be involved in cardiovascular disease. One of the first studies that an infectious agent actually caused atherogenesis was shown in LDL receptor deficient mice infected by C. pneumonia. At the same time, we have also demonstrated that these atherogenic effects of C. pneumonia infection are closely associated with high circulating levels of cholesterol. In addition, in cell culture, C. pneumonia infection of vascular smooth muscle cells induced the expression of cell cycle proteins resulting in a stimulation of cell proiferation. This appears to be achieved through an increased expression of heat shock protein 60. This effect was not observed in endothelial cells. C pneumonia infection of vascular smooth muscle cells can also oxidize LDL in the surrounding medium and this oxidized LDL can augment the atherogenic actions of C. pneumonia. Thus, known risk factors for atherosclerosis can also interact and augment the atherogenic effects of an infectious agent. Our data demonstrates that an infectious agent like C. pneumonia can actively participate in the genesis of an atherogenic plaque by disrupting gene expression in vascular cells in a highly selective manner. This will have deleterious effects on vascular contractile function and serious clinical consequences.
Acknowledgements
This work was supported by a grant from the Canadian Institutes for Health Research.
There has been an exponential increase in the number of coronary heart disease (CHD) candidate genes studied over the past 15 years. The lack of consistency, reproducibility and accuracy for the individual risk of CHD has opened the elementary question of the genetic epidemiology of CHD. As coronary heart disease is multi-factorial, genetic predisposition to hyperlipoproteinemia, hypertension and diabetes represented the first scientific approaches to this field in last decade of the 20th century. We have completed an analysis of more than 20 single nucleotide polymorphisms (SNPs) in different locuses of candidate genes for hypercholesterolemia in a case control study of two groups selected out of 2000 probands. Only one third of analyzed genes displayed a significant difference in genotype frequency between hypercholesterolemic and control individuals but the effect of each single candidate gene was still very low. Use of meta-analysis and combination of several SNPs (gene scores) might be a useful tool to improve final results. Genetic association studies which survey the entire genome have become a common design for uncovering the genetic basis also for CHD. Recently, three genome-wide association scans (GWAS) all identified a single region on chromosome 9p21.3 association with myocardial infarction but surprisingly no one gene of the established CHD risk factors (hyperlipoproteinemia, hypertension, insulin resistance) are located at this region. Continual changes of the most typical pathogenesis of coronary atherosclerosis of myocardial infarction patients during their lifetime and also possible changes of importance of different risk factors in a population over several decades might produce additional complications. Although development of molecular genetic methods is enormously rapid, it is still unable to offer significantly better information compared to classical risk factors and family history.
Acknowledgements
Funded partly through MSMT Project No. 1M0510 and IGA MZ CR Project No. NR 9093-4.
Peroxisome proliferator-activated receptors (PPARs) are transcription factors that belong to the nuclear receptor superfamily regulating expression of genes involved in different aspects of lipid metabolism and energy production (1). Activation of PPAR alpha and gamma isoforms has been found cardioprotective (2), while ischemia/reperfusion (I/R) is associated with dysregulation of PPAR (3). We have recently shown that hypercholesterolemia (H-CH) reduced gene expression of both, PPAR alpha and gamma post-I/R and exacerbated an outcome of I/R injury in the diabetic hearts, while simvastatin (S) restored myocardial ischemic tolerance in these hearts without affecting high levels of CH. Since statins have been reported to increase both mRNA and protein levels of PPAR alpha in some cell types (4), we aimed to explore a potential link between a response to I/R injury and the changes in cardiac PPAR alpha gene expression in S-treated normocholesterolemic rats. After 5 days of treatment with S (10 mg/kg/day, p.o.), Langendorff-perfused hearts were subjected to 30-min regional (occlusion of LAD) or global I followed by 2-h R. Gene expression of PPAR alpha (RT-PCR) was increased by 61% in the hearts of S-treated rats at baseline and was preserved in these hearts after I and R in contrast to significantly reduced mRNA levels of PPAR alpha in the non-treated control (C) group post-I/R. S-treated hearts exhibited smaller size of infarction (TTC) as a percentage of risk area (11.5 ± 0.4% vs. 33.7 ± 4% in C; P<0.05), reduced ectopic activity and duration of ventricular tachycardia during ischemia, as well as improved postischemic contractile recovery and lower severity of reperfusion-induced arrhythmias (arrhythmia score 2.5 ± 0.3 vs. 4.4 ± 0.5 in C; P<0.05). In conclusion, treatment with S may confer an efficient protection against I/R injury independent of lipid-lowering activity. Differential expression of PPAR alpha may potentially account for pleiotropic effects of statins.
Acknowledgements
Grants VEGA SR 2/0173/08, 1/4296/07, APVT 51-027404.
References
- 1.Finck BN. Cardiovasc Res. 2007 doi: 10.1016/j.cardiores.2006.08.023. [DOI] [PubMed] [Google Scholar]
- 2.Smeeths PJH, et al. Acta Physiol. 2007 [Google Scholar]
- 3.Huss JM, Kelly DP. Circ Res. 2004 doi: 10.1161/01.RES.0000141774.29937.e3. [DOI] [PubMed] [Google Scholar]
- 4.Jasinska M, et al. Pharmacol Reports. 2007 [PubMed] [Google Scholar]
Ischaemic postconditioning (PS) was first described by Vinten-Johansen and co. as a very potent and simple method for reducing the reperfusion injury in an experimental cardiac model. We studied the protective effects of PS on ischemia-reperfusion injury of the lower extremities in a rat model of abdominal aortic intervention. We aimed to examine the evoked oxidative stress after revascularisation surgery and the intracellular signalling pathways of postconditioning in different tissues. Anesthetized animals (48 Wistar rats) underwent a 60 min infrarenal aorta cross-clamping. After the ischaemic period, an intermittent 4 times 15 sec reperfusion - 15 sec ischaemic episodes- were applied (ischaemic postconditioning: group PS). Then we started a 120 min reperfusion in the aorta. In untreated group animals underwent a long ischaemia (60 min) and the following reperfusion (group IR). Peripherial blood samples were collected before operation, and in early (5; 10; 15; 30; 60 and 120 min) reperfusion periods. Serum peroxide level, TNF-alpha concentration, myeloperoxidase (MPO) activity and PMA-induced leukocyte ROS production were measured. Tissue samples were taken from femoral quadriceps muscle, heart, lung, kidney, liver and bowel on the end of reperfusion. Akt-1, MAP-kinase, and ERK1/2 were analysed in tissue samples with Western blot. In PS group, plasma peroxide level elevation was significantly lower in very early reperfusion (5–30 min) comparing to non-conditioned IR group. PS also reduced serum TNF-alpha concentration, MPO activity and leukocyte activation detected by PMA-induced leukocyte ROS production. PS increased both Akt-1 and ERK1/2 activation. PS seems to be an effective tool in vascular surgery to reduce reperfusion injuries after revascularization interventions.
Acknowledgements
Supported by OTKA K67731, K48851, K60227.
Endogenous myocardial nitric oxide (NO) may modulate the transition from adaptive to maladaptive hypertrophy leading to heart failure. In rodent models of pressure overload or myocardial infarction, the three NOS isoforms (NOS1, NOS2, NOS3) were shown to play a neutral, protective, or even adverse role in myocardial remodelling, depending on the NOS activity, the location of each NOS and their regulators. More recently, our data, through the analysis of conditions that modulate the expression of NOS1 and NOS3 in the heart according to physiopathological situations, indicated that, beside the level of total NOS activity, unique changes in NO compartmentation secondary to NOS1 or NOS3 subcellular location might be involved in the development of cardiac hypertrophy and failure. Thus, different circuits in NO-signalling pathways in myocardium might be activated and this principle is a key to understand contradictions existing in NO biology in the heart. Unraveling the mechanisms behind the NO, NOS and cardiac function is still an ongoing challenge.
Human cardiac regeneration therapy has been performed by using various sources of stem cell. Myoblasts and bone marrow cells have been injected for patients with ischemic cardiomyopathy in our clinical trial and improved cardiac performance. We had examined the efficacy of cell therapy using tissue engineered sheet technique compared to needle injection. This technique has advantages such as the ability for treatment to large area, and less invasive for host heart such as lethal arrhythmia. In vivo, implantation of autologous myoblast sheet had improved cardiac function of ischemic or dilated cardiomyopathy models using rat, hamster, canine and porcine models. We also showed that myoblast sheets provided various factors inducing angiogenesis, hematopoietic cell recruitment and anti-apoptosis, following anti-remodeling. Thus, after approved by IRB of our institution, we have started the clinical trial of myoblast sheet implantation for DCM patients, and assessed the feasibility and efficacy for the first patient. In this patient, any sequelae including arrhythmia have not occurred after implantation, and the cardiac function showed recovery. Furthermore, we have investigated several improvement of this technology for the layered implantation for ischemic heart, for right heart failure, and another cell source to construct the sheet. Thus, cell sheet implantation could be safe and eligible as cardiac regeneration therapy.
Alterations of hemodynamic loading have been shown to have significant influence on normal cardiac morphogenesis as well as conduction system maturation in chick embryos. The same is expected in the mouse, but the in vivo approach to test this hypothesis is not feasible. Therefore, ex vivo organ culture system has been employed in chick and mouse embryos to test the effect of mechanical unloading. In E3 and E4 chick embryos cultured for 24 h, there was an elongation of the conotruncus with collapse of its lumen, compaction of the trabeculae, delay in conduction system maturation and significant decrease in percentage of hearts with primary ring conduction pathway assessed by optical mapping. Activation pathway utilizing the primary ring or activation from apex to base is dominant in freshly isolated mouse embryonic hearts between ED9.5 and ED11.5, whereas in hearts cultured for 24 hours occurred a regression in normal developmental timeline with appearance of ventricular activation patterns from base to apex. Short-term organ culture of embryonic heart is thus feasible in both chick and mouse, the hearts continue to beat for at least 24 hours. However, heart development and conduction system maturation differs from in vivo situation. We conclude that appropriate mechanical loading is essential during the early phases of CCS formation and maturation.
Acknowledgements
Supported by MSMT VZ 206100-3, AS CR AVOZ50450515, GACR 304/08/0915.
Development of cardiac conduction system is linked with modifications of heart morphology. The focus of this talk will be on the ventricular conduction system, whose main task is to assure rapid and safe spread of electrical activity to the ventricular myocardium to enable coordinated and efficient contractions. During ontogenetic development of higher vertebrates, its function appears when ventricular trabeculae first form; hence, trabeculae are the morphological substrate of nascent ventricular conduction system. Available markers show during further maturation a gradual restriction to definitive conduction tissue, i.e., His bundle, bundle branches, and Purkinje fibers, whose arrangement shows considerable variability among species. It comes as no surprise, then, that equivalent of ventricular conduction system is functionally demonstrable even in lower vertebrates (poikilotherms), whose ventricles also show an apex-to-base activations, considered a hallmark of ventricular conduction system function. However, the only morphological substrate identifiable consists of trabecular strands running from the atrio-ventricular junction to ventricular apex – there are no insulated bundles typical of homoiotherms.
Acknowledgements
Supported by MSMT VZ 206100-3, AS CR AVOZ50450515, GACR 304/08/0915 and the Purkinje Fellowship of the Academy of Sciences of the Czech Republic.
We have addressed the role of structure-function relationships in exerting control over energy metabolism in the normal adult cardiomyocytes (CM) exhibiting regular arrangement of mitochondria and in the HL-1 cardiac cell line characterized by filamentous or granular and dynamically changing pattern of mitochondria. The results show that Ca2+-induced hypercontraction reversibly decreased the apparent Km for ADP in regulation of oxidative phosphorylation (OXPHOS) in permeabilized CM. In HL-1 cells, the mitochondrial adenylate kinase (AK) and cytosolic BB-creatine kinase (CK) were expressed at higher levels than mitochondrial (mi-CK) and cytosolic MM-CK, together with a strong expression of the type 1 and type 2 hexokinases (HK). The HL-1 cells differed from CM by absence of (i) functional coupling (FC) of mi-CK to OXPHOS, (ii) direct channeling of adenine nucleotides (DCA) between mitochondria and ATPases, and (iii) FC of BB-CK to ATPases, but by presence of higher activity of HK and its FC to OXPHOS. We conclude that regulation of OXPHOS in cardiac cells strongly depends on the pattern of intracellular organization of mitochondrial interactions with other cellular structures. In CM, the mitochondria and ATPases with participation of the cytoskeletal proteins form complexes termed as intracellular energy units (ICEUs). Inside the ICEUs, the specialized pathways mediated by CK, AK, and DCA ensure energy transfer and feedback between mitochondria and ATPases with efficiency sufficient to sustain the metabolic stability in conditions of increased workload. The functional coupling of kinases to OXPHOS and ATPases and altered mitochondrial affinity to ADP during sarcomere contraction-relaxation cycle represent the major underlying mechanisms. In contrast to CM, regulation of OXPHOS and intracellular energy transfer is organized differently in HL-1 cells, due to distinct localization pattern of mitochondria and lack of the CK- and DCA-mediated mechanisms.
Neonatal rat cardiomyocytes exposed to medium containing 1 nM angiotensin II undergo apoptosis, an effect prevented by either inhibition of reactive species generation or mitochondrial DNA damage. Because mitochondrial DNA damage reduces the expression of mitochondrial encoded proteins involved in electron transport, DNA damage slows flux through the electron transport chain and facilitates the diversion of electrons to other acceptors, such as oxygen. The resulting formation of superoxide leads to mitochondrial oxidation, activating in the process the mitochondrial permeability transition. Cytochrome c and other pro-apoptotic factors are released and the cell succumbs to apoptosis. A similar sequence of events occurs in cells exposed to medium containing 1 mM palmitate or lacking insulin. Although the toxicity of all three factors is prevented by scavenging reactive oxygen species and reversing mitochondrial DNA damage, differences in the proteins affected by the three factors suggest that the key elements in the initiation of apoptosis are the excessive generation of superoxide by the mitochondria and the formation of the mitochondrial permeability transition pore. Supporting this conclusion is evidence that the mitochondrial DNA damage step can be bypassed, provided that the expression of the mitochondrial encoded proteins is impaired and the level of mitochondrial superoxide generation reaches toxic levels. This occurs in cells containing unconjugated tRNAs, which exhibit impaired mitochondrial protein synthesis. These finding suggest that maintenance of normal electron transport flux prevents the diversion of electrons to oxygen. This coupling not only improves ATP production but also prevents excessive superoxide generation, a trigger of the mitochondrial permeability transition and apoptosis.
Arteriogenesis is the science of adaptation by remodeling of small pre-existent arterioles into full-fledged arteries to compensate for the loss of function of a major artery by atherosclerotic occlusion. Anatomo-pathological observations and in vivo coronary angiography had demonstrated the existence of a collateral circulation capable to limit the damage of arterial occlusion. Animal models were developed to study arteriogenesis in the heart, the limbs and in the brain. The major findings were that remodeling is able to replace about 40% of the maximal conductance of a normal artery. Arteriogenesis proceeds by cell division of endothelial and smooth muscle cells. The primary physical stimulus is increased fluid shear stress caused by the pressure difference between the pre-occlusive and the post-occlusive vascular bed. The shear stress signal is translated into a molecular signal by activation of eNOS and production of NO, of VEGF and by activation of ion channels like TRPV4. The activated endothelium secretes the chemokine MCP-1 that attracts monocytes that adhere and penetrate the intima where they digest the matrix and the elastic lamina enabling the structural dilatation. They also produce the growth factors necessary for the smooth muscle proliferation that proceeds via activation of the ERK-1, -2 MAPKinases. Since no direct junctions exist between the endothelium and the underlying SMCs and since diffusible molecules like NO and VEGF are not growth factors for SMCs, the invading monocytes act as essential “go-betweens”. Many other molecular changes were observed in conjunction with SMC proliferation: differential regulation of the actin binding molecules abra, cofilin, destrin and trb4. The essential role of NO in arteriogenesis should be employed in the design of drugs to stimulate arteriogenesis to treat arterial occlusive diseases.
The ability of the myocardium to mount compensatory response to stress is critical for survival. In mechanically overloaded heart, cardiomyocytes adapt to the work demand through activation of the compensatory hypertrophy program, the hallmark of which is the global increase in RNA synthesis. Positive transcription factor b (P-TEFb), a complex of cdk9 and cyclin T, is critical to stimulation of transcriptional elongation of nascent RNA chains via phosphorylation of RNA polymerase II (pol II). Cardiac lineage protein (CLP-1), a mouse homolog of human HEXIM1, isolated in our laboratory, inhibits the cdk9 kinase activity effectively suggesting that CLP-1 plays a role in regulation of the P-TEFb activity. Using cardiomyocytes in cultures subjected to hypertrophy agents and genetically manipulated animal models rendered hypertrophic by over-expression of cyclin T, we have demonstrated that CLP-1 interacts with the components of P-TEFb complex but is released to facilitate the hypertrophic response. The release of CLP-1 was prevented when Jak2 signaling was inhibited by AG490. We also studied the role of stress protein, aB-Crystallin, which plays a prominent role in preserving the structural integrity of the myofibrillar complements. We have shown that enhanced expression of aB-Crystallin occurs in the mouse heart subjected to trans-aortic constriction to produce pressure-overload cardiac hypertrophy. The stress-induced expression of the Crystallin gene is blocked by AG490 indicating the involvement of Jak/Stat signaling. In addition to Jak/Stat, we have shown the involvement of calcineurin/N-FAT signaling. The stress-induced expression of aB-Crystallin gene is dependent upon the interaction of a complex consisting of transcription factors Nished, N-FAT and Stat3 with the cardiac specific cis-element aBE4 and is regulated by the cross talk between the Jak/Stat and calcineurin/N-FAT signal transduction pathways.
Different cytokines in heart failure are modified in a characteristic manner and the details of their precise role in this condition are not known. In this regard, tumor necrosis factor (TNF) -alpha has been suggested to play a role in the progression of heart failure. Interleukin 10 (IL-10) antagonizes some of the effects of TNF-alpha. We have already shown that heart failure subsequent to MI is associated with the decrease in IL-10/TNF-alpha ratio and that, IL-10 modulates the TNF-alpha induced oxidative stress in isolated cardiomyocytes. However, intracellular signaling events responsible for this are not known. Present study investigates role of p38 and ERK 1/2 MAP kinases in TNF-alpha induced cardiomyocyte apoptosis and its modulation by IL-10. Cardiomyocytes isolated from Sprague Dawley rats were exposed to TNF-alpha (10ng/ml), IL-10 (10ng/ml) and IL-10+TNF-alpha (ratio 1) for 4 hrs. H2O2 (positive control) and antioxidant trolox (20 μmol/L) were used to confirm involvement of oxidative stress. H2O2 (100 μM) increased oxidative stress and apoptosis, TNF-alpha mimicked these effects. Exposure to TNF-alpha increased ROS production, caused cell injury, increased the number of apoptotic cells and Bax/Bcl-xl ratio. This change was associated with an increase in p38 and a decrease in ERK 1/2 phosphorylation. IL-10 by itself had no effect on these MAP kinases, but it prevented TNF-alpha induced changes. Trolox mitigated TNF-alpha induced changes. Pre-exposure of cells to p38 inhibitor SB-203580, prevented TNF-alpha induced changes. Inhibition of ERK pathway with PD98059 attenuated protective role of IL-10 against TNF-alpha induced effects. Present data provide evidence that IL-10 influences the activation of p38 and ERK 1/2 thereby modulating TNF-alpha induced oxidative stress and cardiomyocyte apoptosis.
Acknowledgements
Supported by CIHR.
Translational research is a term largely used by the scientific community with the expectation that numerous relevant biological discoveries can be carried to the clinic. What are the key discoveries at the origin of our knowledge in the biological science, what are the go and back of the ideas, what are the main guiding lines for an efficient biomedicine? The localization of this prestigious meeting in the birth country of Gregor Mendel, is a unique opportunity to discuss several of these aspects by studying the evolution of biological knowledge in the context of a tremendous international effort; we will start with the original document of this so prestigious scientist: Experiments on plant hybrids. In “Verhandlungen des naturforschenden Vereines in Brünn” 3-47 IV, 1865.
Advances in molecular and cellular biology has enabled us to identify the genes responsible for the genesis of various heart diseases but, in most cases, detailed mechanisms by which such genetic defects lead to the signs and symptoms observed at the bedside is left to be elucidated. As an attempt to explore such problems, we have developed a multi-scale, multi-physics heart simulator in which normal or abnormal function of the heart is reproduced based on the molecular mechanisms of cardiac excitation-contraction (E-C) coupling process. This simulator based on the finite element method consists of approximately 670000 solid elements each implemented with the mathematical model of E-C coupling thus functioning as virtual myocyte and the approximately 450000 fluid elements representing the blood in the heart chamber. Because the governing equations for the solid and fluid parts are solved by the strong coupling method, we can obtain the detailed information on the blood flow as well as the electrical and mechanical states of the every myocyte during the cardiac cycle. Accordingly, this simulator can be used as a tool to see any specific molecular abnormality would lead to the development of macroscopic findings. In this presentation, we will show our results on both the healthy and the diseased hearts caused by the reported molecular defects.
Mitochondria are intracellular organelles and play an important role in the cellular energy production. Mitochondria have their own DNA which is different from the nuclear DNA. Human mitochondrial DNA is double stranded and circular consisted of 16,569 base pairs. Each cardiac myocyte contains 2,000 to 3,000 mitochondria and each mitochondrion possesses 2 or 3 circular DNAs. Mitochondrial DNA mutations are maternally inherited. The etiologies of idiopathic cardiomyopathies are still unknown, but recent development in molecular biology have provided suggestive evidences, such as the detection of mutations of the genes for myosin, actin, tropomyosin, troponin, and so on. Myocardial mitochondrial DNA mutations have also been detected in patients with cardiomyopathy. A point mutation that alters adenine to guanine at the position of 3243 within the mitochondrial tRNALeu(UUR) gene is common in patients with the syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and the point mutation is also found in patients with cardiomyopathy or diabetes mellitus. Multiple mitochondrial DNA deletions have been revealed in a pedigree of inherited dilated cardiomyopathy, but the extent to which these mitochondrial DNA mutations are involved in the etiology of idiopathic cardiomyopathy remains to be elucidated.
By a large scale SNP association analysis, we found two SNPs in LTA were associated with the risk of MI. One of the significant SNPs increased expression level of LTA protein, and the other modified functions of this protein. Since two SNPs were in complete linkage disequilibrium, we concluded that increased expression level of functionally modified LTA protein leads to risk of MI. Next, we identified a protein, galectin-2, as a binding partner of LTA protein. Genetic investigation into the gene encoding galectin-2, LGALS2, revealed one SNP in this gene confers risk of MI. This SNP influenced the degree of inflammation by regulating extracellular amount of LTA protein. This result indicated the importance of LTA cascade in the pathogenesis of MI. In total, we have identified five genes that were associated with MI. Although each variation carried modest odds ratio (~1.4), their combination increased odds ratio up to 4.3, indicating multiple genetic factors in combination contribute greatly to its pathogenesis.
Restenosis rate improved remarkably in patients after drug eluting stents implantation in our hospital (18% to 3%). However, increase of late stent thrombosis is the serious problem. Clinically, it is very important to prevent late stent thrombosis using anticoagulation drugs such as ticlopidine or clopidogrel. It is well known that ticlopidine has a relative high incidence of side effects, including liver damage, neutropenia and thrombocytopenia. Side effect of this drug occurred about 10% of Japanese patients who underwent drug eluting stent implantation. This ratio in Japanese patients is very high when compared with the data in Caucasian patients. It is generally accepted that the individuals lacking cytochrome P 2C19 activity is more common in Orientals when compared with Caucasians. Recently, one of human leukocyte antigen allele, HLA-A*3303, that prevalence is higher in Japanese than other races, has been reported to be one of factors to induced liver damage. These data suggested that hepatotoxicity is mediated via an immune-mediated mechanism. They also suggested that it is difficult to explain the mechanism of hepatotoxicity by polymorphism of cytochrome P 2C19 activity. On the other hand, clopidogrel has used to prevent stent thrombosis since last year in Japan and seems reasonable in patients treated with drug eluting stent with ticlopidine intolerance. However, safety and efficacy of clopidogrel use in patients remains unknown. We have to confirm by further investigation with a large number of cases. It will be more important to consider of racial difference about drug metabolism and to investigate the genetic polymorphism before administration of drugs to prevent side effects in patients.
The development of the coronary vasculature begins with the differentiation of cells that migrate from the proepicardium to the epicardium. The initial step is development of a tubular network via vasculogenesis and angiogenesis regulated by a number of temporally expressed transcription (e.g.,SSH, GATA) and growth factors including VEGFs, especially VEGF-B and its Flt-1 receptor, FGFs and angiopoietins. Select FGF receptor splice variants play key roles in the vasculogeneic/angiogenic process, as demonstrated by our embryonic mouse explant experiments using soluble adv-FGF receptors. Moreover, embryonic stem cells promote the proliferation of epicardially-derived endothelial cells. The establishment of coronary ostia and the arterial tree is triggered by closely defined expression of VEGF at the aortic root in the presence of autonomic ganglia and blood islands. Endothelial cells penetrate the aorta forming the 2 coronary ostia. Our data, based on quail in ovo growth factor inhibition, document roles for FGF-2 and PDGF in this process and in the recruitment of smooth muscle cells. VEGF continues to be required for further development of the arterial tree. Coronary anomalies occur frequently if these growth factors are not expressed at the critical time points. These include absence of, or only one, coronary artery stem, failure to develop the compact region of the ventricle, subepicardial and intraseptal accumulation of erythrocytes that arise from epicardial cells.
Hypertriglyceridemia plays a key role in combined hyperlipidemia, diabetes mellitus or metabolic syndrome. The present study was designed to develop rabbit models of hypertriglyceridemia. Through mating a pair with high levels of triglycerides (TG>500 mg/dl) in WHHL rabbits, frequency of rabbits with higher levels of hypertriglyceridemia (TGH offspring) increased with generations and plateaued after 5 generations. Frequency of low levels (TG<250 mg/dl) of hypertriglyceridemia also increased with generations through mating a pair with lower levels of TG. Following crosses of TGH and wild type, 60 mg/dl,±80 and 94±TG and total cholesterol levels were 108 respectively. F2 offspring showed TGH trait in 7 of 33 rabbits, suggesting an autosomal dominant trait. From F2 rabbits, a heritable line of postprandial hypertriglyceridemia was also established. The present three models of hyperlipidemia were vertically transmittable and would be useful for understanding complicated genetic, metabolic and environmental control of TG in human diseases.
Familial hypertrophic cardiomyopathy (FHCM) and dilated cardiomyopathy (FDCM) are mainly caused by mutation(s) in sarcomeric proteins and cytoskeletal proteins, respectively. Mitochondrial gene mutations also induce progressive FDCM. However, pathogenic feature of HCM at the onset followed by the dilated phase with variable courses has not been elucidated yet. Genomic DNA was isolated from peripheral blood or cardiac samples during cardiac transplantation. To expedite search for those genes associated with polygenic diseases, we employed the complementary approaches of nuclear Genechip 600k microarray scan and resequencing of whole mitochondrial genome, combined with nested PCR and the direct sequencing of the relevant genes. We detected homogenous distribution of SNPs throughout mitochondrial genome and four clusters of tentatively pathogenic mutations mainly in the tRNA mutations identified, resulting in DCM in the unrelated four families; 1) FHCM with a single gene mutation of cardiac myosin heavy chain (P731L in MYH7), 2) FDCM with the mild course showing the gene mutation at G15927A in Mt-tRNAthr alone, 3) FDCM with the aggressive course demonstrating G12192A in Mt-tRNAHis solely and 4) HCM with the combination of nuclear plus mitochondrial mutations at the each locus, initially showing HCM followed by the corresponding mild and advanced course to DCM, respectively. We present a novel scheme to explain the clinical features with the complex process applicable for other progressive and/or degenerative disorders in general, intensifying that a part of “genetic penetrance” would be explained by mutual interaction of several responsible genes with aggravating or ameliorating action(s).
At present time, diagnostic and prognostic cardiovascular biomarkers are available but there are no widely accepted and validated clinically useful biomarkers for screening prior to outcome appearance. Our general objective is to search for clinically useful biomarkers focusing on SNPs and other DNA sequence and structural variations alone or combined with existing clinical risk factors. Whole genome association studies (WGA) were performed in French-Canadian population affected by hypertension and dyslipidemia and in type 2 diabetic (T2D) patients with and without complications using Affymetrix (50K, 500K, 5.0 and 6.0) SNP arrays. Ethnicity was ascertained using STRUCTURE 2.0 and principal component analysis with 16K complications-unrelated SNPs. Risk prediction models include SNPs alone or combined to a few uncorrelated linical biomarkers. ROC curves were drawn to yield a predictive value combining sensitivity and specificity. WGA studies identified a number of SNPs which show strong associations with hypertension, C-reactive protein, albuminuria, atrial fibrillation and other outcomes. Odds ratio of being affected increased with the combination of risk alleles. As an example, an area under the ROC curve of 0.8 was obtained with only four SNPs. The predictive power of these multiple biomarkers will be validated in other populations, functionally assessed in rodent models and optimized by adding classical risk factors and complementary epigenomic markers, to the risk model.
Extracellular purines (ATP-ADP) and pyrimidines (UTP-UDP) have major effects on cardiac rhythm and contractile force. They can be released during various physiopathological conditions such as ischemia. Then, despite degradation by ectonucleotidases and creatine kinase, their levels markedly increase in the interstitial space and are clearly associated with arrhythmia. Here, we first show that UV-photorelease of caged-ATP triggers automaticity of rat papillary muscles. Besides, an intraperitoneal injection of creatine, assumed to allow transphophorylation of ATP/UTP to phosphocreatine, reduces ventricular tachycardia and sudden death that generally occurred during the early phase of an experimental infarct induced by coronary ligature in rats. ATP and UTP, applied on ventricular cardiomyocytes isolated from various mammalian species, elicited a sustained, non-selective cationic current, IATP. IATP resulted from the binding of ATP4- to P2Y2 purinoreceptors. IATP was maintained after ATP removal in the presence of GTPgS and was inhibited by U-73122, a phospholipase C inhibitor. ATP markedly increased single channel opening probability, an effect prevented by U-73122. Adult rat ventricular cardiomyocytes expressed TRPC1, 3, 4 and 7 mRNAs and the TRPC3 and TRPC7 proteins that co-immunoprecipitated while the anti-TRPC3 antibody inhibited IATP. Thus, activation of P2Y2 receptors induces the opening of heteromeric TRPC3/7 channels leading to a sustained, non-specific cationic current. Such a depolarising current could induce cell automaticity and trigger arrhythmic events during an early infarct when ATP/UTP release occurs. These results emphasize a new, potentially deleterious, role of TRPC channel. Furthermore, by buffering extracellular ATP/UTP, a protective effect of creatine supplementation on human at risk could be foreseen.
There is increasing evidence that gap junctions (GJ) play an important role in the generation of early ventricular arrhythmias that result from acute myocardial ischaemia. In particular the 1b phase of these arrhythmias, which often terminates in ventricular fibrillation and thus is responsible for sudden cardiac death, is thought to result from the uncoupling of GJs. Ischaemic preconditioning, induced by a brief (5 min) coronary artery occlusion in the anaesthetised canine, markedly suppresses these fatal arrhythmias and preserves GJ function as assessed by changes in tissue electrical impedance and permeability, and in the phosphorylation status of connexin43 (Cx43). Modification of GJ function by pharmacological means prior to ischaemia also influences the occurrence and severity of ischaemia-induced arrhythmias. For example, closing of GJs prior to ischaemia by carbenoxolone, a relatively selective gap junction uncoupler, results in a preconditioning-like antiarrhythmic protection. In contrast, the “antiarrhythmic peptide” rotigaptide, which is supposed to act by opening or by maintaining the open status of these channels, reduces the severity of arrhythmias only when it is infused immediately prior to and also during coronary artery occlusion. Interestingly both drugs attenuate those rapid changes in electrical impedance that just precede the appearance of phase 1b arrhythmias and maintain a better metabolic communication (GJ permeability) between cells, most probably by keeping Cx43 in the phosphorylated form (opened state) even after a 60 min period of ischaemia. A similar effect occurs following the administration of the NO donor sodium nitroprusside. It is tempting to suggest that slowing the rate of GJ uncoupling by whatever means (preconditioning, carbenoxolone, rotigaptide or sodium nitroprusside) during the critical period of ischaemia would result in arrhythmia suppression perhaps by reducing the inhomogeneous impulse conduction.
The regulation of intracellular Ca2+ is key to cardiac function. Deranged Ca2+ regulation linked to contractile dysfunction and certain cardiac arrhythmias is a hallmark of heart failure in humans and some animal models. In part, defective Ca2+ regulation is due to functional alterations of the sarcoplasmic reticulum (SR) Ca2+ pump SERCA2a, the activity of which is negatively regulated by phospholamban (PLB). Therefore, increased expression of functional SR Ca2+ pumps in cardiomyocytes or prevention of SERCA2a inhibition by PLB ablation are thought to be potential therapeutic approaches. In this context, experimental data will be presented demonstrating that overexpression of SERCA2a in the intact myocardium of SERCA2 transgenic rats can protect against both loss of SR Ca2+ pump activity and development of heart failure in settings of secondarily induced chronic left ventricular pressure-overload. In addition, data will be presented indicating efficient and stable PLB gene silencing by RNA interference in primary cultures of neonatal rat cardiomyocytes using an adenoviral vector transcribing short hairpin (sh)RNAs against rat PLB. At submicomolar free [Ca2+], PLB gene silencing was associated with both a marked increase of the SR Ca2+-pumping activity and a loss of SERCA2a responsiveness to protein kinase A-dependent phosphorylation.
Acknowledgements
Supported by DFG, SFB Transregio 19, TP C1.
Reactive oxygen species play an important role in the development of diabetic cardiomyopathy. They can activate matrix metalloproteinases (MMPs) and MMP-2 in particular is known to mediate early consequences of oxidative stress injury in the heart. Therefore, we investigated the role of MMP-2 and the effect of the MMP inhibitor doxycycline on the changes of heart function caused by diabetes. Using streptozotocin-induced diabetic rats, we evaluated the effect of doxycycline on both mechanical and electrical function of isolated hearts, papillary muscle, and cardiomyocytes. Doxycycline abolished the diabetes-induced depression in left ventricular developed pressure and the rates of changes in developed pressure in isolated hearts and normalized the prolongation of the action potential in papillary muscles. In cardiomyocytes isolated from doxycycline-treated diabetic rats the altered kinetic parameters of Ca2+ transients, depressed Ca2+ loading of sarcoplasmic reticulum and basal intracellular Ca2+ level, and the spatio-temporal properties of Ca2+ sparks were significantly restored. Gelatin zymography and Western blot data indicated that the diabetes-induced alterations in MMP-2 activity and protein level, level of tissue inhibitor of matrix metalloproteinase-4, and loss of troponin I were restored to control levels with doxycycline. Our data suggests that these beneficial effects of doxycycline on the mechanical, electrical and biochemical properties of the diabetic rat heart appear, at least in part, to be related to inhibition of MMP activity, implicating a role for MMPs in the development of diabetic cardiomyopathy.
Acknowledgements
Supported by projects TUBITAK-SBAG-107S427, -SBAG-COST-107S304 and COST BM0602.
Ca IX is a cell membrane-bound isoform of carbonic anhydrase IX acting in cell adhesion and pH control. Ca IX is strongly induced by hypoxia (HOX) and was often associated with tumors. Its presence in the myocardium was first time identified in 2005 by ourselves (1). Aims: i) Ascertain whether Ca IX is also expressed in healthy normoxic (NOX) rat cardiomyocytes (CM); ii) Verify whether expression of Ca IX may be also induced in CM exposed either to HOX or to disruption of oxygen sensing by means of some intentionally chosen modulators of the hypoxia-inducible factor (HIF1-α) pathway, such as: dimethyl ester of succinate (DMS), dimethyloxalylglycine (DMOG), diazoxide (DZO) and tempol (TL). Investigations were performed in primary (P) CM isolated from hearts of adult Wistar rats and/or in immortalized (I) H9c2 CM maintained in culture for 3 weeks prior to the experiement. HOX (2% O2) lasted 48 h. Ca IX expression was detected by RT PCR and immunoblotting, its localization by immunofluorescence with M75 monoclonal antibody and FITC-conjugated secondary antibody. In P-CM, Ca IX and VEGF were present already in NOX. HOX induced a 2-fold increase in Ca IX as well as a strong rise in HIF1-α, Glut-1 and iNOS, but only a moderate elevation in VEGF levels. A massive induction of Ca IX gene, in comparison with the VEGF gene, may be ascribed to closer localization of its HIF-binding element on the Ca9 promoter, to the transcription initiation site. In I-CM, the rodent Ca gene (car9) transcription and Ca IX protein expression were also strongly induced by HOX and DMOG, and little less by TL. DMS and DZO proved to be considerably much less effective in induction of car9 expression. Cell surface localization of Ca IX was confirmed in all types of CM investigated. In conclusion, Ca IX is present also in NOX CM and its expression is tightly coupled with molecular responses to HOX as well as to disruption of oxygen sensing.
Acknowledgements
Supported by Grants APVV 51-027404 and 51-024805 as well as by VEGA 2/0173/08 and 02/7126/26.
Reference
- 1.Ziegelhöffer, et al. J Mol Cell Cardiol. 2005;38:1085. Abstract No 240. [Google Scholar]