The transient change in tension development and length in a working cardiac myocyte during the heart beat reflects the integrated effects of signaling cascades regulating mechanisms controlling not only the dynamics and intensity of a transient increase in cytoplasmic Ca2+, but also the responsiveness of the sarcomeres to Ca2+. My focus is on signaling and sarcomeric function. To appreciate the potential role of how signaling through activation of kinases and phosphatases modifies sarcomeric function, it is important to consider the working cardiac myocyte operating in an environment influenced by immediate prevailing mechanical (load and length), neural, endocrine, autocrine, and paracrine control mechanisms, and by short and long term history of this environment. These mechanisms are important in adaptations to cardiovascular stressors that involve altering the balance of multisite phosphorylations of sarcomeric proteins. However, decompensation and maladaptive phosphorylations of sarcomeric proteins also may occur in association with cardiovascular disorders leading to heart failure.
Myocardial infarction (MI) is one of the common diseases that contain both genetic and environmental factors. To reveal genetic backgrounds of MI, we analyzed more than 3,000 patients and 3,000 general populations. By means of high throughput SNP genotyping followed by statistical analyses, we found one gene, LTA, was genetically associated with MI. We then found that lymphotoxin-alpha, encoded by LTA, bound to galectin-2 protein. Galectin-2 was encoded by LGALS2 and further genetic investigation revealed variation in LGALS2 also confers genetic risk of MI. Furthermore, PSMA6, ITIH3 and MIAT were found to be other genetic risk factors of MI. Most of them encode proteins that are related with inflammation, indicating the importance of inflammation in its pathogenesis. Although SNPs in either gene carried modest odds ratio, combination of these five risk factors showed much higher risk of MI, showing the presence of multiple genetic factors that contribute to common diseases.
Atrial and brain natriuretic peptides (ANP, BNP) were cardiac hormones. We first investigated that the secretion sites of NPs in humans, and found that BNP is secreted from the failing ventricles in a large amount. Next, we found that NPs have wide ranges of potent biological effects, including vasodilation, natriuresis, and inhibition of the renin-angiotensin-aldosterone and sympathetic nervous systems. It became clear from discovery of NPs that the heart is a hormone-producing organ. In this line of thinking, we hypothesized that other hormones were possibly synthesized in the heart. By blood sampling in coronary circulation, we found that aldosterone, DHEA and ACTH were secreted form the human heart. The hormone balance in the local heart would also be participating in the pathophysiology of heart failure. Among them, we recently focused on the interaction between aldosterone and NPs on both the whole body and cardiac tissue; these hormones would finely regulate sodium balance.
In cardiac muscle, the gap junction greatly contributes to cell-to-cell impulse propagation. The function of the gap junction depends on kinetic regulation of connexin (Cx) which composes the gap junction channel. In some pathological hearts, tissue Angiotensin II (Ang. II) is raised and affects on expression of Cx43. In the diabetic heart, quantitative decrease in Cx43 protein, scant ~inhomogenous expression of Cx43 at the gap junction area, decrease in conduction velocity and promotion of arrhythmogenesis are observed in association with augmentation of the PKCɛ-mediated phosphorylation of Cx43. It is reported on the other hand that in the rhythmical stretched or the hypertrophied (early stage) heart, quantitative increase in Cx43 protein, extensive expression of Cx43 at the membrane surface rather than at the junction area, increase in conduction velocity and promotion of arrhythmogenesis are observed. These diverse effects of Ang. II may result from the following factors, chronic or acute effects, synthetic concentration of Ang. II or different isoform of PKC activated by Ang. II.
Aging is a major risk factor for heart failure. Suppression of the insulin signaling pathway increases the lifespan of model organisms. We tested the hypothesis if insulin signaling regulates the cardiac aging process. We analyzed young (12 week old) and aged (64–80 week old) wild type (WT) mice, and young and aged transgenic mice that expressed dominant-negative phosphoinositide-3-kinase (dnPI3K) in a heart-specific manner. We analyzed heart function using cardiac catheterization under dobutamine infusion. Max dp/dt in aged dnPI3K mice was higher than that in aged WT mice. The amount of p16, the representative marker of aging, was increased in aged WT mice compared to that in young WT mice, and this increase was attenuated in aged dnPI3K mice. The age associated increases in proinflammatory cytokines and oxidative stress markers were attenuated in dnPI3K mice. Gene expression profiling using microarray analyses identified 157 genes whose expression was decreased in aged WT mice compared to young WT mice. The expression of these down-regulated genes was significantly preserved in aged dnPI3K mice. In conclusion, the insulin signaling is likely to be a conserved mechanism that regulates the aging process in mammals, and the suppression of insulin signaling attenuated an age-associated decline in cardiac function.
Disruption of dystrophin-glycoprotein complex caused by genetic defects of dystrophin or sarcoglycans results in muscular dystrophy and/or cardiomyopathy in humans and animal models. Recently, we focused on the abnormal ionic homeostasis that occurs commonly in such muscle degenerative disorders. Here we report that the plasma membrane Na+/H+ exchanger (NHE) is a critical molecule for generating muscle damage. First, we found that NHE inhibitors prevented cardiopmyopathy and muscle degeneration in delta-sarcoglycan deficient cardiomyopathic hamsters (J2N-k). Surface NHE1 expression was increased in J2N-k cardiac muscles, whereas total cell NHE1 did not differ markedly between normal and J2N-k muscles. NHE1 activity was enhanced in isolated adult cardiomyocytes as well as in myotubes from J2N-k as evidenced by the elevated intracellular pH (pHi) and cytosolic Na+ concentration (Na+i). NHE inhibitor significantly reduced the increase in baseline intracellular Ca2+ as well as Na+i and stretch-induced damage, suggesting that elevation in Na+i may contribute to abnormal Ca2+ homeostatsis and muscle damage by influencing the Na+/Ca2+ exchange activity. Furthermore, we recently found that cardiac-specific overexpression of a constitutively-active form of NHE1 produced dilated cardiomyopathy mainly through activation of CaMKII. These data suggest that enhanced NHE1 activity may be one of common molecular mechanisms to explain the pathogenesis of cardiac and skeletal muscle degeneration.
To examine cardiac metabolism in cardiac hypertrophy and failure, we profiled heart metabolites of rats that showed a distinct transition from compensated hypertrophy to heart failure by metabolomic analyses. Dahl salt-sensitive rats fed a high-salt diet developed hypertension and left ventricular hypertrophy (LVH) at 11 weeks of age and congestive heart failure (CHF) at 17–19 weeks. We administered dichloroacetate (DCA, 80mg/kg/day) or vehicle beginning at 11 weeks. DCA is an activator of pyruvate dehydrogenase and increases glycolysis and glucose oxidation. DCA preserved cardiac function (fractional shortening on echocardiography, CHF 29± 2.2% and DCA 42± 2.1%, p<0.05) and improved the survival of CHF rats (CHF 37% and DCA 65%, p<0.05). 114 metabolites of multiple metabolic pathways were quantified. The amounts of 8 metabolites were different between a control group (11 weeks of age fed a low-salt diet) and LVH, and the amounts of 13 metabolites were different between LVH and CHF. DCA treatment increased the reduced-form of glutathione and the NADPH/NADP+ ratio compared to CHF by 66% and 25%, suggesting that the pentose phosphate pathway is an effector of DCA since NADPH and glutathione reduce oxidative stress. Increased amount of thio-barbitulic acid reactive substances, a marker of oxidative stress, in heart tissue of CHF rats was decreased by DCA treatment (CHF 18.1±3.2 nmol/g and DCA 9.7±2.0 nmol/g; p<0.05). In conclusion, the development of LVH and CHF was associated with distinct changes in the metabolomic profile. DCA may be useful in heart failure treatment and metabolomics appears to be a useful tool for identifying biomarkers and future therapeutic targets in heart failure.
BACKGROUND AND OBJECTIVES
MCP-1 has been shown to be involved in the pathophysiology of ischemic heart diseases; however, its precise role in ischemia/reperfusion (I/R) injury remains unclear. We investigated the role of MCP-1 after global I/R using Langendorff-perfused hearts isolated from cardiac-specific MCP-1 transgenic (MHC/MCP-1) mice.
METHODS AND RESULTDS
Global I/R significantly increased MCP-1 mRNA expression in the wild-type hearts (p<0.01). Cardiac MCP-1 overexpression in MHC/MCP-1 mice remarkably improved LV dysfunction after I/R without affecting coronary blood pressure, especially ameliorated LV diastolic pressure after reperfusion (p<0.05). These effects were not inhibited by either sarcolemmal or mitochondrial KATP channel blocker (HMR1098 or 5-HD) treatment. Cardiac MCP-1 overexpression in the I/R hearts prevented superoxide generation and decreased the expression of NADPH oxidase family, Nox1, gp91phox, and Nox3. Further, superoxide dismutase (SOD) activity in the MHC/MCP-1 hearts was significantly increased compared with that in the wild-type hearts (p<0.001).
CONCLUSION
These findings provide new insight into the beneficial role of cardiac MCP-1 in the pathophysiology of ischemic heart diseases.
BACKGROUND AND OBJECTIVES
Rho-kinase (ROK)-mediated Ca2+-sensitization of vascular smooth muscle (VSM) contraction plays a critical role in abnormal VSM contraction. We previously found that sphingosylphosphorylcholine (SPC) induces the ROK-mediated Ca2+-sensitization, dependently on cholesterol in serum and VSM tissue and through the activation of Fyn, a member of Src family tyrosine kinase. In this study, we examined the involvement of membrane rafts, a cholesterol-enriched membrane microdomain, in the SPC/Fyn/ROK-mediated Ca2+-sensitization.
METHODS
The SPC-induced translocations of Fyn and ROK to membrane rafts were analyzed by immunocytochemistry and sucrose density gradient fractionation.
RESULTS
SPC induced the translocations of Fyn and ROK from cytosol to membrane rafts, which were labeled with caveolin-1 and cholera toxin subunit B. In addition, β-cyclodextrin, which selectively deprived VSM membrane of cholesterol and thereby removed caveolin-1 from membrane rafts, inhibited the SPC-induced translocation of Fyn and ROK and VSM contraction, without affecting Ca2+-induced contraction. In contrast, eicosapentaenoic acid (EPA), an inhibitor of Fyn translocation, inhibited the SPC-induced translocation of Fyn and ROK without affecting the localization of caveolin-1. Moreover, EPA selectively abolished the SPC-induced Ca2+-sensitization, without affecting the Ca2+-induced contraction. These findings suggest that membrane rafts play a pivotal role in abnormal VSM contraction.
BACKGROUND AND OBJECTIVES
In ischemic hearts, a rise of intracellular free Mg2+ concentration ([Mg2+]i) may play a protective role for the myocytes. Although the increase in [Mg2+]i is probably caused by breakdown of MgATP, little is known for the contribution of Mg2+ transport through cell membrane. We studied Mg2+ efflux activities of cardiac myocytes during metabolic inhibition.
METHODS
The rate of Na+-dependent Mg2+ efflux was estimated in ventricular myocytes enzymatically isolated from rat hearts ( 25oC) by measuring [Mg2+]i with the fluorescent indicator mag-fura 2. The cells were treated with a metabolic inhibitor in the Na+ and Ca2+-free conditions, and the initial rate of decrease in [Mg2+]i was analyzed upon introduction of extracellular Na+.
RESULTS
The Mg2+ efflux was largely diminished in FCCP- or KCN-treated cells. The inhibition was not attributed to an increase in [Na+]i or intracellular acidosis caused by metabolic inhibition, because 1) [Na+]i measured with a Na+ indicator SBFI was too low to substantially inhibit the Mg2+ efflux, and 2) normalization of intracellular pH with nigericin, a proton ionophore, did not reverse the inhibition of the Mg2+ efflux.
CONCLUSION
Mg2+ released from ATP may not be effectively extruded from the deenergized cells, which leads to a marked rise of [Mg2+]i.
Screening for cell surface proteins under metabolic stress may lead to understanding of cell-to-cell interaction involved in the pathophysiology of a wide range of cardiac diseases. To search for these genes, we performed a signal sequence trap and found that expression of NCAM, a major regulator of neurite outgrowth in the nervous system, was upregulated in cardiomyocytes treated by oligomycin, a hypoxia mimicking agent. In mouse MI model, expression of NCAM was upregulated at the peri-infracted sites where abnormal sympathetic nerve sprouting was known to cause ventricular arrhythmogenesis.
To investigate the role of NCAM in sympathetic nerve sprouting, we evaluated neurite outgrowth by the use of neuron and cardiomyocyte co-culture system. GFP-positive PC12 cells were cultured on metabolically-stressed or normal caridomyocytes and their neurite length was measured. PC12 cells on the stressed cardiomyocyte expanded longer neurites than control and this effect was abolished when NCAM expression was knocked down in cardiomyocytes. Thus, NCAM upregulated in peri-infarcted zone may be involved in sympathetic remodeling and can be a therapeutic target.
To investigate the diabetes mellitus, an animal model, Tsumura Suzuki Obese Diabetes mouse (TSOD) was developed as a type 2 diabetes model. Diabetes causes cardiomyopathy, thus induces heart failure in human and model animals. There is no report that investigates cardiovascular functions of this model mouse, so far. We studied age-dependent changes of the cardiovascular functions of the model, using 7, 12 and 18 months old TSOD and age matched Tsumura Suzuki Non Obesity (TSNO, non-diabetic control) mice. Cardiovascular and endothelium functions were measured under 1.5% isoflurane anesthesia. As non-invasive parameters, ejection fraction and fractional shortening were measured using echo-cardiograph. A Millar catheter transducer was inserted into left ventricle to measure left ventricular pressure and its 1st derivatives. Lead II ECG was recorded and heart rate was measured. No deterioration, except R wave height, was detected in all cardiovascular parameters measured in the TSOD compared to the TSNO. These findings suggest that TSOD is not a suitable model for investigating the diabetic cardiomyopathy.
BACKGROUND AND OBJECTIVES
Laminin, which is a major component of the extracellular matrix, is known to increase in the myocytes derived from patients of the cardiomyopathy. The laminin gamma 1 chain promoter contains a transcriptional element denoted bcn-1 (Suzuki H et. al., J Biol Chem 271: 1996). To elucidate the molecular pathogenesis of the increase of laminin in the cardiac muscle cells of cardiomyopathy, we have carried out the yeast one-hybrid screen using bcn-1 as a bait, and cloned Smarce1r and MLF1IP proteins as molecular partners (Suzuki H, et. al., Experimental & Clinical Cardiology 9: 2004).
METHODS
The yeast two-hybrid screen analysis was carried out again using MLF1IP protein (amino acids 1-318) as a bait. A human heart cDNA library was screened by the yeast mating method.
RESULTS
We isolated an alternative-RNA splicing form of the human cardiac troponin I (TnI) protein and denoted spliced form of TNI (STNI). The mRNA expression pattern of STNI is heart-specific. The STNI shares several sequence similarities with the human cardiac TnI but lacks troponin T binding portion. The results of the in vivo luciferase binding assay and in vitro pull-down assay show that STNI lacks the binding activity with TnT protein and this result was consistent with the structural character.
CONCLUSION
We report the heart-specific segment of the human cardiac troponin I isoform which lacks troponin T binding portion. These results suggest that STNI might be involved in the molecular pathogenesis of the increase of laminin in the cardiomyopathy.
An osteopontin-derived peptide SVVYGLR (SV) activates the adhesion, migration, and tube formation abilities of endothelial cells in vitro. We investigated the effect of SV treatment on cardiac remodeling following myocardial infarction. SV was injected 5min after left anterior descending coronary (LAD) occlusion (SV group), and the injection site was the ischemic area of myocardium. For control, phosphate buffer saline was injected. Six and nine weeks after LAD ligation, cardiac functions were evaluated by echocardiography, and the hearts were removed and examined histologically. Echocardiography demonstrated that cardiac performance was significantly ameliorated in the SV group at 6 and 9 weeks compared with those of control group. The infarct size was significantly reduced in SV group. The vascular density in the border-zone area was prominently increased in SV group. The average widths of myocytes in the border-zone area and myocardial fibrosis in the normal area were significantly reduced in SV group at 6 and 9 weeks compared with those of control group. These results indicate that SV attenuates cardiac remodeling and improves cardiac performance after myocardial infarction. Thus, SV treatment may be a promising therapy for ischemia caused by myocardial infarction.
BACKGROUND
Homing of cardiac myosin-specific CD4 positive T cells into the myocardium is the initial pathologic event of experimental autoimmune myocarditis (EAM). Subsequently, various bystander inflammatory cells are recruited into the myocardium crossing vascular endothelial cell walls. Sulfated polysaccharide fucoidan binds selectin nonselectively and blocks its function. Therefore, this study was designed to evaluate whether in vivo fucoidan treatment can improve EAM.
METHODS AND RESULTS
A 21-day infusion of fucoidan (Group F, n=10) or vehicle (Group V, n=10) was administrated intraperitoneally to rats 4 weeks after EAM induction. After three weeks, fucoidan treatment improved left ventricular ejection fraction by echocardiogram (p<0.01) with reduced heart-weight-to-body-weight ratio (p<0.05). These beneficial effects of fucoidan were accompanied by decreased myocarditis area and serum level of BNP. Furthermore, fucoidan-treated rats had higher leukocyte count in the systemic circulation and lower counts in the myocardium.
CONCLUSIONS
Fucoindan, a nonselective selectin blocker, treatment attenuates the progression of EAM. This observation may be explained, at least in part, by blocking the infiltration of inflammatory cells into the myocardium.
INTRODUCTION
Phosphorylation of GSK3β by prosurvival kinases, such as Akt, has been reported to alter the opening probability of mitochondrial permeability transition pore (mPTP) and to prevent lethal myocardial damage. Here, we investigated the effect of PKA on the mPTP opening by cytosolic GSK3β.
METHOD
We assessed 1) the opening of the mPTP by measuring calcein leakage from mitochondria, and 2) mitochondrial membrane potential (ΔΨm) with voltage sensitive dye of TMRE in saponin-permeabilized rat cardiomyocytes in a laser scanning confocal microscopy.
RESULTS
(1) Active form (non-phosporylated) of GSK3β (GSK3β: 10 nM) accelerated calcein leakage from mitochondria (82.4±1.0 % of the baseline, p<0.01) in a Cyclosporin A (CsA; an inhibitor of mPTP: 100 nM)-sensitive manner. (2) SB216763 (an inhibitor of GSK3β; 3 μM) and PKA catalytic subunit (PKA; 10 U/ml) inhibited the GSK3β-induced calcein leakage. (3) GSK3β depolarized ΔΨm (77.0±2.1 % of the baseline, p<0.05), whereas CsA, SB216763, and PKA abolished the GSK3β-induced ΔΨm depolarization.
CONCLUSION
We conclude that GSK3β opened mPTP and depolarized ΔΨm, and that these effects were prevented by an inactivation of GSK3β with PKA.
OBJECTIVE
Aim of this study was to examine relationship between the mitochondrial KATP channel (mKATP) and glycogen synthase kinase-3β (GSK-3β) in cardioprotection and in modulation of mitochondrial permeability transition pore (mPTP) complex.
METHODS AND RESULTS
In isolated rat hearts, opening of the mKATP by diazoxide (100 μM) before ischemia reduced infarct size (% risk area) after 20-min global ischemia from 62.5±3.5% to 32.2±8.7%, but combination of a GSK-3β inhibitor (SB216763; 1 μM) with diazoxide failed to afford further protection (28.2±3.8%). Inhibition of GSK-3β activity upon reperfusion limited infarction after 35-min regional ischemia from 40.4±3.3% to 27.7±5.8%, whereas diazoxide infusion commenced 10 min before reperfusion was not protective. Diazoxide infused before ischemia induced GSK-3β Ser9-phosphorylation and reduced binding of cyclophilin-D with adenine nucleotide translocator (ANT), a major subunit of mPTP, upon reperfusion. However, inhibition of GSK-3β activity did not change the cyclophilin-D-ANT complex level.
CONCLUSION
The mKATP functions as a trigger of signal leading to GSK-3β phosphorylation and elevation of threshold for mPTP opening by major subunit of mPTP, upon reperfusion. However, inhibition of GSK-3β activity did not change the cyclophilin-D-ANT complex level.
CONCLUSION
The mKATP functions as a trigger of signal leading to GSK-3β phosphorylation and elevation of threshold for mPTP opening by inhibition of cyclophilin-D-ANT interaction. However, this modulation of ANT-cyclophilin-D interaction is unlikely to be the only mechanism of cardioprotection afforded by inhibitory Ser9-phosphorylation of GSK-3β.
BACKGROUND
The Frank-Starling mechanism of the heart is based on the intrinsic ability of cardiac muscle to produce greater active force in response to stretch (i.e., length-dependent activation). We have reported that the giant elastic protein titin (also known as connectin) performs an important role in length-dependent activation. By using a quasi-complete troponin exchange technique, in the present study we investigated whether or not length-dependent activation is regulated at the thin filament level.
METHODS
Skinned fibers were obtained from porcine left ventricular muscle, and active force was measured. Rabbit fast skeletal muscle troponin complex (sTn) was used for troponin exchange. The rate of force redevelopment (ktr) was quantified as an index of cross-bridge kinetics.
RESULTS
Quasi-complete reconstitution of cardiac thin filaments with sTn increased Ca2+ sensitivity and attenuated length-dependent activation. Cross-bridge kinetics was accelerated upon sTn reconstitution at submaximal levels, indicating enhancement of thin filament cooperative activation. Furthermore, an increase in thin filament cooperative activation by MgADP increased Ca2+ sensitivity and attenuated length-dependent activation in fibers with and without sTn reconstitution. Linear regression analysis revealed that Ca2+ sensitivity and length-dependent activation were strongly correlated in a negative manner (R=0.99; P<0.01); i.e., there was less length-dependent activation at higher Ca2+ sensitivity.
CONCLUSION
Length-dependent activation is modulated at least in part via thin filament cooperative activation. The SL-dependent increase in the fraction of cross-bridges may be less in high cooperative activation states, due to a loss of resting cross-bridges that can potentially produce force.
Phosphatidylinositol 3 (PI3) kinase-dependent phosphorylation of Akt in response to reperfusion protects the heart against ischemic insult. We have recently reported that this signaling pathway is impaired in the streptozotocin (STZ)-induced diabetic heart (Diabetes, 2006). Because adrenomedullin attenuates myocardial ischemia/reperfusion injury, we tested the hypothesis that pretreatment with adrenomedullin immediately before ischemia/reperfusion could restore the depressed Akt activation, and therefore enhances protection against ischemia/reperfusion injury in such a model. Male Sprague-Dawley rats (250–300g) were injected with STZ (60mg/kg). Two weeks after the STZ injection, they were treated with adrenomedullin (0.05μg/kg/min i.v.) or vehicle for 60 min. Thereafter, heart was isolated. We observed the following. 1) Adrenomedullin partially restored Akt activation in response to reperfusion in STZ-induced diabetic heart. 2) In isolated perfused heart experiments, the left ventricular functional recovery during reperfusion in STZ-induced diabetic heart was restored by adrenomedullin. Our results suggest that pretreatment with adrenomedullin could afford protection against ischemia/reperfusion injury in STZ-induced diabetic rats. Enhanced Akt phosphorylation by adrenomedullin may be involved in these processes.
BACKGROUND AND OBJECTIVES
The electrophysiological actions of angiotensin II (Ang II) linked with the genesis of reperfusion arrhythmias were elucidated by clarifying the roles of Ang II and mitochondrial IKATP channel (mitoKATP) on cardiac impulbse propagation.
METHODS
Ventricular papillary muscle (PPM, 3mm length) from the rat heart (WKYIzm) were fixed on the superfusion chamber. Membrane action potential, Dv/Dtmax and activation time (AT) were recorded at the tip of PPM. ATs were measured as the interval from stimulus artifact to Dv/Dtmax. After PPM preparations were superfused with ischemic-like solution for 5 min, they were washed out with oxygenated Tyrode containing Ang II (10–8 M) or Ang II plus 5-HD (mito KATP blocker; 10–6 M), plus CV-11974 (candesartan; 10–7 M) during 5 min. To assess the excitability, the rapid electric stimuli (10 Hz) for 60 sec were applied to the preparations just after reoxygenation.
RESULTS
Ang II accelerated 2:1 conduction block by the rapid pacing and prolonged AT. These actions of Ang II were abolished under 5-HD (p<0.001) and CV-11974 (p<0.001). The amplitude of AP was rapidly recovered during reoxygenation under perfusion of Ang II plus 5-HD (p<0.05).
CONCLUSIONS
We noticed that Ang II can provoke myocardial conduction delay and block during reoxygenation that may be arrhythmogenic properties. The results that mito KATP blocker and Ang II receptor blocker abolished these phenomenon can partly explain the antiarrhythmic action of ARB in instantaneous phase of reperfusion.
BACKGROUND
Although angiotensin II (Ang II) binds to Ang II type 1 (AT1) and type 2 (AT2) receptors, the functions of AT1 and AT2 receptors are antagonistic against blood pressure and cell growth. The molecular mechanisms of this antagonism are not well known.
METHODS AND RESULTS
We examined AT1 and AT2 receptor-induced signal cross-talk in the cytoplasm and the importance of the hetero-dimerization of AT1 receptor with AT2 receptor on the cell surface. We used HepG2 cells and human coronary smooth muscle cells that endogenously expressed both receptors. The AT1 and AT2 receptors showed antagonistic effects toward inositol phosphate production, cell cycle regulatory protein p27kip1 expression and extracellular-signal-regulated kinase 1/2 activity. The AT1 receptors formed mainly homo-dimers but not hetero-dimers with AT2 receptor on the cell surface as determined by immunoprecipitation and induced cell signals. The ratio of the expression levels of homo-dimerized AT1 receptors, homo-dimerized AT2 receptors and hetero-dimerized AT1 and AT2 receptors on the cell surface was 25:20:1. In addition, expression levels of homo-dimerized AT1 receptor or AT2 receptor on the cell surface did not change after 24 hr
BACKGROUND AND OBJECTIVES
Autophagy, a highly conserved cellular mechanism that plays a key role in the turnover of macromolecules, has been implicated in cellular fate in the development of heart failure. However, tools to measure autophagic flux, which represents autophagy more accurately, have been limited. Here, we tried to evaluate cardiac autophagy in concerning with cardiac apoptosis using a newly developed method to measure cardiac autophagic flux.
METHOD
HL-1 cardiac myocytes expressed with GFP-tagged LC3 were prepared for in vitro model , and cardiac-specific expressing mCherry-LC3 transgenic mice were created for in vivo model. After the apoptotic stimulation in both models, autophagic flux was evaluated with chloroquine, an inhibitor of lysosomal activity which was needed for the last step of autophagic flux, using wide field microscopy with Z-stacks.
RESULTS
Oxidative stress, beta-adrenergic stress, or inflammatory stress with lipopolysaccharide increased apoptotic cells in cardiac myocytes. These apoptotic stimuli also increased the number of cells with numerous punctuate GFP-LC3-positive autophagosomes. The accumulation of autophagosomes was inhibited by 3-methylademine, which is known to block autophagic flux at the early phase, and enhanced by concurrent administration of chloroquine. Using transgenic mice expressing mCherry-LC3 exposed to lipopolysaccharide for 4 hour, we observed an increase in mCherry-LC3 labeled autophagosomes in myocardium, which was further increased by concurrent administration of chloroquine.
CONCLUSION
We demonstrated here that autophagic flux was stimulated in cardiac myocytes during apoptosis. Also, our newly developed method for measuring cardiac autophagic flux with chloroquine can be used both in vitro and in vivo.
BACKGROUND
An opening of the mitochondrial permeability transition pore (MPTP), which leads to loss of mitochondrial membrane potential (ΔΨm), is the earliest event that commits cells to death, and this process is potentially a prime target for therapeutic intervention against myocardial infarction. We examined the effects of gene silencing of cyclophilin D (CypD), one of the putative components of MPTP, using RNA interference.
METHODS AND RESULTS
We created an adenovirus carrying short interfering RNA (siRNA) which inactivates CypD. In vitro, transduction of CypD-siRNA in neonatal rat cardiomyocytes had protective effects against oxidant-induced ΔΨm loss and cytotoxicity. In vivo, we monitored the spatio-temporal changes of ΔΨm in perfused rat hearts subjected to ischemia, using the real-time two-photon confocal imaging system. Briefly, adult rats received direct intramyocardial injections of the CypD-siRNA adenovirus. Two-to three days after injection, the rat hearts were removed, perfused in Langendorff mode and underwent two-photon imaging. The progressive loss of ΔΨm induced by myocardial ischemia was significantly suppressed in CypD-siRNA-transduced cells, compared with non-transduced cells. Interestingly, the protective effect of CypD-siRNA was dose-dependent.
CONCLUSION
Targeting CypD by RNA interference protects against oxidant-induced cardiomyocyte injury in vitro, and myocardial ischemia in vivo, implicating CypD as a promising molecular target for anti-ischemia/reperfusion therapy.
BACKGROUND AND OBJECTIVES
The Rho-kinase (ROK)-mediated Ca2+-sensitization of vascular smooth muscle (VSM) contraction contributes to abnormal VSM contraction such as vasospasm. As upstream messengers of ROK, we have identified SPC and Fyn tyrosine kinase. In contrast, as the downstream pathways, the ROK-mediated elevation of myosin light chain (MLC) phosphorylation, and it has been hypothesized that ROK not only directly phosphorylates MLC, but also indirectly increases MLC phosphorylation through the inhibition of MLC phosphatase. Although several molecules are proposed to mediate these downstream pathways of ROK, essential molecule(s) have not been determined yet. In order to clarify this point, in this study we used in vitro motility assay system with highly purified contractile proteins of smooth muscle, but in the complete absence of the MLC kinase and phosphatase.
METHODS
The chicken gizzard MLCs and heavy chain (HMM) were purified as recombinant myosin from the baculovirus expression system and were confirmed by MALDI-TOF MS. The myosin MLC was phosphorylated by ROK, as confirmed by SDS- PAGE with ProQ-Diamond stain.
RESULTS
The in vitro motility assay of the recombinant SM myosin revealed that average velocity of?actin sliding on unphosphorylated and ROK-phosphorylated myosin was 0.0 and 0.3 μm/sec, respectively, the latter of which is comparable with the maximum values of MLCK-phosphorylated myosins (0.27μm/sec).
BACKGROUND AND OBJECTIVES
The integrin-integrin-linked kinase (ILK) signaling plays an important role in cell motility, proliferation and survival. The interaction between ILK and β-parvin (ParvB) is thought to be crucial in regulating the cardiac contractility, involving VEGF expression (Genes Dev 20, 2006). The role of molecular interaction that triggers the ILK-mediated signaling cascades remains unclear.
METHODS
ILK, ParvB and Akt/PKB cDNA were fused either to the N-terminal luciferase or to the C-terminal luciferase cDNA fragment, which was inserted into pcDNA3.1+ expression vector. Using the luciferase complementation imaging on the basis of the protein-protein interaction, the luciferase-derived luminescent signals were detected under various conditions involving Akt/PKB activation and RNA interference-mediated ParvB knockdown.
RESULTS
ParvB bound to Akt/PKB in living cell conditions, more strongly than ILK. The knockdown of ParvB increased expression of VEGF and HIF-1α, suggesting indispensable roles of ParvB in regulating ILK-mediated signaling pathway. This strong interaction between ParvB and Akt/PKB may control the cardiac contractility.
BACKGROUND
Pharmacological interventions for the prevention of arrhythmia with chronic heart failure (CHF) still remain limited. Accumulating evidence implicates increased cardiac expression of T-type Ca2+ channels (TCC) in the progression of CHF. The efficacy of TCC blockade in preventing ventricular arrhythmias associated with CHF has never been tested.
METHODS AND RESULTS
We compared effects of efonidipine or mibefradil, dual T- and L-type Ca2+ channel blockers, with those of nitrendipine, a selective L-type Ca2+ channel blocker, on survival, arrhythmogenicity, and cardiac autonomic nerve activity in transgenic mice expressing dominant-negative mutant of neuron-restrictive silencer factor in a cardiac restricted manner (dnNRSF-Tg), an useful mouse model for CHF with lethal arrhythmias. We revealed that efonidipine but not nitrendipine, corrected imbalance in cardiac autonomic nerve activity. Efonidipine significantly improved survival among dnNRSF-Tg mice by preventing arrhythmias. Arrhythmogenicity, evaluated in an electrophysiological study, was dramatically reduced in dnNRSF-Tg mice treated with efonidipine or mibefradil.
CONCLUSIONS
TCC blockade reduced lethal arrhythmias in the mouse model of CHF with correcting the imbalance in cardiac autonomic function. Our findings suggest therapeutic potential of TCC blockade to prevent sudden death in patients with heart failure.
BACKGROUND
Insulin resistance has been known to occur as a consequence of heart failure (HF). However, the exact mechanisms underlying insulin resistance in this setting remain unknown. Our purpose in the present study is to determine its mechanisms.
METHODS
Myocardial infarction (MI) was created in male mice, and sham operation was also performed. After 4 weeks, fasting serum glucose and plasma insulin concentrations were measured and intraperitorial insulin tolerance tests were performed. Skeletal muscle samples were obtained before and after insulin infusion.
RESULTS
Fasting insulin, but not glucose level, was significantly higher in MI than sham. Peak decrease of plasma glucose after insulin load was significantly smaller in MI than sham. Insulin-stimulated serine phosphoryration of Akt and transition of glucose transporter 4 to plasma membrane were decreased in MI skeletal muscle. Skeletal muscle NAD(P)H oxidase activity measured by lucigenin chemiluminescence assay was significantly increased in MI. The inhibition of NAD(P)H oxidase activity with apocynin ameliorated insulin resistance and improved the impaired insulin signaling in MI skeletal muscle.
CONCLUSIONS
Insulin resistance in HF was associated with the impairment of insulin signaling in skeletal muscle, which was caused by NAD(P)H oxidase-derived oxidative stress.
Intracellular Ca2+ is a key regulator in the generation and/or maintenance of arterial tone. A variety of Ca2+ permeable ion channels, such as receptor-operated channels and voltage-gated Ca2+ channels, participate in the agonist- or depolarization-stimulated Ca2+ entry and contraction in vascular smooth muscle. For vascular relaxation, the entry of Ca2+ must be balanced by extrusion of Ca2+ from the cytosol via Na+/Ca2+ exchanger (NCX) and Ca2+ ATPases. Recently, we have found that phenylephrine-induced cytosolic Ca2+ elevation and vasocontraction are significantly greater in mesenteric arteries from transgenic mice overexpressing NCX1.3 than in those from wild-type mice. On the other hand, NCX1 inhibition suppressed phenylephrine-induced vasocontaction in NCX1.3-transgenic mice, but not in drug-insensitive NCX1.3 mutants, suggesting that α1-agonist-induced vasocontraction may depend on Ca2+ entry mediated by NCX1.3. Interestingly, NCX1 was co-immunoprecipitated with TRPC3, a phospholipase-C-dependent non-selective cation channel, from solubilized vascular smooth muscle cells. Sucrose density gradient fractionation of vascular smooth muscle cells showed that NCX1, TRPC3, and α1 receptor associate with caveolar raft domains. Furthermore, dominant negative TRPC3 transgene reduced NCX1-mediated Ca2+ entry in phenyle-phrine-treated mesenteric arteries. These findings indicate that functional coupling between NCX1 and TRPC3 plays a pivotal role in regulating arterial tone via α1 adrenoceptor.
BACKGROUND AND OBJECTIVES
Hypertensive patients with large BPV have aggravated end-organ damages. We investigated the mechanism whereby large BPV aggravates hypertensive cardiac remodeling and function.
METHODS AND RESULTS
A model of hypertension with large BPV was created by performing bilateral sinoaortic denervation (SAD) in SHR. SAD did not change mean blood pressure. Concentric hypertrophy developed in SHR and, to lesser extent, in WKY+SAD, without changing systolic function. SHR+SAD showed further hypertrophy and myocardial fibrosis associated with systolic dysfunction. In SHR+SAD, the extent of myocyte hypertrophy was additive with those in WKY+SAD and SHR, whereas the extents of myocardial fibrosis and macrophage recruitment were synergistic. MCP-1 was upregulated in SHR+SAD and, to lesser extent, in WKY+SAD, but not in SHR. Angiotensinogen was induced only in SHR+SAD. Subdepressor dose of candesartan abolished the BPV-induced myocyte hypertrophy, myocardial fibrosis, macrophage recruitment, and inductions of MCP-1 and angiotensinogen and subsequently prevented systolic dysfunction in SHR+SAD.
CONCLUSION
Large BPV aggravated hypertensive cardiac remodeling and function by activating the ANGII-mediated inflammation. Superimposing BPV exaggeration on hypertension may be crucial for activation of the angII-mediated changes.
BACKGROUND
Cardiomyocyte stiffness of the longitudinal direction is increased in patients with diastolic heart failure. However, stiffness of the transverse direction has not been elucidated. We measured the stiffness of single cardiomyocytes obtained from rat hearts with hypertrophy induced by the β1-adrenergic agonist isoproterenol (ISO) by use of an atomic force microscope (AFM).
METHODS AND RESULTS
Male Wistar rats (9 weeks old) received vehicle, ISO (2.4 mg· kg–1· day–1 for 7 days) or ISO + β1-blocker, meto-prolol (MET) (24 mg· kg–1· day–1 for 7 days) subcutaneously (each group n=5). After 7 days, compared with the controls and ISO+MET group, ISO only administration significantly increased left ventricular (LV) wall thickness and decreased LV diastolic function. To estimate transverse stiffness, cardiomyocytes were isolated from LV free wall of the rat heart. By pressing the AFM cantilever to the cell surface vertically, the nano/micro-order elasticity was measured by parabolic force curves of cantilever deflection/indentation. Elasticity of cardiomyocytes was significantly higher in ISO group than in control and ISO + MET group (p<0.05, n=10).
CONCLUSIONS
Cardiomyocyte stiffness of the transverse direction was increased in chronic β1-adrenergic stimulated hearts along with hypertrophy and diastolic dysfunction.
BACKGROUND AND OBJECTIVES
Recently, it has been proposed that β-adrenoceptor stimulation (β-ARS) increases Ca2+ leak from cardiac sarcoplasmic reticulum (SR) through the “hyper-phosphorylation” of ryanodine receptor (RyR) (the Ca2+ release channel of the SR), leading to the contractile dysfunction and arrhythmia in heart failure. However, the functional relevance of β-ARS-mediated RyR phosphorylation has not been well understood even in the physiological condition. In the present study, I investigated the effect of β-ARS on Ca2+ leak from SR by using saponin-permeabilized multicellular preparation and clarified the intracellular signal transduction mechanisms involved in this effect.
METHODS
Thin trabeculae, obtained from mouse heart, was treated with 1 μM isoproterenol (Iso), followed by the permeabilization with saponin (50 μg/ml. 30 min). To measure the Ca2+ content, Ca2+ in SR was released by caffeine (50 mM), and was measured with fluo-3 (30 μM). Ca2+ leak was estimated by measuring the remaining Ca2+ in SR after washing SR with the EGTA-contained solution (pCa 20) for various durations (15–300 sec) after Ca2+ loading (pCa 6.2, 120 sec). To evaluate the phosphorylation levels of RyR in the preparations, Western immunoblotting was carried out.
RESULTS
PKA-dependent phosphorylation of RyR at Serine2808 was significantly increased after Iso treatment, whereas CaMKII-dependent phosphorylation of RyR at Serine2814 was not altered in the preparations. The Ca2+ leak in Iso-treated preparation was significantly increased and this increase was blocked by the pretreatment of a PKA inhibitor, 2 μM H-89, but not by a CaMKII-inhibitory peptide, 100 μM AIP.
CONCLUSION
β-ARS increases Ca2+ leak from cardiac SR through PKA-dependent phosphorylation of RyR at Serine2808.
BACKGROUND AND OBJECTIVES
Metabolic syndrome is a major cause of cardiovascular disease, and obese visceral adipose tissue remodeling and malfunction based on chronic inflammation plays a central role.
METHODS
To assess physiological and pathophysiological events that involve dynamic interplay between multiple cell-types, a visualization technique, based on laser confocal microscopy was therefore developed that made it possible to precisely evaluate the three-dimensional structures in living tissue, and the cell dynamics in vivo.
RESULTS
We found close spatial and temporal interrelationships between angiogenesis and adipogenesis, and both were augmented in obese adipose (2007 Diabetes). VEGF-antibody inhibited not only angiogenesis but also the formation of adipogenesis in obesity. We also found increased leukocyte-platelet-endothelial cell interactions in the microcirculation of obese visceral adipose that were indicative of activation of the leukocyte adhesion cascade, a hallmark of inflammation (2008 JCI). Platelets were also activated locally in obese visceral adipose, and upregulated expression of adhesion molecules on macrophages and endothelial cells suggests their increased interactions contribute to local activation of inflammatory processes within visceral obese adipose tissue. Interestingly, the heightened leukocyte-endothelial interactions were not observed in subcutaneous fat pads in the same mice. In addition, cytotoxic T cells infiltrated into obese adipose tissue, contributing to the macrophage recruitment. Our results clearly demonstrated the power of our imaging technique to analyze complex cellular interplays in vivo and to evaluate new therapeutic interventions against them. Results also indicate that visceral adipose tissue obesity is an inflammatory disease.
BACKGROUND
It is important to prevent cardiac remodeling after acute myocardial infarction (AMI), since it causes heart failure and poor prognosis. Although it has been shown that cytokines including G-CSF improve cardiac remodeling and reduce mortality after AMI in animals, little is known about the negative regulation of cytokine function in the development of cardiac remodeling after AMI. In this study, we analyzed phenotype of cardiac-specific SOCS (suppressor of cytokine signaling) deficient mice (SOCS-KO) during AMI.
METHODS AND RESULTS
Western blot analysis revealed that STAT3 and AKT were markedly phosphorylated and SOCS was induced during AMI in the wild-type mice (WT) hearts. Infarct sizes 14 days of coronary ligation were significantly smaller in SOCS-KO than the WT. Duration and intensity of STAT3 phosphorylation was greater in SOCS-KO than the WT. The number of TUNEL-positive cells and caspase3 expression 24 hours after AMI were significantly less in SOCS-KO than the WT.
CONCLUSION
These results suggest that cardiac remodeling is prevented in SOCS-KO via inhibiting cardiomyocyte apoptosis.
BACKGROUND
Endoplasmic reticulum (ER) plays a pivotal role in initiation of apoptosis by unfolded protein responses during cellular stress such as ischemia/reperfusion injury. Sodium 4-phenylbutyrate (4-PBA) has been reported to act as a chemical chaperone reducing the load of unfolded protein. Therefore, we examined if 4-PBA has beneficial effects on cardiac ischemia/reperfusion injury in mice.
METHODS
C57BL/6J mice were subjected to 30 minutes left ascending coronary ischemia followed by reperfusion. ER-stress associated genes were evaluated by microarray analysis in ischemic myocardium at 6 and 24 hours after ischemia. Also 4-PBA (100mg/kg, N=10) or PBS as a control (N=10) was administrated intraperitoneally just before ischemia. Apoptosis was evaluated by TUNEL stein and infarct size was estimated by TTC stein at 2nd day after ischemia. Cardiac function was estimated by UCG at 21st day after ischemia. Survival ratio was compared between the models with and without 4-PBA.
RESULTS
A number of ER-stress associated genes were up-regulated after myocardial ischemia-reperfusion. 4-PBA reduced CPK elevation, apoptotic cells and infarct size after myocardial ischemia-reperfusion. Although ischemia-reperfusion impaired cardiac function in mice with PBS, 4-PBA inhibited worsened cardiac function. While 30% animals treated with PBS died within the observational period of 21 days, all mice treated with 4-PBA survived without optimal congestion.
CONCLUSION
4-PBA, as a chemical chaperon, protected against myocardial ischemia-reperfusion injury and improved survival ratio in mice.
Previous reports showed that transient phosphodiesterase type-3 (PDE-3) inhibition or repetitive transient PKA activation causes cardioprotection after myocardial infarction. We tested whether the intermittent transient PDE-3 inhibition blocks non-ischemic cardiac failure and remodeling. Fifty Wistar-Kyoto rats underwent treatments for 8 weeks with L-NAME or an inactive isomer with/without hydralazine in drinking water. Olprinone was administrated by gavage once a day, 3 times per week. L-NAME increased blood pressure after 4/8 weeks as well as left ventricular weight/body weight ratio (LVW/BW) compared with control, which were totally cancelled by Hydralazine and eliminated by Olprinone (143% vs. 119% of control, P<0.05) after 8 weeks. Olprinone also attenuated myocardial hypertrophy (190% vs. 148%, P<0.05), interstitial fibrosis (11.5% vs. 6.3% of LV area, P<0.05) and wet lung weight/body weight ratio (124% vs. 109%) without modifying increased blood pressure. Furthermore, Olprinone significantly reduced the interstitial infiltration of polymorphic neutrophils induced by L-NAME (520% vs. 279%, P<0.05). Thus, intermittent transient PDE-3 inhibition elicits cardioprotection against non-ischemic heart failure beyond hemodynamic effects, which might be due to inhibition of inflammatory change.
Mitochondrial motility (mito-motility) is regulated by the cytoplasmic [Ca2+] ([Ca2+]c) oscillations to provide the intracellular homeostasis, including Ca2+ buffering, ATP supply, and signal transduction by mito-chondria. We studied the dependence of Ca2+-sensitive mito-motility on Miro (mitochondrial Rho GTPases), which is an integral mitochondrial membrane protein and has two EF-hands (Ca2+-binding site). Mitochondrial YFP-expressed H9c2 cardiac myoblasts were co-transfected with Miros, and then loaded with fura-2 to monitor the mito-motility and [Ca2+]c simultaneously. (1) At the resting [Ca2+]c (<40 nM), the mito-motility was enhanced when cells were overexpressed with Miros regardless of the mutations in EF-hands (MiroWT-EF), whereas was suppressed when Miro was depleted by siRNA. (2) In the elevated [Ca2+]c (0.1–1 mM), the Ca2+-induced mito-motility inhibition was hypersensitive in the Miro-over-expressed cells, whereas was attenuated either in the Miro-siRNA or MiroWT-EF expressed cells. In conclusion, Miro serves as a [Ca2+]c-sensitive bidirectional regulator of the mito-motility, that is, Miro enhances the mito-motility under low [Ca2+]c and suppresses the mito-motility under high [Ca2+]c.
BACKGROUND AND OBJECTIVES
Since AMP-activated protein kinase (AMPK) plays an important role in energy metabolism, we investigated whether the activation of AMPK by metformin attenuates the progression of heart failure in dogs.
METHODS AND RESULTS
Continuous rapid ventricular pacing (230/min for 4 weeks) in dogs caused representative features of chronic heart failure. Treatment with metformin (100mg/kg/day PO, n=8) significantly prevented the progression of heart failure assessed by echocardio-graphic and hemodynamic evaluation compared with the vehicle (n=8). In addition, metformin enhanced phosphorylation of AMPK in the LV myocardium. Furthermore, metformin improved insulin resistance, and increased mRNA expression of endothelial nitric oxide synthase in the LV myocardium and plasma nitric oxide levels, which may be contributable to its beneficial effect.
CONCLUSION
Metformin that activates AMPK prevented the progression of heart failure induced by rapid pacing in dogs. Metformin may be applicable as a novel therapy for heart failure.
AIMS AND METHODS
To elucidate the mechanisms of Stretch-Induced Arrhythmias and the contribution of Stretch-Activated Channels (SACs), we examined the response of myocardium to mechanical stresses at tissue and cellular levels using the fluorescent voltage sensitive or calcium indicator and stretching devices (tissue extension, carbon fiber, and AFM indentation).
RESULTS
The membrane potential of single cardiomyocytes responded to the uniaxial stretch in an amplitude dependent manner. We also found the point indentation of the sarcolemma initiated the local rise in calcium signal. However, at the tissue level, although the uniform stretch was applied, the membrane potential was depolarized heterogeneously and excitation was initiated from the local spot. Morphometry of the tissue revealed that such focal spots were located where the tissue thickness was thin. These results were reproducible by computational simulation studies.
CONCLUSION
Although each myocyte responds to mechanical stress in an amplitude dependent manner, the heterogeneity of the tissue structure leads the focal excitation in response to the globally applied stretch which may be seen in dilatation of ventricle.
Diabetes (DM) contributes to the exacerbation of left ventricle (LV) dysfunction after myocardial infarction (MI). Activation of ERK5 with transcriptional activity inhibits apoptosis and LV dysfunction. SUMOylation has been proposed as a negative regulator of various transcription factors. In the current study, we investigated the role of ERK5-SUMOylation in ERK5 transcriptional activity as well as on DM-mediated exacerbation of LV dysfunction after MI. ERK5 wild type transcriptional activity was inhibited by Ubc9 or PIAS1, but not in the ERK5-SUMOylation-site defective mutant (K6R/K22R). H2O2 and high glucose induced ERK5-SUMOylation, and the K6R/K22R mutant, dominant negative form of Ubc9, and siRNA-PIAS1 reversed H2O2-mediated reduction of ERK5 transcriptional activity in cardiomyocytes, indicating the presence of SUMOylation-dependent ERK5 transcriptional repression. Constitutively active form of MEK5α (CA-MEK5α) inhibited ERK5-SUMOylation independent of kinase activity, but dependent on MEK5-ERK5 association. To investigate the pathological role of ERK5-SUMOylation in DM mice after MI, we utilized cardiac specific CA-MEK5α transgenic mice (CA-MEK5α-Tg). MI was induced in streptozotocin (STZ)-injected (DM + MI group) or vehicle-injected mice (MI group) by ligating the left coronary artery. The ERK5-SUMOylation was increased in the DM + MI. ERK5-SUMOylation, the exacerbation of LV dysfunction, and the number of TUNEL positive cells in DM + MI was significantly inhibited in CA-MEK5α-Tg mice. These results demonstrated that ERK5 transcriptional activity is subject to down regulation by diabetes-dependent SUMOylation, which resulted in a pro-apoptotic condition contributing to poor post-MI LV function.
BACKGROUND AND OBJECTIVES
We have previously reported that apoptosis signal-regulating kinase 1 (ASK1) plays a pivotal role in pathological cardiac remodeling. However, it has not been elucidated the interaction between pathological remodeling cascade and cardiac adaptive responses.
METHODS AND RESULTS
To examined the role of ASK1 in cardiac adaptive responses, mice lacking ASK1 (ASK1−/−) were exercised by swimming. ASK1−/− mice showed exaggerated growth of the heart accompanied by typical characteristics of physiological hypertrophy. Akt, a key molecule of physiological hypertrophy was activated and p38, a downstream kinase of ASK1, was suppressed in the swimming-exercised ASK1−/− mice. Furthermore, inhibition of ASK1 or p38 activity enhanced insulin-like growth factor 1-induced protein synthesis in rat neonatal cardiomyocytes, and the treatment with a specific inhibitor of p38 resulted in enhancement of Akt activation and suppression of protein phosphatase 2A activation. The cardiac-specific p38α-deficient mice developed an exacerbated form of cardiac hypertrophy in response to swimming exercise.
CONCLUSION
ASK1/p38 signaling pathway negatively regulates physiological hypertrophy.
BACKGROUND
Mesenchymal stem cells (MSC) could be of great therapeutic potential after ischemic myocardial injury. However, intolerance and poor cell viability associated with oxidative stress after transplantation has limited the reparative capacity. Under these conditions, Heme Oxygenase-1 (HO-1) plays a pivotal role as anti-oxidative stress molecule.
OBJECTIVE AND METHODS
Transfer of human HO-1 gene in cultured MSC was performed by lipofection method. To evaluate the effect of HO-1 overexpression, MSC or MSCHO-1 were exposed to culture conditions with serum deprivation and hypoxia over different periods of time and characteristics of cell damage were analyzed by flow cytometry. In vitro, cell viability was determined by MTS assay after exposing MSC or MSCHO-1 to H2O2 as an oxidative stress. VEGF level in the supernatant of each cells culture after the load of H2O2 were measured. In rat infarction model, MSC or MSCHO-1 was injected around the infracted border zone, and cardiac examination was performed after cell transplantation.
RESULTS
HO-1 overexpression was prevented MSC from apoptosis. MSCHO-1 was resistant to cell death under condition of oxidative stress and secreted a large amount, more VEGF compared with MSC. Transplantation of MSCHO-1 improved cardiac function. Capillary density was markedly increased in the MSCHO-1 group.
CONCLUSION
These results demonstrate transplantation of MSC with transient overexpression of HO-1 could enhance the reduction of myocardial injury after acute ischemia, probably through suppression of the allogenic reaction and graft loss in early stages.