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Background-Phosphorylative desensitization of G-protein-coupled receptors contributes significantly to post-myocardial infarction (MI) remodeling and heart failure (HF). Here, we determined whether adiponectin receptors (AdipoRs) 1 and 2 (the 7-transmembrane domain-containing receptors mediating adiponectin functions) are phosphorylatively modified and functionally impaired after MI. Methods and Results-Post-MI HF was induced by coronary artery occlusion. Receptor phosphorylation, kinase expression, and adiponectin function were determined via in vivo, ex vivo, and in vitro models. AdipoR1 and AdipoR2 are not phosphorylated in the normal heart. However, AdipoR1 was significantly phosphorylated after MI, peaking at 7 days and remaining significantly phosphorylated thereafter. The extent of post-MI AdipoR1 phosphorylation positively correlated with the expression level of GPCR kinase (GRK) 2, the predominant GRK isoform upregulated in the failing heart. Cardiac-specific GRK2 knockout virtually abolished post-MI AdipoR1 phosphorylation, whereas virus-mediated GRK2 overexpression significantly phosphorylated AdipoR1 and blocked adiponectin metabolic-regulatory/antiinflammatory signaling. Mass spectrometry identified serine-7, threonine-24, and threonine-53 (residues located in the n-terminal intracellular AdipoR1 region) as the GRK2 phosphorylation sites. Ex vivo experiments demonstrated that adenosine monophosphate-activated protein kinase activation and the anti-tumor necrosis factor-α effect of adiponectin were significantly inhibited in cardiomyocytes isolated from nonischemic area 7 days after MI. In vivo experiments demonstrated that acute adiponectin administration-induced cardiac GLUT4 translocation and endothelial nitric oxide synthase phosphorylation were blunted 7 days after MI. Continuous adiponectin administration beginning 7 days after MI failed to protect the heart from adverse remodeling and HF progression. Finally, cardiac-specific GRK2 knockdown restored the cardioprotective effect of adiponectin. Conclusion-AdipoR1 is phosphorylatively modified and desensitized by GRK2 in failing cardiomyocytes, contributing to post-MI remodeling and HF progression. [ABSTRACT FROM AUTHOR]

Expression and activity of cardiac Na+/Ca2+ exchanger (NCX1) are altered in many disease states. We engineered mice in which the phosphomimetic phospholemman S68E mutant (inhibits NCX1 but not Na+-K+-ATPase) was constitutively overexpressed in a cardiac-specific manner (conS68E). At 4-6 wk, conS68E mice exhibited severe bradycardia, ventricular arrhythmias, increased left ventricular (LV) mass, decreased cardiac output (CO), and ~50% mortality compared with wild-type (WT) littermates. Protein levels of NCX1, calsequestrin, ryanodine receptor, and α1- and α2-subunits of Na+-K+-ATPase were similar, but sarco- (endo)plasmic reticulum Ca2+-ATPase was lower, whereas L-type Ca2+ channels were higher in conS68E hearts. Resting membrane potential and action potential amplitude were similar, but action potential duration was dramatically prolonged in conS68E myocytes. Diastolic intracellular Ca2+ ([Ca2+]i) was higher, [Ca2+]i transient and maximal contraction amplitudes were lower, and half-time of [Ca2+]i transient decline was longer in conS68E myocytes. Intracellular Na+ reached maximum within 3 min after isoproterenol addition, followed by decline in WT but not in conS68E myocytes. Na+/Ca2+ exchange, L-type Ca2+, Na+-K+-ATPase, and depolarization- activated K+ currents were decreased in conS68E myocytes. At 22 wk, bradycardia and increased LV mass persisted in conS68E survivors. Despite comparable baseline CO, conS68E survivors at 22 wk exhibited decreased chronotropic, inotropic, and lusitropic responses to isoproterenol. We conclude that constitutive overexpression of S68E mutant was detrimental, both in terms of depressed cardiac function and increased arrhythmogenesis. [ABSTRACT FROM AUTHOR]

Recent data supporting any benefit of stem cell therapy for ischemic heart disease have suggested paracrine-based mechanisms via extracellular vesicles (EVs) including exosomes. We have previously engineered cardiac-derived progenitor cells (CDCs) to express a peptide inhibitor, bARKct, of G protein-coupled receptor kinase 2, leading to improvements in cell proliferation, survival, and metabolism. In this study, we tested whether βARKct-CDC EVs would be efficacious when applied to stressed myocytes in vitro and in vivo. When isolated EVs from βARKct-CDCs and control GFP-CDCs were added to cardiomyocytes in culture, they both protected against hypoxia-induced apoptosis. We tested whether these EVs could protect the mouse heart in vivo, following exposure either to myocardial infarction (MI) or acute catecholamine toxicity. Both types of EVs significantly protected against ischemic injury and improved cardiac function after MI compared with mice treated with EVs from mouse embryonic fibroblasts; however, bARKct EVs treated mice did display some unique beneficial properties including significantly altered pro- and anti-inflammatory cytokines. Importantly, in a catecholamine toxicity model of heart failure (HF), myocardial injections of βARKct-containing EVs were superior at preventing HF compared with control EVs, and this catecholamine toxicity protection was recapitulated in vitro. Therefore, introduction of the bARKct into cellular EVs can have improved reparative properties in the heart especially against catecholamine damage, which is significant as sympathetic nervous system activity is increased in HF. [ABSTRACT FROM AUTHOR]

Myeloid cells, including macrophages, play important roles as first responders to cardiac injury and stress. Epidermal growth factor receptor (EGFR) has been identified as a mediator of macrophage responsiveness to select diseases, though its impact on cardiac function or remodeling following acute ischemic injury is unknown. We aimed to define the role of myeloid cell-specific EGFR in the regulation of cardiac function and remodeling following acute myocardial infarction (MI)-induced injury. Floxed EGFR mice were bred with homozygous LysM-Cre (LMC) transgenic mice to yield myeloid-specific EGFR knockout (mKO) mice. Via echocardiography, immunohistochemistry, RNA sequencing and flow cytometry, the impact of myeloid cell-specific EGFR deletion on cardiac structure and function was assessed at baseline and following injury. Compared with LMC controls, myeloid cell-specific EGFR deletion led to an increase in cardiomyocyte hypertrophy at baseline. Bulk RNASeq analysis of isolated cardiac Cd11b+ myeloid cells revealed substantial changes in mKO cell transcripts at baseline, particularly in relation to predicted decreases in neovascularization. In response to myocardial infarction, mKO mice experienced a hastened decline in cardiac function with isolated cardiac Cd11b+ myeloid cells expressing decreased levels of the pro-reparative mediators Vegfa and Il10, which coincided with enhanced cardiac hypertrophy and decreased capillary density. Overall, loss of EGFR qualitatively alters cardiac resident macrophages that promotes a low level of basal stress and a more rapid decrease in cardiac function along with worsened repair following acute ischemic injury. [ABSTRACT FROM AUTHOR]

Phospholemman (PLM), when phosphorylated at Ser68, inhibits cardiac Na+/Ca2+ exchanger 1 (NCX1) and relieves its inhibition on Na+-K+-ATPase. We have engineered mice in which expression of the phosphomimetic PLM S68E mutant was induced when dietary doxycycline was removed at 5 wk. At 8-10 wk, compared with noninduced or wild-type hearts, S68E expression in induced hearts was ~35-75% that of endogenous PLM, but protein levels of sarco- (endo)plasmic reticulum Ca2+-ATPase, α1- and α2-subunits of Na+- K+-ATPase, α1c-subunit of L-type Ca2+ channel, and phosphorylated ryanodine receptor were unchanged. The NCX1 protein level was increased by ~47% but the NCX1 current was depressed by ~34% in induced hearts. Isoproterenol had no effect on NCX1 currents but stimulated Na+-K+-ATPase currents equally in induced and noninduced myocytes. At baseline, systolic intracellular Ca2+ concentrations ([Ca2+]i), sarcoplasmic reticulum Ca2+ contents, and [Ca2+]i transient and contraction amplitudes were similar between induced and noninduced myocytes. Isoproterenol stimulation resulted in much higher systolic [Ca2+]i, sarcoplasmic reticulum Ca2+ content, and [Ca2+]i transient and contraction amplitudes in induced myocytes. Echocardiography and in vivo close-chest catheterization demonstrated similar baseline myocardial function, but isoproterenol induced a significantly higher +dP/dt in induced compared with noninduced hearts. In contrast to the 50% mortality observed in mice constitutively overexpressing the S68E mutant, induced mice had similar survival as wild-type and noninduced mice. After ischemiareperfusion, despite similar areas at risk and left ventricular infarct sizes, induced mice had significantly higher +dP/dt and dP/dt and lower perioperative mortality compared with noninduced mice. We propose that phosphorylated PLM may be a novel therapeutic target in ischemic heart disease. [ABSTRACT FROM AUTHOR]

Background: Wall stress is a useful concept to understand the progression of ventricular remodeling. We measured cumulative LV wall stress throughout the cardiac cycle over unit time and tested whether this "integrated wall stress (IWS)" would provide a reliable marker of total ventricular workload. Methods and results: We applied IWS to mice after experimental myocardial infarction (MI) and sham-operated mice, both at rest and under dobutamine stimulation. Small infarcts were created so as not to cause subsequent overt hemodynamic decompensation. IWS was calculated over one minute through simultaneous measurement of LV internal diameter and wall thickness by echocardiography and LV pressure by LV catheterization. At rest, the MI group showed concentric LV hypertrophy pattern with preserved LV cavity size, LV systolic function, and IWS comparable with the sham group. Dobutamine stimulation induced a dose-dependent increase in IWS in MI mice, but not in sham mice; MI mice mainly increased heart rate, whereas sham mice increased LV systolic and diastolic function. IWS showed good correlation with a product of peak-systolic wall stress and heart rate. We postulate that this increase in IWS in post-MI mice represents limited myocardial contractile reserve. Conclusion: We hereby propose that IWS provides a useful estimate of total ventricular workload in the mouse model and that increased IWS indicates limited LV myocardial contractile reserve. [ABSTRACT FROM AUTHOR]

Akt2 protein kinase has been shown to promote cell migration and actin polymerization in several cell types, including macrophages. Because migrating macrophages constitute an important inflammatory response after myocardial ischemia, we determined cardiac macrophage expression after ischemia-reperfusion (I/R) injury and cryo-injury in mice lacking Akt2 (Akt2-KO). At 7 days post-I/R, Akt2-KO cardiac tissues showed an increase in immunohistochemical staining for macrophage markers (Galectin 3 and F4/80) compared with wild-type (WT) mice, indicating macrophage density was increased in the injured Akt2-KO myocardium. This change was time dependent because macrophage density was similar between WT and Akt2-KO myocardium at 3 days post-I/R, but by 7 and 14 days post-I/R, macrophage density was significantly increased in Akt2-KO myocardium. Concomitantly, infarct size was larger and cardiac function was reduced in Akt2-KO mice subjected to I/R. However, when cryo-infarction produced similar infarct sizes in the anterior wall in both WT and Akt2-KO mice, macrophage density remained higher in Akt2-KO mouse myocardium, suggesting Akt2 regulates myocardial macrophage density independent of infarct size. Consistently, bone marrow from Akt2-KO mice enhanced myocardial macrophage density in both C57/B6 WT and Akt2-KO recipient mice. Finally, reciprocal ex-vivo coculturing of macrophages and cardiac myocytes showed that activated Akt2-KO peritoneal macrophages had reduced mobility and adhesion when compared with WT littermate controls. Thus, although Akt-2 KO mice did not affect the initial inflammation response after injury and Akt2 deficiency has been shown to impair cell migration or motility in macrophages, our data suggested a novel mechanism in which increasing retention of Akt2-KO macrophages resulted in increasing cardiac Akt2-KO macrophage density in the myocardial space. [ABSTRACT FROM AUTHOR]

AbstractDiabetes exacerbates ischemic heart disease morbidity and mortality viaincompletely understood mechanisms. Although adiponectin (APN) reduces myocardial ischemia/reperfusion (MI/R) injury in nondiabetic animals, whether APN's cardioprotective actions are altered in diabetes, a pathologic condition with endogenously reduced APN, has never been investigated. High-fat diet (HD)–induced diabetic mice and normal diet (ND) controls were subjected to MI viacoronary artery ligation, and given vehicle or APN globular domain (gAPN, 2 μg/g) 10 min before reperfusion. Compared to ND mice (where gAPN exerted pronounced cardioprotection), HD mice manifested greater MI/R injury, and a tripled gAPN dose was requisite to achieve cardioprotective extent seen in ND mice (i.e., infarct size, apoptosis, and cardiac function). APN reduces MI/R injury viaAMP-activated protein kinase (AMPK)–dependent metabolic regulation and AMPK-independent antioxidative/antinitrative pathways. Compared to ND, HD mice manifested significantly blunted gAPN-induced AMPK activation, basally and after MI/R (p< 0.05). Although both low- and high-dose gAPN equally attenuated MI/R-induced oxidative stress (i.e., NADPH oxidase expression and superoxide production) and nitrative stress (i.e., inducible nitric oxide synthase expression, nitric oxide production, and peroxynitrite formation) in ND mice, only high-dose gAPN efficaciously did so in HD mice. We demonstrate for the first time that HD-induced diabetes diminished both AMPK-dependent and AMPK-independent APN cardioprotection, suggesting an unreported diabetic heart APN resistance. Antioxid. Redox Signal.15, 1779–1788. [ABSTRACT FROM AUTHOR]

Stem cell therapy is a potentially effective and promising treatment for ischemic heart disease. Resistin, a type of adipokine, has been found to bind to adipose-derived mesenchymal stem cells (ADSCs). However, the effects of resistin on cardiac homing by ADSCs and on ADSC-mediated cardioprotective effects have not been investigated. ADSCs were obtained from enhanced green fluorescent protein transgenic mice. C57BL/6J mice were subjected to myocardial ischemia-reperfusion (I/R) or sham operations. Six hours after the I/R operation, mice were intravenously injected with resistin-treated ADSCs (ADSC-resistin) or vehicle-treated ADSCs (ADSC-vehicle). Cardiac homing by ADSCs and cardiomyocyte apoptosis were investigated 3 days after I/R. Cardiac function, fibrosis, and angiogenesis were evaluated 4 wk after I/R. Cellular and molecular mechanisms were investigated in vitro using cultured ADSCs. Both immunostaining and flow cytometric experiments showed that resistin treatment promoted ADSC myocardial homing 3 days after intravenous injection. Echocardiographic experiments showed that ADSC-resistin, but not ADSC-vehicle, significantly improved left ventricular ejection fraction. ADSC-resistin transplantation significantly mitigated I/R-induced fibrosis and reduced atrial natriuretic peptide/brain natriuretic peptide mRNA expression. In addition, cardiomyocyte apoptosis was reduced, whereas angiogenesis was increased by ADSC-resistin treatment. At the cellular level, resistin promoted ADSC proliferation and migration but did not affect H2O2-induced apoptosis. Molecular experiments identified the ERK1/ 2-matrix metalloproteinase-9 pathway as a key component mediating the effects of resistin on ADSC proliferation and migration. These results demonstrate that resistin can promote homing of injected ADSCs into damaged heart tissue and stimulate functional recovery, an effect mediated through the ERK1/2 signaling pathway and matrix metalloproteinase-9. [ABSTRACT FROM AUTHOR]

Background: Cell therapy remains the most promising approach against ischemic heart injury. However, the poor survival of engrafted stem cells in the ischemic environment limits their therapeutic efficacy for cardiac repair after myocardial infarction. CTRP9 (C1q/tumor necrosis factor-related protein-9) is a novel prosurvival cardiokine with significantly downregulated expression after myocardial infarction. Here we tested a hypothesis that CTRP9 might be a cardiokine required for a healthy microenvironment promoting implanted stem cell survival and cardioprotection.Methods: Mice were subjected to myocardial infarction and treated with adipose-derived mesenchymal stem cells (ADSCs, intramyocardial transplantation), CTRP9, or their combination. Survival, cardiac remodeling and function, cardiomyocytes apoptosis, and ADSCs engraftment were evaluated. Whether CTRP9 directly regulates ADSCs function was determined in vitro. Discovery-drive approaches followed by cause-effect analysis were used to uncover the molecular mechanisms of CTRP9.Results: Administration of ADSCs alone failed to exert significant cardioprotection. However, administration of ADSCs in addition to CTRP9 further enhanced the cardioprotective effect of CTRP9 (P<0.05 or P<0.01 versus CTRP9 alone), suggesting a synergistic effect. Administration of CTRP9 at a dose recovering physiological CTRP9 levels significantly prolonged ADSCs retention/survival after implantation. Conversely, the number of engrafted ADSCs was significantly reduced in the CTRP9 knockout heart. In vitro study demonstrated that CTRP9 promoted ADSCs proliferation and migration, and it protected ADSCs against hydrogen peroxide-induced cellular death. CTRP9 enhances ADSCs proliferation/migration by extracellular regulated protein kinases (ERK)1/2-matrix metallopeptidase 9 signaling and promotes antiapoptotic/cell survival via ERK-nuclear factor erythroid-derived 2-like 2/antioxidative protein expression. N-cadherin was identified as a novel CTRP9 receptor mediating ADSCs signaling. Blockade of either N-cadherin or ERK1/2 completely abolished the previously noted CTRP9 effects. Although CTRP9 failed to promote ADSCs cardiogenic differentiation, CTRP9 promotes superoxide dismutase 3 expression and secretion from ADSCs, protecting cardiomyocytes against oxidative stress-induced cell death.Conclusions: We provide the first evidence that CTRP9 promotes ADSCs proliferation/survival, stimulates ADSCs migration, and attenuates cardiomyocyte cell death by previously unrecognized signaling mechanisms. These include binding with N-cadherin, activation of ERK-matrix metallopeptidase 9 and ERK-nuclear factor erythroid-derived 2-like 2 signaling, and upregulation/secretion of antioxidative proteins. These results suggest that CTRP9 is a cardiokine critical in maintaining a healthy microenvironment facilitating stem cell engraftment in infarcted myocardial tissue, thereby enhancing stem cell therapeutic efficacy. [ABSTRACT FROM AUTHOR]

Cardiac metabolic remodeling is a central event during heart failure (HF) development following myocardial infarction (MI). It is well known that myocardial glucose and fatty acid dysmetabolism contribute to post-MI cardiac dysfunction and remodeling. However, the role of amino acid metabolism in post-MI HF remains elusive. Branched chain amino acids (BCAAs) are an important group of essential amino acids and function as crucial nutrient signaling in mammalian animals. The present study aimed to determine the role of cardiac BCAA metabolism in post-MI HF progression. Utilizing coronary artery ligation-induced murine MI models, we found that myocardial BCAA catabolism was significantly impaired in response to permanent MI, therefore leading to an obvious elevation of myocardial BCAA abundance. In MI-operated mice, oral BCAA administration further increased cardiac BCAA levels, activated the mammalian target of rapamycin (mTOR) signaling, and exacerbated cardiac dysfunction and remodeling. These data demonstrate that BCAAs act as a direct contributor to post-MI cardiac pathologies. Furthermore, these BCAA-mediated deleterious effects were improved by rapamycin cotreatment, revealing an indispensable role of mTOR in BCAA-mediated adverse effects on cardiac function/structure post-MI. Of note, pharmacological inhibition of branched chain ketoacid dehydrogenase kinase (BDK), a negative regulator of myocardial BCAA catabolism, significantly improved cardiac BCAA catabolic disorders, reduced myocardial BCAA levels, and ameliorated post-MI cardiac dysfunction and remodeling. In conclusion, our data provide the evidence that impaired cardiac BCAA catabolism directly contributes to post-MI cardiac dysfunction and remodeling. Moreover, improving cardiac BCAA catabolic defects may be a promising therapeutic strategy against post-MI HF. [ABSTRACT FROM AUTHOR]

Sphingosine 1-phosphate (S1P) mediates multiple pathophysiological effects in the cardiovascular system. However, the role of S1P signaling in pathological cardiac remodeling following myocardial infarction (MI) remains controversial. In this study, we found that cardiac S1P greatly increased post-MI, accompanied with a significant upregulation of cardiac sphingosine kinase-1 (SphK1) and S1P receptor 1 (S1PR1) expression. In MI-operated mice, inhibition of S1P production by using PF543 (the SphK1 inhibitor) ameliorated cardiac remodeling and dysfunction. Conversely, interruption of S1P degradation by inhibiting S1P lyase augmented cardiac S1P accumulation and exacerbated cardiac remodeling and dysfunction. In the cardiomyocyte, S1P directly activated proinflammatory responses via a S1PR1-dependent manner. Furthermore, activation of SphK1/S1P/ S1PR1 signaling attributed to β1-adrenergic receptor stimulationinduced proinflammatory responses in the cardiomyocyte. Administration of FTY720, a functional S1PR1 antagonist, obviously blocked cardiac SphK1/S1P/S1PR1 signaling, ameliorated chronic cardiac inflammation, and then improved cardiac remodeling and dysfunction in vivo post-MI. In conclusion, our results demonstrate that cardiac SphK1/S1P/S1PR1 signaling plays an important role in the regulation of proinflammatory responses in the cardiomyocyte and targeting cardiac S1P signaling is a novel therapeutic strategy to improve post-MI cardiac remodeling and dysfunction. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-α (TNF-α) antagonism alleviates myocardial ischemia- reperfusion (MI/R) injury. However, the mechanisms by which the downstream mediators of TNF-α change after acute antagonism during MI/R remain unclear. Adiponectin (APN) exerts anti-ischemic effects, but it is downregulated during MI/R. This study was conducted to investigate whether TNF-α is responsible for the decrease of APN, and whether antagonizing TNF-α affects MI/R injury by increasing APN. Male adult wild-type (WT), APN knockout (APN KO) mice, and those with cardiac knockdowns of APN receptors via siRNA injection were subjected to 30 min of MI followed by reperfusion. The TNF-α antagonist etanercept or globular domain of APN (gAD) was injected 10 min before reperfusion. Etanercept ameliorated MI/R injury in WT mice as evidenced by improved cardiac function, and reduced infarct size and cardiomyocyte apoptosis. APN concentrations were augmented in response to etanercept, followed by an increase in AMP-activated protein kinase phosphorylation. Etanercept still increased cardiac function and reduced infarct size and apoptosis in both APN KO and APN receptors knockdown mice. However, its potential was significantly weakened in these mice compared with the WT mice. TNF-α is responsible for the decrease in APN during MI/R. The cardioprotective effects of TNF-α neutralization are partially due to the upregulation of APN. The results provide more insight into the TNF-α-mediated signaling effects during MI/R and support the need for clinical trials to validate the efficacy of acute TNF-α antagonism in the treatment of MI/R injury. [ABSTRACT FROM AUTHOR]

Background Bone mesenchymal stem cell (BMSC) therapy has modest success in ischemic heart disease but has been limited by poor survival in diseased microenvironments. Cellular repressor of E1A-stimulated genes (CREG) can prevent BMSCs from apoptosis in vitro; however, the effects of CREG-modified BMSCs on ischemic heart disease and the related mechanism remain undefined. Therefore, we designed to study the cardioprotective effects of CREG overexpression in BMSCs (CREGBMSCs) after transplantation into infarcted heart of rats. Methods In vivo studies, 50 μl PBS or 1.5 × 106NormBMSCs, GFPBMSCs or CREGBMSCs were implanted intramyocardially in myocardial infarction rat models. 3 or 14 days later, cardiac function, fibrosis, apoptosis and angiogenesis were analyzed by echocardiography, masson, western blot and immunofluorescence staining, respectively. ELISA, western blot and matrigel assay were used in vitro to detect vascular endothelial growth factor (VEGF) secretion, signaling molecule expression, and angiogenic tube formation. Results In vivo, prolonged cardiac function (14d LVEF: 50.87 ± 0.94%; LVFS: 23.41 ± 1.12%), decreased fibrosis (14d Fibrotic area: 27.37 ± 1.03%) and apoptosis and increased angiogenesis were observed in CREGBMSCs, compared with other groups. In vivo and in vitro, VEGF secretion from CREGBMSCs was markedly enhanced. In vitro, angiogenic tube formation in CREGBMSC supernatants significantly increased. Moreover, CREG activated hypoxia-inducible factor-1α (HIF-1α), but not HIF-1β. Knockdown of HIF-1α with siRNA decreased VEGF secretion and angiogenic tube formation. Notably, CREG did not influence HIF-1α mRNA synthesis but inhibited the expression of Von Hippel-Lindau (VHL), a key protein that regulates HIF-1α degradation. Conclusions The CREGBMSC transplantation, directly or indirectly, may promote VEGF's anti-apoptosis and angiogenesis via the inhibition of VHL-mediated HIF-1α degradation, consequently protecting against myocardial infarction. [ABSTRACT FROM AUTHOR]

The adipocyte-secreted hormone adiponectin (APN) exerts protective effects on the heart under stress conditions. Recent studies have demonstrated that APN induces a marked Ca2+ influx in skeletal muscle. However, whether APN modulates [Ca2+]i activity, especially [Ca2+]i transients in cardiomyocytes, is still unknown. This study was designed to determine whether APN modulates [Ca2+]i transients in cardiomyocytes. Adult male wild-type (WT) and APN knockout (APN KO) mice were subjected to myocardial ischemia/reperfusion (I/R, 30min/30min) injury. CaMKII-PLB phosphorylation and SR Ca2+-ATPase (SERCA2) activity were downregulated in I/R hearts of WT mice and further decreased in those of APN KO mice. Both the globular domain of APN and full-length APN significantly reversed the decrease in CaMKII-PLB phosphorylation and SERCA2 activity in WT and APN KO mice. Interestingly, compared with WT littermates, single myocytes isolated from APN KO mice had remarkably decreased [Ca2+]i transients, cell shortening, and a prolonged Ca2+ decay rate. Further examination revealed that APN enhances SERCA2 activity via CaMKII-PLB signaling. In in vivo and in vitro experiments, both APN receptor 1/2 and S1P were necessary for the APN-stimulated CaMKII-PLB-SERCA2 activation. In addition, S1P activated CaMKII-PLB signaling in neonatal cardiomyocytes in a dose dependent manner and improved [Ca2+]i transients in APN KO myocytes via the S1P receptor (S1PR1/3). Further in vivo experiments revealed that pharmacological inhibition of S1PR1/3 and SERCA2 siRNA suppressed APN-mediated cardioprotection during I/R. These data demonstrate that S1P is a novel regulator of SERCA2 that activates CaMKII-PLB signaling and mediates APN-induced cardioprotection. [ABSTRACT FROM AUTHOR]

Background Injury due to myocardial infarction (MI) is largely irreversible. Once an infarct has occurred, the clinical goal becomes limiting remodeling, preserving left ventricular function, and preventing heart failure. Although traditional approaches (e.g., β-blockers) partially preserve left ventricular function, novel strategies are needed to limit ventricular remodeling post-MI. Objectives The aim of this study was to determine the role of glycogen synthase kinase-3α (GSK-3α) in post-MI remodeling. Methods Mice with cardiomyocyte-specific conditional deletion of Gsk3α and littermate controls underwent sham or MI surgery. Heart function was assessed using serial M-mode echocardiography. Results Gsk3α deletion in the heart markedly limits remodeling and preserves left ventricular function post-MI. This is due at least in part to dramatic thinning and expansion of the scar in the control hearts, which was less in the heart of knockout (KO) mice. In contrast, the border zone in the KO mice demonstrated a much thicker scar, and there were more viable cardiomyocytes within the scar/border zone. This was associated with less apoptosis and more proliferation of cardiomyocytes in the KO mice. Mechanistically, reduced apoptosis was due, at least in part, to a marked decrease in the Bax/Bcl-2 ratio, and increased cardiomyocyte proliferation was mediated through cyclin E1 and E2F-1 in the hearts of the KO mice. Conclusions Taken together, these findings show that reducing GSK-3α expression in cardiomyocytes limits ventricular remodeling and preserves cardiac function post-MI. Specifically targeting GSK-3α could be a novel strategy to limit adverse remodeling and heart failure. [ABSTRACT FROM AUTHOR]

The second member of the transient receptor potential-melastatin channel family (TRPM2) is expressed in the heart and vasculature. TRPM2 channels were expressed in the sarcolemma and transverse tubules of adult left ventricular (LV) myocytes. Cardiac TRPM2 channels were functional since activation with H2O2 resulted in Ca2+ influx that was dependent on extracellular Ca2+, was significantly higher in wild-type (WT) myocytes compared with TRPM2 knockout (KO) myocytes, and inhibited by clotrimazole in WT myocytes. At rest, there were no differences in LV mass, heart rate, fractional shortening, and +dP/dt between WT and KO hearts. At 2-3 days after ischemia-reperfusion (I/R), despite similar areas at risk and infarct sizes, KO hearts had lower fractional shortening and +dP/dt compared with WT hearts. Compared with WT I/R myocytes, expression of the Na+/Ca2+ exchanger (NCX1) and NCX1 current were increased, expression of the _1-subunit of Na+-K+-ATPase and Na+ pump current were decreased, and action potential duration was prolonged in KO I/R myocytes. Post-I/R, intracellular Ca2+ concentration transients and contraction amplitudes were equally depressed in WT and KO myocytes. After 2 h of hypoxia followed by 30 min of reoxygenation, levels of ROS were significantly higher in KO compared with WT LV myocytes. Compared with WT I/R hearts, oxygen radical scavenging enzymes (SODs) and their upstream regulators (forkhead box transcription factors and hypoxia-inducible factor) were lower, whereas NADPH oxidase was higher, in KO I/R hearts. We conclude that TRPM2 channels protected hearts from I/R injury by decreasing generation and enhancing scavenging of ROS, thereby reducing I/Rinduced oxidative stress. [ABSTRACT FROM AUTHOR]

: Aims Hypercholesterolaemia and myeloperoxidase (MPO) overexpression are two well-recognized risk factors for ischaemic heart disease. Peroxisome proliferator-activated receptor-γ (PPARγ) agonists have recently been shown to reduce ischaemic heart injury in hypercholesterolaemic animals. However, whether PPARγ agonists may exert their cardioprotective effects by eliminating those risk factors that increase ischaemic injury remains unknown. : Methods and results Male New Zealand rabbits were fed with a normal or a high-cholesterol diet for 8 weeks, treated with vehicle or rosiglitazone (RSG, 3 mg/kg/day for the last 5 weeks) and subjected to myocardial ischaemia/reperfusion (1 h/4 h). MPO expression, activity, and distribution, cardiac caspase-3 activity, and myocardial infarct size were determined. Diet-induced hypercholesterolaemia caused a significant increase in neutrophil MPO expression/activity (7.2-/5.4-fold). Hypercholesterolaemia also tripled MPO activity in ischaemic/reperfused hearts when compared with rabbits fed with a normal diet. Surprisingly, MPO immunostaining was not only observed in perivascular and extracellular spaces in ischaemic/reperfused hearts, but also in cardiomyocytes. This intracardiomyocyte MPO staining was further intensified by hypercholesterolaemia. There is a strong positive correlation between cardiac MPO activity and caspase-3 activity, and treatment with an MPO inhibitor significantly reduced post-ischaemic caspase-3 activation. Treatment with RSG markedly inhibited hypercholesterolaemia-induced leucocyte MPO overexpression and activation, reduced MPO activity in ischaemic/reperfused hearts, decreased caspase-3 activity, and reduced myocardial infarct size (P < 0.01). : Conclusion Our results demonstrated that hypercholesterolaemia and MPO overexpression are causally related and that PPARγ agonists may have great therapeutic value in ischaemic heart disease patients with multiple complications such as hypercholesterolaemia and diabetes. [ABSTRACT FROM AUTHOR]

The receptor for advanced glycation end products (RAGE) and thioredoxin (Trx) play opposing roles in diabetic myocardial ischemia-reperfusion (MI/R) injury. We recently demonstrated nitrative modification of Trx leads to its inactivation and loss of cardioprotection. The present study is to determine the relationship between augmented RAGE expression and diminished Trx activity pertaining to exacerbated MI/R injury in the diabetic heart. The diabetic state was induced in mice by multiple intraperitoneal low-dose streptozotocin injections. RAGE small-interfering RNA (siRNA) or soluble RAGE (sRAGE, a RAGE decoy) was via intramyocardial and intraperitoneal injection before MI/R, respectively. Mice were subjected to 30 min of myocardial infarction followed by 3 or 24 h of reperfusion. At 10 min before reperfusion, diabetic mice were randomized to receive EUK134 (peroxynitrite scavenger), recombinant hTrx-1, nitrated Trx-1, apocynin (a NADPH oxidase inhibitor), or 1400W [an inducible nitric oxide synthase (iNOS) inhibitor] administration. The diabetic heart manifested increased RAGE expression and Nε-(carboxymethyl) lysine (CML, major advanced glycation end product subtype) content, reduced Trx-1 activity, and increased Trx nitration after MI/R. RAGE siRNA or administration of sRAGE in diabetic mice decreased MI/R-induced iNOS and gp91phox expression, reduced Trx nitration, preserved Trx activity, and decreased infarct size. Apocynin or 1400W significantly decreased nitrotyrosine production and restored Trx activity. Conversely, administration of either EUK134 or reduced hTrx, but not nitrated hTrx, attenuated MI/R-induced superoxide production, RAGE expression, and CML content and decreased cardiomyocyte apoptosis in diabetic mice. Collectively, we demonstrate that RAGE modulates the MI/R injury in a Trx nitrative inactivation fashion. Conversely, nitrative modification of Trx blocked its inhibitory effect upon RAGE expression in the diabetic heart. This is the first direct evidence demonstrating the alternative cross talk between RAGE overexpression and nitrative Trx inactivation, suggesting that interventions interfering with their interaction may be novel means of mitigating diabetic MI/R injury. [ABSTRACT FROM AUTHOR]

It is generally accepted that the intercalated disc (ICD) required for mechano-electrical coupling in the heart consists of three distinct junctional complexes: adherens junctions, desmosomes and gap junctions. However, recent morphological and molecular data indicate a mixing of adherens junctional and desmosomal components, resulting in a 'hybrid adhering junction' or 'area composita'. The a-catenin family member αT-catenin, part of the N-cadherin-catenin adhesion complex in the heart, is the only α-catenin that interacts with the desmosomal protein plakophilin-2 (PKP2). Thus, it has been postulated that αT-catenin might serve as a molecular integrator of the two adhesion complexes in the area composita. To investigate the role of αT-catenin in the heart, gene targeting technology was used to delete the Ctnna3 gene, encoding αT-catenin, in the mouse. The αT-catenin-null mice are viable and fertile; however, the animals exhibit progressive cardiomyopathy. Adherens junctional and desmosomal proteins were unaffected by loss of αT-catenin, with the exception of the desmosomal protein PKP2. Immunogold labeling at the ICD demonstrated in the αT-catenin-null heart a preferential reduction of PKP2 at the area composita compared with the desmosome. Furthermore, gap junction protein Cx43 was reduced at the ICD, including its colocalization with N-cadherin. Gap junction remodeling in αT-catenin-knockout hearts was associated with an increased incidence of ventricular arrhythmias after acute ischemia. This novel animal model demonstrates for the first time how perturbation in αT-catenin can affect both PKP2 and Cx43 and thereby highlights the importance of understanding the crosstalk between the junctional proteins of the ICD and its implications for arrhythmogenic cardiomyopathy. [ABSTRACT FROM AUTHOR]

The present study determined the dynamic change of adiponectin (APN, a cardioprotective adipokine), its receptor expression, and their impact upon myocardial ischemia/reperfusion (MI/R) injury during type 1 diabetes mellitus (T1DM) progression, and involved underlying mechanisms. Diabetic state was induced in mice via multiple intraperitoneal injections of low-dose streptozotocin. The dynamic change of plasma APN concentration and cardiac APN receptor-1 and -2 (AdipoR1/2) expression were assessed immediately after diabetes onset (0 wk) and 1, 3, 5, and 7 wk thereafter. Indicators of MI/R injury (infarct size, apoptosis, and LDH release) were determined at 0, 1, and 7 wk of DM duration. The effect of APN on MI/R injury was determined in mice subjected to different diabetic durations. Plasma APN levels (total and HMW form) increased, whereas cardiac AdipoR1 expression decreased early after T1DM onset. With T1DM progression, APN levels were reduced and cardiac AdipoR1 expression increased. MI/R injury was exacerbated with T1DM progression in a time-dependent manner. Administration of globular APN (gAD) failed to attenuate MI/R injury in 1-wk T1DM mice, while an AMP-activated protein kinase (AMPK) activator (AICAR) reduced MI/R injury. However, administration of gAD (and AICAR) reduced infarct size and cardiomyocyte apoptosis in 7-wk T1DM mice. In conclusion, our results demonstrate a dynamic dysfunction of APN/AdipoR1 during T1DM progression. Reduced cardiac AdipoR1 expression and APN concentration may be responsible for increased I/R injury susceptibility at early and late T1DM stages, respectively. Interventions bolstering AdipoR1 expression during early T1DM stages and APN supplementation during advanced T1DM stages may potentially reduce the myocardial ischemic injury in diabetic patients. [ABSTRACT FROM AUTHOR]

The article presents a research that studied the molecular mechanisms for ischemic myocardial injury after nonlethal trauma, with focus on the effect of tumor necrosis factor its downstream effector adinopectin. The response of mice that were given nonlethal mechanical trauma using the Noble-Collip drum followed by myocardial injury were examined. It was found that tumor necrosis factor-induced downregulation of adiponectin is connected to exacerbated posttraumatic ischemic myocardial injury.

Phospholemman (PLM), a member of the FXYD family of regulators of ion transport, is a major sarcolemmal substrate for protein kinases A and C in cardiac and skeletal muscle. In the heart, PLM co-localizes and co-immunoprecipitates with Na+-K+-ATPase, Na+/Ca2+ exchanger, and L-type Ca2+ channel. Functionally, when phosphorylated at serine68, PLM stimulates Na+-K+-ATPase but inhibits Na+/Ca2+ exchanger in cardiac myocytes. In heterologous expression systems, PLM modulates the gating of cardiac L-type Ca2+ channel. Therefore, PLM occupies a key modulatory role in intracellular Na+ and Ca2+ homeostasis and is intimately involved in regulation of excitation–contraction (EC) coupling. Genetic ablation of PLM results in a slight increase in baseline cardiac contractility and prolongation of action potential duration. When hearts are subjected to catecholamine stress, PLM minimizes the risks of arrhythmogenesis by reducing Na+ overload and simultaneously preserves inotropy by inhibiting Na+/Ca2+ exchanger. In heart failure, both expression and phosphorylation state of PLM are altered and may partly account for abnormalities in EC coupling. The unique role of PLM in regulation of Na+-K+-ATPase, Na+/Ca2+ exchanger, and potentially L-type Ca2+ channel in the heart, together with the changes in its expression and phosphorylation in heart failure, make PLM a rational and novel target for development of drugs in our armamentarium against heart failure. Clin Trans Sci 2010; Volume 3: 189–196 [ABSTRACT FROM AUTHOR]

We have produced mice in which expression of the rat cardiac Na+/Ca2+ exchanger (NCX1) transgene was switched on when doxycycline was removed from the feed at 5 wk. At 8 to 10 wk, NCX1 expression in induced (md) mouse hearts was 2.5-fold higher but protein levels of sarco(endo)plasmic reticulum Ca2+ -ATPase, α1- and α2-subunits of Na+-K+-ATPase, phospholamban, ryanodine receptor, calsequestrin, and unphosphorylated and phosphorylated phospholemman were unchanged compared with wild-type (WT) or noninduced (non-md) hearts. There was no cellular hypertrophy since WT, non-md, and md myocytes had similar whole cell membrane capacitance. In md myocytes, NCX 1 current amplitude was ∼42% higher, L-type Ca2+ current amplitude was unchanged, and action potential duration was prolonged compared with WT or non-Ind myocytes. Contraction and intracellular Ca2+ concentration ([Ca2+]i) transient amplitudes in md myocytes were lower at 0.6, not different at 1.8, and higher at 5.0 mM extracellular Ca2+ concentration ([Ca2+]o) compared with WT or non-md myocytes. Despite similar Ca2+ current amplitude and sarcoplasmic reticulum (SR) Ca2+ uptake, SR Ca2+ content at 5.0 mM [Ca2+]o was significantly higher in md compared with non-md myocytes, indicating that NCX1 directly contributed to SR Ca2+ loading. Echocardiography demonstrated that heart rate, left ventricular mass, ejection fraction, stroke volume, and cardiac output were similar among the three groups of animals. In vivo close-chest catheterization demonstrated similar contractility and relaxation among the three groups of mice, both at baseline and after stimulation with isoproterenol. We conclude that induced expression of NCX1 transgene resulted in altered [Ca2+]i homeostasis, myocyte contractility, and action potential morphology. In addition, heart failure did not occur 3 to 5 wk after NCX1 transgene was induced to be expressed at levels found in diseased hearts. [ABSTRACT FROM AUTHOR]

The aim of this study was to explore the effect of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2)— paracrine factors implicated in both cardiac embryogenesis and cardiac repair following myocardial infarction (MI)—on murine bone marrow stem cell (mBMSC) differentiation in an ex vivo cardiac microenvironment. For this purpose, green fluorescent protein (GFP) expressing hematopoietic lineage negative (lin-) c-kit ligand (c-kit) and stem cell antigen-1 (Sca-1) positive (GFP-lin-/c-kit+/sca+) mBMSC were co-cultured with neonatal rat ventricular cardiomyocytes (NVCMs). GFP+ mBMSC significantly induced the expression of BMP-2 and FGF-2 in NVCMs, and approximately 4% GFP+ mBMSCs could be recovered from the co-culture at day 10. The addition of BMP-2 in concert with FGF-2 significantly enhanced the amount of integrated GFP+ mBMSCs by 5-fold (∼20%), whereas the addition of anti-BMP-2 and/or anti-FGF-2 antibodies completely abolished this effect. An analysis of calcium cycling revealed robust calcium transients in GFP+ mBMSCs treated with BMP-2/FGF-2 compared to untreated co-cultures. BMP-2 and FGF-2 addition led to a significant induction of early (NK2 transcription factor related, locus 5; Nkx2.5, GATA binding protein 4; GATA-4) and late (myosin light chain kinase [MLC-2v], connexin 43 [Cx43]) cardiac marker mRNA expression in mBMSCs following co-culture. In addition, re-cultured fluorescence-activated cell sorting (FACS)-purified BMP-2/FGF-2-treated mBMSCs revealed robust calcium transients in response to electrical field stimulation which were inhibited by the L-type calcium channel (LTCC) inhibitor, nifedipine, and displayed caffeine-sensitive intracellular calcium stores. In summary, our results show that mBMSCs can adopt a functional cardiac phenotype through treatment with factors essential to embryonic cardiogenesis that are induced after cardiac ischemia. This study provides the first evidence that mBMSCs with long-term self-renewal potential possess the capability to serve as a functional cardiomyocyte precursor through the appropriate paracrine input and cross-talk within an appropriate cardiac microenvironment. [ABSTRACT FROM AUTHOR]

1: Considerable evidence indicates that calcium plays a critical role in apoptosis. We have previously shown that benidipine, a vasodilatory calcium channel blocker, attenuates postischemia myocardial apoptosis. The present study was designed to determine the mechanisms by which benidipine exerts its antiapoptotic effect. 2: Adult male rats were subjected to 30?min of ischemia followed by 3?h of reperfusion. Rats were randomized to receive either vehicle or benidipine (10?µg?kg-1, i.v.) 10?min before reperfusion. 3: Compared with rats receiving vehicle, those rats treated with benidipine had reduced postischemic myocardial apoptosis as evidenced by decreased TUNEL-positive staining (8.4±1.2 vs 15.3±1.3%, P<0.01) and caspase-3 activity (1.94±0.25 vs 3.43±0.29, P<0.01). 4: Benidipine treatment significantly reduced mitochondrial cytochrome c release and caspase-9 activation, but had no effect on caspase-8 activation, suggesting that benidipine exerts its antiapoptotic effect by inhibiting the mitochondrial-mediated, but not death receptor-mediated, apoptotic pathway. 5: Benidipine treatment not only increased the maximal activity of ERK1/2 at 10?min after reperfusion, but also prolonged the duration of ERK1/2 activation. Benidipine treatment had no significant effect on other apoptotic regulating molecules, such as p38 MAPK. 6: Taken together, our present study demonstrated for the first time the differential regulation of a calcium channel blocker. Benidipine tilted the balance between ERK1/2 and p38 MAPK toward an antiapoptotic state, decreased mitochondrial cytochrome c release, reduced caspase-9 activation, and attenuated subsequent caspase-3 activation and postischemic myocardial apoptosis.British Journal of Pharmacology (2004) 142, 627-634. doi:10.1038/sj.bjp.0705847 [ABSTRACT FROM AUTHOR]