Cardiomyocyte-GSK-3? promotes mPTP opening and heart failure in mice with chronic pressure overload.
ABSTRACT: Chronic pressure-overload (PO)- induced cardiomyopathy is one of the leading causes of left ventricular (LV) remodeling and heart failure. The role of the ? isoform of glycogen synthase kinase-3 (GSK-3?) in PO-induced cardiac remodeling is unclear and its downstream molecular targets are largely unknown. To investigate the potential roles of GSK-3?, cardiomyocyte-specific GSK-3? conditional knockout (cKO) and control mice underwent trans-aortic constriction (TAC) or sham surgeries. Cardiac function in the cKOs and littermate controls declined equally up to 2?weeks of TAC. At 4?week, cKO animals retained concentric LV remodeling and showed significantly less decline in contractile function both at systole and diastole, vs. controls which remained same until the end of the study (6?wk). Histological analysis confirmed preservation of LV chamber and protection against TAC-induced cellular hypertrophy in the cKO. Consistent with attenuated hypertrophy, significantly lower level of cardiomyocyte apoptosis was observed in the cKO. Mechanistically, GSK-3? was found to regulate mitochondrial permeability transition pore (mPTP) opening and GSK-3?-deficient mitochondria showed delayed mPTP opening in response to Ca2+ overload. Consistently, overexpression of GSK-3? in cardiomyocytes resulted in elevated Bax expression, increased apoptosis, as well as a reduction of maximum respiration capacity and cell viability. Taken together, we show for the first time that GSK-3? regulates mPTP opening under pathological conditions, likely through Bax overexpression. Genetic ablation of cardiomyocyte GSK-3? protects against chronic PO-induced cardiomyopathy and adverse LV remodeling, and preserves contractile function. Selective inhibition of GSK-3? using isoform-specific inhibitors could be a viable therapeutic strategy to limit PO-induced heart failure.
Project description:Pressure overload (PO) cardiac hypertrophy and heart failure are associated with generalized insulin resistance and hyperinsulinemia, which may exacerbate left ventricular (LV) remodeling. While PO activates insulin receptor tyrosine kinase activity that is transduced by insulin receptor substrate 1 (IRS1), the present study tested the hypothesis that IRS1 and IRS2 have divergent effects on PO-induced LV remodeling. We therefore subjected mice with cardiomyocyte-restricted deficiency of IRS1 (CIRS1KO) or IRS2 (CIRS2KO) to PO induced by transverse aortic constriction (TAC). In WT mice, TAC-induced LV hypertrophy was associated with hyperactivation of IRS1 and Akt1, but not IRS2 and Akt2. CIRS1KO hearts were resistant to cardiac hypertrophy and heart failure in concert with attenuated Akt1 activation. In contrast, CIRS2KO hearts following TAC developed more severe LV dysfunction than WT controls, and this was prevented by haploinsufficiency of Akt1. Failing human hearts exhibited isoform-specific IRS1 and Akt1 activation, while IRS2 and Akt2 activation were unchanged. Kinomic profiling identified IRS1 as a potential regulator of cardioprotective protein kinase G-mediated signaling. In addition, gene expression profiling revealed that IRS1 signaling may promote a proinflammatory response following PO. Together, these data identify IRS1 and Akt1 as critical signaling nodes that mediate LV remodeling in both mice and humans.
Project description:Numerous studies have proposed that glycogen synthase kinase (GSK)-3beta is a central regulator of the hypertrophic response of cardiomyocytes. However, all of this work has relied on overexpression of GSK-3beta, expression of constitutively active mutants, or small molecule inhibitors with documented off-target effects. Genetic loss of function approaches have not been used in the adult mouse because germ-line deletion of GSK-3beta is embryonic-lethal.This study was designed to define the role played by GSK-3beta in pressure overload (PO)-induced hypertrophy and remodeling following myocardial infarction (MI).We used a mouse model that allows inducible, cardiomyocyte-specific deletion of GSK-3beta in the adult knockout. Surprisingly, we find that knockout mice exposed to PO induced by thoracic aortic constriction exhibit a normal hypertrophic response. Thus, in contrast to virtually all prior published studies, GSK-3beta appears to play at most a minor role in the hypertrophic response to PO stress. However, GSK-3beta does regulate post-MI remodeling because the GSK-3beta knockouts had less left ventricular dilatation and better-preserved left ventricular function at up to 8 weeks post-MI despite demonstrating significantly more hypertrophy in the remote myocardium. Deletion of GSK-3beta also led to increased cardiomyocyte proliferation following PO and MI.Deletion of GSK-3beta protects against post-MI remodeling and promotes stress-induced cardiomyocyte proliferation in the adult heart. These studies suggest that inhibition of GSK-3beta could be a strategy to both prevent remodeling and to promote cardiac regeneration in pathological states.
Project description:Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)-induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase-2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross-clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2-/- ) and wild-type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin-related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N-acetylcysteine (NAC) or PKC-? shRNA treatment on glycogen synthase kinase-3? (GSK-3?) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2-/- mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC-? translocation was lower in ALDH2-/- mice than in WT mice, and PKC-? was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre-treatment under I/R injury. In addition, PKC-? inhibition caused activation of caspase9, caspase3 and Drp1Ser616 in response to I/R stress. Importantly, expression of phosphorylated GSK-3? (inactive form) was lower in ALDH2-/- mice than in WT mice, and both were increased by NAC pre-treatment. I/R-induced mitochondrial translocation of GSK-3? was inhibited by PKC-? shRNA or NAC pre-treatment. In addition, mitochondrial membrane potential (??m ) was reduced in ALDH2-/- mice after I/R, which was partly reversed by the GSK-3? inhibitor (SB216763) or PKC-? shRNA. Collectively, our data provide the evidence that abnormal PKC-?/PKC-? ratio promotes the activation of Drp1 signalling, caspase cascades and GSK-3?-dependent mPTP opening, which results in mitochondrial injury-triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2-/- mice following I/R stress.
Project description:Left ventricular hypertrophy (LVH) is associated with electric remodeling and increased arrhythmia risk, although the underlying mechanisms are poorly understood. In the experiments here, functional voltage-gated (Kv) and inwardly rectifying (Kir) K(+) channel remodeling was examined in a mouse model of pressure overload-induced LVH, produced by transverse aortic constriction (TAC). Action potential durations (APDs) at 90% repolarization in TAC LV myocytes and QT(c) intervals in TAC mice were prolonged. Mean whole-cell membrane capacitance (C(m)) was higher, and I(to,f), I(K,slow), I(ss), and I(K1) densities were lower in TAC, than in sham, LV myocytes. Although the primary determinant of the reduced current densities is the increase in C(m), I(K,slow) amplitudes were decreased and I(ss) amplitudes were increased in TAC LV cells. Further experiments revealed regional differences in the effects of LVH. Cellular hypertrophy and increased I(ss) amplitudes were more pronounced in TAC endocardial LV cells, whereas I(K,slow) amplitudes were selectively reduced in TAC epicardial LV cells. Consistent with the similarities in I(to,f) and I(K1) amplitudes, Kv4.2, Kv4.3, and KChIP2 (I(to,f)), as well as Kir2.1 and Kir2.2 (I(K1)), transcript and protein expression levels were similar in TAC and sham LV. Unexpectedly, expression of I(K,slow) channel subunits Kv1.5 and Kv2.1 was increased in TAC LV. Biochemical experiments also demonstrated that, although total protein was unaltered, cell surface expression of TASK1 was increased in TAC LV. Functional changes in repolarizing K(+) currents with LVH, therefore, result from distinct cellular (cardiomyocyte enlargement) and molecular (alterations in the numbers of functional channels) mechanisms.
Project description:RATIONALE:Glycogen synthase kinase (GSK)-3? upregulates cardiac genes in bone marrow-derived mesenchymal stem cells (MSCs) in vitro. Ex vivo modification of signaling mechanisms in MSCs may improve the efficiency of cardiac cell-based therapy (CBT). OBJECTIVE:To test the effect of GSK-3? on the efficiency of CBT with MSCs after myocardial infarction (MI). METHODS AND RESULTS:MSCs overexpressing either GSK-3? (GSK-3?-MSCs), LacZ (LacZ-MSCs), or saline was injected into the heart after coronary ligation. A significant improvement in the mortality and left ventricular (LV) function was observed at 12 weeks in GSK-3?-MSC-injected mice compared with in LacZ-MSC- or saline-injected mice. MI size and LV remodeling were reduced in GSK-3?-MSC-injected mice compared with in LacZ-MSC- or saline-injected ones. GSK-3? increased survival and increased cardiomyocyte differentiation of MSCs, as evidenced by activation of an Nkx2.5-LacZ reporter and upregulation of troponin T. Injection of GSK-3?-MSCs induced Ki67-positive myocytes and c-Kit-positive cells, suggesting that GSK-3?-MSCs upregulate cardiac progenitor cells. GSK-3?-MSCs also increased capillary density and upregulated paracrine factors, including vascular endothelial growth factor A (Vegfa). Injection of GSK-3?-MSCs in which Vegfa had been knocked down abolished the increase in survival and capillary density. However, the decrease in MI size and LV remodeling and the improvement of LV function were still observed in MI mice injected with GSK-3?-MSCs without Vegfa. CONCLUSIONS:GSK-3? significantly improves the efficiency of CBT with MSCs in the post-MI heart. GSK-3? not only increases survival of MSCs but also induces cardiomyocyte differentiation and angiogenesis through Vegfa-dependent and -independent mechanisms.
Project description:Alteration in endoplasmic reticulum (ER) stress in diabetic hearts and its effect on cytoprotective signaling are unclear. Here, we examine the hypothesis that ER stress in diabetic hearts impairs phospho-glycogen synthase kinase (GSK)-3beta-mediated suppression of mitochondrial permeability transition pore (mPTP) opening, compromising myocardial response to cytoprotective signaling.A rat model of type 2 diabetes (OLETF) and its control (LETO) were treated with tauroursodeoxycholic acid (TUDCA) (100 mg . kg(-1) . day(-1) for 7 days), an ER stress modulator. Infarction was induced by 20-min coronary occlusion and 2-h reperfusion.Levels of ER chaperones (GRP78 and GRP94) in the myocardium and level of nonphoshopho-GSK-3beta in the mitochondria were significantly higher in OLETF than in LETO rats. TUDCA normalized levels of GRP78 and GRP94 and mitochondrial GSK-3beta in OLETF rats. Administration of erythropoietin (EPO) induced phosphorylation of Akt and GSK-3beta and reduced infarct size (% risk area) from 47.4 +/- 5.2% to 23.9 +/- 3.5% in LETO hearts. However, neither phosphorylation of Akt and GSK-3beta nor infarct size limitation was induced by EPO in OLETF rats. The threshold for mPTP opening was significantly lower in mitochondria from EPO-treated OLETF rats than in those from EPO-treated LETO rats. TUDCA restored responses of GSK-3beta, mPTP opening threshold, and infarct size to EPO receptor activation in OLETF rats. There was a significant correlation between mPTP opening threshold and phospho-GSK-3beta-to-total GSK-3beta ratio in the mitochondrial fraction.Disruption of protective signals leading to GSK-3beta phosphorylation and increase in mitochondrial GSK-3beta are dual mechanisms by which increased ER stress inhibits EPO-induced suppression of mPTP opening and cardioprotection in diabetic hearts.
Project description:To investigate whether mitochondria permeability transition pore (mPTP) opening was involved in ginsenoside Rb1 (Gs-Rb1) induced anti-hypoxia effects in neonatal rat cardiomyocytes ex vivo.Cardiomyocytes were randomly divided into 7 groups: control group, hypoxia group (500 micromol/L CoCl(2)), Gs-Rb1 200 micromol/L group (CoCl(2) intervention+Gs-Rb1), wortmannin (PI3K inhibitor) 0.5 micromol/L group, wortmannin+Gs-Rb1 group, adenine 9-beta-D-arabinofuranoside (Ara A, AMPK inhibitor) 500 micromol/L group, and Ara A and Gs-Rb1 group. Apoptosis rate was determined by using flow cytometry. The opening of the transient mPTP was assessed by using co-loading with calcein AM and CoCl(2) in high conductance mode. Expression of GSK-3beta, cytochrome c, caspase-3 and poly (ADP-ribose) polymerase (PARP) was measured by using Western blotting. DeltaGSK-3beta was defined as the ratio of p-Ser9-GSK-3beta to total GSK-3beta.CoCl(2) significantly stimulated mPTP opening and up-regulated the level of DeltaGSK-3beta. There was a statistically significant positive correlation between apoptosis rate and mPTP opening, between apoptosis rate and DeltaGSK-3beta, and between mPTP opening and DeltaGSK-3beta. Gs-Rb1 significantly inhibited mPTP opening induced by hypoxia (41.3%+/-2.0%, P<0.001) . Gs-Rb1 caused a 77.3%+/-3.2% reduction in the expression of GSK-3beta protein (P<0.001) and a significant increase of 1.182+/-0.007-fold (P=0.0001) in p-Ser9-GSK-3beta compared with control group. Wortmannin and Ara A significantly inhibited the effect of Gs-Rb1 on mPTP opening and DeltaGSK-3beta. Gs-Rb1 significantly decreased expression of cytochrome c (66.1%+/-1.7%, P=0.001), caspase-3 (56.5%+/-2.7%, P=0.001) and cleaved poly ADP-ribose polymerase (PARP) (57.9%+/-1.4%, P=0.001).Gs-Rb1 exerted anti-hypoxia effect on neonatal rat cardiomyocytes by inhibiting GSK-3beta-mediated mPTP opening.
Project description:The mechanisms contributing to heart failure remain incompletely understood. d-dopachrome tautomerase (DDT) is a member of the macrophage migration inhibitory factor family of cytokines and is highly expressed in cardiomyocytes. This study examined the role of cardiomyocyte DDT in the setting of heart failure. Patients with advanced heart failure undergoing transplantation demonstrated decreased cardiac DDT expression. To understand the effect of loss of cardiac DDT in experimental heart failure, cardiomyocyte-specific DDT-KO (DDT-cKO) and littermate control mice underwent surgical transverse aortic constriction (TAC) to induce cardiac pressure overload. DDT-cKO mice developed more rapid cardiac contractile dysfunction, greater cardiac dilatation, and pulmonary edema after TAC. Cardiomyocytes from DDT-cKO mice after TAC had impaired contractility, calcium transients, and reduced expression of the sarcoplasmic reticulum calcium ATPase. The DDT-cKO hearts also exhibited diminished angiogenesis with reduced capillary density and lower VEGF-A expression after TAC. In pharmacological studies, recombinant DDT (rDDT) activated endothelial cell ERK1/2 and Akt signaling and had proangiogenic effects in vitro. The DDT-cKO hearts also demonstrated more interstitial fibrosis with enhanced collagen and connective tissue growth factor expression after TAC. In cardiac fibroblasts, rDDT had an antifibrotic action by inhibiting TGF-β-induced Smad-2 activation. Thus, endogenous cardiomyocyte DDT has pleiotropic actions that are protective against heart failure.
Project description:Pressure overload in patients with aortic stenosis (AS) induces an adverse remodeling of the left ventricle (LV) in a sex-specific manner. We assessed whether a sex-specific miR-29b dysregulation underlies this sex-biased remodeling pattern, as has been described in liver fibrosis. We studied mice with transverse aortic constriction (TAC) and patients with AS. miR-29b was determined in the LV (mice, patients) and plasma (patients). Expression of remodeling-related markers and histological fibrosis were determined in mouse LV. Echocardiographic morpho-functional parameters were evaluated at baseline and post-TAC in mice, and preoperatively and 1 year after aortic valve replacement (AVR) in patients with AS. In mice, miR-29b LV regulation was opposite in TAC-males (down-regulation) and TAC-females (up-regulation). The subsequent changes in miR-29b targets (collagens and GSK-3?) revealed a remodeling pattern that was more fibrotic in males but more hypertrophic in females. Both systolic and diastolic cardiac functions deteriorated more in TAC-females, thus suggesting a detrimental role of miR-29b in females, but was protective in the LV under pressure overload in males. Clinically, miR-29b in controls and patients with AS reproduced most of the sexually dimorphic features observed in mice. In women with AS, the preoperative plasma expression of miR-29b paralleled the severity of hypertrophy and was a significant negative predictor of reverse remodeling after AVR; therefore, it may have potential value as a prognostic biomarker.
Project description:Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthases that limits nitric oxide bioavailability. Dimethylarginine dimethylaminohydrolase-1 (DDAH1) exerts a critical role for ADMA degradation and plays an important role in NO signaling. In the heart, DDAH1 is observed in endothelial cells and in the sarcolemma of cardiomyocytes. While NO signaling is important for cardiac adaptation to stress, DDAH1 impact on cardiomyocyte homeostasis is not clear. Here we used the MerCreMer-LoxP model to specifically disrupt cardiomyocyte DDAH1 expression in adult mice to determine the physiological impact of cardiomyocyte DDAH1 under basal conditions and during hypertrophic stress imposed by transverse aortic constriction (TAC). Under control conditions, cardiomyocyte-specific DDAH1 knockout (cDDAH KO) had no detectable effect on plasma ADMA and left ventricular (LV) hypertrophy or function in adult or aging mice. In response to TAC, DDAH1 levels were elevated 2.5-fold in WT mice, which exhibited no change in LV or plasma ADMA content and moderate LV hypertrophy and LV dysfunction. In contrast, cDDAH1 KO mice exposed to TAC showed no increase in LV DDAH1 expression, slightly increased LV tissue ADMA levels, no increase in plasma ADMA, but significantly exacerbated LV hypertrophy, fibrosis, nitrotyrosine production, and LV dysfunction. These findings indicate cardiomyocyte DDAH1 activity is dispensable for cardiac function under basal conditions, but plays an important role in attenuating cardiac hypertrophy and ventricular remodeling under stress conditions, possibly through locally confined regulation of subcellular ADMA and NO signaling.