G protein-coupled receptor kinase 2 promotes cardiac hypertrophy.
ABSTRACT: The increase in protein activity and upregulation of G-protein coupled receptor kinase 2 (GRK2) is a hallmark of cardiac stress and heart failure. Inhibition of GRK2 improved cardiac function and survival and diminished cardiac remodeling in various animal heart failure models. The aim of the present study was to investigate the effects of GRK2 on cardiac hypertrophy and dissect potential molecular mechanisms. In mice we observed increased GRK2 mRNA and protein levels following transverse aortic constriction (TAC). Conditional GRK2 knockout mice showed attenuated hypertrophic response with preserved ventricular geometry 6 weeks after TAC operation compared to wild-type animals. In isolated neonatal rat ventricular cardiac myocytes stimulation with angiotensin II and phenylephrine enhanced GRK2 expression leading to enhanced signaling via protein kinase B (PKB or Akt), consecutively inhibiting glycogen synthase kinase 3 beta (GSK3?), such promoting nuclear accumulation and activation of nuclear factor of activated T-cells (NFAT). Cardiac myocyte hypertrophy induced by in vitro GRK2 overexpression increased the cytosolic interaction of GRK2 and phosphoinositide 3-kinase ? (PI3K?). Moreover, inhibition of PI3K? as well as GRK2 knock down prevented Akt activation resulting in halted NFAT activity and reduced cardiac myocyte hypertrophy. Our data show that enhanced GRK2 expression triggers cardiac hypertrophy by GRK2-PI3K? mediated Akt phosphorylation and subsequent inactivation of GSK3?, resulting in enhanced NFAT activity.
Project description:A clear understanding of the molecular mechanisms underlying hemodynamic stress-initiated cardiac hypertrophy is important for preventing heart failure. Interferon-? (IFN-?) has been suggested to play crucial roles in various diseases other than immunological disorders by modulating the expression of myriad genes. However, the involvement of IFN-? in the pathogenesis of cardiac hypertrophy still remains unclear.In order to elucidate the roles of IFN-? in pressure overload-induced cardiac pathology, we subjected Balb/c wild-type (WT) or IFN-?-deficient (Ifng-/-) mice to transverse aortic constriction (TAC). Three weeks after TAC, Ifng-/- mice developed more severe cardiac hypertrophy, fibrosis, and dysfunction than WT mice. Bone marrow-derived immune cells including macrophages were a source of IFN-? in hearts after TAC. The activation of PI3K/Akt signaling, a key signaling pathway in compensatory hypertrophy, was detected 3 days after TAC in the left ventricles of WT mice and was markedly attenuated in Ifng-/- mice. The administration of a neutralizing anti-IFN-? antibody abrogated PI3K/Akt signal activation in WT mice during compensatory hypertrophy, while that of IFN-? activated PI3K/Akt signaling in Ifng-/- mice. TAC also induced the phosphorylation of Stat5, but not Stat1 in the left ventricles of WT mice 3 days after TAC. Furthermore, IFN-? induced Stat5 and Akt phosphorylation in rat cardiomyocytes cultured under stretch conditions. A Stat5 inhibitor significantly suppressed PI3K/Akt signaling activation in the left ventricles of WT mice, and aggravated pressure overload-induced cardiac hypertrophy.The IFN-?/Stat5 axis may be protective against persistent pressure overload-induced cardiac hypertrophy by activating the PI3K/Akt pathway.
Project description:AMP activated protein kinase (AMPK) plays an important role in regulating myocardial metabolism and protein synthesis. Activation of AMPK attenuates hypertrophy in cultured cardiac myocytes, but the role of AMPK in regulating the development of myocardial hypertrophy in response to chronic pressure overload is not known. To test the hypothesis that AMPKalpha2 protects the heart against systolic overload-induced ventricular hypertrophy and dysfunction, we studied the response of AMPKalpha2 gene deficient (knockout [KO]) mice and wild-type mice subjected to 3 weeks of transverse aortic constriction (TAC). Although AMPKalpha2 KO had no effect on ventricular structure or function under control conditions, AMPKalpha2 KO significantly increased TAC-induced ventricular hypertrophy (ventricular mass increased 46% in wild-type mice compared with 65% in KO mice) while decreased left ventricular ejection fraction (ejection fraction decreased 14% in wild-type mice compared with a 43% decrease in KO mice). AMPKalpha2 KO also significantly exacerbated the TAC-induced increases of atrial natriuretic peptide, myocardial fibrosis, and cardiac myocyte size. AMPKalpha2 KO had no effect on total S6 ribosomal protein (S6), p70 S6 kinase, eukaryotic initiation factor 4E, and 4E binding protein-1 or their phosphorylation under basal conditions but significantly augmented the TAC-induced increases of p-p70 S6 kinase(Thr389), p-S6(Ser235), and p-eukaryotic initiation factor 4E(Ser209). AMPKalpha2 KO also enhanced the TAC-induced increase of p-4E binding protein-1(Thr46) to a small degree and augmented the TAC-induced increase of p-Akt(Ser473). These data indicate that AMPKalpha2 exerts a cardiac protective effect against pressure-overload-induced ventricular hypertrophy and dysfunction.
Project description:In contrast with pathological hypertrophy, exercise-induced physiological hypertrophy is not associated with electrical abnormalities or increased arrhythmia risk. Recent studies have shown that increased cardiac-specific expression of phosphoinositide-3-kinase-? (PI3K?), the key mediator of physiological hypertrophy, results in transcriptional upregulation of ion channel subunits in parallel with the increase in myocyte size (cellular hypertrophy) and the maintenance of myocardial excitability. The experiments here were undertaken to test the hypothesis that Akt1, which underlies PI3K?-induced cellular hypertrophy, mediates the effects of augmented PI3K? signaling on the transcriptional regulation of cardiac ion channels. In contrast to wild-type animals, chronic exercise (swim) training of mice (Akt1(-/-)) lacking Akt1 did not result in ventricular myocyte hypertrophy. Ventricular K(+) current amplitudes and the expression of K(+) channel subunits, however, were increased markedly in Akt1(-/-) animals with exercise training. Expression of the transcripts encoding inward (Na(+) and Ca(2+)) channel subunits were also increased in Akt1(-/-) ventricles following swim training. Additional experiments in a transgenic mouse model of inducible cardiac-specific expression of constitutively active PI3K? (icaPI3K?) revealed that short-term activation of PI3K? signaling in the myocardium also led to the transcriptional upregulation of ion channel subunits. Inhibition of cardiac Akt activation with triciribine in this (inducible caPI3K? expression) model did not prevent the upregulation of myocardial ion channel subunits. These combined observations demonstrate that chronic exercise training and enhanced PI3K? expression/activity result in transcriptional upregulation of myocardial ion channel subunits independent of cellular hypertrophy and Akt signaling.
Project description:Testosterone induces cardiac hypertrophy through a mechanism that involves a concerted crosstalk between cytosolic and nuclear signaling pathways. Nuclear factor of activated T-cells (NFAT) is associated with the promotion of cardiac hypertrophy, glycogen synthase kinase-3? (GSK-3?) is considered to function as a negative regulator, mainly by modulating NFAT activity. However, the role played by calcineurin-NFAT and GSK-3? signaling in testosterone-induced cardiac hypertrophy has remained unknown. Here, we determined that testosterone stimulates cardiac myocyte hypertrophy through NFAT activation and GSK-3? inhibition. Testosterone increased the activity of NFAT-luciferase (NFAT-Luc) in a time- and dose-dependent manner, with the activity peaking after 24 h of stimulation with 100 nM testosterone. NFAT-Luc activity induced by testosterone was blocked by the calcineurin inhibitors FK506 and cyclosporine A and by 11R-VIVIT, a specific peptide inhibitor of NFAT. Conversely, testosterone inhibited GSK-3? activity as determined by increased GSK-3? phosphorylation at Ser9 and ?-catenin protein accumulation, and also by reduction in ?-catenin phosphorylation at residues Ser33, Ser37, and Thr41. GSK-3? inhibition with 1-azakenpaullone or a GSK-3?-targeting siRNA increased NFAT-Luc activity, whereas overexpression of a constitutively active GSK-3? mutant (GSK-3?S9A) inhibited NFAT-Luc activation mediated by testosterone. Testosterone-induced cardiac myocyte hypertrophy was established by increased cardiac myocyte size and [3H]-leucine incorporation (as a measurement of cellular protein synthesis). Calcineurin-NFAT inhibition abolished and GSK-3? inhibition promoted the hypertrophy stimulated by testosterone. GSK-3? activation by GSK-3?S9A blocked the increase of hypertrophic markers induced by testosterone. Moreover, inhibition of intracellular androgen receptor prevented testosterone-induced NFAT-Luc activation. Collectively, these results suggest that cardiac myocyte hypertrophy induced by testosterone involves a cooperative mechanism that links androgen signaling with the recruitment of NFAT through calcineurin activation and GSK-3? inhibition.
Project description:Muscle-specific RING finger protein-1 (MuRF1) is an E3 ligase that inhibits cardiac hypertrophy. However, how MuRF1 regulates cardiac hypertrophy and function during pressure overload (PO) remains poorly understood. We investigated the role of endogenous MuRF1 in regulating cardiac hypertrophy in response to PO in vivo.Transverse aortic constriction (TAC) for 4 weeks significantly reduced expression of MuRF1 in the mouse heart. After 2 and 4 weeks of TAC, MuRF1 knockout (Murf1(-/-)) mice exhibited enhanced cardiac hypertrophy and left ventricular (LV) dysfunction compared with that of nontransgenic (NTg) mice. Histological analyses showed that Murf1(-/-) mice exhibited more severe fibrosis and apoptosis than NTg mice after TAC. TAC-induced increases in the activity of a nuclear factor of activated T cells (NFAT) luciferase reporter were significantly greater in Murf1(-/-) than in NTg mice. TAC-induced increases in calcineurin A (CnA) expression were also significantly enhanced in Murf1(-/-) compared with that in NTg mice. Coimmunoprecipitation assays showed that endogenous MuRF1 and CnA interact with one another. Polyubiquitination of CnA was attenuated in Murf1(-/-) mouse hearts at baseline and in response to TAC, and the protein stability of CnA was enhanced in cardiomyocytes, in which MuRF1 was downregulated in vitro. Furthermore, MuRF1 directly ubiquitinated CnA in vitro. Cardiac-specific overexpression of ZAKI-4?, an endogenous inhibitor of CnA, significantly suppressed the enhancement of TAC-induced cardiac hypertrophy and dysfunction, as well as increases in cardiac fibrosis and apoptosis, in Murf1(-/-) mice.Endogenous MuRF1 negatively regulates cardiac hypertrophy and dysfunction in response to PO through inhibition of the calcineurin-NFAT pathway.
Project description:This study sought to test the efficacy of phosphodiesterase type 5A (PDE5A) inhibition for treating advanced hypertrophy/remodeling caused by pressure overload, and to elucidate cellular and molecular mechanisms for this response.Sildenafil (SIL) inhibits cyclic guanosine monophosphate-specific PDE5A and can blunt the evolution of cardiac hypertrophy and dysfunction in mice subjected to pressure overload. Whether and how it ameliorates more established advanced disease and dysfunction is unknown.Mice were subjected to transverse aortic constriction (TAC) for 3 weeks to establish hypertrophy/dilation, and subsequently treated with SIL (100 mg/kg/day) or placebo for 6 weeks of additional TAC.The SIL arrested further progressive chamber dilation, dysfunction, fibrosis, and molecular remodeling, increasing myocardial protein kinase G activity. Isolated myocytes from TAC-SIL hearts showed greater sarcomere shortening and relaxation, and enhanced Ca(2+) transients and decay compared with nontreated TAC hearts. The SIL treatment restored gene and protein expression of sarcoplasmic reticulum Ca(2+) uptake adenosine triphosphatase (SERCA2a), phospholamban (PLB), and increased PLB phosphorylation (S16), consistent with improved calcium handling. The phosphatase calcineurin (Cn) and/or protein kinase C-alpha (PKCalpha) can both lower phosphorylated phospholamban and depress myocyte calcium cycling. The Cn expression and PKCalpha activation (outer membrane translocation) were enhanced by chronic TAC and reduced by SIL treatment. Expression of PKCdelta and PKCepsilon also increased with TAC but were unaltered by SIL treatment.SIL treatment applied to well-established hypertrophic cardiac disease can prevent further cardiac and myocyte dysfunction and progressive remodeling. This is associated with improved calcium cycling, and reduction of Cn and PKCalpha activation may be important to this improvement.
Project description:RATIONALE:The role of interleukin (IL)-6 in the pathogenesis of cardiac myocyte hypertrophy remains controversial. OBJECTIVE:To conclusively determine whether IL-6 signaling is essential for the development of pressure overload-induced left ventricular (LV) hypertrophy and to elucidate the underlying molecular pathways. METHODS AND RESULTS:Wild-type and IL-6 knockout (IL-6(-/-)) mice underwent sham surgery or transverse aortic constriction (TAC) to induce pressure overload. Serial echocardiograms and terminal hemodynamic studies revealed attenuated LV hypertrophy and superior preservation of LV function in IL-6(-/-) mice after TAC. The extents of LV remodeling, fibrosis, and apoptosis were reduced in IL-6(-/-) hearts after TAC. Transcriptional and protein assays of myocardial tissue identified Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and signal transducer and activator of transcription 3 (STAT3) activation as important underlying mechanisms during cardiac hypertrophy induced by TAC. The involvement of these pathways in myocyte hypertrophy was verified in isolated cardiac myocytes from wild-type and IL-6(-/-) mice exposed to prohypertrophy agents. Furthermore, overexpression of CaMKII in H9c2 cells increased STAT3 phosphorylation, and exposure of H9c2 cells to IL-6 resulted in STAT3 activation that was attenuated by CaMKII inhibition. Together, these results identify the importance of CaMKII-dependent activation of STAT3 during cardiac myocyte hypertrophy via IL-6 signaling. CONCLUSIONS:Genetic deletion of IL-6 attenuates TAC-induced LV hypertrophy and dysfunction, indicating a critical role played by IL-6 in the pathogenesis of LV hypertrophy in response to pressure overload. CaMKII plays an important role in IL-6-induced STAT3 activation and consequent cardiac myocyte hypertrophy. These findings may have significant therapeutic implications for LV hypertrophy and failure in patients with hypertension.
Project description:TAK1 (TGF?-activated kinase-1) signaling is essential in regulating a number of important biological functions, including innate immunity, inflammatory response, cell growth and differentiation, and myocardial homeostasis. The precise role of TAK1 in the adult heart under pathological conditions remains largely unknown. Importantly, we observed that TAK1 is upregulated during compensatory hypertrophy but downregulated in end-stage heart failure. Here we generated transgenic mice with inducible expression of an active TAK1 mutant (TAK1?N) in the adult heart. TAK1?N transgenic mice developed greater cardiac hypertrophy compared with control mice after transverse aortic constriction (TAC), which was largely blocked by ablation of calcineurin A?. Expression of TAK1?N also promoted NFAT (nuclear factor of activated T-cells) transcriptional activity in luciferase reporter mice at baseline, which was further enhanced after TAC. Our results revealed that activation of TAK1 promoted adaptive cardiac hypertrophy through a cross-talk between calcineurin-NFAT and IKK-NF?B pathways. More significantly, adult-onset inducible expression of TAK1?N protected the myocardium from adverse remodeling and heart failure after myocardial infarction or long-term pressure overload, by preventing cardiac cell death and fibrosis. Mechanistically, TAK1 exerts its cardioprotective effect through activation of NFAT/NF?B, downregulation of Bnip3, and inhibition of cardiac cell death.
Project description:G protein-coupled receptor kinases (GRKs) acting in the cardiomyocyte regulate important signaling events that control cardiac function. Both GRK2 and GRK5, the predominant GRKs expressed in the heart, have been shown to be upregulated in failing human myocardium. Although the canonical role of GRKs is to desensitize G protein-coupled receptors via phosphorylation, it has been demonstrated that GRK5, unlike GRK2, can reside in the nucleus of myocytes and exert G protein-coupled receptor-independent effects that promote maladaptive cardiac hypertrophy and heart failure.To explore novel mechanisms by which GRK5 acting in the nucleus of cardiomyocytes participates in pathological cardiac hypertrophy.In this study, we have found that GRK5-mediated pathological cardiac hypertrophy involves the activation of the nuclear factor of activated T cells (NFAT) because GRK5 causes enhancement of NFAT-mediated hypertrophic gene transcription. Transgenic mice with cardiomyocyte-specific GRK5 overexpression activate an NFAT-reporter in mice basally and after hypertrophic stimulation, including transverse aortic constriction and phenylephrine treatment. Complimentary to this, GRK5 null mice exhibit less NFAT transcriptional activity after transverse aortic constriction. Furthermore, the loss of NFATc3 expression in the heart protected GRK5 overexpressing transgenic mice from the exaggerated hypertrophy and early progression to heart failure seen after transverse aortic constriction. Molecular studies suggest that GRK5 acts in concert with NFAT to increase hypertrophic gene transcription in the nucleus via GRK5's ability to bind DNA directly without a phosphorylation event.GRK5, acting in a kinase independent manner, is a facilitator of NFAT activity and part of a DNA-binding complex responsible for pathological hypertrophic gene transcription.
Project description:Factors secreted by the heart, referred to as "cardiokines," have diverse actions in the maintenance of cardiac homeostasis and remodeling. Follistatin-like 1 (Fstl1) is a secreted glycoprotein expressed in the adult heart and is induced in response to injurious conditions that promote myocardial hypertrophy and heart failure. The aim of this study was to investigate the role of cardiac Fstl1 in the remodeling response to pressure overload. Cardiac myocyte-specific Fstl1-KO mice were constructed and subjected to pressure overload induced by transverse aortic constriction (TAC). Although Fstl1-KO mice displayed no detectable baseline phenotype, TAC led to enhanced cardiac hypertrophic growth and a pronounced loss in ventricular performance by 4 wk compared with control mice. Conversely, mice that acutely or chronically overexpressed Fstl1 were resistant to pressure overload-induced hypertrophy and cardiac failure. Fstl1-deficient mice displayed a reduction in TAC-induced AMP-activated protein kinase (AMPK) activation in heart, whereas Fstl1 overexpression led to increased myocardial AMPK activation under these conditions. In cultured neonatal cardiomyocytes, administration of Fstl1 promoted AMPK activation and antagonized phenylephrine-induced hypertrophy. Inhibition of AMPK attenuated the antihypertrophic effect of Fstl1 treatment. These results document that cardiac Fstl1 functions as an autocrine/paracrine regulatory factor that antagonizes myocyte hypertrophic growth and the loss of ventricular performance in response to pressure overload, possibly through a mechanism involving the activation of the AMPK signaling axis.