Targeted deletion of the extracellular signal-regulated protein kinase 5 attenuates hypertrophic response and promotes pressure overload-induced apoptosis in the heart.
ABSTRACT: Mitogen-activated protein kinase (MAPK) pathways provide a critical connection between extrinsic and intrinsic signals to cardiac hypertrophy. Extracellular signal-regulated protein kinase (ERK)5, an atypical MAPK is activated in the heart by pressure overload. However, the role of ERK5 plays in regulating hypertrophic growth and hypertrophy-induced apoptosis is not completely understood.Herein, we investigate the in vivo role and signaling mechanism whereby ERK5 regulates cardiac hypertrophy and hypertrophy-induced apoptosis.We generated and examined the phenotypes of mice with cardiomyocyte-specific deletion of the erk5 gene (ERK5(cko)). In response to hypertrophic stress, ERK5(cko) mice developed less hypertrophic growth and fibrosis than controls. However, increased apoptosis together with upregulated expression levels of p53 and Bad were observed in the mutant hearts. Consistently, we found that silencing ERK5 expression or specific inhibition of its kinase activity using BIX02189 in neonatal rat cardiomyocytes (NRCMs) reduced myocyte enhancer factor (MEF)2 transcriptional activity and blunted hypertrophic responses. Furthermore, the inhibition of MEF2 activity in NRCMs using a non-DNA binding mutant form of MEF2 was found to attenuate the ERK5-regulated hypertrophic response.These results reveal an important function of ERK5 in cardiac hypertrophic remodeling and cardiomyocyte survival. The role of ERK5 in hypertrophic remodeling is likely to be mediated via the regulation of MEF2 activity.
Project description:Stress-induced hypertrophic remodeling is a critical pathogenetic process leading to heart failure. Although many signal transduction cascades are demonstrated as important regulators to facilitate the induction of cardiac hypertrophy, the signaling pathways for suppressing hypertrophic remodeling remain largely unexplored. In this study, we identified p21-activated kinase 1 (Pak1) as a novel signaling regulator that antagonizes cardiac hypertrophy.Hypertrophic stress applied to primary neonatal rat cardiomyocytes (NRCMs) or murine hearts caused the activation of Pak1. Analysis of NRCMs expressing constitutively active Pak1 or in which Pak1 was silenced disclosed that Pak1 played an antihypertrophic role. To investigate the in vivo role of Pak1 in the heart, we generated mice with a cardiomyocyte-specific deletion of Pak1 (Pak1(cko)). When subjected to 2 weeks of pressure overload, Pak1(cko) mice developed greater cardiac hypertrophy with attendant blunting of JNK activation compared with controls, and these knockout mice underwent the transition into heart failure when prolonged stress was applied. Chronic angiotensin II infusion also caused increased cardiac hypertrophy in Pak1(cko) mice. Moreover, we discovered that the Pak1 activator FTY720, a sphingosine-like analog, was able to prevent pressure overload-induced hypertrophy in wild-type mice without compromising their cardiac functions. Meanwhile, FTY720 failed to exert such an effect on Pak1(cko) mice, suggesting that the antihypertrophic effect of FTY720 likely acts through Pak1 activation.These results, for the first time, establish Pak1 as a novel antihypertrophic regulator and suggest that it may be a potential therapeutic target for the treatment of cardiac hypertrophy and heart failure.
Project description:RAS activation is implicated in physiologic and pathologic cardiac hypertrophy. Cross-talk between the Ras and calcineurin pathways, the latter also having been implicated in cardiac hypertrophy, has been suspected for pathologic hypertrophy. Our recent discovery that germ-line mutations in RAF1, which encodes a downstream RAS effector, cause Noonan and LEOPARD syndromes with a high prevalence of hypertrophic cardiomyopathy provided an opportunity to elaborate the role of RAF1 in cardiomyocyte biology. Here, we characterize the role of RAF1 signaling in cardiomyocyte hypertrophy with an aim of identifying potential therapeutic targets. We modeled hypertrophic cardiomyopathy by infecting neonatal and adult rat cardiomyocytes (NRCMs and ARCMs, respectively) with adenoviruses encoding wild-type RAF1 and three Noonan/LEOPARD syndrome-associated RAF1 mutants (S257L, D486N or L613V). These RAF1 proteins, except D486N, engendered cardiomyocyte hypertrophy. Surprisingly, these effects were independent and dependent of mitogen activated protein kinases in NRCMs and ARCMs, respectively. Inhibiting Mek1/2 in RAF1 overexpressing cells blocked hypertrophy in ARCMs but not in NRCMs. Further, we found that endogenous and heterologously expressed RAF1 complexed with calcineurin, and RAF1 mutants causing hypertrophy signaled via nuclear factor of activated T cells (Nfat) in both cell types. The involvement of calcineurin was also reflected by down regulation of Serca2a and dysregulation of calcium signaling in NRCMs. Furthermore, treatment with the calcineurin inhibitor cyclosporine blocked hypertrophy in NRCMs and ARCMs overexpressing RAF1. Thus, we have identified calcineurin as a novel interaction partner for RAF1 and established a mechanistic link and possible therapeutic target for pathological cardiomyocyte hypertrophy induced by mutant RAF1. This article is part of a Special Issue entitled 'Possible Editorial'.
Project description:Reactive oxygen species (ROS) play a pivotal role in the development of pathological cardiac hypertrophy. Delphinidin, a natural flavonoid, was reported to exert marked antioxidative effects. Therefore, we investigated whether delphinidin ameliorates pathological cardiac hypertrophy via inhibiting oxidative stress. In this study, male C57BL/6 mice were treated with DMSO or delphinidin after surgery. Neonatal rat cardiomyocytes (NRCMs) were treated with angiotensin II (Ang II) and delphinidin in vitro. Eighteen-month-old mice were administered delphinidin to investigate the effect of delphinidin on aging-related cardiac hypertrophy. Through analyses of hypertrophic cardiomyocyte growth, fibrosis and cardiac function, delphinidin was demonstrated to confer resistance to aging- and transverse aortic constriction (TAC)-induced cardiac hypertrophy in vivo and attenuate Ang II-induced cardiomyocyte hypertrophy in vitro by significantly suppressing hypertrophic growth and the deposition of fibrosis. Mechanistically, delphinidin reduced ROS accumulation upon Ang II stimulation through the direct activation of AMP-activated protein kinase (AMPK) and subsequent inhibition of the activity of Rac1 and expression of p47phox. In addition, excessive levels of ERK1/2, P38 and JNK1/2 phosphorylation induced by oxidative stress were abrogated by delphinidin. Delphinidin was conclusively shown to repress pathological cardiac hypertrophy by modulating oxidative stress through the AMPK/NADPH oxidase (NOX)/mitogen-activated protein kinase (MAPK) signaling pathway.
Project description:Mitogen-activated protein kinases (MAPKs) are involved in the regulation of cardiac hypertrophy and myocyte survival. Extracellular signal regulated protein kinase 1 and 2 (ERK1/2) are key components in the MAPK signaling pathways. Dysfunction of ERK1/2 in congenital heart diseases (Noonan syndrome and LEOPARD syndrome) leads to cardiac hypertrophy. ERK2 contributes 70% of protein content to total ERK1/2 content in myocardium; however, the specific role of ERK2 in regulating cardiac hypertrophy is yet to be further defined. To investigate the specific role of ERK2 played in the cardiomyocytes, we generated and examined mice with cardiomyocyte-specific deletion of the erk2 gene (ERK2(cko) mice). Following short-term pathological hypertrophic stresses, the mutant mice showed attenuated hypertrophic remodeling characterized by a blunted increase in the cross-sectional area of individual myocytes, downregulation of hypertrophic foetal gene markers (ANP and BNP), and less interstitial fibrosis. However, increased cardiomyocyte apoptosis was observed. Upon prolonged stimulation, ERK2(cko) mice developed deterioration in cardiac function. However, absence of ERK2 did not affect physiological hypertrophy induced by 4weeks of swimming exercise. These results revealed an essential role for ERK2 in cardiomyocytes in the development of pathological hypertrophic remodeling and resistance to cell death.
Project description:Decreased autophagy has been reported to contribute to the progression of cardiac hypertrophy. Our previous research has demonstrated that endophilin A2 (EndoA2) attenuates H2O2-induced cardiomyocyte apoptosis by strengthening autophagy. However, the role of EndoA2 in the regulation of autophagy in cardiac hypertrophy is unknown. In this study, we tested the hypothesis that EndoA2 suppresses cardiac hypertrophy induced by isoproterenol (ISO) by activating autophagy. In vivo, we established a cardiac hypertrophy model by subcutaneous injection of ISO and used intramyocardial delivery of adenovirus vector harboring EndoA2 cDNA (Ad-EndoA2) to overexpress EndoA2. The cardiac hypertrophic response and autophagy level were measured. EndoA2 overexpression suppressed pathological cardiac hypertrophy and enhanced autophagy in rat hearts. In addition, the effects of EndoA2 on cardiac hypertrophy and autophagy were observed in cultured neonatal rat cardiomyocytes (NRCMs) with gain- and loss-of-function approaches to regulate EndoA2 expression. The results were consistent with those of the in vivo study. Furthermore, the involvement of EndoA2-mediated autophagy in the attenuation of ISO-induced cardiac hypertrophy was explored by pharmaceutical inhibition of autophagy. Pretreatment with 3-methyladenine (3-MA) clearly diminished the anti-hypertrophic effects of EndoA2 in ISO-treated NRCMs. The results presented here provide the first evidence that EndoA2 is involved in ISO-induced cardiac hypertrophy. The anti-hypertrophic effects of EndoA2 can be partially attributed to its regulation of autophagy.
Project description:The histone demethylase JMJD family is involved in various physiological and pathological functions. However, the roles of JMJD1A in the cardiovascular system remain unknown. Here, we studied the function of JMJD1A in cardiac hypertrophy. The mRNA and protein levels of JMJD1A were significantly downregulated in the hearts of human patients with hypertrophic cardiomyopathy and the hearts of C57BL/6 mice underwent cardiac hypertrophy induced by transverse aortic constriction (TAC) surgery or isoproterenol (ISO) infusion. In neonatal rat cardiomyocytes (NRCMs), siRNA-mediated JMJD1A knockdown facilitated ISO or angiotensin II-induced increase in cardiomyocyte size, protein synthesis, and expression of hypertrophic fetal genes, including atrial natriuretic peptide (Anp), brain natriuretic peptide (Bnp), and Myh7. By contrast, overexpression of JMJD1A with adenovirus repressed the development of ISO-induced cardiomyocyte hypertrophy. We observed that JMJD1A reduced the production of total cellular and mitochondrial levels of reactive oxygen species (ROS), which was critically involved in the effects of JMJD1A because either N-acetylcysteine or MitoTEMPO treatment blocked the effects of JMJD1A deficiency on cardiomyocyte hypertrophy. Mechanism study demonstrated that JMJD1A promoted the expression and activity of Catalase under basal condition or oxidative stress. siRNA-mediated loss of Catalase blocked the protection of JMJD1A overexpression against ISO-induced cardiomyocyte hypertrophy. These findings demonstrated that JMJD1A loss promoted cardiomyocyte hypertrophy in a Catalase and ROS-dependent manner.
Project description:Pressure overload-induced cardiac stress induces left ventricular hypertrophy driven by increased cardiomyocyte mass. The increased energetic demand and cardiomyocyte size during hypertrophy necessitate increased fuel and oxygen delivery and stimulate angiogenesis in the left ventricular wall. We have previously shown that the transcriptional regulator steroid receptor coactivator-2 (SRC-2) controls activation of several key cardiac transcription factors and that SRC-2 loss results in extensive cardiac transcriptional remodeling. Pressure overload in mice lacking SRC-2 induces an abrogated hypertrophic response and decreases sustained cardiac function, but the cardiomyocyte-specific effects of SRC-2 in these changes are unknown. Here, we report that cardiomyocyte-specific loss of SRC-2 (SRC-2 CKO) results in a blunted hypertrophy accompanied by a rapid, progressive decrease in cardiac function. We found that SRC-2 CKO mice exhibit markedly decreased left ventricular vasculature in response to transverse aortic constriction, corresponding to decreased expression of the angiogenic factor VEGF. Of note, SRC-2 knockdown in cardiomyocytes decreased VEGF expression and secretion to levels sufficient to blunt in vitro tube formation and proliferation of endothelial cells. During pressure overload, both hypertrophic and hypoxic signals can stimulate angiogenesis, both of which stimulated SRC-2 expression in vitro Furthermore, SRC-2 coactivated the transcription factors GATA-binding protein 4 (GATA-4) and hypoxia-inducible factor (HIF)-1? and -2? in response to angiotensin II and hypoxia, respectively, which drive VEGF expression. These results suggest that SRC-2 coordinates cardiomyocyte secretion of VEGF downstream of the two major angiogenic stimuli occurring during pressure overload bridging both hypertrophic and hypoxia-stimulated paracrine signaling.
Project description:Gq-coupled G protein-coupled receptors (GPCRs) mediate the actions of a variety of messengers that are key regulators of cardiovascular function. Enhanced G?(q)-mediated signaling plays an important role in cardiac hypertrophy and in the transition to heart failure. We have recently described that G?(q) acts as an adaptor protein that facilitates PKC?-mediated activation of ERK5 in epithelial cells. Because the ERK5 cascade is known to be involved in cardiac hypertrophy, we have investigated the potential relevance of this pathway in cardiovascular Gq-dependent signaling using both cultured cardiac cell types and chronic administration of angiotensin II in mice. We find that PKC? is required for the activation of the ERK5 pathway by Gq-coupled GPCR in neonatal and adult murine cardiomyocyte cultures and in cardiac fibroblasts. Stimulation of ERK5 by angiotensin II is blocked upon pharmacological inhibition or siRNA-mediated silencing of PKC? in primary cultures of cardiac cells and in neonatal cardiomyocytes isolated from PKC?-deficient mice. Moreover, upon chronic challenge with angiotensin II, these mice fail to promote the changes in the ERK5 pathway, in gene expression patterns, and in hypertrophic markers observed in wild-type animals. Taken together, our results show that PKC? is essential for Gq-dependent ERK5 activation in cardiomyocytes and cardiac fibroblasts and indicate a key cardiac physiological role for the G?(q)/PKC?/ERK5 signaling axis.
Project description:Excessive ?AR stimulation is an independent factor in inducing pathological cardiac hypertrophy. Here, we report miR-145 regulates both expression and localization of GATA6, thereby protecting the heart against cardiomyocyte hypertrophy induced by isoproterenol (ISO). The protective activity of miR-145 was associated with down-regulation of ANF, BNP and ?-MHC expression, a decreased rate of protein synthesis, inhibited cardiomyocyte growth and the modulation of several signaling pathways including ERK1/2, JNK and Akt-GSK3?. The anti-hypertrophic effect was abrogated by exogenous over-expression of transcription factor GATA6 which was further identified as a direct target of miR-145. In addition, GSK3? antagonists, LiCl and TDZD8, restored the nuclear accumulation of GATA6, which was attenuated by miR-145 Finally, we observed a dynamic pattern of miR-145 expression in ISO-treated NRCMs and in the hearts of TAC mice. Together, our results identify miR-145 as an important regulator in cardiac hypertrophy.
Project description:Angiotensin-converting enzyme 3 (ACE3) is a recently defined homolog of ACE. However, the pathophysiological function of ACE3 is largely unknown. Here, we aim to explore the role of ACE3 in pathological cardiac hypertrophy.Neonatal rat cardiomyocytes (NRCMs) with gain and loss of function of ACE3 and mice with global knockout or cardiac-specific overexpression of ACE3 were used in this study. In cultured cardiomyocytes, ACE3 conferred protection against angiotensin II (Ang II)-induced hypertrophic growth. Cardiac hypertrophy in mice was induced by aortic banding (AB) and the extent of hypertrophy was analyzed through echocardiographic, pathological, and molecular analyses. Our data demonstrated that ACE3-deficient mice exhibited more pronounced cardiac hypertrophy and fibrosis and a strong decrease in cardiac contractile function, conversely, cardiac-specific ACE3-overexpressing mice displayed an attenuated hypertrophic phenotype, compared with control mice, respectively. Analyses of the underlying molecular mechanism revealed that ACE3-mediated protection against cardiac hypertrophy by suppressing the activation of mitogen-activated protein kinase kinase (MEK)-regulated extracellular signal-regulated protein kinase (ERK1/2) signaling, which was further evidenced by the observation that inhibition of the MEK-ERK1/2 signaling by U0126 rescued the exacerbated hypertrophic phenotype in ACE3-deficient mice.Our comprehensive analyses suggest that ACE3 inhibits pressure overload-induced cardiac hypertrophy by blocking the MEK-ERK1/2 signaling pathway.