Myocardin-related transcription factor-A induces cardiomyocyte hypertrophy.

Myocardin is a remarkably potent transcriptional coactivator expressed specifically in cardiac muscle lineages and smooth muscle cells during postnatal development. Myocardin shares homology with myocardin-related transcription factor-A (MRTF-A), which are expressed in a broad range of embryonic and adult tissues. Our previous results show that myocardin induces cardiac hypertrophy. However, the effects of MRTF-A in cardiac hypertrophy remain poorly understood. Our present work further demonstrates that myocardin plays an important role in inducing hypertrophy. At the same time, we find that overexpression of MRTF-A in neonatal rat cardiomyocytes might induce cardiomyocyte hypertrophy. Furthermore, MRTF-A expression is induced in phenylephrine, angiotensin-II, and transforming growth factor-ß-stimulated cardiac hypertrophy, whereas a dominant-negative form of MRTF-A or MRTF-A siRNA strongly inhibited upregulation of hypertrophy genes in response to hypertrophic agonists in neonatal rat cardiomyocytes. Our studies indicate that besides myocardin, MRTF-A might play an important role in cardiac hypertrophy. Our findings provide novel evidence for the future studies to explore the roles of MRTFs in cardiac hypertrophy.

VEGF-A Stimulates STAT3 Activity via Nitrosylation of Myocardin to Regulate the Expression of Vascular Smooth Muscle Cell Differentiation Markers.

Vascular endothelial growth factor A (VEGF-A) is a pivotal player in angiogenesis. It is capable of influencing such cellular processes as tubulogenesis and vascular smooth muscle cell (VSMC) proliferation, yet very little is known about the actual signaling events that mediate VEGF-A induced VSMC phenotypic switch. In this report, we describe the identification of an intricate VEGF-A-induced signaling cascade that involves VEGFR2, STAT3, and Myocardin. We demonstrate that VEGF-A promotes VSMC proliferation via VEGFR2/STAT3-mediated upregulating the proliferation of markers like Cyclin D1 and PCNA. Specifically, VEGF-A leads to nitrosylation of Myocardin, weakens its effect on promoting the expression of contractile markers and is unable to inhibit the activation of STAT3. These observations reinforce the importance of nitric oxide and S-nitrosylation in angiogenesis and provide a mechanistic pathway for VEGF-A-induced VSMC phenotypic switch. In addition, Myocardin, GSNOR and GSNO can create a negative feedback loop to regulate the VSMC phenotypic switch. Thus, the discovery of this interactive network of signaling pathways provides novel and unexpected therapeutic targets for angiogenesis-dependent diseases.

Myocardin inhibited the gap protein connexin 43 via promoted miR-206 to regulate vascular smooth muscle cell phenotypic switch.

Myocardin is regarded as a key mediator for the change of smooth muscle phenotype. The gap junction protein connexin 43 (Cx43) has been shown to be involved in vascular smooth muscle cells (VSMCs) proliferation and the development of atherosclerosis. However, the role of myocardin on gap junction of cell communication and the relation between myocardin and Cx43 in VSMC phenotypic switch has not been investigated. The goal of the present study is to investigate the molecular mechanism by which myocardin affects Cx43-regulated VSMC proliferation. Data presented in this study demonstrated that inhibition of the Cx43 activation process impaired VSMC proliferation. On the other hand, overexpression miR-206 inhibited VSMC proliferation. In additon, miR-206 silences the expression of Cx43 via targeting Cx43 3' Untranslated Regions. Importantly, myocardin can significantly promote the expression of miR-206. Cx43 regulates VSMCs' proliferation and metastasis through miR-206, which could be promoted by myocardin and used as a marker for diagnosis and a target for therapeutic intervention. Thus myocardin affected the gap junction by inhibited Cx43 and myocardin-miR-206-Cx43 formed a loop to regulate VSMC phenotypic switch.

NFATc4 and myocardin synergistically up-regulate the expression of LTCC α1C in ET-1-induced cardiomyocyte hypertrophy.

AIMS: Dysregulation of Ca(2+) is a central cause of cardiac hypertrophy. The α1C subunit of L-type Ca(2+) channel (LTCC) is a pore-forming protein which is responsible for the voltage-dependent channel gating and channel selectivity for Ca(2+). Myocardin and nuclear factor of activated T-cells c4 (NFATc4) are two key transcription factors in cardiac hypertrophy. We aimed to investigate the underlying mechanism of the transcriptional regulation of LTCC α1C by myocardin and NFATc4 in hypertrophic cardiomyocytes. MAIN METHODS: Endothelin-1 (ET-1) was used to induce cardiomyocyte hypertrophy. Cyclosporin A (CSA) was used to block the activation of calcineurin/NFATc4 pathway in ET-1-treated cardiomyocytes and the expression of LTCC α1C were examined. Overexpression or RNAi interfering experiments were performed to investigate the effects of NFATc4 or myocardin on the transcriptional regulation of LTCC α1C. Interactions between NFATc4 and myocardin or the association of NFATc4 with myocardin promoter were assessed via Co-IP or ChIP assays respectively. KEY FINDINGS: In the present study, we found that ET-1 stimulated LTCC α1C transcription in neonatal rat cardiomyocytes partially via the activation of calcineurin/NFATc4 pathway. Overexpression of NFATc4 or myocardin promoted LTCC α1C expression in cardiomyocytes. Ca(2+) channel blocker verapamil or knockdown of α1C inhibited myocardin-induced cardiomyocyte hypertrophy. Further studies showed that NFATc4 interacted with myocardin to synergistically activate the expression of LTCC α1C, moreover, NFATc4 activated myocardin expression by binding to its promoter. SIGNIFICANCE: Our results suggest a novel mechanism of the transcriptional regulation of LTCC α1C by synergistic activities of NFATc4 and myocardin in ET-1-induced cardiomyocyte hypertrophy.

MicroRNA-29a-3p attenuates ET-1-induced hypertrophic responses in H9c2 cardiomyocytes.

Transcription factor nuclear factor of activated T cells c4 (NFATc4) is the best-characterized target for the development of cardiac hypertrophy. Aberrant microRNA-29 (miR-29) expression is involved in the development of cardiac fibrosis and congestive heart failure. However, whether miR-29 regulates hypertrophic processes is still not clear. In this study, we investigated the potential functions of miR-29a-3p in endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy. We showed that miR-29a-3p was down-regulated in ET-1-treated H9c2 cardiomyocytes. Overexpression of miR-29a-3p significantly reduced ET-1-induced hypertrophic responses in H9c2 cardiomyocytes, which was accompanied by a decrease in NFATc4 expression. miR-29a-3p targeted directly to the 3'-UTR of NFATc4 mRNA and silenced NFATc4 expression. Our results indicate that miR-29a-3p inhibits ET-1-induced cardiomyocyte hypertrophy via inhibiting NFATc4 expression.

Ca²âº signal-induced cardiomyocyte hypertrophy through activation of myocardin.

Hypertrophic growth of cardiomyocytes in response to pressure overload is an important stage during the development of many cardiac diseases. Ca(2+) overload as well as subsequent activation of Ca(2+) signaling pathways has been reported to induce cardiac hypertrophy. Myocardin, a transcription cofactor of serum response factor (SRF), is a key transducer of hypertrophic signals. However, the direct role of myocardin in Ca(2+) signal-induced cardiomyocyte hypertrophy has not been explained clearly. In the present study, we discovered that embryonic rat heart-derived H9c2 cells responded to the stimulation of calcium ionophore A23187 with a cell surface area enlargement and an increased expression of cardiac hypertrophy marker genes. Increased Ca(2+) also induces an organization of sarcomeres in neonatal rat cardiomyocytes, as revealed by α-actinin staining. Increased Ca(2+) could upregulate the expression of myocardin. Knockdown of myocardin by shRNA attenuates hypertrophic responses triggered by increased intracellular Ca(2+), suggesting that Ca(2+) signals induce cardiomyocyte hypertrophy partly through activation of myocardin. Furthermore, A23187 treatment directly activates myocardin promoter, chelation of Ca(2+) by EGTA inhibits this activation and knockdown of myocardin expression using shRNA also abrogates A23187-induced ANF and SK-α-actin promoter activity. CSA (calcineurin inhibitor) and KN93 (CaMKII inhibitor) inhibit A23187-induced the increase in myocardin expression. These results suggest that myocardin plays a critical role in Ca(2+) signal-induced cardiomyocyte hypertrophy, which may serve as a novel mechanism that is important for cardiac hypertrophy.

NF-κB (p65) negatively regulates myocardin-induced cardiomyocyte hypertrophy through multiple mechanisms.

Myocardin is well known to play a key role in the development of cardiomyocyte hypertrophy. But the exact molecular mechanism regulating myocardin stability and transactivity to affect cardiomyocyte hypertrophy has not been studied clearly. We now report that NF-κB (p65) can inhibit myocardin-induced cardiomyocyte hypertrophy. Then we explore the molecular mechanism of this response. First, we show that p65 can functionally repress myocardin transcriptional activity and also reduce the protein expression of myocardin. Second, the function of myocardin can be regulated by epigenetic modifications. Myocardin sumoylation is known to transactivate cardiac genes, but whether p65 can inhibit SUMO modification of myocardin is still not clear. Our data show that p65 weakens myocardin transcriptional activity through attenuating SUMO modification of myocardin by SUMO1/PIAS1, thereby impairing myocardin-mediated cardiomyocyte hypertrophy. Furthermore, the expression of myocardin can be regulated by several microRNAs, which play important roles in the development and function of the heart and muscle. We next investigated potential role of miR-1 in cardiac hypotrophy. Our results show that p65 can upregulate the level of miR-1 and miR-1 can decrease protein expression of myocardin in cardiac myocytes. Notably, miR-1 expression is also controlled by myocardin, leading to a feedback loop. These data thus provide important and novel insights into the function that p65 inhibits myocardin-mediated cardiomyocyte hypertrophy by downregulating the expression and SUMO modification of myocardin and enhancing the expression of miR-1.