miR-34a Modulates Angiotensin II-Induced Myocardial Hypertrophy by Direct Inhibition of ATG9A Expression and Autophagic Activity

Cardiac hypertrophy is characterized by thickening myocardium and decreasing in heart chamber volume in response to mechanical or pathological stress, but the underlying molecular mechanisms remain to be defined. This study investigated altered miRNA expression and autophagic activity in pathogenesis of cardiac hypertrophy. A rat model of myocardial hypertrophy was used and confirmed by heart morphology, induction of cardiomyocyte autophagy, altered expression of autophagy-related ATG9A, LC3 II/I and p62 proteins, and decrease in miR-34a expression. The in vitro data showed that in hypertrophic cardiomyocytes induced by Ang II, miR-34a expression was downregulated, whereas ATG9A expression was up-regulated. Moreover, miR-34a was able to bind to ATG9A 3′-UTR, but not to the mutated 3′-UTR and inhibited ATG9A protein expression and autophagic activity. The latter was evaluated by autophagy-related LC3 II/I and p62 levels, TEM, and flow cytometry in rat cardiomyocytes. In addition, ATG9A expression induced either by treatment of rat cardiomyocytes with Ang II or ATG9A cDNA transfection upregulated autophagic activity and cardiomyocyte hypertrophy in both morphology and expression of hypertrophy-related genes (i.e., ANP and β-MHC), whereas knockdown of ATG9A expression downregulated autophagic activity and cardiomyocyte hypertrophy. However, miR-34a antagonized Ang II-stimulated myocardial hypertrophy, whereas inhibition of miR-34a expression aggravated Ang II-stimulated myocardial hypertrophy (such as cardiomyocyte hypertrophy-related ANP and β-MHC expression and cardiomyocyte morphology). This study indicates that miR-34a plays a role in regulation of Ang II-induced cardiomyocyte hypertrophy by inhibition of ATG9A expression and autophagic activity.     

Adiponectin up‐regulates the decrease of myocardial autophagic flux induced by β1‐adrenergic receptor autoantibody partly dependent on AMPK

Cardiomyocytes autophagy is essential for maintaining cardiac function. Our previous studies have found that β₁‐adrenergic receptor autoantibody (β₁‐AA) induced the decreased myocardial autophagic flux, which resulted in cardiomyocyte death and cardiac dysfunction. And other studies demonstrated that β₁‐AA induced the decrease of AMPK phosphorylation, the key hub of autophagy pathway, while adiponectin up‐regulated autophagic flux mediated by AMPK. However, it is not clear whether adiponectin improves the inhibition of myocardial autophagic flux induced by β₁‐AA by up‐regulating the level of AMPK phosphorylation. In this study, it has been confirmed that β₁‐AA induced the decrease of AMPK phosphorylation level in both vivo and vitro. Moreover, pretreatment of cardiomyocytes with AMPK inhibitor Compound C could further reduce the autophagic flux induced by β₁‐AA. Adiponectin deficiency could aggravate the decrease of myocardial AMPK phosphorylation level, autophagic flux and cardiac function induced by β₁‐AA. Further, exogenous adiponectin could reverse the decline of AMPK phosphorylation level and autophagic flux induced by β₁‐AA and even reduce cardiomyocyte death. While pretreated with the Compound C, the adiponectin treatment did not improve the decreased autophagosome formation, but still improved the decreased autophagosome clearance induced by β₁‐AA in cardiomyocytes. This study is the first time to confirm that β₁‐AA could inhibit myocardial autophagic flux by down‐regulating AMPK phosphorylation level. Adiponectin could improve the inhibition of myocardial autophagic flux induced by β₁‐AA partly dependent on AMPK, so as to provide an experimental basis for the treatment of patients with β₁‐AA‐positive cardiac dysfunction.     

Cardiac myocyte-specific ablation of follistatin-like 3 attenuates stress-induced myocardial hypertrophy

Transforming growth factor-β family cytokines have diverse actions in the maintenance of cardiac homeostasis. Follistatin-like 3 (Fstl3) is an extracellular regulator of certain TGF-β family members, including activin A. The aim of this study was to examine the role of Fstl3 in cardiac hypertrophy. Cardiac myocyte-specific Fstl3 knock-out (KO) mice and control mice were subjected to pressure overload induced by transverse aortic constriction (TAC). Cardiac hypertrophy was assessed by echocardiography and histological and biochemical methods. KO mice showed reduced cardiac hypertrophy, pulmonary congestion, concentric LV wall thickness, LV dilatation, and LV systolic dysfunction after TAC compared with control mice. KO mice displayed attenuated increases in cardiomyocyte cell surface area and interstitial fibrosis following pressure overload. Although activin A was similarly up-regulated in KO and control mice after TAC, a significant increase in Smad2 phosphorylation only occurred in KO mice. Knockdown of Fstl3 in cultured cardiomyocytes inhibited PE-induced cardiac hypertrophy. Conversely, adenovirus-mediated Fstl3 overexpression blocked the inhibitory action of activin A on hypertrophy and Smad2 activation. Transduction with Smad7, a negative regulator of Smad2 signaling, blocked the antihypertrophic actions of activin A stimulation or Fstl3 ablation. These findings identify Fstl3 as a stress-induced regulator of hypertrophy that controls myocyte size via regulation of Smad signaling.     

Piceatannol attenuates cardiac hypertrophy in an animal model through regulation of the expression and binding of the transcription factor GATA binding factor 6

Piceatannol is found in grapes, passion fruit, and Japanese knotweed. Piceatannol pretreatment suppresses cardiac hypertrophy induced by isoproterenol as assessed by heart weight/body weight ratio, cross-sectional area, and expression of hypertrophic markers. The anti-hypertrophic effect of piceatannol in rat neonatal cardiomyocytes is the same as that in vivo. Piceatannol inhibits lentiviral-GATA6-induced cardiomyocyte hypertrophy. Furthermore, piceatannol reduces the interaction between GATA4 and GATA6 as well as the DNA-binding activity of endogenous GATA6 in the ANP promoter. Our results suggest that piceatannol may be a novel therapeutic agent for the prevention of cardiac hypertrophy.Structured summary of protein interactionsGATA6 physically interacts with GATA4 by anti V5 tag coimmunoprecipitation (View interaction)     

Hypertrophic Preconditioning Attenuates Myocardial Ischaemia‐Reperfusion Injury by Modulating SIRT3‐SOD2‐mROS‐Dependent Autophagy

BackgroundIschaemic preconditioning elicited by brief periods of coronary occlusion and reperfusion protects the heart from a subsequent prolonged ischaemic insult. Here, we test the hypothesis that short‐term non‐ischaemic stimulation of hypertrophy renders the heart resistant to subsequent ischaemic injury.Methods and ResultsTransient transverse aortic constriction (TAC) was performed for 3 days in mice and then withdrawn for 4 days by aortic debanding, followed by subsequent exposure to myocardial ischaemia‐reperfusion (I/R) injury. Following I/R injury, myocardial infarct size and apoptosis were significantly decreased, and cardiac dysfunction was markedly improved in the TAC preconditioning group compared with the control group. Mechanistically, TAC preconditioning markedly suppressed I/R‐induced autophagy and preserved autophagic flux by deacetylating SOD2 via a SIRT3‐dependent mechanism. Moreover, treatment with an adenovirus encoding SIRT3 partially mimicked the effects of hypertrophic preconditioning, whereas genetic ablation of SIRT3 in mice blocked the cardioprotective effects of hypertrophic preconditioning. Furthermore, in vivo lentiviral‐mediated knockdown of Beclin 1 in the myocardium ameliorated the I/R‐induced impairment of autophagic flux and was associated with a reduction in cell death, whereas treatment with a lentivirus encoding Beclin 1 abolished the cardioprotective effect of TAC preconditioning.ConclusionsThe present study identifies TAC preconditioning as a novel strategy for induction of an endogenous self‐defensive and cardioprotective mechanism against cardiac injury. Specifically, TAC preconditioning reduced myocardial autophagic cell death in a SIRT3/SOD2 pathway‐dependent manner.     

Early NADPH oxidase-2 activation is crucial in phenylephrine-induced hypertrophy of H9c2 cells

Reactive oxygen species (ROS) produced by different NADPH oxidases (NOX) play a role in cardiomyocyte hypertrophy induced by different stimuli, such as angiotensin II and pressure overload. However, the role of the specific NOX isoforms in phenylephrine (PE)-induced cardiomyocyte hypertrophy is unknown. Therefore we aimed to determine the involvement of the NOX isoforms NOX1, NOX2 and NOX4 in PE-induced cardiomyocyte hypertrophy. Hereto rat neonatal cardiomyoblasts (H9c2 cells) were incubated with 100 μM PE to induce hypertrophy after 24 and 48 h as determined via cell and nuclear size measurements using digital imaging microscopy, electron microscopy and an automated cell counter. Digital-imaging microscopy further revealed that in contrast to NOX1 and NOX4, NOX2 expression increased significantly up to 4 h after PE stimulation, coinciding and co-localizing with ROS production in the cytoplasm as well as the nucleus. Furthermore, inhibition of NOX-mediated ROS production with apocynin, diphenylene iodonium (DPI) or NOX2 docking sequence (Nox2ds)-tat peptide during these first 4 h of PE stimulation significantly inhibited PE-induced hypertrophy of H9c2 cells, both after 24 and 48 h of PE stimulation. These data show that early NOX2-mediated ROS production is crucial in PE-induced hypertrophy of H9c2 cells.     

HMG-CoA reductase inhibitor fluvastatin prevents angiotensin II-induced cardiac hypertrophy via Rho kinase and inhibition of cyclin D1

HMG-CoA reductase inhibitors, so called statins, decrease cardiac events. Previous studies have shown that HMG-CoA reductase inhibitors inhibit cardiomyocyte hypertrophy in vitro and in vivo by blocking Rho isoprenylation. We have shown that the G1 cell cycle regulatory proteins cyclin D1 and Cdk4 play important roles in cardiomyocyte hypertrophy. However, the relation between Rho and cyclin D1 in cardiomyocyte is unknown. To investigate whether HMG-CoA reductase inhibitors prevent cardiac hypertrophy through attenuation of Rho and cyclin D1, we studied the effect of fluvastatin on angiotensin II-induced cardiomyocyte hypertrophy in vitro and in vivo. Angiotensin II increased the cell surface area and [(3)H]leucine uptake of cultured neonatal rat cardiomyocytes and these changes were suppressed by fluvastatin treatment. Angiotensin II also induced activation of Rho kinase and increased cyclin D1, both of which were also significantly suppressed by fluvastatin. Specific Rho kinase inhibitor, Y-27632 inhibited angiotensin II-induced cardiomyocyte hypertrophy and increased cyclin D1. Overexpression of cyclin D1 by adenoviral gene transfer induced cardiomyocyte hypertrophy, as evidenced by increased cell size and increased protein synthesis; this hypertrophy was not diminished by concomitant treatment with fluvastatin. Infusion of angiotensin II to Wistar rats for 2 weeks induced hypertrophic changes in cardiomyocytes, and this hypertrophy was prevented by oral fluvastatin treatment. These results show that an HMG-CoA reductase inhibitor, fluvastatin, prevents angiotensin II-induced cardiomyocyte hypertrophy in part through inhibition of cyclin D1, which is linked to Rho kinase. This novel mechanism discovered for fluvastatin could be revealed how HMG-CoA reductase inhibitors are preventing cardiac hypertrophy.     

β-Catenin/TCF/LEF1 can directly regulate phenylephrine-induced cell hypertrophy and Anf transcription in cardiomyocytes

β-Catenin/TCF/LEF1 signaling is implicated in cardiac hypertrophy. We demonstrate that knockdown of β-catenin attenuates phenylephrine (PE)-induced cardiomyocyte hypertrophy and the up-regulation of the fetal gene Anf. We explore the mechanism through which β-catenin regulates Anf expression and find a consensus binding sequence on the Anf promoter for TCF/LEF1 family members. LEF1 binds directly to the Anf promoter via this sequence, which shows functional significance, and PE stimulation enhances recruitment of β-catenin onto the Anf promoter. Thus, we document a direct positive role of β-catenin on PE-induced cardiomyocyte hypertrophy and identify a new target gene for β-catenin/TCF/LEF1.