Class III PI3K‐mediated prolonged activation of autophagy plays a critical role in the transition of cardiac hypertrophy to heart failure

Pathological cardiac hypertrophy often leads to heart failure. Activation of autophagy has been shown in pathological hypertrophic hearts. Autophagy is regulated positively by Class III phosphoinositide 3‐kinase (PI3K). However, it is unknown whether Class III PI3K plays a role in the transition of cardiac hypertrophy to heart failure. To address this question, we employed a previously established cardiac hypertrophy model in heat shock protein 27 transgenic mice which shares common features with several types of human cardiomyopathy. Age‐matched wild‐type mice served as control. Firstly, a prolonged activation of autophagy, as reflected by autophagosome accumulation, increased LC3 conversion and decreased p62 protein levels, was detected in hypertrophic hearts from adaptive stage to maladaptive stage. Moreover, morphological abnormalities in myofilaments and mitochondria were presented in the areas accumulated with autophagosomes. Secondly, activation of Class III PI3K Vacuolar protein sorting 34 (Vps34), as demonstrated by upregulation of Vps34 expression, increased interaction of Vps34 with Beclin‐1, and deceased Bcl‐2 expression, was demonstrated in hypertrophic hearts from adaptive stage to maladaptive stage. Finally, administration with Wortmaninn, a widely used autophagy inhibitor by suppressing Class III PI3K activity, significantly decreased autophagy activity, improved morphologies of intracellular apartments, and most importantly, prevented progressive cardiac dysfunction in hypertrophic hearts. Collectively, we demonstrated that Class III PI3K plays a central role in the transition of cardiac hypertrophy to heart failure via a prolonged activation of autophagy in current study. Class III PI3K may serve as a potential target for the treatment and management of maladaptive cardiac hypertrophy.     

gp130 plays a critical role in pressure overload-induced cardiac hypertrophy

gp130, a common receptor for the interleukin 6 family, plays pivotal roles in growth and survival of cardiac myocytes. In the present study, we examined the role of gp130 in pressure overload-induced cardiac hypertrophy using transgenic (TG) mice, which express a dominant negative mutant of gp130 in the heart under the control of alpha myosin heavy chain promoter. TG mice were apparently healthy and fertile. There were no differences in body weight and heart weight between TG mice and littermate wild type (WT) mice. Pressure overload-induced increases in the heart weight/body weight ratio, ventricular wall thickness, and cross-sectional areas of cardiac myocytes were significantly smaller in TG mice than in WT mice. Northern blot analysis revealed that pressure overload-induced up-regulation of brain natriuretic factor gene and down-regulation of sarcoplasmic reticulum Ca(2+) ATPase 2 gene were attenuated in TG mice. Pressure overload activated ERKs and STAT3 in the heart of WT mice, whereas pressure overload-induced activation of STAT3, but not of ERKs, was suppressed in TG mice. These results suggest that gp130 plays a critical role in pressure overload-induced cardiac hypertrophy possibly through the STAT3 pathway.     

The role of post‐translational modifications in cardiac hypertrophy

Pathological cardiac hypertrophy involves excessive protein synthesis, increased cardiac myocyte size and ultimately the development of heart failure. Thus, pathological cardiac hypertrophy is a major risk factor for many cardiovascular diseases and death in humans. Extensive research in the last decade has revealed that post‐translational modifications (PTMs), including phosphorylation, ubiquitination, SUMOylation, O‐GlcNAcylation, methylation and acetylation, play important roles in pathological cardiac hypertrophy pathways. These PTMs potently mediate myocardial hypertrophy responses via the interaction, stability, degradation, cellular translocation and activation of receptors, adaptors and signal transduction events. These changes occur in response to pathological hypertrophy stimuli. In this review, we summarize the roles of PTMs in regulating the development of pathological cardiac hypertrophy. Furthermore, PTMs are discussed as potential targets for treating or preventing cardiac hypertrophy.     

Molecular biomarkers in cardiac hypertrophy

Cardiac hypertrophy is characterized by an increase in myocyte size in the absence of cell division. This condition is thought to be an adaptive response to cardiac wall stress resulting from the enhanced cardiac afterload. The pathogenesis of heart dysfunction, which is one of the primary causes of morbidity and mortality in elderly people, is often associated with myocardial remodelling caused by cardiac hypertrophy. In order to well understand the potential mechanisms, we described the molecules involved in the development and progression of myocardial hypertrophy. Increasing evidence has indicated that micro‐RNAs are involved in the pathogenesis of cardiac hypertrophy. In addition, molecular biomarkers including vascular endothelial growth factor B, NAD‐dependent deacetylase sirtuin‐3, growth/differentiation factor 15 and glycoprotein 130, also play important roles in the development of myocardial hypertrophy. Knowing the regulatory mechanisms of these biomarkers in the heart may help identify new molecular targets for the treatment of cardiac hypertrophy.     

Testin protects against cardiac hypertrophy by targeting a calcineurin‐dependent signalling pathway

Multiple organs express testin (TES), including the heart. Nevertheless, current understanding of the influence of TES on cardiovascular diseases, especially on cardiac hypertrophy and its etiology, is insufficient. This study investigated the influence of TES on cardiac hypertrophy and its etiology. Murine models with excessive TES expression specific to the heart were constructed with an adeno‐associated virus expression system. Cardiac hypertrophy was stimulated through aortic banding (AB). The severity of cardiac hypertrophy was evaluated through molecular, echocardiographic, pathological, and hemodynamic examination. The findings of our study revealed that TES expression was remarkably suppressed not only in failing human hearts but also in mouse hearts with cardiac hypertrophy. It was discovered that excessive TES expression driven by an adeno‐associated viral vector noticeably inhibited hypertrophy triggered by angiotensin II (Ang II) in cultivated cardiomyocytes from newborn rats. It was also revealed that TES knockdown via AdshTES caused the reverse phenotype in cardiomyocytes. Furthermore, it was proved that excessive TES expression attenuated the ventricular dilation, cardiac hypertrophy, dysfunction, and fibrosis triggered by AB in mice. It was discovered that TES directly interacted with calcineurin and suppressed its downstream signalling pathway. Moreover, the inactivation of calcineurin with cyclosporin A greatly offset the exacerbated hypertrophic response triggered by AB in TES knockdown mice. Overall, the findings of our study suggest that TES serves as a crucial regulator of the hypertrophic reaction by hindering the calcineurin‐dependent pathway in the heart.     

Cdc42 plays a critical role in assembly of sarcomere units in series of cardiac myocytes

Cardiomyocyte hypertrophy is observed in various cardiovascular diseases and causes heart failure. We here examined the role of small GTP-binding proteins of Rho family in phenylephrine (PE)-or leukocyte inhibitory factor (LIF)-induced hypertrophic morphogenesis of cultured neonatal rat cardiomyocytes. Both LIF and PE increased cell size of cardiomyocytes. LIF induced an increase in the length/width ratio of cardiomyocytes, while PE did not change the ratio. Adenoviral gene transfer of constitutively active mutants of Cdc42 increased the length/width ratio of cardiomyocytes and dominant negative mutants of Cdc42 conversely inhibited LIF-induced cell-elongation, while mutants of RhoA and Rac1 did not affect the length/width ratio of cardiomyocytes. These results suggest that Cdc42, but not RhoA and Rac1, is involved in LIF-induced sarcomere assembly in series in cardiomyocytes.     

Prevalence and upper limit of cardiac hypertrophy in professional cyclists

The term athlete's heart refers to an increased left ventricular mass. Few studies have assessed the prevalence and normal upper limit of cardiac hypertrophy in highly trained cyclists and this was the aim of this study. A group of 40 professional road cyclists [mean age 26 (SD 3) years] who had participated in European competitions for 3–10 years, were evaluated at the beginning of the 1992–93 season. Evaluation included a clinical history and physical examination, one and two-dimensional echocardiography, 12-lead resting electrocardiogram and a graded exercise test. Determination of the left ventricular mass index (LVMI) was performed using Devereux's formula with correction for the body surface area. Systolic and diastolic blood pressure were measured at rest and at peak exercise. Of the group 23 cyclists (58%) presented a LVMI greater than 130 g · m^–2, 21 cyclists presented a diastolic ventricular thickness equal to or greater than 13 mm, with a superior limit of 19 mm; 3 cyclists presented asymmetrical septum hypertrophy; and the relationship between posterior wall and left ventricular diastolic radius was equal to or greater than 0.45 in 14 cases (35%). Electrocardiographic abnormalities of ST-T segment were seen in only 1 subject. No correlation was found between the degree of ventricular hypertrophy and arterial blood pressure. We concluded that these professional cyclists showed a high prevalence of cardiac hypertrophy (58%). The distribution of this hypertrophy was concentric in 20/33 and asymmetric in 3/23 of the subjects with left ventricular hypertrophy. The electrocardiograms were normal in 98% of the subjects.     

The role of K63‐linked polyubiquitination in cardiac hypertrophy

Ubiquitination, also known as ubiquitylation, is a vital post‐translational modification of proteins that play a crucial role in the multiple biological processes including cell growth, proliferation and apoptosis. K63‐linked ubiquitination is one of the vital post‐translational modifications of proteins that are involved in the activation of protein kinases and protein trafficking during cell survival and proliferation. It also contributes to the development of various disorders including cancer, neurodegeneration and cardiac hypertrophy. In this review, we summarize the role of K63‐linked ubiquitination signalling in protein kinase activation and its implications in cardiac hypertrophy. We have also provided our perspectives on therapeutically targeting K63‐linked ubiquitination in downstream effector molecules of growth factor receptors for the treatment of cardiac hypertrophy.     

Renal dopamine receptors are involved in the development of cardiac hypertrophy

The present study examined the effect of fenoldopam, a known dopamine-1 receptor (DA1) agonist in order to understand its involvement in the cardiac hypertrophic process. Male Sprague-Dawley rats underwent abdominal aortic constriction (AB) with placement of a suprarenal ligature while sham operated animals served as controls. The AB groupsshowed an increase in their heart wt, left ventricular (LV) wt, heart wt/body wt and LV wt/body wt ratio. Furthermore, the length of these hearts, as measured from the auriculoventricular border to the apex, LV wall and interventricular (IV) septal thickness were increased from control levels. Treatment with SCH 23390, a DA1 antagonist, on the other hand, was able to partially regress the cardiac hypertrophic changes. All these parameters were also increased in control animals treated with fenoldopam (F). Such changes were more striking in the F+AB group which showed a significant acceleration of the cardiac hypertrophic process on super-imposing the two treatments. Plasma dopamine and renin activity were increased in all the groups as compared to control. These results indicate that dopamine receptors are implicated in the development of cardiac hypertrophy.     

IGF-1 deficiency resists cardiac hypertrophy and myocardial contractile dysfunction: role of microRNA-1 and microRNA-133a

This study was designed to examine the impact of insulin-like growth factor-1 (IGF-1) deficiency on abdominal aortic constriction (AAC)-induced cardiac geometric and functional changes with a focus on microRNA-1, 133a and 208, which are specially expressed in hearts and govern cardiac hypertrophy and stress-dependent cardiac growth. Liver-specific IGF-1-deficient (LID) and C57/BL6 mice were subject to AAC. Echocardiographic and cardiomyocyte function were assessed 4 wks later. Haematoxylin and eosin staining was used to monitor myocardial morphology. Western blot and real-time PCR were used to detect protein and miR expression, respectively. Neonatal rat cardiomyocytes (NRCMs) were transfected with miRs prior to IGF-1 exposure to initiate cell proliferation. Immunohistochemistry and [(3)H] Leucine incorporation were used to detect cell surface area and protein abundance. C57 mice subject to AAC displayed increased ventricular wall thickness, decreased left ventricular end diastolic and end systolic dimensions and elevated cardiomyocyte shortening capacity, all of which were attenuated in LID mice. In addition, IGF-1 deficiency mitigated AAC-induced increase in atrial natriuretic factor, GATA binding protein 4, glucose transporter 4 (GLUT4) and Akt phosphorylation. In contrast, neither AAC treatment nor IGF-1 deficiency affected glycogen synthase kinase 3b, mammalian target of rapamycin, the Glut-4 translocation mediator Akt substrate of 160 kD (AS160) and protein phosphatase. Levels of miR-1 and -133a (but not miR-208) were significantly attenuated by AAC in C57 but not LID mice. Transfection of miR-1 and -133a obliterated IGF-1-induced hypertrophic responses in NRCMs. Our data suggest that IGF-1 deficiency retards AAC-induced cardiac hypertrophic and contractile changes via alleviating down-regulation of miR-1 and miR-133a in response to left ventricular pressure overload.     

CD38 promotes angiotensin II‐induced cardiac hypertrophy

Cardiac hypertrophy is an early hallmark during the clinical course of heart failure and regulated by various signalling pathways. Recently, we observed that mouse embryonic fibroblasts from CD38 knockout mice were significantly resistant to oxidative stress such as H₂O₂‐induced injury and hypoxia/reoxygenation‐induced injury. In addition, we also found that CD38 knockout mice protected heart from ischaemia reperfusion injury through activating SIRT1/FOXOs‐mediated antioxidative stress pathway. However, the role of CD38 in cardiac hypertrophy is not explored. Here, we investigated the roles and mechanisms of CD38 in angiotensin II (Ang‐II)‐induced cardiac hypertrophy. Following 14 days of Ang‐II infusion with osmotic mini‐pumps, a comparable hypertension was generated in both of CD38 knockout and wild‐type mice. However, the cardiac hypertrophy and fibrosis were much more severe in wild‐type mice compared with CD38 knockout mice. Consistently, RNAi‐induced knockdown of CD38 decreased the gene expressions of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) and reactive oxygen species generation in Ang‐II‐stimulated H9c2 cells. In addition, the expression of SIRT3 was elevated in CD38 knockdown H9c2 cells, in which SIRT3 may further activate the FOXO3 antioxidant pathway. The intracellular Ca²⁺ release induced by Ang‐II markedly decreased in CD38 knockdown H9c2 cells, which might be associated with the decrease of nuclear translocation of NFATc4 and inhibition of ERK/AKT phosphorylation. We concluded that CD38 plays an essential role in cardiac hypertrophy probably via inhibition of SIRT3 expression and activation of Ca²⁺‐NFAT signalling pathway. Thus, CD38 may be a novel target for treating cardiac hypertrophy.     

iNOS in cardiac myocytes plays a critical role in death in a murine model of hypertrophy induced by calcineurin

Transgenic overexpression of calcineurin (CN/Tg) in mouse cardiac myocytes results in hypertrophy followed by dilation, dysfunction, and sudden death. Nitric oxide (NO) produced via inducible NO synthase (iNOS) has been implicated in cardiac injury. Since calcineurin regulates iNOS expression, and since phenotypes of mice overexpressing iNOS are similar to CN/Tg, we hypothesized that iNOS is pathogenically involved in cardiac phenotypes of CN/Tg mice. CN/Tg mice had increased serum and cardiac iNOS levels. When CN/Tg-iNOS(-/-) and CN/Tg mice were compared, some phenotypes were similar: extent of hypertrophy and fibrosis. However, CN/Tg-iNOS(-/-) mice had improved systolic performance (P < 0.001) and less heart block (P < 0.0001); larger sodium current density and lower serum TNF-alpha levels (P < 0.03); and less apoptosis (P < 0.01) resulting in improved survival (P < 0.0003). To define tissue origins of iNOS production, chimeric lines were generated. Bone marrow (BM) from wild-type or iNOS(-/-) mice was transplanted into CN/Tg mice. iNOS deficiency restricted to BM-derived cells was not protective. Calcineurin activates the local production of NO by iNOS in cardiac myocytes, which significantly contributes to sudden death, heart block, left ventricular dilation, and impaired systolic performance in this murine model of cardiac hypertrophy induced by the overexpression of calcineurin.     

Syringic acid mitigates isoproterenol‐induced cardiac hypertrophy and fibrosis by downregulating Ereg

Gallic acid has been reported to mitigate cardiac hypertrophy, fibrosis and arterial hypertension. The effects of syringic acid, a derivative of gallic acid, on cardiac hypertrophy and fibrosis have not been previously investigated. This study aimed to examine the effects of syringic acid on isoproterenol‐treated mice and cells. Syringic acid mitigated the isoproterenol‐induced upregulation of heart weight to bodyweight ratio, pathological cardiac remodelling and fibrosis in mice. Picrosirius red staining, quantitative real‐time polymerase chain reaction (qRT‐PCR) and Western blotting analyses revealed that syringic acid markedly downregulated collagen accumulation and fibrosis‐related factors, including Fn1. The results of RNA sequencing analysis of Ereg expression were verified using qRT‐PCR. Syringic acid or transfection with si‐Ereg mitigated the isoproterenol‐induced upregulation of Ereg, Myc and Ngfr. Ereg knockdown mitigated the isoproterenol‐induced upregulation of Nppb and Fn1 and enhancement of cell size. Mechanistically, syringic acid alleviated cardiac hypertrophy and fibrosis by downregulating Ereg. These results suggest that syringic acid is a potential therapeutic agent for cardiac hypertrophy and fibrosis.     

Long‐term administration of pyridostigmine attenuates pressure overload‐induced cardiac hypertrophy by inhibiting calcineurin signalling

Cardiac hypertrophy is associated with autonomic imbalance, characterized by enhanced sympathetic activity and withdrawal of parasympathetic control. Increased parasympathetic function improves ventricular performance. However, whether pyridostigmine, a reversible acetylcholinesterase inhibitor, can offset cardiac hypertrophy induced by pressure overload remains unclear. Hence, this study aimed to determine whether pyridostigmine can ameliorate pressure overload‐induced cardiac hypertrophy and identify the underlying mechanisms. Rats were subjected to either sham or constriction of abdominal aorta surgery and treated with or without pyridostigmine for 8 weeks. Vagal activity and cardiac function were determined using PowerLab. Cardiac hypertrophy was evaluated using various histological stains. Protein markers for cardiac hypertrophy were quantitated by Western blot and immunoprecipitation. Pressure overload resulted in a marked reduction in vagal discharge and a profound increase in cardiac hypertrophy index and cardiac dysfunction. Pyridostigmine increased the acetylcholine levels by inhibiting acetylcholinesterase in rats with pressure overload. Pyridostigmine significantly attenuated cardiac hypertrophy based on reduction in left ventricular weight/body weight, suppression of the levels of atrial natriuretic peptide, brain natriuretic peptide and β‐myosin heavy chain, and a reduction in cardiac fibrosis. These effects were accompanied by marked improvement of cardiac function. Additionally, pyridostigmine inhibited the CaN/NFAT3/GATA4 pathway and suppressed Orai1/STIM1 complex formation. In conclusion, pressure overload resulted in cardiac hypertrophy, cardiac dysfunction and a significant reduction in vagal discharge. Pyridostigmine attenuated cardiac hypertrophy and improved cardiac function, which was related to improved cholinergic transmission efficiency (decreased acetylcholinesterase and increased acetylcholine), inhibition of the CaN/NFAT3/GATA4 pathway and suppression of the interaction of Orai1/STIM1.     

Cordycepin ameliorates cardiac hypertrophy via activating the AMPKα pathway

Increase of myocardial oxidative stress is closely related to the occurrence and development of cardiac hypertrophy. Cordycepin, also known as 3'‐deoxyadenosine, is a natural bioactive substance extracted from Cordyceps militaris (which is widely cultivated for commercial use in functional foods and medicine). Since cordycepin suppresses oxidative stress both in vitro and in vivo, we hypothesized that cordycepin would inhibit cardiac hypertrophy by blocking oxidative stress‐dependent related signalling. In our study, a mouse model of cardiac hypertrophy was induced by aortic banding (AB) surgery. Mice were intraperitoneally injected with cordycepin (20 mg/kg/d) or the same volume of vehicle 3 days after‐surgery for 4 weeks. Our data demonstrated that cordycepin prevented cardiac hypertrophy induced by AB, as assessed by haemodynamic parameters analysis and echocardiographic, histological and molecular analyses. Oxidative stress was estimated by detecting superoxide generation, superoxide dismutase (SOD) activity and malondialdehyde levels, and by detecting the protein levels of gp91^phox and SOD. Mechanistically, we found that cordycepin activated activated protein kinase α (AMPKα) signalling and attenuated oxidative stress both in vivo in cordycepin‐treated mice and in vitro in cordycepin treated cardiomyocytes. Taken together, the results suggest that cordycepin protects against post‐AB cardiac hypertrophy through activation of the AMPKα pathway, which subsequently attenuates oxidative stress.     

Silibinin attenuates cardiac hypertrophy and fibrosis through blocking EGFR‐dependent signaling

Cardiac hypertrophy is a major determinant of heart failure. The epidermal growth factor receptor (EGFR) plays an important role in cardiac hypertrophy. Since silibinin suppresses EGFR in vitro and in vivo, we hypothesized that silibinin would attenuate cardiac hypertrophy through disrupting EGFR signaling. In this study, we examined this hypothesis using neonatal cardiac myocytes and fibroblasts induced by angiotensin II (Ang II) and animal model by aortic banding (AB) mice. Our data revealed that silibinin obviously blocked cardiac hypertrophic responses induced by pressure overload. Meanwhile, silibinin markedly reduced the increased generation of EGFR. Moreover, these beneficial effects were associated with attenuation of the EGFR‐dependent ERK1/2, PI3K/Akt signaling cascade. We further demonstrated silibinin decreased inflammation and fibrosis by blocking the activation of NF‐κB and TGF‐β1/Smad signaling pathways in vitro and in vivo. Our results indicate that silibinin has the potential to protect against cardiac hypertrophy, inflammation, and fibrosis through blocking EGFR activity and EGFR‐dependent different intracellular signaling pathways. J. Cell. Biochem. 110: 1111–1122, 2010. Published 2010 Wiley‐Liss, Inc.     

Sophocarpine ameliorates cardiac hypertrophy through activation of autophagic responses

ABSTRACT

Mounting evidences indicate that autophagy is an essential homeostatic mechanism to maintain the global cardiac structure function. Sophocarpine (SOP), a major bioactive compound derived from the natural plant Sophora flavescens. However, the role of SOP in cardiac hypertrophy remain to be fully elucidated. In the present study, we tested the hypothesis that SOP protects against Ang II–induced cardiac hypertrophy by mediating the regulation of autophagy. The results demonstrated that SOP attenuated the Ang II–induced cardiac hypertrophy, as assessed by measurements of echocardiography parameters, the ratios of heart weight/body weight and left ventricle weight/body weight, histopathological staining, cross-sectional cardiomyocyte area, and the expression levels of cardiac hypertrophic markers. The anti-hypertrophic effect of SOP was mediated by activating autophagy-related pathway, as revealed by reversal of the increased autophagy marker protein expression. These findings reveal a novel mechanism of SOP attenuating cardiac hypertrophy via activating autophagy-related signaling pathways.