Loss of crossbridge inhibition drives pathological cardiac hypertrophy in patients harboring the TPM1 E192K mutation

Hypertrophic cardiomyopathy (HCM) is an inherited disorder caused primarily by mutations to thick and thinfilament proteins. Although thin filament mutations are less prevalent than their oft-studied thick filament counterparts, they are frequently associated with severe patient phenotypes and can offer important insight into fundamental disease mechanisms. We have performed a detailed study of tropomyosin (TPM1) E192K, a variant of uncertain significance associated with HCM. Molecular dynamics revealed that E192K results in a more flexible TPM1 molecule, which could affect its ability to regulate crossbridges. In vitro motility assays of regulated actin filaments containing TPM1 E192K showed an overall loss of Ca2+ sensitivity. To understand these effects, we used multiscale computational models that suggested a subtle phenotype in which E192K leads to an inability to completely inhibit actin–myosin crossbridge activity at low Ca2+. To assess the physiological impact of the mutation, we generated patient-derived engineered heart tissues expressing E192K. These tissues showed disease features similar to those of the patients, including cellular hypertrophy, hypercontractility, and diastolic dysfunction. We hypothesized that excess residual crossbridge activity could be triggering cellular hypertrophy, even if the overall Ca2+ sensitivity was reduced by E192K. To test this hypothesis, the cardiac myosin–specific inhibitor mavacamten was applied to patient-derived engineered heart tissues for 4 d followed by 24 h of washout. Chronic mavacamten treatment abolished contractile differences between control and TPM1 E192K engineered heart tissues and reversed hypertrophy in cardiomyocytes. These results suggest that the TPM1 E192K mutation triggers cardiomyocyte hypertrophy by permitting excess residual crossbridge activity. These studies also provide direct evidence that myosin inhibition by mavacamten can counteract the hypertrophic effects of mutant tropomyosin.

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Abstract P459: Mavacamten And Danicamtiv Reverse Respective Contractile Abnormalities In Engineered Heart Tissue Models Of Hypertrophic And Dilated Cardiomyopathy

Circulation Research ◽

10.1161/res.129.suppl_1.p459 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Saiti S Halder ◽

Lorenzo R Sewanan ◽

Michael J Rynkiewicz ◽

Jeffrey R Moore ◽

William J Lehman ◽

...

Keyword(s):

Dilated Cardiomyopathy ◽

Calcium Sensitivity ◽

Heart Tissue ◽

Missense Mutations ◽

Wild Type ◽

Twitch Force ◽

In Vitro Motility ◽

Isometric Twitch ◽

Engineered Heart Tissues

Missense mutations in alpha-tropomyosin (TPM1) can lead to development of hypertrophic (HCM) or dilated cardiomyopathy (DCM). HCM mutation E62Q and DCM mutation E54K have previously been studied extensively in experimental systems ranging from in vitro biochemical assays to animal models, although some conflicting results have been found. We undertook a detailed multi-scale assessment of these mutants that included atomistic simulations, regulated in vitro motility (IVM) assays, and finally physiologically relevant human engineered heart tissues. In IVM assays, E62Q previously has shown increased Calcium sensitivity. New molecular dynamics data shows mutation-induced changes to tropomyosin dynamics and interactions with actin and troponin. Human engineered heart tissues (EHT) were generated by seeding iPSC-derived cardiomyocytes engineered using CRISPR/CAS9 to express either E62Q or E54K cardiomyopathy mutations. After two weeks in culture, E62Q EHTs showed a drastically hypercontractile twitch force and significantly increased stiffness while displaying little difference in twitch kinetics compared to wild-type isogenic control EHTs. On the other hand, E54K EHTs displayed hypocontractile isometric twitch force with faster kinetics, impaired length-dependent activation and lowered stiffness. Given these contractile abnormalities, we hypothesized that small molecule myosin modulators to appropriately activate or inhibit myosin activity would restore E54K or E62Q EHTs to normal behavior. Accordingly, E62Q EHTs were treated with 0.5μM mavacamten (to remedy hypercontractility) and E54K EHTs with 0.5 μM danicamtiv (to remedy hypocontractility) for 4 days, followed by a 1 day washout period. Upon contractility testing, it was observed that the drugs were able to reverse contractile phenotypes observed in mutant EHTs and restore contractile properties to levels resembling those of the untreated wild type group. The computational, IVM and EHT studies provide clear evidence in support of the hyper- vs. hypo-contractility paradigm as a common axis that distinguishes HCM and DCM TPM1 mutations. Myosin modulators that directly compensate for underlying myofilament aberrations show promising efficacy in human in vitro systems.

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Cardiomyocyte-Specific RIP2 Overexpression Exacerbated Pathologic Remodeling and Contributed to Spontaneous Cardiac Hypertrophy

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.688238 ◽

2021 ◽

Vol 9 ◽

Author(s):

Jing-jing Yang ◽

Nan Zhang ◽

Zi-ying Zhou ◽

Jian Ni ◽

Hong Feng ◽

...

Keyword(s):

Signaling Pathways ◽

Cardiac Remodeling ◽

Specific Inhibitor ◽

Pressure Overload ◽

Neonatal Rat ◽

Hek293 Cells ◽

Cardiomyocyte Hypertrophy ◽

Rat Cardiomyocytes

This study aimed to investigate the role and mechanisms of Receptor interacting protein kinase 2 (RIP2) in pressure overload-induced cardiac remodeling. Human failing or healthy donor hearts were collected for detecting RIP2 expression. RIP2 cardiomyocyte-specific overexpression, RIP2 global knockout, or wild-type mice were subjected to sham or aortic banding (AB) surgery to establish pressure overload-induced cardiac remodeling in vivo. Phenylephrine (PE)-treated neonatal rat cardiomyocytes (NRCMs) were used for further investigation in vitro. The expression of RIP2 was significantly upregulated in failing human heart, mouse remodeling heart, and Ang II-treated NRCMs. RIP2 overexpression obviously aggravated pressure overload-induced cardiac remodeling. Mechanistically, RIP2 overexpression significantly increased the phosphorylation of TAK1, P38, and JNK1/2 and enhanced IκBα/p65 signaling pathway. Inhibiting TAK1 activity by specific inhibitor completely prevented cardiac remodeling induced by RIP2 overexpression. This study further confirmed that RIP2 overexpression in NRCM could exacerbate PE-induced NRCM hypertrophy and TAK1 silence by specific siRNA could completely rescue RIP2 overexpression-mediated cardiomyocyte hypertrophy. Moreover, this study showed that RIP2 could bind to TAK1 in HEK293 cells, and PE could promote their interaction in NRCM. Surprisingly, we found that RIP2 overexpression caused spontaneous cardiac remodeling at the age of 12 and 18 months, which confirmed the powerful deterioration of RIP2 overexpression. Finally, we indicated that RIP2 global knockout attenuated pressure overload-induced cardiac remodeling via reducing TAK1/JNK1/2/P38 and IκBα/p65 signaling pathways. Taken together, RIP2-mediated activation of TAK1/P38/JNK1/2 and IκBα/p65 signaling pathways played a pivotal role in pressure overload-induced cardiac remodeling and spontaneous cardiac remodeling induced by RIP2 overexpression, and RIP2 inhibition might be a potential strategy for preventing cardiac remodeling.

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Novel PGC-1α/ATF5 Axis Partly Activates UPRmt and Mediates Cardioprotective Role of Tetrahydrocurcumin in Pathological Cardiac Hypertrophy

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/9187065 ◽

2020 ◽

Vol 2020 ◽

pp. 1-21

Author(s):

Bing Zhang ◽

Yanzhen Tan ◽

Zhengbin Zhang ◽

Pan Feng ◽

Wenyuan Ding ◽

...

Keyword(s):

Oxidative Stress ◽

Cardiac Hypertrophy ◽

Test Group ◽

Neonatal Rat ◽

Activation Mechanism ◽

Cardiomyocyte Hypertrophy ◽

Control Group ◽

Pathological Cardiac Hypertrophy

Mitochondrial unfolding protein response (UPRmt) effectively resists the pathological cardiac hypertrophy and improves the mitochondrial function. However, the specific activation mechanism and drugs that can effectively activate UPRmt in the cardiac muscle are yet to be elucidated. The aim of this study was to determine the regulation role of UPRmt on preventing pathological cardiac hypertrophy by tetrahydrocurcumin (THC) and explore its underlying molecular mechanism. Male C57BL/6J wild-type (WT) mice were divided into a control group and subjected to sham treatment for 4 weeks, and a test group which was subjected to transverse aortic constriction (TAC) surgery. Animals in the control and test group were orally administered THC (50 mg/kg) for 4 weeks after TAC procedure; an equivalent amount of saline was orally administered in the control sham-treated group and the TAC group. Subsequently, oxidative stress and UPRmt markers were assessed in these mice, and cardiac hypertrophy, fibrosis, and cardiac function were tested. Small interfering RNA (siRNA) targeting proliferator-activated receptor-gamma coactivator (PGC)-1α and activating transcription factor 5 (ATF5) were used to determine the UPRmt activation mechanism. THC supplement partly upregulated UPRmt effectors and inhibited TAC-induced oxidative stress compared with TAC-operated WT mice, thereby substantially attenuating contractile dysfunction, cardiac hypertrophy, and fibrosis. Furthermore, PGC-1α knockdown blunted the UPRmt activation and the cardioprotective role of THC. The interaction between PGC-1α and ATF5 was tested in neonatal rat cardiac myocytes under normal conditions. The results showed that PGC-1α was an upstream effector of ATF5 and partly activated UPRmt. In vitro, phenylephrine- (PE-) induced cardiomyocyte hypertrophy caused ATF5 upregulating rather than downregulating corresponding to the downregulation of PGC-1α. The PGC-1α/ATF5 axis mediated the UPRmt activation and stress-resistance role of THC in vitro. Collectively, the present study provides the first evidence that PGC-1 and ATF5 can form a signaling axis to partly activate UPRmt that mediates the cardioprotective role of THC in pathological cardiac hypertrophy.

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Hispidulin Attenuates Cardiac Hypertrophy by Improving Mitochondrial Dysfunction

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2020.582890 ◽

2020 ◽

Vol 7 ◽

Author(s):

Yan Wang ◽

Zengshuo Xie ◽

Nan Jiang ◽

Zexuan Wu ◽

Ruicong Xue ◽

...

Keyword(s):

Oxidative Stress ◽

Heart Failure ◽

Cardiac Hypertrophy ◽

Mitochondrial Function ◽

Specific Inhibitor ◽

Cardiomyocyte Hypertrophy ◽

Mitochondrial Morphology ◽

Protective Effects

Cardiac hypertrophy is a pathophysiological response to harmful stimuli. The continued presence of cardiac hypertrophy will ultimately develop into heart failure. The mitochondrion is the primary organelle of energy production, and its dysfunction plays a crucial role in the progressive development of heart failure from cardiac hypertrophy. Hispidulin, a natural flavonoid, has been substantiated to improve energy metabolism and inhibit oxidative stress. However, how hispidulin regulates cardiac hypertrophy and its underlying mechanism remains unknown. We found that hispidulin significantly inhibited pressure overload-induced cardiac hypertrophy and improved cardiac function in vivo and blocked phenylephrine (PE)-induced cardiomyocyte hypertrophy in vitro. We further proved that hispidulin remarkably improved mitochondrial function, manifested by increased electron transport chain (ETC) subunits expression, elevated ATP production, increased oxygen consumption rates (OCR), normalized mitochondrial morphology, and reduced oxidative stress. Furthermore, we discovered that Sirt1, a well-recognized regulator of mitochondrial function, might be a target of hispidulin, as evidenced by its upregulation after hispidulin treatment. Cotreatment with EX527 (a Sirt1-specific inhibitor) and hispidulin nearly completely abolished the antihypertrophic and protective effects of hispidulin on mitochondrial function, providing further evidence that Sirt1 could be the pivotal downstream effector of hispidulin in regulating cardiac hypertrophy.

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Studies on the Dynamic Localization of GFP-Myosin During Cytokinesis in Live Cells

Microscopy and Microanalysis ◽

10.1017/s1431927600007534 ◽

1997 ◽

Vol 3 (S2) ◽

pp. 129-130

Author(s):

James Sabry ◽

Sheri Moores ◽

Shannon Ryan ◽

Ji-Hong Zang ◽

James A. Spudich

Keyword(s):

Fluorescent Protein ◽

Molecular Motor ◽

Myosin Ii ◽

Thick Filament ◽

Live Cells ◽

In Vitro Motility ◽

Multimeric Complex ◽

Ring Constriction ◽

Green Fluorescent

Cell division is thought to be powered by the constriction of an actomyosin containing contractile ring found transiently in the cleavage furrow. Conventional myosin II plays a fundamental role in this process of cytokinesis where, in the form of a multimeric complex known as the bipolar thick filament, it is thought to be the molecular motor that generates the force necessary to cause ring constriction.In order to study the dynamics of this protein in the dividing cell, we have made a fusion protein of the green fluorescent protein (GFP) and the amino terminus of the Dictyostelium myosin heavy chain (GFP-myosin), and imaged the location of this protein in dividing Dictyostelium cells were it is the only myosin II present in the cell. The addition of GFP does not compromise the functioning of the myosin motor as evidenced by the fact that purified GFP-myosin has solution ATPase and in vitro motility kinetics similar to that of non-labelled myosin. In addition, GFP-myosin fully complements the myosin null mutation for both development and cytokinesis in suspension suggesting that GFP-myosin acts as a regulated motor when expressed in cells.

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Amlexanox and Forskolin Prevents Isoproterenol-Induced Cardiomyopathy by Subduing Cardiomyocyte Hypertrophy and Maladaptive Inflammatory Responses

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.719351 ◽

2021 ◽

Vol 9 ◽

Author(s):

Gabriel Komla Adzika ◽

Hongjian Hou ◽

Adebayo Oluwafemi Adekunle ◽

Ruqayya Rizvi ◽

Seyram Yao Adzraku ◽

...

Keyword(s):

Cardiac Hypertrophy ◽

Troponin I ◽

Inflammatory Responses ◽

Myocardial Hypertrophy ◽

Left Ventricular ◽

Cardiomyocyte Hypertrophy ◽

Myocardial Inflammation ◽

Treatment Interventions ◽

Pathological Cardiac Hypertrophy

Chronic catecholamine stress (CCS) induces the occurrence of cardiomyopathy—pathological cardiac hypertrophy (PCH), which is characterized by left ventricular systolic dysfunction (LVSD). Recently, mounting evidence has implicated myocardial inflammation in the exacerbation of pathological cardiac remodeling. However, there are currently no well-defined treatment interventions or regimes targeted at both the attenuation of maladaptive myocardial hypertrophy and inflammation during CCS to prevent PCH. G protein-coupled receptor kinase 5 (GRK5) and adenylyl cyclases (ACs)-cAMP mediates both cardiac and inflammatory responses. Also, GRK5 and ACs are implicated in stress-induced LVSD. Herein, we aimed at preventing PCH during CCS via modulating adaptive cardiac and inflammatory responses by inhibiting GRK5 and/or stimulating ACs. Isoproterenol-induced cardiomyopathy (ICM) was modeled using 0.5 mg/100 g/day isoproterenol injections for 40 days. Alterations in cardiac and inflammatory responses were assessed from the myocardia. Similarities in the immunogenicity of cardiac troponin I (cTnI) and lipopolysaccharide under CCS were assessed, and Amlexanox (35 μM/ml) and/or Forskolin (10 μM/ml) were then employed in vitro to modulate adaptive inflammatory responses by inhibiting GRK5 or activating ACs-cAMP, respectively. Subsequently, Amlexanox (2.5 mg/100 g/day) and/or Forskolin (0.5 mg/100 g/day) were then translated into in vivo during CCS to modulate adaptive cardiac and inflammatory responses. The effects of Amlexanox and Forskolin on regulating myocardial systolic functions and inflammatory responses during CCS were ascertained afterward. PCH mice had excessive myocardial hypertrophy, fibrosis, and aggravated LVSD, which were accompanied by massive CD68+ inflammatory cell infiltrations. In vitro, Forskolin-AC/cAMP was effective than Amlexanox-GRK5 at downregulating proinflammatory responses during stress; nonetheless, Amlexanox and Forskolin combination demonstrated the most efficacy in modulating adaptive inflammatory responses. Individually, the translated Amlexanox and Forskolin treatment interventions were ineffective at subduing the pathological remodeling and sustaining cardiac function during CCS. However, their combination was potent at preventing LVSD during CCS by attenuating maladaptive myocardial hypertrophy, fibrosis, and inflammatory responses. The treatment intervention attained its potency mainly via Forskolin-ACs/cAMP-mediated modulation of cardiac and inflammatory responses, coupled with Amlexanox inhibition of GRK5 mediated maladaptive cascades. Taken together, our findings highlight the Amlexanox and Forskolin combination as a potential therapeutic intervention for preventing the occurrence of pathological cardiac hypertrophy during chronic stress.

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LCZ696 Ameliorates Oxidative Stress and Pressure Overload-Induced Pathological Cardiac Remodeling by Regulating the Sirt3/MnSOD Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/9815039 ◽

2020 ◽

Vol 2020 ◽

pp. 1-15

Author(s):

Shi Peng ◽

Xiao-feng Lu ◽

Yi-ding Qi ◽

Jing Li ◽

Juan Xu ◽

...

Keyword(s):

Oxidative Stress ◽

Heart Failure ◽

Cardiac Hypertrophy ◽

Pressure Overload ◽

Neonatal Rat ◽

Cardiomyocyte Hypertrophy ◽

Rat Cardiomyocytes ◽

Pathological Cardiac Hypertrophy

Aims. We aimed to investigate whether LCZ696 protects against pathological cardiac hypertrophy by regulating the Sirt3/MnSOD pathway. Methods. In vivo, we established a transverse aortic constriction animal model to establish pressure overload-induced heart failure. Subsequently, the mice were given LCZ696 by oral gavage for 4 weeks. After that, the mice underwent transthoracic echocardiography before they were sacrificed. In vitro, we introduced phenylephrine to prime neonatal rat cardiomyocytes and small-interfering RNA to knock down Sirt3 expression. Results. Pathological hypertrophic stimuli caused cardiac hypertrophy and fibrosis and reduced the expression levels of Sirt3 and MnSOD. LCZ696 alleviated the accumulation of oxidative reactive oxygen species (ROS) and cardiomyocyte apoptosis. Furthermore, Sirt3 deficiency abolished the protective effect of LCZ696 on cardiomyocyte hypertrophy, indicating that LCZ696 induced the upregulation of MnSOD and phosphorylation of AMPK through a Sirt3-dependent pathway. Conclusions. LCZ696 may mitigate myocardium oxidative stress and apoptosis in pressure overload-induced heart failure by regulating the Sirt3/MnSOD pathway.

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FNDC5/Irisin inhibits pathological cardiac hypertrophy

Clinical Science ◽

10.1042/cs20190016 ◽

2019 ◽

Vol 133 (5) ◽

pp. 611-627 ◽

Cited By ~ 11

Author(s):

Qing Yu ◽

Wenxin Kou ◽

Xu Xu ◽

Shunping Zhou ◽

Peipei Luan ◽

...

Keyword(s):

Cardiac Hypertrophy ◽

Cardiac Function ◽

Pressure Overload ◽

Protective Role ◽

Cardiomyocyte Hypertrophy ◽

Dependent Manner ◽

Pathological Cardiac Hypertrophy ◽

Adam Family ◽

A Disintegrin And Metalloproteinase

Abstract Cardiac hypertrophy is a common pathophysiological process in various cardiovascular diseases, which still has no effective therapies. Irisin is a novel myokine mainly secreted by skeletal muscle and is believed to be involved in the regulation of energy metabolism. In the present study, we found that irisin expression was elevated in hypertrophic murine hearts and serum. Moreover, angiotension II-induced cardiomyocyte hypertrophy was attenuated after irisin administration and aggravated after irisin knockdown in vitro. Next, we generated transverse aortic constriction (TAC)-induced cardiac hypertrophy murine model and found that cardiac hypertrophy and fibrosis were significantly attenuated with improved cardiac function assessed by echocardiography after irisin treatment. Mechanistically, we demonstrated that FNDC5 was cleaved into irisin, at least partially, in a disintegrin and metalloproteinase (ADAM) family-dependent manner. ADAM10 was the candidate enzyme responsible for the cleavage. Further, we found irisin treatment activated AMPK and subsequently inhibited activation of mTOR. AMPK inhibition ablated the protective role of irisin administration. In conclusion, we find irisin is secreted in an ADAM family-dependent manner, and irisin treatment improves cardiac function and attenuates pressure overload-induced cardiac hypertrophy and fibrosis mainly through regulating AMPK-mTOR signaling.

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Lysosomal-Associated Protein Transmembrane 5 Functions as a Novel Negative Regulator of Pathological Cardiac Hypertrophy

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2021.740526 ◽

2021 ◽

Vol 8 ◽

Author(s):

Lu Gao ◽

Sen Guo ◽

Rui Long ◽

Lili Xiao ◽

Rui Yao ◽

...

Keyword(s):

Angiotensin Ii ◽

Cardiac Hypertrophy ◽

Ventricular Myocytes ◽

Therapeutic Potential ◽

Negative Regulator ◽

Neonatal Rat ◽

Cardiomyocyte Hypertrophy ◽

Pathological Cardiac Hypertrophy

Lysosomal-associated protein transmembrane 5 (LAPTM5) is mainly expressed in immune cells and has been reported to regulate inflammation, apoptosis and autophagy. Although LAPTM5 is expressed in the heart, whether LAPTM5 plays a role in regulating cardiac function remains unknown. Here, we show that the expression of LAPTM5 is dramatically decreased in murine hypertrophic hearts and isolated hypertrophic cardiomyocytes. In this study, we investigated the role of LAPTM5 in pathological cardiac hypertrophy and its possible mechanism. Our results show that LAPTM5 gene deletion significantly exacerbates cardiac remodeling, which can be demonstrated by reduced myocardial hypertrophy, fibrosis, ventricular dilation and preserved ejection function, whereas the opposite phenotype was observed in LAPTM5 overexpression mice. In line with the in vivo results, knockdown of LAPTM5 exaggerated angiotensin II-induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes, whereas overexpression of LAPTM5 protected against angiotensin II-induced cardiomyocyte hypertrophy in vitro. Mechanistically, LAPTM5 directly bound to Rac1 and further inhibited MEK-ERK1/2 signaling, which ultimately regulated the development of cardiac hypertrophy. In addition, the antihypertrophic effect of LAPTM5 was largely blocked by constitutively active mutant Rac1 (G12V). In conclusion, our results suggest that LAPTM5 is involved in pathological cardiac hypertrophy and that targeting LAPTM5 has great therapeutic potential in the treatment of pathological cardiac hypertrophy.

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Abstract 15577: Mst 4, a Novel Stripak Associated Kinase is Upregulated in Human Cardiomyopathy and Regulates Cardiomyocyte Hypertrophy, Contractility and Apoptosis

Circulation ◽

10.1161/circ.142.suppl_3.15577 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Matthias Eden ◽

Marius Leye ◽

Christin Tannert ◽

Norbert Frey

Keyword(s):

Cardiac Fibrosis ◽

Pressure Overload ◽

Adaptor Proteins ◽

Parp Cleavage ◽

Cardiomyocyte Hypertrophy ◽

Adult Rat ◽

Cellular Hypertrophy ◽

Subsequent Loss

Heart failure is one of the major causes of death worldwide. Its pathophysiology is complex and involves alterations in calcium cycling and hypertrophic signaling, as well as subsequent loss of cardiomyocytes due to apoptosis, cardiac fibrosis and progressive contractile dysfunction. The Striatin-interacting phosphatase and kinase (STRIPAK) complex is a mammalian multiprotein complex that consists of phosphatases, kinases and various adaptor proteins. Yet, the STRIPAK complex has so far not been studied in cardiomyocytes. Now we show in cardiomyocytes that the STRIPAK associated kinase MST4 directly interacts with a recently described novel cardiac STRIPAK protein, Myoscape/STRIP2. Multi tissue immunoblot experiments showed that Mst4 abundance is also highly enriched in heart and skeletal muscle. Immunoblot analyses in human biopsy samples form patients with dilated (DCM n=10) or ischemic cardiomyopathy (ICM n=10) revealed that MST4 is strongly upregulated in DCM (12-fold p<0.001) and ICM (9.6 fold p<0.05) vs. controls. Conversely, upon biomechanical stretch we observed a significant downregulation of MST4 in vitro by 30% (p<0.05) as well as in vivo (-45%, n=6; P<0.001) in a murine model for pressure overload (experimental aortic constriction, (TAC) vs. Sham) under Moreover, we also observed downregulation of MST4 in a porcine myocardial infarction model (-48% vs. porcine controls; n=5 p<0.05). Adenoviral overexpression of MST4 in NRVCM results in significant cellular hypertrophy (485μm2 vs 421μm2; p<0.001) compared to LacZ. Overexpression of MST4 also increases cellular and sarcomeric fractional shortening in adult rat cardiac myocytes vs. LacZ (p<0.05). Finally, MST4 overexpression also protects from cardiac apoptosis as shown by 90% reduced PARP cleavage as well as significantly reduced expression of Caspase 3 and 7 (n=3; p<0.05). Taken together we show that MST 4 is a cardiac enriched Kinase that regulates cardiomyocyte hypertrophy, contractility and apoptosis and may thus be a target for future therapeutic intervention

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