Methylation of the hippo signalling effector YAP by SETD7 drives myocardial ischemic injury

Abstract Introduction Myocardial ischemia/reperfusion (I/R) injury is one of the most deleterious cardiovascular conditions and a leading cause of mortality. The Hippo pathway effector YAP critically regulates cardiomyocyte proliferation and survival during myocardial I/R injury. However, the mechanisms regulating YAP activation in this setting remain poorly understood. Post-translational modifications of proteins, namely methylation, modulate pathways implicated in myocardial I/R injury. The methyltransferase SETD7 is emerging as a regulator of cell survival via methylation of histone and non-histone proteins. Whether SETD7 participates to myocardial I/R injury remains elusive. Purpose To investigate the role of SETD7 in regulating Hippo signaling during myocardial I/R injury. Methods Neonatal rat ventricular myocytes (NRVM) were exposed to normal glucose levels or glucose deprivation (GD) for 15 h, in the presence of the selective SETD7 inhibitor [(R)-PFI-2] or its inactive enantiomer [(S)-PFI-2]. Western blot and real time PCR were employed to investigate the effects of energy stress on SETD7 and the Hippo pathway, while apoptosis was assessed by Caspase-3 activity assay. YAP activity was assessed through chromatin immunoprecipitation assay (ChIP), its localization was examined by confocal microscopy while mono-methylation was assessed by immunoblotting. SETD7 knockout (SETD7−/−) mice and wild-type (WT) littermates (male, 8–12 weeks old) underwent 1 h of left anterior descending (LAD) coronary artery ligation followed by 24 h of reperfusion. Infarct size was assessed by TTC staining and shown as infarct size per ventricle surface (I/V). Cardiac function was investigated at 24h by conventional and Tissue Doppler Imaging (TDI) echocardiography. Results GD in NRVMs led to upregulation of SETD7 and physical interaction with the pro-survival transcriptional cofactor YAP, resulting in its direct mono-methylation. Furthermore SETD7-dependent methylation of YAP led to its cytosolic retention and subsequent reduction of YAP binding to the promoter of pro-survival genes. Of note, pharmacological inhibition of SETD7 by (R)-PFI-2 blunted YAP mono-methylation while restoring its nuclear retention. Mechanistically, SETD7 inhibition promoted YAP binding to catalase and superoxide dismutase (SOD) gene promoters, thus preventing GD-induced mitochondrial oxidative stress and apoptosis. In line with our in vitro findings, SETD7−/− mice showed decreased infarct size as compared to WT littermates and preserved cardiac systolic (ejection fraction, fractional shortening) and diastolic function, as assessed by both conventional and TDI echocardiography. Conclusions We show that SETD7-dependent methylation of YAP is required for its inactivation, thus leading to myocyte oxidative stress and apoptosis. Pharmacological modulation of SETD7 by (R)-PFI-2 may represent a new therapeutic approach to prevent myocardial ischemic damage through modulation of the Hippo pathway. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Swiss Heart Foundation

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P5378The methyltransferase SETD7 promotes myocardial ischemic injury by activating Hippo signalling

European Heart Journal ◽

10.1093/eurheartj/ehz746.0339 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

S Ambrosini ◽

F Montecucco ◽

A Akhmedov ◽

S A Mohammed ◽

P Brown ◽

...

Keyword(s):

Confocal Microscopy ◽

Myocardial Ischemia ◽

Cardiac Function ◽

Infarct Size ◽

Cellular Stress ◽

Hippo Pathway ◽

Neonatal Rat ◽

Doppler Imaging ◽

Coronary Artery Ligation ◽

The Hippo Pathway

Abstract Introduction Despite significant advances in coronary revascularization, acute myocardial infarction remains the leading cause of heart failure and death worldwide. The Hippo pathway is a master regulator of cell survival during myocardial ischemia. Upon cellular stress, activation of Hippo signaling leads to cytosolic retention and degradation of the pro-survival transcription factor YAP. Post-translational modifications, namely methylation, critically affect protein functionality in conditions of cellular stress. The SET domain-containing lysine methyltransferase 7 (SETD7) - which induces a specific mono-methylation of both histone and non-histone proteins - has recently emerged as key player in the pathogenesis of vascular disease. However, the role of SETD7 in the heart is largely unknown. Purpose The present study investigates whether SETD7 regulates the Hippo pathway during myocardial ischemia. Methods Neonatal rat ventricular myocytes (NRVM) were exposed to normal glucose levels or glucose deprivation (GD) for 15 h, in the presence of the selective SETD7 inhibitor [(R)-PFI-2] or its inactive enantiomer [(S)-PFI-2]. Western blot and real time PCR were employed to investigate the effects of energy stress on SETD7 and the Hippo pathway, while apoptosis was assessed by Caspase-3 activity assay. YAP localization was examined by confocal microscopy while its mono-methylation was assessed by immunoblotting. SETD7 knockout (SETD7−/−) mice and wild-type (WT) littermates (male, 8–12 weeks old) underwent 1 h of left anterior descending (LAD) coronary artery ligation followed by 24 h of reperfusion. Infarct size was assessed by TTC staining and shown as infarct size per ventricle surface (I/V). Cardiac function was investigated at 24h by conventional and Tissue Doppler Imaging echocardiography (Vevo 3100, Visualsonics). Results GD for 15h in NRVMs led to both YAP phosphorylation and mono-methylation, and subsequent cytosolic retention, as assessed by confocal microscopy. Reduced nuclear content of YAP was confirmed by downregulation of YAP-dependent pro-survival genes, namely Ctgf and Fgf2. GD-induced YAP inactivation was associated with an increase in SETD7 expression. Interestingly, pharmacological inhibition of SETD7 by (R)-PFI-2 blunted YAP mono-methylation, thus restoring nuclear retention of YAP and transcription of pro-survival genes in GD-treated NRVMs. Moreover, (R)-PFI-2 prevented NRVMs apoptosis. In line with our in vitro findings, SETD7−/− mice showed decreased infarct size as compared to WT littermates (I/V: 16.27%±2 vs. 20.54%±3, p<0.005, respectively). Consistently, cardiac function, as assessed by ejection fraction (EF: 46%±2 vs. 38%±5, p<0.001), fractional shortening (FS: 22%±1 vs. 18%±3, p<0.001) as well as by TDI, was preserved in mice lacking SETD7 as compared to WT animals. Conclusions Pharmacological modulation of SETD7 by (R)-PFI-2 may represent a novel therapeutic approach to prevent myocardial ischemic damage.

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Abstract P279: YAP, a Transcriptional Cofactor of the Hippo Pathway, Regulates Cardiomyocyte Growth, Survival, and Proliferation

Circulation Research ◽

10.1161/res.109.suppl_1.ap279 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Yanfei Yang ◽

Noritsugu Nakano ◽

Junichi Sadoshima

Keyword(s):

Cardiac Hypertrophy ◽

Hippo Pathway ◽

Neonatal Rat ◽

Border Zone ◽

Luciferase Reporter ◽

Tunel Staining ◽

H2o2 Treatment ◽

Rat Hearts ◽

The Hippo Pathway

Mst1 and Lats2, components of the mammalian Hippo pathway, stimulate apoptosis and inhibit hypertrophy of cardiomyocytes (CMs), thereby mediating reperfusion injury and heart failure. YAP, a transcription factor co-factor, is negatively regulated by the Hippo pathway, and controls cell survival, proliferation and tissue regeneration. The role of YAP in regulating growth and death of CMs is poorly understood. YAP overexpression in CMs induced cardiac hypertrophy, as indicated by increases in cell size (+1.2 fold, p<0.01), protein content (+1.1 fold, p<0.01) and ANF (luciferase reporter activity +1.7 fold, mRNA +2.2 fold, and staining +2.7 fold, p<0.01). Lats2 phosphorylates YAP at Serine 127, which induces cytoplasmic translocation of YAP, whereas YAP(S127A) is localized constitutively in the nucleus. Expression of YAP(S127A) enhanced hypertrophy in cultured CMs compared to that of wild type YAP (+1.87 fold ANF staining, p<0.05), suggesting that the Mst1/Hippo pathway negatively regulates cardiac hypertrophy through YAP. YAP inhibited cell death induced by H2O2 treatment, as evaluated with TUNEL staining (-65%, p<0.05) and CellTiter Blue assays (+34.9%, p<0.01), indicating that YAP plays an essential role in mediating CM survival. Interestingly, YAP also significantly increased Ki67 positive cells in cultured CMs compared to LacZ (+2.65 fold, p<0.05). We used a mouse model of chronic myocardial infarction (MI) to evaluate the function of YAP in the heart in vivo. Although YAP is diffusely localized both in the nucleus and cytosol in CMs in control hearts, CMs in the border zone of MI exhibited nuclear localization of YAP whereas YAP was excluded from the nucleus in CMs in the remodeling area four days after MI (+6.52 fold and +1.28 fold). Some of the YAP positive CMs in the border zone exhibited positive co-staining with Ki67, suggesting that YAP potentially induces CM proliferation. A significant increase in nuclear YAP and Ki67 positive CMs (+2.95 fold, p<0.01 and +2.18 fold, p<0.05) was also observed in neonatal rat hearts whose apex was surgically resected three days before euthanasia. These results suggest that YAP plays an important role in mediating not only hypertrophy and survival, but also proliferation of CMs in response to myocardial injury.

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Ferroptosis Signaling and Regulators in Atherosclerosis

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.809457 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yuqin Wang ◽

Yajie Zhao ◽

Ting Ye ◽

Liming Yang ◽

Yanna Shen ◽

...

Keyword(s):

Oxidative Stress ◽

Iron Homeostasis ◽

Hippo Pathway ◽

Signal Pathways ◽

Independent Manner ◽

Cellular Iron ◽

New Ideas ◽

Abnormal Lipid Metabolism ◽

The Hippo Pathway ◽

Cellular Iron Homeostasis

Atherosclerosis (AS) is a major cause of cardiovascular diseases such as coronary heart disease, heart failure and stroke. Abnormal lipid metabolism, oxidative stress and inflammation are the main features of AS. Ferroptosis is an iron-driven programmed cell death characterized by lipid peroxidation, which have been proved to participate in the development and progression of AS by different signal pathways. NRF2-Keap1 pathway decreases ferroptosis associated with AS by maintaining cellular iron homeostasis, increasing the production glutathione, GPX4 and NADPH. The p53 plays different roles in ferroptosis at different stages of AS in a transcription-dependent and transcription- independent manner. The Hippo pathway is involved in progression of AS, which has been proved the activation of ferroptosis. Other transcription factors, such as ATF3, ATF4, STAT3, also involved in the occurrence of ferroptosis and AS. Certain proteins or enzymes also have a regulatory role in AS and ferroptosis. In this paper, we review the mechanism of ferroptosis and its important role in AS in an attempt to find a new relationship between ferroptosis and AS and provide new ideas for the future treatment of AS.

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Abstract 357: Activation of Autophagic Flux Blunts Cardiac Ischemia/Reperfusion Injury

Circulation Research ◽

10.1161/res.121.suppl_1.357 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Geoffrey W Cho ◽

Min Xie ◽

Yongli Kong ◽

Dan L Li ◽

Xiang L Luo ◽

...

Keyword(s):

Oxidative Stress ◽

Infarct Size ◽

Reperfusion Injury ◽

Molecular Mechanisms ◽

Contractile Function ◽

Ischemia Reperfusion ◽

Neonatal Rat ◽

Border Zone ◽

Autophagic Flux ◽

Cardiomyocyte Autophagy

Background: Reperfusion injury accounts for a significant portion of myocardial damage in acute coronary syndromes. Autophagy, a process of cell catabolism, plays a vital role in the heart’s response to stress. We have reported that re-induction of ischemia/reperfusion (I/R)-suppressed cardiomyocyte autophagy with histone deacetylase (HDAC) inhibitors affords significant cardioprotection. However, as HDACs govern many processes and may have off-target effects, we set out to modulate autophagy in a manner independent of HDAC activity. Here, we hypothesized that induction of autophagy with a novel agent, Tat-Beclin, at the time of reperfusion, will reduce I/R injury and rescue cardiac function. Methods: Wild type and ATG7 (protein required for autophagic flux) knockout mice were randomized among 3 treatment groups prior to surgical I/R injury [45 min LAD artery ligation; 24h reperfusion]: vehicle control (VC), Tat-Scrambled (TS), or Tat-Beclin (TB). Each agent was delivered at coronary reperfusion. To define molecular mechanisms, cultured adult and neonatal rat ventricular cardiomyocytes (ARVMs/NRVMs) were subjected to simulated I/R. Results: Induction of cardiomyocyte autophagy at reperfusion reduced infarct size 20.1% (±6.3%, n=23, p<0.02 vs VC). This treatment was associated with improved systolic function (declines in fractional shortening: 19.8±3.7% VC; 18.7±2.1% TS; 8.5±1.7% TB, n=11, p<0.01 vs VC). In NRVMs subjected to I/R injury, cell death was reduced 41% (±6%, n=12, p<0.001 vs VC). Improvements correlated with increased autophagic flux measured by the marker LC3-II, particularly at the infarct border zone. Additional data suggested that autophagy rescues I/R injury through reduction of oxidative stress. ATG7 KO mice or NRVM depleted of ATG7 (RNAi) manifested significantly less cardioprotection. Conclusion: Direct induction of cardiomyocyte autophagy reduces infarct size and declines in contractile function. Autophagy rescues I/R injury in part through reduction of oxidative stress. Critically, this cardioprotection was observed when intervention occurred at the time of reperfusion, the clinically relevant context.

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Hippo signaling: A stress response pathway in stem cells

Current Stem Cell Research & Therapy ◽

10.2174/1574888x16666210712100002 ◽

2021 ◽

Vol 16 ◽

Author(s):

Naciye Dilara Zeybek ◽

Eylem Baysal ◽

Ozlem Bozdemir ◽

Esra Buber

Keyword(s):

Oxidative Stress ◽

Stem Cells ◽

Stem Cell ◽

Stress Response ◽

Biological Effects ◽

Energy Levels ◽

Hippo Pathway ◽

Hippo Signaling ◽

Stress Response Pathway ◽

The Hippo Pathway

: The Hippo pathway, with its core components and the downstream transcriptional coactivators, controls the self-renewable capacity and stemness features of stem cells and serves as a stress response pathway by regulating proliferation, differentiation, and apoptosis. The Hippo pathway interaction with other signaling ways plays a vital role in response to various stress stimuli arising from energy metabolism, hypoxia, reactive oxygen species, and mechanical forces. Depending on the energy levels, the Hippo pathway is regulated by AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR), which in turn determines stem cell proliferation (cell survival and growth) and differentiation. Oxidative stress-driven by ROS production also affects the Hippo pathway with transcriptional changes through MST/YAP/FoxO pathway and leads to the activation of pro-apoptotic genes and eventually cell death. HIF1alpha/YAP signaling is critical for the long-term maintenance of mesenchymal stem cells (MSCs) under hypoxia. In this review, we present an overview of stem cell response to stress, including mechanical, hypoxia, metabolic and oxidative stress through the modulation of the Hippo pathway. The biological effects such as autophagy, apoptosis and senescence were discussed in the context of the Hippo pathway in stem cells.

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Abstract 15524: The Methyltransferase Setd7 Drives Myocardial Ischemic Injury by Modulating the Hippo Pathway: A Study in Mice and Humans

Circulation ◽

10.1161/circ.142.suppl_3.15524 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Samuele Ambrosini ◽

Fabrizio Montecucco ◽

Daniela Pedicino ◽

Alexander Akhmedov ◽

Shafeeq Ahmed Mohammed ◽

...

Keyword(s):

Oxidative Stress ◽

Heart Failure ◽

Mononuclear Cells ◽

Hippo Pathway ◽

Ischemic Heart ◽

Ischemic Heart Failure ◽

Peripheral Blood Mononuclear ◽

Myocardial Oxidative Stress ◽

Blood Mononuclear Cells ◽

The Hippo Pathway

Introduction: Ischemic heart disease is a leading cause of death worldwide. Although revascularization strategies significantly reduce mortality after acute myocardial infarction (MI), a significant number of MI patients develop heart failure. Protein methylation is emerging as a key biological signal implicated in the pathophysiology of cardiovascular (CV) disease. In this regard, the methyltransferase SETD7 was recently shown to methylate proteins relevant to CV homeostasis. Hypothesis: To investigate SETD7 role in myocardial ischemia-reperfusion (I/R) injury. Methods: Experiments were performed in neonatal rat ventricular myocytes (NRVM), SETD7 knockout mice (SETD7 -/- ) undergoing myocardial I/R injury, myocardial samples from patients with and without ischemic heart failure as well as peripheral blood mononuclear cells from patients with ST elevation MI (STEMI, n=25) and age-matched healthy controls (n=20). Results: Glucose deprivation (GD) in NRVM led to upregulation of SETD7 and direct mono-methylation of the Hippo signaling effector YAP. SETD7-dependent methylation of YAP led to its cytosolic retention thus impeding YAP binding to the promoter of pro-survival genes. Selective pharmacological inhibition of SETD7 by (R)-PFI-2 blunted YAP mono-methylation thereby restoring its nuclear retention. We show that YAP binds the promoter of antioxidant genes catalase and superoxide dismutase, thus preventing GD-induced mitochondrial oxidative stress, organelle swelling and apoptosis. Consistently, infarct size, myocardial oxidative stress and left ventricular dysfunction were reduced in SETD7 -/- mice undergoing I/R as compared to wild-type littermates. Of clinical relevance, we found that SETD7/YAP signaling was deregulated in myocardial samples from patients with ischemic heart failure as well as in peripheral blood mononuclear cells from STEMI patients. Conclusions: We demonstrate that SETD7-dependent methylation of YAP is an important mechanism underpinning myocardial oxidative stress, mitochondrial damage and apoptosis during ischemia. Pharmacological modulation of SETD7 by (R)-PFI-2 may represent a potential therapeutic approach to prevent myocardial ischemic damage through modulation of the Hippo pathway.

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TAZ inhibits GR and coordinates hepatic glucose homeostasis in normal physiologic states

10.1101/2020.05.12.091264 ◽

2020 ◽

Author(s):

Simiao Xu ◽

Yangyang Liu ◽

Ruixiang Hu ◽

Min Wang ◽

Oliver Stöhr ◽

...

Keyword(s):

Glucose Homeostasis ◽

Blood Glucose Concentration ◽

Hippo Pathway ◽

Glucose Production ◽

Effector Proteins ◽

Binding Motif ◽

Gene Promoters ◽

Hepatic Glucose ◽

The Hippo Pathway

AbstractThe elucidation of the mechanisms whereby the liver maintains glucose homeostasis is crucial for the understanding of physiologic and pathologic states. Here, we show a novel role of hepatic transcriptional co-activator with PDZ-binding motif (TAZ) in the inhibition of glucocorticoid receptor (GR). TAZ interacts via its WW domain with the ligand-binding domain of GR to limit the binding of GR to gluconeogenic gene promoters. Therefore, liver-specific TAZ knockout mice show increases in glucose production and blood glucose concentration. Conversely, the overexpression of TAZ in mouse liver reduces the binding of GR to gluconeogenic gene promoters and glucose production. Thus, our findings demonstrate distinct roles of the hippo pathway effector proteins yes-associated protein 1 (YAP) and TAZ in liver physiology: while deletion of hepatic YAP has little effect on glucose homeostasis, hepatic TAZ protein expression decreases upon fasting and coordinates gluconeogenesis in response to physiologic fasting and feeding.

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