Epicardial YAP/TAZ orchestrate an immunosuppressive response following myocardial infarction

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Abstract

Ischemic heart disease resulting from myocardial infarction (MI) is the most prevalent form of heart disease in the United States. Post-MI cardiac remodeling is a multifaceted process that includes activation of fibroblasts and a complex immune response. T-regulatory cells (Tregs), a subset of CD4⁺ T cells, have been shown to suppress the innate and adaptive immune response and limit deleterious remodeling following myocardial injury. However, the mechanisms by which injured myocardium recruits suppressive immune cells remain largely unknown. Here, we have shown a role for Hippo signaling in the epicardium in suppressing the post-infarct inflammatory response through recruitment of Tregs. Mice deficient in epicardial YAP and TAZ, two core Hippo pathway effectors, developed profound post-MI pericardial inflammation and myocardial fibrosis, resulting in cardiomyopathy and death. Mutant mice exhibited fewer suppressive Tregs in the injured myocardium and decreased expression of the gene encoding IFN-γ, a known Treg inducer. Furthermore, controlled local delivery of IFN-γ following MI rescued Treg infiltration into the injured myocardium of YAP/TAZ mutants and decreased fibrosis. Collectively, these results suggest that epicardial Hippo signaling plays a key role in adaptive immune regulation during the post-MI recovery phase.

Authors

Vimal Ramjee, Deqiang Li, Lauren J. Manderfield, Feiyan Liu, Kurt A. Engleka, Haig Aghajanian, Christopher B. Rodell, Wen Lu, Vivienne Ho, Tao Wang, Li Li, Anamika Singh, Dasan M. Cibi, Jason A. Burdick, Manvendra K. Singh, Rajan Jain, Jonathan A. Epstein

Adaptor proteins NUMB and NUMBL promote cell cycle withdrawal by targeting ERBB2 for degradation

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Abstract

Failure of trabecular myocytes to undergo appropriate cell cycle withdrawal leads to ventricular noncompaction and heart failure. Signaling of growth factor receptor ERBB2 is critical for myocyte proliferation and trabeculation. However, the mechanisms underlying appropriate downregulation of trabecular ERBB2 signaling are little understood. Here, we have found that the endocytic adaptor proteins NUMB and NUMBL were required for downregulation of ERBB2 signaling in maturing trabeculae. Loss of NUMB and NUMBL resulted in a partial block of late endosome formation, resulting in sustained ERBB2 signaling and STAT5 activation. Unexpectedly, activated STAT5 overrode Hippo-mediated inhibition and drove YAP1 to the nucleus. Consequent aberrant cardiomyocyte proliferation resulted in ventricular noncompaction that was markedly rescued by heterozygous loss of function of either ERBB2 or YAP1. Further investigations revealed that NUMB and NUMBL interacted with small GTPase Rab7 to transition ERBB2 from early to late endosome for degradation. Our studies provide insight into mechanisms by which NUMB and NUMBL promote cardiomyocyte cell cycle withdrawal and highlight previously unsuspected connections between pathways that are important for cardiomyocyte cell cycle reentry, with relevance to ventricular noncompaction cardiomyopathy and regenerative medicine.

Authors

Maretoshi Hirai, Yoh Arita, C. Jane McGlade, Kuo-Fen Lee, Ju Chen, Sylvia M. Evans

Cardiac myofibroblast engulfment of dead cells facilitates recovery after myocardial infarction

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Abstract

Myocardial infarction (MI) results in the generation of dead cells in the infarcted area. These cells are swiftly removed by phagocytes to minimize inflammation and limit expansion of the damaged area. However, the types of cells and molecules responsible for the engulfment of dead cells in the infarcted area remain largely unknown. In this study, we demonstrated that cardiac myofibroblasts, which execute tissue fibrosis by producing extracellular matrix proteins, efficiently engulf dead cells. Furthermore, we identified a population of cardiac myofibroblasts that appears in the heart after MI in humans and mice. We found that these cardiac myofibroblasts secrete milk fat globule-epidermal growth factor 8 (MFG-E8), which promotes apoptotic engulfment, and determined that serum response factor is important for MFG-E8 production in myofibroblasts. Following MFG-E8–mediated engulfment of apoptotic cells, myofibroblasts acquired antiinflammatory properties. MFG-E8 deficiency in mice led to the accumulation of unengulfed dead cells after MI, resulting in exacerbated inflammatory responses and a substantial decrease in survival. Moreover, MFG-E8 administration into infarcted hearts restored cardiac function and morphology. MFG-E8–producing myofibroblasts mainly originated from resident cardiac fibroblasts and cells that underwent endothelial-mesenchymal transition in the heart. Together, our results reveal previously unrecognized roles of myofibroblasts in regulating apoptotic engulfment and a fundamental importance of these cells in recovery from MI.

Authors

Michio Nakaya, Kenji Watari, Mitsuru Tajima, Takeo Nakaya, Shoichi Matsuda, Hiroki Ohara, Hiroaki Nishihara, Hiroshi Yamaguchi, Akiko Hashimoto, Mitsuho Nishida, Akiomi Nagasaka, Yuma Horii, Hiroki Ono, Gentaro Iribe, Ryuji Inoue, Makoto Tsuda, Kazuhide Inoue, Akira Tanaka, Masahiko Kuroda, Shigekazu Nagata, Hitoshi Kurose

The H3K9 dimethyltransferases EHMT1/2 protect against pathological cardiac hypertrophy

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Abstract

Cardiac hypertrophic growth in response to pathological cues is associated with reexpression of fetal genes and decreased cardiac function and is often a precursor to heart failure. In contrast, physiologically induced hypertrophy is adaptive, resulting in improved cardiac function. The processes that selectively induce these hypertrophic states are poorly understood. Here, we have profiled 2 repressive epigenetic marks, H3K9me2 and H3K27me3, which are involved in stable cellular differentiation, specifically in cardiomyocytes from physiologically and pathologically hypertrophied rat hearts, and correlated these marks with their associated transcriptomes. This analysis revealed the pervasive loss of euchromatic H3K9me2 as a conserved feature of pathological hypertrophy that was associated with reexpression of fetal genes. In hypertrophy, H3K9me2 was reduced following a miR-217–mediated decrease in expression of the H3K9 dimethyltransferases EHMT1 and EHMT2 (EHMT1/2). miR-217–mediated, genetic, or pharmacological inactivation of EHMT1/2 was sufficient to promote pathological hypertrophy and fetal gene reexpression, while suppression of this pathway protected against pathological hypertrophy both in vitro and in mice. Thus, we have established a conserved mechanism involving a departure of the cardiomyocyte epigenome from its adult cellular identity to a reprogrammed state that is accompanied by reexpression of fetal genes and pathological hypertrophy. These results suggest that targeting miR-217 and EHMT1/2 to prevent H3K9 methylation loss is a viable therapeutic approach for the treatment of heart disease.

Authors

Bernard Thienpont, Jan Magnus Aronsen, Emma Louise Robinson, Hanneke Okkenhaug, Elena Loche, Arianna Ferrini, Patrick Brien, Kanar Alkass, Antonio Tomasso, Asmita Agrawal, Olaf Bergmann, Ivar Sjaastad, Wolf Reik, Hywel Llewelyn Roderick

Hemoglobin S-nitrosylation plays an essential role in cardioprotection

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Abstract

Homeostatic control of tissue oxygenation is achieved largely through changes in blood flow that are regulated by the classic physiological response of hypoxic vasodilation. The role of nitric oxide (NO) in the control of blood flow is a central tenet of cardiovascular biology. However, extensive evidence now indicates that hypoxic vasodilation entails S-nitrosothiol–based (SNO-based) vasoactivity (rather than NO per se) and that this activity is conveyed substantially by the βCys93 residue in hemoglobin. Thus, tissue oxygenation in the respiratory cycle is dependent on S-nitrosohemoglobin. This perspective predicts that red blood cells (RBCs) may play an important but previously undescribed role in cardioprotection. Here, we have found that cardiac injury and mortality in models of myocardial infarction and heart failure were greatly enhanced in mice lacking βCys93 S-nitrosylation. In addition, βCys93 mutant mice exhibited adaptive collateralization of cardiac vasculature that mitigated ischemic injury and predicted outcomes after myocardial infarction. Enhanced myopathic injury and mortality across different etiologies in the absence of βCys93 confirm the central cardiovascular role of RBC-derived SNO-based vasoactivity and point to a potential locus of therapeutic intervention. Our findings also suggest the possibility that RBCs may play a previously unappreciated role in heart disease.

Authors

Rongli Zhang, Douglas T. Hess, James D. Reynolds, Jonathan S. Stamler

Transcription factor ETV1 is essential for rapid conduction in the heart

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Abstract

Rapid impulse propagation in the heart is a defining property of pectinated atrial myocardium (PAM) and the ventricular conduction system (VCS) and is essential for maintaining normal cardiac rhythm and optimal cardiac output. Conduction defects in these tissues produce a disproportionate burden of arrhythmic disease and are major predictors of mortality in heart failure patients. Despite the clinical importance, little is known about the gene regulatory network that dictates the fast conduction phenotype. Here, we have used signal transduction and transcriptional profiling screens to identify a genetic pathway that converges on the NRG1-responsive transcription factor ETV1 as a critical regulator of fast conduction physiology for PAM and VCS cardiomyocytes. Etv1 was highly expressed in murine PAM and VCS cardiomyocytes, where it regulates expression of Nkx2-5, Gja5, and Scn5a, key cardiac genes required for rapid conduction. Mice deficient in Etv1 exhibited marked cardiac conduction defects coupled with developmental abnormalities of the VCS. Loss of Etv1 resulted in a complete disruption of the normal sodium current heterogeneity that exists between atrial, VCS, and ventricular myocytes. Lastly, a phenome-wide association study identified a link between ETV1 and bundle branch block and heart block in humans. Together, these results identify ETV1 as a critical factor in determining fast conduction physiology in the heart.

Authors

Akshay Shekhar, Xianming Lin, Fang-Yu Liu, Jie Zhang, Huan Mo, Lisa Bastarache, Joshua C. Denny, Nancy J. Cox, Mario Delmar, Dan M. Roden, Glenn I. Fishman, David S. Park

Axial tubule junctions control rapid calcium signaling in atria

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Abstract

The canonical atrial myocyte (AM) is characterized by sparse transverse tubule (TT) invaginations and slow intracellular Ca²⁺ propagation but exhibits rapid contractile activation that is susceptible to loss of function during hypertrophic remodeling. Here, we have identified a membrane structure and Ca²⁺-signaling complex that may enhance the speed of atrial contraction independently of phospholamban regulation. This axial couplon was observed in human and mouse atria and is composed of voluminous axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum (SR) that include ryanodine receptor 2 (RyR2) clusters. In mouse AM, AT structures triggered Ca²⁺ release from the SR approximately 2 times faster at the AM center than at the surface. Rapid Ca²⁺ release correlated with colocalization of highly phosphorylated RyR2 clusters at AT-SR junctions and earlier, more rapid shortening of central sarcomeres. In contrast, mice expressing phosphorylation-incompetent RyR2 displayed depressed AM sarcomere shortening and reduced in vivo atrial contractile function. Moreover, left atrial hypertrophy led to AT proliferation, with a marked increase in the highly phosphorylated RyR2-pS2808 cluster fraction, thereby maintaining cytosolic Ca²⁺ signaling despite decreases in RyR2 cluster density and RyR2 protein expression. AT couplon “super-hubs” thus underlie faster excitation-contraction coupling in health as well as hypertrophic compensatory adaptation and represent a structural and metabolic mechanism that may contribute to contractile dysfunction and arrhythmias.

Authors

Sören Brandenburg, Tobias Kohl, George S.B. Williams, Konstantin Gusev, Eva Wagner, Eva A. Rog-Zielinska, Elke Hebisch, Miroslav Dura, Michael Didié, Michael Gotthardt, Viacheslav O. Nikolaev, Gerd Hasenfuss, Peter Kohl, Christopher W. Ward, W. Jonathan Lederer, Stephan E. Lehnart

Optogenetic defibrillation terminates ventricular arrhythmia in mouse hearts and human simulations

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Abstract

Ventricular arrhythmias are among the most severe complications of heart disease and can result in sudden cardiac death. Patients at risk currently receive implantable defibrillators that deliver electrical shocks to terminate arrhythmias on demand. However, strong electrical shocks can damage the heart and cause severe pain. Therefore, we have tested optogenetic defibrillation using expression of the light-sensitive channel channelrhodopsin-2 (ChR2) in cardiac tissue. Epicardial illumination effectively terminated ventricular arrhythmias in hearts from transgenic mice and from WT mice after adeno-associated virus–based gene transfer of ChR2. We also explored optogenetic defibrillation for human hearts, taking advantage of a recently developed, clinically validated in silico approach for simulating infarct-related ventricular tachycardia (VT). Our analysis revealed that illumination with red light effectively terminates VT in diseased, ChR2-expressing human hearts. Mechanistically, we determined that the observed VT termination is due to ChR2-mediated transmural depolarization of the myocardium, which causes a block of voltage-dependent Na⁺ channels throughout the myocardial wall and interrupts wavefront propagation into illuminated tissue. Thus, our results demonstrate that optogenetic defibrillation is highly effective in the mouse heart and could potentially be translated into humans to achieve nondamaging and pain-free termination of ventricular arrhythmia.

Authors

Tobias Bruegmann, Patrick M. Boyle, Christoph C. Vogt, Thomas V. Karathanos, Hermenegild J. Arevalo, Bernd K. Fleischmann, Natalia A. Trayanova, Philipp Sasse

Tyrosine kinase FYN negatively regulates NOX4 in cardiac remodeling

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Abstract

NADPH oxidases (Noxes) produce ROS that regulate cell growth and death. NOX4 expression in cardiomyocytes (CMs) plays an important role in cardiac remodeling and injury, but the posttranslational mechanisms that modulate this enzyme are poorly understood. Here, we determined that FYN, a Src family tyrosine kinase, interacts with the C-terminal domain of NOX4. FYN and NOX4 colocalized in perinuclear mitochondria, ER, and nuclear fractions in CMs, and FYN expression negatively regulated NOX4-induced O₂^– production and apoptosis in CMs. Mechanistically, we found that direct phosphorylation of tyrosine 566 on NOX4 was critical for this FYN-mediated negative regulation. Transverse aortic constriction activated FYN in the left ventricle (LV), and FYN-deficient mice displayed exacerbated cardiac hypertrophy and dysfunction and increased ROS production and apoptosis. Deletion of Nox4 rescued the exaggerated LV remodeling in FYN-deficient mice. Furthermore, FYN expression was markedly decreased in failing human hearts, corroborating its role as a regulator of cardiac cell death and ROS production. In conclusion, FYN is activated by oxidative stress and serves as a negative feedback regulator of NOX4 in CMs during cardiac remodeling.

Authors

Shouji Matsushima, Junya Kuroda, Peiyong Zhai, Tong Liu, Shohei Ikeda, Narayani Nagarajan, Shin-ichi Oka, Takashi Yokota, Shintaro Kinugawa, Chiao-Po Hsu, Hong Li, Hiroyuki Tsutsui, Junichi Sadoshima

Developmental SHP2 dysfunction underlies cardiac hypertrophy in Noonan syndrome with multiple lentigines

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Abstract

Hypertrophic cardiomyopathy is a common cause of mortality in congenital heart disease (CHD). Many gene abnormalities are associated with cardiac hypertrophy, but their function in cardiac development is not well understood. Loss-of-function mutations in PTPN11, which encodes the protein tyrosine phosphatase (PTP) SHP2, are implicated in CHD and cause Noonan syndrome with multiple lentigines (NSML), a condition that often presents with cardiac hypertrophic defects. Here, we found that NSML-associated hypertrophy stems from aberrant signaling mechanisms originating in developing endocardium. Trabeculation and valvular hyperplasia were diminished in hearts of embryonic mice expressing a human NSML-associated variant of SHP2, and these defects were recapitulated in mice expressing NSML-associated SHP2 specifically in endothelial, but not myocardial or neural crest, cells. In contrast, mice with myocardial- but not endothelial-specific NSML SHP2 expression developed ventricular septal defects, suggesting that NSML-associated mutations have both cell-autonomous and nonautonomous functions in cardiac development. However, only endothelial-specific expression of NSML-associated SHP2 induced adult-onset cardiac hypertrophy. Further, embryos expressing the NSML-associated SHP2 mutation exhibited aberrant AKT activity and decreased downstream forkhead box P1 (FOXP1)/FGF and NOTCH1/EPHB2 signaling, indicating that SHP2 is required for regulating reciprocal crosstalk between developing endocardium and myocardium. Together, our data provide functional and disease-based evidence that aberrant SHP2 signaling during cardiac development leads to CHD and adult-onset heart hypertrophy.

Authors

Jessica Lauriol, Janel R. Cabrera, Ashbeel Roy, Kimberly Keith, Sara M. Hough, Federico Damilano, Bonnie Wang, Gabriel C. Segarra, Meaghan E. Flessa, Lauren E. Miller, Saumya Das, Roderick Bronson, Kyu-Ho Lee, Maria I. Kontaridis