Membrane proteins are crucial to heart function and development. Here we combine cationic silica-bead coating with shotgun proteomics to enrich for and identify plasma membrane-associated proteins from primary mouse neonatal and human fetal ventricular cardiomyocytes. We identify Tmem65 as a cardiac-enriched, intercalated disc protein that increases during development in both mouse and human hearts. Functional analysis of Tmem65 both in vitro using lentiviral shRNA-mediated knockdown in mouse cardiomyocytes and in vivo using morpholino-based knockdown in zebrafish show marked alterations in gap junction function and cardiac morphology. Molecular analyses suggest that Tmem65 interaction with connexin 43 (Cx43) is required for correct localization of Cx43 to the intercalated disc, since Tmem65 deletion results in marked internalization of Cx43, a shorter half-life through increased degradation, and loss of Cx43 function. Our data demonstrate that the membrane protein Tmem65 is an intercalated disc protein that interacts with and functionally regulates ventricular Cx43.

Phospholamban (PLN) plays a central role in Ca2+ homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca 2+ -ATPase 2A (SERCA2A) Ca 2+ pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function.phospholamban | proteomic | bioinformatics | heart disease | signaling C ardiovascular diseases (CVDs) leading to systolic/diastolic heart failure (HF), such as hypertensive/diabetic heart disease, stroke, and vascular atherosclerosis, are leading causes of death in the developed world (1). Many CVDs are associated with genetic predispositions. For example, in humans, the arginine to cysteine (R9C) substitution in phospholamban (PLN) has been shown to result in dilated cardiomyopathy (DCM) presenting in adolescence, leading to rapid deterioration of heart function and premature death (2). However, the etiology and molecular mechanisms of progression of DCM and other CVDs leading to HF are complex and still poorly understood, further complicating clinical assessment and management. From a biological and clinical perspective, the identification and characterization of clinically relevant, potentially druggable, pathways driving the maladaptive response in affected heart tissue are key challenges to improved diagnostic and therapeutic tools for earlier detection and preventative treatment of both inherited and chronic CVDs.Cardiac muscle contraction is controlled by Ca 2+ flux and signaling relays, which are perturbed in HF. Internal stores of Ca 2+ required for the proper functioning of cardiomyocytes (CMs) are normally maintained through the function of the sarco(endo)plasmic reticulum Ca 2+ -ATPase 2 ...

BackgroundCardiomyocyte‐specific transgenic mice overexpressing S100A6, a member of the family of EF‐hand calcium‐binding proteins, develop less cardiac hypertrophy, interstitial fibrosis, and myocyte apoptosis after permanent coronary ligation, findings that support S100A6 as a potential therapeutic target after acute myocardial infarction. Our purpose was to investigate S100A6 gene therapy for acute myocardial ischemia‐reperfusion.Methods and ResultsWe first performed in vitro studies to examine the effects of S100A6 overexpression and knockdown in rat neonatal cardiomyocytes. S100A6 overexpression improved calcium transients and protected against apoptosis induced by hypoxia‐reoxygenation via enhanced calcineurin activity, whereas knockdown of S100A6 had detrimental effects. For in vivo studies, human S100A6 plasmid or empty plasmid was delivered to the left ventricular myocardium by ultrasound‐targeted microbubble destruction in Fischer‐344 rats 2 days prior to a 30‐minute ligation of the left anterior descending coronary artery followed by reperfusion. Control animals received no therapy. Pretreatment with S100A6 gene therapy yielded a survival advantage compared to empty‐plasmid and nontreated controls. S100A6‐pretreated animals had reduced infarct size and improved left ventricular systolic function, with less myocyte apoptosis, attenuated cardiac hypertrophy, and less cardiac fibrosis.ConclusionsS100A6 overexpression by ultrasound‐targeted microbubble destruction helps ameliorate myocardial ischemia‐reperfusion, resulting in lower mortality and improved left ventricular systolic function post–ischemia‐reperfusion via attenuation of apoptosis, reduction in cardiac hypertrophy, and reduced infarct size. Our results indicate that S100A6 is a potential therapeutic target for acute myocardial infarction.

BackgroundEndoplasmic reticulum (ER) resident protein 44 (ERp44) is a member of the protein disulfide isomerase family, is induced during ER stress, and may be involved in regulating Ca2+ homeostasis. However, the role of ERp44 in cardiac development and function is unknown. The aim of this study was to investigate the role of ERp44 in cardiac development and function in mice, zebrafish, and embryonic stem cell (ESC)‐derived cardiomyocytes to determine the underlying role of ERp44.Methods and ResultsWe generated and characterized ERp44−/− mice, ERp44 morphant zebrafish embryos, and ERp44−/− ESC‐derived cardiomyocytes. Deletion of ERp44 in mouse and zebrafish caused significant embryonic lethality, abnormal heart development, altered Ca2+ dynamics, reactive oxygen species generation, activated ER stress gene profiles, and apoptotic cell death. We also determined the cardiac phenotype in pressure overloaded, aortic‐banded ERp44+/− mice: enhanced ER stress activation and increased mortality, as well as diastolic cardiac dysfunction with a significantly lower fractional shortening. Confocal and LacZ histochemical staining showed a significant transmural gradient for ERp44 in the adult heart, in which high expression of ERp44 was observed in the outer subepicardial region of the myocardium.ConclusionsERp44 plays a critical role in embryonic heart development and is crucial in regulating cardiac cell Ca2+ signaling, ER stress, ROS‐induced oxidative stress, and activation of the intrinsic mitochondrial apoptosis pathway.