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Cardiomyopathy is a common and lethal complication in patients with limb-girdle muscular dystrophy (LGMD), one of the most prevalent forms of muscular dystrophy. The pathogenesis underlying LGMD-related cardiomyopathy remains unclear. NRIP (gene name DCAF6), a Ca2+-dependent calmodulin binding protein, was reduced in dystrophic muscles from LGMD patients. Mice lacking NRIP exhibit a myopathic phenotype resembling that in LGMD patients, making NRIP deficiency a potential culprit leading to cardiomyopathy. This study aimed to determine if NRIP deficiency leads to cardiomyopathy and to explore the underlying molecular mechanisms. NRIP expression was reduced in both human and mouse failing hearts. Muscle-specific NRIP knockout (MCK-Cre:: Dcaf6 flox/flox) mouse heart and isolated cardiomyocytes exhibited markedly reduced contractility. Transmission electron microscopy revealed abnormal sarcomere structures and mitochondrial morphology in MCK-Cre:: Dcaf6 flox/flox hearts. Protein co-immunoprecipitation and confocal imaging revealed that NRIP interacts with α-actinin 2 (ACTN2) at the Z -disc. We found that NRIP facilitated ACTN2-mediated F-actin bundling, and that NRIP deficiency resulted in reduced binding between Z-disc proteins ACTN2 and Cap-Z. In addition, NRIP-deficiency led to increased mitochondrial ROS and impaired mitochondrial respiration/ATP production owing to elevated cellular NADH/NAD+ ratios. Treatment with mitochondria-directed antioxidant mitoTEMPO or NAD+ precursor nicotinic acid restored mitochondrial function and cardiac contractility in MCK-Cre:: Dcaf6 flox/flox mice. NRIP is essential to maintain sarcomere structure and mitochondrial/contractile function in cardiomyocytes. Our results revealed a novel role for NRIP deficiency in the pathogenesis of LGMD and heart failure. Targeting NRIP, therefore, could be a powerful new approach to treat myocardial dysfunction in LGMD and heart failure patients. • NRIP is reduced in the dystrophic muscles of LGMD patients and in the failing heart. • NRIP deficiency leads to abnormal sarcomere structures and mitochondrial ROS levels. • NRIP binds α-actinin 2 at Z-disc and is required for Cap-Z/α-actinin interaction. • Normalizing mitochondrial ROS and NADH/NAD+ restores NRIP-deficient heart function. • Targeting NRIP could be a powerful new approach for cardiac dysfunction in LGMD. [ABSTRACT FROM AUTHOR]

Background Mobilization of the innate immune response to clear and metabolize necrotic and apoptotic cardiomyocytes is a prerequisite to heart repair after cardiac injury. Suboptimal kinetics of dying myocyte clearance leads to secondary necrosis, and in the case of the heart, increased potential for collateral loss of neighboring non-regenerative myocytes. Despite the importance of myocyte phagocytic clearance during heart repair, surprisingly little is known about its underlying cell and molecular biology. Objective To determine if phagocytic receptor MERTK is expressed in human hearts and to elucidate key sequential steps and phagocytosis efficiency of dying adult cardiomyocytes, by macrophages. Results In infarcted human hearts, expression profiles of the phagocytic receptor MER-tyrosine kinase (MERTK) mimicked that found in experimental ischemic mouse hearts. Electron micrographs of myocardium identified MERTK signal along macrophage phagocytic cups and Mertk −/− macrophages contained reduced digested myocyte debris after myocardial infarction. Ex vivo co-culture of primary macrophages and adult cardiomyocyte apoptotic bodies revealed reduced engulfment relative to resident cardiac fibroblasts. Inefficient clearance was not due to the larger size of myocyte apoptotic bodies, nor were other key steps preceding the formation of phagocytic synapses significantly affected; this included macrophage chemotaxis and direct binding of phagocytes to myocytes. Instead, suppressed phagocytosis was directly associated with myocyte-induced inactivation of MERTK, which was partially rescued by genetic deletion of a MERTK proteolytic susceptibility site. Conclusion Utilizing an ex vivo co-cultivation approach to model key cellular and molecular events found in vivo during infarction, cardiomyocyte phagocytosis was found to be inefficient, in part due to myocyte-induced shedding of macrophage MERTK. These findings warrant future studies to identify other cofactors of macrophage–cardiomyocyte cross-talk that contribute to cardiac pathophysiology. [ABSTRACT FROM AUTHOR]

Cardiac hypertrophy of the left ventricle (LV) in response to dynamic exercise-training (EX) is a beneficial adaptation to increased workload, and is thought to result from genetic reprogramming. We aimed to determine which transcription factors (TFs) are involved in this genetic reprogramming of the LV in swine induced by exercise-training. Swine underwent 3–6 weeks of dynamic EX, resulting in a 16% increase of LV weight/body weight ratio compared to sedentary animals ( P = 0.03). Hemodynamic analysis showed an increased stroke volume index (stroke volume/body weight + 35%; P = 0.02). Microarray-analysis of LV tissue identified 339 upregulated and 408 downregulated genes (false discovery rate < 0.05). Of the human homologues of the differentially expressed genes, promoter regions were searched for TF consensus binding sites (TFBSs). For upregulated and downregulated genes, 17 and 24 TFBSs were overrepresented by > 1.5-fold ( P < 0.01), respectively. In DNA-binding assays, using LV nuclear protein extracts and protein/DNA array, signal intensity changes > 2-fold were observed for 23 TF-specific DNA probes. Matching results in TFBS and protein/DNA array analyses were obtained for transcription factors YY1 (Yin Yang 1), PAX6 (paired box 6) and GR (glucocorticoid receptor). Notably, PAX6 and GR show lower signals in TFBS and protein/DNA array analyses upon exercise-training, whereas we previously showed higher signals for these factors in the remodeled LV of swine post-myocardial infarction (MI). In conclusion, we have identified transcription factors that may drive the genetic reprogramming underlying exercise-training induced LV hypertrophy in swine. PAX6 and GR are among the transcription factors that are oppositely regulated in LV hypertrophy after exercise-training and MI. These proteins may be at the base of the differences between pathological and physiological hypertrophy. [ABSTRACT FROM AUTHOR]

Abstract: We examined pressure overload left ventricular (LV) hypertrophy (H) induced by aortic banding in transgenic mice with cardiac-specific expression of a dominant negative (DN) p38α (TG) and wild type controls (WT). In response to chronic pressure overload, induced by aortic constriction, LV/BW increased more, p <0.05, in female TG (6.4±0.2, n =7) than in WT female (5.1±0.2, n =10), or male TG or WT (5.0±0.2, n =10 vs. 5.5±0.2, n =8). Lung/BW, an index of LV decompensation, was significantly higher, p <0.05, in banded female TG (14 ±1.2 mg/g) than in WT females (9.0±0.8), or male TG or WT (8.2±0.7 vs. 9.3±1.3). This was associated with higher premature mortality, p <0.05, in banded female TG mice (42%) compared with banded WT females (10%), TG males (13%), or WT males (17%). In male, but not female, TG mice, the number of TUNEL-positive cells was smaller, p <0.05, compared with WT. Phospho-Akt kinase activity increased (p <0.05) in female TG after banding, but not in males. After ovariectomy, chronic pressure overload no longer induced greater mortality, greater LVH, or p-Akt levels in female TG mice, and like male TG mice, apoptosis was protected. DN-p38α enhanced estrogen-induced activation of Akt in cultured cardiac myocytes. Thus, inhibition of p38α MAPK paradoxically augments LVH resulting in cardiac decompensation and increased mortality in response to pressure overload more in female mice than male mice, which could be due to increased Akt activation and/or through cross-talk between p38α MAPK and Akt. [Copyright &y& Elsevier]

Abstract: We previously found that a canine model of selective surgical ventricular denervation (VD), which does not permit increased sympathetic tone during the pathogenesis of heart failure (HF), tolerated the development of HF better than controls. To investigate the cellular mechanisms, we examined cellular contraction and L-type Ca2+ channel currents (ICa) and their responses to β-adrenergic receptor (β-AR) stimulation in left ventricular myocytes from 1) control, 2) VD, 3) HF induced by rapid pacing, and 4) HF induced in VD (VD-HF) dogs. The magnitude of myocyte contraction and rate of relaxation in VD were similar to control but were depressed in both HF and VD-HF. These changes were associated with reduced protein expression of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) and protein kinase A phosphorylated phospholamban (PLB), which was reduced in HF, but essentially abolished in VD-HF. β-AR kinase (GRK2) was increased in HF but reduced in VD-HF. Basal ICa density did not differ among control, VD, and HF groups, but VD-HF myocytes showed a markedly reduced ICa density (~40%). Compared to controls, the sensitivity of ICa to isoproterenol (ISO), was significantly higher in VD, but reduced in HF. While ICa responses to ISO in VD-HF were maintained at control levels, the amplitude of the ISO-stimulated ICa was significantly smaller (~50%) compared with HF myocytes. The relative decrease in Ca2+ influx due to downregulation of ICa density may contribute to the cardioprotective effects in VD-HF hearts by preventing Ca2+ overload during the development of HF. These findings, in combination with the virtual abolition of phosphorylated PLB in VD-HF and the decrease in GRK2, may explain, in part, why VD dogs tolerate the development of HF better than control dogs. [Copyright &y& Elsevier]

Mutations in the dystrophin glycoprotein complex, and in particular the sarcoglycan subcomplex, lead to cardiomyopathy and muscular dystrophy. Mice with mutations in γ-sarcoglycan or δ-sarcoglycan develop cardiomyopathy that is characterized by focal regions of tissue damage. These focally damaged regions constitute 0–5% of cardiac tissue. In cardiomyopathy arising from sarcoglycan mutations, we found that endothelial nitric oxide synthase (eNOS) was significantly increased in focally damaged cardiac myocytes. In addition, we noted that nitric oxide (NO) was also increased in regions of tissue damage and altered membrane permeability. In sarcoglycan mutant mice, regionally increased cardiac NO was associated with hypersensitivity to carbachol and decreased sensitivity to adrenergic stimulation. Inhibition of NO production in sarcoglycan mutant mice was associated with improved recovery after carbachol and isoproterenol infusion. These data provide a mechanism where regional, focal cardiac damage creates pathologic gradients of NO. Moreover, inhibition of nitric oxide synthase corrects defects that arise from pathologic NO gradients. [Copyright &y& Elsevier]