calpastatin and Heart-Failure

The precise mechanism of the progression of advanced heart failure is unknown. We assessed a new scheme in two heart failure models: (I) congenital dilated cardiomyopathy (DCM) in TO-2 strain hamsters lacking delta-sarcoglycan (SG) gene and (II) administration of a high-dose of isoproterenol, as an acute heart failure in normal rats. In TO-2 hamsters, we followed the time course of the histological, physiological and metabolic the progressions of heart failure to the end stage. Dystrophin localization detected by immunostaining age-dependently to the myoplasm and the in situ sarcolemma fragility evaluated by Evans blue entry was increased in the same cardiomyocytes. Western blotting revealed a limited cleavage of the dystrophin protein at the rod domain, strongly suggesting a contribution of endogenous protease(s). We found a remarkable up-regulation of the amount of calpain-1 and -2, and no change of their counterpart, calpastatin. After supplementing TO-2 hearts with the normal delta-SG gene in vivo, these pathological alterations and the animals' survival improved. Furthermore, dystrophin but not delta-SG was disrupted by a high dose of isoproterenol, translocated from the sarcolemma to the myoplasm and fragmented. These results of heart failure, irrespective of the hereditary or acquired origin, indicate a vicious cycle formed by the increased sarcolemma permeability, preferential activation of calpain over calpastatin, and translocation and cleavage of dystrophin would commonly lead to advanced heart failure.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cardiomyopathy, Dilated; Cell Membrane Permeability; Cricetinae; Dependovirus; Disease Models, Animal; Dystrophin; Enzyme Activation; Genetic Therapy; Heart Failure; Isoproterenol; Mesocricetus; Models, Biological; Rats; Sarcoglycans; Sarcolemma

Sumoylation is a posttranslational modification that regulates a wide spectrum of cellular activities. Cardiomyopathy is the leading cause of heart failure. Whether sumoylation, particularly SUMO-2/3 conjugation, is involved in cardiomyopathy has not been investigated. We report here that SUMO-2/3 conjugation was elevated in the human failing hearts, and we investigated the impact of increased SUMO-2 conjugation on heart function by using the gain-of-function approach in mice, in which cardiac specific expression of constitutively active SUMO-2 was governed by alpha myosin heavy chain promoter (MHC-SUMO-2 transgenic, SUMO-2-Tg). Four of five independent SUMO-2-Tg mouse lines exhibited cardiomyopathy with various severities, ranging from acute heart failure leading to early death to the development of chronic cardiomyopathy with aging. We further revealed that SUMO-2 directly regulated apoptotic process by at least partially targeting calpain 2 and its natural inhibitor calpastatin. SUMO conjugation to calpain 2 promoted its enzymatic activity, and SUMO attachment to calpastatin mainly promoted its turnover and altered its subcellular distribution. Thus, enhanced SUMO-2 conjugation led to increased apoptosis and played a pathogenic role in the development of cardiomyopathy and heart failure.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cardiomyopathies; Heart Failure; HeLa Cells; Humans; Mice; Protein Binding; Protein Transport; Small Ubiquitin-Related Modifier Proteins; Ubiquitins

The mechanism for the decrease in cardiomyocyte size with mechanical unloading is unknown. The calpain system regulates cardiomyocyte atrophy. We obtained samples from failing human hearts at the time of implantation and explantation of a left ventricular assist device. For mechanical unloading, we also heterotopically transplanted rat or mouse hearts for 1 week. The effect of calpain inhibition on cardiac atrophy was assessed in transplanted hearts overexpressing calpastatin. We measured transcript levels of calpain 1 and 2 in the human and the rodent model, as well as calpain activity, a calpain-specific degradation product and cardiomyocyte size in the two rodent models. Mechanical unloading of the failing human heart significantly increased calpain 2 gene expression. Transcript levels of calpain 1 and 2, calpain activity and a calpain-specific degradation product all significantly increased in the unloaded rat heart. Unexpectedly, in hearts of animals overexpressing calpastatin, cardiomyocyte size also decreased. Mechanical unloading of the mammalian heart activates the calpain system, although other proteolytic systems may compensate for decreased calpain activity when calpastatin is overexpressed.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Size; Disease Models, Animal; Gene Expression Regulation; Heart Failure; Heart Transplantation; Heart-Assist Devices; Humans; Male; Mice; Middle Aged; Myocardium; Myocytes, Cardiac; Rats; Time Factors; Transplantation, Heterotopic

Regulating the balance between synthesis and proteasomal degradation of cellular proteins is essential for tissue growth and maintenance, but the critical pathways regulating protein ubiquitination and degradation are incompletely defined. Although participation of calpain calcium-activated proteases in post-necrotic myocardial autolysis is well characterized, their importance in homeostatic turnover of normal cardiac tissue is controversial. Hence, we evaluated the consequences of physiologic calpain (calcium-activated protease) activity in cultured cardiomyocytes and unstressed mouse hearts. Comparison of in vitro proteolytic activities of cardiac-expressed calpains 1 and 2 revealed calpain 1, but not calpain 2, activity at physiological calcium concentrations. Physiological calpain 1 activation was evident in adenoviral transfected cultured cardiomyocytes as proteolysis of specific substrates, generally increased protein ubiquitination, and accelerated protein turnover, that were each inhibited by coexpression of the inhibitor protein calpastatin. Conditional forced expression of calpain 1, but not calpain 2, in mouse hearts demonstrated substrate-specific proteolytic activity under basal conditions, with hyperubiquitination of cardiac proteins and increased 26S proteasome activity. Loss of myocardial calpain activity by forced expression of calpastatin diminished ubiquitination of 1 or more specific myocardial proteins, without affecting overall ubiquitination or proteasome activity, and resulted in a progressive dilated cardiomyopathy characterized by accumulation of intracellular protein aggregates, formation of autophagosomes, and degeneration of sarcomeres. Thus, calpain 1 is upstream of, and necessary for, ubiquitination and proteasomal degradation of a subset of myocardial proteins whose abnormal accumulation produces autophagosomes and degeneration of cardiomyocytes with functional decompensation.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cardiomyopathy, Dilated; Cells, Cultured; Heart Failure; Homeostasis; Mice; Mice, Transgenic; Microscopy, Electron; Myocardium; Myocytes, Cardiac; Osmolar Concentration; Proteasome Endopeptidase Complex; Protein Isoforms; Proteins; Substrate Specificity; Transfection; Ubiquitin

Changes in proteolytic activity of the myocardium during the development of heart failure after left coronary artery ligation (CAL) of rats were examined. Hemodynamics of the rats at the eighth week (8w-CAL rat), but not at the second week (2w-CAL rat), after CAL showed the symptoms of chronic heart failure. Contents of mu-calpin and m-calpain, but not an intrinsic calpain inhibitor calpastatin, in the viable left ventricular muscle (viable LV) and the right ventricular muscle (RV) of the 2w-CAL and 8w-CAL rats were increased, which was associated with an elevation of intrinsic activities of leupeptin-sensitive, Ca(2+)-activated proteolysis in the cytosolic fractions of the viable LV and RV. Oral administration of 3 mg/kg/d trandolapril or 1 mg/kg/d candesartan from the second to eighth week after CAL improved the hemodynamics of 8w-CAL rats. The drug treatment attenuated the increases in mu-calpain and m-calpain contents and the elevation of the proteolytic activity of the viable LV and RV in the 8w-CAL rat. The drug treatment increased calpastatin content of the RV in the 8w-CAL rat. These results suggest that sustained activation of calpain is involved in the development of chronic heart failure and that trandolapril and candesartan prevent the activation of calpains after CAL.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Calcium-Binding Proteins; Calpain; Cytosol; Enzyme Activation; Heart Failure; Male; Myocardial Infarction; Myocardium; Organ Size; Rats; Rats, Wistar