calpain and Hypertrophy--Left-Ventricular

β-adrenergic activation is able to exacerbate cardiac hypertrophy. Myosin light chain kinase (MLCK) and its phosphorylated substrate, phospho-myosin light chain 2 (p-MLC2), play vital roles in regulating cardiac hypertrophy. However, it is not yet clear whether there is a relationship between β-adrenergic activation and MLCK in the progression of cardiac hypertrophy. Therefore, we explored this relationship and the underlying mechanisms in this work.. Cardiac hypertrophy and cardiomyocyte hypertrophy were induced by pressure overload and isoproterenol (ISO) stimulation, respectively. Echocardiography, histological analysis, immunofluorescence and qRT-PCR were used to confirm the successful establishment of the models. A β-blocker (metoprolol) and a calpain inhibitor (calpeptin) were administered to inhibit β-adrenergic activity in rats and calpain in cardiomyocytes, respectively. The protein expression levels of MLCK, myosin light chain 2 (MLC2), p-MLC2, myosin phosphatase 2 (MYPT2), calmodulin (CaM) and calpain were measured using western blotting. A cleavage assay was performed to assess the degradation of recombinant human MLCK by recombinant human calpain.. The β-blocker alleviated cardiac hypertrophy and dysfunction, increased MLCK and MLC2 phosphorylation and decreased calpain expression in pressure overload-induced cardiac hypertrophy. Additionally, the calpain inhibitor calpeptin attenuated cardiomyocyte hypertrophy, upregulated MLCK and p-MLC2 and reduced MLCK degradation in ISO-induced cardiomyocyte hypertrophy. Recombinant human calpain degraded recombinant human MLCK in vitro in concentration- and time-dependent manners, and this degradation was inhibited by the calpain inhibitor calpeptin.. Our study suggested that β-adrenergic activation may promote the degradation of MLCK through calpain in pressure overload-induced cardiac hypertrophy.

Topics: Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Animals; Calpain; Cardiac Myosins; Cells, Cultured; Cysteine Proteinase Inhibitors; Disease Models, Animal; Enzyme Stability; Hypertrophy, Left Ventricular; Male; Myocytes, Cardiac; Myosin Light Chains; Myosin-Light-Chain Kinase; Phosphorylation; Proteolysis; Rats, Sprague-Dawley; Receptors, Adrenergic, beta; Ventricular Function, Left; Ventricular Remodeling

Topics: Animals; Calpain; Carbamates; Disease Models, Animal; G-Protein-Coupled Receptor Kinase 2; Hypertrophy, Left Ventricular; Isoproterenol; Male; Myocardium; Rats, Sprague-Dawley; Receptors, Adrenergic, beta; Up-Regulation

Cytochrome P-450 2E1 CYP2E1 induction has been linked to oxidative stress in a number of experimental models. The aim of this study was to investigate the relationship between CYP2E1 activity and markers of oxidative stress and cardiac cell apoptosis during the development of alcoholic cardiomyopathy (ACM). Changes in left ventricular morphology were evaluated in 4 groups of chronically instrumented dogs (control; alcohol-receiving; and alcohol-receiving plus treatment with either valsartan or carnitine) after 6 months of treatment. CYP2E1 and calpain-1 protein expression were determined by Western blotting, and apoptosis evaluated by TUNEL and immunohistochemistry. Malonyl dialdehyde levels were assessed as a marker of oxidative stress, while superoxide dismutase and glutathione peroxidase levels were evaluated as markers of antioxidant defense mechanisms. Expression of CYP2E1 was increased in the alcohol-receiving group compared with controls (P<0.05) and was associated with oxidative stress. Similarly, expression of Bad and calpain-1 protein was increased after chronic alcohol exposure, while Bcl-xL protein expression remained at a low level. Bad and calpain-1 protein expressions were significantly inhibited by treatment with valsartan or carnitine, while expression of Bcl-xL protein was increased (P<0.05). Collectively, our results indicate a possibly significant role for CYP2E1 in the oxidative stress associated with chronic alcoholism. The resulting increase in oxidative stress is accompanied by cellular apoptosis and may ultimately contribute to tissue remodeling and ACM. Importantly, these alcohol-induced effects may be abrogated by means such as angiotensin 1 receptor blockade or carnitine supplementation.

Topics: Alcohol Drinking; Animals; Apoptosis; Apoptosis Regulatory Proteins; Calpain; Cardiomyopathy, Alcoholic; Cytochrome P-450 CYP2E1; Dogs; Hypertrophy, Left Ventricular; Oxidative Stress

In hypertension, angiotensin (Ang) II is a critical mediator of cardiovascular remodeling, whose prominent features include myocardial and vascular media hypertrophy, perivascular inflammation, and fibrosis. The signaling pathways responsible for these alterations are not completely understood. Here, we investigated the importance of calpains, calcium-dependent cysteine proteases. We generated transgenic mice constitutively expressing high levels of calpastatin, a calpain-specific inhibitor. Chronic infusion of Ang II led to similar increases in systolic blood pressure in wild-type and transgenic mice. In contrast, compared with wild-type mice, transgenic mice displayed a marked blunting of Ang II-induced hypertrophy of left ventricle. Ang II-dependent vascular remodeling, ie, media hypertrophy and perivascular inflammation and fibrosis, was also limited in both large arteries (aorta) and small kidney arteries from transgenic mice as compared with wild type. In vitro experiments using vascular smooth muscle cells showed that calpastatin transgene expression blunted calpain activation by Ang II through epidermal growth factor receptor transactivation. In vivo and in vitro models of inflammation showed that impaired recruitment of mononuclear cells in transgenic mice was attributable to a decrease in both the release of and the chemotactic response to monocyte chemoattractant protein-1. Finally, results from collagen synthesis assay and zymography suggested that limited fibrogenesis was attributable to a decrease in collagen deposition rather than an increase in collagen degradation. These results indicate a critical role for calpains as downstream mediators in Ang II-induced cardiovascular remodeling and, thus, highlight an attractive therapeutic target.

Topics: Angiotensin II; Animals; Aorta; Blood Pressure; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Disease Models, Animal; Fibrosis; Genetic Therapy; Hypertension; Hypertrophy; Hypertrophy, Left Ventricular; Inflammation; Infusion Pumps, Implantable; Mice; Mice, Transgenic; Muscle, Smooth, Vascular; Myocardium; NF-kappa B; NFATC Transcription Factors; Renal Artery; Time Factors; Ventricular Remodeling

The cysteine proteases calpain and caspase-3 are known mediators of cell death. The aim of this study was to assess their contribution to the tissue damage found in experimental uremia.. Calpain and caspase-3 activities were measured in the hearts of rats that were sham-operated (control), sham-operated and spontaneously hypertensive (SHR), and those rendered uremic by 5/6 nephrectomy (uremic). In an in vitro study, heart myoblasts (Girardi) were incubated with human serum from healthy subjects (control serum conditioned media, CSCM) or uremic patients (uremic serum conditioned media, USCM), in the presence and absence of calpain and caspase-3 inhibitors. After 48 hours the activity of calpain and caspase-3 was measured, and cell injury determined by DNA fragmentation (ELISA) and lactate dehydrogenase (LDH) release. An in situ assay was designed to study how USCM affects calpain activity over time.. In the in vivo study, mean calpain activities were almost identical in the control and SHR groups, but calpain and caspase-3 activities were much elevated in the uremic group (P < 0.01 and 0.001 respectively vs. control). The SHR group had significantly higher mean arterial blood pressure (P < 0.001 vs. control, 0.01 vs. uremic). In the in vitro study calpain activity and DNA fragmentation were markedly higher in USCM treated cells compared to CSCM (both P<0.05). Both were reduced in USCM cells containing calpain inhibitors (E64d, calpastatin, or PD 150606). LDH release was raised also in USCM treated cultures (P < 0.05), which only the E64d treatment could significantly reduce (P < 0.02). Caspase-3 activities were similar in USCM and CSCM groups. The in situ assay showed significant increases in calpain activity in USCM treated cells compared to CSCM after just 3.5 hours (P<0.01).. In vivo results suggest that the increases in calpain and caspase-3 activity in uremic rat hearts were primarily due to uremia and not to hypertension. In vitro data demonstrate that uremia-induced cell injury can be attenuated by calpain inhibition. Therefore, it is likely that calpain is a mediator of uremia-induced myocardial injury.

Topics: Acrylates; Animals; Calcium-Binding Proteins; Calpain; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Disease Models, Animal; Humans; Hypertrophy, Left Ventricular; Leucine; Male; Nephrectomy; Oligopeptides; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Uremia

Calpains are cytoplasmic proteases widely distributed among eucaryotic cells. Low levels of calpain activity were found in hypertrophic hearts from hypertensive rats, but its role in hypertrophic hearts from human hypertensives is unknown. Therefore, calpain activity was investigated in erythrocytes from essential hypertensive patients in relation to their left ventricular mass.. To study the role of calpain activity in the development of left ventricular hypertrophy (LVH) in human essential hypertension.. A total of 115 hypertensives (72 untreated and 43 with treatment interrupted for at least 4 months) were included in the study. Calpain I activity was measured in human erythrocytes and LVH was measured as left ventricular mass index (LVMI) by M-mode echocardiography.. Values are given as mean+/-SEM. The hypertensives (97 men and 18 women) were 43.5+/-0.9 years old with mild to moderate levels of hypertension (systolic/diastolic blood pressure of 147.9+/-1.4/98.7+/-0.9 mmHg) and relatively recent LVH onset (3.5+/-0.5 years). An inverse relation between LVMI and erythrocytic calpain activity was present in all (P = 0.0023, R2 = 7.9%). This relation was still present considering only untreated hypertensives (P = 0.008; R2 = 9.7%), but was lost in the 43 previously treated hypertensives. Moreover, in the untreated hypertensives, after excluding the possible confounding effects of sex, age, body mass index, blood pressure and duration of hypertension, a stepwise regression showed that only two variables remained significantly related to LVMI: calpain (F = 6.23) and mean arterial pressure (F = 4.689). No relations were found between LVMI and calpastatin activity either in the whole population, or in treated or untreated hypertensives.. If we assume that the level of erythrocyte calpain activity mirrors the level in cardiomyocytes, these data seem to suggest that increased protein degradation by calpain may prevent the development of LVH in hypertensive patients. This effect is independent of the duration and severity of hypertension.

Topics: Adult; Biomarkers; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Calpain; Chromatography, Ion Exchange; Echocardiography; Erythrocytes; Female; Heart Ventricles; Humans; Hypertension; Hypertrophy, Left Ventricular; Male

Heart tissue contains large amounts of the Ca(2+)-activated proteinase calpain which has been assigned a specific function in the turnover of muscle protein. The objective of the present study was to determine calpain (E.C. 3.4.22.17)-like activity in homogenates of left ventricle from hypertensive rats that developed ventricular hypertrophy. Calpain activity was assayed using heat-denatured azocasein as a substrate in the presence of 1 mM calcium and corrected by subtraction of the Ca(2+)-independent activities. The latter were measured in the presence of 1 mM EGTA and the products read at 440 nm. Male Wistar rats (225 g) were assigned to control (N = 8, normal drinking water), salt (N = 6, drinking water containing 1% NaCl) and DOCA-salt (N = 6, deoxycorticosterone acetate, 8 mg/kg, sc, twice a week for 20 days plus drinking water containing 1% NaCl) groups. SHR (N = 6, spontaneously hypertensive rats) were also used. The calpain activity of the control group was at 3.90 +/- 0.22 mU/g wet weight tissue. Hypertension induced significant left ventricular hypertrophy in DOCA-salt rats (26%) and in SHR (54%) and a 30% decrease in calpain activity in both groups (P < 0.01). In the high salt load (salt group) calpain activity was also decreased, but this was not accompanied by hypertrophy. In the present indirect measurement of protein degradation capacity of heart tissue homogenates the proteolytic activity was activated (221%) by 1 mM calcium and inhibited (84%) by 1 mM EGTA after a 48-h incubation period, indicating the destruction of the calpain inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calpain; Desoxycorticosterone; Hypertrophy, Left Ventricular; Male; Protein Denaturation; Rats; Rats, Inbred SHR; Rats, Wistar; Tissue Extracts