calpain and Cardiomyopathy--Hypertrophic

Recently we have shown that calpain-1 activation contributes to cardiomyocyte apoptosis induced by hyperglycemia. This study was undertaken to investigate whether targeted disruption of calpain would reduce myocardial hypertrophy and fibrosis in mouse models of type 1 diabetes.. Diabetes in mice was induced by injection of streptozotocin (STZ), and OVE26 mice were also used as a type 1 diabetic model. The function of calpain was genetically manipulated by cardiomyocyte-specific knockout Capn4 in mice and the use of calpastatin transgenic mice. Myocardial hypertrophy and fibrosis were investigated 2 and 5 months after STZ injection or in OVE26 diabetic mice at the age of 5 months. Cultured isolated adult mouse cardiac fibroblast cells were also investigated under high glucose conditions.. Calpain activity, cardiomyocyte cross-sectional areas, and myocardial collagen deposition were significantly increased in both STZ-induced and OVE26 diabetic hearts, and these were accompanied by elevated expression of hypertrophic and fibrotic collagen genes. Deficiency of Capn4 or overexpression of calpastatin reduced myocardial hypertrophy and fibrosis in both diabetic models, leading to the improvement of myocardial function. These effects were associated with a normalization of the nuclear factor of activated T-cell nuclear factor-κB and matrix metalloproteinase (MMP) activities in diabetic hearts. In cultured cardiac fibroblasts, high glucose-induced proliferation and MMP activities were prevented by calpain inhibition.. Myocardial hypertrophy and fibrosis in diabetic mice are attenuated by reduction of calpain function. Thus targeted inhibition of calpain represents a potential novel therapeutic strategy for reversing diabetic cardiomyopathy.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cardiomyopathy, Hypertrophic; Cell Proliferation; Cells, Cultured; Diabetes Mellitus, Type 1; Diabetic Cardiomyopathies; Disease Models, Animal; Fibrosis; Gene Expression Regulation; Heart; Hyperglycemia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Molecular Targeted Therapy; Myocardium; Streptozocin

The first human cardiac troponin I (hcTnI) mutation in the N-terminal 32 residue region, R21C (arginine residue number 21 mutated to cysteine), which has been linked to hypertrophic cardiomyopathy (HCM), has recently been reported. The effect of this mutation on the physiological function of hcTnI was investigated. Human cTnI R21C (in the absence or presence of troponin T and troponin C) was phosphorylated by protein kinase A (PKA) at a significantly slower rate than wild-type hcTnI. In skinned fiber studies, the TnI R21C mutant showed a large increase in Ca(2+)-sensitivity of force development when compared to wild-type TnI (DeltapCa(50)=0.33). Phosphorylation of skinned fibers containing TnI R21C by PKA resulted in a significantly smaller decrease in the Ca(2+)-sensitivity of force development when compared to phosphorylation of fibers containing wild-type TnI. The decreased sensitivity of TnI R21C to PKA is most likely due to a decreased ability of PKA to phosphorylate this TnI rather than conformational problems within this TnI. In addition, skinned fibers were found to contain an endogenous kinase that is capable of phosphorylating wild-type TnI. However, the endogenous kinase activity did not affect the Ca(2+)-sensitivity of force development, the Hill coefficient or maximal force of these skinned fibers. Actomyosin ATPase assays showed that the R21C mutation did not affect the inhibitory properties of TnI or the maximal ATPase activity. TnI R21C was also found to be more susceptible to proteolysis by calpain II than wild-type TnI. These results suggest that this R21C mutation in TnI affects the Ca(2+)-sensitizing effect of Tn, the ability of TnI to be readily phosphorylated by PKA and the stability of TnI to calpain. The results also suggest that the N-terminal region may have important roles such as modulating the Ca(2+)-sensitivity of force-development.

Topics: Actins; Adenosine Triphosphatases; Animals; Calcium; Calpain; Cardiomyopathy, Hypertrophic; Circular Dichroism; Cyclic AMP-Dependent Protein Kinases; Humans; In Vitro Techniques; Mutation; Myocardium; Myosins; Phosphorylation; Swine; Time Factors; Troponin I

To investigate the role of [Ca2+]i from different origins in the course of myocardial hypertrophy mediated by CaN-NFAT3 signal transduction.. The primarily cultured cardiomyocyte were irritated with angiotensin (Ang) II and ryanodine (RY) which cause Ca2+ inflow and release respectively. Then to observe the changes of CaN-NFAT3 pathway were then observed. Western blotting was employed to semi-quantify CaN, NFAT3 and GATA4. The distribution of NFAT3 was shown with immunocytochemistry, (3)H-Leu incorporation was used as an index of myocyte hypertrophy. Cyclosporin A (CsA) was applied to restrain CaN-NFAT3 pathway as a kind of CaN-selective antagonist.. CaN, NFAT3, GATA4 expression significantly increased 1 and 3 days after the stimulation of cardiomyocytes with Ang II and RY (10(-7) mol/L) as compared with that of a control group (P > 0.05) and (3)H-Leu incorporation distinctly increased after Ang II and RY (10(-7) mol/L) stimulation (P > 0.05 versus control group). On the first day of Ang II and RY stimulation, NFAT3 was shown mainly as intra-nuclear expression rather than cytoplasmic expression as seen in the control group. All of the above effects were suppressed by CsA administration, but they were rarely suppressed if CsA was not administered (P > 0.05).. It is shown that both Ca2+ inflow and release may activate CaN-NFAT3 signal pathway, which responds to increase of [Ca2+]i and is independent of its origin, indicating the augment of [Ca2+]i may trigger CaN-NFAT3 signal transduction and consequently induce myocyte hypertrophy. Moreover, CsA may restrain the expression and activation of CaN-NFAT3 and protein synthesis of myocytes in response to Ang II and RY stimulation.

Topics: Angiotensin II; Animals; Animals, Newborn; Blotting, Western; Calcium; Calpain; Cardiomyopathy, Hypertrophic; Cells, Cultured; DNA-Binding Proteins; GATA4 Transcription Factor; Myocytes, Cardiac; NFATC Transcription Factors; Nuclear Proteins; Peptide Fragments; Rats; Rats, Wistar; Signal Transduction; Transcription Factors