The Role Of Calpain/calpastatin System In Cardiac Remodeling After Myocardial Infarction

Background:Myocardial infarction(MI) is due to coronary blood supply drastically reduced or interrupted, causing the corresponding myocardial ischemia, myocardial anoxia, eventually leading to myocardial necrosis. After myocardial infarction mainly as cardiomyocyte hypertrophy, myocardial apoptosis, a large number of fibroblast proliferation, fibroblast replace necrotic myocardial cells, causing cardiac remodeling, and eventually developed into a decompensated heart failure and death. Myocardial infarction is a common cardiovascular disease, and serious threat to human health. Although the development of new drugs and interventional procedures, the complication and the mortality rate is greatly reduced. The rise of myocardial remodeling after myocardial infarction, heart failure is still in a chronic progressive development, eventually lead to end-stage heart failure. Calpains are cytosolic calcium-activated cysteine proteases. Calpains include 14 isoforms. Two major isoforms, calpain μ or 1, which requires micromolar Ca2+ concentrations for activity, and calpain m or 2, which requires millimolar Ca2+concentrations, are ubiquitously expressed. Calpain activity, which is tightly controlled by calpastatin, a specific endogenous inhibitor. Recent studies have shown that, calpains play a key role in cardiac hypertrophy and apoptosis. The more the expression of calpains, the more severe heart failure. However, the role of calpains in cardiac remodeling is not clear, and the role of calpastatin over-expression in post-infarction myocardial remodeling remains poorly understood. Therefore, we propose the following hypothesis: calpains play an important role in the cardiac remodeling after myocardial infarction and over-expressing calpastatin can inhibit the degradation of myocardial skeletal structure, abnormal deposition of fibrous tissue.Objective:The present study established transgenic mice ubiquitously overexpressing calpastatin and used cultured an in vivo model of MI to investigate the role of calpastatin over-expression in post-infarction myocardial remodeling.Method:1. Construction, identification and breeding of calpastatin transgenic mice.Transgenic founder mice were generated on a C57BL/6J genetic background. We established transgenic mice(TG) ubiquitously over-expressing human calpastatin protein. We use Gateway cloning technology to build p RP.EX3d-EF1A-CAST-IRES-e GFP carrier and make it as the DNA solution for microinjection. The female C57BL/6J mice were hormonally superovulated and mated with male C57BL/6J mice. Next morning the fertilized one-cell eggs were collected from the oviducts. The eggs were microinjected with the DNA solution under a microscope. The injected fertilized eggs were transplanted into the oviducts of pseudo-pregnant C57BL/6J mice. We find out the positive transgenic funder mice by PCR. The positive transgenic funder mice were bred with C57BL/6J mice to generate the transgenic mice.2. Weighing the wild-type and transgenic mice weight, measurement of blood glucose and blood lipid.3. Analysis of the expression of calpastatin m RNA in transgenic mice tissues by using PR-PCR method.4. Analysis of the expression of endogenous calpastatin m RNA in wild-type and transgenic mice by using PR-PCR method.5. Analysis of the expression of calpastatin protein in wild-type and transgenic mice by using Western blotting method.6. The wild-type and transgenic mice aged 6–8 weeks were housed under a 12 h/12 h day/night cycle, with ad libitum food and water. They were divided randomly into 4 groups: WT+Sham group(n=20), WT+MI group(n=30), TG+Sham group(n=20), TG+MI group(n=28). MI model was constructed by left anterior descending artery ligation as previous described. Sham operated control mice underwent the same surgical procedures except that the suture placed under the LAD was not tied.7. Twenty-four hours after surgery, surviving mice were monitored and mortality was recorded for 21 days.8. Twenty-one days after surgery, mice were anesthetized by intraperitoneal injection of pentobarbital for evaluation of left ventricular hemodynamics.9. Twenty-one days after surgery, the hearts of mice were harvested and weighed to calculate the heart weight/body weight(HW/BW, mg/g).10. Twenty-four hours after surgery, a part of mice were anesthetized, and myocardial infarct size of hearts was determined by using Evans blue staining. Twenty-one days after surgery, myocardial infarct size of hearts was determined by using HE staining.11. Twenty-one days after surgery, the infarct scar size and fibrotic area was determined by using Masson staining.12. Twenty-one days after surgery, the collagen I and collagen III of hearts were determined by using immunohistochemical staining.13. Twenty-one days after surgery, calpain activity was determined by using a calpain activity assay kit.14. Twenty-one days after surgery, the expression of protein(calpain-1, calpain-2, calpastatin, MMP-2, MMP-9, TFG β, TIMP-1, TIMP-2) in mice was determined by using Western blotting method.Results:A total of 90 injected eggs were implanted into the oviducts of 3 pseudo-pregnant foster mothers, which gave birth to 23 offspring. Two offspring mice were identified to be carrying the CAST c DNA by PCR analysis. Between the TG mice and WT littermates, the FPG, TG, TC, LDL-C and HDL-C were similar(P>0.05). Calpastatin m RNA was expressed in transgenic mice’s genotype heart, lungs, liver, spleen, kidney, skeletal muscle and brain tissue. The m RNA expression of mouse endogenous calpastatin in heart tissue from TG and WT mice was no significant differences(P > 0.05). The calpastatin protein expression was profoundly upregulated in the myocardial tissue of TG mice compared with WT littermates(P < 0.01). Over-expression of calpastatin significantly reduced the infarct size(P < 0.01) and blunted MI-induced interventricular hypertrophy, global myocardial fibrosis and collagen I and collagen III deposition, hypotension and hemodynamic disturbances at 21 days after MI. Moreover, the MI-induced up-regulation and activation of calpains were obviously attenuated in calpastatin TG mice. MI-induced down-regulation of calpastatin was partially reversed in TG mice. Additionally, the MI-caused imbalance of matrix metalloproteinases and their inhibitors was improved in TG mice.Conclusions:Over-expressing human calpastatin gene does not affect endogenous calpastatin gene’s expression. Transgenic over-expression of calpastatin inhibits calpain activation and attenuates post-infarction myocardial remodeling.