| Background:Cardiac hypertrophy is a response of the myocardium to increased workload, regulated by multiple genes. cellular Fas-associated death domain-like interleukin-1β-converting enzyme(FLICE)-like inhibitory protein (cFLIP) is a mammalian homolog of the viral FLICE-inhibitory protein and a natural modulator of tumor necrosis factor signaling.2main forms of cFLIP have been well characterized, cFLIP long form (cFLIPL:55to60kDa) and short form (cFLIPs:26kDa). Both splice variants have death effector domains, with which they bind to Fas-associated death domain at the death-inducing signaling complex and inhibit caspase8activation. It has been well documented that elevated cFLIP expression protects cells from death receptor-mediated apoptosis, whereas downregulation of cFLIP by chemicals or small interfering RNA sensitizes cells to death receptor-mediated apoptosis. A clear reduction in the expression of cFLIP has been demonstrated in the ventricular myocardium of patients with end-stage heart failure and rodents after myocardial infarction. These data suggest that cFLIP plays a vital role in cardiac development and cardiomyocyte survival after stress. Nevertheless, the role of cFLIP in cardiac hypertrophy remains largely unclear. We tested the expression of cFLIP in heart tissues in dilated cardiomyopathy patients with heart failure. We also used cFLIP gene-knock-out mice and cFLIP transgenic mice to identify the roles which cFLIP plays in cardiac hypertrophy established through aortic banding surgery.Methods:Part one:Heart tissues of left ventricle, from healthy donor and DCM patients with heart failure respectively. Testing the expression of cFLIP in both tissues with Western Blot.Part two:cFLIP heterozygote (HZ) male mice and wild-type cFLIP (WT) male mice, whose body weight from25-26g and age of8weeks with CD1background, were divided into sham and AB group. After4weeks, echocardiography was performed in order to test the morphology and function of the mice. Then the hearts and lungs of the sacrificed mice were dissected and weighed to compare the heart weight/body weight (HW/BW, mg/g) and lung weight/body weight (LW/BW, mg/g)ratios. HE,WGA and PSR staining were used to detect the myocyte area and collagen deposition. Real-Time PCR was used to detect the expression of mRNA of hypertrophic and fibrosis markers in different groups.Part three:Cardiac-specific cFLIP-overexpressing transgenic (TG) mice and twenty Non-transgenic(NTG) mice, whose body weight from25-26g and age of8weeks with CD1background, were divided into sham and AB group. After4weeks, echocardiography was performed in order to test the morphology and function of the mice. Then the hearts and lungs of the sacrificed mice were dissected and weighed to compare the heart weight/body weight (HW/BW, mg/g) and lung weight/body weight (LW/BW, mg/g)ratios. HE, WGA and PSR staining were used to detect the myocyte area and collagen deposition. Real-Time PCR was used to detect the expression of mRNA of hypertrophic and fibrosis markers in different groups.Results:Part one:WB showed that the expression of cFLIP in DCM hearts was obviously reduced than that in healthy hearts.Part two:HZ mice that were subjected to AB exhibited more indications of cardiac dysfunction (including decreases in FS and increases in LVEDD and LVESD) than the WT mice that were subjected to AB. The HZ mice that had been subjected to AB showed great increases in HW/BW and LW/BW ratios. The augmentation in cardiac myocyte size and fibrosis area observed after HE, WGA and PSR staining in the HZ mice was also markedly enhanced in comparison to that in WT mice4weeks postoperation. In comparison to HZ mice, the descending expression of cFLIP enhanced the pressure overload-induced changes in the expression of hypertrophic markers ANP, BNP, β-MHC,CTGF, TGF-β1, and collagens I and Ⅳ. However, the sham-operated HZ mice exhibited no significant changes in the expression levels of the ANP, BNP, β-MHC, CTGF, TGF-β1, collagens I and IV genes in comparison to the control mice.Part three:Four independent transgenic lines were established and studied. cFLIP-TG mice that were subjected to AB exhibited fewer indications of cardiac dysfunction (including increases in FS and decreases in LVEDD, LVESD) than the NTG mice that were subjected to AB. The TG mice that had been subjected to AB exhibited an intriguing diminution in cardiac hypertrophy, as measured by the HW/BW and LW/BW ratios. The augmentation in cardiac myocyte size and fibrosis area observed after HE,WGA and PSR staining in the TG mice was also markedly attenuated in comparison to that in NTG mice4weeks postoperation. In comparison to NTG mice, the overexpression of cFLIP attenuated the pressure overload-induced changes in the expression of hypertrophic markers ANP, BNP, P-MHC,CTGF, TGF-β1, and collagens I and Ⅳ. The sham-operated TG mice exhibited no significant changes in the expression levels of the ANP, BNP, β-MHC,CTGF, TGF-β1, and collagens Ⅰ and Ⅳ genes in comparison to the control mice either.ConclusioncFLIP inhibits cardiac hypertrophy and fibrosis, and protects the cardiac function. |
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