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Objective To investigate the role of cathepsin K (CatK) in cardiac dysfunction in alcoholic cardiomyopathy (ACM) mice and its damage mechanism. Methods Forty-eight C57BL/6J male mice (8 weeks old) were randomly divided into control group (n=12, fed with isocaloric maltodextrin control liquid diet) and model group (n=36, fed with 4% ethanol Lieber-DeCarli liquid diet). After 8 weeks, the mice in the model group identified with decreased cardiac systolic function by echocardiography were regarded as ACM mice, and they were randomly divided into ACM group, sh-NC group, and sh-CatK group, with 12 mice in each group. The mice in the sh-NC group and the sh-CatK group were injected with 1×109 vg (100 μL) of recombinant adenoviruses containing scramble-shRNA or CatK-shRNA via tail vein, respectively. At the end of the 12th week, cardiac function was measured by M-mode echocardiography. The expression of CatK and Nox4 was detected by immunohistochemical assay. In in vitro study, H9c2 cardiomyocytes were used to evaluate the effect of CatK silencing on alcohol-induced cardiomyocyte injury. Western blotting was used to analyze the protein expression of AMPK/Nox4 signaling pathway. Flow cytometry was employed to analyze the apoptosis of H9c2 cells. The levels of reactive oxygen species in mouse myocardium and H9c2 cells cultured were measured using fluorescent dye dihydroethidium. Results The surface area of myocardial cells, area of myocardial fibrosis, protein levels of CatK and Nox4 and production of superoxide were significantly increased in the ACM group compared with the control group (P < 0.05), and those indicators in the sh-CatK group were significantly decreased than the ACM group (P < 0.05). Sh-CatK transfection significantly reversed the above changes in ACM mice (P < 0.05). In vitro results showed that the expression of CatK protein in H9c2 cells was increased with the prolonged exposure to ethanol (P < 0.05). CatK inhibition significantly reduced the apoptostic rate and oxidative stress levels (P < 0.05), and up-regulated AMPK phosphorylation in H9c2 cells (P < 0.05), resulting in decreased expression of Nox4 protein, collagen COL-1 and TGF-β (P < 0.05). Conclusion CatK is involved in the pathological process of cardiac dysfunction in the ACM mice, and its mechanism might be related to the promotion of oxidative stress mediated by the AMPK/Nox4 signaling pathway. |
