Gritsyna Yu V, Salmov N N, Bobylev A G, Fadeeva I S, Fesenko N I, Sadikova D G, Kukushkin N I, Podlubnaya Z A, Vikhlyantsev I M
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia.
Biochemistry (Mosc). 2017 Feb;82(2):168-175. doi: 10.1134/S0006297917020080.
Enzymatic activity of Ca2+-dependent calpain proteases as well as the content and gene expression of μ-calpain (activated by micromolar calcium ion concentrations), calpastatin (inhibitor of calpains), and titin (substrate for calpains) were investigated in cardiac muscles of rats subjected to chronic alcoholization for 3 and 6 months. There was no increase in the "heart weight/body weight" parameter indicating development of heart hypertrophy in the alcoholized rats, while a decreasing trend was observed for this parameter in the rats after 6-month modeling of alcoholic cardiomyopathy, which indicated development of atrophic changes in the myocardium. Fluorometric measurements conducted using the Calpain Activity Assay Kit did not reveal any changes in total calpain activity in protein extracts of cardiac muscles of the rats alcoholized for 3 and 6 months. Western blot analysis did not show reliable changes in the contents of μ-calpain and calpastatin, and SDS-PAGE did not reveal any decrease in the titin content in the myocardium of rats after the chronic alcohol intoxication. Autolysis of μ-calpain was also not verified, which could indicate that proteolytic activity of this enzyme in myocardium of chronically alcoholized rats is not enhanced. Using Pro-Q Diamond staining, changes in phosphorylation level of titin were not detected in cardiac muscle of rats after chronic alcoholization during three and six months. A decrease in µ-calpain and calpastatin mRNA content (1.3-fold, p ≤ 0.01 and ~1.9-fold, p ≤ 0.01, respectively) in the myocardium of rats alcoholized for 3 months and decrease in calpastatin mRNA (1.4-fold, p ≤ 0.01) in animals alcoholized for 6 months was demonstrated using real-time PCR. These results indicate negative effect of chronic alcohol intoxication on expression of the abovementioned genes.
在经历3个月和6个月慢性酒精化的大鼠心肌中，研究了钙依赖性钙蛋白酶的酶活性以及微摩尔钙离子浓度激活的μ-钙蛋白酶、钙蛋白酶抑制蛋白（钙蛋白酶的抑制剂）和肌联蛋白（钙蛋白酶的底物）的含量及基因表达。酒精化大鼠的“心脏重量/体重”参数没有增加，这表明未发生心脏肥大，而在酒精性心肌病建模6个月后的大鼠中，该参数呈下降趋势，这表明心肌出现萎缩性变化。使用钙蛋白酶活性检测试剂盒进行的荧光测量未发现酒精化3个月和6个月的大鼠心肌蛋白提取物中总钙蛋白酶活性有任何变化。蛋白质免疫印迹分析未显示μ-钙蛋白酶和钙蛋白酶抑制蛋白含量有可靠变化，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳也未显示慢性酒精中毒大鼠心肌中肌联蛋白含量有任何降低。μ-钙蛋白酶的自溶也未得到证实，这可能表明该酶在慢性酒精化大鼠心肌中的蛋白水解活性未增强。使用Pro-Q Diamond染色，未检测到慢性酒精化3个月和6个月的大鼠心肌中肌联蛋白磷酸化水平的变化。使用实时聚合酶链反应证明，酒精化3个月的大鼠心肌中μ-钙蛋白酶和钙蛋白酶抑制蛋白mRNA含量分别降低了约1.3倍（p≤0.01）和约1.9倍（p≤0.01），酒精化6个月的动物中钙蛋白酶抑制蛋白mRNA降低了约1.4倍（p≤0.01）。这些结果表明慢性酒精中毒对上述基因的表达有负面影响。
