Objective To investigate the role of period 2 (Per2) protein in the death of cardiomyocytes induced by β1-adrenergic receptor autoantibodies (β1-AA). Methods Sixteen male SD rats aged 6~8 weeks were randomly divided into active immunization (model) group and control group (n=8). The model group was immunized with the second extracellular loop of beta1-adrenoceptor (β1-AR-EC Ⅱ), and the control group was injected with Na2CO3 and other solutions. The rat serum was subsequently collected at 8 weeks, followed by the purification of β1-AA. H9c2 cardiomyocyte were selected and randomly divided into control group, β1-AA group, and β1-AR+β1-AA group. Cell viability of each group was detected by CCK-8 assay (n=8). Then H9c2 cells in the control group and β1-AA 1 μmol/L group were synchronized with dexamethasone for 4 h, the expression of Per2 in cardiomyocytes at different circadian time (CT) points was measured by Western blotting, and JTK_CYCLE was used to analyze the circadian rhythm parameters (n=11). Moreover, Per2 in H9c2 cells was knocked down or overexpressed by lentiviral shPer2 and lentiviral Per2, respectively; RT-PCR and Western blotting were performed to detect the changes of Per2 expression (n=6). On the basis of knockdown (n=8) or overexpression (n=10) of Per2, the H9c2 cells were further treated with β1-AA, and the cell survival rate was tested by CCK-8 assay. Results CCK-8 assay showed that the survival rate of H9c2 cells was significantly decreased after β1-AA treatment (P < 0.05). Western blotting demonstrated that β1-AA remarkably inhibited the rhythmic expression of Per2 protein in the cardiomyocytes (JTK_CYCLE, P>0.05), with the decrease at CT8 and CT16 most obviously (P < 0.01). Knockdown of Per2 expression reduced the survival rate of cardiomyocytes, which was further lowered after β1-AA treatment (P < 0.001). However, overexpression of Per2 notably reversed the decline in H9c2 survival rate induced by β1-AA (P < 0.001). Conclusion Per2 protein inhibits β1-AA induced H9c2 cardiomyocyte death.

目的:探讨β1-肾上腺素受体(β1-AR)自身抗体(β1-AA)对大鼠心肌细胞自噬标志物微管相关蛋白1轻链3(LC3)节律表达的影响及其在心肌细胞死亡中的作用.方法:实验材料为Sprague-Dawley(SD)大鼠和H9c2大鼠心肌细胞.将SD大鼠随机分为免疫组(β1-AR组)和对照(control)组,每组6只;将H9c2细胞随机分为control组、β1-AA组、慢病毒(LV)-NC组和LV-shPer2组(n=6);合成β1-AR细胞外第二环抗原肽段,用于主动免疫大鼠,并使用亲和层析法从大鼠血清中提纯β1-AA;β1-AA处理H9c2细胞24 h后使用CCK-8法检测细胞活力;用地塞米松同步化细胞后,再给予β1-AA处理,采用real-time PCR及Western blot法检测LC3的表达情况,采用Western blot法检测生物钟蛋白Per2的表达情况,使用JTK_CYCLE算法分析昼夜节律参数;用LV-shPer2感染H9c2细胞以破坏LC3的节律表达,进而采用CCK-8法检测细胞活力.结果:β1-AR组大鼠血清中β1-AA的A值与control组相比显著升高(P<0.05).β1-AA组H9c2细胞的活力显著低于control组(P<0.05).β1-AA可破坏H9c2细胞LC3和Per2的节律表达(JTK_CYCLE P<0.05).通过LV-shPer2干扰Per2基因而破坏H9c2细胞LC3节律表达(JTK_CYCLE P<0.05)后,细胞活力显著降低(P<0.05).结论:β1-AA破坏H9c2大鼠心肌细胞自噬标志物LC3的节律表达,从而促进细胞死亡.

Cardiomyocytes autophagy is essential for maintaining cardiac function. Our previous studies have found that β 1 ‐adrenergic receptor autoantibody (β 1 ‐AA) induced the decreased myocardial autophagic flux, which resulted in cardiomyocyte death and cardiac dysfunction. And other studies demonstrated that β 1 ‐AA induced the decrease of AMPK phosphorylation, the key hub of autophagy pathway, while adiponectin up‐regulated autophagic flux mediated by AMPK. However, it is not clear whether adiponectin improves the inhibition of myocardial autophagic flux induced by β 1 ‐AA by up‐regulating the level of AMPK phosphorylation. In this study, it has been confirmed that β 1 ‐AA induced the decrease of AMPK phosphorylation level in both vivo and vitro. Moreover, pretreatment of cardiomyocytes with AMPK inhibitor Compound C could further reduce the autophagic flux induced by β 1 ‐AA. Adiponectin deficiency could aggravate the decrease of myocardial AMPK phosphorylation level, autophagic flux and cardiac function induced by β 1 ‐AA. Further, exogenous adiponectin could reverse the decline of AMPK phosphorylation level and autophagic flux induced by β 1 ‐AA and even reduce cardiomyocyte death. While pretreated with the Compound C, the adiponectin treatment did not improve the decreased autophagosome formation, but still improved the decreased autophagosome clearance induced by β 1 ‐AA in cardiomyocytes. This study is the first time to confirm that β 1 ‐AA could inhibit myocardial autophagic flux by down‐regulating AMPK phosphorylation level. Adiponectin could improve the inhibition of myocardial autophagic flux induced by β 1 ‐AA partly dependent on AMPK, so as to provide an experimental basis for the treatment of patients with β 1 ‐AA‐positive cardiac dysfunction.