Period 2 protein inhibits cardiomyocyte death induced by β₁-adrenoceptor autoantibodies

To investigate the role of period 2 (Per2) protein in the death of cardiomyocytes induced by β₁-adrenergic receptor autoantibodies (β₁-AA).

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-ECⅡ), 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.

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).

Per2 protein inhibits β₁-AA induced H9c2 cardiomyocyte death.