To investigate the mechanism by which angiotensin Ⅱ type 1 receptor autoantibody (AT1-AA) promotes phenotypic switch of vascular smooth muscle cells (VSMCs) and vascular fibrosis through abnormal expression of circadian clock protein BMAL1.

Twelve male SD rats (6~8 weeks old, weighing 180~220 g) were randomly divided into (n=6) a control group and an AT1-AA-positive group [established by active immunization of SD rats with AT1R extracellular loop Ⅱ peptide (AT1R-ECL Ⅱ)]. HE and Masson stainings were used to observe structural changes and fibrosis in the thoracic aorta (n=3). Western blotting was performed to detect the expression of Collagen Ⅰ, phenotypic switch-related proteins (SM22, α-SMA, OPN and MMP2) in vascular tissues and primary VSMCs (n=4), as well as the expression of BMAL1 at CT0, CT4, CT8, CT12, CT16, and CT20. Transwell and scratch assays were used to assess the proliferation and migration of VSMCs (n=3). si-RNA was employed to knock down Bmal1, followed by detection of BMAL1, Collagen Ⅰ, and phenotypic conversion-related protein expression (n=3). Additionally, AT1-AA-positive AT1R-knockout (AT1R-KO) rats were constructed to measure BMAL1 expression in thoracic aortic tissues (n=4).

The AT1-AA-positive rats had significantly thickened thoracic aortic vessel wall [(140±9)% vs (120±5)%, P<0.05], badly arranged VSMCs, obvious blue Masson staining, and up-regulated Collagen Ⅰ expression (P<0.05). In the thoracic aorta of AT1-AA-positive rats and AT1-AA-treated VSMCs, the expression of contractile phenotype-related proteins (α-SMA, SM22) was decreased (P<0.05), while the expression of synthetic phenotype-related proteins (OPN, MMP2) was increased (P<0.05). AT1-AA enhanced the scratch healing ability and migration ability of VSMCs. Furthermore, both mRNA and protein levels of Bmal1 were significantly up-regulated at CT12 (P<0.05), and the rhythmicity of Bmal1 was lost. Knockdown of Bmal1 partially ameliorated AT1-AA-induced phenotypic switch of VSMCs. Compared with AT1-AA-positive WT rats, AT1-AA-positive AT1R-KO rats showed significantly reduced BMAL1 expression in the thoracic aorta (1.35±0.06 vs 0.86±0.07, P<0.001). At the cellular level, AT1-AA-induced phenotypic switch and high Collagen Ⅰ expression in VSMCs were partially improved in AT1R-KO VSMCs.

AT1-AA promotes VSMCs phenotypic conversion and vascular fibrosis through the AT1R-Bmal1 axis.

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.