From the Molecular Cardiology Research Laboratory, Department of Cardiology (Q.Z., H.W., Q.W., W.L., G.L., J.-W.H., X.-M.C., J.C., W.-P.X., Y.-G.L., Y.-P.W.) and Department of Pediatrics (S.-S.W.), Affiliated Xinhua Hospital, Shanghai Jiaotong University (SJTU) School of Medicine, China.
Hypertension. 2017 Jun;69(6):1070-1083. doi: 10.1161/HYPERTENSIONAHA.116.08662. Epub 2017 Apr 24.
Cardiac hypertrophy is characterized by increased myofibrillogenesis. Angiotensin II (Ang-II) is an essential mediator of the pressure overload-induced cardiac hypertrophy in part through RhoA/ROCK (small GTPase/Rho-associated coiled-coil containing protein kinase) pathway. FHOD3 (formin homology 2 domain containing 3), a cardiac-restricted member of diaphanous-related formins, is crucial in regulating myofibrillogenesis in cardiomyocytes. FHOD3 maintains inactive through autoinhibition by an intramolecular interaction between its C- and N-terminal domains. Phosphorylation of the 3 highly conserved residues (1406S, 1412S, and 1416T) within the C terminus (CT) of FHOD3 by ROCK1 is sufficient for its activation. However, it is unclear whether ROCK-mediated FHOD3 activation plays a role in the pathogenesis of Ang-II-induced cardiac hypertrophy. In this study, we detected increases in FHOD3 expression and phosphorylation in cardiomyocytes from Ang-II-induced rat cardiac hypertrophy models. Valsartan attenuated such increases. In cultured neonate rat cardiomyocytes, overexpression of phosphor-mimetic mutant FHOD3-DDD, but not wild-type FHOD3, resulted in myofibrillogenesis and cardiomyocyte hypertrophy. Expression of a phosphor-resistant mutant FHOD3-AAA completely abolished myofibrillogenesis and attenuated Ang-II-induced cardiomyocyte hypertrophy. Pretreatment of neonate rat cardiomyocytes with ROCK inhibitor Y27632 reduced Ang-II-induced FHOD3 activation and upregulation, suggesting the involvement of ROCK activities. Silencing of ROCK2, but not ROCK1, in neonate rat cardiomyocytes, significantly lessened Ang-II-induced cardiomyocyte hypertrophy. ROCK2 can directly phosphorylate FHOD3 at both 1412S and 1416T in vitro and is more potent than ROCK1. Both kinases failed to phosphorylate 1406S. Coexpression of FHOD3 with constitutively active ROCK2 induced more stress fiber formation than that with constitutively active ROCK1. Collectively, our results demonstrated the importance of ROCK2 regulated FHOD3 expression and activation in Ang-II-induced myofibrillogenesis, thus provided a novel mechanism for the pathogenesis of Ang-II-induced cardiac hypertrophy.
心肌肥厚的特征是肌原纤维生成增加。血管紧张素 II（Ang-II）是压力超负荷诱导的心肌肥厚的重要介质，部分通过 RhoA/ROCK（小 GTP 酶/Rho 相关卷曲螺旋蛋白激酶）途径。FHOD3（formin 同源 2 结构域包含 3），一种心肌特异性的 diaphanous 相关formin，在调节心肌细胞的肌原纤维生成中至关重要。FHOD3 通过其 C 端和 N 端结构域之间的分子内相互作用保持自身失活。ROCK1 可使 FHOD3 的 C 端（CT）内 3 个高度保守的残基（1406S、1412S 和 1416T）磷酸化，从而使其激活。然而，尚不清楚 ROCK 介导的 FHOD3 激活是否在 Ang-II 诱导的心肌肥厚的发病机制中发挥作用。在这项研究中，我们在 Ang-II 诱导的大鼠心肌肥厚模型的心肌细胞中检测到 FHOD3 表达和磷酸化增加。缬沙坦可减轻这种增加。在培养的新生大鼠心肌细胞中，过表达磷酸化模拟突变体 FHOD3-DDD，但不是野生型 FHOD3，导致肌原纤维生成和心肌细胞肥大。表达磷酸化抗性突变体 FHOD3-AAA 完全消除了肌原纤维生成并减弱了 Ang-II 诱导的心肌细胞肥大。用 ROCK 抑制剂 Y27632 预处理新生大鼠心肌细胞可降低 Ang-II 诱导的 FHOD3 激活和上调，表明 ROCK 活性的参与。在新生大鼠心肌细胞中沉默 ROCK2，而不是 ROCK1，可显著减轻 Ang-II 诱导的心肌细胞肥大。ROCK2 可在体外直接使 FHOD3 的 1412S 和 1416T 磷酸化，其效力比 ROCK1 更强。两种激酶均不能磷酸化 1406S。共表达 FHOD3 与组成型激活的 ROCK2 比共表达与组成型激活的 ROCK1 诱导更多的应激纤维形成。总的来说，我们的结果表明 ROCK2 调节 FHOD3 在 Ang-II 诱导的肌原纤维生成中的表达和激活的重要性，为 Ang-II 诱导的心肌肥厚的发病机制提供了一个新的机制。
