Ca_V1.2 and transient receptor potential canonical channel 3 (TRPC3) are two proteins known to have important roles in pathological cardiac hypertrophy; however, such roles still remain unclear. A better understanding of these roles is important for furthering the clinical understanding of heart failure. We previously reported that Trpc3-knockout (KO) mice are resistant to pathologic hypertrophy and that their Ca_V1.2 protein expression is reduced. In this study, we aimed to examine the relationship between these two proteins and characterize their role in neonatal cardiomyocytes. We measured Ca_V1.2 expression in the hearts of wild-type (WT) and Trpc3^−/− mice, and examined the effects of Trpc3 knockdown and overexpression in the rat cell line H9c2. We also compared the hypertrophic responses of neonatal cardiomyocytes cultured from Trpc3^−/− mice to a representative hypertrophy-causing drug, isoproterenol (ISO), and measured the activity of nuclear factor of activated T cells 3 (NFAT3) in neonatal cardiomyocytes (NCMCs). We inhibited the L-type current with nifedipine, and measured the intracellular calcium concentration using Fura-2 with 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced Ba²⁺ influx. When using the Trpc3-mediated Ca²⁺ influx, both intracellular calcium concentration and calcium influx were reduced in Trpc3-KO myocytes. Not only was the expression of Ca_V1.2 greatly reduced in Trpc3-KO cardiac lysate, but the size of the Ca_V1.2 currents in NCMCs was also greatly reduced. When NCMCs were treated with Trpc3 siRNA, it was confirmed that the expression of Ca_V1.2 and the intracellular nuclear transfer activity of NFAT decreased. In H9c2 cells, the ISO activated- and verapamil inhibited- Ca²⁺ influxes were dramatically attenuated by Trpc3 siRNA treatment. In addition, it was confirmed that both the expression of Ca_V1.2 and the size of H9c2 cells were regulated according to the expression and activation level of TRPC3. We found that after stimulation with ISO, cell hypertrophy occurred in WT myocytes, while the increase in size of Trpc3-KO myocytes was greatly reduced. These results suggest that not only the cell hypertrophy process in neonatal cardiac myocytes and H9c2 cells were regulated according to the expression level of Ca_V1.2, but also that the expression level of Ca_V1.2 was regulated by TRPC3 through the activation of NFAT.

Ca _V 1.2 和瞬时受体电位规范通道 3 (TRPC3) 是已知在病理性心脏肥大中具有重要作用的两种蛋白质；然而，这些作用仍然不明确。更好地了解这些作用对于进一步了解心力衰竭的临床意义很重要。我们之前报道过 Trpc3 基因敲除 (KO) 小鼠对病理性肥大具有抵抗力，并且它们的 Ca _V 1.2 蛋白表达降低。在这项研究中，我们旨在检查这两种蛋白质之间的关系并表征它们在新生儿心肌细胞中的作用。我们测量了野生型 (WT) 和Trpc3 ^-/-心脏中的Ca _V 1.2 表达小鼠，并检查了 Trpc3 敲低和过表达在大鼠细胞系 H9c2 中的影响。我们还比较了从Trpc3 ^-/-小鼠培养的新生儿心肌细胞对代表性肥大引起药物异丙肾上腺素 (ISO)的肥大反应，并测量了新生儿心肌细胞 (NCMC) 中活化 T 细胞 3 (NFAT3) 的核因子的活性. 我们用硝苯地平抑制 L 型电流，并使用 Fura-2 和 1-油酰-2-乙酰基-sn-甘油 (OAG) 诱导的 Ba ²⁺流入测量细胞内钙浓度。当使用 Trpc3 介导的 Ca ²⁺流入时，Trpc3-KO 肌细胞中的细胞内钙浓度和钙流入均降低。不仅是 Ca _V的表达1.2 在 Trpc3-KO 心脏裂解物中大大减少，但NCMC 中Ca _V 1.2 电流的大小也大大减少。当用Trpc3 siRNA处理NCMC时，证实Ca _V 1.2的表达和NFAT的细胞内核转移活性降低。在 H9c2 细胞中，通过 Trpc3 siRNA 处理，ISO 激活的和维拉帕米抑制的 Ca ²⁺流入显着减弱。此外，证实了 Ca _V的表达1.2和H9c2细胞的大小根据TRPC3的表达和激活水平进行调节。我们发现，在用 ISO 刺激后，WT 肌细胞发生细胞肥大，而 Trpc3-KO 肌细胞的大小增加大大减少。这些结果表明，不仅在新生心肌细胞和H9c2细胞的细胞肥大过程是根据Ca的表达水平调节_V 1.2，也认为Ca的表达水平_V 1.2通过TRPC3通过NFAT活化调节。