Molecular Medicine Program, Taiwan International Graduate Program, Institute of Biomedical Sciences, Academia Sinica, 128 Academia Rd Sec 2, Taipei 11529, Taiwan.
Cardiovasc Res. 2013 Nov 1;100(2):222-30. doi: 10.1093/cvr/cvt190. Epub 2013 Aug 8.
The Cav3.2 T-channel plays a pivotal role in inducing calcineurin/nuclear factor of activated T cell (NFAT) signalling during cardiac hypertrophy. Because calcineurin/NFAT signalling is induced early after pressure overload, we hypothesized that Cav3.2 is induced by an early signal. Our aim is to investigate when and how Cav3.2 is induced during cardiac hypertrophy.
The evolutionary conserved promoter Cav3.2-3500 from mouse genome was validated to express the reporter gene as endogenous Cav3.2 in cell lines and transgenic (Tg; Cav3.2-3500-Luc) mice. The early induction of luciferase in Tg mice and Cav3.2 mRNA in wild-type mice after transverse aortic banding (TAB) surgery supported our hypothesis that Cav3.2 is induced early during cardiac hypertrophy. The TAB-responding element [-81 to -41 bp upstream of the transcription start site (TSS) of mouse Cav3.2] was identified by in vivo gene transfer by injecting reporter constructs into the left ventricle followed by TAB surgery. Electrophoresis mobility shift assay and chromatin immunoprecipitation assays revealed that Egr1 bound to the TAB-responding element of Cav3.2. Egr1 level was increased with increased Cav3.2 mRNA level at 3 days after TAB. To demonstrate that Egr1 indeed regulates Cav3.2 expression after hypertrophic stimulation, knockdown of Egr1 with short hairpin RNA prevented the phenylephrine-induced up-regulation of Cav3.2 expression and cellular hypertrophy in neonatal rat ventricular myocytes (NRVMs) and H9c2 cells. Furthermore, overexpression of Cav3.2 in Egr1-knockdown cells restored the phenylephrine-induced hypertrophy.
Cav3.2 is induced early by Egr1 during cardiac hypertrophy and Cav3.2 is an important mediator of Egr1 in regulating cardiac hypertrophy.
Cav3.2 T 通道在心脏肥大过程中诱导钙调神经磷酸酶/活化 T 细胞核因子（NFAT）信号转导中起着关键作用。由于钙调神经磷酸酶/NFAT 信号在压力超负荷后早期诱导，我们假设 Cav3.2 是由早期信号诱导的。我们的目的是研究 Cav3.2 在心脏肥大过程中何时以及如何被诱导。
从鼠基因组中验证了进化保守的启动子 Cav3.2-3500，以在细胞系和转基因（Tg；Cav3.2-3500-Luc）小鼠中表达报告基因作为内源性 Cav3.2。Tg 小鼠中的荧光素酶早期诱导和野生型小鼠在横主动脉缩窄（TAB）手术后的 Cav3.2 mRNA 支持我们的假设，即 Cav3.2 在心脏肥大早期被诱导。通过将报告基因构建体注射到左心室后进行 TAB 手术，在体内基因转移中鉴定了 Cav3.2 的转录起始位点（TSS）上游的早期诱导元件[-81 至-41 bp]。电泳迁移率变动分析和染色质免疫沉淀分析显示，Egr1 与 Cav3.2 的 TAB 反应元件结合。Egr1 水平随着 TAB 后 3 天 Cav3.2 mRNA 水平的增加而增加。为了证明 Egr1 确实在肥大刺激后调节 Cav3.2 的表达，用短发夹 RNA 敲低 Egr1 可防止去甲肾上腺素诱导的新生大鼠心室肌细胞（NRVMs）和 H9c2 细胞中 Cav3.2 表达和细胞肥大的上调。此外，在 Egr1 敲低细胞中过表达 Cav3.2 恢复了去甲肾上腺素诱导的肥大。
Cav3.2 在心脏肥大过程中由 Egr1 早期诱导，Cav3.2 是 Egr1 调节心脏肥大的重要介质。
