Early growth response 1 is an early signal inducing Cav3.2 T-type calcium channels during cardiac hypertrophy

The Ca(v)3.2 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 Ca(v)3.2 is induced by an early signal. Our aim is to investigate when and how Ca(v)3.2 is induced during cardiac hypertrophy. The evolutionary conserved promoter Ca(v)3.2-3500 from mouse genome was validated to express the reporter gene as endogenous Ca(v)3.2 in cell lines and transgenic (Tg; Ca(v)3.2-3500-Luc) mice. The early induction of luciferase in Tg mice and Ca(v)3.2 mRNA in wild-type mice after transverse aortic banding (TAB) surgery supported our hypothesis that Ca(v)3.2 is induced early during cardiac hypertrophy. The TAB-responding element [81 to 41 bp upstream of the transcription start site (TSS) of mouse Ca(v)3.2] 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 Ca(v)3.2. Egr1 level was increased with increased Ca(v)3.2 mRNA level at 3 days after TAB. To demonstrate that Egr1 indeed regulates Ca(v)3.2 expression after hypertrophic stimulation, knockdown of Egr1 with short hairpin RNA prevented the phenylephrine-induced up-regulation of Ca(v)3.2 expression and cellular hypertrophy in neonatal rat ventricular myocytes (NRVMs) and H9c2 cells. Furthermore, overexpression of Ca(v)3.2 in Egr1-knockdown cells restored the phenylephrine-induced hypertrophy. Ca(v)3.2 is induced early by Egr1 during cardiac hypertrophy and Ca(v)3.2 is an important mediator of Egr1 in regulating cardiac hypertrophy.