Abstract 1875: L-type Calcium Channel C-terminus Suppresses L-type Calcium Channel Transcription in Cardiomyocytes
Calcium homeostasis is critical for cardiac myocyte function and must be tightly regulated. In this study we test the hypothesis that a mobile carboxyl-terminal cleavage product (CCt) of the cardiac L-type calcium channel (CaV1.2) auto-regulates expression. Our ongoing in vivo studies showed that persistent CaV1.2 block induced an up-regulation of CaV1.2 mRNA, protein and ICa,L. In the present study we test the hypothesis that CCt contributes to transcriptional signaling associated with cellular hypertrophy. We created eGFP-CCt fusion constructs to explore CCt regulation of the CaV1.2 promoter, and to report sub-cellular localization of CCt in cardiac myocytes. Developing cardiac myocytes were cultured in 10% FBS. Over-expression of native, full-length CCt causes a 34 ± 8% decrease of CaV1.2 promoter activity, and truncated CCt caused 80 ± 3% decrease (n = 12). Wild-type CCt distributes into cytosol and nucleus, with a greater presence in the nucleus than cytosol. A deletion mutant of CCt was also tested. Truncated CCt has a greater relative affinity for the nucleus than wild-type CCt. This is consistent with increased suppression of CaV1.2 promoter activity by truncated CCt. Chromatin immunoprecipitation (ChIP) assay showed that CCt interacts with the CaV1.2 promoter in adult ventricular cardiac myocytes at promoter modules containing NKX2.5/MEF2, C/EBp, and a cis regulatory module identified in silico. We next explored whether fetal cardiac myocyte CaV1.2 was regulated by serum in vitro. We tested ANF promoter activity as a positive control, and measured serum-response, versus serum-withdrawal on CaV1.2 promoter, mRNA, protein, and L-type current (ICa,L) from fetal mouse ventricular myocytes. Serum increased ANF promoter activity and cell size. Serum withdrawal increased CaV1.2 promoter activity, CaV1.2 protein, and ICa,L. Moreover, serum withdrawal decreased the relative nuclear localization of CCt. A combination of promoter deletion mutant analysis, and the response of promoter mutants to serum withdrawal support the conclusion that CCt, a proteolytic fragment of CaV1.2 auto-regulates CaV1.2 expression in cardiac myocytes. These data support the novel mechanism that a mobile segment of CaV1.2 links Ca handling to nuclear signaling.