L-Type Calcium Channel C Terminus Autoregulates Transcription

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Calcium homeostasis is critical for cardiac myocyte function and must be tightly regulated. The guiding hypothesis of this study is that a carboxyl-terminal cleavage product of the cardiac L-type calcium channel (CaV1.2) autoregulates expression. First, we confirmed that the CaV1.2 C terminus (CCt) is cleaved in murine cardiac myocytes from mature and developing ventricle. Overexpression of full-length CCt caused a 34±8% decrease of CaV1.2 promoter activity, and truncated CCt caused an 80±3% decrease of CaV1.2 promoter (n=12). The full-length CCt distributes into cytosol and nucleus. A deletion mutant of CCt has a greater relative affinity for the nucleus than full-length CCt, and this is consistent with increased repression of CaV1.2 promoter activity by truncated CCt. Chromatin immunoprecipitation analysis revealed 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. The next hypothesis tested was that CCt contributes to transcriptional signaling associated with cellular hypertrophy. We explored whether fetal cardiac myocyte CaV1.2 was regulated by serum in vitro. We tested atrial natriuretic factor promoter activity as a positive control and measured the serum response of CaV1.2 promoter, protein, and L-type current (ICa,L) from fetal mouse ventricular myocytes. Serum increased atrial natriuretic factor promoter activity and cell size as expected. Serum withdrawal increased CaV1.2 promoter activity, mRNA, and ICa,L. Moreover, serum withdrawal decreased the relative nuclear localization of CCt. A combination of promoter deletion mutant analyses, and the response of promoter mutants to serum withdrawal support the conclusion that CCt, a proteolytic fragment of CaV1.2, autoregulates 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.

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