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[摘要]：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 (Ca(V)1.2) autoregulates expression. First, we confirmed that the Ca(V)1.2 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 Ca(V)1.2 promoter activity, and truncated CCt caused an 80 +/- 3% decrease of Ca(V)1.2 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 Ca(V)1.2 promoter activity by truncated CCt. Chromatin immunoprecipitation analysis revealed that CCt interacts with the Ca(V)1.2 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 Ca(V)1.2 was regulated by serum in vitro. We tested atrial natriuretic factor promoter activity as a positive control and measured the serum response of Ca(V)1.2 promoter, protein, and L-type current (I-Ca,I-L) from fetal mouse ventricular myocytes. Serum increased atrial natriuretic factor promoter activity and cell size as expected. Serum withdrawal increased Ca(V)1.2 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 Ca(V)1.2, autoregulates Ca(V)1.2 expression in cardiac myocytes. These data support the novel mechanism that a mobile segment of Ca(V)1.2 links Ca handling to nuclear signaling. (Circ Res. 2009; 104: 1373-1381.) |
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