Distinct pathways regulate proapoptotic Nix and BNip3 in cardiac stress

Up-regulation of myocardial Nix and BNip3 is associated with apoptosis in cardiac hypertrophy and ischemia, respectively. To identify mechanisms of gene regulation for these critical cardiac apoptosis effectors, the determinants of Nix and BNip3 promoter activation were elucidated by luciferase reporter gene expression in neonatal rat cardiac myocytes. BNip3 transcription was increased by hypoxia but not by phenylephrine (10 μM), angiotensin II (100 nM), or isoproterenol (10 μM). In contrast, Nix transcription was increased by phenylephrine but not by isoproterenol, angiotensin II, or hypoxia. Since phenylephrine stimulates cardiomyocyte hypertrophy via protein kinase C (PKC), the effects of phorbol myristate acetate (PMA, 10 nM for 24 h) and adenoviral PKC expression were assessed. PMA and PKCα, but not PKCε or dominant negative PKCα, increased Nix transcription. Multiple Nix promoter GC boxes bound transcription factor Sp-1, and basal and PMA- or PKCα-stimulated Nix promoter activity was suppressed by mithramycin inhibition of Sp1-DNA interactions. In vivo determinants of Nix expression were evaluated in Nix promoter-luciferase (NixP) transgenic mice that underwent ischemia-reperfusion (1 h/24 h), transverse aortic coarctation (TAC), or cross-breeding with the G_q overexpression model of hypertrophy. Luciferase activity increased in Gα_q-NixP hearts 3.2 ± 0.4-fold and in TAC hearts 2.8 ± 0.4-fold but did not increase with infarction-reperfusion. NixP activity was proportional to the extent of TAC hypertrophy and was inhibited by mithramycin. These studies revealed distinct mechanisms of transcriptional regulation for cardiac Nix and BNip3. BNip3 is hypoxia-inducible, whereas Nix expression was induced by Gα_q-mediated hypertrophic stimuli. PKCα, a G_q effector, transduced Nix transcriptional induction via Sp1.