Abstract
Mitochondrial protein kinase C isozymes have been reported to mediate both cardiac ischemic preconditioning and ischemia/reperfusion injury. In addition, cardiac preconditioning improves the recovery of ATP levels after ischemia/reperfusion injury. We have, therefore, evaluated protein kinase C modulation of the F(1)F(0) ATPase in neonatal cardiac myocytes. Exposure of cells to 3 or 100 nM 4beta-phorbol 12-myristate-13-acetate induced co-immunoprecipitation of delta protein kinase C (but not alpha, epsilon, or zeta protein kinase C) with the d subunit of the F(1)F(0) ATPase. This co-immunoprecipitation correlated with 40+/-3% and 72+/-9% inhibitions of oligomycin-sensitive F(1)F(0) ATPase activity, respectively. We observed prominent expression of delta protein kinase C in cardiac myocyte mitochondria, which was enhanced following a 4-h hypoxia exposure. In contrast, hypoxia decreased mitochondrial zetaPKC levels by 85+/-1%. Following 4 h of hypoxia, F(1)F(0) ATPase activity was inhibited by 75+/-9% and delta protein kinase C co-immunoprecipitated with the d subunit of F(1)F(0) ATPase. In vitro incubation of protein kinase C with F(1)F(0) ATPase enhanced F(1)F(0) activity in the absence of protein kinase C activators and inhibited it in the presence of activators. Recombinant delta protein kinase C also inhibited F(1)F(0) ATPase activity. Protein kinase C overlay assays revealed delta protein kinase C binding to the d subunit of F(1)F(0) ATPase, which was modulated by diacylglycerol, phosphatidylserine, and cardiolipin. Our results suggest a novel regulation of the F(1)F(0) ATPase by the delta protein kinase C isozyme.
Highlights
The mitochondrial F1F0 ATP synthase produces greater than 90% of cellular ATP in the myocardium, yet relatively little is known regarding how this enzyme complex is regulated [1, 2]
␦protein kinase C (PKC) co-immunoprecipitates with d subunit of the F1F0 ATPase (dF1F0) following Hx conditions, which correlates with inhibition of F1F0 ATPase activity (Fig. 3) in Neonatal Cardiac Myocytes (NCMs)
Our studies suggest a direct binding interaction between the d subunit of the F1F0 ATPase and ␦PKC, which correlates with inhibition of F1F0 ATPase activity
Summary
Primary Neonatal Cardiac Myocytes (NCMs)—NCMs were isolated from the hearts of 1-day-old Sprague-Dawley rats as previously described [22, 23]. The dF1F0 antisera-coupled Affi-gel was blocked for 1 h in IP buffer (13.3 mM Tris-HCl, pH 7.4, 2 mM EDTA, 1.8 mM EGTA, 0.5% Triton X-100 (w/v), 0.5% SDS (w/v), 1.5 mM NaPPi, 0.005% (w/v) bovine serum albumin, 1.5 nM calyculin A, and 9.1 g/ml each of aprotinin, leupeptin, phenylmethylsulfonyl fluoride) before immunoprecipitation. NCP strips were incubated with overlay buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 12 mM -mercaptoethanol, 1.0% polyethylene glycol, and 10 g/ml protease inhibitors (aprotinin, leupeptin, SBTI, phenylmethylsulfonyl fluoride)) containing purified PKC (500 g/ml) and cofactors for 1 h at room temperature. Rat brain PKC (120 units/mg) was added to 50 g of purified F1F0 ATPase for 5 min at room temperature in the presence or absence of DG and PS. Student’s t test or one-way analysis of variance with Bonferroni’s posthoc analyses were used for comparison of differences between groups, and a p value Յ 0.05 was considered to be significant
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