Evidence for calcineurin-mediated regulation of SERCA 2a activity in human myocardium

G. MUNCH, B. BOLCK, P. KARCZEWSXI AND R. H. G. SCHWINGER. Evidence for Calcineurin-mediated Regulation of SERCA 2a Activity in Human Myocardium. Journal of Molecular and Cellular Cardiology (2002) 34, 321-334. Compromised SERCA 2a activity is a key malfunction leading to the Ca2+ cycling alterations in failing human myocardium. SERCA 2a activity is regulated by the Ca2+/calmodulin-dependent protein kinase (CaM-kinase) but alterations of the CaM-kinase pathway regarding SERCA 2a in heart failure are unresolved. Therefore we investigated the CaM-kinase and phosphatase calcineurin mediated regulation of SERCA 2a in failing and non-failing human myocardium. We studied human myocardial preparations from explanted hearts from non-failing organ donors (NF, n = 8) and from patients with terminal heart failure undergoing cardiac transplantation (dilated cardiomyopathy, DCM, n = 8). SERCA 2 a activity was determined using a NADH-coupled enzyme assay [expressed in nmol ATP/(mg protein x min)] and by Ca-45(2+) uptake, Protein expression of SERCA 2a, phospholamban, calsequestrin and calcineurin was assessed by Western blotting (expressed as densitometric units/mug protein); phosphorylation of cardiac proteins was detected with specific phospho-antibodies for phospholamban at threonine-17 (PT17) or by incorporation of [gamma-P-32] (expressed as pmol P-32/mg). Maximal Ca-45(2+) uptake (in pmol/mg/min) (NF: 3402 +/- 174: DCM: 2488 +/- 189) and maximal SERCA 2a activity were reduced in DCM compared to NF (V-max: NF: 125 +/- 9; DCM: 98 +/- 5). The V-max reduction could be mimicked by calcineurin in vitro in NF (NFcontrol: 72.1 +/- 3.7; NF+calcineurin: 49.8 +/- 2.9) and restored in DCM by CaM-kinase in vitro (DCMcontrol: 98 +/- 5; DCM+CaM-kinase: 120 6). Protein expression of SERCA 2a, phospholamban and calsequestrin remained similar, but calcineurin expression was significantly increased in failing human hearts (NF: 11.6 +/- 1.5 v DCM: 17.1 +/- 1.6). Although the capacity of endogenous CaM-kinase to phosphorylate PT17 was significantly higher in DCM (DCMcontrol: 128 +/- 36; DCM+endogenous CAM-kinase: 205 +/- 20) compared to NF myocardium (NFcontrol: 273 +/- 37; NF+endogenous CaM-kinase: 254 +/- 31), net phosphorylation at threonine-17 phospholamban was significantly lower in DCM (DCM 130 +/- 11 v NF 170 +/- 11). A calcineurin-dependent dephosphorylation of phospholamban could be mimicked in vitro by incubation of NF preparations with calcineurin (NFcontrol 80.7 +/- 4.4 v NF+calcuneurin 30.7 +/- 4.1, P<0.05). In human myocardium. the V.. of SERCA 2a and the phosphorylation of phospholamban is modulated by CaM-kinase and calcineurin, at least in vitro, In failing human myocardium, despite increased CaM-kinase activity, calcineurin dephosphorylation leads to decreased net phosphorylation of threonine-17 phospholamban in vivo. Increased calcineurin activity contributes to the impaired V-max of SERCA 2a in failing human myocardium and the disorder in Ca2+-handling in heart failure. (C) 2002 Elsevier Science Ltd. All right reserved.