Article

Evidence for Calcineurin-mediated Regulation of SERCA 2a Activity in Human Myocardium

Laboratory of Muscle Research and Molecular Cardiology, Klinik III für Innere Medizin, University of Cologne, Germany.
Journal of Molecular and Cellular Cardiology (Impact Factor: 5.22). 03/2002; 34(3):321-34. DOI: 10.1006/jmcc.2001.1515
Source: PubMed

ABSTRACT Compromised SERCA 2a activity is a key malfunction leading to the Ca(2+) cycling alterations in failing human myocardium. SERCA 2a activity is regulated by the Ca(2+)/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 2a activity was determined using a NADH-coupled enzyme assay [expressed in nmol ATP/(mg protein x min)] and by(45)Ca(2+) uptake. Protein expression of SERCA 2a, phospholamban, calsequestrin and calcineurin was assessed by Western blotting (expressed as densitometric units/microg protein); phosphorylation of cardiac proteins was detected with specific phospho-antibodies for phospholamban at threonine-17 (PT17) or by incorporation of [gamma -(32)P] (expressed as pmol(32)P/mg). Maximal(45)Ca(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 (NF(control): 72.1+/-3.7; NF(+calcineurin): 49.8+/-2.9) and restored in DCM by CaM-kinase in vitro (DCM(control): 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 (DCM(control): 128+/-36; DCM(+endogenous CaM-kinase): 205+/-20) compared to NF myocardium (NF(control): 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 (NF(control) 80.7+/-4.4 v NF(+calcineurin) 30.7+/-4.1, P<0.05). In human myocardium, the V(max) 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 Ca(2+)-handling in heart failure.

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Available from: Götz Münch, Mar 05, 2014
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    • "Phosphorylation of phospholamban increases sarcoplasmic reticulum Ca 2+ -ATPase activity (Tada et al., 1975). In vitro sarcoplasmic reticulum Ca 2+ -ATPase activity can be depressed by calcineurin-mediated dephosphorylation in non-failing tissue (Münch et al., 2002). When sarcoplasmic reticulum Ca 2+ -ATPase activity is decreased , reuptake of calcium into the sarcoplasmic reticulum is decreased, which result in the Ca 2+ overload in the myocardium cytoplasm and calcineurin as well as initiate hypertrophy in cardiomyocytes . "
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    • "Also observed was a direct interaction between calnexin and Bap31, indicating a role for calnexin and Bap31 communication (Zuppini et al., 2002). In conjunction, calnexin has been observed to interact with both the SERCA transporter (Munch et al., 2002), preventing the uptake of Ca 2+ from the cytoplasm into the ER lumen and the IP 3 R (Joseph et al., 1999), regulating Ca 2+ release via its cytoplasmic tail. Bap31, caspase- 12 and calnexin may be part of the ER stress-induced apoptotic pathway and therefore may also be dependent on Ca 2+ . "
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