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

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Item Type:	Article
Title:	Evidence for calcineurin-mediated regulation of SERCA 2a activity in human myocardium
Creators Name:	Muench, G., Boelck, B., Karczewski, P. and Schwinger, R.H.G.
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 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 2a activity was determined using a NADH-coupled enzyme assay [expressed in nmol ATP/(mg protein×min)] and by45Ca2+ uptake. Protein expression of SERCA 2a, phospholamban, calsequestrin and calcineurin was assessed by Western blotting (expressed as densitometric units/μ g protein); phosphorylation of cardiac proteins was detected with specific phospho-antibodies for phospholamban at threonine-17 (PT17) or by incorporation of [ γ -32P] (expressed as pmol32P/mg). Maximal45Ca2+ uptake (in pmol/mg/min) (NF: 3402±174; DCM: 2488±189) and maximal SERCA 2a activity were reduced in DCM compared to NF (Vmax: NF: 125±9; DCM: 98±5). The Vmax 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 (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 (NFcontrol 80.7±4.4 v NF+calcineurin 30.7±4.1, P<0.05). In human myocardium, the Vmax 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 Vmax of SERCA 2a in failing human myocardium and the disorder in Ca2+-handling in heart failure.
Keywords:	Ca2+-Transporting ATPase, Ca2+/Calmodulin-Dependent Protein Kinase (CaM-kinase), Calcineurin, Calmodulin, Heart Failure, Phospholamban, Sarcoplasmic Reticulum
Source:	Journal of Molecular and Cellular Cardiology
ISSN:	0022-2828
Publisher:	Elsevier
Volume:	34
Number:	3
Page Range:	321-334
Date:	1 January 2002
Official Publication:	https://doi.org/10.1006/jmcc.2001.1515
PubMed:	View item in PubMed

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