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

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

Reduced Ca2+-sensitivity of SERCA 2a in failing human myocardium due to reduced serin-16 phospholamban phosphorylation

It is still a matter of debate, whether decreased protein expression of SERCA 2a and phospholamban (PLB), or alterations in the phosphorylation state of PLB are responsible for the reduced SERCA 2a function in failing human myocardium. Thus, in membrane preparations from patients with terminal heart failure due to idiopathic dilated cardiomyopathy (NYHA IV. heart transplants) and control hearts (NF), SERCA 2a activity was measured with an NADH coupled assay with as well as without stimulation with protein kinase A (PKA). The protein expression of SERCA 2a, PLB and calsequestrin as well as the phosphorylation status of PLB (Back-phosphorylation technique: Serine-16-PLB specific antibody) were analysed using Western blotting technique and specific antibodies. In NF, the maximal activity (Vmax) and the Ca(2+)-sensitivity of SERCA 2a activity were significantly higher compared to NYHA IV. Protein expression of SERCA 2a, PLB and calsequestrin were unchanged, whereas both, the phosphorylation status of PLB as well as serine-16-PLB-phosphorylation, were significantly reduced in NYHA IV. After stimulation with PKA only the Ca(2+)-sensitivity, but not Vmax increased concentration-dependently. Therefore, in human myocardium, the Ca(2+)-sensitivity but not the Vmax of SERCA 2a is regulated by cAMP-dependent phosphorylation of phospholamban at position serine-16. Threonine-17-PLB-phosphorylation or direct phosphorylation of SERCA 2a may be candidates for regulation of maximal SERCA 2a activity in human myocardium