Evidence for calcineurin-mediated regulation of SERCA 2a activity in human myocardium.
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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ABSTRACT: We investigated how exercise training superimposed on chronic hypertension impacted left ventricular remodeling. Cardiomyocyte hypertrophy, apoptosis, and proliferation in hearts from female spontaneously hypertensive rats (SHRs) were examined. Four-month-old SHR animals were placed into a sedentary group (SHR-SED; n = 18) or a treadmill running group (SHR-TRD, 20 m/min, 1 h/day, 5 days/wk, 12 wk; n = 18). Age-matched, sedentary Wistar Kyoto (WKY) rats were controls (n = 18). Heart weight was greater in SHR-TRD vs. both WKY (P < 0.01) and SHR-SED (P < 0.05). Morphometric-derived left ventricular anterior, posterior, and septal wall thickness were increased in SHR-SED relative to WKY and augmented in SHR-TRD. Cardiomyocyte surface area, length, and width were increased in SHR-SED relative to WKY and further increased in SHR-TRD. Calcineurin abundance was increased in SHR-SED vs. WKY (P < 0.001) and attenuated in SHR-TRD relative to SHR-SED (P < 0.05). Protein abundance and mRNA of Akt was not different among groups. The rate of apoptosis was increased in SHR-SED relative to WKY and mitigated in SHR-TRD. The abundance of Ki-67(+) cells across groups was not statistically different across groups. The abundance of cardiac progenitor cells (c-Kit(+) cells) was increased in SHR-TRD relative to WKY. These data suggest that exercise training superimposed on hypertension augmented cardiomyocyte hypertrophy, despite attenuating calcineurin abundance. Exercise training also mitigated apoptosis in hypertension and showed a tendency to enhance the abundance of cardiac progenitor cells, resulting in a more favorable cardiomyocyte number in the exercise-trained hypertensive heart.AJP Heart and Circulatory Physiology 09/2009; 297(4):H1361-8. · 3.71 Impact Factor
Article: Calcineurin inhibition normalizes beta-adrenergic responsiveness in the spontaneously hypertensive rat.[show abstract] [hide abstract]
ABSTRACT: Calcineurin, a Ca(2+)-regulated protein phosphatase, links myocardial Ca(2+) signaling with hypertrophic gene transcription. Calcineurin abundance increases in pressure-overload hypertrophy and may reduce agonist-mediated phospholamban (PLB) phosphorylation to underlie blunted beta-adrenergic receptor (beta-AR) responsiveness in hypertension. This hypothesis was tested by measuring the effects of calcineurin inhibition on changes in cardiac contractility caused by beta-adrenergic stimulation in spontaneously hypertensive rats (SHR). Female SHR (age: 7 mo) and age-matched female Wistar-Kyoto rats (WKY) were studied. Heart weight-to-body weight ratio (P < 0.01) and systolic blood pressure (P < 0.01) were greater in SHR compared with WKY and were associated with increased myocardial calcineurin mRNA (CnAbeta) and activity (P < 0.05). beta-AR stimulation with isoproterenol (Iso) increased calcineurin activity (P < 0.05) in both WKY and SHR hearts, and this activity was suppressed with cyclosporin A (CsA) treatment. In SHR, CsA improved left ventricular whole heart and isolated myocyte beta-AR responsiveness by normalizing PLB phosphorylation at Ser(16) and Thr(17) (P < 0.05). These CsA-induced, PLB-mediated effects were associated with an augmentation in cardiomyocyte peak Ca(2+) and a reduced rate (time constant of isovolumic pressure relaxation, tau) and magnitude of diastolic Ca(2+) during beta-AR stimulation. In conclusion, CsA normalized the blunted beta-AR responsiveness associated with hypertension, in part, by mitigating calcineurin activity while improving PLB phosphorylation and subsequent sarcoplasmic reticulum Ca(2+) regulation.AJP Heart and Circulatory Physiology 11/2007; 293(5):H3122-9. · 3.71 Impact Factor
Article: Ca2+-dependent modulation of single human cardiac L-type calcium channels by the calcineurin inhibitor cyclosporine.[show abstract] [hide abstract]
ABSTRACT: Activity of single L-type calcium channels (LTCC) is enhanced in human failing myocardium (Circulation 98 (1998) 969.), most likely due to impaired dephosphorylation. Protein phosphatase 2B (calcineurin) has recently been shown to be involved in heart failure pathophysiology. We now focus on the regulation of single LTCC by calcineurin that were prevented by Ca(2+)-free experimental conditions in our previous study. Single LTCC currents were recorded in myocytes from human atrium and ventricle. Charge carriers were 70 mM Ba(2+), or a mixture of 30 mM Ca(2+) and 60 mM Ba(2+) to facilitate Ca(2+) permeation through recorded channels. The calcineurin inhibitor cyclosporine (10 microM) was used to reveal a putative role for calcineurin in regulation of LTCC. A mixture of Ca(2+) and Ba(2+) as charge carriers allowed for Ca(2+) permeation through recombinant human embryonic kidney cells and native (atrial and ventricular) human cardiac LTCC. With only Ba(2+) as the charge carrier, activities of both ventricular and atrial LTCC were strongly decreased by cyclosporine. In contrast, channel activity remained constant when Ca(2+) permeation was provided. In the presence of thapsigargin and (S)-BayK 8644, cyclosporine here even increased channel activity. We propose a dual cyclosporine effect on human cardiac LTCC. A non-specific inhibitory effect prevails with Ba(2+) permeation but can be compensated or overcome by a specific Ca(2+)-dependent stimulation with Ca(2+) permeation. More complete restoration of physiological Ca(2+) movements (e.g., Ca(2+) release from sarcoplasmic reticulum) will help to define even more precisely the involvement of calcineurin in regulation of human cardiac LTCC.Journal of Molecular and Cellular Cardiology 03/2004; 36(2):241-55. · 5.17 Impact Factor