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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    ABSTRACT: To observe effects of angiotensin (Ang) II receptor antagonist (AT1) irbesartan and angiotensin-converting enzyme (ACE) inhibitor perindopril on rat myocardium calcineurin expression and sarcoplasmic reticulum Ca(2+)-ATPase activity in the model of pressure-overload cardiac hypertrophy. Forty male adult Sprague Dawley rats were divided into 5 groups. One group was treated by sham operation; four groups were myocardium hypertrophy cases caused by banding aortic above renal artery. Drugs were given one week after operation. Group 1: sham group, rats (n=8) were gavaged with normal saline 2 ml/(kg.d) (ig); Group 2: control group, rats (n=8) were treated with normal saline 2 ml/(kg.d) (ig); Group 3: rats (n=8) were given perindopril 2 mg/(kg.d) (ig); Group 4: rats (n=8) were treated with irbesartan 20 mg/(kg.d) (ig); Group 5: rats (n=8) were given irbesartan 20 mg/(kg.d) plus perindopril 2 mg/(kg.d) (ig). Morphometric determination, calcineurin expression and sarcoplasmic reticulum Ca(2+)-ATPase activity were done at the end of 6 week of drug intervention. Expression of calcineurin in myocardium was detected by immunohistochemistry. Left ventricular mass index (LVMI), transverse diameter of myocardial cell (TDM), calcineurin activity were remarkably decreased after drug intervention and this decrease was most remarkable in the combination drug therapy group. Sarcoplasmic reticulum Ca(2+)-ATPase activity was increased after drug intervention, especially in the combined drug therapy group. Calcineurin expression in myocardium was remarkably decreased after drug intervention. LVMI was positively correlated with TDM and calcineurin, negatively correlated with sarcoplasmic reticulum Ca(2+)-ATPase. These data suggest that irbesartan and perindopril inhibit cardiac hypertrophy through the increased activity of sarcoplasmic reticulum Ca(2+)-ATPase and decreased expression of calcineurin. Their combination had better effects on regressing of ventricular hypertrophy.
    Journal of Zhejiang University SCIENCE B 04/2006; 7(3):228-34. DOI:10.1631/jzus.2006.B0228 · 1.29 Impact Factor
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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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    ABSTRACT: The ER is one of the most important folding compartments within the cell, as well as an intracellular Ca(2+) storage organelle and it contains a number of Ca(2+) regulated molecular chaperones responsible for the proper folding of glycosylated as well as non-glycosylated proteins. The luminal environment of the ER contains Ca(2+) which is involved in regulating chaperones such as calnexin and calreticulin, as well as apoptotic proteins caspase-12 and Bap31, which may play an important role in determining cellular sensitivity to ER stress and apoptosis. The ER quality control system consists of several molecular chaperones, including calnexin, that assist in properly folding proteins and transporting them through the ER as well as sensing misfolded proteins, attempting to refold them and if this is not possible, targeting them for degradation. Accumulation of misfolded protein in the ER leads to activation of genes responsible for the expression of ER chaperones. The UPR mechanism involves transcriptional activation of chaperones by the membrane-localized transcription factor ATF6, in conjunction with the ER membrane kinase IRE1, as well as translational repression of protein synthesis by another ER membrane kinase PERK. When accumulation of misfolded protein becomes toxic, apoptosis is triggered, potentially with IRE1 involved in signaling via caspase-12. Both the extrinsic and intrinsic apoptotic pathways appear to culminate in the activation of caspases and this results in the recruitment of mitochondria in an essential amplifying manner. Bap31 may direct pro-apoptotic crosstalk between the ER and the mitochondria via Ca(2+) in conjunction with caspase-12 and calnexin. Accordingly, ER stress and the resultant Ca(2+) release must be very carefully regulated because of their effects in virtually all areas of cell function.
    Acta biochimica Polonica 02/2005; 52(2):381-95. · 1.39 Impact Factor
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    • "In addition, there have been some hints towards a direct modulation of components involved in cardiac excitation– contraction coupling, such as sarcoplasmic reticulum calcium ATPase (SERCA) or RyR [11] [12]. Since it was reported by Schuhmann et al. [13] that calcineurin directly modulates single human vascular LTCC, we considered it important to test whether or not calcineurin might affect single human cardiac LTCC as well. "
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    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. DOI:10.1016/j.yjmcc.2003.11.013 · 5.22 Impact Factor
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