Phospholemman-Phosphorylation Mediates the β-Adrenergic Effects on Na/K Pump Function in Cardiac Myocytes (original) (raw)
Related papers
Circulation Research, 2006
Because phospholemman (PLM) regulates the Na + /K + pump (NKA) and is a major cardiac phosphorylation target for both protein kinase A (at Ser68) and protein kinase C (PKC) (at both Ser63 and Ser68), we evaluated whether PLM mediates the PKC-dependent regulation of NKA function and protein kinase A/PKC crosstalk in ventricular myocytes. PKC was activated by PDBu (300 nmol/L), and we measured NKA-mediated [Na + ] i decline (fluorescence measurements) and current ( I pump ) (voltage clamp). In wild-type mouse myocytes, PDBu increased PLM phosphorylation at Ser63 and Ser68, I pump (both at 10 and 100 mmol/L Na + in the pipette solution) and maximal NKA-mediated Na + extrusion rate (V max ) from 7.9±1.1 to 12.7±1.9 mmol·L −1 per minute without altering NKA affinity for internal Na + ( K 0.5 ). In PLM knockout mice, PDBu had no effect on either V max or K 0.5 . After pretreatment with isoproterenol (ISO) (1 μmol/L), PDBu still increased the NKA V max and PLM phosphorylation at Ser63 and ...
American Journal of Physiology-Heart and Circulatory Physiology, 2007
Phospholemman (PLM, FXYD1), abundantly expressed in the heart, is the primary cardiac sarcolemmal substrate for PKA and PKC. Evidence supports the hypothesis that PLM is part of the cardiac Na-K pump complex and provides the link between kinase activity and pump modulation. PLM has also been proposed to modulate Na/Ca exchanger activity and may be involved in cell volume regulation. This study characterized the phenotype of the PLM knockout (KO) mouse heart to further our understanding of PLM function in the heart. PLM KO mice were bred on a congenic C57/BL6 background. In vivo conductance catheter measurements exhibited a mildly depressed cardiac contractile function in PLM KO mice, which was exacerbated when hearts were isolated and Langendorff perfused. There were no significant differences in action potential morphology in paced Langendorff-perfused hearts. Depressed contractile function was associated with a mild cardiac hypertrophy in PLM KO mice. Biochemical analysis of crude...
Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+-K+-ATPase
AJP: Heart and Circulatory Physiology, 2008
PLM) regulates cardiac Na ϩ /Ca 2ϩ exchanger (NCX1) and Na ϩ -K ϩ -ATPase in cardiac myocytes. PLM, when phosphorylated at Ser 68 , disinhibits Na ϩ -K ϩ -ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na ϩ -K ϩ -ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca 2ϩ -ATPase levels, and retained near normal contractility, but ␣ 1-subunit of Na ϩ -K ϩ -ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca 2ϩ concentration ([Ca 2ϩ ]i) transients reverted back to those observed in cultured WT myocytes. Both Na ϩ -K ϩ -ATPase current (I pump) and Na ϩ /Ca 2ϩ exchange current (INaCa) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of I NaCa but had no effect on Ipump. Contractility, [Ca 2ϩ ]i transient amplitudes, and sarcoplasmic reticulum Ca 2ϩ contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on I NaCa but decreased Ipump. Contractility, [Ca 2ϩ ]i transient amplitudes, and sarcoplasmic reticulum Ca 2ϩ contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca 2ϩ ]i homeostasis primarily by its direct inhibitory effects on Na ϩ /Ca 2ϩ exchange.
Expression and Phosphorylation of the Na-Pump Regulatory Subunit Phospholemman in Heart Failure
Circulation Research, 2005
Intracellular [Na] is Ϸ3 mmol/L higher in heart failure (HF; in our arrhythmogenic rabbit model; 3 ), and this can profoundly affect cardiac Ca and contractile function via Na/Ca exchange and Na/H exchange. Na/K-ATPase is the primary mechanism of Na extrusion. We examine here in HF rabbits (and human hearts) expression of Na/K-ATPase isoforms and phospholemman (PLM), a putative Na/K-ATPase regulatory subunit that inhibits pump function and is a major cardiac phosphorylation target. Na/K-ATPase ␣1and ␣2-isoforms were reduced in HF in rabbit ventricular homogenates (by 24%) and isolated myocytes (by 30% and 17%), whereas ␣3 was increased (50%) in homogenates and decreased (52%) in myocytes (PϽ0.05). Homogenate Na/K-ATPase activity in left ventricle was also decreased in HF. However, we showed previously that Na/K-ATPase characteristics in intact ventricular myocytes were unaltered in HF. To reconcile these findings, we assessed PLM expression, phosphorylation, and association with Na/K-ATPase. PLM coimmunoprecipitated with Na/K-ATPase ␣1 and ␣2 in control and HF rabbit myocytes. PLM expression was reduced in HF by 42% in isolated rabbit left ventricular (LV) myocytes, by 48% in rabbit LV homogenates, and by 24% in human LV homogenate. The fraction of PLM phosphorylated at Ser-68 was increased dramatically in HF. Our results are consistent with a role for PLM analogous to that of phospholamban for SR Ca-ATPase (SERCA): inhibition of Na/K-ATPase function that is relieved on PLM phosphorylation. So reduced Na/K-ATPase expression in HF may be functionally offset by lower inhibition by PLM (because of reduced PLM expression and higher PLM phosphorylation). (Circ Res. 2005;97:558-565.)
Phospholemman Inhibition of the Cardiac Na+/Ca2+ Exchanger: ROLE OF PHOSPHORYLATION
Journal of Biological Chemistry, 2006
We have previously demonstrated that phospholemman (PLM), a 15 kDa integral sarcolemmal phosphoprotein, inhibits the cardiac Na + /Ca 2+ exchanger (NCX1). In addition, p rotein kinase A phosphorylates serine 68 while protein kinase C phosphorylates both serine 63 and serine 68 of PLM. Using HEK293 cells that are devoid of both endogenous PLM and NCX1, we first demonstrated that the exogenous NCX1 current (I NaCa ) was increased by phorbol 12-myristate 13-acetate (PMA) but not by forskolin. When co-expressed with NCX1, PLM resulted in: (i) decreases in I NaCa ; (ii) attenuation of the increase in I NaCa by PMA; and (iii) additional reduction in I NaCa in cells treated with forskolin. Mutating serine 63 to alanine (S63A) preserved PLM's sensitivity to forskolin in terms of suppression of I NaCa , whereas mutating serine 68 to alanine (S68A) abolished PLM's inhibitory effect on I NaCa . Mutating serine 68 to glutamic acid (phosphomimetic) resulted in additional suppression of I NaCa as compared to wild-type PLM. These results suggest that PLM phosphorylated at serine 68 inhibited I NaCa . The physiological significance of inhibition of NCX1 by phosphorylated PLM was evaluated in PLM-knockout (KO) mice. When compared to wild-type myocytes, I NaCa was significant larger in PLM-KO myocytes. In addition, PMA-induced increase in I NaCa was significantly higher in PLM-KO myocytes. By contrast, forskolin had no effect on I NaCa in wild-type myocytes. We conclude that PLM, when phosphorylated at serine 68 , inhibits Na + /Ca 2+ exchange in the heart.
Journal of Molecular and Cellular Cardiology, 2013
In the heart, Na/K-ATPase regulates intracellular Na + and Ca 2+ (via NCX), thereby preventing Na + and Ca 2+ overload and arrhythmias. Here, we test the hypothesis that nitric oxide (NO) regulates cardiac intracellular Na + and Ca 2+ and investigate mechanisms and physiological consequences involved. Effects of both exogenous NO (via NO-donors) and endogenously synthesized NO (via field-stimulation of ventricular myocytes) were assessed in this study. Field stimulation of rat ventricular myocytes significantly increased endogenous NO (18 ± 2 μM), PKCε activation (82 ± 12%), phospholemman phosphorylation (at Ser-63 and Ser-68) and Na/K-ATPase activity (measured by DAF-FM dye, western-blotting and biochemical assay, respectively; p b 0.05, n = 6) and all were abolished by Ca 2+-chelation (EGTA 10 mM) or NOS inhibition L-NAME (1 mM). Exogenously added NO (spermine-NONO-ate) stimulated Na/K-ATPase (EC50 = 3.8 μM; n = 6/grp), via decrease in K m , in PLM WT but not PLM KO or PLM 3SA myocytes (where phospholemman cannot be phosphorylated) as measured by whole-cell perforated-patch clamp. Field-stimulation with L-NAME or PKC-inhibitor (2 μM Bis) resulted in elevated intracellular Na + (22 ± 1.5 and 24 ± 2 respectively, vs. 14 ±0.6 mM in controls) in SBFI-AM-loaded rat myocytes. Arrhythmia incidence was significantly increased in rat hearts paced in the presence of L-NAME (and this was reversed by L-arginine), as well as in PLM 3SA mouse hearts but not PLM WT and PLM KO. We provide physiological and biochemical evidence for a novel regulatory pathway whereby NO activates Na/K-ATPase via phospholemman phosphorylation and thereby limits Na + and Ca 2+ overload and arrhythmias. This article is part of a Special Issue entitled "Na + Regulation in Cardiac Myocytes".
Pfl�gers Archiv European Journal of Physiology, 1998
The β-agonist isoproterenol (ISO) reduces the Na/K pump current (I p ) via β-adrenergic receptors when the intracellular calcium concentration ([Ca 2+ ] i ) is below 150 nM [8]. In the present study, the intracellular signaling pathway was investigated with whole-cell patch-clamp of isolated guinea pig ventricular myocytes. The inhibitory effect of ISO could be mimicked by external application of the membrane-permeant cAMP analog chlorophenylthio-cAMP (0.5 mM), the phosphodiesterase inhibitor isobutyl-1-methylxanthine (IBMX, 100 µM), or the adenylyl cyclase activator forskolin (50 µM). Intracellular application of the synthetic peptide inhibitor of protein kinase A (PKA), PKI (5 µM), prevented the effect of ISO. These results suggest that the inhibitory effect of ISO on I p is mediated via a phosphorylation step induced by a cAMP-dependent PKA pathway. Neither the non-specific protein kinase inhibitor H 7 (100 µM) nor the protein phosphatase inhibitor calyculin A (0.5 µM) had any effect on I p in the absence of ISO. However, H 7 could increase I p and calyculin A could reduce it in the presence of ISO (1 µM and 12 nM respectively). These results indicate that there is a low basal level of phosphorylation which makes the effects of H 7 and calyculin A difficult to detect in the absence of an ISO-induced increase in phosphorylation level.
2016
Small changes of Na/K pump activity regulate internal Ca release in cardiac myocytes via Na/Ca exchange. We now show conversely that transient elevations of cytoplasmic Ca strongly regulate cardiac Na/K pumps. When cytoplasmic Na is submaximal, Na/K pump currents decay rapidly during extracellular K application and multiple results suggest that an inactivation mechanism is involved. Brief activation of Ca influx by reverse Na/Ca exchange enhances pump currents and attenuates current decay, while repeated Ca elevations suppress pump currents. Pump current enhancement reverses over 3 min, and results are similar in myocytes lacking the regulatory protein, phospholemman. Classical signaling mechanisms, including Ca-activated protein kinases and reactive oxygen, are evidently not involved. Electrogenic signals mediated by intramembrane movement of hydrophobic ions, such as hexyltriphenylphosphonium (C6TPP), increase and decrease in parallel with pump currents. Thus, transient Ca elevation and Na/K pump inactivation cause opposing sarcolemma changes that may affect diverse membrane processes.
Activation of PKC increases Na + -K + pump current in ventricular myocytes from guinea pig heart
Pfl�gers Archiv European Journal of Physiology, 1999
We have previously shown activation of α 1adrenergic receptors increases Na + -K + pump current (I p ) in guinea pig ventricular myocytes, and the increase is eliminated by blockers of phosphokinase C (PKC). In this study we examined the effect of activators of PKC on I p . Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased I P at each test potential without shifting its voltage dependence. The concentration required for a half-maximal response (K 0.5 ) was 6 µM at 15 nM cytosolic [Ca 2+ ] ([Ca 2+ ] i ) and13 nM at 314 nM [Ca 2+ ] i . The maximal increase at either [Ca 2+ ] i was about 30%. Another activator of PKC, 1,2-dioctanoylsn-glycerol (diC 8 ), increased I p similarly. The effect of PMA on I P was eliminated by the PKC inhibitor staurosporine, but not by the peptide PKI, an inhibitor of protein kinase A (PKA). PMA and α 1 -adrenergic agonist effects both were sensitive to [Ca 2+ ] i , blocked by PKC inhibitors, unaffected by PKA inhibition, and increased I p uniformly at all voltages. However, they differed in that α 1 -activation caused a maximum increase of 15% vs 30% via PMA, and α 1 -effects were less sensitive to [Ca 2+ ] i than PMA effects. These results demonstrate that activation of PKC causes an increase in I p in guinea pig ventricular myocytes. Moreover, they suggest that the coupling of α 1 -adrenergic activation to I p is entirely through PKC, however α 1 -activation may be coupled to a specific population of PKC whereas PMA is a more global agonist.