Phospholamban (PLB) can be phosphorylated at Ser 16by cyclic AMP-dependent protein kinase and at Thr 17 by Ca 2؉ -calmodulin-dependent protein kinase during -agonist stimulation. A previous study indicated that mutation of S16A in PLB resulted in lack of Thr 17 phosphorylation and attenuation of the -agonist stimulatory effects in perfused mouse hearts. To further delineate the functional interplay between dual-site PLB phosphorylation, we generated transgenic mice expressing the T17A mutant PLB in the cardiac compartment of the null background. Lines expressing similar levels of T17A mutant, S16A mutant, or wild-type PLB in the null background were characterized in parallel. Phospholamban (PLB)1 is a low molecular weight phosphoprotein in cardiac sarcoplasmic reticulum (SR). Dephosphorylated PLB is an inhibitor of the affinity of SERCA2 for Ca 2ϩ , and phosphorylation of PLB during -adrenergic stimulation relieves its inhibitory effects on SERCA2 (1, 2). The physiological importance of PLB has been elucidated through the generation of genetically engineered mouse models with alterations in cardiac PLB expression levels (3, 4). Ablation of PLB was associated with significantly enhanced Ca 2ϩ affinity of SERCA2 and myocardial performance (3, 5, 6). The elevated basal contractile parameters could be minimally stimulated by -agonists (3, 7), whereas there were no alterations in the -receptor signaling pathway or the phosphorylation states of other major cardiac phosphoproteins (8). On the other hand, overexpression of PLB was associated with significant depression of contractile parameters, which could be reversed upon phosphorylation of PLB during -agonist stimulation (4). These results indicate that PLB is a key regulator of cardiac function and a prominent mediator of the -adrenergic effects in the myocardium.In vitro studies have shown that PLB can be phosphorylated on Ser 10 by protein kinase C, Ser 16 by cAMP-dependent protein kinase (PKA), and Thr 17 by Ca 2ϩ -calmodulin-dependent protein kinase (CaMKII) (1, 9, 10). Each phosphorylation is associated with stimulation of the apparent affinity of SERCA2 for Ca 2ϩ . In vivo studies have shown that only Ser 16 and Thr 17 are phosphorylated in cardiac myocytes or perfused hearts (11, 12), whereas phosphorylation of PLB by protein kinase C has not been detected in vivo. Phosphorylation of PLB by PKA and CaMKII occurs during -agonist exposure, although the relative contribution of each phosphorylation to the cardiac stimulatory effects is not presently clear. Each phosphorylation appears to occur independently of the other (13-16). Some studies have reported additive effects of PKA and CaMKII phosphorylation of PLB on SR Ca 2ϩ transport (13,14,17,18), whereas others (16, 19) have proposed that maximal stimulation of the Ca 2ϩ pump occurs by phosphorylation at a single site, and additional phosphorylation of the other site does not further stimulate the pump activity.Several in vivo studies have shown that Ser 16 phosphorylation or dephosphorylation precede...
IntroductionIn the face of unremitting hemodynamic stress, "adaptive" cardiac hypertrophy inevitably progresses to ventricular dilation and clinical heart failure, which affects an estimated 5 million Americans and has a mortality rate of approximately 50% in 4 years (1). A nearly universal characteristic of hypertrophied and failing myocardium is depressed sarcoplasmic reticulum (SR) Ca 2+ cycling, caused by decreased expression of the cardiac SR Ca 2+ ATPase (SERCA2a), by a relative overabundance of the SERCA2a inhibitory protein phospholamban (PLN), or by both (2, 3). Recent experimental successes have generated enthusiasm for treating heart failure by restoring SR Ca 2+ cycling, either through adenoviral-mediated myocardial-targeted expression of SERCA2a itself (4) or through antisense suppression (5) or genetic ablation of PLN (6), to relieve SERCA2a from endogenous inhibition. The common result of each approach is enhanced SR Ca 2+ cycling, which has improved energetics, survival, and cardiac function at the cellular, organ, and intact animal levels. Furthermore, there is no compromise in exercise performance or longevity in the case of PLN ablation (7,8).To date, PLN ablation or inhibition has improved SR Ca 2+ cycling and/or contractile function in transgenic Cardiac hypertrophy, either compensated or decompensated, is associated with cardiomyocyte contractile dysfunction from depressed sarcoplasmic reticulum (SR) Ca 2+ cycling. Normalization of Ca 2+ cycling by ablation or inhibition of the SR inhibitor phospholamban (PLN) has prevented cardiac failure in experimental dilated cardiomyopathy and is a promising therapeutic approach for human heart failure. However, the potential benefits of restoring SR function on primary cardiac hypertrophy, a common antecedent of human heart failure, are unknown. We therefore tested the efficacy of PLN ablation to correct hypertrophy and contractile dysfunction in two well-characterized and highly relevant genetic mouse models of hypertrophy and cardiac failure, Gαq overexpression and human familial hypertrophic cardiomyopathy mutant myosin binding protein C (MyBP-C MUT ) expression. In both models, PLN ablation normalized the characteristically prolonged cardiomyocyte Ca 2+ transients and enhanced unloaded fractional shortening with no change in SR Ca 2+ pump content. However, there was no parallel improvement in in vivo cardiac function or hypertrophy in either model. Likewise, the activation of JNK and calcineurin associated with Gαq overexpression was not affected. Thus, PLN ablation normalized contractility in isolated myocytes, but failed to rescue the cardiomyopathic phenotype elicited by activation of the Gαq pathway or MyBP-C mutations.This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.
Despite enhanced SR Ca2+ handling and contractility in myocytes, pathological remodeling and defects in intercellular coupling may underlie contractile dysfunction of the calcineurin hearts.
The relative amount of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) and its crucial inhibitor phospholamban (PLN) are closely regulated and play a pivotal role in maintaining muscle function. The functional importance of PLN has been intensively investigated in cardiac muscle. However, little is known about the role of PLN in the slow-twitch skeletal muscle, which expresses a significantly lower level of PLN but a similar level of SERCA2a compared with cardiac muscle. Thus, to define the physiological significance of PLN in slow-twitch skeletal muscle, we generated transgenic mice with PLN-specific overexpression in soleus, which is largely composed of slow-muscle fibers. The PLN protein levels and the PLN/SERCA2a ratio in transgenic soleus were comparable with those in cardiac muscle. Assessment of isometric-twitch contractions indicated that PLN overexpression was associated with depressed rates of contraction and relaxation, which were not linked to reduced SERCA2a abundance, although the levels of other key Ca2+-handling proteins, including ryanodine receptor, FKBP12, and L-type Ca2+ channel, were significantly decreased. However, isoproterenol stimulation reversed the inhibitory effects of PLN on the transgenic soleus twitch kinetics. Furthermore, the PLN-overexpressing soleus had smaller muscle size, mass, and cross-sectional area compared with wild-types. Interestingly, the percentage of slow fibers was increased in PLN-overexpressing soleus. Taken together, these findings indicate that increased PLN expression in slow-twitch skeletal muscle is associated with impaired contractile function and muscle remodeling.
Cardiac hypertrophy, either compensated or decompensated, is associated with cardiomyocyte contractile dysfunction from depressed sarcoplasmic reticulum (SR) Ca(2+) cycling. Normalization of Ca(2+) cycling by ablation or inhibition of the SR inhibitor phospholamban (PLN) has prevented cardiac failure in experimental dilated cardiomyopathy and is a promising therapeutic approach for human heart failure. However, the potential benefits of restoring SR function on primary cardiac hypertrophy, a common antecedent of human heart failure, are unknown. We therefore tested the efficacy of PLN ablation to correct hypertrophy and contractile dysfunction in two well-characterized and highly relevant genetic mouse models of hypertrophy and cardiac failure, Galphaq overexpression and human familial hypertrophic cardiomyopathy mutant myosin binding protein C (MyBP-C(MUT)) expression. In both models, PLN ablation normalized the characteristically prolonged cardiomyocyte Ca(2+) transients and enhanced unloaded fractional shortening with no change in SR Ca(2+) pump content. However, there was no parallel improvement in in vivo cardiac function or hypertrophy in either model. Likewise, the activation of JNK and calcineurin associated with Galphaq overexpression was not affected. Thus, PLN ablation normalized contractility in isolated myocytes, but failed to rescue the cardiomyopathic phenotype elicited by activation of the Galphaq pathway or MyBP-C mutations.
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