Abstract-Activation of the sympathetic nervous system is a common compensatory feature in heart failure, but sustained ␤-adrenergic activation induces cardiomyocyte death, leading to cardiac remodeling and dysfunction. In mouse cardiomyocytes, we recently reported that prolonged exposure to ␤-agonists is associated with transient increases in expression and phosphorylation of a small heat-shock protein, Hsp20. To determine the functional significance of Hsp20, we overexpressed this protein and its constitutively phosphorylated (S16D) or nonphosphorylated (S16A) mutant in adult rat cardiomyocytes. Hsp20 protected cardiomyocytes from apoptosis triggered by activation of the cAMP-PKA pathway, as indicated by decreases in the number of pyknotic nuclei, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling, and DNA laddering, which were associated with inhibition of caspase-3 activity. These protective effects were further increased by the constitutively phosphorylated Hsp20 mutant (S16D), which conferred full protection from apoptosis. In contrast, the nonphosphorylatable mutant (S16A) exhibited no antiapoptotic properties. Immunostaining studies and immunoprecipitations with Hsp20 or actin antibodies demonstrated that Hsp20 translocated to cytoskeleton and associated with actin on isoproterenol stimulation. These findings suggest that Hsp20 and its phosphorylation at Ser16 may provide cardioprotection against ␤-agonist-induced apoptosis. Thus, Hsp20 may represent a novel therapeutic target in the treatment of heart failure.

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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.

Na/K-ATPase (NKA) activity is dynamically regulated by an inhibitory interaction with a small transmembrane protein, phospholemman (PLM). Inhibition is relieved upon PLM phosphorylation. Phosphorylation may alter how PLM interacts with NKA and/or itself, but details of these interactions are unknown. To address this, we quantified FRET between PLM and its regulatory target NKA in live cells. Phosphorylation of PLM was mimicked by mutation S63E (PKC site), S68E (PKA/ PKC site), or S63E/S68E. The dependence of FRET on protein expression in live cells yielded information about the structure and binding affinity of the PLM-NKA regulatory complex. PLM phosphomimetic mutations altered the quaternary structure of the regulatory complex and reduced the apparent affinity of the PLM-NKA interaction. The latter effect was likely due to increased oligomerization of PLM phosphomimetic mutants, as suggested by PLM-PLM FRET measurements. Distance constraints obtained by FRET suggest that phosphomimetic mutations slightly alter the oligomer quaternary conformation. Photon-counting histogram measurements revealed that the major PLM oligomeric species is a tetramer. We conclude that phosphorylation of PLM increases its oligomerization into tetramers, decreases its binding to NKA, and alters the structures of both the tetramer and NKA regulatory complex.The sodium/potassium pump Na/K-ATPase (NKA) 2 is essential to establish the sodium/potassium concentration gradient across the plasma membrane (1). Besides being the foundation for the membrane potential, the sodium/potassium gradient created by NKA is the basis for many other cotransport and exchange processes (2). NKA plays a particularly important role in cardiac function. Disordered sodium/potassium handling is associated with heart disease (3), and targeting NKA with inhibitory drugs is one of the oldest, most effective treatments for the inadequate contractility of the failing heart (4).NKA is functionally regulated by PKA/PKC-dependent signaling pathways (1-6). These pathways impinge on phospholemman (PLM; or FXYD1), a 72-amino acid regulator of NKA in cardiac tissue (6 -9). PLM inhibits NKA activity by reducing its apparent sodium affinity (8, 9). Tonic inhibition of NKA by PLM is relieved upon phosphorylation by PKA or PKC (10 -13). Notably, deletion of PLM abolishes the PKA-or PKC-mediated regulation on NKA (10 -13), emphasizing the central role of this regulatory interaction.Recently, much progress has been made elucidating the structural basis for the functional regulation of NKA by PLM. NMR studies showed that PLM adopts an L-shaped structure with a single membrane span (14). The N-terminal half of the protein consists of an extracellular domain containing the signature Phe-X-Tyr-Asp (FXYD) motif, followed by a transmembrane ␣-helix. Another helical domain on the cytoplasmic side of the plasma membrane contains the phosphate-accepting residues Ser-63 (PKC site) and Ser-68 (PKA/PKC site) (9,14). This positively charged domain appears to associate with the surface of the...