Increases in type 1 phosphatase (PP1) activity have been observed in end stage human heart failure, but the role of this enzyme in cardiac function is unknown. To elucidate the functional significance of increased PP1 activity, we generated models with (i) overexpression of the catalytic subunit of PP1 in murine hearts and (ii) ablation of the PP1-specific inhibitor. Overexpression of PP1 (threefold) was associated with depressed cardiac function, dilated cardiomyopathy, and premature mortality, consistent with heart failure. Ablation of the inhibitor was associated with moderate increases in PP1 activity (23%) and impaired ␤-adrenergic contractile responses. Extension of these findings to human heart failure indicated that the increased PP1 activity may be partially due to dephosphorylation or inactivation of its inhibitor. Indeed, expression of a constitutively active inhibitor was associated with rescue of ␤-adrenergic responsiveness in failing human myocytes. Thus, PP1 is an important regulator of cardiac function, and inhibition of its activity may represent a novel therapeutic target in heart failure.

Abstract-Abnormal calcium cycling, characteristic of experimental and human heart failure, is associated with impaired sarcoplasmic reticulum calcium uptake activity. This reflects decreases in the cAMP-pathway signaling and increases in type 1 phosphatase activity. The increased protein phosphatase 1 activity is partially due to dephosphorylation and inactivation of its inhibitor-1, promoting dephosphorylation of phospholamban and inhibition of the sarcoplasmic reticulum calcium-pump. Indeed, cardiac-specific expression of a constitutively active inhibitor-1 results in selective enhancement of phospholamban phosphorylation and augmented cardiac contractility at the cellular and intact animal levels. Furthermore, the ␤-adrenergic response is enhanced in the transgenic hearts compared with wild types. On aortic constriction, the hypercontractile cardiac function is maintained, hypertrophy is attenuated and there is no decompensation in the transgenics compared with wild-type controls. Notably, acute adenoviral gene delivery of the active inhibitor-1, completely restores function and partially reverses remodeling, including normalization of the hyperactivated p38, in the setting of pre-existing heart failure. Thus, the inhibitor 1 of the type 1 phosphatase may represent an attractive new therapeutic target. Key Words: protein phosphatase 1 Ⅲ protein phosphatase 1 inhibitor 1 Ⅲ heart failure Ⅲ hypertrophy Ⅲ phospholamban Ⅲ gene therapy R eversible protein phosphorylation represents the cellular basis for integration of key signaling pathways, mediating a fine crosstalk between external effector molecules and intracellular events. In the heart, Ca 2ϩ cycling and contractility are controlled by a fine balance of protein kinase and phosphatase activities in response to various second messenger signals. Demands on the heart's pumping action, during fight-or-flight situations, can increase human cardiac output by nearly 5-fold. This is linked to ␤-adrenergic activation of the cAMP dependent protein kinase (PKA). PKA then phosphorylates a set of key regulatory Ca 2ϩ handling proteins that control excitation-contraction coupling cycle, such as phospholamban, the ryanodine receptor, the L-type Ca 2ϩ channel, and troponin I. 1 The protein kinases and their phosphoprotein substrates underlying augmentation of the heart's pumping action have been well characterized. However, similar studies on the protein phosphatases, reversing the increased cardiac contractility, are less well developed. The major Ser/Thr phosphatases [type 1, type 2A, and type 2B (calcineurin)] stem from a common gene family and are highly homologous proteins (40% to 50%) that play critical roles in the control of cardiac contractility and hypertrophy.Overexpression of the catalytic subunit of the protein phosphatase 1 at similar levels observed in human heart failure was associated with dephosphorylation of phospholamban, depressed cardiac function, dilated cardiomyopathy, and premature mortality. 2 Furthermore, PP2A and PP2B (calcineurin) overexpressio...

Background-The conventional protein kinase C (PKC) isoform ␣ functions as a proximal regulator of Ca 2ϩ handling in cardiac myocytes. Deletion of PKC␣ in the mouse results in augmented sarcoplasmic reticulum Ca 2ϩ loading, enhanced Ca 2ϩ transients, and augmented contractility, whereas overexpression of PKC␣ in the heart blunts contractility. Mechanistically, PKC␣ directly regulates Ca 2ϩ handling by altering the phosphorylation status of inhibitor-1, which in turn suppresses protein phosphatase-1 activity, thus modulating phospholamban activity and secondarily, the sarcoplasmic reticulum Ca 2ϩ ATPase. Methods and Results-In the present study, we show that short-term inhibition of the conventional PKC isoforms with Ro-32-0432 or Ro-31-8220 significantly augmented cardiac contractility in vivo or in an isolated work-performing heart preparation in wild-type mice but not in PKC␣-deficient mice. Ro-32-0432 also increased cardiac contractility in 2 different models of heart failure in vivo. Short-term or long-term treatment with Ro-31-8220 in a mouse model of heart failure due to deletion of the muscle lim protein gene significantly augmented cardiac contractility and restored pump function. Moreover, adenovirus-mediated gene therapy with a dominant-negative PKC␣ cDNA rescued heart failure in a rat model of postinfarction cardiomyopathy. PKC␣ was also determined to be the dominant conventional PKC isoform expressed in the adult human heart, providing potential relevance of these findings to human pathophysiology. Conclusions-Pharmacological inhibition of PKC␣, or the conventional isoforms in general, may serve as a novel therapeutic strategy for enhancing cardiac contractility in certain stages of heart failure. (Circulation. 2006;114:574-582.)

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Phospholamban is a phosphoprotein in the cardiac sarcoplasmic reticulum (SR) which regulates the apparent Ca 2؉ affinity of the SR Ca 2؉ -ATPase (SERCA2). To determine the levels of phospholamban which are associated with maximal inhibition of SERCA2, several lines of transgenic mice were generated which expressed increasing levels of a non-phosphorylatable form of phospholamban (S16A,T17A) specifically in the heart. This mutant form of phospholamban was chosen to prevent phosphorylation as a compensatory mechanism in vivo. Quantitative immunoblotting revealed increased phospholamban protein levels of 1.8-, 2.6-, 3.7-, and 4.7-fold in transgenic hearts compared with wild types. There were no changes in the expression levels of SERCA2, calsequestrin, calreticulin, and ryanodine receptor. Assessment of SR Ca 2؉ uptake in hearts of transgenic mice indicated increases in the inhibition of the affinity of SERCA2 for Ca 2؉ with increased phospholamban expression. Maximal inhibition was obtained at phospholamban expression levels of 2.6-fold or higher. Transgenic hearts with functional saturation in phospholamban:SERCA2 (>2.6:1) exhibited increases in ␤-myosin heavy chain expression, associated with cardiac hypertrophy. These findings demonstrate that overexpression of a non-phosphorylatable form of phospholamban in transgenic mouse hearts resulted in saturation of the functional phospholamban:SERCA2 ratio at 2.6:1 and suggest that approximately 40% of the SR Ca 2؉ pumps are functionally regulated by phospholamban in vivo.Phospholamban (PLB), 1 a 52-amino acid phosphoprotein, has been shown to interact with and regulate the apparent Ca 2ϩ affinity of the sarcoplasmic reticulum (SR) Ca 2ϩ -ATPase (1). The mechanism of action and functional significance of PLB have been well characterized in cardiac muscle because of the abundant expression of this protein in cardiac SR (2). Low levels of PLB expression have also been detected in slow twitch skeletal muscle (3, 4), smooth muscle (5), and a non-muscle tissue, the vascular endothelium (6), although the role of PLB in these tissues is not well characterized at present. In cardiac muscle, dephosphorylated PLB inhibits the apparent affinity of the SR Ca 2ϩ -ATPase (SERCA2) for Ca 2ϩ (7-11), and phosphorylation of PLB, in response to ␤-adrenergic stimulation, removes its inhibition of SERCA2 (12,13 concentrations (7,9,11,17). The stimulatory effects of PLB phosphorylation at these two sites can be reversed by a cardiac SR-associated type 1 protein phosphatase, which is also subject to cAMP-dependent phosphorylation of its inhibitor protein (18,19). The apparent affinity of the SERCA2 for Ca 2ϩ is not only regulated by the phosphorylation state of PLB, but is also modulated by changes in the PLB:SERCA2 ratio. Alterations in the stoichiometric ratio of PLB to SERCA2, associated with alterations in SR Ca 2ϩ transport, have been implicated as important determinants of depressed left ventricular function in physiological and pathophysiological conditions. In hypothyroidism, increas...

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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.

Alterations in thyroid hormone levels have a profound impact on myocardial contractility, speed of relaxation, cardiac output, and heart rate. The mechanisms for these changes include altered expression of several key proteins, involved in the regulation of intracellular calcium homeostasis. Most notably, increases in thyroid hormone and the coordinated increases in cardiac contractile parameters are marked by increases in the levels of the sarcoplasmic reticulum (SR) Ca2+-adenosine triphosphatase (ATPase) and decreases in its inhibitor, phospholamban. These changes at the protein level result in enhanced SR calcium transport and myocyte calcium cycling, leading to increases in the force and rates of contraction as well as relaxation rates at the organ level. However, decreases in thyroid hormone levels are associated with opposite alterations in these two proteins, leading to reduced myocyte calcium handling capacity and lower cardiac contractility. Furthermore, changes in the relative ratio of phospholamban/Ca2+-ATPase correlate with changes in the affinity of the SR Ca2+-transport system and relaxation rates in beating hearts. These findings suggest that thyroid hormone directly regulates SR protein levels and thus, cardiac function.