Anand Pathak

PKC-α regulates cardiac contractility and propensity toward heart failure

The protein kinase C (PKC) family of serine/threonine kinases functions downstream of nearly all membrane-associated signal transduction pathways. Here we identify PKC-α as a fundamental regulator of cardiac contractility and Ca2+ handling in myocytes. Hearts of Prkca-deficient mice are hypercontractile, whereas those of transgenic mice overexpressing Prkca are hypocontractile. Adenoviral gene transfer of dominant-negative or wild-type PKC-α into cardiac myocytes enhances or reduces contractility, respectively. Mechanistically, modulation of PKC-α activity affects dephosphorylation of the sarcoplasmic reticulum Ca2+ ATPase-2 (SERCA-2) pump inhibitory protein phospholamban (PLB), and alters sarcoplasmic reticulum Ca2+ loading and the Ca2+ transient. PKC-α directly phosphorylates protein phosphatase inhibitor-1 (I-1), altering the activity of protein phosphatase-1 (PP-1), which may account for the effects of PKC-α on PLB phosphorylation. Hypercontractility caused by Prkca deletion protects against heart failure induced by pressure overload, and against dilated cardiomyopathy induced by deleting the gene encoding muscle LIM protein (Csrp3). Deletion of Prkca also rescues cardiomyopathy associated with overexpression of PP-1. Thus, PKC-α functions as a nodal integrator of cardiac contractility by sensing intracellular Ca2+ and signal transduction events, which can profoundly affect propensity toward heart failure.

Enhancement of Cardiac Function and Suppression of Heart Failure Progression By Inhibition of Protein Phosphatase 1

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 β-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.

Inducible Expression of Active Protein Phosphatase-1 Inhibitor-1 Enhances Basal Cardiac Function and Protects Against Ischemia/Reperfusion Injury

Ischemic heart disease, which remains the leading cause of morbidity and mortality in the Western world, is invariably characterized by impaired cardiac function and disturbed Ca2+ homeostasis. Because enhanced inhibitor-1 (I-1) activity has been suggested to preserve Ca2+ cycling, we sought to define whether increases in I-1 activity in the adult heart may ameliorate contractile dysfunction and cellular injury in the face of an ischemic insult. To this end, we generated an inducible transgenic mouse model that enabled temporally controlled expression of active I-1 (T35D). Active I-1 expression in the adult heart elicited significant enhancement of contractile function, associated with preferential phospholamban phosphorylation and enhanced sarcoplasmic reticulum Ca2+-transport. Further phosphoproteomic analysis revealed alterations in proteins associated with energy production and protein synthesis, possibly to support the increased metabolic demands of the hyperdynamic hearts. Importantly, on ischemia/reperfusion-induced injury, active I-1 expression augmented contractile function and recovery. Further examination revealed that the infarct region and apoptotic as well as necrotic injuries were significantly attenuated by enhanced I-1 activity. These cardioprotective effects were associated with suppression of the endoplasmic reticulum stress response. The present findings indicate that increased I-1 activity in the adult heart enhances Ca2+ cycling and improves mechanical recovery, as well as cell survival after an ischemic insult, suggesting that active I-1 may represent a potential therapeutic strategy in myocardial infarction.

Differential regulation of p38 mitogen-activated protein kinase mediates gender-dependent catecholamine-induced hypertrophy

OBJECTIVE Exogenous catecholamine exposure has been associated with p38 mitogen-activated protein kinase (MAPK) and cardiac hypertrophy. In this study, we investigated the regulation of p38 MAPK in cardiac remodeling elicited by endogenous adrenergic mechanisms. METHODS Transgenic male and female mice with fourfold phospholamban (PLB) overexpression exhibited enhanced circulating norepinephrine (NE), as a physiological compensatory mechanism to attenuate PLBs inhibitory effects. This enhanced noradrenergic state resulted in left ventricular hypertrophy/dilatation and depressed function. RESULTS Male transgenics exhibited ventricular hypertrophy and mortality at 15 months, concurrent with cardiac p38 MAPK activation. Female transgenics, despite similar contractile dysfunction, displayed a temporal delay in p38 activation, hypertrophy, and mortality (22 months), which was associated with sustained cardiac levels of MAP Kinase Phosphatase-1 (MKP-1), a potent inhibitor of p38. At 22 months, decreases in cardiac MKP-1 were accompanied by increased levels of p38 activation. In vitro studies indicated that preincubation with 17-beta-estradiol induced high MKP-1 levels, which precluded NE-induced p38 activation. CONCLUSION These findings suggest that norepinephrine-induced hypertrophy is linked closely with p38 MAP kinase activation, which can be endogenously modulated through estrogen-responsive regulation of MKP-1 expression.

The Presence of Lys27 Instead of Asn27 in Human Phospholamban Promotes Sarcoplasmic Reticulum Ca2+-ATPase Superinhibition and Cardiac Remodeling

Background— Phospholamban (PLN) is an inhibitor of the Ca2+ affinity of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2). The amino acid sequence of PLN is highly conserved, and although all species contain asparagine (Asn), human PLN is unique in containing lysine (Lys) at amino acid 27. Methods and Results— Human PLN was introduced in the null background. Expression of human PLN, at similar levels to mouse wild-type PLN, resulted in significant decreases in the affinity of SERCA2 for Ca2+, attributed to unique spatial conformation of this PLN form and increases in its monomeric active unit compared with mouse PLN. The increased inhibition by human PLN was associated with attenuated cardiac contractility in the intact-animal, organ, and cardiomyocyte levels and with depressed calcium kinetics. These inhibitory effects could not be fully reversed even on maximal isoproterenol stimulation. There were no alterations in the expression levels of SERCA2, calsequestrin, ryanodine receptor, and FKBP12, although the sodium/calcium exchanger and the L-type Ca2+ channel expression levels were upregulated. The depressed function resulted in increased heart/body weight ratios and phosphorylation levels of Akt, p38, and Erk1/2. Conclusions— Human PLN may play a more inhibitory role than that of other species in Ca2+ cycling. Expression of human PLN in the mouse is compensated by alterations in Ca2+-handling proteins and cardiac remodeling in an effort to normalize cardiac contractility. Thus, the unique amino acid sequence of human PLN may be critical in maintaining a high cardiac reserve, which is of paramount importance in the regulation of human cardiac function.

A human polymorphism of protein phosphatase-1 inhibitor-1 is associated with attenuated contractile response of cardiomyocytes to β-adrenergic stimulation

Aberrant β‐adrenergic signaling and de pressed calcium homeostasis, associated with an imbal ance of protein kinase A and phosphatase‐1 activities, are hallmarks of heart failure. Phosphatase‐1 is re strained by its endogenous inhibitor, protein phospha tase inhibitor‐1 (PPI‐1). We assessed 352 normal sub jects, along with 959 patients with heart failure and identified a polymorphism in PPI‐1 (G147D) exclu sively in black subjects. To determine whether the G147D variant could affect cardiac function, we in fected adult cardiomyocytes with adenoviruses expressing D147 or wild‐type (G147) PPI‐1. Under basal con ditions, there were no significant differences in fractional shortening or contraction or relaxation rates. However, the enhancement of contractile parameters after isoproterenol stimulation was significantly blunted in D147 compared with G147 and control myocytes. Similar findings were observed in calcium kinetics. The attenuated β‐agonist response was associ ated with decreased (50%) phosphorylation of phos‐ pholamban (PLN) at serine 16, whereas phosphorylation of troponin I and ryanodine receptor was unaltered. These findings suggest that the human G147D PPI‐1 can attenuate responses of cardiomyo cytes to β‐adrenergic agonists by decreasing PLN phos phorylation and therefore may contribute to deterio rated function in heart failure.— Chen, G., Zhou, X., Nicolaou, P., Rodriguez, P., Song, G., Mitton, B., Pathak, A., Zachariah, A., Fan, G.‐C., Dorn, G. A., II., Kranias, E. G. A human polymorphism of protein phosphatase‐1 inhibitor‐1 is associated with attenuated contractile response of cardiomyocytes to β‐adrenergic stimulation. FASEB J. 22, 1790–1796 (2008)

The Human G147D-Protein Phosphatase 1 Inhibitor-1 Polymorphism Is Not Associated with Altered Clinical Characteristics in Heart Failure

Objectives: A human protein phosphatase inhibitor-1 polymorphism, G147D (c.440G>A, p.147G>D), has been previously demonstrated to blunt the contractile responses of cardiomyocytes to β-adrenergic agonists. The present study sought to examine whether the G147D inhibitor-1 polymorphism may be associated with specific clinical characteristics of heart failure carriers. Methods: Clinical information of 963 heart failure patients was analyzed according to race, inhibitor-1 genotype, treatment with β-blockers and mortality patterns. Results: The G147D inhibitor-1 genetic variant was found almost exclusively in black subjects and its frequency was similar between normals and heart failure patients, indicating that it was not a primary risk factor for developing heart failure. Comparison of the major cardiac functional parameters and transplant-free survival patterns between carrier and noncarrier patients did not reveal any significant differences. Furthermore, echocardiographic evaluation showed similar outcomes of β-blocker treatment between G147D carriers and noncarriers. Conclusions: The present findings indicate that the human inhibitor-1 G147D polymorphism, found almost exclusively in blacks, may act as a modifier rather than risk factor in heart failure development.