Background Alterations in intracellular Ca2+ homeostasis are an important trigger of pathological cardiac remodeling; however, mechanisms governing context-dependent changes in Ca2+ influx are poorly understood. Store-operated Ca2+ entry (SOCE) is a major mechanism regulating Ca2+ trafficking in numerous cell types, yet its prevalence in adult heart and possible role in physiology and disease are each unknown. The Ca2+-binding protein, stromal interaction molecule 1 (STIM1), is a Ca2+ sensor in the sarcoplasmic reticulum (SR), capable of triggering SOCE by interacting with plasma membrane Ca2+ channels. Methods and Results We report that SOCE is abundant and robust in neonatal cardiomyocytes; however, SOCE is absent from adult cardiomyocytes. Levels of STIM1 transcript and protein correlate with the amplitude of SOCE, and manipulation of STIM1 protein levels (via shRNA) or activity (via expression of constitutively active or dominant-negative mutants) reveal a critical role for STIM1 in activating SOCE in cardiac myocytes. In neonatal hearts a recently identified STIM1 splice variant (STIM1L) is predominant but diminishes with maturation, only to reemerge with agonist- or afterload-induced cardiac stress. To test for pathophysiological relevance, we evaluated both in vitro and in vivo models of cardiac hypertrophy, finding that STIM1 expression is re-activated by pathological stress to trigger significant SOCE-dependent Ca2+ influx. STIM1 amplifies agonist-induced hypertrophy via activation of the calcineurin-NFAT pathway. Importantly, inhibition of STIM1 suppresses agonist-triggered hypertrophy, pointing to a requirement for SOCE in this remodeling response. Conclusions Stress-triggered STIM1 re-expression, and consequent SOCE activation, are critical elements in the upstream, Ca2+-dependent control of pathological cardiac hypertrophy.

Abstract-The L-type Ca 2ϩ channel (LTCC) is the major mediator of Ca 2ϩ influx in cardiomyocytes, leading to both mechanical contraction and activation of signaling cascades. Among these Ca 2ϩ -activated cascades is calcineurin, a protein phosphatase that promotes hypertrophic growth of the heart. Coimmunoprecipitations from heart extracts and pulldowns using heterologously expressed proteins provided evidence for direct binding of calcineurin at both the N and C termini of ␣ 1 1.2. At the C terminus, calcineurin bound specifically at amino acids 1943 to 1971, adjacent to a well-characterized protein kinase (PK)A/PKC/PKG phospho-acceptor site Ser1928. In vitro assays demonstrated that calcineurin can dephosphorylate ␣ 1 1.2. Channel function was increased in voltage-clamp recordings of I Ca,L from cultured cardiomyocytes expressing constitutively active calcineurin, consistent with previous observations in cardiac hypertrophy in vivo. Conversely, acute suppression of calcineurin pharmacologically or with specific peptides decreased I Ca,L . These data reveal direct physical interaction between the LTCC and calcineurin in heart. Furthermore, they demonstrate that calcineurin induces robust increases in I Ca,L and highlight calcineurin as a key modulator of pathological electrical remodeling in cardiac hypertrophy. (Circ Res. 2009;105:51-60.) Key Words: action potential remodeling Ⅲ Ca 2ϩ channels Ⅲ calcineurin Ⅲ electrophysiology Ⅲ hypertrophy P erturbation of intracellular Ca 2ϩ signaling and prolongation of the myocyte action potential accompany many forms of heart disease and contribute to the pathogenesis of cardiac hypertrophy and failure. 1 The voltage-gated L-type Ca 2ϩ channel (LTCC) (Ca V 1.2) is the major pathway of Ca 2ϩ influx into cardiomyocytes and an important determinant of action potential morphology. Indeed, the LTCC transduces membrane potential changes into local intracellular calcium transients that initiate a host of physiological events. We and others have reported an increase in LTCC activity in various models of cardiac hypertrophy. 2,3 However, molecular mechanisms responsible for the observed increase in channel function in hypertrophy are unknown, although evidence suggests that it involves an increase in channel availability and open probability. 4 L-type channel function is governed by an array of reversible phosphorylation and dephosphorylation events. Among the best characterized reactions is a robust, transient activation of L-type Ca 2ϩ current (I Ca,L ) by protein kinase (PK)A. 5,6 More complex regulation by PKC has been reported, with both channel activation and inhibition observed. 5,6 Other work has demonstrated I Ca,L inhibition by PKG. 7 However, despite a growing understanding of the role of phosphorylation in the regulation of I Ca,L , less is known regarding the role of protein phosphatases in these reactions. Some reports have described PP2A 8,9 and PP1-mediated 10 channel regulation.Calcineurin (PP2B) is a cytoplasmic Ca 2ϩ /calmodulindependent protein phosphata...

Ca2؉ /calmodulin-dependent protein kinase II (CaMKII) activity is increased in heart failure (HF), a syndrome characterized by markedly increased risk of arrhythmia. Activation of CaMKII increases peak L-type Ca 2؉ current (I Ca ) and slows I Ca inactivation. Whether these events are linked mechanistically is unknown. I Ca was recorded in acutely dissociated subepicardial and subendocardial murine left ventricular (LV) myocytes using the whole cell patch clamp method. Pressure overload heart failure was induced by surgical constriction of the thoracic aorta. I Ca density was significantly larger in subepicardial myocytes than in subendocardial/myocytes. Similar patterns were observed in the cell surface expression of ␣1c, the channel pore-forming subunit. In failing LV, I Ca density was increased proportionately in both cell types, and the time course of I Ca inactivation was slowed. This typical pattern of changes suggested a role of CaMKII. Consistent with this, measurements of CaMKII activity revealed a 2-3-fold increase (p < 0.05) in failing LV. To test for a causal link, we measured frequency-dependentI Ca facilitation.InHFmyocytes,thisCaMKIIdependent process could not be induced, suggesting already maximal activation. Internal application of active CaMKII in failing myocytes did not elicit changes in I Ca . Finally, CaMKII inhibition by internal diffusion of a specific peptide inhibitor reduced I Ca density and inactivation time course to similar levels in control and HF myocytes. I Ca density manifests a significant transmural gradient, and this gradient is preserved in heart failure. Activation of CaMKII, a known pro-arrhythmic molecule, is a major contributor to I Ca remodeling in load-induced heart failure.Patients with heart failure are at increased risk of malignant arrhythmia, which accounts for a substantial component of the mortality associated with this disease (1). Mechanisms underlying these arrhythmias are multifactorial, but they stem, at least in part, from disordered electrical currents arising from prolongation of ventricular action potentials. The resulting delay in the recovery of excitability, a consistent feature of heart failure (2), predisposes to early and delayed after-depolarizations. Heart failure is also associated with myocardial fibrosis, altered electrotonic coupling between cells, slowed conduction, and dispersion of refractoriness, all of which predispose to reentrant mechanisms of arrhythmia. Together, these "electrical remodeling" responses underlie the propensity to arrhythmia, syncope, and sudden death.In recent years, electrical remodeling has emerged as an important pathophysiologic mechanism in many types of cardiac pathology. Whereas considerable progress has been made recently in elucidating the molecular pathogenesis of cardiac hypertrophy and failure (reviewed in Refs. 3 and 4), our understanding of mechanisms underlying disease-related action potential prolongation is limited. As a result, the means of treating heart failure-associated arrhythmias remain disappo...

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The L-type Ca 2+ channel Ca v 1.2 forms macromolecular signaling complexes that comprise the β 2 adrenergic receptor, trimeric G s protein, adenylyl cyclase, and cAMP-dependent protein kinase (PKA1) for efficient signaling in heart and brain. The protein phosphatases PP2A and PP2B are part of this complex. PP2A counteracts increase in Ca v 1.2 channel activity by PKA and other protein kinases, whereas PP2B can either augment or decrease Ca v 1.2 currents in cardiomyocytes depending on the precise experimental conditions. We found that PP2A binds to two regions in the C-terminus of the central, pore-forming α 1 subunit of Ca v 1.2: one region spans residues 1795-1818 and the other residues 1965-1971. PP2B binds immediately downstream of residue 1971. Injection of a peptide that contained residues 1965-1971 and displaced PP2A but not PP2B from endogenous Ca v 1.2 increased basal and isoproterenol-stimulated L-type Ca 2+ currents in acutely isolated cardiomyocytes. Together with our biochemical data, these physiological results indicate that anchoring of PP2A at this site of Ca v 1.2 in the heart negatively regulates cardiac Ltype currents, likely by counterbalancing basal and stimulated phosphorylation that is mediated by PKA and possibly other kinases.Ca 2+ influx through L-type channels controls membrane excitability (1), synaptic plasticity (2-5), and gene expression (6,7) in neurons and triggers myocardial contraction in the heart. L-type Ca 2+ channels are the main targets of so-called organic calcium channel blockers, which include dihydropyridines, phenylalkylamines, and benzothiazepines. Voltage-gated † This work was supported by grant 3020 from the China Scholarship Council (to H.X.), the National Institute of Health research grants R01-NS035563 and R01-AG017502 (to J.W.H.), R01-GM056900 (to M.C.H.), R01-HL075173 (to J.A.H.), , and the American Heart Association grants 0535235N (to D.D.H.) and (0640084N) (to J.A.H.). 1 Abbreviations used are: GST, glutathione-S transferase; ISO, isoproterenol; PAGE, polyacrylamide gel electrophoresis; PKA, cAMP-dependent protein kinase; PP, protein phosphatase; PVDF, polyvinyldifluoride; SDS, sodium dodecylsulphate. * To whom correspondence should be addressed: Johannes W. Hell, Department of Pharmacology, University of California, 451 E. Health Sciences Drive, Davis, Tel: (530) Figure 1 shows that peptide 6-10, which are 10 residues long and cover Peptide 4 and 5, do not inhibit PP2A and PP2B binding to . This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 December 7. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptCa 2+ channels consist of a central ion-conducting pore, the α 1 subunit, and auxiliary α 2 -δ, and β subunits (8). Ca v 1.2 containing the central α 1 1.2 is the main L-type channel in the cardiovascular system, heart, and brain (8).Ca v 1.2 is a point of convergence of multiple regulatory pathways. For...