Myofilament Alterations Associated with Human R14del-Phospholamban Cardiomyopathy.

Phospholamban (PLN) is a major regulator of cardiac contractility, and human mutations in this gene give rise to inherited cardiomyopathies. The deletion of Arginine 14 is the most-prevalent cardiomyopathy-related mutation, and it has been linked to arrhythmogenesis and early death. Studies in PLN-humanized mutant mice indicated an increased propensity to arrhythmias, but the underlying cellular mechanisms associated with R14del-PLN cardiac dysfunction in the absence of any apparent structural remodeling remain unclear. The present study addressed the specific role of myofilaments in the setting of R14del-PLN and the long-term effects of R14del-PLN in the heart. Maximal force was depressed in skinned cardiomyocytes from both left and right ventricles, but this effect was more pronounced in the right ventricle of R14del-PLN mice. In addition, the Ca2+ sensitivity of myofilaments was increased in both ventricles of mutant mice. However, the depressive effects of R14del-PLN on contractile parameters could be reversed with the positive inotropic drug omecamtiv mecarbil, a myosin activator. At 12 months of age, corresponding to the mean symptomatic age of R14del-PLN patients, contractile parameters and Ca2+ transients were significantly depressed in the right ventricular R14del-PLN cardiomyocytes. Echocardiography did not reveal any alterations in cardiac function or remodeling, although histological and electron microscopy analyses indicated subtle alterations in mutant hearts. These findings suggest that both aberrant myocyte calcium cycling and aberrant contractility remain specific to the right ventricle in the long term. In addition, altered myofilament activity is an early characteristic of R14del-PLN mutant hearts and the positive inotropic drug omecamtiv mecarbil may be beneficial in treating R14del-PLN cardiomyopathy.

Isoform changes of action potential regulators in the ventricles of arrhythmogenic phospholamban-R14del humanized mouse hearts.

Arrhythmogenic cardiomyopathy (ACM) is characterized by life-threatening ventricular arrhythmias and sudden cardiac death and affects hundreds of thousands of patients worldwide. The deletion of Arginine 14 (p.R14del) in the phospholamban (PLN) gene has been implicated in the pathogenesis of ACM. PLN is a key regulator of sarcoplasmic reticulum (SR) Ca2+ cycling and cardiac contractility. Despite global gene and protein expression studies, the molecular mechanisms of PLN-R14del ACM pathogenesis remain unclear. Using a humanized PLN-R14del mouse model and human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs), we investigated the transcriptome-wide mRNA splicing changes associated with the R14del mutation. We identified >200 significant alternative splicing (AS) events and distinct AS profiles were observed in the right (RV) and left (LV) ventricles in PLN-R14del compared to WT mouse hearts. Enrichment analysis of the AS events showed that the most affected biological process was associated with "cardiac cell action potential", specifically in the RV. We found that splicing of 2 key genes, Trpm4 and Camk2d, which encode proteins regulating calcium homeostasis in the heart, were altered in PLN-R14del mouse hearts and human iPSC-CMs. Bioinformatical analysis pointed to the tissue-specific splicing factors Srrm4 and Nova1 as likely upstream regulators of the observed splicing changes in the PLN-R14del cardiomyocytes. Our findings suggest that aberrant splicing may affect Ca2+-homeostasis in the heart, contributing to the increased risk of arrythmogenesis in PLN-R14del ACM.

Aberrant PLN-R14del Protein Interactions Intensify SERCA2a Inhibition, Driving Impaired Ca2+ Handling and Arrhythmogenesis.

Phospholamban (PLN), a key modulator of Ca2+-homeostasis, inhibits sarcoplasmic reticulum (SR) calcium-ATPase (SERCA2a) and regulates cardiac contractility. The human PLN mutation R14del has been identified in arrhythmogenic cardiomyopathy patients worldwide and is currently extensively investigated. In search of the molecular mechanisms mediating the pathological phenotype, we examined PLN-R14del associations to known PLN-interacting partners. We determined that PLN-R14del interactions to key Ca2+-handling proteins SERCA2a and HS-1-associated protein X-1 (HAX-1) were enhanced, indicating the super-inhibition of SERCA2a's Ca2+-affinity. Additionally, histidine-rich calcium binding protein (HRC) binding to SERCA2a was increased, suggesting the inhibition of SERCA2a maximal velocity. As phosphorylation relieves the inhibitory effect of PLN on SERCA2a activity, we examined the impact of phosphorylation on the PLN-R14del/SERCA2a interaction. Contrary to PLN-WT, phosphorylation did not affect PLN-R14del binding to SERCA2a, due to a lack of Ser-16 phosphorylation in PLN-R14del. No changes were observed in the subcellular distribution of PLN-R14del or its co-localization to SERCA2a. However, in silico predictions suggest structural perturbations in PLN-R14del that could impact its binding and function. Our findings reveal for the first time that by increased binding to SERCA2a and HAX-1, PLN-R14del acts as an enhanced inhibitor of SERCA2a, causing a cascade of molecular events contributing to impaired Ca2+-homeostasis and arrhythmogenesis. Relieving SERCA2a super-inhibition could offer a promising therapeutic approach for PLN-R14del patients.

Gene editing reverses arrhythmia susceptibility in humanized PLN-R14del mice: modelling a European cardiomyopathy with global impact.

AIMS: A mutation in the phospholamban (PLN) gene, leading to deletion of Arg14 (R14del), has been associated with malignant arrhythmias and ventricular dilation. Identifying pre-symptomatic carriers with vulnerable myocardium is crucial because arrhythmia can result in sudden cardiac death, especially in young adults with PLN-R14del mutation. This study aimed at assessing the efficiency and efficacy of in vivo genome editing, using CRISPR/Cas9 and a cardiotropic adeno-associated virus-9 (AAV9), in improving cardiac function in young adult mice expressing the human PLN-R14del. METHODS AND RESULTS: Humanized mice were generated expressing human wild-type (hPLN-WT) or mutant (hPLN-R14del) PLN in the heterozygous state, mimicking human carriers. Cardiac magnetic resonance imaging at 12 weeks of age showed bi-ventricular dilation and increased stroke volume in mutant vs. WT mice, with no deficit in ejection fraction or cardiac output. Challenge of ex vivo hearts with isoproterenol and rapid pacing unmasked higher propensity for sustained ventricular tachycardia (VT) in hPLN-R14del relative to hPLN-WT. Specifically, the VT threshold was significantly reduced (20.3 ± 1.2 Hz in hPLN-R14del vs. 25.7 ± 1.3 Hz in WT, P < 0.01) reflecting higher arrhythmia burden. To inactivate the R14del allele, mice were tail-vein-injected with AAV9.CRISPR/Cas9/gRNA or AAV9 empty capsid (controls). CRISPR-Cas9 efficiency was evaluated by droplet digital polymerase chain reaction and NGS-based amplicon sequencing. In vivo gene editing significantly reduced end-diastolic and stroke volumes in hPLN-R14del CRISPR-treated mice compared to controls. Susceptibility to VT was also reduced, as the VT threshold was significantly increased relative to controls (30.9 ± 2.3 Hz vs. 21.3 ± 1.5 Hz; P < 0.01). CONCLUSIONS: This study is the first to show that disruption of hPLN-R14del allele by AAV9-CRISPR/Cas9 improves cardiac function and reduces VT susceptibility in humanized PLN-R14del mice, offering preclinical evidence for translatable approaches to therapeutically suppress the arrhythmogenic phenotype in human patients with PLN-R14del disease.

Impaired Right Ventricular Calcium Cycling Is an Early Risk Factor in R14del-Phospholamban Arrhythmias.

The inherited mutation (R14del) in the calcium regulatory protein phospholamban (PLN) is linked to malignant ventricular arrhythmia with poor prognosis starting at adolescence. However, the underlying early mechanisms that may serve as prognostic factors remain elusive. This study generated humanized mice in which the endogenous gene was replaced with either human wild type or R14del-PLN and addressed the early molecular and cellular pathogenic mechanisms. R14del-PLN mice exhibited stress-induced impairment of atrioventricular conduction, and prolongation of both ventricular activation and repolarization times in association with ventricular tachyarrhythmia, originating from the right ventricle (RV). Most of these distinct electrocardiographic features were remarkably similar to those in R14del-PLN patients. Studies in isolated cardiomyocytes revealed RV-specific calcium defects, including prolonged action potential duration, depressed calcium kinetics and contractile parameters, and elevated diastolic Ca-levels. Ca-sparks were also higher although SR Ca-load was reduced. Accordingly, stress conditions induced after contractions, and inclusion of the CaMKII inhibitor KN93 reversed this proarrhythmic parameter. Compensatory responses included altered expression of key genes associated with Ca-cycling. These data suggest that R14del-PLN cardiomyopathy originates with RV-specific impairment of Ca-cycling and point to the urgent need to improve risk stratification in asymptomatic carriers to prevent fatal arrhythmias and delay cardiomyopathy onset.

Phosphorylation of cardiac myosin-binding protein-C contributes to calcium homeostasis.

Cardiac myosin-binding protein-C (cMyBP-C) is highly phosphorylated under basal conditions. However, its phosphorylation level is decreased in individuals with heart failure. The necessity of cMyBP-C phosphorylation for proper contractile function is well-established, but the physiological and pathological consequences of decreased cMyBP-C phosphorylation in the heart are not clear. Herein, using intact adult cardiomyocytes from mouse models expressing phospho-ablated (AAA) and phosphomimetic (DDD) cMyBP-C as well as controls, we found that cMyBP-C dephosphorylation is sufficient to reduce contractile parameters and calcium kinetics associated with prolonged decay time of the calcium transient and increased diastolic calcium levels. Isoproterenol stimulation reversed the depressive contractile and Ca2+-kinetic parameters. Moreover, caffeine-induced calcium release yielded no difference between AAA/DDD and controls in calcium content of the sarcoplasmic reticulum. On the other hand, sodium-calcium exchanger function and phosphorylation levels of calcium-handling proteins were significantly decreased in AAA hearts compared with controls. Stress conditions caused increases in both spontaneous aftercontractions in AAA cardiomyocytes and the incidence of arrhythmias in vivo compared with the controls. Treatment with omecamtiv mecarbil, a positive cardiac inotropic drug, rescued the contractile deficit in AAA cardiomyocytes, but not the calcium-handling abnormalities. These findings indicate a cascade effect whereby cMyBP-C dephosphorylation causes contractile defects, which then lead to calcium-cycling abnormalities, resulting in aftercontractions and increased incidence of cardiac arrhythmias under stress conditions. We conclude that improvement of contractile deficits alone without improving calcium handling may be insufficient for effective management of heart failure.

Phosphorylation of Hsp20 Promotes Fibrotic Remodeling and Heart Failure.

Cardiomyocyte-specific increases in phosphorylated Hsp20 (S16D-Hsp20) to levels similar to those observed in human failing hearts are associated with early fibrotic remodeling and depressed left ventricular function, symptoms which progress to heart failure and early death. The underlying mechanisms appear to involve translocation of phosphorylated Hsp20 to the nucleus and upregulation of interleukin (IL)-6, which subsequently activates cardiac fibroblasts in a paracrine fashion through transcription factor STAT3 signaling. Accordingly, treatment of S16D-Hsp20 mice with a rat anti-mouse IL-6 receptor monoclonal antibody (MR16-1) attenuated interstitial fibrosis and preserved cardiac function. These findings suggest that phosphorylated Hsp20 may be a potential therapeutic target in heart failure.

HAX-1 regulates SERCA2a oxidation and degradation.

Ischemia/reperfusion injury is associated with contractile dysfunction and increased cardiomyocyte death. Overexpression of the hematopoietic lineage substrate-1-associated protein X-1 (HAX-1) has been shown to protect from cellular injury but the function of endogenous HAX-1 remains obscure due to early lethality of the knockout mouse. Herein we generated a cardiac-specific and inducible HAX-1 deficient model, which uncovered an unexpected role of HAX-1 in regulation of sarco/endoplasmic reticulum Ca-ATPase (SERCA2a) in ischemia/reperfusion injury. Although ablation of HAX-1 in the adult heart elicited no morphological alterations under non-stress conditions, it diminished contractile recovery and increased infarct size upon ischemia/reperfusion injury. These detrimental effects were associated with increased loss of SERCA2a. Enhanced SERCA2a degradation was not due to alterations in calpain and calpastatin levels or calpain activity. Conversely, HAX-1 overexpression improved contractile recovery and maintained SERCA2a levels. The regulatory effects of HAX-1 on SERCA2a degradation were observed at multiple levels, including intact hearts, isolated cardiomyocytes and sarcoplasmic reticulum microsomes. Mechanistically, HAX-1 ablation elicited increased production of reactive oxygen species at the sarco/endoplasic reticulum compartment, resulting in SERCA2a oxidation and a predisposition to its proteolysis. This effect may be mediated by NAPDH oxidase 4 (NOX4), a novel binding partner of HAX-1. Accordingly, NOX inhibition with apocynin abrogated the effects of HAX-1 ablation in hearts subjected to ischemia/reperfusion injury. Taken together, our findings reveal a role of HAX-1 in the regulation of oxidative stress and SERCA2a degradation, implicating its importance in calcium homeostasis and cell survival pathways.

Regulation of BECN1-mediated autophagy by HSPB6: Insights from a human HSPB6S10F mutant.

HSPB6/Hsp20 (heat shock protein family B [small] member 6) has emerged as a novel cardioprotector against stress-induced injury. We identified a human mutant of HSPB6 (HSPB6S10F) exclusively present in dilated cardiomyopathy (DCM) patients. Cardiac expression of this mutant in mouse hearts resulted in remodeling and dysfunction, which progressed to heart failure and early death. These detrimental effects were associated with reduced interaction of mutant HSPB6S10F with BECN1/Beclin 1, leading to BECN1 ubiquitination and its proteosomal degradation. As a result, autophagy flux was substantially inhibited and apoptosis was increased in HSPB6S10F-mutant hearts. In contrast, overexpression of wild-type HSPB6 (HSPB6 WT) not only increased BECN1 levels, but also competitively suppressed binding of BECN1 to BCL2, resulting in stimulated autophagy. Indeed, preinhibition of autophagy attenuated the cardioprotective effects of HSPB6 WT. Taken together, these findings reveal a new regulatory mechanism of HSPB6 in cell survival through its interaction with BECN1. Furthermore, Ser10 appears to be crucial for the protective effects of HSPB6 and transversion of this amino acid to Phe contributes to cardiomyopathy.

The antiapoptotic protein HAX-1 mediates half of phospholamban's inhibitory activity on calcium cycling and contractility in the heart.

The antiapoptotic protein HAX-1 (HS-associated protein X-1) localizes to sarcoplasmic reticulum (SR) in the heart and interacts with the small membrane protein phospholamban (PLN), inhibiting the cardiac sarco/endoplasmic reticulum calcium ATPase 2a (SERCA2a) in the regulation of overall calcium handling and heart muscle contractility. However, because global HAX-1 deletion causes early lethality, how much endogenous HAX-1 contributes to PLN's inhibitory activity on calcium cycling is unknown. We therefore generated a cardiac-specific and inducible knock-out mouse model. HAX-1 ablation in the adult heart significantly increased contractile parameters and calcium kinetics, associated with increased SR calcium load. These changes occurred without any changes in the protein expression of SERCA2a, PLN, and ryanodine receptor or in the PLN phosphorylation status. The enhanced calcium cycling in the HAX-1-depleted heart was mediated through increases in the calcium affinity of SERCA2a and reduced PLN-SERCA2a binding. Comparison of the HAX-1 deletion-induced stimulatory effects with those elicited by PLN ablation indicated that HAX-1 mediates ∼50% of the PLN-associated inhibitory effects in the heart. Stimulation with the inotropic and lusitropic agent isoproterenol eliminated the differences among wild-type, HAX-1-deficient, and PLN-deficient hearts, and maximally stimulated contractile and calcium kinetic parameters were similar among these three groups. Furthermore, PLN overexpression in the HAX-1-null cardiomyocytes did not elicit any inhibitory effects, indicating that HAX-1 may limit PLN activity. These findings suggest that HAX-1 is a major mediator of PLN's inhibitory activity and a critical gatekeeper of SR calcium cycling and contractility in the heart.

Phosphorylation of serine96 of histidine-rich calcium-binding protein by the Fam20C kinase functions to prevent cardiac arrhythmia.

Precise Ca cycling through the sarcoplasmic reticulum (SR), a Ca storage organelle, is critical for proper cardiac muscle function. This cycling initially involves SR release of Ca via the ryanodine receptor, which is regulated by its interacting proteins junctin and triadin. The sarco/endoplasmic reticulum Ca ATPase (SERCA) pump then refills SR Ca stores. Histidine-rich Ca-binding protein (HRC) resides in the lumen of the SR, where it contributes to the regulation of Ca cycling by protecting stressed or failing hearts. The common Ser96Ala human genetic variant of HRC strongly correlates with life-threatening ventricular arrhythmias in patients with idiopathic dilated cardiomyopathy. However, the underlying molecular pathways of this disease remain undefined. Here, we demonstrate that family with sequence similarity 20C (Fam20C), a recently characterized protein kinase in the secretory pathway, phosphorylates HRC on Ser96. HRC Ser96 phosphorylation was confirmed in cells and human hearts. Furthermore, a Ser96Asp HRC variant, which mimics constitutive phosphorylation of Ser96, diminished delayed aftercontractions in HRC null cardiac myocytes. This HRC phosphomimetic variant was also able to rescue the aftercontractions elicited by the Ser96Ala variant, demonstrating that phosphorylation of Ser96 is critical for the cardioprotective function of HRC. Phosphorylation of HRC on Ser96 regulated the interactions of HRC with both triadin and SERCA2a, suggesting a unique mechanism for regulation of SR Ca homeostasis. This demonstration of the role of Fam20C-dependent phosphorylation in heart disease will open new avenues for potential therapeutic approaches against arrhythmias.

Human G109E-inhibitor-1 impairs cardiac function and promotes arrhythmias.

A hallmark of human and experimental heart failure is deficient sarcoplasmic reticulum (SR) Ca-uptake reflecting impaired contractile function. This is at least partially attributed to dephosphorylation of phospholamban by increased protein phosphatase 1 (PP1) activity. Indeed inhibition of PP1 by transgenic overexpression or gene-transfer of constitutively active inhibitor-1 improved Ca-cycling, preserved function and decreased fibrosis in small and large animal models of heart failure, suggesting that inhibitor-1 may represent a potential therapeutic target. We recently identified a novel human polymorphism (G109E) in the inhibitor-1 gene with a frequency of 7% in either normal or heart failure patients. Transgenic mice, harboring cardiac-specific expression of G109E inhibitor-1, exhibited decreases in contractility, Ca-kinetics and SR Ca-load. These depressive effects were relieved by isoproterenol stimulation. Furthermore, stress conditions (2Hz +/- Iso) induced increases in Ca-sparks, Ca-waves (60% of G109E versus 20% in wild types) and after-contractions (76% of G109E versus 23% of wild types) in mutant cardiomyocytes. Similar findings were obtained by acute expression of the G109E variant in adult cardiomyocytes in the absence or presence of endogenous inhibitor-1. The underlying mechanisms included reduced binding of mutant inhibitor-1 to PP1, increased PP1 activity, and dephosphorylation of phospholamban at Ser16 and Thr17. However, phosphorylation of the ryanodine receptor at Ser2808 was not altered while phosphorylation at Ser2814 was increased, consistent with increased activation of Ca/calmodulin-dependent protein kinase II (CaMKII), promoting aberrant SR Ca-release. Parallel in vivo studies revealed that mutant mice developed ventricular ectopy and complex ventricular arrhythmias (including bigeminy, trigeminy and ventricular tachycardia), when challenged with isoproterenol. Inhibition of CaMKII activity by KN-93 prevented the increased propensity to arrhythmias. These findings suggest that the human G109E inhibitor-1 variant impairs SR Ca-cycling and promotes arrhythmogenesis under stress conditions, which may present an additional insult in the compromised function of heart failure carriers.

Up-regulation of micro-RNA765 in human failing hearts is associated with post-transcriptional regulation of protein phosphatase inhibitor-1 and depressed contractility.

AIMS: Impaired sarcoplasmic reticulum (SR) Ca(2+) cycling and depressed contractility, a hallmark of human and experimental heart failure, has been partially attributed to increased protein phosphatase 1 (PP-1) activity, associated with down-regulation of its endogenous inhibitor-1. The levels and activity of inhibitor-1 are reduced in failing hearts, contributing to dephosphorylation and inactivation of key calcium cycling proteins. Therefore, we investigated the mechanisms that mediate decreases in inhibitor-1 by post-transcriptional modification. METHODS AND RESULTS: Bioinformatics revealed that 17 human microRNAs may serve as modulators of inhibitor-1. However, real-time PCR analysis identified only one of these microRNAs, miR-765, as being increased in human failing hearts concomitant with decreased inhibitor-1 levels. Expression of miR-765 in HEK293 cells or mouse ventricular myocytes confirmed suppression of inhibitor-1 levels through binding of this miR-765 to the 3'-untranslated region of inhibitor-1 mRNA. To determine the functional significance of miR-765 in Ca(2+) cycling, pri-miR-765 as well as a non-translated nucleotide sequence (miR-Ctrl) were expressed in adult mouse ventricular myocytes. The inhibitor-1 expression levels were decreased, accompanied by enhanced PP-1 activity in the miR-765 cardiomyocytes, and these reflected depressed contractile mechanics and Ca(2+) transients, compared with the miR-Ctrl group. The depressive effects were associated with decreases in the phosphorylation of phospholamban and SR Ca(2+) load. These miR-765 negative inotropic effects were abrogated in inhibitor-1-deficient cardiomyocytes, suggesting its apparent specificity for inhibitor-1. CONCLUSIONS: miR-765 levels are increased in human failing hearts. Such increases may contribute to depressed cardiac function through reduced inhibitor-1 expression and enhanced PP-1 activity, associated with reduced SR Ca(2+) load.

A novel human R25C-phospholamban mutation is associated with super-inhibition of calcium cycling and ventricular arrhythmia.

AIMS: Depressed sarcoplasmic reticulum (SR) Ca(2+) cycling, a universal characteristic of human and experimental heart failure, may be associated with genetic alterations in key Ca(2+)-handling proteins. In this study, we identified a novel PLN mutation (R25C) in dilated cardiomyopathy (DCM) and investigated its functional significance in cardiomyocyte Ca(2+)-handling and contractility. METHODS AND RESULTS: Exome sequencing identified a C73T substitution in the coding region of PLN in a family with DCM. The four heterozygous family members had implantable cardiac defibrillators, and three developed prominent ventricular arrhythmias. Overexpression of R25C-PLN in adult rat cardiomyocytes significantly suppressed the Ca(2+) affinity of SR Ca(2+)-ATPase (SERCA2a), resulting in decreased SR Ca(2+) content, Ca(2+) transients, and impaired contractile function, compared with WT-PLN. These inhibitory effects were associated with enhanced interaction of R25C-PLN with SERCA2, which was prevented by PKA phosphorylation. Accordingly, isoproterenol stimulation relieved the depressive effects of R25C-PLN in cardiomyocytes. However, R25C-PLN also elicited increases in the frequency of Ca(2+) sparks and waves as well as stress-induced aftercontractions. This was accompanied by increased Ca(2+)/calmodulin-dependent protein kinase II activity and hyper-phosphorylation of RyR2 at serine 2814. CONCLUSION: The findings demonstrate that human R25C-PLN is associated with super-inhibition of SERCA2a and Ca(2+) transport as well as increased SR Ca(2+) leak, promoting arrhythmogenesis under stress conditions. This is the first mechanistic evidence that increased PLN inhibition may impact both SR Ca(2+) uptake and Ca(2+) release activities and suggests that the human R25C-PLN may be a prognostic factor for increased ventricular arrhythmia risk in DCM carriers.

Phospholamban interactome in cardiac contractility and survival: A new vision of an old friend.

Depressed sarcoplasmic reticulum (SR) calcium cycling, reflecting impaired SR Ca-transport and Ca-release, is a key and universal characteristic of human and experimental heart failure. These SR processes are regulated by multimeric protein complexes, including protein kinases and phosphatases as well as their anchoring and regulatory subunits that fine-tune Ca-handling in specific SR sub-compartments. SR Ca-transport is mediated by the SR Ca-ATPase (SERCA2a) and its regulatory phosphoprotein, phospholamban (PLN). Dephosphorylated PLN is an inhibitor of SERCA2a and phosphorylation by protein kinase A (PKA) or calcium-calmodulin-dependent protein kinases (CAMKII) relieves these inhibitory effects. Recent studies identified additional regulatory proteins, associated with PLN, that control SR Ca-transport. These include the inhibitor-1 (I-1) of protein phosphatase 1 (PP1), the small heat shock protein 20 (Hsp20) and the HS-1 associated protein X-1 (HAX1). In addition, the intra-luminal histidine-rich calcium binding protein (HRC) has been shown to interact with both SERCA2a and triadin. Notably, there is physical and direct interaction between these protein players, mediating a fine-cross talk between SR Ca-uptake, storage and release. Importantly, regulation of SR Ca-cycling by the PLN/SERCA interactome does not only impact cardiomyocyte contractility, but also survival and remodeling. Indeed, naturally occurring variants in these Ca-cycling genes modulate their activity and interactions with other protein partners, resulting in depressed contractility and accelerated remodeling. These genetic variants may serve as potential prognostic or diagnostic markers in cardiac pathophysiology.

Active inhibitor-1 maintains protein hyper-phosphorylation in aging hearts and halts remodeling in failing hearts.

Impaired sarcoplasmic reticulum calcium cycling and depressed contractility are key characteristics in heart failure. Defects in sarcoplasmic reticulum function are characterized by decreased SERCA2a Ca-transport that is partially attributable to dephosphorylation of its regulator phospholamban by increased protein phosphatase 1 activity. Inhibition of protein phosphatase 1 through activation of its endogenous inhibitor-1 has been shown to enhance cardiac Ca-handling and contractility as well as protect from pathological stress remodeling in young mice. In this study, we assessed the long-term effects of inducible expression of constitutively active inhibitor-1 in the adult heart and followed function and remodeling through the aging process, up to 20 months. Mice with inhibitor-1 had normal survival and similar function to WTs. There was no overt remodeling as evidenced by measures of left ventricular end-systolic and diastolic diameters and posterior wall dimensions, heart weight to tibia length ratio, and histology. Higher phosphorylation of phospholamban at both Ser16 and Thr17 was maintained in aged hearts with active inhibitor-1, potentially offsetting the effects of elevated Ser2815-phosphorylation in ryanodine receptor, as there were no increases in arrhythmias under stress conditions in 20-month old mice. Furthermore, long-term expression of active inhibitor-1 via recombinant adeno-associated virus type 9 gene transfer in rats with pressure-overload induced heart failure improved function and prevented remodeling, associated with increased phosphorylation of phospholamban at Ser16 and Thr17. Thus, chronic inhibition of protein phosphatase 1, through increases in active inhibitor-1, does not accelerate age-related cardiomyopathy and gene transfer of this molecule in vivo improves function and halts remodeling in the long term.

Abnormal calcium cycling and cardiac arrhythmias associated with the human Ser96Ala genetic variant of histidine-rich calcium-binding protein.

BACKGROUND: A human genetic variant (Ser96Ala) in the sarcoplasmic reticulum (SR) histidine-rich Ca(2+)-binding (HRC) protein has been linked to ventricular arrhythmia and sudden death in dilated cardiomyopathy. However, the precise mechanisms affecting SR function and leading to arrhythmias remain elusive. METHODS AND RESULTS: We generated transgenic mice with cardiac-specific expression of human Ala96 HRC or Ser96 HRC in the null background to assess function in absence of endogenous protein. Ala96 HRC decreased (25% to 30%) cardiomyocyte contractility and Ca2+ kinetics compared with Ser96 HRC in the absence of any structural or histological abnormalities. Furthermore, the frequency of Ca2+ waves was significantly higher (10-fold), although SR Ca2+ load was reduced (by 27%) in Ala96 HRC cells. The underlying mechanisms involved diminished interaction of Ala96 HRC with triadin, affecting ryanodine receptor (RyR) stability. Indeed, the open probability of RyR, assessed by use of ryanodine binding, was significantly increased. Accordingly, stress conditions (5 Hz plus isoproterenol) induced aftercontractions (65% in Ala96 versus 12% in Ser96) and delayed afterdepolarizations (70% in Ala96 versus 20% in Ser96). The increased SR Ca2+ leak was accompanied by hyperphosphorylation (1.6-fold) of RyR at Ser2814 by calmodulin-dependent protein kinase II. Accordingly, inclusion of the calmodulin-dependent protein kinase II inhibitor KN93 prevented Ser2814 phosphorylation and partially reversed the increases in Ca2+ spark frequency and wave production. Parallel in vivo studies revealed ventricular ectopy on short-term isoproterenol challenge and increased (4-fold) propensity to arrhythmias, including nonsustained ventricular tachycardia, after myocardial infarction in Ala96 HRC mice. CONCLUSIONS: These findings suggest that aberrant SR Ca2+ release and increased susceptibility to delayed afterdepolarizations underlie triggered arrhythmic activity in human Ala96 HRC carriers.

Constitutive phosphorylation of inhibitor-1 at Ser67 and Thr75 depresses calcium cycling in cardiomyocytes and leads to remodeling upon aging.

The activity of protein phosphatase-1 (PP1) inhibitor-1 (I-1) is antithetically modulated by the cAMP-protein kinase A (PKA) and Ca(2+)-protein kinase C (PKC) signaling axes. ß-adrenergic (ß-AR) stimulation results in PKA-phosphorylation of I-1 at threonine 35 (Thr35) and depressed PP1 activity, while PKC phosphorylation at serine 67 (Ser67) and/or Thr75 increases PP1 activity. In heart failure, pThr35 is decreased while pSer67 and pThr75 are elevated. However, the role of Ser67/Thr75 phosphorylation in vivo and its effects on Ca(2+)-cycling are not known. Thus, our aim was to investigate the functional significance of Ser67 and Thr75 phosphorylation in intact hearts. We generated transgenic mice (TG) with cardiac-specific overexpression of constitutively phosphorylated I-1 at Ser67 and Thr75 (S67D/T75D) and evaluated cardiac function. The S67D/T75D cardiomyocytes exhibited significantly depressed Ca(2+)-kinetics and contractile parameters, compared with wild-type (WT) cells. The decreased Ca(2+)-cycling was associated with a 27 % increase in PP1 activity, no alterations in PP2 activity and impaired phosphorylation of myosin-binding protein-C (MyBPC). Upon aging, there was cardiac remodeling associated with increases in systolic and diastolic left ventricular internal diameter dimensions (at 16 months), compared with WTs. The results indicate that phosphorylation of I-1 at Ser67 and Thr75 is associated with increased PP1 activity and depressed cardiomyocyte Ca(2+)-cycling, which manifests in geometrical alterations over the long term. Thus, hyperphosphorylation of these sites in failing hearts may contribute to deteriorative remodeling.

Regulation of cAMP homeostasis by the efflux protein MRP4 in cardiac myocytes.

Recent studies indicate that members of the multidrug-resistance protein (MRP) family belonging to ATP binding cassette type C (ABCC) membrane proteins extrude cyclic nucleotides from various cell types. This study aimed to determine whether MRP proteins regulate cardiac cAMP homeostasis. Here, we demonstrate that MRP4 is the predominant isoform present at the plasma membrane of cardiacmyocytes and that it mediates the efflux of cAMP in these cells. MRP4-deficient mice displayed enhanced cardiac myocyte cAMP formation, contractility, and cardiac hypertrophy at 9 mo of age, an effect that was compensated transiently by increased phosphodiesterase expression at young age. These findings suggest that cAMP extrusion via MRP4 acts together with phosphodiesterases to control cAMP levels in cardiac myocytes.

The human phospholamban Arg14-deletion mutant localizes to plasma membrane and interacts with the Na/K-ATPase.

Depressed Ca-handling in cardiomyocytes is frequently attributed to impaired sarcoplasmic reticulum (SR) function in human and experimental heart failure. Phospholamban (PLN) is a key regulator of SR and cardiac function, and PLN mutations in humans have been associated with dilated cardiomyopathy (DCM). We previously reported the deletion of the highly conserved amino acid residue arginine 14 (nucleic acids 39, 40 and 41) in DCM patients. This basic amino acid is important in maintaining the upstream consensus sequence for PKA phosphorylation of Ser 16 in PLN. To assess the function of this mutant PLN, we introduced the PLN-R14Del in cardiac myocytes of the PLN null mouse. Transgenic lines expressing mutant PLN-R14Del at similar protein levels to wild types exhibited no inhibition of the initial rates of oxalate-facilitated SR Ca uptake compared to PLN-knockouts (PLN-KO). The contractile parameters and Ca-kinetics also remained highly stimulated in PLN-R14Del cardiomyocytes, similar to PLN-KO, and isoproterenol did not further stimulate these hyper-contractile basal parameters. Consistent with the lack of inhibition on SR Ca-transport and contractility, confocal microscopy indicated that the PLN-R14Del failed to co-localize with SERCA2a. Moreover, PLN-R14Del did not co-immunoprecipitate with SERCA2a (as did WT-PLN), but rather co-immunoprecipitated with the sarcolemmal Na/K-ATPase (NKA) and stimulated NKA activity. In addition, studies in HEK cells indicated significant fluorescence resonance energy transfer between PLN-R14Del-YFP and NKAα1-CFP, but not with the NKA regulator phospholemman. Despite the enhanced cardiac function in PLN-R14Del hearts (as in PLN-knockouts), there was cardiac hypertrophy (unlike PLN-KO) coupled with activation of Akt and the MAPK pathways. Thus, human PLN-R14Del is misrouted to the sarcolemma, in the absence of endogenous PLN, and alters NKA activity, leading to cardiac remodeling.