STIM1 elevation in the heart results in aberrant Ca2 + handling and cardiomyopathy

Stromal interaction molecule 1 (STIM1) is a Ca^2 + sensor that partners with Orai1 to elicit Ca^2 + entry in response to endoplasmic reticulum (ER) Ca^2 + store depletion. While store-operated Ca^2 + entry (SOCE) is important for maintaining ER Ca^2 + homeostasis in non-excitable cells, it is unclear what role it plays in the heart, although STIM1 is expressed in the heart and upregulated during disease. Here we analyzed transgenic mice with STIM1 overexpression in the heart to model the known increase of this protein in response to disease. As expected, STIM1 transgenic myocytes showed enhanced Ca^2 + entry following store depletion and partial co-localization with the type 2 ryanodine receptor (RyR2) within the sarcoplasmic reticulum (SR), as well as enrichment around the sarcolemma. STIM1 transgenic mice exhibited sudden cardiac death as early as 6 weeks of age, while mice surviving past 12 weeks of age developed heart failure with hypertrophy, induction of the fetal gene program, histopathology and mitochondrial structural alterations, loss of ventricular functional performance and pulmonary edema. Younger, pre-symptomatic STIM1 transgenic mice exhibited enhanced pathology following pressure overload stimulation or neurohumoral agonist infusion, compared to controls. Mechanistically, cardiac myocytes isolated from STIM1 transgenic mice displayed spontaneous Ca^2 + transients that were prevented by the SOCE blocker SKF-96365, increased L-type Ca^2 + channel (LTCC) current, and enhanced Ca^2 + spark frequency. Moreover, adult cardiac myocytes from STIM1 transgenic mice showed both increased diastolic Ca^2 + and maximal transient amplitude but no increase in total SR Ca^2 + load. Associated with this enhanced Ca^2 + profile was an increase in cardiac nuclear factor of activated T-cells (NFAT) and Ca^2 +/calmodulin-dependent kinase II (CaMKII) activity. We conclude that STIM1 has an unexpected function in the heart where it alters communication between the sarcolemma and SR resulting in greater Ca^2 + flux and a leaky SR compartment.     

SR-targeted CaMKII inhibition improves SR Ca²+ handling, but accelerates cardiac remodeling in mice overexpressing CaMKIIδC

Cardiac myocyte overexpression of CaMKIIδ_C leads to cardiac hypertrophy and heart failure (HF) possibly caused by altered myocyte Ca²⁺ handling. A central defect might be the marked CaMKII-induced increase in diastolic sarcoplasmic reticulum (SR) Ca²⁺ leak which decreases SR Ca²⁺ load and Ca²⁺ transient amplitude. We hypothesized that inhibition of CaMKII near the SR membrane would decrease the leak, improve Ca²⁺ handling and prevent the development of contractile dysfunction and HF. To test this hypothesis we crossbred CaMKIIδ_C overexpressing mice (CaMK) with mice expressing the CaMKII-inhibitor AIP targeted to the SR via a modified phospholamban (PLB)-transmembrane-domain (SR-AIP). There was a selective decrease in the amount of activated CaMKII in the microsomal (SR/membrane) fraction prepared from these double-transgenic mice (CaMK/SR-AIP) mice. In ventricular cardiomyocytes from CaMK/SR-AIP mice, SR Ca²⁺ leak, assessed both as diastolic Ca²⁺ shift into SR upon tetracaine in intact myocytes or integrated Ca²⁺ spark release in permeabilized myocytes, was significantly reduced. The reduced leak was accompanied by enhanced SR Ca²⁺ load and twitch amplitude in double-transgenic mice (vs. CaMK), without changes in SERCA expression or NCX function. However, despite the improved myocyte Ca²⁺ handling, cardiac hypertrophy and remodeling was accelerated in CaMK/SR-AIP and cardiac function worsened. We conclude that while inhibition of SR localized CaMKII in CaMK mice improves Ca²⁺ handling, it does not necessarily rescue the HF phenotype. This implies that a non-SR CaMKIIδ_C exerts SR-independent effects that contribute to hypertrophy and HF, and this CaMKII pathway may be exacerbated by the global enhancement of Ca transients.     

CaMKIIδ mediates β-adrenergic effects on RyR2 phosphorylation and SR Ca2 + leak and the pathophysiological response to chronic β-adrenergic stimulation

Chronic activation of Ca^2 +/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the deleterious effects of β-adrenergic receptor (β-AR) signaling on the heart, in part, by enhancing RyR2-mediated sarcoplasmic reticulum (SR) Ca^2 + leak. We used CaMKIIδ knockout (CaMKIIδ-KO) mice and knock-in mice with an inactivated CaMKII site S2814 on the ryanodine receptor type 2 (S2814A) to investigate the involvement of these processes in β-AR signaling and cardiac remodeling. Langendorff-perfused hearts from CaMKIIδ-KO mice showed inotropic and chronotropic responses to isoproterenol (ISO) that were similar to those of wild type (WT) mice; however, in CaMKIIδ-KO mice, CaMKII phosphorylation of phospholamban and RyR2 was decreased and isolated myocytes from CaMKIIδ-KO mice had reduced SR Ca^2 + leak in response to isoproterenol (ISO). Chronic catecholamine stress with ISO induced comparable increases in relative heart weight and other measures of hypertrophy from day 9 through week 4 in WT and CaMKIIδ-KO mice, but the development of cardiac fibrosis was prevented in CaMKIIδ-KO animals. A 4-week challenge with ISO resulted in reduced cardiac function and pulmonary congestion in WT, but not in CaMKIIδ-KO or S2814A mice, implicating CaMKIIδ-dependent phosphorylation of RyR2-S2814 in the cardiomyopathy, independent of hypertrophy, induced by prolonged β-AR stimulation.     

Moderate heart dysfunction in mice with inducible cardiomyocyte-specific excision of the Serca2 gene

The sarco(endo)plasmic reticulum calcium ATPase 2 (SERCA2) transports Ca²⁺ from cytosol into the sarcoplasmic reticulum (SR) of cardiomyocytes, thereby maintaining the store of releasable Ca²⁺ necessary for contraction. Reduced SERCA function has been linked to heart failure, and loss of SERCA2 in the adult mammalian heart would be expected to cause immediate severe myocardial contractile dysfunction and death. We investigated heart function in adult mice with an inducible cardiomyocyte-specific excision of the Atp2a2 (Serca2) gene (SERCA2 KO). Seven weeks after induction of Serca2 gene excision, the mice displayed a substantial reduction in diastolic function with a 5-fold increase in the time constant of isovolumetric pressure decay (tau). However, already at 4 weeks following gene excision less than 5% SERCA2 protein was found in myocardial tissue. Surprisingly, heart function was only moderately impaired at this time point. Tissue Doppler imaging showed slightly reduced peak systolic tissue velocity and a less than 2-fold increase in tau was observed. The SR Ca²⁺ content was dramatically reduced in cardiomyocytes from 4-week SERCA2 KO mice, and Ca²⁺ transients were predominantly generated by enhanced Ca²⁺ flux through L-type Ca²⁺ channels and the Na⁺-Ca²⁺ exchanger. Moreover, equivalent increases in cytosolic [Ca²⁺] in control and SERCA2 KO myocytes induced greater cell shortening in SERCA2 KO, suggesting enhanced myofilament responsiveness. Our data demonstrate that SR-independent Ca²⁺ transport mechanisms temporarily can prevent major cardiac dysfunction despite a major reduction of SERCA2 in cardiomyocytes.     

Induced Overexpression of Na+/Ca2+ Exchanger Does Not Aggravate Myocardial Dysfunction Induced by Transverse Aortic Constriction

BackgroundAlterations in expression and activity of cardiac Na⁺/Ca²⁺ exchanger (NCX1) have been implicated in the pathogenesis of heart failure.Methods and ResultsUsing transgenic mice in which expression of rat NCX1 was induced at 5 weeks of age, we performed transverse aortic constriction (TAC) at 8 weeks and examined cardiac and myocyte function at 15–18 weeks after TAC (age 23–26 weeks). TAC induced left ventricular (LV) and myocyte hypertrophy and increased myocardial fibrosis in both wild-type (WT) and NCX1-overexpressed mice. NCX1 and phosphorylated ryanodine receptor expression was increased by TAC, whereas sarco(endo)plasmic reticulum Ca²⁺-ATPase levels were decreased by TAC. Action potential duration was prolonged by TAC, but to a greater extent in NCX1 myocytes. Na⁺/Ca²⁺ exchange current was similar between WT-TAC and WT-sham myocytes, but was higher in NCX1-TAC myocytes. Both myocyte contraction and [Ca²⁺]_i transient amplitudes were reduced in WT-TAC myocytes, but restored to WT-sham levels in NCX1-TAC myocytes. Despite improvement in single myocyte contractility and Ca²⁺ dynamics, induced NCX1 overexpression in TAC animals did not ameliorate LV hypertrophy, increase ejection fraction, or enhance inotropic (maximal first derivative of LV pressure rise, +dP/dt) responses to isoproterenol.ConclusionsIn pressure-overload hypertrophy, induced overexpression of NCX1 corrected myocyte contractile and [Ca²⁺]_i transient abnormalities but did not aggravate or improve myocardial dysfunction.     

L-type Ca current in ventricular cardiomyocytes

L-type Ca²⁺ channels are mediators of Ca²⁺ influx and the regulatory events accompanying it and are pivotal in the function and dysfunction of ventricular cardiac myocytes. L-type Ca²⁺ channels are located in sarcolemma, including the T-tubules facing the sarcoplasmic reticulum junction, and are activated by membrane depolarization, but intracellular Ca²⁺-dependent inactivation limits Ca²⁺ influx during action potential. I_CaL is important in heart function because it triggers excitation-contraction coupling, modulates action potential shape and is involved in cardiac arrhythmia. L-type Ca²⁺ channels are multi-subunit complexes that interact with several molecules involved in their regulations, notably by β-adrenergic signaling. The present review highlights some of the recent findings on L-type Ca²⁺ channel function, regulation, and alteration in acquired pathologies such as cardiac hypertrophy, heart failure and diabetic cardiomyopathy, as well as in inherited arrhythmic cardiac diseases such as Timothy and Brugada syndromes.     

Intracellular [Na<Superscript>+</Superscript>] modulates synergy between Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger and L-type Ca<Superscript>2+</Superscript> current in cardiac excitation–contraction coupling during action potentials

Excitation–contraction coupling (ECC) in cardiac myocytes involves triggering of Ca²⁺ release from the sarcoplasmic reticulum (SR) by L-type Ca channels, whose activity is strongly influenced by action potential (AP) profile. The contribution of Ca²⁺ entry via the Na⁺/Ca²⁺ exchanger (NCX) to trigger SR Ca²⁺ release during ECC in response to an AP remains uncertain. To isolate the contribution of NCX to SR Ca²⁺ release, independent of effects on SR Ca²⁺ load, Ca²⁺ release was determined by recording Ca²⁺ spikes using confocal microscopy on patch-clamped rat ventricular myocytes with [Ca²⁺]_i fixed at 150 nmol/L. In response to AP clamps, normalized Ca²⁺ spike amplitudes (ΔF/F ₀) increased sigmoidally and doubled as [Na⁺]_i was elevated from 0 to 20 mmol/L with an EC₅₀ of ~10 mmol/L. This [Na⁺]_i-dependence was independent of I _Na as well as SR Ca²⁺ load, which was unchanged under our experimental conditions. However, NCX inhibition using either KB-R7943 or XIP reduced ΔF/F ₀ amplitude in myocytes with 20 mmol/L [Na⁺]_i, but not with 5 mmol/L [Na⁺]_i. SR Ca²⁺ release was complete before the membrane repolarized to −15 mV, indicating Ca²⁺ entry into the dyad (not reduced extrusion) underlies [Na⁺]_i-dependent enhancement of ECC. Because I _Ca,L inhibition with 50 mmol/L Cd²⁺ abolished Ca²⁺ spikes, our results demonstrate that during cardiac APs, NCX enhances SR Ca²⁺ release by synergistically increasing the efficiency of I _Ca,L-mediated ECC.     

Histidine-rich calcium binding protein: The new regulator of sarcoplasmic reticulum calcium cycling

The histidine-rich calcium binding protein (HRC) is a novel regulator of sarcoplasmic reticulum (SR) Ca²⁺-uptake, storage and release. Residing in the SR lumen, HRC binds Ca²⁺ with high capacity but low affinity. In vitro phosphorylation of HRC affects ryanodine affinity of the ryanodine receptor (RyR), suggesting a functional role of HRC on SR Ca²⁺-release. Indeed, acute HRC overexpression in isolated rodent cardiomyocytes decreases Ca²⁺-induced Ca²⁺-release, increases SR Ca²⁺-load, and impairs contractility. The HRC effects on RyR may be regulated by the Ca²⁺-sensitivity of its interaction with triadin. However, HRC also affects the SR Ca²⁺-ATPase, as shown by HRC overexpression in transgenic mouse hearts, which resulted in reduced SR Ca²⁺-uptake rates, cardiac remodeling and hypertrophy. In fact, in vitro generated evidence suggests that HRC directly interacts with SR Ca²⁺-ATPase2, supporting a dual role of HRC in Ca²⁺-homeostasis: regulation of both SR Ca²⁺-uptake and Ca²⁺-release. Furthermore, HRC plays an important role in myocyte differentiation and in antiapoptotic cardioprotection against ischemia/reperfusion induced cardiac injury. Interestingly, HRC has been linked with familiar cardiac conduction disease and an HRC polymorphism was shown to associate with malignant ventricular arrhythmias in the background of idiopathic dilated cardiomyopathy. This review summarizes studies, which have established the critical role of HRC in Ca²⁺-homeostasis, suggesting its importance in cardiac physiology and pathophysiology.     

Modification of Cardiac Subcellular Remodeling Due to Pressure Overload by Captopril and Losartan

In view of the activation of renin-angiotensin system under conditions associated with pressure overload on the heart, we examined the effects of captopril, an angiotensin converting enzyme inhibitor, and losartan, an angiotensin II receptor antagonist, on cardiac function, myofibrillar ATPase and sarcoplasmic reticular (SR) Ca²⁺-pump (SERCA2) activities, as well as myosin and SERCA2 gene expression in hypertrophied hearts. Cardiac hypertrophy was induced in rats treated with or without captopril or losartan by banding the abdominal aorta for 8 weeks; sham operated animals served as control. Decrease in left ventricular developed pressure, +dP/dt and -dP/dt as well as increase in left ventricular end diastolic pressure and increased muscle mass due to pressure overload were prevented by captopril or losartan. Treatment of animals with captopril or Icsartan also attenuated the pressure overload-induced depression in myofibrillar Ca₂₊-stimulated ATPase, myosin ATPase, SR Ca₂₊-uptake and SR Ca²⁺-release activities. An increase in β-myosm heavy chain mRNA and a decrease in α-myosin heavy chain mRNA as well as depressed SERCA2 protein and SERCA2 mRNA levels were prevented by captopril or losartan. These results suggest that both captopril and losartan improve myocardial function in cardiac hypertrophy by preventing changes in gene expression and subsequent subcellular remodeling due to pressure overload.     

Ablation of triadin causes loss of cardiac Ca2+ release units,impaired excitation–contraction coupling,and cardiac arrhythmias

Heart muscle excitation–contraction (E-C) coupling is governed by Ca²⁺ release units (CRUs) whereby Ca²⁺ influx via L-type Ca²⁺ channels (Cav1.2) triggers Ca²⁺ release from juxtaposed Ca²⁺ release channels (RyR2) located in junctional sarcoplasmic reticulum (jSR). Although studies suggest that the jSR protein triadin anchors cardiac calsequestrin (Casq2) to RyR2, its contribution to E-C coupling remains unclear. Here, we identify the role of triadin using mice with ablation of the Trdn gene (Trdn^−/−). The structure and protein composition of the cardiac CRU is significantly altered in Trdn^−/− hearts. jSR proteins (RyR2, Casq2, junctin, and junctophilin 1 and 2) are significantly reduced in Trdn^−/− hearts, whereas Cav1.2 and SERCA2a remain unchanged. Electron microscopy shows fragmentation and an overall 50% reduction in the contacts between jSR and T-tubules. Immunolabeling experiments show reduced colocalization of Cav1.2 with RyR2 and substantial Casq2 labeling outside of the jSR in Trdn^−/− myocytes. CRU function is impaired in Trdn^−/− myocytes, with reduced SR Ca²⁺ release and impaired negative feedback of SR Ca²⁺ release on Cav1.2 Ca²⁺ currents (I_Ca). Uninhibited Ca²⁺ influx via I_Ca likely contributes to Ca²⁺ overload and results in spontaneous SR Ca²⁺ releases upon β-adrenergic receptor stimulation with isoproterenol in Trdn^−/− myocytes, and ventricular arrhythmias in Trdn^−/− mice. We conclude that triadin is critically important for maintaining the structural and functional integrity of the cardiac CRU; triadin loss and the resulting alterations in CRU structure and protein composition impairs E-C coupling and renders hearts susceptible to ventricular arrhythmias.     

Calcium signaling regulates ventricular hypertrophy during development independent of contraction or blood flow

In utero interventions aimed at restoring left ventricular hemodynamic forces in fetuses with prenatally diagnosed hypoplastic left heart syndrome failed to stimulate ventricular myocardial growth during gestation, suggesting chamber growth during development may not rely upon fluid forces. We therefore hypothesized that ventricular hypertrophy during development may depend upon fundamental Ca^2 +-dependent growth pathways that function independent of hemodynamic forces. To test this hypothesis, zebrafish embryos were treated with inhibitors or activators of Ca^2 + signaling in the presence or absence of contraction during the period of chamber development. Abolishment of contractile function alone in the setting of preserved Ca^2 + signaling did not impair ventricular hypertrophy. In contrast, inhibition of L-type voltage-gated Ca^2 + influx abolished contraction and led to reduced ventricular hypertrophy, whereas increasing L-type voltage-gated Ca^2 + influx led to enhanced ventricular hypertrophy in either the presence or absence of contraction. Similarly, inhibition of the downstream Ca^2 +-sensitive phosphatase calcineurin, a known regulator of adult cardiac hypertrophy, led to reduced ventricular hypertrophy in the presence or absence of contraction, whereas hypertrophy was rescued in the absence of L-type voltage-gated Ca^2 + influx and contraction by expression of a constitutively active calcineurin. These data suggest that ventricular cardiomyocyte hypertrophy during chamber formation is dependent upon Ca^2 + signaling pathways that are unaffected by heart function or hemodynamic forces. Disruption of Ca^2 +-dependent hypertrophy during heart development may therefore represent one mechanism for impaired chamber formation that is not related to impaired blood flow.     

Vasostatin-1 Stops Structural Remodeling and Improves Calcium Handling via the eNOS-NO-PKG Pathway in Rat Hearts Subjected to Chronic β-Adrenergic Receptor Activation

Purpose

Chronically elevated catecholamine levels activate cardiac β-adrenergic receptors, which play a vital role in the pathogenesis of heart failure. Evidence suggests that vasostatin-1 (VS-1) exerts anti-adrenergic effects on isolated and perfused hearts in vitro. Whether VS-1 ameliorates hypertrophy/remodeling by inducing the chronic activation of β-adrenergic receptors is unknown. The present study aims to test the efficacy of using VS-1 to treat the advanced hypertrophy/remodeling that result from chronic β-adrenergic receptor activation and to determine the cellular and molecular mechanisms that underlie this response.

Methods and Result

Rats were subjected to infusion with either isoprenaline (ISO, 5 mg/kg/d), ISO plus VS-1 (30 mg/kg/d) or placebo for 2 weeks. VS-1 suppressed chamber dilation, myocyte hypertrophy and fibrosis and improved in vivo heart function in the rats subjected to ISO infusion. VS-1 increased phosphorylated nitric oxide synthase levels and induced the activation of protein kinase G. VS-1 also deactivated multiple hypertrophy signaling pathways that were triggered by the chronic activation of β-adrenergic receptors, such as the phosphoinositide-3 kinase (PI3K)/Akt and Ca²⁺/calmodulin-dependent kinase (CaMK-II) pathways. Myocytes isolated from ISO + VS-1 hearts displayed higher Ca²⁺ transients, shorter Ca²⁺ decays, higher sarcoplasmic reticulum Ca²⁺ levels and higher L-type Ca²⁺ current densities than the ISO rat hearts. VS-1 treatment restored the protein expression of sarcoplasmic reticulum Ca²⁺ uptake ATPase, phospholamban and Cav1.2, indicating improved calcium handling.

Conclusions

Chronic VS-1 treatment inhibited the progression of hypertrophy, fibrosis, and chamber remodeling, and improved cardiac function in a rat model of ISO infusion. In addition, Ca²⁺ handling and its molecular modulation were also improved by VS-1. The beneficial effects of VS-1 on cardiac remodeling may be mediated by the enhanced activation of the eNOS-cGMP-PKG pathway.

     

Calcium-calmodulin dependent protein kinase II (CaMKII): a main signal responsible for early reperfusion arrhythmias

To explore whether CaMKII-dependent phosphorylation events mediate reperfusion arrhythmias, Langendorff perfused hearts were submitted to global ischemia/reperfusion. Epicardial monophasic or transmembrane action potentials and contractility were recorded. In rat hearts, reperfusion significantly increased the number of premature beats (PBs) relative to pre-ischemic values. This arrhythmic pattern was associated with a significant increase in CaMKII-dependent phosphorylation of Ser2814 on Ca²⁺-release channels (RyR2) and Thr17 on phospholamban (PLN) at the sarcoplasmic reticulum (SR). These phenomena could be prevented by the CaMKII-inhibitor KN-93. In transgenic mice with targeted inhibition of CaMKII at the SR membranes (SR-AIP), PBs were significantly decreased from 31 ± 6 to 5 ± 1 beats/3 min with a virtually complete disappearance of early-afterdepolarizations (EADs). In mice with genetic mutation of the CaMKII phosphorylation site on RyR2 (RyR2-S2814A), PBs decreased by 51.0 ± 14.7%. In contrast, the number of PBs upon reperfusion did not change in transgenic mice with ablation of both PLN phosphorylation sites (PLN-DM). The experiments in SR-AIP mice, in which the CaMKII inhibitor peptide is anchored in the SR membrane but also inhibits CaMKII regulation of L-type Ca²⁺ channels, indicated a critical role of CaMKII-dependent phosphorylation of SR proteins and/or L-type Ca²⁺ channels in reperfusion arrhythmias. The experiments in RyR2-S2814A further indicate that up to 60% of PBs related to CaMKII are dependent on the phosphorylation of RyR2-Ser2814 site and could be ascribed to delayed-afterdepolarizations (DADs). Moreover, phosphorylation of PLN-Thr17 and L-type Ca²⁺ channels might contribute to reperfusion-induced PBs, by increasing SR Ca²⁺ content and Ca²⁺ influx.     

KChIP2 attenuates cardiac hypertrophy through regulation of Ito and intracellular calcium signaling

Recent evidence shows that the auxiliary subunit KChIP2, which assembles with pore-forming Kv4-subunits, represents a new potential regulator of the cardiac calcium-independent transient outward potassium current (I_to) density. In hypertrophy and heart failure, KChIP2 expression has been found to be significantly decreased. Our aim was to examine the role of KChIP2 in cardiac hypertrophy and the effect of restoring its expression on electrical remodeling and cardiac mechanical function using a combination of molecular, biochemical and gene targeting approaches. KChIP2 overexpression through gene transfer of Ad.KChIP2 in neonatal cardiomyocytes resulted in a significant increase in I_to-channel forming Kv4.2 and Kv4.3 protein levels. In vivo gene transfer of KChIP2 in aortic banded adult rats showed that, compared to sham-operated or Ad.β-gal-transduced hearts, KChIP2 significantly attenuated the developed left ventricular hypertrophy, robustly increased I_to densities, shortened action potential duration, and significantly altered myocyte mechanics by shortening contraction amplitudes and maximal rates of contraction and relaxation velocities and decreasing Ca²⁺ transients. Interestingly, blocking I_to with 4-aminopyridine in KChIP2-overexpressing adult cardiomyocytes significantly increased the Ca²⁺ transients to control levels. One-day-old rat pups intracardially transduced with KChIP2 for two months then subjected to aortic banding for 6–8 weeks (to induce hypertrophy) showed similar echocardiographic, electrical and mechanical remodeling parameters. In addition, in cultured adult cardiomyocytes, KChIP2 overexpression increased the expression of Ca²⁺-ATPase (SERCA2a) and sodium calcium exchanger but had no effect on ryanodine receptor 2 or phospholamban expression. In neonatal myocytes, KChIP2 notably reversed Ang II-induced hypertrophic changes in protein synthesis and MAP-kinase activation. It also significantly decreased calcineurin expression, NFATc1 expression and nuclear translocation and its downstream target, MCiP1.4. Altogether, these data show that KChIP2 can attenuate cardiac hypertrophy possibly through modulation of intracellular calcium concentration and calcineurin/NFAT pathway.     

Ageing-related cardiomyocyte functional decline is sex and angiotensin II dependent

Clinically, heart failure is an age-dependent pathological phenomenon and displays sex-specific characteristics. The renin-angiotensin system mediates cardiac pathology in heart failure. This study investigated the sexually dimorphic functional effects of ageing combined with angiotensin II (AngII) on cardiac muscle cell function, twitch and Ca²⁺-handling characteristics of isolated cardiomyocytes from young (~13 weeks) and aged (~87 weeks) adult wild type (WT) and AngII-transgenic (TG) mice. We hypothesised that AngII-induced contractile impairment would be exacerbated in aged female cardiomyocytes and linked to Ca²⁺-handling disturbances. AngII-induced cardiomyocyte hypertrophy was evident in young adult mice of both sexes and accentuated by age (aged adult ~21–23 % increases in cell length relative to WT). In female AngII-TG mice, ageing was associated with suppressed cardiomyocyte contractility (% shortening, maximum rate of shortening, maximum rate of relaxation). This was associated with delayed cytosolic Ca²⁺ removal during twitch relaxation (Tau ~20 % increase relative to young adult female WT), and myofilament responsiveness to Ca²⁺ was maintained. In contrast, aged AngII-TG male cardiomyocytes exhibited peak shortening equivalent to young TG; yet, myofilament Ca²⁺ responsiveness was profoundly reduced with ageing. Increased pro-arrhythmogenic spontaneous activity was evident with age and cardiac AngII overexpression in male mice (42–55 % of myocytes) but relatively suppressed in female aged transgenic mice. Female myocytes with elevated AngII appear more susceptible to an age-related contractile deficit, whereas male AngII-TG myocytes preserve contractile function with age but exhibit desensitisation of myofilaments to Ca²⁺ and a heightened vulnerability to arrhythmic activity. These findings support the contention that sex-specific therapies are required for the treatment of age-progressive heart failure.     

Intracellular mechanisms of specific beta-adrenoceptor antagonists involved in improved cardiac function and survival in a genetic model of heart failure

β-blockers, as class, improve cardiac function and survival in heart failure (HF). However, the molecular mechanisms underlying these beneficial effects remain elusive. In the present study, metoprolol and carvedilol were used in doses that display comparable heart rate reduction to assess their beneficial effects in a genetic model of sympathetic hyperactivity-induced HF (α_2A/α_2C-ARKO mice). Five month-old HF mice were randomly assigned to receive either saline, metoprolol or carvedilol for 8 weeks and age-matched wild-type mice (WT) were used as controls. HF mice displayed baseline tachycardia, systolic dysfunction evaluated by echocardiography, 50% mortality rate, increased cardiac myocyte width (50%) and ventricular fibrosis (3-fold) compared with WT. All these responses were significantly improved by both treatments. Cardiomyocytes from HF mice showed reduced peak [Ca²⁺]_i transient (13%) using confocal microscopy imaging. Interestingly, while metoprolol improved [Ca²⁺]_i transient, carvedilol had no effect on peak [Ca²⁺]_i transient but also increased [Ca²⁺] transient decay dynamics. We then examined the influence of carvedilol in cardiac oxidative stress as an alternative target to explain its beneficial effects. Indeed, HF mice showed 10-fold decrease in cardiac reduced/oxidized glutathione ratio compared with WT, which was significantly improved only by carvedilol treatment. Taken together, we provide direct evidence that the beneficial effects of metoprolol were mainly associated with improved cardiac Ca²⁺ transients and the net balance of cardiac Ca²⁺ handling proteins while carvedilol preferentially improved cardiac redox state.     

L-type calcium currents in atrial myocytes from patients with persistent and non-persistent atrial fibrillation

Objective. In patients with persistent atrial fibrillation (AF), the atrial myocardium is characterized by a reduced contractile force, by a shortened duration of the action potential and a recently demonstrated reduction of the L-type Ca²⁺ currents. We analyzed potential effects on L-type Ca²⁺ currents of the patients' medication and of the duration of AF. Methods and results. Human atrial myocytes were prepared from the right auricles of patients undergoing open-heart surgery. Three groups of patients were studied: a control group with sinus rhythm (SR, n = 26 patients) and a group with persistent AF (> 3 months duration; n = 10), a group with non-persistent AF (3 patients with SR but with documented episodes of AF in their history). L-type Ca²⁺ currents were measured during depolarizing pulses from a holding potential of −70mV to a test potential of +10mV and are given as mean ±SEM of current densities (currents normalized to the cell capacitance). Ca²⁺ current densities were significantly (p < 0.0001) smaller in cells from patients with persistent AF than in control cells (0.54 ± 0.08 pA/pF vs. 1.96 ± 0.12 pA/pF). No indication was found that these changes were caused by medication with Ca²⁺ channel antagonists, β blockers, or digitalis. Stimulation with the dihydropyridine Bay K 8644 (1 μM) or with isoproterenol (0.1 μM) increased Ca²⁺ currents in control cells 3.5 ± 0.2 and 3.5 ± 0.3-fold. In persistent AF, this increase was significantly larger (6.0 ± 0.5 and 5.2 ± 0.6-fold) but stimulated currents were still significantly lower than in control cells. Patients with non-persistent AF exhibited Ca²⁺ currents well within the control range. Conclusion. A reduction in Ca²⁺ currents, due to a reduction in number as well as a depression of L-type channels, is a characteristic and pathophysiologically important part of the myocardial remodeling during long-lasting atrial fibrillation. It is not present in patients with non-persistent AF and not caused by medication. Received: 27 September 2000, Returned for revision: 9 October 2000, Revision received: 8 November 2000, Accepted: 9 November 2000     

Neonatal gene transfer of Serca2a delays onset of hypertrophic remodeling and improves function in familial hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant genetic disorder linked to numerous mutations in the sarcomeric proteins. The clinical presentation of FHC is highly variable, but it is a major cause of sudden cardiac death in young adults with no specific treatments. We tested the hypothesis that early intervention in Ca²⁺ regulation may prevent pathological hypertrophy and improve cardiac function in a FHC displaying increased myofilament sensitivity to Ca²⁺ and diastolic dysfunction. A transgenic (TG) mouse model of FHC with a mutation in tropomyosin at position 180 was employed. Adenoviral-Serca2a (Ad.Ser) was injected into the left ventricle of 1-day-old non-transgenic (NTG) and TG mice. Ad.LacZ was injected as a control. Serca2a protein expression was significantly increased in NTG and TG hearts injected with Ad.Ser for up to 6 weeks. Compared to TG-Ad.LacZ hearts, the TG-Ad.Ser hearts showed improved whole heart morphology. Moreover, there was a significant decline in ANF and β-MHC expression. Developed force in isolated papillary muscle from 2- to 3-week-old TG-Ad.Ser hearts was higher and the response to isoproterenol (ISO) improved compared to TG-Ad.LacZ muscles. In situ hemodynamic measurements showed that by 3 months the TG-Ad.Ser hearts also had a significantly improved response to ISO compared to TG-Ad.LacZ hearts. The present study strongly suggests that Serca2a expression should be considered as a potential target for gene therapy in FHC. Moreover, our data imply that development of FHC can be successfully delayed if therapies are started shortly after birth.     

Leaky RyR2 trigger ventricular arrhythmias in Duchenne muscular dystrophy

Patients with Duchenne muscular dystrophy (DMD) have a progressive dilated cardiomyopathy associated with fatal cardiac arrhythmias. Electrical and functional abnormalities have been attributed to cardiac fibrosis; however, electrical abnormalities may occur in the absence of overt cardiac histopathology. Here we show that structural and functional remodeling of the cardiac sarcoplasmic reticulum (SR) Ca²⁺ release channel/ryanodine receptor (RyR2) occurs in the mdx mouse model of DMD. RyR2 from mdx hearts were S-nitrosylated and depleted of calstabin2 (FKBP12.6), resulting in “leaky” RyR2 channels and a diastolic SR Ca²⁺ leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the Ca²⁺ channel stabilizer S107 (“rycal”) inhibited the SR Ca²⁺ leak, inhibited aberrant depolarization in isolated cardiomyocytes, and prevented arrhythmias in vivo. This suggests that diastolic SR Ca²⁺ leak via RyR2 due to S-nitrosylation of the channel and calstabin2 depletion from the channel complex likely triggers cardiac arrhythmias. Normalization of the RyR2-mediated diastolic SR Ca²⁺ leak prevents fatal sudden cardiac arrhythmias in DMD.     

Neonatal gene transfer of Serca2a delays onset of hypertrophic remodeling and improves function in familial hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant genetic disorder linked to numerous mutations in the sarcomeric proteins. The clinical presentation of FHC is highly variable, but it is a major cause of sudden cardiac death in young adults with no specific treatments. We tested the hypothesis that early intervention in Ca²⁺ regulation may prevent pathological hypertrophy and improve cardiac function in a FHC displaying increased myofilament sensitivity to Ca²⁺ and diastolic dysfunction. A transgenic (TG) mouse model of FHC with a mutation in tropomyosin at position 180 was employed. Adenoviral-Serca2a (Ad.Ser) was injected into the left ventricle of 1-day-old non-transgenic (NTG) and TG mice. Ad.LacZ was injected as a control. Serca2a protein expression was significantly increased in NTG and TG hearts injected with Ad.Ser for up to 6 weeks. Compared to TG-Ad.LacZ hearts, the TG-Ad.Ser hearts showed improved whole heart morphology. Moreover, there was a significant decline in ANF and β-MHC expression. Developed force in isolated papillary muscle from 2- to 3-week-old TG-Ad.Ser hearts was higher and the response to isoproterenol (ISO) improved compared to TG-Ad.LacZ muscles. In situ hemodynamic measurements showed that by 3 months the TG-Ad.Ser hearts also had a significantly improved response to ISO compared to TG-Ad.LacZ hearts. The present study strongly suggests that Serca2a expression should be considered as a potential target for gene therapy in FHC. Moreover, our data imply that development of FHC can be successfully delayed if therapies are started shortly after birth.