Early effects of Epac depend on the fine-tuning of the sarcoplasmic reticulum Ca2+ handling in cardiomyocytes.

In cardiac muscle, signaling through cAMP governs many fundamental cellular functions, including contractility, relaxation and automatism. cAMP cascade leads to the activation of the classic protein kinase A but also to the stimulation of the recently discovered exchange protein directly activated by cAMP (Epac). The role of Epac in the regulation of intracellular Ca2+ homeostasis and contractility in cardiac myocytes is still matter of debate. In this study we showed that the selective Epac activator, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate (8-CPT), produced a positive inotropic effect when adult rat cardiac myocytes were stabilized at low [Ca2+]o (0.5mM), no changes at 1mM [Ca2+]o and a negative inotropic effect when [Ca2+]o was increased to 1.8mM. These effects were associated to parallel variations in sarcoplasmic reticulum (SR) Ca2+ content. At all [Ca2+]o studied, 8-CPT induced an increase in Ca2+ spark frequency and enhanced CaMKII autophosphorylation and the CaMKII-dependent phosphorylation of SR proteins: phospholamban (PLN, at Thr17 site) and ryanodine receptor (RyR2, at Ser2814 site). We used transgenic mice lacking PLN CaMKII phosphorylation site (PLN-DM) and knock-in mice with an inactivated CaMKII site S2814 on RyR2 (RyR2-S2814A) to investigate the involvement of these processes in the effects of Epac stimulation. In PLN-DM mice, 8-CPT failed to induce the positive inotropic effect at low [Ca2+]o and RyR2-S2814A mice showed no propensity to arrhythmic events when compared to wild type mice myocytes. We conclude that stimulation of Epac proteins could have either beneficial or deleterious effects depending on the steady-state Ca2+ levels at which the myocyte is functioning, favoring the prevailing mechanism of SR Ca2+ handling (uptake vs. leak) in the different situations.

Cardiac expression of the CREM repressor isoform CREM-IbΔC-X in mice leads to arrhythmogenic alterations in ventricular cardiomyocytes.

Chronic ß-adrenergic stimulation is regarded as a pivotal step in the progression of heart failure which is associated with a high risk for arrhythmia. The cAMP-dependent transcription factors cAMP-responsive element binding protein (CREB) and cAMP-responsive element modulator (CREM) mediate transcriptional regulation in response to ß-adrenergic stimulation and CREM repressor isoforms are induced after stimulation of the ß-adrenoceptor. Here, we investigate whether CREM repressors contribute to the arrhythmogenic remodeling in the heart by analyzing arrhythmogenic alterations in ventricular cardiomyocytes (VCMs) from mice with transgenic expression of the CREM repressor isoform CREM-IbΔC-X (TG). Patch clamp analyses, calcium imaging, immunoblotting and real-time quantitative RT-PCR were conducted to study proarrhythmic alterations in TG VCMs vs. wild-type controls. The percentage of VCMs displaying spontaneous supra-threshold transient-like Ca(2+) releases was increased in TG accompanied by an enhanced transduction rate of sub-threshold Ca(2+) waves into these supra-threshold events. As a likely cause we discovered enhanced NCX-mediated Ca(2+) transport and NCX1 protein level in TG. An increase in I NCX and decrease in I to and its accessory channel subunit KChIP2 was associated with action potential prolongation and an increased proportion of TG VCMs showing early afterdepolarizations. Finally, ventricular extrasystoles were augmented in TG mice underlining the in vivo relevance of our findings. Transgenic expression of CREM-IbΔC-X in mouse VCMs leads to distinct arrhythmogenic alterations. Since CREM repressors are inducible by chronic ß-adrenergic stimulation our results suggest that the inhibition of CRE-dependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease.

Phospholamban ablation rescues the enhanced propensity to arrhythmias of mice with CaMKII-constitutive phosphorylation of RyR2 at site S2814.

KEY POINTS: Mice with Ca(2+) -calmodulin-dependent protein kinase (CaMKII) constitutive pseudo-phosphorylation of the ryanodine receptor RyR2 at Ser2814 (S2814D(+/+) mice) exhibit a higher open probability of RyR2, higher sarcoplasmic reticulum (SR) Ca(2+) leak in diastole and increased propensity to arrhythmias under stress conditions. We generated phospholamban (PLN)-deficient S2814D(+/+) knock-in mice by crossing two colonies, S2814D(+/+) and PLNKO mice, to test the hypothesis that PLN ablation can prevent the propensity to arrhythmias of S2814D(+/+) mice. PLN ablation partially rescues the altered intracellular Ca(2+) dynamics of S2814D(+/+) hearts and myocytes, but enhances SR Ca(2+) sparks and leak on confocal microscopy. PLN ablation diminishes ventricular arrhythmias promoted by CaMKII phosphorylation of S2814 on RyR2. PLN ablation aborts the arrhythmogenic SR Ca(2+) waves of S2814D(+/+) and transforms them into non-propagating events. A mathematical human myocyte model replicates these results and predicts the increase in SR Ca(2+) uptake required to prevent the arrhythmias induced by a CaMKII-dependent leaky RyR2. ABSTRACT: Mice with constitutive pseudo-phosphorylation at Ser2814-RyR2 (S2814D(+/+) ) have increased propensity to arrhythmias under ß-adrenergic stress conditions. Although abnormal Ca(2+) release from the sarcoplasmic reticulum (SR) has been linked to arrhythmogenesis, the role played by SR Ca(2+) uptake remains controversial. We tested the hypothesis that an increase in SR Ca(2+) uptake is able to rescue the increased arrhythmia propensity of S2814D(+/+) mice. We generated phospholamban (PLN)-deficient/S2814D(+/+) knock-in mice by crossing two colonies, S2814D(+/+) and PLNKO mice (SD(+/+) /KO). SD(+/+) /KO myocytes exhibited both increased SR Ca(2+) uptake seen in PLN knock-out (PLNKO) myocytes and diminished SR Ca(2+) load (relative to PLNKO), a characteristic of S2814D(+/+) myocytes. Ventricular arrhythmias evoked by catecholaminergic challenge (caffeine/adrenaline) in S2814D(+/+) mice in vivo or programmed electric stimulation and high extracellular Ca(2+) in S2814D(+) /(-) hearts ex vivo were significantly diminished by PLN ablation. At the myocyte level, PLN ablation converted the arrhythmogenic Ca(2+) waves evoked by high extracellular Ca(2+) provocation in S2814D(+/+) mice into non-propagated Ca(2+) mini-waves on confocal microscopy. Myocyte Ca(2+) waves, typical of S2814D(+/+) mice, could be evoked in SD(+/+) /KO cells by partially inhibiting SERCA2a. A mathematical human myocyte model replicated these results and allowed for predicting the increase in SR Ca(2+) uptake required to prevent the arrhythmias induced by a Ca(2+) -calmodulin-dependent protein kinase (CaMKII)-dependent leaky RyR2. Our results demonstrate that increasing SR Ca(2+) uptake by PLN ablation can prevent the arrhythmic events triggered by SR Ca(2+) leak due to CaMKII-dependent phosphorylation of the RyR2-S2814 site and underscore the benefits of increasing SERCA2a activity on SR Ca(2+) -triggered arrhythmias.

Sarcoplasmic reticulum calcium leak and cardiac arrhythmias.

Ventricular arrhythmias deteriorating into sudden cardiac death are a major cause of mortality worldwide. The recent linkage of a genetic form of cardiac arrhythmia to mutations in the gene encoding RyR2 (ryanodine receptor 2) has uncovered an important role of this SR (sarcoplasmic reticulum) calcium release channel in triggering arrhythmias. Mutant RyR2 channels give rise to spontaneous release of calcium (Ca(2+)) from the SR during diastole, which enhances the probability of ventricular arrhythmias. Several molecular mechanisms have been proposed to explain the gain-of-function phenotype observed in mutant RyR2 channels. Despite considerable differences between the models discussed in the present review, each predicts spontaneous diastolic Ca(2+) leak from the SR due to incomplete closure of the RyR2 channel. Enhanced SR Ca(2+) leak is also observed in common structural diseases of the heart, such as heart failure. In heart failure, defective channel regulation in the absence of inherited mutations may also increase SR Ca(2+) leak and initiate cardiac arrhythmias. Therefore inhibition of diastolic Ca(2+) leak through SR Ca(2+) release channels has emerged as a new and promising therapeutic target for cardiac arrhythmias.

Diseases associated with altered ryanodine receptor activity.

Mutations in two intracellular Ca2+ release channels or ryanodine receptors (RyR1 and RyR2) are associated with a number of human skeletal and cardiac diseases. This chapter discusses these diseases in terms of known mechanisms, controversies, and unanswered questions. We also compare the cardiac and skeletal muscle diseases to explore common mechanisms.

Structural determinants of potassium channel blockade and drug-induced arrhythmias.

Cardiac K+ channels play an important role in the regulation of the shape and duration of the action potential. They have been recognized as targets for the actions of neurotransmitters, hormones, and anti-arrhythmic drugs that prolong the action potential duration (APD) and increase refractoriness. However, pharmacological therapy, often for the purpose of treating syndromes unrelated to cardiac disease, can also increase the vul- nerability of some patients to life-threatening rhythm disturbances. This may be due to an underlying propensity stemming from inherited mutations or polymorphisms, or structural abnormalities that provide a substrate allowing for the initiation of arrhythmic triggers. A number of pharmacological agents that have proved useful in the treatment of allergic reactions, gastrointestinal disorders, and psychotic disorders, among others, have been shown to reduce repolarizing K+ currents and prolong the Q-T interval on the electrocardiogram. Understanding the structural determinants of K+ channel blockade might provide new insights into the mechanism and rate-dependent effects of drugs on cellular physiology. Drug-induced disruption of cellular repolarization underlies electrocardiographic abnormalities that are diagnostic indicators of arrhythmia susceptibility.

Congenital and drug-induced long-QT syndrome: an update.

The congenital long-QT syndrome is a potentially life-threatening condition characterised clinically by prolonged QT intervals, syncope and sudden cardiac death. The abnormally prolonged repolarisation is the result of mutations in genes encoding cardiac ion channels. The diagnosis of long-QT syndrome is based on clinical, electrocardiographic, and genetic criteria. Beta-blocking therapy is important in the treatment of long-QT syndrome, although pacemakers and implantable cardioverter defibrillators (ICD) are useful in certain categories of patients. In the near future, mutation-specific treatment will probably become a novel approach to this potentially lethal syndrome. Drug-induced long-QT syndrome has been associated with silent mutations and common polymorphisms in potassium and sodium channel genes associated with congenital long-QT syndrome. Genetic screening for such mutations and polymorphisms may become an important instrument in preventing drug-induced 'torsades de pointes' arrhythmias in otherwise asymptomatic patients.

Effects of experimental lower-limb ischaemia-reperfusion injury on the mesenteric microcirculation.

BACKGROUND: Ischaemia-reperfusion (I-R) of the leg is associated with functional and structural changes in the intestine. This study assessed whether acute hind-limb I-R in rats induced a reduction in perfusion and/or signs of an inflammatory response in the intestine. METHODS: Rats were subjected to 2 h of unilateral hind-limb ischaemia followed by 2 h of reperfusion (I-R group, n = 9) or to a sham procedure (control group, n = 9). Mesenteric microvascular diameters, red blood cell velocity, blood flow and leucocyte-vessel wall interactions during reperfusion were measured using intravital microscopy. RESULTS: Blood pressure and heart rate decreased from 30 min of reperfusion onwards in the I-R group compared with controls. From 15 min after the start of reperfusion, mesenteric arteriolar and venular red blood cell velocity and blood flow decreased by 40-50 per cent. Microvascular diameters and leucocyte-vessel wall interactions did not change. CONCLUSION: Restoration of blood flow to an acutely ischaemic hind limb led to a significant decline in the splanchnic microcirculatory blood flow. There were, however, no signs of an early inflammatory response in the gut.