Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal inherited arrhythmia syndrome in which drug therapy is often ineffective. We discovered that flecainide prevents arrhythmias in a mouse model of CPVT by inhibiting cardiac ryanodine receptor-mediated Ca 2+ release and thereby directly targeting the underlying molecular defect. Flecainide completely prevented CPVT in two human subjects who had remained highly symptomatic on conventional drug therapy, indicating that this currently available drug is a promising mechanism-based therapy for CPVT.CPVT is an inherited arrhythmia syndrome characterized by a normal baseline electrocardiogram (ECG), polymorphic ventricular tachycardia induced by adrenergic stress in the absence of structural heart disease, and a high mortality rate in young individuals1. Treatment with β-adrenergic blockers reduces arrhythmia burden and mortality but is not completely effective1, and implantable cardioverter defibrillators (ICDs) are used for the prevention of sudden death. However, painful appropriate or inappropriate defibrillation shocks can trigger further adrenergic stress and arrhythmias, and deaths have occurred despite appropriate ICD shocks2 , 3. In such instances, stellate ganglionectomy4 or even cardiac transplantation5 have been considered. Two CPVT disease-related genes have been identified: RYR2, encoding the cardiac ryanodine receptor Ca 2+ release channel (RyR2), and CASQ2, encoding cardiac calsequestrin 6,7 . Mutations in these genes destabilize the RyR2 Ca 2+ release complex 8,9 . In mice with CPVT-linked mutations, catecholamines cause spontaneous sarcoplasmic reticulum Ca 2+ release resulting in delayed after depolarizations (DADs), and they produce triggered activity in myocytes and polymorphic ventricular tachycardia in ©2009 Nature America, Inc. All rights reserved NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript vivo 10, 11. Here we identify a therapy that directly targets the underlying arrhythmia mechanisms: we found that flecainide, an approved antiarrhythmic drug known to block sodium channels, showed remarkable efficacy in suppressing spontaneous sarcoplasmic reticulum Ca 2+ release by inhibiting RyR2. Flecainide treatment completely prevented adrenergic stress-induced arrhythmias in a mouse model of CPVT and in humans with CASQ2 or RYR2 mutations that are refractory to standard drug treatment.The local anesthetic tetracaine has been used to inhibit RyR2 and suppress spontaneous sarcoplasmic reticulum Ca 2+ release in isolated myocytes 12 . However, tetracaine causes a rebound increase in sarcoplasmic reticulum Ca 2+ release events during prolonged exposure 13 , effective inhibitory concentrations 14 are too high for clinical use, and systemic administration is contraindicated in humans. We searched among clinically available antiarrhythmic drugs for a more useful RyR2 inhibitor and found that flecainide inhibited RyR2 more potently than tetracaine and by a different mechanism. Whereas tetrac...

Carvedilol is one of the most effective beta-blockers for preventing ventricular tachyarrhythmias (VTs) in heart failure (HF), but the mechanisms underlying its favorable anti-arrhythmic benefits remain unclear. Spontaneous Ca2+ waves, also termed store-overload-induced Ca2+ release (SOICR), are known to evoke VTs in patients with HF. Here we show that carvedilol is the only beta-blocker that effectively suppresses SOICR by directly reducing the open duration of the cardiac ryanodine receptor (RyR2). This unique anti-SOICR activity of carvedilol combined with its beta-blocking activity likely contributes to its favorable anti-arrhythmic effect. To allow individual and optimal titration of these beneficial activities, we developed a novel SOICR-inhibiting, minimally-beta-blocking carvedilol analogue VK-II-86. We found that VK-II-86 alone prevented stress-induced VTs in RyR2 mutant mice, and was more effective when combined with a selective beta-blocker metoprolol or bisoprolol. Thus, SOICR inhibition combined with optimal beta-blockade presents a new, promising and potentially patient-tailorable anti-arrhythmic approach.

Impaired HRT, abnormal TWA, and an EF <0.50 beyond 8 weeks after MI reliably identify patients at risk of serious events. (Assessment of Noninvasive Methods to Identify Patients at Risk of Serious Arrhythmias After a Heart Attack; http://www.clinicaltrials.gov/ct/show/NCT00399503?order=1; NCT00399503).

Tubulointerstitial inflammation and fibrosis are strongly associated with the outcome of chronic kidney disease. We recently demonstrated that the NOD-like receptor, pyrin domain containing-3 (NLRP3) contributes to renal inflammation, injury, and fibrosis following unilateral ureteric obstruction in mice. NLRP3 expression in renal tubular epithelial cells (TECs) was found to be an important component of experimental disease pathogenesis, although the biology of NLRP3 in epithelial cells is unknown. In human and mouse primary renal TECs, NLRP3 expression was increased in response to TGF-β1 stimulation and associated with epithelial–mesenchymal transition (EMT) and the expression of α-smooth muscle actin (αSMA) and matrix metalloproteinase (MMP) 9. TGF-β1–induced EMT and the induction of MMP-9 and αSMA were significantly decreased in mouse Nlrp3−/− renal TECs, suggesting a role for Nlrp3 in TGF-β–dependent signaling. Although apoptosis-associated speck-like protein containing a CARD domain−/− TECs demonstrated a phenotype similar to that of Nlrp3−/− cells in response to TGF-β1, the effect of Nlrp3 on MMP-9 and αSMA expression was inflammasome independent, as IL-1β, IL-18, MyD88, and caspase-1 were dispensable. Smad2 and Smad3 phosphorylation in response to TGF-β1 was attenuated in Nlrp3−/− and apoptosis-associated speck-like protein containing a CARD domain−/− cells, accounting for the dampened EMT and TGF-β1 responsiveness in these cells. Consistent with these findings, overexpression of NLRP3 in 293T cells resulted in increased Smad3 phosphorylation and activity. Taken together, these data support a novel and direct role for NLRP3 in promoting TGF-β signaling and R-Smad activation in epithelial cells independent of the inflammasome.

Caffeine has long been used as a pharmacological probe for studying ryanodine receptor (RyR)-mediated Ca2+ release and cardiac arrhythmias. However, the precise mechanism by which caffeine activates RyRs is elusive. Here we investigated the effects of caffeine on spontaneous Ca2+ release and on the response of single cardiac RyR (RyR2) channels to luminal or cytosolic Ca2+. We found that HEK293 cells expressing RyR2 displayed partial or “quantal” Ca2+ release in response to repetitive additions of submaximal concentrations of caffeine. This quantal Ca2+ release was abolished by ryanodine. Monitoring of endoplasmic reticulum luminal Ca2+ revealed that caffeine reduced the luminal Ca2+ threshold at which spontaneous Ca2+ release occurs. Interestingly, spontaneous Ca2+ release in the form of Ca2+ oscillations persisted in the presence of 10 mM caffeine, and was diminished by ryanodine, demonstrating that unlike ryanodine, caffeine, even at high concentrations, does not hold the channel open. At the single channel level, caffeine markedly reduced the threshold for luminal Ca2+ activation, but had little effect on the threshold for cytosolic Ca2+ activation, indicating that the major action of caffeine is to reduce the luminal, but not the cytosolic, Ca2+ activation threshold. Furthermore, as with caffeine, the clinically relevant, pro-arrhythmic methylxanthines aminophylline and theophylline potentiated luminal Ca2+ activation of RyR2, and increased the propensity for spontaneous Ca2+ release, mimicking the effects of diseased-linked RyR2 mutations. Collectively, our results demonstrate that caffeine triggers Ca2+ release by reducing the threshold for luminal Ca2+ activation of RyR2, and suggest that disease-linked RyR2 mutations and RyR2-interacting pro-arrhythmic agents may share the same arrhythmogenic mechanism.

New findings r What is the central question of this study?Heart failure is associated with persistent sterile inflammation that worsens disease severity; however, the molecular mechanisms behind cytokine recruitment and their relevance in the diseased myocardium remain unknown. r What is the main finding and its importance?We show that interleukin-1β is activated downstream of the Nlrp3 inflammasome in calcineurin-transgene-induced structural heart disease. Genetic deletion of Nlrp3 abrogated inflammasome signalling and interleukin-1β release, improving function. The role of Nlrp3 in non-ischaemic cardiomyopathy and the utility of inflammasome antagonism have not yet been explored, revealing potential for translational application.Heart failure is associated with a low-grade and chronic cardiac inflammation that impairs function; however, the mechanisms by which this sterile inflammation occurs in structural heart disease remain poorly defined. Cardiac-specific heterozygous overexpression of the calcineurin transgene (CNTg) in mice results in cardiac hypertrophy, inflammation, apoptosis and ventricular dilatation. We hypothesized that activation of the Nlrp3 inflammasome, an intracellular danger-sensing pathway required for processing the pro-inflammatory cytokine interleukin-1β (IL-1β), may contribute to myocardial dysfunction and disease progression. Here we report that Nlrp3 mRNA was increased in CNTg mice compared with wild-type. Consistent with inflammasome activation, CNTg animals had increased conversion of procaspase-1 to cleaved and activated forms, as well as markedly increased serum IL-1β. Blockade of IL-1β signalling via chronic IL-1 receptor antagonist therapy reduced cardiac inflammation and myocyte pathology in CNTg mice, resulting in improved systolic performance. Furthermore, genetic ablation of Nlrp3 in CNTg mice reduced pro-inflammatory cytokine maturation and cardiac inflammation, as well as improving systolic performance. These findings indicate that activation of the Nlrp3 inflammasome in CNTg mice promotes myocardial inflammation and systolic dysfunction through the production of pro-inflammatory IL-1β. Blockade of IL-1β signalling with the IL-1 receptor antagonist reverses these phenotypes and offers a possible therapeutic approach in the management of heart failure.

Background-Prior reports on patients with ARVC/D focused on individuals with advanced forms of the disease. There are limited data regarding diagnostic performance of various testing modalities in newly identified individuals suspected of having ARVC/D.

The 12.6-kDa FK506-binding protein (FKBP12.6) is considered to be a key regulator of the cardiac ryanodine receptor (RyR2), but its precise role in RyR2 function is complex and controversial. In the present study we investigated the impact of FKBP12.6 removal on the properties of the RyR2 channel and the propensity for release or store overload-induced Ca 2؉ release (SOICR). FK506 increased the amplitude and decreased the frequency of SOICR in HEK293 cells expressing RyR2 with or without FKBP12.6, indicating that the action of FK506 on SOICR is independent of FKBP12.6. As with recombinant RyR2, the conductance and ligand-gating properties of single RyR2 channels from FKBP12.6-null mice were indistinguishable from those of single wild type channels. Moreover, FKBP12.6-null mice did not exhibit enhanced susceptibility to stress-induced ventricular arrhythmias, in contrast to previous reports. Collectively, our results demonstrate that the loss of FKBP12.6 has no significant effect on the conduction and activation of RyR2 or the propensity for spontaneous Ca 2؉ release and stress-induced ventricular arrhythmias.