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Background: The progression of atrial fibrillation (AF) from paroxysmal to persistent forms remains a major clinical challenge. Abnormal sarcoplasmic reticulum (SR) Ca(2+) leak via the ryanodine receptor type 2 (RyR2) has been observed as a source of ectopic activity in various AF models. However, its potential role in progression to long-lasting spontaneous AF (sAF) has never been tested. This study was designed to test the hypothesis that enhanced RyR2-mediated Ca(2+) release underlies the development of a substrate for sAF and to elucidate the underlying mechanisms., Methods and Results: CREM-IbΔC-X transgenic (CREM) mice developed age-dependent progression from spontaneous atrial ectopy to paroxysmal and eventually long-lasting AF. The development of sAF in CREM mice was preceded by enhanced diastolic Ca(2+) release, atrial enlargement, and marked conduction abnormalities. Genetic inhibition of Ca(2+)/calmodulin-dependent protein kinase II-mediated RyR2-S2814 phosphorylation in CREM mice normalized open probability of RyR2 channels and SR Ca(2+) release, delayed the development of spontaneous atrial ectopy, fully prevented sAF, suppressed atrial dilation, and forestalled atrial conduction abnormalities. Hyperactive RyR2 channels directly stimulated the Ca(2+)-dependent hypertrophic pathway nuclear factor of activated T cell/Rcan1-4, suggesting a role for the nuclear factor of activated T cell/Rcan1-4 system in the development of a substrate for long-lasting AF in CREM mice., Conclusions: RyR2-mediated SR Ca(2+) leak directly underlies the development of a substrate for sAF in CREM mice, the first demonstration of a molecular mechanism underlying AF progression and sAF substrate development in an experimental model. Our work demonstrates that the role of abnormal diastolic Ca(2+) release in AF may not be restricted to the generation of atrial ectopy but extends to the development of atrial remodeling underlying the AF substrate.

Nutrient restriction (NR) prolongs longevity via enhanced mitochondrial function. We tested the hypothesis that NR enhances resistance to ischemia/reperfusion (IR) arrhythmias via preserved calcium (Ca) cycling and mitochondrial function. We examined the protective effects of NR on regional IR in cultured neonatal rat ventricular myocyte monolayers. Optical mapping of intracellular Ca and mitochondrial membrane potential Δψ(m) was performed using Rhod 2-AM and TMRE, respectively. Regional ischemia was mimicked by covering a portion of monolayer with a glass coverslip until loss of Ca propagation, and reperfusion was mimicked by removing the coverslip. NR was mimicked by culture in serum- and glucose-free medium for 24 h. Relative to controls, NR monolayers: (1) sustained Ca oscillations during longer periods of ischemia (19.2 ± 1.8 min vs 10.4 ± 1.4 min, p<0.001); (2) had attenuated increases in Ca transient duration (CaD) and time decay constant (Tau) during ischemia; (3) had preserved conduction velocity (CV) during early reperfusion, leading to protection against reperfusion arrhythmias; (4) had minimal "rebound" decreased CaD and Tau during reperfusion; and (5) had no depolarization of Δψ(m) during IR. NR attenuates IR arrhythmias via (1) stable calcium cycling and (2) prevention of Δψ(m) depolarization during IR. Enhanced mitochondrial resistance to IR arrhythmias may play a role in NR-induced longevity prolongation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A trial fibrillation (AF), the most common human cardiac arrhythmia, is associated with abnormal intracellular Ca2+ handling. Diastolic Ca2+ release from the sarcoplasmic reticulum via "leaky" ryanodine receptors (RyR2s) is hypothesized to contribute to arrhythmogenesis in AF, but the molecular mechanisms are incompletely understood. Here, we have shown that mice with a genetic gain-of-function defect in Ryr2 (which we termed Ryr2R176Q/+ mice) did not exhibit spontaneous AF but that rapid atrial pacing unmasked an increased vulnerability to AF in these mice compared with wild-type mice. Rapid atrial pacing resulted in increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2, while both pharmacologic and genetic inhibition of CaMKII prevented AF inducibility in Ryr2R176Q/+ mice. This result suggests that AF requires both an arrhythmogenic substrate (e.g., RyR2 mutation) and enhanced CaMKII activity. Increased CaMKII phosphorylation of RyR2 was observed in atrial biopsies from mice with atrial enlargement and spontaneous AF, goats with lone AF, and patients with chronic AF. Genetic inhibition of CaMKII phosphorylation of RyR2 in Ryr2S2814A knockin mice reduced AF inducibility in a vagotonic AF model. Together, these findings suggest that increased RyR2-dependent Ca2+ leakage due to enhanced CaMKII activity is an important downstream effect of CaMKII in individuals susceptible to AF induction.

In unexcitable, noncardiac cells, ultrashort (nanosecond) high-voltage (megavolt-per-meter) pulsed electrical fields (nsPEF) can mobilize intracellular Ca2+ and create transient nanopores in the plasmalemma. We studied Ca2+ responses to nsPEF in cardiac cells. Fluorescent Ca2+ or voltage signals were recorded from isolated adult rat ventricular myocytes deposited in an electrode microchamber and stimulated with conventional pulses (CPs; 0.5-2.4 kV/cm, 1 ms) or nsPEF (10-80 kV/cm, 4 ns). nsPEF induced Ca2+ transients in 68/104 cells. Repeating nsPEF increased the likelihood of Ca2+ transient induction (61.8% for <10 nsPEF vs. 80.6% for > or =10 nsPEF). Repetitive Ca2+ waves arising at the anodal side and Ca2+ destabilization occurred after repeated nsPEF (12/29) or during steady-state single nsPEF delivery at 2 Hz. Removing extracellular Ca2+ abolished responses to nsPEF. Verapamil did not affect nsPEF-induced Ca2+ transients, but decreased responses to CP. Tetrodotoxin and KB-R7943 increased the repetition threshold in response to nsPEF: 1-20 nsPEF caused local anodal Ca2+ waves without Ca2+ transients, and > or =20 nsPEF caused normal transients. Ryanodine-thapsigargin and caffeine protected against nsPEF-induced Ca2+ waves and showed less recovery of diastolic Ca2+ levels than CP. Voltage recordings demonstrated action potentials triggered by nsPEF, even in the presence of tetrodotoxin. nsPEF can mobilize intracellular Ca2+ in cardiac myocytes by inducing action potentials. Anodal Ca2+ waves and resistance to Na+ and Ca2+ channel blockade suggest nonselective ion channel transport via sarcolemmal nanopores as a triggering mechanism.

Background: intracellular Na+ accumulation during ischemia and reperfusion leads to cytosolic Ca2+ overload through reverse-mode operation of the sarcolemmal Na+ -Ca2+ exchanger. Cytosolic Ca2+ accumulation promotes mitochondrial Ca2+ (Ca2+ m) overload, leading to mitochondrial injury. We investigated whether limiting sarcolemmal Na+ entry during resuscitation from ventricular fibrillation (VF) attenuates Ca2+ m overload and lessens myocardial dysfunction in a rat model of VF and closed-chest resuscitation., Methods: hearts were harvested from 10 groups of 6 rats each representing baseline, 15 min of untreated VF, 15 min of VF with chest compression given for the last 5 min (VF/CC), and 60 min postresuscitation (PR). VF/CC and PR included four groups each randomized to receive before starting chest compression the new NHE-1 inhibitor AVE4454B (1.0 mg/kg), the Na+ channel blocker lidocaine (5.0 mg/kg), their combination, or vehicle control. The left ventricle was processed for intracellular Na+ and Ca2+ m measurements., Results: limiting sarcolemmal Na+ entry attenuated cytosolic Na+ increase during VF/CC and the PR phase and prevented Ca2+ m overload yielding levels that corresponded to 77% and 71% of control hearts at VF/CC and PR, without differences among specific Na+ -limiting interventions. Limiting sarcolemmal Na+ entry attenuated reductions in left ventricular compliance during VF and prompted higher mean aortic pressure (110 +/- 7 vs. 95 +/- 11 mmHg, P < 0.001) and higher cardiac work index (159 +/- 34 vs. 126 +/- 29 g x m x min(-1) x kg(-1), P < 0.05) with lesser increases in circulating cardiac troponin I at 60 min PR., Conclusions: Na+ -limiting interventions prevented excess Ca2+ m accumulation induced by ischemia and reperfusion and ameliorated myocardial injury and dysfunction.

Background: Mutations in the cardiac ryanodine receptor gene (RyR2) have been recently identified in victims of sudden infant death syndrome. The aim of this study was to determine whether a gain-of-function mutation in RyR2 increases the propensity to cardiac arrhythmias and sudden death in young mice.Methods and Results: Incidence of sudden death was monitored prospectively in heterozygous knock-in mice with mutation R176Q in RyR2 (R176Q/+). Young R176Q/+ mice exhibited a higher incidence of sudden death compared with wild-type littermates. Optical mapping of membrane potentials and intracellular calcium in 1- to 7-day-old R176Q/+ and wild-type mice revealed an increased incidence of ventricular ectopy and spontaneous calcium releases in neonatal R176Q/+ mice. Surface ECGs in 3- to 10-day-old mice showed that R176Q/+ mice developed more ventricular arrhythmias after provocation with epinephrine and caffeine. Intracardiac pacing studies in 12- to 18-day-old mice revealed the presence of an arrhythmogenic substrate in R176Q/+ compared with wild-type mice. Reverse transcription-polymerase chain reaction and Western blotting showed that expression levels of other calcium handling proteins were unaltered, suggesting that calcium leak through mutant RyR2 underlies arrhythmogenesis and sudden death in young R176Q/+ mice.Conclusions: Our findings demonstrate that a gain-of-function mutation in RyR2 confers an increased risk of cardiac arrhythmias and sudden death in young mice and that young R176Q/+ mice may be used as a model for elucidating the complex interplay between genetic and environmental risk factors associated with sudden infant death syndrome. [ABSTRACT FROM AUTHOR]