ryanodine and Ventricular-Fibrillation

Mutations in cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most CPVT RyR2 mutations characterized are gain-of-function (GOF), indicating enhanced RyR2 function as a major cause of CPVT. Loss-of-function (LOF) RyR2 mutations have also been identified and are linked to a distinct entity of cardiac arrhythmia termed RyR2 Ca2+ release deficiency syndrome (CRDS). Exercise stress testing (EST) is routinely used to diagnose CPVT, but it is ineffective for CRDS. There is currently no effective diagnostic tool for CRDS in humans. An alternative strategy to assess the risk for CRDS is to directly determine the functional impact of the associated RyR2 mutations. To this end, we have functionally screened 18 RyR2 mutations that are associated with idiopathic ventricular fibrillation (IVF) or sudden death. We found two additional RyR2 LOF mutations E4146K and G4935R. The E4146K mutation markedly suppressed caffeine activation of RyR2 and abolished store overload induced Ca2+ release (SOICR) in human embryonic kidney 293 (HEK293) cells. E4146K also severely reduced cytosolic Ca2+ activation and abolished luminal Ca2+ activation of single RyR2 channels. The G4935R mutation completely abolished caffeine activation of and [3H]ryanodine binding to RyR2. Co-expression studies showed that the G4935R mutation exerted dominant negative impact on the RyR2 wildtype (WT) channel. Interestingly, the RyR2-G4935R mutant carrier had a negative EST, and the E4146K carrier had a family history of sudden death during sleep, which are different from phenotypes of typical CPVT. Thus, our data further support the link between RyR2 LOF and a new entity of cardiac arrhythmias distinct from CPVT.

Topics: Calcium; Death, Sudden, Cardiac; HEK293 Cells; Humans; Loss of Function Mutation; Ryanodine; Ryanodine Receptor Calcium Release Channel; Ventricular Fibrillation

A strong premature electrical stimulus (S(2)) induces both virtual anodes and virtual cathodes. The effects of virtual electrodes on intracellular Ca(2+) concentration ([Ca(2+)](i)) transients and ventricular fibrillation thresholds (VFTs) are unclear. We studied 16 isolated, Langendorff-perfused rabbit hearts with simultaneous voltage and [Ca(2+)](i) optical mapping and for vulnerable window determination. After baseline pacing (S(1)), a monophasic (10 ms anodal or cathodal) or biphasic (5 ms-5 ms) S(2) was applied to the left ventricular epicardium. Virtual electrode polarizations and [Ca(2+)](i) varied depending on the S(2) polarity. Relative to the level of [Ca(2+)](i) during the S(1) beat, the [Ca(2+)](i) level 40 ms after the onset of monophasic S(2) increased by 36+/-8% at virtual anodes and 20+/-5% at virtual cathodes (P<0.01), compared with 25+/-5% at both virtual cathode-anode and anode-cathode sites for biphasic S(2). The VFT was significantly higher and the vulnerable window significantly narrower for biphasic S(2) than for either anodal or cathodal S(2) (n=7, P<0.01). Treatment with thapsigargin and ryanodine (n=6) significantly prolonged the action potential duration compared with control (255+/-22 vs. 189+/-6 ms, P<0.05) and eliminated the difference in VFT between monophasic and biphasic S(2), although VFT was lower for both cases. We conclude that virtual anodes caused a greater increase in [Ca(2+)](i) than virtual cathodes. Monophasic S(2) is associated with lower VFT than biphasic S(2), but this difference was eliminated by the inhibition of the sarcoplasmic reticulum function and the prolongation of the action potential duration. However, the inhibition of the sarcoplasmic reticulum function also reduced VFT, indicating that the [Ca(2+)](i) dynamics modulate, but are not essential, to ventricular vulnerability.

Topics: Action Potentials; Animals; Calcium Signaling; Disease Models, Animal; Electric Stimulation; Enzyme Inhibitors; Heart Ventricles; Myocytes, Cardiac; Perfusion; Pericardium; Rabbits; Ryanodine; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Time Factors; Ventricular Fibrillation

The role of intracellular calcium (Ca(i)) in defibrillation and vulnerability is unclear.. We simultaneously mapped epicardial membrane potential and Ca(i) during shock on T-wave episodes (n=104) and attempted defibrillation episodes (n=173) in 17 Langendorff-perfused rabbit ventricles. Unsuccessful and type B successful defibrillation shocks were followed by heterogeneous distribution of Ca(i), including regions of low Ca(i) surrounded by elevated Ca(i) ("Ca(i) sinkholes") 31+/-12 ms after shock. The first postshock activation then originated from the Ca(i) sinkhole 53+/-14 ms after the shock. No sinkholes were present in type A successful defibrillation. A Ca(i) sinkhole also was present 39+/-32 ms after a shock on T that induced ventricular fibrillation, followed 22+/-15 ms later by propagated wave fronts that arose from the same site. This wave propagated to form a spiral wave and initiated ventricular fibrillation. Thapsigargin and ryanodine significantly decreased the upper limit of vulnerability and defibrillation threshold. We studied an additional 7 rabbits after left ventricular endocardial cryoablation, resulting in a thin layer of surviving epicardium. Ca(i) sinkholes occurred 31+/-12 ms after the shock, followed in 19+/-7 ms by first postshock activation in 63 episodes of unsuccessful defibrillation. At the Ca(i) sinkhole, the rise of Ca(i) preceded the rise of epicardial membrane potential in 5 episodes.. There is a heterogeneous postshock distribution of Ca(i). The first postshock activation always occurs from a Ca(i) sinkhole. The Ca(i) prefluorescence at the first postshock early site suggests that reverse excitation-contraction coupling might be responsible for the initiation of postshock activations that lead to ventricular fibrillation.

Topics: Action Potentials; Animals; Body Surface Potential Mapping; Calcium; Electric Countershock; Heart Ventricles; Myocardium; Perfusion; Rabbits; Ryanodine; Thapsigargin; Ventricular Fibrillation

The effect of varying the number of preconditioning (PC) episodes on the recovery of cardiac function and on the function of the sarcoplasmic reticulum (SR) was investigated to determine the correlation between the effect of PC and SR function. Isolated rat hearts were subjected to zero to three 5-min episodes of global ischemia with intermittent perfusion (PC0-PC3) followed by 25 min of ischemia (I) and 30 min of reperfusion. The left ventricular (LV) pressure and SR 45Ca2+ uptake in the absence or presence of ryanodine were then measured. The increase in LV end-diastolic pressure and the incidence and duration of ventricular tachyarrhythmias during reperfusion decreased. The recovery of LV developed pressure, LV dP/dtmax and dP/dtmin, increased as the number of episodes of PC increased. The rates of SR 45Ca2+ uptake after PC and after reperfusion were lower in PC3 than in PC0. Conversely, the rate of 45Ca2+ uptake after I did not differ between PC0 and PC3. The ryanodine-sensitive Ca2+ release increased after I, and additional increases were observed during reperfusion in PC0, whereas the release after I and reperfusion decreased progressively in PC3. These observations show that the beneficial effects of PC are associated with a decrease in ryanodine-sensitive SR Ca2+ release.

Topics: Animals; Calcium; Energy Metabolism; Heart; In Vitro Techniques; Ischemic Preconditioning, Myocardial; Male; Myocardium; Rats; Rats, Sprague-Dawley; Ryanodine; Sarcoplasmic Reticulum; Tachycardia, Ventricular; Ventricular Fibrillation; Ventricular Function, Left

Cocaine is a potent cardiac stimulant that can provoke lethal cardiac events, including ventricular fibrillation (VF). The cocaine-induced accumulation of intracellular calcium could contribute significantly to the development of these lethal arrhythmias. To test this hypothesis, VF was induced in 12 mongrel dogs by the combination of cocaine (1.0 mg/kg) and a 2-min coronary occlusion during exercise. This test without cocaine failed to induce arrhythmias. Pretreatment with the intracellular calcium-specific chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM; 1.0 mg/kg iv) prevented VF in 8 of 12 animals (P < 0.001) and delayed the onset of lethal arrhythmias in 3 of the remaining animals. Cocaine induced significant increases in left ventricular (LV) systolic pressure (control 154.7 +/- 8.7, cocaine 167.4 +/- 8.4 mmHg), heart rate (control 195.9 +/- 6.1, cocaine 222.3 +/- 10.6 beats/min), and LV maximum rate of pressure development (dP/dtmax; control 5,251 +/- 317.6, cocaine 6,016 +/- 435.1 mmHg/s). BAPTA-AM attenuated the increase in LV dP/dtmax (BAPTA-AM 4,591 +/- 479.3 mmHg/s) and LV systolic pressure (BAPTA-AM 154.5 +/- 6.8 mmHg). Because vascular muscle relaxation could contribute to the cardioprotection, the cocaine and exercise plus ischemia test was repeated after nitroprusside. The nitroprusside prevented cocaine-induced increases in LV systolic pressure but failed to prevent VF. These data suggest that BAPTA-AM may prevent cocaine-induced VF independently of its vascular actions.

Topics: Animals; Blood Pressure; Chelating Agents; Cocaine; Dogs; Egtazic Acid; Heart Rate; Hemodynamics; Myocardial Ischemia; Physical Exertion; Ryanodine; Ventricular Fibrillation; Ventricular Function, Left

Myocardial ischaemia can provoke a rise in cytosolic calcium which may in turn trigger malignant ventricular arrhythmias. Recently, inhibition of calcium entry has been shown to prevent these lethal arrhythmias. However, the contributions of calcium release from cytosolic stores to these disruptions in cardiac rhythm have not been investigated. This study examines the role of calcium release from the sarcoplasmic reticulum in the initiation of lethal ventricular arrhythmias.. Mongrel dogs were chronically instrumented to measure left ventricular pressure, coronary blood flow, and cardiac electrical activity (ventricular electrocardiogram). The left anterior descending coronary artery was ligated during the surgery to produce a myocardial infarction. In addition, a hydraulic occluder was placed around the left circumflex artery. The susceptibility to ventricular fibrillation was then evaluated by the combination of acute myocardial ischaemia and exercise.. Ventricular fibrillation was induced in 10 animals during the exercise plus ischaemia test. On a subsequent day the exercise plus ischaemia test was repeated after pretreatment with ryanodine (10 micrograms.kg-1, n = 10), a drug which impairs calcium efflux from the sarcoplasmic reticulum. Ryanodine failed to prevent ventricular fibrillation induced by ischaemia. Ryanodine significantly (p < 0.01) increased heart rate [control 115.3(SEM 6.3) v ryanodine 156.4(14.7) beats.min-1] but reduced left ventricular systolic pressure [control 141.8(4.9) v ryanodine 111.1(12.7) mm Hg] and positive left ventricular dP/dt [3312.9(217.4) v ryanodine 1462.9(226.3) mm Hg.s-1] both at rest and during exercise. In contrast, this drug abolished ventricular tachycardia induced by ouabain toxicity (n = 10, 40 micrograms.kg-1 bolus followed by 0.076 microgram.kg-1.min-1 for 1 h, then 20 micrograms.kg-1 bolus, intravenously).. These data suggest that calcium release from ryanodine sensitive channels in the sarcoplasmic reticulum may contribute significantly to the arrhythmias induced by ouabain toxicity but not to ventricular fibrillation provoked by ischaemia.

Topics: Animals; Blood Pressure; Dogs; Electrocardiography; Exercise Test; Heart Rate; Myocardial Ischemia; Ryanodine; Ventricular Fibrillation; Ventricular Pressure

The hypothesis tested was that enhanced entry of calcium into cardiac cells would increase the susceptibility to ventricular fibrillation as measured by the ventricular fibrillation threshold (VFT) of the isolated perfused rat heart. Bay-K-8644 was used as a calcium-channel agonist. There was a biphasic effect with a maximal increase in left ventricular systolic pressure and oxygen uptake at a concentration of 10(-7) M. The same concentration caused a major reduction in the VFT. The bell-shaped pattern of fall of the VFT was inversely related to the effect on LV developed pressure. The changes in VFT could be dissociated from those on myocardial metabolites. Although Bay-K-8644 increased the heart rate, reduction of the VFT could also be obtained in paced hearts. The addition of ryanodine, an agent known to interrupt intracellular recycling of calcium through the sarcoplasmic reticulum, was able to abolish approximately half the effect of Bay-K-8644 on the VFT. Therefore, increased entry of calcium via the calcium channel is able to reduce VFT, acting in part through enhanced recycling of calcium through the sarcoplasmic reticulum.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium; Cardiac Pacing, Artificial; Coronary Circulation; Cyclic AMP; Electric Stimulation; Heart; In Vitro Techniques; Male; Myocardial Contraction; Myocardium; Oxygen Consumption; Rats; Ryanodine; Ventricular Fibrillation

To elucidate the role of changes in [Ca2+]i in the induction of ventricular fibrillation (VF), Ca2+i signals, epicardial electrical potentials, and isovolumic left ventricular pressure were simultaneously recorded in isolated intact ferret hearts loaded with aequorin, a bioluminescent protein. When the preparations were perfused with 3 microM acetylstrophanthidin and 8 mM Ca2+, or with a low Na+ solution (18 mM Na+, 100 mM Li+), spontaneous transitions to the VF state were consistently observed within a short period of time. The initiation of spontaneous VF was preceded by development of a Ca2+i overload state, coincidental with the ascending phase of diastolic Ca2+i oscillations, and was followed by further elevation in Ca2+i levels, which were associated with augmented Ca2+i oscillations of a saw-toothed pattern. Pretreatment with 10 microM ryanodine, which blocked Ca2+i oscillations in the preparation, did not eliminate inducibility of VF by means of AC electrical stimulations; however, VF no longer occurred spontaneously, and the threshold for VF induction increased markedly. In the absence of a state of Ca2+i overload, spontaneous defibrillation occurred within a minute after the initiation of VF. We conclude that 1) VF can be induced in the absence of Ca2+i oscillations; however, 2) Ca2+i oscillations play a crucial role as a trigger for VF and therefore are an important determinant of the vulnerability to VF; and 3) the augmented Ca2+i oscillations after the transition to VF state may support the maintenance of this type of arrhythmia.

Topics: Aequorin; Animals; Calcium; Electric Stimulation; Electrophysiology; Ferrets; In Vitro Techniques; Male; Myocardial Contraction; Myocardium; Perfusion; Ryanodine; Sodium; Strophanthidin; Ventricular Fibrillation

The pathophysiology of the ventricular fibrillation that complicates digitalis intoxication was investigated. In this and other calcium-overload states, oscillations of the intracellular free calcium concentration ([Ca2+]i) have been implicated as the cause of ventricular tachyarrhythmias. We addressed two questions: 1) Are [Ca2+]i oscillations obligatory in the pathogenesis of ventricular fibrillation during digitalis toxicity? 2) What are the metabolic consequences of [Ca2+]i oscillations? Ferret hearts (n = 20) were Langendorff-perfused at constant flow with oxygenated HEPES-buffered Tyrode's solution at 37 degrees C. Isovolumic left ventricular pressure was measured along with the extracellular electrogram or with simultaneous phosphorus nuclear magnetic resonance spectra. When strophanthidin (20 microM) was added during pacing at 3 Hz, the positive inotropic effect soon gave way to a decrease in developed force. The decrease in force was accompanied by an increase in inorganic phosphate concentration, a decrease in phosphocreatine concentration, and a slight acidosis. The rhythm changed to ventricular fibrillation after 12-25 minutes. This change was initially accompanied by further metabolic deterioration, but all metabolites reached steady state within 12-18 minutes of the onset of ventricular fibrillation. Fast Fourier transformation revealed the existence of periodic oscillations at 7-10 Hz in both the extracellular electrogram and the ventricular pressure during ventricular fibrillation. Ryanodine, an inhibitor of [Ca2+]i oscillations, abolished the pressure oscillations but not the voltage oscillations. Exposure to ryanodine significantly decreased the inorganic phosphate concentration and increased the phosphocreatine concentration (p less than 0.05) despite continuing exposure to strophanthidin. The results indicate that oscillations of [Ca2+]i are not required to sustain ventricular fibrillation, but when present, such oscillations contribute importantly to metabolic deterioration.

Topics: Animals; Calcium; Electrocardiography; Ferrets; Fourier Analysis; Ion Channels; Magnetic Resonance Spectroscopy; Ryanodine; Strophanthidin; Ventricular Fibrillation