RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET.

Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca2+-release channels contributes to heart failure and arrhythmias. Reducing the RyR2 activity, particularly during cardiac relaxation (diastole), is a desirable therapeutic goal. We previously reported that the unnatural enantiomer (ent) of an insect-RyR activator, verticilide, inhibits porcine and mouse RyR2 at diastolic (nanomolar) Ca2+ and has in vivo efficacy against atrial and ventricular arrhythmia. To determine the ent-verticilide structural mode of action on RyR2 and guide its further development via medicinal chemistry structure-activity relationship studies, here, we used fluorescence lifetime (FLT)-measurements of Förster resonance energy transfer (FRET) in HEK293 cells expressing human RyR2. For these studies, we used an RyR-specific FRET molecular-toolkit and computational methods for trilateration (i.e., using distances to locate a point of interest). Multiexponential analysis of FLT-FRET measurements between four donor-labeled FKBP12.6 variants and acceptor-labeled ent-verticilide yielded distance relationships placing the acceptor probe at two candidate loci within the RyR2 cryo-EM map. One locus is within the Ry12 domain (at the corner periphery of the RyR2 tetrameric complex). The other locus is sandwiched at the interface between helical domain 1 and the SPRY3 domain. These findings document RyR2-target engagement by ent-verticilide, reveal new insight into the mechanism of action of this new class of RyR2-targeting drug candidate, and can serve as input in future computational determinations of the ent-verticilide binding site on RyR2 that will inform structure-activity studies for lead optimization.

Protocolo clínico asistencial de manejo de la hipertermia maligna / Clinical protocol for the management of malignant hyperthermia

La hipertermia maligna es un síndrome hipermetabólico que ocurre en pacientes susceptibles, tras la exposición a un fármaco anestésico desencadenante (succinilcolina, anestésicos inhalatorios). En España, se presenta en uno de cada 40.000 en adultos, con una mortalidad estimada del 10%. Está inducido por una regulación anormal de los receptores de rianodina, que produce una liberación masiva del calcio del retículo sarcoplasmático del músculo. Las manifestaciones clínicas son variadas y consisten en: elevación del CO2, taquicardia e inestabilidad hemodinámica, acidosis metabólica y respiratoria, sudoración profusa, hiperpirexia, elevación de CPK, mioglobinuria, fallo renal, CID y finalmente parada cardiorrespiratoria. El tratamiento con dantroleno sódico inhibe la liberación de calcio al antagonizar los receptores de rianodina. El diagnóstico definitivo se realiza con el test de contracción de fibra muscular expuesta a cafeína y halotano. Ante este grave evento la protocolización del manejo ayuda a garantizar que el paciente reciba una atención fiable y segura (AU)

Malignant hyperthermia is a hypermetabolic syndrome that appears in susceptible patients after exposure to certain anaesthetic drugs (succinylcholine, inhalation anaesthetics). Its incidence in Spain is 1 in 40,000 adults, with a 10% mortality rate. It is induced by an abnormal regulation of the ryanodine receptors, producing a massive release of calcium from the sarcoplasmic reticulum in the striate muscle. Clinical manifestations include: CO2 increase, tachycardia, haemodynamic instability, metabolic and respiratory acidosis, profuse sweating, hyperpyrexia, CPK increase, myoglobinuria, kidney failure, disseminated intravascular coagulation (DIC), and ending in cardiac arrest. Dantrolene sodium is a ryanodine receptor antagonist, and inhibits the release of intracellular calcium. Definitive diagnosis is achieved by the exposure of muscle fibres to caffeine and halothane. Protocols can help guarantee a reliable and secure management when this severe event occurs (AU)

Axoplasmic reticulum Ca(2+) release causes secondary degeneration of spinal axons.

OBJECTIVE: Transected axons of the central nervous system fail to regenerate and instead die back away from the lesion site, resulting in permanent disability. Although both intrinsic (eg, microtubule instability, calpain activation) and extrinsic (ie, macrophages) processes are implicated in axonal dieback, the underlying mechanisms remain uncertain. Furthermore, the precise mechanisms that cause delayed "bystander" loss of spinal axons, that is, ones that were not directly damaged by the initial insult, but succumbed to secondary degeneration, remain unclear. Our goal was to evaluate the role of intra-axonal Ca(2+) stores in secondary axonal degeneration following spinal cord injury. METHODS: We developed a 2-photon laser-induced spinal cord injury model to follow morphological and Ca(2+) changes in live myelinated spinal axons acutely following injury. RESULTS: Transected axons "died back" within swollen myelin or underwent synchronous pan-fragmentation associated with robust Ca(2+) increases. Spared fibers underwent delayed secondary bystander degeneration. Reducing Ca(2+) release from axonal stores mediated by ryanodine and inositol triphosphate receptors significantly decreased axonal dieback and bystander injury. Conversely, a gain-of-function ryanodine receptor 2 mutant or pharmacological treatments that promote axonal store Ca(2+) release worsened these events. INTERPRETATION: Ca(2+) release from intra-axonal Ca(2+) stores, distributed along the length of the axon, contributes significantly to secondary degeneration of axons. This refocuses our approach to protecting spinal white matter tracts, where emphasis has been placed on limiting Ca(2+) entry from the extracellular space across cell membranes, and emphasizes that modulation of axonal Ca(2+) stores may be a key pharmacotherapeutic goal in spinal cord injury.

Effects of sarcolemmal Ca(2+) entry, ryanodine function, and kinase inhibitors on a rabbit model of heart failure.

QT prolongation may increase the risk of torsades de pointes (TdP). Early afterdepolarizations (EADs) and transmural dispersion of repolarization have been known to serve as physiological substrates and predictors for TdP. Abnormal Ca(2+) cycling is the proximate cause of EADs, and Ca(2+) cycling is abnormal in heart failure (HF). However, the mechanisms for drug-induced TdP in HF are poorly understood. The purpose of this study was to search for torsadogenic-modifying effects of verapamil, ryanodine, KB-R7943, W-7, KN-93, and H-8 on ventricular premature depolarizations (VPD) and TdP in rabbits with HF. Rabbits with HF were pretreated with propranolol followed by test articles before continuous infusion of dofetilide to induce TdP. In the control hearts, VPD and TdP were induced in all rabbits and the onsets of VPD and TdP were 3.6 +/- 1.3 minutes and 10.3 +/- 1.4 minutes, respectively. Dofetilide lengthened RR, QT and QTc. Verapamil, ryanodine and H-8 significantly delayed onset of VPD (P < 0.05) and suppressed TdP (P < 0.01). KB-R7943, W-7, and KN-93 accelerated onset of TdP. Blockades of L-type Ca(2+) channel, ryanodine channel, and protein kinase A prevent dofetilide-induced TdP, suggesting roles for intracellular Ca(2+) overload and Ca(2+) signaling pathways in drug-induced TdP.

[Effects of ryanodine receptors block on spontaneous initiation of atrial fibrillation in the intact canine heart]. / Vliianie blokady rianodinovykh retseptorov na spontannoe vozniknovenie fibrilliatsii predserdii u sobak.

To study the possible role of intracellular Ca2+ overload in initiation of cholinergic-dependent atrial fibrillation (AF), we tested the effects of ryanodine in canine models of AF. In anesthetized open-chest dogs (n=10) AF was induced by two methods: (I) perfusion (9 ml/min) with normal Tyrode solution containing acetylcholine (ACh) into the sinus node artery (SNA) and (II) stimulation of the right vagal nerve (VS, 5 sec train). AF was induced in all dogs: by perfusion with ACh (3.7-/+1.5 mcM) into the SNA in 97-/+3% of attempts and by VS in 78-/+6% of attempts. Intravenous infusion of ryanodine (5 mg/kg) did not prevent induction of AF during ACh perfusion (84-/+5%, NS) but completely prevented the induction of AF by VS (4-/+3%, p<0.001). Atrial activation mapping (112 unipolar electrodes) did not show any significant differences between the beginning of ACh-dependent AF in control and after ryanodine treatment. Ryanodine significantly reduced both systolic and diastolic arterial pressures but had no effect on heart rate, atrial effective refractory period (AERP) and conduction velocity for one hour after infusion. Ryanodine, itself, did not exert antivagal activity, so after ryanodine treatment in the presence of VS (8 Hz) the reduction of AERP and the deceleration of heart rate were similar to that in control. These data suggest that ryanodine can suppress the initiation of AF induced by VS but not AF induced by ACh perfusion. We can conclude that the initiation of AF during ACh perfusion unlikely relates to triggering activity induced by intracellular Ca2+ overload. In addition, we suggest that besides ACh some 'unclear' ryanodine sensitive factor(s) contribute to the initiation of AF induced by VS.

Experimental studies on myocardial stretch and ventricular arrhythmia in hypertrophied and non-hypertrophied hearts.

Hypertension affects about 5% of western populations and in the majority of cases it is of unknown aetiology. It exposes the heart to greater levels of myocardial stretch as a result of increased systolic pressure and peripheral resistance. Under certain circumstances myocardial stretch may trigger arrhythmias but the mechanisms and clinical importance of this phenomenon are unclear. This article outlines the risks of sudden cardiac death conferred by hypertension and left ventricular hypertrophy, presents the results of experiments using an animal model of myocardial stretch and discusses some possible mechanisms underlying stretch-induced arrhythmias which may be important in hypertensive patients.

PKC-dependent preconditioning with norepinephrine protects sarcoplasmic reticulum function in rat trabeculae following metabolic inhibition.

The authors have previously shown that norepinephrine (NE) pretreatment attenuates Ca2+ overloading in cardiac rat trabeculae during metabolic inhibition, and improves contractile function during a subsequent recovery period. The present study investigated: (i) whether protection of sarcoplasmic reticulum (SR) function during metabolic inhibition (MI) is involved in the preconditioning-like effect of NE-pretreatment, and (ii) whether or not this process is PKC-dependent. A 15 min preincubation period was used with 1 micromol/l exogenous NE to precondition isolated, superfused rat trabeculae against contractile dysfunctioning following 40 min of MI in 2 mmol/l NaCN containing Tyrode (gassed with 95% O2/5% CO2; pH 7.4, 24 degrees C) without glucose at 1-Hz stimulation frequency. Contractile recovery was studied during a subsequent 60 min recovery period (RP) in glucose containing Tyrode at 0.2 Hz. Force and intracellular free calcium ([Ca2+]ii) were monitored throughout the experimental protocol. Pretreatment of trabeculae with NE (group NE) substantially diminished the Ca2+ rise from the onset of rigor development during MI, compared to preparations which were pretreated with NE, in the presence of specific PKC blocker chelerythrine (2 micromol/l; group NE+CHEL). After 40 min of MI, resting [Ca2+]i in group NE and NE+CHEL was increased to 0.50+/-0.03 and 2.08+/-0.20 micromol/l, respectively (P<0.05), whereas total intracellular ATP levels were similar in both groups (approximately 0.20 micromol/g dry wt). This corresponded with an increase in active force development (119%) and a decrease in twitch force relaxation time (77%) during subsequent RP in group NE, compared to pre-MI values of the same group. In contrast, a significant decrease in force recovery (54%) and an increase in twitch force relaxation time (123%) was observed in group NE+CHEL. Values for [Ca2+]i, contractile recovery, and twitch force relaxation time in untreated controls as well as CHEL preparations corresponded to those measured in the NE+CHEL group. Rapid cooling contractures (RCCs), which provide information on both SR-Ca2+ loading and Ca2+ re-uptake activity, revealed a 2-fold higher SR Ca2+ content during RP in group NE compared to controls and group NE+CHEL. In addition, kinetic analysis of the RCC rewarming spike (RWS) showed that this was accompanied by greater than a 28% increase in the maximum rate of RWS relaxation (-dF/dt/rws) in group NE compared to group NE+CHEL. The change of -dF/dt/rws in the NE group during RP following MI persisted after SR Ca2+-release channel blockade by ryanodine treatment (100 micromol/l), which suggests involvement of NE-induced, PKC-dependent protection of SR Ca2+-ATPase activity. The results of the present study point to an inverse relationship between the Ca2+ rise during MI and SR functioning, in which PKC appears to play a key role. It is concluded that the preconditioning-like effect of NE-pretreatment on contractile recovery is at least partly mediated by protection of SR function.

The effect of flunarizine and ryanodine on acquired torsades de pointes arrhythmias in the intact canine heart.

INTRODUCTION: Ryanodine, a specific blocker of the Ca2+ release channel of the sarcoplasmic reticulum, and flunarizine, a [Ca2+]i overload blocker, possess antiarrhythmic effects against delayed afterdepolarizations (DADs) and DAD-dependent arrhythmias. In vitro controversy exists about their effect on early after-depolarizations (EADs): no effect was reported on cesium-induced EADs, while ryanodine did prevent EADs induced by isoproterenol. To study the possible role of intracellular Ca2+ overload in acquired EAD-dependent torsades de pointes (TdP) arrhythmias, we tested the effects of flunarizine and ryanodine in our animal model of TdP. METHODS AND RESULTS: Anaesthetized dogs with chronic AV block received d-sotalol or almokalant followed by pacing. A subset of dogs with reproducible TdP (> or = 3 times) were selected to receive flunarizine (2 mg/kg per 2 min) or ryanodine (10 micrograms/kg per 10 min). After d-sotalol, TdP was induced at a mean cycle length of the idioventricular rhythm (CL-IVR) of 2070 +/- 635 msec and a QT(U) interval of 535 +/- 65 msec. Induction of TdP was prevented by flunarizine in all experiments (8/8): electrophysiologically this was associated with a decrease in CL-IVR, QT(U), and QTc interval (390 +/- 100 to 320 +/- 45, P < 0.05). Ryanodine prevented TdP induction in 4 of 5 experiments and decreased the CL-IVR, QT(U), and the QTc interval from 385 +/- 75 to 320 +/- 20 msec (P < 0.05). Both drugs also suppressed the almokalant-induced EADs and related ectopic activity. This antiarrhythmic action corresponded with the inability to reinduce TdP by pacing. CONCLUSIONS: Blockade of the Ca2+ release channel of the sarcoplasmic reticulum by ryanodine or the reduction of [Ca2+]i overload by flunarizine prevents induction of EAD-dependent acquired TdP arrhythmias, suggesting a role for [Ca2+]i overload in acquired TdP.

Mechanisms underlying the genesis of post-extrasystolic potentiation in rat cardiac muscle.

Changes of contractility resulting from changes in stimulation pattern (post-extrasystolic potentiation - PESP) were investigated in right ventricular papillary muscles from female albino rats (EPM strain, 160-200 g). The preparations were superfused with bicarbonate buffered solution at 24 +/- 0.5 degrees C, and stimulated at 0.5 Hz. Maintained paired stimulation was performed at several coupling intervals (360, 500, 660, 770 and 920 ms) with normal Krebs for 30 s. After treatment with ryanodine (1 microM), used as an inhibitor of the release of sarcoplasmic reticulum Ca2+ activity, the same protocol was repeated in the presence of normal Krebs, low Na+ (80 mM, LiCl used as substitute) and low K+ concentrations to change the level of activity of the Na+/Ca2+ exchange mechanism. With normal Krebs, paired pulse stimulation produced a maintained potentiation of the post-extrasystolic beat and an extrasystole with a reduced force generation when compared to the control steady-state contraction. As the interval between the extrasystole and the normal beat was increased the potentiation of the post-extrasystolic beat was reduced and the force of the extrasystole was increased. Ryanodine treatment reduced the force of contraction and increased its duration, and the pattern of the PESP phenomenon was altered. Both the post-extrasystolic and the extrasystolic beats were potentiated compared to the steady-state contraction obtained under ryanodine treatment. The extrasystole displayed a greater potentiation than the post-extrasystolic beat. As the interval between them increased the amplitude of the extrasystolic beat was enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms underlying the genesis of post-extrasystolic potentiation in rat cardiac muscle

Changes of contractility resulting from changes in stimulation pattern (post-extrasystolic potentiation - PESP) were investigated in right ventricular papillary muscles from female albino rats (EPM strain, 160-200 g). The preparations were superfused with bicarbonate buffered solution at 24 + or - 0.5§C, and stimulated at 0.5 Hz. Maintaned paired stimulation was performed at several coupling intervals (360, 500, 660, 770 and 920 ms) with normal Krebs for 30 s. After treatment with ryanodine (1 µM), used as an inhibitor of the release of sarcoplasmic reticulum Ca2+ activity, the same protocol was repeated in the presence of normal Krebs, low Na+ (80 mM, LiCl used as substitute) and low K+ concentrations to change the level of activity of the Na+/Ca2+ exchange mechanism. With normal Krebs, paired pulse stimulation produced a maintained potentiation of the post-extrasystolic beat and an extrasystole and the normal beat was increased the potentiation of the post-extrasystolic beat was reduced and the force of the extrasystole was increased. Rynodine treatment reduced the force of contraction and increased its duration, and the pattern of the PESP...

Protective effects of diltiazem and ryanodine against ischemia-reperfusion injury in neonatal rabbit hearts.

The effects of diltiazem, a sarcolemmal Ca2+ channel blocker, and ryanodine, an inhibitor of sarcoplasmic reticulum function, were investigated in isolated newborn rabbit hearts (2 to 5 days old) subjected to ischemia and reperfusion. After cardioplegic arrest with St. Thomas' Hospital solution, global ischemia was induced at 37 degrees C (normothermia) for 45 minutes or at 20 degrees C (hypothermia) for 180 minutes. The hearts were then reperfused at 37 degrees C for 30 minutes. Diltiazem or ryanodine, at concentrations that have minimal to moderately negative inotropic effects under nonischemic conditions, was added to the cardioplegic solution. After normothermic ischemia, reperfusion of untreated hearts resulted in recovery of left ventricular developed pressure to 52.9% +/- 2.5% of the preischemic level. In hearts treated with diltiazem, recovery of left ventricular developed pressure was significantly improved (84.2% +/- 2.9% at 3 x 10(-8) mol/L; p < 0.01). Comparable improvement was achieved with ryanodine (90.5% +/- 4.1% at 10(-9) mol/L; p < 0.01). Creatine kinase leakage and structural derangement of mitochondria were also reduced by both agents. With hypothermic ischemia, left ventricular developed pressure recovered in untreated hearts to 72.7% +/- 3.3% of preischemic values. Treatment with diltiazem improved the recovery of left ventricular developed pressure to 96.9% +/- 3.5% at 3 x 10(-8) mol/L and reduced creatine kinase leakage and mitochondrial damage. Ryanodine also improved the recovery of left ventricular developed pressure and attenuated ultrastructural damage. These findings suggest that Ca2+ handling by the sarcoplasmic reticulum, like transsarcolemmal Ca2+ influx, plays an important role in the pathogenesis of myocardial ischemia-reperfusion injury in the neonatal heart despite the morphologic and functional immaturity of the sarcoplasmic reticulum in the neonate.

The effects of several pharmacologic agents upon postischemic recovery.

Using an isolated working rat heart model, the effects of DL-verapamil, ryanodine, gabexate mesilate (FOY), recombinant human superoxide dismutase (RH-SOD), and coenzyme Q10 upon myocardial protection were evaluated. Under conditions of normothermic ischemia, all these compounds, except RH-SOD, when added to the St. Thomas' cardioplegic solution at an optimal concentration, showed beneficial effects upon functional recovery and enzyme leakage. In contrast, the above compounds, except ryanodine and FOY, failed to improve the protective properties of the St. Thomas' cardioplegic solution under conditions of hypothermic ischemia. Our results indicate that calcium overload via the calcium channel and calcium-induced calcium release from sarcoplasmic reticulum (SR) may contribute to the onset of ischemic-reperfusion injury. However, under conditions of hypothermic ischemia, calcium-induced calcium release from SR plays a dominant role in calcium overload. Furthermore, intracellular calcium overload may activate proteases and result in the acceleration of myocardial injury.

Analysis of antiarrhythmic effect of ryanodine in guinea-pigs.

The effects of ryanodine on (1) ventricular arrhythmias in guinea-pigs in vivo, (2) delayed afterpotentials and aftercontractions and (3) spontaneous oscillations of the membrane potential (SOP) and of resting tension (SOT) of guinea-pig papillary muscle under ouabain intoxication have been studied. After addition of ouabain (1 microM), the afterpotentials, aftercontractions and the amplitude of SOP and SOT were significantly increased. The power spectra of SOT and SOP under these conditions had a resonance harmonic with a frequency of about 5 Hz. The afterpotentials, aftercontractions, SOP and SOT were abolished 3 to 5 min after ryanodine addition (0.1 to 0.5 microM), suggesting a close relationship between these oscillations and the oscillatory activity of sarcoplasmic reticulum. During in vivo experiments, ouabain-induced (75 to 115 micrograms/kg) ventricular arrhythmias were terminated 4 to 5 min after the intravenous injection of ryanodine (15 micrograms/kg) and within 8 to 10 min, the sinus rhythm was completely restored. We conclude that the antiarrhythmic effect of ryanodine is related to the inhibition of the diastolic fluctuations of the membrane potential.

[Anti-arrhythmic effect of ryanodine in the guinea pig with glycoside poisoning]. / Antiaritmicheskii éffekt rianodina u morskoi svinki pri glikozidnoi intoksikatsii.

The effect of ryanodine on membrane potential oscillations in vitro (in isolated guinea-pig papillary muscle) and on ventricular arrhythmias in vivo (in glycoside intoxication) were studied. 3-5 minutes after ryanodine (0.5 microM) addition the membrane potential oscillations induced by ouabain (1 microM) were abolished. 4-5 minutes after intravenous ryanodine infusion (15 micrograms/kg) ventricular arrhythmias induced by ouabain intoxication (75-115 micrograms/kg) disappeared and 8-10 minutes later sinus rhythm was restored. It is suggested that antiarrhythmic effect of ryanodine is a result of the inhibition of diastolic membrane potential oscillations.