Derek S. Steele

Flecainide inhibits arrhythmogenic Ca2+ waves by open state block of ryanodine receptor Ca2+ release channels and reduction of Ca2+ spark mass

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is linked to mutations in the cardiac ryanodine receptor (RyR2) or calsequestrin. We recently found that the drug flecainide inhibits RyR2 channels and prevents CPVT in mice and humans. Here we compared the effects of flecainide and tetracaine, a known RyR2 inhibitor ineffective in CPVT myocytes, on arrhythmogenic Ca(2+) waves and elementary sarcoplasmic reticulum (SR) Ca(2+) release events, Ca(2+) sparks. In ventricular myocytes isolated from a CPVT mouse model, flecainide significantly reduced spark amplitude and spark width, resulting in a 40% reduction in spark mass. Surprisingly, flecainide significantly increased spark frequency. As a result, flecainide had no significant effect on spark-mediated SR Ca(2+) leak or SR Ca(2+) content. In contrast, tetracaine decreased spark frequency and spark-mediated SR Ca(2+) leak, resulting in a significantly increased SR Ca(2+) content. Measurements in permeabilized rat ventricular myocytes confirmed the different effects of flecainide and tetracaine on spark frequency and Ca(2+) waves. In lipid bilayers, flecainide inhibited RyR2 channels by open state block, whereas tetracaine primarily prolonged RyR2 closed times. The differential effects of flecainide and tetracaine on sparks and RyR2 gating can explain why flecainide, unlike tetracaine, does not change the balance of SR Ca(2+) fluxes. We suggest that the smaller spark mass contributes to flecainides antiarrhythmic action by reducing the probability of saltatory wave propagation between adjacent Ca(2+) release units. Our results indicate that inhibition of the RyR2 open state provides a new therapeutic strategy to prevent diastolic Ca(2+) waves resulting in triggered arrhythmias, such as CPVT.

Na+-Ca2+ Exchange Activity Is Localized in the T-Tubules of Rat Ventricular Myocytes

Abstract— Detubulation of rat ventricular myocytes has been used to investigate the role of the t-tubules in Ca2+ cycling during excitation-contraction coupling in rat ventricular myocytes. Ca2+ was monitored using fluo-3 and confocal microscopy. In control myocytes, electrical stimulation caused a spatially uniform increase in intracellular [Ca2+] across the cell width. After detubulation, [Ca2+] rose initially at the cell periphery and then propagated into the center of the cell. Application of caffeine to control myocytes resulted in a rapid and uniform increase of intracellular [Ca2+]; the distribution and amplitude of this increase was the same in detubulated myocytes, although its decline was slower. On application of caffeine to control cells, there was a large, rapid, and transient rise in extracellular [Ca2+] as Ca2+ was extruded from the cell; this rise was significantly smaller in detubulated cells, and the remaining increase was blocked by the sarcolemmal Ca2+ ATPase inhibitor carboxyeosin. The treatment used to produce detubulation had no significant effect on Ca2+ efflux in atrial cells, which lack t-tubules. Detubulation of ventricular myocytes also resulted in loss of Na+-Ca2+ exchange current, although the density of the fast Na+ current was unaltered. It is concluded that Na+-Ca2+ exchange function, and hence Ca2+ efflux by this mechanism, is concentrated in the t-tubules, and that the concentration of Ca2+ flux pathways in the t-tubules is important in producing a uniform increase in intracellular Ca2+ on stimulation.

TNF-α and IL-1β increase Ca2+ leak from the sarcoplasmic reticulum and susceptibility to arrhythmia in rat ventricular myocytes

Sepsis is associated with ventricular dysfunction and increased incidence of atrial and ventricular arrhythmia however the underlying pro-arrhythmic mechanisms are unknown. Serum levels of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are elevated during sepsis and affect Ca2+ regulation. We investigated whether pro-inflammatory cytokines disrupt cellular Ca2+ cycling leading to reduced contractility, but also increase the probability of pro-arrhythmic spontaneous Ca2+ release from the sarcoplasmic reticulum (SR). Isolated rat ventricular myocytes were exposed to TNF-α (0.05 ng ml−1) and IL-1β (2 ng ml−1) for 3 hr and then loaded with fura-2 or fluo-3 to record the intracellular Ca2+ concentration ([Ca2+]i). Cytokine treatment decreased the amplitude of the spatially averaged Ca2+ transient and the associated contraction, induced asynchronous Ca2+ release during electrical stimulation, increased the frequency of localized Ca2+ release events, decreased the SR Ca2+ content and increased the frequency of spontaneous Ca2+ waves at any given cytoplasmic Ca2+. These data suggest that TNF-α and IL-1β increase the SR Ca2+ leak from the SR, which contributes to the depressed Ca2+ transient and contractility. Increased susceptibility to spontaneous SR Ca2+ release may contribute to arrhythmias in sepsis as the resulting Ca2+ extrusion via NCX is electrogenic, leading to cell depolarisation.

Genetic variation in RYR1 and malignant hyperthermia phenotypes

BACKGROUND Malignant hyperthermia (MH) is associated, in the majority of cases, with mutations in RYR1, the gene encoding the skeletal muscle ryanodine receptor. Our primary aim was to assess whether different RYR1 variants are associated with quantitative differences in MH phenotype. METHODS The degree of in vitro pharmacological muscle contracture response and the baseline serum creatine kinase (CK) concentration were used to generate a series of quantitative phenotypes for MH. We then undertook the most extensive RYR1 genotype-phenotype correlation in MH to date using 504 individuals from 204 MH families and 23 RYR1 variants. We also determined the association between a clinical phenotype and both the laboratory phenotype and RYR1 genotype. RESULTS We report a novel correlation between the degree of in vitro pharmacological muscle contracture responses and the onset time of the clinical MH response in index cases (P<0.05). There was also a significant correlation between baseline CK concentration and clinical onset time (P=0.039). The specific RYR1 variant was a significant determinant of the severity of each laboratory phenotype (P<0.0001). CONCLUSIONS The MH phenotype differs significantly with different RYR1 variants. Variants leading to more severe MH phenotype are distributed throughout the gene and tend to lie at relatively conserved sites in the protein. Differences in phenotype severity between RYR1 variants may explain the variability in clinical penetrance of MH during anaesthesia and why some variants have been associated with exercise-induced rhabdomyolysis and heat stroke. They may also inform a mutation screening strategy in cases of idiopathic hyperCKaemia.

Alternative Splicing of Ryanodine Receptors Modulates Cardiomyocyte Ca2+ Signaling and Susceptibility to Apoptosis

Ca2+ release via type 2 ryanodine receptors (RyR2) regulates cardiac function. Molecular cloning of human RyR2 identified 2 alternatively spliced variants, comprising 30- and 24-bp sequence insertions; yet their role in shaping cardiomyocyte Ca2+ signaling and cell phenotype is unknown. We profiled the developmental regulation and the tissue and species specificity of these variants and showed that their recombinant expression in HL-1 cardiomyocytes profoundly modulated nuclear and cytoplasmic Ca2+ release. All splice variants localized to the sarcoplasmic reticulum, perinuclear Golgi apparatus, and to finger-like invaginations of the nuclear envelope (nucleoplasmic reticulum). Strikingly, the 24-bp splice insertion that was present at low levels in embryonic and adult hearts was essential for targeting RyR2 to an intranuclear Golgi apparatus and promoted the intracellular segregation of this variant. The amplitude variability of nuclear and cytoplasmic Ca2+ fluxes were reduced in nonstimulated cardiomyocytes expressing both 30- and 24-bp splice variants and were associated with lower basal levels of apoptosis. Expression of RyR2 containing the 24-bp insertion also suppressed intracellular Ca2+ fluxes following prolonged caffeine exposure (1 mmol/L, 16 hours) that protected cells from apoptosis. The antiapoptotic effects of this variant were linked to increased levels of Bcl-2 phosphorylation. In contrast, RyR2 containing the 30-bp insertion, which was abundant in human embryonic heart but was decreased during cardiac development, did not protect cardiomyocytes from caffeine-evoked apoptosis. Thus, we provide the first evidence that RyR2 splice variants exquisitely modulate intracellular Ca2+ signaling and are key determinants of cardiomyocyte apoptotic susceptibility.

Cardiac arrhythmia mechanisms in rats with heart failure induced by pulmonary hypertension

Pulmonary hypertension provokes right heart failure and arrhythmias. Better understanding of the mechanisms underlying these arrhythmias is needed to facilitate new therapeutic approaches for the hypertensive, failing right ventricle (RV). The aim of our study was to identify the mechanisms generating arrhythmias in a model of RV failure induced by pulmonary hypertension. Rats were injected with monocrotaline to induce either RV hypertrophy or failure or with saline (control). ECGs were measured in conscious, unrestrained animals by telemetry. In isolated hearts, electrical activity was measured by optical mapping and myofiber orientation by diffusion tensor-MRI. Sarcoplasmic reticular Ca(2+) handling was studied in single myocytes. Compared with control animals, the T-wave of the ECG was prolonged and in three of seven heart failure animals, prominent T-wave alternans occurred. Discordant action potential (AP) alternans occurred in isolated failing hearts and Ca(2+) transient alternans in failing myocytes. In failing hearts, AP duration and dispersion were increased; conduction velocity and AP restitution were steeper. The latter was intrinsic to failing single myocytes. Failing hearts had greater fiber angle disarray; this correlated with AP duration. Failing myocytes had reduced sarco(endo)plasmic reticular Ca(2+)-ATPase activity, increased sarcoplasmic reticular Ca(2+)-release fraction, and increased Ca(2+) spark leak. In hypertrophied hearts and myocytes, dysfunctional adaptation had begun, but alternans did not develop. We conclude that increased electrical and structural heterogeneity and dysfunctional sarcoplasmic reticular Ca(2+) handling increased the probability of alternans, a proarrhythmic predictor of sudden cardiac death. These mechanisms are potential therapeutic targets for the correction of arrhythmias in hypertensive, failing RVs.

Store-operated Ca2+ Entry in Malignant Hyperthermia-susceptible Human Skeletal Muscle

In malignant hyperthermia (MH), mutations in RyR1 underlie direct activation of the channel by volatile anesthetics, leading to muscle contracture and a life-threatening increase in core body temperature. The aim of the present study was to establish whether the associated depletion of sarcoplasmic reticulum (SR) Ca2+ triggers sarcolemmal Ca2+ influx via store-operated Ca2+ entry (SOCE). Samples of vastus medialis muscle were obtained from patients undergoing assessment for MH susceptibility using the in vitro contracture test. Single fibers were mechanically skinned, and confocal microscopy was used to detect changes in [Ca2+] either within the resealed t-system ([Ca2+]t-sys) or within the cytosol. In normal fibers, halothane (0.5 mm) failed to initiate SR Ca2+ release or Ca2+t-sys depletion. However, in MH-susceptible (MHS) fibers, halothane induced both SR Ca2+ release and Ca2+t-sys depletion, consistent with SOCE. In some MHS fibers, halothane-induced SR Ca2+ release took the form of a propagated wave, which was temporally coupled to a wave of Ca2+t-sys depletion. SOCE was potently inhibited by “extracellular” application of a STIM1 antibody trapped within the t-system but not when the antibody was denatured by heating. In conclusion, (i) in human MHS muscle, SR Ca2+ depletion induced by a level of volatile anesthetic within the clinical range is sufficient to induce SOCE, which is tightly coupled to SR Ca2+ release; (ii) sarcolemmal STIM1 has an important role in regulating SOCE; and (iii) sustained SOCE from an effectively infinite extracellular Ca2+ pool may contribute to the maintained rise in cytosolic [Ca2+] that underlies MH.

Diverse mechanisms underlying the regulation of ion channels by carbon monoxide

Carbon monoxide (CO) is firmly established as an important, physiological signalling molecule as well as a potent toxin. Through its ability to bind metal‐containing proteins, it is known to interfere with a number of intracellular signalling pathways, and such actions can account for its physiological and pathological effects. In particular, CO can modulate the intracellular production of reactive oxygen species, NO and cGMP levels, as well as regulate MAPK signalling. In this review, we consider ion channels as more recently discovered effectors of CO signalling. CO is now known to regulate a growing number of different ion channel types, and detailed studies of the underlying mechanisms of action are revealing unexpected findings. For example, there are clear areas of contention surrounding its ability to increase the activity of high conductance, Ca2+‐sensitive K+ channels. More recent studies have revealed the ability of CO to inhibit T‐type Ca2+ channels and have unveiled a novel signalling pathway underlying tonic regulation of this channel. It is clear that the investigation of ion channels as effectors of CO signalling is in its infancy, and much more work is required to fully understand both the physiological and the toxic actions of this gas. Only then can its emerging use as a therapeutic tool be fully and safely exploited.

Characteristics of Prolonged Ca2+ Release Events Associated With the Nuclei in Adult Cardiac Myocytes

Confocal microscopy was used to study the properties of nuclear Ca2+ regulation in adult ventricular myocytes. Prolonged nuclear Ca2+ release (PNCR) events were identified in both intact and permeabilized rat myocytes. PNCR occurred spontaneously and was restricted to localized regions at the ends of the elongated nuclei. Typically, PNCR took the form of a rapid rise in [Ca2+] followed by a maintained plateau. The mean duration of PNCR (1.78±0.19 seconds) was markedly greater than the half decay time for cytosolic Ca2+ sparks (31.2±0.56 ms) obtained under the same conditions. The PNCR width at half maximum amplitude (5.0±0.2 μm) was also significantly greater than that of cytosolic Ca2+ sparks (2.6±0.05 μm) obtained under the same conditions. Experiments involving the use of syto-11 to accurately locate the nuclei demonstrated that PNCR originates from the nuclear envelope or a closely associated structure. The spatial spread of PNCR was asymmetrical, with greater diffusion of Ca2+ toward the center of the nucleus than the cytosol. Both PNCR and Ca2+ sparks were abolished by interventions that deplete SR Ca2+ stores or inhibit RYR activation. Experiments on intact, electrically stimulated cells revealed that diffusion of Ca2+ from the ends of the nucleus toward the center is a prominent feature of the nucleoplasmic Ca2+ transient. The possibility that recruitment of Ca2+ release sites involved in PNCR might influence the temporal and spatial characteristics of the nucleoplasmic [Ca2+] transient is considered.

Effects of cytosolic ATP on spontaneous and triggered Ca2+‐induced Ca2+ release in permeabilised rat ventricular myocytes

The effects of cytosolic ATP on sarcoplasmic reticulum (SR) Ca2+ regulation were investigated in saponin‐permeabilised rat ventricular myocytes. [Ca2+] within the cells was monitored using Fura‐2 or Fluo‐3 fluorescence. Spontaneous cyclic Ca2+ release from the SR was induced by increasing the bathing [Ca2+] to 200–300 nM, in solutions weakly Ca2+ buffered with 0.05 mm EGTA. Alternatively, Ca2+‐induced Ca2+ release (CICR) was triggered by a rapid increase in [Ca2+] induced by flash photolysis of Nitr‐5 (0.08 mm), replacing EGTA in the solution. Stepwise reductions in [ATP] were associated with corresponding decreases in the frequency and increases in the amplitude of spontaneous Ca2+ transients. A decrease from 5 mm to 0.1 mm ATP, reduced the release frequency by 48.6 ± 7 % (n= 7) and almost doubled the amplitude of the Ca2+ transient. Marked prolongation of the spontaneous Ca2+ transient occurred when [ATP] was further reduced to 10 μM, consistent with inhibition of the SR Ca2+ pump. These effects of ATP were compared with other interventions that inhibit Ca2+ uptake or reduce the sensitivity of the SR Ca2+ release mechanism. Inhibition of the SR Ca2+ pump with cyclopiazonic acid (CPA) markedly reduced the spontaneous Ca2+ release frequency, without changing the amplitude. The descending phase of the Ca2+ transient was prolonged in the presence of CPA, while the rising phase was unaffected. In contrast, desensitisation of the SR Ca2+ release mechanism with tetracaine decreased the frequency of spontaneous release, but markedly increased the amplitude. CICR triggered by flash photolysis of Nitr‐5 appeared to be more sensitive to cytosolic [ATP] than spontaneous release and was generally delayed by a decrease to 2.5 mm ATP. In the presence of 0.1‐0.2 mm ATP, release often failed completely or was not consistently triggered. Some preparations exhibited Ca2+ release ‘alternans’, whereby every alternate trigger induced a response. These results suggest that the increase in spontaneous Ca2+ release amplitude and the decrease in frequency that occurs as [ATP] is reduced from 1 mm to 100 μM, is mainly due to desensitisation of the SR Ca2+ release mechanism, which allows the SR Ca2+ content to reach a higher level before release occurs. At very low [ATP], a reduction in the SR Ca2+ uptake rate may also contribute to the decrease in release frequency. CICR triggered by photolysis of Nitr‐5 appeared to be more sensitive to cytosolic [ATP]. The possible underlying mechanisms and the relevance of these results to myocardial ischaemia or hypoxia is considered.