Zhaokang Yang

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.

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.

REGIONAL DIFFERENCES IN THE NEGATIVE INOTROPIC EFFECT OF ACETYLCHOLINE WITHIN THE CANINE VENTRICLE

1. Regional differences in the effects of ACh on sub‐epicardial, mid‐wall and sub‐endocardial cells of the dog left ventricle have been studied. 2. ACh produced a dose‐dependent, atropine‐sensitive negative inotropic effect that was greatest in sub‐epicardial cells and small or absent in sub‐endocardial cells. 3. In sub‐epicardial (but not sub‐endocardial) cells, ACh also resulted in a dose‐dependent decrease in action potential duration. The inotropic effect of ACh on sub‐epicardial cells was primarily the result of the decrease of action potential duration, because during trains of voltage clamp pulses the inotropic effect of ACh was reduced or abolished. At a holding potential of ‐80 mV, 10(‐5)M ACh decreased L‐type Ca2+ current by approximately 8% and this is thought to be responsible for the small inotropic effect during trains of pulses. 4. Although 4‐AP, a blocker of the transient outward current (I(to)), abolished the ‘spike and dome’ morphology of the sub‐epicardial action potential, it had little or no effect on the actions of ACh on sub‐epicardial cells. ACh had no effect on I(to) in sub‐epicardial cells in voltage clamp experiments. 5. ACh activated a Ba(2+)‐sensitive outward current (IK,ACh) in sub‐epicardial cells, but little or no such current in sub‐endocardial cells. In sub‐epicardial cells, ACh also inhibited the inward rectifier current, IK,1. 6. It is concluded that in left ventricular sub‐epicardial cells, ACh activates IK,ACh. This results in a shortening of the action potential and, therefore, a negative inotropic effect. In subendocardial cells, ACh activates little or no IK,ACh and, therefore, it has little or no negative inotropic effect. This may result from a regional variation in the expression of the muscarinic K+ channel.

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.

Effects of phosphocreatine on SR Ca2+ regulation in isolated saponin‐permeabilized rat cardiac myocytes

The effects of phosphocreatine (PCr) on sarcoplasmic reticulum (SR) Ca2+ regulation were investigated in saponin‐permeabilized rat ventricular myocytes. Cells were perfused continuously with weakly Ca2+‐buffered solutions approximating to the intracellular milieu. Ca2+ release from the SR was detected using Fura‐2 or Fluo‐3. Withdrawal of PCr reduced the frequency of spontaneous Ca2+ release by 12.8 ± 3.4 % (n= 9) and the amplitude of the spontaneous Ca2+ transient by 17.4 ± 3.1 % (n= 9). Stepwise reductions in [PCr] progressively increased the time for the spontaneous Ca2+ transient to rise from 25 to 100 % of the maximum value (TP75) and to fall by 75 % of the peak level (DT75). Following complete PCr withdrawal, the TP75 and the DT75 were 147.1 ± 13.2 and 174.8 ± 23.2 % of the control values, respectively. Experiments involving confocal microscopy showed that PCr withdrawal decreased the propagation velocity of spontaneous Ca2+ waves. PCr withdrawal also reduced the frequency and amplitude, but increased the duration of spontaneous Ca2+ sparks. Rapid application of 20 mm caffeine was used to assess the SR Ca2+ content at the point of spontaneous Ca2+ release. In the absence of PCr, the amplitude of the caffeine‐induced Ca2+ transient was 18.4 ± 2.7 % (n= 9) lower than in the presence of 10 mm PCr. This suggests that PCr withdrawal reduces the maximum SR Ca2+ content that can be sustained before spontaneous Ca2+ release occurs. These results suggest that local ADP buffering by PCr is essential for normal Ca2+ regulation by the SR. Prolongation of the descending phase of the spontaneous Ca2+ transient is consistent with a reduction in the efficiency of the SR Ca2+ pump due to ADP accumulation. The fact that spontaneous Ca2+ release occurs at a lower SR Ca2+ content in the absence of PCr suggests that the Ca2+ release mechanism may also be affected. These effects may be of relevance in circumstances where PCr depletion and Ca2+ overload occur, such as myocardial ischaemia or anoxia.

The Golgi apparatus is a functionally distinct Ca2+ store regulated by the PKA and Epac branches of the β1-adrenergic signaling pathway

Calcium released from the Golgi apparatus may promote receptor trafficking to the surface of cardiomyocytes. A distinct calcium store in cardiomyocytes In cardiomyocytes, contraction can be triggered by large influxes of calcium (Ca2+) into the cytoplasm from the sarcoplasmic reticulum. Yang et al. found that Ca2+ released from the Golgi apparatus had a distinct function from the Ca2+ stored in the sarcoplasmic reticulum. Instead of triggering contraction, Golgi-released Ca2+ promoted the trafficking of a receptor to the surface of cardiomyocytes. The adrenaline receptor β1-AR enhanced the release of Ca2+ from the Golgi through a process involving two pathways downstream of the second messenger cAMP. Cardiomyocytes from rats with experimentally induced heart failure showed increased release of Ca2+ from the Golgi, which was associated with decreased abundance of phosphodiesterases in the PDE3 and PDE4 families, which degrade cAMP. Thus, the Golgi apparatus releases Ca2+ in response to changes in cAMP concentrations, such as those that occur during adrenergic stimulation or heart failure. Ca2+ release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. We found that the Golgi apparatus was the source of prolonged Ca2+ release events that originated near the nuclei of primary cardiomyocytes. Golgi Ca2+ release was unaffected by depletion of sarcoplasmic reticulum Ca2+, and disruption of the Golgi apparatus abolished Golgi Ca2+ release without affecting sarcoplasmic reticulum function, suggesting functional and spatial independence of Golgi and sarcoplasmic reticulum Ca2+ stores. β1-Adrenoceptor stimulation triggers the production of the second messenger cAMP, which activates the Epac family of Rap guanine nucleotide exchange factors and the kinase PKA (protein kinase A). Phosphodiesterases (PDEs), including those in the PDE3 and PDE4 families, degrade cAMP. Activation of β1-adrenoceptors stimulated Golgi Ca2+ release, an effect that required activation of Epac, PKA, and the kinase CaMKII. Inhibition of PDE3s or PDE4s potentiated β1-adrenergic–induced Golgi Ca2+ release, which is consistent with compartmentalization of cAMP signaling near the Golgi apparatus. Interventions that stimulated Golgi Ca2+ release appeared to increase the trafficking of vascular endothelial growth factor receptor–1 (VEGFR-1) from the Golgi apparatus to the surface membrane of cardiomyocytes. In cardiomyocytes from rats with heart failure, decreases in the abundance of PDE3s and PDE4s were associated with increased Golgi Ca2+ release events. These data suggest that the Golgi apparatus is a focal point for β1-adrenergic–stimulated Ca2+ signaling and that the Golgi Ca2+ store functions independently from the sarcoplasmic reticulum and the global Ca2+ transients that trigger contraction in cardiomyocytes.

Simvastatin activates single skeletal RyR1 channels but exerts more complex regulation of the cardiac RyR2 isoform: Simvastatin modulates RyR1 and RyR2 channel gating

Statins are amongst the most widely prescribed drugs for those at risk of cardiovascular disease, lowering cholesterol levels by inhibiting 3‐hydroxy‐3‐methylglutaryl (HMG)‐CoA reductase. Although effective at preventing cardiovascular disease, statin use is associated with muscle weakness, myopathies and, occasionally, fatal rhabdomyolysis. As simvastatin, a commonly prescribed statin, promotes Ca2+ release from sarcoplasmic reticulum (SR) vesicles, we investigated if simvastatin directly activates skeletal (RyR1) and cardiac (RyR2) ryanodine receptors.