James E. Kelly

Pacing-induced Heterogeneities in Intracellular Ca2+ Signaling, Cardiac Alternans, and Ventricular Arrhythmias in Intact Rat Heart

Optical mapping studies have suggested that intracellular Ca2+ and T-wave alternans are linked through underlying alternations in Ca2+ cycling-inducing oscillations in action potential duration through Ca2+-sensitive conductances. However, these studies cannot measure single-cell behavior; therefore, the Ca2+ cycling heterogeneities within microscopic ventricular regions are unknown. The goal of this study was to measure cellular activity in intact myocardium during rapid pacing and arrhythmias. We used single-photon laser-scanning confocal microscopy to measure Ca2+ signaling in individual myocytes of intact rat myocardium during rapid pacing and during pacing-induced ventricular arrhythmias. At low rates, all myocytes demonstrate Ca2+ alternans that is synchronized but whose magnitude varies depending on recovery kinetics of Ca2+ cycling for each individual myocyte. As rate increases, some cells reverse alternans phase, giving a dyssynchronous activation pattern, even in adjoining myocytes. Increased pacing rate also induces subcellular alternans where Ca2+ alternates out of phase with different regions within the same cell. These forms of heterogeneous Ca2+ signaling also occurred during pacing-induced ventricular tachycardia. Our results demonstrate highly nonuniform Ca2+ signaling among and within individual myocytes in intact heart during rapid pacing and arrhythmias. Thus, certain pathophysiological conditions that alter Ca2+ cycling kinetics, such as heart failure, might promote ventricular arrhythmias by exaggerating these cellular heterogeneities in Ca2+ signaling.

Variability in timing of spontaneous calcium release in the intact rat heart is determined by the time course of sarcoplasmic reticulum calcium load.

Background: Abnormalities in intracellular calcium (Ca) cycling during Ca overload can cause triggered activity because spontaneous calcium release (SCR) activates sufficient Ca-sensitive inward currents to induce delayed afterdepolarizations (DADs). However, little is known about the mechanisms relating SCR and triggered activity on the tissue scale. Methods and Results: Laser scanning confocal microscopy was used to measure the spatiotemporal properties of SCR within large myocyte populations in intact rat heart. Computer simulations were used to predict how these properties of SCR determine DAD magnitude. We measured the average and standard deviation of the latency distribution of SCR within a large population of myocytes in intact tissue. We found that as external [Ca] is increased, and with faster pacing rates, the average and SD of the latency distribution decreases substantially. This result demonstrates that the timing of SCR occurs with less variability as the sarcoplasmic reticulum (SR) Ca load is increased, causing more sites to release Ca within each cell. We then applied a mathematical model of subcellular Ca cycling to show that a decrease in SCR variability leads to a higher DAD amplitude and is dictated by the rate of SR Ca refilling following an action potential. Conclusions: Our results demonstrate that the variability of the timing of SCR in a population of cells in tissue decreases with SR load and is dictated by the time course of the SR Ca content.

Multiple Defects in Intracellular Calcium Cycling in Whole Failing Rat Heart

Background—A number of defects in excitation-contraction coupling have been identified in failing mammalian hearts. The goal of this study was to measure the defects in intracellular Ca2+ cycling in left ventricular epicardial myocytes of the whole heart in an animal model of congestive heart failure (CHF). Methods and Results—Intracellular Ca2+ transients were measured using confocal microscopy in whole rat hearts from age-matched Wistar-Kyoto control rats and spontaneously hypertensive rats at ≈23 months of age. Basal Ca2+ transients in myocytes in spontaneously hypertensive rats were smaller in amplitude and longer in duration than Wistar-Kyoto control rats. There was also greater variability in transient characteristics associated with duration between myocytes of CHF than Wistar-Kyoto controls. Approximately 21% of CHF myocytes demonstrated spontaneous Ca2+ waves compared with very little of this activity in Wistar-Kyoto control rats. A separate population of spontaneously hypertensive rat myocytes showed Ca2+ waves that were triggered during pacing and were absent at rest (triggered waves). Rapid pacing protocols caused Ca2+ alternans to develop at slower heart rates in CHF. Conclusions—Epicardial cells demonstrate both serious defects and greater cell-to-cell variability in Ca2+ cycling in CHF. The defects in Ca2+ cycling include both spontaneous and triggered waves of Ca2+ release, which promote triggered activity. The slowing of Ca2+ repriming in the sarcoplasmic reticulum is probably responsible for the increased vulnerability to Ca2+ alternans in CHF. Our results suggest that defective Ca2+ cycling could contribute both to reduced cardiac output in CHF and to the establishment of repolarization gradients, thus creating the substrate for reentrant arrhythmias.

Ranolazine Antagonizes the Effects of Increased Late Sodium Current on Intracellular Calcium Cycling in Rat Isolated Intact Heart

Pathological conditions, including ischemia and heart failure, are associated with altered sodium channel function and increased late sodium current (INa,L), leading to prolonged action potential duration, increased intracellular sodium and calcium concentrations, and arrhythmias. We used anemone toxin (ATX)-II to study the effects of increasing INa,L on intracellular calcium cycling in rat isolated hearts. Cardiac contraction was abolished using paralytic agents. Ranolazine (RAN) was used to inhibit late INa. Hearts were loaded with fluo-4-acetoxymethyl ester, and myocyte intracellular calcium transients (CaTs) were measured using laser scanning confocal microscopy. ATX (1 nM) prolonged CaT duration at 50% recovery in hearts paced at a basal rate of 2 Hz and increased the sensitivity of the heart to the development of calcium alternans caused by fast pacing. ATX increased the time required for recovery of CaT amplitude following a previous beat, and ATX induced spontaneous calcium release waves during rapid pacing of the heart. ATX prolonged the duration of repolarization from the initiation of the activation to terminal repolarization in the pseudo-electrocardiogram. All actions of ATX were both reversed and prevented by subsequent or prior exposure, respectively, of hearts to RAN (10 μM). Most importantly, the increased vulnerability of the heart to the development of calcium alternans during rapid pacing was reversed or prevented by 10 μM RAN. These results suggest that enhancement of INa,L alters calcium cycling. Reduction by RAN of INa,L-induced dysregulation of calcium cycling could contribute to the antiarrhythmic actions of this agent in both reentrant and triggered arrhythmias.

Acidosis and ischemia increase cellular Ca2+ transient alternans and repolarization alternans susceptibility in the intact rat heart

Cardiac cellular Ca(2+) transient (CaT) alternans and electrocardiographic T-wave alternans (TWA) often develop in myocardial ischemia, but the mechanisms for this relationship have not been elucidated. Acidosis is a major component of ischemia, but there is no direct evidence linking acidosis-induced cellular CaT alternans to ischemia-induced CaT alternans and TWA in whole heart. We used laser-scanning confocal microscopy to measure intracellular Ca(2+) (Ca(i)(2+)) cycling in individual myocytes of fluo-4 AM-loaded rat hearts and simultaneously recorded pseudo-ECGs to investigate changes in CaTs and late-phase repolarization, respectively, during baseline and rapid pacing under control and either globally acidic or globally ischemic conditions. Acidosis (hypercapnia; pH 6.6) increased diastolic Ca(i)(2+) levels, prolonged CaT duration, and shifted to slower heart rates both the development of pacing-induced acidosis-induced CaT alternans (both concordant and discordant) and of repolarization alternans (RPA, a measure of TWA in rat ECGs). The magnitudes of these shifts were equivalent for both CaT alternans and RPA, suggesting a close association between them. Nearly identical results were found in low-flow global ischemia. Additionally, ischemic preconditioning reduced the increased propensity for CaT alternans and RPA development and was mimicked by preconditioning by acidosis alone. Our results demonstrate that global acidosis or ischemia modifies Ca(i)(2+) cycling in myocytes such that the diastolic Ca(i)(2+) rises and the cellular CaT duration is prolonged, causing spatially concordant as well as spatially discordant cellular CaT alternans to develop at slower heart rates than in controls. Since RPA also developed at slower heart rates, our results suggest that acidosis is a major contributor to CaT alternans, which underlies the proarrhythmic state induced by myocardial ischemia and therefore may play a role in its modulation and prevention.

Characteristics of intracellular Ca2+ cycling in intact rat heart: a comparison of sex differences

Males and females show distinct differences in action potential waveform, ion channel expression patterns, and ECG characteristics. However, it is not known how sex-based differences in Ca2+ cycling might contribute to these differences in electrophysiological activity. The goal of this study was to investigate the differences in cellular Ca2+ transients in males and females and to examine how these might contribute to electrophysiological function. Ca2+ transients were measured in individual myocytes within microscopic regions of the fluo-4 AM-loaded left ventricular epicardium of intact rat heart of both sexes (3 to 5 mo old). Pacing protocols were used to measure transient characteristics at a basic cycle length of 500 ms and during 10-s trains of rapid pacing delivered to the left ventricular apex. Ca2+ transients were smaller in magnitude and longer in duration in females than in males. More importantly, the variability in Ca2+ transient characteristics between myocytes in a microscopic recording site (heterogeneity index) was greater for females than males for characteristics related to transient duration. The rate sensitivity of Ca2+ alternans development in individual myocytes was greater in females than in males, but there was also a greater heterogeneity in cellular responses to the rate dependence of alternans development in females. The longer Ca2+ transients in females were also associated with slower restitution, which was likely to be responsible for the development of Ca2+ and repolarization alternans at slower heart rates. These results demonstrate that there are distinct differences in cellular Ca2+ cycling in male and female rat hearts. Not only is there slower reuptake of Ca2+ in female rats, but there is greater local variability in Ca2+ cycling at the microscopic level. These sex-based differences in Ca2+ cycling could contribute to differences in ECG morphology and in arrhythmia sensitivity in males and females.

Early development of intracellular calcium cycling defects in intact hearts of spontaneously hypertensive rats.

Defects in excitation-contraction coupling have been reported in failing hearts, but little is known about the relationship between these defects and the development of heart failure (HF). We compared the early changes in intracellular Ca(2+) cycling to those that underlie overt pump dysfunction and arrhythmogenesis found later in HF. Laser-scanning confocal microscopy was used to measure Ca(2+) transients in myocytes of intact hearts in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) at different ages. Early compensatory mechanisms include a positive inotropic effect in SHRs at 7.5-9 mo compared with 6 mo. Ca(2+) transient duration increased at 9 mo in SHRs, indicating changes in Ca(2+) reuptake during decompensation. Cell-to-cell variability in Ca(2+) transient duration increased at 7.5 mo, decreased at 9 mo, and increased again at 22 mo (overt HF), indicating extensive intercellular variability in Ca(2+) transient kinetics during disease progression. Vulnerability to intercellular concordant Ca(2+) alternans increased at 9-22 mo in SHRs and was mirrored by a slowing in Ca(2+) transient restitution, suggesting that repolarization alternans and the resulting repolarization gradients might promote reentrant arrhythmias early in disease development. Intercellular discordant and subcellular Ca(2+) alternans increased as early as 7.5 mo in SHRs and may also promote arrhythmias during the compensated phase. The incidence of spontaneous and triggered Ca(2+) waves was increased in SHRs at all ages, suggesting a higher likelihood of triggered arrhythmias in SHRs compared with WKY rats well before HF develops. Thus serious and progressive defects in Ca(2+) cycling develop in SHRs long before symptoms of HF occur. Defective Ca(2+) cycling develops early and affects a small number of myocytes, and this number grows with age and causes the transition from asymptomatic to overt HF. These defects may also underlie the progressive susceptibility to Ca(2+) alternans and Ca(2+) wave activity, thus increasing the propensity for arrhythmogenesis in HF.

Modification of cardiac Na+ channels by batrachotoxin: effects on gating, kinetics, and local anesthetic binding.

The purpose of the present study was to examine the characteristics of Na+ channel modification by batrachotoxin (BTX) in cardiac cells, including changes in channel gating and kinetics as well as susceptibility to block by local anesthetic agents. We used the whole cell configuration of the patch clamp technique to measure Na+ current in guinea pig myocytes. Extracellular Na+ concentration and temperature were lowered (5-10 mM, 17 degrees C) in order to maintain good voltage control. Our results demonstrated that 1) BTX modifies cardiac INa, causing a substantial steady-state (noninactivating) component of INa, 2) modification of cardiac Na+ channels by BTX shifts activation to more negative potentials and reduces both maximal gNa and selectivity for Na+; 3) binding of BTX to its receptor in the cardiac Na+ channel reduces the affinity of local anesthetics for their binding site; and 4) BTX-modified channels show use-dependent block by local anesthetics. The reduced blocking potency of local anesthetics for BTX-modified Na+ channels probably results from an allosteric interaction between BTX and local anesthetics for their respective binding sites in the Na+ channel. Our observations that use-dependent block by local anesthetics persists in BTX-modified Na+ channels suggest that this form of extra block can occur in the virtual absence of the inactivated state. Thus, the development of use-dependent block appears to rely primarily on local anesthetic binding to activated Na+ channels under these conditions.

Characteristics of cocaine block of purified cardiac sarcoplasmic reticulum calcium release channels

We have examined the effects of cocaine on the SR Ca2+ release channel purified from canine cardiac muscle. Cocaine induced a flicker block of the channel from the cytoplasmic side, which resulted in an apparent reduction in the single-channel current amplitude without a marked reduction in the single-channel open probability. This block was evident only at positive holding potentials. Analysis of the block revealed that cocaine binds to a single site with an effective valence of 0.93 and an apparent dissociation constant at 0 mV (Kd(0)) of 38 mM. The kinetics of cocaine block were analyzed by amplitude distribution analysis and showed that the voltage and concentration dependence lay exclusively in the blocking reaction, whereas the unblocking reaction was independent of both voltage and concentration. Modification of the channel by ryanodine dramatically attenuated the voltage and concentration dependence of the on rates of cocaine block while diminishing the off rates to a lesser extent. In addition, ryanodine modification changed the effective valence of cocaine block to 0.52 and the Kd(0) to 110 mM, suggesting that modification of the channel results in an alteration in the binding site and its affinity for cocaine. These results suggest that cocaine block of the SR Ca2+ release channel is due to the binding at a single site within the channel pore and that modification of the channel by ryanodine leads to profound changes in the kinetics of cocaine block.

Modification of cardiac Na+ channels by anthopleurin-A: effects on gating and kinetics

We used the whole cell patch clamp technique to investigate the characteristics of modification of cardiac Na+ channel gating by the sea anemone polypeptide toxin anthopleurin-A (AP-A). Guinea pig ventricular myocytes were isolated enzymatically using a retrograde perfusion apparatus. Holding potential was −140 mV and test potentials ranged from −100 to + 40 mV (pulse duration 100 or 1000 ms). AP-A (50–100 nM) markedly slowed the rate of decay of Na+ current (INa) and increased peak INa conductance (gNa) by 38±5.5% (mean±SEM, P < 0.001, n = 12) with little change in slope factor (n = 12) or voltage midpoint of the gNa/V relationship after correction for spontaneous shifts. The voltage dependence of steady-state INa availability (h∞) demonstrated an increase in slope factor from 5.9±0.8 mV in control to 8.0±0.7 mV after modification by AP-A (P < 0.01, n = 14) whereas any shift in the voltage midpoint of this relationship could be accounted for by a spontaneous time-dependent shift. AP-A-modified INa showed a use-dependent decrease in peak current amplitude (interpulse interval 500 ms) when pulse duration was 100 ms (−15±2%, P < 0.01, n = 17) but showed no decline when pulse duration was 100 ms (−3±1%). This use-dependent effect was probably the result of a decrease in the rate of recovery from inactivation caused by AP-A which had a small effect on the fast time constant of recovery (from 4.1±0.3 ms in control to 6.0±1.1 ms after AP-A, P < 0.05) but increased the slow time constant from 66.2±6.5 ms in control to 188.9±36.4 ms (P< 0.002, n = 19) after exposure to AP-A. Increasing external divalent cation concentration (either Ca2+ or Mg2+) to 10 mM abolished the effects of AP-A on the rate of INa decay. These results demonstrate that modification of cardiac Na+ channels by AP-A markedly slowed INa inactivation and altered the voltage dependence of activation; these alterations in gating characteristics, in turn, caused an increase in gNa presumably by increasing the number of channels open at peak INa. AP-A slows the rate of recovery of INa from inactivation which is probably the basis for a use-dependent decrease in peak amplitude. Finally, AP-A binding is sensitive to external divalent cation concentrations. Thus, increasing [Mg2+]o or [Ca2+]o displaces AP-A from binding, suggesting that they share related binding sites on the external surface of the Na+ channel.