J. Andrew Wasserstrom

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.

Autonomic Remodeling in the Left Atrium and Pulmonary Veins in Heart Failure – Creation of a Dynamic Substrate for Atrial Fibrillation

Background—Atrial fibrillation (AF) is commonly associated with congestive heart failure (CHF). The autonomic nervous system is involved in the pathogenesis of both AF and CHF. We examined the role of autonomic remodeling in contributing to AF substrate in CHF. Methods and Results—Electrophysiological mapping was performed in the pulmonary veins and left atrium in 38 rapid ventricular–paced dogs (CHF group) and 39 control dogs under the following conditions: vagal stimulation, isoproterenol infusion, &bgr;-adrenergic blockade, acetylcholinesterase (AChE) inhibition (physostigmine), parasympathetic blockade, and double autonomic blockade. Explanted atria were examined for nerve density/distribution, muscarinic receptor and &bgr;-adrenergic receptor densities, and AChE activity. In CHF dogs, there was an increase in nerve bundle size, parasympathetic fibers/bundle, and density of sympathetic fibrils and cardiac ganglia, all preferentially in the posterior left atrium/pulmonary veins. Sympathetic hyperinnervation was accompanied by increases in &bgr;1-adrenergic receptor R density and in sympathetic effect on effective refractory periods and activation direction. &bgr;-Adrenergic blockade slowed AF dominant frequency. Parasympathetic remodeling was more complex, resulting in increased AChE activity, unchanged muscarinic receptor density, unchanged parasympathetic effect on activation direction and decreased effect of vagal stimulation on effective refractory period (restored by AChE inhibition). Parasympathetic blockade markedly decreased AF duration. Conclusions—In this heart failure model, autonomic and electrophysiological remodeling occurs, involving the posterior left atrium and pulmonary veins. Despite synaptic compensation, parasympathetic hyperinnervation contributes significantly to AF maintenance. Parasympathetic and/or sympathetic signaling may be possible therapeutic targets for AF in CHF.Background— Atrial fibrillation (AF) is commonly associated with congestive heart failure (CHF). The autonomic nervous system is involved in the pathogenesis of both AF and CHF. We examined the role of autonomic remodeling in contributing to AF substrate in CHF. Methods and Results— Electrophysiological mapping was performed in the pulmonary veins and left atrium in 38 rapid ventricular–paced dogs (CHF group) and 39 control dogs under the following conditions: vagal stimulation, isoproterenol infusion, β-adrenergic blockade, acetylcholinesterase (AChE) inhibition (physostigmine), parasympathetic blockade, and double autonomic blockade. Explanted atria were examined for nerve density/distribution, muscarinic receptor and β-adrenergic receptor densities, and AChE activity. In CHF dogs, there was an increase in nerve bundle size, parasympathetic fibers/bundle, and density of sympathetic fibrils and cardiac ganglia, all preferentially in the posterior left atrium/pulmonary veins. Sympathetic hyperinnervation was accompanied by increases in β1-adrenergic receptor R density and in sympathetic effect on effective refractory periods and activation direction. β-Adrenergic blockade slowed AF dominant frequency. Parasympathetic remodeling was more complex, resulting in increased AChE activity, unchanged muscarinic receptor density, unchanged parasympathetic effect on activation direction and decreased effect of vagal stimulation on effective refractory period (restored by AChE inhibition). Parasympathetic blockade markedly decreased AF duration. Conclusions— In this heart failure model, autonomic and electrophysiological remodeling occurs, involving the posterior left atrium and pulmonary veins. Despite synaptic compensation, parasympathetic hyperinnervation contributes significantly to AF maintenance. Parasympathetic and/or sympathetic signaling may be possible therapeutic targets for AF in CHF.

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.

Heart failure – a challenge to our current concepts of excitation–contraction coupling

Development of novel therapeutic strategies for congestive heart failure (CHF) seems to be hampered by insufficient knowledge of the molecular machinery of excitation‐contraction (EC) coupling in both normal and failing hearts. Cardiac hypertrophy and failure represent a multitude of cardiac phenotypes, and available invasive and non‐invasive techniques, briefly reviewed here, allow proper quantification of myocardial function in experimental models even in rats and mice. Both reduced fractional shortening and reduced velocity of contraction characterize myocardial failure. Only when myocardial function is depressed in vivo can meaningful studies be done in vitro of contractility and EC coupling. Also, we point out potential limitations with the whole cell patch clamp technique. Two main factors stand out as explanations for myocardial failure. First, a basic feature of CHF seems to be a reduced Ca2+ load of the sarcoplasmic reticulum (SR) mainly due to a low phosphorylation level of phospholamban. Second, there seems to be a defect of the trigger mechanism of Ca2+ release from the SR. We argue that this defect only becomes manifest in the presence of reduced Ca2+ reuptake capacity of the SR and that it may not be solely attributable to reduced gain of the Ca2+‐induced Ca2+ release (CICR). We list several possible explanations for this defect that represent important avenues for future research.

Reduction in Hexokinase II Levels Results in Decreased Cardiac Function and Altered Remodeling After Ischemia/Reperfusion Injury

Rationale: Cardiomyocytes switch substrate utilization from fatty acid to glucose under ischemic conditions; however, it is unknown how perturbations in glycolytic enzymes affect cardiac response to ischemia/reperfusion (I/R). Hexokinase (HK)II is a HK isoform that is expressed in the heart and can bind to the mitochondrial outer membrane. Objective: We sought to define how HKII and its binding to mitochondria play a role in cardiac response and remodeling after I/R. Methods and Results: We first showed that HKII levels and its binding to mitochondria are reduced 2 days after I/R. We then subjected the hearts of wild-type and heterozygote HKII knockout (HKII+/−) mice to I/R by coronary ligation. At baseline, HKII+/− mice have normal cardiac function; however, they display lower systolic function after I/R compared to wild-type animals. The mechanism appears to be through an increase in cardiomyocyte death and fibrosis and a reduction in angiogenesis; the latter is through a decrease in hypoxia-inducible factor–dependent pathway signaling in cardiomyocytes. HKII mitochondrial binding is also critical for cardiomyocyte survival, because its displacement in tissue culture with a synthetic peptide increases cell death. Our results also suggest that HKII may be important for the remodeling of the viable cardiac tissue because its modulation in vitro alters cellular energy levels, O2 consumption, and contractility. Conclusions: These results suggest that reduction in HKII levels causes altered remodeling of the heart in I/R by increasing cell death and fibrosis and reducing angiogenesis and that mitochondrial binding is needed for protection of cardiomyocytes.

Ultrastructural and cellular basis for the development of abnormal myocardial mechanics during the transition from hypertension to heart failure

Although the development of abnormal myocardial mechanics represents a key step during the transition from hypertension to overt heart failure (HF), the underlying ultrastructural and cellular basis of abnormal myocardial mechanics remains unclear. We therefore investigated how changes in transverse (T)-tubule organization and the resulting altered intracellular Ca(2+) cycling in large cell populations underlie the development of abnormal myocardial mechanics in a model of chronic hypertension. Hearts from spontaneously hypertensive rats (SHRs; n = 72) were studied at different ages and stages of hypertensive heart disease and early HF and were compared with age-matched control (Wistar-Kyoto) rats (n = 34). Echocardiography, including tissue Doppler and speckle-tracking analysis, was performed just before euthanization, after which T-tubule organization and Ca(2+) transients were studied using confocal microscopy. In SHRs, abnormalities in myocardial mechanics occurred early in response to hypertension, before the development of overt systolic dysfunction and HF. Reduced longitudinal, circumferential, and radial strain as well as reduced tissue Doppler early diastolic tissue velocities occurred in concert with T-tubule disorganization and impaired Ca(2+) cycling, all of which preceded the development of cardiac fibrosis. The time to peak of intracellular Ca(2+) transients was slowed due to T-tubule disruption, providing a link between declining cell ultrastructure and abnormal myocardial mechanics. In conclusion, subclinical abnormalities in myocardial mechanics occur early in response to hypertension and coincide with the development of T-tubule disorganization and impaired intracellular Ca(2+) cycling. These changes occur before the development of significant cardiac fibrosis and precede the development of overt cardiac dysfunction and HF.

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.

Mechanisms Underlying the Formation and Dynamics of Subcellular Calcium Alternans in the Intact Rat Heart

Optical mapping of intact cardiac tissue reveals that, in some cases, intracellular calcium (Ca) release can alternate from one beat to the next in a large-small-large sequence, also referred to as Ca transient (CaT) alternans. CaT alternans can also become spatially phase-mismatched within a single cell, when one part of the cell alternates in a large-small-large sequence, whereas a different part alternates in a small-large-small sequence, a phenomenon known as subcellular discordant alternans. The mechanisms for the formation and spatiotemporal evolution of these phase-mismatched patterns are not known. We used confocal Ca imaging to measure CaT alternans at the sarcomeric level within individual myocytes in the intact rat heart. After a sudden change in cycle length (CL), 2 distinct spatial patterns of CaT alternans emerge. CaTs can form spatially phase-mismatched alternans patterns after the first few beats following the change in CL. The phase mismatch persists for many beats, after which it gradually becomes phase matched via the movement of nodes, which are junctures between phase-mismatched cell regions. In other examples, phase-matched alternans gradually become phase-mismatched, via the formation and movement of nodes. In these examples, we observed large beat-to-beat variations in the cell activation times, despite constant CL pacing. Using computer simulations, we explored the underlying mechanisms for these dynamical phenomena. Our results show how heterogeneity at the sarcomeric level, in conjunction with the dynamics of Ca cycling and membrane voltage, can lead to complex spatiotemporal phenomena within myocytes of the intact heart.

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.

An analysis of lidocaine block of sodium current in isolated human atrial and ventricuiar myocytes

Lidocaine is a Na+ channel blocker that is highly effective for the treatment of ventricular tachyarrhythmias, but is largely ineffective against atrial arrhythmias. If is not known if this differential efficacy is the result of differences in lidocaine inhibition of atrial v ventricular Na+ channels. The purpose of the present study was to characterize lidocaine block of Na+ channels in human atrium and ventricle. We used the whole cell voltage clamp technique with low external and internal Na+ concentrations (5 mM) to study the Na+ current (INa) in single human atrial and ventricular cells isolated enzymatically from specimens obtained during surgery. We found that tonic block of peak INa by lidocaine (200 microM, holding potential = -140 mV, 0.1 Hz, at 17 degrees C) was not voltage dependent in either cell type. Reduction of maximal peak Na+ conductance in 41 atrial cells (19.8 +/- 2.7%) and nine ventricular cells (22.6 +/- 1.7%) was virtually identical. The rate of onset of block development was determined during depolarization to either -80 mV or -20 mV. The time course of onset of block was described by a single exponential at -80 mV and by a double exponential at -20 mV. When the rate of block onset during a single conditioning depolarization was compared to that which developed during conditioning by a train of brief pulses (3 ms, 30 Hz), onset was faster during the pulse train. The results were nearly identical for atrial and ventricular INa. The time constants of recovery from block following either single pulse or multiple-pulse conditioning did not differ. These data suggest that lidocaine binds to both the activated and inactivated states of the human cardiac Na+ channel. Using an analytical method based upon the Guarded Receptor Hypothesis, we calculated apparent rate constants describing lidocaines interaction with the three primary states of the human Na+ channel (resting, activated and inactivated). Rate constants were similar to those reported for other mammalian species. Our results demonstrate that lidocaine block of INa is virtually identical for human atrial and ventricular cells; thus additional mechanisms must be invoked to explain the differential efficacy of lidocaine against ventricular as compared to atrial dysrhythmias.