Cardiac alternans and ventricular fibrillation: a bad case of ryanodine receptors reneging on their duty

Since the first description in 1872 of cardiac alternans by L. Traube in a patient with alcoholic cardiomyopathy,¹ there has been significant progress in understanding the significance of this clinical sign of heart disease. Traube reported this phenomenon before Einthoven invented the electrocardiogram (ECG) in 1903. Thus, he actually described pulsus alternans in his patient, a strong-weak arterial pulse alternation perceptible by unaided finger tact. The patient died shortly after diagnosis, but since he suffered of severe cardiomyopathy, cardiac alternans was not recognized as an ancillary index of disease severity or as a harbinger of mortality. Later, the introduction of ECGs in the routine examination of patients made it clear that cardiac alternans, in the form of T wave alternans (TW-Alt), was a common sign in several cardiomyopathies such as heart failure, coronary artery disease, genetic and acquired channelopathies, and even in electrolyte disturbances of the body. However, an unequivocal association between cardiac alternans and arrhythmia risk was not recognized until the 1990s,² and only recently a multicenter clinical trial established that patients with moderate cardiac dysfunction but lacking TW-Alts may not need an implantable cardiac defibrillator (ICD) to improve their odds of avoiding sudden cardiac death (SCD).³ Thus, from the peculiar weak-strong arterial stroke oscillation detected by Traube to a critical risk stratification factor for SCD, cardiac alternans have come a long way as diagnostic and prognostic manifestation of cardiac disease.

If the pathway of cardiac alternans in the clinical arena has been a smoothly ascending line, explaining its precise cellular mechanisms has resulted in a more tortuous process, reflecting the complexity of the phenomenon.The ability to induce mechanical alternans by rapidly stimulating the heart was recognized early as an inherent ability of all mammalian ventricular muscles (reviewed in ref. 4). Initially, researchers relied on traditional whole heart physiology and explained cardiac alternans based on the Frank-Starling relationship, wherein a strong beat, by expelling more blood, leaves a small residual end-diastolic volume, in turn reducing force development in the next beat. During the weak beat, the end-systolic volume increases due to decreased ejection, leading to a greater end-diastolic volume and thus stronger force in the next beat. However, it was quickly realized that cardiac alternans were more complex than apparently straightforward load-force relationships, as papillary muscles displayed alternans when contracting under a constant load (isotonically) or length (isometrically). Since isometric contractions could be observed in isolated ventricular myocytes,⁵ too, it was therefore inferred that mechanisms intrinsic to the cardiac cell must account for the genesis of cardiac alternans. This conceptual framework was forged before the widespread use of intracellular Ca²⁺ imaging, and when Fura-2 and other fluorescent indicators irrupted in the scene, it became evident that the alternation in the force of contraction was mirrored with amazing faithfulness by alternations in the magnitude of the Ca²⁺ transient, or Ca²⁺ alternans (Ca-Alts). Now in the realm of sub-cellular mechanisms, Ca²⁺ alternans were first explained by a delay of Ca²⁺ transport from reuptake sites to release sites,⁵ but this idea has not gained traction as it has become increasingly evident that Ca²⁺ diffusion between these two compartments in a sarcomere is extremely fast.⁶ Instead, the availability of Ca²⁺in the release sites (through the work of SERCA2a), more than diffusion from reuptake sites, was favored as a likely explanation for Ca-Alts.^7,8 We will discuss now new data indicating that this limitation, SR Ca²⁺ load, is unlikely to be the first critical factor in the generation of Ca-Alts and their progression to ventricular fibrillation.⁹

Since Ca-Alts may be detected in the absence of L-type Ca²⁺ current alternations and are abolished by ryanodine, there is compelling evidence that Ca-Alts are generated by SR behavior.^10,11,12 With the focus squarely on this single organelle, the quest now is to delineate the hierarchical role of cardiac ryanodine receptors (RyR2) and the Ca²⁺-ATPase (SERCA2a) as molecular instigators of Ca-Alts. Thus, in historical terms, we are back to the former question, but with a molecular twist: is an intrinsic dysfunction of RyR2, or an alternating reduction of end-diastolic SR Ca²⁺ load (caused by an insufficient SERCA2a), that first intervenes to generate Ca-Alts? This central problem is elegantly addressed by Wang et al.⁹ in the present issue of Circulation Research. Although studies with isolated ventricular cells may provide detailed examination of SR Ca²⁺ dynamics and RyR2 channel behavior, they are inherently incapable of providing the heterogeneity and complexity that sustains arrhythmogenic behavior such as spatially discordant alternans and other tissue-level phenomena. Thus, Wang et al.⁹ developed a novel and sophisticated dual optical mapping strategy that allowed them to simultaneously measure action potential (AP) and intra-SR Ca²⁺ ([Ca²⁺]_SR)dynamics in epicardial layers of Langendorff-perfused intact rabbit hearts. Paired with electrocardiographic recordings, these technically challenging experiments stretched the physiological limitations of the system: hearts were pharmacologically immobilized to reduce the energetic demand and prevent motion artifacts, and loaded with fluorescent dyes that absorbed light in the same spectral range but that have emission wavelengths apart from each other (RH237 for membrane potential and Fluo-5N for intra-SR Ca²⁺). Because the authors used hearts free from pathological conditions that normally favor the spontaneous appearance of cardiac alternans, the latter had to be induced by rapidly stimulating the apex of the heart, which necessarily short-circuits its normal conduction pathway. Nevertheless, there is much to be learned even from this not entirely physiological system, as hearts were unencumbered from altered protein expression, post-translational modifications, electrical remodeling, etc that are inherent to cardiac pathologies and that complicate interpretation of the role of critical players of cardiac alternans.

Data extracted from the above system both conformed to logical expectations and yielded surprising conclusions. Among the former, fast-pacing of the heart induced alternans both in the action potential duration (APD) and in the magnitude of systolic [Ca²⁺]_SR decay. This was expected from the inherent property of all mammalian hearts to generate alternans once a pacing threshold has been reached, although the virtue here is that now we are presented with windows into [Ca²⁺]_SR and AP dynamics to better understand the subcellular mechanisms underlying this property. Another consistent observation was that the degree of alternation (Spectral Alternans Magnitude) was larger for the intra-SR signals than for the APD-Alts. Moreover, the alternations of [Ca²⁺]_SR decay and APD were in-phase (greater Ca release = longer APD), producing concordant alternans. That a large Ca²⁺ release prolonged the APD was somewhat surprising if we consider that in mammals that have long action potentials, like the rabbit, large SR Ca²⁺ releases are expected to shorten the APD by promoting Ca²⁺ dependent inactivation of L-type Ca²⁺ channels, but the study underscores the preponderant effect of the Na-Ca exchanger in prolonging the APD due to extrusion of the released Ca²⁺.

One of the most profitable advantages of the recording setup of Wang et al.⁹ was its ability to temporally resolve the subcellular events that first led to alternans. Thus, as pacing frequencies increased (and cycle length decreased), [Ca²⁺]_SR alternans preceded APD-Alts and clearly confirmed the SR as chief instigator of this phenomenon. Further, the most critical player in the onset of [Ca²⁺]_SR alternans appears to be the RyR2, as Ca²⁺ release alternans repeatedly proceeded without changes in the end-diastolic [Ca²⁺]_SR.

What causes a pool of RyR2s to default on their duty and appear insensible to triggering signals (SR load, I_Ca) that normally induce their opening? Wang et al.⁹ offers RyR2 refractoriness, an operational term indicating resistance of the RyR2 channel to open after a Ca²⁺ release event, to explain their results. In other words, because the first event is the recalcitrance of RyR2s to open despite triggering stimuli, Wang et al.⁹ propose that “RyR2 refractoriness is the mechanism that is first encroached upon for the development of SR Ca²⁺ alternans”. Because RyR2 refractoriness has been recognized as an outstanding factor in several cardiomyopathies,^{13, 14, but see 15} the results nicely add to the emergent notion that RyR2 refractoriness is a finely regulated process: deleterious consequences emerge when it is too short,^13,14 or too long.^9,16 In fact, continuing with the results of Wang et al.,⁹ at very fast stimulation rates that drive the heart into ventricular fibrillation, there appears to be a steadfast reluctance of RyR2s to release Ca²⁺ despite [Ca²⁺]_SR being high, i.e., RyR2s remain nearly continuously refractory.Thus, a translational value of these results is that, driven to extreme, the persistence absorption of RyR2s into this refractory state may ignite various mechanisms that, in unison, ultimately lead to life-threatening arrhythmias and sudden death. This underscores the importance of defining the molecular underpinnings of RyR2 refractoriness (RyR2 hyperphosphorylation, oxidation, luminal Ca²⁺ desensitization, Ca²⁺-dependent inactivation, etc), which at present are not clearly understood.

The results of Wang et al.⁹ are illuminating but at the same time humbling because they expose the intricacies of cellular Ca²⁺ handling and the necessity to comprehend the hierarchy of multi-check systems in the control of many Ca²⁺-dependent processes. For instance, low doses of caffeine and β-adrenergic stimulation are both clearly arrhythmogenic under conditions where RyR2s are “trigger-ready” (RyR2 gain-of-function mutations)¹⁷ and spontaneously release Ca²⁺ to promote delayed afterdepolarizations (DADs), but caffeine and β-adrenergic stimulation in this study appear to disperse the arrhythmogenic substrate of cardiac alternans by “sensitizing” RyR2s and rescuing them from their refractory state. This pro- or antiarrhythmic paradox, where an exact same maneuver suppresses one type of arrhythmia while promoting another, stands as the most challenging obstacle for the pharmacological treatment of arrhythmias. In the end, a clear understanding of the multi-faceted nature of cardiac pathologies is key to design rationalized therapies. Returning to the results of Wang et al.,⁹ they have shown that RyR2 refractoriness is the first mechanism to be encroached upon in the development of cardiac alternans, making it a potential target for intervention, but they have also made clear that cardiac alternans emerge as a continuum of mechanisms where many cellular processes synergize, support or antagonize each other to produce this macroscopic phenomenon (Fig. 1). From that multi-process viewpoint, if persistent RyR2 refractoriness could be effectively abolished in the face of prolonged cardiac insult, then it is possible that other processes would come to the fore and trigger cardiac alternans. It appears therefore impossible to erect cardiac alternans solely on the legs of RyR2s. Quoting Einstein in his principle of parsimony, “a hypothesis should be kept as simple as possible, but not simpler”. The role of SERCA2a, Na-Ca exchanger, sarcolemmal ion currents, intra SR Ca²⁺ binding proteins, etc., cannot be disregarded. Finally, these novel results will surely invite the evaluation of RyR2 behavior in in silico frameworks to determine if the detailed single channel kinetic information already available for RyR2 (Fig. 1) may reproduce the conclusive experimental evidence presented in this paper.

Fig. 1. Ca-Alts and their subcellular origins.

(A) Alternans promoted by insufficient SR Ca²⁺ load. An increase in the heart rate prevents the end-diastolic [Ca²⁺]_SR to reach the previous beat diastolic level (1a). The reduced SR Ca²⁺ then reduces RyR2 Ca²⁺ release, leading to a smaller SR Ca²⁺ decay (2a) and a reduction in [Ca²⁺]_i transients (3a). Smaller [Ca²⁺]_i transient promotes a decrease in the activation of NCX forward mode (4a). The reduction of Na⁺ influx through the NCX (4a) leads to a faster AP repolarization (5a). (B) Alternans promoted by slow RyR2 recovery from inactivation. In this case, an increase in the heart rate does not allow RyR2 to recover from inactivation. A distinct signature of RyR2 behavior is it modal gating.¹⁸ This modal gating allows the channel to open in a high open probability (P_o) mode (HP_o, C3-C4-O1-O2) or in a low P_o mode (LP_o, C3-O3). For a fast change in the cytosolic [Ca²⁺], the probability of visiting the HP_o is higher than visiting the LP_o. After populating the O2 state of the HP_o the channel will decrease its P_o by visiting a Ca²⁺-dependent inactivated state (C7) or a set of “adapted” closed states (C5-C6). The process of coming back from these closed states (C5, C6 and C7) defines the intrinsic refractoriness of the RyR2 channel. If the backward pathways (green and yellow lines on the Markovian scheme) that lead to the initial closed states (C1, C2, C3) are slow in comparison with the heart rate, the channel will be partially refractory for a new cytosolic Ca²⁺ stimulus. This refractoriness will finally reduce the mean P_o of RyR2 and the Ca²⁺ flux (J_RYR) through the RyR2 (1b). The reduction of the Ca²⁺ efflux from the SR will lead to a smaller [Ca²⁺]_SR decay with no changes in the end-diastolic [Ca²⁺]_SR (2b). This will subsequently reduce both the amplitude of the cytosolic Ca²⁺ transient (3b) and the size of the Na⁺ influx through the NCX (4b) leading to a shorter AP (5b) . Finally, if the recovery from inactivation is highly cooperative this can lead to a non-linear refractoriness that can set a substrate for Ca²⁺ alternanses.