Mutant cardiac ryanodine receptors and ventricular arrhythmias: is ?gain-of-function? obligatory?

See article by Thomas et al.[1](pages 52–60) in this issue. In this issue of Cardiovascular Research, Thomas et al. [1] evidence functional differences among sudden cardiac death (SCD)-linked ryanodine receptors (RyR2) mutants. They challenge the conventional view that arrhythmogenic disorders associated with RyR2 mutations result exclusively from ‘gain-of-function’ channelopathies generating aberrant spontaneous Ca2+ release [2–4]. Thomas et al. [1] now report a ‘loss-of-function’ phenotype of one RyR2 mutant associated with SCD. The RyR2 is primarily involved in cardiac contractile function. At each heartbeat, membrane depolarization during an action potential (AP) activates voltage-dependent L-type Ca2+ channels (L-VDCC), generating transmembrane Ca2+ influx (ICaL). ICaL triggers Ca2+ release via sarcoplasmic reticulum (SR) Ca2+ release channels (RyR2). This Ca2+-induced Ca2+ release (CICR) amplification step provides the amount of Ca2+ required for contraction. Hence, RyR2 are a key element in the control of cardiac output. However, RyR2-released Ca2+ can, in turn, modify membrane potential by modulating Ca2+-sensitive sarcolemmal ion channels or transporters. Potential targets include, among others, L-VDCC, Ca2+-activated Cl− channels, IK1 channels, and the Na+/Ca2+ exchanger. The RyR2 contributes to the setting of normal sinusal automaticity [5], but disordered RyR2 activity generates ventricular arrhythmia [2–4,6]. Normal heart beating rate is fixed by the sinus node. Slow diastolic depolarization brings cellular membrane potential to a threshold, allowing the firing of an AP. RyR2 activity contributes to accelerate diastolic depolarization [7,8]. Indeed, the Ca2+ released during diastole by the RyR2 activates the Na+/Ca2+ exchanger in the forward mode. The resulting inward current accelerates diastolic depolarization and, thereby, promotes automaticity. The RyR2 has been reported to be essential in determining cardiac rhythm under normal conditions [9,10] and during catecholamine stimulation [11]. Although the extent of its contribution, relative to other ionic currents, has evoked some controversy [12], RyR2 clearly plays a significant role [5]. RyR2-released Ca2+ is also involved in triggered arrhythmia. Diastolic RyR2 ‘gain-of-function’ is involved in the genesis of ventricular arrhythmia, mainly by activating an inward Na+/Ca2+ exchanger current. Systolic RyR2 ‘gain-of-function’ might result in AP prolongation, which favors arrhythmogenicity. Modulation of both automatic activity and AP duration by RyR2-released Ca2+ may also involve channels or transporters other than the Na+/Ca2+ exchanger. For example, the role of L-VDCC has been established in both cases [13,14]. The Ca2+ released by RyR2 terminates Ca2+ entry through L-VDCC, because these channels are inactivated by Ca2+[15], which shortens the AP plateau. When the amount of Ca2+ released by RyR2 is reduced, as in heart failure (HF) or at high stimulation rate [16], ICaL inactivates slowly (known as frequency-dependent facilitation of ICaL[17]), resulting in AP prolongation in rat ventricular cells [14]. This regulation occurs on a beat-to-beat basis and potentially provides a substrate for early afterdepolarizations (EADs) in pathological conditions or in the presence of drugs lengthening AP duration [18]. Therefore, a depression of Ca2+ release from the SR can certainly increase the incidence of EADs, commonly associated with long AP duration [15]. Therefore, systolic RyR2 ‘loss-of-function’ might also be arrhythmogenic. In HF, at stage IV of the NYHA, 50% of deaths are sudden and related to tachycardia degenerating into ventricular fibrillation. At this stage, diastolic [Ca2+]i is increased and systolic [Ca2+]i is depressed, which involves disordered activity of the RyR2. However, SCD can also occur in non-failing hearts. Although it has been proposed that alteration of RyR2 function have only transient effects [19], several mutations in the RyR2 with major functional consequences have been identified in at least two different kinds of ventricular arrhythmia causing juvenile SCD: (i) Catecholamine-induced polymorphic ventricular tachycardia (CPVT), induced by stress and exercise, and (ii) arrhythmogenic right ventricular dysplasia/cardiomyopathy type 2 (ARVD2), characterized by structural alteration of the myocardium [20–23]. At least 21 single amino acid mutations (15 CPVT and 6 for ARVD2) have been related to arrhythmia. These mutations are localized in 3 highly conserved regions of the RyR2: the FKBP12.6-binding region and the cytosolic N- and C-terminals. Mutations in the FKBP12.6-RyR2 binding region are expected to disturb the binding of FKBP12.6 to RyR2. Because FKBP12.6 deficiency promotes ‘leaky RyR2’ [24,25], it has been proposed that ‘leaky RyR2’ underlies arrhythmia and SCD [2], which seems to be confirmed by the finding that FKBP12.6-deficient mice exhibit exercise-induced ventricular arrhythmias—mostly DADs—similar to those observed in CPVT [2]. However, George et al. [4] have shown that RyR2 mutants related to CPVT maintain normal binding to FKBP12.6. Furthermore, basal activity of mutant RyR2 was similar to wild-type RyR2, although Ca2+ release induced by caffeine or isoproterenol was increased [4]. These analyses suggested that not only diastolic ‘gain-of-function’ but also systolic ‘gain-of-function’ under stress is related to CPVT. In CPVT [22,23] and ARVD2 [21], RyR2 mutations have been found at sites similar to that of skeletal RyR1 mutants involved in malignant hyperthermia and central core disease. Hence, it has been hypothesized that, as in these skeletal muscle diseases, RyR2 mutations would involve RyR2 ‘gain-of-function’ [6]. This hypothesis was supported by the findings of Jiang et al. [3], who analyzed the function of one CPVT mutation. These authors found that the corresponding RyR2 mutant has higher activity at low [Ca2+]i and enhanced sensitivity to caffeine. These properties are expected to promote SR Ca2+ leak during diastole and DADs, triggering arrhythmogenic disorders. The findings by Thomas et al. [1] further support this conclusion for 3 ARDV2 mutants, suggesting that ‘gain-of-function’ phenotype can be found both in CPVT and ARDV2. Thomas et al. analyzed the function of 4 RyR2 mutants linked to SCD (ARVD2). They expressed the wild-type and mutant RyR2 in human embryonic kidney cells lacking endogenous RyR2 and accessory proteins. Since these cells do not exhibit EC coupling, they used caffeine to assess Ca2+ sensitivity of the various RyR2 mutants. Caffeine greatly enhances Ca2+ sensitivity of the RyR2 that are activated at resting [Ca2+]i, allowing measurable Ca2+ release. After verification of RyR2 expression levels, the amplitude and kinetics of caffeine-induced Ca2+ release were compared among mutants and wild-type RyR2 to get an insight into their function. The caffeine concentration–Ca2+ release curves revealed different behaviors. Three mutants were more sensitive to caffeine than the wild-type (lower EC50), exhibiting a ‘gain-of-function’ phenotype. In contrast, one mutant, L433P, showed a higher EC50, suggesting that this mutant is less sensitive to Ca2+ than other mutants and than the wild-type. Assuming that the intrinsic caffeine sensitivity was similar among these RyR2 subtypes, the shift in the EC50 would account for a shift in the Ca2+ sensitivity of mutants RyR2. Therefore, the finding of SCD-related RyR2 mutant with likely less Ca2+ sensitivity challenges the current perception that RyR2-related arrhythmias are dependent on enhanced diastolic activity or so-called ‘gain-of-function’, and extends the current therapeutic perspectives [26]. Not only an excess of Ca2+ release, but also a depression of Ca2+ release by the SR could induce arrhythmia—as mentioned above for ICaL. Although these data were obtained from a RyR2 heterologous expression system in cells lacking RyR2 accessory proteins and normal EC coupling, they argue against the concept of unicity in RyR2-related arrhythmia. It is interesting to note that a depression of RyR2 expression associated with atrial fibrillation has been reported [27]. Now, analysis of the biophysical properties of single RyR2 (incorporated in lipid bilayers) and of their functional properties (E–C coupling, Ca2+ sparks) in native cardiac cells is highly desirable. Moreover, the ionic currents linking RyR2 ‘loss-of-function’ to electrical disorders need to be identified. Transgenic animals will probably provide a unique means to address these important issues. In conclusion, the observations by Thomas et al. [1] open new perspectives, suggesting that mechanistic analysis is mandatory for each SCD-linked RyR2 mutant. The stake would be to design adequate therapeutic strategies on ‘a case-by-case’ basis for each identified mutation.