The release of calcium ions from sarcoplasmic reticulum (SR) during cardiac action potentials is essential for cardiac muscle contraction. In cardiac excitation–contraction coupling, a small calcium influx through the plasmalemma activates the SR Ca2+ release channel, known as type-2 ryanodine receptors (RYR2s), by the mechanism of cytosolic Ca2+ activation, thereby Ca2+-induced Ca2+ release (Bers, 2002). Dysfunctional RYR2 regulations are known to associate with multiple cardiomyopathies, including various types of arrhythmogenesis as well as cardiac hypertrophy and failure. Over 200 mutations in human RYR2 gene have been reported to associate with catecholaminergic polymorphic ventricular tachycardia (CPVT), in which patients suffer life-threatening arrhythmias triggered by emotional or exercise stresses. One well-known SR Ca2+ release aberrancy with these RYR2 mutations is observed as a diastole Ca2+ leak and spontaneous Ca2+ release, driving electrogenic Na+–Ca2+ exchanger, thereby, delayed- or early-afterdepolarizations (Wehrens et al., 2005). Molecular mechanisms underlying such gain-of-function RYR2 mutations are, however, controversial (Fig. 1). Chen and colleagues proposed that CPVT-associated RYR2 mutations increase sensitivity of RYR2 to luminal Ca2+ activation, and therefore lower the luminal Ca2+ threshold for spontaneous Ca2+ release from SR. During β-adrenergic stimulation, more Ca2+ are taken up into SR, which facilitates the spontaneous Ca2+ release (Chen et al., 2014; Jiang et al., 2005; Jiang et al., 2004). On the other hand, it is also conceivable that CPVT mutations render RYR2 easily open by cytosolic ligands, e.g., cytosolic Ca2+ at the diastole conditions. Thus, upon β-adrenergic stimulation, locally spilled Ca2+ from SR activates the mutant RYR2 from the cytosolic side, resulting in the spontaneous Ca2+ release and oscillation. In this issue of JGP, Kurebayashi et al. (2022) demonstrated systematic examinations that showed tight correlation between RYR2 regulation by resting cytosolic Ca2+ concentration (∼0.1 µM) and the occurrence of spontaneous Ca2+ release and oscillation, as well as a threshold of luminal Ca2+ concentration for spontaneous Ca2+ release. They tackled the controversial cytosolic Ca2+ versus luminal Ca2+ issue using two parallel experimental techniques: (1) simultaneous fluorescence measurements of cytosolic and luminal Ca2+ of heterologous HEK293 cells expressing recombinant RYR2 mutant proteins, and (2) in vitro RYR2 activity measurements by [3H]ryanodine binding methods to determine RYR2 activity at 0.1 µM cytosolic Ca2+ concentration.
Authors defined Arest as an index for RYR2 activity in the resting cells with 0.1 µM cytosolic Ca2+. They calculated Arest using parameters obtained from [3H]ryanodine binding assay which determined the bell-shaped cytosolic Ca2+-dependent activities of RYR2 (Fig. 2 A). They proposed that an Arest value depend not only on a dissociation constant for activating Ca2+ (KACa) but on maximal gain of Ca2+ activated RYR2 channels, Amax, and a dissociation constant for inhibiting Ca2+ (KICa), which is usually >10,000 times higher than resting Ca2+ concentration, therefore, minimally affects Arest (see Eqs. 1–3 in Kurebayashi et al., 2022). They also pointed out that, in some earlier studies (Jiang et al., 2005; Jiang et al., 2004), mutant RYR2 activation by cytosolic Ca2+ in [3H]ryanodine binding studies was normalized to the peak activity at ∼100 μM Ca2+, and the apparent activating Ca2+ affinities of RYR2 mutants were compared. Under this data analysis, possible differences of Amax values among the RYR2 mutants were not considered, which may be one of the reasons for the controversy. In the present study, the authors examined wild type and 10 different CPVT-linked RYR2 mutants and found tight correlation between Arest values and luminal Ca2+ thresholds for spontaneous Ca2+ release in CPVT-linked RYR2 mutants. In detail, they found that high RYR2 activity at resting cytosolic Ca2+ (high Arest values) reduced the luminal Ca2+ threshold, resulting in a high rate of Ca2+ oscillation even with low SR Ca2+ load (Fig. 2 B), supporting an idea that CPVT mutations affect cytosolic Ca2+ regulation. On the other hand, the RYR2 mutants with low Arest indexes required a high SR Ca2+ load, and thereby more local Ca2+ spills to trigger the Ca2+ release and oscillation (Fig. 2 C). Their wet-lab findings are also supported by mathematical simulation in which the authors took not only cytosolic Ca2+ and luminal Ca2+ affinity but also SR store-operated Ca2+ entry to the cytosol into their considerations. In addition, they found that a human patient harboring a CPVT mutation showing high Arest value in their experiments tended to exhibit onset of arrhythmogenic symptoms at a younger age, indicating pathological relevance of the experimental Arest index. Overall, the induction of the Arest value is a key point in this article to address controversial issues.
This article advanced our understanding of Ca2+ signaling mechanisms underlying CPVT pathology; however, their intriguing findings also open a door for other challenging questions as the authors discussed and partly addressed. The article showed quantitative evidence that spontaneous Ca2+ release through CPVT RYR2 mutants was caused by their enhanced activity in cytosolic Ca2+ regulation. However, this does not rule out a luminal Ca2+ regulatory mechanism in RYR2. Ca2+-dependent regulation from both the cytosolic and luminal side may work synergically. The authors partly addressed this question by constructing RYR2 carrying double mutations, one on the CPVT site (R2474S) and the other on the previously reported luminal Ca2+ sensing site (E4872; Chen et al., 2014). The double mutations R2474S/E4872Q slightly increased the spontaneous Ca2+ oscillation frequency and cytosolic Ca2+-dependent RYR2 activity compared with E4872Q mutation only, suggesting that increased spontaneous Ca2+ oscillation in R2474S CPVT mutant does not require the putative luminal Ca2+ binding site. One problem in this experiment is that the E4872Q mutation greatly reduced cytosolic Ca2+-dependent RYR2 activities, thereby ∼0 Arest. With this loss-of-function property of E4872Q mutation, it is difficult to quantitatively evaluate and compare the Arest values of the double mutants; thus, a synergetic role of luminal Ca2+ activation for the spontaneous Ca2+ release cannot be excluded.
Recently, the cryo-EM–based structural mapping identified a cytosolic Ca2+ binding site in RYRs (des Georges et al., 2016; Gong et al., 2019). Mutations on this site attenuated cytosolic Ca2+-dependent activation of RYRs (Chirasani et al., 2019; Guo et al., 2020; Murayama et al., 2018). Thus, we may test inversely whether CPVT mutations together with cytosolic Ca2+ site mutations still increase luminal Ca2+ activation of the mutant RYR2. However, this will require an experimental platform where luminal Ca2+ is controlled, such as single channel bilayer recording. Another interesting question is about a global structural impact by CPVT mutations. Since RYR2 mutations examined in this study spread all over the large protein complex, these mutations may allosterically alter the conformation of cytosolic or luminal Ca2+ sensing site (or their surrounding domains). Does the structural rearrangement of the Ca2+ binding site also correlate with the Arest value? Investigators have started providing high-resolution structures of the recombinant mutant RYR proteins (Iyer et al., 2020); therefore, this challenging question may be answered in the near future.
The paper uses heterologous cell expression of the CPVT mutant RYR2. This is an advantage to focus solely on the intrinsic properties of RYR2 proteins, but can be a drawback because the system excludes the possibility that CPVT mutations on RYR2 alter protein interactions or post-translation modifications. The authors reported that the dissociation constant of activating Ca2+ in wild type RYR2 is >10 µM and even the highest affinity CPVT mutant (H4762P-RYR2) is ∼3 µM. These values are somewhat beyond the threshold of the cytosolic Ca2+ concentration triggering Ca2+-induced Ca2+ release in cardiomyocytes. This discrepancy may have been caused by the difference between heterologous cell and cardiac cell environments. It also should be noted that one well-characterized CPVT pathology is that RYR2 mutations causes RYR2 hyperphosphorylation at Ser2809, resulting in the dissociation of FKBP12.6, thereby causing leaky Ca2+ release (Wehrens et al., 2003). In this regard, it is important to evaluate the CPVT mechanism proposed by authors in the cardiac cell environment with adrenergic stimulation. Recent advances in stem cell technology and genetic techniques are helpful in creating a series of CPVT mutant cardiac muscle cell lines by introducing point mutations in RYR2 gene in the pluripotent stem cells using a gene editing technique and then differentiating them into cardiomyocytes (Wei et al., 2018).
Lastly, most of the CPVT-linked RYR2 mutations in human patients are heterozygote, expressing various types of heterotetrameric RYR2, while the authors’ experiments with transfection of the mutant RYR2 cDNA mimic the homozygote situation. I noticed that the homozygous mutant RYR2 expression is likely to exhibit significant functional differences from wild type, which is valuable information as a phenotypic characterization of the gene mutations; however, one interesting experiment would be coexpression of wild type and mutant RYR2 cDNAs in the HEK293 cells. The authors assume partial cooperative regulation of tetrameric RYR2 by cytosolic-activating Ca2+ (a Hill coefficient is 2); thus, Arest values would be different in the presence of large populations of heterotetrameric mutant RYR2.
Eduardo Ríos served as editor.
Support by National Institutes of Health grants HL147054 and HL153504 is gratefully acknowledged.
The author declares no competing financial interests.
This work is part of a special issue on excitation–contraction coupling.