Do CPVT-linked mutations alter RYR2 regulation by cytosolic Ca²⁺ in cardiomyocytes?

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 A_rest as an index for RYR2 activity in the resting cells with 0.1 µM cytosolic Ca²⁺. They calculated A_rest 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 A_rest value depend not only on a dissociation constant for activating Ca²⁺ (K_ACa) but on maximal gain of Ca²⁺ activated RYR2 channels, A_max, and a dissociation constant for inhibiting Ca²⁺ (K_ICa), which is usually >10,000 times higher than resting Ca²⁺ concentration, therefore, minimally affects A_rest (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 A_max 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 A_rest 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 A_rest 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 A_rest 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 A_rest value in their experiments tended to exhibit onset of arrhythmogenic symptoms at a younger age, indicating pathological relevance of the experimental A_rest index. Overall, the induction of the A_rest 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 A_rest. With this loss-of-function property of E4872Q mutation, it is difficult to quantitatively evaluate and compare the A_rest 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 A_rest 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, A_rest values would be different in the presence of large populations of heterotetrameric mutant RYR2.