Can cardiomyocytes bypass the ‘Ca2+-induced Ca2+-release’ mechanism?

This editorial refers to ‘Point mutations in RyR2 Ca2+-binding residues of human cardiomyocytes cause cellular remodelling of cardiac excitation contraction-coupling’ by Y. Xia et al., https://doi.org/10.1093/cvr/cvad163. The Ca2+ release channel, ryanodine receptor type 2 (RyR2), is key in the excitation–contraction coupling (ECC) mechanism, providing Ca2+ for cardiac contraction in each heartbeat. During the ECC, the RyR2 releases Ca2+ from the sarcoplasmic reticulum (SR) when activated by Ca2+ influx through the L-type Ca2+ channel (LTCC), a process known as the Ca2+-induced Ca2+ release (CICR) mechanism.1 Thus, the Ca2+ sensitivity of the RyR2 is fundamental for the ECC and heart function. With the identification of a point mutation in the RyR2 linked to sudden cardiac death within the arrhythmogenic disease named catecholaminergic polymorphic ventricular tachycardia (CPVT),2 the involvement of RyR2 in fatal arrhythmias drove attention. Since then, ∼350 mutations have been identified, indicating impaired RyR2 function as a cause of inherited cardiac arrhythmias, mostly CPVT. The general mechanism involves the appearance of post-depolarizations, triggered activity, and ectopic foci, attributed to the generation of inward current generated by Na+/Ca2+ exchanger, as the result of spontaneous SR Ca2+ release. To elucidate the specific mechanisms leading to these arrhythmias, several approaches have been undertaken. Mutated channels have been expressed in heterologous systems such as HEK293 cells (e.g. Jiang et al.3) and in knock-in mouse models (e.g. Yin et al.4). Over the last two decades, to overcome some limitations related to these models, cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) became an important additional tool to study pathophysiology in human cellular context, despite the still recognized immaturity of the hiPSC-CMs. Hence, this in vitro model has been used to recapitulate complex physiological phenotypes of several specific RyR2 mutations, providing a more relevant representation of human cardiac function compared with traditional animal models (Table 1). The hiPSC-CMs model used in these studies come from different sources, including patient carriers compared with either their healthy relatives or a commercial control line, or obtained by knocking-in the mutation in control ‘WT’ hiPSC clones. RyR2 mutations investigated in hiPSC-CM Het and Homo, analysis conducted on heterozygous and homozygous mutation, respectively. Origin: cells originated from CPVT patients (CPVT1), polymorphic ventricular tachycardia (PMVT) patients, or for control commercial lines with knock-in mutation introduced by CRISPR/Cas9. Loc, location; NT, N-terminal portion; Ctr, central domain; CT, C-terminal domain. RyR2 mutations investigated in hiPSC-CM Het and Homo, analysis conducted on heterozygous and homozygous mutation, respectively. Origin: cells originated from CPVT patients (CPVT1), polymorphic ventricular tachycardia (PMVT) patients, or for control commercial lines with knock-in mutation introduced by CRISPR/Cas9. Loc, location; NT, N-terminal portion; Ctr, central domain; CT, C-terminal domain. Xia et al.5 employed CRISPR/Cas9 technology for gene editing hiPSC-CMs. The use of CRISPR/Cas9 technology allows researchers to introduce the specific mutation into control cells, so avoiding the confounding patient genetic context, which may carry other genetic variants. In addition, to closely monitor SR Ca2+ variations, the hiPSC-CMs were infected by genetically encoded SR-targeted low-affinity Ca2+ indicator, GCaMP6. Xia et al.5 explored the effect of a sudden death-related mutation, RyR2Q3925E compared with RyR2E3848A, both being related to the Ca2+ activating region of the RyR2 channel. The E3848 site is one of the five RyR2 Ca2+ binding sites, whereas the Q3925 is an indirect Ca2+-binding site. Using an elegant approach, monitoring simultaneous cytosolic and intra-SR Ca2+ as well as the Ca2+ influx, the study provided an interesting view of the ECC remodelling in hiPSC-CMs due to alteration of RyR2 Ca2+-binding sites. Their results showed that both mutations share the same functional defects, with loss of the CICR mechanism, defining them as loss-of-function (LOF) mutations. Surprisingly, the mutant hiPSC-CMs with loss of CICR mechanism are still able to produce spontaneous [Ca2+]i transients of almost normal amplitude, without SR contribution. Most of the investigated RyR2 mutations to date are characterized as gain-of-function, resulting in a hyperactive channel, but rare LOF variants, leading to hypo-active RyR2, have been found6 as seen in the newly described calcium release deficiency syndrome.7,8 While it has been proposed that the arrhythmogenic mechanism associated with LOF could be related to the induced overload of the SR Ca2+ content and favouring proarrhythmogenic Ca2+ waves,6 the results presented by Xia et al. are puzzling. Their data in homozygous conditions demonstrate that the mutation completely losses the CICR mechanism and strongly suggests that mutations in RyR2 Ca2+ activation sites impair caffeine activation. In heterozygous conditions, they observed a decrease in CICR and a slightly increase in the SR Ca2+ load. Thus, the pathogenic mechanism of RyR2Q3925E found on a patient who suffered sudden death may well be similar to the one proposed in other LOF mutations. However, data collected in homozygous conditions are of great interest too. On the one hand, they demonstrate that when all RyR2 monomers are mutated, the cell completely loses the CICR mechanism. Thus, the partial maintenance of CICR in heterozygous conditions is due to the mixture of WT and mutated RyR2 monomers and different combinations of them to form the homotetramers. On the other hand, these findings evidence that the hiPSC-CM show remarkable plasticity, being able to remodel themselves to compensate the loss of CICR. Similar to WT, monolayers of homozygous mutant hiPSC-CMs are able to produce spontaneous oscillations with robust [Ca2+]i transients in the absence of CICR and without significant SR contribution. Various inhibitors were tested to identify mechanisms underlying these spontaneous [Ca2+]i transients that operate in the absence of CICR. The findings reveal that Ca2+ influx through LTCC is fundamental, as evidenced by the completely abolition of [Ca2+]i transients in the presence of nifedipine and Cd2+. Other channels, including TRP and hemichannels, as well as possible involvement of IP3R and mitochondria, also contribute, but in a cell-dependent fashion, pointing out the cell-to-cell variability of cellular remodelling in hiPSC-CMs. In conclusion, Xia et al.’s study is a step forward in cardiac research. On the one hand, it provides a mechanism in a patient-related RyR2 mutation. On the other hand, it advances the knowledge of the interplay between Ca2+ and caffeine sensitivity of the RyR2, and demonstrates that cardiac myocytes may produce [Ca2+]i transients of normal amplitude without CICR. This work was supported by the Agence nationale de la recherche (ANR-19-CE14-0031-01, ANR-23-CE14-0009-02), European commission, Horizon 2020 Framework Programme, Research and Innovation Staff Exchange (RISE) Call Marie Skłodowska-Curie Research and Innovation Staff Exchange (H2020-MSCA-RISE-2016, Project number: 734931), National Institute of Health (2R01HL055438-22). S.L. is a recipient of the Chinese Scholarship Council (Chinese Scholarship Council—202208320083).