RyR2 gain-of-function and not so sudden cardiac death

Gain-of-function of the cardiac ryanodine receptor (RyR2), the major Ca²⁺ release channel of the sarcoplasmic reticulum (SR), can be catastrophic, and is associated with sudden cardiac death in inherited arrhythmogenic disorders. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by gain-of-function mutations in RyR2, or loss-of-function mutations in accessory proteins that bind to or modulate RyR2 channel activity, such as calsequestrin (CSQ2) and triadin (TRDN) [1, 2]. It manifests as polymorphic or bidirectional ventricular tachycardia during exercise or emotional stress, typically in the absence of structural heart disease. In CPVT, mutations in proteins of the RyR2 complex cause untimely channel activity, promoting arrhythmogenic spontaneous Ca²⁺ release in cardiomyocytes that can precipitate into delayed afterdepolarizations and ventricular arrhythmias [2]. While this straightforward explanation remains an attractive way to describe dysfunction underlying CPVT, molecular mechanisms are apparently far more complex. The clinical manifestation of the disease is highly heterogeneous, the penetrance of RyR2 mutations is highly variable, and the vast list of CPVT-associated mutations reported in patients (>200) continues to grow, including loss-of-function mutations [3, 4]. Functional heterogeneity of RyR2 mutations linked to CPVT has long been suspected [5], and several molecular mechanisms underlying defective RyR2 channel activity have been proposed, including altered Ca²⁺ sensitivity, disruption of interdomain interactions, dissociation of accessory proteins and posttranslational modifications [2].

In this issue of Circulation Research, Yin et al. comprehensively characterize arrhythmogenic mechanisms caused by the RyR2^R420Q mutation, in both a genetic mouse model and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from family members with the mutation [6]. They report dysfunction not only at the single RyR2 channel level, but importantly, at the ultrastructural level too. This new mechanism challenges the notion that CPVT is strictly a channelopathy, without apparent secondary remodeling in the heart. Is sudden cardiac death from CPVT therefore not so sudden after all?

Mutations of RyR2 preferentially map to three ‘hotspots’ within the human RyR2 polypeptide – the N-terminal domain (NTD), the central domain, and the C-terminal domain [3]. The RyR2^R420Q mutation characterized in this paper maps to the NTD, and has since been reported in several families [6]. Additionally, other pathogenic mutations of the same amino acid have been identified, suggestive that this region is critical to channel function. Although [³H] ryanodine binding assays did not demonstrate significant differences in Ca²⁺ sensitivity, single channel studies of Yin et al. in the present manuscript revealed that RyR2^R420Q channels dwell longer in the open state at low [Ca²⁺]_i vs. WT channels, and are predominantly in an open, subconductance state, which could not be attributed to phosphorylation at Serine-2808 or Serine-2814 [6]. Yin et al. hypothesized that such alterations in biophysical properties of the RyR2^R420Q channel were caused by defective interdomain interactions within the channel tetramer. The NTD forms an interface with core solenoid of the central domains, as well as the NTD of a neighboring monomer. Ikemoto and Matsuzaki first postulated that CPVT mutations of RyR2 induce diastolic Ca²⁺ leak by disrupting and weakening interdomain interactions, including that of the NTD-central domain [7]. Conversely, here Yin et al. report that RyR2^R420Q causes a tighter interdomain interaction between the NTD and core solenoid, increasing it two-fold, and postulate this mutation may cause conformational changes within the channel structure [6]. Importantly, this defect may explain altered channel binding to accessory protein junctophilin (JHP2), and links dysfunction at the single channel level to intriguing changes that occur at the ultrastructural level.

Hints of CPVT-associated subcellular structural remodeling first appeared in studies utilizing models of RyR2 accessory protein knockout to recapitulate the disease phenotype. In a series of works from the Knollmann laboratory, mice lacking RyR2 accessory protein CSQ2 presented with a drastic increase in SR volume, in parallel with RyR2 dysfunction and severe CPVT phenotype [8]. Given that CSQ2 is a major SR Ca²⁺ buffering protein, Knollmann et al. suggested that the increase in SR volume in this knockout model is an adaptation to offset the decrease in SR Ca²⁺ buffering capacity. In the present manuscript, authors noted longer lasting RyR2-mediated Ca²⁺ sparks in RyR2^R420Q mouse cardiomyocytes, in parallel with enlarged junctional SR (jSR) width, likely causing a delay in local Ca²⁺ depletion at Ca²⁺ release units (CRUs) and Ca²⁺ spark termination [6]. Yin et al. attempted to reverse jSR expansion in this model by manipulating intracellular osmolarity to promote SR swelling. However, this did not result in measurable functional improvements of RyR2-mediated SR Ca²⁺ release, suggesting that while increased width of jSR may indeed contribute to RyR2^R420Q-associated dysfunction, it cannot explain it completely.

It was previously demonstrated that knockout of TRDN, an accessory protein involved in RyR2 regulation by luminal Ca²⁺, caused loss of contacts between the SR and T-tubules in mouse cardiomyocytes [9]. Accessory protein of RyR2, JHP2, also plays a key structural role in cardiomyocytes by coordinating the position of plasmalemmal L-type Ca²⁺ channels (LTCCs) in close proximity to jSR RyR2s in the dyad. This ensures the high fidelity of conversion of membrane depolarization into highly synchronized SR Ca²⁺ release. Loss of JHP2 in acquired cardiac disease leads to reduced T-tubule density and organization, as well as redistribution of LTCCs across the plasmalemma [10]. Data from JHP2 overexpression and knockdown studies suggest this ultrastructural reorganization contributes to asynchronous Ca²⁺ release and promotes pro-arrhythmic Ca²⁺ alternans [11]. Here, Yin et al. show that CPVT mutation RyR2^R420Q leads to a loss of channel ability to bind JHP2, leading to nano-disturbances in jSR width, dyadic structure and thus CRUs (RyR2 clusters), which is expected to contribute to Ca²⁺ mishandling and arrhythmogenesis.

In general, persistent RyR2-mediated SR Ca²⁺ leak affects numerous intracellular processes and signaling pathways. Indeed, recent studies showed that RyR2 gain-of-function affects mitochondrial function, thereby excitation-contraction-bioenergetics coupling and redox homeostasis, which can further exacerbate pro-arrhythmic Ca²⁺ mishandling [12]. Moreover, it appears that genetic CPVT-linked gain-of-RyR2-complex-function can lead to organ-level structural remodeling of the heart after all [13]. Of note, a number of RyR2 gain-of-function mutations are associated with arrhythmogenic right ventricular cardiomyopathy (ARVC), and one was recently linked to autosomal dilated cardiomyopathy [14], both cardiac disorders associated with significant cardiac structural abnormalities. This underscores “the power of secondary remodeling” in disease phenotype.

Since the condition was first described [1], β-blockers have remained the frontline therapy for CPVT, yet a significant number of patients still experience treatment failures including syncope or cardiac arrest. This could further indicate the functional heterogeneity of CPVT-associated mutations. The need for new drugs to stabilize RyR2 complex activity in affected patients is paramount. The production and testing of highly selective RyR2 inhibitors is currently underway, and is a very welcome development [15]. However, given the accumulative work of now several laboratories, how do we treat presentations of CPVT where secondary remodeling is apparent, and perhaps too severe to overcome with RyR2 inhibitors alone? In the case of data presented here by Yin et al., where RyR2^R420Q mutation prevents correct association with JHP2 and promotes substantial ultrastructural remodeling [6], targeting RyR2 gain-of-function alone will not rescue subcellular heterogeneity. Moving forward, the research community must take into account how to address such multifactorial problems, and as we consider ‘personalized medicine’ for the future, we need to finally shed the assumption that all gain-of-function CPVT-associated mutations of RyR2 are equal.

Figure 1. Schematic demonstrating the mechanisms of dysfunction caused by CPVT-associated mutation RyR2^R420Q.

R420Q mutation causes tighter RyR2 interdomain interaction, SR expansion and derangement of LTCC-RyR2 coupling due to the loss of RyR2 association with JPH2. Created with Biorender.com.