Fifty Shades of Calpain: Couplon Breakdown in Catecholaminergic Polymorphic Ventricular Tachycardia and Beyond

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic syndrome that primarily affects children and young adults and is characterized by potentially fatal ventricular arrhythmias triggered by physical or emotional stress in a structurally normal heart.¹ CPVT is caused by aberrant sarcoplasmic reticulum (SR) calcium (Ca) release due to genetic variants in ryanodine receptor 2 (RyR2), cardiac calsequestrin (Casq2) and associated proteins, which are the key components of the cardiomyocyte junctional SR complex (couplon) responsible for coordinating SR Ca release.² While RyR2 mutations account for most cases,³ Casq2 mutations are associated with a less common but often more severe form of CPVT.⁴ Notably, Casq2-mutant mouse models exhibit reduced levels of Casq2 and its protein partners triadin and junctin, along with abnormal Ca release and triggered electrical activity.⁵ However, the mechanisms driving this coordinated loss of couplon proteins and its role in CPVT pathogenesis remain incompletely understood.

In this issue, Ramos-Hernández et al.⁶ provide a new perspective on how disruption of the couplon proteins contributes to Casq2-associated CPVT. Using the homozygous Casq2^R33Q/R33Q mouse model that recapitulates the human CPVT phenotype, the authors address a longstanding observation: mutant hearts exhibit markedly reduced Casq2, triadin (TRDN), and junctin (JCTN) protein despite preserved RNA transcript expression.⁷ Through proteomic profiling and complementary pharmacological studies, they demonstrate that these proteins are not lost through a single pathway. Mutant Casq2 is targeted by endoplasmic reticulum (ER)-associated degradation, the ubiquitin-proteasome system, and autophagy, whereas TRDN emerges as a direct substrate of the Ca-activated protease calpain.⁶ Importantly, TRDN degradation occurs more rapidly than Casq2 loss, and restoring TRDN by modified RNA in cardiomyocytes or AAV-mediated expression in vivo increases Casq2 abundance, placing TRDN destabilization upstream of at least part of the subsequent loss of Casq2.⁶

The most intriguing aspect of this work is the link between Ca mishandling and proteostasis. Casq2^R33Q/R33Q hearts show activation of ER stress and unfold protein response, along with increased calpain activity.⁶ Additionally, attenuating RyR2-mediated Ca leak with flecainide partially restores Casq2 and TRDN expressions,⁶ suggesting that degradation of the couplon proteins is not merely a primary consequence of defective Casq2 but is enhanced by the pathologic Ca environment it produces. These observations suggest a feedforward model: pathological SR Ca release activates calpain, which promotes TRDN degradation, and reduction in TRDN further destabilizes Casq2 and the integrity of the couplon. The reduction of triadin as a molecular driver of couplon disintegration has previously been documented by ablation of the gene encoding cardiac triadin.⁸ The resulting deterioration in SR Ca regulation exacerbates Ca leak and calpain activation, establishing a self-sustained, arrhythmogenic cycle. Moreover, this concept adds a crucial dimension to the established model of CPVT pathogenesis. Previous studies showed that reduced Casq2 expression itself increases SR Ca leak and arrhythmia susceptibility, and pathogenic loss of TRDN can independently produce a severe inherited arrhythmia phenotype.^5,8 The current study confirms that their roles should not necessarily be considered separately: destabilization of one couplon protein, i.e. TRDN or Casq2 can alter the stability of another, which is consistent with the previous study by Chopra et al.⁸ Particularly compelling is the observation that preventing Casq2 degradation through proteasomal or autophagic inhibition does not restore TRDN, whereas restoring TRDN increases Casq2.⁶ This hierarchy places TRDN at a mechanistic intersection between Ca dysregulation and proteostasis. The Ca–calpain–proteolysis axis likely extends beyond CPVT and represents a broader mechanism linking Ca dysregulation to cardiac disease progression. Recent studies in heart failure (HF) models have identified junctophilin-2 (JPH2), an essential organizer of the junctional membrane complex, as a functionally important calpain substrate.⁹ Remarkably, preventing site-specific calpain cleavage of JPH2 preserves junctional membrane architecture and Ca homeostasis and attenuates stress- and ischemia-induced HF.⁹ A similar paradigm has emerged in atrial fibrillation (AF), where increased calpain activity is associated with JPH2 loss, Ca leak, and atrial remodeling, whereas genetic calpain inhibition preserves JPH2 and reduces AF susceptibility.¹⁰ Together with the current report by Ramos-Hernández et al., these studies suggest that calpain-mediated degradation of proteins responsible for maintaining junctional SR Ca-handling proteins may constitute a shared feed-forward mechanism across arrhythmogenic heart diseases, as illustrated in the Figure.

Figure: A Ca-Calpain-Proteolysis Feed-Forward Cycle in Arrhythmogenic Heart Diseases.

The proposed Ca-calpain-proteolysis feed-forward model begins with (1) an initial insult, such as a pathogenic Casq2 mutation in CPVT, that disrupts normal SR Ca handling and promotes pathological Ca leak through RyR2. (2) The resulting increase in cytosolic Ca activates calpain, which cleaves key proteins responsible for maintaining specialized Ca-handling domains, including triadin in Casq2-associated CPVT and junctophilin-2 in heart failure and atrial fibrillation. (3) Loss of these scaffold proteins destabilizes their respective Ca-handling complexes; in CPVT, triadin degradation promotes Casq2 destabilization and couplon disruption, whereas junctophilin-2 cleavage compromises the junction between the T-tubule and junctional SR. (4) Disruption of these Ca-handling domains further impairs regulation of SR Ca release, resulting in additional Ca leak that can sustain calpain activation and protein degradation, thereby establishing a self-reinforcing cycle of Ca dysregulation, calpain activation, and proteolysis that may contribute to arrhythmogenesis and progression of cardiac disease.

The in vivo experiments by Ramos-Hernández et al. further strengthen this interpretation. Pharmacological inhibition of calpain restored Casq2, TRDN, and JCTN levels and reduced markers of ER stress, and these molecular effects were translated into the antiarrhythmic effects in Casq2^R33Q/R33Q mice.⁶ Importantly, the improvement in Ca handling persisted after cardiomyocytes were isolated and studied in the absence of the inhibitor,⁶ arguing against a purely acute electrophysiological action of the drug. This distinction is important because current guideline-directed therapies for CPVT either target RyR2 directly or the sympathetic activation upstream of SR Ca mishandling.¹¹ The present findings raise the possibility that targeting the proteolytic response to Ca dysregulation could instead stabilize the molecular machinery responsible for controlling SR Ca release. Viewed in the context of similar calpain-dependent remodeling in other cardiac diseases, these results raise the broader possibility that breaking the Ca–calpain–proteolysis feed-forward loop may represent a therapeutic principle extending beyond CPVT.

Several questions nevertheless remain before calpain inhibition can be considered a therapeutic strategy for CPVT. The experiments were performed predominantly in a single homozygous Casq2^R33Q/R33Q mouse model, and whether calpain-mediated TRDN degradation represents a general mechanism across other Casq2 variants remain unknown. More broadly, an emerging theme across arrhythmogenic heart diseases is that Ca dysregulation can activate calpain, which subsequently degrades proteins essential for maintaining specialized Ca-handling domains, thereby further disrupting Ca homeostasis. Whether TRDN degradation in CPVT represents a disease-specific event or another example of this more general Ca–calpain–proteolysis feed-forward mechanism will be important to establish. The authors acknowledge that additional disease models and controls will be needed before these findings can be translated clinically.

The framework of a Ca-Calpain-Proteolysis Feed-Forward Cycle (Figure) also suggests a more selective therapeutic strategy: Rather than chronically suppressing calpain itself, it may be possible to identify and protect the disease-relevant substrates through which calpain amplifies Ca dysfunction. In CPVT, stabilizing TRDN or rendering its critical calpain-sensitive sites resistant to cleavage could preserve the couplon while leaving physiological calpain signaling largely intact. Combined with established therapies that reduce RyR2-mediated Ca leak, such a strategy could interrupt the proposed feed-forward loop at both ends: reducing the Ca signal that activates pathological proteolysis while preserving the molecular machinery responsible for SR Ca release. By connecting defective Ca handling with selective degradation of the couplon, Ramos-Hernández et al. therefore provide not only a new potential therapeutic avenue for CPVT, but also another example of an emerging principle in cardiac disease: when Ca activates calpain, degradation of the machinery controlling Ca may itself become a driver of disease progression.