Catecholaminergic polymorphic ventricular tachycardia-linked ryanodine receptor variants exhibit domain-specific...

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited stress-induced arrhythmogenic disease often caused by point variants in the cardiac ryanodine receptor (RyR2) that enhance diastolic sarcoplasmic reticulum (SR) Ca²⁺ leak. These RyR2 variants cluster in three hot-spots (N-terminal, central and C-terminal domains). We previously demonstrated a pathologic diastolic RyR2 conformation in heart failure, oxidative stress and CaMKII phosphorylation exhibiting a trilogy of effects: (1) reduced calmodulin (CaM)-RyR2 affinity, (2) enhanced unzipping peptide (DPc10) binding and (3) elevated diastolic SR Ca²⁺ leak that are dantrolene-sensitive. Here we test whether this pathological trilogy occurs in CPVT knock-in (KI) mice bearing N-terminal (R176Q/+), central (R2474S/+) and C-terminal variants (R4496C/+). Isolated saponin-permeabilized ventricular myocytes from all three KI mice exhibited unaltered baseline CaM and DPc10 binding to RyR2 versus wild-type (WT) myocytes. However cAMP-induced protein kinase A (PKA)-dependent RyR2 phosphorylation revealed the pathological trilogy, but only in R176Q and R2474S KI myocytes. That contrasts with WT and R4496C KI myocytes where cAMP enhanced SR Ca²⁺ leak without altering either CaM- or DPc10-RyR2 affinity. We conclude that CPVT-linked RyR2 variants at baseline may not exhibit the pathological diastolic trilogy above, but that trilogy is induced by PKA activation only in N-terminal and central domain CPVT variants. Thus the mechanism of arrhythmogenic RyR2 leak in pore domain variants (e.g. R4496C) may not involve the same pathological conformational changes as in N-terminal and central domain variants. This may inform which specific CPVT-linked RyR2 variants may benefit from therapeutic strategies that target this pathological trilogy conformation. KEY POINTS: We tested whether CPVT-linked RyR2 variants in the N-terminal (R176Q/+), central (R2474S/+) and C-terminal/pore (R4496C/+) domains mimic a pathologic trilogy of reduced CaM affinity, enhanced DPc10 peptide binding and arrhythmogenic leaky RyR2. At baseline, none of these RyR2 variant knock-in ventricular myocytes exhibited the above trilogy that is seen with heart failure, oxidative stress and CaMKII activation. However PKA activation by cAMP promotes that pathological trilogy, but only in the N-terminal and central domain variant myocytes, not the pore domain variant. Targeted therapies may need to be different for different CPVT-linked variants.