Abstract Body: Introduction:
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a life-threatening inherited arrhythmia in which adrenergic stress triggers ventricular tachyarrhythmias in otherwise healthy individuals. Although β-blockers and flecainide reduce risk, they do not eliminate arrhythmias, and the core mechanism—CaMKII-driven RyR2 hyperphosphorylation and diastolic calcium leak—remains without a direct therapeutic target.
Hypothesis:
We tested the hypothesis that Hesperadin directly engages CaMKIIδ, reduces RyR2-mediated calcium leak, and suppresses stress-induced ventricular arrhythmias in CPVT.
Methods:
Hesperadin–CaMKIIδ binding was modeled using Boltz protein–ligand co-folding, with kinome-wide selectivity profiled by KinomePro-DL. Biochemical assays confirmed CaMKIIδ inhibition, and direct binding was validated by binding assay and co-crystal structure determination. In vivo target engagement was assessed in wild-type mice treated with Hesperadin (0.5 mg/kg IP daily ×5) by measuring phospholamban Thr17 phosphorylation (p-PLN-Thr17). Disease efficacy was evaluated in RyR2-H2464D CPVT mice (n=8–9/group) following acute Hesperadin (0.5 mg/kg IP) or vehicle, with epinephrine (2 mg/kg) and caffeine (120 mg/kg) challenge and continuous ECG recording. Translational efficacy was assessed in CPVT (n=3 lines) and LMNA iPSC-cardiomyocytes using optical calcium mapping.
Results:
AI modeling predicted high-affinity binding of Hesperadin within the CaMKIIδ catalytic pocket, confirmed by co-crystal structure and biochemical assay (EC50 = 51.69 nM). Hesperadin reduced p-PLN-Thr17 in vivo, confirming CaMKIIδ inhibition. In CPVT mice, Hesperadin reduced ventricular ectopy compared to vehicle following adrenergic challenge. In human iPSC-cardiomyocytes, Hesperadin (100 nM) reduced abnormal calcium transients and arrhythmic events under stress.
Conclusions:
Hesperadin directly targets CaMKIIδ, suppresses RyR2-mediated calcium leak, and reduces stress-induced arrhythmias in CPVT, supporting CaMKIIδ inhibition as a mechanism-based antiarrhythmic strategy.