Abstract Body: Background: SIRT5, a mitochondrial NAD⁺-dependent deACYLase, is implicated in arrhythmia through hypomorphic P114T variants identified in small families with sudden cardiac death. Cardiac Na⁺ current (I_Na), predominantly through Na_V1.5, governs cardiac action potential. We previously demonstrated elevated mitochondrial reactive oxygen species (ROS) and late I_Na in P114T-SIRT5 hearts.

Hypothesis: ROS-driven late I_Na augmentation drives Na⁺ and Ca²⁺ overload, triggering DADs and arrhythmias in SIRT5-loss-of-function hearts under adrenergic stress.

Aim: To define the arrhythmia substrate downstream of mitochondrial ROS-late I_Na dysregulation in P114T-Sirt5 knock-in and Sirt5^-/- mice.

Methods: Patch-clamp in isolated ventricular cardiomyocytes, confocal Ca²⁺ spark/wave imaging, in vivo epinephrine/caffeine ECG challenge, co-immunoprecipitation for Na_V1.5 ACYLation, and cardiac RNA-seq.

Results: Mitochondrial ROS scavenger mitoTEMPO normalized late I_Na in P114T cardiomyocytes (Fig. A), establishing causal ROS→late I_Na coupling, while antioxidant GSH reversed spontaneous Ca²⁺ waves and triggered activity. Sirt5^−/− hearts showed elevated Ca²⁺ spark frequency (Fig. B) and isoproterenol-induced DADs and triggered activity. In vivo, P114T mice exhibited genotype-dependent arrhythmias under epinephrine/caffeine challenge, including ventricular tachycardia (VT; Fig. C). At the molecular level, the Na⁺/Ca²⁺ exchanger NCX1 expression was upregulated, and Na_V1.5 succinylation and acetylation were elevated without changes in protein expression, consistent with impaired SIRT5 deACYLase activity. RNA-seq identified 86 differentially expressed genes including Scn3b and Cybrd1.

Conclusion: SIRT5 loss-of-function hearts exhibit mitochondrial ROS-driven late I_Na augmentation, Ca²⁺ mishandling, and arrhythmias under stress. The mechanisms linking SIRT5 loss to late I_Na elevation and proarrhythmic Na⁺ and Ca²⁺ overload warrant further investigation.