Abstract Body: Background: The cardiac Na⁺ channel Na_V1.5 (encoded by SCN5A) governs inward Na⁺ current (I_Na), playing a crucial role in the fast upstroke and plateau phases of the cardiac action potential. Mutations in SCN5A are implicated in inherited and acquired arrhythmias, including ~20% of Brugada Syndrome (BrS) cases. Altered I_Na impact Ca²⁺ handling and excitation-contraction coupling. Previously, we demonstrated that SIRT1-mediated deacetylation of Na_V1.5 at K1479 enhances peak I_Na. Recently, potential mutations in SIRT5—including P114T—were identified in small families with BrS. SIRT5, another NAD⁺-dependent deACYLase from the Sirtuin family, is primarily localized to mitochondria.
Hypothesis: Sirt5 dysfunction leads to arrhythmogenesis through Na⁺ and Ca²⁺ mishandling in an oxidative stress- dependent manner in mouse hearts.
Aims: To investigate SIRT5’s role in BrS using heterologous expression systems, homozygous P114T-Sirt5 knock-in (P114T-KI) and Sirt5^-/- mice.
Methods: Protein expression and physical interactions were assessed by immunoprecipitation and immunoblot. Patch-clamp electrophysiology in HEK cells and mouse ventricular cardiac myocytes evaluated I_Na. Reactive oxygen species (ROS) levels and Ca²⁺ imaging were measured by confocal microscopy.
Results: Both WT and P114T-SIRT5 co-immunoprecipitate with Na_V1.5, but only WT enhanced peak I_Na in K1479-Na_V1.5 dependent manner in HEK cells. P114T-SIRT5 failed to do so. The late I_Na of cardiomyocytes was elevated (Figure, A), and QTc was prolonged in both Sirt5^-/- and P114T mice. P114T-KI hearts showed increased Na⁺/Ca²⁺ exchanger (NCX1) expression, elevated phosphorylation of phospholamban at Thr17 (p-PLN-Thr17, Figure, B). Live-cell staining using DCFDA or mitoSOX revealed that ROS levels were higher in P114T-KI hearts at baseline compared to WT littermates and were further exacerbated by H₂O₂. Aberrant Ca²⁺ handling and arrhythmias in Langendorff-perfused hearts of P114T mice were mitigated by the mitochondrial ROS scavenger MitoTEMPO (Figure, C).
Conclusion: SIRT5 dysfunction in P114T-SIRT5 disrupts Na⁺ and Ca²⁺ homeostasis and induces arrhythmias in a ROS-dependent manner, suggesting potential mechanisms underlying BrS. These findings highlight SIRT5 as a promising therapeutic target for BrS.