Sirtuins Positively Regulate KATP Channels, Which Contributes to their Cardioprotective Role

Sirtuins are NAD +-dependent enzymes that deacetylate proteins specific lysine(K) residues and are involved in aging, energy metabolism, inflammation, and stress resistance. We hypothesized that sirtuins positively regulate K ATP channels, thereby contributing to stress resistance. We increased cytosolic NAD + by preincubation with the NAD + precursor nicotinamide-mononucleotide (NMN) (2 mM). After 24 hours, K ATP channel currents were recorded in excised patches from HEK293 cells expressing Kir6.2/SUR2A, rat cardiomyocytes, or INS-1 cells. In all cell types, K ATP current density was increased by NMN and the inhibitory response of ATP was decreased. We confirmed that NMN increases cytosolic NAD + levels and tested the roles of the cytosolic sirtuins (SIRT1 and SIRT2). Inhibiting their activities with EX5720 and AGK2 abolished the ability of NMN to increase K ATP channel density. We performed biotinylation assays and found that the increased channel density was due (at least in part) to elevated surface expression. To determine if K ATP channel subunits are K-acetylated, we immunoprecipitated K-acetylated proteins with anti-K-Ac antibodies and immunoblotted with K ATP channel antibodies. Kir6.2 and SUR2A were both constitutively K-acetylated, and the K-acetylated levels were decreased by NMN in a sirtuin-dependent manner. Kir6.2ΔC36, expressed in the absence of SUR2A, was also K-acetylated, but not functionally modulated by NMN, suggesting a key role for SUR2A. We are in the process of identifying specific K-residues responsible for function. To determine the pathophysiological relevance, we preformed cellular ischemia assays using isolated rat ventricular myocytes. Cardiomyocyte contracture in an oil-overlay ischemia assay was mitigated by pharmacological preconditioning with phenylephrine or by NMN. The protective effect of NMN was reversed by inhibiting sirtuins or by K ATP channel block with glibenclamide. In summary, sirtuin-dependent K-deacetylation positively regulates K ATP channels and has a cardioprotective role that depends on K ATP channels (Supported by TUBITAK-SBAG117S386)