Establishing a direct interaction between the 19,20-EDP analog SA-22 and SIRT3: impact on cardiac mitochondrial...

Despite extensive study, the structural, metabolic, and mechanistic heterogeneity amongst polyunsaturated fatty acids (PUFA) have confounded identification of their molecular targets and roles in cardiovascular diseases. Previously our group demonstrated that the cardioprotective properties of both 19,20-epoxydocosapentaenoic acid (EDP), a CYP450-derived metabolite of docosahexaenoic acid (DHA), and a synthetic structural analog SA-22, were SIRT3-dependent. Thus, we explored the impact of this signaling on mitochondrial homeostasis in the context of hypoxic myocardial injury. SA-22 ligand binding was confirmed via SYPRO Orange thermal shift assay.

SIRT3 catalytic activity was measured using an acetylated HDAC fluorogenic substrate assay. Point mutagenesis experiments confirmed the involvement of residue SER149. H9c2 cells were used as an in vitro model of hypoxia/reoxygenation (HR) injury. Cells were deprived of oxygen for 24 h followed by a 6-h reoxygenation period wherein cells were treated with either vehicle, 19,20-EDP (1 µM), or SA-22 (1 µM), either with the pan-sirtuin inhibitor nicotinamide (NAM) (30 µM), or the SIRT3-selective inhibitor 3-(1H-1,2,3-triazol-4-yl)-pyridine (3-TYP) (50 µM). Mitophagy was assessed via the pH-dependent fluorescent mitochondrial autophagy reporter protein (mito-Keima). Mitochondrial respiration was measured using high-resolution respirometry (Oroboros-O2K).

Addition of SA-22 altered SYPRO Orange fluorescence and improved catalytic activity in vitro but was abrogated by SER149 substitution, indicating that SA-22 is a positive allosteric modulator of SIRT3. Lastly, SA-22 protected cardiac cells against HR-induced changes in mitophagy and mitochondrial respiration in a SIRT3-dependent manner.

In conclusion, SA-22 directly binds and enhances the activity of SIRT3, preserving cardiac mitochondrial homeostasis despite myocardial hypoxia-reoxygenation injury.