Cardioprotective Action of a Novel Synthetic 19,20-EDP Analog Is Sirt Dependent

Abstract

Mounting evidence suggests that cytochrome P450 epoxygenase-derived metabolites of docosahexaenoic acid, called epoxydocosapentaenoic acids (EDPs), limit mitochondrial damage after cardiac injury. In particular, the 19,20-EDP regioisomer has demonstrated potent cardioprotective action. Thus, we investigated our novel synthetic 19,20-EDP analog SA-22 for protection against cardiac ischemia-reperfusion (IR) injury. Isolated C57BL/6J mouse hearts were perfused through Langendorff apparatus for 20 minutes to obtain baseline function, followed by 30 minutes of global ischemia. Hearts were then treated with vehicle, 19,20-EDP, SA-22, or SA-22 with the pan-sirtuin inhibitor nicotinamide or the SIRT3-selective inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP) at the start of 40 minutes reperfusion (N = 5-8). We assessed IR injury-induced changes in recovery of myocardial function, using left ventricular developed pressure and systolic and diastolic pressure change. Tissues were assessed for electron transport chain function, SIRT1 and SIRT3, optic atrophy type 1, and caspase-1. We also used H9c2 cells in an in vitro model of hypoxia/reoxygenation injury (N = 3-6). Hearts perfused with SA-22 had significantly improved postischemic left ventricular developed pressure, systolic and diastolic recovery (64% of baseline), compared with vehicle control (15% of baseline). In addition, treatment with SA-22 led to better catalytic function observed in electron transport chain and SIRT enzymes. The protective action of SA-22 resulted in reduced activation of pyroptosis in both hearts and cells after injury. Interestingly, although nicotinamide cotreatment worsened functional outcomes, cell survival, and attenuated sirtuin activity, it failed to completely attenuate SA-22-induced protection against pyroptosis, possibly indicating EDPs exert cytoprotection through pleiotropic mechanisms. In short, these data demonstrate the potential of our novel synthetic 19,20-EDP analog, SA-22, against IR/hypoxia-reoxygenation injury and justify further development of therapeutic agents based on 19,20-EDP.

Figure 1.. SA-22 is a structural 19,20-EDP mimetic.

Chemical structures of an endogenously abundant metabolite of DHA, 19,20-EDP (A) as well as the metabolically more stable, synthetic analog SA-22 (B). DHA: Docosahexaenoic acid, EDP: Epoxydocosapentaenoic acid

Figure 2.. SA-22 enhanced postischemic functional recovery.

Hearts were assessed for contractile function at baseline (B₂₀), during ischemia (10, 20, 30), and after reperfusion (R₁₀, R₂₀, R₃₀, and R₄₀). Measurement parameters included LVDP (A,D), rate of contraction (dP/dt max) (B), and rate of relaxation (dP/dt min) (C). Heart rate was assessed as beats per minute (BPM) (E). Values represent mean ± SEM; # *p<0.05 vs. vehicle IR; *p<0.05 vs. SA-22 (n = 5–8 per group). LVDP; left ventricular developed pressure.

Figure 3.. SA-22 treatment preserved ETC function following IR-injury.

Mitochondria extract from perfused hearts were assessed for RC activity (nmol/mg/ml) at complex(s) I (A), II (B), III (D), and IV (E). Representative immunoblots and densitometric quantification for SDH-A (C), COX IV (F), and Long OPA-1 isoforms (I). Enzymatic activity as well as representative immunoblots and densitometric quantification of citrate synthase (G-H). Values represent mean ± SEM, *p<0.05 vs. aerobic control; # p<0.05 vs. vehicle IR (n = 4–6 per group). OPA-1; optic atrophy type-1. ETC; electron transport chain. VDAC; voltage-dependent anion channel. SDH-A; succinate dehydrogenase subunit A. NADH; reduced nicotinamide adenine dinucleotide. COX IV; cytochrome c oxidase

Figure 4.. SA-22 administration preserved sirtuin activity against IR injury.

SIRT3 and −1 deacetylase activity assayed with fluorescent kit (RLU) in isolated mitochondrial and cytosolic fractions respectively (A,D). Representative immunoblots and densitometric quantification of SIRT3 (B), acetyl MnSOD₂ (C), SIRT1 (E), and acetyl lysine (F). Values represent mean ± SEM, *p<0.05 vs. aerobic vehicle; # p<0.05 vs. vehicle IR (n = 4–5 per group). RLU; relative luminescence units. VDAC; voltage-dependent anion channel. MnSOD₂; manganese superoxide dismutase. SIRT1/3; silent mating type information regulation 2 homolog 1/3.

Figure 5.. SA-22 perfusion attenuated IR-induced pyroptosis.

Caspase-1 proteolytic activity assayed with a fluorescent kit (RLU) in isolated cytosol fraction (A). Representative immunoblots and densitometric quantification of cytosolic cleaved IL-1β normalized to non-cleaved IL-1β (B), and cleaved GSDMD resolubilized from plasma membranes (C). Values represent mean ± SEM, *p<0.05 vs. aerobic control; # p<0.05 vs. vehicle IR (n = 4–5 per group). RLU; relative luminescence units. IL-1β; interleukin-1 beta. GSDMD; gasdermin D.

Figure 6.. SA-22 ameliorated HR injury in H9c2 cells.

Retinoic acid-induced cell differentiation was confirmed by immunoblotting and densitometric quantification of cTnT normalized to β-actin (A). General cell viability after 24 hours of hypoxia followed by 6 hours of reoxygenation was assayed via trypan blue cell counting (B). Caspase-1 proteolytic activity assayed with a fluorescent kit (RLU) (C). Representative immunoblots and densitometric quantification of acetyl MnSOD₂ (D), SIRT1 (E), SIRT3 (F). Values represent mean ± SEM, *p<0.05 vs aerobic control, #p<0.05 vs hypoxic control (n = 3–6 per group). MnSOD₂; manganese superoxide dismutase. cTnT; cardiac Troponin T. SIRT1/3; silent mating type information regulation 2 homolog 1/3.