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

Abstract

Mounting evidence suggests that CYP epoxygenase-derived metabolites of docosahexaenoic acid, called epoxydocosapentaenoic acids (EDPs), limit mitochondrial damage following 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 IR injury. Isolated C57BL/6J mouse hearts were perfused via Langendorff apparatus for 20 minutes to obtain baseline function followed by 30 minutes of global ischemia. Hearts were then treated with either vehicle, 19,20-EDP, SA-22, or SA-22 with the pan-sirtuin inhibitor nicotinamide (NAM), 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, systolic and diastolic pressure change. Tissues were assessed for ETC function, SIRT-1 and −3, optic atrophy type-1, and caspase-1. We also utilized H9c2 cells in an in vitro model of hypoxia/reoxygenation injury (N=3–6). Hearts perfused with SA-22 had significantly improved postischemic LVDP, systolic and diastolic recovery (64% of baseline), compared to 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 or cells following injury. Interestingly, while NAM co-treatment 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/HR injury and justifies further development of therapeutic agents based upon 19,20-EDP.

INTRODUCTION

Morbidity and mortality due to ischemic heart disease continues to be a major health issue worldwide.^{1; 2} The gold standard treatment for ischemia remains the immediate restoration of myocardial blood-flow.^{3; 4} Paradoxically, reperfusion triggers a secondary wave of myocardial cell death known as ischemia-reperfusion (IR) injury.^{5; 6} As cardiomyocytes have a limited regenerative capacity, the myocardium is especially vulnerable to IR injury.^{7; 8} During ischemia, cardiomyocytes metabolically shift towards anaerobic glycolysis, which subsequently lowers cytosolic pH and causes an accumulation of intracellular calcium. Upon restoration of physiological pH, calcium triggers mitochondria depolarization via the mitochondrial permeability transition pore. ^9–11 Research demonstrates mitochondrial dysfunction contributes to adverse cardiac outcomes following IR injury, as approaches to preserve mitochondrial integrity limit cardiac damage.^{5; 12}

Sirtuins (SIRT1–7) are a class of nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylase enzymes known to regulate a multitude of critical cellular processes, such as inflammation, mitobiogenesis, cell death, and transcription.^13–15 While sirtuins localize to a wide range of cellular compartments, they all possess a highly conserved NAD⁺ binding domain.¹⁶ Thus, sirtuins can act as a metabolic switch, regulating cellular metabolism across many locales.¹⁷ Notably, SIRT1 and SIRT3 possess strong deacetylase activity.¹⁸ Due to high pH, most mitochondrial proteins are acetylated under physiological conditions and as such can be activated by SIRT3.^{19; 20} Subsequently, SIRT3 has been shown to be involved in mitochondrial biology, from dynamic fission/fusion and respiration to antioxidant processes, and is critical for adaptive cellular responses to various stresses, such as ischemia.^{15; 18; 21; 22} Growing evidence indicates pyroptosis plays a central role in the pathogenesis of myocardial IR injury, where enhanced sirtuin activity can blunt the response.^23–26

N-3 polyunsaturated fatty acids (PUFAs) are abundant in the human body and have been correlated with positive cardiovascular outcomes.^{27; 28} PUFAs are readily metabolized by cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) enzymes to a plethora of bioactive lipid mediators, however, their mechanisms of action remain elusive. Recent research has shown CYP epoxygenase generated metabolites of docosahexaenoic acid (DHA) are cytoprotective.^29–31 Currently, the therapeutic potential of epoxy fatty acid (EpFA) molecules is limited by several issues regarding their bioavailability including (i) autoxidation, (ii) β-oxidation, (iii) esterification, and (iv) metabolism by other pathways of the eicosanoid cascade, which led to the development of novel pharmacophores.^{27; 32–34} Our group has also demonstrated that synthetic analogs based upon the structure of 16,17-epoxydocosapentaenoic acid (16,17-EDP) were capable of exerting cardioprotective effects against IR injury.³⁵ Previous data indicated that EDP-induced cardioprotection is in part dependent upon SIRT1 and SIRT3 activity.^{22; 35} The current study demonstrates the cardioprotective effect of a novel 19,20-EDP EpFA analog.

METHODS

Synthesis of 19,20-EDP Analog SA-22

(10Z,16Z)-18-(3-Ethyloxiran-2-yl)octadeca-10,16-dienoic acid (SA-22) (Figure 1) was synthesized by the Falck lab at ≥95% purity using established synthetic methods and characterized using ¹H/ ¹³C Nuclear Magnetic Resonance (NMR) and mass spectroscopy (see Supplemental Digital Content 1, http://links.lww.com/AIE/A1).^{36; 37}

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

Animals

All experiments used young, male wildtype (WT) C57BL/6J mice weighing 25–35g, aged between 2–6 months. The mouse colony was housed and maintained at the University of Alberta under stable conditions with regards to temperature and humidity. Mice were given standard rodent chow ad libitum (fat 11.3%, fiber 4.6%, protein 21% (w/w)). Dietary composition included linolenic acid (0.27%), linoleic acid (2.12%), arachidonic acid (0.01%), omega-3 fatty acid (0.45%), total SFA (0.78%), and total MSFA (0.96%) (PicoLab^®Rodent Diet 20 Cat. No 5053, LabDiets, Inc., St. Louis, MO, USA). All animal experimental protocols were approved by the University of Alberta Health Sciences Welfare Committee (University of Alberta Animal Welfare, ACUC, study ID#AUP330) and conducted according to strict guidelines provided by the Guide to the Care and Use of Experimental Animals (Volume. 1, 2nd ed., 1993, from the Canadian Council on Animal Care).

Isolated Heart Perfusions

Mice were anesthetized via intraperitoneal injection with sodium pentobarbital (Euthanyl, 100mg/kg). Mice were then monitored for complete non-responsiveness to external stimulation before the hearts were excised and perfused in Langendorff mode with Krebs-Henseleit buffer (pH 7.4) (in mM) 120 NaCl, 25 NaHCO₃, 10 Dextrose, 1.75 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 4.7 KCL, 2 Sodium Pyruvate and bubbled with 95% O₂, and 5% CO₂ at 37°C.^{29; 38} Following hanging of the heart, the left atrium was excised, and a water-filled, plastic saran-wrap balloon was inserted into the left ventricle via the mitral valve. Connection of the balloon to a pressure transducer allowed for continuous monitoring of LVDP (cm H₂O) and heart rate (HR) (beats/min). Hearts were omitted from experimental analysis if they failed to sustain an LVDP greater than 80 cm H₂O or had a persistent arrhythmia. Perfusions were done in retrograde at a constant flow rate for 20 minutes at baseline before 30 minutes of global no flow ischemia followed by 40 minutes of reperfusion. Starting at reperfusion hearts were perfused with vehicle (final concentration contained less than 0.1% DMSO), SA-22 (1 μM), or 19,20-EDP (1 μM). Some hearts were perfused with SA-22 (1 μM) together with either the pan-sirtuin inhibitor nicotinamide (NAM, 30 μM) (Sigma-Aldrich, N3376–100G) or the SIRT3-selective inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP, 50 μM) (Selleck, S8628). Previous studies with CYP-derived n-3 EpFAs demonstrating cardioprotective benefits informed our choice of concentrations.^{39; 40} Hearts were immediately snap frozen in liquid nitrogen for tissue analysis following reperfusion. Postischemic functional recovery was assessed by taking the percentage of left ventricular developed pressure (LVDP) at 40min reperfusion as a percentage of baseline LVDP. Software from ADI (AD Instruments, 4360 Arrowswest Drive, Colorado Springs, Colorado) was used to acquire and analyze haemodynamic parameters.

Cell Culture and Treatment Protocols

H9c2 rat embryonic myoblast cells (Sex Unspecified) were cultivated in standard DMEM media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cell differentiation was induced by supplementation of media with 10 nM all-trans retinoic acid for 14 days. Differentiation of cells was confirmed by densitometric blot analysis of cardiac Troponin T (cs5593s), comparing cells that did or did not receive retinoic acid⁴¹. Cells were plated at a density of 1.0 × 10⁶ cells/mL and culture conditions were maintained at 37°C, 5% CO₂ and 95% air. Cell viability was assessed with a trypan blue exclusion test. H9c2 cells subjected to hypoxia-reoxygenation (HR) or normoxia were treated with the following agents: SA-22 (1 μM) and/or the pan-sirtuin inhibitor nicotinamide (NAM) (Sigma-Aldrich, N3376–100G) (30 μM). Stock solutions of both compounds were prepared in 100% ethanol. Final concentrations of both solvents were less than 0.01% of the treatment solutions.

Hypoxia-Reoxygenation Exposure

All hypoxic treatments used deoxygenated medium. H9c2 cells were placed in a computer-controlled humidified hypoxic chamber (0.9% O2, 5% CO2, and 94% N2) for 24 h followed by reoxygenation under normoxic conditions for 6 h. Control cells were exposed to 30 h of normoxia. The hypoxic chamber and controller were custom-designed and assembled in the instrumentation workshop at the Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada.

Immunoblotting

Subcellular fractions were isolated from frozen heart tissues following grinding with a mortar and pestle on dry ice and homogenization in ice-cold buffer (20 mmol/L Tris-HCL, 50 mmol/L NaCl, 50 mmol/L NaF, 5 mmol/L sodium pyrophosphate, 1 mmol/L EDTA, and 250 mmol/L sucrose, pH 7.0) as previously described.⁴² Cellular debris was separated via centrifugation at 800x g and 4°C for 10 minutes. Plasma membrane associated proteins such as Gasdermin D contained in the debris pellet were re-solubilized with homogenization buffer containing 8M urea. Subsequent centrifugation of the supernatant at 10,000x g for 20 minutes produced mitochondrial-enriched pellets. Mitochondrial pellets were resuspended in homogenization buffer for analysis. Ultracentrifugation of the supernatant at 105,000g for 60 minutes separated microsomal membranes from the cytosol. Total protein concentration was assessed via Bradford assay, and Western blotting was carried out as previously described.²⁹ In short, sample proteins (15–50ug) were resolved on polyvinylidene difluoride (PVDF) membranes (BioRad Laboratories) whereupon they were probed with antibodies against SDHA-1 (cs5839s, 1/2000), COX IV (cs11967s, 1/5000), Citrate Synthase (ab129095, 1/5000), SIRT3 (cs5490s, 1/1000), SIRT1 (cs9475s, 1/1000) total MnSOD₂ (ab13533, 1/1000), acetyl-MnSOD₂ (ab137037, 1/1000), acetyl-Lysine (cs9441, 1/1000), OPA-1 (bd612606, 1/1000), IL-1β (ab9722, 1/500), GSDMD (af4012, 1/500), NLRP3 (cs15101s, 1/500), VDAC (ab14734, 1/2000), β-Actin (cs4967, 1/2000), α-Tubulin (ab4074, 1/2000), and cardiac Troponin T (cs5593s, 1/500). After washing, membranes were incubated with corresponding secondary antibodies. Blots were visualized with SuperSignal^™ West Pico PLUS (Thermo Scientific, Ref# 34577), and band intensities were assessed using Image J (NIH, USA).

Enzymatic Assays

Assessment of Mitochondrial Enzyme Activity

Spectrophotometry was employed to assess mitochondrial respiratory chain enzymatic function as previously described.⁴³ Mitochondrial heart samples were assessed for NADH:ubiquinone oxidoreductase (complex I), succinate dehydrogenase (SDH, complex II), decyl-ubiquinol cytochrome C oxidoreductase (complex III), cytochrome C oxidase (complex IV), and citrate synthase (CS) activities. Complex activity was normalized to volume and protein concentration.

Sirtuin Activity

Mitochondrial SIRT3 and cytosolic SIRT1 activity was detected according to the manufacturer’s instructions using a fluorescent assay kit (50088 and 50081 respectively, BPS Bioscience, San Diego, CA, USA). This assay involves mixing samples with a specific fluorogenic HDAC substrate, bovine serum albumin, NAD⁺, and proprietary assay buffer. Upon initial deacetylation by SIRT and subsequent exposure to the assay developer solution, the HDAC substrate produces a fluorescent product that can be measured using a fluorometric plate reader at 350–380/440–460 nm excitation/emission wavelengths. SIRT activity in heart mitochondrial lysates was expressed as a fold of the aerobic vehicle group in relative luminescence units (RLU).

Caspase Activity

Cytosolic Caspase-1 activity was assessed as a marker of NLRP3 inflammasome complex activation. Activity in heart tissue lysate is proportional to the generation of a fluorescent signal following cleavage and release of 7-amino-4-methylcoumarin (AMC) from a tagged caspase-1 peptide substrate, Ac-YVAD-AMC (Enzo Life Sciences, ALX-260–024-M005) over time. Fluorescence was measured using a fluorometric plate reader at 380/460 nm excitation/emission wavelengths. Activity was normalized to protein concentration then expressed as a fold of the aerobic vehicle treatment group in relative luminescence units (RLU).

Statistics

Values represent mean ± standard error of mean (SEM). Statistical significance was determined by one-way ANOVA with a Bonferroni post hoc test when comparing differences between groups; p < 0.05 was considered statistically significant. All replicates presented are individual biological replicates.

RESULTS

Postischemic Functional Recovery is Improved by SA-22 Treatment

Initial assessment demonstrated SA-22 had similar cardioprotective effects when compared to 19,20-EDP (Figure 2). Importantly, pre-ischemic myocardial function was comparable across all treatment groups (Figure 2). Hearts perfused with SA-22 had significantly improved postischemic left ventricular developed pressure (LVDP) as well as rates of contraction (dP/dt max) and relaxation (dP/dt min) compared to the vehicle IR group (Figure 2A–D). Perfusion with the pan-sirtuin inhibitor NAM as well as the SIRT3-selective inhibitor 3-TYP markedly reduced SA-22-induced recovery of LVDP, dP/dt max, and dP/dt min (Figure 2A–D). Lastly, there were no notable differences in heart rate (HR) between all study groups (Figure 2E). Altogether, these data suggest that SA-22 can improve postischemic functional recovery.

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.

SA-22 Preserves Respiratory Chain Function Following IR-Injury

Preserving mitochondrial respiration is critical to ameliorating myocardial injury in post-myocardial infarction and postischemic contexts.⁴⁴ Thus, we assessed the catalytic activity of NADH:ubiquinone oxidoreductase (complex I), succinate dehydrogenase (SDH, complex II), cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), and citrate synthase (CS), an aerobic metabolism biomarker due to its role as the rate-limiting factor for Krebs cycle entry. IR injury significantly decreased enzymatic activity in complexes I, II, III and IV when compared to the aerobic vehicle group but not CS activity (Figure 3A,B,D,E,G). Perfusion with SA-22 consistently preserved catalytic activity of all 4 complexes following the myocardial insult (Figure 3A,B,D,E). Interestingly, NAM co-treatment abrogated analog-induced protection of activity at complexes I and II but not III or IV, suggesting sirtuins are potentially important mediators for the benefits evoked by EpFAs (Figure 3A,B,D,E,G). Protein expression levels of succinate dehydrogenase subunit A, a major catalytic subunit of complex II, were unchanged across all groups (Figure 3C). Intriguingly, while SA-22 and vehicle IR treatment did not appear to change COX IV expression, treatment with NAM alongside SA-22 significantly reduced protein levels when compared to SA-22 alone (Figure 3F). SA-22 treatment protected citrate synthase when compared to the vehicle IR group. NAM treatment did not affect CS levels compared to SA-22 alone (Figure 3H).

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

OPA-1 is a structural protein vital for proper mitochondrial cristae organization, respiration, and metabolic adaptation.^{45; 46} OPA-1 oligomerization within the mitochondrial inner membrane is critical for proper stabilization of cristae and ATP synthase.⁴⁷ Following mitochondrial depolarization during IR injury, OPA-1 dissociates from ATP synthase and is subsequently proteolytically cleaved, exacerbating mitochondrial injury. As such, protecting respiratory chain function may help to curtail IR-induced dysregulations in mitochondrial structure. Interestingly, perfusion with SA-22 partially ameliorated the accumulation of shorter OPA-1 isoforms stimulated by IR-injury (Figure 3I). Treatment with NAM did not appear to reduce this effect.

Sirtuin Function is Maintained in Post-IR Hearts Perfused with SA-22

SIRT3 is capable of deacetylating and activating several targets involved with cellular stress responses, including the antioxidant enzyme manganese superoxide dismutase (MnSOD₂), whose ability to scavenge ROS is greatly enhanced upon deacetylation by SIRT3.^48–50 Importantly, over 90% of the cellular ROS produced during IR injury arises from electrons escaping disrupted ETC subunits, which are known SIRT3 substrates. Thus, we theorized that analogs of 19,20-EDP may limit mitochondrial dysfunction, ROS production, and oxidative damage following IR injury by preserving sirtuin activity.^51–54 From our results, both SIRT1 and SIRT3 activity were significantly reduced when compared to the aerobic vehicle group (Figure 4A,D). However, perfusion with SA-22 significantly attenuated the decline in sirtuin activity caused by IR injury (Figure 4A,D). While levels of SIRT1 and SIRT3 protein expression showed no significant differences between treatment groups (Figure 4B,E). The changes in SIRT3 activity were reflected in the acetylation levels of two known SIRT3 substrates, MnSOD₂ and total lysine, where SA-22 prevented an increase in acetylation of both markers induced by IR injury (Figure 4C,F). NAM co-perfusion attenuated SA-22-mediated protection of sirtuin activity, reducing SIRT1 and −3 activity levels and enhancing acetylation of MnSOD₂ and lysine residues (Figure 4A–F).

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.

Perfusion with SA-22 Attenuates IR injury-induced Pyroptosis

Caspase-1 is a key mediator of pyroptotic cell death, transducing inflammatory signals by proteolytically cleaving the pyroptotic pore protein gasdermin D (GSDMD) as well as secreted cytokine interleukin 1 beta (IL-1β) following IR injury.^{24; 55; 56} We tested SA-22 for potential attenuation of pyroptosis. Caspase-1 activity levels were significantly increased in the vehicle IR group compared to aerobic control, with SA-22 treatment returning them to aerobic vehicle levels (Figure 5A). Secondly, SA-22 reduced cleavage of IL-1β when compared to vehicle IR, where virtually all IL-1β was cleaved/active (Figure 5B). Once activated via proteolytic cleavage, GSDMD localizes to the cellular plasma membrane (PM) where it functions as a pore for pyroptotic signals such as IL-1β to be secreted.⁵⁷ Across all treatment groups, only the PMs of the vehicle IR hearts possessed any notable levels of cleaved GSDMD. SA-22 administration reduced the PM-localization of cleaved GSDMD (Figure 5C). Unexpectedly, despite the ability of NAM to prevent the benefits induced by SA-22 regarding myocardial function and respiration, there was no effect on the activation of pyroptosis when compared to treatment with SA-22 alone (Figure 5A–C). Cumulatively, these data suggest that maintenance of sirtuin activity is important to the cardioprotective actions exerted by EpFAs in the context of myocardial IR injury.

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.

SA-22 Protects H9c2 Cells Against Hypoxia/Reoxygenation Injury

Previous work has demonstrated EDPs are protective against HR injury in an atrial cell line, HL-1 cardiac cells.²² In the current study, we differentiated the ventricular cardiac myoblast cell line, H9c2 cells, to assess the cytoprotective effect of SA-22. First, successful differentiation of H9c2 cells using retinoic acid (RA) was confirmed by densitometric blot analysis of cardiac troponin T levels (cTnT) compared to cells that did not receive RA (Figure 6A). Exposing H9c2 cells to HR injury caused a significant decrease in cell viability (Figure 6B). Notably, SA-22 treatment attenuated a significant increase in caspase-1 activity elicited by HR (Figure 6C). NAM treatment prevented SA-22 from limiting caspase-1 activity, suggesting that sirtuins have a role in the cytoprotective response. HR injury also stimulated robust increases in acetylation of MnSOD₂, indicating a reduction in SIRT3 activity, which was reversed by addition of SA-22 (Figure 6D). SIRT1 levels were relatively unchanged across treatment groups (Figure 6E). HR cells also possessed a marked reduction in SIRT3 levels compared to aerobic baseline, which SA-22 treatment rescued (Figure 6F). Notably, NAM treatment did not affect SIRT levels (Figure 6E–F).

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.

DISCUSSION/CONCLUSION

This study presents evidence for the cardioprotective action of a novel synthetic 19,20-EDP analog, SA-22. One of the largest hindrances to the translation of therapeutic interventions from experimental models to clinical applications is the unpredictability of MI onset.⁵⁸ Fortunately, here we have demonstrated a novel pharmacological agent that remains efficacious even when administered post-ischemically. Analysis of our data suggests that SA-22 attenuates postischemic injury by ensuring sirtuins remain active despite insult, which subsequently preserves mitochondrial function and blunts activation of pyroptosis. Co-perfusion with sirtuin inhibitors abrogated the benefits of SA-22 treatment. These findings are supported by repetition in an in vitro model of hypoxia-reoxygenation injury using H9c2 cardiac cells. Notably, EDPs appear capable of penetrating and protecting cardiac tissues in the absence of any special delivery mechanism or formulation. Theoretically, these compounds could be administered alongside existing reperfusion protocols (e.g., thrombolytic agents) upon hospitalization. These data demonstrate how a synthetic 19,20-EDP analog exhibits cardioprotective effects against IR injury.

Sirtuins are a family of NAD⁺-dependent deacetylase enzymes, consisting of seven members, which can regulate a vast array of mitochondrial proteins via deacetylation activity, including many involved in respiration, oxidative stress responses, metabolism, and mitochondrial dynamics.^18–20 The relative pH and abundance of acetyl-CoA contribute to greater than 65% of mitochondrial proteins being acetylated under normal physiological conditions. The mitochondrial antioxidant ROS-scavenger MnSOD₂ has its activity significantly upregulated upon deacetylation by SIRT3.⁵⁰ Complexes I and II have been demonstrated to be direct targets for binding and subsequent deacetylation by SIRT3.⁵⁹ In agreement with the broader literature, this study demonstrated hearts perfused with SA-22, a potential sirtuin-potentiating compound, improved respiratory chain function and lowered mitochondrial protein acetylation. In addition, SA-22 administration lowered proteolytic cleavage of OPA-1, a protein critically important for proper mitochondrial function and cristae organization, in response to IR injury; possibly because of potentiated sirtuin activity.⁶⁰ A rate-limiting factor for sirtuin activity is NAD⁺; as such, decreased SIRT3 activity following IR injury could be caused by a reduction in the NAD⁺/NADH ratio. Previously, we demonstrated hearts perfused with a synthetic 16,17-EDP analog possessed a much higher NAD⁺/NADH ratio than hearts treated with vehicle.³⁵ Broader literature indicates deleterious cardiac effects occur following depletion of sirtuin activity, thus the observed SA-22 mediated preservation of sirtuin activity correlates with significantly enhanced postischemic functional recovery. Moving forward it will be important to consider possible sex differences, specifically as they relate to sirtuins. While our group has not observed significant differences between young male and female mice regarding the efficacy of DHA and 19,20-EDP, we have yet to substantively test synthetic epoxylipid analogs in female mice.²⁹ Furthermore, emerging evidence seems to suggest potential significant sex differences in SIRT expression.^{61; 62} Future exploration of these topics may help to rationalize prior observations regarding sexually dimorphic susceptibility to ischemic heart disease.⁶³

Activation of pyroptosis following reperfusion exacerbates tissue injury and contributes to adverse outcomes. Cleavage of GSDMD by caspase-1 leads to semi-permeabilization of the outer plasma membrane and secretion of cell death promoting factors such as IL-1β. These factors actively polarize and recruit neighboring macrophages to the site of injury, perpetuating inflammation, and aggravating cell death.^{23; 24; 64} Research has shown activation of sirtuin activity may be a viable strategy for attenuation of pyroptotic cell death.^{25; 26} Our results demonstrate SA-22 attenuated the activation of pyroptotic mediators such as caspase-1, GSDMD, and IL-1β. Intriguingly, despite perfusion with NAM greatly worsening myocardial function recovery, these hearts exhibited protection against pyroptosis comparable to hearts perfused with SA-22 alone. Further work is necessary to identify and characterize potential pleiotropic mechanisms by which SA-22s preserve heart function and protect cardiomyocytes against IR-induced pyroptosis.

While we presume that bioactivity of EDPs specifically within the cardiomyocyte mitochondrium is vital to their cardioprotective properties in the context of ischemic heart disease, mounting evidence indicates that EpFAs are active in several constituents of the myocardium, including endothelial and smooth muscle cells across a plethora of injury models.^{27; 30; 34; 65} It is imperative to investigate the role of other cell types in EpFA-mediated cardioprotection. However, severe limitations regarding the bioavailability of endogenous EpFAs have hindered their therapeutic utilization until now.^{33; 66; 67} In addition to rapid metabolic conversion into diol compounds by soluble epoxide hydrolase, a disadvantage of PUFAs is their strong tight binding interactions with plasma proteins.^{68; 69} Despite this problem however, eicosanoids seem to readily transfer to cells.^{27; 34} Further elucidation of EDP pharmacokinetics involving off-target interactions and their relationship to experimental efficacy is necessary for the future consideration of EDPs as therapeutic agents. SA-22 is a first-generation mimetic, intended solely for proof-of-concept studies. Subsequent generations of analogs will feature modifications that obviate metabolism by other eicosanoid pathways (e.g., COX and LOX) and reduce auto-oxidation. Additional work characterizing the efficacy of EDPs at various concentrations and how this is affected by chemical modification will be critical for discovering potential candidates for clinical trials. While all our experiments here were conducted using 1 μM epoxide, a concentration informed by previous studies, significant bioactivity of EpFA at nanomolar concentrations has been reported.⁷⁰ Furthermore, exploration of higher concentrations may yield greater benefits than what has been observed previously.

Here we demonstrated that SA-22, a synthetic analog of 19,20-EDP, could improve postischemic functional recovery in murine hearts when compared with vehicle alone. Secondly, our data suggests that SA-22-induced protection is dependent on maintaining sirtuin activity, as pharmacological inhibition of sirtuins abrogated many of the benefits evoked by SA-22 treatment. Furthermore, SA-22 preserved ETC complex I activity, which corresponded with a reduction in the cleavage of the mitochondria cristae organizer OPA-1. Crucially, SA-22 attenuated activation of key pyroptosis markers after insult when compared to the ischemic vehicle group. Although the exact molecular mechanisms of action for SA-22/EpFAs requires further elucidation, this study provides further evidence for the viability of synthetic EpFA analogs as a therapeutic agent for IR injury.

LIST OF ABBREVIATIONS AND ACRONYMS

3-TYP

3-(1H-1,2,3-triazol-4-yl) pyridine

COX

Cyclooxygenase

COX IV

Cytochrome c oxidase subunit 4

CS

Citrate Synthase

CYP

Cytochrome p450

DHA

Docosahexaenoic acid

EDPs

Epoxydocosapentaenoic acids

EpFA

Epoxy Fatty Acid

ETC

Electron Transport Chain

HR

Hypoxia-Reoxygenation

IL-1β

Interleukin 1 Beta

IR

Ischemia-Reperfusion

LVDP

Left ventricular developed pressure

MnSOD₂

Manganese superoxide dismutase

NAD+

Nicotinamide adenine dinucleotide

NAM

Nicotinamide

OPA-1

Optic Atrophy Type-1

PUFAs

Polyunsaturated fatty acids

PVDF

Polyvinylidene difluoride

RA

Retinoic acid

RC

Respiratory Chain

ROS

Reactive oxygen species

SDH-A

Succinate dehydrogenase subunit A

SIRT1

Sirtuin-1

SIRT3

Sirtuin-3

VDAC

Voltage-dependent anion channel