Sex Differences in Cardioprotection in the sEH/EET Signaling Pathway

1. Sex Differences in Cardioprotection in the sEH/EET Signaling Pathway Matthias J Merkel, MD PhD1,2; William Packwood, BS; Liu Lijuan, DVM2; Cao Zhiping, PhD2; Donna M Van Winkle, PhD1,2 1Anesthesiology, OHSU, Portland, OR; 2Anesthesia & Research Services, OHSU, Portland, OR RESULTS BACKGROUND Soluble epoxidehydrolase (sEH) is the primary enzyme that metabolizes epoxyeicosatrienoic acids (EETs). We previously showed that sEH inhibition reduces infarct size in male mice after temporary left coronary artery occlusion (LCAO), in part by a STAT3-dependent pathway (1). However, it is unknown if sEH inhibition can protect the female heart against myocardial ischemia-reperfusion (I/R) injury. HYPOTHESIS CONCEPT We tested the hypothesis that pharmacologic sEH inhibition reduces myocardial ischemic injury independent of sex. METHOD Figure 1: Effect of sEH inhibition with t-AUCB (A) and 14,15-EET (B) on infarct size in female and male mice There is a sex-specific dose-response difference with t-AUCB with males being protected at the lower dose (10 mg/kg IP) and females at the higher dose (30 mg/kg IP) tested 14,15-EET (2.5 mg/kg IV) significantly protected hearts from male mice against I/R-induced injury, but not females • Animals. • Female and male C57BL\6 mice, 16-24 weeks old (in vivo) and 1 week old (in vitro) • All animals received care in accordance with the Guide and Use of Laboratory Animals (Institute of Laboratory Animal Research, National Research Council, National Academy Press 1996) and IACUC approval • Regional Myocardial Ischemia-Reperfusion Injury. • Anesthetized with isoflurane and intubated with a 20G plastic IV catheter and ventilated. • ECG and rectal temperature (maintained at 37ºC) were monitored. • A PE-10 catheter was inserted into the jugular vein for drug infusion. • Left-sided thoracotomy (4th intercostal space) • Ligature placed around the Left Coronary Artery (LCA) • Re-occlusion and fluorescent microspheres infusion via needle puncture of the LV apex • Ventricles were sliced into seven sections (d=1 mm) for imaging and staining • 1% 2,3,5 triphenyltetrazolium chloride (TTC) followed by 10% formalin bath overnight • In Vitro simulation of Ischemia-Reperfusion Injury. • 3-4 hearts from same-gender mice • Dissociation with 0.625% wt/voltrypsin in Ca2+ and Mg2+ free HBSS • Centrifugation and re-suspension in FBS-M199 • Supplementation with estrogen-free 15% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 µM cytosine arabinoside, • Plated at 37°C for 1 hr under a water-saturated atmosphere of 5% CO2 -95% O2 • Suspended cells were collected and plated at 1.0 × 105 cells/cm2 incubated as above for 24 hr • Oxygen-Glucose Deprivation (OGD) • Custom-made Plexiglas hypoxia chamber • OGD 100% N2 at 37 °C and glucose-free medium (MEM/HBSS) • Reoxygenation: in 21% O2 (5% CO2)and glucose-replete FBS-M199 medium • Determination of Cell Viability • Cell death assessment via propidium iodide (PI, 5 µM) • >300 cells/sample • Data are expressed as percentage of dead cells (mean ± SEM); • Western Blot • 4-20% linear gradient SDS–polyacrylamide gels (Bio-Rad), transfer to PVDF membrane • incubation overnight at 4C with primary rabbit anti-sEH antibody (Santa Cruz, 1:2000 in 5% dry milk) • enhanced chemiluminescence (ECL-plus WB detection kit, Amersham) (1:2000 in 5% dry milk) • 1-way ANOVA and Newman-Keuls post test 1) t-AUCB (10 or 30 mg/kg IP) 2) 14,15-EET (2.5 mg/kg IV) 20-30 min 40 min 2 hours 15’ Surgical preparation Reperfusion Ischemia Figure 3: Similar Expression of sEH in cardiomyocytes from male and female mice hearts Figure 2: Cardiomyocytes from female hearts showed increased viability after pretreatment with 14,15-EET or AUDA and STAT3 inhibition abolished 14,15-EET, but not AUDA induced cytoprotection STATTIC (10 µM) 5 min before AUDA (2 µM) CONCLUSION merkelm@ohsu.edu 24 hours 90 min 3 hours 1h • The sEH/EET pathway can be utilized in both male and female hearts to protect against I/R injury. • There is a sex-specific difference in the dose-response which will need to be considered in the future before moving sEH inhibitors into clinical practice. • The role of STAT3 in 14,15-EET-mediated protection in female is similar to our previous findings in males (1) Surgical preparation Reperfusion OGD REFERENCES: 1) Merkel et al. Inhibition of soluble epoxidehydrolase preserves cardiomyocytes: Role of STAT3 signaling. Am J Physiol Heart Circ Physiol. 2009 ; 298(2): H679-H687 T-AUCB generous gift from Bruce Hammock, UC Davis, CA