Activation of orexin 2 receptor (OX2R) in cardiac myocytes protects from hypoxia-induced cell death

Aim and Hypothesis: Coronary artery disease (CAD) is the leading cause of death globally, and while coronary revascularization has decreased mortality in CAD, the incidence of heart failure (HF) from ICM continues to rise. Revascularization can lead to severe cardiac myocyte (CM) dysfunction largely driven by mitochondrial damage and oxidative stress. Therapeutically, reducing mitochondrial damage and oxidative stress in CMs remains an unmet challenge. Orexins (OR-A and OR-B) are neuropeptides derived from orexin-containing neurons in the lateral hypothalamus of the brain. Orexins exert their physiological effects through G-protein coupled receptors, orexin receptors 1 and 2 (OX1R and OX2R). Low OR-A concentrations or mutations in the OX2R gene are associated with higher risk for adverse cardiovascular outcomes, particularly after myocardial infarction and HF. Based on these findings, we hypothesize OX2R signaling attenuates hypoxia-induced cell death in CMs, leading to less ischemic injury. Approach: Expression of prepro-orexin, OX1R, OX2R was quantified by qRT-PCR using cDNA libraries prepared from individually isolated adult CMs. To assess the effects of orexin receptor signaling to attenuate cell death induced by ischemic injury, we employed an in vitro model of hypoxia-reoxygenation (H/R) in which cultured CMs (from male and female murine hearts) were exposed to 3 hours of hypoxia (hypoxic medium bubbled with 94% N2, 5% CO 2 , and 1% O 2 for 30 minutes; hypoxic chamber pressurized for 5 minutes with hypoxic gas), and then 17 hours of reoxygenation in normoxic medium. CMs were pretreated with orexin receptor ligands for 3 hours prior to H/R, and treatments were maintained for the 20 hour H/R protocol. Compounds included the endogenous ligands OR-A or OR-B, and/or OX1R- or OX2R-specific agonists (OX1Ra, OX2Ra), (Cayman Chemical). After 17 hours, cell death was quantified by counting live versus dead cells. Percent death was calculated as (hypoxia treated – hypoxia untreated)/hypoxia untreated. Results: A high proportion of individual CMs express prepro-orexin (97%), OX1R (81.5%), and OX2R (92.3%), while 78.1% of CMs express all three. In CMs subjected to H/R, OR-A and OR- B attenuated H/R-induced cell death, although OR-A was more efficacious (OR-A, 100nM: 97.9% ± 4.7%, n=8, P< 0.0001; OR-B, 100nM: 54% ± 7.1%, n=7, P=0.0112) Further, the OX1R- and OX2R-specific agonists attenuated H/R-induced cell death, although OX2Ra was more efficacious (OX1Ra, 100nM: 53% ± 9.8%, n=6, P=0.083; OR2Ra, 100nM: 107.7% ± 12.6%, n=3, P=0.002). Taken together, OR-A and the OX2R-specific agonist conferred a greater protection from hypoxia-induced cell death than OR-B or OX1R-specific agonist. Conclusion: Our data indicate that in CMs, OX2R signaling is more efficacious than OX1R signaling in attenuating H/R-induced cell death. This potentially identifies OX2R as a novel therapeutic target to prevent CM death in ICM. Funding: Project supported by IBP T32 Training Fellowship (T32 HL16642), NIH HLR01152215 (TDO) and 1R01DK134468-A1 (CMK) This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.