Endocrine Abstracts

Oxytocin (OT) treatment improves heart functional recovery in rat model of myocardial infarct. We investigated in the H9c2 cell line (cardiomyocytes model), mechanism of OT action in simulated ischemia - reperfusion (sI-R). H9c2 cells were suspended in warm ischemic buffer and placed inside an anoxic chamber for 2 h at 37 °C, then ‘reperfused’ under normal nutrients and oxygen conditions for 2 h. OT presence during ischemia increased cell viability by 9.7±2.5% (P<0.05) and OT at reperfusion increased cell viability further by 15.8±3.5% (P<0.001) as measured by MTT cell proliferation assay, OT treatment before ischemia had no effect. The significant cellular protection started at a concentration of 1 nM of OT, with an optimal protection at 62.5–125 nM. OT antagonist dose-dependently blocked the protective effect of OT. OT treatment of H9c2 cells transfected with OT receptor (OTR) siRNA increased cell apoptosis threefold in comparison to the control. OT increase in cell mortality in OTR siRNA cells could be mediated by vasopressin (AVP) receptors. Indeed, blockade of AVP receptors by conivaptan increased cell viability in sI-R conditions. OT treatment reduced fluorescence of CM-H₂DCFDA products in sI-R-treated cells indicating decrease of reactive oxidative species (ROS). Interestingly, these experiments revealed that under normoxic conditions, OT treatment alone is sufficient to trigger a short-lived burst in intracellular ROS. The OT protection of sI-R was blocked by the PI3K–Akt inhibitor, Wortmannin. Using confocal microscopy, we noted that cells treated with OT displayed increased Akt (Thr308) phosphorylation and specifically, Akt was accumulated in ring-like structures associated with mitochondria and nuclei. Demonstration that KT5823, inhibitor of protein kinase G (PKG) and ODQ, inhibitor of soluble guanylyl cyclase, reduced OT-mediated protection in sI-R, indicated the role of cGMP-dependent protein kinase. Consequently, the confocal microscopy demonstrated the increase of eNOS phosphorylation and its nuclear accumulation in cells treated with OT. OT protection in sI-R was also inhibited by ANP receptor antagonist, A71915. These data suggest that OT provides protection to the injured heart by inhibition of ROS, cardiomyocyte apoptosis and stimulation of cell protective PI-3K–Akt–PKG signaling.