oCOm-21 as a Cardioprotective Inotrope Through S-Nitrosylation
Patients with severe ischaemic heart disease (IHD) often undergo cardiac surgery to alleviate symptoms and improve survival outcomes. However, such interventions can inevitably induce episodes of ischaemia reperfusion (IR). IR generates large amounts of reactive oxygen species (ROS) which irreversibly oxidises proteins and results in cardiomyocyte death. Due to the heart’s inability to replenish dead cardiomyocytes, there is an imperative to investigate prophylactic interventions to attenuate IR injury. Our lab group previously showed that the novel organic CO releasing molecule, oCOm-21, preconditioned isolated rat hearts subjected to IR injury. From these studies it was noted that oCOm-21 produced an inotropic effect. Inotropy is the result of increased [Ca2+]i and Ca2+ handling proteins which modulate [Ca2+]i are extremely susceptible to ROS modification. Therefore, this study aimed to 1) confirm the inotropic effects of oCOm-21 and 2) determine whether oCOm-21 S-nitrosylated the ryanodine 2 receptor (RyR2) to induce inotropy. oCOm-21 was confirmed to be an inotrope in a concentration-dependent manner in isolated rat hearts. This effect was found to be dependent on a nitric oxide (NO) synthase signalling pathway. Attempts to further elucidate the contribution of ROS through the use of H2DCF-DA and NO though H2DAF-FM DA and spin trapping was performed. oCOm-21 did not alter H2DCF-DA or H2DAF-FM DA fluorescence. Spin trapping also failed to sensitively detect any cellular changes in NO due to oCOm-21 treatment. These findings suggest that low basal ROS and NO contribute to the inotropic and preconditioning effect seen with oCOm-21. RyR2 gating was examined in order to investigate the effects of oCOm-21 on myocardial function. oCOm-21 did not increase the number of HEK293/RyR2 cells experiencing store overload induced Ca2+ release (SOICR); suggesting that RyR2 release threshold was not altered. In isolated rat cardiomyocytes, changes in [Ca2+]i fluorescence due to oCOm-21 was observed but only at physiological extracellular [Ca2+]. Unfortunately, the present study could not detect S-nitrosylation of RyR2 due to oCOm-21 infusion through western blotting. Interpretation of these findings indicate that oCOm-21 may change Ca2+ reuptake or myofilament Ca2+ sensitivity. To explore the clinical relevance of oCOm-21 as an inotrope in human myocardium, oCOm-21 was perfused onto cardiac trabeculae isolated from clinical right atrial appendage biopsies. oCOm-21 only exhibited an inotropic effect in patients presenting with higher body weights, increased body surface area, larger right atrial area and greater right atrial end systolic volume but lower left ventricular posterior wall thickness. This suggests that oCOm-21 can be effective in the clinical setting but further evidence is needed to confirm oCOm-21’s ability to S-nitrosylate RyR2. If so, oCOm-21 would be a novel pharmacological agent where both inotropy and cardioprotection are achieved through the same mechanism of action. This evidence would also address how a preconditioning agent may also protect against non-specific ROS damage which the current literature surrounding preconditioning fails to explain.
Advisor: Sammut, Ivan
Degree Name: Master of Science
Degree Discipline: Pharmacology and Toxicology
Publisher: University of Otago
Keywords: Carbon monoxide; inotropy; ryanodine receptor 2; calcium handling; reactive oxygen species; nitric oxide; S-nitrosylation; human trabeculae
Research Type: Thesis