Abstract
Episodes of ischaemia reperfusion are paradoxically imposed during prolonged cardiac surgical interventions resulting in perioperative complications, such as myocardial injury, arrhythmias, and end organ injury. The presence of pre-existing cardiac pathologies, such as hypertrophic cardiomyopathy, can potentiate these perioperative complications and result in reduced outcome benefits. Various prophylactic cardioprotective strategies to reduce ischaemia reperfusion injury and improve outcomes have been trialled with limited success. A new class of carbon monoxide (CO) delivery molecules (oCOms), selected on the basis of established pleiotropic effects, have been developed as potential anti-ischaemic agents. The studies within this thesis examined the “fast” CO releasing oCOm-21 and the “slower” CO releasing oCOm- 54 utilising a variety of rodent models and strains. The overall aims of the thesis was to examine if these oCOms are effective pre-ischaemic cardioprotective agents in normotrophic and hypertrophic hearts, and further to demonstrate the pharmacological effects, and safety of the compounds following in vivo administration.
In a follow up to earlier studies on oCOm-21, oCOm-54 was investigated as a pre-ischaemic cardioprotective agent in isolated perfused non-diseased Sprague-Dawley rat hearts subjected to 30 minutes of warm global ischaemia, followed by a 60 minute reperfusion period. Infusion of oCOm-54 (10 and 30 μM), but not the inactive desbromo analogue, DB-54 into the physiological media, resulted in a positive inotropic response and significantly improved left ventricular (LV) haemodynamic recovery which was sustained during reperfusion.
Subsequent, systemic administration of oCOm-21 and oCOm-54, in male Sprague-Dawley rats resulted in rapid but short-term changes in mean arterial pressure. The distribution of oCOm- 21 in biological tissues was established using LC-MS, and the presence of the by-product in the heart, liver, kidney, and urine confirmed the release of CO. Assessment of blood carboxyhaemoglobin levels, plasma alanine aminotransferase and creatinine data ascertained appropriate safe pharmacologically effective concentrations of oCOms for in vivo administration.
A clinically relevant model of pressure overload cardiomyopathy was re-established to preserve normotypical phenotype in a Cyp1a1-Ren2 transgenic rat colony. Indole-3-carbinol (0.167% w/w) administration in the diet of the male rats at 10 weeks of age, resulted in increased; systolic blood pressure, heart weight, LV wall thickness, and LV fibrosis.
Systemic administration of one of the CO-releasing analogues, oCOm-21 (3 μmol/kg) or oCOm-54 (30 μmol/kg), prior to temporary LAD coronary artery ligation in normotensive animals with normotrophic hearts from Cyp1a1-Ren2, reduced myocardial injury during the 30 minute ischaemic period and throughout the 48 hour reperfusion period. Surprisingly, administration of DB-54 (30 μmol/kg) also provided a similar cardioprotective effect suggesting that the DB analogue of oCOm-54 may be enzymatically cleaved in vivo to release CO. Subsequent studies conducted in the hypertensive Cyp1a1-Ren2 rats with hypertrophic hearts, showed that oCOm-21 (3 μmol/kg) provided a degree of cardioprotective, however it was attenuated in comparison to normotensive animals with normotrophic hearts. The reduction in myocardial injury with oCOms was observed as reduced ventricular arrhythmias during ischaemia, a reduction in the cTnI biomarker of myocardial injury four hours following reperfusion, reduced tissue injury, and preservation of LV haemodynamic function.
The studies in this thesis have consistently demonstrated that the prophylactic administration of oCOms, as pre-ischaemic agents both ex vivo and in vivo, provides significant protection against ischaemia reperfusion injury. Importantly, oCOm-21 was able to provide a degree of protection even in disease burdened hearts. Overall, this thesis provides valuable evidence supporting the clinical use of oCOms as pre-ischaemic conditioning agents in acute cardiovascular interventions.