Abstract
INTRODUCTION Low dose carbon monoxide (CO), a byproduct of heme oxygenase, has been shown to cause vasodilatation, modify platelet adherence, reduce inflammation and inhibit apoptosis. Cardiac ischaemia-reperfusion (IR) injury occurs in patients with coronary artery disease and is also a common side effect of cardiac surgery. Our aims were to study the effects of low dose inhaled CO administered continuously for 1 day prior to and 1 or 28 days after a cardiac IR injury, specifically carboxyhaemoglobin (COHb) levels, haemodynamics, markers of inflammation and mitochondrial electron transfer chain (ETC) function.
METHODS Lewis rats were acclimatised and maintained within a custom built humidity, temperature controlled chamber. Rats inhaled gaseous CO at 20 parts per million (ppm) or air for 1 day prior to either a 30 minute left anterior descending artery (LAD) ligation and reperfusion or sham ligation surgery. CO delivery was continued for 1 or 28 days post reperfusion until termination. An additional group received CO for 1 day following reperfusion and were then allowed to breathe room air without CO for 27 days. Arterial blood was sampled for COHb concentration and the rats sacrificed. Haemodynamics were immediately measured while the hearts were perfused ex-vivo in the Langendorff mode. Left ventricular peri-infarct tissue was sampled and analysed for interleukin (IL)1a, IL1b, IL2, IL4, IL6, IL10, tumour necrosis factor (TNF)-α, and granulocyte macrophage colony stimulating factor concentrations. Analysis of left ventricular mitochondrial ETC I, II,III and IV enzyme activities was performed in isolated freeze-fractured mitochondrial fractions.
RESULTS There was a significant improvement in maximum left ventricular developed pressure (LVDPmax) at 1 day and 28 days following IR injury in the continuous CO treatment groups versus control (p = 0.004 and p = 0.005 respectively). CO significantly attenuated the elevated heart rate observed in the air treated group 1 day following IR injury group (p = 0.002). Coronary blood flow was reduced in the air treated IR injury group at 28 days compared to sham intervention (p = 0.05). This injury induced flow reduction was significantly attenuated by continual CO treatment (p = 0.07). CO also had an amplifying effect on early release of IL2, IL6 and IL10 at one day post IR injury (p = 0.005, p = 0.009, p = 0.0008 respectively), and significantly increased the IL10 / TNF-α ratio at 1 day (p = 0.0006). While complex I activity was increased by CO treatment in the 1 day group there were no other significant differences in mitochondrial ETC in this model as a consequence of treatment or IR injury .
DISCUSSION We have shown inhaled CO at 20ppm reduces cardiac IR injury as measured by heart rate, LVDPmax and coronary artery flow in a rat heart model of IR injury. Chronic intervention with CO gas following IR injury maintained cardioprotection over the healing phase and is superior to air exposure alone and also short term CO exposure (1 day before and 1 day after IR injury) when outcomes were measured at 28 days.
We have demonstrated a CO-mediated modulation of the cytokine response to injury by massive upregulation of IL10 and an increase in the IL10 / TNF-α ratio which have beneficial roles in modifying the detrimental effects of inflammation in cardiac IR injury. Cardiac energetics were difficult to analyse within the peri-infarct tissue sampled, however CO tended to increase Complex I activity in sham ligation groups at 1 and 28 days.
We postulate that low dose CO may offer important cardioprotective benefits to patients undergoing cardiac surgery associated with an ischaemic insult.