CO in the low-doses tested in this model results in a more favorable energy metabolic condition in that glycolysis is decreased in spite of maintained energy charge. Further work is warranted to clarify the possible mechanistic role of energy metabolism for CO protection.

During ischaemia, ATP depletion leads to insufficient fuelling for Na(+) /K(+) ATPase, decreased electrochemical potential and increased influx of calcium ions. This study demonstrated a means to assess the effects of ischaemic preconditioning (IP) on the free intracellular Ca(2+) pool during prolonged ischaemia. In a porcine myocardial ischaemia model, microdialysis (MD) was used for sampling of metabolic and injury markers in IP and non-IP (control) groups. (45) Ca(2+) was delivered in microperfusate locally to ischaemic myocardium, with distribution and uptake assessed by (45) Ca(2+) recovery in microdialysate. Cardiomyocytes in vitro were exposed to a Ca(2+) ionophore and tested for (45) Ca(2+) uptake. An accentuated myocardial calcium ion influx (observed as an increased microdialysate (45) Ca(2+) recovery in the extracellular milieu) was noted in control pigs compared with IP pigs during ischaemia. Suspended cardiomyocytes preincubated with a Ca(2+) ionophore to increase the intracellular calcium ion pool and subsequently incubated with (45) Ca(2+) , displayed lower (45) Ca(2+) uptake in cells compared with control cells not exposed to the ionophore, corroborating the idea of a strong relationship between degree of intracellular calcium overload and microdialysate (45) Ca(2+) recovery. The ischaemic insult was differentially verified by metabolic and injury markers. We introduce an in vivo method for serial assessment of myocardial calcium overload during ischaemia, using a MD technique and (45) Ca(2+) inclusion. IP leads to relatively less calcium overload as assessed by this new method, and we interpret this to mean that reduction in calcium overload is an important part of the IP protective effect.

Carbon monoxide is thought to be cytoprotective and may hold therapeutic promise for mitigating ischaemic injury. The purpose of this study was to test low-dose carbon monoxide for protective effects in a porcine model of acute myocardial ischaemia and reperfusion. In acute open-thorax experiments in anaesthetised pigs, pretreatment with low-dose carbon monoxide (5% increase in carboxyhaemoglobin) was conducted for 120 min before localised ischaemia (45 min) and reperfusion (60 min) was performed using a coronary snare. Metabolic and injury markers were collected by microdialysis sampling in the ventricular wall. Recovery of radio-marked calcium delivered locally by microperfusate was measured to assess carbon monoxide treatment effects during ischaemia/reperfusion on the intracellular calcium pool. Coronary occlusion and ischaemia/reperfusion were analysed for 16 animals (eight in each group). Changes in glucose, lactate and pyruvate from the ischaemic area were observed during ischaemia and reperfusion interventions, though there was no difference between carbon monoxide-treated and control groups during ischaemia or reperfusion. Similar results were observed for glycerol and microdialysate ⁴⁵Ca(2+) recovery. These findings show that a relatively low and clinically relevant dose of carbon monoxide did not seem to provide acute protection as indicated by metabolic, energy-related and injury markers in a porcine myocardial ischaemia/reperfusion experimental model. We conclude that protective effects of carbon monoxide related to ischaemia/reperfusion either require higher doses of carbon monoxide or occur later after reperfusion than the immediate time frame studied here. More study is needed to characterise the mechanism and time frame of carbon monoxide-related cytoprotection.

This association between increased adenosine turnover and decreased glycolytic flow during prolonged ischaemia in response to IP can possibly be explained by the competitive effect for the metabolites from both glucose and adenosine metabolism for entering glycolysis. We conclude that this study provides support for an energy-metabolic explanation for the protective mechanisms of IP.