The current study tested the hypothesis that second-window protection would result in reduced LCFA oxidation with improved regional myocardial function and mechanical performance, during coronary stenosis. Surprisingly, the metabolic changes induced by the second window of preconditioning run counter to these notions, as we now demonstrate that SWOP elevated the contribution of LCFA to oxidative metabolism during stenosis, to similar levels in the non-ischaemic, normal myocardium, but still improved regional function and mechanical performance despite ischaemia. The changes induced by the SWOP indicate normalization in the reliance on LCFA oxidation, despite levels of non-oxidative glycolysis similar to the ischaemic myocardium, during persistent coronary stenosis and reduced regional blood flow.
We have previously shown during coronary stenosis in pigs that the capacity of mitochondria for oxidation of fuels through β-oxidation and the citric acid cycle actually exceeds the regulated entry of LCFA across the mitochondrial membrane during the hypoxia induced by a 40% reduction in regional blood flow, as implemented similarly in the current work.22
In the prior study, the hypoperfused myocardium preferentially oxidized short-chain fatty acids (SCFA) over endogenous LCFA compared with normal hearts under baseline conditions. Although an indirect assessment, the results of the previous study suggest a regulatory mechanism at the level of LCFA transport into the mitochondria via the carnitine palmitoyltransferase 1 (CPT-1). Since SCFA do not require CPT, this may explain the increased percentage of SCFA oxidized by the hypoperfused myocardium. Importantly, our previous work demonstrated that LCFA oxidation appears to be inhibited during hypoperfusion by factors other than the availability of residual tissue oxygenation, as the SCFA bypassed the mechanism for restricting LCFA oxidation and were amply oxidized in mitochondria despite the 40% reduction in coronary blood flow. The findings of the current study, which directly monitored LCFA contributions to mitochondrial oxidation, are consistent with these previous results. While hypoperfusion reduced the contribution of LCFA to oxidative metabolism, preconditioning revealed an improved capacity for LCFA oxidation during similar reductions in coronary blood flow. It is of interest to note that the tissue lactate levels in the SWOP + stenosis group, although not significantly different, were elevated relative to the stenosis group, a finding which would be consistent with inhibition of the pyruvate dehydrogenase complex secondary to an increase in fatty acid oxidation, i.e. the Randle cycle effect.
Importantly, the SWOP also produced a significant improvement in wall function during the coronary stenosis. Whereas numerous studies have demonstrated cardioprotection and reduced infarct size with the second window of preconditioning29,30
and, correspondingly, improved recovery of cardiac function during reperfusion following a complete coronary occlusion of sufficient duration to induce infarct,43
these studies could not demonstrate protection of regional function in the absence of infarction.29,30
This is likely because the prior studies utilized models involving complete coronary occlusion. However, Sun et al
demonstrated reduced stunning after repetitive brief coronary occlusions. In the current investigation, we employed a model of sublethal coronary stenosis, which permitted the observation of protected regional function during ischaemia induced by coronary stenosis. These data suggest that mechanical performance is improved in the preconditioned heart, compared with the control myocardium subjected to ischaemia, since the blood flow reduction was similar in the two groups, yet regional contractile function was better after preconditioning. Whether this can be translated to an improvement in cardiac efficiency requires a measurement of venous oxygen, which was not possible in our study. It is likely that oxygen extraction was not affected by preconditioning, since ischaemia induces near maximal extraction of oxygen in the heart.
Interestingly, the reduction in LCFA oxidation in the mitochondria during coronary stenosis in the absence of preconditioning was independent of the regional, transmural gradient of myocardial blood flow. As expected, during coronary stenosis in the normal pig heart, the contribution of palmitate to mitochondrial oxidation declined. However, the reduced LCFA oxidation occurred transmurally, despite the lack of any decrease in blood flow within the subepicardium. This reduced oxidation of palmitate coincided with elevated production of end products of non-oxidative glycolysis, lactate and alanine, which also occurred transmurally despite no blood flow reduction to the subepicardium. Thus, the regional metabolic profile responded to coronary stenosis, even in the subepicardium where blood flow was not reduced.
The finding that LCFA oxidation was reduced similarly in both subendocardial and subepicardial tissue during coronary stenosis, despite maintenance of subepicardial blood flow, is consistent with our previous finding of transmural changes in SCFA oxidation.22
This disassociation between transmural blood flow and metabolism indicates that oxygen availability alone is not the limiting factor that produces the relative shifts in glycolytic activity and LCFA oxidation during coronary stenosis, with or without preconditioning. Transmural differences in blood flow were previously shown not to correlate with changes in creatine phosphate levels in the presence of coronary stenosis in open-chest dogs.44–46
Coronary stenosis in the absence of preconditioning in the current study was associated with a significant reduction in tissue malonyl-CoA content. This was not unexpected, as any reduced production of acetyl-CoA from fatty acid oxidation by mitochondria would then reduce the production of malonyl-CoA by acetyl-CoA carboxylase-2 (ACC-2).45–48
Ischaemia also increases AMPK-activated protein kinase activity, which can phosophorylate and inhibit ACC-2, thereby lowering malonyl-CoA levels during ischaemia.46,47
Malonyl-CoA plays a key role in control of fatty acid oxidation by inhibiting CPT-1 and mitochondrial oxidation of LCFA.47–49
However, the current data sets, showing reduced malonyl-CoA in the hypoperfused myocardium with low palmitate oxidation as well as increased malonyl-CoA in the preconditioned myocardium showing normalized palmitate oxidation during hypoperfusion, do suggest a complex relationship, or perhaps even compartmentation issues, when examined in light of the widely accepted notion of malonyl-CoA inhibition of CPT-1 activity.2,50
As mentioned, AMPK phosphorylates and inhibits ACC-2 activity,49
serving to decrease malonyl-CoA levels during ischaemia. Therefore, an unexpected result was that SWOP resulted in increases in both LCFA contributions to mitochondrial oxidation and tissue malonyl-CoA content during coronary stenosis when compared with during coronary stenosis in the absence of preconditioning. Indeed, malonyl-CoA content and the contribution of LCFA to oxidation produced in the preconditioned, ischaemic myocardium were both similar to levels observed in the non-ischaemic myocardium. These data are in conflict with the proposed inhibitory role of malonyl-CoA on CPT-1 transport and LCFA oxidation, but are not inconsistent with similar findings in the in vivo
Although the inverse association between malonyl-CoA content and LCFA oxidation is widely accepted, very rarely are any discrepancies to this theory mentioned. Studies in isolated mitochondria suggest that malonyl-CoA is a prime factor in reducing LCFA oxidation, but in some studies of intact hearts, this is not the case. For example, an increase in cardiac workload induced by aortic constriction and dobutamine infusion in open-chest pigs resulted in a 2.5-fold increase in LCFA oxidation and malonyl-CoA content.51
The authors of this study proposed a possible compartmentalization of malonyl-CoA that was masked by the total tissue content which was reported (i.e. malonyl-CoA inhibits CPT-1 on the cytosolic side of the enzyme and the increase in tissue malonyl-CoA in this study may have been a selective increase in malonyl-CoA inside the mitochondrial matrix). Whether the decrease in malonyl-CoA content during coronary stenosis in the absence of preconditioning in the current study resulted from a selective decrease in mitochondrial matrix malonyl-CoA is not known, since cellular malonyl-CoA distribution is difficult, if not impossible to measure. However, the increase in malonyl-CoA content in the preconditioned, ischaemic myocardium was clearly the product of the elevated LCFA oxidation in response to SWOP. In any case, the data on myocardial malonyl-CoA content alone measured in our current study do not offer particular insight into the mechanism by which SWOP normalized the contributions of palmitate to oxidative metabolism, despite the presence of coronary stenosis and regional hypoperfusion. Importantly though, the ratio malonyl-CoA to acetyl-CoA (Table
), which serves as an index for ACC-2 activity, was significantly elevated in the presence of ischaemia plus SWOP when compared with control hearts. This difference, despite near identical proportions of LCFA entering β-oxidation in both groups (Table
), suggests not only a different level of ACC-2 activity in response to SWOP but also a different mechanism for regulating the entry of fatty acids into the mitochondria through CPT-1 due to preconditioning and ischaemia.