Pharmacological preconditioning with diazoxide slows energy metabolism during sustained ischemia

Abstract

Ischemic preconditioning (PC) is associated with slower destruction of the adenine nucleotide pool ( summation operatorAd) and slower rate of anaerobic glycolysis during ischemic stress. These changes are concordant with the preconditioned state, supporting an essential role of lowered energy demand in the cardioprotective mechanism of PC. Although pharmacological PC induced by the activation of mitochondrial K(ATP) channels also limits infarct size, its effect on energy metabolism during sustained ischemia is unknown. Using metabolite levels found at baseline and after a 15 min test episode of regional ischemia, the effect of a cardioprotective dose of diazoxide on metabolic features associated with PC was tested in barbital-anesthetized, open-chest dogs. Diazoxide (3.5 mg/kg at an intravenous rate of 1 mL/min) infused before a test episode of ischemia had no effect on baseline metabolic indices. However, during ischemic stress, treated hearts exhibited less destruction of ATP, less degradation of the summation operatorAd into nucleosides and bases, as well as less lactate production than control hearts subjected only to ischemic stress. Thus, diazoxide mimics the metabolic alterations observed in PC tissue. This supports the hypothesis that a reduction in energy demand, which is now equated with an increased ATP to ADP ratio in the sarcoplasm, is a critical component of the mechanism of cardioprotection in preconditioned myocardium. It is hypothesized that during PC or diazoxide treatment, the passage of the summation operatorAd into and out of the mitochondria is slowed, limiting the level of ATP available to the mitochondrial ATPase and preserving ATP and the total summation operatorAd. Altered ischemic mitochondrial metabolism plays an important role in establishing and maintaining the preconditioned state.

Keywords: Energy metabolism; Infarction; Ischemia; KATP channel; Preconditioning.

Figure 1

Relationship between infarct size and collateral blood flow for control, preconditioned and diazoxide groups. Each symbol represents one dog. Overlapping points in the control group are indicated by the number 2 within the symbol. Both preconditioned (n=7) and diazoxide (n=7) treatment protocols resulted in much smaller infarcts than that of the control group (n=6), as assessed by ANCOVA (P≤0.05)

Figure 2

Hemodynamic response to diazoxide or saline infusion. Arterial pressure decreased in all animals during the diazoxide infusion and stabilized during the equilibration (washout) phase. Within 1 min of the onset of ischemia, mean blood pressure decreased approximately 15 mmHg in both the saline and the diazoxide groups, returning approximately to the level observed at the onset of ischemia in both groups. Bars represent SEMs

Figure 3

Plasma glucose before (Pre) and after (Post) treatment with either saline (A) or diazoxide (B). Diazoxide treatment significantly increased plasma glucose levels after 10 min of completing infusion. Bars represent SEMs

Figure 4

Baseline adenylate pool (A), creatine phosphate (B) and glucose (C) levels within the myocardium of saline- and diazoxide-treated groups. Baseline data were obtained from nonischemic regions of the left ventricle outside the area at risk. Diazoxide did not affect the supply of these energy sources in the absence of coronary artery occlusion, and differences in diazoxide effect are therefore not attributable to an enhanced supply of high-energy phosphates at the onset of ischemia

Figure 5

ATP loss (A), the corresponding production of nucleosides and bases (B), and the lactate produced (C) at the end of 15 min of ischemia in saline- and diazoxide-treated hearts. Values were calculated as the difference between the content in the nonischemic ventricle and that found at the end of the 15 min ischemic test within the ischemic region. The ATP lost during the test episode was reduced by approximately 16% in the diazoxide-treated group, which was paralleled by attenuated production of nucleosides and bases, as well as lactate. *Indicates significantly different from saline-treated myocardium

Figure 6

The hypothetical role played by mitochondrial metabolism during an episode of ischemia in generating the metabolic changes found in myocardium that has been preconditioned with diazoxide, or by an episode of ischemia and reperfusion. In both circumstances, the mitochondrial K_ATP channels are open. As a consequence, when preconditioned hearts are made ischemic, they exhibit preservation of the adenine nucleotide pool (∑Ad), a higher level of ATP and less lactate production than equivalently ischemic reversibly injured nonpreconditioned myocardium. In hearts with the channels open, it was proposed that the rate of transport of ATP and ADP via the voltage-dependent ion-selective channel (VDAC) in the outer mitochondrial membrane was depressed, perhaps secondarily to the effects of reactive oxygen species released into the mitochondria of tissue treated with a preconditioning episode of ischemia and reperfusion, or by diazoxide treatment, while the myocardium is aerobic. The depression of the transport rate slows the exit of ATP and ADP from the mitochondria, as well as the entry of ATP produced by anaerobic glycolysis into the mitochondria. This results in a higher ambient sarcoplasmic ATP concentration, which slows glycolysis, resulting in less lactate being produced. Also, the rate of destruction of the ∑Ad is slowed, because there is less ADP for sarcoplasmic adenylate kinase to convert into ATP and AMP. Providing less AMP for dephosphorylation by 5′nucleotidase slows the destruction of the ∑Ad. It should be noted that these metabolic changes are present only when the tissue is in the preconditioned state. They disappear when preconditioning is dissipated by prolonged reflow, at which time cardioprotection is lost (6). This suggests that the VDAC is repaired quickly under aerobic conditions. It is of interest that oligomycin has exactly the same metabolic effects during ischemia (45), ie, preservation of the ∑Ad and less glycolysis. Again, the explanation is a presumed increase in sarcoplasmic ATP secondary to the oligomycin block of the ATPase