Cytochrome P450 metabolites of arachidonic acid play a role in the enhanced cardiac dysfunction in diabetic rats following ischaemic reperfusion injury

Abstract

1 This study examined the contribution of cytochrome P450 metabolites of arachidonic acid in mediating ischaemia/reperfusion (I/R)-induced cardiac dysfunction in normal and diabetic rats. 2 We first compared the metabolism of arachidonic acid in microsomes prepared from the hearts of control rats and rats treated with streptozotocin (55 mg kg(-1)) to induce diabetes. The production of dihydroxyeicosatrienoic acids and epoxyeicosatrienoic acids (EETs) were similar in microsomes prepared from the hearts of control and diabetic rats, but the production of 20-hydroxyeicosatetraenoic acid (20-HETE) was two-fold higher in diabetic hearts than in control animals. 3 We then compared the change in left ventricular pressure (P(max)), left ventricular end-diastolic pressure, coronary flow and coronary vascular resistance in isolated perfused hearts obtained from control and diabetic animals after 40 min of global ischaemia (I) followed by 30 min of reperfusion (R). The decline in cardiac function was three- to five-fold greater in the hearts obtained from diabetic vs. control animals. 4 Pretreatment of the hearts with N-hydroxy-N'-(4-butyl-2-methyl-phenyl)-formamidine (HET0016, 1 microm), a selective inhibitor of the synthesis of 20-HETE, for 30 min before I/R resulted in significant improvement in the recovery of cardiac function in the hearts obtained from diabetic but not in control rats. Perfusion with an inhibitor of soluble epoxide hydrolase, 1-cyclohexyl-3-dodecyl urea (CDU), before I/R improved the recovery of cardiac function in hearts obtained from both control and diabetic animals. Perfusion with both HET0016 and CDU resulted in significantly better recovery of cardiac function of diabetic hearts following I/R than that seen using either drug alone. Pretreatment of the hearts with glibenclamide (1 microm), an inhibitor of ATP-sensitive potassium channels, attenuated the cardioprotective effects of both CDU and HET0016. 5 This is the first study to suggest that acute blockade of the formation of 20-HETE and/or reduced inactivation of EETs could be an important strategy to reduce cardiac dysfunction following I/R events in diabetes.

Figure 1

Cytochrome P-450 ω-hydroxylase and epoxygenase activity in microsomes prepared from the hearts of diabetic and non-diabetic rats: (a) 20-HETE formation was significantly elevated in microsomes prepared from the hearts of diabetic rats as compared to the non-diabetic animals; (b) DiHETEs and EETs formation were not different between diabetic and non-diabetic rats. Mean ± SEM (n = 5). *Significantly different compared to untreated control, P < 0.05.

Figure 2

Effect of CDU (0.01, 0.1 and 1.0 μM) and combination treatment with CDU (1.0 μM) and glibenclamide (1 μM) on (a) % recovery in P_max and (b) LVEDP in perfused hearts of control and diabetic rats. Mean ± SEM (n = 5). *Significantly different compared to untreated control, #Significantly different compared to untreated-STZ, †Significantly different compared to control-CDU, §Significantly different compared to STZ-CDU, P < 0.05.

Figure 3

Effect of CDU (0.01, 0.1 and 1.0 μM) and combination treatment with CDU (1.0 μM) and glibenclamide (1 μM) on (a) % recovery in CF and (b) CVR in perfused hearts of control and diabetic rats. Mean ± SEM (n = 5). *Significantly different compared to untreated control, #Significantly different compared to untreated-STZ, †Significantly different compared to control-CDU, §Significantly different compared to STZ-CDU, P < 0.05.

Figure 4

Effect of HET0016 (0.01, 0.1 and 1.0 μM) on (a) % recovery in P_max and (b) LVEDP in perfused hearts of control and diabetic rats. Effect of combination treatment with HET0016 (0.1 μM) and glibenclamide (1 μM) was investigated in hearts from diabetic rats only. Mean ± SEM (n = 5). *Significantly different compared to untreated control, #Significantly different compared to untreated-STZ, §Significantly different compared to STZ-HET0016, P < 0.05.

Figure 5

Effect of HET0016 (0.01, 0.1 and 1.0 μM) on (a) % recovery in CF and (b) CVR in perfused hearts of of control and diabetic rats. Effect of combination treatment with HET0016 (0.1 μM) and glibenclamide (1 μM) was investigated in hearts from diabetic rats only. Mean ± SEM (n = 5). *Significantly different compared to untreated control, #Significantly different compared to untreated-STZ, §Significantly different compared to STZ-HET0016, P < 0.05.

Figure 6

Effect of combined treatment with CDU (0.1 μM) and HET0016 (0.01 μM) on (a) % recovery in P_max and (b) LVEDP in perfused hearts of control and diabetic rats. Mean ± SEM (n = 5). *Significantly different compared to untreated control, #Significantly different compared to untreated-STZ, P < 0.05.

Figure 7

Effect of combined treatment with CDU (0.1 μM) and HET0016 (0.01 μM) on (a) % recovery in CF and (b) CVR in perfused hearts of of control and diabetic rats. Mean ± SEM (n = 5). *Significantly different compared to untreated control, #Significantly different compared to untreated-STZ, P < 0.05.