Insulin preconditioning elevates p-Akt and cardiac contractility after reperfusion in the isolated ischemic rat heart

Abstract

Insulin induces cardioprotection partly via an antiapoptotic effect. However, the optimal timing of insulin administration for the best quality cardioprotection remains unclear. We tested the hypothesis that insulin administered prior to ischemia provides better cardioprotection than insulin administration after ischemia. Isolated rat hearts were prepared using Langendorff method and divided into three groups. The Pre-Ins group (Pre-Ins) received 0.5 U/L insulin prior to 15 min no-flow ischemia for 20 min followed by 20 min of reperfusion. The Post-Ins group (Post-Ins) received 0.5 U/L insulin during the reperfusion period only. The control group (Control) was perfused with KH buffer throughout. The maximum of left ventricular derivative of pressure development (dP/dt(max)) was recorded continuously. Measurements of TNF-α and p-Akt in each time point were assayed by ELISA. After reperfusion, dP/dt(max) in Pre-Ins was elevated, compared with Post-Ins at 10 minutes after reperfusion and Control at all-time points. TNF-α levels at 5 minutes after reperfusion in the Pre-Ins were lower than the others. After 5 minutes of reperfusion, p-Akt was elevated in Pre-Ins compared with the other groups. Insulin administration prior to ischemia provides better cardioprotection than insulin administration only at reperfusion. TNF-α suppression is possibly mediated via p-Akt leading to a reduction in contractile myocardial dysfunction.

Figure 1

Experimental protocol. The Pre-Ins group received 0.5 U/L insulin in KH buffer prior to 15 min no-flow ischemia for 20 min followed by 20 min of reperfusion. The Post-Ins group received 0.5 U/L insulin during the reperfusion period only. The control group was perfused with KH buffer throughout.

Figure 2

Time-course changes of heart rate before and after ischemia in the three groups (n = 12 in each group). The data are presented as means ± SD. HR: heart rate (bpm).

Figure 3

Time-course changes of coronary flow before and after ischemia in the three groups (n = 12 in each group). Coronary flow (mL/min) was measured by timed collection of the perfusate (baseline, after 20 min of preconditioning and after 5, 10, 15, and 20 minutes of reperfusion). The data are presented as means ± SD.

Figure 4

Time-course changes of LV dP/dt max before and after ischemia in the three groups (n = 12 in each group). The data are presented as means ± SD. *P < 0.05 versus Control; ^† P < 0.05 versus Post-Ins. In Pre-Ins, LV dP/dt max increased after administration of insulin prior to ischemia (PC 20′) compared to both Post-Ins and Control. After reperfusion, dP/dt max in Pre-Ins was elevated, compared with Post-Ins at 10 minutes after reperfusion (Rep 10′) and Control at all-time points (Rep 5′, 10′, 15′, and 20′). dP/dt max (mmHg/sec): maximum of left ventricular derivative of pressure development.

Figure 5

Time-course changes of TNF-α before and after ischemia in the three groups (n = 12 in each group). Coronary efferent fluid samples for TNF-α measurements were drawn after 20 min of preconditioning (preischemia) and after 1, 5, and 10 minutes of reperfusion. Measurements of TNF-α in the coronary effluent were normalized to 1 min volume of coronary flow (pg/mL). The data are presented as means ± SD. *P < 0.05 versus Control; ^† P < 0.05 versus Post-Ins. A significant increase in TNF-α was observed in Control at the beginning of reperfusion (Rep 1′ and 5′). The TNF-α at 5 minutes after reperfusion (Rep 5′) in the Pre-Ins was lower than both Post-Ins and Control.

Figure 6

Time-course changes of p-Akt before and after ischemia in the three groups. Muscle samples for p-Akt concentrations (ng/gram of dry heart weight) were taken after 20 min of preconditioning (preischemia, n = 12 in each group) and after 5 min of reperfusion (n = 12 in each group) and 20 min of reperfusion (n = 12 in each group). The data are presented as means ± SD. *P < 0.05 versus Control; ^† P < 0.05 versus Post-Ins. In Pre-Ins, p-Akt increased after administration of insulin before ischemia (preischemia). After 5 minutes of reperfusion, p-Akt was elevated in the Pre-Ins when compared with Control and Post-Ins (Rep 5′). The p-Akt levels in the Pre-Ins were still higher than Control after 20 minutes of reperfusion (Rep 20′).