Activated protein C has a protective effect against myocardial I/R injury by improvement of endothelial function and activation of AKT1

Abstract

Objectives: Activated protein C (APC) has a protective efficacy against ischemia-reperfusion (I/R) injury in several organs. The objective of this study was to investigate effect of APC in myocardium with possible mechanism.

Methods: We used regional and global myocardial I/R injury models of rats. They consisted of I/R injuries (1) by ligation of left coronary artery, or (2) using Langendorff apparatus. Langendorff was used to focus the mechanism of APC excluding coagulation cascade in a working heart. Each experiment had an APC group (n=10) and a control group with normal saline (n=10). Injections of these solutions into rats were performed 30 minutes before the planned-I/R injury. Cardiac performance after the procedure was evaluated by echocardiography or indices with Langendorff apparatus. Coronary flow (CF) was measured in the global I/R injury model. Western blotting was performed to detect the change of AKT1 signal in myocardium after global I/R injury.

Results: LV FUNCTION IMPROVED SIGNIFICANTLY IN THE APC GROUP: %EF at 2 weeks after procedure, 70.8%± 4.5% vs. 56.5%± 0.7%; APC vs. control; p<0.01. Percent LV development pressure (LVDP) also improved in the APC group significantly, 88.8%± 45.3% vs. 28.1%± 15.4%; APC vs. control; p<0.01. In APC group, %CF improved significantly, 88.5%± 15.8% vs. 65.0%± 13.4%; APC vs. control; p<0.01. It was enhanced significantly when acetylcholine was administered; % CF: 103.5%± 9.9% vs. 87.0%± 12.1%; APC vs. control; p<0.05. Western blotting revealed that APC significantly induced activation of phosphorylated AKT1 in myocardium (p<0.05).

Conclusions: APC has a novel effect to protect myocardium and cardiac performance against I/R injury through improvement of endothelial function and activation of AKT1.

Figure 1. Assessment of cardiac performance in the regional ischemia/reperfusion injury model.

We evaluated cardiac function by ultrasound cardiography at 1 and 2 weeks after the procedure in controls and the activated protein C (APC) group (n = 10/group). The parameters were percent ejection fraction (EF), percent fractional shortening (FS), left ventricular end diastolic area (LVEDA) and left ventricular end systolic area (LVESA). Significant differences were observed between the two groups at 2 weeks after the procedure. Open circle indicate APC group and closed circle indicate controls. (*P<0.05; APC group versus controls, **P<0.01; APC group versus controls).

Figure 2. Assessment of myocardial infarct size after regional ischemia/reperfusion injury.

We stained slices of left ventricular (LV) tissue with hematoxylin and eosin and Sirius red 2 weeks after inducing transient ischemia. The size of the infarct area was assessed by calculating the percentage of total LV area (% infarction) using Image J. The bar graph shows that the percent infarction in the activated protein C (APC) group was significantly smaller than the controls (* p<0.05; APC versus controls).

Figure 3. Representative Western blots for AKT1 and p-AKT1.

The photograph shows Western blots of myocardium for β-actin, whole AKT1, and p-AKT1in the global ischemia/reperfusion injury model during the time course. AKT1 was strongly detectable 30 min after reperfusion in the activated protein C (APC) group (p = 0.08, vs. control). The bar graph shows that p-AKT1 in the APC group was strongly and significantly expressed 30, 60, and 120 min after reperfusion, (* p<0.05; APC group versus controls).