Phosphomimetic modulation of eNOS improves myocardial reperfusion and mimics cardiac postconditioning in mice

Abstract

Objective: Myocardial infarction resulting from ischemia-reperfusion injury can be reduced by cardiac postconditioning, in which blood flow is restored intermittently prior to full reperfusion. Although key molecular mechanisms and prosurvival pathways involved in postconditioning have been identified, a direct role for eNOS-derived NO in improving regional myocardial perfusion has not been shown. The objective of this study is to measure, with high temporal and spatial resolution, regional myocardial perfusion during ischemia-reperfusion and postconditioning, in order to determine the contribution of regional blood flow effects of NO to infarct size and protection.

Methods and results: We used myocardial contrast echocardiography to measure regional myocardial blood flow in mice over time. Reperfusion after myocardial ischemia-reperfusion injury is improved by postconditioning, as well as by phosphomimetic eNOS modulation. Knock-in mice expressing a phosphomimetic S1176D form of eNOS showed improved myocardial reperfusion and significantly reduced infarct size. eNOS knock-out mice failed to show cardioprotection from postconditioning. The size of the no-reflow zone following ischemia-reperfusion is substantially reduced by postconditioning and by the phosphomimetic eNOS mutation.

Conclusions and significance: Using myocardial contrast echocardiography, we show that temporal dynamics of regional myocardial perfusion restoration contribute to reduced infarct size after postconditioning. eNOS has direct effects on myocardial blood flow following ischemia-reperfusion, with reduction in the size of the no-reflow zone. These results have important implications for ongoing clinical trials on cardioprotection, because the degree of protective benefit may be significantly influenced by the regional hemodynamic effects of eNOS-derived NO.

Figure 1. eNOS S1176 phosphorylation protects against I/R injury in vivo.

Wild-type, S1176D and eNOS knockout mice were subjected to 45 minutes of myocardial ischemia (LAD ligation) followed by traditional reperfusion (MIR) or postconditioned reperfusion (MIPc: 6 cycles of 10sec reperfusion, 10 sec ischemia). A. Percentage of left ventricle area at risk (AAR), (P = NS). B. Quantitative analysis of infarct size over AAR, *P<0.05 compared to wild-type control. C. Representative heart sections perfused with 1% Evans blue and stained with 2% TTC; infarct areas are outlined in black. MIR: Myocardial ischemia with reperfusion, MIPc: Myocardial ischemia with postconditioning, AAR: Area at Risk, LV: Left Ventricle. n = 6–9 mice per group. Data are expressed as the mean±SD.

Figure 2. Postconditioning activates Akt and eNOS.

A. Western blot of total eNOS and GADPH in WT (C57BL6/J), S1176D mice (S1176ki), and eNOS ko mice. B. Western blot demonstrating phosphorylation (Ser1176) and total protein levels of eNOS in wild-type mice under conditions of control (CTL), MIR, and MIPc. C. Representative Western blot demonstrating phosphorylated (Ser473) and total protein levels of Akt. Densities (arbitrary units, AU) show that MIPc phosphorylates Akt in WT and eNOS ko mice. n = 5 per group. Data are expressed as the mean±SD. *P<0.05.

Figure 3. MCE of regional blood flow following I/R.

A. The left ventricular septum in the parasternal long axis view was divided into three regions of interest: apical septum (black), mid septum (blue) and basal septum (red). RV: Right ventricle, RA: Right atrium, LV: Left ventricle. Representative MCE image taken at baseline, B. Representative MCE image during ischemia. C. Representative MCE image 30 minutes after reperfusion. D. Analysis of regional change in myocardial perfusion. Representative region-specific replenishment curves 30 minutes post-reperfusion are shown: apical septum (black), mid septum (blue), and basal septal regions (red). Replenishment curves are characterized by the myocardial blood flow parameters A (plateau intensity) and β (flow velocity).

Figure 4. Temporal myocardial contrast echocardiography.

Myocardial blood flow (Aβ) profiles in the apical region for A. C57BL/6J mice. B. eNOS knockout mice, and C. S1176D knockin mice. Levels of myocardial myocardial blood flow were normalized to baseline values and measured at 2, 10 and 30 minutes after reperfusion. D. Apical myocardial blood flow 30 minutes after reperfusion. MIR: Traditional myocardial ischemia with reperfusion, MIPc: Myocardial ischemia with postconditioning. n = 5–6 per group. Data are expressed as the mean±SD. *P<0.05.

Figure 5. Effect of postconditioning and S1176D mutation on no-reflow zones.

A. Representative images 30 minutes after reperfusion. Superimposed areas (blue) indicate regions with ≤20% residual blood flow. B. Composite graph showing areas of the myocardium with ≤20% (black) and ≤30% (white) residual blood flow compared to preischemic baseline. MBF: myocardial blood flow. n = 5–6 per group. Data are expressed as the mean±SD. *P<0.05.