Enhanced Na+/H+ exchange during ischemia and reperfusion impairs mitochondrial bioenergetics and myocardial function

Abstract

Inhibition of Na+/H+ exchange (NHE) during ischemia reduces cardiac injury due to reduced reverse mode Na+/Ca2+ exchange. We hypothesized that activating NHE-1 at buffer pH 8 during ischemia increases mitochondrial oxidation, Ca2+ overload, and reactive O2 species (ROS) levels and worsens functional recovery in isolated hearts and that NHE inhibition reverses these effects. Guinea pig hearts were perfused with buffer at pH 7.4 (control) or pH 8 +/- NHE inhibitor eniporide for 10 minutes before and for 10 minutes after 35- minute ischemia and then for 110 minutes with pH 7.4 buffer alone. Mitochondrial NADH and FAD, [Ca2+], and superoxide were measured by spectrophotofluorometry. NADH and FAD were more oxidized, and cardiac function was worse throughout reperfusion after pH 8 versus pH 7.4, Ca2+ overload was greater at 10-minute reperfusion, and superoxide generation was higher at 30-minute reperfusion. The pH 7.4 and eniporide groups exhibited similar mitochondrial function, and cardiac performance was most improved after pH 7.4+eniporide. Cardiac function on reperfusion after pH 8+eniporide was better than after pH 8. Percent infarction was largest after pH 8 and smallest after pH 7.4+eniporide. Activation of NHE with pH 8 buffer and the subsequent decline in redox state with greater ROS and Ca2+ loading underlie the poor functional recovery after ischemia and reperfusion.

Figure 1

Changes in NADH (A) and FAD (B) (autofluorescence units, afu), during perfusion with HEPES buffer at pH 7.4 (control; n=7), pH 8 (n=7), pH 7.4+eniporide (ENI, 10 µM) (n=6), or pH 8+ENI (10 µM) (n=6) 10 min before and 10 min after 35 min no flow, global ischemia. A non-ischemia, pH 7.4 time control group (n=4) is also displayed for all variables. For P <0.05: * pH 8 vs. 7.4; # pH 8+ENI vs. pH 8; † pH 7.4+ENI vs. pH 7.4. Attenuated Na⁺/H⁺ exchange by the lower pH and or ENI led to a less oxidized redox state.

Figure 2

Changes in mt[Ca²⁺] in nM (A) and superoxide (O₂^−•) in afu (B), during perfusion with HEPES buffer at pH 7.4 (control; n=8 each variable), pH 8 (n=7 each variable), pH 7.4+ENI (10 µM) (n=7 each variable), or pH 8+ENI (10 µM) (n=7 each variable) 10 min before and 10 min after 35 min no flow, global ischemia. For P <0.05: * pH 8 vs. 7.4; # pH 8+ENI vs. pH 8; † pH 7.4+ENI vs. pH 7.4. Attenuated Na⁺/H⁺ exchange by the lower pH and or ENI led to a smaller increase in mt[Ca²⁺] during ischemia and smaller increases in both mt[Ca²⁺] and O₂^−• during early reperfusion.

Figure 3

Changes in developed left ventricular pressure (systolic-diastolic LVP or devLVP in mmHg; A), and diastolic left ventricular pressure (diaLVP in mmHg; B) during perfusion with HEPES buffer at pH 7.4 (control; n=12), pH 8 (n=12), pH 7.4+ENI (10 µM) (n=12), or pH 8+ENI (10 µM) (n=12) 10 min before and 10 min after 35 min no flow, global ischemia. For P <0.05: * pH 8 vs. 7.4; # pH 8+ENI vs. pH 8; † pH 7.4+ENI vs. pH 7.4. Attenuated Na⁺/H⁺ exchange by the lower pH and or ENI led to better cardiac muscle function throughout reperfusion.

Figure 4

Changes in coronary flow (A) during perfusion with HEPES buffer at pH 7.4 (control; n=12), pH 8 (n=12), pH 7.4+ENI (10 µM) (n=12), or pH 8+ENI (10 µM) (n=12) 10 min before and 10 min after 35 min no flow, global ischemia. For P <0.05: * pH 8 vs. 7.4; # pH 8+ENI vs. pH 8; † pH 7.4+ENI vs. pH 7.4. Infarct size (B) as a percentage of total ventricular weight measured after 120 min reperfusion. For P <0.05: * pH 8 vs. 7.4; # pH 8+ENI vs. pH 8; † pH 7.4+ENI vs. pH 7.4. Attenuated Na⁺/H⁺ exchange by ENI at both pH’s led to a higher coronary flow on reperfusion and less infarction.