Abstract 186: NHE-1 Inhibition Reduces Rigor and Hypercontracture in Isolated Cardiomyocytes During Ischemia and Reperfusion

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Background: During ischemia, anaerobic metabolism prompts intracellular acidosis with activation of the sodium hydrogen exchanger isoform-1 (NHE-1), resulting in intracellular Na+ overload consequent to reductions in Na+-K- ATPase activity. The Na+ excess prompts Ca2+ entry through the sodium-calcium exchanger, leading to Ca+ overload. Ca+ overload, predisposes cells to hypercontracture upon reoxygenation. NHE-1 inhibition reduces Na+ overload and the subsequent Ca+ overload. We developed a programmable system to model ischemia and reperfusion by dynamically blending O2, CO2, and N2 and used it to model changes in gas tension at the tissue levels during ischemia and reperfusion. We validated the model by comparing the effects of reoxygenation with and without the NHE-1 inhibitor zoniporide in isolated adult rat cardiomyocytes. Methods: The set-up included: (i) a programmable dynamic gas blender developed by us, (ii) equilibration of ischemic buffer with anoxic gas mixture at 3-5 LPM and perfusion within an imaging chamber at 2.5 ml/min, (iii) a glass reservoir and stainless steel tubing to avoid gas diffusion, (iv) an acrylic hood, constantly flushed with anoxic gas mixture, covering the chamber on the stage of the microscope, and (v) an O2 microelectrode to continuously monitor PO2 in the chamber. The set-up resulted in PO2 of 0 mmHg during ischemia. The cells were subjected to 15 min of baseline stabilization, 45 min of ischemia, and 120 min of reperfusion. Zoniporide (at a final concentration of 3 μM) was administered starting at 10 min of ischemia and was maintained throughout reperfusion. Results: Zoniporide significantly attenuated development of rigor and subsequent hypercontracture (P=0.001, Gehan Breslow survival analysis). Conclusion: The newly develop system reproduced at the cell level effects of ischemia and reperfusion. These effects were attenuated by NHE-1 inhibition as it has been reported in isolated organs and in the in situ heart.