Abstract 11475: A Genetically Modified pH Resistant NCX1 Mouse Prevents Calcium Overload During Acidosis and is Cardioprotective

Abstract

Acidosis inhibits the cardiac sodium(Na)/calcium(Ca) exchanger (NCX1), allegedly contributing to elevated cytoplasmic Na and Ca concentrations during ischemia. Upon reperfusion and restoration of normal pH, a reactivated NCX1 abruptly shifts into reverse (Ca influx) mode because of the abnormally high cellular Na concentration, thus producing massive Ca overload and cell damage. To test the dependence of ischemia/reperfusion (I/R) injury on NCX1’s pH sensitivity, we used CRISPR/Cas9 technology to create mice with a pH-resistant NCX1 by substituting Alanine for Histidine at position 165 (H165A). These mice live into adulthood with slightly reduced LV function (EF% H165A: 50 ± 5; Wildtype (WT): 67 ± 2; p<0.025, N=4-6). In enzymatically isolated adult ventricular myocytes, we used the patch clamp technique and voltage ramps from +80 to -120 mV to confirm that NCX1 current in H165A mice is resistant to pH reduction (from 7.4 to ~pH 6.4 by substituting 80 mM Na-Acetate for equimolar NaCl) when compared to WT animals (% reduction at +80mV: WT 77.7± 2.8, P<0.027 in 3 cells from 2 mice; H165A 0.21 ± 11.35, P=0.31 in 7 cells from 3 mice). We then performed I/R studies in explanted hearts perfused in Langendorff fashion with oxygenated Tyrode’s containing (in mM): 136 NaCl, 1.8 CaCl 2 , 5.4 KCl, 0.33 NaH 2 PO 4 , 1.0 MgCl 2 , 10 HEPES, and 10 glucose, pH 7.4, 37 o C. 20 minutes of stop-flow ischemia were followed by reperfusion for 120 minutes. Necrosis area (pixels 2 ) in WT was 26 ± 3 (N=11), compared to only 9 ± 1 (N=7) in H165A mice (P<0.0004). To compare effects of lower pH on isolated cardiomyocyte Ca, we used a photometric epifluorescence detection system to measure cytosolic Ca in enzymatically isolated ventricular myocytes loaded with the Ca indicator fura-2 AM (2 μM). When pH was lowered to ~6.4, Ca transients (ΔF340/F380) increased in WT from 0.6 to 0.9 (P < 0.0004, N=19 cells from 3 mice) but notably did not change in H165A (0.5 ± 0.03 @ pH 7.4, 0.5 ± 0.04 @ pH 6.4, P=0.307, N=21 cells from 3 mice). pH changes were verified in separate cells using the pH indicator SNARF-AM (10 μM). We conclude that eliminating pH sensitivity in NCX1 has major effects on intracellular Ca regulation, is cardioprotective, and a potential strategy for therapies in patients with prolonged ischemia prior to reperfusion.