Cardioprotection Conferred by a CRISPR/Cas9 Single Amino Acid Substitution of NCX1 (H165A): the PH Insensitive NCX Mouse

Abstract

The cardiac sodium calcium-exchanger (NCX1), the dominant calcium efflux mechanism of cardiomyocytes, is strongly regulated by pH. This pH regulation is thought to contribute to ischemia/reperfusion injury by allowing Na ions to accumulate to very high levels during ischemia when NCX is disabled by low pH, resulting in massive Ca overload via reverse NCX when reperfusion suddenly restores normal pH and NCX function. We recently found in a heterologous system that substitution of a single Histidine for Alanine at position 165 (H165A) of NCX1 drastically decreases its pH sensitivity (John S et al., J Gen Physiol, 2018). We then used CRISPR/Cas9 technology (Ma X et al, Sci Reports, 2017) to produce the same single amino acid substitution in mice. We confirmed the mutation by sequencing and RT-qPCR. The mice live into adulthood and are fertile with reduced LV function. In enzymatically isolated adult ventricular myocytes, we used the patch clamp technique with voltage ramps to measure nickel sensitive NCX current. At normal pH, NCX current was the same in control and H165A mice. However, when pH was lowered to 6.4 using NH4Cl, NCX current in H165A mice was minimally inhibited, while NCX in control mice was reduced severely. In voltage mapping studies of control mouse hearts, acidosis to pH 6.4 resulted in the onset of abnormal cardiac rhythm, characterized by repetitive bursts of depolarization alternating with pauses. However, in H165A mice, rhythm was unaffected by lowering the pH. In preliminary whole heart ischemia/reperfusion studies using Langendorff perfusion, pH mice had a 58% reduction in the area of necrosis compared to WT. These results suggest that a pH insensitive NCX may be able to reduce ischemia/reperfusion injury and arrythmia.