Prolongation of Beating Rate Caused by Reduction of NCLX, a Mitochondrial Na+-Ca2+ Exchanger, in Hl-1 Cardiomyocytes

Abstract

NCLX/NCKX6 is a recently identified gene responsible for a mitochondrial Na+-Ca2+ exchanger (NCXmit), a Ca2+ extrusion system from mitochondria. Although there have been accumulated data regarding roles of NCLX in various kinds of cells, little is known about the roles in cardiomyocytes, where repetitive Ca2+ transients occur and where a huge amount of ATP is generated and utilized. In the present study, we carried out a combination study of NCLX knockdown in spontaneously beating atrial cell line HL-1 and mathematical simulations. In HL-1 cells, NCLX knockdown by siRNA reduced the protein expression by ∼50%. A cytosolic Na+-dependent mitochondrial Ca2+ efflux was decelerated by knocking down NCLX, confirming that NCLX is a gene responsible for NCXmit in cardiomyocytes. Interestingly, the cycle length of spontaneous Ca2+ oscillation and action potential generation was significantly prolonged by knocking down NCLX. Detailed inspection revealed that the rate of initial membrane depolarization and upstroke of Ca2+ rise were markedly slower in the NCLX knockdown cells. Furthermore Ca2+ content in sarcoplasmic reticulum (SR) was lower and SR Ca2+ reuptake was slower in the NCLX knockdown cells. A mathematical model of HL-1 cells showed that an automaticity of HL-1 cells are driven by a spontaneous Ca2+ leak from SR, called “Ca2+ clock”. Analyses using the model demonstrated that blocking NCXmit reduced SR Ca2+ content and SR Ca2+ leak, resulting in the prolongation of beating rate. Taken together, the NCLX has an important role in regulating cardiac automaticity by modulating “Ca2+ clock”.