Expression and Regulation of Shark NCX Gene in Transgenic Mouse Heart

Abstract

Inward currents generated by the mammalian cardiac Na+-Ca2+-exchanger (NCX1.1) have arrhythmogenic potential, especially in the failing heart where expression of NCX is up-regulated. We hypothesize that arrhythmogenesis might be alleviated if NCX were subject to the same cAMP mediated regulation (suppression of Ca2+-influx, but enhancement of Ca2+-efflux on NCX) as found in the native shark ventricle. To test this hypothesis, we created heterozygous transgenic mice that express the shark NCX protein with a myc-tag, under the control of the alpha-myosin heavy chain promoter (α-MHC). The construct was evaluated by expression and functionality prior to production of transgenic lines. The expression of the transgene was confirmed by immunocytochemistry staining using DAPI and myc-FITC antibody on transfected HL-1 cells. Using dual laser confocal microscopy, the pattern of staining was consistent with NCX expression at the protein level. To determine the functionality of the transgene, HL-1 cells were co-transfected with the shark NCX transgene and GFP. GFP positive cells were incubated with Fluo-4 AM and imaged confocally. These cells showed NCX activity in response to withdrawal and readmission of [Na+]o consistent with electrophysiological data of native shark myocytes.Echocardiography and ECG studies on transgenic mice showed no remarkable cardiac phenotype, but analysis of initial voltage-clamp and western blot studies verify robust exchanger currents and expression levels. Our findings show that shark NCX is functional in the transgenic mouse producing no discernable cardiac pathology, but it remains to be determined as yet whether shark NCX can confer anti-arrhythmic properties to the mammalian heart.