Sodium‐calcium exchanger (NCX‐1) and calcium modulation: NCX protein expression patterns and regulation of early heart development

Ouabain‐induced inhibition of early heart development indicated that Na/K‐ATPase plays an important role in maintaining normal ionic balances during differentiation of cardiomyocytes (Linask and Gui [1995] Dev Dyn 203:93–105). Inhibition of the sodium pump is generally accepted to affect the activity of the Na+‐Ca++ exchanger (NCX) to increase intracellular [Ca++]. These previous findings suggested that Ca++ signaling may be an important modulator during differentiation of cardiomyocytes. In order to identify a connection between heart development and NCX‐mediated Ca++ regulation, we determined the embryonic spatiotemporal protein expression pattern of NCX‐1 during early developmental stages. In both chick and mouse embryos, NCX‐1 (the cardiac NCX isoform) is asymmetrically expressed during gastrulation; in the right side of the Hensen's node in the chick, in the right lateral mesoderm in the mouse. At slightly later stages, NCX‐1 is expressed in the heart fields at comparable stages of heart development, in the chick at stage 7 and in the mouse at embryonic day (ED) 7.5. By ED 8 in the mouse, the exchanger protein displays a rostrocaudal difference in cardiac expression and an outer curvature‐inner curvature ventricular difference. By ED 9.5, cardiac expression has increased from that seen at ED8 and NCX‐1 is distributed throughout the myocardium consistent with the possibility that it is important in regulating initial cardiac contractile function. Only a low level of expression is detected in inflow and outflow regions. To substantiate a role for the involvement of calcium‐mediated signaling, using pharmacologic approaches, ionomycin (a Ca++ ionophore) was shown to perturb cardiac cell differentiation in a manner similar to ouabain as assayed by cNkx2.5 and sarcomeric myosin heavy chain expression. In addition, we show that an inhibitor of NCX, KB‐R7943, can similarly and adversely affect early cardiac development at stage 4/5 and arrests cardiac cell contractility in 12‐somite embryos. Thus, based upon NCX‐1 protein expression patterns in the embryo, experimental Ca++ modulation, and inhibition of NCX activity by KB‐R7943, these results suggest an early and central role for calcium‐mediated signaling in cardiac cell differentiation and NCX's regulation of the initial heartbeats in the embryo. © 2001 Wiley‐Liss, Inc.