Abstract 1089: Functional Expression and Regulation of the Embryonic Na+/Ca2+ Exchanger
The Na+/Ca2+ exchanger (NCX) is one of the earliest functional genes and is currently assumed to compensate for the rudimentary sarcoplasmatic reticulum (SR) in the developing mouse heart. To date little is known about the functional expression and regulation of NCX during development. This prompted us to investigate the NCX current (INCX) in very early (E8.5-E9.5 post coitum), early (E10.5-E11.5), middle (E13.5) and late (E16.5) stage mouse embryonic cardiomyocytes. At very early stages of development a high INCX density was observed (+60 mV: 4.6±0.7 pA/pF, n=14). Likewise, we found a strong Ca2+ outward mode of INCX (-120 mV: -3.9 ± 0.7 pA/pF, n = 14). At later stages, however, INCX Ca2+ inward mode was reduced by 54±6% (n=15, p<0.0001) in ventricular and 68±10% (n=9, p<0.0006) in atrial cells. For the outward mode a reduction by 43±10% (n=15, p<0.01) in ventricular and 62±11% (n=9, p<0.004) in atrial cardiomyocytes was observed. NCX isoform expression and the reversal potential did not significantly change during development. In late stage ventricular cardiomyocytes application of the catalytic subunit of PKA (cPKA) increased INCX approximately 3-fold (-6.3±1.5 pA/pF, p=0.006, 6.4±1.5 pA/pF, n=10, p=0.008) as compared to control cells (-1.7±0.4 pA/pF; 2.1±0.5 pA/pF, n=11). Likewise, PMA, a direct activator of PKC, induced a 2- to 3-fold increase of INCX (-6.5±1.7 pA/pF, p=0.01; 5.8±1.3 pA/pF, n=12, p=0.02). Despite these effects in late stage cardiomyocytes we could not detect a significant impact of cPKA and PMA on INCX in early stage cardiomyocytes. However, application of the alkaline phosphatase led to an approx 80% reduction of INCX in early stages (-0.8±0.2 pA/pF, p=0.004; 0.7±0.2 pA/pF, n=13, p=0.002) but did not affect INCX density in late stage ventricular cardiomyocytes. In summary, NCX plays a prominent role in transsarcolemmal Ca2+ fluxes especially during early embryonic heart development and may thereby compensate for the rudimentary SR. Phosphorylation processes are critically involved in the high intrinsic INCX density at the early stage and regulation of NCX activity at later stages of murine embryonic heart development.