Regulation of the Na+/Ca2+ exchanger (NCX) in the murine embryonic heart
Abstract
OBJECTIVE: The Na+/Ca2+ exchanger (NCX) is involved in embryonic heart development and function demonstrated by the abnormal myofibrillar organization, defects in heartbeat, and early embryonic death of NCX-null embryos. It was therefore the aim of our study to identify key functional regulators of the embryonic NCX.
METHODS: NCX current (I(NCX)) density was measured as the Ni2+ (5 mM)-sensitive current applying the whole-cell patch-clamp technique in early (EDS, E10.5V) and late developmental stage (LDS, E16.5V) mouse ventricular cardiomyocytes.
RESULTS: Compared to LDS, cardiomyocytes derived from EDS showed a significantly higher basal I(NCX) density for the I(NCX) forward (-120 mV: -4.1+/-1 pA/pF, n=15 versus -1.7+/-0.4, n=11, p<0.05) and reverse modes (+60 mV: 4.0+/-0.9 pA/pF, n=15 versus 1.8+/-0.4, n=11, p<0.05). There was 2-3-fold elevation of forward and reverse current in LDS on application of ATP-gamma-S (2 mM) together with forskolin (1 microM) as well as intracellular application of the catalytic subunit of cAMP-dependent protein kinase (cPKA, 200 U/mL), cAMP (200 microM), phorbol 12-myristate-13-acetate (PMA), a direct activator of protein kinase C (PKC), and 8-Br-cGMP, a membrane permeable analog of cGMP. The specific PKC inhibitor Ro 31-8220 significantly reduced I(NCX) by 70%. Co-application of 20 microM PKA inhibitor Fragment 14-22 (PKI), a specific inhibitor of PKA, and cAMP significantly reduced the exchanger activity by approx 60%. Despite these obvious…
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