Regulation of the Na⁺/Ca²⁺ exchanger (NCX) in the murine embryonic heart

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-γ-S (2 mM) together with forskolin (1 μM) as well as intracellular application of the catalytic subunit of cAMP-dependent protein kinase (cPKA, 200 U/mL), cAMP (200 μM), 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 μM PKA inhibitor Fragment 14-22 (PKI), a specific inhibitor of PKA, and cAMP significantly reduced the exchanger activity by approx 60%. Despite these obvious effects in LDS we could not detect a significant impact of these compounds on I_NCX in EDS-derived cardiomyocytes. Application of the alkaline phosphatase to test for constitutive phosphorylation of NCX did not affect I_NCX density in LDS but led to an approx 80% reduction of I_NCX in EDS. Conclusion: In EDS cardiomyocytes I_NCX density is upregulated, at least in part by the high phosphorylation of the exchanger protein. At LDS, embryonic cardiomyocytes showed a strong increase of I_NCX density upon stimulation by PKC- and PKA-dependent signalling pathways.