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

doi:10.1016/j.cardiores.2007.03.018

Cardiovascular Research 2007 75(1):99-108; doi:10.1016/j.cardiores.2007.03.018

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

Michael Reppel^a^,b^,*, Bernd K. Fleischmann^c, Hannes Reuter^d, Philipp Sasse^c, Heribert Schunkert^b and Jürgen Hescheler^a

^aInstitute of Neurophysiology, University of Cologne, Cologne, Germany
^bMedizinische Klinik II, Universität Schleswig-Holstein, Campus Lübeck, Lübeck, Germany
^cInstitute of Physiology I, Life and Brain Center, University of Bonn, Bonn, Germany
^dDepartment of Internal Medicine III, University of Cologne, Germany

^* Corresponding author. University of Cologne, Institute of Neurophysiology, Robert-Koch-Str. 39, D-50931 Cologne, Germany. Tel.: +49 221 478 6960; fax: +49 221 478 3834. akp72{at}uni-koeln.de

Objective The Na⁺/Ca²⁺ exchanger (NCX) is involved in embryonicheart development and function demonstrated by the abnormalmyofibrillar organization, defects in heartbeat, and early embryonicdeath of NCX-null embryos. It was therefore the aim of our studyto 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-clamptechnique in early (EDS, E10.5V) and late developmental stage(LDS, E16.5V) mouse ventricular cardiomyocytes.

Results Compared to LDS, cardiomyocytes derived from EDS showeda 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 currentin LDS on application of ATP- ${gamma}$ -S (2 mM) together with forskolin(1 µM) as well as intracellular application of thecatalytic 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 inhibitorRo 31-8220 significantly reduced I_NCX by 70%. Co-applicationof 20 µM PKA inhibitor Fragment 14-22 (PKI), a specificinhibitor of PKA, and cAMP significantly reduced the exchangeractivity by approx 60%. Despite these obvious effects in LDSwe could not detect a significant impact of these compoundson I_NCX in EDS-derived cardiomyocytes. Application of the alkalinephosphatase to test for constitutive phosphorylation of NCXdid 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 exchangerprotein. At LDS, embryonic cardiomyocytes showed a strong increaseof I_NCX density upon stimulation by PKC- and PKA-dependent signallingpathways.

KEYWORDS NCX; Cardiac development; Embryonic heart; Calcium; PKA; PKC; Cyclic nucleotides

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