Na+/Ca2+ exchange in neonatal rat heart cells: antisense inhibition and protein half-life

Cardiac Na+/Ca2+ exchanger (NCX) protein half-life (t1/2) and antisense knockdown were studied in primary cultured neonatal rat cardiomyocytes. Protein t1/2 was determined using [35S]methionine with a pulse-chase protocol. The 35S signal in NCX was identified by immunoprecipitation and Western blotting. The t1/2 of NCX protein was 33 h. Low concentrations (0.5 microM) of chimeric, phosphorothioated antisense oligodeoxynucleotides (AS-oligos) targeted to the region around the start codon of NCX1 transcript were used to knock down NCX protein and activity. Control myocytes (no oligos or scrambled oligos for at least 4 days) exhibited spontaneous Ca2+ transients (measured with fura 2). The sustained ("diastolic") Ca2+ concentration in the cytosol ([Ca2+]cyt) of control cells was unaffected by cyclopiazonic acid (CPA) plus caffeine (Caf), which promote depletion of sarcoplasmic reticular Ca2+ stores, but [Ca2+]cyt rose in control cells when external Na+ was removed. In contrast, approximately 60% of cells treated with AS-oligos for at least 4 days did not exhibit spontaneous Ca2+ transients or respond to Na+-free medium; however, CPA + Caf did induce a prolonged elevation in [Ca2+]cyt in these cells. In all cells, 50 mM K+ increased [Ca2+]cyt. NCX protein was reduced by approximately 50% in cells treated with AS-oligos for 7 days but was not reduced after only 2 days. These biochemical data are consistent with the physiological evidence of NCX knockdown in approximately 60% of cells.