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

Cardiac Na⁺/Ca²⁺ exchanger (NCX) protein half-life (t( 1/4 )) and antisense knockdown were studied in primary cultured neonatal rat cardiomyocytes. Protein t( 1/4 ) was determined using [³⁵S]methionine with a pulse-chase protocol. The ³⁵S signal in NCX was identified by immunoprecipitation and Western blotting. The t( 1/4 ) of NCX protein was 33 h. Low concentrations (0.5 μM) 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, ~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 ~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 ~60% of cells.