Antisense oligodeoxynucleotide inhibition of sodium-calcium exchange in cardiac and arterial myocytes
AuthorSlodzinski, Martin Konrad
AdvisorBlaustein, Mordecai P.
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AbstractThe cardiac NCX protein half-life and the effects of chimeric phosphorothioated antisense oligodeoxynucleotides (AS-oligos) targeted against the cardiac and arterial Na - Ca2+ exchanger (NCX) were studied in rat myocytes. Low concentrations (0.5 muM) of chimeric, phosphorothioated AS-oligos targeted to the NCX1 transcript were used to knock down NCX protein and activity. Control cardiac myocytes incubated without oligos or with scrambled (NS-) oligos exhibited spontaneous Ca2+ transients (measured with fura-2). The cytosolic free Ca2+ concentration ([Ca2+]i) rose in control cells when external Na (Nao) was removed. In contrast, 60% of cells treated with AS-oligos did not exhibit spontaneous Ca2+ transients or respond to Na-free medium. In all cells, 50 mM K increased [Ca2+]i. NCX protein was reduced by 50% in cells treated with AS-oligos for 7 days, but was not reduced after only 2 days. The biochemical and protein half-life are consistent with the physiological evidence of about 60% knock down of cardiac NCX activity after 4 days. Arterial myocytes proliferated normally in the presence of NS-, AS-, or no oligos, without detectable quantitative or morphological differences. Reduction of Nao to 5 mM, which promotes the Nai-dependent component of Ca2+ influx caused [Ca2+]i to rise in controls and in NS-oligo treated cells, but not in AS-oligos treated cells. Incubation with 1 mM ouabain to raise Nai augmented Nai-dependent component of Ca2+ influx in controls and NS-oligo treated cells, but the AS-oligo treated cells still did not respond with a rise in [Ca2+]i. Nevertheless, all three groups of cells exhibited increases in [Ca2+]i in response to 10 muM serotonin (5-HT). With La3+ inhibition of the PMCA pump and Na-free media (to inhibit NCX), CAF and CPA elevated [Ca2+]i; the Nao-dependent component of Ca2+ efflux was selectively inhibited by AS-oligos, but not in sense (S-), or NS-oligos treated cells or controls. The knock down of NCX activity in arterial myocytes was reversible. The La3+-sensitive (PMCA pump) component of Ca2+ efflux was unaffected in AS-oligos treated cells. The transient [Ca2+]i elevations by 5-HT (10 muM, 30 s) at low frequency (15 min) in AS-oligo treated cells were no different from those in controls and NS-oligos treated cells in terms of [Ca2+]i amplitude, and rates of rise and recovery. [Ca2+]i recovery in AS-oligo treated cells was markedly slowed when the PMCA pump was inhibited by La3+. When cells were stimulated at higher frequency (3 min), however, the peak amplitudes of the 2nd and 3rd responses were larger, and [Ca2+]cyt returned to baseline more slowly in AS-oligo treated cells than controls. Peak 5-HT evoked responses in controls, but not AS-oligo treated cells, were augmented >2-fold in Na-free media. The response to 5-HT was tested immediately after removal of external Ca2+ before and after 10 min incubation with 1 muM ouabain. Ouabain significantly augmented the 5-HT induced rise in [Ca2+]cyt in controls, but had no effect on AS-oligos treated cells. The latter observation implies that the arterial myocytes NCX is involved in Na+ gradient modulation of SR Ca2+ stores and cell responsiveness, while the results of the repetitive stimulation experiments suggest that the NCX may be important during tonic activation.
DescriptionUniversity of Maryland, Baltimore. Physiology. Ph.D. 1998
Biology, Animal Physiology