Effects of NCX1 Antisense Oligodeoxynucleotides on Cardiac Myocytes and Primary Neurons in Culture a

The molecular characterization of the Nat-Ca2+ protein NCXl has led to a wealth of information about the structural properties of the transporter, the multiplicity of splice variants, and the chromosomal localization of the gene for the exchanger. 1-3 Nevertheless, there is still a great deal of argument and ambiguity regarding the functional properties of the exchanger in various tissues, particularly in excitable cells such as cardiac myocytes and brain neurons. In efforts to probe the contributions of exchanger activity to Ca2+-regulated events in intact cells, we designed antisense (AS) oligodeoxynucleotides for NCXl to assess the effects of decreasing expression of this exchanger on primary cells in culture. In one series of studies, we prepared cardiac myocytes from day-18 embryonic rats. In these cultures -90% of the myocytes contracted spontane~us ly .~ Twentyfour hours after plating, a 20-mer AS oligo complementary to a region near the 3' end of NCXl was added to the cultures at a final concentration of 10 pM. A second addition (5 pM) was made 20 h later, and the assays were carried out 4 h after the second addition. Control cultures were treated with vehicle only (lipofectamine) or with the 20-mer sense (S) oligo. Exchanger activity was assessed in myocytes using fura-2 to monitor Na'mediated Ca2+ influx. Beating rates of cultured myocardial cells were measured at 23°C with an inverted phase-contrast micro~cope .~ Essentially similar conditions were used to treat rat primary cortical neurons (embryonic day 18), and exchanger activity was measured as described previously with neuronal culture^.^ Neuronal cell viability was monitored via the mitochondria1 dehydrogenase assay (MTT). In cardiac myocytes the AS treatment described above led to an -30% decrease in exchanger activity in 5 culture preparations. AS-treated cells also consistently exhibited higher beating rates than control or S-treated cells. Mean beating rates in 6 culture preparations were: control cells, 40 * 2 (n = 142); S-treated cells, 41 1 (n = 285); and AS-treated cells, 69 -+ 1 (n = 309). Enhancement of the mean beating rate in AS-treated cells was statistically significant ( p < 0.01),