Regulation of Volume-Sensitive Chloride Current in Cardiac HL-1 Myocytes

HL-1 cells derived from mouse atrial myocytes retain many features of differentiated adult cardiomyocytes, continuously divide, and are emerging as a useful experimental tool. Features of several HL-1 cation channels have been described, but the characteristics of its Cl− channels are unknown. We studied regulation of volume-sensitive Cl− current, ICl,swell, under conditions that isolate anion currents. Modest osmotic swelling (0.85T; T, times-isosmotic) elicited robust outwardly-rectifying Cl− currents in virtually every HL-1 cell (typically, 15 - 30 pA/pF at +60 mV; ECl = −40 mV). As expected for ICl,swell, Cl− current in 0.85T was fully inhibited by DCPIB (10 μM) and was outwardly rectifying in both physiological and symmetrical Cl− gradients. Regulation of HL-1 ICl,swell matched that in enzymatically dissociated adult cardiomyocytes. In 0.85T, HL-1 ICl,swell was fully blocked by both the NADPH oxidase inhibitor gp91ds-tat (500 nM) and the mitochondrial ETC inhibitor rotenone (10 μM), and in isosmotic bath solution (1T), DCPIB fully suppressed H2O2-induced (100 μM) ICl,swell. Furthermore, as in adult cardiomyocytes, endothelin-1 (ET-1; 10 nM) activated a DCPIB-sensitive current in 1T that was outwardly-rectifying in HL-1 cells with physiological and symmetrical Cl− gradients. ET-1-induced HL-1 ICl,swell was suppressed by the ETA receptor blocker BQ123 (1 μM) and by blocking ROS production with gp91ds-tat. HL-1 ICl,swell also was activated by bacterial sphingomyelinase (0.03 U/mL) that produces ceramide. These findings in HL-1 cells recapitulated the biophysical and pharmacological features of ICl,swell and its regulation by ROS, endothelin, and ceramides in adult myocytes. Our data indicate that HL-1 cells are a useful tool for dissecting the regulation and role of ICl,swell in cardiac myocytes.