Ceramide Activates ICl,swell in Rabbit Ventricular Myocytes via Mitochondrial ROS Production

We previously showed that ICl,swell is activated by ROS generation via NADPH oxidase (NOX) and the mitochondrial electron transport chain (ETC). Sphingolipid signaling is implicated in channel regulation. Here we examined the role of ceramide in the modulation of ICl,swell. Under isosmotic conditions, the addition of exogenous C2-ceramide (C2-Cer, 2 ∝M) increased Cl− current density by 0.7 ± 0.1 pA/pF at +60 mV after 10 min (n = 11, P<0.01). DCPIB (10 ∝M), a highly selective ICl,swell antagonist, inhibited C2-Cer-induced ICl,swell by 76 ± 8% (n = 6, P<0.01). The inactive analogue C2-H2Cer (2 ∝M) failed to stimulate ICl,swell (n = 6). Bacterial sphingomyelinase (SMase, 0.03 U/mL) was used to elicit endogenous ceramide production. SMase increased ICl,swell by 1.1 ± 0.1 pA/pF after 14 min (n = 30, P<0.01). This activation was inhibited by DCPIB (78 ± 6%, 10 ∝M, n = 7 and 81 ± 6%, 30 ∝M, n = 4) and tamoxifen (116 ± 16%, 10 ∝M, n = 5). Next we identified the source of ROS. Exposure to the NOX-specific inhibitor apocynin (500 ∝M, 10 min) failed to suppress SMase-induced ICl,swell (n = 9), whereas we previously showed apocynin blocks activation of ICl, swell on swelling and stretch. Diphenyleneiodonium (60 ∝M), a flavoprotein oxidase antagonist that suppresses both NOX and ETC Complex I, fully inhibited SMase-induced ICl,swell after 20 min (100 ± 14%, n = 4, P<0.01). Rotenone (10 ∝M), a specific ETC Complex I inhibitor, also abrogated the SMase-induced activation of ICl,swell after 20 min (110 ± 18%, n = 5, P<0.05). These data indicate that there is a ceramide-sensitive component of ICl,swell, and it is regulated by mitochondrial ROS. Ceramide signaling may modulate ICl,swell in cardiac disease.