Sphingolipid metabolite ceramide causes metabolic perturbation contributing to HERG K+ channel dysfunction.
Ceramide, a sphingolipid metabolite, has emerged as a key second messenger molecule that mediates multiple cellular functions. Its de nova synthesis and accumulation in ischemic myocardium, congestive heart failure and diabetic cardiomyopathy is associated with the abnormalities such as abnormal QT prolongation and increased risk of arrhythmias. To investigate how ceramide is involved in modulating cardiac repolarization, we performed whole-cell patch-clamp studies on HERG current (I(HERG)), a critical determinant of cardiac repolarization, expressed in HEK293 cells. Acute application (superfusion for 25 min) of membrane permeable ceramide (C2, 5 microM) did not alter I(HERG). Prolonged incubation with C2 for 10 hrs caused pronounced I(HERG) inhibition in a concentration-dependent and voltage-independent fashion and positive shift of voltage-dependent HERG activation. The IC(50) for I(HERG) suppression was 19.5 microM. C2 did not affect the inactivation property and time-dependent kinetics of I(HERG). Similar effects were observed with production of endogenous ceramide catalyzed by sphingomyelinase. Tyrosine kinase inhibitors failed to reverse C2-induced suppression of HERG function, and PKA and PKC inhibitors only slightly reversed the I(HERG) depression. Western blotting and immunocytochemical analyses indicate that C2 does not alter HERG protein expression on the cytoplasmic membrane. The inhibitory effect of C2 on I(HERG) was reversed by antioxidants vitamin E or MnTBAP. C2 caused considerable production of intracellular reactive oxygen species (ROS), which was prevented by vitamin E or MnTBAP. We conclude that ceramide depresses I(HERG) mainly via ROS overproduction and ceramide-induced I(HERG) impairment may contribute to QT prolongation in prolonged myocardial ischemia, heart failure and diabetic cardiomyopathy.
Bai
Yunlong
Y
Department of Pharmacology (State-Province Key Laboratory of China), Harbin Medical University, Harbin, Heilongjiang, China.
Wang
Jingxiong
J
Shan
Hongli
H
Lu
Yanjie
Y
Zhang
Ying
Y
Luo
Xiaobin
X
Yang
Baofeng
B
Wang
Zhiguo
Z
D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism* / Q000494:pharmacology*; D000072237:ERG1 Potassium Channel; D004594:Electrophysiology; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D018408:Patch-Clamp Techniques; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013107:Sphingolipids / Q000378:metabolism*; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D014810:Vitamin E / Q000494:pharmacology
D002518:Ceramides; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D013107:Sphingolipids; D014810:Vitamin E; D011494:Protein Kinases; D013108:Sphingomyelin Phosphodiesterase