Involvement of TREK-1 Channel in Cell Viability of H9c2 Rat Cardiomyoblasts Affected by Bupivacaine and Lipid Emulsion

Abstract

Lipid emulsion (LE) therapy has been used to reduce overdose of bupivacaine (BPV)-induced cardiotoxicity. The TWIK-related potassium channel-1 (TREK-1) is inhibited by BPV and activated by polyunsaturated fatty acids, which are the main component in LE. These pharmacological properties inspired us to investigate whether the TREK-1 channel is associated with cell viability of H9c2 cardiomyoblasts affected by BPV and LE. Consistent with previous studies, BPV-induced cell death was reduced by LE treatment. The reduction in the TREK-1 expression level by BPV was alleviated by LE. The BPV cytotoxicity highly decreased in TREK-1 overexpressed cells but was the opposite in TREK-1 knocked-down cells. TREK-1 channel activators and inhibitors increased and decreased cell viability, respectively. BPV-induced depolarization of the plasma and mitochondrial membrane potential and increase in intracellular Ca2+ level were blocked by LE treatment. BPV-induced depolarization of membrane potential was reduced in TREK-1 overexpressed cells, indicating that TREK-1 channels mediate setting the resting membrane potentials as a background K+ channel in H9c2 cells. These results show that TREK-1 activity is involved in the BPV cytotoxicity and the antagonistic effect of LE in H9c2 cells and suggest that TREK-1 could be a target for action of BPV and LE.

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“…Cell viability of Chang liver and RAW264.7 cells was determined using a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent (5 mg/mL in phosphate buffered saline (PBS), Duchefa Biochemie, Haarlem, The Netherlands). As previously mentioned, the MTT assay procedure was performed [ 51 ].…”

“…Chang liver cells at a density 3 × 10 3 /well were seeded in a poly-L-lysine-coated glass-bottomed culture dish (SPL, Pocheon, Korea) and incubated at 37 °C and 5% CO 2 for 48 h. Then, the cells were treated with 2% EtOH and 100 µg/mL OH for 24 h. As previously mentioned, the staining procedure was performed [ 51 ].…”

“…Chang liver cells at a density of 3 × 10 3 /well were seeded in a poly-L-lysine-coated confocol image dish for recoding MMP. As previously mentioned, the measurement of MMP was performed [ 51 ].…”

“…Chang liver cells cultured on the poly-L-lysine-coated confocol image dish were incubated with 5 μM Fluo-3AM (Thermo Fisher Scientific, Rockford, IL, USA) in serum-free DMEM media for 30 min and washed three times with PBS. As previously mentioned, the measurement of [Ca 2+ ] i was performed [ 51 ].…”

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“…Bupivacaine also inhibits the aerobic respiration of the mitochondria; speci cally, decoupling of oxidative phosphorylation alters MMP and inhibits mitochondrial respiration, resulting in the production of reactive oxygen species (ROS) in the mitochondria and triggering the mitochondrial pathway of apoptosis [11]. Lipid emulsion is widely used to alleviate bupivacaine-induced myocardial toxicity because this technique can enhance mitochondrial metabolism and improve myocardial contractility [12,13]. Similarly, puerarin isolated from the traditional Chinese medicine radix puerariae has demonstrated a protective effect on myocardial infarction by targeting the mitochondria [14].…”