A Xanthine-Derivative K⁺-Channel Opener Protects against Serotonin-Induced Cardiomyocyte Hypertrophy via the Modulation of Protein Kinases

Abstract

This study investigated whether KMUP-1, a xanthine-derivative K+ channel opener, could prevent serotonin-induced hypertrophy in H9c2 cardiomyocytes via L-type Ca2+ channels (LTCCs). Rat heart-derived H9c2 cells were incubated with serotonin (10 μM) for 4 days. The cell size increased by 155.5%, and this was reversed by KMUP-1 (≥1 μM), and attenuated by the LTCC blocker verapamil (1 μM) and the 5-HT2A antagonist ketanserin (0.1 μM), but unaffected by the 5-HT2B antagonist SB206553. A perforated whole-cell patch-clamp technique was used to investigate Ca2+ currents through LTCCs in serotonin-induced H9c2 hypertrophy, in which cell capacitance and current density were increased. The LTCC current (ICa,L) increased ~2.9-fold in serotonin-elicited H9c2 hypertrophy, which was attenuated by verapamil and ketanserin, but not affected by SB206553 (0.1 μM). Serotonin-increased ICa,L was reduced by KMUP-1, PKA and PKC inhibitors (H-89, 1 μM and chelerythrine, 1 μM) while the current was enhanced by the PKC activator PMA, (1 μM) but not the PKA activator 8-Br-cAMP (100 μM), and was abolished by KMUP-1. In contrast, serotonin-increased ICa,L was blunted by the PKG activator 8-Br-cGMP (100 μM), but unaffected by the PKG inhibitor KT5823 (1 μM). Notably, KMUP-1 blocked serotonin-increased ICa,L but this was partially reversed by KT5823. In conclusion, serotonin-increased ICa,L could be due to activated 5-HT2A receptor-mediated PKA and PKC cascades, and/or indirect interaction with PKG. KMUP-1 prevents serotonin-induced H9c2 cardiomyocyte hypertrophy, which can be attributed to its PKA and PKC inhibition, and/or PKG stimulation.

The Xanthine Derivative KMUP-1 Attenuates Serotonin-Induced Vasoconstriction and K+-Channel Inhibitory Activity via the PKC Pathway in Pulmonary Arteries

“…To elucidate the role of PKA and/or PKC in 5-HT inhibition of K + channels and in KMUP-1's effects on this channel, PKA and PKC activators (8-Br-cAMP, PMA) and inhibitors (KT5720, chelerythrine) were used in rat PASMCs. 5-HT-inhibited Kv1.5 protein was unaffected by the PKA inhibitor KT5720 but significantly affected by the PKC inhibitor chelerythrine, suggesting that 5-HT is not involved in cross-talk with AC/PKA cascades 19 , 28 but exclusively involved in the PKC pathway in rat PASMCs. PKC plays a central role inhibiting K + efflux and depolarizing membrane potential, and accordingly results in PA vasoconstriction.…”

“…Although 5-HT inhibition of Kv channels in PASMC has been explored, so far no K + channel opener has ever used to control PAH. In the present study, we further established the functional relationship between 5-HT and K + channels in PAs and sought to determine if the K + channel opener KMUP-1 16 , 19 could be beneficial in PAH.…”

Effects of Trans, Trans-2,4-decadienal on the Ions Currents of Cardiomyocytes: Possible Mechanisms of Arrhythmogenesis Induced by Cooking-oil Fumes

“…We used H9c2 cells (ATCC CLR-1446; Rockville, MD, USA) derived from rat embryonic myoblasts as an in vitro model of cardiomyocytes biology, since they show similar ions currents responses to primary adult and neonatal cardiomyocytes. The cells were plated onto collagen-coated culture dishes and cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO 2 and 95% air at 37 °C 14,18 . Cells were used under the 20th passage.…”

“…To measure the I Ca,L through L-type Ca 2+ channels, perforated whole-cell patch clamp electrophysiology was used in H9c2 cells treated with (tt-DDE group) or without (control group) tt-DDE-containing media (1 µM) for 18 hours. This technique was previously described in detail 18 . In brief, H9c2 cells were placed in a recording dish and perfused with a bath solution containing (in mM): 135 tetraethylammonium (TEA)-Cl, 1.8 CaCl 2 , 2 MgCl 2 , 10 glucose, and 10 HEPES (pH 7.4, Tris) using a fire polished glass pipette.…”

“…The protein levels of Kv2.1 and Cav1.2 in the H9c2 cells were analyzed using western blotting. The methods used in western blotting have previously been described in detail 14,18 . In brief, total protein content was extracted using a Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, USA) and subsequently separated using a 10% denaturing acrylamide gel.…”