Hong Han

Choline produces cytoprotective effects against ischemic myocardial injuries: evidence for the role of cardiac m3 subtype muscarinic acetylcholine receptors.

Background/Aims: Accumulating evidence indicates the presence of functional M3 subtype of acetylcholine muscarinic receptors (M3-mAChR), in addition to the well-recognized M2-mAChR, in the heart of various species including man. However, the pathophysiological role of the cardiac M3-mAChR remain undefined. This study was designed to explore the possible role of M3-mAChR in cytoprotection of myocardial infarction and several related signaling pathways as potential mechanisms. Methods: Studies were performed in a rat model of myocardial infarction and in isolated myocytes. Results: We found that choline relieved myocardial injuries during ischemia or under oxidative stress, which was achieved by correcting hemodynamic impairment, diminishing ventricular arrhythmias and protecting cardiomyocytes from apoptotic death. The beneficial effects of choline were reversed by the M3-selective antagonists but not by the M2-selective antagonist. Choline/M3-mAChR activated several survival signaling molecules (antiapoptotic proteins Bcl-2 and ERKs), increased endogenous antioxidant reserve (SOD), and reduced apoptotic mediators (proapoptotic proteins Fas and p38 MAPK) and intracellular Ca2+ overload. Conclusion: Choline improves cardiac function and reduces ischemic myocardial injuries via stimulating the cardiac M3-mAChRs which in turn result in alterations of multiple signaling pathways leading to cytoprotection. The findings suggest M3-mAChR as a new target for drug development for improving cardiac function and preventing cardiac injuries during ischemia/reperfusion.

Normal function of HERG K+ channels expressed in HEK293 cells requires basal protein kinase B activity

The potential role of protein kinase B (PKB), a serine/threonine protein kinase, in regulating HERG (human ether‐a‐go‐go related gene) K+ channel function was investigated. Wortmannin (a phosphoinositide 3‐kinase (PI3K) inhibitor) caused ∼30% reduction of HERG current (I HERG) stably expressed in HEK293 cells. Transient transfection with the constitutively active PI3K in HERG‐expressing HEK293 cells slightly increased (∼7%) I HERG while a dominant negative PI3K significantly reduced I HERG (∼25%) relative to results in vehicle‐transfected cells. I HERG was ∼35% greater in cells transfected with the constitutively activated PKB (caPKB), whereas it was ∼47% smaller in cells transfected with dominant negative PKB (dnPKB). Basal activation of PKB was detected by immunocytochemistry. PKB activity was significantly enhanced in caPKB‐transfected cells and nearly abolished in dnPKB‐transfected cells. We conclude that normal HERG function in HEK293 cells requires basal activity of PKB. Our data represent the first evidence that PKB phosphorylation regulates K+ channels.

HERG K+ Channel Conductance Promotes H2O2-Induced Apoptosis in HEK293 Cells: Cellular Mechanisms

The human ether-a-go-go-related gene (HERG) encodes a delayed rectifier K+ channel, which is expressed in a variety of tissues and cells. Besides its well-recognized function in cellular electrophysiology, HERG channels have also been implicated in neuronal differentiation and cell cycle regulation. We have recently found that HERG regulates apoptosis. To elucidate the signaling pathways, we performed studies in HEK293 cells stably expressing HERG channels. ELISA was used to quantify DNA fragmentation, a biochemical hallmark of apoptosis. In HERG-transfected HEK cells, the degree of DNA fragmentation was found consistently higher (ñ4-times) than in non-transfected cells. Correspondingly, remarkable activation of caspase 3, caspase 9 and cleavage of PARP were seen in HERG-expressing cells, which were otherwise minimal in non-transfected cells. Exposure of cells to H2O2 (10 hrs) at concentrations up to 1 mM, which is known to induce apoptosis in a variety of cells, caused minimal DNA fragmentation in non-transfected cells. HERG expression facilitates DNA fragmentation induced by H2O2 at a concentration-dependent fashion, starting at 200 µM and reaching maximum at 1 mM. Selective HERG channel inhibitors, dofetilide or E-4031 (5 µM) prevented DNA fragmentation. Inhibition of p38 by SB-203580 alleviated DNA-F and PD-98059, which inhibited activation of ERKs, nearly abolished DNA-F. Immunoblotting analysis demonstrated that p38, SAPKs and ERKs MAP kinases were all substantially activated (>10-fold higher) in HERG-expressing cells vs. non-transfected cells. Akt activity was ñ4-fold lower in HERG cells vs. non-transfected cells in the absence of H2O2 and was slightly increased (ñ2-fold) after H2O2 exposure. We conclude that HERG channels facilitate cellular DNA fragmentation in HEK cells via concomitant activation of MAP kinases and inactivation of Akt.

Opposite Cell Density-Dependence between Spontaneous and Oxidative Stress-Induced Apoptosis in Mouse Fibroblast L-Cells

Background/Aims: Spontaneous apoptosis in culture is cell density-dependent. Yet, little is known about the density-dependence of apoptosis induced by oxidative stress and the underlying signaling mechanisms. Methods: We compared apoptosis occurring spontaneously and induced by H₂O₂ in mouse fibroblast L-cells seeded at three different densities, using ELISA and TUNEL to measure DNA fragmentation, Western blot and pharmacological probes to evaluate the roles of signaling molecules. Results: The high-density (HD, 2.5x10⁵/cm²) culture had ñ1.5- and ñ3-times more spontaneous apoptosis than medium-density (MD, 5x10⁴/cm²) and low-density (LD, 1x10⁴/cm²) cultures, respectively. The opposite was seen with H₂O₂ (500 µM)-induced apoptosis: more apoptosis in LD than in MD and HD. Caspase inhibitor (Z-VAD-fmK) diminished the density-dependence of both spontaneous and H₂O₂-induced apoptosis. Activation of caspase 3 by H₂O₂ was more pronounced in LD than in MD and HD. Inhibition of PI3K/Akt increased spontaneous apoptosis more in LD and H₂O₂-induced apoptosis more in HD, diminishing the density-dependence of apoptosis. p38 MAPK inhibition reduced H₂O₂-induced apoptosis more in LD, diminishing the density-dependence of H₂O₂-induced apoptosis without altering that of spontaneous apoptosis. Akt level and activity and Hsp72 level were increased in HD, but decreased in MD and LD cells, by H₂O₂, and the active p38 MAPK was enhanced by H₂O₂ in all groups. Conclusion: Spontaneous apoptosis is cell density-dependent whereas H₂O₂-induced apoptosis is reverse density-dependent. PI3K/Akt, p38 MAPK and Hsp72 pathways are critical for the opposite density-dependence of apoptosis.

Potential mechanisms for the enhancement of HERG K+ channel function by phospholipid metabolites

Phospholipid metabolites lysophospholipids cause extracellular K+ accumulation and action potential shortening with increased risk of arrhythmias during myocardial ischemia. Here we studied effects of several lysophospholipids with different lengths of hydrocarbon chains and charged headgroups on HERG K+ currents (IHERG) expressed in HEK293 cells and the potential mechanisms using whole‐cell patch‐clamp techniques. Only the lipids with 16 hydrocarbons such as 1‐palmitoyl‐lysophosphatidylcholine (LPC‐16) and 1‐palmitoyl‐lysophosphatidylglycerol (LPG‐16) were found to produce significant enhancement of IHERG and negative shifts of HERG activation, although the voltage dependence of the effects was different between LPC‐16 and LPG‐16 which have differently charged headgroups. The lipid with 18 hydrocarbons modestly increased IHERG. The lipids with 6 or 24 hydrocarbons had no effect or slightly decreased IHERG. Inhibition or activation of protein kinase C did not alter the effects of LPC‐16 and LPG‐16. Participation of phosphatidylinositol‐4,5‐bisphosphate in IHERG enhancement by LPC‐16/LPG‐16 was also excluded. Vitamin E augmented the effects of LPC‐16/LPG‐16 whereas xanthine/xanthine oxidase reduced IHERG: indicating that LPC‐16/LPG‐16 produced dual effects on IHERG: direct enhancement of IHERG and indirect suppression via production of superoxide anion. We conclude that enhancement of HERG function by lysophospholipids is specific to the lipids with 16‐hydrocarbon chain structure and the pattern of voltage dependence is determined by the polar headgroups. The increase in IHERG is best described by direct interactions between lipid molecules and HERG proteins, which is consistent with lack of effects via membrane destabilization or modulation by intracellular signaling pathways.