Jae Boum Youm

Stretch-activated and background non-selective cation channels in rat atrial myocytes

1 Stretch‐activated channels (SACs) were studied in isolated rat atrial myocytes using the whole‐cell and single‐channel patch clamp techniques. Longitudinal stretch was applied by using two patch electrodes. 2 In current clamp configuration, mechanical stretch of 20 % of resting cell length depolarised the resting membrane potential (RMP) from ‐63·6 ± 0·58 mV (n= 19) to ‐54·6 ± 2·4 mV (n= 13) and prolonged the action potential duration (APD) by 32·2 ± 8·8 ms (n= 7). Depolarisation, if strong enough, triggered spontaneous APs. In the voltage clamp configuration, stretch increased membrane conductance in a progressive manner. The current‐voltage (I–V) relationship of the stretch‐activated current (ISAC) was linear and reversed at ‐6·1 ± 3·7 mV (n= 7). 3 The inward component of ISAC was abolished by the replacement of Na+ with NMDG+, but ISAC was hardly altered by the Cl− channel blocker DIDS or removal of external Cl−. The permeability ratio for various cations (PCs:PNa:PLi= 1·05:1:0·98) indicated that the SAC current was a non‐selective cation current (ISAC,NC). The background current was also found to be non‐selective to cations (INSC,b); the permeability ratio (PCs:PNa:PLi= 1·49:1:0·70) was different from that of ISAC,NC. 4 Gadolinium (Gd3+) acted on INSC,b and ISAC,NC differently. Gd3+ inhibited INSC,b in a concentration‐dependent manner with an IC50 value of 46·2 ± 0·8 μM (n= 5). Consistent with this effect, Gd3+ hyperpolarised the resting membrane potential (‐71·1 ± 0·26 mV, n= 9). In the presence of Gd3+ (0·1 mM), stretch still induced ISAC,NC and diastolic depolarisation. 5 Single‐channel activities were recorded in isotonic Na+ and Cs+ solutions using the inside‐out configuration. In NMDG+ solution, outward currents were abolished. Gd3+ (100 μM) strongly inhibited channel opening both from the inside and outside. In the presence of Gd3+ (100 μM) in the pipette solution, an increase in pipette pressure induced an increase in channel opening (21·27 ± 0·24 pS; n= 7), which was distinct from background activity. 6 We concluded from the above results that longitudinal stretch in rat atrial myocytes induces the activation of non‐selective cation channels that can be distinguished from background channels by their different electrophysiology and pharmacology.

Blockade of the HERG human cardiac K+ channel by the antidepressant drug amitriptyline

Amitriptyline has been known to induce QT prolongation and torsades de pointes which causes sudden death. We studied the effects of amitriptyline on the human ether‐a‐go‐go‐related gene (HERG) channel expressed in Xenopus oocytes and on the rapidly activating delayed rectifier K+ current (IKr) in rat atrial myocytes. The amplitudes of steady‐state currents and tail currents of HERG were decreased by amitriptyline dose‐dependently. The decrease became more pronounced at more positive potential, suggesting that the block of HERG by amitriptyline is voltage dependent. IC50 for amitriptyline block of HERG current was progressively decreased according to depolarization: IC50 values at −30, −10, +10 and +30 mV were 23.0, 8.71, 5.96 and 4.66 μM, respectively. Block of HERG by amitriptyline was use dependent: exhibiting a much faster block at higher activation frequency. Subsequent decrease in frequency after high activation frequency resulted in a partial relief of HERG blockade. Steady‐state block by amitriptyline was obtained while depolarization to +20 mV for 0.5 s was applied at 0.5 Hz: IC50 was 3.26 μM in 2 mM [K+]o. It was increased to 4.78 μM in 4 mM [K+]o, suggesting that the affinity of amitriptyline on HERG was decreased by external K+. In rat atrial myocytes bathed in 35°C, 5 μM amitriptyline blocked IKr by 55%. However, transient outward K+ current (Ito) was not significantly affected. In summary, the data suggest that the block of HERG currents may contribute to arrhythmogenic side effects of amitriptyline.

Non-genomic effect of glucocorticoids on cardiovascular system

Glucocorticoids (GCs) are essential steroid hormones for homeostasis, development, metabolism, and cognition and possess anti-inflammatory and immunosuppressive actions. Since glucocorticoid receptor II (GR) is nearly ubiquitous, chronic activation or depletion of GCs leads to dysfunction of diverse organs, including the heart and blood vessels, resulting predominantly from changes in gene expression. Most studies, therefore, have focused on the genomic effects of GC to understand its related pathophysiological manifestations. The nongenomic effects of GCs clearly differ from well-known genomic effects, with the former responding within several minutes without the need for protein synthesis. There is increasing evidence that the nongenomic actions of GCs influence various physiological functions. To develop a GC-mediated therapeutic target for the treatment of cardiovascular disease, understanding the genomic and nongenomic effects of GC on the cardiovascular system is needed. This article reviews our current understanding of the underlying mechanisms of GCs on cardiovascular diseases and stress, as well as how nongenomic GC signaling contributes to these conditions. We suggest that manipulation of GC action based on both GC and GR metabolism, mitochondrial impact, and the action of serum- and glucocorticoid-dependent kinase 1 may provide new information with which to treat cardiovascular diseases.

A mathematical model of pacemaker activity recorded from mouse small intestine.

The pacemaker activity of interstitial cells of Cajal (ICCs) has been known to initiate the propagation of slow waves along the whole gastrointestinal tract through spontaneous and repetitive generation of action potentials. We studied the mechanism of the pacemaker activity of ICCs in the mouse small intestine and tested it using a mathematical model. The model includes ion channels, exchanger, pumps and intracellular machinery for Ca2+ regulation. The model also incorporates inositol 1,4,5-triphosphate (IP3) production and IP3-mediated Ca2+ release activities. Most of the parameters were obtained from the literature and were modified to fit the experimental results of ICCs from mouse small intestine. We were then able to compose a mathematical model that simulates the pacemaker activity of ICCs. The model generates pacemaker potentials regularly and repetitively as long as the simulation continues. The frequency was set at 20 min−1 and the duration at 50% repolarization was 639 ms. The resting and overshoot potentials were −78 and +1.2 mV, respectively. The reconstructed pacemaker potentials closely matched those obtained from animal experiments. The model supports the idea that cyclic changes in [Ca2+]i and [IP3] play key roles in the generation of ICC pacemaker activity in the mouse small intestine.

Inhibition of acetylcholine-activated K+ currents by U73122 is mediated by the inhibition of PIP2-channel interaction

We have investigated the effect of U73122, a specific inhibitor of phospholipase C (PLC), on acetylcholine‐activated K+ currents (IKACh) in mouse atrial myocytes. In perforated patch clamp mode, IKACh was activated by 10 μM acetylcholine. When atrial myocytes were pretreated with U73122 or U73343, IKACh was inhibited dose‐dependently (half‐maximal inhibition at 0.12±0.0085 and 0.16±0.0176 μM, respectively). The current‐voltage relationships for IKACh in the absence and in the presence of U73122 showed that the inhibition occurred uniformly from −120 to +40 mV, indicating a voltage‐independent inhibition. When U73122 was applied after IKACh reached steady‐state, a gradual decrease in IKACh was observed. The time course of the current decrease was well fitted to a single exponential, and the rate constant was proportional to the concentration of U73122. When KACh channels were directly activated by adding 1 mM GTPγS to the bath solution in inside‐out patches, U73122 (1 μM) decreased the open probability significantly without change in mean open time. When KACh channels were activated independently of G‐protein activation by 20 mM Na+, open probability was also inhibited by U73122. Voltage‐activated K+ currents and inward rectifying K+ currents were not affected by U73122. These findings show that inhibition by U73122 and U73343 of KACh channels occurs at a level downstream of the action of Gβγ or Na+ on channel activation. The interference with phosphatidylinositol 4,5‐bisphosphate (PIP2)‐channel interaction can be suggested as a most plausible mechanism.

Modulation by melatonin of the cardiotoxic and antitumor activities of adriamycin.

In this study, we investigated the effects of melatonin on adriamycin-induced cardiotoxicity both in vivo in rats and in vitro, and on the antitumor activities of adriamycin on MDA-231 and NCI breast cancer cells. Rats that received a single intraperitoneal injection of 25 mg/kg adriamycin showed a mortality rate of 86%, which was reduced to 20% by melatonin treatment (10 mg/kg, SC for 6 days). Melatonin attenuated adriamycin-induced body-weight loss, hemodynamic dysfunction, and the morphologic and biochemical alterations caused by adriamycin. Melatonin also reduced adriamycin-induced nuclear DNA fragmentation, as assessed by the comet assay. In addition, the antitumor activity of adriamycin could be maintained using lower doses of this drug in combination with melatonin. Melatonin treatment in the concentration range of 0.1-2.5 mM inhibited the growth of human breast cancer cells. In terms of oncolytic activity, the combination of adriamycin and melatonin improved the antitumor activity of adriamycin, as indicated by an increase in the number of long-term survivors as well as decreases in body-weight losses resulting from adriamycin treatment. These results indicate that melatonin not only protects against adriamycin-induced cardiotoxicity but also enhances its antitumor activity. This combination of melatonin and adriamycin represents a potentially useful regimen for the treatment of human neoplasms because it allows the use of lower doses of adriamycin, thereby avoiding the toxic side effects associated with this drug.

Blockade of HERG Channels Expressed in Xenopus laevis Oocytes by External Divalent Cations

We have investigated actions of various divalent cations (Ba2+, Sr2+, Mn2+, Co2+, Ni2+, Zn2+) on human ether-a-go-go related gene (HERG) channels expressed in Xenopus laevis oocytes using the voltage clamp technique. All divalent cations inhibited HERG current dose-dependently in a voltage-dependent manner. The concentration for half-maximum inhibition (Ki) decreased at more negative potentials, indicating block is facilitated by hyperpolarization. Ki at 0 mV for Zn2+, Ni2+, Co2+, Ba2+, Mn2+, and Sr2+ was 0.19, 0.36, 0. 50, 0.58, 2.36, and 6.47 mM, respectively. The effects were manifested in four ways: 1) right shift of voltage dependence of activation, 2) decrease of maximum conductance, 3) acceleration of current decay, and 4) slowing of activation. However, each parameter was not affected by each cation to the same extent. The potency for the shift of voltage dependence of activation was in the order Zn2+ > Ni2+ >/= Co2+ > Ba2+ > Mn2+ > Sr2+, whereas the potency for the decrease of maximum conductance was Zn2+ > Ba2+ > Sr2+ > Co2+ > Mn2+. The kinetics of activation and deactivation were also affected, but the two parameters are not affected to the same extent. Slowing of activation by Ba2+ was most distinct, causing a marked initial delay of current onset. From these results we concluded that HERG channels are nonselectively blocked by most divalent cations from the external side, and several different mechanism are involved in their actions. There exist at least two distinct binding sites for their action: one for the voltage-dependent effect and the other for reducing maximum conductance.

Dual Modulation of the Mitochondrial Permeability Transition Pore and Redox Signaling Synergistically Promotes Cardiomyocyte Differentiation From Pluripotent Stem Cells

Background Cardiomyocytes that differentiate from pluripotent stem cells (PSCs) provide a crucial cellular resource for cardiac regeneration. The mechanisms of mitochondrial metabolic and redox regulation for efficient cardiomyocyte differentiation are, however, still poorly understood. Here, we show that inhibition of the mitochondrial permeability transition pore (mPTP) by Cyclosporin A (CsA) promotes cardiomyocyte differentiation from PSCs. Methods and Results We induced cardiomyocyte differentiation from mouse and human PSCs and examined the effect of CsA on the differentiation process. The cardiomyogenic effect of CsA mainly resulted from mPTP inhibition rather than from calcineurin inhibition. The mPTP inhibitor NIM811, which does not have an inhibitory effect on calcineurin, promoted cardiomyocyte differentiation as much as CsA did, but calcineurin inhibitor FK506 only slightly increased cardiomyocyte differentiation. CsA‐treated cells showed an increase in mitochondrial calcium, mitochondrial membrane potential, oxygen consumption rate, ATP level, and expression of genes related to mitochondrial function. Furthermore, inhibition of mitochondrial oxidative metabolism reduced the cardiomyogenic effect of CsA while antioxidant treatment augmented the cardiomyogenic effect of CsA. Conclusions Our data show that mPTP inhibition by CsA alters mitochondrial oxidative metabolism and redox signaling, which leads to differentiation of functional cardiomyocytes from PSCs.

Endothelin-1 inhibits inward rectifier K+ channels in rabbit coronary arterial smooth muscle cells through protein kinase C.

We studied inward rectifier K+ (Kir) channels in smooth muscle cells isolated from rabbit coronary arteries. In cells from small- (<100 μm, SCASMC) and medium-diameter (100 ∼ 200 μm, MCASMC) coronary arteries, Kir currents were clearly identified (11.2 ± 0.6 and 4.2 ± 0.6 pA pF−1 at -140 mV in SCASMC and MCASMC, respectively) that were inhibited by Ba2+ (50 μM). By contrast, a very low Kir current density (1.6 ± 0.4 pA pF−1) was detected in cells from large-diameter coronary arteries (>200 μm, LCASMC). The presence of Kir2.1 protein was confirmed in SCASMC in a Western blot assay. Endothelin-1 (ET-1) inhibited Kir currents in a dose-dependent manner. The inhibition of Kir currents by ET-1 was abolished by pretreatment with the protein kinase C (PKC) inhibitor staurosporine (100 nM) or GF 109203X (1 μM). The PKC activators phorbol 12,13-dibutyrate (PDBu) and 1-oleoyl-2-acetyl-sn-glycerol (OAG) reduced Kir currents. The ETA-receptor inhibitor BQ-123 prevented the ET-1-induced inhibition of Kir currents. The amplitudes of the ATP-dependent K+ (KATP), Ca2+-activated K+ (BKCa), and voltage-dependent K+ (KV) currents, and effects of ET-1 on these channels did not differ between SCASMC and LCASMC. From these results, we conclude that Kir channels are expressed at a higher density in SCASMC than in larger arteries and that the Kir channel activity is negatively regulated by the stimulation of ETA-receptors via the PKC pathway.

Blockade of HERG channels expressed in Xenopus oocytes by external H

Abstract We have investigated the effect of external H+ concentration ([H+]o)on the human-ether-a-go-go-related gene (HERG) current (IHERG), the molecular equivalent of the cardiac delayed rectifier potassium current (IKr), expressed in Xenopus oocytes, using the two-microelectrode voltage-clamp technique. When [H+]o was increased, the amplitude of the IHERG elicited by depolarization decreased, and the rate of current decay on repolarization was accelerated. The activation curve shifted to a more positive potential at lower external pH (pHo) values (the potential required for half-maximum activation, V1/2, was: –41.8 mV, –38.0 mV, –33.7 mV, –26.7 mV in pHo 8.0, 7.0, 6.6, 6.2, respectively). The maximum conductance (gmax) was also affected by [H+]o: a reduction of 7.9%, 14.6%, and 22.8% was effected by decreasing pHo from 8.0 to 7.0, 6.6, and 6.2, respectively. We then tested whether this pH effect was affected by the external Ca2+ concentration, which is also known to block HERG channels. When the extracellular Ca2+ concentration was increased from 0.5 mM to 5 mM, the shift in V1/2 caused by increasing [H+]o was attenuated, suggesting that these two ions compete for the same binding site. On the other hand, the decrease in gmax caused by increasing [H+]o was not significantly affected by changing external Ca2+ levels. The results indicate that HERG channels are inhibited by [H+]o by two different mechanisms: voltage-dependent blockade (shift of V1/2) and the decrease in gmax. With respect to the voltage-dependent blockade, the interaction between H+ and Ca2+ is competitive, whereas for the decreasing gmax, their interaction is non-competitive.