Evaluation of electrocardiograms recorded in cynomolgus monkeys with short- and long-term intracardiac lead implantations.

INTRODUCTION: minimally invasive placement of intracardiac (IC) ECG leads in monkeys has greatly improved signal quality and the ability to interpret these ECGs. However, information on characteristics of the ECGs recorded using the IC lead is not available in the literature. There are concerns about the potential impact of IC lead placement on the ECG waveform and cardiac function as a result of potential irritation or trauma resulting from the placement and/or long term residence of the IC lead. The purposes of this study were to characterize IC ECG morphology, to obtain information on the recovery processes after IC ECG lead implantation, and to evaluate the IC ECG model application to safety pharmacology studies. METHODS: the telemetry transmitter, arterial blood pressure catheter and IC ECG lead were implanted in 40 cynomolgus monkeys, two of which were also implanted with subcutaneous (SC) ECG leads. The data of IC ECG, heart rate (HR) and mean arterial blood pressure (MABP) were collected telemetrically for a period of 1-12 months after implantation, and measured using computer softwares. RESULTS: the IC ECG waveforms varied greatly from those of SC ECG. There was no clearly identifiable S-T segment, and T waves were biphasic in the majority of IC ECGs. The morphology of IC ECG was diversified among animals, progressively changed in the first 2 weeks post-surgery and stabilized approximately 3 weeks post-surgery. MABP and HR were elevated after implant surgery, but recovered to the levels comparable to those of SC in approximately 1 and 4 weeks, respectively. The IC ECG values obtained during week 8 to 10 (HR=134+/-25 bpm, PR interval=87+/-13 ms, QRS interval=40+/-7 ms, and QT interval=246+/-30 ms, QTcF=318+/-28 ms) were comparable to those from SC ECG. DISCUSSION: the IC ECG provides a clear ECG signal with values comparable to, and waveforms different from, SC recordings. The complicated surgical procedure with long substantial recovery time, high incidence of IC lead malfunction, and high costs for IC leads may limit application of the IC ECG model in safety pharmacology studies.

Dimethyl sulfoxide effects on hERG channels expressed in HEK293 cells.

INTRODUCTION: Dimethyl sulfoxide (DMSO) is widely used as a solvent to facilitate formulation of test substances in cell perfusion solutions. However, DMSO concentration in bath (extracellular) solution is usually limited to 0.1-0.3% to avoid DMSO-induced changes in cell morphology and membrane properties due to elevation of osmolality. The purpose of this study was to examine whether DMSO-induced hyperosmotic effects on hERG expressing cells could be compensated by adding an equivalent amount of DMSO in pipette (intracellular) solution, to investigate DMSO effects on hERG channels, and to determine the impact of DMSO on the potency of hERG channel blockers. METHOD: Whole-cell patch clamp method was used to record hERG currents in HEK293 cells. DMSO at concentrations of 0.1% to 2% was applied to bath and pipette solutions. Various voltage protocols were used to examine DMSO effects on hERG channel properties and to evaluate DMSO impacts on the potency of terfenadine and E-4031. RESULTS: When DMSO was added simultaneously in bath and pipette solutions, normal cell morphology and the proper current recording conditions could be maintained with application of up to 2% DMSO. DMSO slightly shifted the current-voltage relationship, activation curve, and inactivation curve of the hERG channel to more positive voltages. DMSO had little effect on the concentration-response relationship of hERG channel blockers we assessed. The IC50 for terfenadine and E-4031 were not significantly changed in the presence of 0.3, 0.5, 1 and 2% DMSO. DISCUSSION: Our results demonstrate that changes in cell morphology induced by extracellular DMSO can be prevented by application of DMSO in pipette solution. By utilizing this approach, we successfully performed hERG current recordings using bath solution containing up to 2% DMSO. DMSO-induced shifts of the voltage-dependence of hERG channel gating had little impact on the potency of hERG channel blockers.

Estimation of potency of HERG channel blockers: impact of voltage protocol and temperature.

INTRODUCTION: The HERG channel is widely used for the assessment of proarrhythmic risk for new drugs. HERG channel blockers obstruct channel functions through various mechanisms, which usually show time dependence, voltage dependence, and state dependence. The voltage protocol and temperature may affect the estimation of drug potency, but limited information is available in this regard. The purpose of this study was to evaluate the influence of voltage protocol and temperature on predicting the potency of HERG channel blockers, and to determine electrophysiological approaches for new drugs screening studies. METHOD: Whole-cell patch-clamp electrophysiology was carried out by utilizing different voltage step protocols to examine the potency of compounds known to preferentially block the channel in the closed (ketoconazole and BeKm-1), open, and/or inactivated states (E-4031, astemizole, and terfenadine) in HEK293 cells transfected with HERG cDNA at room temperature and near-physiological temperature. RESULTS: Drug potency determined using different voltage protocols varied dependent on the mechanisms of drug actions. For most compounds, the IC(50) values obtained with a long pulse step protocol at room temperature were close to those determined with the voltage protocols designed to disclose their intrinsic potency. Relative to room temperature, the potency of E-4031, terfenadine, and ketoconazole was not changed at approximately 35 degrees C, but potency of astemizole was reduced. DISCUSSION: The long pulse step protocol with room temperature can be selected for HERG channel safety screening studies. Alternative voltage protocols or temperatures should be considered if HERG study results are not consistent with other cardiac safety assessments.

Cell coupling between ventricular myocyte pairs from connexin43-deficient murine hearts.

Mice with cardiac-restricted inactivation of the connexin43 gene (CKO mice) have moderate slowing of ventricular conduction and lethal arrhythmias. Mechanisms through which propagation is maintained in the absence of Cx43 are unknown. We evaluated gap junctional conductance in CKO ventricular pairs using dual patch clamp methods. Junctional coupling was reduced to 4+/-2 nS (side-to-side) and 11+/-2 nS (end-to-end), including 21% of cell-pairs with no detectable coupling, compared with 588+/-104 nS (side-to-side) and 558+/-92 nS (end-to-end) in control cell-pairs. Voltage dependence of control gap junctions was characteristic of Cx43. CKO conductance showed increased voltage dependence, suggesting low-level expression of other connexin isoforms. From theoretical models, this degree of CKO coupling is not expected to support levels of conduction persisting in vivo, suggesting the possibility that there are additional mechanisms for maintained propagation when gap junctional conductance is severely reduced.

Remodeling of gap junctional channel function in epicardial border zone of healing canine infarcts.

The epicardial border zone (EBZ) of canine infarcts has increased anisotropy because of transverse conduction slowing. It remains unknown whether changes in gap junctional conductance (Gj) accompany the increased anisotropy. Ventricular cell pairs were isolated from EBZ and normal hearts (NZ). Dual patch clamp was used to quantify Gj. At a transjunctional voltage (Vj) of +10 mV, side-to-side Gj of EBZ pairs (9.2+/-3.4 nS, n=16) was reduced compared with NZ side-to-side Gj (109.4+/-23.6 nS, n=14, P<0.001). Gj of end-to-end coupled cells was not reduced in EBZ. Steady-state Gj of both NZ and EBZ showed voltage dependence, described by a two-way Boltzmann function. Half-maximal activation voltage in EBZ was shifted to higher Vj in positive and negative directions. Immunoconfocal planimetry and quantification showed no change in connexin43 per unit cell volume or surface area in EBZ. Decreased side-to-side coupling occurs in EBZ myocytes, independent of reduced connexin43 expression, and is hypothesized to contribute to increased anisotropy and reentrant arrhythmias.