Jules C. Hancox

Cultured adult cardiac myocytes : Future applications, culture methods, morphological and electrophysiological properties

Isolated adult cardiac myocytes maintained in primary culture have been used as a model of the adult myocardium for 20 years. With the recent advances and current interest in using molecular biological techniques to investigate cardiac physiology, culturing myocytes is becoming an increasingly important technique. Acutely isolated myocytes do not remain viable for the time needed for the changes in gene expression to occur, and therefore it is necessary to maintain myocytes in culture. The aims of this review are: (1) To describe a method for isolating and culturing myocytes in serum-free medium. This section is targeted at new researchers in the field, with particular emphasis on aspects of the isolation procedure which are important for optimising myocyte culture. (2) To review current knowledge of how contractile, electrophysiological and morphological properties of adult myocytes are preserved in culture. Over the past 5 to 10 years significant advances have been made in developing novel techniques which help maintain the in-vivo properties of myocytes in culture. Efficient methods for transporting exogenous genes and anti-sense oligonucleotides into adult myocytes are now available. We anticipate that in future these advances will make cultured myocytes more attractive for use in biophysical and molecular investigations of cardiac physiology.

Psychotropic drugs, cardiac arrhythmia, and sudden death

A variety of drugs targeted towards the central nervous system are associated with cardiac side effects, some of which are linked with reports of arrhythmia and sudden death. Some psychotropic drugs, particularly tricyclic antidepressants (TCAs) and antipsychotic agents, are correlated with iatrogenic prolongation of the QT interval of the electrocardiogram (ECG). In turn, this is associated with the arrhythmia torsades de pointes (TdP). This review discusses the association between psychotropic agents, arrhythmia and sudden death and, focusing on TCAs and antipsychotics, considers their range of cellular actions on the heart; potentially pro-arrhythmic interactions between psychotropic and other medications are also considered. At the cellular level TCAs, such as imipramine and amitriptyline, and antipsychotics, such as thioridazine, are associated with inhibition of potassium channels encoded by HERG. In many cases this cellular action correlates with ECG changes and a risk of TdP. However, not all psychotropic agents that inhibit HERG at the cellular level are associated equally with QT prolongation in patients, and the potential for QT prolongation is not always equally correlated with TdP. Differences in risk between classes of psychotropic drugs, and between individual drugs within a class, may result from additional cellular effects of particular agents, which may influence the consequent effects of inhibition of repolarizing potassium current.

Blockade of HERG potassium currents by fluvoxamine: incomplete attenuation by S6 mutations at F656 or Y652

Pharmacological blockade of the Human ether‐a‐go‐go related gene (HERG) potassium channel is commonly linked with acquired long QT syndrome and associated proarrhythmia. The objectives of this study were (i) to identify and characterise any inhibitory action on HERG of the selective‐serotonin re‐uptake inhibitor fluvoxamine, (ii) to then determine whether fluvoxamine shared the consensus molecular determinants of HERG blockade of those drugs so far tested. Heterologous HERG potassium current (IHERG) was measured at 37°C, using the whole‐cell patch‐clamp technique, from a mammalian cell line (Human embryonic kidney 293) expressing HERG channels. IHERG tails, following repolarisation from +20 to −40 mV, were blocked by fluvoxamine with an IC50 of 3.8 μM. Blockade of wild‐type HERG was of extremely rapid onset (within 10 ms) and showed voltage dependence, with fluvoxamine also inducing a leftward shift in voltage‐dependent activation of IHERG. Characteristics of block were consistent with a component of closed channel (or extremely rapidly developing open channel) blockade and dependence on open and inactivated channel states. The attenuated‐inactivation mutation S631A partially reduced the blocking effect of fluvoxamine. The S6 mutations, Y652A and F656A, and the pore helix mutant S631A only partially attenuated blockade by fluvoxamine at concentrations causing profound blockade of wild‐type HERG. All HERG‐blocking pharmaceuticals studied to date have been shown to block F656 mutant channels with over 100‐fold reduced potency compared to their blockade of the wild‐type channel. Fluvoxamine is therefore quite distinct in this regard from previously studied agents.

Familial And Acquired Long QT Syndrome And The Cardiac Rapid Delayed Rectifier Potassium Current

1. Long QT syndrome (LQTS) is a cardiac disorder characterized by syncope, seizures and sudden death; it can be congenital, idiopathic, or iatrogenic.

Inhibition of the current of heterologously expressed HERG potassium channels by flecainide and comparison with quinidine, propafenone and lignocaine

The inhibition of the cardiac ‘rapid’ delayed rectifier current (IKr) and its cloned equivalent HERG mediate QT interval prolonging effects of a wide range of clinically used drugs. In this study, we investigated the effects of the Class Ic antiarrhythmic agent flecainide (FLEC) on ionic current (IHERG) mediated by cloned HERG channels at 37°C . We also compared the inhibitory potency of FLEC with other Class I agents: quinidine (QUIN, Class Ia); lignocaine (LIG, Class Ib) and propafenone (PROPAF, Class Ic). Whole cell voltage clamp recordings of IHERG were made from an HEK293 cell line stably expressing HERG. FLEC inhibited IHERG ‘tails’ following test pulses to +30 mV with an IC50 of 3.91±0.68 μM (mean±s.e.mean) and a Hill co‐efficient close to 1 (0.76±0.09). In experiments in which IHERG tails were monitored following voltage commands to a range of test potentials, IHERG inhibition by FLEC was observed to be voltage‐dependent and to be associated with a ∼−5 mV shift of the activation curve for the current. Voltage‐dependence of inhibition was greatest over the range of potentials corresponding to the steep portion of the IHERG activation curve. The time‐course of IHERG tail deactivation was not significantly altered by FLEC. In experiments in which 10 s depolarizing pulses were applied from −80 to 0 mV, the level of current inhibition by FLEC did not increase between 1 and 10 s. Some time‐dependence of inhibition was observed during the first 200 – 300 ms of depolarization. This observation and the voltage‐dependence of inhibition are collectively consistent with FLEC exerting a rapid open channel state inhibition of IHERG. Under similar recording conditions QUIN inhibited IHERG with an IC50 of 0.41±0.04 μM and PROPAF inhibited IHERG with an IC50 of 0.44±0.07 μM. Similar to FLEC, both QUIN and PROPAF showed voltage‐dependence of inhibition and blockade developed rapidly during a sustained depolarization. LIG showed little effect on IHERG at low micromolar concentrations, but could inhibit the current at higher concentrations; the observed IC50 was 262.90±22.40 μM. Our data are consistent with FLEC, PROPAF and QUIN exerting IHERG blockade at clinically relevant concentrations. The rank potency as HERG blockers of the Class I drugs tested in this study was QUIN=PROPAF>FLEC>>LIG.

Inhibition of the current of heterologously expressed HERG potassium channels by imipramine and amitriptyline.

Tricyclic antidepressants (TCAs) are associated with cardiovascular side effects including prolongation of the QT interval of the ECG. In this report we studied the effects of two TCAs (imipramine and amitriptyline) on ionic current mediated by cloned HERG potassium channels. Voltage clamp measurements of HERG currents were made from CHO cells transiently transfected with HERG cDNA. HERG‐encoded potassium channels were inhibited in a reversible manner by both imipramine and amitriptyline. HERG tail currents (IHERG) following test pulses to +20 mV were inhibited by imipramine with an IC50 of 3.4±0.4 μM (mean±s.e.mean) and a Hill coefficient of 1.17±0.03 (n=5). 3 μM amitriptyline inhibited IHERG by 34±6% (n=3). The inhibition showed only weak voltage dependence. Using an ‘envelope of tails’ comprised of pulses to +20 mV of varying durations, the τ of activation was found to be 155±30 ms for control and 132±26 ms for 3 μM imipramine (n=5). Once maximal channel activation was achieved after 320 ms (as demonstrated by maximal tail currents), further prolongation of depolarization did not increase imipramine‐mediated HERG channel inhibition. Taking current measurements every second during a 10 s depolarizing pulse from −80 mV to 0 mV, block was observed during the first pulse in the presence of imipramine and the level of IHERG block was similar throughout the pulse (n=5). A three pulse protocol (two depolarizing pulses to +20 mV separated by 20 ms at −80 mV) revealed that imipramine did not significantly alter the kinetics of IHERG inactivation. The τ of inactivation was 8±2 ms and 5.6±0.4 ms (n=5) in the absence and presence of 3 μM imipramine, respectively, and currents inactivated to a similar extent. Our data are consistent with TCAs causing components of block of the HERG channel in both the closed and open states. Any component of open channel block occurs rapidly upon depolarization. Inhibition of IHERG by the prototype TCAs imipramine and amitriptyline may suggest a mechanism for QT prolongation associated with risks of arrhythmia and sudden death that accompany high concentrations of TCAs following overdose.

A New Perspective in the Field of Cardiac Safety Testing through the Comprehensive In Vitro Proarrhythmia Assay Paradigm

For the past decade, cardiac safety screening to evaluate the propensity of drugs to produce QT interval prolongation and Torsades de Pointes (TdP) arrhythmia has been conducted according to ICH S7B and ICH E14 guidelines. Central to the existing approach are hERG channel assays and in vivo QT measurements. Although effective, the present paradigm carries a risk of unnecessary compound attrition and high cost, especially when considering costly thorough QT (TQT) studies conducted later in drug development. The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative is a public-private collaboration with the aim of updating the existing cardiac safety testing paradigm to better evaluate arrhythmia risk and remove the need for TQT studies. It is hoped that CiPA will produce a standardized ion channel assay approach, incorporating defined tests against major cardiac ion channels, the results of which then inform evaluation of proarrhythmic actions in silico, using human ventricular action potential reconstructions. Results are then to be confirmed using human (stem cell–derived) cardiomyocytes. This perspective article reviews the rationale, progress of, and challenges for the CiPA initiative, if this new paradigm is to replace existing practice and, in time, lead to improved and widely accepted cardiac safety testing guidelines.

VEGF-Mediated Elevated Intracellular Calcium and Angiogenesis in Human Microvascular Endothelial Cells In Vitro are Inhibited by Dominant Negative TRPC6

Objective: Vascular endothelial growth factor (VEGF)‐induced vascular permeability has been shown to be dependent on calcium influx, possibly through a transient receptor potential cation channel (TRPC)‐mediated cation channel with properties of the TRPC3/6/7 subfamily. To investigate further the involvement of this subfamily, we determined the effects of dominant negative TRPC6 overexpression on VEGF‐mediated changes of human microvascular endothelial cell (HMVEC) calcium, proliferation, migration, and sprouting. Methods: Cytoplasmic calcium concentration was estimated by fura‐2 fluorescence spectrophotometry, migration by Boyden chamber assay, sprouting by immunofluorescence imaging of stimulated endothelial cells, and proliferation by flow cytometry. Results: Overexpression of a dominant negative TRPC6 construct in HMVECs inhibited the VEGF‐mediated increases in cytosolic calcium, migration, sprouting, and proliferation. In contrast, overexpression of a wild‐type TRPC6 construct increased the proliferation and migration of HMVECs. Conclusions: TRPC6 is an obligatory component of cation channels required for the VEGF‐mediated increase in cytosolic calcium and subsequent downstream signaling that leads to processes associated with angiogenesis.

Inhibitory actions of the selective serotonin re-uptake inhibitor citalopram on HERG and ventricular L-type calcium currents

Using whole‐cell patch clamp recording of heterologous HERG‐mediated currents in transfected mammalian cells, we observed that the selective serotonin re‐uptake inhibitor citalopram blocks HERG with an IC50 of 3.97 μM. This is slightly less potent than fluoxetine in our system (IC50 of 1.50 μM). In isolated guinea pig ventricular cardiomyocytes citalopram inhibited L‐type calcium current (I Ca,L). The voltage dependence of I Ca,L inactivation in the presence of 100 μM citalopram was shifted significantly leftward. As a result, the I Ca,L ‘window’ in citalopram was found to be (a) smaller and (b) leftward‐shifted compared to control. The effects of citalopram on both calcium current amplitude and the I Ca,L ‘window’ may help to explain citaloprams good cardiac safety profile, given its propensity to block HERG at excessive dosages.

ACTION POTENTIALS, ION CHANNEL CURRENTS AND TRANSVERSE TUBULE DENSITY IN ADULT RABBIT VENTRICULAR MYOCYTES MAINTAINED FOR 6 DAYS IN CELL CULTURE

Adult rabbit ventricular myocytes were cultured in a basic medium (Medium 199) for up to 6 days to assess preservation of morphology and ion channel currents. In culture, cells remained rod shaped and striated but their ends became progressively rounded. Cell cross-sectional area declined slightly (by 14%) over the first 24 h, in contrast, whole-cell capacitance (which reflects external surface membrane plus membrane infoldings) decreased by 42% over the same time. Using whole-cell patch-clamp, we observed that the typical “N” shape steady-state current-voltage (I-V) relation became flattened after 24 h in culture. L-type Ca channel density was assessed as barium flux (IBa,L) via the channel. IBa,L (normalised to cell capacitance) declined by 50% after 24 h and recovered partially by days 4 and 6. The density of inward rectifier K current declined by 54% after 24 h and showed no recovery subsequently. In contrast, there was no significant decline in the density of transient outward K current after 24 h, but it declined subsequently by 65% after 6 days. We speculate that the time course of change in each ion channel density may reflect a change in pattern of ion channel expression, or differential membrane loss since the density of transverse tubules decreased by 57% after 6 days in culture. These results suggest that even by 24 h in culture, ion channel density in myocytes has changed substantially from the acutely isolated state.