Antonio E. Lacerda

The action potential and comparative pharmacology of stem cell-derived human cardiomyocytes

INTRODUCTION The cardiac action potential (CAP) of stem cell-derived human cardiomyocytes (SC-hCMs) is potentially the most powerful preclinical biomarker for cardiac safety and efficacy in humans. Our experiments tested this hypothesis by examining the CAP and relevant pharmacology of these cells. METHODS The electrophysiological and pharmacological profiles of SC-hCMs were compared to rabbit and canine Purkinje fibers (PFs). Ventricular SC-hCMs provided the dominant electrophysiological phenotype (approximately 82%) in a population of ventricular, atrial and nodal cardiomyocytes (CMs). The effects of reference compounds were measured in SC-hCMs using perforated patch, current clamp recording. Selective inhibitors of I(Kr), I(Ks), I(Ca,L), and I(Na), and norepinephrine (NE), were tested on SC-hCM action potentials (APs). RESULTS AP prolongation was observed upon exposure to hERG channel blockers (terfenadine, quinidine, cisapride, sotalol, E-4031 and verapamil), with significantly shorter latencies than in PF assays. For the torsadogenic compounds, terfenadine and quinidine, SC-hCM AP prolongation occurred at significantly lower concentrations than in canine or rabbit PF APs. Moreover, the I(Ks) blocker chromanol 293B prolonged APs from SC-hCMs, whereas both rabbit and canine PF assays are insensitive to I(Ks) blockers in the absence of adrenergic preconditioning. Early afterdepolarizations (EADs) were induced by 100 nM E-4031 and 100 nM cisapride in the SC-hCM assay, but not in the canine or rabbit PF assay. Selective inhibition of I(Na) and I(Ca,L) slowed V(max) and shortened AP duration, respectively. NE prolonged the AP duration of SC-hCMs. DISCUSSION The CAP of SC-hCMs has been validated as a powerful preclinical biomarker for cardiac safety and efficacy. In addition to its human nature, the SC-hCM AP assay removes diffusion delays, reduces test compound consumption, demonstrates an overall pharmacological sensitivity that is greater than conventional rabbit or canine PF assays, and accurately predicts cardiac risk of known torsadogenic compounds.

Alfuzosin Delays Cardiac Repolarization by a Novel Mechanism

The United States Food and Drug Administration (FDA) uses alfuzosin as an example of a drug having QT risk in humans that was not detected in nonclinical studies. FDA approval required a thorough clinical QT study (TCQS) that was weakly positive at high doses. The FDA has used the clinical/nonclinical discordance as a basis for mandatory TCQS, and this requirement has serious consequences for drug development. For this reason, we re-examined whether nonclinical signals of QT risk for alfuzosin were truly absent. Alfuzosin significantly prolonged action potential duration (APD)60 in rabbit Purkinje fibers (p < 0.05) and QT in isolated rabbit hearts (p < 0.05) at the clinically relevant concentration of 300 nM. In man, the QT interval corrected with Fridericias formula increased 7.7 ms, which exceeds the 5.0-ms threshold for a positive TCQS. Effects on hKv11.1, hKv4.3, and hKv7.1/hKCNE1 potassium currents and calcium current were not involved. At 300 nM, ∼30× Cmax, alfuzosin significantly increased whole-cell peak sodium (hNav1.5) current (p < 0.05), increased the probability of late hNav1.5 single-channel openings, and significantly shortened the slow time constant for recovery from inactivation. Alfuzosin also increased hNav1.5 burst duration and number of openings per burst between 2- and 3-fold. Alfuzosin is a rare example of a non-antiarrhythmic drug that delays cardiac repolarization not by blocking hKv11.1 potassium current, but by increasing sodium current. Nonclinical studies clearly show that alfuzosin increases plateau potential and prolongs APD and QT, consistent with QT prolongation in man. The results challenge the FDA grounds for the absolute primacy of TCQS based on the claim of a false-negative, nonclinical study on alfuzosin.

Endothelin increases single-channel calcium currents in coronary arterial smooth muscle cells.

Endothelin (ET), a newly identified vasoconstrictor peptide produced by endothelial cells, depends on extracellular calcium for its action [(1988) Nature 332, 411–415]. It is not yet known whether the increase in calcium influx induced by ET results from a direct effect on the Ca2+ channels or is secondary to a reduction in membrane potential. To address this question, we studied the effects of ET on single‐channel calcium currents of freshly dissociated porcine coronary artery smooth muscle cells using the cell‐attached mode of the patch‐clamp technique. We show that ET increases Cal+‐channel activity with no effect on channel open time or conductance. The ability of bath‐applied ET to increase single‐channel calcium currents in the cell‐attached mode is evidence that the peptide acts via a second messenger system.

T-type and N-type calcium channels of Xenopus oocytes: evidence for specific interactions with beta subunits.

We used amplifying effects of calcium channel beta subunits to identify endogenous calcium channels in Xenopus oocytes. Expression of rat brain beta 4 increased macroscopic endogenous current magnitude with a small effect on kinetics. In contrast, expression of rat brain/cardiac beta 2 produced a much larger increase in current magnitude and dramatically slowed current decay. Low concentrations of omega-conotoxin GVIA irreversibly blocked currents in both uninjected and beta 2-injected oocytes. Single channel recordings revealed both T- and N-type calcium channels with conductances of 9 and 18 pS, respectively, in uninjected oocytes and in oocytes expressing either beta subunit. Expression of either beta subunit slowed average current decay of T-type single channels. Slowing of T-type current decay by expression of beta 2 was due to reopening of the channels. N-type single channel average current decay showed little change with expression of beta 4, whereas expression of beta 2 slowed average current decay.

Voltage-dependent inactivation in a cardiac-skeletal chimeric calcium channel.

The loci for inactivation in calcium channel proteins are unknown. Mechanisms for inactivation may be distributed across Ca2+ channel subunits and appear to be complex, multiple and interacting. We took advantage of the properties of chimeras, constructed between cardiac (H4) and skeletal muscle (Sk4) calcium channel α 1 subunits to study the molecular mechanism of inactivation in L‐type calcium channels. Sk1H3, a chimeric construct of these two L‐type calcium channels, was expressed in Xenopus oocytes in the absence of auxiliary subunits. Sk1H3 incorporated repeat I from skeletal muscle α 1, and repeats II, III, IV from heart α 1, subunit. Sk1H3 inactivated faster (τ ≈ 300 ms) and more fully than the wild‐type H4 with Ba2+ ions as the charge carrier. Thus, inactivation of Sk1H3 was 90% complete after a 5‐s conditioning pulse at +20 mV while inactivation of H4 was only 37% complete. Sk1H3 inactivation also developed at more negative potentials with E 0.5 = −15 mV as compared to E 0.5 = −5 mV for H4. In the presence of external calcium ions, the extent of inactivation significantly increased from 37 to 83% for H4 while inactivation of Sk1H3 was only slightly increased. Inactivation with Ba2+ as the charge carrier was confirmed at the single‐ channel level where averaged single‐channel ensembles showed a similar rate of inactivation. Collectively, these observations demonstrate that Sk1H3 inactivation appears to have a prominent voltage‐dependent component. Whether Sk1H3 inactivation involves interactions within repeat I alone or interactions between repeat I and site(s) located in the three other repeats of the α 1 subunit has yet to be determined.

[3H]PN200-110 Binding in a fibroblast cell line transformed with the α1 subunit of the skeletal muscle L-type Ca2+ channel

We examined the binding of the 1,4-dihydropyridine (DHP) [3H]PN200-110 to membranes from a fibroblast cell line transfected with the alpha 1 subunit (DHP receptor) of the L-type Ca2+ channel from rabbit skeletal muscle. Binding site affinity (KD) and density (Bmax) were 1.16 +/- 0.31 nM and 142 +/- 17 fmoles/mg protein, respectively. This affinity corresponded closely with that observed in native skeletal muscle. The Ca2+ channel antagonists diltiazem and MDL 12,330A stimulated [3H]PN200-110 binding in a dose-dependent manner while flunarizine, quinacrine and trifluoperazine inhibited binding. Surprisingly, D600 also stimulated [3H]PN200-110 binding in a dose-dependent and stereoselective manner. It is concluded that the fibroblast cells used in this study provide a unique system for interactions of the Ca2+ channel ligands with the alpha 1 subunit of the skeletal muscle L-type Ca2+ channel.

Na and Ca Channels in the Heart

Thanks to the development of the gigaseal patch-clamp method (34) cardiac membrane electrophysiology has entered the molecular phase thus allowing single- Na- and Ca-channel proteins to be studied in the living state. The application of this approach has been particularly fortuitous because the development of viable isolated adult cell preparations (48,49,78,79,93) so important for adequate voltage clamping involved enzymatic dispersion which also prepared the cell membranes in such a way as to allow gigaseals to be made. Although the single-channel method has the highest resolution of any electrophysiological method presently available, data analysis is tedious and the method itself suffers from bandwidth limitation. Therefore, application of the voltage-clamp method to single cells (11) has been very important and provides a necessary reference for interpreting the single-channel results.