Q: Are SC-hCM cells true cardiomyocytes?

A: SC-hCMs share many characteristics with native cardiomyocytes. SC-hCMs stain positive for a variety of cardiac biomarkers (tropomyosin, alpha-actinin, troponin I and troponin T, sarcomeric myosin, NKX2.5, GATA4, ANF and connexin 43) spontaneously contract, and differentiate into each of the three major cell types of the human heart (atrial, nodal, and ventricular).

Q: Compared with Purkinje fiber data, the SC-hCMs show greater sensitivity to pharmacological modulators of the cardiac action potential. Have you observed any false positives?

A: We have not observed any false positives. The action potential duration (APD) is predictive of cardiac risk for all compounds tested to date. For known torsadogenic compounds (e.g., sotalol, terfenadine, cisapride, and quinidine) the SC-hCM AP assay accurately predicts cardiac risk, and non-torsadogenic compounds (e.g., verapamil and nifedipine) are predicted to be safe.

Q: You perform the SC-hCM assay using manual patch-clamp, which is a relatively low throughput technique – why not use automated instruments?

A: Higher-throughput automated electrophysiology instruments are not current-clamp capable, meaning the action potential cannot be measured, so the SC-hCM action potential assay cannot be performed using the higher-throughput instruments. Moreover, the manual patch-clamp technique is the “gold standard” for electrophysiology assays, producing high quality data that would likely not be possible using automated instruments, especially given the technical difficulty of the SC-hCM assay. We are currently evaluating the individual ion channel currents that underlie the action potential on both manual and automated platforms for higher-throughput screens. We have observed each of the major currents (IK, INa, and ICa).

Q: At what developmental stage are the SC-hCMs – mature or immature?

A: The cells appear to be functionally and structurally embryonic or fetal, rather than adult-like. Compared to adult myocytes, the SC-hCM resting membrane potential is slightly more depolarized, the upstroke velocity is slower, and the cells spontaneously contract. However, the cells demonstrate expected pharmacological responses to modulators of the action potential, and accurately predict cardiac risk. The SC-hCMs have the additional advantage that they are human cells, thereby avoiding species variability.

Q: Is there variability of the action potential between cells, and does this impact the interpretation of action potential experiments?

A: The population of SC-hCMs includes ventricular, atrial and nodal cell types, and the action potential duration at 90% repolarization (APD90) varies as would be expected for a mixed population. For pharmacological experiments we select ventricular-type cells with APD90 values ranging from ~150 - 500 ms. The variability in baseline values is avoided by calculating and reporting percent changes (delta values).

Q: What is the position of the FDA on the SC-hCM assay?

A: Data from the SC-hCM assay was presented to members of the FDA and the feedback was very positive. In addition, the ICH S7B guidelines recommend the use of cardiomyocytes:

2.2 Considerations for Selection and Design of Studies

Non-clinical methodologies can address the following: • Ionic currents measured in isolated animal or human cardiac myocytes, cultured cardiac cell lines, or heterologous expression systems for cloned human ion channels;

3.1.2 In Vitro Electrophysiology Studies

• In vitro electrophysiology studies can provide valuable information concerning the effect of a test substance on action potential duration and/or cardiac ionic currents. These assays have an important role in assessing the potential for QT interval prolongation and elucidating cellular mechanisms affecting repolarization. In vitro electrophysiology studies employ either single cell (e.g., heterologous expression systems, disaggregated cardiomyocytes) or multicellular (e.g., Purkinje fiber; papillary muscle; trabeculae; perfused myocardium; intact heart) preparations.
• Tissue and cell preparations for in vitro assays are obtained from different laboratory animal species including rabbit, ferret, guinea pig, dog, swine, and occasionally from humans.
• Species differences in terms of which cardiac ion channels contribute to cardiac repolarization and to the duration of the action potential should be considered in selecting a test system.

Q: How does the SC-hCM assay fit into a preclinical safety testing program?

A: The SC-hCM assay serves an important role in a thorough preclinical safety assessment. ChanTest offers a Thorough Preclinical Safety Package, that includes evaluation of compound effects on in vitro systems (individual ion channel block including hERG, ion channel trafficking, SC-hCM action potentials) and in vivo telemetered dog. This comprehensive cardiac safety evaluation provides a complete safety profile for IND submissions, and avoids the significant costs associated with drug failure in the clinic.

Q: Can the SC-hCM cells be used for automated patch clamp experiments?

A: We have performed preliminary experiments on Molecular Devices PatchXpress®, IonWorks Quattro™ and Sophion QPatch that indicate that these experiments are feasible but require optimization. Notably, these instruments are capable of recording ion channel currents, but not action potentials. Sodium (INa) and calcium (ICa,L) currents are readily observed, whereas IKr is relatively small (~50 – 100 pA).