Cardiac Kinetic Image Cytometry®

Single-cell high content screening to predict drug toxicity and efficacy

Vala’s Kinetic Image Cytometry® high content screening platform captures sensitive single-cell readouts of cardiomyocyte energetics, including voltage, calcium, contractility, and mitochondrial/ER health. Our hiPSC-cardiomyocyte drug discovery in a dish strategy predicts clinical effects with greater than 90% accuracy, revolutionizing preclinical drug discovery.

The holy grail of in vitro drug discovery is better clinical predictivity than in vivo animal studies, both to reduce/eliminate them and to improve clinical trial success rates.1,2 With Vala’s cardiac Kinetic Image Cytometry® high content screening platform, this holy grail is within reach.

Drug candidates for all indications often fail because preclinical animal studies do not accurately predict drug-induced human cardiotoxicity. Cardiac arrhythmia and cardiomyopathy side effects also dominate post-marketing drug withdrawals.3,4

Drug discovery for cardiac conditions suffers from similar failures. Heart failure drug candidates predicted to improve life expectancy based on animal studies have instead often reduced survival.5 Many antiarrhythmic drugs have narrow therapeutic windows, and off-target proarrhythmic effects limit their clinical use.6 Increasing the accuracy of preclinical in vitro drug discovery screens will drive the development of safer and more effective therapies.

Kinetic Image Cytometry® (KIC®) is the first and only in vitro preclinical screening system to exceed animal study predictivity for any indication. KIC® has been featured in four FDA Investigational New Drug (IND) applications by pharmaceutical partners for whom Vala has screened >1,200 compounds on hiPSC-cardiomyocytes.

Along with our partners, Vala uses KIC® of hiPSC-cardiomyocyte and adult cardiomyocyte models to predict drug toxicity in humans, to identify new drug targets and mechanisms of action for cardiac diseases, and to develop safer and more effective drug candidates.

The Kinetic Image Cytometry® strategy

Multiplexed readouts of cardiomyocyte energetics

KIC® is highly predictive because it produces comprehensive phenotypic readouts of cardiomyocyte energetics by measuring the kinetics of action potentials, calcium transients, and contraction along with endoplasmic reticulum and mitochondrial health.

Action potential-induced cardiomyocyte membrane depolarization that opens L-type calcium channels and allows calcium to enter the cytoplasm. This calcium influx activates ryanodine type 2 (RyR2) in the sarcoplasmic reticulum membrane, releasing large amounts of calcium to promote cross-bridge formation and contraction in the sarcomeres. ATP supplied by the mitochondria provides the energy required for sarcomere contraction and restoration of resting membrane voltage and calcium concentrations.

The Kinetic Image Cytometer® captures single-cell cardiomyocyte kinetics from up to 1,500 cells at once. CyteSeer® reports more than 30 kinetic measurements that quantify each contractile cycle. 

The cardiomyocyte contractile cycle (action potential > calcium transient > contraction > relaxation) depends on mitochondrial ATP energetics and calcium homeostasis (see diagram). Calcium also regulates apoptosis, transcription, and endoplasmic reticulum (ER) protein translation and posttranslational modification. In turn, the ER buffers and regulates mitochondrial calcium. Cardiac disease and drug-induced toxicity impact these cardiomyocyte energetic pathways.7

Sensitive single-cell  measurements

Because cardiomyocyte energetics are very sensitive to heart disease and toxicity,7 the most effective phenotypic preclinical readouts detect kinetic changes that occur at the lowest compound doses in the fewest cells.

Like all cellular characteristics, individual cardiomyocytes vary considerably in voltage, calcium, and contraction kinetics. This cardiomyocyte heterogeneity extends to drug treatments and disease-associated mutations. By recording single-cell kinetics, Kinetic Image Cytometry® captures the widest range of this variability, providing an unrivaled description of cardiomyocyte phenotypes and detecting effects that other preclinical screening strategies miss.

The Kinetic Image Cytometer® captures millisecond-resolution time series images of hundreds of cardiomyocytes loaded with fluorescent indicators of membrane voltage and/or intracellular calcium. CyteSeer®, our automated image analysis software, identifies each cardiomyocyte nucleus in the field of view, assigns each one a unique cell ID, and estimates the plasma membrane border. CyteSeer® then derives single-cell kinetic traces for voltage, calcium, and contraction from the fluorescent videos.

hiPSC-cardiomyocyte drug discovery in a dish

Vala’s strategy for accurate clinical prediction with cost-effective cardiac drug discovery:

  1. Generate hiPSCs from genetically diverse donors to model genotypic effects on drug responses
  2. Differentiate each hiPSC line into cardiomyocytes
  3. Culture hiPSC-cardiomyocytes in 96- or 384-well plates compatible with fluorescence microscopy then treat cells with drug candidates
  4. Perform automated, high content imaging of cardiomyocyte phenotypes with KIC®
  5. Analyze KIC® data of cardiomyocyte voltage action potentials, calcium transients, contraction, and/or mitochondrial and ER health
  6. Use single-cell KIC® readouts to identify safer, more effective drugs

Vala’s hiPSC-cardiomyocyte drug discovery in a dish workflow. Figure designed with Biorender.com.

Fully automated, unbiased, high-speed, high-resolution cardiomyocyte KIC® imaging in a 384-well format can evaluate the therapeutic potential and toxicity of large sets of drug candidates at multiple doses and in multiple genetic backgrounds.8,9

Single-cell, data-rich KIC® readouts provide increased statistical power to detect both population-level average drug effects and rare, life-threatening drug reactions, predicting the clinical effect of a drug before it reaches patients.10

References

  1. Ekert, J.E., et al. Recommended Guidelines for Developing, Qualifying, and Implementing Complex In Vitro Models (CIVMs) for Drug Discovery. SLAS Discov 25, 1174-1190 (2020)
  2. Ballard, P., et al. The right compound in the right assay at the right time: an integrated discovery DMPK strategy. Drug Metab Rev 44, 224-252 (2012)
  3. Valentin, J.-P. & Delaunois, A. Developing solutions to detect and avoid cardiovascular toxicity in the clinic (S24-02). in 54th EUROTOX Annual Congress ( Belgian Society of Toxicology and Ecotoxicology (Beltox), Brussels, Belgium, 2018).
  4. Onakpoya, I.J., Heneghan, C.J. & Aronson, J.K. Post-marketing withdrawal of 462 medicinal products because of adverse drug reactions: a systematic review of the world literature. BMC Med 14, 10 (2016)PMC4740994.
  5. Ahmad, T., et al. Why has positive inotropy failed in chronic heart failure? Lessons from prior inotrope trials. Eur J Heart Fail 21, 1064-1078 (2019)PMC6774302.
  6. Mankad, P. & Kalahasty, G. Antiarrhythmic Drugs: Risks and Benefits. Med Clin North Am 103, 821-834 (2019)
  7. Zhang, D., Wang, F., Li, P. & Gao, Y. Mitochondrial Ca(2+) Homeostasis: Emerging Roles and Clinical Significance in Cardiac Remodeling. Int J Mol Sci 23(2022)PMC8954803.
  8. McKeithan, W.L., et al. An Automated Platform for Assessment of Congenital and Drug-Induced Arrhythmia with hiPSC-Derived Cardiomyocytes. Front Physiol 8, 766 (2017)PMC5641590.
  9. Del Alamo, J.C., et al. High throughput physiological screening of iPSC-derived cardiomyocytes for drug development. Biochim Biophys Acta 1863, 1717-1727 (2016)PMC4885786.
  10. Hnatiuk, A.P., Briganti, F., Staudt, D.W. & Mercola, M. Human iPSC modeling of heart disease for drug development. Cell Chem Biol 28, 271-282 (2021)PMC8054828.

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