High-Throughput Phenotyping Toolkit for Characterizing Cellular Models of Hypertrophic Cardiomyopathy in Vitro

Mosqueira, Diogo; Lis-Slimak, Kataryzna; Denning, Chris

Abstract

Hypertrophic cardiomyopathy (HCM) is a prevalent and complex cardiovascular disease characterised by multifarious hallmarks, a heterogeneous set of clinical manifestations, and several molecular mechanisms. Various disease models have been developed to study this condition, but they often show contradictory results, due to technical constraints and/or model limitations. Therefore, new tools are needed to better investigate pathological features in an unbiased and technically refined approach, towards improving understanding of disease progression. Herein, we describe three simple protocols to phenotype cellular models of HCM in vitro, in a high-throughput manner where technical artefacts are minimized. These are aimed at investigating: (1) Hypertrophy, by measuring cell volume by flow cytometry; (2) HCM molecular features, through the analysis of a hypertrophic marker, multinucleation, and sarcomeric disarray by high-content imaging; and (3) mitochondrial respiration and content via the Seahorse™ platform. Collectively, these protocols comprise straightforward tools to evaluate molecular and functional parameters of HCM phenotypes in cardiomyocytes in vitro. These facilitate greater understanding of HCM and high-throughput drug screening approaches and are accessible to all researchers of cardiac disease modelling. Whilst HCM is used as an exemplar, the approaches described are applicable to other cellular models where the investigation of identical biological changes is paramount.

Citation

Mosqueira, D., Lis-Slimak, K., & Denning, C. (2019). High-Throughput Phenotyping Toolkit for Characterizing Cellular Models of Hypertrophic Cardiomyopathy in Vitro. Methods and Protocols, 2(4), 1-26. https://doi.org/10.3390/mps2040083

• Journal Article Type: Article
• Acceptance Date: Oct 24, 2019
• Online Publication Date: Oct 26, 2019
• Publication Date: Oct 26, 2019
• Deposit Date: Oct 31, 2019
• Publicly Available Date: Oct 31, 2019
• Journal: Methods and Protocols
• Electronic ISSN: 2409-9279
• Publisher: MDPI
• Peer Reviewed: Peer Reviewed
• Volume: 2
• Issue: 4
• Article Number: 83
• Pages: 1-26
• DOI: https://doi.org/10.3390/mps2040083
• Keywords: disease modelling; hypertrophic cardiomyopathy; phenotyping; high-throughput; hypertrophy; high-content imaging; mitochondrial respiration; cardiomyocytes; cellular models; drug screening
• Public URL: https://nottingham-repository.worktribe.com/output/2999234
• Publisher URL: https://www.mdpi.com/2409-9279/2/4/83
• Contract Date: Oct 31, 2019