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

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.

An Important Role for DNMT3A-Mediated DNA Methylation in Cardiomyocyte Metabolism and Contractility

“…Cardiomyocyte mitochondrial and glucose metabolism was analyzed with the Seahorse XF96 extracellular flux analyzer using the Glycolysis and Mito Stress Test kits (Agilent) as previously described. 35 …”

Mitochondrial DNA: Hotspot for Potential Gene Modifiers Regulating Hypertrophic Cardiomyopathy

“…Real-time qPCR reactions were performed via TaqMan ® Gene Expression assays (Applied Biosystems, Foster City, CA, USA), as previously described [ 24 ]. Briefly, 50 ng of genomic DNA was added to Taqman mastermix containing the probe of interest (NT-ND1: Hs02596873_s1, MT-ND2: Hs02596874_g1, ACTB: Hs03023880_g1).…”

“…The Seahorse XF96 extracellular flux analyzer (Agilent Technologies, Santa Clara, CA, USA) was used to evaluate cardiomyocyte metabolism, as previously described [ 24 ], with the values of oxygen consumption rate (OCR) being normalized to total cell number, quantified by Hoechst33342 (Sigma #BSS61) staining using fluorescence at 355 nm excitation and 460 nm emission in an automated imaging platform (CellaVista, Synentec, Elmshorn, Germany).…”

Transfection of hPSC-Cardiomyocytes Using Viafect™ Transfection Reagent

“…hPSC-CMs were generated following previously published methods achieving >95% purity [ 29 ], quantified by high-content imaging of α-actinin ( Supplementary Figure S1a–c ), as detailed in [ 32 ]. Cardiomyocytes were then transfected with a pmaxGFP reporter plasmid and the transfection efficiency was evaluated by measuring the percentage of cells expressing GFP using fluorescent imaging ( Figure 2 a).…”

“…2D hPSC-CM phenotyping assays rely mostly on cell clusters or monolayers [ 32 , 33 ]. Thus, different hPSC-CM densities were tested to ensure compatibility with those assays.…”