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

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

Diogo Mosqueira *, Katarzyna Lis-Slimak and Chris Denning Department of Stem Cell Biology, Centre of Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, UK; [email protected] (K.L.-S.); [email protected] (C.D.) * Correspondence: [email protected]

Received: 4 October 2019; Accepted: 24 October 2019; Published: 26 October 2019

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.

Keywords: disease modelling; hypertrophic cardiomyopathy; phenotyping; high-throughput; hypertrophy; high-content imaging; mitochondrial respiration; cardiomyocytes; cellular models; drug screening

1. Introduction Hypertrophic cardiomyopathy (HCM) is an intricate disease affecting 1:500 individuals where heart dysfunction often leads to sudden cardiac death [1]. The complexity of HCM encompasses a wide range of disease hallmarks as well as clinical manifestations and outcomes, hampering the development of efficient pharmacological treatment options [2]. Modeling HCM in vitro leads to a better understanding of disease progression towards unveiling novel molecular mechanisms and paving the way for targeted drug therapy [3]. Various models based on tissue and cellular explants, skinned myofibrils, and actin/myosin preparations have been generated, greatly contributing to uncover disease hallmarks and facilitating the development of new drugs, with some reaching clinical trials [4]. However, these disease models frequently produce contradictory results due not only to the recognized disease complexity (e.g., mutation-specific effects), but also owing to fundamental differences and limitations between models, such as low availability and demanding logistics of heart explants, over-simplicity of protein preparations, and species-differences relative to animal models [4].

Methods Protoc. 2019, 2, 83; doi:10.3390/mps2040083 www.mdpi.com/journal/mps Methods Protoc. 2019, 2, 83 2 of 26

Remarkably, cellular models enabled multi-parametric evaluation of numerous features of HCM, not only in cardiomyocytes derived from biopsies, but mostly in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), which can be generated in unlimited numbers and recreate the patient’s genome [5]. These have paved the way for high-throughput drug screening approaches aimed at identifying new compounds with therapeutic potential [6]. Moreover, genome editing technologies such as CRISPR/Cas9 resulted in a more profound comprehension of genetic causation of HCM in hPSC-CMs, as mutations in sarcomeric genes often underlie disease progression in HCM patients [7,8]. This is a growing field, and recent isogenic cellular models enabled detailed description of disease progression in vitro, envisioning the development of new therapeutics [9–14]. Thus, there is a need for establishing a simple and straightforward toolkit for phenotyping HCM in vitro, to accelerate the characterization of disease hallmarks of different cellular models and expedite high-throughput drug screening approaches. Herein, we describe in detail three protocols to phenotype 2D cellular models of HCM in vitro, focused on the analysis of hypertrophy, molecular features of disease, and bioenergetics. Altogether, these protocols consist of high-throughput and non-subjective tools to evaluate HCM disease hallmarks [9,14], indispensable for in-depth investigation of molecular mechanisms of disease underlying pharmacological intervention strategies. They are applicable to any (cardiac) cell-type but were optimized for the purpose of modelling HCM by using hPSC-CMs, available either commercially or via in-house differentiation protocols. The first protocol is aimed at evaluating hypertrophy (i.e., increase in cell size) using label-free flow cytometry of live cells. The overwhelming majority of hypertrophy analysis in vitro relies on the measurement of cell area, which is greatly affected by several parameters of 2D culture such as substrate properties [15], time in culture [16], and serum supplementation [17]. This method of flow volumetry relies on defining forward scatter (FSC) values of spherical beads of known diameters towards establishing a calibration curve. The FSC of freshly dissociated cardiomyocytes in suspension can then be analysed to determine their volume, in a high-throughput (100,000–250,000 cells/sample), unbiased, and statistically powerful flow cytometry method. This technique overcomes the pitfalls of 2D analysis and time-consuming imaging modalities of 3D stacking where the analysis of a limited number of cells requires acquisition of a high number of images [18]. Although this protocol demands immediate processing of freshly dissociated cardiomyocytes, it is inexpensive and only requires access to a standard flow cytometer. The second protocol consists of quantifying known molecular hallmarks of HCM by automated high-content imaging, enabling high-throughput evaluation of pathological markers by immunofluorescence. While several methods exist to undertake such analysis [19–21], they vary widely on sample preparation procedures [22], and often lack detailed methodological information needed to accurately acquire, and mostly quantify, fluorescent signal. Importantly, we have developed powerful algorithms to determine: (i) Multinucleation, (ii) expression of hypertrophic marker brain natriuretic peptide (BNP), and (iii) sarcomeric disarray. These require a high-content imaging system and computationally demanding processing software, which are commonplace in any modern research institute. We provide detailed step-by-step guidance to recreate our algorithms in order to accurately measure the above-mentioned hallmarks of HCM. This facilitates deep molecular characterisation of HCM phenotypes in cellular models by staining only three proteins and the cell nucleus. The final protocol is based on the functional evaluation of mitochondrial respiration in cardiomyocytes, known to be affected in HCM (termed the ‘energy depletion model’ [23]). Cardiomyocyte culture conditions (e.g., density, substrate, time in culture) required to accurately determine normalised oxygen consumption values in the Seahorse™ platform [24] were optimized. In addition, a method for measuring mitochondrial content by quantifying mitoDNA (ratiometric mitochondrial/nuclear DNA qPCR) in cardiomyocytes was adapted from other cell types [25]. These techniques require standard laboratory equipment such as a routine real-time qPCR system, a fluorescent cell imager, and a Seahorse™ analyzer (launched 13 years ago and used in over 5000 publications since Methods Protoc. 2019, 2, 83 3 of 26 then). With the extensive optimization we explore herein, they enable complementary assessment of compensatory responses in cardiomyocytes, associated with energetic imbalance in HCM. The information gained by these tools can be harnessed to narrow down the number of conditions (i.e., genetic mutants and/or drugs) meriting further investigation, based on the analysis of cardiomyocyte contractility, calcium handling, and voltage transients. The established technologies to evaluate these functional parameters (e.g., patch clamp electrophysiology, multi-electrode arrays, optical imaging, and traction force microscopy) typically rely on low- to medium-throughput assays using single cells, 3D organoid, and/or engineered heart tissues [5,26]. However, recent developments have enabled measurement of contractility across all these configurations, based on quantification of pixel displacement in high-speed movies using a publicly available software [27]. Remarkably, it is now even possible to measure action potentials, cytosolic calcium flux, and contractility simultaneously [28]. These tools have achieved 44%–78% drug predictivity scores in hPSC-CMs [29]. Moreover, despite the existence of challenges such as incomplete hPSC-CM maturity and lack of multicellular complexity, these approaches have been used to investigate the effects of genetic mutations in HCM progression, towards clarifying genotype-phenotype relationships [8]. Altogether, our three protocols are aimed at providing a fundamental characterisation of the main HCM phenotypes in cellular models (Figure 8, expected results), creating a platform for more in-depth studies into the molecular mechanisms governing disease progression, towards its treatment.

2. Experimental Design Accurate phenotyping of cellular models of HCM via this toolkit typically requires dissociation of 2D monolayers or 3D constructs into single-cell suspensions. Efficient isolation of cardiomyocytes from heart tissue biopsies has been the subject of numerous reports and typically consists of enzymatic bulk digestion [30], Langendorff method [31] or mechanical disruption procedures [32]. A recent protocol optimized this process in five main steps: 1) Myocardium dissection into 200 µm-thick slices; 2) slice perfusion with a Ca2+-free solution; 3) enzymatic digestion using collagenase II and protease XXIV; 4) filtration of cardiac tissue extract with a 100 µm mesh (to break cell clumps and minimize cell sampling biases [33]); and 5) in vitro culture in 5% foetal bovine serum-containing medium [34]. This method resulted in up to 65% viable cardiomyocyte isolation yield and enabled phenotypic studies of electrophysiology, Ca2+ imaging, and Seahorse™ analysis. Alternatively, cardiomyocytes can be efficiently sourced in high numbers through cardiac differentiation of hPSCs [35] followed by their dissociation into single cells using a collagenase II digestion protocol [36]. While these cell sources and their culture methods vary between laboratories, baseline conditions such as serum supplementation and time in culture should be kept constant between the different groups being compared (e.g., cell lines or treatments), to minimize technical artefacts. All cell culture manipulation is performed in a routine type II Biological Safety Cabinet, and cells are cultured in a humidified incubator, at 37 ◦C and 5% CO2. Once a single cell suspension is obtained, each of these 3 protocols can be performed (Figure1). While the hypertrophy analysis consists of direct assessment of freshly dissociated cardiomyocytes, the other 2 protocols require further cell culture upon replating. All protocols have built-in quality control steps (e.g., exclusion of cell debris and non-cardiomyocytes) for the cells being used, as well as normalization methods to account for differences in cell numbers. All the exemplary data reported in this manuscript (Figure 8) are based on the comparison between a hPSC-CM cell line bearing the R453C-β-myosin heavy-chain mutation (HCM line) and its isogenic wild-type control (healthy). hPSC lines were derived in-house [37] from material harvested under ethical approval from Nottingham Research Ethics Committee 2 (09/H0408/74) with informed patient consent. Methods Protoc. 2019, 2, 83 4 of 26 Methods Protoc. 2019, 2, 83 4 of 26

FigureFigure 1. Workflow 1. Workflow showing showing toolkit toolkit for phenotypingfor phenotyping hypertrophic hypertrophic cardiomyopathy cardiomyopathy (HCM) (HCM) in in cellular models.cellular Cardiomyocytes models. Cardiomyocytes can be obtained can be obtained from di fromfferent diff sourceserent sources (biopsies (biopsies or cell or cell lines) lines) and and require dissociationrequire dissociation into a single into cell a suspensionsingle cell suspension prior to phenotyping. prior to phenotyping. Evaluation Evaluation of in vitro of inhallmarks vitro of HCMhallmarks is done of via: HCM (1) is Flow done volumetry via: 1) Flow tovolumetry investigate to investigate hypertrophy; hypertrophy; (2) high-content 2) high-content imaging imaging to assess multinucleation,to assess multinucleation, brain natriuretic brain peptidenatriuretic (BNP) peptide hypertrophic (BNP) hypertrophic marker marker expression, expression, and sarcomeric and sarcomeric disarray; 3) Seahorse assay and qPCR to assess mitochondrial respiration and content, disarray; (3) Seahorse assay and qPCR to assess mitochondrial respiration and content, respectively. respectively. 2.1. Materials 2.1. Materials 2.1.1. Hypertrophic Analysis 2.1.1. Hypertrophic Analysis Polystyrene Particle Size Standard Kit, Flow Cytometry Grade (Spherotech, Lake Forest, IL, USA; • • Polystyrene Particle Size Standard Kit, Flow Cytometry Grade (Spherotech, Lake Forest, IL, Cat. no.:USA; PPS-6K). Cat. no.: PPS-6K). Phosphate• Phosphate Buffer Buffer Saline Saline (PBS, (PBS, Gibco Gibco™,™, Fisher Fisher Scientific, Scientific, Loughborough, Loughborough, UK; UK; Cat.Cat. No: 14190- 14190-094). • 094). 2.1.2. High-Content Imaging 2.1.2. High-Content Imaging CellCarrier-96 well plates (PerkinElmer, Waltham, MA, USA; cat. No.: 6005550) • • Phosphate CellCarrier-96 Buffer Saline well (PBS, plates Gibco (PerkinElmer,™, Fisher Waltham, Scientific, MA, Loughborough, USA; cat. No.: 6005550) UK; Cat. No: 14190-094). • • Phosphate Buffer Saline (PBS, Gibco™, Fisher Scientific, Loughborough, UK; Cat. No: 14190- Vitronectin (VTN-N) Recombinant Human Protein, Truncated (500 µg/mL in PBS) (Gibco™, Fisher • 094). Scientific,• Vitronectin Loughborough, (VTN-N) UK;Recombinant Cat. No: Human A14700). Protein, Truncated (500 μg/mL in PBS) (Gibco™, RPMI 1640Fisher medium Scientific, (Gibco Loughborough,™, Fisher Scientific,UK; Cat. No: Loughborough, A14700). UK; Cat. No: 21875034). • B-27•™ SupplementRPMI 1640 medium (50X), (Gibco™, custom Fisher (Gibco Scientific,™, Fisher Loughborough, Scientific, Loughborough,UK; Cat. No: 21875034). UK; Cat. No: • 0080085SA).• B-27™ Supplement (50X), custom (Gibco™, Fisher Scientific, Loughborough, UK; Cat. No: Endothelin-10080085SA). (10 µ M in cell culture grade water) (ET1, Sigma-Aldrich, St. Louis, MO, USA; Cat. • • Endothelin-1 (10 μM in cell culture grade water) (ET1, Sigma-Aldrich, St. Louis, MO, USA; No.: E7764). Store at 80 C for 1 year. Cat. No.: E7764).− Store◦ at –80 °C for 1 year. Bosentan• (100 µM in DMSO) (BOS, Sigma-Aldrich, St. Louis, MO, USA; Cat. No.: SML1265). • Bosentan (100 μM in DMSO) (BOS, Sigma-Aldrich, St. Louis, MO, USA; Cat. No.: SML1265). Store at 80 C for 1 year. Store− ◦at –80 °C for 1 year. Brefeldin• Brefeldin A (1 mg A/ mL(1 mg/mL in methanol) in methanol) (BFA, (BFA, Sigma-Aldrich, Sigma-Aldrich, St. Louis,St. Louis, MO, MO, USA; USA; Cat. Cat. No.: No.: B7651). • Store atB7651).20 C Store for 1at year. –20 °C for 1 year. − ◦ 4% Parafolmadehyde (PFA, VWR International, Leicester, UK; Cat. No.: J61899.AP). • Parafilm (VWR International, Leicester, UK; Cat. No.: HS234526A). • Triton™-X-100 (0.1% v/v in PBS) (Sigma-Aldrich, St. Louis, MO, USA; Cat. No.: T8787). Store at • room temperature for 1 year. Methods Protoc. 2019, 2, 83 5 of 26

Tween™-20 (0.1% v/v in PBS) (Fisher Scientific, Loughborough, UK; Cat. No: 10113103) • Store at room temperature for 1 year. • Goat Serum (4% v/v in PBS) (Sigma-Aldrich, St. Louis, MO, USA; Cat. No.: G9023). • Anti α-actinin antibody raised in mouse (Sigma-Aldrich, St. Louis, MO, USA; Cat. No.: A7811). • Anti-cardiac troponin-T antibody raised in rabbit (Abcam, Cambridge, UK; Cat. No.: 45932). • Anti-proBNP antibody raised in mouse (Abcam, Cambridge, UK; Cat. No.: 13115). • Alexa Flour-488 secondary antibody Goat anti-mouse (Thermo Fisher Scientific, Waltham, MA, • USA; cat. No.: A11001). Alexa Flour-647 secondary antibody Goat anti-rabbit (Thermo Fisher Scientific, Waltham, MA, • USA; cat. No.: A21244). HCS CellMask Deep Red Stain (Invitrogen, Carlsbad, CA, USA, Cat. No.: H32721). • 4 ,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA; Cat. No.: D9542). • 0 2.1.3. Mitochondrial Respiration and Content Seahorse XF Cell Mito Stress Starter Pack 96-well format (Agilent, Santa Clara, CA, USA, Cat No.: • 103708-100). VWR Signature™ 200 µL Pipet Tips, Graduated (VWR International, Leicester, UK; Cat. No.: • 37001-532). bisBenzimide H 33,342 trihydrochloride (Hoechst 33342, Sigma-Aldrich, St. Louis, MO, USA; Cat. • No.: 14533). 1.7 mL microtubes (Axygen, Corning, Amsterdam, The Netherlands; Ctat. No.: MCT-175C). • DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany; Cat. No.: 69504). • Molecular-grade water (Sigma-Aldrich, St. Louis, MO, USA; Cat. No.: W4502). • TaqMan™ Gene Expression Master Mix (Fisher Scientific, Loughborough, UK; Cat. No.: 4369016). • MicroAmp™ Fast Optical 96-Well Reaction Plate, 0.1 mL (Applied Biosystems, Foster City, CA, • USA, Cat. No.: 4346907). MicroAmp™ Optical Adhesive Film (Applied Biosystems, Foster City, CA, USA, Cat. No.: • 4311971). TaqMan™ Gene Expression Assay (FAM) - MT-ND1 probe (Applied Biosystems, Foster City, CA, • USA, Cat. No.: 4331182, Assay ID: Hs02596873_s1). TaqMan™ Gene Expression Assay (FAM) - MT-ND2 probe (Applied Biosystems, Foster City, CA, • USA, Cat. No.: 4331182, Assay ID: Hs02596874_g1). TaqMan™ Gene Expression Assay (FAM) - ACTB probe (Applied Biosystems, Foster City, CA, • USA, Cat. No.: 4331182, Assay ID: Hs03023880_g1).

2.2. Equipment

2.2.1. Hypertrophic Analysis FC500 Flow Cytometer (Beckman Coulter, Indianapolis, IN, USA), or equivalent. • 2.2.2. High-Content Imaging Operetta™ High-Content Imaging System (PerkinElmer, Waltham, MA, USA; Cat. No.: • HH12000000), or equivalent. Nikon’s Eclipse TE2000 Inverted Research Microscope (Nikon, Minato, Tokyo, Japan), • or equivalent.

2.2.3. Mitochondrial Respiration and Content Non-CO oven set at 37 C (Stuart Scientiﬁc, Staﬀordshire, UK; Cat. No.: S120H). • 2 ◦ Methods Protoc. 2019, 2, 83 6 of 26 Methods Protoc. 2019, 2, 83 6 of 26

2.2.3. Mitochondrial Respiration and Content Seahorse XFe96 Analyzer (Agilent, Santa Clara, CA, USA, Cat No.: S7800B). • • CellaVistaNon-CO2 oven™ Automated set at 37 °C plate(Stuart imager Scientific, (Synentec, Staffordshire, Elmshorn, UK; Cat. Germany), No.: S120H). or equivalent. • • Seahorse XFe96 Analyzer (Agilent, Santa Clara, CA, USA, Cat No.: S7800B). 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA, Cat. No.: 4351106), • • CellaVista™ Automated plate imager (Synentec, Elmshorn, Germany), or equivalent. • or7500 equivalent. Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA, Cat. No.: Nikon’s Eclipse TE2000 Inverted Research Microscope (Nikon, Minato, Tokyo, Japan), • 4351106), or equivalent. • orNikon’s equivalent. Eclipse TE2000 Inverted Research Microscope (Nikon, Minato, Tokyo, Japan), or NanoDropequivalent.™ Microvolume Spectrophotometer (ND-2000, Fisher Scientiﬁc, Loughborough, UK). • • NanoDrop™ Microvolume Spectrophotometer (ND-2000, Fisher Scientific, Loughborough, 2.3. SoftwareUK).

2.3.1. Hypertrophic Analysis 2.3. Software Kaluza analysis v2.1 (Beckman Coulter, Indianapolis, IN, USA), or equivalent. • 2.3.1. HypertrophicMicrosoft Excel Analysis™ (Microsoft, Redmond, WA, USA). • • GraphPadKaluza analysis Prism v2.1 v8.2.0 (Beckman (La Jolla, Coulter, CA, Indianapolis, USA). IN, USA), or equivalent. • • Microsoft Excel™ (Microsoft, Redmond, WA, USA). 2.3.2.• GraphPad High-Content Prism Imaging v8.2.0 (La Jolla, CA, USA). Harmony High-Content Imaging and Analysis Software (PerkinElmer, Waltham, MA, USA; 2.3.2.• High-Content Imaging cat. No.: HH17000001), with PhenoLOGIC™ machine-learning technology (required only for • Harmonysarcomeric High-Content disarray analysis). Imaging and Analysis Software (PerkinElmer, Waltham, MA, USA; cat. No.: HH17000001), with PhenoLOGIC™ machine-learning technology (required only for GraphPad Prism v8.2.0 (La Jolla, CA, USA). • sarcomeric disarray analysis). • GraphPad Prism v8.2.0 (La Jolla, CA, USA). 2.3.3. Mitochondrial Respiration and Content 2.3.3. MitochondrialSeahorse Wave Respiration Desktop and Software Content (Agilent, Santa Clara, CA, USA, https://www.agilent.com/en/ • • productsSeahorse/ cell-analysisWave Desktop/cell-analysis-software Software (Agilent,/data-analysis Santa/wave-desktop-2-6 Clara, CA, ). USA, 7500https://www.agilent.com/en/products/cell- Real-Time PCR Software (v2.3,analysis/cell-analys Applied Biosystems,is-software/data- Foster City, CA, USA, • https:analysis/wave-desktop-2-6).//www.thermofisher.com /uk/en/home/technical-resources/software-downloads/applied- • 7500 Real-Time PCR Software (v2.3, Applied Biosystems, Foster City, CA, USA, biosystems-7500-real-time-pcr-system.html), or equivalent. https://www.thermofisher.com/uk/en/home/technical-resources/software- Microsoft Excel™ (Microsoft, Redmond, WA, USA). • downloads/applied-biosystems-7500-real-time-pcr-system.html ), or equivalent. • GraphPadMicrosoft Excel™ Prism (Microsoft, v8.2.0 (La Jolla,Redmond, CA, USA).WA, USA). • • GraphPad Prism v8.2.0 (La Jolla, CA, USA). 3. Procedure 3. Procedure 3.1. Hypertrophy Analysis. Time for Completion: 1 Day 3.1. Hypertrophy Analysis. Time for Completion: 1 day 3.1.1. Data Acquisition. Time for Completion: 2 h 3.1.1. Data Acquisition. Time for Completion: 2 h 1. Resuspend freshly dissociated cardiomyocytes in 500 µL Phosphate Buffer Saline (PBS) at 1 × 1. 10Resuspend5–2.5 10 freshly5 cells dissociated per sample cardiomyocytes and place them in 500 on μ iceL Phosphate prior to flow Buffer cytometry Saline (PBS) analysis. at 1×105-2.5x10× 5 cells per sample and place them on ice prior to flow cytometry analysis. CRITICALCRITICAL STEP STEP PriorPrior culture culture of ofcardiomyocytes cardiomyocytes in serum-containing in serum-containing medium medium may may mask hypertrophicmask hypertrophic phenotypes phenotypes [17], [17], so exposure so exposure to serumto serum should should be be minimized minimized or or fully fully eliminated. eliminated. 2. Add 2 drops of bead solution of each size to a flow cytometer tube, mix well by flicking it, and 2. Add 2 drops of bead solution of each size to a flow cytometer tube, mix well by flicking it, measure FSC and side scatter (SSC) values (area, width, and height) at 488 nm laser wavelength and measure FSC and side scatter (SSC) values (area, width, and height) at 488 nm laser (areawavelength and height). (area and height). CRITICAL STEP Use of 100 μM flow cytometer nozzle size is highly recommended to CRITICAL STEP Use of 100 µM flow cytometer nozzle size is highly recommended to minimize sampling biases [33]. Methodsminimize Protoc. 2019 sampling, 2, 83 biases [33]. 7 of 26 3. Run cell samples in the flow cytometer, using the same operating settings as those used for the 3. Run cell samples in the flow cytometer, using the same operating settings as those used for calibration beads. the calibration beads. PAUSEPAUSE STEPSTEP OnceOnce data data from from flow flow cytometry cytometry is acquired, is acquired, it can it be can analysed be analysed at any point. at any point. 3.1.2. Data Analysis. Time for Completion: 3 h 4. In the Kaluza software (or any equivalent flow cytometry analysis software), gate to remove debris and duplets/triplets (exclude events with very low FSC and SSC Area values) (Figure 2A,B). 5. Gate to define FSC-A for each bead used for the calibration. Export FSC-A values relative to each gate (corresponding to each bead) to an excel file. Ensure there is consistency between the units being used for beads and samples (i.e., linear or log) (Figure 2C,D). 6. In the Excel software, plot the FSC-A median values (y axis, obtained from each gate in the Kaluza software) relative to the known bead diameters (x axis). Add trendline of linear regression and display equation (𝐹𝑆𝐶𝐴 = 𝑠𝑙𝑜𝑝𝑒 × 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 +𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡) and R-square value determining the calibration curve (Figure 2E). CRITICAL STEP If the R-squared value (measure of how much the variation of the FSC- A is explained by variation of the bead size) is below 0.95, then the bead solutions must be thoroughly mixed to disrupt clumps. 7. Gate samples to remove debris and duplets/triples. In cell samples, SSC-width is used in the x axis instead of FSC-A due to higher heterogeneity of the cardiomyocytes relative to the beads (Figure 2 F–H). 8. Export data from each sample from Kaluza to Excel, as done in 5. 9. Using the calibration equation determined in 6, convert the FSC-A values measured for ( ) each sample into cell diameter: 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 = . 10. Determine the cell volume by applying the formula of the volume of a sphere: 𝑉 = × 𝑑, where d is the cell diameter determined in 9. This assumes the approximation of each cell in suspension to a sphere, which is also applicable to the calibration beads. 11. Import the cell volume values into GraphPad software, as a column table (whereby each column represents a separate sample). 12. Choose “violin plot only” in the “Box and plot” options to graphically represent the data distribution and the main statistical parameters (median and quartiles). Adjust colour as desired (Figure 8A). 13. Perform statistical analysis (depending on the experimental design) to compare volume distribution between samples. OPTIONAL STEP Plotting such large sample sizes in GraphPad can be computationally demanding. Alternatively, a boxplot can be generated by inputting only 7 cardiomyocyte volume values per condition: Minimum, maximum; 25th percentile, 75th percentile, and the median three times. These values can be easily determined using Excel™ software (Figure 8B).

Methods Protoc. 2019, 2, 83 6 of 26

2.2.3. Mitochondrial Respiration and Content

• Non-CO2 oven set at 37 °C (Stuart Scientific, Staffordshire, UK; Cat. No.: S120H). • Seahorse XFe96 Analyzer (Agilent, Santa Clara, CA, USA, Cat No.: S7800B). • CellaVista™ Automated plate imager (Synentec, Elmshorn, Germany), or equivalent. • 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA, Cat. No.: 4351106), or equivalent. • Nikon’s Eclipse TE2000 Inverted Research Microscope (Nikon, Minato, Tokyo, Japan), or equivalent. • NanoDrop™ Microvolume Spectrophotometer (ND-2000, Fisher Scientific, Loughborough, UK).

2.3. Software

2.3.3. Mitochondrial Respiration and Content • Seahorse Wave Desktop Software (Agilent, Santa Clara, CA, USA, Methodshttps://www.agilent.com/en/products/cell- Protoc. 2019, 2, 83 analysis/cell-analysis-software/data- 7 of 26 analysis/wave-desktop-2-6). • 7500 Real-Time PCR Software (v2.3, Applied Biosystems, Foster City, CA, USA, 3.1.2.https://www.thermofisher.com/uk/en/ Data Analysis. Time for Completion:home/technical-resources/software- 3 h 4. Indownloads/applied-biosystems-7500-real-time-pcr-system.html the Kaluza software (or any equivalent flow cytometry ), or analysis equivalent. software), gate to remove • Microsoft Excel™ (Microsoft, Redmond, WA, USA). debris and duplets/triplets (exclude events with very low FSC and SSC Area values) (Figure2A,B). • GraphPad Prism v8.2.0 (La Jolla, CA, USA). 5. Gate to define FSC-A for each bead used for the calibration. Export FSC-A values relative to each 3. Proceduregate (corresponding to each bead) to an excel file. Ensure there is consistency between the units being used for beads and samples (i.e., linear or log) (Figure2C,D). 3.1.6. HypertrophyIn the Excel Analysis. software, Time for plotCompletion: the FSC-A 1 day median values (y axis, obtained from each gate in the Kaluza software) relative to the known bead diameters (x axis). Add trendline of linear regression 3.1.1. Data Acquisition. Time for Completion: 2 h and display equation (FSCA = slope diameter + intercept) and R-square value determining the × 1. calibrationResuspend freshly curve (Figuredissociated2E). cardiomyocytes in 500 μL Phosphate Buffer Saline (PBS) at 1×105-2.5x105 cells per sample and place them on ice prior to flow cytometry analysis. CRITICALCRITICAL STEP STEP PriorIf the culture R-squared of cardiomyocytes value (measure in serum-containing of how much the medium variation may of the FSCA is explainedmask hypertrophic by variation phenotypes of the bead [17], size)so exposure is below to 0.95, serum then should the bead be minimized solutions mustor fully be thoroughly mixedeliminated. to disrupt clumps. 2. Add 2 drops of bead solution of each size to a flow cytometer tube, mix well by flicking it, 7. Gate samples to remove debris and duplets/triples. In cell samples, SSC-width is used in the x and measure FSC and side scatter (SSC) values (area, width, and height) at 488 nm laser axiswavelength instead (area of FSC-A and height). due to higher heterogeneity of the cardiomyocytes relative to the beads (Figure CRITICAL2F–H). STEP Use of 100 μM flow cytometer nozzle size is highly recommended to 8. Exportminimize data sampling from eachbiases sample [33]. from Kaluza to Excel, as done in 5. 9. Using the calibration equation determined in 6, convert the FSC-A values measured for each (FSCA intercept) − sample into cell diameter: diameter = slope . 10. Determine the cell volume by applying the formula of the volume of a sphere: V = Π d3, 6 × where d is the cell diameter determined in 9. This assumes the approximation of each cell in suspension to a sphere, which is also applicable to the calibration beads. 11. Import the cell volume values into GraphPad software, as a column table (whereby each column represents a separate sample). 12. Choose “violin plot only” in the “Box and plot” options to graphically represent the data distribution and the main statistical parameters (median and quartiles). Adjust colour as desired (Figure 8A). 13. Perform statistical analysis (depending on the experimental design) to compare volume distribution between samples. OPTIONAL STEP Plotting such large sample sizes in GraphPad can be computationally demanding. Alternatively, a boxplot can be generated by inputting only 7 cardiomyocyte volume values per condition: Minimum, maximum; 25th percentile, 75th percentile, and the median three times. These values can be easily determined using Excel™ software (Figure 8B). Methods Protoc. 2019, 2, 83 8 of 26 Methods Protoc. 2019, 2, 83 8 of 26

FigureFigure 2. 2.ProtocolProtocol for for evaluating evaluating hypert hypertrophyrophy by by flow ﬂow cytometry. cytometry. (A (–AC–)C Forward) Forward (FSC) (FSC) and and side side (SSC) (SSC) scatterscatter values values of ofcalibration calibration beads beads of ofknown known sizes sizes are are measured, measured, and and debris debris and and clumps clumps are are excluded excluded byby gating. gating. (D (,DE), EEach) Each bead bead has has a distinctive a distinctive FSC FSC value value that that is used is used to toplot plot a calibration a calibration curve curve (FSC (FSC vs. vs. size).size). (F– (FH–)H Cardiomyocyte) Cardiomyocyte samples samples are are analysed analysed and and gate gatedd similarly similarly to tocalibrat calibrationion beads beads to tomeasure measure FSCFSC values, values, which which are are used used to todetermine determine cell cell volume. volume.

3.2.3.2. High High Content Content Imaging. Imaging. Time Time for for Completion: Completion: 7 days 7 Days 3.2.1. Replating Cardiomyocytes. Time for Completion: 2 h 3.2.1. Replating Cardiomyocytes. Time for Completion: 2 h 1. Prepare a Vitronectin solution at 5 µg/mL in PBS (1:100 dilution from stock). 1. Prepare a Vitronectin solution at 5 μg/mL in PBS (1:100 dilution from stock). µ µ 2. 2. CoatCoat a a CellCarrier-96 CellCarrier-96 well well plate plate with with 5050 μLL/well/well of of Vitronectin Vitronectin at at 55 μgg/mL./mL. Incubate Incubate forfor 11 hh at roomat room temperature temperature (RT). (RT). 3. 3. SupplementSupplement RPMI RPMI 1640 1640 medium medium by addingby adding B27 B27 supplement supplement (RB27, (RB27, 50x dilution), 50x dilution), aliquot aliquot volume neededvolume and needed let it reachand let RT. it reach RT. OPTIONALOPTIONAL STEP STEPOther Other extracellular extracellular matrix matrix proteins proteins and and culture culture media media could could be used be insteadused ofinstead Vitronectin of Vitronectin and RB27, and provided RB27,they provided were previouslythey were testedpreviously for the tested specific for cardiomyocyte the specific sourcecardiomyocyte in culture. source in culture. 4. 4. AspirateAspirate Vitronectin Vitronectin and and add add 50 50µL μ ofL of PBS PBS per per well. well. Aspirate Aspirate PBS PBS and and add add 75 µ75L μ ofL RB27of RB27 per per well. 5. Platewell. cardiomyocytes at approximately 110,000 cells/cm2 (35,000 cells per well). 5. OPTIONALPlate cardiomyocytes STEP Allow at approximately the cells to settle 110,000 for 0.5 cells/cm h at room2 (35,000 temperature cells per before well). placing them in theOPTIONAL incubator, STEP to minimize Allow the edge cells eff ectsto settle due for to the0.5 formationh at room temperature of convection before currents placing because them of temperaturein the incubator, differences to minimize between edge the effects medium dueand to the the formation incubator. of convection currents because of temperature differences between the medium and the incubator. 3.2.2. Culturing and Fixing Cardiomyocytes for Immunostaining. Time for Completion: 4 Days 3.2.26. . CulturingReplace cardiomyocyte and Fixing Cardiomyocytes medium (100 µforL/ well)Immunostaining. every 2 days Time for 4 days.for Completion: 4 days 7. 6. (a)Replace For multinucleation cardiomyocyte andmedium sarcomeric (100 μL/well) disarray every quantification, 2 days for 4 days. fix cells by incubating with 7. 50a)µ ForL/well multinucleation 4% PFA for 15 and min sarcomeric at RT, after disarray washing quantification, cardiomyocytes fix withcells PBS.by incubating Wash again with after fixation50 μL/well and add4% PFA 200 µforL PBS 15 min/well. at RT, after washing cardiomyocytes with PBS. Wash again (b)after To determinefixation and expression add 200 μ ofL hypertrophicPBS/well. marker BNP, replace medium with either 100 µL of RB27b) To (untreated determine control),expression RB27 of hypertrophic supplemented marker with 10BNP, nM replace Endothelin-1 medium (ET1, with 1,000X either dilution),100 μL orof RB27 RB27 supplemented (untreated control), with 100 RB27 nM supplemented Bosentan (BOS, with 1000X 10 dilution)nM Endothelin-1 and incubate (ET1, for 1,000X 15 h at 37dilution),◦C, 5% CO or 2RB27. Prepare supplemented a 5 µg/mL BFAwith solution100 nM Bosentan in RB27 (200X (BOS, dilution 1000X dilution) from the and stock) incubate and add 25forµ L15/well h at to 37 the °C, remaining 5% CO2. Prepare 100 µL ofa 5 RB27 μg/mL (+/ -BFA ET1 solution or BOS). in Incubate RB27 (200X cells dilution for 3 h at from 37 ◦C, the 5% COstock)2 followed and add by 25 fixation μL/well in PFAto the as remaining described 100 in 7a. μL of RB27 (+/- ET1 or BOS). Incubate cells for 3 h at 37 °C, 5% CO2 followed by fixation in PFA as described in 7a.

Methods Protoc. 2019, 2, 83 6 of 26

2.2.3. Mitochondrial Respiration and Content

2.3. Software

3. Procedure

3.1. Hypertrophy Analysis. Time for Completion: 1 day

3.1.1. Data Acquisition. Time for Completion: 2 h Methods Protoc. 2019, 2, 83 9 of 26 1. Resuspend freshly dissociated cardiomyocytes in 500 μL Phosphate Buffer Saline (PBS) at 1×105-2.5x105 cells per sample and place them on ice prior to flow cytometry analysis. CRITICAL STEP STEP PriorAs culture controls of forcardiomyocytes immunostaining, in serum-containing some wells medium should bemay included for stainingmask hypertrophic with the secondary phenotypes antibody [17], so exposure only to detect to serum unspecific should bindingbe minimized (secondary-only or fully control). Alternatively,eliminated. a cell-type control based on culturing non-cardiomyocytes (e.g., fibroblasts) in 2. Add 2 drops of bead solution of each size to a flow cytometer tube, mix well by flicking it, the same plate type can also be used and immunostained the same way as cardiomyocytes and measure FSC and side scatter (SSC) values (area, width, and height) at 488 nm laser (Figurewavelength A1). (area and height). CRITICAL STEP Use of 100 μM flow cytometer nozzle size is highly recommended to Methods Protoc.CRITICAL 2019, 2, 83 STEP BFA treatment prevents BNP secretion in cardiomyocytes, reaching7 of 26 maximal minimize sampling biases [33]. levels at 15 h post ET1 treatment. ET1 and BOS treatments are required to threshold BNP signal 3. Run cell samples in the flow cytometer, using the same operating settings as those used for intensity (Figure A2). the calibration beads. PAUSEPAUSE STEPSTEP FixedOnce data cell platesfrom flow can cytometry be stored is at acquired, 4 ◦C for up it can to 2be months analysed prior at any to immunostaining point. 3.1.2.and Data imaging. Analysis. Wrap Time platesfor Completion: in parafilm 3 h to prevent PBS evaporation and consequent plate drying.

3.2.3.4. Immunostaining In the Kaluza software Cardiomyocytes. (or any equivalent Time for flow Completion: cytometry analysis 2 Days software), gate to remove debris and duplets/triplets (exclude events with very low FSC and SSC Area Reagentvalues) volume (Figure per 2A,B). well: 50 µL. 5. Gate to define FSC-A for each bead used for the calibration. Export FSC-A values relative to 8. Washeach ﬁxed gate cells (corresponding with PBS. Permeabilize to each bead) cellsto an byexcel treating file. Ensure with there 0.1% isv/ vconsistencyTriton-Xand between incubating at RT forthe15 units min. being used for beads and samples (i.e., linear or log) (Figure 2C,D). 9. Wash6. In 3the times Excel with software, PBS. plot the FSC-A median values (y axis, obtained from each gate in the 10. AddKaluza 4% v/v software)goat serum relative in PBS to the (blocking known bead solution). diameters Incubate (x axis). for Add 1 h trendline at RT. of linear regression and display equation (𝐹𝑆𝐶𝐴 = 𝑠𝑙𝑜𝑝𝑒 × 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 +𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡) and R-square 11. Replace blocking solution by primary antibodies pertaining each staining: value determining the calibration curve (Figure 2E). CRITICAL STEP If the R-squared value (measure of how much the variation of the FSC- a. Multinucleation and sarcomeric disarray: Anti α-actinin antibody (1:800 in blocking A is explained by variation of the bead size) is below 0.95, then the bead solutions must be thoroughlysolution). mixed to disrupt clumps. b.7. GateHypertrophic samples to remove marker: debris Anti and cardiac duplets/triples. troponin-T In and cell antisamples, proBNP4 SSC-width (both is at used 1:500 in in blocking thesolution). x axis instead of FSC-A due to higher heterogeneity of the cardiomyocytes relative to the CRITICALbeads (Figure STEP 2 F–H).The secondary-only controls should be incubated with blocking solution instead.8. Export Cell-type data from controls each sample should from be Kaluza treated to the Excel, same as done way in as 5. cardiomyocytes. 9. Using the calibration equation determined in 6, convert the FSC-A values measured for 12. Incubate for 15 h at 4 ◦C. ( ) each sample into cell diameter: 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 = . 13. Wash 3 times in 0.1% v/v Tween-20. 10. Determine the cell volume by applying the formula of the volume of a sphere: 𝑉 = × 14. Replace washing solution with secondary antibodies pertaining each staining. Incubate for 1 h 𝑑, where d is the cell diameter determined in 9. This assumes the approximation of each at RT: cell in suspension to a sphere, which is also applicable to the calibration beads. 11. Import the cell volume values into GraphPad software, as a column table (whereby each a. Multinucleation and sarcomeric disarray: Alexa Fluor-488 goat anti-mouse (1:400 in column represents a separate sample). 12. Chooseblocking “violin solution). plot only” in the “Box and plot” options to graphically represent the data b. distributionHypertrophic and the marker: main statistical Alexa Fluor-488 parameters goat (median anti-mouse and quartiles). and Alexa Adjust Fluor-647 colour as anti-rabbit desired(both (Figure at 1:400 8A). in blocking solution). 13. Perform statistical analysis (depending on the experimental design) to compare volume 15. Washdistribution 3 times with between 0.1% samples.v/v Tween-20 and incubate for 5 min at RT. 16. AddOPTIONAL nuclei/cytoplasm STEP Plotting counterstains. such large Incubate sample forsizes 30 in min GraphPad at RT: can be computationally demanding. Alternatively, a boxplot can be generated by inputting only 7 cardiomyocyte a. volumeMultinucleation values per condition: and sarcomeric Minimum, disarray: maximum; HCS 25th Cell percentile, Mask Deep 75th Redpercentile, (1:10,000 and in the PBS) and medianDAPI three (1:500 times. in PBS). These values can be easily determined using Excel™ software (Figure b. 8B).Hypertrophic marker: DAPI (1:500 in PBS).

17. Wash 2x with PBS. Add 200 µL PBS per well prior to analysis or storage.

PAUSE STEP Immunostained cell plates can be stored at 4 ◦C for up to 2 weeks prior to imaging. Wrap plates in aluminium to prevent PBS evaporation and consequent plate drying, and to protect from light.

MethodsMethods Protoc. Protoc. 20192019, 2, 83, 2, 83 6 of 26 10 of 26

2.2.3. Mitochondrial Respiration and Content 3.2.4. Imaging Immunostained Cardiomyocytes. Time for Completion: 2 h per Plate • Non-CO2 oven set at 37 °C (Stuart Scientific, Staffordshire, UK; Cat. No.: S120H). • 18. InitiateSeahorse Operetta XFe96 Analyzer™, light (Agilent, source, Santa and Harmony Clara, CA, softwareUSA, Cat forNo.: image S7800B). acquisition. • 19. SelectCellaVista™ “96 PerkinElmer Automated plate CellCarrier” imager (Synentec, as plate Elmshorn, type, “20x Germany), long WD” or asequivalent. Objective, “Non-confocal” • 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA, Cat. No.: as Optical Mode, and 100% excitation intensity. 4351106), or equivalent. • OPTIONALNikon’s Eclipse STEP TE2000These Inverted settings Research maximize Microscope signal and (Nikon, image Minato, quality Tokyo, without Japan), producing or excessive amountsequivalent. of images/data but could be adapted to different machine specifications. Other high • contentNanoDrop™ screening Microvolume systems Spectrophotometer from PerkinElmer (ND-2000, (e.g., Fisher Opera Scientific, Phenix) Loughborough, are compatible with the HarmonyUK). software used herein. 20. Add channels for image capture, using predefined settings:

2.3. Software a. DAPI (excitation 360–400 nm; emission 410–480 nm); 2.3.1. Hypertrophicb. Alexa Analysis 488 (excitation 460–490 nm; emission 500–550 nm); c. Alexa 647 and HCS Cell Mask Deep Red (excitation 620–640 nm; emission 650–760 nm). • Kaluza analysis v2.1 (Beckman Coulter, Indianapolis, IN, USA), or equivalent. 21.• PerformMicrosoft a Excel™ z-stack (Microsoft, image acquisition Redmond, in WA, a field USA). of a well in order to determine the right focus plane • andGraphPad time ofPrism exposure v8.2.0 for(La eachJolla, channelCA, USA). (i.e., to input the right “Time” and “Height” variables for each channel). Confirm these settings are correct by taking snapshots in other wells of the plate. 2.3.2. High-Content Imaging 22. Define plate layout (typically 10–20 fields distributed in several regions of the well). • Harmony High-Content Imaging and Analysis Software (PerkinElmer, Waltham, MA, USA; 23. Initiate automated image capture. cat. No.: HH17000001), with PhenoLOGIC™ machine-learning technology (required only for sarcomericPAUSE STEP disarrayOnce analysis). images are acquired, they can be analysed at any point. Store plates at 4 ◦C • GraphPadwrapped Prism in aluminium v8.2.0 (La Jolla, foil, inCA, case USA). additional imaging is needed.

2.3.3.3.2.5. Mitochondrial Image Analysis. Respiration Time and for Content Completion: 1–2 h per Plate, per Analysis • 3.2.5.1. Seahorse Multinucleation Wave AnalysisDesktop Sequence,Software Time (Agilent, for Completion: Santa 1Clara, h per PlateCA, (Figure USA, 3) https://www.agilent.com/en/products/cell-analysis/cell-analysis-software/data- 1. Inputanalysis/wave-desktop-2-6). image and select “Advanced” in option Flatfield correction (Figure3A–D). 2.• Insert7500 Real-Time “Find Nuclei” PCR buildingSoftware block. (v2.3, PickApplied Channel Biosystems, “DAPI” andFoster select City, Method CA, USA, “C”. In the inset menu,https://www.thermofisher.com/uk/en/ define Common threshold athome/technical-resources/software- 0.40; Area > 30 µm2; Split factor: 27.5; Individual Threshold: 0.40;downloads/applied-biosystems-7500-real-time-pcr-system.html Contrast > 0.10. Output Population: “Nuclei” (Figure ),3 E).or equivalent. • Microsoft Excel™ (Microsoft, Redmond, WA, USA). 3. Insert “Calculate Morphology Properties” building block. Select Population “Nuclei”; Region: • GraphPad Prism v8.2.0 (La Jolla, CA, USA). “Nucleus”; Method “Standard”, tick “Area” and Roundness” in the inset menu. Define Output 3. ProcedureProperties as “Nuclei Properties” (Figure3F). 4. Insert “Select Population” building block. Select Population “Nuclei”; Method “Filter by Property” 3.1. Hypertrophyand enter Analysis. as conditions Time for Completion: “Nuclei properties 1 day Area [µm2] > 80” and “Nuclei properties Roundness” > 0.6. Define Output Population as “Nuclei Selected” (Figure3G). 3.1.1. Data Acquisition. Time for Completion: 2 h 5. Insert “Select Population” building block. Select Population “Nuclei Selected”; Method “Common 1. Filters”Resuspend and freshly tick “Remove dissociated Border cardiomyocytes Objects” box.in 500 Output μL Phosphate Population: Buffer Saline “Nuclei (PBS) Final” at (Figure3H). 1×105-2.5x105 cells per sample and place them on ice prior to flow cytometry analysis. CRITICALCRITICAL STEP STEP PriorThese culture steps of ensure cardiomyocytes exclusion ofin debris,serum-containing cell clumps, medium and cells may in the border ofmask the hypertrophic field from the phenotypes subsequent [17], analysis. so exposure The to exact serum values should were be optimizedminimized foror fully hPSC-CMs and mayeliminated. need to be adapted to different cardiomyocyte source being investigated. 2. Add 2 drops of bead solution of each size to a flow cytometer tube, mix well by flicking it, 6. Insert “Calculate Morphology Properties” building block. Select Population “Nuclei Final”; and measure FSC and side scatter (SSC) values (area, width, and height) at 488 nm laser Region:wavelength “Nucleus”; (area and Method:height). “Standard”. Tick “Area” and “Roundness” boxes in the inset menu. Output CRITICAL properties: STEP “NucleusUse of 100 Morphology”.μM flow cytometer This nozzle block size does is highly the same recommended operation asto in 3 but for theminimize population sampling “Nuclei biases Final”.[33]. 7. Insert “Modify Population” building block. Select Population “Nuclei Final”; Region “Nucleus”; 2 Method “Cluster by Distance”. Enter in the inset menu Distance “5 µm”; Area > “80 µm ”. Output population: “Total population” (Figure3I). Methods Protoc. 2019, 2, 83 6 of 26

2.2.3. Mitochondrial Respiration and Content

2.3. Software

3. Procedure

3.1. Hypertrophy Analysis. Time for Completion: 1 day

3.1.1. Data Acquisition. Time for Completion: 2 h Methods Protoc. 2019, 2, 83 11 of 26 1. Resuspend freshly dissociated cardiomyocytes in 500 μL Phosphate Buffer Saline (PBS) at 1×105-2.5x105 cells per sample and place them on ice prior to flow cytometry analysis. CRITICAL STEP STEP PriorThese culture steps of enable cardiomyocytes the identification in serum-containing of several medium nuclei belonging may to the samemask cell,hypertrophic other than phenotypes being part [17], of theso exposure different to cells, serum by inferringshould be onminimized their proximity. or fully The values pertainingeliminated. “Distance” and “Area” may need to be changed when using cardiomyocyte sources 2. Add 2 drops of bead solution of each size to a flow cytometer tube, mix well by flicking it, other than hPSC-CMs (i.e., displaying different morphology properties). Methodsand Protoc. measure 2019, 2FSC, 83 and side scatter (SSC) values (area, width, and height) at 488 nm laser 11 of 26 8. Insertwavelength “Calculate (area and Properties” height). building block. Population “Total population”; Method “By Related 9.Population”. InsertCRITICAL “Select In STEP the Population” insetUse of menu: 100 buildingμM Related flow cytometerblock. Population Popula nozzle “Nucleition size “Total is final”; highly population”; tick recommended box: “Number Method to “Filter of Nuclei by Final”.minimizeProperty”. Output sampling Inset Properties: biases menu: [33]. “per “Number Cell”. Thisof Nuclei step will Final eff ectively– per Cell calculate = 1”. the Output number Population of nuclei belonging“Mononucleated” to each cell. (Figure 3J). 9. 10.Insert Insert “Select “Select Population” Population” building building block. block. Popula Populationtion “Total “Total population”; population”; Method Method “Filter “Filter by byProperty”. Inset Inset menu: menu: “Number “Number of of Nuclei Nuclei Final – perper CellCell == 1”.2”. OutputOutput PopulationPopulation “Mononucleated”“Binucleated” (Figure (Figure 33K).J). 10. Insert “Select Population” building block.block. Popu Populationlation “Total “Total population”; Method “Filter byby Property”.Property”. Inset Insetmenu: menu: “Number “Number of Nuclei of Nuclei Final Final – per – Cell per Cell> 2”.= Output2”. Output Population Population “Multinucleated” “Binucleated” (Figure(Figure 3L). 3K).

Figure 3. Multinucleation Analysis Sequence by high-content imaging. Representative micrographs of Figure 3. Multinucleation Analysis Sequence by high-content imaging. Representative micrographs image analysis of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). After inputting of image analysis of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). After the image (A–D), the software excludes debris and cell clumps (E–H) and identifies different degrees of inputting the image (A–D), the software excludes debris and cell clumps (E–H) and identifies different multinucleation based on the distance between nuclei in cardiomyocytes (I–L). Scale bar = 100 µm. degrees of multinucleation based on the distance between nuclei in cardiomyocytes (I–L). Scale bar = Insert100 μm. “Select Population” building block. Population “Total population”; Method “Filter by Property”. Inset menu: “Number of Nuclei Final – per Cell > 2”. Output Population “Multinucleated” 11. In the default “Define Results” building block select Method ”List of Outputs” in the drop (Figure3L). down menu, then tick the box “Number of Objects” for the options: “Population: Total 11. In thepopulation”, default “Define “Population: Results” buildingMononucleated”, block select Population: Method ”List Binucleated”, of Outputs” and in the “Population: drop down menu,Multinucleated”. then tick the box “Number of Objects” for the options: “Population: Total population”, 12.“Population: In the same Mononucleated”, drop-down menu Population: within “Define Binucleated”, Results”, and define “Population: three sections Multinucleated”. of “Method— 12. In theFormula same drop-down Output”. In menu each within of these “Define sections Results”, in the define inset three menu, sections enter of “Formula: “Method—Formula (a/b)×100; Output”.Variable A: In “Mononucleated—Number each of these sections in of theObje insetcts”, “Binucleated—Number menu, enter “Formula: of Objects”, (a/b) 100; or “Multinucleated—Number of Objects”. Enter as Variable B “Total population—Number× of Objects”. The Output Names for each section are “%mononucleated”, “%binucleated”, and “%multinucleated”, respectively. 13. Copy the percentages pertaining to each proportion of mono-, bi-, or multinucleated cells to a GraphPad “Grouped” Table, by inserting “% mononucleated”, “% binucleated”, and “% multinucleated” in columns and different cell lines/conditions in rows. 14. Create a “Stacked bars” Graph Type. Colour each bar and perform statistical analysis according to the experimental design (Figure 8C).

Methods Protoc. 2019, 2, 83 12 of 26

Variable A: “Mononucleated—Number of Objects”, “Binucleated—Number of Objects”, or “Multinucleated—Number of Objects”. Enter as Variable B “Total population—Number of Objects”. The Output Names for each section are “%mononucleated”, “%binucleated”, and “%multinucleated”, respectively. 13. Copy the percentages pertaining to each proportion of mono-, bi-, or multinucleated cells to a GraphPad “Grouped” Table, by inserting “% mononucleated”, “% binucleated”, and “% multinucleated” in columns and different cell lines/conditions in rows. 14. Create a “Stacked bars” Graph Type. Colour each bar and perform statistical analysis according to the experimental design (Figure 8C). OPTIONAL STEP As a quality control measure, the cardiomyocyte purity in the cell population being analysed can be determined in a separate analysis script, as follows (Figure4): 15. After repeating building blocks in steps 1–5 of Section 3.2.5.1, insert “Find Cytoplasm” building block. Channel “DRAQ5” (same excitation/emission parameters as HCS Cell Mask DeepRed); Nuclei “Nuclei Final”; Method “F”. In the inset menu, define Membrane Channel “DRAQ5”; Individual Threshold: 0.04. This step delineates the cytoplasm of cardiomyocytes by using the Cell Mask counterstain (Figure4A–C). 16. Insert “Calculate Intensity Properties” building block. Channel “Alexa 488”; Population: “Nuclei Final”; Region “Cytoplasm”; Method “Standard”, tick box “Mean” in the inset menu. Output Properties “Intensity alpha-actinin”. 17. Insert “Select Population” building block. Population “Nuclei Final”; Method “Filter by Property”. In the inset menu, insert “Intensity alpha-actinin Mean” and set it > the value of the secondary only or cell-type control (Figures4D and A1). Output population “Cardiomyocytes”. Confirm if the highlighted cells display positive α-actinin signal (Figure4E). 18. Insert “Select Population” building block. Population “Nuclei Final”; Method “Filter by Property”. In the inset menu, insert “Intensity alpha-actinin Mean” and set it the value of the secondary ≤ only or cell-type control. Output population “Non-Cardiomyocytes”. Confirm if the highlighted cells display negative alpha-actinin signal (Figure4F). 19. In the default “Define Results” building block, select Method ”List of Outputs”, then tick the box “Number of Objects” for the options “Population: Nuclei Final” and “Population: Cardiomyocytes”. 20. In the same drop-down menu within “Define Results”, define “Method—Formula Output”, and in the inset menu, enter “Formula: (a/b)*100; Variable A: “Cardiomyocytes—Number of Objects”; Variable B “Nuclei Final—Number of Objects”. Output Name “% Cardiomyocyte Purity”. 21. Import purity values to GraphPad (e.g., “Column” table, “Scatter plot” graph) and perform statistical analysis according to the experimental design (Figure 8D). Cardiomyocyte purities should typically be >90% using directed differentiation protocols from hPSCs [35]. Methods Protoc. 2019, 2, 83 13 of 26 Methods Protoc. 2019, 2, 83 13 of 26

Figure 4. Cardiomyocyte purity analysis sequence. Representative micrographs of image analysis Figure 4. Cardiomyocyte purity analysis sequence. Representative micrographs of image analysis of of hPSC-CMs. After inputting the image (A), the software excludes debris and cell clumps (B) and hPSC-CMs. After inputting the image (A), the software excludes debris and cell clumps (B) and uses uses the cell mask staining to define cell cytoplasm (C). The α-actinin signal intensity is quantified in the cell mask staining to define cell cytoplasm (C). The α-actinin signal intensity is quantified in the the cytoplasm and a positive signal threshold is determined by comparing with cell-type control (e.g., cytoplasm and a positive signal threshold is determined by comparing with cell-type control (e.g., fibroblasts) (D), enabling the distinction of cardiomyocytes (E) from non-cardiomyocytes (F), required fibroblasts) (D), enabling the distinction of cardiomyocytes (E) from non-cardiomyocytes (F), required to calculate purity. Scale bar = 100 µm. to calculate purity. Scale bar = 100 μm. 3.2.5.2. Hypertrophic Marker BNP Analysis Sequence, Time for Completion: 2 h per Plate (Figure 5) 3.2.5.2. Hypertrophic Marker BNP Analysis Sequence, Time for Completion: 2 h per plate (Figure 5). 1. Input image and select “Advanced” in option Flatfield correction (Figure5A–C).

2. 1.RepeatInput steps image 2–5 and from select Section “Advanced” 3.2.5.1 to in ensure option exclusion Flatfield of correction debris, cell (Figure clumps, 5A–C). and cells in the

2.borderRepeat of the steps field. 2–5 Output from Section Population: 3.2.5.1 “Nuclei to ensure Final” exclusion (Figure of5 D,E)debris, cell clumps, and cells in the border of the field. Output Population: “Nuclei Final” (Figure 5D–E) 3. Insert “Find Cytoplasm” building block. Pick Channel “DRAQ5” (same excitation/emission 3. Insert “Find Cytoplasm” building block. Pick Channel “DRAQ5” (same excitation/emission parameters as Alexa Fluor 647) and select Method “F”. In the inset menu, define Membrane parameters as Alexa Fluor 647) and select Method “F”. In the inset menu, define Membrane Channel “DRAQ5”; Individual Threshold: 0.04 (Figure5F). The sarcomeric signal is ubiquitous Channel “DRAQ5”; Individual Threshold: 0.04 (Figure 5F). The sarcomeric signal is in cardiomyocytes so can be used to define cytoplasm, similar to the Cell Mask. ubiquitous in cardiomyocytes so can be used to define cytoplasm, similar to the Cell Mask. 4. Insert “Calculate Intensity Properties” building block. Select Population “Nuclei Final”; Region: 4. Insert “Calculate Intensity Properties” building block. Select Population “Nuclei Final”; “Cytoplasm”; Method “Standard”, tick “Mean” and “Standard Deviation” in the inset menu. Region: “Cytoplasm”; Method “Standard”, tick “Mean” and “Standard Deviation” in the Define Output Properties as “Intensity cardiac Troponin T”. inset menu. Define Output Properties as “Intensity cardiac Troponin T”. 5. Insert “Select Population” building block. Population “Nuclei Final”; Method “Filter by Property”. 5. Insert “Select Population” building block. Population “Nuclei Final”; Method “Filter by In the inset menu, insert “Intensity cardiac Troponin T” and set it > the value of the secondary Property”. In the inset menu, insert “Intensity cardiac Troponin T” and set it > the value of only or cell-type control (e.g., fibroblasts). Output population “Cardiomyocytes”. Confirm if the the secondary only or cell-type control (e.g., fibroblasts). Output population highlighted cells display positive cardiac Troponin T signal. This is important so the subsequent “Cardiomyocytes”. Confirm if the highlighted cells display positive cardiac Troponin T analysis is performed only in cardiomyocytes. It can also be used for calculating purity (Figures A1 signal. This is important so the subsequent analysis is performed only in cardiomyocytes. It and5G). can also be used for calculating purity (Figures A1 and 5G). 6. 6.Insert Insert “Select “Select Region” Region” building building block. block. Select Select Population Population “Nuclei“Nuclei Final”;Final”; Region “Nucleus”; “Nucleus”; MethodMethod “Resize “Resize Region Region [%]”. [%]”. In the In insetthe inset menu, menu enter, enter “-75%” “-75%” in the in section the section Outer Outer Border. Border. Leave “InnerLeave Border” “Inner section Border” empty. section Output empty. Population: Output Po “Cytoplasmicpulation: “Cytoplasmic ring”. This ring”. step This defines step a perinucleardefines a ringperinuclear where BNP ring expressionwhere BNP is expression maximal (Figure is maximal5H). (Figure 5H).

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2.3. Software

2.3.1. Hypertrophic Analysis • Kaluza analysis v2.1 (Beckman Coulter, Indianapolis, IN, USA), or equivalent. • FigureMicrosoft 5. Hypertrophic Excel™ (Microsoft, marker Redmond, BNP analysis WA, sequence. USA). Representative micrographs of image analysis Figure 5. Hypertrophic marker BNP analysis sequence. Representative micrographs of image analysis • GraphPad Prism v8.2.0 (La Jolla, CA, USA). of hPSC-CMs.of hPSC-CMs. After After inputting inputting the the image image (A (–AC–),C), the the software software excludes excludes debris debris and and cell cell clumps clumps ((DD,,EE)) and uses the cardiac troponin T (cTnT) staining to define cell cytoplasm (F). The cTnT signal intensity is 2.3.2. High-Contentand uses the Imaging cardiac troponin T (cTnT) staining to define cell cytoplasm (F). The cTnT signal intensity quantifiedis quantified anda and positive a positive signal sign thresholdal threshold is determined is determined by comparing by comparing with cell-typewith cell-type control control enabling • Harmonyexclusionenabling High-Content of exclusion non-cardiomyocytes of Imagingnon-cardiomyocytes fromand theAnalysis analysis from Software the (G ).analysis A perinuclear(PerkinElmer, (G). A ringperinuclear Waltham, is defined ring MA, in is cardiomyocytes definedUSA; in cat.(H )cardiomyocytesNo.: and HH17000001), BNP intensity (H) iswithand quantified BNPPhenoLOGIC™ intensity in this is region. machine-learningquantified Comparison in this region.technology of BNP Comparison signal (required intensity of onlyBNP to thatfor signal of ET1- sarcomericor BOS-treatedintensity disarray to that cardiomyocytes of analysis). ET1- or BOS-treated enables cardiomy distinctionocytes between enables high distinction vs. medium between BNP-expressing high vs. medium cells • GraphPad(I–LBNP-expressing). BNP—brain Prism v8.2.0 cells natriuretic (La(I–L Jolla,). BNP—brain peptide; CA, USA). cTnT—cardiac natriuretic peptide; troponin cTnT T;—cardiac scale bar troponin= 100 µ T;m. scale bar = 100 μm. 2.3.3.7. MitochondrialInsert “Calculate Respiration Intensity and Content Properties” building block. Select Channel “Alexa 488”; Population • “Cardiomyocytes”;Seahorse7. Insert Wave“Calculate Region:Desktop Intensity “Cytoplasmic Software Properties” Ring”;(Agilent, buil Methodding Santablock. “Standard”, SelectClara, tickChannelCA, “Mean” USA,“Alexa and “Standard488”; Deviation”https://www.agilent.com/en/products/cell-Population in the “Cardiomyocytes”; inset menu. Define Region: Outputanalysis/cell-analys Properties“Cytoplasmic asis-software/data- “IntensityRing”; Method BNP perinuclear”.“Standard”, tick “Mean” and “Standard Deviation” in the inset menu. Define Output Properties as “Intensity 8. Insertanalysis/wave-desktop-2-6). “Select Population” building block. Population “Cardiomyocytes”; Method “Filter by • 7500 BNPReal-Time perinuclear”. PCR Software (v2.3, Applied Biosystems, Foster City, CA, USA, Property”. In the inset menu, insert “Intensity BNP perinuclear” and set it > the value of the https://www.thermofisher.com/uk/en/8. Insert “Select Population” buildinghome/technical-resources/software- block. Population “Cardiomyocytes”; Method “Filter by bosentan-treated cardiomyocytes (Figure A2). Output population “BNP positive cardiomyocytes”. downloads/applied-biosystems-7500-real-time-pcr-system.htmlProperty”. In the inset menu, insert “Intensity BNP perinuclear” ), or equivalent. and set it > the value of the • ConfirmMicrosoftbosentan-treated ifExcel™ the highlighted (Microsoft, cardiomyocytes cellsRedmond, display WA, positive(Figure USA). BNPA2). signal. Output This population signal should “BNP always positive be higher • thanGraphPad thatcardiomyocytes”. ofPrism secondary-only v8.2.0 (La Confirm Jolla, or CA, cell-typeif the USA). highlighted controls (Figurecells display5I). positive BNP signal. This signal 9. Insertshould “Select always Population” be higher building than that block. of secondary-only Population or “Cardiomyocytes”; cell-type controls (Figure Method 5I). “Filter by 3. ProcedureProperty”.9. Insert “Select In the Population” inset menu, building insert “Intensityblock. Popu BNPlation perinuclear”“Cardiomyocytes”; and setMethod it “Filterthe value by of ≤ the bosentan-treatedProperty”. In the cardiomyocytesinset menu, insert (same “Intensity value BNP as inperinuclear” 8). Output and population set it ≤ the “BNPvalue of negative the 3.1. Hypertrophy Analysis. Time for Completion: 1 day cardiomyocytes”.bosentan-treated Confirm cardiomyocytes if the highlighted (same value cells doas in not 8). display Output positive population BNP “BNP signal. negative cardiomyocytes”. Confirm if the highlighted cells do not display positive BNP signal. 3.1.1. DataOPTIONAL Acquisition. STEP Time Furtherfor Completion: division 2 h of positive BNP signal into medium vs. high intensity can be accuratelyOPTIONAL done STEP provided Further ET1 division and BOS of posi treatmenttive BNP of signal cardiomyocytes into medium hasvs. high been intensity performed, 1. Resuspendcan befreshly accurately dissociated done cardiomyocytes provided ET1 in and 500 BOSμL Phosphate treatment Buffer of cardiomyocytes Saline (PBS) at has been as follows. 1×105-2.5x10performed,5 cells peras follows. sample and place them on ice prior to flow cytometry analysis. CRITICALCRITICAL CRITICAL STEP STEP PriorSTEPPrior culturePrior culture cultureof ofcardiomyocytes cardiomyocytes of cardiomyocytes in serum-containing in serum-containing in serum-containing medium medium medium may may may mask hypertrophicmask hypertrophicmask hypertrophic phenotypes phenotypes phenotypes [17], [17], so exposure so [17], exposure so toexposu serumto serumre should to shouldserum be beshould minimized minimized be minimized or or fully fully eliminated. or fully eliminated. 10. Inserteliminated. “Select Population” building block. Population “Cardiomyocytes”; Method “Filter by 2. Add 2 drops of bead solution of each size to a flow cytometer tube, mix well by flicking it, Property”. In the inset menu, insert “Intensity BNP perinuclear” and set it > the value of the and measure FSC and side scatter (SSC) values (area, width, and height) at 488 nm laser wavelength (area and height). CRITICAL STEP Use of 100 μM flow cytometer nozzle size is highly recommended to minimize sampling biases [33].

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2.3. Software

2.3.3. Mitochondrial Respiration and Content • Seahorse Wave Desktop Software (Agilent, Santa Clara, CA, USA, https://www.agilent.com/en/products/cell-analysis/cell-analysis-software/data- analysis/wave-desktop-2-6). • 7500 Real-Time PCR Software (v2.3, Applied Biosystems, Foster City, CA, USA, https://www.thermofisher.com/uk/en/home/technical-resources/software- Methodsdownloads/applied-biosystems-7500-real-time-pcr-system.html Protoc. 2019, 2, 83 ), or equivalent. 15 of 26 • Microsoft Excel™ (Microsoft, Redmond, WA, USA). • GraphPad Prism v8.2.0 (La Jolla, CA, USA). ET1-treated cardiomyocytes. Output population “BNP high cardiomyocytes”. Confirm if the 3. Procedurehighlighted cells display positive and high BNP signal (Figure5J). 11. Insert “Select Population” building block. Population “Cardiomyocytes”; Method “Filter 3.1. Hypertrophyby Property”. Analysis. InTime the for inset Completion: menu, 1 day insert “Intensity BNP perinuclear” and set it the value ≤ of the endothelin-treated cardiomyocytes. Add an additional row and set “Intensity BNP 3.1.1. Data Acquisition. Time for Completion: 2 h perinuclear” to the value of bosentan-treated cardiomyocytes. Output population “BNP medium ≥ 1. cardiomyocytes”.Resuspend freshly dissociated Confirm if cardiomyocytes the highlighted in cells500 μ displayL Phosphate positive Buffer BNP Saline signal (PBS) (Figure at 5K). 1×105-2.5x105 cells per sample and place them on ice prior to flow cytometry analysis. CRITICALCRITICAL STEP STEP PriorDefining culture the of threshold cardiomyocytes for positive in serum-containing BNP signal should medium be done may carefully and accordingmask hypertrophic to the controls phenotypes used. [17], The so control exposure where to serum cardiomyocytes should be minimized were treated or withfully ET1 should typicallyeliminated. display >90% BNP-positive cardiomyocytes, so the intensity of BNP signal should be 2. Add 2 drops of bead solution of each size to a flow cytometer tube, mix well by flicking it, higher in those wells (this should be selected as the BNP-medium/high signal threshold). In and measure FSC and side scatter (SSC) values (area, width, and height) at 488 nm laser contrast,wavelength>90% (area BOS-treated and height). cardiomyocytes should display negative BNP signal, so the signal threshold CRITICAL should STEP distinguish Use of 100 between μM flow negative cytometer and nozzle medium size /ispositive highly BNPrecommended signal (Figure to A2). The thresholdminimize sampling for medium biases/high [33]. signal intensity is typically 4 times higher than that of negative/medium signal intensity (Figure5L).

12. In the default “Define Results” building block, select Method ”List of Outputs” in the drop down menu, then tick the box “Number of Objects” for the options: “Population: Cardiomyocytes”, “Population: Nuclei Final”, “Population: BNP positive Cardiomyocytes”, “Population: BNP negative Cardiomyocytes”, “Population: BNP medium Cardiomyocytes”, and “Population: BNP high Cardiomyocytes”. 13. In the same drop-down menu within “Define Results”, define five sections of “Method—Formula Output”. In each of these sections in the inset menu, enter “Formula: (a/b)*100. 14. In the first Menu define Variable A: “Cardiomyocytes—Number of Objects”, and Variable B: “Nuclei Final—Number of Objects”. Output Name: “% Cardiomyocyte Purity”. 15. In the second menu: VariableA: “BNP positive cardiomyocytes—Number of Objects”, and Variable B “Cardiomyocytes—Number of Objects”. Output Name: “%BNP positive cardiomyocytes”. 16. In the third menu: Variable A: “BNP high cardiomyocytes—Number of Objects”, and Variable B “Cardiomyocytes—Number of Objects”. Output Name: “%BNP high cardiomyocytes”. 17. In the fourth menu: Variable A: “BNP medium cardiomyocytes—Number of Objects”, and Variable B “Cardiomyocytes—Number of Objects”. Output Name: “%BNP medium cardiomyocytes”. 18. In the fifth menu: Variable A: “BNP negative cardiomyocytes—Number of Objects”, and Variable B “Cardiomyocytes—Number of Objects”. Output Name: “%BNP negative cardiomyocytes”. 19. Check that the percentages pertaining to each Output add up. If not, confirm that the intensity thresholds between building blocks are consistent and that the comparison operators are complementary.

%BNP positive cardiomyocytes + %BNP negative cardiomyocytes = 100% %BNP positive cardiomyocytes = %BNP high cardiomyocytes + %BNP medium cardiomyocytes % BNP high cardiomyocytes + %BNP medium cardiomyocytes + %BNP negative cardiomyocytes = 100%

20. Import purity and %BNP positive values to GraphPad and perform statistical analysis according to the experimental design. 21. Copy the percentages pertaining to each proportion of BNP high, BNP medium, or BNP negative cardiomyocytes cells to a GraphPad “Grouped” Table, by inserting “% high”, “% medium”, and “% negative” in columns and diﬀerent cell lines/conditions in rows. 22. Create a “Stacked bars” Graph Type. Colour each bar and perform statistical analysis according to the experimental design (Figure 8E). Methods Protoc. 2019, 2, 83 6 of 26 Methods Protoc. 2019, 2, 83 16 of 26 2.2.3. Mitochondrial Respiration and Content

3.2.5.3.• Non-CO Sarcomeric2 oven Disarrayset at 37 °C Analysis (Stuart Scientific, Sequence, Staffordshire, Time for Completion: UK; Cat. No.:2.5 S120H). h per Plate (Figure 6) • Seahorse XFe96 Analyzer (Agilent, Santa Clara, CA, USA, Cat No.: S7800B). 1. • InputCellaVista™ image Automated and select plate “Advanced” imager (Synentec, in option Elmshorn, Flatﬁeld Germany), correction or (Figure equivalent.6A–C). 2.• Insert7500 Fast “Filter Real-Time Image” PCR building System block. (Applied Pick Bios Channelystems, “Alexa Foster 488”;City, MethodCA, USA, “Sliding Cat. No.: Parabola”. In the4351106), inset menu,or equivalent. enter value “10” in Curvature. Output Image: “Alpha-actinin sharp” (Figure6D). • OPTIONALNikon’s Eclipse STEP TE2000The Inverted rest of thisResearch script Microscope is performed (Nikon, using Minato, the sharpened Tokyo, Japan), alpha-actinin-Alexa or 488equivalent. signal, but the same principle is applicable to cardiac troponin T, as both indicate sarcomeric • proteins.NanoDrop™ This Microvolume can be used Spectrophotometer to conﬁrm if the results(ND-2000, obtained Fisher Scientific, in one channel Loughborough, are corroborated by UK). the other channel/sarcomeric protein.

3. Repeat steps 2–5 from Section 3.2.5.1 to ensure exclusion of debris, cell clumps, and cells in the 2.3. Softwareborder of the field. Output Population: “Nuclei Final” (Figure6E,F) 4. Insert “Find Cytoplasm” building block. Pick Channel “DRAQ5” (same excitation/emission 2.3.1. Hypertrophicparameters Analysis as Alexa Fluor 647) and select Method “F”. In the inset menu, define Membrane • ChannelKaluza analysis “DRAQ5”; v2.1 (Beckman Individual Coulter, Threshold: Indianapolis, 0.04 (Figure IN, USA),6G). or equivalent. • 5. InsertMicrosoft “Calculate Excel™ (Microsoft, Intensity Properties”Redmond, WA, building USA). block. Select Population “Nuclei Final”; Region: • “Cytoplasm”;GraphPad Prism Method v8.2.0 (La “Standard”, Jolla, CA, USA). tick “Mean” and “Standard Deviation” in the inset menu. Define Output Properties as “Intensity alpha-actinin”. 2.3.2. High-Content Imaging 6. Insert “Select Population” building block. Population “Nuclei Final”; Method “Filter by Property”. • HarmonyIn the inset High-Content menu, insert Imaging “Intensity and Analysis alpha-actinin” Software and (PerkinElmer, set it > the Waltham, value of theMA, secondary USA; only or cat. No.: HH17000001), with PhenoLOGIC™ machine-learning technology (required only for cell-type control. Output population “Cardiomyocytes”. Confirm if the highlighted cells display sarcomeric disarray analysis). • GraphPadpositive alpha-actininPrism v8.2.0 (La signal. Jolla, CA, This USA). is important so the subsequent analysis is performed only in cardiomyocytes. It can also be used for calculating purity (Figures A1 and6H). 2.3.3.7. MitochondrialInsert “Calculate Respiration Morphology and Content Properties” building block. Select Population “Nuclei Final”; • RegionSeahorse “Cytoplasm”; Wave Desktop Method “STAR”.Software In the(Agilent, inset menu, Santa tick ChannelClara, “A488CA, sharp”USA, and tick all thehttps://www.agilent.com/en/products/cell- boxes in that section. In the subsectionanalysis/cell-analys Profile Inner Region,is-software/data- select “Cytoplasm” and tick the box;analysis/wave-desktop-2-6). set Profile Width to 4 px. Output Properties: “Sarcomere Morphology” (Figure6I). 8.• Insert7500 Real-Time “Calculate PCR Texture Software Properties” (v2.3, buildingApplied block.Biosystems, Select ChannelFoster City, “A488 CA, sharp”; USA, Population “Nucleihttps://www.thermofisher.com/uk/en/ Final”; Region “Cytoplasm”;home/technical-resources/software- Method “SER features”. In the inset menu, set Scale to “0.5 px”,downloads/applied-biosystems-7500-real-time-pcr-system.html Normalization by “Kernel”. Tick all the SER boxes below. ), or equivalent. Output Properties: “Sarcomere • Microsoft Excel™ (Microsoft, Redmond, WA, USA). Texture SER”. • GraphPad Prism v8.2.0 (La Jolla, CA, USA). 9. Insert “Calculate Texture Properties” building block. Select Channel “A488 sharp”; Population 3. Procedure“Nuclei Final”; Region “Cell”; Method “Haralick features”. In the inset menu, set Distance to “1 px”, and tick all the Haralick boxes below. Output Properties: “Sarcomere Texture Haralick”. 3.1.10. HypertrophyInsert “Calculate Analysis. Time Texture for Completion: Properties” 1 day building block. Select Channel “A488 sharp”; Population “Nuclei Final”; Region “Cell”; Method “Gabor features”. In the inset menu, set Scale to “4 px”, 3.1.1. Data Acquisition. Time for Completion: 2 h Wavelength 2, Number of angles: 6, and Normalization by “Kernel”. Tick all the Gabor boxes 1. below.Resuspend Output freshly Properties: dissociated “Sarcomere cardiomyocytes Texture in 500 Gabor”. μL Phosphate Buffer Saline (PBS) at 1×105-2.5x105 cells per sample and place them on ice prior to flow cytometry analysis. CRITICAL STEP STEP PriorSteps culture 7–10 of are cardiomyocytes performed in in order serum-containing for the Harmony medium software may to detect dimaskfferences hypertrophic in morphology phenotypes and [17], texture so exposure between to cardiomyocytes. serum should be Texture minimized information or fully is extracted fromeliminated. each image by using different filtering strategies. The parameters for each approach were 2. Add 2 drops of bead solution of each size to a flow cytometer tube, mix well by flicking it, optimized for hPSC-CMs and should be adapted for other cardiomyocyte sources. and measure FSC and side scatter (SSC) values (area, width, and height) at 488 nm laser wavelength (area and height). Gabor: Filtering by frequency and orientation of pixels in an image; • CRITICAL STEP Use of 100 μM flow cytometer nozzle size is highly recommended to Haralick: Not based on pixel-wise filtering; each feature is a statistical characteristic of the •minimize sampling biases [33]. whole image/region of interest; SER: Gaussian derivative filtering. • 11. Insert “Select Population” building block. Population: “Cardiomyocytes”; Method “Linear Classifier”. In the inset menu, set Number of Classes to 2 and tick all the boxes below. Output Population A: “Organised”; Output Population B: “Disarrayed”. Methods Protoc. 2019, 2, 83 6 of 26

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2.3. Software

2.3.3. Mitochondrial Respiration and Content • Seahorse Wave Desktop Software (Agilent, Santa Clara, CA, USA, https://www.agilent.com/en/products/cell-analysis/cell-analysis-software/data- analysis/wave-desktop-2-6). • 7500 Real-Time PCR Software (v2.3, Applied Biosystems, Foster City, CA, USA, https://www.thermofisher.com/uk/en/home/technical-resources/software- downloads/applied-biosystems-7500-real-time-pcr-system.html ), or equivalent. • Microsoft Excel™ (Microsoft, Redmond, WA, USA). • GraphPad Prism v8.2.0 (La Jolla, CA, USA). Methods Protoc. 2019, 2, 83 17 of 26 3. Procedure Methods Protoc. 2019, 2, 83 17 of 26 11. Insert “Select Population” building block. Population: “Cardiomyocytes”; Method “Linear 3.1. HypertrophyClassifier”. Analysis. In Time the insetfor Completion: menu, set 1Number day of Classes to 2 and tick all the boxes below. Output 12. ProvidePopulation a training A: “Organised”; set in the option Output “Training” Population byB: “Disarrayed”. picking cells belonging to each class (either 3.1.1. Data Acquisition. Time for Completion: 2 h organized12. Provide or a disarrayed),training set in inthe independent option “Training” fields, by wells,picking or cells plates. belonging Select to only each cells class that(either belong to organized or disarrayed), in independent fields, wells, or plates. Select only cells that belong 1. oneResuspend of these freshly categories dissociated clearly cardiomyocytes (Figure6J). in 500 μL Phosphate Buffer Saline (PBS) at 1×105to-2.5x10 one of5 cells these per categories sample andclearly place (Figure them 6J).on ice prior to flow cytometry analysis. CRITICALCRITICAL CRITICAL STEP STEP STEP PriorDue Due culture to theto the heterogeneityof heterogeneity cardiomyocytes ofof hPSC-CMs,hPSC-CMs in serum-containing, aa large large training training medium setset ofmay at of least at least 100 cellsmask100 forhypertrophic eachcells for category each phenotypes category is desired. [17],is desired. Phenotypicso exposure Phenot controlstoypic serum controls suchshould such as be cardiomyocytes asminimized cardiomyocytes or fully with with disrupted disrupted sarcomeres (genetic knockouts or pharmacologically induced) greatly support this sarcomereseliminated. (genetic knockouts or pharmacologically induced) greatly support this distinction. 2. Add distinction.2 drops of bead solution of each size to a flow cytometer tube, mix well by flicking it, 13. Evaluate the validity of the training performed by checking that the “Goodness” Parameter is >1, and13. measureEvaluate FSC the validityand side of scatter the training (SSC) values perfor (area,med by width, checking and height)that the at “Goodness” 488 nm laser Parameter indicatingwavelengthis >1, indicating a (area good and separation a height). good separation between between the 2 classes, the 2 classes, and which and which texture texture or morphology or morphology properties more CRITICALproperties accurately moreSTEP define accuratelyUse those of 100 didefine μffMerences flow thos cytometere (Figuresdifferences nozzle A3 (Figure and size6K,L). A3 is highlyand 6K,L). recommended to minimize sampling biases [33].

FigureFigure 6. 6. SarcomericSarcomeric disarray disarray analysis analysis sequence sequence.. Representative Representative micrographs micrographs of image ofanalysis image of analysis ofhPSC-CMs. hPSC-CMs. After After inpu inputtingtting the theimage image (A–C (A–C), the), software the software uses usesmathematical mathematical transformation transformation algorithmsalgorithms to to sharpen sharpen sarcomeric sarcomeric signalsignal ( D),), followed followed by by exclusion exclusion of ofdebris debris and and cell cell clumps clumps (E,F ().E,F ). The cellThe mask cell channelmask channel is used is toused define to define cell cytoplasm cell cytoplasm (G) and(G) theandα the-actinin α-actinin signal signal intensity intensity is quantified is andquantified compared and againstcompared that against of a cell-typethat of a cell-type control control to exclude to excl non-cardiomyocytesude non-cardiomyocytes from from the the analysis (Hanalysis). Morphologic (H). Morphologic and texture-based and texture-based information information is extracted is extracted from from the the sharpened sharpened α-actinin signal displayedsignal displayed by cardiomyocytes by cardiomyocytes (I), followed (I), followed by a machine-learningby a machine-learni procedureng procedure where where the the user user manually selectsmanually cardiomyocytes selects cardiomyocytes displaying displaying organised organised vs. disarrayed vs. disarrayed sarcomeres sarcomeres (J). The (J). software The software then applies then applies this algorithm to the rest of the images being analysed to distinguish organised vs. this algorithm to the rest of the images being analysed to distinguish organised vs. disarrayed disarrayed cardiomyocytes (K,L). Scale bar = 100 μm, except for L where it is 20 μm. cardiomyocytes (K,L). Scale bar = 100 µm, except for L where it is 20 µm. 14. In the default “Define Results” building block, select Method ”List of Outputs” in the drop 14. In the default “Define Results” building block, select Method ”List of Outputs” in the drop down down menu, then tick the box “Number of Objects” for the options: “Population: menu,Cardiomyocytes”, then tick the box “Population: “Number Nuclei of Objects” Final”. for the options: “Population: Cardiomyocytes”, “Population:15. In the same Nuclei drop-down Final”. menu within “Define Results”, define two menus of “Method— 15. In theFormula same drop-down Output”, and menu in the within inset menu, “Define enter Results”, “Formula: define (a/b)×100. two menus of “Method—Formula Output”, and in the inset menu, enter “Formula: (a/b) 100. × 16. In the first Menu define Variable A: “Cardiomyocytes—Number of Objects”; Variable B “Nuclei Final—Number of Objects”. Output Name “% Cardiomyocyte Purity”. 17. In the second Menu, define Variable A: “Disarrayed—Number of Objects” and Variable B: “Cardiomyocytes—Number of Objects. Output Name “% Cardiomyocyte Disarray”. Methods Protoc. 2019, 2, 83 6 of 26

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2.3. Software

2.3.2. High-Content Imaging • Harmony High-Content Imaging and Analysis Software (PerkinElmer, Waltham, MA, USA; cat. No.: HH17000001), with PhenoLOGIC™ machine-learning technology (required only for sarcomeric disarray analysis). • GraphPad Prism v8.2.0 (La Jolla, CA, USA). Methods Protoc. 2019, 2, 83 18 of 26 2.3.3. Mitochondrial Respiration and Content 18.• InSeahorse the third Wave Menu, Desktop deﬁne VariableSoftware A: “Organized—Number(Agilent, Santa Clara, of Objects”CA, USA, and Variable B: “Cardiomyocytes—Numberhttps://www.agilent.com/en/products/cell- of Objects.analysis/cell-analys Output Name “%is-software/data- Cardiomyocyte Organized”. Conﬁrm thatanalysis/wave-desktop-2-6). % organized + % disarrayed = 100% • 7500 Real-Time PCR Software (v2.3, Applied Biosystems, Foster City, CA, USA, 19. Import percentages to a GraphPad “Column” Table, create a “Box-plot” graph, and perform https://www.thermofisher.com/uk/en/home/technical-resources/software- statisticaldownloads/applied-biosystems-7500-real-time-pcr-system.html analysis according to experimental design (Figure ), or 8F). equivalent. • Microsoft Excel™ (Microsoft, Redmond, WA, USA). 3.3. Mitochondrial Respiration and Content. Time for Completion: 5 Days • GraphPad Prism v8.2.0 (La Jolla, CA, USA).

3. 3.3.1.Procedure Evaluation of Mitochondrial Respiration by Seahorse™ Assay. Time for Completion: 5 Days

3.1.3.3.1.1. Hypertrophy Replating Analysis. Cardiomyocytes. Time for Completion: Time 1 day for Completion: 2 h 1. Coat a Seahorse XF96 cell culture microplate (cell plate) with Vitronectin following steps 1–4 from 3.1.1. Data Acquisition. Time for Completion: 2 h Section 3.2.1. 2. 1. PlateResuspend cardiomyocytes freshly dissociated at approximately cardiomyocytes 5000 in cells500 μ/mmL Phosphate2 (50,000 Buffer cells perSaline well). (PBS) at 1×105-2.5x105 cells per sample and place them on ice prior to flow cytometry analysis. CRITICALCRITICAL STEP STEP PriorThis culture cell density of cardiomyocytes is aimed at covering in serum-containing the well completely medium withoutmay forming clumpsmask hypertrophic or cell aggregates phenotypes (crucial [17], for so anexposure accurate to Seahorseserum should assay be and minimized subsequent or fully normalization). Iteliminated. may need to be adjusted to different cardiomyocyte sources (e.g., tissue-derived or resulting 2. Add 2 drops of bead solution of each size to a flow cytometer tube, mix well by flicking it, from various differentiation protocols that produce cells with varying sizes). and measure FSC and side scatter (SSC) values (area, width, and height) at 488 nm laser wavelengthCRITICAL (area STEP and height).Prior culture of cardiomyocytes in serum-containing medium may mask CRITICAL STEP Use of 100 μM flow cytometer nozzle size is highly recommended to hypertrophic phenotypes [17], so exposure to serum should be minimized or fully eliminated. minimize sampling biases [33]. OPTIONAL STEP Allow the cells to settle for 0.5 h at room temperature before placing them in the incubator, to minimize edge effects due to the formation of convection currents because of

temperature diﬀerences between the medium and the incubator.

3.3.1.2. Preparation for Seahorse Assay. Time for Completion: 4 Days 3. Replace cardiomyocyte medium (100 µL/well) every 2 days for 4 days. 4. On the day prior to the assay, add 200 µL of XF calibrant per well to a sensor cartridge plate (utility plate) and incubate overnight in the non-CO2 oven at 37 ◦C, to hydrate the sensors.

3.3.1.3. Performing Seahorse Mitostress Test. Time for Completion: 4 h 5. Prepare XF assay medium following the kit’s instructions: 97 mL XF DMEM medium + 1 mL XF 1.0 M Glucose + 1 mL XF 100 mM Pyruvate + 1 mL XF 200 mM Glutamine. Adjust pH to 7.4. This is enough for 1 plate.

6. Warm medium in water bath (37 ◦C). Aspirate cardiomyocyte medium and wash twice with 200 µL of XF assay medium per well. Add 175 µL medium/well. Incubate cells in non-CO2 oven at 37 ◦C for 1 h. 7. Prepare injection compounds in XF assay (3 mL per compound):

Oligomycin: 12 µM to achieve a final concentration (in well) of 1.5 µM (8X dilution); • FCCP: 4.5 µM to achieve a final concentration (in well) of 0.5 µM (9X dilution); • Rotenone: 10 µM to achieve a final concentration (in well) of 1 µM (10X dilution). •

CRITICAL STEP These concentrations have been optimized for hPSC-CMs and may need to be adjusted to diﬀerent cardiomyocyte sources 8. Load the injection compounds (25 µL/port) into the cartridge plate ports using the ultraﬁne 200 µL tips and the corresponding port guides (port A—oligomycin; port B—FCCP; port C—rotenone; port D—XF assay medium). Methods Protoc. 2019, 2, 83 6 of 26

2.2.3. Mitochondrial Respiration and Content

2.3. Software

2.3.1. Hypertrophic Analysis • Kaluza analysis v2.1 (Beckman Coulter, Indianapolis, IN, USA), or equivalent. • Microsoft Excel™ (Microsoft, Redmond, WA, USA). • GraphPad Prism v8.2.0 (La Jolla, CA, USA). Methods Protoc. 2019, 2, 83 19 of 26 2.3.2. High-Content Imaging • Harmony High-Content Imaging and Analysis Software (PerkinElmer, Waltham, MA, USA; 9. cat.Initiate No.: HH17000001), Seahorse analyser with PhenoLOGIC™ and Wave software machine-learning and set up technology experimental (required protocol only based for on 3 basal sarcomericrate measurements disarray analysis). prior to the first injection, followed by 3 measurements after each injection. • GraphPadMix-Wait-Measure Prism v8.2.0 times (La Jolla, per CA, measurement USA). are 3 min–0 min–3 min (Figure7A). 10. Insert cartridge into the XF analyser for calibration. When prompted by the software, replace 2.3.3. Mitochondrialutility plate Respiration with cell plate, and Content and run the assay to determine oxygen consumption rate (OCR). Save • analysisSeahorse fileWave upon completion.Desktop Software (Agilent, Santa Clara, CA, USA, https://www.agilent.com/en/products/cell-analysis/cell-analysis-software/data- 3.3.1.4.analysis/wave-desktop-2-6). Normalization of OCR Values. Time for Completion: 2 h • 7500 Real-Time PCR Software (v2.3, Applied Biosystems, Foster City, CA, USA, 11. Aspirate XF medium, wash with 50 µL/well PBS and incubate with 50 µL/well Hoechst 33,342 https://www.thermofisher.com/uk/en/home/technical-resources/software- (1:400 in PBS) for 30 min at 37 ◦C. Wash with PBS once and add 200 µL/well PBS. Methodsdownloads/applied-biosystems-7500-real-time-pcr-system.html Protoc. 2019, 2, 83 ), or equivalent. 7 of 26 • OPTIONALMicrosoft Excel™ STEP (Microsoft,Alternatively, Redmond, fix the WA, cardiomyocytes USA). by aspirating XF medium, washing with • 50 µL/well PBS and adding 100 µL of 4% PFA per well. Incubate for 15 min at RT, wash with PBS, GraphPad3. Run cell Prism samples v8.2.0 (Lain theJolla, flow CA, cytometer, USA). using the same operating settings as those used for and add 200 µL PBS/well. 3. Procedure the calibration beads. PAUSEPAUSE STEP STEPFixed Once cellsdata canfrom be flow stored cytometry at 4 ◦C is for acquired, up to 2 monthsit can be with analysed plates at wrapped any point. in 3.1. Hypertrophy Analysis. Time for Completion: 1 day 3.1.2.parafilm, Data Analysis. prior to Time staining for andCompletion: imaging. 3 h 12. Image plates in Cellavista plate reader by optimizing Hoechst signal intensity and focus in a 3.1.1. Data Acquisition. Time for Completion: 2 h single4. Infield the Kaluza followed software by scanning (or any the equivalent whole plate flow across cytometry the entire analysis well (Figuresoftware),7B). gate to 13.1. QuantifyResuspendremove number freshly debris dissociated of and cell duplets/triplets nuclei cardiomyocytes in the “Evaluation” (exclude in 500 μeventsL mode. Phosphate with Export very Buffer data low Saline toFSC an (PBS) and Excel atSSC file. Area 1×105values)-2.5x105 (Figurecells per 2A,B).sample and place them on ice prior to flow cytometry analysis. 5. CRITICALCRITICALGate to define STEP STEP FSC-A PriorOther culturefor normalization each of bead cardiomyocytes used techniques for th ein basedcalibration. serum-containing on protein Export content medium FSC-A should mayvalues be relative avoided to asmask aeach hypertrophichypertrophic gate (corresponding phenotypes phenotype [17],to characteristic each so exposure bead) to of toan HCMserum excel file.should is typically Ensure be minimized there associated is consistency or fully with increased between proteineliminated.the/ DNAunits ratiobeing [ 38used]. for beads and samples (i.e., linear or log) (Figure 2C,D). 2. Add 2 drops of bead solution of each size to a flow cytometer tube, mix well by flicking it, OPTIONAL6. In the Excel STEP software,Using Cellavistaplot the FSC-A ensures median high-throughput values (y axis, quantification obtained from of each cell nucleigate in butthe and measure FSC and side scatter (SSC) values (area, width, and height) at 488 nm laser Kaluza software) relative to the known bead diameters (x axis). Add trendline of linear otherwavelength instruments (area and (automated height). or not) can be used for the same purpose. regression and display equation (𝐹𝑆𝐶𝐴 = 𝑠𝑙𝑜𝑝𝑒 × 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 +𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡) and R-square CRITICAL STEP Use of 100 μM flow cytometer nozzle size is highly recommended to 3.3.1.5. Data analysis. Time for Completion: 2 h minimizevalue sampling determining biases the [33]. calibration curve (Figure 2E). 14. Paste the CRITICAL cell count numberSTEP If the of each R-squared well in value the normalization (measure of how tab ofmuch the Analysisthe variation file createdof the FSC- in A is explained by variation of the bead size) is below 0.95, then the bead solutions must be the Wave software upon the conclusion the Seahorse assay. thoroughly mixed to disrupt clumps. 15. Export data as Excel and transform into OCR values per 30,000 cells (to enable comparisons 7. Gate samples to remove debris and duplets/triples. In cell samples, SSC-width is used in between experimental replicates and with the literature) (Figure7C). the OPTIONAL STEP Alternatively, input OCR and cell number values into the Excel Macro x axis instead of FSC-A due to higher heterogeneity of the cardiomyocytes relative to the provided by Agilent for the analysis (https://www.agilent.com/en/products/cell-analysis/cell- beads (Figure 2 F–H). analysis-software/data-analysis/seahorse-xf-cell-mito-stress-test-report-generators). 8. Export data from each sample from Kaluza to Excel, as done in 5. 16. Calculate parameters of mitochondrial respiration as follows (Figure7C): 9. Using the calibration equation determined in 6, convert the FSC-A values measured for ( ) each sample into cell diameter: 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 = . Basal respiration = average OCR [0 15min] average OCR [80 95min] − − − 10. MaximalDetermine respiration the cell =volumeaverage by OCRapplying[40 the55min formula] average of the OCR volume[average of a OCRsphere:60 𝑉75 min = ] × − − − 𝑑, whereATP d is production the cell diameter= average determined OCR [0 in15 min9. This] averageassumes OCR the [approximation20 35min] of each − − − cell in suspension to a sphere, which is also applicable to the calibration beads. OPTIONAL11. Import the STEP cell volumeAdditional values parameters into GraphPad can also software, be reported as a column although table these(whereby are each not as informativecolumn forrepresents phenotyping a separate HCM: sample). 12. Choose “violin plot only” in the “Box and plot” options to graphically represent the data distribution andSpare the capacity main statistical= average parameters OCR [40 (median55min] andbasal quartiles). respiration Adjust colour as − − desired (FigureNon 8A).mitochondrial respiration = average OCR [60 75min] − − 13. Perform statisticalProton analysis leak = (dependingOCR [20 35 onmin the] experimentalaverage OCR design)[60 75 minto compare] volume − − − distribution between samples. OPTIONAL STEP Plotting such large sample sizes in GraphPad can be computationally demanding. Alternatively, a boxplot can be generated by inputting only 7 cardiomyocyte volume values per condition: Minimum, maximum; 25th percentile, 75th percentile, and the median three times. These values can be easily determined using Excel™ software (Figure 8B).

Methods Protoc. 2019, 2, 83 20 of 26 Methods Protoc. 2019, 2, 83 20 of 26

FigureFigure 7. 7.Evaluation Evaluation of of mitochondrial mitochondrial respiration respiration using using Seahorse Seahorse platform. platform. (A ()A Protocol) Protocol summary summary for for mitostressmitostress test test using using the the Seahorse Seahorse assay assay™.™.(B )(B Representative) Representative micrographs micrographs detailing detailing normalisation normalisation byby automated automated quantification quantification of of cell cell nuclei nuclei in in Cellavista Cellavista™™ plate plate imager imager ((i) ((i) Hoechst Hoechst staining staining of of nuclei; nuclei; (ii)(ii) quantification quantification of of individual individual nuclei; nuclei; (iii)(iii) overlay).overlay). ( (CC)) Sample Sample graph graph for for oxygen oxygen consumption consumption rate rate(OCR) (OCR) over over time time following following the the Seahorse mitostressmitostress kit,kit, highlightinghighlighting the the di differentfferent mitochondrial mitochondrial respirationrespiration parameters. parameters.

17. Import data into GraphPad as an “XY Table”. Plot and amend the colour of the lines/bar graphs 17. Import data into GraphPad as an “XY Table”. Plot and amend the colour of the lines/bar according to the experimental design and indicate in the graph when compounds were added graphs according to the experimental design and indicate in the graph when compounds (Figure8G–J). were added (Figure 8G–J). 18. Perform18. Perform statistical statistical analysis analysis (depending (dependi onng the on experimental the experimental design). design).

3.3.2.3.3.2. Evaluation Evaluation of of Mitochondrial Mitochondrial Content Content by by qPCR. qPCR. Time Time for for Completion: Completion: 1 Day1 day. 3.3.2.1. DNA Extraction from Cardiomyocytes. Time for Completion: 1 h 3.3.2.1. DNA Extraction from Cardiomyocytes. Time for Completion: 1 h 1. Pellet cardiomyocyte single-cell suspension by centrifugation at 100 xg for 15 min in a 1.7 mL tube. 1. Pellet cardiomyocyte single-cell suspension by centrifugation at 100 xg for 15 min in a 1.7 mL 2. Aspirate supernatant. Wash with 1 mL PBS and pellet again. tube. 3. Extract DNA using Qiagen Blood and Tissue kit, following manufacturers’ instructions. 2. Aspirate supernatant. Wash with 1 mL PBS and pellet again. 4. Determine3. Extract DNA DNA concentration using Qiagen and Blood purity and using Tissue NanoDrop kit, following or equivalent. manufacturers’ instructions. 4. Determine DNA concentration and purity using NanoDrop or equivalent. 3.3.2.2. Quantitative Real-Time PCR for Mitochondrial/Nuclear DNA Ratio. Time for Completion: 4 h 5. 3.3.2.1.Dilute Quantitative DNA samples Real-Time to 25 ng PCR/µL for in molecular-grade mitochondrial/nuclear water. DNA ratio. Time for completion: 4 h 6. Prepare5. Dilute qPCR DNA mastermixes samples to as 25 follows ng/μL (perin molecular-grade reaction): water. 6. Prepare qPCR mastermixes as follows (per reaction): Mastermix: 5 µL; •• Mastermix: 5 μL; • Probe (ACTB, MT-ND1 or MT-ND2): 0.5 µL; • Probe (ACTB, MT-ND1 or MT-ND2): 0.5 μL; • Molecular grade water: 3.5 µL. • Molecular grade water: 3.5 μL. 7. Reverse-pipette 9 μL of each reaction mastermix into the MicroAmp™ Fast Optical 96-Well Reaction Plate.

Methods Protoc. 2019, 2, 83 6 of 26

2.2.3. Mitochondrial Respiration and Content

2.3. Software

3. Procedure

3.1.Methods Hypertrophy Protoc. 2019 Analysis., 2, 83 Time for Completion: 1 day 21 of 26

3.1.1. Data Acquisition. Time for Completion: 2 h 7. Reverse-pipette 9 µL of each reaction mastermix into the MicroAmp™ Fast Optical 96-Well 1. Resuspend freshly dissociated cardiomyocytes in 500 μL Phosphate Buffer Saline (PBS) at Reaction Plate. 1×105-2.5x105 cells per sample and place them on ice prior to flow cytometry analysis. CRITICALCRITICAL STEP STEP PriorReverse-pipetting culture of cardiomyocytes is to ensure in high serum-containing accuracy of pipetting medium technique, may due to themask very hypertrophic high sensitivity phenotypes of the [17], qPCR so technique.exposure to serum should be minimized or fully eliminated. 8. Add 1 µL of DNA template to each respective well of the 96-well plate. 2. Add 2 drops of bead solution of each size to a flow cytometer tube, mix well by flicking it, 9. Stickand measure the MicroAmp FSC and ™sideOptical scatter Adhesive(SSC) values Film (area, to thewidth, 96-well and height) plate. at 488 nm laser 10. Initiatewavelength the (area 7500 and Real-Time height). PCR equipment and its respective software. Deﬁne experimental setup CRITICAL as: Instrument STEP Use “7500 of 100 Fast μM (96 flow Wells); cytometer Type ofnozzle Experiment size is highly “Quantitative recommended – Comparative to CT ® (minimize∆∆CT); Reagents sampling “TaqManbiases [33]. Reagents”; Ramp Speed “Standard (~2 h to complete a run). 11. Enter plate setup according to experimental design. Set Run Method as:

Holding stage: 50.0 C for 20 s, followed by 95.0 C for 10 min; • ◦ ◦ Cycling stages: 95.0 C for 15 s, followed by 60.0 C for 1 min—40 cycles. • ◦ ◦ 12. Run qPCR reaction. Save data upon completion. 13. Analyse data using the ∆∆CT method, where the average of healthy cardiomyocytes is used for calculating relative quantiﬁcation of mtDNA content (i.e., used as the control sample) [39]:

∆CTmitoDNAgene, sampleX = CTmitoDNAgene, sampleX CTnuclearDNAgene, sampleX − ∆∆CTmitoDNAgene, sampleX = ∆CTmitoDNAgene, sampleX ∆CTmitoDNAgene, control sample − Expression f old change mitoDNAgene, sampleX = 2∆∆CTmitoDNAgene,sampleX

14. Import data into GraphPad. Perform statistical analysis according to the experimental design (Figure8K).

4. Expected Results HCM is a complex and intractable disease that requires further characterization to facilitate therapeutic intervention [2]. Several cellular and molecular mechanisms of disease have been described [38], but they have shown to be mutation-specific and/or restricted to particular patient backgrounds [10]. Thus, uncovering novel molecular mechanisms of HCM progression in cellular models where the genetic causation has been elucidated is a promising strategy to address the lack of effective treatment in order to restore cardiac function [4,40]. So as to address the complex in vitro characterization of HCM, we developed a set of phenotypic assays that can be used to quantify disease hallmarks. Our toolkit consists of three straightforward protocols with varying degrees of logistic requirement (e.g., equipment and cost) that are not only accessible to any research groups investigating HCM, but also applicable to other fields evaluating identical changes in cellular responses. The data generated from this characterization encompass analysis of (i) hypertrophy, (ii) HCM molecular features, and (iii) mitochondrial respiration and content (Figure8). While cardiomyocytes cultured in 2D conditions do not fully replicate pathophysiological disease features [29,41], the information acquired from such a multi-parametric evaluation produces a significant level of understanding of HCM characteristics with directly quantifiable variables. Remarkably, these can be harnessed in high-throughput drug screening approaches aimed at identifying new compounds with therapeutic potential, which are integrated in early stages of drug discovery pipelines where fast and inexpensive assays are paramount [6]. In this regard, this toolkit can be used to detect attenuation or rescue of HCM phenotypes upon pharmacological of genetic intervention. This is useful to exclude drug candidates early on, before more complex, lower-throughput, and often time-consuming and labour-intensive assays are required [5]. This greatly expedites subsequent functional assaying of cardiomyocytes (e.g., investigating calcium handling and contractile force), typically performed in 3D organoids or engineered heart tissues [26–28,42–44]. Methods Protoc. 2019, 2, 83 22 of 26

Overall, it is expected that disseminating this toolkit will greatly support disease modelling research ﬁelds, accelerating the discovery of new mechanisms of disease and ultimately paving the way for more eﬃcient therapeutics. Methods Protoc. 2019, 2, 83 22 of 26

FigureFigure 8. 8.Example Example of application of of phenotypin phenotypingg toolkit toolkit to HCM to HCM cellular cellular model. model. (A,B) Hypertrophy (A,B) Hypertrophy analysisanalysis by by flowflow volumetry volumetry indicating indicating increased increased median median volume volume in HCM in cardiomyocytes HCM cardiomyocytes relative to relative healthy counterparts. (C–F) High-content imaging evaluation of HCM cardiomyocytes showing to healthy counterparts. (C–F) High-content imaging evaluation of HCM cardiomyocytes showing increased proportion of multinucleation, expression of hypertrophic marker BNP, and sarcomeric increased proportion of multinucleation, expression of hypertrophic marker BNP, and sarcomeric disarray relative to healthy controls. (G–J) Assessment of mitochondrial respiration in Seahorse™ disarrayassay revealing relative higher to healthy energy controls. production (G– Jin) AssessmentHCM cardiomyocytes of mitochondrial in comparis respirationon with healthy in Seahorse ones, ™ assay revealingshowing higher(K) similar energy mitochondrial production DNA in HCM content cardiomyocytes (determined by inratiometric comparison mito/nuclear with healthy DNA ones, qPCR showing (Kanalysis).) similar Data mitochondrial represent mean DNA +/- content SEM of (determinedfive independent by ratiometricexperiments mitocomparing/nuclear R453C- DNAβ-myosin qPCR analysis). Dataheavy-chain represent hPSC-CMs mean +/- SEM(HCM) of fiveto isogenic independent wild-type experiments control (h comparingealthy), with R453C- 100,000–250,000β-myosin cells heavy-chain hPSC-CMsanalysed per (HCM) sample. to isogenicStatistical wild-typeanalysis was control performed (healthy), by unpaired with 100,000–250,000 parametric Student’s cells analysedt test per sample.between Statistical HCM and analysis Healthy was conditions, performed with by the unpaired exception parametric of Panel E, Student’s where unpaired t test between one-way HCM and HealthyANOVA conditions, test was used with with the Dunnett’s exception post of hoc Panel test for E, wherecorrection unpaired of multiple one-way comparisons ANOVA relative test to was used withHealthy Dunnett’s condition post (n. hoc s., non-significant; test for correction *P < 0.05; of multiple **P < 0.01; comparisons ***P < 0.005; **** relativeP < 0.0001, to Healthy colour-coded condition (n. by inter-compared category—black asterisks apply to all categories). s., non-significant; *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001, colour-coded by inter-compared

Authorcategory—black Contributions: asterisks Conceptualization, apply to all D.M. categories). and C.D.; writing—original draft preparation, D.M.; data acquisition, D.M.; methodology and software, D.M. and K.L-S.; supervision and funding acquisition, C.D.; writing—review and editing, D.M., K.L-S. and C.D. Author Contributions: Conceptualization, D.M. and C.D.; writing—original draft preparation, D.M.; data acquisition,Funding: This D.M.; research methodology was funded and by software, the British D.M. Heart and Foundation K.L-S.; supervision(BHF) [grant andnumbers: funding SP/15/9/31605, acquisition, C.D.; writing—reviewPG/14/59/31000, andRG/14/1/30588, editing, D.M., RM/13/30157, K.L-S. and P47352/CRM], C.D. Britain Israel Research and Academic Exchange Funding:PartnershipThis (BIRAX) research [04BX14CDLG], was funded Medical by the BritishResearch Heart Council Foundation (MRC) [MR/M017354/1], (BHF) [grant numbers:Engineering SP and/15 /9/31605, PGPhysical/14/59/31000, Sciences RG Research/14/1/30588, Council RM (EPSRC)/13/30157, [DM’s P47352 Doctor/alCRM], Prize Research Britain IsraelFellowship], Research National and Centre Academic for the Exchange PartnershipReplacement, (BIRAX) Refinement, [04BX14CDLG], and Reduction Medical of An Researchimals in CouncilResearch (MRC) (NC3Rs) [MR [CRACK-IT:35911-259146,/M017354/1], Engineering and PhysicalNC/K000225/1, Sciences NC/S001808/1], Research Council Heart Research (EPSRC) UK, [DM’s German Doctoral Research Prize Foundation Research [DFG-Es-88/12-1, Fellowship], HA3423/5- National Centre for1], the European Replacement, Research Refinement, Council [ERC-A andG-IndivuHeart], Reduction of European Animals Commission in Research [FP7- (NC3Rs) Biodesign], [CRACK-IT:35911-259146, German Centre NCfor/K000225 Cardiovascular/1, NC/S001808 Research/1], (DZHK), Heart Research German UK, Minist Germanry of ResearchEducation Foundationand Research, [DFG-Es-88 and the /12-1,Freie HA3423und /5-1], EuropeanHansestadt Research Hamburg Council for funding. [ERC-AG-IndivuHeart], European Commission [FP7- Biodesign], German Centre for Cardiovascular Research (DZHK), German Ministry of Education and Research, and the Freie und Hansestadt HamburgAcknowledgments: for funding. The authors would like to thank James Hutt (Imaging Specialist at PerkinElmer UK) for his support and advice in validating the high-content imaging analysis sequences in the Harmony software. Acknowledgments: The authors would like to thank James Hutt (Imaging Specialist at PerkinElmer UK) for his supportConflicts and of advice Interest: in The validating authors thedeclare high-content no conflict imagingof interest. analysis sequences in the Harmony software. Conflicts of Interest: The authors declare no conflict of interest. Appendix A

Methods Protoc. 2019, 2, 83 23 of 26

Methods Protoc. 2019, 2, 83 23 of 26 MethodsAppendix Protoc. A 2019, 2, 83 23 of 26

Figure A1. Cell-type control for α-actinin staining. Comparing signal intensities of α-actinin between Figure A1. Cell-type control for α-actininα staining. Comparing signal intensities of α-actinin betweenα fibroblastsFigure or A1. anyCell-type cell typecontrol that does for not-actinin express staining. the cardiac Comparing marker signal(cell-type intensities control) of with-actinin fibroblastsbetween ﬁbroblastsor any cell or type any cellthattype does that not does expr notess expressthe cardiac the cardiacmarker marker(cell-type (cell-type control) control) with cardiomyocytes enables determination of positive signal threshold required for high-content imaging. cardiomyocyteswith cardiomyocytes enables enables determination determination of positive of positivesignal threshold signal threshold required fo requiredr high-content for high-content imaging. Scale bar = 100 μm. Scaleimaging. bar = Scale100 μ barm. = 100 µm.

Figure A2.Figure Controls A2. Controlsfor BNP for signal BNP signalthresholding thresholding.. In order In order to categorize to categorize signal signal intensities intensities of Figure A2. Controls for BNP signal thresholding. In order to categorize signal intensities of cardiomyocytescardiomyocytes expressing expressing BNP, endothelin-1 BNP, endothelin-1 treatment treatment can canbe used be used to todetermine determine high-intensity high-intensity thresholdcardiomyocytesthreshold as it maximises as expressing it maximises BNP expression BNP, BNP expressionendothelin-1 relative relative to tr untreatedeatment to untreated cancells, cells,be whereas used whereas to bosentan determine bosentan treatment treatment high-intensity has has µ the oppositethresholdthe oppositeeffect as it maximises(defining eﬀect (deﬁning low/nonexistent BNP expression low/nonexistent signal).relative signal). Scale to untreated Scale bar bar= 100= cells,100 μm. whereas m. bosentan treatment has the opposite effect (defining low/nonexistent signal). Scale bar = 100 μm.

Methods Protoc. 2019, 2, 83 24 of 26 Methods Protoc. 2019, 2, 83 24 of 26

Figure A3. Parameters for machine learning step in the sarcomericsarcomeric disarraydisarray analysisanalysis sequence.sequence. The PhenoLOGIC™PhenoLOGIC™ trainingtraining reliesrelies on on manual manual assignment assignment of of cardiomyocytes cardiomyocytes to to two two di ffdifferenterent categories categories (A, (A,organised; organised; B, disarrayed).B, disarrayed). The The parameters parameters of of algorithm algorithm using using the the training trainingset set providedprovided byby the user indicate goodness of fit fit factor ((>1)>1) as as well as th thee texture/ texture/ morphological properties evaluated by the software that didifferffer the mostmost betweenbetween thethe twotwo categories.categories.

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