hiPSC culture. Control hiPSCs were provided by the Stanford Cardiovascular Institute (Stanford University Cardiovascular Institutes Biobank, Stanford, CA, USA; Hypertrophic Cardiomyopathy/MYH7/Control iPSC line) (4). All hiPSCs were maintained in StemFlex media (Thermo Fisher Scientific) according to the manufacturer’s protocol. Cryopreserved hiPSCs were thawed and added to StemFlex media supplemented with 5 μM Y-27632 (BD Biosciences). hiPSCs were then plated onto 8.7 μg/cm2 Matrigel-coated (GFR, BD Biosciences) 6-well dishes. hiPSCs were subsequently cultured at 37°C at 5% CO2 until they were 70%–90% confluent with daily media changes before passaging. For passaging, hiPSCs were dissociated using Versene (Thermo Fisher Scientific), resuspended in StemFlex media, and replated onto Matrigel-coated plates (Corning).
Differentiation of hiPSCs into CMs. hiPSCs from the Stanford Cardiovascular Institutes (SCVI) WT line were differentiated into CMs using a small molecule–directed protocol as previously described (42). In short, hiPSCs in culture were dissociated and seeded onto Matrigel-coated 6-well plates at 1.5 × 106 to 2.0 × 106 cells/well in StemFlex media. Cells were cultured for approximately 3–5 days in StemFlex media (1 day after 100% confluence, at which time differentiation was initiated [day 0]). On day 0, StemFlex media were replaced with RPMI supplemented with B27 without insulin (Thermo Fisher Scientific) with a 9 μM CHIR99021 addition (Tocris Bioscience). Exactly 24 hours later (day 1), media were changed to 3 mL/well RPMI + B27 without insulin and cells were cultured in this media for 48 hours (day 3). On day 3, the media were changed to 3 mL/well RPMI + B27 without insulin supplemented with 5 μM IWP-2 (Stemgent). Forty-eight hours later (day 5), the media were changed to 3 mL RPMI + B27 without insulin. The media were changed to RPMI + B27 complete supplement (with insulin) (Thermo Fisher Scientific) on day 7, and the differentiated cells were maintained in this media until day 15, with media changes every 48–72 hours. On day 15, cells from wells containing ≥ 80% contracting cells by visual inspection were dissociated with 10× TrypLE (Thermo Fisher Scientific) according to the manufacturer’s protocol. Cells were then cryopreserved using a 1:9 ratio of DMSO (MilliporeSigma) and FBS (Thermo Fisher Scientific).
hiPSC-CM purification. After thawing and suspending in EB20 media (2), cells were replated on Synthemax-coated (Corning) 6-well plates. Each hiPSC-CM batch (5 batches of 15 million cells) was matched for both lactate and MACS purifications. For the lactate-purified hiPSC-CMs, 48 hours after replating, hiPSC-CMs were purified using CDM3L media, made with RPMI 1640 no glucose (Invitrogen), 500 μg/mL recombinant human albumin, 213 μg/mL L-ascorbic acid 2-phosphate, and 4 mM L-lactic acid (Sigma-Aldrich) (16) for 7 days, with media changes every 48–72 hours. Following selection, hiPSC-CMs were maintained in RPMI with B27 supplement until day 30, at which point hiPSC-CMs were combined with hiPSC-CFs to generate ECTs. For the MACS-selected cells, thawed cells were maintained in RPMI with B27 supplement until day 30, which is the day of ECT generation. Cells were dissociated from the plate using 10× TrypLE (Thermo Fisher Scientific) and purified with the MACS system (Miltenyi Biotec) according to the manufacturer’s protocol. In summary, the cell suspension was quenched with EB20, passed through a 100 μM cell strainer (Thermo Fisher Scientific), counted, and centrifuged (200g at 4°C for 5 minutes). The remaining cell pellet was then resuspended (80 μL/5 × 106 cells) in “Miltenyi” buffer containing 0.5% w/v BSA (MilliporeSigma), DPBS, and 2 mM EDTA (Thermo Fisher Scientific). Non-Cardiomyocyte Depletion co*cktail (Miltenyi Biotec) was added (20 μL/5 × 106 cells) and incubated at 4°C for 5 minutes. The mixture was then washed with additional Miltenyi buffer (0.5% BSA w/v [MilliporeSigma] with 4% EDTA 0.5M solution [pH 8.0; Thermo Fisher Scientific] in DPBS, no Ca or Mg [Thermo Fisher Scientific]) and centrifuged (200g at 4°C for 5 minutes). The cell pellet was then resuspended (80 μL/5 × 106 cells) in Miltenyi buffer, and anti-biotin MicroBeads (Miltenyi Biotec) were added in a 20 μL/5 × 106 cell ratio and incubated at 4˚C for 10 minutes. Additional buffer was added to bring the final concentration to 500 μL/5 × 106 cells. A total of 500 μL of the cell suspension was loaded into the LS Column/MACS Separation apparatus (Miltenyi Biotec). Flow-through of the pure hiPSC-CMs was centrifuged (200g at 4°C for 5 minutes), resuspended, and taken for either ECT generation or flow cytometry analysis. Images of hiPSC-CMs were taken using 40× Olympus microscope objective via DSLR camera mounted to a cell culture microscope. Images were not digitally altered in any way.
Differentiation of hiPSCs into fibroblasts. Isogenic hiPSC-CFs were generated as previously described (48). In brief, hiPSCs were dissociated with Versene solution (Thermo Fisher Scientific) and seeded on Matrigel-coated 6-well plates at the density of 1.5 × 106 to 2.0 × 106 cells/well in mTeSR1 media (WiCell) supplemented with 10 μM Y-27632. Cells were maintained in mTeSR1 media for approximately 5 days with daily changes until they reached 100% confluency (day 0). On day 0, the cells were treated with 2 mL RPMI + B27 without insulin and supplemented with 12 μM CHIR99021 for 24 hours. After 24 hours, the media were changed to 2 mL RPMI + B27 without insulin for 24 hours. After 24 hours, the media were changed to 2 mL of the CFBM media with 75 ng/mL bFGF (WiCell). Subsequently, cells were maintained with CFBM + 75 ng/mL bFGF every other day until day 20. On day 20, cells were either taken for flow cytometry analysis or cryopreserved using a 1:9 ratio of DMSO (MilliporeSigma) and FBS (Thermo Fisher Scientific). Once thawed, the hiPSC-CFs were maintained in FibroGRO-LS media (MilliporeSigma) in uncoated 6-well culture plates (Corning) and passaged every 5–6 days. Low passage number (<12 passages) were used for hiPSC-ECT generation.
Flow cytometry. hiPSC-CMs and hiPSC-CFs were analyzed as previously described (42, 48). Briefly, dissociated cells were vortexed to disrupt the aggregates; neutralization followed, as equal volume of EB20 media were added. Cells were counted to designate 1 million cells for labeling. Cells were fixed in 1% paraformaldehyde, washed with FACS buffer (DPBS, 0.5% BSA, 0.1% NaN3), centrifuged (200g at 4°C for 5 minutes), and resuspended in about 50 μL FACS. Primary antibodies, including monoclonal anti–α-actinin (IgG1, MilliporeSigma, A7732, 1:500 dilution) and monoclonal anti-cTnT (IgG1, Thermo Fisher Scientific, A-21121, 1:200 dilution), were incubated with the cells according to the manufacturer’s instructions. Afterward, cells were washed with FACS buffer plus 0.1% Triton X-100 and centrifuged (200g at 4°C for 5 minutes), and all but 50 μL supernatant was discarded. Secondary antibody appropriate to the primary IgG isotype (IgG, Thermo Fisher Scientific, MS-113-P1; IgG1, Thermo Fisher Scientific, MS295P) and was diluted at 1:1,000 in FACS buffer plus 0.1% Triton X-100. Samples were incubated for 30 minutes at room temperature, washed in FACS buffer, and resuspended in FACS buffer for analysis. Data were collected on a FACSCalibur (Beckton Dickinson) and Attune Nxt (Thermo Fisher Scientific) flow cytometers and were analyzed using FlowJo.
hiPSC-ECT generation. Day 30 lactate-purified hiPSC-CMs were dissociated with 10× TrypLE and counted using a hemocytometer. MACS-purified hiPSC-CMs were taken straight from purification and counted using hemocytometry. hiPSC-CMs were subsequently resuspended at 2 × 106 CM/mL in fibrin ECT media (60.3% high-glucose DMEM; 20% F12 nutrient supplement; 1 mg/mL gentamicin; 8.75% FBS; 6.25% horse serum; 1% HEPES; 1× nonessential amino acid co*cktail; 3 mM sodium pyruvate; 0.004% [wt/vol] NaHCO3; 1 μg/mL insulin; 400 μM tranexamic acid; and 17.5 μg/mL aprotinin) (13) and incubated for at least 1.5 hours on a rotating platform at 37°C. Low-passage isogenic hiPSC-CFs were dissociated using 1× TrypLE (Thermo Fisher Scientific) and counted using a hemocytometer. After rotational culture, hiPSC-CMs were mixed with hiPSC-CFs in a 10:1 ratio per hiPSC-ECT, as previously described (69). In total, 1.25 mg/mL fibrinogen and 0.5 unit of thrombin were added to the cell mixture, and the cell suspension was quickly loaded onto a 20 × 3 mm cylindrical mold of FlexCell Tissue Train silicone membrane culture plate. Following polymerization of the fibrin matrix, ECTs were maintained with fibrin ECT media at 37°C with 5% CO2 for 4 weeks, with media changes every 2–3 days. Images of hiPSC-ECTs were taken using 5× Olympus microscope objective via microscope attachment on Sony digital camera. Images were not digitally altered in any way.
Twitch force and Ca2+TR measurements. Isometric twitch force and Ca2+TR were measured in hiPSC-ECT using protocols similar to those previously described (13, 70). In brief, each hiPSC-ECT construct was attached using sutures to a model 801B small intact fiber test apparatus (Aurora Scientific) in Krebs-Henseleit buffer (119 mmol/L NaCl, 12 mmol/L glucose, 4.6 mmol/L KCl, 25 mmol/L NaHCO3, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgCl2, 1.8 mmol/L CaCl2, gassed with 95% O2 to 5% CO2 [pH 7.4]) at 37°C. Krebs-Henseleit buffer flowed throughout the experiments at a rate of 1 mL/min. Force readouts were performed on a model 403A force transducer (Aurora Scientific). Stimulation was initiated on each hiPSC-ECT at 1 Hz (2.5 ms, 12.5 V). Constructs were stretched incrementally by 0.12 mm until plateau TF was achieved. Each construct was stretched to optimal length (until maximal twitch force was achieved based on Frank-Starling law; approximately 2.2 μm sarcomere length). Constructs were left to equilibrate for 20 minutes at 1 Hz. Following equilibration, twitch force production was measured with pacing at a frequency of 1.5 Hz both at baseline and following 5-minute preincubation with 1 μM isoproterenol. Automaticity was captured after pacing. Functional data generated from ECTs producing > 250 μN of raw twitch force were included in the analysis. After functional analyses, hiPSC-ECTs were washed with DPBS, flash frozen, and stored at –80˚C until proteomic analysis.
hiPSC-ECTs were then introduced to a Fura-2 loading solution consisting of Krebs-Henseleit buffer supplemented with 5 μM Fura2-AM (Invitrogen) and 1% (vol/vol) Chremophor EL (MilliporeSigma) with constant oxygenation (95% O2, 5% CO2) for 30 minutes at 37°C. Following Fura-2 loading, ECTs were left to equilibrate for 40 minutes with perfusion with Krebs-Henseleit buffer at a rate of 1 mL/min and paced at 1 Hz. Both twitch force and Ca2+TR data were recorded at pacing frequency 1.5 Hz. Fura-2 fluorescence was measured by alternately illuminating the preparation with 340 and 380 nm light (at a frequency of 250 Hz) while measuring the emission at 510 nm using IonOptix hardware and software (IonOptix Corporation). The emitted fluorescence and force data were stored as the 340 and 380 nm counts and as the ratio R = F340/F380. Data were analyzed using IonWizard 6.0 software (IonOptix). Under each condition, 40–60 successive contractions were collected and averaged. These data were exported to Microsoft Excel for parameter calculations. Statistical significances were determined by normal 2-tailed t test with α = 0.05 with 2-sided analysis. All data represented as data mean ± SEM.
Cryopreservation, sectioning, and IHC. hiPSC-ECTs were rinsed in DPBS (Thermo Fisher Scientific) in the well and incubated in 30 mM 2,3-butanedione monoxime in DPBS for 5 minutes. hiPSC-ECTs were then exposed to a filtered 30% sucrose (in DPBS) solution for 1 hour at room temperature, followed by a 1-hour incubation in a 1:1 mixture of optimal cutting temperature (OCT) compound (Tissue-Tek) and 30% sucrose solution in DPBS. The sucrose solution was aspirated from the well and hiPSC-ECTs were covered with OCT compound in the well before freezing the wells on a metal plate on dry ice. Cryopreserved hiPSC-ECTs were then stored at −80°C. The cryopreserved hiPSC-ECT disks were taken out of the plate before sectioning. Cryopreserved hiPSC-ECTs were then sectioned lengthwise at 6 μm thickness (Leica CM 1950UV), mounted onto charged slides (Superfrost +, Thermo Fisher Scientific), and fixed in 100% acetone for 15 minutes at 4°C. After drying, slides were placed in a vertical washer and rinsed with water for 10 minutes. Slides were then rehydrated in PBS and incubated in blocking buffer (0.15% Triton-X-100, 5% normal goat serum [NGS], 2 mg/mL BSA in PBS) for 1 hour at room temperature. Sections were incubated with primary antibodies overnight in a humidified chamber at 4°C with α-actinin (1:1,000, MilliporeSigma, A7811). Slides were incubated with secondary antibody (Alexa Fluor Plus 488; Invitrogen) at 5–8 μg/mL in blocking buffer for 1 hour at room temperature in a humidified chamber. Following labeling, sections were coverslipped using Prolong Gold Antifade Reagent (Invitrogen) with DAPI to label nuclei. Imaging was performed using Leica SP8 Confocal WLL STED Microscope using the 100× objective and Leica imaging software. Multiple sections from each hiPSC-ECT were imaged, and 100 sarcomeres were measured using manual annotation. The Z disc interval given by α-actinin staining was quantified via ruler function on the Leica Imaging software using the repeating fluorescence pattern of the α-actinin staining. Statistical significances were determined by normal 2-tailed t test with α = 0.05 with 2-sided analysis. All data represented as data mean ± SEM.
Global bottom-up proteomics. Global proteomics was performed similarly as previously described (71). Samples were randomized and blindly prepared to avoid bias. Cryopreserved hiPSC-ECTs or hiPSC-CMs were gently thawed on ice and hom*ogenized in 40 μL of Azo (46) buffer (0.1% w/v Azo, 25 mM ammonium bicarbonate, 10 mM L-methionine, 1 mM dithiothreitol (DTT), and 1× HALT protease and phosphatase inhibitor) using a handheld Teflon hom*ogenizer (Thomas Scientific). Samples were centrifuged (21,000g at 4°C for 15 minutes), and the supernatant was normalized to 1 mg/mL using 0.1% Azo buffer by the Bradford assay (Bio-Rad). Samples were reduced with 30 mM DTT for 1 hour and subsequently alkylated with 30 mM iodoacetamide for 1 hour. Trypsin Gold (Promega) was added in a 50:1 ratio to the samples and incubated for 24 hours. After 24 hours, trypsin was quenched with formic acid. Azo was then degraded at 305 nm for 5 minutes (Analytik Jena). Samples were centrifuged (21,000g at 4°C for 15 minutes), and the resulting supernatant was desalted using 100 μL Pierce C18 tips (Thermo Fisher Scientific) according to the manufacturer’s protocol. The peptides were dried in a vacuum centrifuge (21,000g at room temperature for 2 hours) and resuspended in 0.1% FA. The peptide concentrations were determined using Nanodrop One Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific).
LC–trapped ion mobility spectrometry–tandem MS (LC-TIMS-MS/MS) was performed using a nanoElute nanoflow LC system (Bruker Daltonics) coupled to the timsTOF Pro (Bruker Daltonics). In total, 200 ng of each peptide sample was loaded on an Aurora Elite capillary C18 column (IonOpticks). Peptides were separated using a 120-minute gradient at a flow rate of 400 nL/min (mobile phase A [MPA]: 0.1% FA; MPB: 0.1% FA in acetonitrile). For the first 60 minutes, a gradient of 2%–17% MPB was applied, then 17%–25% MPB was applied for the next 30 minutes, 25%–37% MPB for 10 minutes, 37%–85% MPB for 10 minutes, and 85% MPB for an additional 10 minutes. The column utilized nanoESI source for sample passage to the mass spectrometer. MS spectra were captured with a Bruker timsTOF Pro quadrupole-TOF (Q-TOF) mass spectrometer (Bruker Daltonics) operating in diaPASEF mode, using 32 windows ranging from m/z 400 to 1,200 and 1/K0 0.6–1.42. Data processing occurred similarly as previously described (72). LC-MS data were processed using DIA-Neural Network (DIA-NN) (73) using the default parameters unless noted in the following: 1% FDR, library-free search enabled; minimum fragment m/z, 200; maximum fragment m/z, 1,800; minimum precursor m/z, 400; maximum precursor m/z, 1,200; minimum precursor charge, 2; maximum precursor charge, 4; minimum peptide length, 7; maximum peptide length, 30; maximum missed cleavages, 2; MS1/MS2 mass accuracy, 10 ppm; quantification strategy, Robust LC (High Precision); NN classifier, souble-pass mode. All other analyses were performed in R. Protein-level quantification data were filtered using the “DAPAR” package (74) to include proteins identified in 2 of 3 runs in at least 1 sample group. Values were then median normalized, and missing values were imputed via ssla for partially observed values within a condition or set to the 2.5% quantile of observed intensities for observations that were missing entirely within a condition. The “DEP” R package was used to perform a Limma test between all specified contrasts, and the “IHW” R package was used to adjust all P values, using the number of quantified peptides per protein as a covariate. A Padj threshold of 0.05 and a log2 fold change threshold of 0.6 were set to identify significant changes to protein abundance. Subsequent data were visualized and plotted using ggplot2 (74). All data represented as data mean ± SEM.
Myofilament top-down proteomics. Myofilament proteomics was performed similarly as previously described (51). All steps were kept at 4°C to minimize artifactual modifications (52). In summary, cryopreserved hiPSC-ECTs were gently thawed on ice and hom*ogenized in 50 μL of HEPES extraction buffer (25 mM HEPES [pH 7.4], 60 mM NaF, 1 mM L-methionine, 1 mM DTT, 1 mM PMSF in isopropanol, 1 mM Na3VO4 containing protease and phosphatase inhibitors) using a handheld Teflon hom*ogenizer. The cell hom*ogenate was centrifuged (21,000g at 4°C for 30 minutes), and the supernatant containing cytosolic proteins was discarded. The resulting pellet was washed again in HEPES extraction buffer and centrifuged (21,000g at 4°C for 30 minutes), and the supernatant was discarded. The pellet was then hom*ogenized in 40 μL of TFA extraction buffer (1% TFA, 5 mM TCEP, 5 mM L-methionine) using a handheld Teflon hom*ogenizer. The hom*ogenate was centrifuged (21,000g at 4°C for 30 minutes), and the resulting supernatant was desalted with 5 volumes of MPA (0.1% formic acid in HPLC grade water) using a 10 kDa molecular weight cutoff filter (Amicon). Protein normalization was performed using a bovine serum standard curve and Bradford assay.
A NanoAcquity LC system (Waters) was used as part of the reverse phase chromatography (RPC) system. In total, 500 ng of the protein extracts was run through a home-packed PLRP-S capillary column (200 mm long, 0.25 mm Inner diameter (i.d.), 5 μm particle size, 1,000 Å pore size; Agilent Technology). The column was heated to 60°C at an 8 μL/min flow rate. The gradient is as follows, in terms of MPB (0.1% formic acid in 50:50 acetonitrile/ethanol): 10% MPB at 0–5 minutes, slow increase to 65% at 5–65 minutes, 95% at 70–75 minutes, back to 10% at 75.1 minutes, and steadily at 10% until 80 minutes. The eluted proteins were analyzed using a Bruker Impact II Q-TOF mass spectrometer (Bruker). The mass spectrometer parameters were as follows: end plate offset,500 V; capillary voltage, 4500 V; nebulizer, 0.3 bar; dry gas flow rate, 4.0 L/min at 200°C; quadrupole low mass, 650 m/z; scan rate, 1 Hz; m/z range, 200–3,000 m/z. Three technical replicates were collected for 1 sample across instrument run time to ensure data reproducibility and stability.
DataAnalysis 4.3 software (Bruker Daltonics) was used for all MS data analysis. A smoothing width of 2.01 using the Gaussian algorithm was applied to each chromatogram. The Maximum Entropy algorithm within the DataAnalysis 4.3 software was implemented to deconvolute spectra for proteins of interest at resolving power of 50,000. The sophisticated numerical annotation procedure (SNAP) algorithm was used to determine the monoisotopic masses of all deconvoluted ions. Relative quantification of protein phosphorylation is reported from deconvoluted spectra, in a relative abundance of a particular proteoform (Ptotal). Ptotal is equivalent to the ratio of the peak intensity of the proteoform (mol Pi) to the total sum of peak intensities of all proteoforms (mol protein) of the same protein. Statistical significances were determined by normal 2-tailed t test with α = 0.05 with 2-sided analysis. All data represented as data mean ± SEM.
Statistics. All statistical analyses were performed as 2-tailed Student’s t tests with assumed normal distribution, unless otherwise stated, with α = 0.05. Thus, a P value less than 0.05 was considered significant. All data are represented as data mean ± SEM, unless otherwise stated.
Study approval. Protocols for the generation of hiPSCs were approved by the Stanford University Human Subjects Research IRB, and written consent was obtained from all study participants.
Data availability. Source data for this manuscript available via MassIVE repository at massive.ucsd.edu with identifier: MassIVE MSV000091869. All raw data values are reported in the Supporting Data Values file. Any additional information is available by request from the corresponding authors.
