Smooth muscle and hiPSC-CM co-culture preparation and live-cell fluorescence imaging

Vascular smooth muscle cells were isolated using previously described methods shown to yield highly pure CD31/CD45/lineage marker-negative and α-smooth muscle actin-positive cells⁵⁵. Thoracic and abdominal aortae were excised and placed in ice-cold Dulbecco’s Modified Eagle Media (DMEM) containing Fungizone™▯ (1:1000; Thermo Fisher Scientific). Vessels were cleaned of connective tissue, minced into 1 mm segments, and transferred to a sterile 15 ml tube and washed (3x) with cold Tyrode’s solution containing (in mM): 126 NaCl, 44 KCl, 17 mM NaHCO₃, 1 MgCl₂, 10 Glucose, and 4 mM HEPES, pH 7.4. After washing, the solution was replaced with 1 ml of Tyrode’s solution containing 1 mg/ml collagenase (Type 2, Worthington) and 20 μM CaCl₂. The tissue was incubated with intermittent agitation (4–5 h, 37 °C, 5% CO₂), centrifuged at 300g, and resuspended in DMEM containing 10% fetal bovine serum and 1% penicillin streptomycin. The cells were plated in 35 mM Primaria™ plates (Corning) for 5 days prior to use (P0-3) in imaging experiments.

Human-induced pluripotent stem cell-derived cardiomyocytes used for this study were purchased as differentiated myocytes (iCell Cardiomyocytes², FujiFilm Cellular Dynamics International; 01434; manufacturer’s protocols) or derived from iPSCs in-house (iPSC line #SCVI15; Joseph Wu Laboratory, Stanford University). The SCVI15 iPSC line is previously established and deidentified; thus, the use of iPSCs in this study did not require IRB approval. Cryopreserved iPSCs were reprised and plated on Matrigel (Corning)-coated tissue culture grade dishes. The iPSCs were subsequently propagated in a feeder-free environment using StemFlex Medium (Thermo Fisher Scientific), replaced every two days, and maintained under standard incubation conditions (37 °C with 5% atmospheric CO₂). When approaching 85% confluency, iPSCs underwent clump cell passaging using Versene dissociation agent (Thermo Fisher Scientific). Cells were then differentiated using the iPSC Cardiomyocyte Differentiation Kit (Thermo Fisher) following the manufacturer’s instructions. Briefly, at ~75–80% confluency, cells were stimulated to differentiate by the addition of pre-warmed Cardiomyocyte Differentiation Medium A (day 1). On differentiation day 3, medium was replaced with pre-warmed Cardiomyocyte Differentiation Medium B. At differentiation day 5, medium was replaced with pre-warmed Cardiomyocyte Maintenance Medium and replenished every other day until differentiation day 10. At this time, differentiated cells were subjected to metabolic selection using cardiomyocyte enrichment medium (48.1 mL of glucose-free RPMI 1640 Medium, 1.7 mL of Bovine Albumin Fraction V 7.5% w/v, 0.4 mL of 1 M sodium lactate, and 0.13 mL of 250x ascorbic acid solution). This solution was then replaced every other day for 5 days, after which, cultures were replenished with kit-supplied Cardiomyocyte Maintenance Medium with replacement every other day until use.

Plasmids for peredox-mCherry were obtained via Addgene (pcDNA3.1-Peredox-mCherry, #32383) and Ad-peredox-mCherry adenovirus was generated by insertion to an adenoviral backbone (Type 5, dE1/E3; Vector Biolabs). After reaching 50–80% confluency, arterial myocytes were treated with Ad-peredox-mCherry (6.4 × 10⁷ PFUs, 50–100 MOI; 4 h at 37 °C). During infection and thereafter, cells were maintained in antibiotic-free DMEM supplemented with 2% FBS. For arterial myocyte-iPSC-CM co-cultures, Ad-peredox-mCherry-treated (24 h) arterial myocytes were seeded onto iPSC-CM monolayers, and the co-culture was maintained in DMEM supplemented with 5% FBS for 48 h prior to imaging.

Calibration of peredox-mCherry fluorescence for quantification of cytosolic NADH:NAD⁺ was performed as previously described²¹. Briefly, cells were perfused with a baseline extracellular solution consisting of (in mM): 121.5 NaCl, 2 KCl, 25 NaHCO₃, 1.25 NaH₂PO₂, 1 MgCl₂, and 2 CaCl₂ (pH 7.4, maintained by aeration with 5% CO₂). Bath temperature was monitored in all experiments throughout with a thermistor probe and maintained at 36.5–37.5 °C. Green (ex 405, em 525) and red (ex 560, em 630) fluorescence was monitored during sequential application (10–15 min each) of lactate:pyruvate at ratios of 500, 160, 50, 20, and 6. At the end of each experiment, maximum and minimum green:red fluorescence was recorded in the presence of 10 mM lactate and 10 mM pyruvate, respectively. For experiments testing the effects of hypoxia and electrical stimulation of co-cultures, cells were bathed in DMEM/5% FBS in an enclosed stage-top incubation system (Warner Harvard Apparatus) to allow equilibration of the bath solution to controlled O₂ levels (1–5%). Co-cultures and arterial myocytes alone (i.e., in the absence of iPSC-CMs) were electrically paced from 1–3 Hz using platinum electrodes connected to a MyoPacer field stimulator (IonOptix). Fluorescence images were acquired using a Keyence BZ-X800 epifluorescence imaging system with BZ-X800 Viewer software (version 01.01.01.03) in time-lapse mode with a 4X objective lens. Images were captured every 20 s in brightfield, green (T-sapphire), and red (mCherry) channels. Image series were converted to.avi files in Keyence Analyzer software (version 1.1.1.8) and analyzed using FIJI software (2.0.0-rc-69/1.53i; NIH).