Ls004174

NCM and NCF were isolated from heart ventricles of one- to three-day old Wistar rats (Janvier Labs, Le Genest-Saint-Isle, France) as previously described [41 (link)]. Briefly, minced ventricular tissue was digested with type II collagenase (LS004174, Worthington, Lakewood, NJ, USA) and pancreatin (P3292, Sigma-Aldrich, St. Louis, MO, USA), and dissociated cells were pelleted by centrifugation at 800× g. NCMs were separated from NCFs via discontinuous Percoll gradient (bottom 58.5%, top 40.5% [v/v], P4937, Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 1600× g. NCMs were seeded at 2.5 × 10⁶ cells per 100 mm collagen-coated culture dish and were cultured in a 4:1 mixture of DMEM (D1152, Sigma Aldrich, St. Louis, MO, USA) and Medium 199 (M4530 Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% horse serum (HS) (16050122, Thermo Fisher Scientific, Waltham, MA, USA), 5% fetal calf serum (FCS) (30-2020, ATCC, Manassas, VA, USA), and 1% penicillin-streptomycin (PS) (15140-122, Gibco, Waltham, MA, USA) for 5 days at 37 °C under 5% CO₂ atmosphere. NCFs were seeded at 2 × 10⁶ cells per 100 mm culture dish and were cultured in DMEM (31966-02, Gibco, Waltham, MA, USA), 10% FCS, and 1% PS for 48 h at 37 °C under 5% CO₂ atmosphere. NCF at passage 1 were used for experiments.

CSPs were isolated as previously described [21 (link)]. The minced cardiac tissue from freshly isolated mouse hearts was digested using collagenase type 2 (LS004174, Worthington Biochemical Corp, Lakewood, NJ, USA) and dispase (17105–041, Life Technologies). The cells were resuspended at a density of 1.0 × 10⁶/mL in PBS containing 3% fetal bovine serum. The cells were incubated in 1 μg/mL Hoechst 33342 dye (H3570, Molecular Probes) for 60 min at 37°C in the dark, with or without 50 μM verapamil. Following incubation, the cells were analyzed using Altra flow cytometric analysis (Beckman Coulter, Fullerton, CA, USA). Hoechst 33342 dye was excited at 350 nm using an ultraviolet laser. Fluorescent emission was detected using 450-nm bandpass (Hoechst blue) and 675-nm longpass (Hoechst red) filters, respectively. Isolated CSPs were used for in vitro incubation experiments or directly centrifuged onto slides using a CytoSpin Cytocentrifuge (Thermo Fisher Scientific, Waltham, MA, USA) at 71.8 × g for 5 min. For the β-galactosidase expression analysis, isolated CSPs and residual cardiomyocytes were fixed with 0.25% glutaraldehyde for 10 min and stained in X-gal staining solution for 1 h. For normal immunofluorescence staining, CSPs were fixed with 4% paraformaldehyde for 10 min and stained using the method described for fresh-frozen section staining.

EVs were isolated by ultracentrifugation from HeLa conditioned culture media (media from 4 dishes/sample), human plasma (250 µL/sample) and human cardiac biopsies (200 mg/sample were minced and incubated in 6 mL PBS 1X containing 1 mg/mL type II collagenase (LS004174, Worthington) at 37 °C for 30 min with agitation). Conditioned culture media, plasma and cardiac minced tissue-containing PBS were centrifuged at 3000 g to remove debris and dead cells. The supernatants were ultracentrifuged at 20,000g (70 min, 4 °C) to pellet large EVs (lEVs), then at 100,000g (70 min, 4 °C) to pellet small EVs (sEVs). Rotors used were either Beckman 50.2 Ti or SW-32.1 (337,901/369651 Beckman Coulter France, Villepinte, France).

Mouse VSMCs were derived from thoracic-abdominal aortas isolated from 3–4-week-old mice by removing the adventitia and endothelial layer using an enzyme mixture containing collagenase Type II (Worthington Biochemical, LS004174), soybean trypsin inhibitor (Worthington Biochemical, LS003570), and elastase (Worthington Biochemical, LS002279). They were maintained in DMEM/F12 media (Gibco, 11320) containing 20% FBS, L-glutamine, and P/S. Cells were used at passages under 10 for the experiments.

Coronary PVAT was obtained from 4 patients undergoing heart transplantation and digested as described previously.^{28 (link)} Briefly, coronary PVAT was collected in 10 mL DMEM (11885084; Gibco) containing 10% fetal bovine serum (10091148; Gibco) on ice. Samples were washed by PBS to remove the remaining blood cells and then cut into small pieces. These small pieces were washed by PBS again and digested in DPBS (14040133; Gibco) containing 0.1% (w/v) collagenase (LS004174; Worthington) at 37 °C for 60 minutes, followed by gently shaking. The cell suspension was centrifuged at 300g at 21 °C for 5 minutes. A vigorous shaking was performed for 10 s to ensure that individual cells are released from the strands of fibrous tissue. Then a second centrifugation (300g) was performed and supernatant was discarded, and the cell pellets were resuspended in 10 mL DMEM, followed by a third centrifugation (300g). Finally, the supernatant was discarded, and the cell pellets were resuspended in 10 mL fibroblast growth medium (C-23010; Sigma) containing 10% fetal bovine serum and 1% penicillin and streptomycin and culture in 5% CO₂ at 37 °C. Early passage (P2 and P3) cultures were used for the cell migration assay and cell culture immunostaining.