Isolation and Culture of Murine Cardiomyocytes

All animal work was approved by the University of Pittsburgh Division of Laboratory Animal Resources. Primary cardiomyocytes were isolated from Swiss Webster or Black 6 mouse neonates (P1–P3) as described (Ehler et al., 2013 (link); Wickline et al., 2016 (link)). For protein isolation, Swiss Webster-derived cardiomyocytes were plated onto 35 mm dishes (1×10⁶ cells/dish) coated with Collagen Type I (Millipore). For immunostaining, cardiomyocytes were plated onto 35 mm MatTek dishes with 10 mm insets coated with Collage Type I. Cardiomyocytes were plated in plating media: 65% high-glucose DMEM (Thermo Fisher Scientific), 19% M-199 (Thermo Fisher Scientific), 10% horse serum (Thermo Fisher Scientific), 5% FBS (Atlanta Biologicals) and 1% penicillin-streptomycin (Thermo Fisher Scientific). Media was replaced 16 h after plating with maintenance media: 78% high-glucose DMEM, 17% M-199, 4% horse serum, 1% penicillin-streptomyocin, 1 µM AraC (Sigma) and 1 µM isoproternol (Sigma). Cells were cultured in maintenance media for 2–4 days until lysis or fixation.