Isolation and Stimulation of Cardiac Cell Types

Hearts of wild-type and ΔPAR1 embryos (embryonic day [E]14.5) were isolated and incubated overnight in 0.25% trypsin/EDTA (Sigma-Aldrich). Cell suspensions were plated for 90 min to allow adherence of the CFs^{5 (link),16 (link),41 (link)}. Non-adherent meCMs were isolated by adherence overnight. MEFs were derived from wild-type embryos (E14.5) as described^{41 (link)}. In addition, rat neonatal (rn) CFs and CMs were isolated from 1 day old rat pups as described^{2 (link)}. The addition of 0.1 mM BrdU to the CM culture maintenance medium prevented the proliferation of any contaminating CFs. Equal distribution of both sexes within each litter was assumed. Cells were stimulated with Poly I:C (25 µg/mL, γ-radiated; Sigma-Aldrich), thrombin (IIa, 5–10 nM, human α-thrombin, Enzyme Research Laboratories, South Bend, IN, USA) or PAR1 agonist peptide (PAR1 AP) (TFLLR, 200 µM, Abgent, San Diego, CA, USA) in serum-free media (SFM)^{5 (link),30 (link),41 (link)}.