Isolation of Ventricular Myocytes from Mouse Hearts

Myocytes were isolated from the LV of adult (10–20-wk-old) male and female WT, Fgf13 floxed (female Fgf13^fl/fl and male Fgf13^fl/y), cFgf13KO (female cFgf13^−/− and male cFgf13^−/y), and Fgf12KO mice by enzymatic and mechanical dissociation using previously described methods (Xu et al. 1999 (link); Brunet et al., 2004 (link)). In addition, LV myocytes were isolated from cFgf13KO mice (4–6 wk) after retro-orbital (eGFP- + FGF12B-expressing) AAV9 injections. Briefly, hearts were quickly removed from avertin-anesthetized mice and perfused retrogradely through the aorta with collagenase-containing solution (Type II, Worthington) at 37°C. After 15–20 min perfusion, the LV was separated, minced, and dispersed by gentle trituration. The resulting cell suspension was filtered and resuspended in serum-free medium-199 (M-199; Sigma-Aldrich). Isolated myocytes were plated on laminin-coated coverslips and maintained in a 95% air-5% CO₂ incubator at 37°C until used (within 24–48 h) in electrophysiological experiments.