Isolation of adult mouse ventricular cardiomyocytes.

Adult (12- to 14-week-old) male C57BL/6 mice (The Jackson Laboratory, 000664) were injected with heparin (100 IU, i.p.) 20–30 minutes before dissection. Mice were anesthetized with isoflurane, cervical dislocation was performed, and hearts were removed rapidly and immersed in ice-cold calcium-free perfusion buffer. Hearts were retrogradely perfused through the aorta and digested with collagenase II (2 mg/ml) according to a previously established method (64). The ventricular tissue was then gently dispersed into cell suspension and centrifuged at 40 g for 3 minutes. Damaged myocytes and nonmyocytes were removed by a series of washes in buffer containing, sequentially, 100, 400, or 900 μmol/l CaCl₂. Cardiomyocytes were pelleted by centrifugation at 40 g for 3 minutes after each wash. After the final wash, cardiomyocytes were resuspended in cardiomyocyte culture medium, plated onto dishes precoated with 10 μg/ml laminin, and maintained for 2 hours in 37°C incubator with 5% CO₂ to allow them to adhere.