Culture of Adult Mouse Cardiac Myocytes

We next defined conditions to sustain myocytes in culture. Unfortunately, mouse myocytes, unlike rat and rabbit myocytes, are highly susceptible to contracture in culture, which causes rod-shaped myocytes to become rounded. While the cause of this contracture is unknown, it presents a significant obstacle to culturing adult mouse myocytes. Based on the methods of Zhou et al.(15), we developed a protocol for the culture of adult mouse cardiac myocytes that consistently maintains rod-shaped myocytes for 24 hours (Fig. 1). At 0 hours, myocytes were 90% rod-shaped. At 24 hours, cultured myocytes were 80% rod-shaped, and cultures maintained 88% of the viable, rod-shaped myocytes originally plated (Table 3). Therefore, combined with our ability to isolate myocytes from several hearts, sufficient numbers of myocytes could be cultured to perform large-scale signaling experiments.

Fig. 1. Adult mouse cardiac myocytes cultured for 24 hours. Myocytes were photographed under both phase contrast (10X and 20X) and differential interference contrast (60X) microscopy after (A) 0 hr and (B) 24 hr in culture.

We identified additional factors that improved the maintenance of viable, rod-shaped myocytes in culture (summarized in Table 1). The pH of the culture medium was the most critical factor. Myocytes were cultured in Modified Eagle's Medium (MEM) with Hanks' Balanced Salt Solution, supplemented with 0.1 mg/ml bovine serum albumin and penicillin 100 U/ml at 37 °C in a 2% CO2 incubator. This resulted in medium with pH 6.9 to 7.0 (compared to normal medium with pH 7.4), similar to medium used by Zhou et al.(15), which provided a protective effect on the myocytes, possibly by reducing spontaneous contractile activity.