Long-term Culture of Adult Mouse Myocytes

A goal of the AfCS is to develop methods to interfere selectively with cell signaling proteins in cultured myocytes, using either vector-based RNA interference (RNAi) or the expression of dominant-negative signaling proteins. Using the AfCS protocol described herein, many of the cultured adult mouse myocytes did not maintain their rod-shaped morphology for longer than 24 hours*, which is not long enough to allow for the manipulation of gene expression. Therefore, we developed an alternative culture system that maintained viable, rod-shaped myocytes in culture for 72 hours.

Several agents were tested to improve the maintenance of viable, rod-shaped myocytes in culture (listed in Table 1). Viable, rod-shaped myocytes were best sustained in medium containing 2, 3-butanedione monoxime (BDM), a contractile inhibitor, as well as insulin, transferrin, and selenium (ITS). Myocytes cultured with 10 mM BDM and 10 mg/ml (1.7 mM) insulin, 5.5 mg/ml transferrin, and 5 ng/ml selenium showed no significant loss of rod-shaped morphology at 48 hours. At 72 hours, the number of rod-shaped myocytes was reduced by 30% (Fig. 5). Myocytes cultured without BDM and ITS showed a 20% loss of rod-shaped myocytes at 24 hours, but by 48 hours rod-shaped morphology was lost almost completely (Fig. 5). Similar losses of rod-shaped myocytes were observed with either BDM or ITS alone.

View larger image

Fig. 5. Myocytes cultured with 2, 3-butanedione monoxime (BDM) and insulin, transferrin, and selenium (ITS) for 72 hours. (A) Myocytes were plated at a density of 50,000 rod-shaped myocytes per 35mm culture dish and were cultured for 72 hr in medium with or without 10 mM BDM, 10 mg/ml insulin, 5.5 mg/ml transferrin, and 5 ng/ml selenium. Myocytes (both rod-shaped and round myocytes) were counted at 0, 24, 48, and 72 hr. Data show total myocyte number (rod + round, closed symbols) and rod-shaped myocyte number (open symbols) in culture medium alone (squares) or culture medium with BDM and ITS (circles). Data are mean ± S.E.M. for n = 11; * P < 0.05 for myocytes in medium alone vs. myocytes with 10 mM BDM and ITS at each time point; † P < 0.05 for myocytes at each time point vs. 0 hr for each group. (B) The percentage of rod-shaped myocytes at 24, 48, or 72 hr compared to 0 hr calculated by the formula: (number of rod shaped myocytes at X hrs)/(number of rod shaped myocytes at 0 hrs). Data are mean ± S.E.M. for n = 11; * P < 0.05 vs. 0 hr for each group. Myocytes cultured in (C) medium alone or (D) medium supplemented with BDM and ITS were photographed under phase contrast microscopy (10X) after 0, 24, 48, and 72 hr in culture.

Because the insulin concentration used (0.17 mM) activates insulin-dependent signaling pathways, we tested lower concentrations of ITS. A 1000-fold dilution of the ITS supplement (10 ng/ml, 1.7 nM, insulin, 5.5 ng/ml transferrin, 5 pg/ml selenium) maintained myocyte rod-shaped morphology almost as well. Compared to myocytes at 0 hours, 99% of myocytes were rod-shaped at 24 hours in medium with the reduced ITS concentration, 83% at 48 hours, and 66% at 72 hours.

The preservation of myocyte morphology by BDM might involve one or more previously proposed mechanisms, including the regulation of calcium entry through the L-type calcium channel(22) and Na⁺/Ca²⁺ exchanger(23), the regulation of calcium content in the sarcoplasmic reticulum(24), and the inhibition of contraction through dephosphorylation of troponin I and phospholamban(25). Regardless of the mechanism, at the concentration used, BDM appeared to have a minimal effect on cultured myocytes. It was previously shown that contraction is not inhibited by the presence of BDM in isolated myocytes(24). Furthermore, preliminary experiments suggested that ligand-induced changes in cAMP or phosphoprotein phosphorylation were not altered qualitatively by the presence of BDM in the culture medium. The effect of the ITS medium supplement to maintain rod-shaped myocyte morphology was most likely related to the known metabolic effects of insulin to stimulate glucose and amino acid uptake. However, at higher concentrations, insulin might also inhibit apoptosis by stimulation of the PI3-kinase signaling pathway. Therefore, the long-term culture of myocytes in medium supplemented with BDM and ITS appears to provide a stable platform for signaling experiments, and additional experiments are underway to characterize further myocytes in long-term culture.

*Although myocytes did not maintain their rod-shaped morphology past 24 hours, the myocytes were still viable as assessed by staining with vital dyes.