Adenoviral Infection of Adult Mouse Myocytes

An important requirement for developing methods to investigate cell signaling is a system for introducing exogenous DNA to myocytes. Adenoviral infection has been used for high-efficiency gene transduction in both rat and mouse cultured myocytes (> 90% efficiency)(15,26,27). To test the capacity for adenoviral-mediated gene transduction in long-term cultures of adult mouse cardiac myocytes, myocytes were infected with an adenovirus-b-galactosidase ( b-gal) reporter gene. Low viral titers showed variable expression and a lower percentage of infected cells at 24 hours (Fig. 6A). Both the level of b-gal expression and the percentage of infected cells increased with increased viral titer (multiplicity of infection [MOI] 30, 39% blue myocytes; MOI 100, 74%; MOI 300, 90%; MOI 1000, 96%). After 72 hours (Fig. 6B), b-gal expression was extensive even at the lowest titer measured (MOI 30, 96% blue myocytes). Myocyte survival was decreased at the highest titer (MOI 1000, 51% survival), possibly due to adenovirus-induced toxicity. The effective expression of this reporter gene for 72 hours suggests that the expression of dominant-negative signaling proteins or vector-based RNAi for selective protein knock-downs should be possible in cultured adult mouse myocytes.

Fig. 6. Adenovirus infection and b-galactosidase expression in cardiac myocytes. Myocytes were cultured for 72 hr in medium supplemented with 10 mM BDM, 10 g/ml insulin, 5.5 g/ml transferrin, and 5 ng/ml selenium. Myocytes were infected at 0 hr with increasing titers (MOI 30 to 1000) of adenovirus containing a b-galactosidase transgene (b-gal). b-gal activity was assayed at (A) 24 and (B) 72 hr and myocytes were photographed under phase contrast microscopy (10X).

In summary, we present a protocol for reproducibly isolating large numbers of viable, rod-shaped myocytes and maintaining them in short-term culture (24 hours). Under these conditions, both ligand-induced signaling responses and excitation-contraction coupling remain intact. Further, we present a long-term culture system (72 hours), using medium supplemented with BDM and ITS to maintain rod-shaped myocytes for extended duration in culture. Under these conditions, 95% to 100% of cultured myocytes can be transduced with adenovirus to express exogenous DNA for 72 hours. Therefore, this protocol presents the opportunity for developing methods to interfere with cell signaling.