Primary culture of cardiomyocytes and adenovirus infection. Calcium-tolerant adult rat ventricular myocytes were obtained from 10- to 14-week-old male Sprague–Dawley rats (240–260 g) using previously published methods with minor modifications [14]. Animals were used for the present study in accordance with our institutional and the NIH Guidelines for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996). The isolated myocytes were maintained in 1% BSA-Ringer solution (125 mM NaCl, 5 mM KCl, 2 mM NaH2PO4, 1.2 mM MgSO4, 25 mM HEPES, 5 mM sodium pyruvate, 11 mM glucose,
Walrycin B 7.4 adjusted with NaOH) containing 1.8 mM extracellular Ca2+. This procedure usually yielded more than 50% viable rod-shaped ventricular myocytes with clear sarcomere striations. Myocytes with obvious sarcolemmal blebs or spontaneous contraction were not used. Myocytes were plated on laminin-coated coverslips and incubated at 37 °C, 5% CO2 in Dulbeco’s modified Eagle’s medium (DMEM) containing 5% fetal bovine serum, 50 U/ml penicillin, 50 μg/ml streptomycin, 5 mM taurine, 5 mM carnitine, and 5 mM creatine. After 2 h incubation, unattached
cells were removed by washing with serum-free DMEM and incubated with same media containing appropriate adenovirus (MOI 100) for 1 h followed by incubation replaced with serum-free DMEM at 37 °C, 5% CO2 for 24 h. The infection efficiency of cardiomyocytes infected was monitored using LacZ staining method in Ad-LacZ infected myocytes [15]. For biochemical analysis, cultured myocytes were washed two times with pre-warmed PBS and harvested quickly using cell scraper after adding cold PBS followed by centrifugation at 90g for 3 min. Only pellets were quickly frozen and stored at ?80 °C until use.
