FIGURE 2.

Adenoviral expression of cMyBP-C^C10mut in cultured isolated rat cardiomyocytes. Isolated rat cardiomyocytes were cultured without infection (uninfected, UI), with adenovirus and without protein coding sequence (empty vector, EV), with cMyBP-C^WT, or with cMyBP-C^C10mut. Adenovirus-derived cMyBP-C (WT and C10^mut) are N-terminally cMyc-tagged to distinguish AV-derived cMyBP-C from endogenous cMyBP-C. Low (L) and high (H) MOI of cMyBP-C^WT and cMyBP-C^C10mut were used to achieve dose-dependent protein expression. Cells were harvested 48 h postinfection. A, transcript levels of cMyBP-C relative to GAPDH expression and UI controls show a significant increase in cMyBP-C transcripts in a MOI-dependent manner. B, representative Coomassie-stained gel of total lysates of cultured cardiomyocytes show no apparent differences in overall protein levels. C, immunoblotting for cMyc (virus-derived cMyBP-C), cMyBP-C (endogenous + virus-derived cMyBP-C), and α-TPM as a loading control. D, quantification of cMyc levels, showing that similar levels of cMyBP-C^WT and cMyBP-C^C10mut were expressed 48 h postinfection. E, quantification of cMyBP-C levels, normalized with the levels of α-TPM, showed no significant increases in total cMyBP-C levels except cMyBP-C^C10mut high MOI expression. The data in A, C, and D were averages ± S.E. from three independent experiments. *, p < 0.01 versus empty vector/uninfected; #, p < 0.01 versus respective low MOI virus titers (n = 4–6).