Hypertrophic Stimulation Increases β-actin Dynamics in Adult Feline Cardiomyocytes
Figure 2
β-actin is localized to adult cardiomyocyte cytoskeleton and contributes to contractility of cardiomyocytes.
A) Adult cardiomyocytes cultured on laminin coated cover slips were fixed and stained for β-actin (red) along with α−actinin (green), and the image was recorded using a confocal microscope. B) Adult cardiomyocytes cultured on laminin coated cover slips were infected with β-actin-GFP adenovirus (MOI = 10). After 24–36 h of infection, the cells were fixed and stained using α−actinin antibody. The GFP fluorescence corresponding to β-actin and red fluorescence corresponding to α−actinin staining were recorded by confocal microscopy. C) Cells were processed as in B above except for they were costained for vinculin localization (red). D) Adult cardiomyocytes were infected with an equal MOI of β-gal (Control) or β-actin-GFP adenovirus and the proteins were allowed to express for 24–36 h. After this period, the cells were subjected to electrical stimulation, and cell shortening measurement was performed as described under Materials and Methods. The percent shortening is recorded for 15 cells in each condition and the experiment was repeated at least thrice. E) Adult cardiomyocytes were transduced with either β-actin antibody or control IgG using HVJ as described under Materials and Methods. After overnight incubation with the transduction mixture, cells were then subjected to cell shortening measurement as described under Materials and Methods. The percent shortening is recorded for 15 cells in each condition and the experiment was repeated at least thrice. F) Adult feline cardiomyocytes grown on laminin-coated cover slips were transduced with either β-actin antibody or control IgG using HVJ envelope as described under Materials and Methods. After overnight incubation with the transduction mixture, the cells were fixed and incubated with rabbit α−actinin primary antibody. Subsequently the cells were immunostained with Alexa 568– labeled anti-rabbit IgG to stain for α−actinin and, Alexa 488– labeled anti-mouse IgG to detect β-actin antibodies that were transduced into the cells by HVJ envelope. Scale bar = 5 µm.