Figure 4. Reduction of TrpC3 does not prevent the increase in cell size associated with hypertrophy.

(A) Cell size measurement of ventricular myocytes expressing either TrpC3 or a negative control shRNA and stimulated with 20 µM PE for 48 hours. Size is shown as percent increase compared to the size of unstimulated myocytes expressing negative control shRNA (the left column). Cell size was measured using an automated fluorescence microscope and analysis program. For each condition, n >1000 transfected myocytes analyzed. Columns 2–4 were each significantly different than column 1, P<0.001. Columns 2 and 4 (PE-stimulated myocytes expressing negative control shRNA or TrpC3-shRNA, respectively) are not significantly different from each other. (B) The results in A were confirmed by using manual-measurements of myocyte cell size in cells stimulated with 20 µM PE for 60 hours. Cells expressing TrpC3-shRNA were not significantly different in size than those expressing LacZ-shRNA. n = 125 cells per condition. (C) Examples of myocytes expressing negative control shRNA (left) and TrpC3-shRNA (right) used in the manual measurement of cell size. Myocytes were stained with anti-α-actinin antibody (green), and cells that were expressing shRNAs are red. (D) Myocytes expressing LacZ- or TrpC3-shRNAs were stimulated with 20 µM PE for 60 hours and then fixed, immunostained with anti-ANP antibody, and measured simultaneously for cell size and ANP-immunofluorescence. TrpC3-shRNA had no effect on cell size (columns 2 and 3), but significantly decreased ANP expression (data displayed in Fig. 2F). Average n = 31 cells per condition. * indicates statistically significant difference compared to control, P<0.0001. For all bar graphs, data are represented as mean±SEM.