TrpC3 Regulates Hypertrophy-Associated Gene Expression without Affecting Myocyte Beating or Cell Size (original) (raw)
Figure 5
TrpC3 regulates baseline [Ca2+]i and the PE-induced [Ca2+]i elevation but does not affect beat frequency.
(A) Mean [Ca2+]i (normalized) obtained by fura-2 measurements in uninfected myocytes. Myocytes maintained in media containing 2 mM Ca2+ and the CaV1.2 blocker verapamil were then stimulated with 20 µM PE (in addition to verapamil) in the presence of 2 mM extracellular Ca2+ (left panel, n = 58 cells) or 0 mM extracellular Ca2+ (center panel, n = 101 cells) for the time indicated by the bar at the top of the panels.. Right panel (n = 58 cells), myocytes previously stimulated with PE and incubated in media containing 2 mM Ca2+ and verapamil were then perfused with depolarizing media containing 65 mM K+ and no verapamil. (B-C) [Ca2+]i measurements in myocytes infected with lentiviruses expressing shRNAs targeting LacZ or TrpC3. Ratiometric fura-2 [Ca2+]i measurements were performed on myocytes treated with verapamil and then stimulated with 20 µM PE (in addition to verapamil) for the time indicated by the bar at the top of the panels. Representative traces from single myocytes are shown in B (top panel depicts examples of individual cells expressing LacZ-shRNA; bottom panel, TrpC3-shRNA). Average traces are shown in C and represent the mean of 82 cells expressing LacZ-shRNA (blue) and 117 cells expressing TrpC3-shRNA (r_ed_). (D) Mean baseline, peak, and peak minus baseline [Ca2+]i of cells depicted in C. Average baseline [Ca2+]i sampled at 5 time-points before treatment with PE (columns 1 and 2), average amplitude of the [Ca2+]i peak triggered by PE stimulation (columns 3 and 4), and peak minus baseline calculated for each individual myocyte and then averaged across the population (columns 5 and 6). (E) Spontaneous beat frequency of myocytes infected with lentiviruses expressing either LacZ- or TrpC3-shRNAs and stimulated for 48 hours with PE to induce hypertrophy. Beat frequency was measured by phase contrast microscopy in 20 wells per condition. TrpC3 shRNAs had no effect on myocyte beat frequency. (F) Fluo-4 measurements of [Ca2+]i in spontaneously-beating myocytes expressing LacZ- or TrpC3-shRNAs and stimulated for 72 hours with PE. Calcium traces are displayed as Fluo-4 intensity values normalized by the minimum Fluo-4 value recorded from the given cell. Displayed traces are from single representative myocytes expressing either LacZ-shRNA (top panel) or TrpC3-shRNA (bottom panel). Comparing Fluo-4 calcium traces for myocytes expressing LacZ-shRNA (n = 23 cells) and TrpC3-shRNA (n = 23 cells) did not detect any statistical difference in beat frequency, peak:baseline ratio, or decay constants. * indicates statistically significant difference compared to control, P<0.0001. ** indicates P<0.005. For all bar graphs and the traces in B, data are represented as mean±SEM.