Figure 4: Effects of Chronic (24 h) Cyclic Stretch on [Ca

(A) Control (top panel) and stretched (bottom panel) AE cells were stained with Fluo-4 following administration of hyperforin to activate TRPC-6. In control cells, strong and continuous Ca2+ influx through the evenly distributed TRPC-6 channels at the cell surface is indicated (white arrows, top panel inset). In chronically stretched cells, Ca2+ influx at the cell membrane is restricted to small, localized areas that form a green punctate pattern (white arrows, bottom panel inset). (B) Bar graph showing the effects of chronic stretch, hyperforin, and 1-oleoyl-2-acetyl-sn-glycerol (OAG), a mechanosensitive channel activator, on the [Ca2+]i (respective cell numbers = 26, 30, 32, 25, 33, 40, in the order shown in graph). Ca2+ uptake by TRPC-6 and mechanosensitive Ca2+ channels, in general, is lower in the 24-h stretched AE cells compared with the non-stretched (control) cells. (C) Representative TRPC-6 immunoblot of biotin-labeled/streptavidin-immunoprecipitated cell membrane fraction (M) and total cell lysate (T) from 0-, 1-, and 24-h stretched AE cells, showing depleted levels of TRPC-6 in the cell membrane fraction of the 24-h stretched cells. Bar graph shows quantification of TRPC-6 levels from 3 separate experiments. β-tubulin immunolabeling was used as loading control. See Supplemental Figure 7C for full Western blots. All values are mean ± SEM. *p < 0.001 versus control cells. See Supplemental Videos 2, 3, and 4. Abbreviations as in Figures 1, 2, and 3.