Table 1.
Channel names, and gene names.
Fig 1.
Mechanosensitive ion channel activity in human aortic valve interstitial cells.
A: Representative recording of a cation non-selective mechanosensitive channel (MSCNS) in cell-attached patch configuration (C.A.), activated in response to 255 ms long negative pressure pulses, from 0 to −80 mm Hg, applied in increments of −10 mm Hg, at a holding potential of -80 mV. Each trace shows the mechanically-induced changes in membrane current in response to applied pressure. B: Current-voltage relation obtained from MSCNS single channel activity at a pressure of -20 mm Hg in the cell-attached configuration for a representative cell. C: Representative recording of a potassium-selective MSC (MSCK) in cell-attached and in inside-out configurations, in response to 500 ms long negative pressure pulses, from 0 to -80 mmHg, applied in increments of -20 mm Hg, at a holding potential of 0 mV. D: Current-voltage relation obtained from MSCK single channel activity at a pressure of -20 mm Hg in the inside-out configuration for a representative cell. E: The typical progressive activation of MSCK following patch excision. F: Percentage of cells in which the two types of MSC were recorded, MSCNS = 156 cells (from 8 patients), MSCK = 64 cells (from 5 patients).
Fig 2.
Protein levels of mechanosensitive ion channels in phenotypically different VIC populations.
A: Western blots showing expression of MSC in cultures of VICFB, VICMB, and VICOB. B: Quantification of relative expression of individual channel proteins in cells of each phenotype, normalized to GAPDH levels (n = 4 patients; * = p<0.05).
Fig 3.
Protein expression of mechanosensitive ion channels in non-calcified and calcified aortic valve tissue from patients.
A: Western blots showing expression of various MSC, and of markers of calcification in cusp tissue from calcified and non-calcified valves. B: Quantification of relative expression of individual channel proteins and calcification markers in non-calcified and calcified valves (n = 6 patients; * = p<0.05, ** = p<0.01).
Fig 4.
Representative images of immunohistochemical localisation of mechanosensitive ion channel in non-calcified and calcified aortic valve cusp tissue.
Tissue sections were stained with antibodies against MSCK TREK-1 and Kir6.1, and against MSCNS TRPM4, TRPV4, and TRPC6. Signal (brown staining) from each antibody was assessed on sections from 8–10 individual valves. The sections are orientated with the ventricularis at the top of each section.
Fig 5.
Role of TREK-1, TRPV4 and TRPC6/3 channels in stretch-induced changes in collagen mRNA expression in VICFB.
Changes in mRNA expression (relative to 18S) for COL I and COL III in VICFB under static conditions (white bars), after cyclic stretch (grey bars), and after stretch in the presence of either the TREK-1 channel inhibition by spadin, the general MSCNS blocker streptomycin, the TRPV4 inhibitor RN-9893 or the TRPC6/3 inhibitor GSK417651A (hatched bars) after 24 h of stretch. Gene expression was measured by RT-PCR and normalized to the housekeeping gene (n = 4 and 6 patients for COL I and III respectively; * = p<0.05, ** = p<0.01, *** = p<0.001).
Fig 6.
Effect of streptomycin on VICFB migration in a cell-culture scratch assay.
Phase-contrast microscopy image of scratch assays, showing VICFB and scratch edge on Day-0 (top row) and Day-3 (bottom row) in the absence (control) and presence of 100 μM streptomycin. Scratch edge in each panel is indicated by the white dotted-line. Quantification of migration (day-3) in the control and streptomycin treated cells is shown the lower panels (n = 6 patients; *** = p<0.001).
Fig 7.
Effect of inhibitors of TRPV4 and TRPC6/3 on VICFB migration in a cell-culture scratch assay.
Phase-contrast microscopy image of scratch assays, showing VICFB and scratch edge at Day-0 (top row) and Day-3 (bottom row) in the absence (control) and presence of different concentrations of (A) the TRPV4 inhibitor RN-9883 and (B) the TRPC6/3 inhibitor GSK417651A. Scratch edge in each panel is indicated by the white dotted-line. Quantification of migration (day 3) as a function of different concentrations of RN-9883 and GSK417651A is shown in the lower panels of A and B, respectively (n = 6 patients; * = p<0.05).