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First published online 30 September 2008
doi: 10.1242/jcs.024521

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Myofibroblast communication is controlled by intercellular mechanical coupling

¹ Laboratory of Cell Biophysics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Bâtiment SG–AA-B143, Station 15, CH-1015 Lausanne, Switzerland
² CNRS-UMR 6543, Centre A. Lacassagne, 33 Avenue Valombrose, Nice 06189, France

View larger version (33K): [in this window] [in a new window]   Fig. 1. Myofibroblasts and fibroblasts exhibit distinct periodic Ca2+[i] oscillations. Spontaneous Ca2+[i] oscillations in cultures of myofibroblasts (A-D) and of fibroblasts (E-H) (see also supplementary material Movie 1) were compared between contacting (continuous lines) and isolated cells (dotted lines). The Em340/Em380 fluorescence ratios of Fura-2-loaded cells were recorded every 3-5 seconds over regions of interest including the entire cell (A and E show Em380 fluorescence image) and are plotted against time (B,F). The dominant periods of spontaneous Ca2+[i] oscillations were determined by Fast Fourier Transform (Supplementary material Fig. S1A) of each profile and summarised in histograms for myofibroblasts (C, nexp=25, n=193) and for fibroblasts (G, nexp=18, n=184). (D,H) The dominant periods of Ca2+[i] oscillations were separately analysed for contacting cell (blue histogram fit, myofibroblasts, n=97; fibroblasts, n=83) and isolated cells (red histogram fit, myofibroblasts, n=96; fibroblasts, n=101). This revealed no influence of the physical cell contact on the mean oscillation period within each group (Table 1). Scale bar: 50 µm.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Period comparison of cell pairs quantifies intercellular coupling. (A) To quantify intercellular coordination we compared over time the periods (defined as the time intervals between two consecutive Ca2+[i] peaks) of cell pairs, here exemplified for two cells (cell 1, red; cell 2, blue). Fura-2 Em340/Em380 fluorescence ratios (solid line) were band-pass filtered to eliminate noise (A, dashed lines) (supplementary material Fig. S1A, inset). First, all peaks (symbols) were automatically detected in the filtered curves, and the periods were determined. Second, over the whole observation time (here 1200 seconds), a computer algorithm matched for every peak i of cell 1 the closest peak on the time scale of cell 2 (first match connected with green dashed line). (B) Third, the periods T1i and T2i following these matched peaks are displayed in a scatter plot to illustrate the degree of correlation between these two variables. In such diagrams, every data point represents the matched periods of two cells at a given time point (first matched periods indicated with dotted lines). All values are reported to one side of the diagonal (where T1i equals T2i) by plotting the longer period versus the shorter period (all points above the diagonal as in B) or vice versa (all points below the diagonal). This allows representation of two different experimental conditions in one scatter plot to facilitate direct comparison. The mean of all points (T1i, T2i) of two compared cells is represented by the centre of an ellipse of whose semi-major and semi-minor axes indicate s.d. The smaller the ellipse and the closer it is located with respect to the diagonal, the better the two cells are coordinated.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Ca2+[i] oscillations are coordinated between contacting myofibroblasts but random between fibroblasts and non-contacting cells. The periods between every two consecutive Ca2+[i] peaks in the Fura-2 Em340/Em380 fluorescence ratios of myofibroblasts (A) and fibroblasts (B) were compared over time for every possible cell pair combination within one image field (myofibroblasts, nexp=25 and ncells=193; fibroblasts, nexp=18 and ncells=184). All matching period pairs (square data points) obtained from cells that are not in physical contact are plotted as longer period versus shorter period above the diagonal (non-contacting pairs in myofibroblasts, n=324 and in fibroblasts, n=482). Period pairs obtained from all physically contacting cells are plotted as shorter period versus longer period below the diagonal (contacting pairs in myofibroblasts, n=60 and in fibroblasts, n=55). The occurrence of identical period pairs is colour-coded and normalised to the total number of data points in each condition (scale in %). The deviation of a period pair (one example encircled), i.e. its orthogonal distance to the diagonal, is demonstrated with a yellow line in A. By dividing the deviation by the distance z of the intersection point between the orthogonal and the diagonal to the origin (red section of diagonal) the relative deviation can be obtained (see Materials and Methods). The mean relative deviation is the average of all relative deviations and is represented as a dotted line.

View larger version (92K): [in this window] [in a new window]   Fig. 4. Intercellular junctions differ between myofibroblasts and fibroblasts. (A) Expression of myofibroblast marker -SMA, gap junction marker Cx43 and adherens junction markers pan-cadherin and β-catenin was assessed together with loading control vimentin, by western blotting. The cytoskeletal fractions are compared after 1-7 days culture between control fibroblasts (lanes labelled C) and fibroblasts treated with TGFβ1 to induce myofibroblast differentiation (lanes labelled T). (B) Band signal strength in western blots was analysed by optical densitometry and related to loading control vimentin (n=3). After 4 days of growth, Triton-X-100-extracted myofibroblasts (C) and fibroblasts (D) were immunostained for β-catenin (green), Cx43 (red) and -SMA (blue). Scale bars, 25 µm.

View larger version (83K): [in this window] [in a new window]   Fig. 5. Quantification of gap junction coupling by intercellular small molecule diffusion. To demonstrate gap junction coupling, individual myofibroblasts (A) and fibroblasts (B) in confluent monolayer were microinjected with Lucifer Yellow (yellow, *) and stained after diffusion for 5 minutes for cell nuclei (blue). Scale bar: 25 µm. (C) To quantify diffusion of small molecules through gap junctions, monolayers of myofibroblasts and fibroblasts were scrape-loaded with Lucifer Yellow (green) and neurobiotin (red) in control conditions (n=34) and in the presence of gap junction uncoupling agents palmitoleic acid (PA; n=11) and carbenoxolone (CBX; n=11). Scale bar: 100 µm. The number of cell layers receiving Lucifer Yellow (D) and neurobiotin (E) from the scrape wound was quantified; shown are means ±s.d. per experiment. *P0.001 compared with the respective control.

View larger version (26K): [in this window] [in a new window]   Fig. 6. Synchronisation of periodic Ca2+[i] oscillations between fibroblasts is mediated by gap junction coupling. Spontaneous Ca2+[i] oscillations in cultures of Fura-2-loaded myofibroblasts (A,C) and fibroblasts (B,D) were compared between contacting cells that were preselected for exhibiting coordinated periods. (A-B) Fura-2 Em340/Em380 ratios were recorded every 3 seconds over regions of interest including the entire cell and are here displayed for two contacting cells. During recording, gap junctions were uncoupled by adding 50 µM palmitoleic acid (PA) at the indicated time point. Arrows on top of the fluorescence profiles indicate how the position of Ca2+[i] peaks of cell 2 (grey) develops over time in respect to the matched peak of cell 1 (black); dots indicate simultaneous oscillations of both cells and changing arrow lengths over time demonstrate desynchronisation of the two cells. (C,D) For statistical analysis, all matched period pairs obtained before addition of palmitoleic acid are displayed as longer period versus shorter period (above diagonal) and all data obtained after palmitoleic acid treatment are plotted as shorter period versus longer period (below diagonal). The mean of all period pairs obtained from two contacting cells is represented by the centre of an ellipse of which semi-major and semi-minor axes indicate s.d. Each cell pair is represented by one colour. Note that ellipses become larger and more distant from the diagonal in fibroblasts after gap junction inhibition, indicating uncoupling of Ca2+[i] oscillations.

View larger version (27K): [in this window] [in a new window]   Fig. 7. Synchronisation of periodic Ca2+[i] oscillations between myofibroblasts implies adherens junction coupling, cell contraction and MS channels. Spontaneous Ca2+[i] oscillations in cultures of Fura-2-loaded myofibroblasts (A,C,E) and fibroblasts (B,D,F) were compared between contacting cells that were preselected for exhibiting coordinated periods. The corresponding Fura-2 Em340/Em380 ratios of two contacting cells are shown in supplementary material Fig. S3. (A,B) adherens junctions were disassembled by adding a mixture of anti-N- and anti-OB-cadherin peptides at 0.5 mg/ml for 45 minutes. (C,D) Cell contraction was inhibited by adding 1 mM BDM. (E,F) MS channels were inhibited by adding 300 µM Gd3+. All matched period pairs are reported above the diagonal before addition of the drug and below the diagonal after addition of the drug. The mean of all period pairs obtained from two contacting cells is represented by the centre of an ellipse of which semi-major and semi-minor axes indicate s.d.; each cell pair is represented by one colour. Note that ellipses become larger and more distant from the diagonal in myofibroblasts upon drug treatment, indicating uncoupling of Ca2+[i] oscillations after inhibition of adherens junctions, of contraction and of MS channels.

View larger version (37K): [in this window] [in a new window]   Fig. 8. Myofibroblasts and fibroblasts exhibit different kinetics of intercellular Ca2+[i] wave propagation. Myofibroblasts (A-C) and fibroblasts (D-F) were grown between 50-µm-wide non-adhesive lines, created on glass by means of microcontact printing (A,D, Fura-2 Em380 fluorescence image, scale bar: 25 µm) (see also supplementary material Movie 3). Individual cells were locally stimulated by touching gently with a micropipette (*); Ca2+[i] transients were analysed in the stimulated cell (yellow outline) and all following cells in the chain (blue to red to green; grey traces depict non-contacting control cells). Fura-2 Em340/Em380 fluorescence ratios were recorded at 1 frame/second over the indicated cell outlines and plotted over time with the respective colour (B,E); arrows indicate time delay of Ca2+[i] transient initiation after stimulation of the first cell (black line). A slow fluid flow applied perpendicularly to the lines excluded the possibility that released soluble factors induce a Ca2+ response. For quantitative analysis, fluorescence ratio intensity values were recorded along a line drawn across all cells in the chain (dotted line; A, 250 µm; D, 175 µm) and are displayed over time in a kymograph. In the kymograph image, delays of Ca2+[i] wave propagation at cell junctions appear as clear steps along the time axis (t=40 seconds) in the case of myofibroblasts (B) but are hardly detectable between fibroblasts (E). All time delays at cell junctions were measured on kymograph images and summarised in histograms for myofibroblasts (C, nexp=17, npairs=34) and for fibroblasts (F, nexp=24, npairs=55).

View larger version (53K): [in this window] [in a new window]   Fig. 9. Model of mechanical communication between contacting myofibroblasts. (A) The -SMA-positive stress fibres (green) of contacting myofibroblasts are connected to the ECM at sites of focal adhesions (FA, grey) and intercellularly at sites of OB-cadherin-type adherens junctions (AJ; light green). MS channels (violet) in the plasma membrane are closed in relaxed cells (inset A) and do not permit entrance of Ca2+ ions (orange). Cytoskeleton-mediated or extracellular signalling events trigger a Ca2+[i] transient (indicated by orange cytoplasm). (B) This rise in Ca2+[i] leads to stress fibre contraction of the left cell (indicated by thicker fibres and cell shortening) that is transmitted to the right myofibroblast at sites of adherens junctions. The induced stretch leads to the opening of MS channels (inset B). (C) The resulting influx of Ca2+ through open MS channels then triggers a contractile event in the right cell that again feeds back to the left myofibroblast. At this point, the cycle can start again.

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