Research ArticleBIOPHYSICS

Intercellular communication controls agonist-induced calcium oscillations independently of gap junctions in smooth muscle cells

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Science Advances  05 Aug 2020:
Vol. 6, no. 32, eaba1149
DOI: 10.1126/sciadv.aba1149
  • Fig. 1 Effect of matrix stiffness on agonist-induced Ca2+ oscillations in SMCs.

    (A) To study the role of altered matrix stiffness on the time period of agonist-induced Ca2+ oscillations, we micropatterned a 2D approximation of the in situ organization of SMCs. Cells were also cultured on nonpatterned surfaces in three different densities: (B) isolated, (C) sparse, and (D) confluent. The colors in Fig. 1 (A to D) correspond to cytosolic [Ca2+] concentrations as indicated by the color bar in Fig. 1A. Scale bars, 200 μm. (E) Increasing matrix stiffness from 0.3 to 13 kPa caused a significant decrease in Ca2+ oscillation period in SMC rings (t test, P < 0.001; N = 4 each). (F and G) The periods of SMC Ca2+ oscillations were not affected by matrix stiffness in both isolated (soft, N = 14; stiff, N = 12) and sparse (N = 4 each) conditions (t test, P = 0.93 and P = 0.481, respectively). (H) Confluent cells behaved like those patterned in a ring, with cells plated on stiff matrix exhibiting significantly faster Ca2+ oscillations in response to 10−5 M histamine compared to those on a soft matrix (soft, N = 5; stiff, N = 7; Mann-Whitney rank sum test, P = 0.003). These results demonstrate that matrix stiffness can modulate the agonist-induced Ca2+ response of confluent SMCs but not that of isolated cells.

  • Fig. 2 Effect of matrix stiffness and confluence on the correlated nature of Ca2+ oscillations.

    (A) The cross-correlation coefficients (ρi,j) range in values from 1 (positively correlated, pink) to 0 (uncorrelated, white) to −1 (negatively correlated, green), as shown in a representative 24 × 24 cell matrix. (B) Examples of Ca2+ oscillations measured in two cells with high pairwise correlation (ρ = 0.78) and low pairwise correlation (ρ = 0.02). A.U., arbitrary units. (C) In isolated cells, the Ca2+ oscillations were generally uncorrelated with a probability density function centered around zero. ECM stiffness did not affect pairwise correlations in isolated SMCs (soft, N = 5; stiff, N = 4; P = 0.733, two-way ANOVA). (D) In confluent cells, there is a statistically significant increase in ρ, indicating that the Ca2+ oscillations were more synchronized on stiffer ECM (soft, N = 4; stiff, N = 5; two-way ANOVA, P = 0.007). (E and F) To evaluate the time it takes after agonist addition for Ca2+ oscillations to become synchronized, we calculated ρi,j(τ) within a 120-s moving window. (E) ρi,j(τ) in isolated cells does not change over time (soft, N = 5; stiff, N = 4; P = 0.274) or with stiffness (P = 0.303) (two-way ANOVA). (F) However, in confluent cells on stiff matrix, ρi,j(τ) shows a significant increase after 30 s (soft, N = 4; stiff, N = 5) (two-way ANOVA, P < 0.001). Error bars indicate SD.

  • Fig. 3 Effect of matrix stiffness on intercellular communication via gap junctions.

    Confluent SMCs on soft (A) and stiff (B) matrix were stained for the gap junction protein Cx43, actin, and the nucleus. (C) Gap junctions were counted for each SMC in individual frames on soft (N = 5) and stiff (N = 5) matrix, represented by the mean and SD of these measurements, and we found no statistical difference due to matrix stiffening (t test, P = 0.977). (D) Diffusion through gap junctions was quantified by bleaching a small fluorescent molecule within a cell and measuring signal recovery due to intercellular diffusion, a technique called gap-FRAP. (E) A representative recovery curve for one cell shows a recovery of ~40% its initial fluorescence. These values were used to calculate mobile fraction, a measure of diffusion efficiency through gap junctions. (F) Matrix stiffness had no effect on mobile fraction (t test; soft, N = 16; stiff, N = 18; P = 0.532). The gap junction blocker βGA significantly reduced the recovery (D) and mobile fraction (F) (N = 18, Mann-Whitney rank sum test). (G) Despite blocking gap junctions with βGA, there was no effect on Ca2+ oscillation periods in confluent cells on either soft (N = 6) or stiff (N = 4) matrix (two-way ANOVA, P = 0.784 treatment within stiffness). Scale bars, 30 μm.

  • Fig. 4 Role of mechanical force in Ca2+ wave propagation through multicellular ensembles of SMCs.

    (A) SMCs in confluent layers form organized clusters of cells, with certain cells aligned end-to-end along their contractile axis (parallel), and others branching off at an angle (perpendicular). Scale bars, 250 μm. Insets (a) and (b) show cells 2 and 4 parallel to the contractile axis of cell 1, whereas cells 3 and 5 are perpendicular. Inset scale bars, 50 μm. The conditional probability for a localized increase in Ca2+ in cell 1 to be followed by an increase in a parallel or perpendicular neighbor is plotted in (B) with mean and SD. Ca2+ waves were statistically more likely to propagate along the contractile axis (t test, N = 10). (C) Histamine caused a significantly greater increase in traction stress in confluent cells on stiff matrix (N = 21) rather than on soft matrix (N = 12, t test). (D) The faster agonist-induced Ca2+ oscillations on stiff ECM were systematically abrogated in a dose-dependent manner by preincubating the SMCs with the ROCK inhibitor Y-27632 for 1 hour before histamine exposure (N = 3 and 4; P < 0.001, one-way ANOVA). Identical results can be obtained with MLCK inhibition by preincubating with increasing doses of ML-7 for 5 min (N = 3 each; P < 0.001, one-way ANOVA).

  • Fig. 5 Effect of confining SMCs in a line using micropatterning.

    (A) When SMCs were patterned in lines, (B) the probability of finding SMCs with high time periods decreased and the variance of the time periods from aligned cells was significantly smaller than confluent cells (F test, P < 0.001). Matrix stiffness still affected the oscillation period (soft, N = 8; stiff, N = 6; Mann-Whitney rank sum test). To probe the limits of the SMC cluster’s collective matrix sensing abilities, we simulated localized ECM stiffening by (C) patterning SMCs in lines spanning a dual-stiffness PDMS substrate (E = 13 kPa highlighted in green, left, and E = 0.3 kPa, right). (D) The Ca2+ oscillation time period of cells along lines is plotted as a function of the distance from the stiff matrix. Binning cells in 400-μm intervals, only those over 800 μm from stiff matrix had different Ca2+ oscillation time periods from cells directly in contact (t test, N = 4). Scale bars, 250 μm.

Supplementary Materials

  • Supplementary Materials

    Intercellular communication controls agonist-induced calcium oscillations independently of gap junctions in smooth muscle cells

    S. E. Stasiak, R. R. Jamieson, J. Bouffard, E. J. Cram, H. Parameswaran

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