Figure 1.
Calcium oscillations depend on the extracellular calcium concentration and mechanical microenvironment.
(A) Cytosolic FRET-based calcium sensor shows the intracellular calcium dynamics with high spatiotemporal resolutions in hMSC. Red and blue colors represent the high and low FRET ratios, respectively. Images were captured in 10 second interval. Scale bar, 20 µm. (B) Frequency of calcium oscillations (the number of peaks per minute) is measured in cells subjected to various extracellular calcium concentrations (0 mM to 10 mM, n = 14–32). (C) Time courses of calcium signals as imaging medium is replaced with physiological calcium concentration (2 mM). (D) Estimated percentage of hMSCs showing spontaneous calcium oscillations on different substrates. (n = 18–33). (E) Bar graphs show that the strength of Integrin-mediated cell adhesion with increased fibronectin (FN) concentration affects the frequency of calcium oscillations. The frequency of calcium oscillations on FN 40 (40 µg/ml)-coated gel are significantly higher than that from FN 5 (5 µg/ml) (**P<0.01, n = 7).
Figure 2.
Prolonged mechanical stretch (PMS) triggers the calcium oscillations in hMSCs without spontaneous activities.
(A) A schematic drawing of PMS application. Cells were seeded on polyacrylamide (PA) gels with 20 kPa of elastic modulus and a glass capillary probe tip can be placed near a cell and inserted in the gel to pull the gel and cause the stretch of the target cell. The right images show typical displacement and strain maps during prolonged mechanical stretch. The cold and hot colors represent the small and large relative displacement (top, 0–15 µm) and strain index (bottom, 0–0.3). (B) Mechanical stretch was applied to the cytosolic FRET biosensor-transfected hMSC and intracellular calcium dynamics were visualized by the FRET ratio. High FRET ratio (Red color) denotes high concentration of calcium. White arrow points to the direction of mechanical stretch. ON: calcium increase, OFF: calcium decrease. Scale bar, 20 µm. (C) The time course shows that PMS triggers the calcium oscillations in a cell without spontaneous activities. DIC image shows the cell and the probe tip on the PA gel.
Figure 3.
Calcium oscillations triggered by the PMS depend on calcium entry via the plasma membrane, cytoskeleton and actomyosin contractility.
(A) PMS evokes calcium oscillations in a subpopulation of hMSCs without spontaneous activities (Ctr; control). (B, C) PMS-induced calcium oscillations were inhibited in GdCl3- or BAPTA-pretreated cells, suggesting the involvement of calcium entry at the plasma membrane. (D, E, F) PMS-induced calcium oscillations were also inhibited in CytoD-, Noc-, or ML-7 pretreated cells, suggesting the involvement of cytoskeleton and actomyosin contractility in the PMS-induced calcium oscillations. Amplitude (G) and frequency (H) of PMS-induced calcium oscillations are inhibited in the presence of inhibitors. PMS, control group with prolonged mechanical stretch only (n = 9); PMS+GdCl3 (5 µM), an inhibitor of stretch-activated calcium channel was applied 1 hr before stretch (n = 4); PMS+BAPTA (20 µM in the calcium free medium), a calcium chelator was applied before stretch (n = 5); PMS+CytoD (1 µM), an inhibitor of actin filament was applied 1 hr before stretch (n = 5); PMS+Noc (5 µM), an inhibitor of microtubule was applied 1 hr before stretch (n = 4); PMS+ML-7 (10 µM), an inhibitor of MLCK was applied 1 hr before stretch (n = 4). * indicates statistically significant difference from all other groups by the Bonferroni multiple comparison test with p<0.05.
Figure 4.
The effect of PLC inhibitor on the PMS-induced calcium oscillations.
(A) In U73122 (5 µM, 30 min) pretreated hMSCs, PMS does not induce sustainable calcium oscillations. (B–C) Both amplitude and frequency of PMS-calcium oscillations are inhibited in the presence of U73122 (PMS, n = 4; PMS+U73122, n = 4, **P<0.01 and ***P<0.001).
Figure 5.
PMS-induced calcium oscillation in a subpopulation of hMSCs lacking the spontaneous calcium oscillation.
A schematic drawing represents that PMS-induced calcium oscillations depend on stretch-activated channels, cytoskeletal supports, actomyosin contractility and PLC activity.