SummaryMyofibroblasts promote tissue contractures during fibrotic diseases. To understand how spontaneous changes in the intracellular calcium concentration, [Ca 2+ ] i , contribute to myofibroblast contraction, we analysed both [Ca 2+ ] i and subcellular contractions. Contractile events were assessed by tracking stress-fibre-linked microbeads and measured by atomic force microscopy. Myofibroblasts exhibit periodic (~100 seconds) [Ca 2+ ] i oscillations that control small (~400 nm) and weak (~100 pN) contractions. Whereas depletion of [Ca 2+ ] i reduces these microcontractions, cell isometric tension is unaffected, as shown by growing cells on deformable substrates. Inhibition of Rho-and ROCK-mediated Ca 2+ -independent contraction has no effect on microcontractions, but abolishes cell tension. On the basis of this two-level regulation of myofibroblast contraction, we propose a single-cell lock-step model. Rho-and ROCKdependent isometric tension generates slack in extracellular matrix fibrils, which are then accessible for the low-amplitude and highfrequency contractions mediated by [Ca 2+ ] i . The joint action of both contraction modes can result in macroscopic tissue contractures of ~1 cm per month.

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Myofibroblast contraction is fundamental in the excessive tissue remodeling that is characteristic of fibrotic tissue contractures. Tissue remodeling during development of fibrosis leads to gradually increasing stiffness of the extracellular matrix. We propose that this increased stiffness positively feeds back on the contractile activities of myofibroblasts. We have previously shown that cycles of contraction directly correlate with periodic intracellular calcium oscillations in cultured myofibroblasts. We analyze cytosolic calcium dynamics using fluorescent calcium indicators to evaluate the possible impact of mechanical stress on myofibroblast contractile activity. To modulate extracellular mechanics, we seeded primary rat subcutaneous myofibroblasts on silicone substrates and into collagen gels of different elastic modulus. We modulated cell stress by cell growth on differently adhesive culture substrates, by restricting cell spreading area on micro-printed adhesive islands, and depolymerizing actin with Cytochalasin D. In general, calcium oscillation frequencies in myofibroblasts increased with increasing mechanical challenge. These results provide new insight on how changing mechanical conditions for myofibroblasts are encoded in calcium oscillations and possibly explain how reparative cells adapt their contractile behavior to the stresses occurring in normal and pathological tissue repair.