Regulatory volume increase in single mouse soleus muscle fibres assessed simultaneously using intracellular fluorescence and fibre width

Mammalian skeletal muscle cells have the ability to regulate volume in response to increases or decreases in extracellular osmolarity. In the present study we measured the time course of change in single fibre intracellular calcein fluorescence (volume indicastor) and width in response to varied 200 mosmol/L increase in extracellular osmolarity using NaCl or sucrose. Adult mouse EDL single fibres were isolated using collagenase and incubated in DMEM prior to and during experimentation. Fibres were loaded with calcein‐AM for 30 min, and triple‐rinsed with calcein‐free DMEM. After obtaining baseline images NaCl or sucrose solution was added. Fibre images were obtained at 3–6 s intervals for up to 60 min. Fibre images were analyzed for intensity and width at 2–3 sites. Increased osmolarity resulted in a rapid increase in fibre fluorescence and decrease in fibre width. Both variables gradually recovered to baseline values within ~45 min. Bumetanide, an inhibitor of the sodium‐potassium‐2 chloride cotransporter (NKCC) impaired recovery. There was a linear relationship between increases in fibre fluorescent intensity and decreases in fibre width. It is concluded that the NKCC is involved in regulatory volume increase in skeletal muscle, and that changes in fluorescence intensity can be used as an indicator of changes in cell volume. Supported by NSERC of Canada.