Reply from M. I. Lindinger, M. Leung, K. E. Trajcevski and T. J. Hawke

Dr Fraser's letter follows his review on the topic of muscle volume regulation (Usher-Smith et al. 2009) in which the authors take the view that muscle cells do not regulate their volume in response to changes in the tonicity of their extracellular environment. This view is puzzling in view of the fact that most animal cells studied to date display volume regulation characteristics (O’Neill, 1999; Lang, 2007). The fact that mammalian skeletal muscle cells also regulate volume has also been demonstrated (Sitdikov et al. 1989; Urazaev, 1998; Lindinger et al. 2011). To refute the likelihood that mammalian skeletal muscle cells regulate cell volume, Dr Fraser presents some interesting results on amphibian muscle cells, and raises some good points regarding experimental design. He shows that in single Rana pectoris muscle fibres the volume responses occurring after addition of either 100 mm DMSO or 100 mm glycerol result from an initial osmotic loss of volume by muscle, followed by volume recovery. The mechanism by which the cells recover volume is not mentioned, but it may be implied that volume recovery is due to the influx of DMSO or glycerol into the cell interior, and that volume recovery is solely due to achieving complete chemical equilibration of the solute across the sarcolemma. There is no evidence presented that the vacuolation is not an occurrence that occurs with fibres under various non-DMSO conditions in his experimental set-up. It is also unknown if factors other than DMSO, or in combination with DMSO, contributed to the vacuolation or, indeed, if this is a normal response of muscle (Launikonis & Stephenson, 2004). It also appears that some of the methodology and results presented in our paper (Lindinger et al. 2011) were misinterpreted. Regarding methodology, we did not simultaneously add NaCl and remove DMSO as these tasks, perhaps fortunately, cannot be performed simultaneously. In his letter, Fraser referred to procedures performed using single muscle fibres. However, Fig. 4 is in reference to experiments performed on intact muscle. Even when using single fibres, the ‘50 s delay’ is the time after completion of the triple rinsing procedure and the acquisition of new images. The total time from first removal of the bumetanide-containing solution was closer to 5 min. Regardless, when baseline images were obtained prior to imposing the osmotic challenge, no volume changes occurred. When performing experiments in which muscle was incubated with bumetanide with DMSO, the solution containing bumetanide was first removed and flushed, and replaced with solution that was free of bumetanide and DMSO. This required about 5 min; baseline measurements were then obtained over the next 5–10 min – during this period no volume changes occurred. Then extracellular osmolarity was increased, resulting in cell shrinkage, followed by volume recovery. The observed responses cannot be explained by a DMSO-induced phenomenon. We have never observed volume transients during baseline acquisition periods following bumetanide treatments. Furthermore, we have observed the same volume responses reported in these experiments in hundreds of single cells treated with step increases in extracellular osmolarity that have not been exposed to DMSO, i.e. not calcein loaded and not incubated in bumetanide (M. Leung, J Moynes & MI Lindinger, unpublished and presented at several meetings). Dr Fraser's pilot data do raise concerns regarding use of DMSO in physiological experiments and the timing of experiments when using muscle treated with DMSO (also see Santos et al. 2003; Velasco et al. 2003). Further investigation is required to definitively and rigorously demonstrate whether a vacuolation response to DMSO treatment occurs in all such treated frog muscle fibres, as well as cells of other species using the experimental conditions employed by other labs, and then the physiological impact can be determined.

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