Intracellular [H⁺]: a determinant of cell volume in skeletal muscle

It has long been recognized that skeletal muscle function and contractility is affected by alterations in the acid–base status of the intracellular environment (Meyerhof & Lohman, 1926; Furasawa & Kerridge, 1927; Aikin & Thomas, 1977). For example, reduced muscle force development (fatigue) in the presence of increased intracellular [H+] has been associated with inhibition of high affinity Ca2+-binding sites on troponin C (thus impairing actin–myosin interactions), decreased activity of the Ca2+-ATPases in the SR and sarcolemma responsible for lowering myoplasmic [Ca2+], and decreased activity of myosin-ATPase (for review see Fitts, 1994). These combined effects thus include impairment of both the force generation and the relaxation processes. Cell volume may now also be considered a variable that is dependent on intracellular acid–base status. Fraser et al. (2005), in this issue of The Journal of Physiology, report that increases in intracellular acidity of resting frog skeletal muscle cells predictably decreases cell volume without change in resting membrane potential. While it is evident that intracellular acid–base state is an important determinant of muscle cell function, the regulation of muscle cell acid–base status has not been extensively studied and is not well understood. The regulation of cell volume in skeletal muscle has also received very little attention and the mechanisms for determining muscle cell volume are poorly understood (Gosmanov et al. 2003). Amongst the reasons for this are (1) the inherent difficulty in studying the intracellular environment, and the level of difficulty is increased when muscle cells are made to contract, (2) not all of the factors that determine intracellular acid–base status and cell volume have been identified, and (3) there is a complex interplay amongst factors that determine acid–base status and cell volume. The regulation of intracellular acid–base status and cell volume each involves (1) complex interactions amongst intracellular variables that define the intracellular milieu in physical and chemical terms (Stewart, 1981, 1983), (2) the activities of metabolic pathways within the cells, and (3) transmembrane exchanges of ions and gases between cells and the extracellular environment. In an elegant series of experiments, using an innovative fluorescence technique to measure cell volume changes, Fraser et al. (2005) manipulated cell volume by adding and removing extracellular solutions containing NH4Cl. In separate experiments, intracellular H+ activity and membrane potential were measured using intracellular microelectrodes. After a period of incubation of muscle cells in solutions containing NH4Cl, removal of NH4Cl produced predictable decreases in cell volume proportionate to the quantity of NH4+ loaded into and subsequently lost from the cells. Because NH4+ is a weak base, it is expected that its loss from muscle will be associated with an increase in intracellular [H+]. The critical and important finding, however, was that this increase in [H+] was ultimately responsible for the decrease in cell volume. What do these results mean in terms of how skeletal muscle cells function? The primary function of skeletal muscle cells is to provide power for locomotion. The rest-to-work transition in skeletal muscle is associated with increased cell volume due to the accumulation of osmotically active metabolites within muscle cells (Hill, 1930, Lundvall et al. 1972). This initial period of exercise is also associated with an increase in intracellular acidification (Spriet et al. 1987). The rate and magnitude of increases in the accumulation of intracellular osmolytes (Lundvall et al. 1972) and H+ (Aikin & Thomas, 1977) is proportionate to the intensity of muscular contraction. The increase in cell volume appears to be beneficial with respect to muscle contractility because the accompanying decrease in intracellular ionic strength has been directly associated with an increase in force generated per actin–myosin crossbridge (Bressler & Matsuba, 1991; Rapp et al. 1998). There is, however, a limit to which the muscle can increase in volume, beyond which protein–protein interactions and cell integrity are impaired. While all cells appear to have mechanisms for reducing cell volume in the face of cell swelling (Lang et al. 1998), such ‘regulatory volume decrease’ mechanisms have yet to be identified in skeletal muscle. The net losses of K+, lactate and inorganic phosphate from contracting muscle, together with increased activity of the Na+,K+-ATPase (Gosmanov et al. 2003), helps reduce intracellular osmolality and cell volume, but it is likely that other specific mechanisms are also activated. In most cell types studied these include a KCl cotransport system that moves K+ and Cl− out of cells, thus reducing intracellular osmolality and cell volume (Lang et al. 1998). A role for intracellular acidification as a means for decreasing cell volume in contracting muscle cells is provocative and warrants consideration. One may speculate that the intracellular acidification associated with moderate to high intensity muscle contraction may provide an additional, albeit indirect, means by which excessive increases in cell volume can be prevented. The binding of protons to fixed, negatively charged and osmotically active sites within the cells (Fraser et al. 2005) provides a means for reducing intracellular osmolality and hence volume. An important and attractive aspect about this mechanism is the apparent absence of a further depolarizing effect on resting membrane potential which would contribute to impairment of muscle excitability. It yet remains to be determined if results similar to those of Fraser et al. (2005) can be obtained in mammalian muscle, but it appears likely that this physical mechanism would be conserved in mammalian muscle. Consideration of what the functional importance of this cell volume determining mechanism during and following contractile activity could be should provide hypotheses deserving of further research.

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