Experimental work in amphibian skeletal muscle and modeling studies have demonstrated that intramuscular fluid volume is an important determinant of the passive force that develops during lengthening. However, this effect has yet to be investigated in mammalian skeletal muscle. Therefore, we exposed isolated mouse soleus and extensor digitorum longus (EDL) muscles to a graded series of hypotonic solutions to promote fluid uptake while measuring passive force development, muscle mass, and 2D projected muscle area. Normalized to the tension measured at 1.2 L₀ in isotonic Ringer’s solution, the relative passive forces in the soleus were 1.14, 1.31, 1.52, and 1.92 in 70%, 60%, 55%, and 50% relative tonicity, respectively. Comparable values for the EDL in relative tonicities of 70%, 60%, and 55% were 1.13, 1.78, and 2.10, respectively. In both muscles, increases in passive force were accompanied by increases in mass and projected area. We also investigated the effect of muscle tension on fluid uptake. Soleus muscles left slack and allowed to shorten when exposed to a hypotonic solution gained much more mass compared to muscles held at the predicted length for maximal active force production, which suggests that at this length water uptake is limited by the buildup of hydrostatic pressure. Our findings support the hypothesis that in mammalian muscle, intramuscular fluid volume is an important determinant of passive force development. These results could have implications for human movement performance, where muscle volume change has been observed in vivo.

Animals

Female CD-1 Mice (Mus musculus [16]) were acquired from Charles River Laboratories in Wilmington, MA and were 10 - 20 weeks of age when used. All animal use was approved by the Brown University Institutional Animal Care and Use Committee.

Muscle dissection and in vitro setup for force measurements

Muscles were isolated from both hindlimbs of each animal (soleus, N=6 animals; EDL, N=7 animals). For the EDL, only the head to the fifth toe was used. One muscle from each pair was used to acquire passive tension measurements, and the contralateral muscle was used as a control to obtain a measure of untreated muscle mass. Immediately following dissection, isolated muscles were placed into oxygenated mammalian Ringer’s solution [17]consisting of 144 mmol 1^-1 NaCl, 6 mmol 1^-1 KCl, 2 mmol 1^-1 CaCl₂, 1 mmol 1^-1 MgSO₄, 1 mmol 1^-1 NaH₂PO₄ H₂O, 10 mmol 1^-1glucose, and 10 mmol 1^-1 Hepes, and adjusted to a pH of 7.4 using 1 molar Tris base. Assuming complete ionization, this Ringer’s solution had an osmotic concentration of 332 mOsm, which is within the range of osmolalities found for mouse plasma [18]. Hypotonic solutions were produced by changing only the NaCl concentration to decrease the overall osmolality of each solution relative to the isotonic (100%) solution (70%, 232 mOsm; 60%, 199 mOsm; 55%, 183 mOsm; and 50%, 166 mOsm).

The contralateral control muscles were pinned at approximately resting length by removing any slack and they remained in isotonic Ringer’s solution at room temperature for the duration of the experiment.

Foil clips were attached to the proximal and distal tendons of the experimental muscles. Muscles were then positioned vertically in a chamber filled with isotonic Ringer’s solution maintained at 37 ˚C by pumping oxygenated Ringer’s through the chamber from a reservoir in a water bath. One end of the muscle was attached via the foil clip to a hook at the bottom of the chamber and the other end of the muscle attached via the foil clip and a light-weight silver chain to a servo motor lever (Cambridge Instruments Model 300B). Force and length outputs from the servo system were collected using a 16-bit A-D system (PowerLab Model 16/35) using LabChart v8.1.30 running on a Macintosh laptop computer.

Active length-tension

To reference the measured passive tension to the same functional range of muscle lengths for both muscles, we determined the optimal length for active isometric force production (L₀) for each experimental muscle. To elicit active contractions, platinum foil electrodes were placed on either side of the muscle and 0.2-ms stimuli were delivered from a high current stimulus isolation unit (Hugo Sachs Elektronik - Harvard Apparatus Model 263) controlled by a Grass stimulator (Astro-Med Inc Model S48H). Following a 30-minute rest period, a series of twitch contractions were performed to identify the supramaximal voltage to be used in subsequent contractions. Active length-tension curves were constructed using the results of tetanic contractions over a series of lengths. Stimulation frequencies were 300 and 400 Hz for the soleus and EDL, respectively. A 3-minute rest period occurred between tetanic contractions. The length at which maximal isometric force was produced (L₀) was determined by visual inspection of the active force versus length plots (Supplementary Fig. S1). Photographs were taken of the muscles at the length that resulted in maximum tetanic force. For the EDL, fiber length at L₀ was measured along the fibers between the proximal and distal aponeuroses using Image J. For the soleus, the fiber trajectory between the aponeuroses was not always visible because of the muscle shape. For this muscle, the length of the muscle belly was measured from the photographs and fiber L₀ was calculated as 85% of belly length [19]. (The belly length is the length of the fleshy part of the muscle including the aponeuroses but excluding the external tendons.)

Passive Length-Tension

The starting lengths for the passive length tension measurements were chosen so the muscles had enough passive tension at the beginning of passive lengthening to see noticeable changes in passive tension in the first lengthening step. For the soleus, the starting length was set at L₀. However, the EDL was slack at L₀ and therefore we set the starting length at L₀ + 0.5 mm. For each individual muscle tested (6 muscles for the soleus and 7 muscles for the EDL), passive force-length data were collected first under isotonic (100%) conditions, followed by 70%, 60%, 55%, and 50% relative tonicity (osmotic concentration) solutions. Early experiments showed signs of cell lysis in the EDL in the 50% tonicity solution, indicating instability. Thus the 50% dilution was omitted for the EDL. For each dilution, the chamber was drained and refilled with the appropriate solution and then passive lengthening was initiated at 5, 10, and 20 min after immersion. Thus, for each individual soleus muscle we collected 15 passive curves and for each individual EDL muscle we collected 12 passive curves. Additionally, images were taken every minute in each solution when the muscle was not undergoing passive lengthening (for a total of 16 images per solution) using a Nikon D90 camera. After an experimental muscle completed the passive lengthening protocol for all solutions, it was removed from the chamber, blotted to remove excess fluid, and weighed using a laboratory balance (B 120 S, Sartorius Co.). The control muscle was also removed from isotonic solution and weighed.

Passive lengthening was performed using a step function (Fig. 1A) coded in IgorPro v. 9 and delivered to the servo length control using a 16-bit D-A output (National Instruments Model USB-6218). The choice of the lengthening protocol was determined by balancing two objectives. First, avoiding large and rapid lengthening that might induce large viscous effects, and second, avoiding prolonged lengthening times that might induce water movement out of the muscle. The total lengthening time was 1.6 min accomplished in 8 steps (details of the length steps are shown in Supplemental Fig. S2). After each lengthening step there was an approximately 6 s pause, during which rapid stress-relaxation occurred. During this brief pause the force typically declined to within 10% or less of its asymptotic value (Supplemental Fig. S3). The total lengthening across all steps was 2 mm for the soleus and 1 mm for the EDL. This lengthening brought the relative length of the fibers to a mean of approximately 1.21 L₀ for both muscles.

To account for any change in the passive tension resulting from the repeated stretching, we also subjected 4 soleus and 4 EDL muscles to the same protocol but each time the solution was replaced with isotonic Ringer’s. We refer to these preparations as isotonic time controls.

Passive Force Analysis

The recorded length and force data were transferred to IgorPro v. 9 for analysis. Force measurements were corrected to account for the small resting force offset of the servo motor lever during lengthening, and for the weight of the chain attaching the muscle to the servo motor lever. Force for each step in lengthening was measured as the mean value during the final 1 s of the plateau in length. These forces measured in isotonic Ringer’s were plotted as a function of relative length (fiber length divided by L₀). Using the nonlinear curve fitting algorithm in Igor Pro the isotonic data were fitted with a power function: F = F₀ + aL^b, where F is force, L is relative length, and F₀, a, and b are constants. The starting force before the first lengthening was omitted because the initial increase in force in the first step was somewhat larger than would be predicted from the power function (Supplemental Fig. S3). Previous work has noted a larger than expected increase in passive force in the first of a series of small rapid length steps [20]. Using the fitted curve in isotonic solution, the value at 1.2 L₀ was interpolated (Fig. 1C, Fig. 2). The passive forces at all tonicities were then normalized by dividing by this predicted isotonic 1.2 L₀ force value; plotted as a function of relative length; and fitted with power functions (Fig. 1C, Fig. 2). The predicted normalized forces at 1.2 L₀ were determined from the fitted curves (Fig. 2) and these values used to analyze the effects of time and tonicity.

The passive forces for the isotonic time controls were measured in the same manner as the experimental muscles. These values, expressed as fractions of the initial 20 min value (trial 3), were plotted as a function of lengthening-trial number and fitted with regressions to quantify the average effect of repeated stretches. The values for the experimental muscles in hypotonic solutions were adjusted to correct for the small decline in force expected from repeated stretches.

Muscle Area and Mass

Measurements of muscle mass for the contralateral limb (control muscle) were compared to the mass of the experimental muscle after exposure to the final dilution (55% for EDL, 50% for the soleus). (For one of the seven EDL prep pairs there was damage to the isotonic control muscle in the pair. Therefore, the masses from this muscle and its hypotonic partner were excluded from our final mass comparison.) The mass of the control muscle and its fiber length were used to calculate the cross-sectional area of the fibers, and this value used to calculate the active and passive stresses as a basic description of the muscle preparations.

Because the experimental muscles remained in the chamber and attached to the lever, it was not possible to measure mass for each of the serial dilutions. Instead, measures of projected area taken from photos were used as an indicator of change in muscle volume. For both the soleus and the EDL muscles, images taken during initial isotonic exposure and during the twentieth minute of exposure to each of the hypotonic dilutions were used to measure projected area. In ImageJ [21], the outline of approximately the middle third of the muscle belly muscle was traced, and the outlined area was quantified (Supplemental Figure S4). The ratio of areas between isotonic and hypotonic solutions was compared for each soleus and EDL muscle to determine swelling.

Effect of Length and Resulting Passive Tension on Swelling

In this experiment, soleus muscles were isolated from both legs of all animals (n=10). For five of the animals (10 muscles total) both muscles were pinned out in petri dishes at a muscle belly length of 11.1 mm. This value corresponds to the mean belly length at L₀ of the muscles used in the passive force measurements. One muscle from each animal was submerged in isotonic Ringer’s solution as a control while the other was submerged in 50% hypotonic Ringer’s solution for 20 minutes. At 20 minutes, both muscles were removed from their respective dishes, blotted, and weighed on a laboratory balance (Mettler model MS105DU). For the remaining five animals (10 muscles total), five of the muscles were pinned at 11.1 mm length and exposed to isotonic ringers as a control. For the contralateral muscles only one tendon was pinned, with the other tendon remaining free. This arrangement resulted in the unpinned muscle being slack and allowed to shorten as it swelled. The slack muscle was submerged in 50% hypotonic Ringer’s solution for 20 minutes. At 20 minutes, both muscles in each pair were removed from their respective dishes and their masses were measured.

Statistics

Statistical analyses were run using SPSS, v.31 for Macintosh Computers. Means are presented ± standard errors (SE). For the passive forces analyzed by ANOVA, the SEs reported are those resulting from the analyses. The ANOVA and ANCOVA models were run using SPSS’s general linear model. The effect of the measurement time after exposure to the hypotonic solutions (5, 10, and 20 min) on passive force was investigated using ANOVAs. The two muscles and the individual dilutions were analyzed separately with time as a fixed factor and the individual preparation as a random factor. Post-hoc comparisons were corrected with the Bonferroni method. The effect of the Ringer’s dilution on the fractional increase in passive force after 20 min for both muscles was examined using ANCOVA with muscle entered as a fixed factor and dilution entered as a covariate. Similarly, the effect of dilution on the projected area of the muscles was analyzed by ANCOVA with muscle entered as a fixed factor and dilution entered as a covariate. The relations of the log of normalized passive force versus length were compared separately for each muscle using ANCOVA with tonicity as a fixed factor, length as the covariate, and individual preparation as a random factor. The difference between the masses of the muscles maintained in isotonic solution and the masses after exposure to the most dilute solutions was examined with a paired t-test. The fractional increases in mass between the soleus muscles pinned at predicted optimal length in hypotonic solution and those allowed to be slack were compared with a two-sample t-test.