Incubating Isolated Mouse EDL Muscles with Creatine Improves Force Production and Twitch Kinetics in Fatigue Due to Reduction in Ionic Strength

Creatine supplementation can improve performance during high intensity exercise in humans and improve muscle strength in certain myopathies. In this present study, we investigated the direct effects of acute creatine incubation on isolated mouse fast-twitch EDL muscles, and examined how these effects change with fatigue. muscle from mice aged 12–14 weeks was isolated and stimulated with field electrodes to measure force characteristics in 3 different states: (i) before fatigue; (ii) immediately after a fatigue protocol; and (iii) after recovery. These served as the control measurements for the muscle. The muscle was then incubated in a creatine solution and washed. The measurement of force characteristics in the 3 different states was then repeated. In un-fatigued muscle, creatine incubation increased the maximal tetanic force. In fatigued muscle, creatine treatment increased the force produced at all frequencies of stimulation. Incubation also increased the rate of twitch relaxation and twitch contraction in fatigued muscle. During repetitive fatiguing stimulation, creatine-treated muscles took 55.1±9.5% longer than control muscles to lose half of their original force. Measurement of weight changes showed that creatine incubation increased EDL muscle mass by 7%. sensitivity of contractile proteins as a result of ionic strength decreases following creatine incubation

Twitch kinetics of EDL muscles before and after creatine treatment.

<p><i>A</i>, Rate of relaxation. Creatine treatment resulted in significantly faster relaxation in fatigued muscle. <i>B</i>, Rate of contraction. Creatine treatment resulted in significantly faster contraction in fatigued muscle. Statistically significant differences are indicated by “*” (<i>P</i><0.05).</p

Fatigue run in EDL muscles before and after creatine treatment.

<p><i>A</i>, time taken for force to fall to 50% of original force. Creatine incubation resulted in a significant increase in this parameter. <i>B</i>, pattern of force decline during the fatigue run, showing the effect of creatine treatment in improving the resistance of muscles to fatigue. It is evident that creatine incubation is associated with an increase in the time taken for force to fall to 50%, as well as a reduction in the total force decline over the whole fatigue run. Statistically significant differences are indicated by “*” (<i>P</i><0.05).</p

Half-frequency and Hill coefficient of force-frequency curves.

<p>Sigmoidal curves were fitted to the force-frequency data to obtain the half-frequency (<i>A</i>) and Hill coefficient (<i>B</i>). Creatine treatment resulted in a significant reduction in the half-frequency in fatigued muscle. Statistically significant differences are indicated by “*” (<i>P</i><0.05).</p

Changes in EDL muscle mass and FDB fibre diameter following creatine incubation.

<p>Creatine incubation resulted in a significant increase in the wet weight of whole EDL muscle (<i>A</i>) and a significant increase in the diameter of single FDB fibres (<i>B</i>), demonstrating the uptake of creatine by these muscles and fibres. Statistically significant differences are indicated by “*” (<i>P</i><0.05).</p

Force-frequency curves for EDL muscles before and after creatine treatment.

<p>Each graph shows the force-frequency curves under control and creatine-treated conditions. In the pre-fatigue state (<i>A</i>), creatine significantly increased the force produced at 100 Hz. In the fatigued state (<i>B</i>), creatine incubation significantly increased the force produced at every frequency. In the recovered state (<i>C</i>), creatine had no significant effect on the force at any frequency. Statistically significant differences are indicated by “*” (<i>P</i><0.05). Tests for significance at each frequency were conducted using Bonferroni multiple comparison post-tests following two-way repeated measures ANOVA (GraphPad Prism).</p

Twitch and tetanus in EDL muscles before and after creatine treatment.

<p><i>A</i>, twitch to tetanus ratios expressed relative to the pre-fatigue twitch to tetanus ratio. Before creatine incubation, the twitch to tetanus ratio in fatigued muscle was similar to that in the pre-fatigue state. After creatine incubation however, the twitch to tetanus ratio in the fatigued state was significantly higher than in the pre-fatigue state. Statistically significant differences are indicated by “*” (<i>P</i><0.05). <i>B</i>, force tracings of twitches and 100-Hz tetani obtained from one EDL muscle. In the fatigued muscle, creatine incubation increases the twitch force and tetanic force. In the recovered muscle however, creatine incubation makes little difference to the twitch and tetanic forces.</p