To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.
To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.
The protocol is performed with the approval of the UCSF Institutional Animal Care and Use Committee (IACUC).
1. Dissection of the Mouse Extensor Digitorum Longus (EDL) Muscle
2. Mounting of the Mouse EDL in the Muscle Strip Myograph
3. Myo-mechanical Analysis
A. Twitch tension
B. Tetanus tension
C. Force-frequency
D. Fatigue
E. Additional data collection at end of protocol
4. Calculations
5. Representative Results
Figure 1. Dissection of EDL muscle. A, Exposure of hindlimb muscles.TA, tibialis anterior. B, Exposure of EDL (extensor digitorum longus) muscle.C, Attachment of sutures to EDL tendons.D, Tension transducer bath (view from side). E, EDL mounted in bath (view from above). The muscle in incompletely immersed in the buffer for illustrative purposes; in practice, the muscle should be completely immersed to prevent from drying out.
Figure 2. Example of tension curves. A, Example of twitch tension curve illustrating maximal twitch tension (Pt), contraction time (CT) and half-relaxation time (HRT).Bar, 1s. B, Example of tetanic tension curve showing maximal tetanic tension (Po) and half-relaxation tetanic tension (HRTT). Bar, 1s.
Figure 3. Example of force-frequency relationship analysis. A, Tensions generated by incremental stimulation frequencies.B, Example of pulse train at 30MHz. Bar, 80ms. C, Example of pulse train at 140MHz. Bar, 80ms. D, Example of force-frequency curve derived from data shown in A. The shape of the force-frequency curve is characteristic of muscle strength, and can be compared between muscles from different animals.
Figure 4. Example of low frequency fatigue analysis. A, Decremental tensions generated over period of low frequency stimulation.Examples of pulse trains at indicated time points (B,C,D) are shown below. E, Example of low frequency fatigue curve derived from data shown in A. The shape of the low frequency fatigue curve is characteristic of muscle strength, and can be compared between muscles from different animals.
We describe a detailed approach to evaluating myo-mechanical properties in explanted hindlimb muscle from the mouse. The EDL, while more difficult to dissect because of its posterior position behind the tibialis anterior muscle, is easier to evaluate than the tibialis anterior because of its prominent tendinous attachments to the ankle and knee joints. These tendons facilitate mounting in the muscle strip myograph. In contrast, the more easily accessed tibialis anterior has a broad, almost atendinous attachment at the knee joint, making it exceptionally difficult to both dissect without compromising the muscle, and mount securely in the myograph. We also point out that rapidly mounting the muscle in an oxygenated bath in physiological buffer and temperature is essential to preserving the muscle’s mechanical properties. We have found that we can repeat this analysis for up to 30 minutes without significant changes in muscle response under these conditions. Finally, it is essential that the muscle fibers not be touched during the dissection and mounting procedures, as this can have adverse effects on muscle function, and result in underestimation of myo-mechanical force. By following these procedures, this analysis provides a robust quantitative approach to evaluating the effects of genetic modification on muscle function 6,7,8,9, as well as comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5.
The authors have nothing to disclose.
This work was supported by a Public Health Service Grant (HL086513) from NHLBI to P.E.O, and a Comprehensive Research Grant from the California Institute for Regenerative Medicine (RC1-00104), a Public Health Service Grant (HL085377) from NHLBI, and a gift from the Pollin Foundation to H.S.B.
S.C. was supported by a California Institute for Regenerative Medicine Bridges to Stem Cell Research Award (TB1-01194) to San Francisco State University.
Material Name | Typ | Company | Catalogue Number | Comment |
5-0 silk sutures | Oasis | MV682 | General surgery | |
Dupont #5 forceps | WPI | 500233 | General surgery | |
Hemostat, straight | WPI | 501241 | General surgery | |
Iris forceps | WPI | 15914 | General surgery | |
Lab Chart software | ADInstruments | Version 7 | Data analysis | |
Muscle Strip Myograph | DMT | 820MS | Tension transduction | |
Operating scissors | WPI | 501754 | General surgery | |
Oscilloscope | EZ | OS-5020 | Tension stimulation | |
Pentobarbital, sodium salt | Sigma | P3761 | Euthanasia | |
PowerLab | ADInstruments | 8/30 | Data acquisition | |
Square Pulse Stimulator | Grass Tech. | S48 | Tension stimulation | |
Vannas spring scissors | WPI | 14003 | General surgery |
Solutions and Media
Lactated Ringer’s solution
Krebs Henseleit solution
Pentobarbital