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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.
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.
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.
No conflicts of interest declared.
The protocol is performed with the approval of the UCSF Institutional Animal Care and Use Committee (IACUC).