Intact extensor digitorum longus (EDL) muscles from mice were used to test skeletal muscle contraction force as described previously (9 (link)). Briefly, freshly isolated EDL muscles were attached to an isometric force transducer (model 407A; Aurora Scientific) between platinum stimulating electrodes in a glass chamber containing modified Tyrode buffer. A force-frequency relationship was determined by measuring muscle contraction force at a range of frequencies (1–200 Hz; model 701C; Aurora Scientific) to obtain a maximum tetanic force plateau. Dynamic Muscle Control software and Dynamic Muscle Analysis software (dmc version 5.300, dma version 5.010; Aurora Scientific) were used for stimulation and analysis. The specific force at various stimulating points from individual EDL muscle were analyzed and averaged by group.
Dynamic muscle analysis software
Manufactured by Aurora Scientific
Sourced in Canada
The Dynamic Muscle Analysis software is a tool designed for the analysis of muscle performance data. It provides a platform for the collection, visualization, and interpretation of various muscle properties, including force, power, and fatigue characteristics. The software offers a comprehensive suite of analysis tools to assist researchers and clinicians in understanding muscle function and dynamics.
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7 protocols using dynamic muscle analysis software
1
Measuring Mouse Skeletal Muscle Contraction
2
Isometric Torque-Frequency Profiling of Ankle Plantar Flexors
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Beginning at 24 months of age, six isometric contractions were used to determine the baseline in vivo isometric torque–frequency profile of the ankle plantar flexor muscles (gastrocnemius, plantaris, and soleus) as previously described.7 Briefly, while anaesthetized with isoflurane, the left hindfoot of each rat was secured to a footplate attached to an Aurora Scientific 300B servomotor, and the plantar flexor muscles were stimulated by two needle electrodes inserted proximally to the peroneal nerve. Torque was measured at stimulation frequencies of 20, 40, 60, 80, 100, and 125 Hz. Each contraction was 200 ms in duration with 45 s of rest between contractions. Data acquisition and analysis were completed using Dynamic Muscle Control and Dynamic Muscle Analysis software (Aurora Scientific). Torque was not measured in the rats that received D2O.
3
In vivo Isometric Torque Measurement of Ankle Plantar Flexors
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To measure muscle function in vivo, six isometric contractions were used to determine the baseline in vivo isometric torque–frequency profile of the ankle plantar flexor muscles (gastrocnemius, plantaris, and soleus) as previously described [24 ]. Briefly, while anaesthetized with isoflurane, the left hindfoot of each rat was shaved using electric clippers, and later cleaned with alcohol swabs. The foot was secured to a footplate attached to an Aurora Scientific 300C servomotor, and the plantar flexor muscles were stimulated by two needle electrodes inserted proximally to the peroneal nerve. Torque was measured at stimulation frequencies of 20, 40, 60, 80, 100, and 125 Hz. Each contraction was 200ms in duration with 15s of rest between contractions. For twitch measurement, a single electric impulse was delivered 3 times with 20 seconds between contraction. The peak response was used for the analysis. Data acquisition and analysis were completed using Dynamic Muscle Control and Dynamic Muscle Analysis software (Aurora Scientific).
4
Force-Frequency Contractility of Extensor Digitorum Longus Muscles
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Skeletal muscle contraction force studies using intact extensor digitorum longus muscles from four groups of mice were performed as described previously,10 (link) as the EDL is commonly studied during cancer-induced muscle dysfunction, particularly in affected glycolytic muscles.69 (link),70 (link) Briefly, at the end of experiment (∼28-week-old mice), intact isolated extensor digitorum longus muscles from four groups of mice were attached to an isometric force transducer (model 407A; Aurora Scientific) between platinum stimulating electrodes in a glass chamber containing modified Tyrode solution. A force-frequency relationship was determined by measuring muscle contraction force at a range of frequencies (1–200 Hz; model 701C; Aurora Scientific) to obtain a maximum tetanic force plateau. An endurance protocol to measure fatigue was run by inducing a tetanic response (70 Hz, 300 ms) and repeating for 50 cycles (EDL). Stimulation was controlled by Dynamic Muscle Control software and analyzed using Dynamic Muscle Analysis software ( dmc version 5.300, dma version 5.010; Aurora Scientific). The specific force (millinewtons) was calculated at various stimulating points from individual EDL muscle and averaged by group.
5
Measuring Sepsis-Induced Muscle Weakness
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After 5 days of sepsis, the timeframe required to develop sepsis-induced muscle weakness50 (link), immediately after euthanasia, the EDL muscle was isolated and suspended in a temperature controlled and continuously perfused organ bath8 (link). The maximal isometric tetanic force was measured by tetanic stimuli and the specific maximal isometric tetanic force was determined according the muscle cross-sectional area. Data collection was done with use of the Dynamic Muscle Analysis software (Aurora scientific, Ontario, Canada). More details are available in the online supplement.
6
Assessing Plantar Flexor Muscle Function
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Plantar flexor muscle function was assessed as described previously [43 (link)]. Briefly, mice were anesthetized by inhalation of a mixture of 97% oxygen and 3% isoflurane gas and placed supine on a plate heated to 37°C to maintain body temperature. The hindlimb was then immobilized with the ankle positioned at 90° flexion and secured to the footplate of the dynamometer (model 6350*358; Cambridge Technology, Aurora Scientific, Aurora, ON, Canada). Subcutaneous platinum electrodes were placed on either side of the tibial nerve to activate plantar flexor muscles. Maximal force was measured by seven sequential electrical impulses (single pulse, 10, 20, 50, 75, 100, and 120 Hz) with five min of rest between each contraction. Contractile data were analyzed off-line (Dynamic Muscle Analysis software; Aurora Scientific). The maximum tension generated by the CTX injured leg was normalized to the sham PBS injected leg of the same animal and recorded as percent recovery (i.e., (CTX tension / PBS tension) *100%). The tension generated by mdx mice was normalized to body mass to account for differences in mouse strains.
7
In vivo Plantarflexor Muscle Torque Assessment
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In vivo maximal isometric torque of the plantarflexor muscle group (gastrocnemius and soleus muscles) was assessed at the various time points of chronic exercise activity as described previously [31] . Mice were anaesthetized via inhalation (≃4% isoflurane and 1.5% O 2 l/min) and placed on a thermostatically controlled table; anesthesia was maintained via a nose cone (≃2% isoflurane and 1.5% O 2 l/min). The right hind limb was shaved and aseptically prepared and the foot was placed on the pedal connected to a servomotor (model 300C-LR; Aurora Scientific, Aurora, ON, Canada). Contractions were elicited by percutaneous electrical stimulation of the tibial nerve via needle electrodes (Chalgren Enterprises) connected to a stimulator (model 701B; Aurora Scientific) to induce contraction of the group of plantar flexor muscles. The current was adjusted from 30 to 50 mA until maximal isometric torque was achieved, and a series of stimulations was then performed at increasing frequencies [31] . Data were analyzed using Dynamic Muscle Analysis software (DMAv5.201; Aurora Scientific) to obtain torque, which was normalized to mouse BW. Normalized values were used to construct torque-frequency curves.
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