Effects of Myofiber Isolation Technique on Sarcolemma Biomechanics

Figures

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  Figure 1.. Methods for obtaining single isolated fibers. (A) The extensor digitorum longus muscle is shown in place (left), then after removal the muscle is tied at both tendons (middle) and myofibers are dissected away until a single myofiber remains (right). (B) The flexor digitorum brevis is shown in place (left). Once removed (middle), the flexor digitorum brevis muscle is placed into solution where isolated myofibers are obtained via enzymatic dissociation (right). Note that the dissection methods yield only one or two myofibers per muscle, whereas the enzymatic dissociation provides hundreds of isolated myofibers (which can be diluted as needed). Adapted with permission from [4].

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  Figure 2.. Elastimetry. (A) Scanning electron microscopy image shows examples of normal myofibers and branched myofibers with abnormal morphology of MDX mice. (B) Light microscopy shows further examples of such myofibers after enzymatic dissociation. Obtaining isolated myofibers with branched abnormal morphology is improbable, if not impossible, and therefore not able to be studied by the myofiber dissection technique. (C) Light microscopy image showing how an open-mouthed pipette is attached to a single myofiber to assess sarcolemma mechanical properties. Suction pressure (P) applied through a pipette to the sarcolemma form a bleb; bleb height increases with increasing P. Higher P causes the separation of the sarcolemma from myofibrils and eventual bursting of the sarcolemmal bleb. A sarcolemmal bleb inside a pipette can be obtained using either the dissection technique or enzymatic dissociation (far right panel). (D) Effect of dissected versus enzymatically dissociated myofibers in wild-type and MDX myofibers. Mean sarcomere length was 3.6 ± 0.1 and 3.2 ± 0.1 μm in dissected and dissociated myofibers, respectively. Observe that the values from enzymatic dissociation are up to six-times…