To examine the effects of numerous complex branched fibers (CBF) on whole muscle contractile properties, we established a model of myopathic muscles containing a large number of CBF by repeated local injection of bupivacaine hydrochloride (Marcaine) into the plantaris (PLA) muscle. Marcaine injections were administered once weekly for 10 weeks into the right PLA muscles of Wistar male rats. The in situ contractile properties of Marcaine-injected PLA muscles (I-PLA) were examined under urethane anesthesia, and compared with the contralateral (control) PLA muscle (C-PLA). Numerical and morphological examination using the modified nitric acid fiber digestion method and scanning electron microscopy showed that Marcaine resulted in an 8-fold increase in the number of branched fibers in the I-PLA muscles and about 70% of these fibers were CBF. The latter were composed of ten or more muscle fibers fused together along with many thin and thick, long and short daughter branches. The time to peak tension of twitch and tetanus, and 1/2 relaxation time were significantly longer in I-PLA muscles, representing a shift to slow muscle characteristics. However, the total area of slow fibers/muscle cross-sectional area was similar in I-PLA and C-PLA muscles. Aggregates of same-type fibers (slow fibers) with small and large diameters were observed, reflecting an expected cross-sectional property of CBF. Our results suggest that the appearance of several CBF in a muscle is associated with a shift towards slower contractile properties in the affected muscle.

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