Adherent Primary Cultures of Mouse Intercostal Muscle Fibers for Isolated Fiber Studies

Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene Street, Baltimore, MD 21201, USA.
BioMed Research International (Impact Factor: 2.71). 08/2011; 2011(1):393740. DOI: 10.1155/2011/393740
Source: PubMed


Primary culture models of single adult skeletal muscle fibers dissociated from locomotor muscles adhered to glass coverslips are routine and allow monitoring of functional processes in living cultured fibers. To date, such isolated fiber cultures have not been established for respiratory muscles, despite the fact that dysfunction of core respiratory muscles leading to respiratory arrest is the most common cause of death in many muscular diseases. Here we present the first description of an adherent culture system for single adult intercostal muscle fibers from the adult mouse. This system allows for monitoring functional properties of these living muscle fibers in culture with or without electrical field stimulation to drive muscle fiber contraction at physiological or pathological respiratory firing patterns. We also provide initial characterization of these fibers, demonstrating several common techniques in this new model system in the context of the established Flexor Digitorum Brevis muscle primary culture model.

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Available from: Erick O Hernández-Ochoa
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    • "Our initial observation that NFATc1 showed abnormally high resting activation in cultured ItC fibers [24] leads us to examine the activity of NFATc1 in these fibers more closely. Fibers derived from both the soleus (Figure 2D-F) and FDB (Figure 2G-I) show increased nuclear import of NFATc1 characteristic of the canonical activation pathway following 90 minutes of repetitive electrical field stimulation. "
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    ABSTRACT: The NFATc transcription factor family is responsible for coupling cytoplasmic calcium signals to transcription programs in a wide variety of cell types. In skeletal muscle, these transcription factors control the fiber type in response to muscle activity. This excitation-transcription (E-T) coupling permits functional adaptation of muscle according to use. The activity dependence of these transcription programs is sensitive to the firing patterns of the muscle, not merely the period of activity, enabling a nuanced adaptation to various functional tasks. Isolated skeletal muscle fibers expressing exogenous fluorescent NFATc1 were studied by confocal microscopy under stimulation both with and without pharmacological inhibitors. Western blots of whole muscle lysates were also used. This study investigates the activity dependent response of NFATc1 skeletal muscle fibers cultured from mice, comparing fibers of respiratory origin to muscles responsible for limb locomotion. Using patterns of stimulation known to strongly activate NFATc1 in the commonly cultured flexor digitorum brevis and soleus muscles, we have observed significant deactivation of NFATc1 in cultured intercostal muscle fibers. This effect is at least partially dependent on the action of JNK and CaMKII in intercostal fibers. Our findings highlight the role of lineage in the NFAT pathway, showing that the respiratory intercostal muscle fibers decode similar E-T coupling signals into NFAT transcriptional programs in a different manner from the more commonly studied locomotor muscles of the limbs.
    Full-text · Article · Jan 2014 · Skeletal Muscle
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    ABSTRACT: Background Myofibers with an abnormal branching cytoarchitecture are commonly found in muscular dystrophy and in regenerated or aged nondystrophic muscles. Such branched myofibers from dystrophic mice are more susceptible to damage than unbranched myofibers in vitro, suggesting that muscles containing a high percentage of these myofibers are more prone to injury. Little is known about the regulation of myofiber branching. Methods To gain insights into the formation and fate of branched myofibers, we performed in-depth analyses of single myofibers isolated from dystrophic and nondystrophic (myotoxin-injured or aged) mouse muscles. The proportion of branched myofibers, the number of branches per myofiber and the morphology of the branches were assessed. Results Aged dystrophic mice exhibited the most severe myofiber branching as defined by the incidence of branched myofibers and the number of branches per myofiber, followed by myotoxin-injured, wild-type muscles and then aged wild-type muscles. In addition, the morphology of the branched myofibers differed among the various models. In response to either induced or ongoing muscle degeneration, branching was restricted to regenerated myofibers containing central nuclei. In myotoxin-injured muscles, the amount of branched myofibers remained stable over time. Conclusion We suggest that myofiber branching is a consequence of myofiber remodeling during muscle regeneration. Our present study lays valuable groundwork for identifying the molecular pathways leading to myofiber branching in dystrophy, trauma and aging. Decreasing myofiber branching in dystrophic patients may improve muscle resistance to mechanical stress.
    Full-text · Article · May 2014 · Skeletal Muscle