Muscle Physiology, Membrane Structure, and Susceptibility to Injury in Mice Lacking Intermediate Filaments
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Intermediate filaments, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. Our previous results show that the tibialis anterior muscles (TAs) of mice lacking keratin 19 (K19) lose costameres, accumulate mitochondria under the sarcolemma, and generate lower specific tension than controls. PURPOSE: Here we compare the physiology and morphology of TA muscles of mice lacking K19 with muscles lacking desmin or both proteins (DKO). METHODS: The TA was isolated in situ to measure contractile characteristics (i.e., twitch, tetany, and fatigue). Dorsiflexor torque was measured before and at several time points after animals sustained an injury induced by lengthening contractions. We evaluated membrane damage by measuring serum CK and counting Evans blue dye labeled fibers, assessed structural changes with confocal and electron microscopy, and determined functional changes on a treadmill. RESULTS: The absence of desmin caused a larger change in specific tension (40%) than the absence of K19 (19%), and played the predominant role in the DKO (40%). By contrast, the absence of both proteins was required to obtain a significantly greater loss of contractile torque after injury (48%), compared to wild type (39%), or a significant decrease in the tolerance to exercise. Although the loss of organization of costameres was much greater in the desmin-null TA muscle, the absence of both K19 and desmin was required for near complete disruption of costameres. By contrast, the subsarcolemmal accumulation of mitochondria was only seen in K19-null TA muscles, and the absence of both K19 and desmin yielded a milder phenotype. CONCLUSIONS: Our results suggest that keratin filaments containing K19 and desmin-based intermediate filaments play distinct but complementary roles in the physiology and morphology of fast-twitch skeletal muscle. Supported by grants form the Muscular Dystrophy association (MDA, grant 4278) and NIH-NIAMS (K01AR053235) to RML and grants from the MDA and the NIH (RO1AR055928) to RJBKeywords:
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INTRODUCTION Titin is a protein that spans the length of a half sarcomere in skeletal muscle myofibrils. It behaves like a molecular spring within the myofibril, playing a role in stabilizing sarcomeres and regulating passive force [1, 2]. Isolated titin has been shown to be essentially elastic if immunoglobulin (Ig) domain unfolding/refolding is prevented [3]. In its native, sarcomeric environment, it has been suggested that stretching and holding a myofibril at very long lengths produces a time-dependent unfolding of all Ig domains, thus, allowing titin’s elastic behavior to be exhibited [4]. Experiments on active myofibrils showed a decrease in force and a persistent hysteresis throughout a stretch-shortening (SS) protocol, suggesting a time-dependent unfolding of Ig domains [5]. Holding active myofibrils at long lengths prior to stretch-shortening cycles should allow most (all) of the Ig domains to unfold thus reducing (eliminating) force loss and hysteresis. The goal of this study was to test the hypothesis that holding myofibrils at long lengths prior to small stretch-shortening cycles would result in essentially elastic properties of myofibrils, compared to the highly visco-elastic properties for conditions without holding. METHODS Rabbit psoas muscle myofibrils (n = 5) with clear striation patterns were tested. Single myofibrils were attached at one end to a glass needle (to control length) and at the other end to a nanolever (to quantify force). Myofibrils were activated at an average sarcomere length of 2.7 µm, and then stretched to a length of 5.2 µm/sarcomere, where they were held for 2 minutes to allow for Ig domain unfolding to occur. The myofibril then underwent a SS protocol with amplitude of ± 0.25 µm (10 cycles) before being shortened to its original length. Myofibril length, diameter, and force were quantified. Diameter was used to calculate cross-sectional area, which accommodated the calculation of myofibril stress from force. Hystereses were calculated as the difference in area under the loading and unloading curves for each SS cycle of the force-length plots. RESULTS Peak stress throughout the 10 cycles remained approximately constant, averaging 102 % relative to the first cycle (Fig 1a). Hysteresis did not follow a specific trend throughout the 10 SS cycles (Fig 1b). DISCUSSION AND CONCLUSIONS The “constant” peak forces are indicative of elastic recoil of myofibrils during the SS cycles. However, the persistent and random hystereses are indicative of viscous properties. If Ig domains were still unfolding during the SS cycles, peak stresses should also decrease. Since this is not observed, we suggest that all Ig domains are unfolded in this experiment, and that the viscous behaviour producing the hystereses must come from a source other than titin. At this point, any proposition as to the origin of the remnant hystereses is highly speculative but might be associated with titin binding-unbinding to another structural (titin) or contractile (actin) protein that is forming and breaking continuously during the SS cycles.
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