Growing old too early, automated assessment of skeletal muscle single fiber biomechanics in ageing R349P desmin knock-in mice using the MyoRobot technology
Charlotte MeyerMichael HaugBarbara ReischlGerhard PrölßThorsten PöschelStefan J. RupitschChristoph S. ClemenRolf SchröderOliver Friedrich
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Abstract Muscle biomechanics is determined by active motor-protein assembly and passive strain transmission through cytoskeletal structures. The extrasarcomeric desmin filament network aligns myofibrils at the z-discs, provides nuclear-sarcolemmal anchorage and may also serve as memory for muscle repositioning following large strains. Our previous analyses of R349P desmin knock-in mice, an animal model for the human R350P desminopathy, already depicted pre-clinical changes in myofibrillar arrangement and increased fiber bundle stiffness compatible with a pre-aged phenotype in the disease. Since the specific effect of R349P desmin on axial biomechanics in fully differentiated muscle fibers is unknown, we used our automated MyoRobot biomechatronics platform to compare passive and active biomechanics in single fibers derived from fast- and slow-twitch muscles from adult to senile mice hetero- or homozygous for this desmin mutation with wild-type littermates. Experimental protocols involved caffeine-induced Ca 2+ -mediated force transients, pCa-force curves, resting length-tension curves, visco-elasticity and ‘slack-tests’. We demonstrate that the presence of R349P desmin predominantly increased single fiber axial stiffness in both muscle types with a pre-aged phenotype over wild-type fibers. Axial viscosity was unaffected. Likewise, no systematic changes in Ca 2+ -mediated force properties were found. Notably, mutant single fibers showed faster unloaded shortening over wild-type fibers. Effects of ageing seen in the wild-type always appeared earlier in the mutant desmin fibers. Impaired R349P desmin muscle biomechanics is clearly an effect of a compromised intermediate filament network rather than secondary to fibrosis.Keywords:
Desmin
Myofibril
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Abstract Human movement occurs through contraction of the basic unit of the muscle cell, the sarcomere. Sarcomeres have long been considered to be arranged end-to-end in series along the length of the muscle into tube-like myofibrils with many individual, parallel myofibrils comprising the bulk of the muscle cell volume. Here, we demonstrate that striated muscle cells form a continuous myofibrillar matrix linked together by frequently branching sarcomeres. We find that all muscle cells contain highly connected myofibrillar networks though the frequency of sarcomere branching goes down from early to late postnatal development and is higher in slow-twitch than fast-twitch mature muscles. Moreover, we show that the myofibrillar matrix is united across the entire width of the muscle cell both at birth and in mature muscle. We propose that striated muscle force is generated by a singular, mesh-like myofibrillar network rather than many individual, parallel myofibrils.
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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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