Growing old too early, automated assessment of skeletal muscle single fiber biomechanics in ageing R349P desmin knock-in mice using the MyoRobot technology

Abstract

Growing old too early, automated 1 assessment of skeletal muscle single 2 fiber biomechanics in ageing R349P 3 desmin knock-in mice using the 4 MyoRobot technology 5Abstract Muscle biomechanics is determined by active motor-protein assembly and passive 28 strain transmission through cytoskeletal structures. The extrasarcomeric desmin filament network 29 aligns myofibrils at the z-discs, provides nuclear-sarcolemmal anchorage and may also serve as 30 memory for muscle repositioning following large strains. Our previous analyses of R349P desmin 31 knock-in mice, an animal model for the human R350P desminopathy, already depicted pre-clinical 32 changes in myofibrillar arrangement and increased fiber bundle stiffness compatible with a 33 pre-aged phenotype in the disease. Since the specific effect of R349P desmin on axial 34 biomechanics in fully differentiated muscle fibers is unknown, we used our automated MyoRobot 35 biomechatronics platform to compare passive and active biomechanics in single fibers derived 36 from fast-and slow-twitch muscles from adult to senile mice hetero-or homozygous for this 37 desmin mutation with wild-type littermates. Experimental protocols involved caffeine-induced 38 Ca 2+ -mediated force transients, pCa-force curves, resting length-tension curves, visco-elasticity and 39 'slack-tests'. We demonstrate that the presence of R349P desmin predominantly increased single 40 fiber axial stiffness in both muscle types with a pre-aged phenotype over wild-type fibers. Axial 41 viscosity was unaffected. Likewise, no systematic changes in Ca 2+ -mediated force properties were 42 found. Notably, mutant single fibers showed faster unloaded shortening over wild-type fibers. 43 Effects of ageing seen in the wild-type always appeared earlier in the mutant desmin fibers. 44 Impaired R349P desmin muscle biomechanics is clearly an effect of a compromised intermediate 45 filament network rather than secondary to fibrosis. 46 47 48 Skeletal muscle is the largest organ system of the body and under constant mechanical stress, either 49 due to passive strain or through active contraction producing axial and lateral stresses. While lateral 50 forces are distributed between single fibers across anchorage points in the extracellular matrix 51 (ECM) to the intracellular cytoskeleton via the dystrophin-glycoprotein complex (DGC) Ramaswamy 52 et al. (2011) and focal adhesion complexes Ra et al. (1999), axial forces are distributed through 53 contractile (active) and non-contractile (passive) elements. Apart from the giant, roughly 1.5 m 54 long elastomeric protein titin, being responsible for the visco-elastic properties of single muscle 55 fibers through unfolding of globular domains under strain Mártonfalvi and Kellermayer (2014); 56 Powers et al. (2018), connecting proteins of the extra-sarcomeric intermediate filament (IF) family 57 may also be a vital determinant of axial elasticity. An important member of the IFs is the type 58 III filament protein desmin, transversely linking adjacent myofibri...