Levosimendan's Effects on Length-Dependent Activation in Murine Fast-Twitch Skeletal Muscle.

Levosimendan's calcium sensitizing effects in heart muscle cells are well established; yet, its potential impact on skeletal muscle cells has not been evidently determined. Despite controversial results, levosimendan is still expected to interact with skeletal muscle through off-target sites (further than troponin C). Adding to this debate, we investigated levosimendan's acute impact on fast-twitch skeletal muscle biomechanics in a length-dependent activation study by submersing single muscle fibres in a levosimendan-supplemented solution. We employed our MyoRobot technology to investigate the calcium sensitivity of skinned single muscle fibres alongside their stress-strain response in the presence or absence of levosimendan (100 µM). While control data are in agreement with the theory of length-dependent activation, levosimendan appears to shift the onset of the 'descending limb' of active force generation to longer sarcomere lengths without notably improving myofibrillar calcium sensitivity. Passive stretches in the presence of levosimendan yielded over twice the amount of enlarged restoration stress and Young's modulus in comparison to control single fibres. Both effects have not been described before and may point towards potential off-target sites of levosimendan.

Myofibrillar Lattice Remodeling Is a Structural Cytoskeletal Predictor of Diaphragm Muscle Weakness in a Fibrotic mdx (mdx Cmah-/-) Model.

Duchenne muscular dystrophy (DMD) is a degenerative genetic myopathy characterized by complete absence of dystrophin. Although the mdx mouse lacks dystrophin, its phenotype is milder compared to DMD patients. The incorporation of a null mutation in the Cmah gene led to a more DMD-like phenotype (i.e., more fibrosis). Although fibrosis is thought to be the major determinant of 'structural weakness', intracellular remodeling of myofibrillar geometry was shown to be a major cellular determinant thereof. To dissect the respective contribution to muscle weakness, we assessed biomechanics and extra- and intracellular architecture of whole muscle and single fibers from extensor digitorum longus (EDL) and diaphragm. Despite increased collagen contents in both muscles, passive stiffness in mdx Cmah-/- diaphragm was similar to wt mice (EDL muscles were twice as stiff). Isometric twitch and tetanic stresses were 50% reduced in mdx Cmah-/- diaphragm (15% in EDL). Myofibrillar architecture was severely compromised in mdx Cmah-/- single fibers of both muscle types, but more pronounced in diaphragm. Our results show that the mdx Cmah-/- genotype reproduces DMD-like fibrosis but is not associated with changes in passive visco-elastic muscle stiffness. Furthermore, detriments in active isometric force are compatible with the pronounced myofibrillar disarray of the dystrophic background.

Effects of Adjuvant Exercise and Nutrition Therapy on Muscle Fibre Biomechanics in Gastrointestinal Cancer Patients.

Patients with aggressive cancer, e.g., gastrointestinal cancer, are prone (≥50% chance) to developing cancer cachexia (CC). Little is known about the effects of CC on the biomechanical function of muscle. A promising prevention strategy was found in the form of a multi-modal therapy combining mild resistance exercise (e.g., whole-body electro-myostimulation, WB-EMS) and a protein-rich diet. In a previous study of ours, this was effective in counteracting the loss of muscle mass, yet a systematic and comprehensive assessment of active and passive single muscle fibre functions was so far absent. This pilot study investigated the biomechanical function of single muscle fibres (rectus abdominis) from the biopsies of conventionally treated (pre-)cachectic cancer ((pre-)CC) patients (m = 9), those receiving the multi-modal therapy comprising WB-EMS training and protein-rich nutrition (m = 3), and a control group (m = 5). Our findings not only align with previous findings showing the absolute force loss in CC that is accelerated by atrophy but also speak in favour of a different, potentially energy- and Ca2+-homeostasis-related effect that compromises muscle contraction (F ~0.9 mN vs. F ~0.6 mN in control patients). However, myofibrillar Ca2+ sensitivity and the quality of contraction were unaltered (pCa50: 5.6-5.8). Single fibres from the (pre-)CC patients receiving WB-EMS training and protein supplementation were significantly more compliant (p < 0.001 at ≥130% of resting length L0). Those fibres displayed a similar softness to the ones from the control patients (axial compliance ~15 m/N at ≥130% L0), while single fibres from the patients with (developing) cachexia were significantly stiffer (axial compliance ~7 m/N, p < 0.001 at ≥130% L0). Adjuvant multi-modal therapy (WB-EMS training and nutritional support) contributes to maintaining the axial compliance of single fibres and potentially improves the quality of life for patients at risk of developing CC.

Structure-Function Relationships in Muscle Fibres: MyoRobot Online Assessment of Muscle Fibre Elasticity and Sarcomere Length Distributions.

OBJECTIVE: Muscle biomechanics is set by the spacing of repetitive striation patterns of individual sarcomeres within single muscle fibres of stacked myofibrils. Sarcomere lengths (SL) are rather unequally distributed than of equal distance. This non-uniformity may affect both, force production as well as passive-elastic deformation. However, online recording of SL during axially imposed strains is cumbersome due to a lack of compact technologies. METHODS: To fuse SL pattern recognition with restoration force assessments during quasi-static axial stretch, we implemented live tracking of SL distributions simultaneous to voice-coil actuated stretch and restoration force recordings in our MyoRobot 2.0 automated biomechatronics platform. Both were obtained online during stretch-relaxation cycles of murine single muscle fibres. RESULTS: Under quasi-static stretch conditions (  âˆ¼ 1 µm/s fibre length changes), almost no apparent hysteresis was detected in single fibres. SL showed a non-uniform distribution. While mean SL varied between 2.6 µm and 3.4 µm upon 140% stretch, two populations of fibres were noticed: one showing a minor change in SL distribution with stretch, and one becoming more equally distributed upon stretch. CONCLUSION: A roughly 5% SL variability under rest either diminishes or remains almost unaltered upon elastic axial deformation. This may reflect differential impact of mostly extra-sarcomeric components to stretch in this stretch range. SIGNIFICANCE: The augmented functionality of the MyoRobot 2.0 towards online sarcomere analyses within single fibres shall provide a valuable tool for the muscle community to study the contribution of serial elastic and force producing elements in health and disease models.

Redox Balance Differentially Affects Biomechanics in Permeabilized Single Muscle Fibres-Active and Passive Force Assessments with the Myorobot.

An oxidizing redox state imposes unique effects on the contractile properties of muscle. Permeabilized fibres show reduced active force generation in the presence of H2O2. However, our knowledge about the muscle fibre's elasticity or flexibility is limited due to shortcomings in assessing the passive stress-strain properties, mostly due to technically limited experimental setups. The MyoRobot is an automated biomechatronics platform that is well-capable of not only investigating calcium responsiveness of active contraction but also features precise stretch actuation to examine the passive stress-strain behaviour. Both were carried out in a consecutive recording sequence on the same fibre for 10 single fibres in total. We denote a significantly diminished maximum calcium-saturated force for fibres exposed to ≥500 µM H2O2, with no marked alteration of the pCa50 value. In contrast to active contraction (e.g., maximum isometric force activation), passive restoration stress (force per area) significantly increases for fibres exposed to an oxidizing environment, as they showed a non-linear stress-strain relationship. Our data support the idea that a highly oxidizing environment promotes non-linear fibre stiffening and confirms that our MyoRobot platform is a suitable tool for investigating redox-related changes in muscle biomechanics.

MyoBio: An Automated Bioreactor System Technology for Standardized Perfusion-Decellularization of Whole Skeletal Muscle.

OBJECTIVE: Decellularizing solid organs is a promising top-down process to produce acellular bio-scaffolds for 'de novo' regrowth or application as tissue 'patches' that compensate, e.g., large volumetric muscle loss in reconstructive surgery. Therefore, generating standardized acellular muscle scaffolds marks a pressing area of need. Although animal muscle decellularization protocols were established, those are mostly manually performed and lack defined bioreactor environments and metrologies to assess decellularization quality in real-time. To close this gap, we engineered an automated bioreactor system to provide chemical decellularization solutions to immersed whole rat gastrocnemius medialis muscle through perfusion of the main feeding arteries. RESULTS: Perfusion control is adjustable according to decellularization quality feedback. This was assessed both from (i) ex situ assessment of sarcomeres/nuclei through multiphoton fluorescence and label-free Second Harmonic Generation microscopy and DNA quantification, along with (ii) in situ within the bioreactor environment assessment of the sample's passive mechanical elasticity. CONCLUSION: We find DNA and sarcomere-free constructs after 72 h of 0.1% SDS perfusion-decellularization. Furthermore, passive elasticity can be implemented as additional online decellularization quality measure, noting a threefold elasticity decrease in acellular constructs. SIGNIFICANCE: Our MyoBio represents a novel and useful automated bioreactor environment for standardized and controlled generation of acellular whole muscle scaffolds as a valuable source for regenerative medicine.