Transcriptomic profiles of muscular dystrophy with myositis (mdm) in extensor digitorum longus, psoas, and soleus muscles from mice.

BACKGROUND: Titinopathies are inherited muscular diseases triggered by genetic mutations in the titin gene. Muscular dystrophy with myositis (mdm) is one such disease caused by a LINE repeat insertion, leading to exon skipping and an 83-amino acid residue deletion in the N2A-PEVK region of mouse titin. This region has been implicated in a number of titin-titin ligand interactions, hence are important for myocyte signaling and health. Mice with this mdm mutation develop a severe and progressive muscle degeneration. The range of phenotypic differences observed in mdm mice shows that the deletion of this region induces a cascade of transcriptional changes extending to numerous signaling pathways affected by the titin filament. Previous research has focused on correlating phenotypic differences with muscle function in mdm mice. These studies have provided understanding of the downstream physiological effects resulting from the mdm mutation but only provide insights on processes that can be physiologically observed and measured. We used differential gene expression (DGE) to compare the transcriptomes of extensor digitorum longus (EDL), psoas and soleus muscles from wild-type and mdm mice to develop a deeper understand of these tissue-specific responses. RESULTS: The overall expression pattern observed shows a well-differentiated transcriptional signature in mdm muscles compared to wild type. Muscle-specific clusters observed within the mdm transcriptome highlight the level of variability of each muscle to the deletion. Differential gene expression and weighted gene co-expression network analysis showed a strong directional response in oxidative respiration-associated mitochondrial genes, which aligns with the poor shivering and non-shivering thermogenesis previously observed. Sln, which is a marker associated with shivering and non-shivering thermogenesis, showed the strongest expression change in fast-fibered muscles. No drastic changes in MYH expression levels were reported, which indicated an absence of major fiber-type switching events. Overall expression shifts in MYH isoforms, MARPs, and extracellular matrix associated genes demonstrated the transcriptional complexity associated with mdm mutation. The expression alterations in mitochondrial respiration and metabolism related genes in the mdm muscle dominated over other transcriptomic changes, and likely account for the late stage cellular responses in the mdm muscles. CONCLUSIONS: We were able to demonstrate that the complex nature of mdm mutation extends beyond a simple rearrangement in titin gene. EDL, psoas and soleus exemplify unique response modes observed in skeletal muscles with mdm mutation. Our data also raises the possibility that failure to maintain proper energy homeostasis in mdm muscles may contribute to the pathogenesis of the degenerative phenotype in mdm mice. Understanding the full disease-causing molecular cascade is difficult using bulk RNA sequencing techniques due to intricate nature of the disease. The development of the mdm phenotype is temporally and spatially regulated, hence future studies should focus on single fiber level investigations.

Comparative analysis of the transcriptomes of EDL, psoas, and soleus muscles from mice.

BACKGROUND: Individual skeletal muscles have evolved to perform specific tasks based on their molecular composition. In general, muscle fibers are characterized as either fast-twitch or slow-twitch based on their myosin heavy chain isoform profiles. This approach made sense in the early days of muscle studies when SDS-PAGE was the primary tool for mapping fiber type. However, Next Generation Sequencing tools permit analysis of the entire muscle transcriptome in a single sample, which allows for more precise characterization of differences among fiber types, including distinguishing between different isoforms of specific proteins. We demonstrate the power of this approach by comparing the differential gene expression patterns of extensor digitorum longus (EDL), psoas, and soleus from mice using high throughput RNA sequencing. RESULTS: EDL and psoas are typically classified as fast-twitch muscles based on their myosin expression pattern, while soleus is considered a slow-twitch muscle. The majority of the transcriptomic variability aligns with the fast-twitch and slow-twitch characterization. However, psoas and EDL exhibit unique expression patterns associated with the genes coding for extracellular matrix, myofibril, transcription, translation, striated muscle adaptation, mitochondrion distribution, and metabolism. Furthermore, significant expression differences between psoas and EDL were observed in genes coding for myosin light chain, troponin, tropomyosin isoforms, and several genes encoding the constituents of the Z-disk. CONCLUSIONS: The observations highlight the intricate molecular nature of skeletal muscles and demonstrate the importance of utilizing transcriptomic information as a tool for skeletal muscle characterization.

Effects of a titin mutation on force enhancement and force depression in mouse soleus muscles.

The active isometric force produced by muscles varies with muscle length in accordance with the force-length relationship. Compared with isometric contractions at the same final length, force increases after active lengthening (force enhancement) and decreases after active shortening (force depression). In addition to cross-bridges, titin has been suggested to contribute to force enhancement and depression. Although titin is too compliant in passive muscles to contribute to active tension at short sarcomere lengths on the ascending limb and plateau of the force-length relationship, recent evidence suggests that activation increases titin stiffness. To test the hypothesis that titin plays a role in force enhancement and depression, we investigated isovelocity stretching and shortening in active and passive wild-type and mdm (muscular dystrophy with myositis) soleus muscles. Skeletal muscles from mdm mice have a small deletion in the N2A region of titin and show no increase in titin stiffness during active stretch. We found that: (1) force enhancement and depression were reduced in mdm soleus compared with wild-type muscles relative to passive force after stretch or shortening to the same final length; (2) force enhancement and force depression increased with amplitude of stretch across all activation levels in wild-type muscles; and (3) maximum shortening velocity of wild-type and mdm muscles estimated from isovelocity experiments was similar, although active stress was reduced in mdm compared with wild-type muscles. The results of this study suggest a role for titin in force enhancement and depression, which contribute importantly to muscle force during natural movements.

Muscle Function from Organisms to Molecules.

Gaps in our understanding of muscle contraction at the molecular level limit the ability to predict in vivo muscle forces in humans and animals during natural movements. Because muscles function as motors, springs, brakes, or struts, it is not surprising that uncertainties remain as to how sarcomeres produce these different behaviors. Current theories fail to explain why a single extra stimulus, added shortly after the onset of a train of stimuli, doubles the rate of force development. When stretch and doublet stimulation are combined in a work loop, muscle force doubles and work increases by 50% per cycle, yet no theory explains why this occurs. Current theories also fail to predict persistent increases in force after stretch and decreases in force after shortening. Early studies suggested that all of the instantaneous elasticity of muscle resides in the cross-bridges. Subsequent cross-bridge models explained the increase in force during active stretch, but required ad hoc assumptions that are now thought to be unreasonable. Recent estimates suggest that cross-bridges account for only ∼12% of the energy stored by muscles during active stretch. The inability of cross-bridges to account for the increase in force that persists after active stretching led to development of the sarcomere inhomogeneity theory. Nearly all predictions of this theory fail, yet the theory persists. In stretch-shortening cycles, muscles with similar activation and contractile properties function as motors or brakes. A change in the phase of activation relative to the phase of length changes can convert a muscle from a motor into a spring or brake. Based on these considerations, it is apparent that the current paradigm of muscle mechanics is incomplete. Recent advances in our understanding of giant muscle proteins, including twitchin and titin, allow us to expand our vision beyond cross-bridges to understand how muscles contribute to the biomechanics and control of movement.

Case Study: A Bio-Inspired Control Algorithm for a Robotic Foot-Ankle Prosthesis Provides Adaptive Control of Level Walking and Stair Ascent.

Powered ankle-foot prostheses assist users through plantarflexion during stance and dorsiflexion during swing. Provision of motor power permits faster preferred walking speeds than passive devices, but use of active motor power raises the issue of control. While several commercially available algorithms provide torque control for many intended activities and variations of terrain, control approaches typically exhibit no inherent adaptation. In contrast, muscles adapt instantaneously to changes in load without sensory feedback due to the intrinsic property that their stiffness changes with length and velocity. We previously developed a "winding filament" hypothesis (WFH) for muscle contraction that accounts for intrinsic muscle properties by incorporating the giant titin protein. The goals of this study were to develop a WFH-based control algorithm for a powered prosthesis and to test its robustness during level walking and stair ascent in a case study of two subjects with 4-5 years of experience using a powered prosthesis. In the WFH algorithm, ankle moments produced by virtual muscles are calculated based on muscle length and activation. Net ankle moment determines the current applied to the motor. Using this algorithm implemented in a BiOM T2 prosthesis, we tested subjects during level walking and stair ascent. During level walking at variable speeds, the WFH algorithm produced plantarflexion angles (range = -8 to -19°) and ankle moments (range = 1 to 1.5 Nm/kg) similar to those produced by the BiOM T2 stock controller and to people with no amputation. During stair ascent, the WFH algorithm produced plantarflexion angles (range -15 to -19°) that were similar to persons with no amputation and were ~5 times larger on average at 80 steps/min than those produced by the stock controller. This case study provides proof-of-concept that, by emulating muscle properties, the WFH algorithm provides robust, adaptive control of level walking at variable speed and stair ascent with minimal sensing and no change in parameters.