Effects of Genetic Variability on Muscle Adaptation to Mechanical Unloading

Introduction: Extended periods of mechanical unloading, such as during spaceflight or bedrest, can cause significant changes to the musculoskeletal system, particularly reducing the volume and strength of the affected bone and muscle by up to 20% in the first month. Genetics are known to play a large role in the proportion of lean mass and risk for sarcopenia with age, though the role of genetic variability in muscle adaptation to mechanical unloading is unknown. Diversity outbred (DO) mice are a genetically heterogeneous population of mice that can be used as a model to understand how genetic variability influences the response of the musculoskeletal system subjected to mechanical unloading. The traditional method for studying unloading, hindlimb unloading, requires a separate group for controls whereas our model of single limb immobilization (SLI) is a less harsh treatment and allows for the opposite leg to be used as a control. In addition to understanding the phenotypical changes that occur, gene expression analysis can be used to understand the underlying pathways and genetic contributions that occur in the adaptation to mechanical unloading. The objective of our study is to understand the mechanisms of musculoskeletal response to unloading and the effect of genetic variability on these responses, which will allow researchers to develop treatments to combat unloading-induced muscle loss. We hypothesize that genetic variation will determine the extent of muscle morphology and strength performance changes in the hind limb as a result of mechanical unloading via SLI.

Materials and

Methods: All animals were purchased and procedures performed with the approval of the Virginia Commonwealth University (VCU) Institutional Animal Care and Use Committee (IACUC). Thirty-nine male and thirty-eight female 16-week-old DO mice were used to simulate mechanical unloading via SLI of the right leg. The left leg served as a contralateral control for each mouse. After three weeks of immobilization, mice were sacrificed. Low resolution (60µm) in vivo µCT scans of the hind limbs were taken at day 0 and after 21 days to analyze hind limb muscle morphology. Muscle morphology were quantified by mean cross-sectional muscle area and muscle volume. Muscle performance was measured at the end of the study using an in vivo muscle lever system where electrical stimulation of the tibial nerve causes plantar flexion of the foot. Muscle performance were quantified by twitch and tetanus torque. At sacrifice, hind limb muscles were extracted and preserved for RNA sequencing. Total RNA from the gastrocnemius tissue was removed using a Qiagen RNeasy Fibrous Tissue Kit. RNA integrity was quantified using an Agilent Bioanalyzer and only samples with an RNA integrity number (RIN) of greater than 5.0 were used for analysis. Samples were analyzed through the VCU Genomics Core. Raw RNA sequencing data were processed using BioJupies to generate gene counts and differentially expressed gene lists. Ingenuity Pathway Analysis (IPA) was performed on RNAseq data to elucidate the underlying pathways, upstream regulators and cell functions causing the morphological or physiological changes that occur during mechanical unloading.

Results, Conclusions, and Discussions: Three weeks of right limb immobilization caused a significant decrease in body mass, gastrocnemius mass and quadriceps mass for both the control and immobilized limb (Fig. 1). These results are consistent with previous literature showing a decrease in muscle mass in both the treatment and control limbs due to systemic effects of the treatment. Interesting changes in muscle morphology were observed due to immobilization; specifically, a sex-dependent increase in mean cross-sectional leg muscle area and muscle volume in males, but not females (Fig. 2A, B). These results are consistent with visual observations made during the study, including increased swelling in the casted leg of males compared to females. Similar results were observed for muscle volume, where there was a sex-dependent increase in muscle volume for males, but not females (Fig 2C, D). In both of these cases, the sex-dependent nature of the results is supported by the fact that males are statistically larger than females at baseline, though all the casts were the same size. For muscle performance, it was found that immobilization caused an increase in twitch torque for both limbs (Fig. 3A, B). These results suggest that immobilization suppresses slow-twitch fibers and activates fast-twitch fibers, which results in an increased twitch torque production. Immobilization had no effect on tetanus torque production, though it is unclear whether or not the treatment had an overall impact on the magnitude of tetanus torque production given baseline measurements were not taken. Pathway analysis of gene expression data from gastrocnemius muscle of control and treatment limbs revealed increased activation of pathways involving inflammation and fibrosis (Fig. 3A). The most significant upstream regulators found are inflammatory cytokines that regulate the regeneration of muscle and formation of connective tissue. The top diseases and cell functions were found to be cell migration, specifically the infiltration of immune cells and tumorigenic cells. These gene expression results validate the morphology changes that were measured, showing that increased muscle volume and area is caused by the infiltration of immune cells and fibrotic tissue as a response to muscle atrophy and chronic damage to the tissue.

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