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Transcriptome analysis of tenotomy immobilized rat soleus muscle

ABSTRACT: Tenotomy is the release of muscle preload that causes abrupt shortening of the muscle and models atrophy and fibrosis without inflammatory response. Fibrosis in the skeletal muscle is known to be triggered by TGF-β, which is activated by mediators of inflammatory events. As these were lacking, tenotomy provided an opportunity to investigate transcriptional events on a background without inflammation. An unbiased look at the transcriptome of tenotomy-immobilized soleus muscle revealed that the majority of the transcriptional changes took place in the first four weeks. The transcriptome provided clear-cut evidence for the upregulation of collagens and several extracellular matrix components that defined fibrotic remodeling of the skeletal muscle architecture as well as activation of the fibro-adipogenic precursors.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE73510 | GEO | 2016/05/23

SECONDARY ACCESSION(S): PRJNA297188

REPOSITORIES: GEO

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Project description:Skeletal muscle atrophy is a consequence of many diseases, environmental insults, inactivity, age and injury. Atrophy is characterized by active degradation and removal of contractile proteins and a reduction in fiber size. Animal models have been extensively used to identify pathways leading to atrophic conditions. Here we have used genome-wide expression profiling analysis and quantitative PCR to identify the molecular changes that occur in two clinically relevant animal mouse models of muscle atrophy, hindlimb casting and Achilles tendon laceration (tenotomy). Gastrocnemius muscle samples were collected 2, 7 and 14 days after insult. The total amount of muscle loss as measured by wet weight and muscle fiber size was equivalent between models, although tenotomy resulted in a more rapid induction of muscle atrophy. Furthermore, tentomy resulted in the regulation of significantly more mRNA transcripts then casting. Analysis of the regulated genes and pathways suggest that the mechanism of atrophy is distinct between these models. The degradation following casting appears ubiquitin-proteasome-mediated while degradation following tenotomy appears lysosomal and matrix-metalloproteinase (MMP)-mediated. This data suggests that there are multiple mechanisms leading to muscle atrophy and that specific therapeutic agents may be necessary to combat the atrophy seen under different conditions. Muscle atrophy was induced through two methods; unloading induced by tendon laceration (Tenotomy) and immobilization induced by hindlimb casting (Casting). For the tenotomy, eight week old male C57/B6 mice were anesthetized by ether inhalation and subsequent intramuscular injection of ketamine-xylazine (0.1 ml/g). The right Achilles tendon just proximal to the calcaneus was severed with a sterile scalpel. The mice were allowed to recover and move freely about their cage. An additional cohort of animals was sham operated (Sham) which involved isolation and manipulation of the Achilles tendon without laceration. For the casting, the right hindlimbs of eight week old male C57Bl/6 mice were immobilized through casting. After 2, 7 or 14 days, animals were sacrificed and the left and right gastrocnemius muscles were carefully removed, flash frozen in liquid nitrogen, and stored at -80°C for further processing. The left untreated legs served as controls. A group of naïve animals that received no treatment or manipulation was included as an additional control. Each group consisted of between 5 and 10 samples for a total of 111 individual samples.

Project description:Skeletal muscle regeneration is primarily accomplished through the concerted action of the myoblasts as well as the supporting stromal cells. Following accomplishment of injury repair, activated stromal cells disappear via apoptosis. In the case of muscle dystrophies where in a chronic inflammatory environment is present, activated stromal cells are transformed into a myofibroblastic state with the deposition of extracellular matrix components. Excessive accumulation of ECM leads to irreversible fibrosis and tissue deterioration. A distinct stromal cell population named fibro-adipogenic precursors (FAPs) are known to be responsible for contribution to fibrosis and fatty infiltration. Fibrosis is also evident in disuse atrophy and sarcopenia without any significant inflammation. This study aims to analyze the heterogeneous population of FAPs comparatively in the course of acute damage and immobilization models. We isolated and analyzed the mononuclear cell population of skeletal muscles following tenotomy and denervation models in mice. Immunophenotyping of the FAPs was conducted based on exclusion of CD45, CD31 and CD11b and a comparative analysis was accomplished on three different subpopulations of i) CD140a(+), Sca1(+) ii) CD140a(+), Sca1(-) iii) CD140a(-), Sca1(+). High-throughput transcriptome analysis was employed to further characterize the transcriptome of these three cell populations and compare their quiescent and activated states. Results: Investigated sub-populations exhibited diverse activation properties and kinetics for each studied model. While CD140a(+), Sca1(+) was the predominating activated population along with tenotomy, denervation majorly stimulated the proliferation of CD140a(+), Sca1(+) cells. Both immobilization models lacked myofiber damage and exhibited a minute inflammatory response compared to the acute damage. Transcriptome analysis supports commonalities in upregulation of ECM components in diverse subpopulation of cells as well as discriminating candidate cell surface markers. Skeletal muscle regeneration is primarily accomplished through the concerted action of the myoblasts as well as the supporting stromal cells. Following accomplishment of injury repair, activated stromal cells disappear via apoptosis. In the case of muscle dystrophies where in a chronic inflammatory environment is present, activated stromal cells are transformed into a myofibroblastic state with the deposition of extracellular matrix components. Excessive accumulation of ECM leads to irreversible fibrosis and tissue deterioration. A distinct stromal cell population named fibro-adipogenic precursors (FAPs) are known to be responsible for contribution to fibrosis and fatty infiltration. Fibrosis is also evident in disuse atrophy and sarcopenia without any significant inflammation. This study aims to analyze the heterogeneous population of FAPs comparatively in the course of acute damage and immobilization models. We isolated and analyzed the mononuclear cell population of skeletal muscles following tenotomy and denervation models in mice. Immunophenotyping of the FAPs was conducted based on exclusion of CD45, CD31 and CD11b and a comparative analysis was accomplished on three different subpopulations of i) CD140a(+), Sca1(+) ii) CD140a(+), Sca1(-) iii) CD140a(-), Sca1(+). High-throughput transcriptome analysis was employed to further characterize the transcriptome of these three cell populations and compare their quiescent and activated states. Results: Investigated sub-populations exhibited diverse activation properties and kinetics for each studied model. While CD140a(+), Sca1(+) was the predominating activated population along with tenotomy, denervation majorly stimulated the proliferation of CD140a(+), Sca1(+) cells. Both immobilization models lacked myofiber damage and exhibited a minute inflammatory response compared to the acute damage. Transcriptome analysis supports commonalities in upregulation of ECM components in diverse subpopulation of cells as well as discriminating candidate cell surface markers.

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Transcriptome analysis of tenotomy immobilized rat soleus muscle

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