Cellular Mechanisms Involved in Muscle Pathology

Study Overview
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Brief Summary
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The overall purpose of this proposed study is the understanding of cellular mechanisms involved in the pathologic fatty degeneration of muscle. Fatty infiltration in skeletal muscle is observed following sports injuries such as muscle strain injuries and Achilles tendon rupture. It is also observed in the degenerative state after rotator cuff tears as well as in the aging process. While fatty degeneration of skeletal muscle occurs in many different conditions and is known to decrease muscle function, the cellular processes involved in fatty infiltration have not been investigated in human muscle. Hypotheses: There is a high amount of fibro-adipogenic progenitors (FAPs) with an adipogenic pattern in pathologic skeletal muscle following a muscle strain injury and a full Achilles tendon rupture. We hypothesize that there is an increased number of FAPs with an adipogenic signature already in the acute phase after a strain injury, but a significantly higher number in the chronic stage as well as in the muscle following an Achilles tendon rupture. Mechanical cues are a major driver of the phenotypic drift of FAPs. The lack of mechanical stimuli initiates the adoption of an adipogenic pathway in naïve FAPs, whereas naïve FAPs exposed to mechanical stimuli will maintain their undifferentiated phenotype. The adherence of FAPs to a soft material will activate the adoption of an adipogenic phenotype, whereas a stiff material will favor a more fibrotic phenotype in naïve FAP's isolated from healthy skeletal muscle.

Detailed Description
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Skeletal muscle strain injury is one of the most common sports injuries, which is caused by excessive tensile strain of the muscle and affects the region where muscle fascicles insert into the connective tissue (aponeurosis). It is therefore not an injury solely affecting skeletal muscle, but the skeletal muscle-aponeurosis interface. Having sustained one strain injury increases the risk of re-injury substantially, changes the biomechanics of the involved muscle and leads to a significantly enlarged aponeurosis. Recently, the investigators have discovered the accumulation of ectopic fat in the intra-fascicular space and within myofibrils. The built-up of ectopic fat in skeletal muscle is a degenerative state and has mostly been described for rotator cuff injuries where it is associated with worse shoulder functional scores. At the same time, the investigators have observed a high cellularity in the pathologic muscle-aponeurosis following strain injuries, but it is not known what cell types these cells are. It seems therefore likely that at least some of the cells are Fibro/adipogenic progenitors (FAPs), which contribute to the fat accumulation in strain injured muscles and that fat accumulation might be a factor contributing to the high risk of recurrent injuries, as well as other medical problems such as pain and decreased function often described long time after a strain injury. Another common injury of the lower leg is the full Achilles tendon rupture. Recent data show that patients have long-term impairments in the injured leg when compared to the non-injured side. Also after full Achilles tendon ruptures, fatty degeneration of the muscles in the calf has been reported and has been linked to poor patient outcome. Recently, the investigators have observed fatty infiltration of calf muscles in patients following Achilles tendon rupture, which might lead to a decrease in muscle function and thereby to a poor patient outcome. Despite the high prevalence of muscle strain injuries, very little is known about the cellular mechanisms following a strain injury. In addition, the literature on the cellular composition and alterations following Achilles tendon ruptures is scarce. A decade ago, a group of cells named fibro/adipogenic progenitors (FAPs) have been identified in the interstitial space of skeletal muscle based on the presence of stem cell antigen 1+, CD34+, Platelet Derived Growth Factor Receptor α (PDGFR α)+ . Aside from their supporting activity in muscle regeneration, FAPs have the potential to adopt an adipogenic or profibrotic phenotype. Despite the high incidence, it has not been studied whether the traumatic injury and the subsequent biomechanical changes in the skeletal muscle and associated connective tissue following strain injuries or Achilles tendon rupture activate FAPs, and how these cells contribute to the fatty accumulation in the skeletal muscle. Hypotheses: There is a high amount of fibro-adipogenic progenitors (FAPs) with an adipogenic pattern in pathologic skeletal muscle following a muscle strain injury and a full Achilles tendon rupture. The investigators hypothesize that there is an increased number of FAPs with an adipogenic signature already in the acute phase after a strain injury, but a significantly higher number in the chronic stage as well as in the muscle following an Achilles tendon rupture. Mechanical cues are a major driver of the phenotypic drift of FAPs. The lack of mechanical stimuli initiates the adoption of an adipogenic pathway in naïve FAPs, whereas naïve FAPs exposed to mechanical stimuli will maintain their undifferentiated phenotype. The adherence of FAPs to a soft material will activate the adoption of an adipogenic phenotype, whereas a stiff material will favor a more fibrotic phenotype in naïve FAP's isolated from healthy skeletal muscle. To test this, the investigators will take small tissue samples from the injured area in either the acute phase (subject group A) or the chronic phase (subject group B) after a strain injury as well as from the muscle tissue of individuals who had a full Achilles tendon rupture at least a year prior to inclusion (subject group C). In all three groups, the investigators will also take a small tissue sample from the same muscle as the injured one on the healthy side. In addition, a larger muscle biopsy from the contralateral healthy vastus lateralis muscle is taken to compare the cellular composition and the cellular profile between the pathologic skeletal muscle-aponeurosis and a healthy skeletal muscle. To test the importance of mechanical stimuli, isolated muscle cells will be sorted with Fluorescence Activated Cell Sorting (FACS) and cultured in vitro thereafter. The investigators particularly focus on culture under static and dynamic tension and in the de-tensioned state. An alpha level of 0.05 will be considered significant. Based on previous data, 10 participants will be recruited for each group, enabling the investigators to detect an estimated doubling of FAPs in the injured muscles (primary outcome). Power analysis for the primary outcome resulted in n=4 for each group (α=0.05, β=0.80, CV=72.4%), which the investigators consider to be clinically relevant. The investigators plan to include 10 participants in each group to have sufficient biopsy material from 5 individuals for single nuclei RNA sequencing and 5 biopsies for fluorescence-activated cell sorting and the subsequent in vitro studies.

Official Title
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Cellular Mechanisms Involved in Pathology Following Musculo-tendinous Injuries

Conditions
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Muscle Strain, Lower Leg, Muscle Strained Thigh, Achilles Tendon; Laceration, Fatty Degeneration

Intervention / Treatment
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* Procedure: Biopsies


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: 18 years and older, male and female, with an acute muscle strain injury (<14 days post injury) in the calf or hamstring muscles (group 1). Pathological changes visible on an ultrasound scan as hypoechoic areas. 18 years and older, male and female, with a chronic muscle strain injury (> 6 months prior to inclusion) in the calf or hamstring muscles (group 2), sequela including recurrent strain injuries, pain and/ or decrease in muscle function. Pathological changes visible on an ultrasound scan as hypo-/ hyperechoic areas. 18 years and older, male and female, with a full Achilles tendon rupture (> 1 year prior to inclusion) with pathological changes: Fatty degeneration visible on an ultrasound scan (group 3). Exclusion Criteria: Type I and II Diabetes, Connective tissue and/or rheumatic diseases, or any observed organ dysfunctions Daily smoking Persons with daily intake of non-steroidal anti-inflammatory drugs (NSAIDs) within three months prior to time of contact Allergic reactions to local anesthesia Use of anticoagulant treatment Needle phobia Any drug or alcohol abuse now or in the past The absence of any pathological changes visible on an ultrasound scan as hypo-/ hyperechoic areas (group 1 and 2) The absence of any pathological changes (fatty degeneration) in either the gastrocnemius or the soleus muscle visible on an ultrasound scan (Subject group 3)

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Acute muscle strain<br>Subjects with an acute muscle strain (within 14 days post injury) in either the hamstrings or the calf muscles | Procedure: Biopsies<br>* Biopsies from the pathological muscle and healthy, unaffected muscle<br>|
| Chronic muscle strain<br>Subjects with a chronic muscle strain more than 6 months prior to inclusion in either the hamstrings or the calf muscles | Procedure: Biopsies<br>* Biopsies from the pathological muscle and healthy, unaffected muscle<br>|
| Achilles tendon rupture<br>Subjects with a full Achilles tendon rupture more than 12 months prior to inclusion | Procedure: Biopsies<br>* Biopsies from the pathological muscle and healthy, unaffected muscle<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Presence and number of Fibro-adipogenic progenitors | Determination of the number and the phenotypic appearance of fibro-adipogenic progenitors in the injured compared to the healthy muscle | 2 years: Sampling and analyses | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Cell cluster analysis | Analysis of cell clusters present in the injured compared to the healthy muscle by single nuclei RNA sequencing | 2 years: Sampling and analyses | 
| Importance of mechanical stimuli for Fibro-adipogenic progenitors | Importance of mechanical stimuli on the phenotypic drift of fibro-adipogenic progenitors (FAPS. Isolated FAP´s will be subjected to either mechanical stimulation (by using the FlexCell system) or unloading (FAPs embedded in a free-floating matrix). FAPs will also be cultured on stiff versus soft surfaces to study thereafter the effect of the substrate properties on the phenotypic appearance of FAPs. The phenotypic appearance of FAPs will be tested by qRT-PCR, protein analysis and compared to the well-described effect on phenotypic apprearance of FAPs when subjected to pro-fibrotic or pro-adipogenic differentiation medium. | 2 years: Sampling and analyses |