Cellular Mechanisms Involved in Muscle Pathology

Overview

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: 1. 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. 2. 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. 3. 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.

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: 1. 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. 2. 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. 3. 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.