Identification of underexplored mesenchymal and vascular-related cell populations in human skeletal muscle.

Skeletal muscle repair and maintenance are directly and indirectly supported by interstitial cell populations such as vascular cells and fibro-adipogenic progenitors (FAPs), a subset of which express Twist2 and possess direct myogenic potential. Furthermore, work in rodents has highlighted the potential of pericytes to act as progenitor cells, giving rise to muscle cells and transdifferentiating into endothelial cells. However, less is understood about these populations in human skeletal muscle. Here, we performed single-cell RNA sequencing (scRNAseq) on ∼2,000 cells isolated from the human semitendinosus muscle of young individuals. This demonstrated the presence of a vascular-related cell type that expressed pericyte and pan-endothelial genes that we localized to large blood vessels within skeletal muscle cross sections and termed endothelial-like pericytes (ELPCs). RNA velocity analysis indicated that ELPCs may represent a "transition state" between endothelial cells and pericytes. Analysis of published scRNAseq data sets revealed evidence for ELPCs in trunk and heart musculature, which showed transcriptional similarity. In addition, we identified a subset of FAPs expressing TWIST2 mRNA and protein. Human TWIST2-expressing cells were anatomically and transcriptionally comparable to mouse Twist2 cells as they were restricted to the myofiber interstitium, expressed fibrogenic genes but lacked satellite cell markers, and colocalized with the FAPs marker PDGFRα in human muscle cross sections. Taken together, these results highlight the complexity of stromal cells residing in human skeletal muscle and support the utility of scRNAseq for discovery and characterization of poorly described cell populations.

Twist2-expressing cells reside in human skeletal muscle and are responsive to aging and resistance exercise training.

Skeletal muscle is maintained and repaired by sub-laminar, Pax7-expressing satellite cells. However, recent mouse investigations have described a second myogenic progenitor population that resides within the myofiber interstitium and expresses the transcription factor Twist2. Twist2-expressing cells exclusively repair and maintain type IIx/b muscle fibers. Currently, it is unknown if Twist2-expressing cells are present in human skeletal muscle and if they function as myogenic progenitors. Here, we perform a combination of single-cell RNA sequencing analysis and immunofluorescence staining to demonstrate the identity and localization of Twist2-expressing cells in human skeletal muscle. Twist2-expressing cells were identified to be anatomically and transcriptionally comparable to fibro-adipogenic progenitors (FAPs) and lack expression of typical satellite cell markers such as Pax7. Comparative analysis revealed that human and mouse Twist2-expressing cells were highly transcriptionally analogous and resided within the same anatomical structures in vivo. Examination of young and aged skeletal muscle biopsy samples revealed that Twist2-positive cells are more prevalent in aged muscle and increase following 12-weeks of resistance exercise training (RET) in humans. However, the quantity of Twist2-positive cells was not correlated with indices of muscle mass or muscle fiber cross-sectional area (CSA) in young or older muscle, and their abundance was surprisingly, negatively correlated with CSA and myonuclear domain size following RET. Taken together, we have identified cells expressing Twist2 in human skeletal muscle which are responsive to aging and exercise. Further examination of their myogenic potential is warranted.

The effect of anterior cruciate ligament graft orientation on rotational knee kinematics.

PURPOSE: To determine whether coronal graft orientation and tunnel placement for single-bundle anterior cruciate ligament (ACL) reconstruction is associated with tibial rotation excursion during functional activities. METHODS: Eighty-four patients who had undergone ACL reconstruction over a ten-year time span had tibial rotation measured during level walking, using a three-dimensional motion analysis system. Fifty-two patients also had measures taken during the more dynamic task of single-limb landing. During the 10-year period, the position of the graft was deliberately changed from a vertical to more horizontal orientation in the coronal plane. Post-operative radiographs were analysed for the coronal graft orientation and femoral and tibial tunnel positions. Radiographic measurements of graft orientation and tunnel position were then correlated with the amount of tibial rotational excursion recorded during the walking and landing tasks. RESULTS: For the single-limb landing task, a significant positive correlation was observed between the coronal graft angle and rotational excursion (R = 0.35, R (2)  = 0.12, p = 0.01). This indicated greater rotational excursion was associated with vertical graft orientation, but only explained 12 % of the variance. No correlations were found between coronal graft angle and tibial rotation during level walking. CONCLUSIONS: These findings support the notion that ACL graft orientation may play a role in rotational kinematics of the ACL-reconstructed knee, particularly during higher impact activities. LEVEL OF EVIDENCE: IV.