Distinct human stem cell subpopulations drive adipogenesis and fibrosis in musculoskeletal injury.

Fibroadipogenic progenitors (FAPs) maintain healthy skeletal muscle in homeostasis but drive muscle degeneration in chronic injuries by promoting adipogenesis and fibrosis. To uncover how these stem cells switch from a pro-regenerative to pro-degenerative role we perform single-cell mRNA sequencing of human FAPs from healthy and injured human muscles across a spectrum of injury, focusing on rotator cuff tears. We identify multiple subpopulations with progenitor, adipogenic, or fibrogenic gene signatures. We utilize full spectrum flow cytometry to identify distinct FAP subpopulations based on highly multiplexed protein expression. Injury severity increases adipogenic commitment of FAP subpopulations and is driven by the downregulation of DLK1. Treatment of FAPs both in vitro and in vivo with DLK1 reduces adipogenesis and fatty infiltration, suggesting that during injury, reduced DLK1 within a subpopulation of FAPs may drive degeneration. This work highlights how stem cells perform varied functions depending on tissue context, by dynamically regulating subpopulation fate commitment, which can be targeted improve patient outcomes after injury.

Defining Endogenous Mitochondrial Transfer in Muscle After Rotator Cuff Injury.

BACKGROUND: Rotator cuff muscle degeneration leads to poor clinical outcomes for patients with rotator cuff tears. Fibroadipogenic progenitors (FAPs) are resident muscle stem cells with the ability to differentiate into fibroblasts as well as white and beige adipose tissue. Induction of the beige adipose phenotype in FAPs has been shown to improve muscle quality after rotator cuff tears, but the mechanisms of how FAPs exert their beneficial effects have not been fully elucidated. PURPOSE: To study the horizontal transfer of mitochondria from FAPs to myogenic cells and examine the effects of ß-agonism on this novel process. STUDY DESIGN: Controlled laboratory study. METHODS: In mice that had undergone a massive rotator cuff tear, single-cell RNA sequencing was performed on isolated FAPs for genes associated with mitochondrial biogenesis and transfer. Murine FAPs were isolated by fluorescence-activated cell sorting and treated with a ß-agonist versus control. FAPs were stained with mitochondrial dyes and cocultured with recipient C2C12 myoblasts, and the rate of transfer was measured after 24 hours by flow cytometry. PdgfraCreERT/MitoTag mice were generated to study the effects of a rotator cuff injury on mitochondrial transfer. PdgfraCreERT/tdTomato mice were likewise generated to perform lineage tracing of PDGFRA+ cells in this injury model. Both populations of transgenic mice underwent tendon transection and denervation surgery, and MitoTag-labeled mitochondria from Pdgfra+ FAPs were visualized by fluorescent microscopy, spinning disk confocal microscopy, and 2-photon microscopy; overall mitochondrial quantity was compared between mice treated with ß-agonists and dimethyl sulfoxide. RESULTS: Single-cell RNA sequencing in mice that underwent rotator cuff tear demonstrated an association between transcriptional markers of adipogenic differentiation and genes associated with mitochondrial biogenesis. In vitro cocultures of murine FAPs with C2C12 cells revealed that treatment of cells with a ß-agonist increased mitochondrial transfer compared to control conditions (17.8% ± 9.9% to 99.6% ± 0.13% P < .0001). Rotator cuff injury in PdgfraCreERT/MitoTag mice resulted in a robust increase in MitoTag signal in adjacent myofibers compared with uninjured mice. No accumulation of tdTomato signal from PDGFRA+ cells was seen in injured fibers at 6 weeks after injury, suggesting that FAPs do not fuse with injured muscle fibers but rather contribute their mitochondria. CONCLUSION: The authors have described a novel process of endogenous mitochondrial transfer that can occur within the injured rotator cuff between FAPs and myogenic cells. This process may be leveraged therapeutically with ß-agonist treatment and represents an exciting target for improving translational therapies available for rotator cuff muscle degeneration. CLINICAL RELEVANCE: Promoting endogenous mitochondrial transfer may represent a novel translational strategy to address muscle degeneration after rotator cuff tears.

Multitissue responses to exercise: a MoTrPAC feasibility study.

We assessed the feasibility of the Molecular Transducers of Physical Activity Consortium (MoTrPAC) human adult clinical exercise protocols, while also documenting select cardiovascular, metabolic, and molecular responses to these protocols. After phenotyping and familiarization sessions, 20 subjects (25 ± 2 yr, 12 M, 8 W) completed an endurance exercise bout (n = 8, 40 min cycling at 70% V̇o2max), a resistance exercise bout (n = 6, ∼45 min, 3 sets of ∼10 repetition maximum, 8 exercises), or a resting control period (n = 6, 40 min rest). Blood samples were taken before, during, and after (10 min, 2 h, and 3.5 h) exercise or rest for levels of catecholamines, cortisol, glucagon, insulin, glucose, free fatty acids, and lactate. Heart rate was recorded throughout exercise (or rest). Skeletal muscle (vastus lateralis) and adipose (periumbilical) biopsies were taken before and ∼4 h following exercise or rest for mRNA levels of genes related to energy metabolism, growth, angiogenesis, and circadian processes. Coordination of the timing of procedural components (e.g., local anesthetic delivery, biopsy incisions, tumescent delivery, intravenous line flushes, sample collection and processing, exercise transitions, and team dynamics) was reasonable to orchestrate while considering subject burden and scientific objectives. The cardiovascular and metabolic alterations reflected a dynamic and unique response to endurance and resistance exercise, whereas skeletal muscle was transcriptionally more responsive than adipose 4 h postexercise. In summary, the current report provides the first evidence of protocol execution and feasibility of key components of the MoTrPAC human adult clinical exercise protocols. Scientists should consider designing exercise studies in various populations to interface with the MoTrPAC protocols and DataHub.NEW & NOTEWORTHY This study highlights the feasibility of key aspects of the MoTrPAC adult human clinical protocols. This initial preview of what can be expected from acute exercise trial data from MoTrPAC provides an impetus for scientists to design exercise studies to interlace with the rich phenotypic and -omics data that will populate the MoTrPAC DataHub at the completion of the parent protocol.

Human adipose and skeletal muscle tissue DNA, RNA, and protein content.

The purpose of this project was to provide a profile of DNA, RNA, and protein content in adipose tissue, which is relatively understudied in humans, to gain more insight into the amount of tissue that may be required for various analyses. Skeletal muscle tissue was also investigated to provide a direct comparison into potential differences between these two highly metabolically active tissues. Basal adipose and skeletal muscle tissue samples were obtained from 10 (7 M, 3 W) recreationally active participants [25 ± 1 yr; 84 ± 3 kg, maximal oxygen consumption (V̇o2max): 3.5 ± 0.2 L/min, body fat: 29 ± 2%]. DNA, RNA, and protein were extracted and subsequently analyzed for quantity and quality. DNA content of adipose and skeletal muscle tissue was 52 ± 14 and 189 ± 44 ng DNA·mg tissue-1, respectively (P < 0.05). RNA content of adipose and skeletal muscle tissue was 46 ± 14 and 537 ± 72 ng RNA·mg tissue-1, respectively (P < 0.05). Protein content of adipose and skeletal muscle tissue was 4 ± 1 and 177 ± 10 µg protein·mg tissue-1, respectively (P < 0.05). In summary, human adipose had 28% of the DNA, 9% of the RNA, and 2% of the protein found in skeletal muscle per mg of tissue. This information should be useful across a wide range of human clinical investigation designs and various laboratory analyses.NEW & NOTEWORTHY This investigation studied DNA, RNA, and protein contents of adipose and skeletal muscle tissues from young active individuals. A series of optimization steps were investigated to aid in determining the optimal approach to extract high-yield and high-quality biomolecules. These findings contribute to the knowledge gap in adipose tissue requirements for molecular biology assays, which is of increasing importance due to the growing interest in adipose tissue research involving human exercise physiology research.

Influence of low-dose aspirin, resistance exercise, and sex on human skeletal muscle PGE2 /COX pathway activity.

Prostaglandin (PG) E2  has been linked to increased inflammation and attenuated resistance exercise adaptations in skeletal muscle. Nonaspirin cyclooxygenase (COX) inhibitors have been shown to reduce these effects. This study examined the effect of low-dose aspirin on skeletal muscle COX production of PGE2 at rest and following resistance exercise. Skeletal muscle (vastus lateralis) biopsies were taken from six individuals (4 M/2 W) before and 3.5 hr after a single bout of resistance exercise for ex vivo PGE2 production under control and low (10 µM)- or standard (100 µM)-dose aspirin conditions. Sex-specific effects of aspirin were also examined by combining the current findings with our previous similar ex vivo skeletal muscle investigations (n = 20, 10 M/10 W). Low-dose aspirin inhibited skeletal muscle PGE2 production (p < 0.05). This inhibition was similar to standard-dose aspirin (p > 0.05) and was not influenced by resistance exercise (p > 0.05) (overall effect: -18 ± 5%). Men and women had similar uninhibited skeletal muscle PGE2 production at rest (men: 1.97 ± 0.33, women: 1.96 ± 0.29 pg/mg wet weight/min; p > 0.05). However, skeletal muscle of men was 60% more sensitive to aspirin inhibition than women (p < 0.05). In summary, the current findings 1) confirm low-dose aspirin inhibits the PGE2 /COX pathway in human skeletal muscle, 2) show that resistance exercise does not alter aspirin inhibitory efficacy, and 3) suggest the skeletal muscle of men and women could respond differently to long-term consumption of low-dose aspirin, one of the most common chronically consumed drugs in the world.

Betalain-rich concentrate supplementation improves exercise performance and recovery in competitive triathletes.

We aimed to determine the effects of a betalain-rich concentrate (BRC) of beetroots, containing no sugars or nitrates, on exercise performance and recovery. Twenty-two (9 men and 13 women) triathletes (age, 38 ± 11 years) completed 2 double-blind, crossover, randomized trials (BRC and placebo) starting 7 days apart. Each trial was preceded by 6 days of supplementation with 100 mg·day-1 of BRC or placebo. On the 7th day of supplementation, exercise trials commenced 120 min after ingestion of 50 mg BRC or placebo and consisted of 40 min of cycling (75 ± 5% maximal oxygen consumption) followed by a 10-km running time trial (TT). Subjects returned 24 h later to complete a 5-km running TT to assess recovery. Ten-kilometer TT duration (49.5 ± 8.9 vs. 50.8 ± 10.3 min, p = 0.03) was faster with the BRC treatment. Despite running faster, average heart rate and ratings of perceived exertion were not different between treatments. Five-kilometer TT duration (23.2 ± 4.4 vs 23.9 ± 4.7 min, p = 0.003), 24 h after the 10-km TT, was faster in 17 of the 22 subjects with the BRC treatment. Creatine kinase, a muscle damage marker, increased less (40.5 ± 22.5 vs. 49.7 ± 21.5 U·L-1, p = 0.02) from baseline to after the 10-km TT and subjective fatigue increased less (-0.05 ± 6.1 vs. 3.23 ± 6.1, p = 0.05) from baseline to 24 h after the 10-km TT with BRC. In conclusion, BRC supplementation improved 10-km TT performance in competitive male and female triathletes. Improved 5-km TT performances 24 h after the 10-km TT and the attenuated increase of creatine kinase and fatigue suggest an increase in recovery while taking BRC.