Human skeletal muscle CD90⁺ fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients

Background

Obesity and obesity-related disorders are a global epidemic (for review see Blüher, 2019). People with obesity have increased risk of morbidity and mortality mainly due to comorbidities associated with excessive weight gain, hyperglycemia and metabolic impairment (Piché et al., 2020). Therefore, obesity negatively impacts the quality of life of obese individuals and represents a major burden on our ageing society. Type 2 diabetes (T2D) is a progressive condition and the risk of developing T2D dramatically increases with obesity, insufficient physical activity and ageing. In T2D our cells become resistant to the effects of insulin (a peptide hormone which reduces the levels of glucose in the blood) and/or the pancreas loses its competence to produce insulin, which results in hyperglycemia. Additionally, obese individuals with T2D develop muscle atrophy – a condition where muscles are smaller than normal – and fibro-fatty infiltration, both of which negatively affect muscle contractile function (Hilton et al., 2008; Tam et al., 2014).

The adult mammalian skeletal muscles are composed of many cell types, and therefore their niche composition is highly complex and dynamic, especially upon damage. Muscle cells communicate by physically interacting and/or secreting a plethora of factors to maintain muscle homeostasis. When muscle homeostasis is acutely disrupted following injury, adult muscle stem cells (MuSCs) (also known as satellite cells) activate, proliferate, self-renew, and differentiate to fuse with pre-existent myofibers to effectively restore the lost muscle to its previous condition, a phenomenon called adult skeletal muscle regeneration (Joe et al., 2010). However, during chronic degenerative conditions, such as obesity, T2D, and ageing, the muscle milieu is gradually disrupted, leading to a pathophysiological condition that triggers exacerbated accumulation of extracellular matrix (ECM) and adipose tissue. This associates with muscle metabolic profile dysregulation, increased tissue stiffness and reduced contraction of the affected muscles (Buras et al., 2019; Collao et al., 2020; Teng et al., 2019). Fibro-adipogenic progenitors (FAPs) are the fibroblasts of mammalian muscles with multipotency towards all the mesenchymal cell lineages (Contreras et al., 2019; Eisner et al., 2020; Kopinke et al., 2017; Scott et al., 2019; Uezumi et al., 2010, 2014). FAPs are diverse and dynamic cells required for adult muscle maintenance and effective muscle regeneration (De Micheli et al., 2020; Giordani et al., 2019; Malecova et al., 2018; Marinkovic et al., 2019; Wosczyna et al., 2019). However, FAPs can contribute to muscle pathology, where chronic injury and inflammation blunt muscle regeneration and lead to progressive tissue degeneration (Contreras et al., 2016; Hogarth et al., 2019; Reggio et al., 2020). Although important progress has been made in understanding the heterogeneity of the stromal compartment in muscle health, regeneration, and disease (Oprescu et al., 2020; Rubenstein et al., 2020; Scott et al., 2019), the cellular and molecular responses of human skeletal muscle FAPs to gradual degenerative diseases are underexplored.

In this preprint, Farup and colleagues unveiled a subpopulation of human muscle-resident FAPs that associates with progressive muscle degeneration in T2D patients. They suggested that a subset of muscle FAPs (Lin^–CD56^–CD82^–CD34⁺CD90⁺) associates with enhanced fibrosis and ectopic adipose tissue in degenerative T2D settings, shedding new light on the role of fibro-adipogenic progenitors underpinning skeletal muscle degenerative fibro-fatty remodelling in the obese and T2D population.

Obese individuals with T2D develop muscle atrophy – a condition where muscles are smaller than normal – and fibro-fatty infiltration, both of which negatively affect muscle contractile function.

Key findings

First, using skeletal muscle biopsies, the authors profiled bulk transcriptomic changes from 3 separate groups: individuals with obesity, T2D, or insulin-treated T2D (itT2D), which represented a disease progression-type model that associates with the severity of insulin resistance (obese<T2D<itT2D). Farup et al. found increased ECM remodelling gene signatures in itT2D, and therefore, hypothesised that FAPs influence muscle pathology in hyperglycemic patients. To further test this hypothesis, the authors isolated and characterized human FAPs from muscle biopsies based on CD90 cell-surface protein expression using fluorescent-activated cell sorting (FACS). This because neither of the tested antibodies against PDGFRA (also known as CD140a) worked -even when PDGFRA is highly expressed in human FAPs- nor Sca-1/Ly6a antigen is expressed in human cells, which are two well-characterized FAP markers in mouse skeletal muscles. The authors also corroborated that human FAPs display clonal expansion (4% for FAPs compared to 17% for MuSCs) and are distinct to other muscle-resident populations of mononuclear cells, which confirm previous findings in mice. Platelet-derived growth factors (PDGFs) bind to PDGFRα and PDGFRβ to regulate key molecular and cellular processes including proliferation, migration and gene expression. Furthermore, PDGF signaling regulates the fate of skeletal muscle FAPs (Contreras et al., 2019; Mueller et al., 2016). Farup et al. described that PDGFRA expression correlates with COLLAGEN 1A1 expression, as previously proposed (Contreras et al., 2019). Owing to this relationship, they asked whether PDGF signalling could impact the fate of FAPs. PDGF-AA treatment increased the expression of fibrillar collagen in FAPs, whereas it reduced their adipogenic differentiation. The authors showed that the PDGF-AA-mediated fibrogenic activation of FAPs associates with a metabolic switch that favours an enhanced consumption of glucose in these cells compared to non-treated cells. This increased glycolytic flux present in TGF-b-induced fibrogenic conditions seems to be required for enhanced ECM synthesis and deposition by FAPs.

The expression of the cell-surface protein CD90 has been widely used to identify tissue-resident fibroblasts and/or mesenchymal stromal/stem cells in different tissues and organs. In this preprint, combining FACS with single-cell RNA-seq, the authors showed that CD90 expression was restricted to a particular FAPs subpopulation. Remarkably, CD90 positive (CD90⁺) FAPs and CD90 negative (CD90^–) FAPs exhibited distinct phenotypic and molecular signatures. CD90⁺ FAPs were bigger in size, proliferated faster, and displayed higher expression of extracellular matrix genes compared to CD90- FAPs. Glycolytic flux and maximal oxygen consumption were also higher in CD90⁺ FAPs compared to CD90- FAPs. These results suggest that at least two distinct FAP subpopulations are present in skeletal muscle and are distinguished by the expression of CD90/THY1. Since evidence of fibrosis was detected in skeletal muscle biopsies from patients with T2D, the authors compared the content of the CD90+, pro-fibrotic FAP subpopulation between patients with T2D and healthy controls. They found that the CD90+ FAP subpopulation was higher in muscles of individuals with T2D compared to non-T2D controls. Further, CD90+ FAPs from patients with T2D proliferated faster and had higher expression of COLLAGEN 1 compared to muscleCD90+ FAPs isolated from non-diabetic individuals. And lastly, the authors sought to pharmacologically target FAPs to prevent their excessive accumulation and fibro-fatty infiltration in muscles of T2D patients. Metformin is a usual first-line pharmacological approach to counteract T2D. Metformin treatment reduced the proliferation, oxygen consumption, and adipogenic differentiation of CD90+ FAPs but increased the glycolytic flux of these cells, suggesting a novel therapeutically targetable cellular mechanism to reduce intra/intermuscular adipose tissue deposition in T2D. Taken together, these findings describe two distinct FAP subsets in human skeletal muscles that participate in modulating the fibro-fatty infiltration of muscles in T2D, a highly prevalent condition in western countries.

Figure. Two major FAP populations described by Farup et al., 2020. CD90 positive (CD90⁺) FAPs and CD90 negative (CD90^–) FAPs exhibited distinct phenotypic and molecular signatures. These phenotypic differences shed new light on the role of these intriguing cells in modulating muscle degenerative fibro-fatty remodelling in the obese and T2D population.

What we liked of this preprint

The highlight of this preprint is the study of FAP heterogeneity in human muscle and how this diversity might impact skeletal muscle homeostasis. Since most of our understanding of tissue-resident FAPs comes from murine models, this study fills an important gap in our knowledge related to the role of FAPs in human degenerative disorders. Therefore, novel therapies targeting FAPs represent a promising strategy for preventing, testing and treating muscle degeneration in chronic metabolic disorders.

Future directions and questions to the authors

1. The authors described that CD90 discriminates at least two subpopulations of FAPs. How is the expression of CD90 regulated in FAPs following injury and ultimately what is the role of CD90 in controlling FAP fate?
2. The work is lacking scRNA-seq data sets from obese, T2D and insulin-resistant T2D patients. Perhaps this should help to understand the dynamics and heterogeneity of FAPs in progressive human chronic pathologies and to unveil the molecular and cellular responses of FAPs to weight gain and/or T2D. Are you thinking to perform these experiments?
3. From what population of endogenous FAPs are CD90+ expressing FAPs descending from. Are you planning to do lineage tracing or fate-mapping experiments (in the mouse) to clarify the hierarchy and clonality of this CD90+ subpopulation of cells and their lineage origin?
4. It would be interesting to know the factors that participate in the metabolic changes of muscle FAPs associated with T2D and insulin resistance over time.
5. Since FAPs from healthy muscles support muscle stem cell-dependent regeneration through the secretion of trophic signals, how would be the cross-talk between CD90+ FAP and MuSCs affected in T2D? And finally, what is the behavior of MuSCs in the degenerative microenvironment of T2D patients?
6. Does insulin downregulate the expression of PDGFRA? Or does the reduction of PDGFRA you observed in insulin-treated T2D patients compared to non-treated T2D patients secondary to the improved metabolic phenotype in the insulin-treated T2D individuals? What muscle-resident cells express the insulin receptor at the single-cell level? Can FAPs become resistant to insulin?

Acknowledgements

The authors are grateful to Dr Michael De Lisio (University of Ottawa) for proofreading the highlight and Dr Mate Pálfy for helpful suggestions.

References

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Tags: diet, extracellular matrix, faps, fibro-adipogenic progenitors, fibroblasts, fibrosis, obesity, skeletal muscle, type 2 diabetes

doi: https://doi.org/10.1242/prelights.24354

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