(I-350) Effects of Brown Adipose Tissue on Muscle Regeneration

Introduction:: Evidence supports beneficial crosstalk between brown/beige adipose tissue (BAT) and skeletal muscle in rotator cuff muscle regeneration [1, 2]. This has potential translational impact because the adipose tissue that accumulates in the torn rotator cuff is a beige phenotype capable of “browning,” making it a novel therapeutic target [3]. However, muscle regeneration is a complex process involving muscle satellite cells (SCs) that differentiate into myocytes and merge with damaged myotubes during muscle regeneration and fibroadipogenic progenitors (FAPs) that coordinate this process by recruiting macrophages and other cells to the injury site. Given their close proximity and simultaneous activation during muscle regeneration, we propose that BAT could regulate a communication network between these cell populations through paracrine signaling. Uncovering the temporal dynamics of this network during regeneration in the presence of local BAT will help define the mechanism(s) of cross-talk. Therefore, we conducted a study using a mouse model where brown adipose tissue (BAT) was transplanted into the rotator cuff to investigate the changes in the transcriptional profiles of FAPs and SCs. Our goal was to identify key factors and pathways secreted or mediated by brown adipose tissue that contribute to the promotion of muscle regeneration.


Materials and Methods:: 12-week-old male C57BL6/J or UCP1-DTA mice were given intermuscular transplants of intrascapular BAT from 4-week-old male donor mice, as previously described [1]. To induce acute muscle injury and regeneration, the recipient mice were injected with cardiotoxin to the supraspinatus (SSP) muscle bilaterally. After 3, 7, and 28 days post-surgery (dps), the SSP muscles were collected and weighed, and digested. At 3 and 7 dps, two antibody panels were utilized to isolate SCs and FAPs by fluorescence activated cell sorting and profile macrophages and neutrophils by flow cytometry: (1) SCs and FAPs: α-7 integrin-PE, Sca-1– FITC, CD31&CD45-eFluor 450; (2) macrophages and neutrophils: CD45, CD206-APC, Ly6G-FITC, F4/80-PE, CD11b-PerCP-Cy5.5, and MHCII-BV605. The RNA of FAPs and SCs was extracted utilizing the RNeasy Mini Kit, followed by the preparation of cDNA libraries using the SAMRTer RNA kit for Illumina Sequencing. The transcriptional profiles of FAPs and SCs were obtained using Novaseq. DESeq2 was employed to analyze the sequencing data for differential expression, and IPA (Ingenuity Pathway Analysis) was used for pathway and network analysis. At 28 dps, SSPs of mice with similar surgical preparation were harvested for contractility and histology as previously described [1]. Briefly, peak tetanic tension was measured in isolated SSPs and the contralateral SSP was flash frozen for hematoxylin & eosin, Oil Red O and Sirius Red staining for gross morphology, fatty infiltration and fibrosis, respectively. Fiber areas by fiber type were also assessed with immunostaining for myosin heavy chain isoforms.


Results, Conclusions, and Discussions:: At 7 dps, regenerating SSP transplanted with BAT had more muscle mass compared to non-transplanted controls (CTL) in C57BL6/J (A). No difference was observed on 3 and by 28 dps, mass had fully recovered in BAT and CTL groups. FACS showed a temporal shift in the relative ratio of FAPs and SCs during regeneration (B), but no difference in population density was found between transplant groups. Similarly, while macrophage and neutrophil populations changed between 3 and 7 dps, no differences were observed between groups (C). RNA sequencing found more differentially expressed genes in FAPs at 3 dps and in SCs at 7 dps, consistent with the population dynamics (D). Pathways upregulated with BAT transplant include inflammatory response pathways (F) that converge on upstream cytokine regulators that impact adipogenesis and fibrogenesis in FAPs (E). However, we did not find any differences between transplant groups in fatty infiltration or fibrosis at 28 dps (data not shown). To determine whether the minor effect of BAT transplant was due to efficient regeneration enabled by endogenous BAT in wildtype C57BL6/J mice, we also assessed the effects of BAT transplant in mice lacking brown fat (UCP1-DTA). BAT transplant in UCP1-DTA mice did not impact muscle mass (G), but did improve contractility (H) suggesting that BAT may be more important for maintaining contractile performance than mass in regeneration.



Our results indicate that while BAT transplant does have a transient effect on muscle mass and cellular transcriptional dynamics, it does not meaningfully impact muscle regeneration long-term in wildtype mice. However, contractile deficits in mice lacking BAT are rescued by BAT transplant suggesting BAT plays a role in maintaining muscle contractility.


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References (Optional): : [1] Bryniarski, AR and Meyer, GA J Orthop Res 2019, [2] Davies, MR et al. Am. J Sports Med. 2022, [3] Meyer, GA et al., Stem Cells Transl Med 2015.