Maresin 1 repletion improves muscle regeneration after volumetric muscle loss

  1. Jesus A Castor-Macias
  2. Jacqueline A Larouche
  3. Emily C Wallace
  4. Bonnie D Spence
  5. Alec Eames
  6. Pamela Duran
  7. Benjamin A Yang
  8. Paula M Fraczek
  9. Carol A Davis
  10. Susan V Brooks
  11. Krishna Rao Maddipati
  12. James F Markworth
  13. Carlos A Aguilar  Is a corresponding author
  1. Department of Biomedical Engineering, University of Michigan, United States
  2. Biointerfaces Institute, University of Michigan, United States
  3. Department of Molecular & Integrative Physiology, University of Michigan, United States
  4. Department of Pathology, Lipidomics Core Facility, Wayne State University, United States
  5. Department of Animal Sciences, Purdue University, United States
  6. Program in Cellular and Molecular Biology, University of Michigan, United States
5 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
In vivo comparison of degenerative and regenerative volumetric muscle loss (VML) injuries.

(A) Schematic of experiment, whereby young (3 mo) mice were administered 1-mm (administered to left leg) or 2-mm (administered to right leg) biopsy punches to their tibialis anterior muscle to …

Figure 1—figure supplement 1
Functional assessment of response to healing or degenerative volumetric muscle loss injuries.

(A, B) Force frequency of muscle (A) and nerve stimulation (B) of uninjured, 1-mm and 2-mm groups uninjured (blue) tibialis anterior muscle at 28 d post injury (dpi) (1 mm [black] or 2 mm [gray]). …

Figure 2 with 1 supplement
Lipidomic profiling after volumetric muscle loss (VML) injuries reveals stronger and sustained lipid mediator response in degenerative injuries.

(A) Mice were administered a bilateral defect to tibialis anteriors (TAs) (left leg received a 1-mm defect and right leg received a 2-mm defect). Tissues were harvested at 0, 3, 7, and 14 d post …

Figure 2—figure supplement 1
Dynamics of lipid mediators after volumetric muscle loss injury.

(A) Dirichlet Process Gaussian Process (DPGP) mixture model-based clustering of mean analyte abundance fold change z-scores across injury timepoints. Black lines are cluster means, and the gray bars …

Figure 3 with 3 supplements
Repletion of Maresin 1 after degenerative volumetric muscle loss (VML) injury results in reductions in inflammation and fibrosis.

(A) Mice were administered bilateral 2-mm VML defects to their tibialis anterior (TA) muscles. One limb received an intramuscular injection of Maresin 1 and the other limb received vehicle (saline + …

Figure 3—figure supplement 1
Immune profiling of response to volumetric muscle loss (VML) injury after treatment with Maresin 1.

(A) Complete gating strategy for neutrophils and macrophages. Values indicate percentage of parent. (B) Maresin 1 treatment significantly reduced neutrophil abundance and reduced F4/80+ macrophage …

Figure 3—figure supplement 2
Regenerative response to volumetric muscle loss (VML) injury after treatment with Maresin 1.

(A) Representative cross-sections from muscles isolated 7 d post injury (dpi) with vehicle (top) or Maresin 1 (bottom) treatment. Sections are immunostained for embryonic myosin heavy chain (eMyHC) …

Figure 3—figure supplement 3
Changes in muscle force after volumetric muscle loss (VML) injury are enacted with treatment with Maresin 1.

(A, B) Force frequency of nerve (A) and muscle stimulation (B) of uninjured (blue) tibialis anterior muscle at 28 d post injury (dpi) following treatment with saline (black) or Maresin 1 (gray). …

Figure 4 with 1 supplement
Maresin 1 impacts muscle stem cell (MuSC) proliferation through Lgr6.

(A) Lgr6 is highly upregulated in proliferating myoblasts by RT-qPCR. Graph shows mean ± SEM, ****p<0.0001 by one-way ANOVA and Benjamini–Hochberg (BH) post hoc analysis. n = 3–4 wells per …

Figure 4—figure supplement 1
Experimental evaluation of parameters for Maresin 1 treatment of muscle stem cells (MuSCs) and their progeny in vitro.

(A) Maresin 1 dose–response curve showed the most impact on proliferation with treatment using 10 nM. There was no statistically significant effect with higher doses. *p<0.05 by one-way ANOVA with …

Figure 5 with 1 supplement
Cellular and molecular response to Maresin 1 repletion after degenerative volumetric muscle loss supports enhanced myogenic response and reduction of fibrotic macrophages at 7 d post injury (dpi).

(A) Uniform Manifold Approximation and Projection (UMAP) plots colored by clusters at a resolution of 0.2 and annotated into cell types. (B) UMAP plots split by treatment and colored by cell type. (C

Figure 5—figure supplement 1
Quality control metrics for single-cell sequencing after volumetric muscle loss (VML) injury and treatment with Maresin 1.

(A) Uniform Manifold Approximation and Projection (UMAP) plots colored by the number of genes per cell (left) and the number of unique molecular identifiers per cell (right). (B) Dot plot of known …

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Biological sample (Mus musculus)C57BL/6J wild-type miceJackson LabsJackson Stock 000664Female (2–3 mo)
Biological sample (M. musculus)Pax7CreERT2/+;Rosa26Tcf/Lef-LSL-H2B-GFP miceUniversity of MichiganJackson stock 017763 crossed with Jackson stock 032577Female (2–3 mo)
Biological sample (M. musculus)Pax7CreERT2 – Rosa26-TdTomatoUniversity of MichiganJackson stock 017763 crossed with Jackson stock 007914Female (2–3 mo)
AntibodyAF488 anti-mouse monoclonal F4/80, clone: BM8, isotype: Rat IgG2a, κBioLegend123119; RRID:AB_893491FC (1:200)
AntibodyAPC-Cy7 anti-mouse monoclonal Ly-6G, clone: 1A8, isotype: Rat IgG2a, κBioLegend127624; RRID:AB_10640819FC (1:400)
AntibodyAPC anti-mouse monoclonal Ly-6A/E (Sca-1), clone: D7, isotype: Rat IgG2a, κBioLegend108112; RRID:AB_313349FC (1:400)
AntibodyAPC anti-mouse monoclonal CD45, clone: 30-F11, isotype: Rat IgG2b, κBioLegend103112; RRID:AB_312977FC (1:400)
AntibodyAPC anti-mouse monoclonal TER-119, clone: TER-119, isotype: Rat IgG2b, κBioLegend116212; RRID:AB_313713FC (1:400)
AntibodyAPC anti-mouse monoclonal CD31, clone: 390, Isotype: Rat IgG2a, κBioLegend102410; RRID:AB_312905FC (1:400)
AntibodyAPC anti-mouse/human monoclonal CD11b, clone: M1/70. Isotype: Rat IgG2b, κBioLegend101212; RRID:AB_312795FC (1:400)
AntibodyPE anti-mouse/rat monoclonal CD29 (B1 int), clone: HMβ1-1, isotype: Armenian Hamster IgGBioLegend102208; RRID:AB_312885FC (1:200)
AntibodyBiotin Rat AntiMouse monoclonal CD184, clone: 2B11/CXCR4 (RUO), isotype: Rat IgG2b, κBD Biosciences551968; RRID:AB_394307FC (1:200)
AntibodyAnti-mouse monoclonal CD68, clone: FA-11, isotype: IgG2aBio-RadMCA1957IF (1:50)
AntibodyMouse monoclonal Anti-human MYH3DHSBF1.652IF (1:20)
AntibodyChicken polyclonal Anti-mouse GFPAbcamab13970; RRID:AB300798IF (1:1000)
AntibodyRabbit anti-mouse laminin 1+2, Isotype: Polyclonal IgGAbcamab7463; RRID:AB_305933IF (1:500)
AntibodyRabbit polyclonal Anti-RFPRockland600-401-379; RRID:AB_2209751IF (1:50)
AntibodyRabbit polyclonal Anti-Beta catenin polyclonal (CAT-15)Thermo Fisher71-2700; RRID:AB_2533982IF (1:100)
AntibodyGoat polyclonal Anti-rabbit IgG (H+L), Alexa Fluor 647 conjugateThermo FisherA27040; RRID:AB_2536101IF (1:500)
AntibodyGoat polyclonal Anti-rat (H+L), Alexa Fluor 647 conjugateThermo FisherA21247; RRID:AB_141778IF (1:500)
AntibodyGoat polyclonal Anti-mouse (H+L), Alexa Fluor 488 conjugateThermo FisherA28175; RRID:AB_2536161IF (1:500)
AntibodyGoat polyclonal Anti-chicken IgY (H+L), Alexa Fluor 488 conjugateThermo FisherA32931TR; RRID:AB_2866499IF (1:500)
AntibodyGoat polyclonal Anti-rabbit (H+L), Alexa Fluor 555 conjugateThermo FisherA21247; RRID:AB_141778IF (1:500)
Sequence-based reagentPrimeTime Mouse GAPDH PrimerIntegrated DNA TechnologiesMm.PT.39a1
Sequence-based reagentPrimeTime Mouse Lgr6 PrimerIntegrated DNA TechnologiesMm.PT.58.9348010
Sequence-based reagentMouse Lgr6 DsiRNA #1Integrated DNA Technologiesmm.Ri.Lgr6.13.2
Sequence-based reagentMouse Lgr6 DsiRNA #2Integrated DNA Technologiesmm.Ri.Lgr6.13.1
Sequence-based reagentCell Multiplexing Oligos10x Genomics1000261
Peptide, recombinant proteinBovine serum albuminFisher ScientificBP9703-100
Peptide, recombinant proteinFibroblast growth factor basicGibco-InvitrogenPHG0263
Peptide, recombinant protein0.25% Trypsin EDTAGibco-Invitrogen25200072
Peptide, recombinant proteinHALT Protease Inhibitor CocktailThermo Fisher87786
Chemical compound, drugDispase II (activity ≥ 0.5 units/mg solid)SigmaD4693-1GDigestion of tissue to extract MuSCs
Chemical compound, drugCollagenase type II (654 U/mg, non-specific proteolytic activity 487 U/mg)Life Technologies17101015Digestion of tissue to extract MuSCs
Chemical compound, drugDMEM, high glucose, pyruvateLife Technologies11995065
Chemical compound, drugHam’s F-10 Nutrient MixLife Technologies11550043
Chemical compound, drugTissue Plus O.C.T CompoundFisher Scientific23-730-571
Chemical compound, drugMagnesium sulfate heptahydrateSigma-Aldrich63138-250G
Chemical compound, drugSodium bicarbonateSigma-AldrichS5761
Chemical compound, drugSafeClear IIFisher Scientific23-044192
Chemical compound, drugPicric acidSigma-AldrichP6744-1GA
Chemical compound, drugGlacial acetic acidSigma-AldrichBP2401-500
Chemical compound, drugXylenesSigma-Aldrich534056-4L
Chemical compound, drugPermountFisher ScientificSP15-100
Chemical compound, drug0.5 M EDTAInvitrogen15575-038
Chemical compound, drugSodium azideSigma-Aldrich71289
Chemical compound, drug4% paraformaldehyde in PBSThermo FisherJ19943-K2
Chemical compound, drugPenicillin streptomycinGibco-Invitrogen15640055
Chemical compound, drugMaresin 1Cayman Chemicals10878
Chemical compound, drugTween-20Sigma-AldrichP1379
Chemical compound, drugTritonX-100Sigma-AldrichT8787
Chemical compound, drugTamoxifenSigma-AldrichT2859-1G
Chemical compound, drugCorn OilSigma-AldrichC8267-2.5L
Commercial assay or kitSatellite Cell Isolation Kit, mouseMiltenyi130-104-268
Commercial assay or kitTGF beta 1 DuoSet ELISA KitR&D SystemsDY1679
Commercial assay or kitPierce BCA Assay KitThermo Fisher23227
Commercial assay or kitEdU ClickIt Reaction KitThermo FisherC10340
Commercial assay or kitSuperScript III First-Strand Synthesis KitThermo Fisher18080051
Commercial assay or kitQIAGEN RNeasy Mini KitQIAGEN74104
Commercial assay or kitQubit RNA HS AssayThermo FisherQ32852
Commercial assay or kitSingle cell 3’ Library & Gel Bead Kit10x Genomics120267
Commercial assay or kitcAMP-Glo AssayPromegaV1501
Software, algorithmCellRanger v7.0.010x Genomicshttps://support.10xgenomics.com/single-cell-gene-expression/software/downloads
Software, algorithmR v4.2.1The R Foundation for Statistical Computinghttps://www.r-project.org/; RRID:SCR_001905
Software, algorithmPythonhttps://www.python.org/
Software, algorithmMetaboDiff v0.9.5Mock et al., 2018; Mock, 2020https://github.com/andreasmock/MetaboDiff
Software, algorithmDPGP v0.1McDowell et al., 2018a; McDowell et al., 2018bhttps://github.com/PrincetonUniversity/DP_GP_cluster
Software, algorithmMATLAB_R2020aMathWorkshttps://www.mathworks.com/products/matlab.html
Software, algorithmSeurat v4.2.1Stuart et al., 2019https://satijalab.org/seurat/; RRID:SCR_007322
Software, algorithmggplot2 v3.2.1Wickham et al., 2016https://ggplot2.tidyverse.org; RRID:SCR_014601
Software, algorithmFlowJo v10https://www.flowjo.com; RRID:SCR_008520
Software, algorithmImageJ v2.1.0https://imagej.net/ImageJ; RRID:SCR_002285
Software, algorithmCellposeStringer et al., 2021RRID:SCR_021716
Software, algorithmLabelsToROIsWaisman et al., 2021https://labelstorois.github.io/
Software, algorithmBioinformatics analysis codeThis paperhttps://github.com/AguilarLab/Maresin1 (copy archived at Castor-Macias et al., 2023)Algorithm used to generate Figure 5, Figure 5—figure supplement 1. Refer to data processing and analysis under single-cell RNA sequencing
OtherStreptavidin PE-Cyanine7Thermo Fisher25-4317-82; RRID:AB_10116480FC (1:100)
OtherscRNA-seq datasetsThis paperGSE215808Refer to data processing and analysis under single-cell RNA sequencing.
OtherLS ColumnsMiltenyi130-042-401MACS isolation
OtherUltraComp eBeadsFisher Scientific01-2222-42Single color controls for Flow Cytometry
OtherRIPA BufferThermo Fisher89900Tissue homogenization reagent in ELISA assays
OtherLipofectamine RNAiMAXInvitrogen13-778-030Transfection reagent
OtherHoechst 33342Thermo FisherH3570IF (1:1000) nuclear stain
OtherSYBR Green PCR MasterMixThermo Fisher4309155PCR reagent
OtherMatrigelBD Biosciences356234Cell culture coating reagent
OtherHorse SerumGibco-Invitrogen26050088Cell culture
OtherProlong DiamondThermo FisherP36965Mounting media
OtherDirect Red 80Fisher ScientificAAB2169306Picrosirius red stain
OtherHematoxylinRicca Chemical Company3530-16H&E stain
OtherEosinEMD-Millipore588X-75H&E stain
OtherFetal bovine serumLife Technologies10437028Tissue digestion reagent
OtherNormal goat serumAbcamAb7481; RRID:AB_2716553IHC stain
OtherPropidium iodide – 1.0 mg/mL solution in WaterLife TechnologiesP3566FC (1:400)
Other7-AADBioLegend420403FC (1:200)
OtherMouse on Mouse blocking reagentVector LabsMKB-2213IHC stain

Additional files

MDAR checklist
https://cdn.elifesciences.org/articles/86437/elife-86437-mdarchecklist1-v2.docx
Supplementary file 1

Metabolite concentration (pmol/g) for different pathways in uninjured, 1 mm VML, and 2 mm (TA) tissue homogenates.

https://cdn.elifesciences.org/articles/86437/elife-86437-supp1-v2.xlsx

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