DOI: 10.5061/dryad.j0zpc86qw
title: Spatial-proteomics reveals recombinant human laminin-111 restores adhesion-signaling and metabolic function to laminin-α2 deficient muscle

description: Laminin-α2-related Congenital Muscular Dystrophy (LAMA2-CMD) is a severe
 neuromuscular disorder caused by mutations in the LAMA2 gene, leading to
 loss of heterotrimers laminin-211/221, key components of the skeletal
 muscle extracellular matrix. Their absence disrupts adhesion between the
 cytoskeleton and extracellular matrix, resulting in progressive muscle
 wasting. Laminin-211/221 interacts with adhesion complexes such as the
 dystrophin/utrophin glycoprotein complexes and α7β1-integrins. However,
 the regulatory mechanisms of these laminin-binding complexes and the
 broader role of laminin’s influence on the formation of the macromolecular
 network in skeletal muscle remain unclear. We previously demonstrated that
 delivering mouse laminin-111 to the dyW⁻/⁻ mouse model of LAMA2-CMD
 prevented disease progression, improved strength, and extended survival.
 We hypothesize that laminin-111, the embryonic laminin isoform, restores
 key adhesion-signaling networks. Using spatial proteomics on patient and
 mouse muscle, we identified loss of essential signaling components: heat
 shock proteins 27 and 70, c-Jun N-terminal kinase, and glucose transporter
 1 in laminin-α2-deficient muscle. Treatment with recombinant human
 laminin-111 (rhLAM-111) restored protein localization, reduced ROS, and
 promoted glycolytic, pro-survival signaling. These findings highlight
 laminin’s role in maintaining muscle homeostasis and metabolism and
 support the therapeutic potential of rhLAM-111 for treating LAMA2-CMD by
 restoring adhesion and intracellular signaling in dystrophic muscle.
description: # Spatial-proteomics reveals recombinant human laminin-111 restores
 adhesion-signaling and metabolic function to laminin-α2 deficient muscle
 [https://doi.org/10.5061/dryad.j0zpc86qw](https://doi.org/10.5061/dryad.j0zpc86qw) ## Description of the data and file structure ### **Supplemental tables used for data analysis** * **Quality Control and Normalization:** Data normalization was performed using NanoString’s GeoMx DSP analysis suite, utilizing the geometric mean of negative control proteins (IgG variants) for normalization. Column A lists the experimental group used in this study (control/unaffected human versus LAMA2-CMD), and Column B contains the ROI identification associated with each skeletal muscle biopsy from the patients. Columns C to VJ then list the proteins from the GeoMx IPA panel (row 1) and the normalized GeoMx IPA panel "counts" found for each ROI. Sheet 1 has the values for the Area ROIs, and sheet 2 contains the data for the single-fiber ROIs.  * These values were then used for differential protein expression calculation.s * Table S1 * **Differential Protein Expression Analysis:** * Processed using the DESeq2 package in R. Column A lists all the proteins from the GeoMX IPA panel. Column B shows that the calculations were done with LAMA2-CMD patients compared to unaffected controls. Column C contains the Log2 fold-change values associated with each protein, followed by column D, which includes the associated p-value. Column E = failed discovery rate (FDR) and column F = the significance classification based on the FDR. Log10(p) (column G) and log10 FDR (column H) are also included in this dataset. Sheet 1 contains area ROI values, and sheet 2 contains single-fiber ROI values. * Table S2 * Differentially expressed proteins (DEPs) were identified using a threshold of Log2FC ≥ 0.2 and p &lt; 0.1 due to exploratory study constraints. * These values were then separated and sorted into individual Excel files to generate volcano plots. Table S2 was split between differentially expressed proteins that fit within the previous threshold, Table S3 = younger patient ROIs, and Table S4 = older patient ROIs. Sheet 1 contains area ROI values, and sheet 2 contains single-fiber ROI values. * Table S3-4 * **Pathway Enrichment Analysis:** Conducted using R packages, Gene Ontology (GO) annotation, and clusterProfiler, to identify Biological Process for upregulated and downregulated DEPs. * Used to generate dot plots ### **Immunofluorescence** * **Tissue Processing:** Tibialis anterior muscles were harvested, flash-frozen, sectioned, and fixed in PFA or acetone. * **Imaging and Analysis:** Tissues were imaged at 40X on a Leica Stellaris 8 STED microscope and processed in ImageJ. ### **NADH Enzyme Histochemistry** * **Protocol:** The NADH staining followed an established histological protocol to visualize enzymatic activity in dyᵂ-/- and WT tibialis anterior muscles. Images were taken at 60X on a Zeiss Axiocam 305 light microscope. * Sheehan, D. C. (1987). *Theory and practice of histotechnology*. Battelle Press. ### Files and variables #### File: TableS1-HumanDataAllCounts.xlsx **Description:** Protein counts from human ROIs generated by Nanostring GeoMx Digital Spatial Profiling * Normalized using negative control counts in Nanostring Data Analysis Suite #### File: TableS2-HumanDEPs.xlsx **Description:** Differentially expressed protein (DEP) counts with corresponding p-values between all unaffected controls and LAMA2-CMD patients. Table S1 was utilized to generate this data. #### File: TableS3-YoungerDEPs.xlsx **Description:** Table S1 was split between younger and older patients, and DEPs were generated similarly to Table S2.  #### File: TableS4-OlderDEPs.xlsx **Description: **Table S11 was split between younger and older patients, and DEPs were generated similarly to Table S2.  #### File: TableS5-MouseDataAllCounts.xlsx **Description:** Protein counts from mouse ROIs generated by Nanostring GeoMx Digital Spatial Profiling * Normalized using negative control counts in Nanostring Data Analysis Suite #### File: SupportingDataValues.xlsx **Description: All supporting data values are displayed on the figures within the manuscript. Each tab is listed with the associated figure number.** * Figure 2E: Differentially expressed protein values between unaffected and LAMA2-CMD patient muscle biopsies. Log2 fold-change, p-value, FDR, log10p, and log10FDR for each antigen (column A) were calculated using spatial-proteomics counts from area ROIs.  * Figure 2F: Differentially expressed protein values between unaffected and LAMA2-CMD patient muscle biopsies. Log2 fold-change, p-value, FDR, log1,  and log10FDR for each antigen (column A) were calculated using spatial-proteomics counts from single-fiber ROIs.  * Figure 3A: Differentially expressed protein values between unaffected and LAMA2-CMD patient muscle biopsies. Log2 fold-change, p-value, FDR, log10p, and log10FDR for each antigen (column A) were calculated using spatial-proteomics counts from younger area ROIs.  * Figure 3B: Differentially expressed protein values between unaffected and LAMA2-CMD patient muscle biopsies. Log2 fold-change, p-value, FDR, log10p, and log10FDR for each antigen (column A) were calculated using spatial-proteomics counts from older area ROIs.  * Figure 3C: Differentially expressed protein values between unaffected and LAMA2-CMD patient muscle biopsies. Log2 fold-change, p-value, FDR, log10p, and log10FDR for each antigen (column A) were calculated using spatial-proteomics counts from younger single-fiber ROIs. * Figure 3D: Differentially expressed protein values between unaffected and LAMA2-CMD patient muscle biopsies. Log2 fold-change, p-value, FDR, log10p, and log10FDR for each antigen (column A) were calculated using spatial-proteomics counts from older single-fiber ROIs.   * Figure 5B: Differentially expressed protein values between wild-type and dyW-/- mouse tissue. Log2 fold-change, p-value, FDR, log10p, and log10FDR for each antigen (column A) were calculated using spatial-proteomics counts from area ROIs. * Figure 5C: Differentially expressed protein values between wild-type and dy^W^-/- mouse tissue. Log2 fold-change, p-value, FDR, log10p, and log10FDR for each antigen (column A) were calculated using spatial-proteomics counts from single-fiber ROIs. * Figure 7B: Band intensity values for each protein indicated from wild-type, dy^W^-/-, and dy^W^-/- +rhLAM-111 treated mice. Values were derived from iBright analysis software and normalized to total protein.  * Figure 7C: Differentially expressed protein values between dy^W^ and dy^W^-/- +rhLAM-111 mouse tissue. Log2 fold-change, p-value, FDR, log10p, and log10FDR for each antigen (column A) were calculated using spatial-proteomics counts from area ROIs. * Figure 7D: Differentially expressed protein values between dy^W^ and dy^W^-/- +rhLAM-111 mouse tissue. Log2 fold-change, p-value, FDR, log10p, and log10FDR for each antigen (column A) were calculated using spatial-proteomics counts from single-fiber ROIs. * Figure 8B: Band intensity values for HSP70 from wild-type, dy^W^-/- and dy^W^-/- +rhLAM-111 treated mice. Values were derived from iBright analysis software and normalized to total protein.  TIFF files for Western Blot images used to calculate protein expression levels normalized to total protein: * aDG_TotalProtein_01.tif * aDG_TotalProtein_02.tif * CHEMI_aDG_1_02.tif * CHEMI_aDG_02.tif * ITGa7_aSG_TotalProtein_01.tif * ITGa7_aSG_TotalProtein_02.tif * FL_ITGa7_01.tif * FL_ITGa7_02.tif * CHEMI_aSG_01.tif * CHEMI_aSG_02.tif * HSP70_TotalProtein_01_composite.tif * SEPT_HSP70_TotalProtein_02_composite.tif * CHEMI_HSP70_01_composite.tif * CHEMI_HSP70_02_composite.tif * CHEMI_GLUT1_01_composite.tif * CHEMI_GLUT1_02_composite.tif * SEPT_ITGb1_TotalProtein_02.tif * SEPT_ITGb1_TotalProtein_01.tif * SEPT_ITGb1_02.tif * SEPT_ITGb1_01.tif **Supplemental Data:** * All files downloaded to Zenodo are immunofluorescent images used to generate panels found in the manuscript. - Biorender publication license has been uploaded for the use of the graphical abstract.