# Intracellular Membrane Repair Dysregulation and Accumulation of Mature Myostatin Protein are Novel Markers of Muscle Pathophysiology in Pompe Disease

Candice Babarit, Sabrina Jagot, Cindy Schleder, Johan Deniaud, Tiffany Bourgeton, Lydie Lagalice, Lucie Vezzana, Isabelle Leroux, Aurélie Lardenois, Bertrand Evrard, et al.

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Candice Babarit, Sabrina Jagot, Cindy Schleder, Johan Deniaud, Tiffany Bourgeton, et al. Intra-cellular Membrane Repair Dysregulation and Accumulation of Mature Myostatin Protein are Novel Markers of Muscle Pathophysiology in Pompe Disease. 2026. hal-05487367

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# Intracellular Membrane Repair Dysregulation and Accumulation of Mature Myostatin Protein are Novel Markers of Muscle Pathophysiology in Pompe Disease

**Candice BABARIT**
Oniris, INRAE, PAnTher

**Sabrina JAGOT**
Oniris, INRAE, PAnTher

**Cindy SCHLEDER**
Oniris, INRAE, PAnTher

**Johan DENIAUD**
Oniris, INRAE, PAnTher

**Tiffany BOURGETON**
Oniris, INRAE, PAnTher

**Lydie LAGALICE**
Oniris, INRAE, PAnTher

**Lucie VEZZANA**
Oniris, INRAE, PAnTher

**Isabelle LEROUX**
Oniris, INRAE, PAnTher

**Aurélie LARDENOIS**
Rennes University, Inserm, EHESP, Irset - UMR_S 1085

**Bertrand EVRARD**
Rennes University, Inserm, EHESP, Irset - UMR_S 1085

**Laurence DUBREIL**
Oniris, INRAE, PAnTher

**Jérôme AMIAUD**
Inserm UMR 1307, CNRS UMR 6075, Nantes Université, Université d'Angers, CRCI2NA

**Franck VERRECCHIA**
Inserm UMR 1307, CNRS UMR 6075, Nantes Université, Université d'Angers, CRCI2NA

**Chantal THORIN**
Oniris, INRAE, PAnTher

**Alicia Mayeuf-Louchart**
Inserm INSERM UMR-S 1172, University of Lille
**Karl ROUGER**
Oniris, INRAE, PAnTher

**Marie-Anne COLLE**
marie-anne.colle@inrae.fr

Oniris, INRAE, PAnTher

## Research Article

**Keywords:** Pompe disease, skeletal muscle, membrane repair, satellite cell, lysosome, autophagosome, T-tubule, myostatin

**Posted Date:** January 30th, 2026

**DOI:** https://doi.org/10.21203/rs.3.rs-8603735/v1

**License:** © Ⓟ This work is licensed under a Creative Commons Attribution 4.0 International License.

Read Full License

**Additional Declarations:** No competing interests reported.
1 Intracellular Membrane Repair Dysregulation and Accumulation of Mature Myostatin Protein are Novel Markers of Muscle Pathophysiology in Pompe Disease

2 Myostatin Protein are Novel Markers of Muscle Pathophysiology in Pompe Disease

3 Authors

4 Candice Babarit¹*, Sabrina Jagot¹,²*, Cindy Schleder¹, Johan Deniaud¹, Tiffany Bourgeton¹, Lydie Lagalice¹, Lucie Vezzana¹, Isabelle Leroux¹, Aurélie Lardenois¹, Bertrand Evrard³, Laurence Dubreil¹, Jérôme Amiaud⁴, Franck Verrecchia⁴, Chantal Thorin¹, Alicia Mayeuf-Louchart⁵, Karl Rouger¹** and Marie-Anne Colle¹**#

5 Bertrand Evrard³, Laurence Dubreil¹, Jérôme Amiaud⁴, Franck Verrecchia⁴, Chantal Thorin¹, Alicia Mayeuf-Louchart⁵, Karl Rouger¹** and Marie-Anne Colle¹**#

6 Thorin¹, Alicia Mayeuf-Louchart⁵, Karl Rouger¹** and Marie-Anne Colle¹**#

7 Bertrand Evrard³, Laurence Dubreil¹, Jérôme Amiaud⁴, Franck Verrecchia⁴, Chantal Thorin¹, Alicia Mayeuf-Louchart⁵, Karl Rouger¹** and Marie-Anne Colle¹**#

8 Consortium

9

10 ¹ Oniris, INRAE, PAnTher, 44307 Nantes, France.

11 ² CNRS, INMG, MeLiS, UMR5284, UCBL, 69008 Lyon, France.

12 ³ Rennes University, Inserm, EHESP, Irset - UMR_S 1085, F-35000 Rennes, France.

13 ⁴ Inserm UMR 1307, CNRS UMR 6075, Nantes Université, Université d'Angers, CRCI2NA, F-44035 Nantes, France

14 ⁵ Inserm UMR-S 1172, University of Lille, F-59045 Lille, France

15

16 *, ** Authors contributed equally to this work.

17 # Correspondence should be addressed to Marie-Anne Colle, E-mail: marie-anne.colle@inrae.fr, Phone number: +33 (0)2.40.68.78.07; INRAE/Oniris UMR 703, Oniris, Route de Gachet, CS.40706, F-44307 Nantes, France

18 LYSAUMI Consortium members are presented in Appendix A.

19

20

21

22
26 **Abstract**

27 Pompe disease is an autosomal recessive metabolic disorder caused by acid alpha-
28 glucosidase deficiency, characterized by progressive skeletal muscle weakness and
29 respiratory insufficiency. Affected muscles exhibit glycogen-filled lysosomes, autophagic
30 build-up, and mitochondrial abnormalities. Despite global myofibrillar disorganization,
31 satellite cells (SCs) fail to activate, due to mechanisms that remain unclear. This study
32 sought to comprehensively characterize the phenotypic features of affected muscles in
33 Pompe disease, focusing in particular on membrane repair processes, as membrane
34 damage is a primary trigger for SC activation. Longitudinal transcriptomic analysis of
35 muscle from a Pompe disease mouse model, combined with immunohistochemical and
36 biochemical analyses, showed early and sustained overexpression of dysferlin (DYSF),
37 annexin A2 (ANXA2), and AHNAK2, proteins involved in membrane repair. Abnormal
38 localization of these proteins was observed throughout the disease course, as evidenced
39 by sarcoplasmic accumulation at lysosomes, autophagosomes, and T-tubules,
40 respectively. These alterations suggest a compensatory mechanism to preserve the
41 integrity of intracellular structures. Analysis of muscle biopsies from patients with late-
42 onset Pompe disease (LOPD) suggested sarcoplasmic localization of DYSF, ANXA2 and
43 AHNAK2 that correlated with the severity of the histological phenotype. Moreover, in
44 the mouse model, we observed persistent post-transcriptional accumulation of mature
45 myostatin, a key negative regulator of muscle growth, which may contribute to impaired
46 SC activation and the absence of muscle regeneration despite extensive tissue damage. In
47 conclusion, our findings identified differential expression of proteins associated with
48 intracellular membrane repair and dysregulation of myostatin as key markers in the course
49 of Pompe disease. These insights provide new perspectives on the underlying
pathophysiology and point to novel therapeutic avenues for limiting disease-associated
damage.

## Keywords

Pompe disease, skeletal muscle, membrane repair, satellite cell, lysosome, autophagosome, T-tubule, myostatin

55

## Introduction

Pompe disease, also known as glycogen storage disease type II (OMIM #232300), is a lysosomal storage disorder resulting from mutations in the gene encoding acid alpha-glucosidase (GAA) [19]. GAA is essential for glycogen breakdown within lysosomes, and its deficiency leads to abnormal glycogen accumulation, particularly in skeletal and cardiac muscles, causing progressive muscle weakness and atrophy [6, 23]. Histopathological hallmarks include enlarged and damaged glycogen-filled lysosomes, accumulation of autophagic debris and mitochondrial abnormalities [18, 51, 58]. They are associated with impaired fusion between lysosomes and autophagosomes in muscle fibers, resulting in autophagic build-up and defective autophagic flux [16]. Furthermore, accumulation of dysfunctional mitochondria exacerbates oxidative stress and disrupts calcium homeostasis [30]. Collectively, these structural changes contribute to progressive and profound muscle tissue remodeling in both patients and Gaa⁻/⁻ mice, the primary mouse model of the disease. This remodeling is characterized by vacuolized fibers, myofibril disarray, anisocytosis, nuclear internalization, and fiber splitting [25, 30, 53].

Glycogen overload can damage lysosomal membranes, as evidenced by increased expression of galectin-3, a cytosolic lectin known to regulate lysosomal repair [1, 2, 22, 36, 47]. Galectin-3 can also coordinate the degradation of damaged lysosomes via autophagy and their replacement through biogenesis [22]. Autophagosomes, double-
membrane vesicles that sequester cytoplasmic components or organelles, fuse with lysosomes to facilitate their elimination [37]. Specific repair mechanisms are initiated following damage to both lysosomal and plasma membranes. Lysosomal membrane integrity is primarily maintained by the endosomal sorting complexes required for transport (ESCRT), which are recruited prior to galectin-3 upregulation and the activation of the lysophagy machinery [40, 41, 44]. In cases of severe lysosomal membrane damage, annexins 1 and 2 are involved via an ESCRT-independent mechanism [61]. Annexin A2 (ANXA2) also supports plasma membrane integrity via active repair processes triggered by calcium influx. This influx leads to the recruitment of intracellular vesicles and associated proteins, which form a membrane repair patch that reseals the rupture site. Dysferlin (DYSF), a transmembrane protein with Ca²⁺-binding C2 domains located on both the plasma membrane and the transverse tubule (T-tubule) membrane, plays a critical role in this process [34]. Autosomal recessive mutations in the dysferlin gene impair cell membrane repair, contributing to the development of muscular dystrophies [42]. Compensatory overexpression of DYSF is also observed in Duchenne muscular dystrophy (DMD) [56]. DYSF interacts with AHNAK, S100 calcium binding protein A10 (S100A10), and ANXA2 [21, 28]. AHNAK is a large scaffolding protein involved in sub-sarcolemma cytoarchitecture and calcium homeostasis [20]. It is also a defining feature of enlargeosomes, vesicles that facilitate the reduction of cell surface tension that is required for membrane repair [12, 62]. Like DYSF, AHNAK expression is significantly reduced in the muscles of patients with dysferlinopathy but remains unchanged in patients with other muscular dystrophies, such as DMD [21]. ANXA2 expression is elevated in dysferlinopathies and is strongly correlates with the severity of the clinical phenotype [8]. Despite the various architectural changes observed at the muscle fiber level, which are supposed to stimulate satellite cell (SC) activation to repair tissue damage, SCs remain
100 quiescent and muscle fiber regeneration does not occur in both patients and Gaa⁻/⁻ mice
101 [25, 50]. This is particularly perplexing given that SC number and function are preserved,
102 suggesting the presence of specific, yet unidentified, pathophysiological processes that
103 impair muscle repair in Pompe disease. Given that membrane injury is a major stimulus
104 for SC activation [48], we hypothesized that adaptive membrane repair mechanisms may
105 be activated in muscle fibers in Pompe disease to keep the intensity of damage below this
106 threshold for SC quiescence exit.

107 In this study, we performed a comprehensive transcriptomic analysis of the biological
108 processes (BP) involved in the pathophysiology of Pompe disease, focusing primarily on
109 the global membrane repair mechanism and the failed activation of SCs, two events that
110 may be linked. To this end, we characterized the gene expression signature over the
111 disease course in Gaa⁻/⁻ mouse muscle to provide a broad overview of the genetic
112 alterations that underlie the pathophysiological changes associated with Pompe disease.
113 We then correlated these transcriptional profiles with histological and biochemical
114 findings in Gaa⁻/⁻ mice and patients suffering from late-onset Pompe disease (LOPD).
115 These data provide new insights into the involvement of membrane repair-associated
116 proteins and regulation of SC activation in Pompe disease pathogenesis. Notably, DYSF,
117 ANXA2, and AHNAK2 have been identified as potential biomarkers of disease
118 progression.

119

120 **Materials and Methods**

121 *Human samples*

122 Human samples were obtained from three patients with a molecular diagnosis of Pompe
disease (2 women and 1 man aged 31, 38 and 46 years, respectively). A control muscle
sample was also obtained from a 30-year-old male subject free of known genetic muscle disease despite a history of muscle pain. The study was approved by the Ethics Committee of the relevant institutions in compliance with the Helsinki Declaration. Patients signed a consent form allowing their biopsy to be used for research, according to French regulation (Ref: RC22-0249, ID-RCB: 2022-A00971-42; Committee for the Protection of Individuals: 22.01632.000094). Patients were categorized into three groups based on age of disease onset and disease severity. A 7.5-year-old DMD patient was also added to the cohort (tissue sample obtained from the Brest Biological Resources Center BB-0033-00037 ["CRB Santé du CHU de Brest"], France). For LOPD patients (Table 1), deltoid muscle samples (10 µm-thick cryosections) were processed for conventional histochemical staining (hematoxylin and eosin [HE] and periodic acid-Schiff [PAS]) (Additional file 1). Histologically, mild, moderate and severe LOPD patients were characterized by various degrees of muscle fiber size variability, vacuolation (top panel) and glycogen overload defined by dark pink PAS-positive material (bottom panel).

138

<caption>**Table 1.** Summary of histopathological and clinical criteria for Pompe disease patients.</caption>

140

141

142

143

<table><thead><tr><th>Patient ID</th><th>Histological grading</th><th>Age (year-old)</th><th>Gender</th><th>Disease severity and evolution</th></tr></thead><tbody><tr><td>1</td><td>Mild</td><td>31</td><td>Woman</td><td>Under ERT for more than 17 years with improvement in overall health</td></tr><tr><td>2</td><td>Moderate</td><td>46</td><td>Man</td><td>Under ERT for more than 3 years with deterioration in overall health</td></tr><tr><td>3</td><td>Severe</td><td>38</td><td>Woman</td><td>Axial and proximal deficit; significant fatigue. Stabilization of overall health under ERT</td></tr></tbody></table>
<figure><img src="image_3.png" /><figcaption>**Additional file 1:** Hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) staining of muscle from late-onset Pompe disease patients. Scale bars = 50 µm.</figcaption></figure>

**Mouse Model**

Gaa⁻/⁻ mice (GAA KO 6ⁿeo/6ⁿeo mouse model; RRID: IMSR_JAX:004154) with a targeted disruption of exon 6 in the *Gaa* gene were used. Breeding heterozygous 6ⁿeo/6ⁿeo mice were kindly provided by Dr. Nina Raben (NIH, Bethesda, MD, USA) and maintained in the specific pathogen-free animal facility at Oniris (Nantes, France). Gaa⁻/⁻ homozygous breeding was performed, and wild-type (WT) littermates were bred as controls. Mice were provided with standard mouse chow and water *ad libitum* and housed under a 12:12 h dark:light cycle at 22°C. All animals shared the same 129xC57BL/6 genetic background and were exclusively male to limit the potential risk of variability linked to hormonal regulation in female mice. All experiments were conducted in accordance with European guidelines for the care and use of laboratory animals and were reviewed and approved by the Ethics Committee on Animal Experimentation of the Pays de la Loire region, France (permit number APAFIS #1267).
161 **Experimental Design**

162 The experimental design incorporated both the Pompe disease patients and the murine
163 model for Pompe disease. A longitudinal pathophysiological study was designed to
164 identify dysregulated biological pathways in the tibialis anterior (TA) muscle of Gaa<sup>-/-</sup>
165 mice. This study was based on microarray gene expression analysis, supplemented by
166 histological and biochemical investigations. Four time points were selected to compare
167 muscle from Gaa<sup>-/-</sup> and WT mice, coinciding with a pre-symptomatic age (1.5 months
168 [mo]), the age at the onset of clinical symptoms (4 mo), a lesion establishment (6 mo),
169 and the presence of advanced lesions (9 mo). Four Gaa<sup>-/-</sup> mice and four WT mice were
170 included per time point. Muscles collected at each age were used to compare the overall
171 molecular signatures of the mouse groups. To specifically investigate changes associated
172 with disease progression, muscles from Gaa<sup>-/-</sup> mice at different ages were compared,
173 focusing on the differentially expressed genes (DEGs) identified in the initial analysis to
174 exclude age-related genes. In addition, deltoid muscle biopsies from LOPD patients with
175 mild, moderate, or severe disease form were subjected to histopathological examination.
176

176

177 **Tissue Collection**

178 TA muscles from mice were harvested and divided into three samples. One sample, for
179 transcriptomic study, was collected and immediately placed in RNAlater® (#R0901,
180 Merck, Saint-Quentin-en-Yvelines, France) overnight (ON) at 4°C. The RNAlater® was
181 then removed, and tissue samples were stored at -80°C until processing. For histology,
182 another sample was positioned on tragacanth gum, snap-frozen in isopentane prechilled
183 in liquid nitrogen, and stored at -80°C until sectioning. Finally, the remaining sample, for
184 biochemical study, was directly frozen in liquid nitrogen and stored at -80°C until
185 processing.
186
186 RNA Isolation

187 Total RNA was extracted from tissue (approximately 20 mg) preserved in RNAlater®,
188 using the Nucleozol RNA extraction kit (#740406, Macherey-Nagel, Hœrdt, France)
189 according to the manufacturer's instructions. Each sample was treated with DNAse I
190 (#AM2235, Life technologies, Saint-Aubin, France). RNA was quantified using a
191 NanoDrop spectrophotometer (Thermo Fisher Scientific, Illkirch, France), and its
192 integrity was determined using the Agilent Eukaryote Total RNA Nano kit (#5067,
193 Agilent Technologies, Les Ulis, France) and the 2100 Bioanalyzer (Agilent
194 Technologies).

195

196 Microarray Gene Expression

197 RNA (100 ng) was used to generate double-strand cDNA, which was fragmented and
198 biotin-labeled using the Genechip WT Plus Reagent Kit (#900720, Applied Biosystems,
199 Warrington, UK). Fragmented cDNA (2.3 µg) was hybridized to a mouse Clariom S array
200 (#902931, Applied Biosystems) for 16 h at 45°C. Microarrays were washed and stained
201 with phycoerythrin-conjugated streptavidin in an Affymetrix Fluidics Station 450
202 (Affymetrix, Santa Clara, CA, USA). Fluorescent images were captured using a gene
203 Array Scanner 2500 (Affymetrix). The resulting .CEL files were processed using the oligo
204 Bioconductor package (version 1.56.0) from R software (version 4.1.0). Data were
205 normalized and corrected with the RMA method. Raw data were deposited in the NCBI
206 Gene Expression Omnibus (GEO) repository under accession number GSE293648. CEL
207 files were processed using the oligo Bioconductor package (version 1.56.0) in R (version
208 4.1.0). Data were preprocessed using the standard RMA algorithm, including background
209 correction, quantile normalization and probe summarization. Arrays from different age
210 groups were processed in two batches (1.5/4 months and 6/9 months). Because batch was
confounded with age, differential expression analyses were performed using age-specific
211

contrasts (`Gaa`\textasciitilde\textasciitilde vs. WT mice sample at each age). Within each age group, WT and
212

`Gaa`\textasciitilde\textasciitilde mice samples were balanced and processed in the same batch, thereby avoiding
213

batch confounding of genotype effects. DEGs were identified using the *Limma* R package
214

(version 3.48.3) with an absolute fold-change ≥2 and a Benjamini-Hochberg (BH)
215

adjusted p-value <0.05.
216

217

218

**Gene Set Enrichment Analysis**

Gene Ontology (GO) enrichment analysis was performed using the topGO R package
219

(version 2.44.0) with the Biological Process (BP) and Cellular Component (CC)
220

annotation databases (BP and CC with org.Mm.eg.db; version 3.13.0). P-values were
221

computed using the “classic” and “elim” method algorithm parameters and “Fisher” as
222

the statistical parameter, according to the reference manual.
223

224

225

**Western Blot**

Muscle samples were lysed with a Precellys® homogenizer (#P000062-PEVO0-A, Bertin
226

Technologies, Montigny Le Bretonneux, France) in RIPA buffer (#9806S, Ozyme, Saint-
227

Cyr-l'École, France) containing a protease/phosphatase inhibitor cocktail (#11836153001
228

and #P0044 respectively, Merck, Saint-Quentin-en-Yvelines, France). Total protein
229

lysates were centrifuged at 13,000 rpm (30 min, 4°C), and supernatants were collected in
230

2X Laemmli buffer (#LC2676, Thermo Fisher) containing 5% v/v 2-mercaptoethanol.
231

Samples (20 µg) were loaded onto a TGX Mini-PROTEAN SDS-PAGE gel (Bio-Rad,
232

Marnes-la-coquette, France). Separated proteins were transferred onto nitrocellulose
233

membranes (#GE10600001, Amersham, Velizy-Villacoublay, France). Membranes were
234

then treated with blocking buffer (5% nonfat milk), incubated ON with primary antibodies
235
236 (Abs), washed, and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary Abs at 1:5,000 dilution (polyclonal donkey anti-rabbit [#711-035-152, Interchim, Montluçon, France] or polyclonal rabbit anti-goat [#A5420, Merck]).

239 HRP-chemiluminescence was developed using the Clarity Western Substrate kit (#1705060, Bio-Rad, Marnes-la-coquette, France). Membranes were scanned on an imager (LI-COR Biosciences, Lincoln, NE, USA) and quantified using ImageJ Software.

242 Band intensity (number of positive pixels per unit area) of the target protein was measured and normalized to the total loaded protein per lane. Uncropped images of blots are provided in Additional file 2. The primary Abs used and their dilutions are listed in Table 2.

246

<table><caption>247 **Table 2.** List of primary antibodies used for western blot experiments.</caption><thead><tr><td>Antibody</td><td>Host species</td><td>Clonality</td><td>Source</td><td>Reference</td><td>Dilution</td></tr></thead><tbody><tr><td>Pro-Myostatin (Pro-MSTN)</td><td>Goat</td><td>PC</td><td>R&D</td><td>AF788</td><td>1:300</td></tr><tr><td>Myostatin (MSTN)</td><td>Rabbit</td><td>PC</td><td>Sigma-Aldrich</td><td>AB3239-I</td><td>1:500</td></tr><tr><td>SMAD2/3</td><td>Rabbit</td><td>PC</td><td>Cell signaling technology</td><td>5678</td><td>1:1,000</td></tr><tr><td>P- SMAD1/2/3/5</td><td>Rabbit</td><td>MC (EP823Y)</td><td>Abcam</td><td>ab52903</td><td>1:2,000</td></tr><tr><td>Dysferlin (DYSF)</td><td>Rabbit</td><td>MC (JAI-1-49-3)</td><td>Abcam</td><td>ab124684</td><td>1:1,000</td></tr><tr><td>Annexin A2 (ANXA2)</td><td>Rabbit</td><td>PC</td><td>Abcam</td><td>ab154113</td><td>1:1,000</td></tr><tr><td>Galectin-3 (LGALS3)</td><td>Mouse</td><td>MC (B2C10)</td><td>Santa Cruz Biotechnology</td><td>sc-32790</td><td>1:500</td></tr><tr><td>AHNAK2 (KIS)</td><td>Rabbit</td><td>PC</td><td>Gentil et al. [17]</td><td></td><td>1:1,000</td></tr><tr><td>GAPDH</td><td>Rabbit</td><td>PC</td><td>Santa Cruz Biotechnology</td><td>sc-25778</td><td>1:5,000</td></tr></tbody></table>

248 MC, monoclonal; PC, polyclonal

249
<figure><img src="image_4.png" /><figcaption>Figure 2b: uncropped western blot images</figcaption></figure>

Additional file 8 : uncropped western blot images

<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /><figcaption>Figure 6b: uncropped western blot images</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Figure 6d: uncropped western blot images</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Figure 8b: uncropped western blot images</figcaption></figure>

<figure><img src="image_9.png" /></figure>

251

**Additional file 2:** Uncropped scans of western blots from main figures. Uncropped full scans of western blots from the corresponding cropped western blots shown within the main text. Molecular weight (MW) markers are indicated. Figure subpanel is indicated for each respective blot.
258 *Immunohistochemistry*

259 Cryosections (10 µm-thick) were fixed for 10 min with the appropriate buffer according
to the specific Ab used (Table 3) and saturated in blocking buffer containing 5% goat
260 serum in 0.1 M phosphate-buffered saline (PBS; Sigma-Aldrich) for 30 min at room
temperature (RT). Sections were then incubated with primary Ab diluted in blocking
261 buffer (ON, 4°C). After washing in PBS, sections were incubated for 1 h at RT with
262 Alexa® 555 or Alexa® 488 secondary Abs (1:300; #A21429; #A21240; #A21434,
263 #A11008, Invitrogen, Carlsbad, CA, USA). After rinsing, nuclei were stained with DAPI
264
265 (#D1306, Invitrogen) followed by mounting slides with Mowiol (#475904, VWR,
266 Pennsylvania, PA, USA). For some experiments, Alexa® 647-conjugated wheat germ
267 agglutinin (WGA; #W32466, Invitrogen) was used to visualize muscle fiber outlines.
268

269 Images were acquired using a confocal laser microscope (Nikon, Champigny-sur-Marne,
270 France) with NIS software (Nikon) or with the Axioscan slide scanner (Zeiss,
271 Oberkochen, Germany) using ZenBlue software (Zeiss). For human samples,
272 immunolabeling protocols were the same as those used for the mouse samples, except for
273 DYSF, which required antigen retrieval pretreatment in citrate buffer (10 min, 99°C) pH6
274 (#ZUC028, Zytomed, Berlin, Germany). Macrophage infiltration was quantified on
275 scanned slides and analyzed blindly using QuPath software (version 0.4.2).
276 Colocalization analysis were performed in Fiji by calculating the Pearson's correlation
277 coefficient, using the colocalization threshold plugin.

278

279

280

281
<table><caption>282 **Table 3.** List of primary antibodies used for immunohistochemistry experiments.</caption><thead><tr><th>Antibody</th><th>Host species</th><th>Clonality</th><th>Source</th><th>Reference</th><th>Fixation</th><th>Dilution</th></tr></thead><tbody><tr><td>LC3</td><td>Mouse</td><td>MC (E5Q2K)</td><td>Cell Signaling Technology</td><td>83506</td><td>/</td><td>1:100</td></tr><tr><td>LAMP1 (CD107a)</td><td>Rat</td><td>MC (1D4B)</td><td>BD Biosciences</td><td>553792</td><td>PFA 4%, RT</td><td>1:50</td></tr><tr><td>LAMP1</td><td>Mouse</td><td>MC (H4A3)</td><td>Santa Cruz Biotechnology</td><td>555800</td><td>PFA 4%, RT</td><td>1:50</td></tr><tr><td>CD68</td><td>Rat</td><td>MC (FA-11)</td><td>BioRad</td><td>MCA1957</td><td>PFA 4%, RT</td><td>1:400</td></tr><tr><td>MRC1 (CD206)</td><td>Rabbit</td><td>PC</td><td>Abcam</td><td>ab64693</td><td>PFA 4%, RT</td><td>1:600</td></tr><tr><td>devMHC</td><td>Mouse</td><td>MC (RNMy2/9D2)</td><td>Novocastra</td><td>NCL-MHCd</td><td>/</td><td>1:20</td></tr><tr><td>Type IIA Myosin</td><td>Mouse</td><td>MC (SC-71)</td><td>DSHB</td><td>SC-71</td><td>/</td><td>1:100</td></tr><tr><td>Type IIb Myosin</td><td>Mouse</td><td>MC (BF-F3)</td><td>DSHB</td><td>BF-F3</td><td>/</td><td>1:50</td></tr><tr><td>Type I Myosin</td><td>Mouse</td><td>MC (BA-F8)</td><td>DHSB</td><td>BA-F8</td><td>/</td><td>1:50</td></tr><tr><td>Dystrophin (DYS2)</td><td>Mouse</td><td>MC (DyS8/6C5)</td><td>Novocastra</td><td>NCL-DYS2</td><td>PFA 4%, RT</td><td>1:50</td></tr><tr><td>Dysferlin</td><td>Rabbit</td><td>MC (JAI-1-49-3)</td><td>Abcam</td><td>ab124684</td><td>METOH, -20°C</td><td>1:200</td></tr><tr><td>DHPR (Cav1.1)</td><td>Mouse</td><td>MC (1A)</td><td>Invitrogen</td><td></td><td>METOH, -20°C</td><td>1:100</td></tr><tr><td>Annexin A2 (ANXA2)</td><td>Rabbit</td><td>PC</td><td>Abcam</td><td></td><td>PFA 4%, RT</td><td>1:200</td></tr><tr><td>AHNAK2 (KIS)</td><td>Rabbit</td><td>PC</td><td>Gentil et al. [17]</td><td></td><td>/</td><td>1:100</td></tr></tbody></table>

283 DSHB, Developmental Studies Hybridoma Bank; MC, monoclonal; PC, polyclonal; PFA, paraformaldehyde; RT, room temperature

284

285

286 Statistical Analysis

288 For western blot analysis, differences between the two groups for each protein were analyzed with the Wilcoxon test and GraphPad Prism v.6.0 (GraphPad Software, La Jolla, CA, USA). For quantification of macrophages, the effects of age and group (WT/Gaa⁻/⁻) on CD68⁺ cells/mm², type M1 cells/mm², and type M2 cells/mm² were analyzed by a two-way analysis of variance followed by multiple comparisons with Tukey's post-hoc test when the independence and normality of the residuals were validated. Data analysis

289

290

291

292

293
294 was performed using R-Software. All data are reported as the mean ± standard deviation (SD). The p-value correction for multiple testing was assessed by the « multcomp » package for Tukey's test. Otherwise, a Kruskal-Wallis test was performed and followed by multiple testing with Wilcoxon pairwise test and Benjamini-Hochberg correction. For all statistical tests, a p-value of 0.05 or less was considered significant.

299

300 **Results**

301 **Pompe disease mice muscle exhibits a molecular signature rapidly established,**
302 **selective and stable in time**

303 To comprehensively identify cellular processes disrupted in Pompe disease and to
304 determine how these processes evolve over time, we performed a longitudinal
305 transcriptomic analysis on TA muscle from Gaa⁻/⁻ and WT mice at 1.5, 4, 6, and 9 months
306 of age. Principal component analysis (PCA) of the gene expression dataset (6,021 genes
307 with SD ≥ 0.3) showed global differences in baseline transcriptomes with Dimension 1
308 (Dim1) and Dim2 explaining 26.9% and 18.8% of the variance, respectively (Fig. 1a).
309 This analysis demonstrated a clear genotype-specific separation. Dim1 distinguished the
310 transcriptome of Gaa⁻/⁻ mice from that of WT mice. Furthermore, the overall molecular
311 signature of WT mice from 1.5 to 6 months of age was distinct from that of 9-mo-old WT
312 mice and all Gaa⁻/⁻ mice, regardless of age (Fig. 1a). The cluster corresponding to 1.5-
313 mo-old Gaa⁻/⁻ mice was distinct from the other Gaa⁻/⁻ mouse groups, indicating a specific
314 transcriptomic signature for this age. Biplot visualization, using the 25 genes with the
315 highest contribution to the PCA, highlighted specific gene expression patterns correlated
316 with genotype (Fig. 1b): The expression of acid alpha-glucosidase (Gaa),
317 methyltransferases (*Mettl21c* and *Mettl21e*), aquaporin-4 (*Aqp4*), nexilin F-actin binding
318 protein (*Nexn*), and myostatin (*Mstn*) was negatively correlated with the Gaa⁻/⁻ genotype.
Conversely, the expression of metallothioneins (MT1, MT2, and MT3), serine peptidase inhibitor clade B member 1a (Serpinb1a), serine peptidase inhibitor clade A member 3N (Serpina3n), TIMP metallopeptidase inhibitor 1 (Timp1), and Ahnak nucleoprotein 2 (Ahnak2) was positively correlated with the Gaa⁻/⁻ genotype. DEGs were identified using the Limma R package (BH-corrected p-value < 0.05 and absolute log2 fold change > 1).

Comparisons between Gaa⁻/⁻ and WT mice were performed at each age. A set of 414 DEGs, including 116 downregulated and 298 upregulated genes in Pompe disease, was identified and represented in a heatmap (Fig. 1c, Additional file 3). GO analysis was performed to identify functional enrichment and cellular localization of all DEGs (Padj < 0.01), revealing significant enrichment in 183 biological processes (BPs) and 26 cellular components (CCs) (Additional files 4 and 5). After excluding duplications and pathways involved in general biological mechanisms, 11 BPs and 6 CCs were specifically identified (Fig 1d). The most represented BPs included metabolic processes including sphingolipid metabolism (GO: 0006665, p = 0.0006 and GO: 0030148, p = 0.00896), glycogen metabolic process (GO: 0005977, p = 0.0002), ceramide metabolism (GO: 0006672; p = 0.00082), skeletal muscle tissue regeneration (GO: 0043403, p = 0.0002), calcium homeostasis (GO: 0070296, p = 0.0019), oxidative stress (GO: 0010273, p = 0.0002, GO: 0006979, p = 0.0090), and inflammatory response (GO: 0006954, p = 0.0002). The most represented CCs included Z-disc (GO: 0030018, p = 7e-06), T-tubule (GO: 0030315, p = 6e-05), secretory granule (GO: 0030141, p = 0.0036), plasma membrane organization (GO: 0005886, p = 0.00329), neuromuscular junction (GO: 0031594, p = 0.0007), and lysosome (GO: 0005764, p = 0.0096) (Fig. 1d).

Among the 414 DEGs, 58 genes displayed changes in expression across disease stages within Gaa⁻/⁻ mice (Fig. 1e, Additional file 6), indicating that the specific molecular signature of Pompe disease mice muscle is acquired early and remains relatively stable.
GO term analysis on these 58 DEGs revealed significant enrichment in 8 BPs (Fig. 1f, Additional file 7), primarily related to the regulation of axonogenesis (GO: 0050770, p = 0.0003; GO: 0050767, p = 0.002; GO: 0030516, p = 0.002) and neurogenesis (GO: 0050767, p = 0.00184), potentially reflecting progressive central nervous system involvement in Pompe disease. Another major BP was circadian rhythm. In summary, these results demonstrate rapid alterations in the molecular profile of Pompe disease mice muscle, with a distinct intermediate state during the first month of life. This alteration involves a restricted set of genes linked to metabolism, inflammation, muscle regeneration and organization as well as the neuromuscular system, and remains relatively stable over the subsequent course of disease progression.
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354

355 **Fig. 1:** Molecular Profile of the tibialis anterior muscle of Gaa⁻/⁻ mice. (a) Principal

356 component analysis (PCA) plot illustrating global differences in baseline transcriptomes.

357 Dimension (Dim) and percent variance indicate the amount of variability between

358 subjects explained by each component. (b) Biplot showing the top 25 genes that most

359 contribute to the variability of the data, highlighting specific gene expression patterns
360 correlated to genotype. (c) Heatmap of the 414 differentially expressed genes (DEGs)
361 identified between WT and Gaa⁻/⁻ mice across all ages. (d) Bubble plot depicting enriched
gene ontology (GO) terms for cellular component (CC) and biological process (BP) that
differ in Gaa⁻/⁻ vs WT mice. (e) Heatmap of the 58 DEGs for which changes in expression
362 were observed during the disease course, revealing a relatively stable molecular
363 signature in Pompe disease mice muscle. Red, blue, and white indicate upregulation,
364 down-regulation, and no significant change, respectively. (f) Bubble plot depicting GO
terms for BP that are altered during the disease course, potentially reflecting progressive
365 involvement of specific biological processes.
366

369

370 **Sarcoplasmic Dysferlin Accumulation Reflects the Severity of Muscle Damage in**
371 **both Pompe Disease Mice and Patients**

372 We sought to understand the significance of the enrichment of "plasma membrane" and
"plasma membrane organization" terms in the BPs and CCs identified by the GO analysis.
373

374 Among the DEGs identified was dysferlin (Dysf), expression of which was significantly
375 increased in Gaa⁻/⁻ mice from 1.5 months of age and remained stable over the subsequent
disease course (Fig. 2a). Increase of the protein level was observed at 1.5 and 9 months
376 of age (3- and 10-fold, respectively; Fig. 2b). DYSF was predominantly localized at the
377 plasma membrane in WT mouse muscle (Fig. 2c, top). By contrast, Gaa⁻/⁻ mouse muscle
378 showed a much more pronounced and diffuse expression pattern (Fig. 2c, bottom): while
379 DYSF was sometimes observed exclusively in the plasma membrane of certain
380 myofibers, it was predominantly localized in the sarcoplasm in Gaa⁻/⁻ mouse muscles.
381
382 Some fibers showed DYSF expression in both locations. Muscles from 1.5-mo-old
383 Gaa⁻/⁻ mice showed mosaic pattern of DYSF expression in myofibers while those from
384 9-mo-old mice showed generalized sarcoplasmic accumulation in all myofibers, with
greater immunolabeling intensity. We next investigated the potential link between this
abnormal DYSF expression and the T-tubules identified as CCs in the GO analysis. To
detect T-tubules corresponding to characteristic invaginations of the plasma membrane,
dihydropyridine receptors (DHPR) expression was investigated in addition to DYSF on
longitudinal sections. Marked expression of DYSF was observed in the T-tubules of
*Gaa*<sup>-/-</sup> mice, in contrast to the exclusively peripheral expression profile seen in WT mice
(Fig. 2d).

<figure><img src="image_16.png" /></figure>
**Fig. 2:** Dysferlin content and localization in the tibialis anterior muscle of Gaa⁻/⁻ mice. (a) Boxplot depicting dysferlin (Dysf) transcript expression as determined by microarray analysis, showing significant upregulation in Gaa⁻/⁻ versus WT mice. Adjusted p-values correspond to the mean differential expression between the two groups as established by multiple testing. ****p < 0.0001. (b) Western blot analysis of dysferlin (DYSF) protein levels at 1.5-mo and 9-mo of age, confirming increased expression in Gaa⁻/⁻ versus WT mice. First and third quartile range (top and bottom of the box), median (central band) and individual data points are visualized. AU = Arbitrary Unit. Statistics: Wilcoxon test; *p < 0.05. (c) Representative cross-sections of tibialis anterior (TA) muscles from 1.5- and 9-mo-old WT and Gaa⁻/⁻ mice immunolabeled with anti-DYSF antibody (green), showing abnormal sarcoplasmic accumulation of DYSF in Gaa⁻/⁻ mice. Scale bars = 25 µm. (d) Colocalization of DYSF with T-tubules in Gaa⁻/⁻ mice. Longitudinal sections of TA muscle from 1.5-mo-old WT and Gaa⁻/⁻ mice immunolabeled with anti-DYSF antibody (red) and anti-DHPR antibody (green), demonstrating the association of DYSF with T-tubules in Gaa⁻/⁻ mice. Scale bars = 10 µm.

Furthermore, the presence of DYSF in the myofiber sarcoplasm did not seem to correlate with glycogen overload, as observed on by PAS-stained serial sections of 1.5- and 9-mo-old mice (Fig. 3a). To determine whether DYSF accumulation is associated with autophagic defects and with muscle fiber metabolism, co-immunolabeling against LC3/LAMP1 (canonical autophagosomal and lysosomal markers, respectively) and against fiber-type-specific myosin heavy chain (MHC) were performed (Fig. 3b). This analysis showed that, at 1.5 months, DYSF⁺ myofibers predominantly correspond to fibers exhibiting autophagic debris and to fast-twitch enriched with type IIb fibers (as type IIX MHC was not evaluated here): Specifically, 59.0% ± 8.8% of the DYSF⁺ myofibers were LC3⁺ (given that LC3⁺ structures are barely visible in the muscles of WT
mice) and 85.8% ± 14.8% were type IIb MHC-enriched fibers, with the remaining fibers
expressing MHC isoforms other than types I and IIa (Fig. 3c). At 9 months of age, while
similar results were obtained for the detection of DYSF⁺/LC3⁺ myofibers (57.3% ±
14.5%), the proportion of DYSF⁺/type IIb MHC⁺ myofibers was lower (51.6% ± 25.5%)
(Fig. 3c). Additionally, DYSF⁺ myofibers positive for type IIa (28.8% ± 20.0%) and non-
type I MHCs (19.6% ± 21.7%) were also detected, indicating less restrictive expression
of DYSF in type IIb MHC-enriched fibers at this later stage. Together, these data
demonstrate DYSF increased levels in type IIb MHC-enriched myofibers containing
accumulated autophagic debris, with preferential localization in T-tubules.

To further investigate the role of DYSF in the pathophysiology of Pompe disease, we
examined its expression in LOPD patients, specifically those with low, moderate, and
high glycogen levels (Additional file 1). DYSF was localized mainly in the plasma
membrane of myofibers in mildly affected patients, similar to healthy subjects. In
moderately and severely affected patients, DYSF was detected in the plasma membrane
and in the sarcoplasm (Fig. 3d). This abnormal accumulation of DYSF was also observed
in muscle sections from DMD patients. Severely affected LOPD patients exhibited much
more intense sarcoplasmic labeling than those with mild or moderate lesions, particularly
in small myofibers, although labeling was also detected in some large myofibers.
Furthermore, myofibers exhibiting sarcoplasmic accumulation of DYSF did not
correspond to those expressing the developmental isoform of MHC (devMHC), which
represent less than 0.2% of total fibers, even in advanced stages of the disease. This
contrasts with the labeling observed in regenerating fibers in DMD patients (Fig. 3d,
bottom panel). Overall, these results demonstrate increased levels of DYSF and
sarcoplasmic accumulation in Pompe disease mice muscle, particularly in type IIb fibers
with autophagic debris. This abnormal DYSF accumulation seems to correlate with the
severity of histopathological lesions in LOPD patients but is distinct from muscle

regeneration.

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445

Fig. 3: Fiber-specific dysferlin expression in Gaa⁻/⁻ mice and Pompe disease patients. (a)

Serial cross-sections of tibialis anterior (TA) muscle from 1.5- and 9-mo-old WT and

Gaa⁻/⁻ mice immunolabeled with anti-dysferlin (DYSF) antibody (green) and stained with
periodic acid-Schiff (PAS), showing no correlation between DYSF sarcoplasmic accumulation and glycogen overload. Scale bars = 20 µm. (b) Serial TA muscle cross-sections from 1.5-mo and 9-mo Gaa⁻/⁻ mice submitted to antibodies against DYSF (left panel), LC3 (green) / LAMP1 (red, middle panel), and type I (purple) / type IIa (red) / type IIb (green) myosin heavy chain (MHC, right panel). Wheat germ agglutinin (WGA, blue) allows visualization of the muscle fiber outlines and shows that DYSF⁺ myofibers mainly correspond to fibers containing a build-up of autophagic debris and to fast-twitch enriched-type IIb fibers. Scale bars = 50 µm. (c) Characterization of DYSF⁺ muscle fibers. Bar graph illustrates the fiber type composition of DYSF⁺ muscle fibers at 1.5 and 9 months of age. For fiber typing, myosin heavy chain (MHC) expression was used; fibers referred to as type IIb should therefore be considered IIb-enriched rather than purely type IIb, as type IIX MHC fibers were not detected. Values represent mean percentages ± SD (left panel) or relative distributions (right panel). Four individuals and 368 DYSF⁺ fibers were considered per group. (d) Immunolabeling for DYSF (green, upper panel) and developmental isoform of MHC (devMHC, red, lower panel) in serial cross-sections of deltoid muscle biopsies from late-onset Pompe disease (LOPD) patients. WGA (blue) allows visualization of the muscle fiber outlines, and shows that myofibers with sarcoplasmic overexpression of DYSF do not correspond to regenerating fibers. Scale bars = 50 µm.

**Accumulation of Annexin 2 in Pompe Disease Mice and Patient Muscles could preserve the lysosomal membrane integrity**

Transcriptomic analysis revealed a significant increase, evident from 1.5 months of age, of *Anxa2* transcript expression in muscles from Gaa⁻/⁻ mice (Fig. 4a). This increase was also observed at the protein level in muscle extracts from 1.5- and 9-mo-old Gaa⁻/⁻ mice,
474 which showed 2.0- and 2.4-fold increases, respectively, relative to WT counterparts (Fig. 4b). In WT mice muscle, ANXA2 was expressed in the plasma membrane and, to a lesser extent, in the sarcoplasm in the form of faint punctiform deposits (Fig. 4c, top). By contrast, these deposits were larger and more abundant in *Gaa*⁻/⁻ muscle (Fig. 4c, bottom), in which they colocalized with lysosomes, as shown by LAMP1 immunolabeling (Fig. 4c, bottom). Based on pooled muscle fibers (individuals not distinguished), a Pearson correlation coefficient of 0.64 ± 0.13 (range: 0.54 ± 0.13 to 0.71 ± 0.08) indicated a moderate to strong correlation between ANXA2 and LAMP1 localization (violin plot, Fig. 4d). Furthermore, a 5.0-fold and 8.4-fold increase in galectin-3 protein (LGALS3) expression levels was observed in muscle samples from 1.5-mo-old and 9-mo-old *Gaa*⁻/⁻ mice, respectively, compared to their WT counterparts (Additional file 8).

485 In addition, determination of ANXA2 expression in biopsies from LOPD patients showed comparable expression levels in mild LOPD patients and healthy subjects, with plasma membrane and diffuse sarcoplasmic localization (Fig. 4e, left panel). Moderate LOPD patients showed a more pronounced punctiform sarcoplasmic expression pattern, which was defined by LAMP1⁺ labeling (Fig. 4e, middle panel). Interestingly, in patients with severe LOPD, sarcoplasmic ANXA2 labeling was not associated with LAMP1 labeling (Fig. 4e, right panel). Instead, LAMP1 immunolabeling was mainly identified in the center of fibers, where it often associated with autophagosomes (LC3⁺ structures), reflecting exacerbated autophagic build-up (Fig. 4f). Taken together, these findings show that ANXA2 accumulates in lysosomal membranes, where it could be mobilized to maintain their integrity in the face of damage early in the course of Pompe disease.
<figure><img src="image_21.png" /><figcaption>**Fig. 4:** Annexin A2 content and localization in the tibialis anterior muscle of Gaa<sup>-/-</sup> mice.</figcaption></figure>

(a) Boxplot depicting annexin A2 (AnxA2) transcript expression as determined by microarray analysis, showing significant upregulation in Gaa<sup>-/-</sup> versus WT mice. P-values correspond to the mean differential expression between the two groups as established by multiple testing: **** p < 0.0001. (b) Western blot analysis of annexin A2 (ANXA2)

496

497

498

499

500

501
protein levels at 1.5-mo and 9-mo of age, confirming increased expression in Gaa⁻/⁻ mice.

Interquartile range (top and bottom of the box), median (central band) and individual data points are visualized. AU = Arbitrary Unit. Statistics: Wilcoxon test; * p < 0.05. (c)

Representative cross-sections of tibialis anterior muscle from 1.5-mo-old WT and Gaa⁻/⁻ mice immunolabeled with anti-ANXA2 (green) and LAMP1 (red) antibodies, showing colocalization of ANXA2 with lysosomes in Gaa⁻/⁻ mice. Scale bars = 10 µm. (d) Violin plot showing the distribution of Pearson's correlation coefficients between ANXA2 and LAMP1 localization calculated for individual muscle fibers and pooled across all individuals. Each dot represents a single muscle fiber, and the violin shape reflects the density distribution of correlation values. (e) Annexin A2 expression profile in Pompe disease patients. Immunolabeling of annexin A2 (ANXA2, green) and LAMP1 (red) in serial cross-sections from deltoid muscle biopsies of late-onset Pompe disease (LOPD) patients. Wheat germ agglutinin (WGA, blue) allows visualization of the muscle fiber outlines, showing distinct expression patterns depending on disease severity. Scale bars = 50 µm. (f) Immunolabeling of LAMP1 (red) and LC3 (green) in serial muscle cross-sections from a severely affected LOPD patient, demonstrating exacerbated autophagic build-up. Scale bars = 50 µm.

519

520

521
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**Additional file 8:** Western blot analysis of galectin 3 (LGALS3) protein levels at 1.5-mo and 9-mo of age, confirming lysosomal membrane damage in Gaa⁻/⁻ mice. Interquartile range (top and bottom of the box), median (central band) and individual data points are visualized. Statistics: Wilcoxon test; \* p < 0.05

## Pompe Disease Mice and Patient Muscle Accumulates AHNAK2 in Autophagosomes

Furthermore, based on our PCA findings, which identified genes that most contributed to the Pompe disease phenotype, we focused our analysis on the nucleoprotein AHNAK2 [24]. Although the specific function of this giant protein is unclear, roles as a scaffold protein [5, 26] and in plasma membrane repair [10] have been proposed. *Ahnak2* transcript expression was significantly increased in *Gaa*-/- versus WT mouse muscle from 1.5 months of age (Fig. 5a). This expression profile was maintained across all age groups studied. Moreover, this effect was replicated at the protein level in 1.5- and 9-month-old *Gaa*-/- mice (Fig. 5b). In *Gaa*-/- muscle, AHNAK2 was expressed at the plasma membrane and in the sarcoplasm, specifically in autophagosomes identified by LC3 immunolabeling (Fig. 5c, bottom panel). By contrast, AHNAK2 expression was restricted to the plasma membrane in WT muscle, regardless of age (Fig. 5c, upper panel).

In human muscle biopsies, similar plasma membrane expression of AHNAK2 was observed in healthy subjects and mild LOPD patients (Fig. 5d). In moderate LOPD
542 patients, AHNAK2 expression was observed both in the plasma membrane and in autophagosomes, in line with findings in *Gaa*-/- mouse muscle (Fig. 5d). Interestingly, in 543 severe LOPD patients, AHNAK2 no longer colocalized with autophagosomes, similar to 544 the findings for ANXA2 with lysosomes. Overall, these results highlight that AHNAK2 545 accumulates in autophagosomes at the early stage of the Pompe disease. 546

547

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548
**Fig. 5:** AHNAK2 expression profile in the tibialis anterior muscle of Gaa⁻/⁻ mice. (a) Boxplot depicting Ahnak2 transcript expression as determined by microarray analysis, showing significant upregulation in Gaa⁻/⁻ versus WT mice. P-values correspond to the mean differential expression between the two groups as established by multiple testing: ****p < 0.0001. (b) Western blot analysis of AHNAK2 protein levels at 1.5-mo and 9-mo of age, confirming increased expression in Gaa⁻/⁻ mice vs WT mice. Interquartile range (top and bottom of the box), median (central band) and individual data points are visualized. AU = Arbitrary Unit. Statistics: Wilcoxon test; *p < 0.05. (c) Immunolabeling of AHNAK2 (red) and LC3 (green) in tibialis anterior muscle cross-sections from 9-mo-old WT and Gaa⁻/⁻ mice, showing localization of AHNAK2 in autophagosomes in Gaa⁻/⁻ mice. Scale bars = 20 µm. (d) AHNAK2 expression profile in Pompe disease patients. Immunolabeling of AHNAK2 (red) and LC3 (green) in cross-sections from deltoid muscle of late-onset Pompe disease (LOPD) patients. Wheat germ agglutinin (WGA, blue) allows visualization of the muscle fiber outlines, showing distinct expression patterns depending on disease severity. Scale bars = 100 µm.

## Accumulation of Mature Myostatin Protein and Disruption of SMAD-Dependent Signaling Pathway Characterize Pompe Disease Mice Muscle

Among the genes most strongly discriminating Gaa⁻/⁻ from WT muscles in the PCA, myostatin (Mstn) emerged as a key DEG associated with skeletal muscle tissue regeneration (GO:0043403, p = 0.0002). Mstn encodes a major negative regulator of muscle growth acting notably through the TGF-β/activin signaling pathway [35]. Mstn transcript expression was significantly downregulated in Gaa⁻/⁻ mice compared to WT mice at all ages studied and also decreased over time (Fig. 6a, p < 0.0001). At the protein level, while the immature pro-MSTN form remained unchanged (Fig. 6b, left panel), that
of mature MSTN was significantly increased at each age, with respective fold changes of 574 2.0, 2.9, 2.9, and 3.9 (Fig. 6b, right panel). This observation shows an increasing 575 divergence in protein expression as the disease progresses. To determine whether the 576 increased levels of mature MSTN resulted from enhanced proteolytic activation, the 577 expression of enzymes involved in its processing was examined. Furin, BMP-1, and TLL- 578 1 transcripts, which are proteolytic enzymes involved in the production of the active form 579 of MSTN [27, 59], exhibited no significant differences in the transcriptomic analysis that 580 was confirmed by RT-qPCR (data not shown). 581

Given the central role of MSTN in regulating muscle growth, the increased levels of 582 mature MSTN protein in *Gaa*⁻/⁻ mice muscle prompted us to examine the potential 583 involvement of an atrophic program in Pompe disease. An upregulation of the muscle- 584 specific E3 ubiquitin ligase Trim63 and the translational repressor Eif4ebp1 transcript 585 expression, indicative of muscle atrophy, was observed at all ages in *Gaa*⁻/⁻ mice (p < 586 0.0001; Additional file 9). Next, we investigated the downstream SMAD signaling 587 cascade to determine whether MSTN dysregulation was associated with altered 588 transduction of the TGF-β/activin signaling pathway. A decreased expression of the 589 inhibitory protein SMAD7, through which MSTN typically regulates itself by inducing 590 its own expression [14], was observed at all ages in *Gaa*⁻/⁻ mice (Fig. 6c, left panel). It 591 was significant from 4 months of age onwards with fold changes ranging from 2.2 to 2.6 592 (p < 0.05). SMAD3, which regulates SMAD7 transcription in conjunction with SMAD4 593 directly binding to its promoter [38], was increased at all four time points in *Gaa*⁻/⁻ mice 594 (Fig. 6c and Additional file 10). Interestingly, a decrease in the phosphorylated form of 595 SMAD3 was observed at each age leading to a significantly reduced phosphorylated 596 SMAD3 (P-SMAD3)/SMAD3 ratio in *Gaa*⁻/⁻ mice (p < 0.05) (Fig. 6c, right panel). 597
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<figure><img src="image_35.png" /><figcaption>**Fig. 6:** Abnormal expression profile of Myostatin and profound disruption of SMAD 7 and 3 protein expression levels from the transforming growth factor beta (TGF-β)/activin signaling pathway in Gaa⁻/⁻ mice. (a) Boxplot depicting myostatin (Mstn) transcript</figcaption></figure>
expression as determined by microarray analysis, showing significant down-regulation
in Gaa⁻/⁻ versus WT mice. P-values correspond to the mean differential expression
between two groups as established by multiple testing; \*p < 0.05, \*\*p < 0.01, \*\*\*p <
0.0001. (b) Western blot analysis of pro-myostatin (PRO-MSTN) and mature myostatin
(MSTN) protein levels in muscle lysates from WT and Gaa⁻/⁻ mice. First and third quartile
range (top and bottom of the box), median (central band) and individual data points are
visualized. AU = Arbitrary Unit. Statistics: Wilcoxon test; \*p < 0.05. (c) Western blot
analysis of SMAD7, SMAD2/3 and phosphorylated-SMAD3 (P-SMAD3) in muscle lysates
from WT and Gaa⁻/⁻ mice. First and third quartile range (top and bottom of the box),
median (central band) and individual data points are visualized. AU = Arbitrary Unit.
Statistics: Wilcoxon test; \*p < 0.05.

613

<figure><img src="image_36.png" /></figure>

614

Additional file 9: Altered expression of atrophy-related genes in the tibialis anterior
muscle of Gaa⁻/⁻ mice. Boxplots depicting Trim63 and Eif4ebp1 transcript expressions as
determined by microarray analysis, showing significant upregulation in Gaa⁻/⁻ versus WT
mice. Adjusted p-values correspond to the mean differential expression between the two
groups as established by multiple testing. \*\*\*p < 0.0001.

620

621
<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

**Additional file 10:** Western blot analysis of SMAD2/3 and phosphorylated-SMAD3 (P-SMAD3) in muscle lysates from WT and Gaa⁻/⁻ mice. First and third quartile range (top and bottom of the box), median (central band) and individual data points are visualized.
AU = Arbitrary Unit. Statistics: Wilcoxon test; \* p < 0.05.

In parallel, transcriptomic analysis also revealed significant enrichment of genes involved in the inflammatory response (GO: 0006954, p = 0.0002), particularly those associated with a pro-inflammatory state. Cell density in the connective tissue surrounding muscle fibers was significantly higher in 6-mo-old Gaa⁻/⁻ versus WT mice (Fig. 7a), with an increasing trend observed at earlier time points. Most of these cells were macrophages, as demonstrated by CD68 immunolabeling (Fig. 7b). The absence of MRC1 expression by CD68⁺ macrophages indicated an over-representation of pro-inflammatory M1 macrophages, which are known to predominate during the activation and expansion of SCs following muscle injury (Fig. 7 c, d) [15, 54]. However, no increased expression of the pro-inflammatory cytokines IL-6 and IL-10 was detected by RT-qPCR (data not shown). Collectively, these data showed an abnormal regulation of the expression of the
negative myogenic regulator MSTN coupled with a profound disruption of the TGF-
β/activin signaling pathway in Pompe disease mice muscle characterized by an
enrichment of pro-inflammatory macrophages, an effect conducive to SC activation.

<figure><img src="image_40.png" /><figcaption>**Fig. 7:** Macrophage expression profile in the tibialis anterior muscle of Gaa⁻/⁻ mice. (a) Representative cross-sections of tibialis anterior (TA) muscles stained with hematoxylin-eosin-saffron (HES), showing increased cell density in the connective tissue surrounding muscle fibers in Gaa⁻/⁻ mice. Scale bars = 30 µm. (b) Immunolabeling of TA muscle cross-sections with anti-CD68 (red) and anti-MRC1 (green) antibodies, showing the presence of macrophages in the muscle tissue. Dystrophin antibody (DYS2; grey) allows</figcaption></figure>
649 visualization of the muscle fiber outlines. Nuclei are counterstained with DAPI (blue).
650 Scale bars = 25 µm. (c) Quantification of CD68⁺ cells/ mm², showing increased
651 macrophage infiltration in Gaa⁻/⁻ mice. (d) Quantification of CD68⁺MRC1⁻ cells (type M1
652 macrophages) and CD68⁺MRC1⁺ cells (type M2 macrophages), indicating a
653 predominance of pro-inflammatory M1 macrophages in Gaa⁻/⁻ mice. Statistics: Two-way
654 ANOVA and Tukey's post-hoc test for CD68⁺ cells/mm² and CD68⁺MRC1⁻ ;
655 ***p < 0.001.

656

657 Discussion

658 To our knowledge, the present work is the first longitudinal transcriptomic analysis
659 performed in Pompe disease and describes the molecular expression profile of skeletal
660 muscle in the Gaa⁻/⁻ mouse model from 1.5 to 9 months of age. The altered biological
661 pathways identified in Gaa⁻/⁻ mouse muscle included those involved in cell metabolism,
662 calcium homeostasis, oxidative stress, inflammation, and muscle regeneration, consistent
663 with transcriptional alterations described in previous transcriptomic studies in mouse
664 models and Pompe disease patients [7, 11, 29, 39, 57]. Our findings also provide
665 compelling evidence of profound dysregulation of membrane repair processes in muscle
666 fibers in Pompe disease, and highlights marked membrane remodeling in intracellular
667 organelles implicated in autophagy. Affected muscles exhibit accumulation of DYSF,
668 ANXA2, and AHNAK2 in T-tubules, lysosomes, and autophagosomes, respectively. This
669 abnormal sarcoplasmic build-up of these different proteins is an early-onset phenomenon
670 that progresses to advanced stages of the disease and correlates with other classically
671 described histopathological alterations, making these proteins potentially valuable
672 markers of Pompe disease progression.
673 DYSF plays a crucial role in plasma membrane repair and T-tubule integrity [43].
674 Abnormal sarcoplasmic accumulation of DYSF protein was established in myofibers of
675 Gaa⁻/⁻ mice, consistent with a previous finding in quadriceps and triceps muscle from 4-
676 mo-old Gaa⁻/⁻ mice [60]. That study reported a significant reduction in such accumulation
677 following ERT with ATB200/AT221, suggesting that DYSF accumulation is directly
678 linked to the degree of autophagic flux impairment. Interestingly, we found that
679 sarcoplasmic expression of DYSF primarily corresponded to myofibers containing
680 accumulated autophagic debris and to type IIb-enriched myofibers, which are the most
681 resistant to ERT [13]. Furthermore, we observed that a correlation between abnormal
682 sarcoplasmic accumulation of DYSF and severity of the histological phenotype seems to
683 exist in LOPD patients. Similar DYSF expression pattern has been described in various
684 forms of limb-girdle muscular dystrophy where it is presented as a direct consequence of
685 defects in proteins required for effective membrane targeting [31, 42]. We can
686 hypothesize that DYSF, like ANXA2 and AHNAK2, fails to reach the plasma membrane
687 in Pompe disease due to sarcoplasmic accumulation of autophagic aggregates that disrupt
688 intracellular trafficking. Thus, well-established autophagic flux impairment in Pompe
689 disease could be at least partially involved in the sarcoplasmic accumulation observed in
690 these proteins. It could prevent their degradation via lysosomal or proteasomal
691 dysfunction, but also limit their bioavailability.

692 Upregulation of galectin-3 was observed as early as 1.5 months in Gaa⁻/⁻ mice, showing
693 early lysosomal membrane damage in Pompe disease, consistent with previous findings
694 in the gastrocnemius muscle of 4-month-old Gaa⁻/⁻ mice [36]. In addition, the
695 accumulation of ANXA2 in lysosomes aligns with the previous results demonstrating its
696 role in repairing severe damaged lysosomal membranes [61]. The colocalization of
697 ANXA2 and LAMP1 observed as early as 1.5 months of age in Gaa⁻/⁻ mice suggests
already severe lysosomal membrane damages. In LOPD patients, it thus offers a valuable means of categorizing patients according to the severity of myoarchitectural disarray. Its loss in severely affected patients could mean that the level of intracellular remodeling prevents ANXA2 from reaching and repairing damaged lysosomal membranes. Finally, our findings provide evidence that AHNAK2, a component of the costameric network [32], is also dysregulated in Pompe disease. Although its biological function is not completely defined, it has been suggested to play a significant role in plasma membrane assembly and repair [4, 32]. In *Gaa*-/- mouse muscle, we found that AHNAK2 is localized to the sarcolemma and to autophagosomes. This expression profile is identical to that observed in moderate LOPD patients, suggesting that AHNAK2 may be mobilized in Pompe disease to compensate for autophagosome damage. By contrast, this autophagosome localization of AHNAK2 is absent in severely affected patients. AHNAK2 may thus be involved in autophagic vesicle dynamics, possibly via its cytoskeletal or membrane repair-related functions, although further functional studies will be required to clarify this hypothesis. Of note, knockdown of ANXA2 is associated with reduced expression and sarcoplasmic localization of AHNAK [4], while an interaction between AHNAK and DYSF has also been reported in myopathic patients [21]. Taken together, the present findings strongly support a link between the expression profiles of AHNAK2, DYSF, and ANXA2. Future research should focus on elucidating the regulatory pathways controlling their interactions.

Another key finding of our study in *Gaa*-/- mice is profoundly disrupted expression of myostatin, a well-known negative regulator of muscle growth [3, 9, 33]. *Mstn* gene was one of the most dysregulated genes in our study, displaying a significant and consistent reduction in expression. Also, MSTN, the mature form of the encoded protein, showed increased expression at all analyzed ages. Given that the levels of pro-MSTN, the latent
precursor form without biological activity, remain unchanged in Gaa⁻/⁻ mice compared to that observed in WT mice, a plausible explanation for this discrepancy would be an increased protein stability or reduced degradation in diseased muscle. This is reinforced by the fact that no significant differences were observed in the expression level of transcripts for enzymes furin and tolloid-like metalloproteinases (BMP-1 and TLL-1/2) involved in proteolytic cleavages [27, 59]. Since MSTN regulation occurs at the protein level, further characterization of their protein profile and activity would be informative.

The permanent overexpression of Trim63 and Eif4ebp1 transcripts observed in Gaa⁻/⁻ mice support the notion that MSTN dysregulation could be associated with the activation of an atrophic process rather than representing an isolated alteration. An in-depth study of muscle mass in the mouse model would be useful to consolidate this hypothesis and support the findings on the involvement of MSTN in the pathophysiology of Pompe disease. Considering that SMAD7 is a transcriptional repressor of MSTN, its consistent down-regulation is intriguing in light of the reduction in *mstn* RNA levels. Furthermore, the accumulation of MSTN should be accompanied by phosphorylation of SMAD3, which is not observed. These data suggest, first, that MSTN is either not bioactive or available for example by being unable to bind to receptors already occupied by other factors, and, second, that the TGF-β/activin signaling pathway is significantly disrupted in Pompe disease. Given the key roles of MSTN and, more broadly, the TGF-β/activin signaling pathway in the regulation of myogenesis, the alterations in MSTN and SMAD3/7 described here should encourage further investigation. This is all the more important as a continuous increase in mature MSTN protein levels may partially explain why SCs are not activated in Pompe disease [25, 49][50]. Indeed, studies using models of chronic MSTN overexpression have demonstrated impaired SC activation capacity and decreased proliferation [33, 45, 46, 52, 55]. Pharmacologic inhibition assays using
748 follistatin, Fsl3 or anti-MSTN antibodies could also provide useful information to
749 elucidate the role of MSTN in the SC behavior in Pompe disease. Finally, increased
750 MSTN may also reflect accumulation due to impaired autophagic flux, which would
751 prevent its degradation but also limit functional effects, particularly on SC.

752

753 **Conclusions**

754 Our findings demonstrate that Pompe disease is characterized by early build-up of DYSF,
755 ANXA2, and AHNAK2, which are involved in membrane repair. Their respective
756 localization in the T-tubule, lysosomes, and autophagosomes suggests the establishment
757 of an adaptive mechanism to preserve the integrity of intracellular structures. The
758 abnormal sarcoplasmic accumulation of ANXA2 and AHNAK2 seems to correlate with
759 the severity of tissue damage in LOPD patients. Proteins involved in membrane repair
760 processes may therefore constitute candidate pathophysiological markers to monitor
761 Pompe disease progression and evaluate the treatment impact. Finally, a global alteration
762 in the TGF-β/activin signaling pathway, as reflected by a continuous increase in mature
763 MSTN protein levels and modulation of SMAD 3 and 7 expressions, could contribute to
764 the defective SC activation observed in Pompe disease in response to accumulating
765 muscle damage.

766

767 **List of abbreviations**

768 ANXA2: Annexin A2

769 BP: Biological process

770 CC: Cellular component

771 DHPR: Dihydropyridine receptor

772 DMD: Duchenne muscular dystrophy
773 DEG: Differentially expressed gene

774 devMHC: Developmental isoform of myosin heavy chain

775 DYSF: Dysferlin

776 ERT: Enzyme replacement therapy

777 ESCRT: Endosomal sorting complexes required for transport

778 GAA: Acid alpha glucosidase

779 GAPDH: Glyceraldehyde 3-phosphate dehydrogenase

780 GO: Gene ontology

781 HES: Hematoxylin-eosin-saffron

782 HRP: Horseradish peroxidase

783 IOPD: Infantile-onset Pompe disease

784 LAMP1: Lysosome-associated membrane protein-1

785 LGALS3: Galectin-3

786 LGMD2B: Limb-girdle muscular dystrophy type 2B

787 LOPD: Late-onset Pompe disease

788 M1: Type M1 macrophage

789 M2: Type M2 macrophage

790 METOH: methanol

791 MHC: Myosin heavy chain

792 mo: Month

793 MSTN: Myostatin

794 PAS: Periodic acid-Schiff

795 PBS: Phosphate buffered saline

796 PCA: Principal component analysis

797 PFA: Paraformaldehyde
798 RT: Room temperature

799 S100A10: S100 calcium binding protein A10

800 SC: Satellite cell

801 SR: Sarcoplasmic reticulum

802 TA: Tibialis anterior

803 TGF-β: Transforming growth factor beta

804 WGA: Wheat germ agglutinin

805 WT: Wild type

806

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1028 61. Yim WWY, Yamamoto H, Mizushima N (2022) Annexins A1 and A2 are
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1034

1035 **Declarations**

1036 **Ethics approval and consent to participate**

1037 All experiments were carried out according to European guidelines for the care and use

1038 of experimental animals. The study was reviewed and approved by the Regional Ethics

1039 Committee (CEEA Pays de la Loire, permit number APAFIS #1267).

1040

1041 **Consent for publication**

1042 Not applicable.

1043

1044 **Availability of data and materials**

1045 Microarray data have been deposited on the NCBI GEO portal under the accession

1046 number GSE293648. All data are publicly available.

1047

1048 **Competing interests**

1049 The authors declare that they have no competing interests.

1050

1051 **Funding**

1052 This work was supported by the associations Vaincre les Maladies Lysosomales (VML)

1053 #2020-3 and the Association Française contre les Myopathies (AFM) #23775, and the

1054 "Investissement d'Avenir-ANR-11-INBS-0011" - "NeurATRIS: A Translational

1055 Research Infrastructure for Biotherapies in Neurosciences".

1056

1057

1058
1059 **Authors' contributions**

1060 CB, SJ, FV, AML, MAC and KR wrote the paper and prepared the figures. CB and BE
1061 conducted the transcriptomic experiments. CB, SJ and AL analyzed the transcriptomics
data. JD and LL were responsible for animal sacrifice and tissue sampling. CB, CS, LV,
1062 JA and FV performed the Western Blot experiments. CB, SJ, TB, LD and IL performed
1063 the immunohistochemistry experiments and related quantitative analyses. CT supervised
1064 the statistical analyses. MAC and KR designed, supervised and coordinated the study. All
1065 authors read and approved the final manuscript.
1066

1067

1068 **Appendix A**

1069 **Members of the LYSAUMI Consortium:** Françoise Bouhour (Lyon, France), Marie
1070 Csanyi (Lille, France), Jean-Baptiste Davion (Lille, France), Teresinha Evangelista
1071 (Paris, France), Pascal Laforêt (Garches, France), Armelle Magot (Nantes, France),
1072 Pascale Marcorelles (Brest, France), Claude-Alain Maurage (Lille, France), Yann Péréon
1073 (Nantes, France), Nathalie Streichenberger (Lyon, France), Céline Tard (Lille, France).

1074

1075 **Acknowledgements**

1076 We thank the technical staff of Oniris rodent facility (Nantes, France) for animal care
1077 services and the Brest Biological Resources Center BB-0033-00037 ("CRB Santé du
1078 CHU de Brest") for providing high-quality annotated samples. We are also grateful to all
1079 members of the LYSAUMI Consortium for their support and work in the inclusion and
1080 follow-up of LOPD patients.

1081

1082

1083
1084 **Figure legends**

1085 **Fig. 1:** Molecular Profile of the tibialis anterior muscle of *Gaa*-/- mice. (a) Principal component analysis (PCA) plot illustrating global differences in baseline transcriptomes.

1086 Dimension (Dim) and percent variance indicate the amount of variability between
1087 subjects explained by each component. (b) Biplot showing the top 25 genes that most
1088 contribute to the variability of the data, highlighting specific gene expression patterns
1089 correlated to genotype. (c) Heatmap of the 414 differentially expressed genes (DEGs)
1090 identified between WT and *Gaa*-/- mice across all ages. (d) Bubble plot depicting enriched
gene ontology (GO) terms for cellular component (CC) and biological process (BP) that
differ in *Gaa*-/- vs WT mice. (e) Heatmap of the 58 DEGs for which changes in expression
were observed during the disease course, showing a relatively stable molecular signature
in Pompe disease mice muscle. Red, blue, and white indicate upregulation, down-
regulation, and no significant change, respectively. (f) Bubble plot depicting GO terms
for BP that are altered during the disease course, potentially reflecting progressive
involvement of specific biological processes.

1099 **Fig. 2:** Dysferlin content and localization in the tibialis anterior muscle of *Gaa*-/- mice.

1100 (a) Boxplot depicting dysferlin (*Dysf*) transcript expression as determined by microarray
1101 analysis, showing significant upregulation in *Gaa*-/- versus WT mice. Adjusted p-values
1102 correspond to the mean differential expression between the two groups as established by
1103 multiple testing. **** p < 0.0001. (b) Western blot analysis of dysferlin (DYSF) protein
1104 levels at 1.5-mo and 9-mo of age, confirming increased expression in *Gaa*-/- versus WT
1105 mice. First and third quartile range (top and bottom of the box), median (central band)
1106 and individual data points are visualized. AU = Arbitrary Unit. Statistics: Wilcoxon test;
1107 * p < 0.05. (c) Representative cross-sections of tibialis anterior (TA) muscles from 1.5-
1108 and 9-mo-old WT and *Gaa*-/- mice immunolabeled with anti-DYSF antibody (green),
1109 showing abnormal sarcoplasmic accumulation of DYSF in Gaa⁻/⁻ mice. Scale bars = 25 µm. (d) Colocalization of DYSF with T-tubules in Gaa⁻/⁻ mice. Longitudinal sections of TA muscle from 1.5-mo-old WT and Gaa⁻/⁻ mice immunolabeled with anti-DYSF antibody (red) and anti-DHPR antibody (green), demonstrating the association of DYSF with T-tubules in Gaa⁻/⁻ mice. Scale bars = 10 µm.

1114 **Fig. 3:** Fiber-specific dysferlin expression in Gaa⁻/⁻ mice and Pompe disease patients. (a) Serial cross-sections of tibialis anterior (TA) muscle from 1.5- and 9-mo-old WT and Gaa⁻/⁻ mice immunolabeled with anti-dysferlin (DYSF) antibody (green) and stained with periodic acid-Schiff (PAS), showing no correlation between DYSF sarcoplasmic accumulation and glycogen overload. Scale bars = 20 µm. (b) Serial TA muscle cross-sections from 1.5-mo and 9-mo Gaa⁻/⁻ mice submitted to antibodies against DYSF (left panel), LC3 (green) / LAMP1 (red, middle panel), and type I (purple) / type IIa (red) / type IIb (green) myosin heavy chain (MHC, right panel). Wheat germ agglutinin (WGA, blue) allows visualization of the muscle fiber outlines and shows that DYSF⁺ myofibers mainly correspond to fibers containing a build-up of autophagic debris and to fast-twitch enriched-type IIb fibers. Scale bars = 50 µm. (c) Characterization of DYSF⁺ muscle fibers. Bar graph illustrates the fiber type composition of DYSF⁺ muscle fibers at 1.5 and 9 months of age. For fiber typing, myosin heavy chain (MHC) expression was used; fibers referred to as type IIb should therefore be considered IIb-enriched rather than purely type IIb, as type IIX MHC fibers were not detected. Values represent mean percentages ± SD (left panel) or relative distributions (right panel). Four individuals and 368 DYSF⁺ fibers were considered per group. (d) Immunolabeling for DYSF (green, upper panel) and developmental isoform of MHC (devMHC, red, lower panel) in serial cross-sections of deltoid muscle biopsies from late-onset Pompe disease (LOPD) patients. WGA (blue) allows visualization of the muscle fiber outlines, and shows that myofibers with
1134 sarcoplasmic overexpression of DYSF do not correspond to regenerating fibers. Scale
1135 bars = 50 µm.

1136 **Fig. 4:** Annexin A2 content and localization in the tibialis anterior muscle of *Gaa*-/- mice.
1137 (a) Boxplot depicting annexin A2 (*AnxA2*) transcript expression as determined by
1138 microarray analysis, showing significant upregulation in *Gaa*-/- versus WT mice. P-values
1139 correspond to the mean differential expression between the two groups as established by
1140 multiple testing: **** p < 0.0001. (b) Western blot analysis of annexin A2 (*ANXA2*)
1141 protein levels at 1.5-mo and 9-mo of age, confirming increased expression in *Gaa*-/- mice.
1142 Interquartile range (top and bottom of the box), median (central band) and individual data
1143 points are visualized. AU = Arbitrary Unit. Statistics: Wilcoxon test; * p < 0.05. (c)
1144 Representative cross-sections of tibialis anterior muscle from 1.5-mo-old WT and *Gaa*-/-
1145 mice immunolabeled with anti-*ANXA2* (green) and *LAMP1* (red) antibodies, showing
1146 colocalization of *ANXA2* with lysosomes in *Gaa*-/- mice. Scale bars = 10 µm. (d) Violin
1147 plot showing the distribution of Pearson's correlation coefficients between *ANXA2* and
1148 *LAMP1* localization calculated for individual muscle fibers and pooled across all
1149 individuals. Each dot represents a single muscle fiber, and the violin shape reflects the
1150 density distribution of correlation values. (e) Annexin A2 expression profile in Pompe
1151 disease patients. Immunolabeling of annexin A2 (*ANXA2*, green) and *LAMP1* (red) in
1152 serial cross-sections from deltoid muscle biopsies of late-onset Pompe disease (LOPD)
1153 patients. Wheat germ agglutinin (WGA, blue) allows visualization of the muscle fiber
1154 outlines, showing distinct expression patterns depending on disease severity. Scale bars
1155 = 50 µm. (f) Immunolabeling of *LAMP1* (red) and *LC3* (green) in serial muscle cross-
1156 sections from a severely affected LOPD patient, demonstrating exacerbated autophagic
1157 build-up. Scale bars = 50 µm.
**Fig. 5:** AHNAK2 expression profile in the tibialis anterior muscle of *Gaa*-/- mice. (a) Boxplot depicting *Ahnak2* transcript expression as determined by microarray analysis, showing significant upregulation in *Gaa*-/- versus WT mice. P-values correspond to the mean differential expression between the two groups as established by multiple testing: **** p < 0.0001. (b) Western blot analysis of AHNAK2 protein levels at 1.5-mo and 9-mo of age, confirming increased expression in *Gaa*-/- mice vs WT mice. Interquartile range (top and bottom of the box), median (central band) and individual data points are visualized. AU = Arbitrary Unit. Statistics: Wilcoxon test; * p < 0.05. (c) Immunolabeling of AHNAK2 (red) and LC3 (green) in tibialis anterior muscle cross-sections from 9-mo-old WT and *Gaa*-/- mice, showing localization of AHNAK2 in autophagosomes in *Gaa*-/- mice. Scale bars = 20 µm. (d) AHNAK2 expression profile in Pompe disease (LOPD) patients. Immunolabeling of AHNAK2 (red) and LC3 (green) in cross-sections from deltoid muscle of late-onset Pompe disease patients. Wheat germ agglutinin (WGA, blue) allows visualization of the muscle fiber outlines, showing distinct expression patterns depending on disease severity. Scale bars = 100 µm.

**Fig. 6:** Abnormal expression profile of Myostatin and profound disruption of SMAD 7 and 3 protein expression levels from the transforming growth factor beta (TGF-β)/activin signaling pathway in *Gaa*-/- mice. (a) Boxplot depicting myostatin (*Mstn*) transcript expression as determined by microarray analysis, showing significant down-regulation in *Gaa*-/- versus WT mice. P-values correspond to the mean differential expression between two groups as established by multiple testing; * p < 0.05, ** p < 0.01, **** p < 0.0001. (b) Western blot analysis of pro-myostatin (PRO-MSTN) and mature myostatin (MSTN) protein levels in muscle lysates from WT and *Gaa*-/- mice. First and third quartile range (top and bottom of the box), median (central band) and individual data points are visualized. AU = Arbitrary Unit. Statistics: Wilcoxon test; * p < 0.05. (c) Western blot
1183 analysis of SMAD7, SMAD2/3 and phosphorylated-SMAD3 (P-SMAD3) in muscle
1184 lysates from WT and Gaa<sup>-/-</sup> mice. First and third quartile range (top and bottom of the
1185 box), median (central band) and individual data points are visualized. AU = Arbitrary
1186 Unit. Statistics: Wilcoxon test; * p < 0.05.

1187 **Fig. 7:** Macrophage expression profile in the tibialis anterior muscle of Gaa<sup>-/-</sup> mice. (a)
1188 Representative cross-sections of tibialis anterior (TA) muscles stained with hematoxylin-
1189 eosin-saffron (HES), showing increased cell density in the connective tissue surrounding
1190 muscle fibers in Gaa<sup>-/-</sup> mice. Scale bars = 30 µm. (b) Immunolabeling of TA muscle cross-
1191 sections with anti-CD68 (red) and anti-MRC1 (green) antibodies, showing the presence
1192 of macrophages in the muscle tissue. Dystrophin antibody (DYS2; grey) allows
1193 visualization of the muscle fiber outlines. Nuclei are counterstained with DAPI (blue).
1194 Scale bars = 25 µm. (c) Quantification of CD68<sup>+</sup> cells/ mm<sup>2</sup>, showing increased
1195 macrophage infiltration in Gaa<sup>-/-</sup> mice. (d) Quantification of CD68<sup>+</sup>MRC1<sup>-</sup> cells (type M1
1196 macrophages) and CD68<sup>+</sup>MRC1<sup>+</sup> cells (type M2 macrophages), indicating a
1197 predominance of pro-inflammatory M1 macrophages in Gaa<sup>-/-</sup> mice. Statistics: Two-way
1198 ANOVA and Tukey's post-hoc test for CD68<sup>+</sup> cells/mm<sup>2</sup> and CD68<sup>+</sup>MRC1<sup>-</sup> ;
1199 *** p < 0.001.

1200

1201 **Additional Files**

1202 Additional file 1: (ppt) Hematoxylin and eosin (HE) and periodic acid-Schiff (PAS)
1203 staining of muscle from late-onset Pompe disease (LOPD) patients. Scale bars = 50 µm.

1204 Additional file 2: (ppt) Uncropped scans of western blots from main figures. Uncropped
1205 full scans of western blots from the corresponding cropped western blots shown within
1206 the main text. Molecular weight (MW) markers are indicated. Figure subpanel is indicated
1207 for each respective blot.
1208 Additional file 3: (xls) Expression_matrix: Gaa<sup>-/-</sup> and WT mice. Panel of 414
differentially expressed genes (DEGs) between groups.
1209

1210 Additional file 4: (xls) GO_analysis_ Gaa<sup>-/-</sup> vs WT_BP processes.

1211 Additional file 5: (xls) GO_analysis_ Gaa<sup>-/</sup> vs_WT_CC processes.

1212 Additional file 6 : (xls) Expression_matrix: Gaa<sup>-/</sup> mice. Panel of 58 differentially
1213 expressed genes (DEGs) evolving over the course of the disease in Gaa<sup>-/</sup> mice muscle.

1214 Additional file 7: (xls) GO_analysis_ Gaa<sup>-/</sup> vs Gaa<sup>-/</sup> BP processes.

1215 Additional file 8: (ppt) Western blot analysis of galectin 3 (LGALS3) protein levels at
1216 1.5-mo and 9-mo of age, confirming lysosomal membrane damage in Gaa<sup>-/-</sup> mice.

1217 Interquartile range (top and bottom of the box), median (central band) and individual data
1218 points are visualized. Statistics: Wilcoxon test; * p < 0.05

1219 Additional file 9: (ppt) Altered expression of atrophy-related genes in the tibialis anterior
1220 muscle of Gaa<sup>-/-</sup> mice. Boxplots depicting Trim63 and Eif4ebp1 transcript expressions as
determined by microarray analysis, showing significant upregulation in Gaa<sup>-/-</sup> versus WT
1221 mice. Adjusted p-values correspond to the mean differential expression between the two
1222 groups as established by multiple testing. **** p < 0.0001.
1223

1224 Additional file 10: (ppt) Western blot analysis of SMAD2/3 and phosphorylated-SMAD3
1225 (P-SMAD3) in muscle lysates from WT and Gaa<sup>-/-</sup> mice. First and third quartile range
1226 (top and bottom of the box), median (central band) and individual data points are
1227 visualized. AU = Arbitrary Unit. Statistics: Wilcoxon test; * p < 0.05.
1228

1228
**Supplementary Files**

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* Additionalfile3.xls

* Additionalfile4.xls

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* Additionalfile6.xls

* Additionalfile7.xls