CO:08:1 | Aquaporin-4 downregulation in dysphagic idiopathic inflammatory myopathies: association with Type 2 fiber damage and inflammation

Marco Fornaro¹, Margherita Giannini², Dario D'Abbicco³, Maria Luisa Fiorella⁴, Antonio Frigeri⁵, Francesco Girolamo⁶, Florenzo Iannone¹. | ¹Unit of Rheumatology, Department of Precision and Regenerative Medicine and Ionian Area, Bari, Italy; ²Physiologie et Explorations Fonctionnelles Musculaires, University Hospital of Strasbourg, Strasburgo, France; ³Institute of General Surgery G Marinaccio, Department of Precision and Regenerative Medicine and Ionian Area, Bari; ⁴Unit of Otolaryngology, Department of Translational Biomedicine and Neuroscience DiBraiN, Bari; ⁵Unit of Human Physiology, Department of Translational Biomedicine and Neuroscience DiBraiN, Bari; ⁶Unit of Human Anatomy and Histology, Department of Translational Biomedicine and Neuroscience DiBraiN, Bari, Italy

Background. To assess whether Aquaporin-4 (AQP4) expression in skeletal muscle is altered in idiopathic inflammatory myopathies (IIMs), particularly in dysphagic patients, and to explore its association with muscle fiber type, regeneration, inflammation, and tissue damage.

Methods. Muscle biopsies from 54 IIM patients (25 dysphagic: 11 DM, 7 PM, 2 IBM, 2 IMNM, 3 OM; 29 normophagic: 9 DM, 4 ASyS, 4 PM, 4 IBM, 8 IMNM) and 6 non-inflammatory controls were analyzed using immunofluorescence and confocal microscopy. AQP4 immunolocalization was assessed alongside markers of fiber type (MHCfast), regeneration (neonatal myosin), inflammation (CD68), and matrix remodeling (MMP2). Morphometric parameters (fiber diameter, area, atrophy factor, variability coefficient) were quantified. Clinical correlations were examined using MMT-8 and the Myositis Damage Index (MDI).

Results. A significant reduction in AQP4-positive myofibers was observed in dysphagic IIM patients compared to normophagic patients and controls, predominantly affecting type 2 fibers. Dysphagic muscles showed marked hypotrophy, with reduced fiber diameter and area, and increased variability and atrophy indices. These changes were mainly driven by AQP4-negative type 2 fibers (Figure 1A-B). Dysphagic patients showed a significantly higher percentage of AQP4-negative fibers co-expressing neonatal myosin (p<0.001 vs normophagic IIMs and healthy controls), indicating impaired regeneration. AQP4-negative fibers were more frequently surrounded by CD68-positive macrophages and MMP2-positive vessels (p<0.001 vs healthy controls). The proximity of inflammatory and vascular cells to AQP4-negative sarcolemmas suggests inflammation-induced AQP4 loss. Anti-HMGCR-positive IMNM cases (red circles and triangles in Figure 1A) exhibited the most severe AQP4 reduction and fiber damage. In the overall IIM cohort, the percentage of AQP4-positive type 1 myofibers correlated positively with MMT-8 scores (r:0.383, p<0.01), while AQP4-negative type 1 and type 2 fibers correlated inversely (r:-0.389, p<0.01; r:-0.288, p<0.05, respectively). No significant correlation emerged with CK levels. MDI was negatively correlated with AQP4-positive type 1 (r:-0.433, p<0.01) and type 2 (r:–0.265, p=0.05) fibers, and positively correlated with AQP4-negative type 1 (r:0.442, p<0.01) and type 2 (r:0.265, p=0.05) fibers.

Conclusions. AQP4 loss in type 2 myofibers is a distinctive feature of dysphagic IIM patients and reflects myofiber damage, inflammation, and regenerative failure. AQP4 downregulation may represent a novel histopathological marker of disease severity and tissue vulnerability in IIMs.