Optoacoustic molecular signatures of muscle involvement in facioscapulohumeral dystrophy compared to conventional imaging

Background and Objectives: Facioscapulohumeral muscular dystrophy (FSHD) is characterised by asynchronous muscle degeneration and marked pathological heterogeneity. While conventional MRI can detect fatty replacement and oedema-like abnormalities, it provides limited insight into other relevant biological processes such as extracellular matrix remodelling and tissue perfusion. This study investigated whether multispectral optoacoustic tomography (MSOT), an emerging imaging technique that non-invasively interrogates tissue composition through endogenous optical absorbers, can provide complementary molecular information on muscle involvement in FSHD. Methods: MSOT was performed on 568 muscles from 15 genetically confirmed FSHD patients and 9 healthy volunteers using a standardized protocol. The analysis included single-wavelength signal intensity and spectrally unmixed signals related to lipid, collagen, deoxygenated haemoglobin, oxygenated haemoglobin, total haemoglobin, and tissue oxygen saturation. Patients also underwent conventional muscle MRI together with clinical strength and disease severity assessments. Feasibility, reproducibility, discrimination between patients and controls, and associations with MRI and clinical measures were evaluated. Results: MSOT acquisition was well tolerated and highly reproducible (intra- and inter-rater ICC = 0.88). FSHD muscles displayed a distinct optoacoustic profile characterised by reduced signal intensity at wavelengths below 900 nm, increased lipid signal and reduced oxygenated and total haemoglobin signals. Lipid signal discriminated FSHD from control muscles (AUC 0.830, p = 0.008) and increased with MRI-defined fatty replacement (p < 0.0001). Short tau inversion recovery (STIR)-positive muscles on MRI exhibited a distinct molecular phenotype, with higher collagen-associated and lipid signals together with increased deoxygenated and total haemoglobin signals, consistent with active tissue remodelling. Although FSHD muscles classified as normal by conventional imaging showed group-level optoacoustic profiles comparable to controls, 13 of 141 (9%) demonstrated at least one abnormal MSOT-derived parameter. Reduced haemoglobin-related signals correlated with greater global disease severity, whereas increased lipid signal was associated with lower muscle strength. Discussion: MSOT is a feasible and reproducible imaging technique in FSHD that provides complementary molecular information beyond conventional structural imaging. Its ability to detect signatures related to fatty replacement, STIR positivity, functional impairment, and isolated abnormalities in radiologically normal muscles supports further longitudinal studies of MSOT-derived readouts as potential biomarkers of disease activity, progression, and therapeutic response.