86P Muscle 3D models to investigate LGMDD2 transportin 3 related deficiency: insights into myogenic processes and contractile dysfunction

• Published in: Neuromuscular disorders : NMD 2024-10, Vol.43, p.104441, Article 104441.260
• Main Authors: Pacilio, S., Costa, R., Rodia, M., Lombardi, S., Di, L., Banos, G., Didier, N., Malfatti, E., Focarete, M., Cenacchi, G.
• Format: Article
• Language: English
• ISSN: 0960-8966
• DOI: 10.1016/j.nmd.2024.07.269

Limb Girdle Muscular Dystrophy D2, transportin 3 (TNPO3) related deficiency, is a very rare autosomal dominant myopathy, characterized by muscle fiber degeneration and progressive weakness. We cultured immortalized human myoblasts from LGMDD2 patients in two distinct 3D scaffolds mimicking muscle tissue components: a 3D collagen bio-printed hydrogel, faithfully recapitulating the native SKM tissue, proved valuable for investigating myogenic processes and understanding the role of the TNPO3 protein in disease development; a 3D micropillar system, resembling the SKM structure, facilitated real-time monitoring of cell functionality, particularly through the study of cell contractility. We found that TNPO3 is dislocalized, present in both the nucleus and cytoplasm, moreover we observed a reduction in expression at both gene and protein levels in LGMDD2. TNPO3 dysfunction altered SRSF1 (Serine and Arginine Rich Splicing Factor 1) activity, affecting muscle myogenesis and muscle development. Early myogenin expression and late myosin 2, 3, and 8 increased in LGMDD2 compared to controls, indicating diminished fiber maturation. Reduced fiber diameter, sparse a-actinin striation and branched fibers in LGMDD2, alongside elevated Murf-1 and p62, indicative of atrophy and autophagy respectively, mirrored muscle biopsy findings. Electrical stimulation (5V, 0.5 Hz for twitch contraction; 5V, 10 Hz for tetanic contraction) evaluated cell contractility, with live imaging tracking 3D micropillar deflections. LGMDD2 exhibited greater contractile force but with irregularities in frequency, amplitude, and declining force over time, suggested abnormal functionality due to altered myogenesis. Real time calcium analysis during electrostimulation revealed a peak at contraction onset in LGMDD2, confirming altered calcium flux. As a whole, our research highlights the role of TNPO3 dysfunction in LGMDD2 pathogenesis, shedding light on altered myogenesis, muscle maturation, and contractile irregularities, providing key insights for potential therapeutic strategies. Our 3D in vitro models are an innovative approach for modelling muscular dystrophies, offering a viable alternative to conventional 2D cell cultures and animal models that could serve as a versatile tools for preclinical investigations across a spectrum of pathologies, presenting an ethical and cost-effective alternative to traditional models and minimizing reliance on animal testing. Further analysis and electron microscop