In vivo imaging of skeletal muscle in mice highlights muscle defects in a model of myotubular myopathy

Abstract

Skeletal muscle structure and function are altered in different myopathies. However, the understanding of the molecular and cellular mechanisms mainly rely on in vitro and ex vivo investigations in mammalian models. In order to monitor in vivo the intracellular structure of the neuromuscular system in its environment under normal and pathological conditions, we set-up and validated non-invasive imaging of ear and leg muscles in mice. This original approach allows simultaneous imaging of different cellular and intracellular structures such as neuromuscular junctions and sarcomeres, reconstruction of the 3D architecture of the neuromuscular system, and video recording of dynamic events such as spontaneous muscle fiber contraction. Second harmonic generation was combined with vital dyes and fluorescentcoupled molecules. Skin pigmentation, although limiting, did not prevent intravital imaging. Using this versatile toolbox on the Mtm1 knockout mouse, a model for myotubular myopathy which is a severe congenital myopathy in human, we identified several hallmarks of the disease such as defects in fiber size and neuromuscular junction shape. Intravital imaging of the neuromuscular system paves the way for the follow-up of disease progression or/and disease amelioration upon therapeutic tests. It has also the potential to reduce the number of animals needed to reach scientific conclusions.

Multi-omics comparisons of different forms of centronuclear myopathies and the effects of several therapeutic strategies

“…Dysregulation of NMJ components (CHRNA1, CHRND, CHRNG) were recently reported in the MTM1 dog, and alteration of the NMJ function was suggested in a mtm1 knockdown zebrafish and in Mtm1 mouse models. 46,50,51 We found a high increase in Sln expression in the three CNM mouse models, and upregulation of Sln was previously reported following microarray analysis of the Mtm1 À/y mouse. 49 In addition, longitudi-nal analyses of the muscle transcriptome and proteome of Mtm1 À/y mice at pre-symptomatic (E18.5), early (2 weeks), and late (7 weeks) disease stages highlighted the same dysregulated pathways, although the same dysregulated genes/proteins were not necessarily found (Figures 3 and 4).…”

“…Finally, CHRNA1 (MIM: 601462) and CHRND (MIM: 616322) are mutated in congenital myasthenic syndromes, correlating with the defect in NMJ found in MTM1-CNM models. 50,51,62 Common therapeutic targets for CNMs…”

Transcription Factor Movement and Exercise-Induced Mitochondrial Biogenesis in Human Skeletal Muscle: Current Knowledge and Future Perspectives

“…To directly analyse the movement of proteins following contraction, confocal microscopy has been employed within various skeletal muscle systems [55][56][57]. To detect a specific protein, a fluorescent tag is inserted into the coding region of the gene leading to the expression of a protein containing a conjugated tag that is detectable via absorption and emission of specific wavelengths of light [58].…”

Dissection, in vivo imaging and analysis of the mouse epitrochleoanconeus muscle

“…Consequently, it is important to be able to study the intact neuromuscular system in its natural environment. Due to accessibility restrictions, intravital NMJ studies have been performed in only relatively few muscles, including the thin and flat sternomastoid muscle in the neck (Li et al, 2011 ; Lichtman et al, 1987 ; Turney & Lichtman, 2012 ) and at superficial synapses of hind (pelvic) limb muscles (Bruusgaard et al, 2003 ; Hill & Robbins, 1991 ; Martineau et al, 2018 ; Mercier et al, 2016 ; Yampolsky et al, 2010 ). Unfortunately, protocols outlining these in vivo imaging procedures are rare (Blanco & Ribchester, 2012 ; Turney et al, 2012 ).…”