A Novel Strategy to Restore Meaningful Function to Permanently Denervated Skeletal Muscles

Abstract

Skeletal muscles rely on neuromuscular junctions (NMJs) and motoneuron control for function. Permanent denervation, caused by spinal cord injuries (SCIs), peripheral nerve injuries (PNIs), or motoneuron diseases like Amyotrophic Lateral Sclerosis (ALS), leads to muscle paralysis and atrophy, with the inability to regenerate or reinnervate. Despite advances in neuroprosthetics, restoring movement in fully denervated muscles remains a challenge due to the lack of neural connections. This thesis explores new techniques to regenerate and restore function in permanently denervated skeletal muscles. First, a protocol was developed to differentiate mouse embryonic stem (ES) cells into functional skeletal muscle cells in vitro. These myofibres expressed muscle markers, and developmental analysis revealed the progression of cells from a quiescent to a differentiated state. The cells were engineered to express external genes, including tdTomato or channelrhodopsin-2 (ChR2), facilitating their identification or optical control post-transplantation in vivo. Additionally, ChR2 gene expression also was achieved using adeno-associated virus (AAV) vectors, allowing ChR2-expressing myofibres to be used for optical control of muscles. In the second study, irradiated soleus muscles followed by muscle fibres degradation using notexin, a myotoxic agent, were transplanted with ES cell-derived myoblasts expressing ChR2 or tdTomato. The transplanted myoblasts successfully engrafted and regenerated the muscles. tdTomato-expressing muscles showed increased mass but smaller fibres, and ChR2-expressing muscles responded to light stimulation, producing contractile forces comparable to neural stimulation, demonstrating the effectiveness of optogenetics in restoring function in denervated muscles. The next study focused on ex vivo anatomical and physiological analysis of transplanted muscles. Regenerated tdTomato-expressing muscles were innervated by endogenous motor axons, and showed normal responses to nerve stimulation, with largely normal synaptic transmission. The innervation rate between endogenous motor axons and exogenous muscle fibres was more 80% with newly formed NMJs at their original sites. Interestingly, the regenerated muscles contained only fast-twitch fibres, unlike the typical slow-twitch composition of the soleus muscle. Lastly, in search of alternative methods to introduce external genes into muscle cells, I explored lipid nanoparticles (LNPs) as non-viral vectors for gene delivery into ES cell-derived muscle cells. LNPs efficiently transfected muscle cells with minimal cytotoxicity and high specificity for muscle cells, with apolipoprotein E (ApoE) enhancing transfection efficiency. This work underscores the potential of LNPs in muscle transfection and paves the way for future in vivo experiments.