Galectin-3 and N-acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy

The muscle membrane, sarcolemma, must be firmly attached to the basal lamina. The failure of proper attachment results in muscle injury, which is the underlying cause of Duchenne muscular dystrophy (DMD), where mutations in the dystrophin gene disrupts the firm adhesion. In DMD patients, even moderate contraction causes damage, leading to progressive muscle degeneration. The damaged muscles are repaired through myogenesis. Consequently, myogenesis is highly active in DMD patients, and the repeated activation of myogenesis leads to the exhaustion of the myogenic stem cells. Therefore, approaches to reducing the risk of the exhaustion are to develop a treatment that strengthens the interaction between the sarcolemma and the basal lamina, and increases the efficiency of myogenesis. Galectin-3 is an oligosaccharide-binding protein and known to be involved in cell–cell interactions and cell–matrix interactions. Galectin-3 is expressed in myoblasts and skeletal muscle while its function in muscle remains elusive. In this study, we found evidence that galectin-3 and the monosaccharide N-acetylglucosamine, which increases the ligands (oligosaccharides) of galectin-3, promotes myogenesis in vitro. Moreover, in the mdx mouse model of DMD, treatment with N-acetylglucosamine increased the muscle force production. Our results demonstrate that treatment with N-acetylglucosamine can mitigate the burden of DMD

Injectable Immunomodulatory Strategies to Enhance Muscle Recovery Following Injury

Although skeletal muscle displays an astonishing regenerative capacity, injuries or diseases that resulted in bedridden or chronic muscle wasting can overwhelm this intrinsic feature of skeletal muscle and lead to functional deficit (range of motion and/or strength) and overall reduction in quality of life. Microenvironmental cues within injured skeletal muscle dictate regenerative and repair process which are tightly coordinated interplay among resident cells, cells recruitment and immune response following an assault in the muscle extracellular matrix (ECM). The successful regeneration of functional tissues requires both appropriate modulation of the inflammatory response, and activation of a variety of cell populations. Biomaterials offer unique opportunities to spatiotemporally control cytokine delivery and may provide significant benefits in the modulation of immune cells and muscle-immune micro-environment (MIME) to promote regeneration. The aim of this dissertation is to create injectable therapeutics that are capable of both modulating the inflammatory response and directly promoting muscle regeneration. In aim 1, we investigated the safety and therapeutic potential of human muscle derived decellularized ECM to promote muscle regeneration in mouse disuse and reloading hindlimb injury model and rabbit chronic rotator cuff tear muscle degenerative injury model. In aim 2, we investigated the therapeutic potential of delayed delivery of IL-10 to boost muscle regeneration when combined with progenitor cells repair (mined muscle grafts) in rat volumetric muscle loss injury model. Together, the findings of these two aims further our understanding of the role that pro-regenerative immune cells play in promoting endogenous mechanisms of tissue repair after muscle injury and present potential therapeutic avenues to modulate the MIME in aiding muscle repair and recovery

Injectable Immunomodulatory Strategies to Enhance Muscle Recovery Following Injury

Although skeletal muscle displays an astonishing regenerative capacity, injuries or diseases that resulted in bedridden or chronic muscle wasting can overwhelm this intrinsic feature of skeletal muscle and lead to functional deficit (range of motion and/or strength) and overall reduction in quality of life. Microenvironmental cues within injured skeletal muscle dictate regenerative and repair process which are tightly coordinated interplay among resident cells, cells recruitment and immune response following an assault in the muscle extracellular matrix (ECM). The successful regeneration of functional tissues requires both appropriate modulation of the inflammatory response, and activation of a variety of cell populations. Biomaterials offer unique opportunities to spatiotemporally control cytokine delivery and may provide significant benefits in the modulation of immune cells and muscle-immune micro-environment (MIME) to promote regeneration. The aim of this dissertation is to create injectable therapeutics that are capable of both modulating the inflammatory response and directly promoting muscle regeneration. In aim 1, we investigated the safety and therapeutic potential of human muscle derived decellularized ECM to promote muscle regeneration in mouse disuse and reloading hindlimb injury model and rabbit chronic rotator cuff tear muscle degenerative injury model. In aim 2, we investigated the therapeutic potential of delayed delivery of IL-10 to boost muscle regeneration when combined with progenitor cells repair (mined muscle grafts) in rat volumetric muscle loss injury model. Together, the findings of these two aims further our understanding of the role that pro-regenerative immune cells play in promoting endogenous mechanisms of tissue repair after muscle injury and present potential therapeutic avenues to modulate the MIME in aiding muscle repair and recovery

Myonuclear dynamics in muscle plasticity and the transcriptional regulation of resistance training induced hypertrophy.

Skeletal muscle is highly responsive to changes in mechanical load or activity and can adjust its morphological, metabolic, and contractile properties accordingly. The remodeling of these characteristics is controlled by the reprogramming of the transcriptional output of the myonuclei along the length of the muscle fiber. To meet the transcriptional demands of growth and increased activity, myonuclei can be added to the existing cytoplasm through the fusion of satellite cells, to support synthetic activity. This project utilises improved methodologies including automated, high-throughput immunohistochemical analysis and bulk RNA-sequencing of skeletal muscle. With these techniques, we define the temporal patterns of myonuclear dynamics and how they correspond to fiber-type specific adaptations in response to loading, unloading, reloading and changes in activity and how the acute transcriptional response is altered, dependent on the training status of the muscle. To induce these modalities of activity or inactivity, we utilised in-vivo models from our lab including, (1) programmed exercise delivered through miniature implanted pulse generators (IPGs) to induce muscle hypertrophy or metabolic adaptation and (2) disuse by means of tetrodotoxin-induced nerve silencing to induce muscle atrophy. We report that the genes that most closely track with changes in muscle mass are controlled centrally by the basic-helix-loop-helix transcription factor Myc, that functions to bind to E-box containing DNA sequences. In addition, we identify 10 other genes that appear as important regulators across species and modalities of exercise that warrant further investigation. Lastly, we investigate a promising marker for specifically identifying myonuclei, pericentriolar material-1 (PCM1), which would allow for deconvolution of mRNA signals from bulk skeletal muscle mRNA analysis, allowing for identification of myogenic and non-myogenic transcriptional changes. In summary, our aim is to provide key mechanistic insights into myonuclear dynamics and how adaptation of skeletal muscle is regulated at the transcriptional level

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin