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Author ORCID Identifier

https://orcid.org/0000-0002-8879-7864

Date Available

3-9-2026

Year of Publication

2026

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Medicine

Department/School/Program

Physiology

Faculty

John McCarthy

Faculty

Lance Johnson

Abstract

Muscle stem cells (MuSCs) are a unique stem cell population that can activate in response to both injury and growth stimuli. Studies on muscle regeneration have shown MuSC activation follows a uniform progression through proliferation, differentiation, and fusion. Therefore, to determine whether MuSC replication is a prerequisite for differentiation under hypertrophic stimulus, I used a lineage-tracking mouse model (Pax7rtTA; TRE-H2B-GFP) to label MuSC nuclei and simultaneously tracked DNA synthesis via EdU incorporation. This dual-labeling approach allowed us to not only distinguish MuSC-derived nuclei but also demarcate which MuSC had proliferated prior to fusion. I then employed a mechanical overload (MOV) model of synergist ablation on the hindlimb muscles; this model has been shown to stimulate robust activation and fusion of the MuSC population in the plantaris muscle.

Using immunohistochemistry, I quantified GFP+ MuSC-derived myonuclei within myofibers in concert with the EdU labeling. My analysis showed that a significant fraction of GFP+ MuSC-derived myonuclei were negative for EdU. This unexpected finding showed that, in response to hypertrophic stimulus, cell division is not obligatory for MuSC fusion during muscle growth. Further, trajectory analysis of our scRNA-seq data revealed a bifurcation in MuSC fate: one branch progressed to a proliferative state prior to fusion, whereas the other routed directly to differentiation and fusion without proliferation. Together, these findings provide strong evidence that the MuSC dynamics are different with hypertrophy compared to previous studies in the muscle damage and regeneration context, and that MuSC are capable of fusion into the muscle fibers independent of proliferation.

Next, I wanted to examine the fate of MuSC organelles other than the nucleus when fusion occurs. Previously, tracking the fusion of MuSC to myofibers during muscle hypertrophy has been exclusively done by assessing myonuclear abundance. These nuclear-centric methods have directed the focus of study on myonuclear accretion. As it stands in the field, the increase in transcriptional capacity is thought to be the primary functional benefit of MuSC fusion. However, little is known about the fate of other organelles such as mitochondria, ribosomes, and lysosomes during myofiber fusion. While it is presumed that these organelles are also transferred and retained within muscle fibers post-fusion, there is no direct evidence to support this presumption.

To address this gap in knowledge, I focused on mitochondria because they are a highly abundant organelle in activated MuSCs. Alongside, there is a well-established mitochondrial marker, mito-Dendra2, which will allow us to track the fate of MuSC-derived mitochondria following myofiber fusion.  I crossed a MuSC-specific driver mouse to the fluorescent reporter mouse to generate a MuSC-specific mitochondrial labeled mouse. I then subjected the mice to the same (MOV) model to stimulate MuSC fusion. Plantaris muscles were collected after 3-, 7-, and 14- days of MOV. This experimental design allowed us to evaluate whether mitochondrial transfer precedes fusion and to assess MuSC temporal dynamics. I observed an increase in Dendra2⁺ myofibers across the MOV time course. Super-resolution imaging captured the simultaneous transfer of mitochondria and nuclei during MuSC fusion, visualizing direct evidence of mitochondrial transfer into muscle fibers in response to a hypertrophic stimulus. EdU incorporation, to track MuSC fusion, showed early MuSC fusion was primarily independent of proliferation and preferentially occurred with oxidative Type 2A fibers.

Thus, with this study, I provide for the first time definitive evidence that under a hypertrophic stimulus, mitochondria are transferred into muscle fibers through MuSC fusion. Furthermore, the sensitivity of our model allowed us to characterize the dynamics of early MuSC fusion; I show that MuSCs are fusing earlier than 3 days and that this fusion is largely preferential to 2A fibers in the plantaris.

In my final study, I investigated the transcriptomic impact of MuSC fusion on muscle in the context of two key variables: adaptation to a hypertrophic stimulus and the effects of aging. Our lab previously showed that at least in the short term, adult muscle is able to grow at a similar level in the absence or presence of MuSCs; but on the opposite end, growth response of aged muscle is blunted irrespective of MuSC presence. Therefore, I wanted to perform sequencing on muscle under these conditions. Because the size of the myofiber (muscle cell) prohibits us from using single cell isolation, I instead opted to focus on the myonucleus for sequencing. To achieve this, I used a mouse model in which MuSCs could be selectively depleted and allow us to simultaneously label myonuclei. I also utilized the same synergist ablation model to induce a hypertrophic stimulus via MOV on the adult and aged mice of this mouse model.

This allowed me to compare the transcriptome of murine muscle in the presence or absence of MuSCs, in resting or (MOV) models, and both in adult and aged conditions; thereby isolating the transcriptional consequences of MuSC-derived fusion across age and MOV adaptation. After integrating all datasets, my comparisons revealed four significant findings: 1) Adult muscle can adapt to mechanical overload in the absence of MuSCs,2) MOV adaptation was blunted in aged muscle, and the blunting is amplified with MuSC depletion,3) Aging muscle has many additional myonuclear clusters that exhibit non-canonical expression of genes, 4) MOV rejuvenates aged muscle transcriptome, but only in the presence of MuSCs.

Overall, I showed that MuSCs play a much bigger role in regulating adaptation in aged muscle, while adult muscle can compensate without MuSCs. Based on the additional myonuclear clusters in aged samples, our data suggests that with aging, transcriptional dysregulation occurs. Finally, while previous studies have shown the benefits of exercise in attenuating aging effects, this study provides the first detailed characterization of how MOV remodels aged muscle transcriptome and highlight the MuSC contribution to this response.

Overall, my work has extended our understanding of MuSC fusion by demonstrating that MuSCs can contribute to myofiber growth through a proliferation-independent route. Beyond nuclear accretion, my findings establish for the first time that MuSCs also donate mitochondria during fusion and a preference for early MuSC fusion with oxidative Type 2A fibers. I show that adult muscle retains the ability to compensate for MuSC loss during hypertrophy and that this adaptive capacity is diminished with aging due to a loss in myonuclear transcriptional programs. Although the single nuclear RNA sequencing project is descriptive and based solely on bioinformatic data at a transcriptional level, the results provide evidence that MOV-specific rejuvenation of muscle occurs through the reprogramming of aged myonuclei. This suggests that resistance exercise in the presence of MuSCs might restore aged muscle to a more youthful profile.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2026.29

Archival?

Archival

Funding Information

J.G was supported by the National Institutes of Health grants from the National Institute of Aging (RO1 AG069909 to J.J.M.)

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