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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session A

Poster I5 - Determining the mechanism of myonuclear loss in skeletal muscle fibers with Lmna mutations

Thursday, October 24, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Jun Ni

Undergraduate Research Assistant
Cornell University
Ithaca, New York, United States

Co-Author(s)

HZ

Hind Zahr

Postdoctoral Research
Cornell University, United States

Primary Investigator(s)

Jan Lammerding, PhD (he/him/his)

Professor
Cornell University
Ithaca, New York, United States

Introduction: Lamins A and C are nuclear envelope proteins, encoded by the LMNA gene, that play an important role in maintaining nuclear structural stability. Mutations in the LMNA gene cause many human diseases, such as dilated cardiomyopathy, that mainly affect striated muscle tissues. The molecular mechanisms for the muscle-specific defects caused by LMNA mutations remain unclear. Previous findings from our lab showed that lamin loss in skeletal muscle fibers leads to structurally impaired myonuclei, characterized by nuclear envelope rupture and DNA damage (Earle et al. Nature Materials 2020). Nuclear damage also correlated with myonuclear loss in single muscle fibers from Lmna knockout (KO) mice. Based on these findings, we asked what caused the myonuclear loss in the Lmna KO mice. Myonuclear loss can potentially result from reduced replenishment from satellite cells, apoptosis, which is often mediated by p53, and potentially other, not yet well characterized, mechanisms. Satellite cells are muscle stem cells that can replenish myonuclei when they sense injury. Deficient satellite cells may fail to replenish myonuclei, thus leading to myonuclear loss. We further hypothesize that apoptosis in muscle fibers, which is different from apoptosis in typical single nucleated cells where the whole cell dies, may cause the impaired nuclei to be degraded. Therefore, to confirm their role in myonuclear loss, we aimed to test whether deleting p53 can reverse myonuclear loss and track the fusion of newly acquired myonuclei from satellite cells.

Materials and

Methods: To determine the role of apoptosis in myonuclear loss in lamin KO mice, we generated mice carrying deletion of the Trp53 gene, which encodes for p53, a major regulator of apoptosis, and the Lmna gene (Lmna KO), alone or in combination. We performed single muscle fiber isolation (SFI) from these mice at 3 or 6 weeks of age using an established method (Fig. 1). Single muscle fibers were collected and fixed, and fibers were stained for DNA. After staining was completed, fibers were mounted onto slides. Slides were imaged using confocal microscopy and acquiring Z stacks to capture the whole fiber. Image analysis was performed in ZEN Blue for myonuclear count per 100 um in both body and myotendinous junction (MTJ) of fibers, and average myonuclear counts were determined for each mouse. To answer whether myonuclear loss is caused by impaired myonuclei replenishment, we used EdU incorporation to track the fusion of newly acquired myonuclei from muscle stem cells. Satellite cells proliferate before fusing intro myofiber, and EdU incorporates into newly synthesized DNA. Therefore, EdU incorporation can be used to specifically label stem-cell derived myonuclei. EdU was administered 2 weeks via water prior to mice sacrifice, and SFI was performed on 6-weeks Lmna KO and 10-weeks Lmna N195K mice. Fibers were stained for DNA and EdU and imaged. We quantified the number of EdU positive (EdU+) nuclei per 100 um in the body and MTJ of muscle fibers, and average EdU+ nuclei counts were calculated for each mouse.

Results, Conclusions, and Discussions: In the myonuclear apoptosis study, the average myonuclear count did not change in either Lmna WT or KO mice when Trp53 was deleted (Fig. 2). We observed the same trend in the body and MTJ of muscle fibers from 3- and 6-week-old male and female mice (Fig. 3). This indicates that myonuclear loss does not arise from p53-mediated myonuclear apoptosis.

In the myonuclei replenishment analysis, the average EdU+ nuclear counts either decreased or remained unchanged in the body and MTJ of male Lmna KO muscle fibers, while EdU+ nuclear counts remained unchanged or increased in the body and MTJ of female Lmna KO muscle fibers (Fig. 4A). Meanwhile, average EdU+ nuclear counts remained unchanged or increased in male Lmna N195K muscle fibers, and EdU+ nuclear counts remained unchanged in female Lmna N195K muscle fibers (Fig. 4B). This result indicates sex-specific and genotype-specific differences for myonuclei incorporation, which is consistent with our previous result.

In Lmna KO mice, the decreased or unchanged nuclear incorporation was associated with myonuclear loss in male, and the increased or unchanged nuclear incorporation was associated with unchanged myonuclear counts in females (Fig. 5A). In Lmna N195K mice, the increased or unchanged nuclear incorporation was associated with unchanged myonuclear counts in males, and the unchanged nuclear incorporation is associated with myonuclear loss in females (Fig. 5B). This indicates that myonuclear loss is also not a result of impaired replenishment from satellite cells. Satellite cells appear to increase their effort to replenish damaged nuclei to prevent nuclear loss.

Our findings provide insights into the underlying mechanisms of muscle degeneration in laminopathies. Myonuclear loss may result from other not yet well characterized mechanisms such as autophagy, and our research opens new avenues for future studies to explore other cellular mechanisms that cause muscle degeneration. Understanding the mechanisms could ultimately lead to the development of targeted therapies aiming at preserving myonuclei in patients.

Acknowledgements (Optional):