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Orthopedic and Rehabilitation Engineering

Orthopedic and Rehabilitation Engineering - Poster Session C & D

Y42 - Transcriptomic Profiling of Skeletal Muscle in Lamin A/C-Deficient Mice

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Kanita Hrustanovic

Student
Colorado State University, College of Engineering
PEYTON, Colorado, United States

Last Author(s)

Soham Ghosh

Associate Professor
Colorado State University
Timnath, Colorado, United States

Primary Investigator(s)

Soham Ghosh

Associate Professor
Colorado State University
Timnath, Colorado, United States

Co-Author(s)

SS

Stephanie Ellyse Schneider, PhD

Research Associate
University of Colorado Boulder
Boulder, Colorado, United States
Corey P. Neu

Endowed or Named Professor, Director-Faculty, Professor
University of Colorado Boulder, United States

Introduction: : The nuclear lamina provides mechanical integrity to the eukaryotic cell nucleus and serves as a scaffold at the nuclear envelope for lamina-associated domains of chromatin, which are essential for maintaining chromatin structure and fidelity of gene expression. Lamin A/C, a key component of the nuclear lamina, is compromised in several clinical conditions, including hereditary laminopathies and aging. Lamin A/C deficiency, characterized by reduced levels and mislocalization, leads to altered cardiac and musculoskeletal phenotypes, including severe muscle atrophy in both humans and mice. Our previous in vivo work showed that multiscale mechanics of the tissue and nucleus is disrupted by Lamin A/C deficiency. However, the biological pathways and their interactions that lead to the altered phenotype resulting from Lamin A/C deficiency are not well understood. The goal of this study is to provide a comprehensive transcriptomic analysis of the effects of Lamin A/C deficiency, in which the Lamin A/C protein fails to integrate into the nuclear lamina. We used a LMNA mutant mouse model to investigate the effect of Lamin A/C deficiency on the quadriceps muscle transcriptome. Homozygous Lamin A/C deficient (Lmna -/- , LD ), heterozygous Lamin A/C deficient (Lmna +/-, HZ) and wild type (Lmna +/+, WT) mice were used for the investigation to elucidate potential differences in transcriptional regulation between groups.

Materials and

Methods: : Lamin A/C deficient mice: B6129S1(Cg)-Lmnatm1Stw/BkknJ mice were purchased from Jackson Laboratory (#009125). Mice were housed in a temperature-controlled environment with a 12-hour light and dark cycle and allowed to graze for both food and water ad libitum. The targeted allele expresses a truncated Lamin A/C protein. Tissues were obtained from Lmna+/+ (WT), (HZ) Lmna+/- (HZ), Lmna-/- (LD) mice. The WT and HZ groups each included two male and two female mice, while the LD group consisted of three males and one female.

Tissue collection and RNA sequencing: Mice were euthanized under approved IACUC protocols at the University of Colorado Boulder. Skeletal tissues were dissected, rinsed in DPBS, flash frozen and stored at -80°C until for processing by Novogene (n = 4/group). Total RNA was extracted and subjected to mRNA enrichment using poly-T oligo-attached magnetic beads. The enriched mRNA was fragmented and reverse transcribed into cDNA using random hexamer primers. Second-strand synthesis was followed by end repair, A-tailing, adapter ligation, size selection, PCR amplification, and purification to construct sequencing libraries. Library quality was assessed using Qubit, qPCR, and a Bioanalyzer. Libraries were sequenced on an Illumina NovaSeq X Plus platform to generate 150 bp paired end reads. Raw sequencing data underwent quality control to remove reads containing adapters, excessive unknown bases (N > 10%), or >50% low-quality bases (Q ≤ 5). Clean reads were used for downstream analysis.

Results, Conclusions, and Discussions:: Transcriptomic profiling of WT, HZ, and LD mice revealed clear genotype-driven clustering patterns via principal component analysis (PCA) (Fig. 1A). LD samples formed a tight cluster along one side of PC1, while WT and HZ samples clustered together on the opposite side. PC2 separated samples by sex, with females clustering higher and males lower, indicating sex as a secondary factor influencing transcriptomic variation after genotype.

Volcano plots comparing gene expression between genotypes further highlighted these differences (Fig. 1B). LD mice exhibited extensive transcriptomic disruption, with 473 genes upregulated and 206 downregulated compared to WT. In contrast, HZ mice showed no significant changes, suggesting that biallelic Lamin A/C loss is required to disrupt transcriptomic regulation and that one functional allele is sufficient to maintain near-normal expression.

Heatmaps of genes from key functional categories revealed striking similarities between HZ and WT profiles. Several muscle structure–related genes were downregulated in LD mice, but genes involved in structural integrity, contractility, and extracellular matrix (ECM) remodeling exhibited elevated Z-scores, suggesting a compensatory response to preserve muscle function (Fig. 1C, D). Immune response and inflammation-related genes were also upregulated in LD mice but suppressed in WT and HZ, reinforcing the transcriptional divergence of LD and similarity of WT and HZ. These trends persisted in the top 20 genes ranked by log2 fold change (Fig. 2B), highlighting the distinct transcriptomic signature of LD muscle.

To identify disrupted biological pathways, GO Biological Process enrichment analysis was performed on differentially expressed genes between LD and WT (Fig. 2A). No enriched pathways were detected in the HZ vs WT comparison. In LD muscle, pathways related to muscle structure, contraction, and ECM remodeling were significantly enriched (FDR < 0.05), confirming broad transcriptional dysregulation linked to Lamin A/C deficiency. Overall, these findings demonstrate that complete Lamin A/C loss severely disrupts muscle transcriptomics, while partial loss preserves a transcriptional landscape similar to WT.

Acknowledgements and/or References (Optional): :