The investigators reanalyzed RNA-sequencing data from the plantaris muscle of male mice subjected to synergist-ablation mechanical overload. The procedure removes part of the gastrocnemius–soleus complex, increasing the loading experienced by the plantaris. Sham-operated animals provided controls, and plantaris muscles were collected 72 hours after surgery. This acute time point represents the early phase of a hypertrophic response, before detectable changes in muscle-fiber size. The study therefore focused on molecular responses that preceded measurable growth, rather than comparing fully developed hypertrophic muscle.

Reanalysis of the previously published bulk RNA-sequencing data suggested that genes in the KEGG spliceosome pathway responded more strongly to overload in adult muscle than in aged muscle. A composite splicing score was higher in adult overloaded muscle but not in aged overloaded muscle, with individual spliceosome modules showing smaller responses in older animals. Aged sham muscle also had a higher splicing score than adult sham muscle, consistent with prior reports that alternative splicing becomes dysregulated with aging. The team paired the short-read data with Oxford Nanopore long-read sequencing to examine transcript-level changes more directly and to distinguish isoform usage from coordinated changes in overall transcript abundance.

Short-read sequencing generated 150-base-pair reads, with 60 million read pairs per sample and six or seven mice per group in the adult and aged comparisons. Using the IsoformSwitchAnalyzeR workflow, the researchers defined significant switching as an absolute difference in isoform fraction greater than 10% with an adjusted P value below 0.05. The most common forms of switching during overload were alternative transcription start sites, accounting for about 25% of events, alternative transcriptional termination sites, at about 23%, and exon skipping, at roughly 20–22%. Seventy-five genes showed significant isoform switching with aging alone when sham-operated adults and aged animals were compared.

Some switches occurred in genes whose total expression also changed, including Runx1, a transcriptional regulator associated in earlier studies with ribosome biogenesis, prevention of myofibrillar disorganization and attenuation of atrophy during muscle denervation. Two Runx1 isoforms increased during overload in both adult and aged muscle, but significant switching was detected only in adults. The fraction of the longer canonical isoform fell from about 75% to 46%, while a shorter noncanonical isoform lacking exons 1, 2 and 6 rose from 23% to 37%. Isoform-specific PCR followed by gel electrophoresis confirmed that both transcripts were present, and the shorter transcript appeared more abundant during overload. Its specific function during hypertrophy was not tested.

Cacnb1, which encodes the calcium voltage-gated channel auxiliary subunit beta 1, was another gene with a strong switching signal. It was downregulated at the total gene level during overload but underwent one of the most substantial isoform switches, with an adjusted P value below 0.001. In sham muscle, one transcript accounted for 72–78% of the gene’s isoform fraction; overload shifted usage toward another transcript. Cacnb1 has previously been studied in the context of isoform switching during muscle denervation, regeneration and aging, when embryonic or neonatal isoforms can be preferentially expressed over the mature adult isoform. The present experiment did not establish whether the calcium-channel switch improves or impairs muscle function.

A second group of genes switched isoforms without a statistically significant change in total gene expression, demonstrating that gene-level RNA analysis can miss part of the overload response. In adult muscle after overload, Sh3kbp1 shifted by roughly 36–38% from a non-protein-coding transcript to a transcript containing an SH3 domain, with an adjusted P value of 1.3 × 10−8. The corresponding shift was smaller in aged muscle, at 19–24%. SH3KBP1 has been linked in experimental systems to myonuclear organization during myofiber formation, myonuclear movement in mature myotubes, T-tubule formation, excitation–contraction coupling and autophagy. Prosaposin, or Psap, and the nascent polypeptide-associated complex subunit alpha gene Naca also showed significant adult-specific switching without differential total gene expression, with adjusted P values below 1.96 × 10−19 and 7.1 × 10−6, respectively.

The Naca switch involved inclusion of a large exon that produces skNAC, a skeletal-muscle-specific variant of a protein described as a ribosomal chaperone involved in assembling and transporting newly synthesized proteins to intracellular compartments. skNAC is also among the most highly phosphorylated proteins during acute aerobic and resistance exercise. The authors stated that the absence of an increase in this variant in aged muscle could conceivably contribute to blunted muscle adaptability, but that interpretation remains a hypothesis because the study did not manipulate the isoform, measure its phosphorylation or test its contribution to hypertrophy. The Psap transcripts encode four saposin domains and have been investigated in oxidative stress and several disease models, yet their roles in adaptation to mechanical loading remain unknown.

Long-read sequencing broadly supported the short-read findings while providing a stricter test of whether coordinated changes in all transcripts had been mistaken for isoform switching. The researchers aligned reads to the GRCm39 mouse genome and used IsoQuant, which identified 25,093 novel transcripts alongside the reference annotation. Adult overload produced 992 upregulated and 1,029 downregulated transcripts, representing 1,820 unique genes. Only two aged-overload transcripts passed the adjusted P-value threshold of 0.05: isoforms of Mustn1 and Trem2. At the more permissive adjusted threshold of 0.10, 638 aged-overload transcripts were differentially expressed, associated with 615 unique genes, and the enriched pathway was cytokine–cytokine receptor interaction, with normalized enrichment score 2.42 and adjusted P value 1.47 × 10−7.

After distinguishing genuine switching from coordinated regulation, the long-read analysis identified 19 switching genes in adult overloaded muscle and none in aged overloaded muscle. Nine were also detected by short-read sequencing: Cacnb1, Rtn4, Capzb, Gsn, Macroh2a1, Mars1, Naca, Psap and Slc25a3. Long reads confirmed the adult-specific shift toward the skNAC variant of Naca and greater abundance of a switched Psap transcript. The smaller number of long-read events may reflect lower statistical power: long-read libraries were prepared from pooled RNA with two or three replicates per condition, compared with six or seven individual mice in the short-read experiment. The switch predictions were also not validated at the protein or functional level.

Overall, the study shows that acute mechanical overload elicits extensive transcript-isoform remodeling in adult mouse muscle and that this response is attenuated in aged muscle. The findings complement earlier evidence that aging and mechanical loading each alter the muscle transcriptome, but they separate isoform usage from total gene expression and show that a substantial part of the overload response can remain invisible to gene-level analysis. The results provide candidate transcripts for mechanistic studies, including Naca, Sh3kbp1, Psap and Cacnb1. They do not yet establish the causes or physiological consequences of the age-related difference, and the single 72-hour sampling point cannot distinguish permanently impaired switching from a response that is merely delayed.

Subject of Research: Age-dependent transcript isoform switching during mechanical overload in mouse skeletal muscle

Article Title: A Hypertrophic Stimulus Drives Age‐Dependent Transcript Isoform Switching With and Without Differential Gene Expression in Skeletal Muscle

Article References: Koopmans, P. J., Stokes, A. L., Nelson, C. E., Wen, Y., Ismaeel, A., & Murach, K. A. (2026). A Hypertrophic Stimulus Drives Age‐Dependent Transcript Isoform Switching With and Without Differential Gene Expression in Skeletal Muscle. Aging Cell, 25(10), Article e70761. https://doi.org/10.1111/acel.70761

Image Credits: AI Generated

DOI: 10.1111/acel.70761

Keywords: skeletal muscle, aging, mechanical overload, isoform switching, alternative splicing, RNA sequencing, transcriptomics, mouse model, long-read sequencing, plantaris muscle, Hypertrophic, Stimulus