Alternative Splicing–Derived Muscle Isoforms as Biomarkers of Muscle Function and Aging

Abstract

Exercise serves as a potent physiological stimulus that induces gene expression and post-transcriptional regulation in skeletal muscle. Among these mechanisms, alternative splicing (AS) plays a pivotal role in enhancing the functional diversity and adaptability of muscle tissue by generating multiple protein isoforms from a single gene. However, previous studies investigating exercise-induced AS have largely been limited to transcriptome-level observations, and the functional impact of specific isoforms on muscle health and performance remains poorly understood. Although numerous computational algorithms identify a large number of statistically significant AS events, many of these events may not be biologically meaningful, highlighting the need for biologically informed prioritization strategies. In this study, we prioritized genes belonging to major regulatory families involved in muscle function (kinases, transcription factors, cytokines, membrane receptors) based on Human Protein Atlas (HPA) classification. Mouse exercise RNA-seq data (GSE198266, GSE222163) demonstrated selective regulation of specific isoforms following exercise, and isoforms showing significant exercise-induced changes were further filtered based on their expression levels in Genotype-Tissue Expression (GTEx) skeletal muscle and Consensus Coding Sequence (CCDS) annotation status, through which Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1B (DYRK1B) was selected. Single-nucleus RNA-seq analysis further confirmed its expression in cell populations relevant to skeletal muscle function, particularly type II myofiber nuclei and the myotendinous junction (MTJ). We focused on DYRK1B and examined molecular signatures associated with the relative expression differences between the DYRK1B-b (isoform with amino acids 426–465 deleted) and DYRK1B-a (isoform containing amino acids 426–465) using GTEx transcriptomic data. Histological analysis of GTEx hematoxylin and eosin (H&E)-stained muscle tissue revealed that the DYRK1B-b–dominant group exhibited significantly greater myofiber cross-sectional area (CSA) compared with the DYRK1B-a–dominant group. Pathology-language Image Pretraining (PLIP) analysis further indicated that the DYRK1B-b group exhibited lower similarity to sarcopenia-related terms such as fibrosis, atrophy, and sarcopenia. Independent validation using an external human cohort (GSE111017) confirmed that greater expression of the DYRK1B-b isoform was significantly associated with favorable clinical phenotypes, including greater muscle mass, strength, and physical performance. Additionally, in vitro validation using electrical pulse stimulation (EPS) in differentiated C2C12 myotubes confirmed the preferential induction of the DYRK1B-b isoform through RT-PCR. Collectively, this study demonstrates that a biologically filtered, exercise-responsive splice isoform is quantitatively associated with both tissue morphology and functional indicators of muscle health. These findings highlight the translational potential of alternative splicing events as precision targets for muscle aging and degenerative disease.