The Age‐Dependent Resident Myonuclear Multi‐Omic Response to an Acute Skeletal Muscle Hypertrophic Stimulus in Mice

ABSTRACT A detailed analysis of how muscle fiber nuclei (myonuclei) respond to a hypertrophic stimulus could provide a critical step toward understanding compromised skeletal muscle plasticity with age. We used recombination‐independent doxycycline‐inducible myonucleus‐specific fluorescent labelling, tissue RNA‐sequencing, myonuclear DNA methylation analysis, multi‐omic integration, and single myonucleus RNA‐sequencing (smnRNA‐seq) to define the molecular characteristics of adult (6–8 month) and aged (24 month) murine skeletal muscle after acute mechanical overload (MOV). In adult and aged MOV muscles, we found that: 1) similarities in the transcriptional response to loading—specifically in metabolism genes – were partly explained by a post‐transcriptional microRNA‐mediated mechanism that we corroborated using an inducible muscle fiber‐specific miR‐1 knockout model, 2) differences in age‐dependent transcriptional responses were linked to the magnitude and location of differential DNA methylation in resident myonuclei, specifically around genes such as Myc , Runx1 , Mybph , Ankrd1 , collagen ( Col ) genes, and minichromosome maintenance ( Mcm ) genes, 3) adult and aged resident myonuclear transcriptomes had differing enrichment for innervation‐related transcripts as well as unique transcriptional profiles in an Atf3+ “sarcomere assembly” population after MOV, and 4) cellular deconvolution analysis and smnRNA‐seq supports a role for neuromuscular junction regulation in age‐specific hypertrophic adaptation. These data are a roadmap for uncovering molecular targets to enhance aged muscle adaptability.