The paradox of hnRNPK: both absence and excess impair skeletal muscle function in mice.

Abstract

Background: The RNA-binding protein hnRNPK is essential for animal growth and development, with a particular emphasis in myogenesis. Despite its importance, the precise mechanisms by which hnRNPK influences skeletal muscle physiology and development remain inadequately characterized.

Methods: To explore its regulatory function, we developed a Myf5-cre-mediated myoblast precursor-specific knockout mouse model (Hnrnpk mKO), an Acta1-CreEsr1-mediated myofiber-specific inducible knockout mouse model (Hnrnpk aKO), and an AAV9-mediated skeletal muscle-specific overexpression mouse model (AAV9-hnRNPK). Morphological alterations in skeletal muscle were assessed using hematoxylin and eosin (HE) staining subsequent to hnRNPK knockout or overexpression. Global gene expression changes in the tibialis anterior (TA) muscle were assessed via RNA sequencing (RNA-seq). Furthermore, reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blot analysis, immunofluorescence, immunohistochemistry, co-immunoprecipitation (Co-IP), dual luciferase analysis, and reactive oxygen species (ROS) detection were utilized to elucidate the molecular mechanisms by which hnRNPK contributes to skeletal muscle development.

Results: Our findings indicate that the ablation of hnRNPK in myoblast precursors significantly impairs muscle development, disrupts fetal myogenesis, and results in embryonic lethality. In adult mice, both the loss and gain of hnRNPK function led to reduced muscle mass, decreased fiber size, and compromised skeletal muscle homeostasis. Importantly, the knockout of hnRNPK had a more substantial impact on skeletal muscle development compared to its overexpression, with myofiber-specific knockout leading to mortality within two weeks. Mechanistically, hnRNPK deficiency was associated with increased apoptosis and muscle atrophy, characterized by elevated expression of genes involved in apoptosis, muscle atrophy, and protein catabolism, along with impaired muscle contraction and extracellular matrix (ECM) organization. Conversely, hnRNPK overexpression was correlated with enhanced ferroptosis pathway and improved ECM organization, but was also associated with reduced oxidative phosphorylation and protein synthesis. The overexpression likely promotes ferroptosis via the hnRNPK/P53/Slc7a11/Gpx4 pathway, thereby accelerating muscle aging and reducing muscle mass.

Conclusion: In conclusion, our findings underscore the critical importance of precise hnRNPK expression levels in maintaining skeletal muscle health. Both deficiency and overexpression of hnRNPK disrupt skeletal muscle development, highlighting its pivotal role in muscle physiology.

Clinical trial number: Not applicable.