Musclin ‐ a Novel Exercise Factor
Authors: Subbotina Ekaterina , Sierra Ana , Zhu Zhiyong , Gao Zhan , Koganti Sivaramakrishna , Stepniak Elizabeth , Hodgson‐Zingman Denice , Zingman Leonid 
Published: November 2023

Exercise remains the most effective way to promote physical and metabolic wellbeing, but molecular mechanisms underlying exercise tolerance are only partially understood.This study is focused on the recently discovered myokine musclin. Musclin expression has been linked to insulin‐induced activation of protein kinase B (Akt) that phosphorylates the forkhead box O1 transcription factor (FOXO1), thus releasing musclin encoding gene inhibition. Musclin contains two putative serine protease cleavage sites and a region homologous to members of the natriuretic peptide (NP) family. Musclin does not have two cysteine residues needed to form the W‐like structure characteristic for NPs. In line with these characteristics musclin binds to the NP clearance receptor, NPRC, with affinity comparable to NPs, but exhibits only weak binding to NPRA and NPRB without activating the linked guanylyl cyclase. The physiological role of musclin production in skeletal muscles has remained elusive.Here we show that musclin production and secretion into the systemic circulation are stimulated by exercise. Mice subjected to daily treadmill exercise demonstrate an ~100% increase in skeletal muscle musclin protein and mRNA. Plasma levels of circulating musclin are increased from 27.71±5.54 pg/ml in sedentary control mice, to 46.24±4.69 pg/ml in the post‐exercise group.Furthermore, we demonstrate that in our exercise model that Akt is phosphorylated and FOXO1 exported from nuclei in response to exercise. We also show that musclin production is stimulated by Ca2+‐ionophore in human and murine myoblast cell culture in the presence of Ca2+ and is nonresponsive to ionophore in a Ca2+‐free environment. Application of an Akt inhibitor eliminates the stimulatory effect of Ca2+‐ionophore in cell culture establishing that musclin production is driven by Ca2+‐dependent activation of Akt.To establish the physiological role of systemically secreted musclin we created an Ostn (musclin‐encoding gene)‐KO mouse model. Disruption of Ostn and elimination of musclin secretion in mice results in reduced exercise tolerance that can be rescued by treatment with synthetic musclin. Specifically, Ostn‐KO mice demonstrate a significant deficit in exertional tolerance with respect to duration (71±6 vs. 91±6 min) and distance (769±102 vs. 1147±121 meters) on the treadmill. Similarly, when mice are offered the opportunity for voluntary exercise on running wheels, Ostn‐KO mice demonstrate significantly lower mean velocity (35±4 vs. 52±4 rotations/5 min), duration (303±38 vs. 383±64 min) and distance (1505±231 vs.2218±253 meters). Musclin delivered by osmotic pump equalizes running wheel activity between Ostn‐KO and WT mice.In agreement with the previously established musclin ability to interact with NP clearance receptors, Ostn‐KO mice show a trend toward higher plasma ANP levels compared to WT (140.4±19.9 pg/ml vs. 98.2±4.5) and significantly lower cGMP concentrations in muscle (23.13±.88 fmol/mg vs. 20.62±.60 skeletal muscle tissue). Experiments in primary myoblast culture show that induction of cGMP by the combination of musclin and ANP is associated with a significant increase in intracellular cGMP.In summary, this study defines musclin as an exercise‐responsive factor promoting exercise endurance.Support or Funding InformationThis work was supported by the National Institutes of Health [HL113089 to D.H‐Z., HL093368 and DK092412 to L.Z.]; the VA Merit Review Program [1I0BX000718 to L.Z.]; and the Fraternal Order of Eagles Diabetes Research Center.