Musclin is an activity-stimulated myokine that enhances physical endurance

Abstract

Exercise remains the most effective way to promote physical and metabolic wellbeing, but molecular mechanisms underlying exercise tolerance and its plasticity are only partially understood. In this study we identify musclin-a peptide with high homology to natriuretic peptides (NP)-as an exercise-responsive myokine that acts to enhance exercise capacity in mice. We use human primary myoblast culture and in vivo murine models to establish that the activity-related production of musclin is driven by Ca(2+)-dependent activation of Akt1 and the release of musclin-encoding gene (Ostn) transcription from forkhead box O1 transcription factor inhibition. Disruption of Ostn and elimination of musclin secretion in mice results in reduced exercise tolerance that can be rescued by treatment with recombinant musclin. Reduced exercise capacity in mice with disrupted musclin signaling is associated with a trend toward lower levels of plasma atrial NP (ANP) and significantly smaller levels of cyclic guanosine monophosphate (cGMP) and peroxisome proliferator-activated receptor gamma coactivator 1-α in skeletal muscles after exposure to exercise. Furthermore, in agreement with the established musclin ability to interact with NP clearance receptors, but not with NP guanyl cyclase-coupled signaling receptors, we demonstrate that musclin enhances cGMP production in cultured myoblasts only when applied together with ANP. Elimination of the activity-related musclin-dependent boost of ANP/cGMP signaling results in significantly lower maximum aerobic capacity, mitochondrial protein content, respiratory complex protein expression, and succinate dehydrogenase activity in skeletal muscles. Together, these data indicate that musclin enhances physical endurance by promoting mitochondrial biogenesis.

Keywords: exercise; mitochondria; natriuretic peptide; osteocrin; skeletal muscle.

Fig. 1.

Musclin expression is exercise-responsive. Musclin expression was tested in muscles of WT mice after 5 d of treadmill exercise vs. no exercise (control). (A) Representative Western blots for musclin and GAPDH in protein extracts from gastrocnemius. (B–D) Summary statistics for musclin protein expression normalized to GAPDH by densitometry of Western blots of protein extracts from gastrocnemius (B), tibialis anterior musclin mRNA normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT) by quantitative RT-PCR (qRT-PCR) (C), and musclin peptide expression in plasma by custom ELISA (the y axis range begins at the lower limit for detection for this assay of 20 pg/mL; D). (E) Representative immunohistochemical stains of gastrocnemius cross-sections imaged by confocal microscopy. Red, musclin; green, nuclei. *P < 0.05 vs. control.

Fig. 2.

Exercise promotes skeletal muscle Akt phosphorylation and FOXO1 nuclear export. Gastrocnemius of WT mice were assayed after 5 d of treadmill exercise vs. no exercise (control). (B–D) Representative Western blots of GAPDH and Akt phosphorylated at residue 473 (A), residue 308 (B), and total Akt (C) in muscle and representative Western blots of TBP and FOXO1 in nuclear extracts from muscle (D). (E–H) Summary statistics for expression of Akt phosphorylated at residue 473 (E), residue 308 (F), and total Akt normalized to GAPDH in muscle (G) and FOXO1 normalized to TBP in nuclear extracts from muscle by densitometry of Western blots (H). TBP, anti-TATA binding protein. *P < 0.05 vs. control.

Fig. S1.

Exercise increases FOXO1 phosphorylation. (A and B) Representative Western blots of phosphorylated FOXO1 (p-FOXO1) (A) and total FOXO1 (B), as well as corresponding GAPDH in gastrocnemius muscle of exercised and nonexercised (control) mice. (C and D) Summary statistics for phosphorylated FOXO1 (C) and total FOXO1 (D) normalized to GAPDH. *P < 0.05 vs. sedentary control.

Fig. 3.

Musclin production is stimulated by Ca²⁺-dependent Akt phosphorylation. (A) Representative Western blots of Akt, FOXO1, and GAPDH from cultured murine primary myoblasts in 1 mM Ca²⁺ without ionophore (control) vs. with 1 µM ionophore A23187 (Sigma-Aldrich). (B and C) Summary statistics for phosphorylated Akt (pAkt) and phosphorylated FOXO1 (pFOXO1) normalized to GAPDH (B) and total Akt and total FOXO1 (C), respectively, with (gray) and without (white; control) 1 µM ionophore. *P < 0.05 vs. control. (D and E) Summary statistics for musclin mRNA normalized to HPRT in murine cultured primary myoblasts exposed to various concentrations of ionophore, Ca²⁺ and Akt inhibitor-viii (D) and human cultured primary myoblasts exposed to no Ca²⁺ vs. 1.0 mM Ca²⁺ and various doses of ionophore, by densitometry of Western blots (E). *P < 0.05 vs. no ionophore (D) or vs. Ca²⁺-free (E).

Fig. 4.

Musclin supports physical performance. (A) Schematic of the modified Ostn gene indicating excision of the ATG-containing exon 2 to create the Ostn-KO mouse model. (B) Representative Western blot of musclin from gastrocnemius of WT and Ostn-KO mice. (C) Schematic of the treadmill exercise protocol. Vertical lines indicate mean time points of exercise dropout. (D–F) Summary statistics for treadmill exercise tolerance in terms of duration (D), distance (E), and tolerated workload: E_k + E_p (F). *P < 0.05 Ostn-KO vs. WT. (G) Schematic of a running wheel. (H–J) Summary statistics for voluntary running-wheel exercise performance at day and night in terms of mean velocity (H), duration (I), and distance (J). *P < 0.05 Ostn-KO vs. WT. (K) Schematic of a running wheel and an osmotic pump loaded with musclin (m) or saline (s). (L–N) Summary statistics for voluntary running wheel performance at night in terms of mean velocity (L), duration (M), and distance (N). *P < 0.05 vs. WT saline. KO, Ostn-KO.

Fig. S2.

Skeletal structure is grossly intact in Ostn-KO. Representative projections of high-resolution CT 3D reconstruction of the skeletons of WT (A) and Ostn-KO (B) mice, with enlarged views of the lower extremities of WT (C) and Ostn-KO (D) mice.

Fig. 5.

Musclin augments ANP signaling in skeletal muscle. Mice were exercised on a treadmill for 5 d before undergoing assessment of ANP signaling. (A) Summary statistics for plasma ANP as assessed by ELISA. (B) Summary statistics for cGMP in gastrocnemius by enzyme immunoassay. *P < 0.05 Ostn-KO vs. WT. (C) Summary statistics for cGMP production in a culture of murine primary myoblasts exposed to various concentrations of ANP and musclin. *P < 0.05 vs. columns indicated by bar. (D) Summary statistics for percent increase in peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1-α) mRNA over baseline in response to exercise in tibialis anterior by qRT-PCR. *P < 0.05 vs. WT. (E) Summary statistics for primary myoblast culture mRNA of PGC1-α normalized to HPRT in response to musclin/ANP or to 8-Br-c-GMP. (F) Summary statistics for primary myoblast culture relative mRNA of TFAM, NRF1, and NRF2 with and without musclin/ANP. KO, Ostn-KO.

Fig. 6.

Musclin signaling improves aerobic capacity and prompts mitochondrial biogenesis. (A) Summary statistics for trend of oxygen consumption over time of exercise-trained mice upon initiation of treadmill exercise at time 0. *P < 0.05 vs. Ostn-KO. (B) Representative electron micrographs of longitudinal tibialis anterior sections from exercised mice. White arrows indicate mitochondria. (C) Summary statistics for mitochondrial content by weight in gastrocnemius isolates of exercised mice. *P < 0.05 vs. WT. (D and E) Representative Western blots of respiratory chain enzymes and GAPDH (D) and summary statistics for respiratory complex expression normalized to GAPDH in gastrocnemius of exercise-trained mice (E). *P < 0.05 vs. WT. (F and G) Representative stains for SDH activity of tibialis anterior cross-sections (F) and summary statistics for percent area of cross-sections stained for SDH activity (G) in exercise-trained mice. *P < 0.05 vs. WT. KO, Ostn-KO.

Fig. S3.

Oxygen consumption of Ostn-KO is rescued by musclin infusion. The oxygen consumption, VO₂, over time upon initiation of treadmill exercise was compared in WT and Ostn-KO mice treated with 3 wk of saline or musclin delivered by osmotic pump. Oxygen consumption of WT mice treated with musclin was slightly better for several time points than that of WT mice treated with saline. Oxygen consumption of Ostn-KO mice treated with musclin is equivalent to that of WT mice treated with musclin and was better than WT treated with saline once steady state was achieved. *P < 0.05 for WT + musclin vs. WT + saline; ^§P < 0.05 for Ostn-KO + musclin vs. WT + saline.

Fig. S4.

Musclin controls skeletal muscle fiber type. (A) Representative immunohistochemical stains for fiber type in cross-sections of biceps femoris (BI) and tibialis anterior (TA) of exercise-trained WT and Ostn-KO mice. Red, IIB; green, IIA; blue, I; black, IIX staining. (B) Summary statistics for fiber type as assessed by counting the number of stained fibers per field. *P < 0.05 vs. WT.

Fig. S5.

Differences in aerobic capacity and markers of mitochondrial biogenesis are minimal in sedentary WT and Ostn-KO mice. (A) Summary statistics for trend of oxygen consumption over time of sedentary WT and Ostn-KO mice upon initiation of treadmill exercise at time 0 (all points NS for WT vs. Ostn-KO). (B) Representative electron micrographs of longitudinal tibialis anterior sections from sedentary mice. (C) Summary statistics for mitochondrial content by weight in gastrocnemius isolates of sedentary mice. *P < 0.05 vs. WT. (D and E) Representative Western blots of respiratory chain enzymes and GAPDH (D) and summary statistics for respiratory complex expression normalized to GAPDH in gastrocnemius (E) of sedentary WT and Ostn-KO mice. (F and G) Representative stains for SDH activity of tibialis anterior cross-sections (F) and summary statistics for percent area of cross-sections (G) stained for SDH activity in sedentary WT and Ostn-KO mice. *P < 0.05 vs. WT. KO, Ostn-KO; Sed, sedentary.

Fig. S6.

Differences in skeletal muscle fiber type are less marked in sedentary WT vs. Ostn-KO mice. Summary statistics for fiber type as assessed by counting number of stained fibers per field in tibialis anterior (TA; Left) and biceps femoris (BI; Right) of sedentary WT and Ostn-KO mice. *P < 0.05 vs. WT.