Loss-of-JNK enhances endurance remodeling in muscle

Our previous work using models of high and low adaptive response to endurance exercise demonstrated that skeletal muscle JNK activation during exercise was negatively correlated to the adaptive response to endurance exercise16. Based on those data16, we hypothesized that inhibition of JNK during exercise would enhance adaptive remodeling in response to endurance exercise in skeletal muscle. To test this hypothesis, an endurance exercise training study was performed in muscle-specific JNK1/2 knockout (mJNKKO; Mapk8LoxP/LoxP Mapk9LoxP/LoxP MCK-Cre−/+) mice and wild-type (WT; MCK-Cre−/+) controls. Mice were placed in cages with access to a running wheel, and running activity was electronically monitored for 10 week (Exercise-trained). A separate group of mice was kept in static cages for the experimental period (Sedentary). Total running distance was similar over the training duration in WT and mJNKKO mice (Supplementary Figure 1). Following the 10-week training period, endurance capacity was measured using a graded treadmill running test in all experimental groups as a physiological marker of endurance adaptation. Endurance capacity in trained mJNKKO was 45% higher compared to wild-type controls (Fig. 1a). Endurance capacity was similar in untrained WT and mJNKKO animals, demonstrating that increased endurance capacity in mJNKKO was due to enhanced adaptation to training, rather than intrinsic differences between genotypes (Fig. 1a). High endurance capacity, also known as cardiorespiratory fitness, is the gold-standard measure for the adaptive response to endurance training and has emerged as one of the best clinical markers for metabolic health and longevity in humans. These data demonstrate that skeletal muscle JNK activation during exercise is a negative regulator of improvements to endurance capacity.

Fig. 1 Loss of JNK in skeletal muscle enhances adaptation to endurance training. Muscle-specific JNK knockout mice (mJNKKO) and controls (WT; MCK-Cre−/+) were placed in voluntary wheel-running cages for 10 weeks (Exercise-trained; EXT). A separate cohort of mice remained in cages without running wheels (Sedentary; SED) and acted as controls. a Exercise capacity was similar in WT and mJNKKO mice that were sedentary, but was higher in mJNKKO mice after training. b, c Histological analysis was performed on the red gastrocnemius muscle from sedentary and exercise-trained mJNKKO mice and controls. Staining with antibodies against CD31 (red) (b) was used to calculate capillary density (c). d–f Sections were stained with wheat germ agglutinin (WGA; green) and myosin heavy chain type I (red) antibodies (f) to determine fiber type (e) and cross-sectional area (f). *P < 0.05 vs SED of the same genotype by two-way ANOVA and Sidak’s post hoc testing. Scale bars represent 100 µm. Each data point represents the results from an individual animal. Bar plots indicate mean ± SEM for all data Full size image

Consistent with a phenotype of high-endurance capacity, mJNKKO had a higher capillary density (Fig. 1b, c) and a greater proportion of oxidative (type I) fibers in the red gastrocnemius muscle (Fig. 1d, e). In addition, exercise training caused a significant decrease in myofiber cross-sectional area in mJNKKO mice, but not in WT controls (Fig. 1f). Smaller myofiber cross-sectional area contributes to enhanced endurance capacity by allowing for better diffusion of oxygen and nutrients to working muscle18. In humans, small fiber cross-sectional area, higher capillary density, and increased oxidative myosin heavy chain expression characterize the muscle of elite endurance athletes18. Therefore, mJNKKO mice have a muscle phenotype that mimics that of endurance trained athletes. These experiments demonstrate that JNK is a negative regulator of endurance remodeling in muscle, and identify inhibition of JNK as a strategy to improve endurance adaptations.

JNK is necessary for muscle hypertrophy

It has been proposed that the signaling mechanisms regulating the remodeling programs for endurance or hypertrophic adaptations in muscle are antagonistic9,10. Indeed, concurrent training regimens employing both endurance and resistance exercise modalities can blunt muscle adaptation in humans11. Since JNK was a negative regulator of endurance training adaptations (Fig. 1), we hypothesized that JNK may be a positive regulator of hypertrophic adaptations in muscle. Functional overload (i.e., synergist ablation) is a surgical procedure that consistently induces muscle hypertrophy in mice, mimicking gains in muscle mass due to resistance training in humans. Functional overload of the plantaris muscle was induced by partial removal of the gastrocnemius muscle in mJNKKO mice and WT controls to determine the effect of JNK knockout on muscle hypertrophy. Overload surgery was performed on one leg from each animal, and the contralateral leg served as a control. Two weeks of overload increased plantaris muscle weight by 2.5-fold in WT mice (Fig. 2a). However, the overload-induced increase in muscle weight was significantly blunted in mJNKKO mice (Fig. 2a). In addition, muscle fiber cross-sectional area was increased by 50% in WT animals, but JNK knockout prevented overload-induced increases in muscle cross-sectional area (Fig. 2b, c). These data demonstrate that JNK is necessary for increased muscle mass and myofiber hypertrophy in response to increased load, supporting the hypothesis that JNK is a positive regulator of hypertrophic adaptation in muscle.

Fig. 2 JNK is necessary for muscle hypertrophy with overload. The distal portion of the gastrocnemius muscle was surgically removed from one limb in muscle JNK knockout (mJNKKO) mice and wild-type controls (WT; MCK-Cre−/+) to induce functional overload (OVL) of the plantaris muscle. The contralateral limb served as control (CON). a Fourteen days following overload surgery, the plantaris muscles were removed and wet weight was recorded. b Sections from plantaris muscles were stained with wheat germ agglutinin (green) for the calculation of fiber cross-sectional area (CSA). Scale bar represents 100 µm. c, d Average CSA was calculated (c) from the Control and Overload muscles from both genotypes, and fiber size distribution was plotted (d). *P < 0.05, **P < 0.01 vs Control from the same genotype by two-way ANOVA and Sidak’s post hoc testing. Main effects of genotype are displayed with P-values. Each data point represents the results from an individual animal. Bar plots indicate mean ± SEM for all data Full size image

Muscle JNK activation coincides with SMAD2 phosphorylation

Our data demonstrate that JNK acts as a positive regulator of hypertrophic adaptations and a negative regulator of endurance adaptations in muscle (Figs. 1 and 2). We next aimed to determine the mechanism by which JNK mediates muscle adaptive remodeling. Our previous work identified a novel exercise-activated phosphorylation site on the transcription factor, SMAD2, at a predicted JNK consensus sequence in its linker region (Ser245/250/255)16. As SMAD2 is a known regulator of muscle remodeling19,20, we hypothesized that phosphorylation of SMAD2 may be a mechanism for JNK-mediated muscle remodeling with exercise. To determine whether JNK activation tracks with SMAD2-linker (SMAD2-L) phosphorylation with exercise, we performed treadmill running and in situ contraction experiments in ICR (outbred) mice. SMAD2-L phosphorylation tracked closely with activation of JNK with both treadmill running and in situ contraction (Fig. 3a, b). However, treadmill running activated JNK/SMAD2-L phosphorylation to a relatively small extent (Fig. 3a) compared to in situ contraction, which more consistently and robustly induced JNK/SMAD2-L phosphorylation (Fig. 3b). While treadmill running induces metabolic stress in muscle via the utilization of energy substrates and ATP, in situ contraction elicits strong, eccentric contractions that induce both metabolic and mechanical stress. To determine whether mechanical stress alone is able to activate JNK/SMAD2-L signaling, we performed muscle stretch experiments. Passive stretch activated JNK and SMAD2-L robustly, while the metabolic stress indicator, AMPK, was minimally activated (Fig. 3c). Thus, JNK/SMAD phosphorylation is preferentially activated by contractions that elicit mechanical stress, rather than those producing metabolic stress (i.e., endurance exercise). Although other mitogen-activated protein kinases, p38 and ERK, were activated by muscle contraction and stretch; only JNK was activated in a similar pattern to pSMAD2-L. Therefore, our results identify JNK as a putative upstream kinase for SMAD2-L phosphorylation in muscle.

Fig. 3 SMAD-linker and JNK phosphorylation with exercise and muscle contraction in mice. a ICR mice underwent moderate intensity treadmill running for 15, 30, or 60 min and gastrocnemius muscles were collected. Control (rest) mice did not undergo treadmill running. Western blotting was used to determine exercise-induced signal transduction. Data from N = 3 mice/group are shown. b Electrodes were used to stimulate the lower hindlimb muscles from ICR mice [C; contracted]. The contralateral limb was unstimulated and acted as a control [B; basal]. Data from N = 3 mice/group are shown. c Both soleus muscles were rapidly removed from mice and attached to a force tranducer in oxygenated Kreb’s Henseleit Buffer. One muscle from each mouse was left at resting tension and acted as a basal control [Basal; B], while contralateral muscle was stretched for 10 min at a force of 0.12 N [Stretched; S]. N = 6 independent experiments were performed, and individual results from N = 3 are shown. pSMAD2L, linker region phosphorylated SMAD2; pJNK, phosphorylated (active) C-Jun N-terminal Kinase; pAMPK, phosphorylated (T172) AMP-activated protein kinase; pERK, phosphorylated extracellular signal regulated kinase; pP38, phosphorylated P38 Mitogen-Activated Protein Kinase; SMAD2, Total SMAD2. Images obtained using stain-free gel technology (Bio-Rad) that allows for total protein visualization and quantification are shown as a loading control (Loading) Full size image

JNK phosphorylates the SMAD2-linker region in muscle

Muscles from WT and mJNKKO mice following in situ (tibialis anterior; TA) or in vitro (extensor digitorum longus; EDL) contraction were analyzed to directly test the hypothesis that JNK is the upstream kinase for SMAD2-linker phosphorylation with exercise. In line with that hypothesis, SMAD2-L phosphorylation with muscle contraction was severely blunted in muscle-specific JNK1/2 knockout mice compared to WT controls (Fig. 4a–c). Next, C2C12 myoblasts were treated with the JNK activator Anisomycin (5 μM), to determine whether JNK activation is sufficient to induce SMAD2-L phosphorylation in muscle. Thirty minutes of Anisomycin treatment resulted in robust increases in JNK activation and SMAD2-linker phosphorylation (Fig. 4d). Skeletal muscle expresses two independent JNK isoforms, JNK1 and JNK2. Therefore, fusion plasmids for active JNK1 or JNK2 were expressed in C2C12 to determine whether JNK-associated SMAD2-L phosphorylation is isoform specific. Active JNK1 and JNK2 isoforms independently induced SMAD2-L phosphorylation in cultured muscle (Fig. 4e). SMAD2-linker phosphorylation was also increased following in vitro incubation of recombinant SMAD2 protein with purified immunoprecipitates of either active JNK1 or JNK2 (Fig. 4f). In contrast, we were unable to detect JNK-mediated SMAD3 phosphorylation in its linker region at the Ser208 site using a commercially available antibody. In vitro phosphorylation indicates that JNK can directly interact with SMAD2 to phosphorylate its linker region in the absence of other cellular proteins. Taken together, these experiments (Fig. 4) demonstrate that JNK activation is both necessary and sufficient for SMAD2-L phosphorylation in muscle. Furthermore, we identify JNK as the primary upstream kinase for SMAD2-linker phosphorylation during exercise.

Fig. 4 JNK is the upstream kinase for SMAD2 linker phosphorylation in muscle. a–c EDL and TA muscles from wild-type control (WT; MCK-Cre−/+) and muscle-specific JNK1/2 knockout mice (KO) were stimulated via in vitro (EDL) and in situ (TA) contraction. Immunoblotting of phosphorylated and total SMAD2 and JNK was performed (a). SMAD2-linker (SMAD2L) phosphorylation in response to in vitro contraction (b) and in situ contraction (c) was blunted in muscle JNK knockout mice. Each data point represents the basal and contraction results from an individual animal joined by a line. *P < 0.05, ***P < 0.01 vs Basal from the same genotype by repeated measures two-way ANOVA and Sidak’s post hoc testing. Main effects of genotype are displayed with P-values. d C2C12 myoblasts were treated with the JNK activator anisomycin (5 μM) for 30 min. e C2C12 myoblasts were transfected with plasmids expressing constitutively active JNK1, JNK2, or a combination of JNK1 and 2. Empty vector (EV) transfected cells were used as a control. f The ability of JNK to directly phosphorylate SMAD2 was assessed using an in vitro kinase assay. C2C12 lysates expressing empty vector (EV), or constitutively active JNK1 or JNK2 were purified by FLAG immunoprecipitation and incubated with recombinant SMAD2 and SMAD3 proteins. For tissue culture experiments (d–f), three independent experiments were performed and the data from one representative experiment, including all replicates is displayed. In all experiments, JNK activation (pJNK) and SMAD2 linker phosphorylation (pSMAD2L) were assessed by immunoblotting. Images obtained using stain-free gel technology (Bio-Rad) that allows for total protein visualization and quantification are shown as a loading control (Loading). pJNK phosphorylated (active) C-Jun N-terminal Kinase, pSMAD2L, linker-region phosphorylated SMAD2; SMAD2, total SMAD2; pP38, phosphorylation P38 MAPK; pERK, phosphorylated extracellular signal regulated kinase Full size image

JNK inhibits SMAD activity

A JNK/SMAD signaling axis represents a novel exercise-activated pathway in skeletal muscle; introducing the possibility that JNK mediates hypertrophic vs. endurance remodeling via SMAD2 regulation. SMAD2 is a mediator of the transforming growth factor (TGF)-β family of ligands, which are critical regulators of remodeling in many tissues21. One of the most important TGFβ ligands for skeletal muscle remodeling is myostatin- a master regulator of muscle mass and metabolism in humans and animals that signals via SMAD222. Therefore, we determined whether JNK-mediated SMAD2-linker phosphorylation regulates signaling or transcriptional activity in response to myostatin. To measure SMAD transcriptional activation, HEK293 cells were transfected with a SMAD binding element luciferase reporter (SBE4) in the presence or absence 20 nM myostatin. Myostatin induced a ~5-fold increase in luciferase accumulation in control (Empty Vector) cells (Fig. 5a). However, cells expressing active JNK1 had severely blunted basal (Vehicle) and myostatin-mediated SMAD transcriptional activity, demonstrating that JNK activation has an inhibitory effect on SMAD activity. Impaired SMAD transcriptional activity was associated with increased SMAD2-L phosphorylation in cells expressing active JNK (Fig. 5b). Thus, our data demonstrate that JNK activation initiates non-canonical phosphorylation of SMAD2 in its linker region, which has an inhibitory effect on SMAD2 activity.

Fig. 5 JNK inhibits myostatin/SMAD activity. a HEK293 cells were transfected with a SMAD-binding element (SBE4) luciferase reporter construct in the absence or presence of active JNK. 16 h following transfection, the cells were treated with 20 nM myostatin or Vehicle control for 4 h. Cells were lysed and analyzed for luciferase activity. b Western immunoblotting analysis was performed on whole-cell lysates from the luciferase experiment to assess levels of SMAD2 and JNK phosphorylation. c C2C12 myoblasts were transfected with empty vector (Control) or plasmids expressing active JNK fusion proteins. 24 h following transfection, the cells were treated with 20 nM myostatin or Vehicle for 30 min. Cytosolic and nuclear fractions were collected and immunoblotting was performed to assess the localization of total and phosphorylated SMAD2 and JNK. d,e Nuclear accumulation of phosphorylated SMAD2 (SMAD2C) (d) and total SMAD2 (e) were quantified. Data are representative of three independent experiments (a, c) and the data displayed are replicates from one representative experiment. pSMAD2-C, C-terminus phosphorylated SMAD2; pJNK, phosphorylated (active) c-Jun N-terminal Kinase; pSMAD2L, linker-region phosphorylated SMAD2; SMAD2/3, total SMAD2/3; GAPDH is a cytosolic loading control; Lamin A/C is a nuclear loading control. Stain-free gel images (Bio-Rad) are also shown as a loading control (Loading). *P < 0.05, **P < 0.001, ***P < 0.0001 vs vehicle treatment by two-way ANOVA with Sidak’s post hoc testing. Main effects of genotype (JNK activity) are displayed. Bar plots indicate mean ± SEM for all data Full size image

JNK inhibits myostatin-induced SMAD nuclear translocation

In canonical SMAD signaling, TGFβ ligands (e.g., myostatin) bind to cell surface receptors, which initiate receptor-mediated phosphorylation of SMAD2 on its C-terminus (pSMAD2-C), followed by SMAD2 translocation to the nucleus and transcriptional activation23,24. Active JNK reduced SMAD2-C phosphorylation in response to myostatin by ~20%, without altering SMAD2 protein levels (Fig. 5b). Therefore, impaired receptor-mediated phosphorylation may partly explain reduced SMAD transcriptional activity in the presence of active JNK. In some cellular contexts, linker-region phosphorylation of SMADs may prevent their translocation to the nucleus, and therefore transcriptional activity, even in the presence of receptor-mediated C-terminus phosphorylation23. Cytosolic and nuclear levels of SMAD2 were measured in C2C12 myoblasts to determine whether JNK-mediated SMAD linker phosphorylation prevents nuclear translocation in response to myostatin. Treatment with 20 nM myostatin for 30 min induced a threefold higher accumulation of pSMAD2-C and total SMAD2 in the nucleus of myoblasts (Fig. 5c–e). However, in cells overexpressing active JNK, the accumulation of SMAD2 in the nucleus was significantly blunted. Using an antibody that detects both SMAD2 and SMAD3 isoforms, we also demonstrate that SMAD3 accumulation in the nucleus was impaired in cells expressing active JNK. This indicates that reduced SMAD transcriptional activity with JNK overexpression (Fig. 5a), is likely due to effects on both the SMAD2 and SMAD3 isoforms. The effects of JNK activity on SMAD3 translocation may be due to direct phosphorylation at an unknown site, or through its complex formation with SMAD225. Consistent with a role for SMAD linker phosphorylation in preventing nuclear translocation, the immunoblotting signal for pSMAD2-L was much higher in the cytosolic fraction of myocytes, while pSMAD2-C was enriched in the nuclear fraction (Fig. 5c). Thus, our results demonstrate that JNK-mediated phosphorylation of SMAD2 in its linker region inhibits transcriptional activity in response to myostatin by impairing both C-terminus phosphorylation and nuclear translocation of SMAD2/3 (Fig. 5).

JNK suppresses myostatin activity in vivo

Myostatin acts to suppress muscle growth, while myostatin inhibition via genetic or pharmacological means leads to muscle fiber hypertrophy in humans and animals26. Consistent with a role for JNK in myostatin inhibition, mJNKKO mice display lower body weight and lean mass compared to WT (Fig. 6a, b). Expression of myostatin mRNA in skeletal muscle was similar between WT and mJNKKO mice (Fig. 6c). However, expression of the myostatin target genes, MuRF1 and MAFBX, were elevated in mJNKKO mice (Fig. 6c). Reduced lean mass and increased myostatin target gene expression in mJNKKO mice are consistent with evidence demonstrating JNK is a negative regulator of myostatin activity (Fig. 5).

Fig. 6 Lean mass and myogenic gene expression in mJNKKO mice. a, b Body weight was measured (a) and Lean mass (b) was calculated by DEXA in muscle-specific JNK1/2 knockout (mJNKKO) mice and wild-type (WT; MCK-Cre−/+) controls. N = 8 per group. c mRNA was extracted from the gastrocnemius muscle of WT and mJNKKO mice and expression levels of myogenic regulators and myostatin target genes were measured using RT-PCR. mRNA expression is expressed relative to β2-microglobulin housekeeping gene. Each data point represents the results from an individual animal. Bar plots indicate mean ± SEM for all data. P-values derived from unpaired t-tests are shown for each measurement. NS, not significant (P > 0.05) Full size image

Resistance exercise activates JNK/SMAD signaling in humans

Our data identify JNK activation as an inhibitor of TGFβ/myostatin signaling and a key mediator of muscle hypertrophy. To determine whether a JNK/SMAD2 signaling axis is activated in response to endurance or hypertrophic stimuli in humans, biopsies were taken from the vastus lateralis muscles of healthy young men at multiple time points before and after completion of a standard bout of acute endurance (cycling) or resistance (weighted leg extension) exercise. Resistance exercise robustly increased SMAD2-L phosphorylation ~4-fold in human skeletal muscle (Fig. 7a, b), with peak activation occurring immediately post-exercise. A secondary peak in SMAD2-L phosphorylation occurred 60 min post-exercise (Fig. 7b). JNK activation followed a very similar pattern to SMAD2-L phosphorylation with exercise (Fig. 7c). In contrast to resistance exercise, SMAD2-L and JNK were phosphorylated to a much smaller degree (<2-fold; not statistically significant) in response to endurance exercise in healthy humans (Fig. 7a–c). These data demonstrate that skeletal muscle JNK/SMAD2L signaling is upregulated by exercise in humans, and activation of this novel signaling axis is more consistent and robust following resistance exercise. Resistance exercise is the most effective modality for inducing muscle hypertrophy in humans. Thus, selective activation of JNK/SMAD signaling with resistance exercise is in line with data demonstrating JNK is necessary for overload-induced muscle hypertrophy in mice (Fig. 2).

Fig. 7 SMAD2-linker phosphorylation with exercise humans. a Healthy young men underwent a standard session of endurance exercise (cycling; N = 8 per time point) or resistance exercise (weighted leg extension; N = 7 per time point). Skeletal muscle biopsies were taken before exercise (R; rest), immediately post-exercise (Time 0), and 15, 30, and 60 min post-exercise. b, c The time course of SMAD2-linker phosphorylation (pSMAD-L) (b) and JNK activation (pJNK) (c) was determined. Mean ± SEM are shown for (b) and (c). d Individual levels of pSMAD2-L in resting (Rest) and immediately post-exercise (Exercise) biopsies for each subject that underwent endurance exercise are shown with values from the same subject joined by a line. e Linear regression analysis identified a significant correlation between JNK phosphorylation and pSMAD2-linker phosphorylation. *P < 0.05 vs Rest by two-way ANOVA and Tukey’s Multiple Comparison Testing. pSMAD2L, linker-region phosphorylated SMAD2; pJNK, phosphorylated (active) C-Jun N-terminal Kinase. Different exposures are shown for the P54 and P46 splice forms of JNK, as the P54 band appeared darker in human skeletal muscle Full size image

Although mean JNK/SMAD signaling was not significantly activated by endurance exercise in humans, the response was varied among subjects, with some individuals displaying no activation and others having >2-fold increases in phosphorylation (Fig. 7d). Heterogeneous activation of SMAD2-L phosphorylation with endurance exercise also occurs in animal models, and high-JNK/SMAD2-L activation predicts a poor adaptation to endurance training16. When data from all human muscle biopsies were pooled, patterns of SMAD2L phosphorylation mirrored JNK activation with exercise in humans, and linear regression analysis showed a significant correlation between pSMAD2-L and pJNK (Fig. 7e). Thus, we identify JNK-induced phosphorylation of SMAD2 in its linker region as a novel signaling axis in human skeletal muscle that is primarily activated by hypertrophic stimuli, such as resistance exercise. When considered collectively, our data support the hypothesis that activation of a JNK/SMAD2 signaling axis with resistance exercise induces muscle hypertrophy via TGFβ/myostatin inhibition; while suppression of JNK/SMAD signaling enhances endurance adaptations (Fig. 8).