Myostatin Knockout Affects Mitochondrial Function by Inhibiting the AMPK/SIRT1/PGC1α Pathway in Skeletal Muscle

Simple Summary Myostatin (Mstn) is a negative regulator of skeletal muscle mass, and its deletion leads to reduced mitochondrial function. However, the exact regulatory mechanism remains unclear. In this study, we used CRISPR/Cas9 to generate myostatin-knockout (Mstn-KO) mice via pronuclear microinjection. The skeletal muscle of Mstn-KO mice significantly increased, and the basal metabolic rate, muscle ATP synthesis, mitochondrial respiratory chain complex activity, tricarboxylic acid cycle (TCA), and thermogenesis decreased. In the muscle tissue of Mstn-KO mice, the expression of SIRT1 and pAMPK decreased, and the acetylation modification of PGC-1α increased. Furthermore, the treatment of isolated muscle cells from Mstn-KO and wild-type mice with AMPK activator (AICAR) and AMPK inhibitor (Compound C) found that Compound C down-regulated the expression of pAMPK and SIRT1 and the activity of citrate synthase (CS), isocitrate dehydrogenase (ICDHm) and α-ketoglutarate acid dehydrogenase (α-KGDH) similar to that of Mstn-KO. However, AICAR partially reversed the inhibitory effect of Mstn-KO on the expression of pAMPK and SIRT1 and activity of three enzymes. Thus, Mstn-KO affects mitochondrial function by inhibiting the AMPK/SIRT1/PGC1α signaling pathway. Abstract Myostatin (Mstn) is a major negative regulator of skeletal muscle mass and initiates multiple metabolic changes. The deletion of the Mstn gene in mice leads to reduced mitochondrial functions. However, the underlying regulatory mechanisms remain unclear. In this study, we used CRISPR/Cas9 to generate myostatin-knockout (Mstn-KO) mice via pronuclear microinjection. Mstn-KO mice exhibited significantly larger skeletal muscles. Meanwhile, Mstn knockout regulated the organ weights of mice. Moreover, we found that Mstn knockout reduced the basal metabolic rate, muscle adenosine triphosphate (ATP) synthesis, activities of mitochondrial respiration chain complexes, tricarboxylic acid cycle (TCA) cycle, and thermogenesis. Mechanistically, expressions of silent information regulator 1 (SIRT1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) were down-regulated, while peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) acetylation modification increased in the Mstn-KO mice. Skeletal muscle cells from Mstn-KO and WT were treated with AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR), and the AMPK inhibitor Compound C, respectively. Compared with the wild-type (WT) group, Compound C treatment further down-regulated the expression or activity of pAMPK, SIRT1, citrate synthase (CS), isocitrate dehydrogenase (ICDHm), and α-ketoglutarate acid dehydrogenase (α-KGDH) in Mstn-KO mice, while Mstn knockout inhibited the AICAR activation effect. Therefore, Mstn knockout affects mitochondrial function by inhibiting the AMPK/SIRT1/PGC1α signaling pathway. The present study reveals a new mechanism for Mstn knockout in regulating energy homeostasis.

In addition, the lack of Mstn leads to the decline of ATP synthesis capacity [12,13]. Mitochondria are the main energy-converting organelles in eukaryotic cells, producing adenosine triphosphate (ATP) through the tricarboxylic acid cycle (TCA cycle) and oxidative phosphorylation (OXPHOS), which is the basic energy molecule of the cell [14]. The OXPHOS system is embedded in the inner mitochondrial membrane and consists of five complexes, namely complex I (CI), complex II (CII), complex III (CIII), complex IV (CIV), and complex V (CV). These enzymes catalyze the oxidation of biological substrates and the synthesis of ATP [15]. It is possible to directly or indirectly represent the respiratory function of mitochondria through the respiratory chain complex enzyme activity [16]. It has also been reported that the mitochondrial membrane potential (∆Ψm) represents the energy stored in the mitochondrial electric field for the conversion of ADP to ATP [17]. There are reports that mice with deletion of the Mstn gene exhibit a marked decrease in mitochondria content and disturbance in respiratory function [18,19]. However, the signaling mechanisms by which Mstn regulates mitochondria activity are still unknown.
In this study, we built Mstn-KO mice by CRISPR/Cas9 to explore the mechanism that how Mstn regulated mitochondrial activity. We found that basal metabolic rate, mitochondrial electron transport chain complexes, mitochondrial membrane potential, and TCA cycle were inhibited in Mstn-KO mice. Meanwhile, the expression of SIRT1 and pAMPK was down-regulated and increased PGC-1α acetylation in the Mstn-KO mice. These results indicated that Mstn knockout suppressed mitochondrial function probably by inhibiting the AMPK/SIRT1/PGC1alpha pathway.

Generation of Mstn-KO Mice by CRISPR/Cas9 System
To generate Mstn-KO mice by CRISPR/Cas9 techniques, we designed four guide RNAs to target exon 2 and exon 3 of the mouse MSTN gene, respectively (Figure 1a). Three transgenic founders were generated by pronuclear injection. To produce obvious phenotype Mstn-KO mice (F2), we mated only exon 3 deletion mutant F1 mice. A total of 10 mice (59%) among 17 F2 mice were identified as Mstn mutants, showing nine different genotypes with deletions ranging from 5 to 8 nt (Figure 1b). Phenotypic analysis showed that the Mstn-KO exhibited muscle hypertrophy in the skeletal muscle of Mstn-KO mice ( Figure 1c). We isolated single muscle fibers from mice gastrocnemius and found that the Mstn-KO mice were significantly thicker than the WT mice ( Figure 1d). Furthermore, MSTN protein expression was significantly decreased in the Mstn-KO mice compared with the WT mice ( Figure 1e).
To generate Mstn-KO mice by CRISPR/Cas9 techniques, we designed four guide RNAs to target exon 2 and exon 3 of the mouse MSTN gene, respectively (Figure 1a). Three transgenic founders were generated by pronuclear injection. To produce obvious phenotype Mstn-KO mice (F2), we mated only exon 3 deletion mutant F1 mice. A total of 10 mice (59 %) among 17 F2 mice were identified as Mstn mutants, showing nine different genotypes with deletions ranging from 5 to 8 nt (Figure 1b). Phenotypic analysis showed that the Mstn-KO exhibited muscle hypertrophy in the skeletal muscle of Mstn-KO mice (Figure 1c). We isolated single muscle fibers from mice gastrocnemius and found that the Mstn-KO mice were significantly thicker than the WT mice ( Figure 1d). Furthermore, MSTN protein expression was significantly decreased in the Mstn-KO mice compared with the WT mice ( Figure 1e).

Growth Performance and Phenotypic Traits
We compared and analyzed growth performances and phenotypic characteristics between Mstn-KO and WT mice (from 3 to 10 weeks). The average body weights of Mstn-KO and WT mice increased continuously from 3 to 10 weeks (Figure 2a,b). After 6 weeks, the average body weight of Mstn-KO male mice was significantly higher than WT mice (Figure 2a). However, the average body weight of Mstn-KO female mice was continuously higher than WT mice from 7 weeks (Figure 2b). Moreover, compared with the WT mice, the weights of the liver, spleen, lungs, thyroid, pancreas, brain, testis, and ovary were decreased by 0.55%, 33.51%, 10.01%, 0.26%, 14.51%, 24.23%, 30.79%, and 14.51%, respectively, in Mstn-KO. The heart and kidney weight of Mstn-KO mice was higher at 22.13% and 2.59% (Table 1). We investigated the expression of Mstn in the skeletal muscle and internal organs of WT and Mstn-KO mice using real-time quantitative PCR. Mstn mRNA expression in the heart, liver, lungs, kidneys, pancreas, brain, and muscle was significantly lower in Mstn-KO mice than in WT mice, while no signal was detected in the spleen (Figure 2c). the weights of the liver, spleen, lungs, thyroid, pancreas, brain, testi decreased by 0.55%, 33.51%, 10.01%, 0.26%, 14.51%, 24.23%, 30.79%, a tively, in Mstn-KO. The heart and kidney weight of Mstn-KO mice wa and 2.59% (Table 1). We investigated the expression of Mstn in the s internal organs of WT and Mstn-KO mice using real-time quantitative expression in the heart, liver, lungs, kidneys, pancreas, brain, and m cantly lower in Mstn-KO mice than in WT mice, while no signal was de ( Figure 2c).  All values are presented as mean ± SD (n = 6). KO, Mstn-KO mice; WT, WT mi . All data are presented as mean ± SD. * p < 0.05, ** p < 0.01; t-tests were used to calculate the p-values. All values are presented as mean ± SD (n = 6). KO, Mstn-KO mice; WT, WT mice.

Effect of Mstn Knockout on Basal Metabolic Rate and Body Temperature
Next, we evaluated the basal metabolic rate (BMR) in Mstn-KO and WT mice. The levels of VO 2 , VCO 2 , and respiratory quotient (CO 2 release/O 2 consumption, RQ) of the resting state were examined in the groups. Compared to the WT mice, Mstn-KO mice consumed less O 2 (2.40 ± 0.47 ml/min for Mstn-KO and 2.57 ± 0.25 ml/min for WT) and released less CO 2 (1.65 ± 0.34 ml/min for Mstn-KO and 1.95 ± 0.26 ml/min for WT) ( Table 2).
RQ was reduced in Mstn-KO mice. Mstn-KO mice have a lower BMR (basal metabolic rate) compared with the WT mice (Figure 3a). In a resting state, the two major components of energy expenditure are the basal metabolic rate and maintaining body temperature. The body temperature of mice was monitored in real time over seven consecutive days. We found that body temperature was slightly lower in either male or female Mstn-KO mice compared to WT mice, and females had slightly higher body temperatures than males within the same group. (Figure 3b). However, the body temperature of these groups was maintained in the normal range. These results suggest that Mstn knockout reduced energy expenditure in a resting state.

Effect of Mstn Knockout on Basal Metabolic Rate and Body Temperature
Next, we evaluated the basal metabolic rate (BMR) in Mstn-KO and WT mice. The levels of VO2, VCO2, and respiratory quotient (CO2 release/O2 consumption, RQ) of the resting state were examined in the groups. Compared to the WT mice, Mstn-KO mice consumed less O2 (2.40 ± 0.47 ml/min for Mstn-KO and 2.57 ± 0.25 ml/min for WT) and released less CO2 (1.65 ± 0.34 ml/min for Mstn-KO and 1.95 ± 0.26 ml/min for WT) ( Table 2). RQ was reduced in Mstn-KO mice. Mstn-KO mice have a lower BMR (basal metabolic rate) compared with the WT mice (Figure 3a). In a resting state, the two major components of energy expenditure are the basal metabolic rate and maintaining body temperature. The body temperature of mice was monitored in real time over seven consecutive days. We found that body temperature was slightly lower in either male or female Mstn-KO mice compared to WT mice, and females had slightly higher body temperatures than males within the same group. (Figure 3b). However, the body temperature of these groups was maintained in the normal range. These results suggest that Mstn knockout reduced energy expenditure in a resting state.

Mstn Knockout Reduced Mitochondria Activity
It is well-established that mitochondria are the center of cellular energy metabolism. The mitochondrial TCA cycle and the electron transport chain are the two main components that determine mitochondrial energy metabolism. Firstly, we measured the total ATP content of muscle tissues. The results were that ATP synthesis was significantly decreased in the Mstn-KO mice (Figure 4a). To further explore the effects of Mstn on the main processes of energy metabolism, we examined the individual activities of mitochondrial electron transport chain complexes I-V. The data showed that the activities of complexes I to V were reduced 0.6-fold, 0.74-fold, 0.77-fold, 0.53-fold, and 0.7-fold in Mstn-KO with Mstn-KO male and female mice (n = 1). All data are presented as mean ± SD. * p < 0.05; t-tests were used to calculate the p-values.

Mstn Knockout Reduced Mitochondria Activity
It is well-established that mitochondria are the center of cellular energy metabolism. The mitochondrial TCA cycle and the electron transport chain are the two main components that determine mitochondrial energy metabolism. Firstly, we measured the total ATP content of muscle tissues. The results were that ATP synthesis was significantly decreased in the Mstn-KO mice (Figure 4a). To further explore the effects of Mstn on the main processes of energy metabolism, we examined the individual activities of mitochondrial electron transport chain complexes I-V. The data showed that the activities of complexes I to V were reduced 0.6-fold, 0.74-fold, 0.77-fold, 0.53-fold, and 0.7-fold in Mstn-KO mice, respectively (Figure 4b-f). Furthermore, Mstn knockout decreased mitochondrial membrane potential compared with the WT mice (Figure 4g). We further investigated the mRNA levels of the mitochondrial activity gene by qPCR. Lower transcript levels of Tfam, Nrf, and CIpp genes were found in Mstn-KO mice (Figure 4h).  . All data are presented as mean ± SD. * p < 0.05, ** p < 0.01; t-tests were used to calculate the p-values.

Mstn Knockout Inhibited the TCA Cycle
We further examined key enzymes and metabolites in the TCA cycle. Citrate synthase and citrate acids are enzymes and a product of the initial step in the TCA cycle. As shown in Figure 5a,b, citrate content and citrate synthase activity were reduced in the Mstn-KO mice compared to the WT mice. In addition, isocitrate dehydrogenase activity and α-ketoglutarate content were decreased in the Mstn-KO mice (Figure 5c,d). Isocitrate dehydrogenase converts isocitrate to α-ketoglutarate in the TCA cycle. These results indicated that Mstn knockout decreased mitochondrial function whether electron transport chain or TCA cycle. . All data are presented as mean ± SD. * p < 0.05, ** p < 0.01; t-tests were used to calculate the p-values.

Mstn Knockout Inhibited the TCA Cycle
We further examined key enzymes and metabolites in the TCA cycle. Citrate synthase and citrate acids are enzymes and a product of the initial step in the TCA cycle. As shown in Figure 5a,b, citrate content and citrate synthase activity were reduced in the Mstn-KO mice compared to the WT mice. In addition, isocitrate dehydrogenase activity and α-ketoglutarate content were decreased in the Mstn-KO mice (Figure 5c,d). Isocitrate dehydrogenase converts isocitrate to α-ketoglutarate in the TCA cycle. These results indicated that Mstn knockout decreased mitochondrial function whether electron transport chain or TCA cycle. . All data are presented as mean ± SD. * p < 0.05, ** p < t-tests were used to calculate the p-values.

Mstn Knockout Inhibited AMPK/SIRT1/PGC1alpha Pathway
Previous studies have demonstrated that AMPK regulates mitochondrial fu [27]. Thus, we next determined whether the phosphorylation level AMPK chan Mstn-KO mice skeletal muscle. As expected, compared to the WT mice, the expres pAMPK was significantly downregulated (Figure 6a,b). We further examined the e sion of SIRT1 of the downstream molecule of AMPK by Western blot. Mstn-KO mi significantly decreased levels of SIRT1 (Figure 6a,c). Moreover, to investigate the a tion level of PGC1α protein in Mstn-KO and WT mice, we detected the acetylated P protein in Mstn-KO and WT mice. Since no commercial acetylation antibody of P protein was available, the pan acetyllysine antibody was used to assess the acet level of PGC1α by IP analysis. Briefly, PGC1α was pulled down with the anti-PGC tibody, and an IP/Western blot assay was carried out to analyze the acetylation of P using the previously reported method [28,29]. Mstn knockout resulted in PGC1α a tion increase, suggesting that PGC-1α activity was decreased (Figure 6d,e). This f supports previous findings that PGC-1α expression is subject to auto-regulation in oration with SIRT1, which activates PGC-1α through deacetylation. Taken together findings show that Mstn knockout inhibited the AMPK/SIRT1/PGC1alpha pathwa . All data are presented as mean ± SD. * p < 0.05, ** p < 0.01; t-tests were used to calculate the p-values.

Mstn Knockout Inhibited AMPK/SIRT1/PGC1alpha Pathway
Previous studies have demonstrated that AMPK regulates mitochondrial function [27]. Thus, we next determined whether the phosphorylation level AMPK changes in Mstn-KO mice skeletal muscle. As expected, compared to the WT mice, the expression of pAMPK was significantly downregulated (Figure 6a,b). We further examined the expression of SIRT1 of the downstream molecule of AMPK by Western blot. Mstn-KO mice had significantly decreased levels of SIRT1 (Figure 6a,c). Moreover, to investigate the acetylation level of PGC1α protein in Mstn-KO and WT mice, we detected the acetylated PGC1α protein in Mstn-KO and WT mice. Since no commercial acetylation antibody of PGC1α protein was available, the pan acetyllysine antibody was used to assess the acetylation level of PGC1α by IP analysis. Briefly, PGC1α was pulled down with the anti-PGC1α antibody, and an IP/Western blot assay was carried out to analyze the acetylation of PGC1α using the previously reported method [28,29]. Mstn knockout resulted in PGC1α acetylation increase, suggesting that PGC-1α activity was decreased (Figure 6d,e). This finding supports previous findings that PGC-1α expression is subject to auto-regulation in collaboration with SIRT1, which activates PGC-1α through deacetylation. Taken together, these findings show that Mstn knockout inhibited the AMPK/SIRT1/PGC1alpha pathway.

Expression of pAMPK and SIRT1 Following Treatment with AICAR and Compound C
To further explore the relationship between MSTN and the AMPK/SIRT1/PGC pathway in skeletal muscle mitochondrial function, cells of Mstn-KO and WT mice w treated with AMPK activator AICAR and the AMPK inhibitor Compound C. AICAR i AMP analog. Similar to AMP, AICAR binds to the γ subunit of AMPK, allosterically a vates the enzyme, stimulates phosphorylation at Thr 172 by liver kinase B1 (LKB1), protects against pThr 172 dephosphorylation [30]. Compound C is an ATP-competitive hibitor and binds to the highly conserved active site of AMPK [31]. SIRT1 and pAM protein expression and activity of citrate synthase (CS), isocitrate dehydrogen (ICDHm), and α-ketoglutarate acid dehydrogenase (α-KGDH) were determined by W ern blotting analysis and biochemical detection methods, respectively. AICAR-trea cells exhibited increased pAMPK and SIRT1 expression compared to AICAR-untrea cells (Figure 7a-c). The expressions of pAMPK and SIRT1 proteins were decreased in cells compared with WT cells (Figure 7a-c). Meanwhile, the activity of CS, ICDHm, α-KGDH in the treated and untreated cells obtained similar results (Figure 7d-f). AICAR effects were inhibited by the Mstn knockout (Figure 7a-f). Furthermore, Mstncells showed dramatically decreased pAMPK and SIRT1 protein levels and activity of three enzymes compared to WT cells, and the same results were also obtained in treated group compared with the untreated group (Figure 7g-l). The SIRT1 and pAM expression and activity of the three enzymes of Mstn-KO cells were reduced furthe response to compound c stimulation (Figure 7g-l). These results suggest that Mstn kno out reduces mitochondrial function by inhibiting AMPK-SIRT1 signaling. . All data are presented as mean ± SD. ** p < 0.01; t-tests were used to calculate the p-values.

Expression of pAMPK and SIRT1 following Treatment with AICAR and Compound C
To further explore the relationship between MSTN and the AMPK/SIRT1/PGC1α pathway in skeletal muscle mitochondrial function, cells of Mstn-KO and WT mice were treated with AMPK activator AICAR and the AMPK inhibitor Compound C. AICAR is an AMP analog. Similar to AMP, AICAR binds to the γ subunit of AMPK, allosterically activates the enzyme, stimulates phosphorylation at Thr 172 by liver kinase B1 (LKB1), and protects against pThr 172 dephosphorylation [30]. Compound C is an ATP-competitive inhibitor and binds to the highly conserved active site of AMPK [31]. SIRT1 and pAMPK protein expression and activity of citrate synthase (CS), isocitrate dehydrogenase (ICDHm), and α-ketoglutarate acid dehydrogenase (α-KGDH) were determined by Western blotting analysis and biochemical detection methods, respectively. AICAR-treated cells exhibited increased pAMPK and SIRT1 expression compared to AICAR-untreated cells (Figure 7a-c). The expressions of pAMPK and SIRT1 proteins were decreased in MT cells compared with WT cells (Figure 7a-c). Meanwhile, the activity of CS, ICDHm, and α-KGDH in the treated and untreated cells obtained similar results (Figure 7d-f). The AICAR effects were inhibited by the Mstn knockout (Figure 7a-f). Furthermore, Mstn-KO cells showed dramatically decreased pAMPK and SIRT1 protein levels and activity of the three enzymes compared to WT cells, and the same results were also obtained in the treated group compared with the untreated group (Figure 7g-l). The SIRT1 and pAMPK expression and activity of the three enzymes of Mstn-KO cells were reduced further in response to compound c stimulation (Figure 7g-l). These results suggest that Mstn knockout reduces mitochondrial function by inhibiting AMPK-SIRT1 signaling.

Discussion
Mstn is a potent inhibitor of skeletal muscle mass [32]. Mstn knockout animals all showed a skeletal muscle hypertrophy phenotype [7,[33][34][35][36][37][38]. The Mstn-knockout mice we obtained also showed a hypertrophic phenotype. In addition to the characteristic effects on the skeletal muscle, Mstn knockout can affect other organs. Currently, the weight of organs has previously been reported on cattle, mice, and piglets of Mstn deficiency. At 15 or 20 months of age, the weights of the heart, liver, spleen, and lungs of Charolais double muscle cattle decreased by 20%, 20%, 30%, and 10%, respectively [39]. Moreover, at 4, 8, and 12 weeks of age, the kidneys and liver of Mstn-deficient mice were lighter than those of WT mice, while the weight of the heart and lungs were similar [40]. In MSTN-KO piglets, the weights of the heart, liver, lungs, kidneys, and stomach were decreased by 21.4%, 21.3%, 29.8%, 16.7%, and 20.0% relative to body weight, respectively [41]. In the current study, the weights of the liver, spleen, lungs, thyroid, pancreas, brain, testis, and ovary were decreased by 0.55%, 33.51%, 10.01%, 0.26%, 14.51%, 24.23%, 30.79%, and 14.51%, respectively, whereas the heart and kidney weight of Mstn-KO mice was higher in 22.13% and 2.59%. We reasoned that Mstn might exert a different effect on organ weight in different species.
Mstn knockout is closely related to skeletal muscle metabolism [42,43]. Several studies have reported that Mstn knockout decreases ATP production during exercise [12,44]. Moreover, Li et al. found that knockout Mstn in loach significantly decreased ATP synthesis by directly measuring the total ATP content of loach muscle tissue [13]. In this study, we demonstrated that Mstn knockout muscle decreased ATP synthesis in the resting state of Mstn-KO mice. Interestingly, we also found that the basal metabolic rate and body temperature were significantly decreased in Mstn-KO mice correlating with the reduced ATP synthesis capacity. Indeed, mitochondria produce most of the ATP in cells [45]. Our present study confirmed that mitochondrial electron transport chain complexes, mitochondrial membrane potential, and TCA cycle were reduced in Mstn-KO mice muscle. These results further reveal that Mstn knockout impact mitochondrial function, as studied previously in Mstn KO animals [13,18,19].
AMPK, SIRT1 and PGC1α are all involved in regulating mitochondrial function [20][21][22]. According to Price and his colleagues [46], resveratrol improves mitochondrial biogenesis and function by activating SIRT1. SIRT1 activates PGC1α by deacetylation [47]. Melatonin prevents mitochondrial fission through the SIRT1-PGC1α pathway [48]. Meanwhile, salidroside may treat diabetic nephropathy in mice through SIRT1-PGC1α mediated mitochondrial biogenesis [49]. In addition, AMPK regulates SIRT1 activity by modulating intracellular NAD + levels and thereby influencing PGC1α deacetylation [50]. We have found, for the first time, that Mstn knockout down-regulated the expressions of SIRT1 and pAMPK, enhancing PGC-1α acetylation. Skeletal muscle cells from Mstn-KO and WT were treated with AMPK activators AICAR and the AMPK inhibitor Compound C, respectively. Compared with WT mice, Compound C treatment further down-regulated the expression of pAMPK and SIRT1 expression and activity of CS, ICDHm, and α-KGDH in Mstn-KO mice, while Mstn knockout inhibited the AICAR activation effect. Therefore, Mstn knockout inhibited mitochondrial function via the AMPK/SIRT1/PGC1α signaling pathway.

Mstn-KO Mouse Production and Validation
The Mstn-KO mice were generated by pronuclear microinjection. The sgRNA oligos were synthesized and cloned into the pCas-Guide-EF1α-GFP plasmid downstream at the BamHI and BsmBI restriction sites to generate the pCas-Guide-EF1α-GFP-sgRNA recombinant plasmid. The positive clones were confirmed by Sanger sequencing. The purified transgene was microinjected into the male pronuclei of fertilized eggs from superovulated female mice and transferred to recipient pseudopregnant females. The mouse genotypes were determined by PCR-based assays; the primers used for genotyping are listed in Table 3. Table 3. Primers used for real-time PCR and genotyping PCR.

Body Temperature Measurements
The body temperature of the animals was measured daily by a subcutaneously located temperature chip.

Metabolic Measurements
Mice were individually housed in the metabolic cages (Oxylet) and acclimatized for 24 h before recording. Their 24 h oxygen consumption (VO 2 ), carbon dioxide production (VCO 2 ), and respiratory quotient (RQ) were measured every hour for 3 min in each cage. Mice were maintained on their normal diet or water throughout the detection process.

Characterization and Analysis of Organs
Healthy mice from each group (Mstn-KO and WT) were euthanized and tissues were collected for experimental purposes. The organs analyzed were the spleen, brain, lungs, pancreas, heart, liver, kidney, thyroid, testicle, and ovary. Body weight and organ weight were calculated.

Real-Time PCR
Real-time PCR was performed referring to our previous reported [51]. Primer sequences were as tabulated in Table 3.

Co-Immunoprecipitation
Lysates of mice skeletal muscle tissue generated under the addition of proteinase inhibit cocktail Complete Mini (Thermo, Waltham, MA, USA) and phosphatase inhibitor cocktail PhosSTOP (Thermo, Waltham, MA, USA). The total protein of the lysates was measured by the Pierce BCA Protein Assay Kit (Thermo, Waltham, MA, USA). Co-immunoprecipitation (co-IP) was completed using the Thermo Scientific Pierce co-IP kit (#26149) following the manufacturer's protocol. Ten micrograms of the antibody were incubated with the deliv-Institutional Review Board Statement: The animal study protocol was approved by the Institutional Animal Care and Use Committee of Inner Mongolia University (No. IMU-MICE-2020-037, 20 December 2020).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.