Myostatin Knockout Limits Exercise-Induced Reduction in Bovine Erythrocyte Oxidative Stress by Enhancing the Efficiency of the Pentose Phosphate Pathway

Simple Summary Myostatin (MSTN) is mainly expressed in skeletal muscle and is involved in the regulation of skeletal muscle growth and development. Loss of MSTN results in muscle hypertrophy. In this study; red blood cells were used as materials to study the effect of MSTN on the antioxidant capacity of bovine erythrocyte after exhaustive exercise. The findings suggest that knockdown of MSTN accelerates the pentose phosphate pathway; thereby enhancing the antioxidant capacity of erythrocytes. Abstract Moderate exercise can strengthen the body, however, exhaustive exercise generates large amounts of reactive oxygen species (ROS). Although erythrocytes have antioxidant systems that quickly eliminate ROS, erythrocytes become overwhelmed by ROS when the body is under oxidative stress, such as during exhaustive exercise. Myostatin (MSTN) has important effects on muscle hair development. Individuals lacking myostatin (MSTN) exhibit increased muscle mass. The purpose of this study was to investigate the mechanism by which MSTN affects erythrocyte antioxidant changes after exhaustive exercise in cattle. Antioxidant and metabolite detection analysis, western blotting, immunofluorescence, and fatty acid methyl ester analysis were used to assess exercise-associated antioxidant changes in erythrocytes with or without MSTN. Knockdown of MSTN enhances Glucose-6-phosphate dehydrogenase (G6PD) activity after exhaustive exercise. MSTN and its receptors were present on the erythrocyte membrane, but their levels, especially that of TGF-β RI, were significantly reduced in the absence of MSTN and following exhaustive exercise. Our results suggest that knockout of MSTN accelerates the pentose phosphate pathway (PPP), thereby enhancing the antioxidant capacity of erythrocytes. These results provide important insights into the role of MSTN in erythrocyte antioxidant regulation after exhaustive exercise.


Introduction
Erythrocytes (i.e., red blood cells) are the primary transporter of oxygen in the blood in vertebrates [1]. This critical function can be impaired by cellular damage caused by reactive oxygen species (ROS). Major cellular ROS include superoxide anion (O 2 -), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (OH). The two primary sources of cellular ROS are the mitochondrial electron transport chain [2], and substrate oxidation by NADPH oxidase (NOX) and other oxidases [3]. Mature mammalian erythrocytes are anucleate (i.e., have no DNA) and lack mitochondria [4], instead producing energy by anerobic glycolysis [5].
In this study, we investigated how MSTN knockout affects erythrocyte oxidative damage and antioxidant capacity. Specifically, using an exhaustive exercise model in cattle, we assessed the effect of MSTN knockout on exercise-induced oxidative damage in erythrocytes. We found that exhaustive exercise increased oxidative damage in bovine erythrocytes, and MSTN knockout enhanced the erythrocyte antioxidant capacity by enhancing metabolism through the PPP. These results provide insight into the antioxidant capacity of erythrocytes of MSTN after exhaustive exercise.

Ethics Statement
All experimental procedures used in this research were in accordance with the the Regulation on the Administration of Laboratory Animals (2017, China State Council). All protocols were approved by the Animal Ethics Committee of Inner Mongolia University. This experiment was carried out with the approval of the ethics committee of experimental animals of Inner Mongolia University (No. IMU-CATTLE-2020-033, 1 April 2020).

Animals
As in our previous report, generation of MSTN-knockout Luxi cattle using CRISPR/ Cas9 [31,32]. Wild-type and MSTN knockout cattle (10 animals each), 24 months of age, were used in this study. All cattle were raised in the same field and were fed under the same conditions.
The weight, height and body length of MSTN knockout cattle and wild cattle are shown in the Supplementary Tables (Tables S1-S3). Red blood cells were collected from the neck of cattle, and 5 mL EDTA K 2 blood vessels were collected. The obtained blood was added into a 50 mL centrifuge tube and PBS (PBS: blood = 9:1) was added. Centrifugation at 4 • C for 30 min could obtain red blood cells.

Experimental Design
Before exhaustive exercise, cattle were fasted for 12 h, at which point resting-state (RS) blood samples were collected from the jugular vein. For exercise to failure, the cow ran on the ground for 3 h at a moderate speed, covering a distance of 10 km. Cattle were considered exhausted when they would no longer run, even when prodded. After a short rest (10 min), an exhaustive-exercise-state (EE) blood sample was collected from the neck vein. Cattle were subjected to exhaustive exercise once a week for 3 weeks ( Figure 1) [33][34][35].

Blood Sample Preparation
Blood samples were poured into EDTA-containing tubes and serum-separator tubes immediately following collection. The EDTA-tubes samples were washed three times with PBS by centrifugation (1500 rpm/min, 15 min, 4 • C), after which the plasma and white blood cells were removed, and the packed erythrocytes were collected. The sera from samples in serum-separator tubes were centrifuged (4000× g, 5 min, 4 • C) and supernatants were collected for analysis.

Antioxidant and Metabolite Detection Analysis
Erythrocytes were analyzed by commercial kits following the manufacturer's instructions. Briefly, 5 × 10 5 cells were lysed in erythrocyte lysis buffer by sonication (30 cycles, 3 s pulses, 20% power, 10 s intervals). The supernatant was added to a 96-well plate containing the reagents, and well absorbances were measured using a spectrophotometer (Thermo, Waltham, MA, USA). Absorbance values were used to calculate the activities of various enzymes, including SOD (SOD-1

Fatty Acid (FA) Methyl Ester Analysis
Erythrocytes were washed twice with PBS and mixed with 1 mL of 2.5% (v/v) H 2 SO 4 in methanol. Samples were incubated at 80 • C for 90 min. After cooling to room temperature (RT), 1.5 mL of 0.9% NaCl was added. The samples were mixed, vortexed (5 min), and then centrifuged (2000 rpm/min, 5 min) to isolate the FA-containing organic phase. After transferring the supernatants to fresh tubes, 0.4 mL of a saturated KOH solution in methanol was added, and samples were mixed, vortexed (5 min), and centrifuged (2000 rpm/min, 10 min). The supernatant FA were analyzed using gas chromatography-mass spectroscopy (Shimadzu, Kyoto, Japan).

MSTN Protein Was Detected by ELISA
The isolated erythrocytes were tested according to the kit is a bovine MSTN ELISA Kit (CSB-EL015057BO, CUSABIO, Wuhan, China) instructions. Remove each reagent to room temperature for 30 min; Add 100 µL standard or sample to each well, and incubate at 37 • C for 2 h. 100 µL biotin-labeled antibody working solution, incubated at 37 • C for 1 h; Wash 3 times, soak for 2 min each, 200 µL/ well, dry by swing; Add 100 µL of pepper root peroxidase labeled avidin working solution to each well, and incubate at 37 • C for 1 h. Wash solution 5 times, soak 2 min each, 200 µL/ well, spin-dry; Add 90 µL substrate solution to each well, and shade at 37 • C for 18 min. Add 50 µL stop solution to each well to stop the reaction. Within 5 min after the termination of the reaction, a microplate reader with a wavelength of 450 nm was used. The optical density (OD) of each well was measured, and the MSTN protein content in the sample was calculated according to the standard curve. The kit detects the mature peptide of MSTN.

Immunofluorescence Staining
Erythrocytes were rinsed three times with PBS, fixed with 4% paraformaldehyde 10 min at RT, permeabilized with 0.01% Triton X-100 for 30 min at RT, and then thoroughly washed with 0.3% BSA in PBS. Fixed samples were blocked with 3% BSA, in PBS at 37 • C for 1 h and then incubated with anti-Band 3 antibody overnight at 4 • C. Samples were then washed several times in PBS and incubated for 1 h at 37 • C with CoraLite594-conjugated Goat Anti-Rabbit IgG (H + L) secondary antibodies (Proteintech, Wuhan, China). Stained erythrocytes were imaged on a laser scanning microscope (Nikon, Tokyo, Japan).

Calculations and Statistical Analyses
All data are expressed as the mean ± standard error of the mean of at least three independent experimental replicates. In graphs, bars represent means, and error bars represent one standard error. Statistical significance was evaluated by two-way ANOVA and Welch's two-tailed t-test with Bonferroni correction for post hoc analysis to adjust for multiple comparisons; p-values < 0.05 were considered statistically significant (*, p < 0.05; **, p < 0.01).

Effect of Exhaustive Exercise on Bovine Hematological Parameters
The effects of exhaustive exercise on bovine hematological parameters are shown in Table 1 and Figure 2. There were no significant differences between Wild-type (WT) and MSTN knockou (MT) cattle for any parameter at either resting-state (RS) or Exhaustive exercise (EE). EE caused slight decreases in red cell distribution width, mean corpuscular volume, and haemoglobin (HGB) levels, and a slight increase in hematocrit in both groups (Table 1). Additionally, plasma lactic acid (LA) levels increased dramatically after exhaustive exercise in both MT cattle (RS vs. EE, 5.64 ± 0.24 vs. 15.05 ± 1.62 µg/mL) and WT cattle (RS vs. EE, 5.49 ± 0.24 vs. 12.44 ± 0.84 µg/mL; Figure 2a). The concentration of Methemoglobin (MHB) in MT group was significantly lower than that in WT group at RS (12.54 ± 0.138 vs. 16.73 ± 0.37 µg/mL) and EE (33.62 ± 0.29 vs. 49.83 ± 1.72 µg/mL), and the MHB increased significantly after exhaustive exercise in both MT cattle and WT cattle group (Figure 2b). The Methemoglobin Reductase (MHBR) is closely related to the content of MHB, and was significantly higher in MT group than in WT group both at RS (344.97 ± 11.37 vs. 256.97 ± 5.8 U/L) and EE (927.13 ± 11.522 vs. 488.80 ± 21.65 U/L; Figure 2c). These results indicated that the cattle model of exhaustive exercise was effective, it also indicates that MSTN knockout affects the oxidation of red blood cells.
We next examined if the elevated exercise-induced ROS levels caused erythrocyte protein or lipid damage. Protein carbonyl levels were elevated significantly in WT-EE erythrocytes (RS vs. EE, 6.79 ± 0.29 vs. 9.38 ± 0.41 µmol/10 8 cells) and increased less markedly in WT-EE erythrocytes (RS vs. EE, 6.59 ± 0.  FA is an important structural component of erythrocyte membrane and can be destroyed by ROS. After exercise, total FA concentrations in MT and WT erythrocytes decreased, respectively ( Figure S1A). MSTN knockdown slightly decreased the SFA content of RS and significantly decreased the SFA content of EE. For UFA, there was no significant difference between MT and WT erythrocytes before and after exercise, and exercise did not affect UFA levels ( Figure S1B). However, UFAs include monounsaturated FA (MUFA) and polyunsaturated FA (PUFA), and MUFA concentrations in MT erythrocytes and WT erythrocytes were significantly reduced upon EE. There was no significant difference in PUFA between MT and WT erythrocytes before and after exercise ( Figure S1C). C18:3n6 and C20:3n6 concentrations were low in all groups and showed little variation ( Figure S1E).

MSTN Knockout Affects Erythrocyte Band 3
Band 3 is a key protein regulating erythrocyte metabolism. Exhaustive exercise reduced the expression of Band3, there was no difference in Band3 expression between WT-RS and MT-RS, while the expression of Band3 in MT-EE was lower than WT-EE (Figure 7a-d,f). Homozygous (MSTN −/− ), heterozygotes (MSTN +/− , MT) and wild-type (WT) Band 3 were detected in the resting state, and the results showed that homozygous (MSTN −/− ) Band3 protein expression was lower than that of heterozygotes (MSTN +/− , MT) and wild type (WT) ( Figure S2A). We used ELISA to detect the expression of MSTN in erythrocytes of MT cattle and WT cattle before and after exhaustive exercise. The results showed that the MSTN protein content of MT-RS cattle was significantly lower than that of the control group (22.102 ± 1.497 vs. 37.9 ± 1.954 ng/10 7 cell, Figure 7e). And it also showed the same trend after exhaustive exercise (12.159 ± 1.576 vs. 21.42 ± 0.333 ng/10 7 cell, Figure 7e). Exhaustive exercise can decreased expression of MSTN receptor Act RII and TGF-βRI. Knockout of MSTN did not affect the expression of ActRII, MT-RS and WT-RS showed no difference in ActRII content in resting state, MT-EE and WT-EE showed the same results after exhaustive exercise. In the resting state, the TGF-βRI, MT-RS receptor is significantly higher than WT-RS (Figure 7f). Exhaustive exercise made TGF-βRI expression is decreased, but MT-EE is significantly higher than WT-EE (Figure 7f).

Discussion
Exhaustive exercise significantly decreased bovine erythrocyte antioxidant capacity and increased oxidative damage, indicating that exhaustive exercise disrupts redox balance in bovine erythrocytes, resulting in a more oxidized state. LA is released from muscles into the blood based on the type, intensity, and duration of exercise, and intensity exercise increases the level of lactic acid in the blood [36]. In this study, lactic acid content increased significantly after exhaustive exercise. Erythrocyte HCT will be increased by exercise [34]. According to the blood routine results of this study, we found that red blood cell HCT showed an upward trend after exhaustive exercise. Exhaustive exercise decreased membrane levels of FA, especially SFA and MUFA, resulting in decreased membrane fluidity. However, C20:4n-6 levels, which correlate with erythrocyte oxalate levels [37], C20:4n-6 gives rise to prostaglandins and leukotrienes that are proinflammatory in a context-dependent fashion [38]. Surprisingly, SOD, POD, and GSH-Px activities decreased after exercise, as did GSH levels. However, exhaustive exercise did not significantly decrease protein sulfhydryl levels, despite that GSH functions to keep protein thiols in a reduced state, thereby preserving erythrocyte function. Our results suggest that, in an attempt to quickly restore GSH levels after exhaustive exercise, erythrocyte glycolysis is inhibited and the PPP is activated, thereby increasing NADPH synthesis and accelerating GSH transformation.
Under normal conditions, erythrocytes maintain redox balance, and MSTN knockout does not affect erythrocyte oxidative damage. However, exhaustive exercise disrupts erythrocyte redox balance, resulting in oxidative damage; it is under these conditions that MSTN knockout has evident effects. Compared with WT erythrocytes, MT erythrocytes harbor less ROS (H 2 O 2 and O 2 − ), oxidative damage, MDA, PC, and nonprotein sulfhydryls. Additionally, the levels of C20:4n-6, a proinflammatory factor, was significantly increased in MT erythrocytes after exercise, although the mechanism underlying this increase remains unclear. According to the measured activities of SOD, CAT, and POD, MSTN has little effect on erythrocyte enzymatic antioxidants. Instead, MSTN knockout enhances ROS scavenging via the nonenzymatic antioxidant GSH. There are two sources of GSH in erythrocytes: GSH synthesis from amino acids, and the conversion of GSSG to GSH by GR. NADPH, which is produced by G6PD, is a crucial CR cofactor [39]. The high GSH concentration observed in MT erythrocytes is explained by the high GR and G6PD activities in these cells, as increased G6PD activity within the PPP provides erythrocytes with additional NADPH. Overall, MSTN primarily affects erythrocyte antioxidant capacity via the PPP and, consequently, via GSH levels.
In mature erythrocytes, energy production depends on glycolysis, which uses glucose as a substrate. Following glucose uptake from the plasma, HK converts glucose to G6P, of which~5-10% enters the PPP; the remaining 90% is metabolized via glycolysis to PA and LA. HK, PFK, and PK are the key glycolytic enzymes, and decreases in their activities result in diminished glycolysis rates in erythrocytes. Band 3 is the most abundant protein in the erythrocyte membrane and, in conjunction with Band 4.1, Band 4.2, ankyrin, and spectrin, maintains the structural stability of the membrane [40]. Band 3 also regulates blood HCO 3− /CO 2− metabolism [41] and erythrocyte glucose metabolism [42]. In the study, Bnad3 expression decreased after exhaustive exercise, which was associated with decreased Band3 levels and increased aggregation, consistent with previously observed changes in Band3 in human erythrocytes [43]. The content of end-products of glycolysis, pyruvate and lactate, increased after MSTN knockout exercise. Knockout of MSTN reduced the activity of the glycolytic rate-limiting enzyme PFK. Band 3 is a transmembrane protein of erythrocytes, the cytoplasmic portion of which regulates the pentose phosphate pathway (PPP) by competitively binding to the central lumen of the deoxygenated hemoglobin β chain and some glycolytic enzymes [44]. Glycolytic enzymes bind to Band 3 proteins with greater advantage. When the glucose molecule enters the erythrocyte, it turns to the PPP because glycolytic enzymes have been involved with the Band 3 protein, which reduces cytosolic glycolysis within the erythrocyte [44,45].

Conclusions
Erythrocytes are of interest regarding exercise-induced oxidative damage due to their important roles in free radical scavenging and redox balance maintenance in the body. Here, we showed that MSTN knockout alleviates exercise-induced oxidative stress by increasing the efficiency of the PPP, thereby increasing erythrocyte GSH content and antioxidant potential.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ani12070927/s1, Table S1: Body weight of cattle at different age (kg); Table S2: Body length of cattle at different age (cm); Table S3: Wither length of cattle at different age (cm). Figure   Institutional Review Board Statement: The animal study protocol was approved by the Institutional Animal Care and Use Committee of Inner Mongolia University (No. IMU-CATTLE-2020-033, 1 April 2020).

Data Availability Statement:
The data generated and analyzed during this study are available upon reasonable request from the corresponding author.