Loss of MuRF1 in Duroc pigs promotes skeletal muscle hypertrophy

Muscle mass development depends on increased protein synthesis and reduced muscle protein degradation. Muscle ring-finger protein-1 (MuRF1) plays a key role in controlling muscle atrophy. Its E3 ubiquitin ligase activity recognizes and degrades skeletal muscle proteins through the ubiquitin–proteasome system. The loss of Murf1, which encodes MuRF1, in mice leads to the accumulation of skeletal muscle proteins and alleviation of muscle atrophy. However, the function of Murf1 in agricultural animals remains unclear. Herein, we bred F1 generation Murf1+/− and F2 generation Murf1−/− Duroc pigs from F0 Murf1−/− pigs to investigate the effect of Murf1 knockout on skeletal muscle development. We found that the Murf1+/− pigs retained normal levels of muscle growth and reproduction, and their percentage of lean meat increased by 6% compared to that of the wild type (WT) pigs. Furthermore, the meat color, pH, water-holding capacity, and tenderness of the Murf1+/− pigs were similar to those of the WT pigs. The drip loss rate and intramuscular fat decreased slightly in the Murf1+/− pigs. However, the cross-sectional area of the myofibers in the longissimus dorsi increased in the adult Murf1+/− pigs. The skeletal muscle proteins MYBPC3 and actin, which are targeted by MuRF1, accumulated in the Murf1+/− and Murf1−/− pigs. Our findings show that inhibiting muscle protein degradation in MuRF1-deficient Duroc pigs increases the size of their myofibers and their percentage of lean meat without influencing their growth or pork quality. Our study demonstrates that Murf1 is a target gene for promoting skeletal muscle hypertrophy in pig breeding.


Background
Improving production is always an important goal in the pork industry, and the identification of genes that affect muscle growth facilitates effective breeding. Previous studies have shown that the number of myofibers in mammals does not change after birth (Du et al. 2013). The development of skeletal muscle is divided into three stages: embryonic, fetal, and adult. The number of myofibers only increases before birth (hyperplasia). After birth, the number of myofibers remains constant, and muscle growth depends on an increase in the size of the myofibers (hypertrophy) (Du et al. 2013;Thornton 2019). The formation and number of myofibers are important to pork production at the prenatal development stage. Piglets with low birth weight have low myofiber differentiation rates, owing to maternal and genetic factors; they exhibit comparatively low growth performance and lean meat percentage at slaughter (Rehfeldt and Kuhn 2006). Therefore, improving skeletal muscle mass after birth is achieved by regulating the size of the myofibers. A delicate balance between protein synthesis and degradation is important for muscle production. Myofibers and consequently, skeletal muscles grow when the synthesis of muscle protein increases or degradation decreases (Gumucio and Mendias 2013).
Muscle ring-finger protein-1 (MuRF1), also known as E3 ubiquitin-protein ligase, is a classical muscle atrophy factor that plays an important role in protein degradation. It was first identified in skeletal muscle in 2001 (Centner et al. 2001). Studies using a Lesliemouse model of skeletal muscle atrophy have revealed the function of MuRF1. In dexamethasone-induced muscle atrophy models, the deletion of Murf1, which encodes MuRF1, alleviates muscle atrophy and increases the cross-sectional area (CSA) of the myofibers and the tension output of the gastrocnemius muscle (Leslie et al. 2011). MuRF1 deficiency also relieves age-related muscle atrophy in mice. Proteasome activity, especially that of the stand-alone proteasome 20S, decreases significantly in the skeletal muscle of aging wild type (WT) mice. In contrast, there is no decrease in 20S activity, and only a slight decrease in 26S B5 activity in Murf1 knockout (KO) mice (Hwee et al. 2014). In a mouse model of protein degradation induced by amino acid deprivation, the Murf1 KO mice were less prone to muscle atrophy, in both the myocardium and skeletal muscle. Muscle protein synthesis was reduced in the WT mice, while the Murf1 KO mice maintained non-physiologically high levels of skeletal muscle protein synthesis (Polge et al. 2011).
MuRF1 contains the unique RING domain of E3 ubiquitin ligase and degrades skeletal muscle proteins in vivo via the ubiquitin-proteasome degradation pathway (UPS) . In 2007, Clarke et al. discovered that myosin heavy chain protein (MYH) is a substrate of MuRF1, and MuRF1 causes skeletal muscle atrophy when dexamethasone is injected into the hind limbs of mice (Gumucio and Mendias 2013;Clarke et al. 2007). MuRF1 also plays a key role in cardiac protein degradation. Studies have shown that MuRF1 indirectly regulates the degradation of the downstream protein, cardiac myosin-binding protein C3 (cMYBPC3), via MYH interaction (Fielitz et al. 2007). α-Actin is a major protein of skeletal muscle and is a UPS substrate that is rapidly degraded during catabolic stimulation. MuRF1 interacts directly with α-actin, both in vitro and in vivo, to further induce its polyubiquitination and subsequent degradation (Polge et al. 2011). Therefore, MuRF1 has a positive regulatory effect on skeletal muscle atrophy and protein degradation.
However, it is unclear whether the loss of MuRF1 affects skeletal muscle growth in agricultural animals. In the present study, we used Duroc pigs as our research model to investigate the effect of MuRF1 deficiency on skeletal muscle. Duroc pigs grow rapidly, with an average daily weight gain of more than 900 g in finishing pigs. Therefore, they are used globally as the major sire line in current pork production Zhang et al. 2018). Genetic modification of genes involved in muscle protein degradation in Duroc and other breeds has great potential for improving pork production. We induced MuRF1 deficiency in Duroc boars to study the effect of MuRF1 deletion on skeletal muscle and meat yield. We used the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/ Cas9) nicking system to establish Murf1 deletion in Duroc founders (Hu 2017). We bred F1 generation Murf1 +/− and F2 generation Murf1 −/− pigs based on Murf1 −/− F0 generation Duroc pigs and compared their lean meat percentage and meat quality traits with those of WT pigs. We further examined skeletal muscle protein degradation caused by the expression of MuRF1. Our findings suggest that MuRF1 plays a role in myofiber hypertrophy and skeletal muscle protein degradation in pigs.

Animal studies
The experimental protocols were reviewed and approved by the laboratory animal welfare and animal experimental ethical council of China Agricultural University (AW01217102-3-1) and the 948 Program of the Ministry of Agriculture of China (2012-G1(4)). All animal experiments were performed according to the guide for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology in China.
The experimental pigs were housed under standard conditions and had free access to water and food. Their environment was maintained at 20-26 °C, 40-60% humidity, and a 9 h light/15 h dark cycle. The pigs were euthanized using ketamine before sample collection.

Polymerase chain reaction (PCR) analysis
We collected ear skin from each newborn piglet and stored it in 75% ethanol at 4 °C. The ear skin was digested and the DNA extracted through the DNeasy blood and tissue kit (69,504; QIAGEN). A forward primer (5′-TCT TTC AGG CTT GGA GGA AA-3′) and a reverse primer (5′-GTG CGT CAT GGA GAA GGA AT-3′) were used to amplify Murf1 by PCR. The PCR reaction mix included Taq™ 2X Master Mix (10 µL), forward primer (0.4 µL), reverse primer (0.4 µL), DNA (150 ng), and water. The PCR program was performed according to the manufacturer's instructions for the use of TaKaRa R004A, and the PCR products were detected by agarose gel electrophoresis. The WT and Murf1 −/− PCR product comprised 629 and 712 bp, respectively.
Total RNA extraction and reverse-transcription polymerase chain reaction (RT-PCR) analysis A 100 mg sample of thawed longissimus dorsi (LD) from the pigs was placed in a 1.5 mL microcentrifuge tube, which was placed in a low temperature automatic grinding machine at 4 °C for 10 min. RNA was extracted according to the manufacturer's instructions (RC112-01; Vazyme). Complementary DNA (cDNA) was reverse-transcribed from the total RNA (500 ng) using the PrimeScript™ RT Reagent Kit with a genomic DNA eraser (RR047A; TaKaRa), and stored at − 20 °C. The cDNA was diluted five times with water, and RT-PCR was performed using the forward primer (5′-TTA GAG CAG GTG AAG GAG GC-3′) and reverse primer (5′-TGT CAA TGA TGT TCT CCA CCA-3′) of Murf1, and the forward primer (5′-GTC GGA GTG AAC GGA TTT GGC-3′) and reverse primer (5′-CAC CCC ATT TGA TGT TGG CG-3′) of GAPDH.

Performance testing, slaughter, and sampling
To determine the growth traits of the pigs, we measured their daily food intake and weight increase during the growth fattening stage starting at 70 days of age until their average body weight reached 100 kg (Cabling et al. 2015). The pigs were then euthanized and exsanguinated at a commercial abattoir. The head, skin, forelimbs, hindlimbs, and viscera were eliminated. The carcass, skeletal muscle, and skin were weighed, and the dressing percentage was collected and calculated. The carcass length and backfat thickness were measured. The skeletal muscle from the left half of the carcass was selected and weighed to calculate the lean meat percentage Cabling et al. 2015).

Meat quality trait measurement
The freshly cut surface of the LD from the thoracolumbar of the left half of each carcass was examined 45 min after sacrifice. Meat color values, i.e., lightness (L*), redness (a*), and yellowness (b*), were measured three times at 1 h and 24 h using a colorimeter (NR20XE; Shenzhen 3NH Technology Co. Ltd). The pH values of the LD, on the last rib, were measured three times at 1 h (pH1) and 24 h (pH24) at 4 °C using a portable pH meter (PH-STAR; Matthaus Co. Ltd.). LD samples from the twelfth to thirteenth lumbar vertebrae were suspended from the lid of a plastic tube at 4 °C for 24 h to determine the drip loss rate. Intramuscular fat was detected in the lumbar vertebrae LD samples using the petroleum ether extraction method and a Soxtec™ fat tester. The water holding-capacity of each lumbar vertebrae LD sample was measured in an oven at 0.25 MPa and 60 °C, and at 0.20 MPa and 65 °C. LD samples without fascia, aponeuroses, or fat were taken 72 h after slaughter, and the tenderness of each sample was determined five times using a shear device ).

Histological analysis
The LD samples were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. The paraffin blocks were cut into 5 mm sections and stained with hematoxylin and eosin (H&E) or used for immunofluorescence (IF) staining. The thickness of LD tissue sections from F1 generation pigs used for H&E staining was 5 µm. H&E staining was performed as follows: the sections were soaked in xylene twice for 2 min each, and then in a 1:1 xylene to absolute ethanol solution for 2 min. Next, the sections were sequentially washed in a graded series of alcohol (100-50%) for 3 min each and then in water for 2 min. Thereafter, they were incubated with hematoxylin for 10 min, washed with water and 95% ethanol, and incubated with eosin for 30 s. Next, they were sequentially washed in 95% and 100% ethanol and xylene, respectively, twice for 2 min each. Finally, they were placed on slides and covered with cover slides for observation under a microscope. The LD tissue sections from F2 generation pigs were blocked with goat serum for 50 min at room temperature to prepare them for IF staining. The samples were incubated with anti-MYH1 (sc-376157; Santa Cruz Biotechnology; 1:500), anti-WGA (L4895; Sigma-Aldrich; 1:500), anti-MYBPC3 (sc-32920; Santa Cruz Biotechnology), and anti-α-actin (sc-58670; Santa Cruz Biotechnology; 1:500) at 4 °C overnight. Next, they were incubated with the secondary antibodies (1:200) for 1 h. The IF signals were visualized using a fluorescence microscope. Mean CSA of the LD was quantified using the ImageJ 2.0 software (National Institutes of Health).

Results
MuRF1 protein was successfully deleted in the gene-edited pigs We obtained F0 generation Murf1 knockout pigs using the CRISPR/Cas9n system to prematurely terminate translation by inserting either an 83 bp insertion or a marker-free neomycin (Nm)-resistance gene (neo) in the first exon of Murf1 (Hu 2017). A Cas9 nickase (Cas9n) was used in combination with two sgRNAs designed to direct staggered cleavage of the positive and negative strands of DNA, respectively, allowing deletion of the intervening sequence which included the MuRF1 transcription start site. Repair of such breaks by non-homologous end joining typically results in small insertions or deletions, and in this instance the clone selected as a donor for somatic cell nuclear transfer contained an 83 bp insertion (Fig. 1A). We mated F0 Murf1 −/− pigs with WT Duroc pigs to produce the first batch of F1 generation. F1 pigs were identified as heterozygous through PCR analysis, which amplified 712 and 629 bp products from the gene-edited Murf1 and WT pigs, respectively (Fig. 1B). The second batch of F1 generation pigs was identified as heterozygous through PCR analysis, which amplified the marker-free neomycin insertion from the geneedited Murf1 pigs and a 629 bp product from the WT allele (S1A, B). The Murf1 +/− pigs were mated with each other to produce Murf1 −/− F2 generation littermates (Fig. 1C). We identified the 83 bp insertion in exon 1 of the F2 Murf1-deficient pigs using Sanger sequencing (S1C). We used 7 and 8-monthold F1 and 2-month-old F2 generation pigs for subsequent experiments.
To detect the expression of Murf1, RNA was extracted from the LD and reverse-transcribed into cDNA for RT-PCR. The transcripts of Murf1 in the skeletal muscles of Murf1 −/− pigs and WT pigs were 712 and 629 bp, respectively (Fig. 1D). LD samples from the 7 to 8-month-old F1 and 2-month-old F2 generation pigs were collected and subjected to protein analysis by western blot. In the F1 generation pigs, the MuRF1 protein level in the Murf1 +/− pigs was lower than that in the WT pigs (Fig. 1E, F). In the F2 generation pigs, the MuRF1 protein was not detected in the Murf1 −/− pigs, and the MuRF1 protein levels in the Murf1 +/− pigs were also lower than those in the WT pigs (Fig. 1G). These data indicated that the MuRF1 protein was deficient in the gene-edited pigs.
Meat productivity and quality are important factors in Duroc pig farming (Zhang et al. 2018;Cabling et al. 2015). We found that in the Murf1 +/− pigs, the lean meat percentage increased by 6% without influencing the meat quality. After the F1 generation pigs had grown and fattened, we examined their food intake and weight increase ( Fig. 2A, B) (Cabling et al. 2015). There were no significant changes in food intake or weight increase after Murf1 deletion. Compared to that in WT pigs, the backfat thickness between the fifth and sixth ribs and scapula area of the Murf1 +/− pigs decreased by 0.422 cm and 0.411 cm, respectively (Fig. 2D). The carcass percentage was similar between the Murf1 +/− and WT pigs (F2E). Furthermore, the lean percentage increased by 6% in the Murf1 +/− pigs (Fig. 2F).
We also determined if the meat quality traits changed in the Murf1 +/− pigs. As shown in Fig. 3, the color (as determined by the a, b, and L values), water-holding capacity, pH, and tenderness of the meat from the Murf1 +/− pigs were similar to those of the meat from the WT pigs ( Fig. 3A-D) Zhang et al.2018). Moreover, the drip loss rate and intramuscular fat of the Murf1 +/− pigs were slightly lower than those of the WT pigs (Fig. 3E, F). These results indicated that there was no deterioration in the quality, taste, or nutritional value of pork from the MuRF1-deficient pigs (Zhang et al. 2018;Cabling et al. 2015).
The CSA of the myofibers in the LD increased in the adult Murf1 +/− pigs compared to the CSA of the myofibers in WT pigs To further explore the effect of Murf1 deficiency on muscle growth, we collected LD samples from the F1 pigs to perform H&E staining. H&E staining and CSA analysis revealed that the myofibers were larger in the Murf1 +/− pigs than in the WT pigs (Fig. 4A, B, D) (Fielitz et al. 2007). CSA analysis of the F2 generation pigs by IF staining revealed that the CSA of LD also increased in the Murf1 −/− pigs (Fig. 4C, E). These findings indicated that Murf1 deficiency results in large myofibers.
The protein levels of MYBPC3 and α-actin increased in the MuRF1-deficient pigs We also determined whether the metabolism of the skeletal muscle was altered in the Murf1 KO pigs. The proteins that participate in MuRF1 degradation were detected by western blot. In the F1 pigs, we found that the protein levels of α-actin, MYBPC3, and MYH7 increased in the 7-and 8-month-old Murf1 +/− pigs compared to the levels in the WT pigs ( Fig. 5A-D) (Polge et al. 2011;Clarke et al. 2007;Mearini et al. 2010). In the F2 pigs, the protein levels of MYBPC3 increased in the Murf1 −/− and Murf1 +/− pigs compared to levels in the WT pigs (Fig. 5E, F). We also determined the structures of the myofibers in the F2 pigs by immunohistochemistry using anti-MYBPC3 and anti-α-actin antibodies. The results showed that the structures did not change in the Murf1-deficient pigs (S2). These results further demonstrated that MuRF1 deficiency in pigs leads to the accumulation of sarcomeric proteins without muscle atrophy.

Discussion
It is unclear whether skeletal muscle is affected by MuRF1 deficiency in pigs. Herein, we examined meat production and quality in MuRF1-deficient Duroc pigs. To this end, we bred F1 and F2 generation Duroc pigs with Murf1 loss-of-function mutation by mating Murf1 −/− pigs with WT pigs to produce an F0 generation. PCR analysis and Sanger sequencing confirmed that the sizes and positions of the insertion fragments in the F1 Murf1 +/− and F2 Murf1 −/− pigs were as stable as they were in the F0 Murf1 −/− pigs. This led to the premature termination of protein translation and failure to produce intact MuRF1 (Hu 2017).
We found that food intake and increase in body weight did not change in the MuRF1-deficient pigs, indicating that MuRF1 KO does not affect the general growth of an animal. Furthermore, we demonstrated that backfat thickness decreased by 0.4 cm and that carcass percentage remained the same in the Murf1 +/− pigs compared to those in the WT pigs. This suggests that MuRF1 deficiency reduces backfat thickness but does not affect the growth of pigs.
However, as compared to that in the WT pigs, the lean meat percentage increased by 6% in the Murf1 +/− pigs, indicating that MuRF1 deletion improves meat production. MuRF1 specifically targets and degrades sarcomeric proteins through E3 ubiquitin ligase via the UPS (Bodine and Baehr 2014). The UPS is a classical pathway for protein catabolism and is involved in many biological events, such as cell cycle regulation, inflammatory responses, immune responses, and degradation of misfolded proteins (Hirner et al. 2008;Koyama et al. 2008;Nandi et al. 2006). The operation of UPS mainly depends on three types of enzymes: ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin-protein ligases (E3s). The process starts with the ATP-dependent activation of ubiquitin by E1s. The activated ubiquitin is then transferred to E2s. In the final step, E3s specially recognize and recruit target proteins and transfer activated ubiquitin from the E2s to the substrate. This results in protein modification and degradation (Metzger et al. 2012;Navon and Ciechanover 2009;Passmore and Barford 2004). MuRF1 belongs to the group of RING-related E3s that act as molecular bridges connecting the E2-ubiquitin complex with  (Metzger et al. 2012). It transfers the activated ubiquitin to lysine residues in the substrate, forming K48-and K29-linked polyubiquitin chains that are recognized and degraded by the 26S proteasome or K63-linked mono-ubiquitin-modified proteins (Navon and Ciechanover 2009;Cohen et al. 2009). When the protein substrate is α-actin or MHC, MuRF1 specifically combines with the E2 ubiquitin-conjugating enzyme UBE2L3 to degrade the protein via the UPS, and the affinity of MuRF1 for filamentous F-actin is higher than that for monomeric G-actin (Peris-Moreno et al. 2021). Recent studies also found that MuRF1 deficiency increased mitochondrial content in skeletal muscle and regulated lipid metabolism. Compared to those in WT mice, energy expenditure and fat metabolism increased in 3-month-old Murf1 −/− mice, and the body weight and fat mass were lower in Murf1 −/− mice aged over 7 months. MuRF1 deficiency led to increased PDK4 content in the mitochondria of skeletal muscle and Fig. 2 Identification of production traits in F1 generation pigs during their growth and fattening period. A The total food intake was measured in the WT (n = 4) and Murf1 +/− (n = 7) pigs during their growth and fattening period. B The weight increase was calculated by comparing the beginning and end of growth and the fattening period. C The backfat thickness was measured from the 5th to 6th ribs, and decreased by 0.422 cm in the Murf1 +/− (n = 7) pigs compared to that in the WT (n = 4) pigs. D The backfat thickness was measured from the scapula area, and decreased by 0.411 cm in the Murf1 +/− (n = 7) pigs compared to that in the WT (n = 4) pigs. E The carcass percentage did not change in the Murf1 +/− pigs (n = 7). F The average lean meat percentage increased by 6% in the Murf1 +/− (n = 7) pigs compared with that in the WT (n = 4) pigs. All data are presented as the mean +/− SD. (WT Wild type, MuRF1 Muscle ring-finger protein-1) improved lipid metabolism (Sugiura et al. 2022). In MuRF1-KO and MuRF2-KO mice, the levels of glucose and triglyceride were higher than those in WT mice. However, the developed progressive muscle weakness was relieved in type 2 diabetic mice with MuRF1 and MuRF2 deficiency (Labeit et al. 2021). Therefore, Murf1 is increasingly being widely studied in mice and humans. As previous studies have shown, deletion or mutation of Murf1 causes skeletal muscle hypertrophy. Deficiency of MuRF1 and Fig. 3 Evaluation of meat quality traits in F1 generation Murf1 +/− pigs. A The color of the LD meat was similar in the Murf1 +/− (n = 7) and WT (n = 4) pigs. B-D The water-holding capacity, pH, and tenderness did not change in the Murf1 +/− pigs (n = 7). E The drip loss rate decreased slightly in the Murf1 +/− (n = 7) pigs compared to that in the WT (n = 4) pigs. F The intramuscular fat decreased slightly in the Murf1 +/− (n = 7) pigs compared to that in the WT (n = 4) pigs. All data are presented as the mean +/− SD. (MuRF1 Muscle ring-finger protein-1, LD Longissimus dorsi, WT Wild type) MuRF3 results in hypertrophy of the skeletal and cardiac muscles of mice (Fielitz et al. 2007). In cardiac cachexia-induced skeletal muscle atrophy mice, the atrophy of the soleus and TA muscles reduced in MuRF1-and MuRF2-KO mice compared with that in WT mice, the constrictive function was protected, and metabolic enzyme levels were not reduced (Nguyen et al. 2020). MuRF1 deficiency-induced muscle loss was reduced, and the size of myofibers was increased, but muscle force decreased in nemaline myopathy mouse models (Lindqvist et al. 2022). Patients with homozygous Murf1 nonsense mutations combined with heterozygous Murf3 mutations exhibit hypertrophy in skeletal and cardiac muscles, including left ventricular dilation (Olive et al. 2015). However, the above studies found that MuRF1 deficiency alleviated muscle atrophy in models of muscle atrophy caused by external stimulation or disease. In this study, we knocked out MuRF1 in healthy pigs without stimulation of muscle atrophy. We found that there was a significant difference in backfat thickness and lean meat percentage between the Murf1 +/− and WT pigs, and the muscle quality was good. These results are consistent with Murf1 regulation of muscle atrophy in mice and humans. This indicated that the loss of MuRF1 results in a reduction in skeletal muscle degradation via the UPS. In addition, we speculate that the loss of MuRF1, as well as dexamethasone and other external stimulations, could also alleviate muscle atrophy in pigs suffering from muscle atrophy. This hypothesis may be verified using small-molecule compounds targeting MuRF1 in pigs.
As MuRF1 has a classic regulatory effect on muscle atrophy, several small-molecule compounds targeting Murf1 have also attracted attention. After treatment with the small-molecule compound MyoMed-205 to disturb the recognition of the substrate of MuRF1 in heart failure HFpEF mice, the myocardial fibrosis and skeletal muscle disease were alleviated, muscle function was enhanced, MuRF1 content in skeletal muscle was decreased, mitochondrial complex activity was increased, and the symptoms of heart failure were improved (Adams et al. 2022). In a myocardial infarction mouse model induced by ligation of the left anterior descending coronary artery, the degree of myofiber atrophy of the diaphragm was decreased, the upregulation of MuRF1 and MuRF2 was decreased, and the activity of mitochondrial enzyme was reduced after treating mice with the MuRF1 inhibitor ID#704,946 (Adams et al. 2019). However, it is unclear whether these small-molecule compounds also play a role in pigs, and the MuRF1-knockout pigs generated herein may be a good reference for further research. Our results are consistent with those obtained with the Duroc × Meishan Hybrid Population with MSTN KO. MSTN is a well-known inhibitor of muscle growth. To improve the meat production of livestock animals, MSTN has become widely studied in pigs. Compared to WT pigs, MSTN +/− pigs showed no significant change in average daily weight gain and body weight in Meishan and Duroc hybrid populations and carcass percentage remained normal, while lean meat rate increased and average backfat thickness decreased (Li et al. 2020). However, Bama pigs with MSTN biallelic KO showed significant weight gain and increased number of myofibers at 3-6 months after birth but exhibited no significant change in myofiber size compared to WT pigs (Zhu et al. 2020). The different phenotypes of MSTN KO in pigs may be related to pig breed.
An evaluation of meat quality revealed that the a, b, and L meat color values, water-holding capacity, pH, and tenderness of the Murf1 +/− pigs were similar to those of the WT pigs. The drip loss rate of the Murf1 +/− pigs was slightly reduced, which demonstrated the superior water-holding capacity of their pork (Rehfeldt and Kuhn 2006). The amount of intramuscular fat in the Murf1 +/− pigs was also slightly reduced, which further illustrated that the muscle mass increased in the Murf1-deficient pigs. Furthermore, the CSAs of the myofibers in the LD increased significantly in the 7-month-old F1 generation Murf1 +/− pigs, and there was a similar increase in the 2-month-old F2 generation Murf1 −/− pigs. However, these results differed from those for mice and humans. Previous studies reported no morphological changes or muscle atrophy in the heart and skeletal muscles of Murf1 −/− mice (Bodine et al. 2001). However, there has been a report of skeletal muscle hypertrophy in Murf1 −/− Murf3 −/− double-KO mice (Fielitz et al. 2007). In humans, hypertrophic cardiomyopathy, caused by mutated MuRF1, is a rare autosomal recessive genetic disease that is characterized by moderate to severe hypertrophy, ventricular arrhythmias, extensive fibrosis, and frequent left ventricular systolic dysfunction; it causes significant disruption to daily life (Salazar-Mendiguchia et al. 2020). The loss of MuRF1 in pigs causes changes in myofibers and muscle mass in contrast to that of MSTN which increases muscle production. A more pronounced muscle mass, distinct intermuscular sulci in the hind legs, and wider back and rump were observed in MSTN −/− Erhualian pigs than in WT pigs (Wang et al. 2017), and FSI-I-I knock in pigs by editing the MSTN antagonist FST domain also promoted skeletal muscle hypertrophy, with an increase in LD and myofiber size . It has been shown that homozygous MSTN KO mutant pigs develop umbilical hernias and tippy toe standing pathology and have more severe urinary tract bleeding symptoms, which may be due to decreased collagen in the tendons and linea alba fibroblasts (Paek et al. 2021). MSTN exon 3-deletion large white (LW) piglets showed symptoms of hind limb weakness and a large number of broken myofibers in the biceps femoris. In addition, the average daily standing time of MSTN KO pigs was shorter than that of WT pigs, and the meat color values of MSTN KO Meishan pigs and LW pigs were significantly reduced (Fan et al. 2022). However, F1 generation Murf1 +/− and F2 generation Murf1 −/− pigs moved freely, had healthy body and strong hind limbs, and did not show pathological symptoms like MSTN KO pigs. At the same time, lean meat percentage of MuRF1 KO pigs was increased and their meat color, pH, and other meat quality traits were maintained at normal levels, further indicating that MuRF1 is a key factor in the regulation of skeletal muscle growth.
In the present study, we discovered that, as compared to that in the WT pigs, the protein levels of MYBPC3 increased in both the F2 generation Murf1 −/− pigs and the F1 generation Murf1 +/− pigs. Similarly, the levels of α-actin and MYH7 increased in the F1 generation Murf1 +/− pigs. However, MuRF1 deficiency had no significant effect on the structures of the myofibers. Studies on mice have revealed that during denervation-induced and fast-induced muscle atrophy, the levels of MYBPC and MYLC2 decrease significantly and are preferentially degraded in Murf1 knock-in mice. Furthermore, those levels do not decrease in mice after the RING domain deletion of MuRF1 (Cohen et al. 2009). Other researchers have enriched and purified myofiber proteins using recombinant glutathione-S-transferase-MuRF1 and discovered that actin is polyubiquitinylated by MuRF1 (Polge et al. 2011). Actin and MYHC levels are also reduced by MuRF1 degradation in murine cancer cachexia (Cosper and Leinwand 2012). Consistent with the results in mice, in the present study, the loss of MuRF1 caused the accumulation of target proteins to further promote skeletal muscle hypertrophy.

Conclusions
In the present study, Murf1 knockout increased the lean meat percentage without affecting the meat quality of Duroc pigs. Our study thus provides important reference information on the role of MuRF1 in agricultural animals for the improvement of meat yield. Fig. 5 The expression of MYBPC3, α-actin, and MYH7 proteins. A A western blot of the LD samples revealed that expression of α-actin increased in the 7-month-old Murf1 +/− pigs compared to expression in the 7-month-old WT pigs. B Analysis of the protein levels in the 7-month-old pigs using the ImageJ 2.0 software. The results are shown as the means +/− SDs. The asterisk indicates a significant difference versus the control (p ≤ 0.05). C Western blot of the LD samples showing the expression of MYBPC3, α-actin, and MYH7 proteins in the Murf1 +/− and WT pigs. D Analysis of the protein levels in the 8-month-old pigs using the ImageJ 2.0 software. The results are shown as the means +/− SDs. E Western blot of the LD samples showing that the expression of MYBPC3 protein increased in the 2-month-old Murf1 −/− pigs compared to in the 2-month-old WT pigs. F Analysis of the protein levels in the 2-month-old pigs using the ImageJ 2.0 software. The results are shown as the means +/− SDs. The asterisk indicates significant difference versus the control (p ≤ 0.05). (LD Longissimus dorsi, MuRF1 Muscle ring-finger protein-1, WT Wild type) ◂