Transgenic Res (2015) 24:837–845 DOI 10.1007/s11248-015-9896-2

ORIGINAL PAPER

Functional verification of a porcine myostatin propeptide mutant Dezun Ma . Shengwang Jiang . Pengfei Gao . Lili Qian . Qingqing Wang . Chunbo Cai . Gaojun Xiao . Jinzeng Yang . Wentao Cui

Received: 7 June 2015 / Accepted: 7 July 2015 / Published online: 15 July 2015 Ó Springer International Publishing Switzerland 2015

Abstract Myostatin is a member of TGF-b superfamily that acts as a key negative regulator in development and growth of embryonic and postnatal muscles. In this study, the inhibitory activities of recombinant porcine myostatin propeptide and its mutated form (at the cleavage site of metalloproteinases of BMP-1/TLD family) against murine myostatin was evaluated in vivo by intraperitoneal injection into mice. Results showed that both wild type and mutated form of porcine propeptide significantly inhibited myostatin activity in vivo. The average body weight of mice receiving wild type propeptide or its mutated form increased by 12.5 % and 24.14 %, respectively, compared to mice injected with PBS, implying that the in vivo efficacy of porcine

propeptide mutant is greater than its wild type propeptide. Transgenic mice expressing porcine myostatin propeptide mutant were generated to further verify the results obtained from mice injected with recombinant porcine propeptide mutant. Compared with wild type (non-transgenic) mice, relative weight of gastrocnemius, rectusfemoris, and tibialis anterior increased by 22.14 %, 34.13 %, 25.37 %, respectively, in transgenic male mice, and by 19.90 %, 42.47 %, 45.61 %, respectively, in transgenic female mice. Our data also demonstrated that the mechanism by which muscle growth enhancement is achieved by these propeptides is due to an increase in fiber sizes, not by an increase in number of fiber cells. Keywords Myostatin  Propeptide  Mutation  Muscle mass  Transgenic mice

Dezun Ma and Shengwang Jiang have contributed equally to this work. D. Ma  S. Jiang  P. Gao  Q. Wang  G. Xiao  W. Cui (&) State Key Laboratory for Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, People’s Republic of China e-mail: [email protected]; [email protected] L. Qian  C. Cai College of Biological Sciences, China Agricultural University, Beijing 100193, People’s Republic of China J. Yang Department of Human Nutrition, Food and Animal Sciences, University of Hawaii, Honolulu, HI 96822, USA

Introduction Myostatin, also known as growth differentiation factor-8 (GDF-8), is an important member of the transforming growth factor b (TGF-b) superfamily that regulate muscle development in a dominant negative manner (Lee 2004). Myostatin knockout mice show a large and widespread in skeletal muscle mass result from a combination of muscle cell hyperplasia and hypertrophy (McPherron et al. 1997). Myostatin gene inactivating mutations are

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responsible for increased muscling in cattle (Grobet et al. 1997; Kambadur et al. 1997; McPherron and Lee 1997;Grobet et al. 1998), sheep (Clop et al. 2006), dog (Mosher et al. 2007) and human (Schuelke et al. 2004). All these studies demonstrate that myostatin acts to limit skeletal muscle growth, and thus, it is practicable to increase skeletal muscle by inhibiting myostatin activity. Like other TGF-b family members, myostatin is a secreted protein that is cleaved by furin proteases to generate an N-terminal propeptide and a biologically active C-terminal dimer. The N-terminal propeptide remains bound to C-terminal dimer to form latent complex, which can be cleaved by members of the BMP-1/TLD family of metalloproteinase at 76th aspartate residue (75th in porcine propeptide) to release the active mature peptide (Thies et al. 2001; Lee and McPherron 2001; Wolfman et al. 2003). Injection of a murine myostatin propeptide mutant with a point mutation (D76A) in mice resulted in an increase in muscle mass (Hu et al. 2010). Lee et al. transformed myostatin mutant gene (D76A) into mouse embryonic stem cells to obtain transgenic mice that were completely the same as those of the knockout myostatin mice (Lee 2008). These previous results clearly demonstrate that the point mutation D76A of myostatin propeptide can inhibit myostatin activity and thus to increase muscle mass. Pig is a key livestock animal and is the major source of meat in China. Therefore, it is very important to improve the quality (such as lean yield and fat content) of pork by either genetically engineering the structure of myostatin or by intervention of myostatin’s function with specific inhibitors. Myostatin gene is extraordinarily well conserved among different species, the amino acid sequences of the mature and active myostatin pepetide from humans, mice, rats, pigs, and chickens are identical (Lee 2004), and the the amino acid sequences between murine and porcine propeptides are about 95 % identical. In this study, the in vivo effects of wild type porcine myostatin propeptide and its mutant, D75A, on muscle growth were investigated in mice by direct administration of these two recombinant proteins and also by transgene expression of porcine propeptide mutant gene. Our results not only verified that porcine propeptide and its mutant indeed can enhance muscle growth in mice by increasing fiber sizes, with the mutant being more effective than wild type propeptide, but also provide

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useful insight and basic theory to future studies on improving pork quality by regulating myostatin activity using propeptide and its mutant.

Materials and methods Expression and purification of porcine propeptide and its mutant In this study, a prokaryotic expression system was used to produce recombinant porcine myostatin propeptide and its mutant at position 75 (the cleavage site of metalloproteinases of BMP-1/TLD family) where aspartate residue is replaced with alanine (D75A). Expression and purification of these two recombinant propeptides were performed as previously described (Sheng-Wang et al. 2012). Intraperitoneal injection of porcine propeptide and its mutant One Kunming white male mouse mated with 4 female mice, respectively. Then, four litters of half sib mice (32 mice) were bred. Eighteen male mice with similar weight and of the same age were selected and divided into three groups: control group, wild type porcine propeptide, and porcine propeptide mutant. There were 6 mice in each group. Each mouse in the experimental groups were injected intraperitoneally with propeptide or its mutant at a dose of 30 mg/kg (the injection volume per mouse was adjusted to 1 ml). Mice in the control group were injected with the same volume of PBS. The intraperitoneal injection of recombinant porcine propeptide or its mutant into mice was given at ages of 11, 18, 25 and 32 days. Each mouse was weighed prior to each injection. Following the last injection, each mouse was weighed once every 7 days. At day 60, mice reached mature stage, and the growth and development maintained basically as stable. Each mouse was weighed for the last time. Generation of transgenic mice expressing propeptide mutant (D75A) Not I site was added to both ends of porcine propeptide mutant gene during PCR amplification using specific primers. Sleeping beauty transposon carrier pT2-HB

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was digested with Not I and then ligated with the above PCR product using T4 ligase to generate a vector containing porcine propeptide mutant D75A. This plasmid is then linearized with Sca I and mixed with SB100 transposase at 1:1 ratio. The mixture was then injected into the male pronuclear of fertilized C57BL/6 J mouse eggs. All mice used in this study were F1 offsprings generated by breeding male F0 transgenic mice with female wild type C57BL/6 J. All F1 mice were from the same litter with an age of 30 weeks. Analysis of porcine myostatin propeptide mutant (D75A) expression in transgenic mice DNA samples were taken from tails of 4 weeks-old mice and PCR was performed using primers designed to amplify SV40 promoter and MLC1 enhancer. The sequences of primers are: 50 -CACTGCATTCTAGTT GTGGTTTGT-30 and 50 -AAGCATGATGTCTGTGC GGT-30 . The PCR condition was 95 °C for 5 min, 35 cycles including 30 s at 95 °C, 30 s at 64 °C, 40 s at 72 °C and 10 min at 72 °C. All PCR products were sequenced to confirm their identity. In addition, RT-PCR was performed to confirm transgene expression at RNA level in transgenic mice. Based on difference in DNA sequences between murine propeptide and porcine propeptide mutant, specific primers were designed to amplify only transgenic porcine propeptide mutant D75A without amplification of endogenous murine propeptide. Sequences of these specific primers are: 50 -AGAACAGCGAGCAAAAGGAAA-30 and50 -TCCACTTGC ATTAGAAGATCAGA -30 . PCR conditions are: 95 °C for 5 min, 35 cycles including 30 s at 95 °C; 30 s at 60 °C; 20 s at 72 °C and 72 °C for 10 min. We also measure the mRNA expression level of myostatin, which include endogenous and exogenous myostatin of transgenic mice and control non-transgenic mice, by real-time quantitative PCR. We extract RNA from gastrocnemius of transgenic mice and nontransgenic mice using Trizol (Invitrogen) according to the manufacturer’s protocol. The cDNA was reversetranscribed from total RNA using a First Strand cDNA Synthesis Kit (Fermentas), a SYBR Green kit (Applied Biosystems) and an ABI-7500 Real-Time PCR system (Applied Biosystems). Gene expression levels were calculated according to the 2-44CT method as previously described (Wang et al. 2014). Primers that can

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amplify the transgene and endogenous myostatin gene were as follow: 50 -AGTGATGGCTCCTTGGA AGA-30 and 50 -TGTAGGAGTCTTGACGGGTC-30 . Primer that amplify the GAPDH were as follows: 50 ACCCAGAAGACTGTGGATGG-30 and 50 -CACAT TGGGGGTAGGAACAC-30 . Animal care All experimental mice were maintained under the following conditions: temperature of 25 ± 1 °C, relative humidity of 70 ± 4 %, air change 10 times/h, automatic light control, rearing density less than or equal to 4 per cage. Mice were provided food and water ad libitum. All animal experiments were approved by Institutional Animal Care and Use Committee of Chinese Academy of Agricultural Sciences. Histological and morphometric analysis Total body weight of each mouse was measured. All mice were humanely euthanized, then Longissimus dorsi, gastrocnemius, pectoralis, rectusfemoris, and tibialis anterior (TA) were dissected and weighed. Gastrocnemius was dissected, fixed in 4 % paraformaldehyde, and embedded in paraffin 5 min after each mouse was euthanized. Transverse sections of gastrocnemius were stained with hematoxylin and eosin, and pictures were taken from four random fields at 4009 magnifications. Image J software was used to calculate the muscle cross sectional area and fiber number. Statistical analysis Analysis of diagram and data by SAS program 9.2. The differences between 2 groups and[2 groups were analyze by Student’s t test. Values of P \ 0.05 and P \ 0.01 were labeled as *, **, respectively.

Results In vivo effect of recombinant porcine wild type propeptide and its mutant (D75A) on muscle growth in mice To evaluate effects of recombinant porcine propeptide and its mutant in mice, three groups of mice were

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Fig. 1 Comparison of body weight and weight of three tissue muscles among three groups of mice injected with PBS, wild type propeptide (WTP) and mutated propeptide (MP). There were 6 male mice in each group. a Changes in the increase of body weight of the mice. b Changes in weight of different tissue muscles. c Photos of muscles from mice treated with PBS, wild type propeptide and propeptide mutant.*P \ 0.05; **P \ 0.01

treated by intraperitoneal Injection with PBS or recombinant propeptides and their body weights were monitored during the study period. Statistical analysis was performed on weight data collected from mice intraperitoneally injected with PBS, wild type propeptide and its mutant D75A during different time periods by using SAS software. There was a significant difference on 25th day between mice injected with porcine propeptide mutant and PBS (P \ 0.05), and this difference became even more significant (P \ 0.01) on day 39. There was a significant difference on day 32 between mice injected with wild type propeptide and PBS, and this became more

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significant on day 53. In addition, there was a tremendously significant difference on day 39 (P \ 0.01) between mice injected with porcine wild type propeptide and its mutant (D75A), with the exception on day 53 day (P \ 0.05) (Fig. 1a). Body weight was 24.14 % greater in mice injected with porcine propeptide mutant and 12.5 % greater in mice injected with porcine wild type propeptide, respectively, than that of mice injected with the PBS. As illustrated in Fig. 1b, c, there was a tremendously significant difference in three types of muscle tissues between three mouse groups (P \ 0.01). For example, the main skeletal muscle from mice injected

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Fig. 2 Morphometric analysis of gastrocnemius muscles. There were 4 male mice in each group. a Paraffin sections of transverse section of male mouse gastrocnemius. b The average area of muscle fiber of the gastrocnemius from mice of various groups. c The average number of muscle fibers of muscle bundles from three different groups. PBS PBS injection; WTP Wild-type propeptide injection; MP mutated propeptide injection. *P \ 0.05; **P \ 0.01

with propeptide mutant and mice injected with wild type propeptide was 24.45 % and 10.51 % greater, respectively, than in PBS group. We also measured sizes and numbers of muscle fibers collected from mice injected with propeptide mutant, wild-type propeptide, and PBS by HE-stained section of gastrocnemius. Results demonstrated that significant difference (P \ 0.05) was observed among three groups, but the difference is much more significant (P \ 0.01) between mice injected with propeptide mutant and PBS (Fig. 2a–c). No significant change was observed in the number of myofibers in mice treated with PBS, porcine propeptide or its mutant (Fig. 2a–c). Generation of transgenic mice expressing porcine propeptide mutant D75A Transgenic mice were generated from pronuclear injection of Sleeping Beauty transposon vector

(pT2-HB) and SB100 transposase to express porcine propeptide myostatin mutant D75A under a skeletal muscle specific promoter (MLC1). PCR analysis confirmed the identity of all transgenic mice (Fig. 3). Based on difference in DNA sequences between murine propeptide and porcine propeptide mutant, specific primers were designed to amplify transgenic porcine propeptide mutant D75A without amplification of endogenous murine propeptide (Fig. 4a). Result from real-time quantitative PCR analysis of relative mRNA expression level of myostatin, which includes endogenous murine myostatin and exogenous porcine propeptide mutant D75A in transgenic mice and non-transgenic control mice, showed that mRNA level of myostatin is much greater in transgenic mice than in wild type mice (Fig. 4b). These data clearly show that transgenic mice were successfully generated and the exogenous porcine propeptide myostatin mutant was expressed.

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Fig. 3 PCR analysis of transgenic mice and non-transgenic mice. Lane M marker; lanes 1-6 and 8 transgenic mice; lane 7 non-transgenic mouse as a negative control; lane 9 plasmid pT2HB containing myostatin propetide mutation (D76A) as the positive control; lane 10 H2O

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leads to an increase in muscle mass, weights of major skeletal muscles from hind limb containing gastrocnemius, rectusfemoris, and tibialis anterior (TA) were measured at week 30. Results show that weights of all three skeletal muscle tissues from transgenic male mice expressing the D75A mutant increased 22.14 %, 34.13 %, 25.37 %, respectively, compared to nontransgenic mice littermates (Fig. 5a and c). Similarly, weights of all three skeletal muscle tissues from transgenic female mice expressing the D75A mutant increased 19.90 %, 42.47 %, 45.61 % respectively, compared to non-transgenic mice littermates (Fig. 5b). Similarly, whole body weight and weights of gastrocnemius and rectusfemoris were significantly greater (P \ 0.05) in transgenbic mice than in wild type mice at week 5 (data not shown). In order to determine if there is an increase in myofiber size in transgenic mice, we performed analysis of HE-stained gastrocnemius muscle from both transgenic mice and wild type mice. Our results indicated that the diameter of myofibers is 13.58 % and 27.90 % greater in transgenic male and female mice, respectively, than in wild type mice (Fig. 6a–c). On the other hand, there was no change in total number of myofibers between transgenic and non-transgenic mice (Fig. 6d). It is thus clear that transgene expression of porcine propeptide mutant D75A in mice inhibits myostatin activity and enhances muscle growth by increasing sizes of myofibers.

Discussion

Fig. 4 Analysis of porcine propetide mutatant (D76A) expression in transgenic mice and non-transgenic control group. a RTPCR analysis of transgenic mice and non-transgenic control group. b Real-time quantitative PCR analysis of relative mRNA expression level of myostatin, which includes endogenous murine myostatin and exogenous propeptide mutant D75A in transgenic mice and non-transgenic control mice. Three mice from each group were used for PCR analysis. Lane M marker; lanes 1-6 and 8 transgenic mice; lane 7 non-transgenic mouse as a negative control; lane 9 H2O. *P \ 0.05; **P \ 0.01

Effect of porcine propeptide mutant on muscle growth in transgenic mice In order to determine if porcine propeptide mutant can inhibit the activity of endogenous myostatin and thus

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In recent years, myostatin attracts more attention by scientists since it was found by McPherron and Lee (1997) that it acts as a key negative regulator of skeletal muscle growth (McPherron et al. 1997). Myostatin exerts its activity by binding to receptor type IIB (Acvr2b) to inhibit muscle mass, therefore, inactivation or inhibition of myostatin activity by genetic mutations or by its inhibitors could lead to an increase in muscle mass (Zhu et al. 2000; Lee 2007). In previous research, Li et al. expressed a mutated porcine myostatin propeptide (D75A) by using an insect expression system to obtain purified recombinant mutant protein, which was then intraperitoneally injected into neonatal mice on days 11 and 18 at a dose of 10 mg/kg. Their results showed that the body weight in neonatal mice injected with a mutant protein

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Fig. 5 Analysis of muscle weight between transgenic mice and non-transgenic control littermates as a percentage of body weight. a Comparison of skeletal muscle mass between transgenic male mice (n = 3) and control non-transgenic littermates (n = 7). b Comparison of skeletal muscle between

transgenic female mice (n = 5) and control non-transgenic littermates (n = 7). c Comparison of transgenic mice that expressed the porcine propetide mutatant and non-transgenic control group. TA anterior tibialis. *P \ 0.05; **P \ 0.01

was 12–15 % higher than those mice injected with PBS (Li et al. 2010). In our study, the body weight change is more dramatic (16.5–24.14 %) compared to results of Li et al. This maybe due to fact that we gave four injections at a dose of 30 mg/kg, while two injections were gaven at 10 mg/kg in the protocol described by Li et al. 2010. Although the recombinant mutated porcine propeptide obtained from eukaryotic expression system had been demonstratred to have good biological activity, the yield was relatively low and thus the production cost is very high. Therefore, the prokaryotic expression system, Escherichia coli, was used in this study to generate recombinant wild type and mutated porcine propeptides. In our current study, we administered wild type and mutant propeptides into mice at a dose of 30 mg/kg on days 11, 18,

25, and 32. Our results showed that E. coli produced wild type porcine propeptide and its mutant D75A indeed possessed good biological activity in inhibiting myostatin activity and in promoting muscle growth. However, the efficacy of the mutant was greater than the wild type propeptide, probably due to the fact the mutated propeptide is resistant to the BMP-1/tolloid family of metalloproteinases and thus may have a longer half life than wild type propeptide which is sensitive to the BMP-1/tolloid family of metalloproteinases (Wolfman et al. 2003). Wang et al. previously reported that transgenic mice expressing porcine myostatin propeptide. They noted that, at the age of 9 weeks, weights of carcass, fore limb and hind limb increased by 6.0 %, 9.0 %, 8.7 % respectively in transgenic male mice, and by

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Fig. 6 Comparison of muscle fiber size between transgenic mice and their control littermates. Gastrocnemius were dissected from transgenic male mice (n = 3) and control littermates (n = 7). a Muscle cross-sections of transgenic female mice. b Muscle cross-sections of non-transgenic control female littermate mice. c Average muscle fiber size was compared

between transgenic mice (male, n = 3; female, n = 5) and nontransgenic control group (male, n = 7; female n = 5). d Number of myofibers of gastrocnemius muscles from non-transgenic control group (male, n = 7; female, n = 5) and transgenic mice (male, n = 3; female, n = 5). Scale bars 50 lm. *P \ 0.05

11.4 %, 14.5 % and 14.5 % respectively in transgenic female mice, compared to wild-type controls (Wang et al. 2013). In our current study, weights of gastrocnemius, rectusfemoris, and tibialis anterior isolated from mutated porcine propeptide (D75A) transgenic male mice are heavier 22.14 %, 34.13 %, 25.37 %, respetviely, than in those from WT male mice. Similarly, weights of gastrocnemius, rectusfemoris, and tibialis anterior isolated from mutated porcine propeptide (D75A) transgenic female mice are 19.90 %, 42.47 %, 45.61 % heavier than those from WT female mice. It is clear that the effect of mutated porcine myostatin propeptide on muscle growth is much more greater than its corresponding wild type

proppetide. Although one potential reason may be that mutated porcine myostatin propeptide may have a longer half life in vivo than its corresponding wilt type propeptide, further investigation is needed to understand the actual mechanism why mutated propeptide is more effective than wild type propeptide. Based on our in vivo results using recombinant propeptide and its mutant D75A, we employed the Sleeping Beauty transposon technology to generate transgenic mice expressing porcine propeptide mutant D75A to further verify the efficacy of this mutant in enhancing muscle growth in vivo. The data from this experiment showed that murine myostatin function is inhibited in transgenic mice, and muscles from

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transgenic mice were much heavier than their wild type littermates, implying that transgene expression of porcine propeptide mutant is a practical approach to inhibit myostatin activity and to promote mass growth. Our data clearly demonstrate that muscle growth could be enhanced by injection of recombinant porcine propeptide or by transgene expression. In both approaches, the mechanism by which these propeptides promote muscle growth in mice is due to an increase in myofiber size, not due to an increase in total number of myofibers. In summary, our current study paved the way for producing high quality pork (higher lean yield and less fat) using genetically engineered pigs that express myostatin propeptide mutant. Acknowledgments This study was support by the National Natural Science Foundation of China (Grant No. 30901022) and by the Agricultural Science and Technology Innovation Program (ASTIP-IAS05). We thank Chengyi Song for providing plasmids for ‘‘Sleeping Beauty’’ transposon (pT2-HB) carrier and SB100 transposase. We also thank for Jinan Jiao for editing the manuscript and valuable comments and suggestions on the manuscript. Compliance with Ethical Standards Conflict of interest The authors have declared that no competing interest exists.

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Functional verification of a porcine myostatin propeptide mutant.

Myostatin is a member of TGF-β superfamily that acts as a key negative regulator in development and growth of embryonic and postnatal muscles. In this...
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