Porcine congenital splayleg is characterised by muscle fibre atrophy associated with relative rise in MAFbx and fall in P311expression
© Ooi et al; licensee BioMed Central Ltd. 2006
Received: 11 May 2006
Accepted: 25 July 2006
Published: 25 July 2006
Porcine congenital splayleg (PCS) is the most important congenital condition of piglets, associated with lameness and immobility, of unknown aetiology and pathogenesis, hence the need to better understand the condition by defining, in the first instance, its histopathology and molecular pathology.
Semitendinosus, longissimus dorsi, and gastrocnemius muscles were removed from 4 sets of 2-day-old splayleg piglets, each with a corresponding normal litter mate. Based on immunohistochemistry and histological image analysis, PCS piglets showed significantly smaller fibre size without any accompanying sign of inflammation. Although there was no dramatic change in fibre type composition in affected muscles, several structural myosin heavy chain genes were significantly down-regulated. MAFbx, a major atrophy marker, was highly up-regulated in nearly all PCS muscles, in comparison with controls from normal litter mates. In contrast, P311, a novel 8 kDa protein, was relatively down-regulated in all the PCS muscles. To investigate a functional role of P311 in skeletal muscle, its full-length cDNA was over-expressed in murine C2C12 muscle cells, which resulted in enhanced cell proliferation with reduced myotube formation. Hence, reduced P311 expression in PCS piglets might contribute to atrophy through reduced muscle cell proliferation. P311, predictably, was down-regulated by the over-expression of calcineurin, a key signalling factor of muscle differentiation.
We demonstrated that PCS is a condition characterised by extensive fibre atrophy and raised fibre density, and propose that the combined differential expression of MAFbx and P311 is of potential in the diagnosis of subclinical PCS.
Porcine congenital splayleg (PCS), also known as straddlers, and myofibrillar hypoplasia, is a clinical condition of newborn piglets, characterised by muscle weakness, resulting in the inability to properly stand and walk, with affected limbs extended sideways or forwards . It is arguably the most important congenital defect of commercial piglets and causes significant economic loss to pig farmers . The prevalence of PCS can range from less than 1% in most farms to over 8% in some establishments .
PCS can be found at or a few hours after birth in up to 2 to 3 piglets in an affected litter. Arising from long periods of recumbency, abrasions and ulceration may develop, which could predispose the affected individual to secondary arthritis, pododermatitis, and osteomyelitis of the digits. Mortality rate can reach 50% due to starvation or crushing by the sow. Affected piglets can recover after a week if supportive treatment is provided. PCS is prevalent in Landrace, Large White breed, and in other heavily muscular breeds [4–6]. Male and female piglets appear susceptible to PCS in similar proportion [1, 5, 6]. There are, however, conflicting reports on the association of PCS with litter size or birth weight [5, 7].
PCS appears to be a multifactorial condition of unknown aetiology. The predisposing factors are thought to include genetics and environmental factors, such like nutrition, management, pharmacological administration, and mycotoxins. Pregnant sows treated with glucocorticoids , and experimentally fed Fusarium (F-2) toxin (zearalenone) contaminated grain  showed higher incidence of PCS. However, histological changes in glucocorticoid induced or mycotoxin-induced PCS are distinct from naturally occurring PCS . The role of dietary choline appears unimportant in the prevention of PCS [11, 12]. Administration of pyrimethamine, an antibiotic often used in combination with a sulphonamide, to pregnant Goettingen minipigs , and of prostaglandins to induce parturition in late pregnancy  have been reported to lead to higher incidence of PCS.
A range of pathological lesions has been described for PCS, the most common feature being the presence of myofibrillar hypoplasia, often interpreted as an immaturity of the muscle [1, 10]. Myofibrillar hypoplasia ranges from a slight reduction of myofibrillar content to severe myofibrillar deficiency, with vacuolation, focal degeneration and necrosis. However, myofibrillar hypoplasia is not exclusive to PCS as the condition is also found in clinically normal piglets. The descriptive finding of myofibrillar hypoplasia is therefore not be diagnostic of PCS . Furthermore, subjective microscopic and ultrastructural muscle examination had found no significant qualitative changes between normal and PCS piglets [3, 15]. Presently, there is a lack of objective morphological information available on PCS muscles. The term hypoplasia refers to underdevelopment of a tissue or organ that results in a decrease in cell number. Atrophy refers to a decrease in the size of a tissue or organ caused by disease or disuse. It is not certain if clinical PCS is related to a reduction of fibre size (atrophy), number (hypoplasia) or both. Additionally, no histochemical, biochemical, or molecular changes have been reported that are characteristic of PCS. Therefore, there is a need to better define the histopathological, biochemical and molecular changes that take place in muscles of PCS. The development of objective cellular and molecular parameters to assess clinical PCS could add valuable information to our understanding of the pathogenesis of the disease. With recent advances in imaging technology and quantitative PCR, morphological analysis and relative muscle gene expression can now be conducted with greater precision. We describe here the histopathological and molecular characterisation of PCS. We found that PCS piglets had smaller fibre size and higher fibre density in the semitendinosus (ST), longissimus dorsi (LD) and gastrocnemius (G) muscles. Muscle Atrophy F-box (MAFbx), a marker of muscle atrophy, was more highly expressed in all PCS muscles. Conversely, expression of P311, a novel 8 kDa protein with a conserved PEST domain, was down-regulated in all PCS muscles tested.
Muscle fibre atrophy in PCS muscles
Selective reduction of MYH expression in PCS muscles
Up-regulation of MAFbx and down-regulation of P311 in PCS muscles
P311 over-expression in C2C12 cells by adenovirus infection and stable transfection
P311 over-expression increased C2C12 cell proliferation and reduced differentiation
PCS is associated with extensive muscle fibre atrophy
PCS is a well-recognised, commercially important clinical congenital condition of piglets. However, woefully little is known about its aetiology, pathogenesis or pathology. In this study, we demonstrated that PCS muscles (LD, ST and G) consistently showed fibre atrophy with concomitant increase in fibre density (Fig. 1B). It appears that PCS is an extensive muscle condition that affects several muscle groups. Indeed, previous work has shown that PCS affects different muscles [1, 15]. We found that the ST and LD were more severely affected than the G (Fig. 2A). PCS associated atrophy was not accompanied by significant changes in fibre type composition (Fig. 2B), but some affected muscles showed significant reduction in the expression of MYHslow, MYH2x or MYH2b gene (Fig. 3). Hence PCS is a condition associated with muscle fibre atrophy ostensibly connected to reduced protein accretion, and is associated with reduction in the expression of structural muscle genes. At present, it is unclear if the atrophic fibre change observed in PCS muscles is a primary pathology of the condition or is a secondary outcome of disuse atrophy. In the present work, due to technical difficulty in obtaining complete cryostat cross-sections representative of total muscle areas, total fibre number of each muscle could not be reliably determined. At present, it is not certain if PCS-associated fibre atrophy is also accompanied by fibre hypoplasia.
PCS is associated with muscle wasting
MAFbx, an E3 ubiquitin ligase enzyme, was identified as an early marker of atrophy through differential expression screening studies in multiple models of skeletal muscle atrophy [16, 17]. E3 ubiquitin ligase is one of three enzymatic components of the ubiquitin-proteasome pathway , a major protein degradation route known to be responsible for skeletal muscle atrophy . We demonstrated high levels of MAFbx mRNA in all 4 atrophic PCS piglets from 4 litters (presented as individual and combined litters) (Fig. 4), in contrast with basal levels found in normal muscles pre- and post-natally (Fig. 6A). The elevated expression of MAFbx would suggest that the PCS muscles were subjected to a process of muscle wasting, possibly as a consequence of disuse atrophy. It remains possible that PCS is primary condition of gestational muscle under development.
Functional significance of P311
P311, first identified in murine embryonic neurons , is a small 8 kDa 68-amino acid protein with a short half-life of around 5 minutes . It is characterised by the presence of a conserved PEST domain (sequences rich in proline, glutamic acid, serine and threonine) a targeted site for degradation by the ubiquitin-proteasome system . We found rising levels of P311 expression throughout gestation, which was maintained in post-natal normal muscles (Fig. 6B). By contrast, in PCS muscles, P311 was detected at much reduced levels (Fig. 5 and 6B). Expression of P311 was previously reported to be down-regulated in atrophic murine muscles . In conditions of muscle wasting, arising from denervation or disuse, the process of protein degradation exceeds the rate of protein synthesis, resulting in net protein loss . In PCS muscles, reduced P311 expression could be a consequence of net protein loss. On the other hand, given that P311 promoted cell proliferation (Fig. 8A), it may have an active role in promoting muscle growth through raised myoblast number.
P311 does not belong to any known family of proteins, and its cellular function remains largely unclear. To investigate the function of P311 in skeletal muscle, it is necessary to ascertain its effects in muscle in the context of cell proliferation, differentiation and phenotype determination. We established that P311 over-expression led to raised C2C12 cell proliferation and reduced myotube formation (Fig. 8A and 8C). Consistent with reduced myotube formation, expression of several muscle genes (MYHembryonic, MYH2b, α-actin and myf-5) was down-regulated in late differentiation of P311-over-expressed C2C12 cells (Fig. 9). Myf-5, like MyoD, is transcriptionally active in proliferating myoblasts; its exogenous expression can cause non-myogenic cells to differentiate and fuse into myotubes . Previously, P311 was shown to be involved in glioblastoma cell migration and fibroblast cell proliferation [28, 29]. Moreover, differentiation of neural cells was related to loss of P311 expression . Hence, P311 may play an active part in the determination of muscle mass through the promotion of myoblast proliferation. The endogenous expression of P311 was suppressed by Cn over-expression (Fig. 10). As Cn is a key mediator of muscle differentiation [20, 21] it would suggest that P311 could also have an effect of limiting muscle differentiation. In PCS muscles, reduced P311 expression could conceivably mediate fibre atrophy through reduced cell proliferation, leading to reduced availability of total myoblast number in the formation of muscle fibres. Moreover, reduced myoblast number could potentially lead to the formation of less muscle fibres, hence fibre hypoplasia. Future work will require detailed total fibre counting to determine if fibre hypoplasia is also a feature of PCS muscles.
In conclusion, we demonstrated that the PCS is a condition characterised by extensive fibre atrophy and raised fibre density with no significant difference in fibre type composition. At the molecular level, PCS muscles showed reduced expression of a number of sarcomeric genes, elevated MAFbx and reduced P311 expression. It seems likely that the development of clinical PCS is a function of fibre size and, possibly, fibre number at birth, such that below a certain fibre threshold, the newborn would no longer be able to properly support its own weight. The differential expression patterns of MAFbx and P311 between muscles of normal and affected litter mates indicate their potential for use as biomarkers in the diagnosis of subclinical PCS. Clearly, more work is needed to evaluate this potential.
Immunohistochemistry and image analysis
ST, LD and G muscles were removed from four 2-day-old splayleg male piglets, along with 4 corresponding normal litter mates. Ten μm thick serial sections were immunostained with antibody for β-dystroglycan used at 1:200 dilution (VisionBiosystems), for MYH slow used at 1:50 dilution (M-32, Sigma) and for MYH fast used at 1:50 dilution (NOQ7.5.4D, Sigma), as previously described . For each muscle sample, 6 fields with at least 500 fibres each were randomly selected for morphometric analysis under × 20 magnification using the KS300 image analysis software (Carl Zeiss Ltd).
Full length porcine P311 cDNA and a constitutively active murine calcineurin A (Cn) (from Dr. S. Williams, University of Texas) were cloned into an adenovirus vector, using the Adeno-X Expression System 2 (BD Biosciences). The starting plasmid vector pDNR-CMV for the insertion of P311 and Cn housed an expression cassette that was derived from the plasmid pAAV-IRES-hrGFP (Stratagene) which comprised a multiple cloning site for the creation of a 3'-end FLAG fusion gene and a GFP reporter gene, with an internal ribosomal entry site (IRES) between the two. For time course studies, infection with P311 and Cn-adenovirus constructs were used at multiplicity of infection (MOI) of 5. For stable transfection, full length P311 cDNA was cloned into a neomycin resistant pBK-CMV expression plasmid vector (Stratagene), where transcription was driven by a cytomegalovirus immediate early promoter.
Cell culture, transfections and expression assays
C2C12 cells (CRL-1772) were grown in proliferation medium (PM) (10% foetal bovine serum in DMEM with 100 units/ml penicillin and 100 μg/ml streptomycin). At 80% confluence, PM was replaced by differentiation medium (DM) (4% horse serum in DMEM with penicillin and streptomycin). For stable transfections, C2C12 cells at 35% confluence in T25 culture flasks were transfected with 6.0 μg P311 plasmid, by the use of lipofectamine and Opti-MEM, according to manufacturer's instructions (Invitrogen). Next day, the transfection medium was replaced with PM for 1 day, followed by G418 (Invitrogen) selection at 2000 μg/ml over several days until a day after all cells in the control untransfected flask had been eliminated by G418. All subsequent experiments on stably transfected cells were conducted in the absence of G418.
C2C12 cells, infected with P311 or GFP control adenovirus, were grown for 3 days in PM, followed by 8 days in DM. To improve P311 stability, o-phenanthorline and lactacystin were added to the DM, at final concentrations of 1.26 mM and 10 mM respectively, 2 hour prior to fixation. Cells were fixed in 4% paraformaldehyde and incubated with the primary anti-FLAG M2 monoclonal antibody (Sigma), at a dilution of 1/2000, overnight at 4°C. A goat anti-mouse-TRITC (Southern Biotech) at 1/200 was used as the secondary antibody in a 45 minute incubation at 37°C. It was mounted using Vectashield (Vector) hard set mounting medium with DAPI. Images were captured by a CCD camera mounted on an inverted Olympus fluorescence microscope.
BrdU, fusion index and cell cytotoxicity assays
BrdU assays on C2C12 cells, infected with P311-adenovirus and stably transfected with P311 plasmid construct, were performed as previously described . Briefly, cells were incubated with BrdU for 2 hours and immunostained with a rat anti-BrdU monoclonal antibody. BrdU positive cells were counted. For each construct, three independent experiments were conducted.
For fusion index assays, after 9 days in DM, cells were fixed and incubated with a primary antibody (anti-desmin 1:200, Dako, or anti-MYH fast 1:200) at 4°C overnight. Secondary FITC conjugated sheep anti-mouse antibody (Sigma) was used at 1/200 dilution. For each sample, 5 fields were randomly chosen for counting. Fusion index was defined as the number of DAPI stained nuclei within myotubes in a given field divided by the total number of DAPI stained nuclei in the same field. Three independent experiments were conducted.
Cell cytotoxicity assay, a resazurin-based reduction assay that measured the metabolic capacity of cells, was performed according to manufactururer's protocol, normalised to unit weight of protein (CellTiter-Blue, Promega).
Quantitative real-time RT-PCR
Sequences of primers and TaqMan probes
Sequence 5' → 3'
GAG GGA AGG CCT AAG GG
CGG TCT CGC CAT CCT TCT T
ACT TCC CAT CCC AAA GGA AGT GAA CCG
GCC TGG GCT TAC CTC TCT ATC AC
CTT CTC AGA CTT CCG CAG GAA
CGT TTG AGA ATC CAA GGC TCA
CAG CTG CAC CTT CTC GTT TG
CCC GAA AAC GGC CAT CT
TGA GTT CAG CAG TCA TGA G
GGA CCC ACG GTC GAA GTT G
GGC TGC GGG CTA TTG GTT
CTA AAG GCA GGC TCT CTC ACT GGG CTG
CAA TCA GGA ACC TTC GGA ACA C
GTC CTG GCC TCT GAG AGC AT
TGC TGA AGG ACA CAC AGC TGC ACC T
CAG CCC CAC CTC CAA CTG
GCA GCA CAT GCA TTT GAT ACA TC
TGT CTG GTC CCG AAA GAA CAG CAG CTT
TCC GAC AAC GCC TAC CAG TT
CCC GGA TTC TCC GGT GAT
ATG CTG ACT GAT CGT GAG AAC CAG TCT ATC CT
GGA GGC CAG GGT ACG TGA A
GAG CAC ATT CTT GCG GTC TTC
AGG AAC TTA CCT ACC AGA CTG
GAG CGT GGC TAT TCC TTC GT
CAC ATA GCA CAG CTT CTC TTT GAT
CGC GCA CAA TCT CAC GTT CAG CTG
CGT GAA AAG ATG ACC CAG ATC A
CAC AGC CTG GAT GGC TAC GT
TTG AGA CCT TCA ACA CCC CAG CCA TG
SAS software (SAS procedure mixed, SAS Institute) was used. Fibre size, fibre density, real-time PCR data for PCS (Fig. 3 to 5) were analyzed by mixed model variance analysis using muscle type (ST, LD, and G), disease status (normal, PCS) as fixed effects with litters and replicate as random effects. BrdU and fusion index data were analyzed by mixed model variance analysis, using treatment (P311 and GFP control) as fixed effects with experiments and replicate as random effects. The standard errors of the difference between sample means were calculated using the error mean square from the ANOVA. Differences between pair-wise combinations of the least square means were tested for significance (p < 0.05). Unless specifically stated, quantitative real-time RT-PCR data were expressed as mean ± standard deviation from triplicate samples within the same experiment.
This study was supported by the Ministry of Science, Technology and Innovation Malaysia (MOSTI), the University of Putra Malaysia (UPM), through a postgraduate scholarship to PTO, and by DEFRA and BBSRC through the part funding of NDC and JE. We are grateful to Prof. Michael Stear, for assistance in the statistical analyses.
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