Porcine congenital splayleg is characterised by muscle fibre atrophy associated with relative rise in MAFbx and fall in P311expression

Muscle fibre atrophy in PCS muscles

Morphological and fibre type analyses were conducted on 4 sets of muscles from 4 separate litters of pigs (3 muscles per pig: ST, LD and G); each set comprised one 2-day-old affected PCS piglet and a normal litter mate. Histological examination of all PCS muscles showed no evidence of significant inflammatory change (data not shown). The average fibre cross-sectional area of affected PCS piglets was consistently smaller than their normal counterparts in all 3 muscles (Fig. 1). Consequently, fibre density (fibre number per unit area) from affected individuals was higher in the 3 muscles (Fig. 1B). PCS piglets showed higher distribution of fibre number in the smaller fibre range than normal piglets (Fig. 2A). The contrast in fibre size distribution was particularly evident in the ST and LD of PCS piglets, which showed a narrow and steep distribution pattern of smaller fibres (Fig. 2A). Hence fibre atrophy was a feature of PCS muscles. However, based on immunostaining for myosin heavy chain (MYH) slow and MYH fast fibres, no significant difference was found in fibre type composition between normal and affected muscles (Fig. 2B).

Selective reduction of MYH expression in PCS muscles

To further assess the phenotypic differences between muscles of normal and PCS piglets, TaqMan quantitative real-time PCR was performed on the muscles from the 4 sets of litter mates to determine the relative mRNA expression levels of MYHembryonic, MYHperinatal, MYHslow, MYH2a, MYH2x, MYH2b and α-actin. PCS piglets showed significantly lower expression of MYHslow in LD and G muscles (Fig. 3). Expression of MYH2x and MYH2b in ST muscle was also statistically lower in PCS piglets (Fig. 3). No significant difference was found between normal and PCS piglets for MYHembryonic, MYHperinatal, MYH2a and α -actin (data not shown).

Up-regulation of MAFbx and down-regulation of P311 in PCS muscles

The ubiquitin-proteasome proteolysis pathway is a principal route of muscle atrophy in which MAFbx, an E3 ubiquitin ligase, was found to be consistently up-regulated in a variety of muscle atrophic conditions [16, 17]. Because of the close association of MAFbx expression with muscle atrophy, we determined the relative expression of porcine MAFbx in normal and PCS muscles (Fig. 4). We found that in almost all muscle samples examined, with the exception of ST muscle in litter 2, MAFbx expression was clearly up-regulated in PCS piglets (Fig. 4). Another growth-related gene associated with muscle atrophy, P311, is a novel gene reported to be down-regulated in a number of muscle atrophic conditions [17] but up-regulated under moderate dietary restriction [18]. In contrast to MAFbx, P311 expression in PCS muscles was consistently down-regulated in comparison with normal controls (Fig. 5). It is worthy to note that the significant differences in MAFbx and P311 expression between normal and affected muscles of litter 2 were not as great as in the other 3 litters, which might be a reflection of differing gradation of the disease process (Fig. 4 and 5). Moreover, the wide variation in expression levels of MAFbx and P311 between litters, were not entirely surprising. Endogenous gene expression is known to vary widely between individual pigs of the same genetic background and age, reared under identical husbandry conditions [19].

As little is known about the normal developmental expression pattern of MAFbx and P311, we examined their embryonic and muscle foetal expression at different stages of gestation, and their expression in adult muscle (Fig. 6). Real-time PCR results showed that MAFbx expression remained at basal levels throughout gestation and in adult muscle (Fig. 6A). In relation to normal muscles, MAFbx expression in PCS LD muscles was significantly elevated (Fig. 6A). Real-time PCR results showed that P311 expression in normal animals increased with gestation and remained raised in newborn and adult muscles. However, the expression of P311 in 2-day-old PCS piglets was considerably down-regulated (Fig. 6B).

P311 over-expression in C2C12 cells by adenovirus infection and stable transfection

To assess the function of P311 in muscle cells, we over-expressed P311 in C2C12 muscle cells by stable transfection as well as by infection with a P311-adenovirus construct (Fig. 7). Expression of both constructs could be demonstrated by P311 mRNA detection (Fig. 7A), and by P311-FLAG fusion protein detection by immunofluorescence (Fig. 7B). Cell cytotoxicity assays that measured metabolic capacity showed no significant difference between infected/transfected cells, and control cells over an extended period of 10 days differentiation, which indicated similar cell viability (Fig. 7C). This finding also indicates that over-expression of P311 is not detrimental to C2C12 cells.

P311 over-expression increased C2C12 cell proliferation and reduced differentiation

To determine the effect of P311 on cell proliferation, 5'-bromo-2'-deoxyuridine (BrdU) assays were performed on P311-adenovirus infected and stably transfected C2C12 cells. Both constructs promoted proliferation compared with control GFP cells (Fig. 8A). C2C12 cells stably transfected with P311 gave rise to less myotubes as visualised by immunostaining for desmin, and MYH fast proteins (Fig. 8B). This observation could be appreciated quantitatively by determining the fusion index of the transfected myotubes (Fig. 8C), which showed that P311 over-expression produced less myotube formation than control.

No obvious differences in MYH expression were detected between P311-infected and control C2C12 cells during the early stages (3I and 3I 3D) of development (Fig 9). However, at the later stage of differentiation (3I 6D), there was the suggestion of reduced expression of MYHembryonic, MYH2b, Myf-5 and α-actin in P311-infected C2C12 myotubes (Fig. 9), with the notable exception of MYHslow which showed raised expression. The overall results of extended P311 over-expression indicated a trend towards reduction in the expression of a number regulatory and structural muscle genes, which was consistent with reduced muscle differentiation. Since P311 appeared to associate with increased proliferation (Fig. 8A), and reduced differentiation and myotube formation (Fig. 8B to 9), we examined whether it might also be down-regulated by the over-expression of constitutively active calcineurin (Cn), a key mediator of skeletal muscle differentiation [20, 21]. Indeed, endogenous expression of P311 was down-regulated a few days after Cn infection, regardless of the developmental stage of the C2C12 cells (Fig. 10). Hence, in the promotion of muscle differentiation, Cn down-regulated P311 expression.

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 [22], a major protein degradation route known to be responsible for skeletal muscle atrophy [23]. 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 [24], is a small 8 kDa 68-amino acid protein with a short half-life of around 5 minutes [25]. 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 [26]. 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 [17]. 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 [17]. 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 [27]. 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 [25]. 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.

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 [30]. 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).

Expression constructs

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.

Immunofluorescence

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 [31]. 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

Statistical analyses

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.