Porcine congenital splayleg is characterised by muscle fibre atrophy associated with relative rise in MAFbx and fall in P311 expression

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Disparity of fibre size and density between PCS piglets and their normal litter mates. ST, LD and G muscles were removed from 4 sets of 2-day-old splayleg male piglets, each comprised an affected and a normal litter mate. Muscles were immunostained for dystroglycan, myosin heavy chain (MYH) slow and MYH fast. For each muscle sample, 6 microscopic fields were randomly selected for morphometric analysis. (A) Typical muscle fields of normal and PCS (ST muscles) immunostained for dystroglycan. PCS fibres were clearly smaller than those in the normal counterpart. (B) Fibre cross-sectional area was determined in PCS and normal muscles. PCS piglets showed smaller fibre size and higher fibre density in all three muscles. Differences between pair-wise combinations of the least square means were tested for significance (*p < 0.05). Asterisks indicated statistical significance between normal and PCS muscle. The error bars indicate standard error.

Figure 2

Distribution of muscle fibre cross-sectional areas in four sets of 2-day-old PCS piglets, each with a corresponding normal litter mate. At least 10,000 fibres were measured per muscle section and grouped by size in 25 μm² increments (A). ST and LD muscles of PCS piglets showed a narrow and steep pattern of distribution. In ST muscles, more than 50% of PCS fibres were less than 100 μm², compared with normal fibres where only 35% were less than 100 μm². In LD muscles, more than 50% of PCS fibres were less than 75 μm²; compared with normal fibres only about 35% were less than 75 μm². G muscles of PCS piglets showed a similar but smaller fibre size distribution than their normal litter mates. (B) However, no significant difference in slow or fast fibre type composition was found between normal and affected muscles. The error bars indicate standard error. Norm = normal; PCS = porcine congenital splayleg.

Figure 3

Comparison of muscle gene expression between PCS affected and normal muscles. Quantitative expression of MYHslow, MYH2x and MYH2b in ST, LD and G muscles, presented as combined results of 4 sets of 2-day-old PCS piglets. Differences between pair-wise combinations of the least square means were tested for significance (*p < 0.05). PCS piglets showed significantly lower expression of MYHslow in LD and G muscles, and lower expression of MYH2x and MYH2b in ST muscles. Asterisks indicate statistical significance between normal and PCS muscles. Error bars indicate standard error.

Figure 4

Quantitative PCR was performed for MAFbx expression. With the exception of ST muscles in litter 2, MAFbx was more highly expressed in all PCS muscle samples compared with the corresponding normal muscles. Results of each litter are expressed as mean ± standard deviation from triplicate samples within the same experiment. Combined results of 4 sets of PCS piglets also tested for significance (*p < 0.05) in differences between pair-wise combinations of the least square means. Norm = normal; PCS = porcine congenital splayleg.

Figure 5

Quantitative PCR was performed for P311 expression. In contrast to MAFbx, P311 expression was down-regulated in all PCS muscle samples. Results of each litter are expressed as mean ± standard deviation from triplicate samples within the same experiment. Combined results of 4 sets of PCS piglets also tested for significance (*p < 0.05) in differences between pair-wise combinations of the least square means. Norm = normal; PCS = porcine congenital splayleg.

Figure 6

Developmental expression of MAFbx and P311 . Expression of (A) MAFbx and (B) P311 at different stages of embryonic/foetal development (14, 21, 35, 49, 63, 77 and 91 days of gestation), in 24-week-old (adult) pigs, in 2-day-old PCS piglets and corresponding normal litter mates (Norm) were determined. Embryonic/foetal and adult cDNAs were pooled samples from 3 individuals. PCS and Norm cDNAs were pooled from the LD muscles of the 4 sets of litter mates. Expression of MAFbx was detected at basal levels throughout gestation and into adulthood but was elevated in PCS muscles. P311 expression showed rising levels throughout development. However, its expression in PCS piglets was much reduced. Results expressed as mean ± standard deviation from triplicate samples within the same experiment. Norm = normal; PCS = porcine congenital splayleg.

Figure 7

Over-expression of P311 in C2C12 cells. (A) P311 expression, through infection with an adenoviral construct and through stable transfection with an expression plasmid, was determined over a time course. Proliferating cells were infected for 3 days in proliferation medium [PM] (3I), followed by 3 days in differentiation medium [DM] (3I 3D) or 6 days in DM (3I 6D). Stably transfected cells were incubated for 3 days in PM (PM), followed by 3 days in DM (DM 3D) or 6 days in DM (DM 6D). Results expressed as mean ± standard deviation from triplicate samples within the same experiment. (B) Adenovirus-mediated (at 0.5 MOI) expression of P311 in C2C12 cells (infected for 3 days during proliferation followed by 6 days of differentiation), viewed under fluorescence for GFP and immunostained for FLAG. P311-infected cells showed co-localisation of both proteins. (C) The viability of P311-infected and P311-transfected C2C12 cells was similar to control cells over an extended culture period, as demonstrated using a CellTiter-Blue kit (Promega). For P311 adenovirus, cells were infected in PM for 3 days (3I), followed by 3 day DM (3I 3D), or by 6 days in DM (3I 6D), or 10 days in DM (3I 10D). Stably transfected cells were grown in PM for 3 days (PM), followed by 3 day in DM (DM 3D), or 6 days in DM (DM 6D), or 10 days in DM (DM 10D). Results expressed as mean ± standard deviation from triplicate samples within the same experiment.

Figure 8

Effects of P311 on C2C12 cell proliferation and differentiation. (A) BrdU assay was performed on P311-infected and adenovirus-GFP control cells, as well as P311-transfected and vector only-transfected cells. For both approaches, P311 over-expression significantly increased cell proliferation. (B)P311-stably transfected C2C12 myotubes (differentiated for 9 days) were separately immunostained for desmin and MYH fast, along with DAPI nuclei staining. Under immunofluorescence, myotubes appeared less abundant in P311-transfected cells. (C) Fusion index quantification on immunostained images showed that P311-transfected cells resulted in less mytoube formation than control cells. Asterisks indicate statistical significance. BrdU assay and fusion index determination were analysed with SAS software, differences between pair-wise combinations of the least square means were tested for significance (*p < 0.05). Error bars were standard error, n = 3 replicates from 3 independent experiments.

Figure 9

Muscle gene expression in P311-adenovirus infected C2C12 cells. Real-time PCR was performed on cDNAs derived from infected cells over a time course as detailed in Fig. 4. No dramatic difference in MYH expression between P311-infected and control C2C12 cells was seen during the early infection time points (3I and 3I 3D). Reduction in expression of MYHembryonic, MYH2b, Myf-5 and α-actin in P311-infected C2C12 myotubes was noticeable at the last time point (3I 6D). Results expressed as mean ± standard deviation from triplicate samples within the same experiment.

Figure 10

Effect of calcineurin over-expression on endogenous P311 expression in C2C12 cells. (A) Cells were infected with a constitutively active calcineurin (Cn) or GFP-control adenoviral construct in PM for 2 days (2I), or for 3 days (3I), followed by 1 day DM (3I 1D), or 3 days in DM (3I 3D), or 6 days in DM (3I 6D). (B) Cells were also infected as myotubes (at 4 days in DM) for 2 days, followed by a further 2 days in DM (4+4), or 5 days in DM (4+7), or 7 days in DM (4+9). In both series (A and B), extended expression of Cn led to a reduction of P311 expression. Results expressed as mean ± standard deviation from triplicate samples within the same experiment.