Muscle transcriptome analysis reveals molecular pathways and biomarkers involved in extreme ultimate pH and meat defect occurrence in chicken

Abstract

The processing ability and sensory quality of chicken breast meat are highly related to its ultimate pH (pHu), which is mainly determined by the amount of glycogen in the muscle at death. To unravel the molecular mechanisms underlying glycogen and meat pHu variations and to identify predictive biomarkers of these traits, a transcriptome profiling analysis was performed using an Agilent custom chicken 8 × 60 K microarray. The breast muscle gene expression patterns were studied in two chicken lines experimentally selected for high (pHu+) and low (pHu-) pHu values of the breast meat. Across the 1,436 differentially expressed (DE) genes found between the two lines, many were involved in biological processes related to muscle development and remodelling and carbohydrate and energy metabolism. The functional analysis showed an intensive use of carbohydrate metabolism to produce energy in the pHu- line, while alternative catabolic pathways were solicited in the muscle of the pHu+ broilers, compromising their muscle development and integrity. After a validation step on a population of 278 broilers using microfluidic RT-qPCR, 20 genes were identified by partial least squares regression as good predictors of the pHu, opening new perspectives of screening broilers likely to present meat quality defects.

Figure 1

Serial light micrographs of the pHu+ (A,C,E,G) and pHu− (B,D,F,H) pectoralis major muscles. Periodic Acid Schiff histochemical staining (A,B), immunohistochemical staining of the adult (C,D), both embryonic and neonatal (E,F), and neonatal (G,H) isoforms of the fast myosin heavy chain. The adult and neonatal isoforms of the chicken fast myosin heavy chain were detected using the MF14 (C,D) and 2E9 (G,H) antibodies, respectively; the embryonic and neonatal isoforms were simultaneously revealed using the B103 antibody (E,F). All primary antibodies were purchased from the Developmental Studies Hybridoma Bank (Iowa City, IA). Scale bar: 100 µm.

Figure 2

Heat map from the hierarchical clustering of differentially expressed genes between the pHu− and pHu+ individuals. The scaled expression by row (gene) is shown as a heat map and is reordered by a hierarchical clustering analysis (Pearson’s distance and Ward’s method) on both rows and columns.

Figure 3

Top 10 enriched biological process GO terms. A functional enrichment analysis of the biological process GO terms was performed using the topGo R package on the genes that were differentially expressed in the breast pectoralis major muscle from the two lines of broilers divergently selected for their breast meat ultimate pH. The levels of significance are indicated by ***p-value ≤1 × 10⁻⁵, **p-value ≤0.001, and *p-value ≤0.01.

Figure 4

Correlation between the log2 fold-change (pHu−/pHu+) of gene expression from microarray and quantitative RT-PCR microfluidic technologies.

Figure 5

Final PLS model for the pectoralis major pHu prediction. The final PLS model was fitted on the mRNA expression of 20 genes (measured using RT-qPCR microfluidic technology) on a population of 278 male and female individuals from the pHu+ and pHu− lines in order to predict the pectoralis major pHu. (a) Score plot of the two first components (t1, t2) from the PLS model. Points correspond to chicken tags coloured according to their measured pHu value in the pectoralis major muscle. The cumulative explanatory and predictive performance characteristics of the model are R²(cum) = 0.65 and Q²(cum) = 0.62. The root mean square error of the estimation (RMSEE) was 0.16. (b) Scatterplot of the observed pHu vs. predicted pHu. (c) Variable Importance in Projection (VIP) of each variable (genes) in the final PLS model (the genes from the fsPLS model are highlighted in green, those with an extreme FC based on microarray analysis are in orange, and those whose function is directly related to carbohydrate and muscle metabolism are in blue).

Figure 6

Molecular actors of glycogen turnover. Function of the studied enzymes in the regulation of muscle glycogen metabolism. AMPK = adenosine monophosphate-activated protein kinase; PHK = phosphorylase kinase; GYS = glycogen synthase; PYG = glycogen phosphorylase; PP1 = protein phosphatase-1; PPP1R3A = protein phosphatase-1 regulatory subunit 3A.