Biomarkers related to postmortem lamb meat quality and their molecular mechanism

Meat quality is an essential prerequisite for consumer acceptability and industrial profitability, which includes meat tenderness, color, water holding capacity (WHC) and flavor. Meat quality deterioration mainly consists of discoloration, high drip loss and texture deterioration of meat in postmortem or retail displays. However, the loss or waste of meat caused by deterioration of meat quality traits accounts for approximately 20% per year, according to FAO's report, which leads to a huge waste for meat resources, environmental pollution, and economic loss. Fresh meat quality is greatly determined through biochemical changes occurring in the muscle during its conversion to meat. These changes are key to imparting a unique set of characteristics on fresh meat, including its appearance, ability to retain moisture, and texture. Biomarkers are molecular compositions of biological processes that reveal the differential expression associated with the phenotype of a specific trait. Therefore, it is very important to discover the key biomarkers that could characterize and supervise the budding deterioration of meat quality, aiming to evaluate and regulate meat quality in postmortem conditioning. The objective of present study was to investigate and confirm the biomarkers of lamb meat quality and elaborate the molecular mechanism of their characterizing the postmortem meat quality. Firstly, proteomics was used to identify the dysregulated proteins related to three differential lamb meat quality traits groups (n=6) based on tenderness parameters (shear force, myofibrillar fragmentation index (MFI)), color parameters (a* value, R630/580) and water holding capacity parameters (drip loss and cooking loss). A total of 2176 proteins were quantified in these samples using of TMT-proteomics. Among them, 317, 233 and 69 showed the differential abundance in three different meat quality groups, respectively. Subsequently, 8 dysregulated proteins (phosphoglycerate kinase 1 (PGK1), pyruvate kinase M2 (PKM2), phosphoglucomutase 1 (PGM1), β-enolase (ENO3), myosin-binding protein C (MYBPC1), myosin regulatory light chain 2 (MYLPF), Troponin C 1 (TNNC1) and Troponin I 1 (TNNI1)) were confirmed that they were associated with lamb meat quality traits by parallel reaction monitoring (PRM) targeted proteomics and western blotting combined with Pearson correlation analysis. These proteins may be used as potential biomarkers to assess and characterize the postmortem lamb meat quality. Secondly, aiming at the 8 potential biomarkers, the different meat quality traits groups of Small Tailed Han sheep and different muscle types (Longissimus thoracis (LT) and Quadriceps femoris (QF) muscles) were selected from 100 lamb carcasses to verify them by analyzing their relative abundance and enzymatic activity. The results found that the relative abundance of PKM2, PGK1, PGM1, and ENO3 with the significant difference in different breeds and muscle types groups, while the relative abundance of MYBPC1, MYLPF, and TNNI1 was significantly different between LT and QF muscle groups. Moreover, the activity of PKM2 and PGK1 showed a significant difference in different breeds and muscle types groups. Overall, the relative abundance of PKM2 and PGK1 as well as PKM2 and PGK1 activity had a remarkably difference in various meat quality groups of different breeds and muscle types. Therefore, it was suggested that PKM2 and PGK1 can be used as biomarkers of lamb meat quality. Subsequently, in order to further confirm the ability of PKM2 and PGK1 as robust biomarkers and their relationship with postmortem muscle metabolism and physiochemical changes. The activity of PGK1 and PKM2 was regulated by their inhibitors and activators to construct the postmortem glycolysis vitro model and then incubated at 4°C for 24 h. The results revealed that compared to PGK1 and PKM2 inhibitors groups, the addition of PGK1 and PKM2 activators could accelerate glycogen consumption, ATP and lactate production, while declining pH value. Moreover, the addition of PGK1 and PKM2 activators could increase Desmin degradation, μ-calpain activity, and caspase-3 abundance. Interestingly, Troponin-T degradation was significantly increased both in PKM2 inhibitor and activator groups. It was suggested that PGK1 and PKM2 could be used as robust biomarkers to regulate meat quality by affecting the glycolysis, myofibrillar proteins, μ-calpain and apoptosis pathways in postmortem muscle. Finally, to clarify the precise molecular mechanism of PGK1 and PKM2 affecting and regulating the development of meat quality, the high-activity and low-activity groups (n=10) were selected from 60 lamb muscles at 24 h postmortem based on the activity levels of PGK1 and PKM2. The metabolomic, proteomic, and transcriptomic analysis combined with deeply integrated multi-omics analysis were used to reveal the mechanisms by which PGK1 and PKM2 characterize meat quality. The results indicated that glycolysis was a critical biological pathway that could affect the activity of PGK1 and PKM2 at the metabolome, proteome, and transcriptome levels. In glycolysis, the differential metabolites (ATP, ADP, glucose-6-phosphate, NADP, fructose-6-phosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, NAD+, lactate and pyruvate), different abundance proteins (LDHB and ALDOB), and different expressed genes (HK and FBP1) closely related to PGK1 and PKM2 activity were identified. It was concluded that PGK1 and PKM2 may affect the formation of lamb meat quality by regulating these critical substrates. Additionally, PGK1 and PKM2 could also affect the TCA cycle, oxidative phosphorylation, and muscle contraction to regulate the postmortem meat quality. In summary, the present study has identified PGK1 and PKM2 as robust biomarkers associated with lamb meat quality. They could regulate meat quality by influencing glycolysis, myofibrillar proteins, μ-calpain, and apoptosis pathways in postmortem muscle. Additionally, the differential metabolites (ATP, ADP, glucose-6-phosphate, NADP, fructose-6-phosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, NAD+, lactate, and pyruvate), different abundant proteins (LDHB and ALDOB), and differently expressed genes (HK and FBP1) in the glycolytic pathway, which may be regulated by the activity of PGK1 and PKM2 and then affect postmortem lamb meat quality. This study offers valuable insight into unraveling the molecular mechanisms underlying postmortem meat quality development and provides a reference for developing non-destructive detection technology for meat quality.