meat science: from proteomics to integrated omics towards...

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Review

Meat science: From proteomics to integrated omics towardssystem biology

Angelo D'Alessandro, Lello Zolla⁎

Department of Ecological and Biological Sciences, University of Tuscia, Largo dell'Università, snc, 01100 Viterbo, Italy

A R T I C L E I N F O

⁎ Corresponding author at: Tuscia University,E-mail address: [email protected] (L. Zolla).

1874-3919/$ – see front matter © 2012 Elseviehttp://dx.doi.org/10.1016/j.jprot.2012.10.023

A B S T R A C T

Article history:Received 22 August 2012Accepted 26 October 2012Available online 6 November 2012

Since the main ultimate goal of farm animal raising is the production of proteins for humanconsumption, research tools to investigate proteins play a major role in farm animal andmeat science. Indeed, proteomics has been applied to the field of farm animal science tomonitor in vivo performances of livestock animals (growth performances, fertility, milkquality etc.), but also to further our understanding of the molecular processes at the basis ofmeat quality, which are largely dependent on the post mortem biochemistry of the muscle,often in a species-specific way. Post mortem alterations to the muscle proteome reflect thebiological complexity of the process of “muscle to meat conversion,” a process that, despitedecades of advancements, is all but fully understood.This is mainly due to the enormous amounts of variables affecting meat tenderness per se,including biological factors, such as animal species, breed specific-characteristic, muscleunder investigation. However, it is rapidly emerging that the tender meat phenotype is notonly tied to genetics (livestock breeding selection), but also to extrinsic factors, such as therearing environment, feeding conditions, physical activity, administration of hormonalgrowth promotants, pre-slaughter handling and stress, post mortem handling.From this intricate scenario, biochemical approaches and systems-wide integrated in-vestigations (metabolomics, transcriptomics, interactomics, phosphoproteomics, mathe-matical modeling), which have emerged as complementary tools to proteomics, havehelped establishing a few milestones in our understanding of the events leading frommuscle to meat conversion. The growing integration of omics disciplines in the field ofsystems biology will soon contribute to take further steps forward.

© 2012 Elsevier B.V. All rights reserved.

Keywords:Farm animalSystem biologyMuscle to meat conversionProteomicsIntegrated omics

Contents

1. Proteomics and farm animal science: a special focus on meat science . . . . . . . . . . . . . . . . . . . . . . . . . . 5591.1. Proteomics and swine science and meat investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559

1.1.1. Breed and age or gender-related investigations . . . . . . . . . . . . . . . . . . . . . . . . . . 5601.1.2. Pig meat leanness and fat accumulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5601.1.3. Pig meat quality: color, water holding capacity and PSE . . . . . . . . . . . . . . . . . . . . . 560

1.2. Proteomics and bovine science and meat investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561

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1.2.1. Proteomics and bovine meat tenderness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5611.3. Proteome mapping of poultry and rabbit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562

2. Technological innovation in omics disciplines and farm animal science . . . . . . . . . . . . . . . . . . . . . . . . 5622.1. miRNAomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5622.2. Post translational modification “omics”—PTMomics in farm animal science . . . . . . . . . . . . . . . . . . 5632.3. Metabolomics and lipidomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563

3. From “Omics” to system biology in farm animal and meat science . . . . . . . . . . . . . . . . . . . . . . . . . . . 5634. Muscle to meat conversion: an overview through integrated omics . . . . . . . . . . . . . . . . . . . . . . . . . . . 565

4.1. Step I: Blood supply loss: oxygen and nutrient levels decline . . . . . . . . . . . . . . . . . . . . . . . . . . 5654.2. Step II: Glycolysis ensues, pH drops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5664.3. Step III: Apoptosis ensues: caspases and heat shock proteins . . . . . . . . . . . . . . . . . . . . . . . . . . 5674.4. Step IV: Energyless muscle and onset of rigor: control is lost over Calcium reservoirs, kinases are activated

and proteins are phosphorylated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5694.5. Step V: Calcium-dependent and independent proteases cleave myofibrils at the Z-disk . . . . . . . . . . . 5694.6. Step VI: the controversial role of oxidative stress in the promotion of myofibril degradation . . . . . . . . . 5704.7. Step VII: Muscle structure is altered by proteolysis and ion homeostasis dysregulation . . . . . . . . . . . . 570

5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572

1. Proteomics and farm animal science: aspecial focus on meat science

Research tools to investigate proteins play a major role in meatscience, since the ultimate end objective of farm livestockraising is the production of proteins for human consumption.Within the framework of protein-oriented investigations,proteomics has achieved a leading role over the last twodecades, owing to a long series of technological innovations inthe fields of protein separation (through chromatography andelectrophoresis), mass spectrometry and bioinformatics andtheir application to farmanimal science-relevant issues [1–4]. Inaddition, the more extensive publication of species genomes ismaking the proteomic approach in animal science more viable[1,2], especially as far as protein identification through massspectrometry and database interrogation are concerned.

The flourishing of the field of farm animal proteomics is alsoconfirmed by the constant growth and spread of internationalinitiatives. The European Cooperation in Science and Technol-ogy (COST) farm animal proteomics (Action FA1002) [2] is aninitiative that has been promoted by the EU with the goal toapply proteomics in animal science in order to reach a deeperunderstanding of the phenotype, physiology, pathophysiologyand productivity of land and water raised farm animals.

To date, proteomics has been applied to the field of farmanimal science to monitor in vivo performances of livestockanimals (growth performances, fertility, milk quality etc. [1–7]),but also to further our understanding of themolecular processesat the basis of meat quality, which are largely dependent on thepost mortem biochemistry of the muscle, often in a species-specific way [8]. Indeed, one of the major goals of proteomics inthe field of farm animal science is to shed light on skeletalmuscle biochemistry [9] and to deepen our understanding of thephysiological changes taking place at the protein level followingharvest. Post mortem alterations to the muscle proteome reflectthe biological complexity of the process often referred to as“muscle to meat conversion” [8].

1.1. Proteomics and swine science and meat investigations

The pig (Sus scrofa domesticus) is one of the most importantdomestic animals, with global populations estimated to be1 billion pigs (www.thepigsite.com and www.zoosavvy.com),while pig meat represents an essential protein source to thehuman diet, although its consumption is often hampered byreligious constraints, as it happens for example in Muslimcountries. Pig has been selectively bred to fulfill differentpurposes, including features like growth performances, prolifi-cacy (Chinese breeds such as theMeishan), backfat thickness forthe production of lard and smoked ham production (Casertanaand Iberian/Alentejano pig), meat leanness (commercial linesbased on Large White and Landrace breeds), feed conversionrate, muscle growth development and size, adaptation to harshrearing environments and stress, flavor and taste-affectingtraits, like boar taint and the suitability for biomedical research(Yucatan or the Göttingen minipigs) [10].

Investigations in the field of swine farming range frominsemination to slaughter and industrial pork production [10].

The control of pig reproduction and the extensive use ofartificial inseminationhavebeen a key feature of pig productionover the last decades. Several experimental articles and reviewsaddress the post-fecundation events: from zygote formationand implantation, to embryo development and birth, thereexists a collection of complex physiological processes, depen-dent onvariables and conditionings both inherent to the animaland to environmental factors (the interested reader is referredto the reviews by Oestrup et al. [11],Waclawik [12] and Croy andco-workers [13]). Briefly, proteomics has been successfullyapplied to several aspects of bothmale and female reproductionas well as the interaction between the sperm and oocytes [14].Spermatozoa's surface proteins play amajor role in the processof fecundation of the oocyte. In a recent study, sperm surfaceproteins were purified, identified and their changes weremonitored during the different stages of maturation in theepididymis [15]. Proteomics approaches have been also applied

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to investigate the modification to the porcine oocytes during invitro maturation [16]. More recently, Powell and colleagues [17],using ExacTag™ labeling kit proteomics, have identified bio-markers of oocyte quality and developmental, reprogrammingpotential by comparing high and low quality oocytes.

Pork meat is the ultimate goal in pig production andmuscle is themajor component of meat and derived products.Many proteomic investigations have been carried out over theyears to the end of gathering information relevant to improveporkmeat quality in an efficient and cost-effective productionsystem. In this view, proteomics technologies have been alsoapplied to monitor breed-specific differences [18–22], genderdifferences [23], growth performances (meat leanness, backfatthickness [24–28]) in response to differential rearing condi-tions (e.g. intensive farming, feeding regime) and environ-ment (outdoor vs. indoor) [23,24] and, in general, to further ourunderstanding of pig meat quality [10,18–34] and of itsderivative products, such as dry-cured [35–37] and cookedham [38].

1.1.1. Breed and age or gender-related investigationsIn 2010, Kim and colleagues [18] investigated the pig muscleproteome and transcriptome by means of 2DE and microarrayanalyses, respectively. The proteomes of white and redskeletal muscles (longissimus dorsi and soleus, respectively)were compared in Large White, Landrace and Duroc crossedanimals. Significant breed-related differences were highlight-ed, including differential expression of heat shock proteins(HSPs) and metabolism-related proteins. Breed differenceswere also investigated by Mach and colleagues in a compar-ative proteomic profiling on 2 muscles from 5 different purepig breeds [21]. From this study it emerged that there existpotential biomarkers for breed classification. These proteinscan be used as suitable biomarkers to tackle the pigtraceability, an economy-relevant pig farming issue.

Kwasiborski et al. [23,24] used 2DE to study proteomechanges as a result of the original sire breed, rearing environ-ment and gender. Potential gender specific markers wereidentified, including an actin isoform, a myosin light chain 2isoform and cytochrome Bc1. Over the last decade, Hollung'sgroup played a key role in the field of proteomics application tomeat science [39], also in the field of pigmuscle research [25]. Inlike fashion to gender-related protein biomarkers, Hollung andcolleague suggested that protein-wide signatures also exist inrelation to pig age, including structural (actin) and metabolicproteins (enolase or triosephosphate isomerase), whichare overexpressed in older and younger animals, respectively [25].

Besides pure meat science research, proteomics has beenso far used also to address the effects of animal handling priorto slaughter, including the analysis of proteomics profiles inthe animals transported to the abattoir by comparison toanimals under fattening conditions [40]. From the study byYamane and colleagues [40], it emerged that stress levels inpigs transported to the abattoir resulted in a significantincrease in the levels of the pig major acute-phase protein(Pig-MAP).

1.1.2. Pig meat leanness and fat accumulationMarbling is the deposition of fat within the skeletal muscleand is a major indicator of the juiciness and flavor of pig and

beef. Recently, we performed a proteomics, transcriptomics andmetabolomics comparison of the longissimus lumborum musclesfrom the high fat-depositing Casertana pig in comparison to thelean meat Large White pigs [26]. Combining proteomics totranscriptomics profiling, we evidenced that Casertana wascharacterized by a greater amount of proteins involved inglycolytic metabolism and thus mainly relied on energy sourcesthat can be rapidlymobilized. On the other hand, in LargeWhitewedetected the over-expression of cell cycle and skeletalmusclegrowth related genes. In a second study [27], we thus combinedproteomic and metabolomics to understand whether higherlevels of glycolytic enzymes were utterly related to actualmetabolic changes (such as lactate accumulation). As a result,we observed that a slow pHdrop inCasertana pigs, albeit not therapid pH lowering in LW counterparts, significantly correlatedwith the alterationof enzyme levels to someextent, and thiswasalso reflected in the relative quantities of specific metabolites,including glycolysis intermediates and end-product metabolites[26,27].

In the study by Liu et al. [28] on the longissimus dorsi fromseveral species, it emerged that interindividual variability inintramuscular fat content might arise essentially from differ-ences in early adipogenesis.

Indeed, Cagnazzo et al. [41] showed that these differencesarise yet at the 14 days embryo stage.

1.1.3. Pig meat quality: color, water holding capacity and PSEPork meat quality is the ultimate goal in pig production.Changes in the organoleptic properties of the meat can bemonitored through assaying of specific parameters, includingwater holding capacity (WHC), pH drop and Minolta values –lightness, redness and yellowness – which are related to meatcolor. These values are intrinsically related to muscle proteincomposition, if we consider for example that red muscles relyon oxygen for their metabolism and display higher concentra-tions of the heme group-carrying protein myoglobin. WHC is amajor parameter for pork quality and, when inadequate, maylead to PSE (Pale, Soft and Exudative) orDFD (Dark FirmandDry)meat. The PSE zones are characterized by a reduction ofproteolysis rate of three proteins, troponin T, myosin lightchain and α-crystallin, and the total absence of heat shockprotein 27 [8]. Development of PSE zones in muscle affectsnegatively coherence of the meat and represents a well-knownissuewith texture in cookedhamproduction. PSEporkmeat, forexample, has been proposed to be primarily caused by anaccelerated rate of post mortem glycolysis resulting in lowmuscle pH while carcass temperature remains high, thuscausing protein denaturation, which is usually correlated topoor WHC [42,43].

In a comparative investigation on Casertana (fat meat) andLarge White (lean meat) pigs [26,27], we could assess thatbreed-specific differences at the protein level were not onlyrelated to growth performances and fat accumulation ten-dency in vivo, but they also affected post mortem perfor-mances through a direct influence on the forcedly anaerobicbehavior of pig muscles after slaughter. In particular, higherlevels of the enzyme glycerol-3-phosphate dehydrogenaseand glycerol 3-phosphate and glycerol metabolites wereindividuated in Casertana than in Large White, which couldbe related to the higher backfat thickness in the former and


lean meat in the latter. The differential individuation ofglycolytic enzymes in Casertana was related to higher lactateaccumulation in this breed, although it did not produce lowerultimate pH in respect to Large White. On the other hand,alterations of the levels of glycolytic enzymes, heat shockproteins (HSPB6) andanti-oxidant enzymes (SOD1, glutaredoxinand lipoxygenase) were correlated to a lower chewing timevalue, higher proteolysis and moderately lower WHC inCasertana in comparison to Large White.

The centrifugal drip proteome was recently tested throughproteomics by Di Luca and colleagues [34] in order to correlatethe differential abundance of specific proteins to anomalousWHC. As a result, HSPs appear to be altered in the centrifugaldrip proteomeof those animals displaying poorWHCvalues [34].

1.2. Proteomics and bovine science and meat investigations

Cattle farming represents a leading economic sector, withglobal populations estimated to be 1.3 billions of cattle (www.cattletoday.com). Bovine meat quality and traceability haveincreasingly become two pivotal issues in the internationalagenda both in developing and industrialized countries. Overthe years, proteomics tools have been applied in bovineresearch both as far as it concerns dairy cattle (milk [44–47],metabolism [48], nutrition [49], fertility [50], health [51]) andbeef cattle [52–71].

As for pig meat, bovine meat quality is largely influencedby three main attributes: flavor, juiciness and tenderness [72].

1.2.1. Proteomics and bovine meat tendernessBovine meat tenderness biochemistry is a long-debated issue[73]. Despite decades of advancements in the field [73–76],bovine meat tenderness still remains a hot topic. A handful ofkey biochemical aspects underpinning bovine meat tender-ness have been known since decades [73]. A role has beenconfirmed for the alterations of sarcoplasmic proteins,myofibrillar proteins (actomyosin complex, cleavage of disul-fide linkages, depolymerization of F-actin filaments, cleavageof myosin filaments, digestion of desmin, disorganization ofZ-bands and the troponin–tropomyosin complex) and sarco-lemma [19,54–56,58,60,62,67–69]. Other structural alterationstackle the connective tissue elements (collagen fibrils, groundsubstance). In parallel, post mortem (forcedly anaerobic)metabolism triggers accumulation of lactic acid (and acidifi-cation of the muscle), both at the intra- and extracellular level[63]. Degradation enzymes are involved in the process ofmuscle protein fragmentation/digestion, a process in whichlysosomal cathepsins and calcium-dependent calpains play amajor role, while the proteasome is involved to some extent[74–76]. In the last few years, it has been recognized asubstantial overlap between the process of meat conversionto muscle and apoptosis [58,76].

Most of the proteomics investigations on bovine meattenderness have been so far performed on the longissimusdorsi, which is a valuable cut for steak (ribeye, striploin ort-bone steaks).

Over the last two decades, proteomics investigation hashelped delineating a role for structural proteins, glycolyticenzymes [54,63] and heat shock proteins (HSPs)/chaperones[60] in the frame of muscle tenderization after slaughter.

Most of the above-mentioned proteomics papers on bovinemeat tenderness [51–68] have been recently reviewed [1,3,8,9].In two recent investigations on Chianina and Maremmanalongissimus dorsi tenderness [70,71], we integrated tenderness-related parameters (Warner Bratzler Shear force—WBS; colla-gen insolubility as an indicator of intramuscular connectivetissue; myofibril degradation at 48 h and 10 days after slaugh-ter; and sarcomere length) with the results obtained fromproteomics (2DE andMALDI-TOF TOF identification of differen-tial proteins between tender and tough meat; titanium dioxideenrichment and CID–ETD determination of phosphorylationsites; protein–protein interactionmodeling and gene ontology –GO – term enrichment) and results obtained through HPLC–MSmetabolomics.

The puzzle resulting from the integration of the above-mentioned proteomics analyses on bovine meat tenderness,along with the ones recently reviewed by Paredi and col-leagues [8], also fueled the debate about the likely benefits oflow-voltage electrical stimulation to improve carcass qualityand contributed to the realization of valuable studies on thistopic [52].

1.2.1.1. Fat accumulation. Besides meat tenderness, juici-ness and flavor constitute two main parameters affectingmeat palatability. Both are largely dependent on intramuscu-lar fat content, which influences fatty acid composition of themuscle and contributes to modifying meat flavor. Indeed, thevarying fatty acid compositions of adipose tissue and muscledetermine the firmness/oiliness of adipose tissue and theoxidative stability of muscle, which in turn affects flavor andmuscle color [77,78].

In Korean cattle steers, triosephosphate isomerase andsuccinate dehydrogenase are highly expressed in the earlyfattening stage and qualify as potential candidate biomarkersfor intramuscular fat content of beef [79].

In the study by Zhao et al. [80], the differences in proteinexpression detected in subcutaneous adipose tissues betweentwo breeds showing different backfat thickness accumulationtendencies (Hereford and Angus breeds versus ContinentalCharolais), indicated that annexin 1 expression was respon-sible for variation in adipogenesis in different breeds.

In a recent study [53], Zhang and colleagues concluded thatcarbonic anhydrase 2 andmyosin light chain 3, which expresseddown during adipogenic differentiation could be indicativemarkers for negative regulation of IMF development.

In a similar investigation, Aldai and colleagues observed thatTudanca beef had a better fatty acid profile than Limousincounterparts, especially in terms of the content of polyunsat-urated (P<0.05), long-chain polyunsaturated fatty acids (P<0.05)[81].

1.2.1.2. Beef color. Meat color is an economy-relevant param-eter, since it affects meat quality and consumers' appraisal, inlikewise fashion to the PSE and DFD meat discussed above forpig meat.

Changes in the sarcoplasmic proteomesmay lie at the basisof beef color-stable (longissimus lumborum) and color-labile (psoasmajor) muscles [82]. In particular, aldose reductase, creatinekinase, andβ-enolase positively correlatedwith redness values,while peroxiredoxin-2, dihydropteridine reductase, and heat

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shock protein-27 kDa correlatedwith surface color stability [82].On the other hand, mitochondrial aconitase exhibited anegative correlation with redness values.

1.3. Proteome mapping of poultry and rabbit

Despite the fact that chicken meat is a food commonly andintensively consumed worldwide, very few investigationshave been aimed at characterizing chicken muscle and meatproteomics and, in most of these studies, chickens aremainly regarded as “animal models” [83–89]. Notably, thechicken meat tryptic peptidome is characterized by someunique entries, which easies detection of chicken traces inmeat mixes containing as low as 1% chicken in pork meat[90,91].

Generally, protein profiles in chicken are strongly influencedby growth and diet. In a recent investigation [88], Doherty et al.reported that the weight of chicken pectoralis muscle increasedapproximately 44-fold from day 1 to 27 days. Since substantialenergy derived from the glycolytic enzymes is required tomaintain this mass of tissue, chickens showed a dramaticchange in the relative expression levels of glicolytic enzyme, inparticular of two enolase isoforms, triosophosphate isoforms,creatine kinase isoforms and tubulin isoforms and their post-translational modifications [88]. Upon comparison of Thaichicken meat to commercial broiler chicken meat, all thespecies showed a specific protein profile that changed duringgrowth [85]. Also investigated was the higher quality of Thaichicken meat with respect to commercial broiler chickenmeat, the former being characterized by a firmer texture andimproved flavor, although it grows slowly and contains less fat.Higher meat quality of Thai chicken was largely attributed tothe expression and activity of glycolytic enzymes [85].

Proteomic tools also helped evaluating the effects of dietarymethionine on breast-meat accretion and protein expression inthe skeletal muscles of broiler chickens [92]. A total of 190individual proteinswere identified in the pectoralis majormuscletissue, out of which three resulted to correlate withmethioninedeficiency in the diet.

Chicken breed proteomics produced encouraging results,as it was possible to discriminate three different chickenbreeds (Pépoi, Padovana and Ermellinata di Rovigo) on thebasis of breed-specific protein profiles [84]. Such an applica-tion might pave the way for new scenarios in the field ofchicken meat traceability and authenticity certification, still acompelling issue just a few years later than the avian fluinternational alarm.

Fewer reports are present in the literature about turkeymeat proteomics [93], although this investigation representsonly a preliminary breakthrough in turkey muscle proteomicsprofiles during early development.

Analogously, rabbit (Oryctolagus cuniculus) meat has beenlargely underinvestigated through proteomics [94–96], althoughthe beneficial properties of rabbit meat are widely recognized,as it has been recently reviewed [97]. Again, most of the pro-teomics investigation on rabbit muscles in the literatureaddress this species in the perspective of a “model” for humandiseases (especially at the cardiocirculatory level), and thusonlycover but marginal aspects of food science-relevant aspects ofrabbit muscles/meat.

2. Technological innovation in omicsdisciplines and farm animal science

In this section, we will highlight how the fields of farmanimal, and in particular, of meat science are increasinglytaking advantage of other non-proteomics “omics” disciplinesor, as in the case of post translational modifications, of newlyintroduced omics that stemmed from the main proteomicsworkflow. This brief discussion is a mandatory step to betaken before introducing the concept of the integration ofmultiple omics, envisaged by systems biology (see Section 3).

Recent trends in farmanimal research involve the applicationof high-throughput technologies to alternative omics disciplines,such as the study of miRNAs [98–100], protein post-translationalmodification—PTM (PTMomics [101]), mass spectrometry andNMR-based metabolomics [102,103], quantitative proteomics[104] and lipidomics [105].

2.1. miRNAomics

Questions still arise and persist about the reasons underpin-ning the scarce overlap among transcriptomics and proteo-mics datasets [106]. It is still a matter of debate whether theseinconsistencies only result from intrinsic technical bias ofboth approaches, such as in the case of déjà vu proteins inproteomics, which might either result from technical bias[107] or rather reflect an actual biological phenomenon (as inthe case of “balancer proteins” [108]).

On the other hand, it is often easily forgotten thatmany levelsof control do exist in between gene transcription regulation andactual translation ofmRNAs into functional proteins.Micro RNAs(miRNA) are noncoding small RNA, 18 to 26 nucleotides long,that regulate gene expression by altering translation of protein-encoding transcripts, on the basis of multiple mechanisms thatinvolve i) degradation of target mRNA, ii) blocking of initiation ofmRNA translation and iii) blocking of translocation of mRNA toprocessing bodies [99]. Micro RNAs represent one key intermedi-ate actor in the genome-to-proteome cascade, and almost athousands ofmiRNAs have been resolved over the last few yearswhich display 1165 either direct or indirect relationships between270 miRNAs and 581 genes [109]. The miRNAome (the wholemiRNA complement of a given cell/tissue) is now one of thosefields attracting the bulk of attention of researchers worldwide[98–100,109–111], also in the fieldof farmanimal science [112,113].Indeed,miRNAshave beendemonstrated to play a role in porcinepre- and post-natal development [112], intestinal development[113], growth performances and fat accumulation [114]. In 2006,Clop et al. reported that a muscle-specific miRNA regulated agene that directly affects economic traits in livestock animals,myostatin [115]. In detail, the authors reported that amutation inthe myostatin gene of heavily muscled Belgian Texel sheepcreates a target site for miR-1 and miR-206 containing RISCcomplexes in the exon encoding the 3′UTR of the transcript. As aresult, the Clop et al. evidenced a decreased translation of themyostatin protein and a consequent increase in muscle mass.

However, miRNAs seem not only to be involved in biologicalphenomena of living farm animal, but they also play a roleafter slaughter, as they have been related to meat tendernessand acute stress responses in Angus cattle [116], mainly by


modulating the expression of metabolism-related proteins,thereby expanding transcriptomics observations on the samebreed [116].

Recently, McDaneld reviewed the role of miRNA in generegulation of livestock animals [99]. AlsomiRNAs play a role incell cycle regulation and apoptosis [111], a biological processunderpinning post mortem muscle biochemistry. SpecificmiRNAs affect protein expression of glycolytic enzymes andresult in suppression of their activity, a phenomenon whichhas significant pitfalls in the frame of cancer cells [110], and itmight also be relevant in post mortemmuscles, which rely onglycolysis for energy production.

2.2. Post translational modification “omics”—PTMomicsin farm animal science

Whether post-transcriptional control utterly modulates mRNAtranslation into proteins, post-translational regulation is a keyprocess which influences protein functionality. Among post-translational modifications, phosphorylation [117–120], glycosyl-ation/glycation [121], oxidation [122,123] and ubiquitinylation/sumoylation [124] seem to play the major roles in animal andmeat science.

Phosphorylation ofmuscle proteins has also been suggestedto play a role in the post-mortem process and hence in meatquality, both in cattle [117–119] and pigs [33]. During the last fewyears, protein phosphorylation has been related to intramus-cular fat content [137] and meat tenderness [119] in cattle.

Phosphorylation of the myosin regulatory light chainduring rigor mortis has been demonstrated to be involved inmuscle contraction and thus meat tenderness in the bovinelongissimus muscle [118].

Recently, through TiO2 preliminary enrichment of phos-phopeptides, followed by mass spectrometry analysis viacollision induced dissociation (CID) and electron transferdissociation (ETD), we observed that, in the longissimus dorsiof Chianina and Maremmana cattle, higher levels of phos-phorylation of structural proteins were related to a lowermyofibrillary degradation index, while higher phosphoryla-tion of glycolytic enzymes resulted in a reduction in theirenzymatic activity [70,71]. The “phosphorylation-inducedenzyme inhibition” hypothesis in muscle conversion totender meat is further underpinned by recent observationthat the process of meat tenderization induced throughelectrical stimulation appears to be related to phosphoryla-tion levels [119], which is consistent with the activation ofcalcium-sensitive kinases resulting from Ca2+-release fromthe sarcoplasmic reticulum duringmuscle conversion to meat[5,75], in an apoptosis-like fashion [76].

2.3. Metabolomics and lipidomics

One of the most widely investigated aspects of the process of“muscle to meat” conversion is post mortem metabolism. Asthe animal dies and the heartbeat stops, blood flow arrests andmuscle oxygenation drops. Therefore, post mortem musclemetabolism is forcedly anaerobic and thus mainly lies uponglycolysis and creatine-phosphocreatine shuttle to provideenergy before rigor occurs [124]. Glycolysis is probably the beststructurally characterized pathway in enzymology and it is

considered the archetype of a universal metabolic pathway[125]. Under anaerobic conditions, pyruvate is converted intolactate in muscle tissues, which results in pH drop andacidification of the muscle [126].

Metabolomics represents one founding pillar of SystemsBiology [102,103]. However, despite decades of biochemicalinvestigations, it is but in recent years that actual metabolomeand lipidome-wide technologies have become available andreadily applicable to the field of muscle/meat research. Nuclearmagnetic resonance (NMR) [127–130] and mass spectrometry-basedmetabolomics have been applied tomeat-relevant issues,such as fat accumulation and tenderness and WHC [127],though only in recent years. This is mainly due to the ratherrecent advancements in both technological platforms (such asthe introduction and broader diffusion of triple quadrupoles,quadruopole-time of flight, Fourier transform ion cyclotronresonance and orbitrap MS instruments) and, most important-ly, to the growing completeness of accurate metabolite spectradatabases and availability of bioinformatic tools to interrogatethem, such as XCMS, MetLine, MetaboSearch, the HumanMetabolome Database and KEGG [131–134]. Co-accumulationof Omics data and integrated interpretation has also beensimplified by the diffusion of ad hoc browsing tools, such asKaPPA-View4, which is based upon the KEGG pathway repos-itory [135].

In parallel, meat science metabolomics and lipidomics stilltake advantage from the application of classic biochemical,spectrophotometry and fluorescence-based assays, whichallow monitoring oxygen consumption and mitochondrialrespiration rate [136,137].

Metabolism is often considered to be “one-step closer to thephenotype” in comparison to other omics, in that proteinexpression is not necessarily tied to enzymatic activity, owingto the tuning effect of PTMs and, above all, phosphorylation[136]. Glycolytic enzymes are amongst the most abundantproteins in the muscle [124] and, although phosphorylation islong known to play a role inmodulating their activity [137], it isbut in recent times that mass spectrometry-based proteomicshas allowed identifying phosphorylated aminoacid residuesand, more recently, to relate them to actual metabolic modu-lation in post mortemmuscles and meat tenderness.

Modern lipidomics almost relies on the same technologicalapproaches which have contributed to the rapid growth of thefield of metabolomics [138,139]. Implications of fatty acids andlipid metabolism in the determination of meat organolepticproperties (fat accumulation, flavor and taste) have beenmentioned above and recently reviewed [77].

3. From “Omics” to system biology in farmanimal and meat science

Recent trends in the scientific community have promptedreconsidering proteomics as one of the essential components ofsystemsbiology, rather thana separated science. Systemsbiologyis a methodological and holistic approach that integrates, as awhole, the results gathered through multiple “omics” disciplines[140–145]. Indeed, systems biology is a multi-faceted approachthat tackles the biological complexity of the sample underinvestigation from several perspectives, from the genome to the


transcriptome, the proteome and the metabolome, and thenintroduces mathematics and statistical modeling in order tointerpret biological phenomena in the light of high-throughputresults [140]. An interesting definition of systems biology isprovided by Woelders and colleagues, according to which“systems biology can be described as the study of the emergenceof functional properties that arepresent in abiological systembutnot in its individual components” [140], and these properties areinvestigated through the “analysis, filtering, combining andintegration of mass “omics” data” [140]. For, as Lucretius wasaware of, almost two thousand years ago, “rerum summa novatursemper, et inter se mortales mutua vivunt” (Lucretius – De rerumnaturae; Book II, line 54 – “Thus the sum of things is ever beingrenewed, and mortals live dependent one upon another”). Inmore simplistic terms, biological systems are more than a merecollection of molecules, cells or organs (the parts of the system),as we need to investigate how these parts work together in orderto understand the “emerging properties” of the system. “Emerg-ing properties” imply those characteristics that are not present inthe independent components but they emerge from their properintegration. To put it simply, you canhave all the pieces to build abike, but you cannot ride it unless you have put all the pieces inthe right place. In biological terms, youhave to look at the systemas a whole in order to understand how the orchestra of geneticsand regulatory interactions and the environmental factors endup affecting animal development, aging, response to diseasesand, utterly, higher quality meat [140–145].

The main goal of systems biology is to produce informationtomake biologypredictive (throughmathematicalmodels [144])at thewhole organism level. Such prediction at the animal levelwould result in many practical applications, including theimprovement of phenotypic traits, especially those with lowheritability, among which there are meat quality traits [143].

The integration of omics disciplines (see previous sections)envisages a close future of newdiscoveries in the areas of animalgrowth, development, and production and in related infectiousdiseases. In order to fulfill this ambitious agenda, it is essential tobridge the gap from genome-centric and transcriptome-centricinvestigations, by complementing results with informationabout the production of proteins in a cell, tissue or fluid withthe metabolic processes and functions [2]. This integration isachieved by means of mathematical descriptions of theinteractions (either chemical or physical) among molecules inliving systems [144]. Amongst the variouspossiblemathematicaldescriptions, the simplest one is based upon graph theory [144].A graph is a set of objects called nodes or vertices that areconnected by links, called lines or edges. Nodes can representdifferent biomolecular species (either proteins, metabolites,exogenous compounds), while edges are representative of theinteractions between thesemolecules [144]. Graphs can be eitherdirected or undirected: in an undirected graph, a line (edge) froma node A to node B is considered equal to a line from B to A,which isnot true indirected graphs,where the twodirections arecounted as distinct arcs or directed edges [144].

While graph representations are often merely qualitative,quantitative mechanistics models imply that the descriptionsof the interactions among molecules are represented in termsof mass action or Michaelis–Menten kinetics [144]. Whetherreal thermodynamics are included in the equation describingthe reaction rates in a kinetic model, the model is also called a

(nonequilibrium) thermodynamic model [141]. Such a repre-sentation is more informative, though most of the researchersin the field tend to limit quantitative representations to subsetsof the whole organism as a system, such as single organs orcompound classes. Depending on the extent of variablesconsidered, a model can be described as simplified or detailed(please, refer to Ref. [144] for further details).

Finally, in systems biological models there are threestrategies to build the link between the layer of interactingbiomolecules (e.g. genes, enzymes, metabolites) and the sys-temic functioning of the organism emerging from these in-teractions [144]:

i) bottom-up strategies, that describe the mechanisms interms of mathematical equations on the basis of theproperties of the components, in order to deduce systemsfunctions as a consequence; themodel undergoes testingand verification through the comparison of the simulatedbehavior upon changing a variable, against the behaviorof the real system;

ii) top-down approaches, where the systemic behavior is thestarting point and, only subsequently, on the basis ofdata-driven hypotheses, the system is perturbed andinvestigated as a whole to understand how the singlecomponents react onagenome, proteomeormetabolome-scale.

iii) middle-out approaches, which imply that a sub-systemis complex enough to exhibit its own emergent proper-ties (metabolic networks, for example), which in turnintegrate with other sub-systems in order to form morecomplex systems (for example, cells can be seen assmall albeit complex systems, which are integrated in atissue and, utterly, in more complex systems such asorgans). In this approach, fragmentary knowledge ofsmaller systems can be further integrated in order tounderstand, in a step by step process, the system as awhole.

As anticipated above, the final goal of these models is todeliver a robust in silico platform that eases biological datainterpretation and supports experimental design by anticipat-ing the likely effect of a perturbation on a whole system or,conversely, by suggesting the most likely working solution forthe improvement of a peculiar trait. A paradigmatic example isE-Cell, amodeling and simulation environment for biochemicaland genetic processes that allows defining functions of pro-teins, protein–protein interactions, protein–DNA interactions,regulation of gene expression and other features of cellularmetabolism, as a set of reaction rules [146].

Despite a significant theoretical background, systems biolo-gy is a young field [145], especially its application to livestockscience [140–144]. Although some genetic quantitative traitshave been successfully related to organoleptic properties (forexample, DNAJA1 and PRKAG1 in the case of meat tenderness[147–152]), heritability estimate for carcass traits is often poor[153]. Therefore, many authors have indicated that otherparameters, such as ante mortem stress [154] and birth andrearing environment and conditions [155] end up influencingnot only growth performances, but also meat quality, and canbe possibly used to predict the latter more effectively than


genome-wide signatures. While it is inopportune to indicatethis as a failure of genomics, it is to be considered that genes(encrypted in breed specificities and animal sex) mainly affectin vivo characteristics in the long term (growth rate, foodconversion) other than post mortem events. The processesleading to muscle conversion to meat are thus both tied togenome fingerprints, but also (and more closely) to post mortemspecific transcripts [156] and utterly translated proteins. This iseven more intriguing when considering that transcriptomicsand proteomics results on the same biological matrix oftenscarcely overlap [157,158]. On the other hand, where poor or nodirect overlap among transcriptomics and proteomics datasetsis found, integration through pathway analysis often results inhighlighting the same biological pathways being altered (eitherover-activated or down-regulated) both at the transcript andthe protein level, a phenomenon described as “indirect overlap”[106,158]. From this standpoint, the integration of huge datasetsfrom multiple “omics” approaches should be preferred toindependent single-omic investigations. However, it should bealso pointed out that the integration of multiple omicsstrategies can be a double-edged sword, in that results from asingle approach might be either confirmed or confuted byintegrated assays. In other terms, a jigsaw puzzle gets morecomplicated proportionally to the number of pieces it is madeup of; thus, analogously, pages and pages of high-throughputdata from integrated platforms become more difficult tointerpret, although they better help elucidating the schemebehind and unraveling thewhole picture (or, in other terms, thecomplexity of biological phenomena).

Onemajor advantage of farm animal research is that, owingto centuries of livestock breeding selection, farmanimal geneticdiversity is narrower than in the human population. This is anon-secondary aspect when performing “omics” approaches inhuman research, where statistically significant and biologicallymeaningful results are often achievable only when screeninglarge cohorts of patients [159], which help coping with thepresence of outliers and the biological variability issue. On theother hand, species diversity further complicates the analysis ofhigh-throughput data from proteomics and other “omics”approaches, which results in delays in the uptake of relevantapplications. Nevertheless, characterization of species-specificmuscle protein compositions enables compositional analysis ofmeatmixtures, which is pivotal to detect fraudwhere either theless expensive meat is mixed in bovine and pig meat ormechanical methods are applied to recover cheap meat versusthe more costly hand-made recovery upon deboning [90,91].Indeed, peptide sequences are resistant to food processing, andhigh sensitivity mass spectrometry or targeted quantitativeapproaches for peptide analysis, such as multiple reactionmonitoring (MRM), also overcome the issue of heat-inducedepitope modification, hampering reliable antibody-based rec-ognition approaches.

Omics technologies have also paved the way for newstrategies in the identification of illegal growth promoters inbiological fluids of farm animals [160]. To understand theimportance of this issue, it is worth mentioning that, in 2007,the official monitoring of residues in cattle throughout theEU found <0.2% non-compliance for the use of illegal growth-promoters (GPs), including sex steroids, corticosteroids andβ-agonists [160].

4. Muscle to meat conversion: an overviewthrough integrated omics

Muscle to meat conversion is a multi-factorial process, whichhas been largely reviewed by expert zootechnicians andbiochemists over the last decades [8,29,73–76]. What we arehereby proposing is a brief summary, which attempts tointroduce the main novelties in the field, as they emergedfrom proteomics and recent multiple omics integrated studies(see previous paragraphs).

In this section, we will not discuss into details the effects ofpre-slaughter stress for simplicity, despite its critical relevancein the field of meat quality. Indeed, proper “quality assurance”(QA) programs should be designed as to aim for a “whole ofchain” approach, in order to implement the system “from thepaddock to the plate,” each step in the process corresponding toa critical control point in the production chain [161]. As it hasbeen recently reviewed, animal management, transportationand the slaughter process itself at the abattoir level significantlyinfluence the conversion of muscle to meat, its tenderness andultimately meat quality [162,163]. Pre-slaughter variables affectmeat quality of cattle [163], pigs [164–169] and ovines [170–172].Pre-slaughter stresses range from physical such as highambient temperature, vibration and changes in acceleration,confinement, noise, and crowding; to psychological such as thebreakdown of social groupings and mixing with unfamiliaranimals, unfamiliar or noxious smells and novel environment[162]. The effects of these stresses on meat quality range fromweight loss to impaired tenderness or WHC, reduced carcassyield or carcass contamination, as it has been extensivelyreviewed [162]. The interested reader is referred to thesereviews for further details [162,163].

It is also worthwhile to stress that muscle glycogencontent not only varies in a species-specific fashion (higherin pigs than in cattle, for example), but also in a breed-specificand muscle-specific (fast twitch, slow twitch) fashion,vanishing any attempt to cursorily over-generalize theprocess of “muscle to meat conversion” [173–175]. Neverthe-less, decades of research in this field have helped individu-ating a few cornerstones in the post mortem biology ofanimal muscles.

Previously, Ouali and colleagues had schematized thisprocess by considering three main phases: the pre-rigor step,the rigor step and the tenderizing step [74]. For the sake of clarity,we will dissect the process of “muscle to meat conversion” inseven steps and briefly discuss what is known from basicbiochemistry and recent omics investigations. However, omicsinvestigations have suggested that the boundaries among thesesteps might be more labile than previously thought.

4.1. Step I: Blood supply loss: oxygen and nutrient levelsdecline

After slaughter, animals are dressed, deboned and musclesare stored at refrigerated temperature for one week or moredepending on the current national practice and/or regulationsbefore selling. This period largely affects the organolepticqualities of the final product, meat, and largely depends onspecies and breed characteristics.


Postmortem changes involve several biochemical pathwaysand protein metabolism, because muscle remains functionaland metabolically active for several days after slaughteralthough depleted of the circulating blood that supplies oxygenand removes metabolic end products (i.e. lactate) [125–127].

Residual oxygen in the muscle is related to the concentra-tions of hemoglobin andmyoglobinmolecules. Myoglobin levelsalso affect meat color, since oxygenated myoglobin gives anappealing light red coloredmeat and a dark red colormeat in theabsence of oxygen. In Large White pigs, residual myoglobinlevels positively correlatedwith the rednessMinolta value a [27].Oxidation of the heme iron from a ferrous (Fe2+) to a ferric (Fe3+)state gives rise to the brown color and is often associated to therelease of oxygen radical [74,176]. Meat color has an importanteconomic value, because it largely affects consumers' appraisalwhen purchasing the meat, since brown color is often consid-ered as a synonymous to bad hygienic quality [72].

Blood flow also exerts a key role in nutrient delivery andend-product removal. The interruption of this process triggersconsumption of the local energy stores and accumulation ofmetabolic end products.

4.2. Step II: Glycolysis ensues, pH drops

Under aerobic conditions, the enzymatic conversion of glucoseto pyruvate is followed by the reactions of the citric acidcycle and oxidative phosphorylation [125,127]. In parallel, aminor role in skeletal muscles is also played by the creatine/phosphocreatine shuttle, which provides an eligible substratefor rapid reconstitution of ATP reservoir through dephosphor-ylation of phosphocreatine [125,127]. Within the framework ofpostmortemmetabolism,whenphosphocreatine stores rapidlytend towards exhaustion, energy is mainly produced throughdegradation of glycogen by glycolysis. Therefore, initial phos-phocreatine stores only affect to a lesser extent the continua-tion of the “muscle to meat” conversion process.

Muscle glycogen content is the subsequent parameter,affecting the theoretical extent of glycolysis, of which itrepresents the main fuel in the muscle after slaughter [29].

Fast twitch and slow twitch muscles have their peculiarglycolytic enzyme levels [173,177], although most of theproteomics papers published so far indicated a positivecorrelation between glycolytic enzyme levels (especially ofphosphoglucomutase 1, aldolase, glyceraldehyde 3-phosphatedehydrogenase, and triose-phosphate isomerase, enolase, py-ruvate kinase and lactate dehydrogenase) andmeat tenderness[58] (Fig. 1). On the other hand, it has been also proposed thatthis might reflect a technical bias of the 2DE approach inresponse to altered protein solubility resulting from lower pH inpost mortem muscles [59,178]. It is also worth mentioning thatonly some glycolytic enzyme isoforms have been shown tocorrelate with meat tenderness (such as enolase 3 rather thanenolase 1 [179]). Therefore glycolytic rate largely depends on thetype of muscle considered, although in every case it ismodulated by a negative feedback on enzyme activities inresponse to acidic pH resulting from prolonged glycolysis.

In porcine muscles, which are characterized by higherglycolytic rates, a rapid fall of pH levels is related to compromisedtenderness and juiciness [32,180], although 45 min post mortempH might not be as much indicative as ultimate pH [27]. On the

other hand, in cattle, amoderately accelerated rate of pH declinemight favor the tenderization process, as suggested by earlystudies on electrical stimulation [181]. In our studies on bovinemeat tenderness, pH 1 h and pH 24 h postmortem, albeit not pH48 h, were good predictors of meat tenderness [70,71].

The relationbetween theultimatepHand tenderness ofmeatis somehowconfounding. Daszkiewicz et al. [182] concluded that“there is no need to divide normal-qualitymeat (with pH 5.5–6.0)into technological subgroups based on its acidity.” On the otherhand, Purchas [183] individuated a positive correlation betweenmeat tenderness and ultimate pH in between 5.5 and 6.0 pHunits, while the correlation became negative when pH exceeded6.0. Analogously, Whalgren et al. [184] also indicated that,unrelated to the pH drop rate, a positive correlation can beobserved between WBS and pH levels.

In this paragraph, we summarize a simplistic overview ofpost mortem pH drop and its pitfalls on cell biochemistry.However, the interested reader is referred to Ouali et al. [74]and Huff-Lonergan et al. [185] for further details about postmortem pH drop and its intertwinement with other biochem-ical/structural changes, influencing meat quality parameterssuch as tenderness and WHC. While glycolysis progresses inpost mortemmuscles, it should be expected a constant rate ofpH drop and lactate accumulation over storage duration ofmeat. This is not what is usually observed, since there exist adiscontinuity in pH fall which has been explained by areduced capability of muscle cells to cope with bufferingcapacity and charge distribution [74]. It has been proposedthat this phenomenon might result from the replacement ofacidic components (phosphatidylserine) by basic components(phosphatidylcholine and phosphatidylethanolamine) in theintracellular compartment (altered membrane potential),which is accompanied by a redistribution of ions, that couldexplain the existence of these transient pH stability steps [74].This would reflect the ongoing of apoptosis-like phenomena(externalization of phosphatidylserine, for example, is a door-way to apoptosis [74]), and also supports the alteration of WHCvalues in response to altered pH. Indeed, the increasedconcentration of hydrogen ions reduces electrostatic repulsionbetween the myofibrillar proteins, which decreases the repul-sion between the filaments and contributes to lateral shrinkageof the muscle fibers, sarcomere extension and, utterly, tendermeat [34,42,127]. Altered ion homeostasis in post mortemmusclesmight be further exacerbated bydifferential expressionor post-translational control over specific ion transporters andchannels, such as carbonic anhydrase 3 and ATP sensitiveinward rectifier potassium channel 15 [70,71].

A transient pH stability occurs between 1 and 8 h post-mortem (and apoptosis is thought to ensue within fewminutes to 1 h after animal slaughter [74,76]), inversion ofpolarity of the plasmatic membrane probably takes placeduring the first 8 h postmortem when pH ranged between 6.4and approximately 6.8 [74]. Another tentative explanationinvolves protein PTMs and, in particular, phosphorylation ofglycolytic enzymes, a phenomenon that we reported to ensuein post mortem muscles yielding altered glycolytic enzymeactivity and, in the end, tough meat. Indeed, tough meatmuscles displayed lower levels of glycolytic enzymes, andthese proteins were more phosphorylated than in tendermeat counterparts [70,71]. Meat tenderness correlated with

Fig. 1 – After slaughter, blood flow stops and the muscle becomes anoxic. Therefore, energy is produced through glycolysis bymobilizing glycogen and consuming local sugars, while the creatine/phosphocreatine represents an alternative pathway toprovide ATP for rapid consumption. Metabolites are enlisted in the figure along with the abbreviated UniProt names for eachspecific enzyme catalyzing the production of the subsequent metabolite in the column. Enzymes highlighted in red have beenassociated to tender meat in proteomics investigations in the literature. Red and green ATP circles are indicated next to thosereaction characterized by the consumption or production of ATP, respectively. Finally, in the left panel we briefly summarizethemain effects of ATP deprivation inmuscle cells. Abbreviations (in alphabetical order): ALDO (aldolase), CKM (creatine kinaseM), ENO (enolase), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), HEX (hexokinase), LDH (lactate dehydrogenase), PCr(phosphocreatine), PFK (phosphofructokinase), PGAM (phosphoglycerate mutase), PGM (phosphoglucose mutase), PGK(phosphoglycerokinase), PYGM (glycogen phosphorylase), TPI (triose phosphate isomerase).


phosphorylation of many metabolic enzymes that regulateanaerobicmetabolism (including glycogen phosphorylase, pyru-vate kinase and phosphofructokinase—Fig. 1) and, therefore, itmay be directly linked to post-mortem pH decline. However, itremains unclear whether low pH leads to differential phosphor-ylation of sarcoplasmic proteins, or whether differential phos-phorylation leads to an extended pH decline.

Furthermore, it has been recently reported that electricalstimulation, which promotes meat tenderization, also altersthe phosphorylation pattern of metabolic enzymes [119].

4.3. Step III: Apoptosis ensues: caspases and heat shockproteins

In the previous paragraph, we have mentioned the phenom-enon of phosphatidylserine externalization in tenderizingmeat (Fig. 2). This phenomenon is also linked to prothrombinrelease in the extracellular space (Fig. 3), where the enzyme iscleaved in its activated form, thrombin, and it is thought tocontribute to neural plaque degeneration [74]. This holdsrelevant consequences in the frame of post slaughter han-dling of carcasses, since a compromised neuromuscularsynapsis translates into electrical stimulation being lesseffective if performed later during storage [74].

Most of the studies about meat proteomics indicated astrict correlation between tenderness and a series of heatshock proteins (HSPs), including DNAJA1 (HSP40) [104], HSPB1(HSP27) [60], HSP70, HSPA8, α-crystallin (CRYAB) and otherchaperone proteins, as it has been recently reviewed byGuillemin and colleagues [178]. HSPs are known to play apivotal role in apoptosis, in that they protect HSP-interactingproteins (including transcription factors, for example) fromdenaturation/digestion through direct binding [74] (Figs. 2–3).HSPs might contribute to tenderness-related phenomenathrough i) the modulation of caspase activities (initiators oreffectors) through direct interaction; ii) the direct binding ofprotease cleavable substrate, thus preventing their degrada-tion; iii) a chaperone role in refolding those proteins whichunfold/denaturate in the harsh environment of post mortemmuscles, where pH drop and protease activities end updramatically altering protein integrity and native conforma-tion; iv) the promotion of apoptosis when stress levels becomeunbearable for muscle cells [70,74].

Other than glycolytic enzymes, we could report higher levelsof phosphorylated HSPs in tender meat, thereby modulatingtheir multimerization state and thus their functions, through amechanisms that might involve changing of steric interactorsof HSPs (an example is reported for HSPB1 [186]) (Figs. 2–3).


In relation to DNAJA1 zinc finger domain, it is worthwhileto stress that proteomics investigation highlighted a signifi-cant correlation of certain zinc finger protein (zinc fingerprotein 197, for example [71]) to bovine meat tenderness(Fig. 3).

Calcium dysregulation, low pH and altered levels/PTM-modulation of HSP activity end up triggering apoptotic cascades[74,76]. However, apoptosis in muscle cells might also followalternative pathways, as the one promoted by the caspase-triggered poly ADP ribose polymerase (PARP) fragmentation [71],

Fig. 2 – An overview of the intricate pathways leading to tenderizblood flow arrest (causing anoxia, accumulation of oxidized irondeprivation) is linked to the increase in reactive oxygen species (mitochondrial metabolic activity, while the cells end up relying onegative feedback on glycolysis triggered by pH lowering and lacsubsequently, accumulation of AMP (activation of AMPK and aut(blockade of ion pumps and calcium release from mitochondriaaccumulation activates calpains, which activate Bax and promot(release of cytochrome C, activation of caspases). In turn, activatpromotes transcription of calpains. A role for zinc finger proteinssuggested, as discussed in the text. Kinase activation triggers dobinding-partners (hampering their protective role against proteaprotein resistance to degradation (please, refer to the main bodycontribute to tenderness through modulation of glycolyte enzym

amechanismwhich is known to trigger caspase 3 activation andskeletal muscle apoptosis, other than being indirectly related tocalpain over-expression via PARP-mediated transcriptional con-trol [187] (Fig. 3).

Another mechanism through which apoptosis mightunfold in muscle cells could involve the protein 14-3-3gamma (YWHAG) (overexpressed in tender meat in Chianina[71]), which is a mediator of signal transduction playing a rolein apoptosis, through binding to MDM and thus affecting theactivity of p53 (Fig. 2).

ation of the muscle through apoptosis. The interplay betweenand of byproducts of metabolism, such as lactate and nutrientROS) within the cell. Altered metabolism leads to the arrest ofn glycolysis and the phosphocreatine shuttle. However, thetate accumulation result in ATP consumption and,ophagic pathways), alteration of ion homeostasis modulationand sarcoplasmic reticulum). Calcium intracellulare apoptosis through the mitochondrial intrinsic pathwayed caspases cleave PARP, which relocates in the nucleus andand alternative histone 2 proteins (H2AFX and Y) is

wnstream phosphorylation which affects Heat shock proteinses), enzymatic activity of glycolytic enzymes and structuralof the article for further details). Finally, miRNA appears toe expression.

image of Fig.�2

Fig. 3 – An overview of the interplay amongmuscle proteases (in bold font) in the frame of meat tenderization.While activationof calpains is triggered by calcium ions and sustained at the transcriptional level by PARP-mediated pro-apoptotic signaling,calpains themselves trigger release of cathepsins from the lysosome. In the frame of the apoptotic process, caspase areactivated via the intrinsic mitochondrial pathway of apoptosis. In addition, a role is proposed for the proteasome (inhibited byacidic pH of longer stored meat), metalloproteinases (activated by extracellular serin peptidases) and matrixmetalloproteinases. Each one of these proteases ends up attacking the structure of myofibrils, especially at the Z-disk level,promoting tenderization.


Finally, as predicted through bioinformatic by Guilleminand colleagues [178] and subsequently confirmed elsewhere[70], specific histone protein isoforms might take part in theprocess of programmed muscle cell death, such as histoneH2AF isoforms (e.g. H2AFY and H2AFX) (Fig. 2).

4.4. Step IV: Energyless muscle and onset of rigor: controlis lost over Calcium reservoirs, kinases are activated andproteins are phosphorylated

As a consequence of the slaughter, glycogen levels in themuscle decrease, and so does the energy available to keep themuscle in a relaxed state. Indeed, ATP is a fundamental energytoken to trigger dissociation of the actin–tropomyosin complex[188]. This process is also influenced by free Ca2+ concentra-tions. Muscle acidification triggers dysregulation in calciumrelease and, along with ATP exhaustion, it leads to theformation of cross-bridges betweenmyosin and actin filamentsand thus to the onset of rigor [74,185] (Fig. 2). Being a secondaryintracellularmessenger, calcium release results in downstreamcascades leading to the activation of calcium-dependentkinases (such as PKC) and phosphorylation of target proteins[120]. Energy consumption and an increased AMP/ATP ratio

might in turn activate the kinase AMPK in an apoptotic-likefashion, triggering downstream cascades leading to proteinphosphorylation and utterly to apoptosis [120] (Fig. 2). This isalso consistent with the role of AMPK in PSE meat [120].Phosphorylation of glycolytic enzymes might be modulated bythis mechanism [189]. On the other hand, protein phosphoryla-tion events might occur early after death (1 h post mortem, forexample [119]), when high energy phosphate-rich metabolites(such asATP) are still abundant, andphosphorylation of proteinscould parallel or participate in apoptotic cascades. The drop ofpH can cause the development of PSE zones in muscle, whichproduces zones that are characterized by alteration in texture,color and exudation, related to a reduction of proteolysis rate ofthree proteins, troponin T, myosin light chain and α-crystallin,and the total absence of heat shock protein 27 [8,34].

4.5. Step V: Calcium-dependent and independent proteasescleave myofibrils at the Z-disk

Although it has been long thought that cathepsins and calpainswere the only proteases responsible formeat tenderization, it isnow clearly emerging that the tenderization process is amultienzymatic phenomenon implying the activation of

image of Fig.�3


cathepsin and calpains, along with other proteolytic systems(proteasomes, caspases, metalloproteases, metallopeptidases,serine proteases—Fig. 3) [74].

In most unsupervised proteomics studies, calpains andcathepsins (at the protein level) do not usually emerge asmajor players in the tenderization process (no statisticallysignificant differential protein concentrations are usuallyobserved in proteomics studies), though targeted quantita-tive approaches (multiple reaction monitoring—MRM; west-ern blot) might have bypassed the probable technical biasof certain experimental tools such as 2DE, which are morelikely to reveal major fluctuations or differences in mostabundant species (i.e. glycolytic enzymes, structural proteinsand HSPs in muscles). Also, it is worthwhile to stress thatfluctuations of protein activities are not necessarily tied toover-expression of those specific enzymes, since activitymight be influenced by the alteration of the biochemicalmilieu (e.g. higher free calcium level, lower pH) and posttranslational modifications.

Despite technical difficulties encountered by proteomicsinvestigation in elucidating the role of calpains, cathepsinand the proteasome in meat tenderness, biochemical evi-dences have been accumulated over the years about theircentral role in meat protein digestion after slaughter[190–193] (Fig. 3). Protease activity is either boosted by pHlowering or calcium accumulation (such as in the case ofμ-calpains) [29,74,193]. Calcium-binding proteins, such ascalmodulin and calsequestrin, which are involved in calciumsignaling modulation in the living animal [194], after slaugh-ter contribute to lowering the levels of free Ca2+ and thus inreduced calpain activity. Calpastatin exerts an inhibitoryaction on calpains by directly binding to multiple calpainproteins through specific subdomains [193]. Calpain inhibi-tion utterly influences meat tenderness and WHC.

Another factor contributing to reducing the extent ofprotease-mediated degradation ofmyofibrillary proteins is phos-phorylation of structural proteins, especially of Z-disk-relatedproteins, thereby preventing proteases from attacking fibers andthus resulting in tough meat. Z-disk proteins regulate musclefunctions through reciprocal interaction. Disruption of theseinteractions results in muscle disorders. Phosphorylationsof Z-disk proteins increase interactions of myotilin, myozenin1 and troponin at the Z-disk line [195], andmodulate actomyosincomplex formation [196,197], increasing sarcomere cohesionand reducing accessibility to proteases. Phosphorylation ofsynaptopodin also affects its compartmentalization, mediatingits release from the Z-line and nuclear import [198].

While proteomics is probably not suited to directlyinvestigate the correlation of protease levels to myofibrilfragmentation, indirect hints of the extent of proteaseactivity (the utterly relevant biological phenomenon, ratherthan differential protease expression) appear to emerge fromfragmentation levels and the overall number of protein spotsthat could be discriminated through electrophoretic ap-proaches. Studies over the years have indicated how specificprotein fragments arise after slaughter and how the levels ofspecific proteins (including glycolytic enzymes, HSPs andantioxidant enzymes) do correlate with myofibrillar degra-dation indexes, either at 24 h and 48 h post mortem[19,70,71].

4.6. Step VI: the controversial role of oxidative stress in thepromotion of myofibril degradation

Antioxidant enzymes are perhaps the most interesting catego-ries of proteins, though their correlation to bovine meattenderness is controversial. Indeed, myofibril protein fragmen-tation is not only a protease-mediated process, since oxidativestress might play a role as well (Fig. 4). The effect of proteinoxidation in muscle food has been recently reviewed by Lundand colleagues [142]. Both in pigs [27] and cattle [70,71], meatquality parameters such as WHC and tenderness significantlycorrelated with oxidative stress accumulation (GSH/GSSG ratios,altered levels of anti-oxidant stress enzymes, suchas superoxidedismutase [199], glutaredoxin, lipoxygenase and ascorbatecarrier [26]) (Fig. 4). Residual myoglobin and non-heme iron[176], along with altered mitochondrial activity in the anaerobicenvironment of post mortem muscles lead to the formation ofreactive oxygen species (ROS) that cause oxidative damages toproteins (Fig. 4). During the first 4 days a sharp increase (57%) inoxidation levels in sarcoplasmic proteins takes place [200].

According to Rowe and colleagues [200], protein carbonylcontent positively correlated with WBS values in beef, which issuggestive that increased oxidation of muscle proteins earlypost mortem could have negative effects on fresh meat colorand tenderness. SOD1 (free radical scavenger) positively corre-lated with meat tenderness also in the report by Guillemin andcolleagues [178], although opposite correlation trends wereobtained for peroxiredoxin 6 (tackling hydrogen peroxide) [57].

A tentative explanation of the process is provided by Roweand colleagues [201], which indicates that antioxidant enzymesmight protect proteases (such as μ-calpain) from oxidation andinactivation, thus indirectly favoringmeat tenderization. Often,overexpression of antioxidant enzymes is more frequent inglycolyticmuscles (such as in the semitendinosus, as in Ref. [178])or in more “glycolytic-breeds” [26,27] (Fig. 4). However, weshould also report that controversial results have been obtainedwhen studying cattle beef tenderness [70], although in this caseoxidative stress was related to promotion of apoptosis and thusimprovement of meat tenderness.Cursorily summarizing therole of oxidative stress in the process of muscle to meatconversion, it could be concluded that the presence of higherlevels of anti-oxidant enzymes (andvitaminE [202]) protects themuscle from early oxidative stress and thus reduces the extentof protein carbonylation; it protects proteases from oxidativestress-induced inactivation and thus prevents yielding oftougher and darker meat (Fig. 4).

4.7. Step VII: Muscle structure is altered by proteolysis andion homeostasis dysregulation

Myofibrillar degradation is one fundamental phenomenonduring meat tenderization [19,177,189].

Degradation of troponin T isoforms during postmortemmuscles aging is known to progress simultaneously with thepost-mortem tenderization of beef meat [203]. Despite rathercomplex and multi-factorial processes at the basis of meattenderization events, all the nine fast-type and the twoslow-type isoforms present in the bovine muscle are cleavedduring post-mortem aging primarily in the glutamic acid-richamino-terminal region to generate basic fragments that are

Fig. 4 – A brief summary of the controversial role of oxidative stress and reactive oxygen species (ROS) with respect to meatquality. While it has always been thought that ROS promote toughness by attacking and inhibitin μ-calpain and thuspreventing its pro-tenderization activity, ROS might also play a positive role in meat tenderization through direct triggering ofROS-mediated fragmentation of myofibrillary proteins. In this view, ROS-mediated damage to DNA favors apoptosis and thustenderization processes. However, it has been proposed that antioxidant defenses might improve tenderness by preventingROS-triggered calpain inhibition. On the other hand, ROS negatively influence meat appearance by contributing to theoxidation of hemoglobin andmyoglobin iron from Fe2+ to Fe3+, which results inmeat acquiring a brownish, undersirable color.


likely good predictivemarkers for beef aging and developmentof tenderness [204]. In this view, Bauchart and colleagues [204]performed a characterization of low molecular weightpeptides (lower than 5 kDa) generated in bovine pectoralisprofundus muscle during meat aging and cooking in order toreveal post mortem proteolytic degradation of muscle whichoccurs as part of cellular death and meat aging process.

However, the disruption of myofibrillar structure integrityresults both in improved tenderness and impaired WHC. Thisis the main reason why increasing storage length will beprofitable for tenderness and flavor, although it will have arather deleterious effect on juiciness and color [74].

5. Conclusion

Despite decades of investigations, the mechanisms underpin-ning meat tenderness are only partially understood. This ismainly due to the enormous amounts of variables affectingmeat tenderness per se, including biological factors, such asanimal species, breed specific-characteristic, muscle underinvestigation. However, it is rapidly emerging that the tendermeat phenotype is not only tied to genetics (livestock breedingselection), but also to extrinsic factors, such as the rearingenvironment, feeding conditions, physical activity, administra-tion of hormonal growth promotants, pre-slaughter handling

and stress. Finally, post mortem handling plays a role as well,including storage temperature and duration, hanging method,delays in transfer to cold tunnel, electrical stimulation and, lastbut not the least, cooking methods.

From this intricate scenario, biochemical approaches andomics investigations have helped establishing a fewmilestonesin our understanding of the events leading frommuscle tomeatconversion. While some groups are already at that point whenintegration and mathematical modeling are being optimized,most of proteomics investigators still rely on single omicsapproaches. Therefore, the growing need for integration ofomics disciplines in the field of systems biology will sooncontribute to take further step forward also within the contextof meat science. Soon enough, we could end up realizing that,by looking at each independent pathway separately in theframework of meat tenderization, we could not see the forestfor the trees.

Acknowledgments

ADA and LZ are supported by

– the “GENZOOT” research program, funded by theItalian Ministry of Agricultural, Food and Forestry Policies(Ministero delle Politiche Agricole, Alimentari e Forestali).

image of Fig.�4


– the “Nutrigenomicamediterranea: dalla nutrizione mole-colare alla valorizzazione dei prodotti tipici della dietamediterranea—NUME” project, funded by the Italian Minis-try ofAgricultural, Food andForestry Policies (MinisterodellePolitiche Agricole, Alimentari e Forestali).

– the PRIN-20087ATS57 “Food Allergenes” research program.

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