Expression of genes involved in meat tenderization of bovine and caprine muscles

From a technological and commercial perspective, the scope of meat quality takes into consideration several aspects including colour, pH, water losses, tenderness, chemical composition, and hygienic conditions. Among the sensory aspects, meat tenderness is the most important eating factor that influences consumer’s preference. Meat tenderness is related to many animal characteristics, such as breed, age, gender, feeding and management, but also to specific properties of the muscle, such as the collagen and fat content, the metabolic and fibre type, the myofibrillar structure, the pH, etc. The myofibrillar structure of the muscle and the destructuration that occurs during meat ageing, seem to be the most important factors that affect meat tenderization. The degradation of the muscle is the result of the actions of many enzymatic and protein systems of the muscle cell, e.g. calpains/calpastatin, cathepsins, proteasome, caspases and heat shock proteins. Calpain-1 and 2 are calcium-dependent proteases and calpastatin is their competitive inhibitor. Calpain/calpastatin system is considered the most important enzymatic system involved in degradation of myofibrillar structure of the muscle and the calpain/calpastatin ratio is considered as the best predictor of meat tenderness. More recently, apoptosis has been recognized to cause the death of muscle cell during the slaughter of the animal. Caspases are the effectors of apoptosis, and in particular, caspase 3 and 9 are involved in the intrinsic pathway of apoptosis that is the most important in the muscle cell. Caspase 3 is an effector caspase, which can degrade many proteins in the sarcoplasm, while caspase 9 is an initiator enzyme, which can activate caspase 3. Based on these roles, caspases are investigated in relation to their possible involvement in meat tenderization process, as apoptosis effectors and/or as proteolytic enzymes. Heat shock proteins (Hsp) comprise factors that are constitutively expressed and are involved in protein assembly and transport, folding and unfolding, and in the refolding of damaged proteins. Hsp include also resistance factors that are expressed in cells subjected to various types of stress. For their protective function on myofibrillar proteins and their anti-apoptotic properties, Hsp are also taken into account for a possible role in meat tenderization. AlphaB-crystallin and Hsp27 are small Hsp that can inhibit the apoptosis pathways, acting on various actors of the death chains. Furthermore, αB-crystallin is involved in the protection of myofibrillar proteins, like desmin and titin, acting against their denaturation and degradation. Hsp27 is also involved in the stabilization of myofibrillar proteins and in particular, it regulates polymerization and protect actin filaments, which constitute the base of cell muscle structure. Hsp40 is a co-chaperon of the large Hsp70 and it assists the latter in the folding and compartmentalization of nascent proteins. Moreover, the complex Hsp70-40 have been identified to directly inhibit the intrinsic and extrinsic apoptotic pathways and Hsp70 is known to have a cellular role in resistance against heat stress. In the present thesis, we made an attempt at investigating the role of calpain/calpastatin system, some caspases and Hsp proteins in meat tenderization process in different experimental conditions: two different muscle types in the bovine species, the same muscle (Longissimus dorsi, LD), in two different bovine species and the same muscle (LD) in animals of different ages, in caprine species. In general, we confirmed the primary role of the calpain/calpastatin system in meat tenderization process, showing its positive relatedness to meat tenderness in the experiments conducted. In particular, in mature animals, higher expression levels (both at mRNA and protein level) and activity of calpain-1 and lower levels of calpastatin mRNA are associated with higher levels of tenderness. Still before, the calpain system confirmed its involvement in the turnover of the muscle proteins, with a degradative action, being the calpain-1 and 2 less expressed, at least at mRNA level, in the phase of higher muscle growth in young animals. This degradative function is the basis of the role that this enzymatic system has at the post-mortem, during the muscle to meat transformation. Regarding caspases, our results confirmed the action of these proteases in early post-mortem, as characteristic of the apoptotic process, but seems to negate a fundamental importance of these enzymes in meat tenderization. In fact, even though we confirmed the expression of caspases 3/9 mRNAs and the activity of caspase 3 in post-mortem period, it seems these facts were not in relation with the different levels of tenderness in muscles. Moreover, we observed that caspase 3 is active only in the early post-mortem (the first few hours after slaughter) and at lower levels compared to calpain-1, which confirms its primary role in meat tenderization. However, similarly to calpains, during the phase of great muscle growth in young animals, caspase 3 mRNA seems to be espressed at a lower level probably because of the necessity of a low degradation of myofibrillar proteins in this phase of life. With respect to Hsp, we hightlighted a correlation between αB-crystallin, Hsp27 and Hsp70 and meat tenderization; instead, on the contrary of some other studies, Hsp40 seems to be not related to the process. The expression (at least at mRNA level) of Hsp27, Hsp70 and αB-crystallin are in relation with meat tenderness, showing that higher expression levels of these Hsp correspond to higher levels of toughness of meat. Basing on their functions, high grades of protection of myofibrillar proteins and/or high levels of inhibition of apoptosis lead to a slowing-down of the maturation process of the meat, producing tougher meat. Furthermore, in mature animals, this process of myofibrillar proteins protection seems to overhang the degradative potential of calpains. We observed a growing level of Hsp with age, which could be due to a progressive adaptation of animal to environmental stresses, probably in addition to a physiological hormonal change. Hsp70 has also confirmed its importance in thermoprotection of muscle cells. In conclusion, in this thesis, we confirmed the importance of calpain/calpastatin system in meat tenderization process, underlining the lesser importance of caspases. Regarding αB-crystallin, Hsp27 and 70, we pointed out the relationship of their higher presence with lower tenderness of meat, suggesting their possible utilization as new, negative markers of tenderness

La tesi di dottorato ha riguardato lo studio dell’espressione di alcuni geni (codificanti per calpaine, caspasi e heat shock proteins - Hsp) nella specie bovina e caprina, in diverse condizioni sperimentali, per evidenziare il ruolo di tali geni nel processo di maturazione post-mortem del muscolo e quindi nell’intenerimento della carne. In particolare le prove sperimentali condotte hanno voluto esaminare il ruolo dei geni elencati di seguito nel processo post-mortem di maturazione della carne: calpaina 1, calpaina 2, calpastatina, caspasi 3, caspasi 9, alfaB-cristallina, Hsp27, Hsp40 e Hsp70. E’ stata valutata l’espressione in mRNA dei suddetti geni, ma anche l’attività o la presenza delle proteine da essi codificate, nelle diverse situazioni sperimentali. I casi sperimentali affrontati sono stati i seguenti: il confronto tra un muscolo di tipo “slow” e un muscolo di tipo “fast” in bovino; lo stesso muscolo (Lungissimus dorsi) in due tipi genetici diversi di bovino, Bos taurus e Bos indicus x Bos taurus; lo stesso muscolo (Lungissimus dorsi) nella specie caprina in età diverse della crescita giovanile. Il lavoro di tesi ha confermato il ruolo primario del sistema calpaine/calpastatina nel processo di maturazione della carne, e in particolare l’importanza della calpaina 1, che è correlata positivamente alla tenerezza della carne. In ogni caso, però, durante la fase di notevole sviluppo muscolare negli animali più giovani, questo sistema può essere inibito per ridurre la degradazione miofibrillare. E’ stato inoltre evidenziato il ruolo secondario delle caspasi nella maturazione della carne, anche se è stata verificata la loro attività nell’immediato post-mortem, momento in cui avviene il processo apoptotico della cellula muscolare. Inoltre, similmente alle calpaine, l’espressione delle caspasi può essere limitata nella fase di massima crescita del muscolo, negli animali giovani. Infine, i risultati più interessanti sono stati quelli riguardanti il ruolo delle Hsp nell’intenerimento del muscolo. E’ stata infatti evidenziata una costante relazione negativa tra l’espressione delle Hsp e la tenerezza della carne. In particolare Hsp27 e 70, ma anche αB-cristallina, si sono rivelate come maggiormente espresse nella carne più dura, e la loro espressione cresce con l’età dell’animale. Hsp70 ha anche confermato il suo ruolo nella capacità di termo-tolleranza degli animali, mentre Hsp40 non ha mostrato l’importanza evidenziata nei lavori di altri autori. Da questi studi si può dedurre che le Hsp possono essere dei buoni candidati per futuri studi su marker (negativi) della tenerezza, utilizzabili nella selezione degli animali da carne