Calpains and capsases are two families of cytosolic proteases essential for a proper skeletal muscle function, which recognize specific target proteins for degradation. Moreover, μ-calpain is believed to play a major role in post mortem proteolysis and meat tenderization, which in turn depends on fibre type composition. Recent data also support the involvement of caspases in the development of tender meat. In fact, it is plausible that the hypoxic conditions occurring in post mortem may activate these proteases, which function as vital executioners of apoptosis in hypoxia/ischemia and degrade structural components of the cytosckeleton. The goal of this work was to evaluate the activities of μ-calpain and caspases in two different muscle types from bulls, immediately after slaughtering, by immunodetection of their specific proteolytic products and to compare such activities to their related mRNA expression level. In particular, caspases 9 and 3 have been analysed, which are an initiating and an executer caspase, respectively, to follow the entire cascade of events occurring during a hypoxic stress.
Samples of Longissimus dorsi (white/type IIB muscle) and Infraspinatus (red/type I muscle) muscles were collected within 20 minutes from slaughtering of 16 Italian Simmental young bulls, and stored at -80° until the analysis. By SDS-PAGE the alpha II spectrin and its degradation products 145kDa (from μ-calpain degradation) and 150kDda and 120kDda (from caspases degradation) were separated and then quantified in relative terms. Total RNA was extracted from the two muscles and cDNA was produced; the DNA was amplified by q-PCR technic. The genes analyzed were CAPN1 (coding for μ-calpain), CASP3 (caspase 3), CASP9 (caspase 9). Genes coding for GAPDH, β-Actin, RPLP0 and Cyclophilin was used as reference genes.
Both caspases and μ-calpain were found active, having been recognized their target degradation products. In particular no difference in the level of alpha II spectrin fragment, derived from the caspase-3 activity, was found between the Longissimus dorsi and Infraspinatus muscles, in agreement with gene expression results. On the other hand, the μ-calpain activity was influenced by the type of muscles.
