The role of Calpain 3 in sheep skeletal muscle growth and post-mortem meat quality

Abstract

Calpain 3 is a tissue specific calpain, and its mRNA is the most expressed calpain isoform in skeletal muscles. Many mutations and polymorphisms within the human calpain 3 gene have been reported and related to limb-girdle muscular dystrophy. In livestock the proteolytic activity of calpain 3 is implicated in aspects of meat quality such as tenderness and meat yield. One of the most distinctive characteristics of calpain 3 is its very rapid autolysis. In skeletal muscle, calpain 3 is stabilised by binding titin but it is spontaneously autolysed during extraction. Due to its rapid autolysis, purification and characterisation of calpain 3 has been extremely difficult. However the pattern of autolysis can be used to identify the activity and physiological functions of calpain 3. Calpain 3 autolysis can be observed via banding patterns on Western blotting using calpain 3 specific antibodies and relative amounts of calpain 3 can be determined from band density. The Western blotting protocol to detect calpain 3 from skeletal muscle samples were optimised in this study. The objective of this PhD project was to investigate the role of calpain 3 in meat quality, focusing on tenderness and meat yield. The role of calpain 3 in tenderness was tested by muscle stretching expected to disrupt calpain 3/titin interactions and enhance calpain 3 autolysis and proteolytic activity. Lamb carcasses were hung by two different methods, conventional and bar-hanging. The degree of muscle stretching was determined by sarcomere length measurements. Carcasses treated by the bar-hanging method showed longer sarcomere length than conventionally hung samples, and the muscle stretching showed an obvious impact on initial tenderness during rigor mortis development. Enhanced titin degradation has been observed in stretched muscle, regulated from the disruption of the calpain 3/titin interaction. However, strong evidence of enhanced calpain 3 activation was not obtained from this study. Roles of calpain 3 in muscle yield were analysed on the basis of a calpain 3 gene marker study in livestock. A correlation between specific allele variants in exon 10 of ovine calpain 3 and the yield of fat trimmed meat cuts has been reported. This research aimed to investigate the biochemical significance of polymorphic variation in calpain 3. Muscle samples were collected from lambs which were homozygous for each of the three alleles within calpain 3 exon-10. Calpain 3 mRNA sequences were obtained from these homozygous muscle samples to identify any base substitutions. Four single base substitutions were found besides those in exon-10, but none of them, including variations within exon-10, caused an amino acid sequence difference. Therefore they were unlikely to influence calpain 3 protein structure and function. Expression of calpain 3 mRNA and the amounts of calpain 3 protein were also compared among genotypes, and there were no significant differences found. These results suggest that the reported phenotype variations related to specific allele variants within calpain 3 exon-10 were not direct effects of calpain 3 polymorphisms. Calpain 3 has important roles in the maintenance and development of skeletal muscle but how much it contributes toward meat quality is still uncertain.