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Title: Muscle type influences u-calpain mediated troponin-T proteolysis in bovine myofibrils in vitro

Author
item WEAVER, AMANDA - SOUTH DAKOTA STATE UNIV
item Bowker, Brian
item GERRARD, DAVID - PURDUE UNIV

Submitted to: American Meat Science Association Conference Reciprocal Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 4/11/2008
Publication Date: 6/22/2008
Citation: Weaver, A., Bowker, B.C., Gerrard, D. 2008. Muscle type influences u-calpain mediated troponin-T proteolysis in bovine myofibrils in vitro [abstract]. American Meat Science Association Reciprocal Meat Conference Proceedings. Paper No. 45.

Interpretive Summary:

Technical Abstract: The influence of muscle type on postmortem proteolysis remains largely unexplored. Previous attempts to classify muscle tenderness based on ‘muscle type’ indicated differences in tenderness were due to variations in proteolytic enzyme levels, but failed to account for the influence of sarcomere length on the extent of protein degradation. The objective of this study was to define the role that muscle type plays in troponin-T (TnT) degradation at constant sarcomere lengths and protease levels. Two market steers were harvested and samples with similar sarcomere lengths were generated from muscles containing vastly different fiber type compositions, namely the masseter (MS), semitendinosus (ST) and cutaneous trunci (CT) muscles. Pre-rigor muscle samples were dissected into strips (0.5 cm wide and 5-10 cm long parallel to muscle fiber orientation. Pre-rigor muscle samples were then stretched to varying degrees and fastened to wooden applicator sticks to maintain stretch and generate a wide range of post-rigor sarcomere lengths. Samples were immersed in buffer with 0.1 mM phenylmethylsulfonyl fluoride, to inhibit endogenous proteolytic activity, and held at 4°C overnight with stirring. Upon completion of rigor, myofibrils were isolated for sarcomere length determination and proteolytic digestion. Overall sarcomere lengths were slightly different between the two animals (2.73 ± 0.4 µm and 2.95 ± 0.04 µm respectively, means ± SE) but were not different among the three muscles within either animal (P=0.49 and 0.98 respectively). Myofibrils were incubated with excess µ–calpain for 0, 2, 60, 1440 and 2880 min and subjected to SDS-PAGE and western blotting using TnT antibodies. Immunoblotting revealed that ST and CT contain three intact fast TnT isoforms while the MS expressed two intact slow TnT isoforms. Additionally, six degradation products of varying molecular weights were identified in ST and CT samples including the well documented 30 kDa TnT fragment. The 30 kDa fragment was also detected in MS samples; however, no other degradation products were observed. Quantitative analysis of intact TnT degradation demonstrated that degradation of TnT was greater (P<0.05) in CT compared with ST and MS after 1440 and 2880 min of incubation with µ-calpain suggesting that slow TnT isoforms are less susceptible to proteolysis. Data suggest that differences in the degradation of TnT between various muscle types is due to factors other than sarcomere length and calpain concentration or activity and may be partially due to conformational changes in the isoforms that alter access to calpain cleavage sites.