Study On Expression Of Plectin In Pork During Postmortem Aging And Its Correlation With Water Holding Capacity

Changes in water-holding-capacity(WHC)during the conversion of muscle to meat have always been a hot topic in meat science research.Recent research has shown that the degradation of several key structural cytoproteins could contribute to the changes in WHC during postmortem aging.Plectin,an IF-associated protein which is expressed widely in muscle fiber,plays an important role in sustaining and strengthening the cytoarchitecture integrity.The aim of this study is to investigate the distribution and degradation pattern of plectin in pork,and to study the correlations between plectin-desmin degradation and changes in WHC during postmortem aging,providing a better explanation of the formation of WHC during postmortem aging.1.Distribution of pork plectin in muscle fibersThis chapter was designed to study the distribution of plectin in pork at 0 h postmortem aging by immunofluorescence analysis.LD muscles(between the 10th and 14th thoracic vertebra)were collected from the right side of carcasses.Serial cross and longitudinal sections(10?m)were obtained on a cryostat microtome.Anti-plectin,anti fast-MyHC,anti-desmin,anti-?-calpain and anti m-calpain were subjected to sections for either single or double staining.The results showed that plectin was distributed in a honeycomb pattern in cross sections and in a highly regular cytoplasmic striated pattern in the longitudinal sections.Furthermore,plectin was preferentially distributed in fast muscle fibers,while was hardly detected in slow muscle fibers.Co-localization of plectin and desmin appeared both in the cytosol and beneath the sarcolemma on the cross and longitudinal sections in a highly-ordered striated pattern.Besides,plectin was found co-localized with ?-calpain both in the cytosol and beneath the sarcolemma,while no co-localization with m-calpain was found.This indicated that pork plectin might be degraded during postmortem aging.2.Degradation of pork plectin during postmortem agingThis chapter was designed to explore the postmortem degradation pattern of plectin by immunoblot analysis,the calpain and caspase inhibitor were used to find the potential endogenous enzyme(s)on account of plectin degradation,and the immunofluorescence analysis was performed to vertify these results.LD muscles(between the 10th and 14th thoracic vertebra)were collected from the right side of carcasses and aged at 4? for 0 h,6 h,12 h,1 d,3 d,7 d and 13 d.Samples for inhibitor-incubation were cut into pieces and immersed into inhibitor solutions at 1:1(w/v).The results showed that the amount of intact plectin was rapidly reduced during the early postmortem aging(P<0.05)and almost disappeared at day 3.The degraded 240 kDa plectin accumulated fast and was further cleaved after 3 days of aging(P<0.05).The plectin degradation could be significantly blocked by calpain inhibitor MDL-28170 rather than caspase-3 inhibitor Ac-DEVD-CHO(P<0.05).Double immunostaining of ?-calpain and plectin showed a large amount of overlap at 0 h and 3 days of postmortem,indicating that plectin postmortem degradation might be attributed to ?-calpain.3.Correlations between plectin degradation and WHC during postmortem agingThis chapter was designed to analyze the correlations between IF-protein degradation and changes in WHC during postmortem aging.LD muscles(between the 10th and 14th thoracic vertebra)from a total of 44 samples were collected from the right side of carcasses and aged at 4? for 0 h,6 h,12 h,1 d,3 d,7 d and 13 d.The samples were subjected to LF-NMR T2 analysis,drip loss determination and the immunoblot analysis of plectin,desmin and ?-calpain.The results of correlation analysis showed that the intensity of intact plectin and desmin were significantly and negatively correlated with P21,T21 and T22,while positively correlated with P22.Furthermore,the correlation were established between the intensity of plectin and desmin in the early postmortem and the LF-NMR T2 parameters in the late period.In addition,plectin and desmin have similar degradation patterns during postmortem aging.During the 13-day postmortem aging,correlations between the intensity of 80 kDa and 78 kDa subunits and P21 were negative,while positive with P22.These results indicated that ?-calpain was the endogenous enzyme which led to the postmortem degradation of plectin and desmin,therefore contributed to the changes in water distribution.Finally,a hypothesis concerning the structural proteins'degradation and changes in WHC with the emphasis on the plectin-desmin intermediate filament networks was proposed.