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Studies on the correlation between expression and/or autolysis of calpain and postmortem proteolysis in muscle have provided conflicting evidence regarding the possible role of calpain 3 in postmortem tenderization of meat. Thus, the objective of this research was to test the effect of postmortem storage on proteolysis and structural changes in muscle from normal and calpain 3 knockout mice. Knockout mice (n = 6) were sacrificed along with control mice (n = 6). Hind limbs were removed and stored at 4 degrees C; muscles were dissected at 0, 1, and 3 d postmortem and subsequently analyzed individually for degradation of desmin. Pooled samples for each storage time and mouse type were analyzed for degradation of nebulin, dystrophin, vinculin, and troponin-T. In a separate experiment, hind-limb muscles from knockout (n = 4) and control mice (n = 4) were analyzed for structural changes at 0 and 7 d postmortem using light microscopy. As an index of structural changes, fiber detachment, cracked or broken fibers, and the appearance of space between sarcomeres were quantified. Cumulatively, the results of the first experiment indicated that postmortem proteolysis of muscle occurred similarly in control and in calpain 3 knockout mice. Desmin degradation did not differ (P > 0.99), and there were no indications that degradation of nebulin, dystrophin, vinculin, and troponin-T were affected by the absence of calpain 3 in postmortem muscle. Structural changes were affected by time postmortem (P < 0.05), but not by the absence of calpain 3 from the muscles. In conclusion, these results indicate that calpain 3 plays a minor role, if any, in postmortem proteolysis in muscle.

To identify the mechanisms underlying muscle aging, we have undertaken a high-resolution differential proteomic analysis of gastrocnemius muscle in young adults, mature adults, and old LOU/c/jall rats. Two-dimensional gel electrophoresis and subsequent MALDI-ToF mass spectrometry analyses led to the identification of 40 differentially expressed proteins. Strikingly, most differences characterized old (30-month) animals, whereas young (7-month) and mature (18-month) adults exhibited similar patterns of expression. Important modifications in contractile (actin, myosin light-chains, troponins-T) and cytoskeletal (desmin, tubulin) proteins, and in essential regulatory proteins (gelsolin, myosin binding proteins, CapZ-beta, P23), likely account for dysfunctions in old muscle force generation and speed of contraction. Other features support decreases in cytosolic (triose-phosphate isomerase, enolase, glycerol-3-P dehydrogenase, creatine kinase) and mitochondrial (isocitrate dehydrogenase, cytochrome-c oxidase) energy metabolisms. Muscle aging is often associated with increased oxidative stress. Accordingly, we observed differential regulation of molecular chaperones (hsp20, hsp27, reticuloplasmin ER60) and of proteins implicated in reactive aldehyde detoxification (aldehyde dehydrogenase, glutathione transferase, glyoxalase). We further noticed up-regulation of proteins involved in transcriptional elongation (RNA capping protein) and RNA-editing (Apobec2). Most of these proteins were previously unrecognized as differentially expressed in old muscles, and they represent novel starting points for elucidating the mechanisms of muscle aging.

This study was conducted to determine whether the application of high hydrostatic pressure could modify the enzymatic activity and membrane integrity of lysosomes in muscle. Several combinations of pressure (0-600 MPa) and time (0-300 s) were applied to two types of samples: purified enzymes (cathepsin D and acid phosphatase) in buffer solution and intact muscle (biceps femoris). The enzymes studied showed varying degrees of susceptibility depending on the level of pressure, holding time, and environment. Acid phosphatase activity was minimally affected by pressure in buffer solution, whereas cathepsin D was modulated significantly by the pressure and time applied. The activities of the enzymes extracted from meat increased with pressure. The cytochemical observations showed the presence of primary and secondary lysosomes in muscles. After pressurization, the membrane integrity of the lysosomes was modified. A correlation could be established between lysosomal enzymatic activities and the lysosome membrane breakdown.

These experiments were conducted to examine ultrastructural changes in longissimus from normal and callipyge lamb during 14 d of postmortem storage at 4 degrees C. Six crossbred ewe lambs (1/2 Dorset x 1/2 Romanov) were grain-fed and slaughtered at approximately 250 d of age. Leg conformation score was the basis for classifying carcasses into normal and callipyge. The normal and callipyge longissimus had mean Warner-Bratzler shear force of 2.8 (2.7, 2.4, and 3.4) and 9.0 (12.2, 6.9, and 7.9) kg, respectively, after 14 d of postmortem storage. The results of transmission electron microscopy demonstrated ultrastructural changes, including sarcolemma detachment, loss of myofibril lateral attachments, and I-band breaks in normal longissimus. Detachment of sarcolemma from myofibrils occurred in both phenotypes, but it was delayed by several days in callipyge longissimus. Thus, the sarcolemma detachment seems not to contribute significantly to postmortem tenderization. The endomysium of both phenotypes did not change with postmortem storage. In normal longissimus, the percentage of fractured I-bands increased from 0% at d 1 to 11% at d 3 (P<.05) and did not change between 3 and 14 d (15%) postmortem (P>.05). However, postmortem storage did not affect (0 to 3%) the frequency of the I-band breaks in the callipyge longissimus (P>.05). Therefore, the break in the I-band region in postmortem muscle is a change that is associated with postmortem tenderization. We conclude that the major factor responsible for the toughness of meat from callipyge longissimus is the postmortem stability of myofibrils.

Striated muscle cytoskeleton was studied by ultrastructure and electrophoresis. Treatment of sea bass white muscle myofibrils and glycerinated fibres with calpain caused disruption of costameres, intermediate filaments, and Z-line, without altering sarcomeres. V8 protease also caused loss of costameres and Z-line, and disrupted sarcomeres without affecting the intermediate filaments. Recombinant lipase caused loss of Z-lines and also sarcolemma detachment, without changing sarcomeres or intermediate filaments. DNase-1 removed thin filaments and partially removed Z-lines while leaving intact the sarcolemma attachments and intermediate filaments. Calpain, V8 protease, lipase and DNase-1 treatments induced extensive loss of alpha-actinin from the Z-line, which could be related to titin cleavage (calpain, V8), phosphoinositide hydrolysis (lipase), and actin depolymerisation (DNase-1). These results show that the cytoskeletal components are independent of intact thin filaments.

The effect of hepatocyte growth factor (HGF) on the activation of quiescent rat skeletal muscle satellite cells was evaluated in vitro. Satellite cells from 9-month-old adult rats are quiescent in vivo and when cultured, display a protracted lag phase prior to division that is not present in satellite cells from neonatal or regenerating muscle. Under normal growth conditions, satellite cells divide for the first time between 42 and 60 hr. Hepatocyte growth factor increased proliferation in a dose-dependent fashion prior to 48 hr with half-maximal stimulation at approximately 3 ng/ml; in addition, heparin enhanced this activity. The time course of cyclin-D1 and proliferating cell nuclear antigen (PCNA) expression was accelerated in HGF-treated satellite cells, indicating that cells entered the cell cycle earlier. No significant effects on muscle-derived fibroblast proliferation was observed. The signalling receptor for HGF is the product of the c-met protooncogene, and rtPCR analysis of satellite cells 0-72 hr in culture demonstrated the presence of this message throughout this time period. The presence of c-met in quiescent satellite cells, the ability of HGF to stimulate precocious entry into the cell cycle, and the previously described localization of HGF message in regenerating muscle (Jennische et al., 1993) indicate that HGF could act as an activator of quiescent satellite cells in vivo.

A number of studies have suggested that Z-disk degradation is a major factor contributing to postmortem tenderization. These conclusions seem to have been based largely on experimental findings showing that the calpain system has a major role in postmortem tenderization, and that when incubated with myofibrils or muscle strips, purified calpain removes Z-disks. Approximately 65 to 80% of all postmortem tenderization occurs during the first 3 or 4 d postmortem, however, and there is little or no ultrastructurally detectable Z-disk degradation during this period. Electron microscope studies described in this paper show that, during the first 3 or 4 d of postmortem storage at 4 degrees C, both costameres and N2 lines are degraded. Costameres link myofibrils to the sarcolemma, and N2 lines have been reported to be areas where titin and nebulin filaments, which form a cytoskeletal network linking thick and thin filaments, respectively, to the Z-disk, coalesce. Filamentous structures linking adjacent myofibrils laterally at the level of each Z-disk are also degraded during the first 3 or 4 d of postmortem storage at 4 degrees C, resulting in gaps between myofibrils in postmortem muscle. Degradation of these structures would have important effects on tenderness. The proteins constituting these structures, nebulin and titin (N2 lines); vinculin, desmin, and dystrophin (three of the six to eight proteins constituting costameres); and desmin (filaments linking adjacent myofibrils) are all excellent substrates for the calpains, and nebulin, titin, vinculin, and desmin are largely degraded within 3 d postmortem in semimembranosus muscle. Electron micrographs of myofibrils used in the myofibril fragmentation index assay show that these myofibrils, which have been assumed to be broken at their Z-disks, in fact have intact Z-disks and are broken in their I-bands.