Epinephrine-induced MMP expression is different between skeletal fibroblasts and myoblasts.

Skeletal fibroblasts and myoblasts are among the cell types currently being considered in cell therapy for ischaemic heart disease. To investigate whether the expression of the tissue-remodelling proteolytic enzymes matrix metalloproteinases (MMPs) and the cellular energy regulator AMP-activated protein kinase (AMPK) is comparable between the two cell lines in response to epinephrine treatment, mouse skeletal fibroblasts (NOR-10) and myoblasts (C2C12) were treated with or without a low (11 nmol·l(-1) ) or high (55 nmol·l(-1) ) dose of epinephrine for 2 or 6 h. Cellular MMP-3 expression was increased by the high-dose epinephrine at both treatment periods in both cell lines. Cellular MMP-2 and MMP-13 expressions were amplified by the 2- or 6-h epinephrine incubation in fibroblasts. However, in myoblasts, such an increase was only seen at the longer treatment time. An elevated AMPKα expression was observed after a 2-h presence of epinephrine in both cell lines, which matches temporally with the early increased cellular MMP-2 and MMP-13 expression in fibroblasts. Activity of secreted MMP-2 increased only after 6-h epinephrine treatment in both cell types. Our data suggest that skeletal fibroblasts respond earlier to epinephrine application in terms of endogenous synthesis of the proteolytic and the energy homeostasis enzymes, whereas such response occurs later and to a milder dose of the beta adrenergic agonist in myoblasts.

Matrix metalloproteinases are less essential for the in-situ gelatinolytic activity in heart muscle than in skeletal muscle.

In order to determine whether the contribution of matrix metalloproteinases (MMPs) to the tissue gelatinolytic activity is similar between myocardium and skeletal muscle tissue, in-situ zymography was applied to myoblasts originated from myocardium or skeletal muscle of rodents as well as tissue sections of heart and soleus muscles of rats. Gelatinolyic activity was observed in cytoplasm and nucleus of both heart and skeletal myoblasts. The chelating agent EDTA blocked much of the gelatinolytic activity and the organomercurial activator of MMPs increased the activity in cells of both muscle origins. However, the inhibition of gelatinolytic activity by a broad spectrum MMP inhibitor was less profound in heart myoblasts than that in skeletal myoblasts. Gelatinolytic activity was also expressed in the endomysium and perimysium of tissue sections of heart and soleus muscles. Similar with findings in the cell studies, the gelatinase activity was increased by the MMP activator, mostly blocked by EDTA and partially inhibited by the MMP inhibitor. In the presence of the MMP inhibitor, the remaining gelatinolytic activity in the tissue sections was again higher in myocardium than that in soleus muscle. This observation was further supported by the gelatinolytic activity examined in tissue homogenates. Our findings suggest that other proteinases, in addition to MMPs, are more responsive for the tissue gelatinolytic activity in heart muscle as compared with that in skeletal muscle.

Calcium determines the supramolecular organization of fibrillin-rich microfibrils.

Microfibrils are ubiquitous fibrillin-rich polymers that are thought to provide long-range elasticity to extracellular matrices, including the zonular filaments of mammalian eyes. X-ray diffraction of hydrated bovine zonular filaments demonstrated meridional diffraction peaks indexing on a fundamental axial periodicity (D) of approximately 56 nm. A Ca2+-induced reversible change in the intensities of the meridional Bragg peaks indicated that supramolecular rearrangements occurred in response to altered concentrations of free Ca2+. In the presence of Ca2+, the dominant diffracting subspecies were microfibrils aligned in an axial 0.33-D stagger. The removal of Ca2+ caused an enhanced regularity in molecular spacing of individual microfibrils, and the contribution from microfibrils not involved in staggered arrays became more dominant. Scanning transmission electron microscopy of isolated microfibrils revealed that Ca2+ removal or addition caused significant, reversible changes in microfibril mass distribution and periodicity. These results were consistent with evidence from x-ray diffraction. Simulated meridional x-ray diffraction profiles and analyses of isolated Ca2+-containing, staggered microfibrillar arrays were used to interpret the effects of Ca2+. These observations highlight the importance of Ca2+ to microfibrils and microfibrillar arrays in vivo.

Modelling quality variations in commercial Ontario pork production.

This study explores the interactions of sensory and nutritional environment with genotype occurring in current commercial pork production in Ontario, Canada, which may interact to result in poor quality meat. The study focussed on identifying factors and signalling mechanisms that contribute to poor meat quality, in order to develop strategies to reduce the incidence of unacceptable product quality. In the first phase of the work reported here, animal behaviour and muscle metabolism studies were related to meat colour, tenderness and water-holding capacity measurements from commercially-produced pigs killed in a commercial packing plant. A partial least squares analysis was used to determine the most important of the principal production variables, peri-mortem biochemical measures and post-mortem carcass condition variables studied, in terms of their influence on water-holding, toughness and colour (L*-value). Variations between producer and kill day at the slaughterhouse were very strong contributors to variability in these three meat quality parameters, followed by pH variations. A second phase of the study is currently underway to characterize patterns of gene expression related to extremes of end-product quality and to reduce quality variations by nutritional and behavioural management strategies.

The structural basis of cooking loss in beef: Variations with temperature and ageing.

Meat loses fluid during cooking, resulting in textural changes and loss in cook yield. To understand the structural basis of cooking losses, this work used 10 bovine semitendinosus muscles and two ageing periods (1 vs 14days) to examine micro- and macro-level dimensional changes in muscle during heating. Muscle blocks, muscle fibre fragments and myofibrils all showed similar maximum shrinkage in cross sectional area (20-24%) but maximum length shrinkage was less in myofibrils (15%) than muscle blocks and fibre fragments (25%). Dimensional changes were dominated by shrinkage in individual muscle fibres and myofibrils, indicating that connective tissue does not play a major role. Transverse shrinkage predominantly occurred over 50-65°C whereas the longitudinal shrinkage predominantly occurred over 70-75°C; we attribute these two separate shrinkage events to denaturation of myosin and actin respectively. Higher cook losses in samples aged for 14days versus 1day suggests that desmin, nebulin and titin denaturation are not major drivers of fluid expulsion as these proteins are degraded during ageing. We postulate that proteolysis during ageing produces protein fragments which are more easily lost from the structure during cooking, along with water.

Biomechanical and biochemical study of a standardized wound healing model.

Standardized protocols were developed for use in a detailed investigation into the biomechanical and biochemical properties of a dermal wound healing model in the rat. The use of a rapid freezing method at -80 degrees C minimized the detrimental effects of freezing on the biomechanical properties of the tissue and also allowed for convenient inter-laboratory collaboration to be performed. The methodology described allowed for the simultaneous and reproducible measurement of tensile strength, collagen cross-linking and proteolytic enzyme activity. Increases in the tensile properties of the tissue with time were consistent with an active process of remodelling process as indicated by changes in the cross-link and enzyme profiles. Initially the granulation tissue was comparatively rich in the keto-imine cross-link hydroxylysino-keto-norleucine, which was later replaced by the aldimine cross-link dehydro-hydroxy-lysinonorleucine. The mature cross-link histidino-hydroxy-lysinonorleucine was not observed within the granulation tissue at any stage and was also absent in aged control skin. A peak of matrix metalloproteinase-9 activity was observed at early timepoints (48 hr) and then decreased rapidly to normal levels and is consistent with an acute inflammatory response. In contrast matrix metalloproteinase-2 activity peaked later (3 days) and then decreased gradually, consistent with its role as one of the predominant enzymes involved in the remodelling process. The results described validate the animal model used and emphasize its potential for use in combined biomechanical and biochemical studies of acute wound healing.

Fibrillin-rich microfibrils: an X-ray diffraction study of the fundamental axial periodicity.

Microfibrils are ubiquitous matrix polymers which are thought to provide elastic properties in all extracellular matrix structures. The major component of the elastic microfibrils is the protein fibrillin; its molecular structure is unknown. In electron microscopy, microfibrils appear as beaded structures exhibiting a variable periodicity, indicating that they may be elastomeric. The X-ray diffraction of fibrillin-rich microfibrils in the form of zonular filaments from bovine eyes exhibits meridional diffraction peaks indexing on a fundamental periodicity of 55 nm in the relaxed state. The application of a 40% extension produced a lengthening of the periodicity by 3% as judged by alteration of the D spacing of the principal peaks. This effect was shown to be reversible. Changes in the periodicity of the meridional reflections indicate changes in the fundamental structure of the microfilaments, but cannot account for all long range elastomeric properties of fibrillin-containing microfibrils.

Epinephrine upregulates calpain activity in cultured C2C12 muscle cells.

C2C12 cells were grown to confluence at 37 degrees C under a continuous 5% CO(2) stream and myotube formation was stimulated. The cultures were then incubated with or without 2 microg/mL epinephrine for 18 h prior to harvesting and calpain extraction. Epinephrine treatment resulted in a three-fold increase in extractable mu-calpain activity (P < 0.05), a three-fold increase in extractable m-calpain activity (P < 0.05), a 36% increase in calpastatin activity (P < 0.001), and a 16% decrease (P < 0.05) in the total protein content in the C2C12 cell homogenate. These results suggest that calpains may play a role in protein metabolism and that the hormone epinephrine may be directly involved in the regulation of their cellular expression.

Age related compliance of the lamina cribrosa in human eyes.

AIMS: To investigate changes in the mechanical compliance of ex vivo human lamina cribrosa with age. METHODS: A laser scanning confocal microscope was used to image the surface of the fluorescently labelled lamina cribrosa in cadaver eyes. A method was developed to determine changes in the volume and strain of the lamina cribrosa created by increases in pressure. The ability of the lamina cribrosa to reverse its deformation on removal of pressure was also measured. RESULTS: Volume and strain measurements both demonstrated that the lamina cribrosa increased in stiffness with age and the level of pressure applied. The ability of the lamina cribrosa to regain its original shape and size on removal of pressure appeared to decrease with age, demonstrating an age related decrease in resilience of the lamina cribrosa. CONCLUSIONS: The mechanical compliance of the human lamina cribrosa decreased with age. Misalignment of compliant cribriform plates in a young eye may exert a lesser stress on nerve axons, than that exerted by the rigid plates of an elderly lamina cribrosa. The resilience of the lamina cribrosa also decreased with age, suggesting an increased susceptibility to plastic flow and permanent deformation. Such changes may be of importance in the explanation of age related optic neuropathy in primary open angle glaucoma.

Positional variations in fracture toughness, stiffness and strength of descending thoracic pig aorta.

The tunica media and intima of descending thoracic aortas from commercially killed pigs showed variations in mechanical properties along their length in a region extending from just in front of the first intercostal artery to the sixth intercostal artery. Along this region circumferential toughness, measured as work of fracture from a tear test, increased, longitudinal and circumferential ultimate tensile strength increased and longitudinal and circumferential stress-strain gradients of excised strips increased further away from the heart. These increasing mechanical properties are positively correlated with an increase in collagen mass fraction and an increase of radius/thickness ratio away from the heart over this region.

Strain-induced reorientation of an intramuscular connective tissue network: implications for passive muscle elasticity.

The most abundant intramuscular connective tissue component, the perimysium, of bovine M. sternomandibularis muscle was shown to be a crossed-ply arrangement of crimped collagen fibres which reorientate and decrimp on changing muscle fibre sarcomere length. Reorientation of perimysial strands was observed by light microscopy and identification of these strands as collagen fibres was confirmed by high-angle X-ray diffraction. Mean collagen fibre direction with respect to the muscle fibres ranged from approximately 80 degrees at sarcomere length = 1.1 micron to approximately 20 degrees at 3.9 microns. This behaviour was well described by a model of a crimped planar network surrounding a muscle fibre bundle of constant volume but varying length. Modelling of the mechanical properties of the perimysium at different sarcomere lengths produced a load-sarcomere length curve which was in good agreement with the passive elastic properties of the muscle, especially at long sarcomere lengths. It is concluded that the role of the perimysial collagen network is to prevent over-stretching of the muscle fibre bundles.

Functional morphology of the endomysium in series fibered muscles.

Many skeletal muscles, including the feline biceps femoris, are composed of short, tapered myofibers arranged in an overlapping longitudinal series. The endomysium of such muscles transfers tension between overlapping myofibers, and is thus an elastic element in series with them. The endomysium of the cat biceps femoris contains curvilinear collagen fibrils in an approximately isotropic (random) array. The collagen fibrils undergo only a modest reorientation as the myofibers shorten or lengthen within the physiological range. A geometrical model predicts no change in the thickness of the endomysium on changing muscle fiber length and quantifies the expected collagen fibril reorientation in the endomysium as a function of muscle extension. It is also demonstrated that a high proportion of the collagen fibrils will be curvilinear at all sarcomere lengths. The organization of endomysial collagen is appropriate for the transfer of loads between myofibers by means of shear.

Fibrillin-rich microfibrils of the extracellular matrix: ultrastructure and assembly.

Fibrillin-rich microfibrils are a unique class of extensible connective tissue macromolecules. Their critical contribution to the establishment and maintenance of diverse extracellular matrices was underlined by the linkage of their principal structural component fibrillin to Marfan syndrome, a heritable connective tissue disorder with pleiotropic manifestations. Microscopy and preparative techniques have contributed substantially to the understanding of microfibril structure and function. The supramolecular organisation of microfibrillar assemblies in tissues has been examined by tissue sectioning and X-ray diffraction methods. Published findings are discussed and new information reported on the organisation of microfibrils in the ciliary zonular fibrils by environmental scanning electron microscopy. This review summarises microscopy and X-ray diffraction studies that are informing current understanding of the ultrastructure of fibrillin-rich microfibrils.

Dietary-induced changes of muscle growth rate in pigs: effects on in vivo and postmortem muscle proteolysis and meat quality.

The effects of various growth rates in pigs induced by four different feeding strategies on the activity of the calpain system and on postmortem (PM) muscle proteolysis and tenderness development were studied. An increased growth rate may be caused by an increased protein turnover, which results in up-regulated levels of proteolytic enzymes in vivo that, in turn, possibly will affect PM tenderness development. It can be hypothesized that increased proteolytic activity pre-slaughter will increase the PM tenderization rate. From postnatal d 28 to d 90 (phase 1) the pigs were divided into two groups, given either ad libitum (A) or restricted (R, 60% of ad libitum) access to feed. The two groups were then divided into two subgroups, given either restricted or ad libitum access to feed from d 91 to slaughter at d 165 (phase 2). Measurements of the activity of mu-calpain, m-calpain, and calpastatin; concentrations of total collagen and the percent of soluble collagen; and RNA, DNA, and elongation factor-2 where made at slaugther. Myofibrillar fragmentation index (MFI) was determined at slaughter and 24 h PM. Warner-Braztler shear force was determined 1 d and 4 d PM. Pigs fed restricted diets in phase 1 and fed ad libitum in phase 2 (RA pigs) had increased growth rates in the last phase compared to pigs fed ad libitum during both phase 1 and phase 2 (AA pigs). The increased growth rate (compensatory growth) was followed by an increased proteolytic potential (mu-calpain:calpastatin ratio), increased MFI values, and higher tenderization rates. There was a positive correlation between the activities of m-calpain and growth rates (r = 0.35, P = 0.03), and between RNA levels and growth rates (r = 0.43, P = 0.006). The proposed hypothesis is largely supported by the results. The activities of both mu- and m-calpain at slaughter were highest in fast-growing pigs. The calpain activity was highest in RA pigs, which in turn also had the fastest growth rates prior tslaughter among the four groups. This implies that the synthesis of these enzymes was up-regulated during the second feeding period to a larger extent in RA pigs. The proteolytic potential and the MFI values indicate that the up-regulated in vivo calpain activity had an effect on PM protein degradation, which also is supported by the higher tenderization rate in RA pigs.

The physical basis of meat texture: Observations on the fracture behaviour of cooked bovine M. Semitendinosus.

The fracture behaviour of cooked strips of beef M. semitendinosus was studied by qualitative observation of the manner in which fracture occurred and by quantitative measurements of ultimate tensile strength, work of fracture and notch sensitivity. Qualitative observations showed that fracture started in the perimysial connective tissue in all test configurations used, resulting initially in the separation of intact muscle fibre bundles. The ultimate tensile strength along and across the fibres was ∼-300kNm(-2) and ∼-25kNm(-2), respectively. The qualitative aspects of fracture were explained on the basis of a uniaxial fibrous composite of strong muscle fibre bundles in a weak connective tissue 'matrix', with poor interfacial strength. Work of fracture through the perimysium was in the range 0·4 to 1·8kJm(-2). The difficulty in propagating fracture across the muscle fibre bundles was explained in terms of the material's complete insensitivity to notches running across the fibres. The results imply that the muscle fibre bundle is an important level of structural organisation as far as fracture is concerned and that the strength of the perimysium, or perimysium/muscle fibre bundle interface, is likely to have a major influence on the toughness of'the cooked meat.

A comparison between tensile and shear adhesive strength of meat-myosin junctions.

A model meat-myosin gel junction was used to compare the adhesive strength of binding junctions when subject to tensile or shear forces. This comparison was made on junctions cooked to 80°C with three different alignments of muscle fibres with respect to the junction plane. Tensile adhesive strength (TAS) and shear adhesive strength (SAS) did not differ significantly when fibres in both of the bound pieces of meat were perpendicular to the plane of the myosin gel function (90°/90° junction). However, SAS was higher than TAS if the muscle fibres in one or both of the meat pieces were parallel to the plane of the junction. This suggests that tensile failure of binding junctions is the more likely mode of failure. Differences between SAS and TAS in any one junction orientation were small compared to the effect of muscle fibre orientation with respect to the junction; both TAS and SAS were highest for 90°/90° junctions and lowest when muscle fibres in both meat pieces were parallel to the junction (0°/0° junctions).

The strength and stiffness of perimysial connective tissue isolated from cooked beef muscle.

Thick transverse slices of bovine M. semitendinosus were cooked for 1 h at 50°-90° and then cooled. Perimysial connective tissue was dissected from the cooked meat and subjected to mechanical testing in a small-scale device. The initial 'toe' region of the J-shaped load-extension curve was progressively lost with increasing temperature, the curve becoming more nearly linear after cooking at 90°C. These effects are explained on the basis of the progressive straightening out of the crimps from the collagen fibres, the crimps becoming finally lost at approximately 70°C. The final stiffness of the perimysium at greater extensions was unchanged at higher temperatures. Breaking strength increased from raw to cooked at 50°C, thereafter decreasing at cooking temperatures up to 90°C. it is proposed that this technique of testing isolated perimysium gives a valuable means of directly measuring the effects of cooking, or other treatments, on the intrinsic properties of perimysial collagenous material. Quantitative knowledge of these will help to determine its contribution to the overall mechanical properties, and hence eating quality, of cooked meat.

The effect of ageing on the water-holding capacity of pork: role of cytoskeletal proteins.

The water-holding capacity (WHC) of pork decreases post-mortem but has been shown to increase during subsequent ageing. In order to test a hypothesis that water-holding capacity increases during ageing due to degradation of the cytoskeleton, WHC was followed 10 days post-mortem and related to the extent of proteolysis of cytoskeletal proteins. A fast method for measuring WHC in small meat samples was developed by the use of centrifugation. The WHC of fresh pork decreases in the first part of post-mortem storage after which it increases to the level of 1 day PM. No changes in total water content of the meat were observed which could explain changes in WHC during ageing. Vinculin and desmin degrade gradually during ageing while talin degrades rapidly. These observations are consistent with the hypothesis that degradation of the cytoskeleton slowly removes the linkage between lateral shrinkage of myofibrils and shrinkage of entire muscle fibres, so removing the force that causes flow into the extracellular space. Inflow of previously expelled water is then possible, so increasing WHC as observed in later periods of storage.

Mechanical and structural characteristics of single muscle fibres and fibre groups from raw and cooked pork longissimus muscle.

The aim of this study was to analyse the mechanisms that determine why small groups of muscle fibres may have different mechanical properties than single muscle fibres. The method used combined light microscopy and tensile testing on single fibres and small groups of fibres from raw and cooked (80 °C) meat, from both conditioned and unconditioned porcine longissimus muscle. The results showed that small groups of fibres had different breaking properties than constituent single fibres in raw muscle, but that these differences diminished on cooking. Raw groups of fibres showed a more uniform lengthening along their entire length and a higher extension to rupture than single fibres. Conditioning increased maximum strains in both single fibres and small fibre groups. In unconditioned cooked meat, single fibres and fibre groups showed comparable breaking stresses and extensions. Conditioning resulted in a lower strength in fibre groups than in single fibres. These results show that (endomysial) connective tissue linkages between adjacent muscle fibres in a small group significantly alter the breaking behaviour of single fibres. The effects of these connective tissue linkages are not reduced by conditioning alone, but are largely diminished by cooking to 80 °C.

Connective tissue differences in the strength of cooked meat across the muscle fibre direction due to test specimen size.

Systematic variations in the tensile strenght of cooked beef M. semitendinosus across the muscle fibre direction due to the cross-sectional size of specimens are demonstrated in specimens from (a) longitudinal and (b) transverse slices. The strength perpendicular to the fibre direction of longitudinal slices of thickness 0·25-5·75 mm varied by a factor of 2, thicker slices being stronger. This factor of 2 is in approximate agreement with the difference in strength of transverse versus longitudinal slices across the fibre direction. These variations of strength due to specimen geometry are explained on the basis of the increasing likelihood of including a ribbon of the perimysial connective tissue network which is continuous along the whole length of the test piece in larger samples. The breaking strength of small cross-sectional area specimens is likely to be dominated by the strength of the endomysial-perimysial junction. Larger cross-sectioned specimens, by including continuous strands of the perimysial network, have higher strengths resulting from the necessity to break these strands. These findings highlight the need to specify specimen dimensions in tensile test results. They also show that by manipulating specimen geometry, the relative magnitude of the two mechanisms of connective tissue fracture (endomysial-perimysial separation and perimysial strand fracture) may be assessed.