Degradation Of Myofibrils Research Articles

Induction of metamorphosis with potassium ions requires development of competence and an anterior signalling centre in the ascidian Herdmania momus.

Increased K+ concentration in seawater induces metamorphosis in the ascidian Herdmania momus. Larvae cultivated at 24°C exhibit highest rates of metamorphosis when treated with 40 mM KCl-elevated seawater at 21°C. At 24°C, H. momus larvae develop competence to respond to KCl-seawater and initiate metamorphosis approximately 3 h after hatching. Larval trunks and tails separated from the anterior papillae region, but maintained in a common tunic at a distance of greater than 60 μm, do not undergo metamorphosis when treated with KCl-seawater; normal muscle degradation does not occur in separated tails while ampullae develop from papillae-containing anterior fragments. Normal programmed degradation of myofibrils occurs when posterior fragments are fused to papillae-containing anterior fragments. These data indicate that H. momus settlement and metamorphosis only occurs when larvae have attained competence, and suggest that an anterior signalling centre is stimulated to release a factor that induces metamorphosis.

Principal forms of acute damage to cardiomyocytes according to polarization microscopy data

The principal forms of acute cardiomyocyte damage of metabolic and ischemic genesis are accompanied by changes in the contractile apparatus, which is reflected in double refraction of myofibrils. Thus, polarization microscopy is the most sensitive method allowing one to detect the early stages of cardiomyocyte damage. Individual types of cardiomyocyte lesions are identified on the basis of parallel histological studies and polarization and electron microscopy of prenecrotic alterations and myocardial infarction using experimental and autopsy material. They are: I, II, and III degree myofibrillar contracture, intracellular myocytolysis, primary lumpy degradation of myofibrils, and cytolysis. These types of damage form the morphological basis of acute myocardial pathology with a possible outcome of coagulative or colliquative necrosis.

Effect of heating rate on shortening, ultrastructure, and fracture behavior of prerigor beef muscle

Rapidly heated prerigor beef is tender because of incompletely described myofibrillar disruption and tissue fracture. This study was designed to evaluate the effects of heating rate on heat-induced myofibrillar shortening, ultrastructural changes, and fracture behavior in prerigor triceps brachii muscle. Rapid heating (2°C/2 min) to 53°C caused (P < 0·05) more severe myofibrillar shortening in a shorter time and at higher muscle pH and temperature, less muscle weight loss, and shorter sarcomeres than slow heating (2°C/12 min) to 47 or 53°C. Rapid heating caused more extensive degradation of A and I bands, greater loss of the tridimensional pattern of myofibrils, more fragmentation and melting of myofibrils, widened intermyofibrillar spaces, and maximum separation of fiber bundles as compared to slow heating. Slow heating caused extensive shortening but not extensive degradation and disruption of myofibrils. Muscles slowly heated to 53°C sustained greater loss of structural integrity than those slowly heated to 47°C, but fracture behavior was similar. Separation and fracture occurred near the perimysial/endomysial junction in all heated samples, but the perimysium remained affixed to the endomysium at one side of the interface in many rapidly heated samples. Longitudinal fractures showed a granular endomysium and large numbers of supercontraction nodes alternating with areas of sarcolemmal membrane fragmentation and fiber tearing in rapidly heated samples. Alterations of myofibrillar ultrastructure and fiber structure, and separation of bundles, may account for enhanced tenderness of rapidly heated prerigor muscle.

Purification and characterization of a proteinase identified as cathepsin D from tilapia muscle (Tilapia nilotica .times. Tilapia aurea)

For investigating which pepstatin-sensitive proteinases are involved in post-mortem myofibril degradation, the cathepsin D was first purified and characterised

Role of pepstatin-sensitive proteases on the postmortem changes of tilapia (Tilapia nilotica .times. Tilapia aurea) muscle myofibrils

To investigate the pepstatin-sensitive proteases in fish muscle tenderization, tilapia myofibrils (G-I), myofibrils with crude proteases added (G-II), and myofibrils with crude proteases and pepstatin added (G-III) at pH 5.5, 6.0, and 6.5 were incubated at 4 o C for 2.3-3.5 days. Evidence from phase micrographs indicated that the proteolysis of myofibrils of G-I and G-II was much more severe than that of G-III. The dispersed myosin heavy chain on SDS-PAGE profiles of all samples during incubation at pH 6.5 suggested that the activity of pepstatin-sensitive proteases decreased markedly at this pH condition and did not contribute to the myofibril degradation

Alterations in postmortem degradation of myofibrillar proteins in muscle of lambs fed a beta-adrenergic agonist.

Dietary administration of 4 ppm of the beta-agonist L-644,969 (Merck Sharpe and Dohme Research Laboratories) to finishing lambs induced a decrease (10 to 14%, P less than .05) in extractable calpain I activity in the longissimus muscle (LD) at death (d 0). At 4 d postmortem (d 4), extractable calpain I levels in the LD of both control and treated lambs were reduced (P less than .001) from those present at d 0, but the extractable activity in the LD was reduced to a greater extent in control than in treated lambs. Calpain II activity was increased 42% (P less than .005) in LD of treated lambs; however, no significant differences were observed between d 0 and d 4 calpain II activity within treated or control LD samples (P greater than .1). Calpastatin activity was higher in the LD of treated lambs (74% on d 0, P less than .001 and 430% on d 4, P less than .001) than in the LD of control lambs. Measurable cathepsin B activity was decreased (29% on d 0, P less than .05) and measurable cathepsin H activity was increased (10% on d 0, P less than .05 and 10% on d 4, P less than .05) in the LD of treated lambs compared with controls. On d 2, 4 and 6 postmortem, degradation in myofibrils isolated from the LD was lower for treated than for control lambs. Warner-Bratzler shear values for loin chops from treated lambs were higher on both d 3 (111%) and 6 (108%) postmortem than for chops from control lambs (P less than .001). L-644,969-induced decreases in muscle proteolytic capacity may limit postmortem myofibril degradation and contribute to the reduced tenderness observed. This decreased proteolytic capacity may contribute to increased muscularity of L-644,969-treated lambs.

Degradation of myofibrils from rabbit, chicken and beef by cathepsin l and lysosomal lysates

The degradation of rabbit, chicken and beef myofibrils by cathepsin L or lysosomal lysates was studied by SDS-polyacrylamide-gel electrophoresis and electron microscopy (EM). Similar degradation patterns were observed for each myofibrillar preparation incubated with cathepsin L, except that myosin heavy chain and tropomyosin of beef were more susceptible than those of rabbit and chicken. Otherwise, troponin T, troponin in I and C-protein were rapidly degraded with slower degradation of titin, nebulin, myosin heavy chain, α-actinin, α-tropomyosin, actin and myosin light chains, LC1 and LC2. However, the component of 30 000 Mr was found to be further degraded to smaller peptides. Degradation at pH 5·5 (approximate post-mortem limit value) was faster than at pH 6·0 but slower than at pH 5·0. A number of new protein bands were identified (130 000, 120 000, 90 000, 85 000, 80 000, 31 000 and 30 000 Mr). The degradation patterns of rabbit myofibrils by rabbit muscle lysosomal lysates were similar to that of myofibrils incubated with purified cathepsin L except for the retention of the 30 000 Mr component and reduced degradation of actin, due presumably to the reduced amount or stability of cathepsin L in the crude enzyme preparations. Electron micrographs revealed that myofibrillar degradation by cathepsin L occurred preferentially at the Z-lines leading to removal of the Z-line proteins and fracturing of the myofibrils at these sites. Catheptic damage was seen to be most rapid in chicken myofibrils and least rapid in beef myofibrils consistent with the more rapid conditioning process in chicken.

Analysis of structural proteins from biopsied human myocardium with special emphasis on methodology.

An analytical procedure for the quantitative determination of the structural proteins from a biopsied human cardiac muscle weighing approximately 1 mg was described to be applicable in clinical studies in 20 patients with various heart diseases. The principle of the method is glycerinization of heart muscle and analysis by SDS gel electrophoresis. In 6 control heart muscles obtained from patients having almost normal hearts, the pattern of the structural proteins was similar to that of the normal canine heart. Myosin heavy chain-actin ratio ranged 1.26 +/- 0.44. In 5 cases with secondary cardiac hypertrophy, the pattern of the structural proteins was the same as that of the control heart. In 4 cases with hypertrophic cardiomyopathy, an increase in myosin heavy chain was observed in 2 cases, while myosin heavy chain and alpha-actinin decreased in another 2 cases. Hypertrophy and severe disarray of myofibrils were noted in the former, and atrophy and degradation were done in the latter in electron microscopy. In 5 cases with dilated cardiomyopathies, the relative contents of myosin heavy chain and alpha-actinin was reduced in all cases together with atrophy and degradation of myofibrils.

Endogenous proteolytic degradation of cardiac myofibrils of diabetic rats: A.N. Belcastro and I. Maclean, Appl. Biochem. Lab., Department of Physical Education, University of Alberta, Edmonton, Alberta, Canada, T6G 2H9

Endogenous proteolytic degradation of cardiac myofibrils of diabetic rats: A.N. Belcastro and I. Maclean, Appl. Biochem. Lab., Department of Physical Education, University of Alberta, Edmonton, Alberta, Canada, T6G 2H9

Quantitative ultrastructural differences in the development of normal and dystrophic muscle

Ultrastructural alterations in developing dystrophic muscle, which can be clues to the initiation and timing of the dystrophic lesion, have not yet been carefully studied. We studied ultrastructural changes in the developing pectoral muscle of a line of dystrophic chickens and closely related normal chickens using stereological methods. These methods provided a quantitative estimate of the relative volume and surface area of cell structures and can often detect subtle changes. We found that most ultrastructural differences between normal and dystrophic fibers were not apparent unitl between 18 days in ovo and 1 week posthatching, but that differences in myofibril development were apparent as early as 10 days in ovo. This relative lack of myofibril development may be related to the increased myosin and myofibril degradation that has been shown in young chicks.

Immunocytochemical localization of a calcium-activated protease in skeletal muscle cells

The 80 000-D subunit of a calcium-activated protease from skeletal muscle was purified to homogeneity using preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and was used to elicit antibody production in rabbits. Antiserum was purified using affinity chromatography to yield a monospecific antibody fraction (anti-80K) directed against the 80 000-D subunit. Localization studies showed that the 80 000-D subunit is present in or near the sarcolemma of cultured myoblasts and sectioned muscle tissue, in discrete areas of the cytoplasm of myoblasts, and in the Z disks of the myofibril. The location of the calcium-activated protease in the cell suggests that the enzyme may be involved in myofibril degradation and in membrane alterations in developing and mature muscle cells.

Mode of degradation of myofibrillar proteins by an endogenous protease, cathepsin L

The mode of degradation of myofibrils and their constituent proteins by cathepsin L (EC 3.4.22.15) of rabbit skeletal muscle was studied. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that cathepsin L degraded myosin heavy chain, α-actinin, actin, troponin T and troponin I assembled in myofibrils and produced mainly fragments of 160 000 and 30 000 daltons in the acidic pH region. This degradation was most intense around pH 4. Degradation of myosin in the isolated state by cathepsin L resulted in the disappearance of the heavy chain and the decrease of light chains 1, 2 and 3, producing fragments of 160 000, 92 000, 83 000 and 60 000 daltons. The degradation of the heavy chain was most severe at pH 4.2. Cathepsin L degraded actin into fragments of 40 000, 37 000 and 30 000 daltons. This action was most intense at pH 4.7. Tropomyosin was not degraded. Troponin T and troponin I were degraded into fragments of 30 000 and 13 000 daltons at pH 3.7-6.7, which were degraded further into smaller fragments. Troponin C was not degraded. α-Actinin was degraded into several fragments, the major one of which showed an M r of 80 000. This degradation was most intense at pH 3.0–3.5.

Degradation of myofibrils and formation of premer omyosin by a neutral protease produced by Pseudomonas fragi

Rabbit muscle myofibrils were treated with a neutral protease (200: 1-protein: enzyme) isolated from Pseudomonas fragi (P. fragi) at 0° C in 100 m m KCl, 5·0 m m CaCl 2, 0·2 m m DTT (dithiothreitol) and 20 m m TRIS. HCl (pH 7·50) for 0–60 min. SDS-polyacrylamide disc gel electrophoresis showed extensive fragmentation of the myofibrillar proteins. Three of the major products correspond in molecular weight to heavy meromyosin (HMM), light meromyosin (LMM) and the recently discovered premeromyosin (PMM). Results demonstrated that the neutral protease produced by P. fragi is capable of hydrolysing myosin and probably plays an important role in microbial breakdown of the muscle proteins during meat spoilage.

Isolation from porcine cardiac muscle of a Ca 2+-activated protease that partially degrades myofibrils

A protein fraction displaying Ca 2+-activated proteolytic activity has been isolated from porcine cardiac muscle. The crude enzyme was purified approximately 2000 fold by isoelectric precipitation followed by gel permeation chromatography and by ion exchange chromatography. The partially purified enzyme exhibited optimal activity against either cardiac myofibril or casein substrates between pH 7.5 and 8.0, and in the presence of 1 m m Ca 2+ and at least 2 m m 2-mercaptoethanol. The enzyme removes Z-discs from skeletal and cardiac myofibrils and also removes the density from intercalated discs of cardiac myofibrils. The enzyme hydrolyzes troponin-T and troponin-I of both cardiac and skeletal muscle myofibrils in vitro. In its proteolytic effect on either cardiac or skeletal myofibrils and in all other properties examined, the Ca 2+-activated, cardiac protease is similar to a Ca 2+-activated protease (CAF) recently purified from porcine skeletal muscle (Dayton, W. R., Reville, W. J., Goll, D. E. and Stromer, M. H. (1976) Biochemistry 15, 2159–2167). It is possible that the Ca 2+-activated, cardiac protease plays a role in degradation of myofibrils in injured myocardial cells.

Degradation of rat cardiac myofibrils and myofibrillar proteins by a myosin-cleaving protease.

The degradation of rat cardiac myofibrils and their constituent proteins with a myosin-cleaving protease was studied. Electrophoretograms of the digestion products of myofibrils showed that myosin,M-protein, C-protein, and troponin were degraded, but actin and tropomyosin were not. Degradation of these constituents resulted in losses of the Mg2+-ATPase activity and its Ca2+-sensitivity of myofibrils. Incubation of myofibrils with the protease induced the release of alpha-actinin without degradation. Susceptibilities of myosin, actin, troponin, and alpha-actinin purified from rat and pig hearts to the protease were essentially identical to those of the assembled forms in myofibrils. Although the purified tropomyosin was readily degraded into five fragments with the protease, the tropomyosin assembled in myofibrils and actin-tropomyosin complex were insusceptible to the protease. Digestion of myosin in the filamentous state with the protease resulted in the disappearance of myosin heavy chain and light chain 2, producing two fragments having molecular weights of 130,000 and 94,000 which originated from the degradation of heavy chain. The Ca2+- and EDTA-ATPase activities of the degradation products remained unchanged during incubation for 22 h. The actin-activated ATPase activity of myosin was reduced by 30% during incubation for 6 h, and recovered to the original level on adding actin to give a ratio of actin to myosin of 2:1. The pH optima for degradation of myosin in the soluble and filamentous states were 8.5 and 7.0, respectively. The results indicate that cardiac myosin in the filamentous state was more readily degraded with the protease than the myosin in the soluble state.

The action of caffeine in promoting ultrastructural damage in frog skeletal muscle fibres

1. Caffeine at concentrations above 5 mM was shown to cause rapidly extensive ultrastructural damage to the myofibrils of frog skeletal muscle. 2. The effect was promoted at lower temperatures, whereas the myofibrils were protected by prior exposure to procaine. 3. It is argued that caffeine causes a Ca2+-induced release of Ca2+ (the CROC) from the S.R. and that the consequent rise in [Ca2+]i promotes the ultrastructural damage observed. 4. Myofibril digradation is also produced by treatment of the muscle with the divalent cation ionophore A23187; this effect is not protected by either procaine or Dantrolene sodium. 5. It is suggested that A23187 causes the release of Ca2+ from the S.R. by a mechanism that differs from both excitation and the CROC; the resultant rise in [Ca2+]i again causes myofibril degradation. 6. The ways in which a marked rise in [Ca2+]i could cause muscle damage and the possible relevance of these findings to the sequence of events in the development of myopathies of human skeletal muscle are discussed.

Ultrastructural changes of the dog myocardium in extracorporeal circulation.

The effects of the cardiopulmonary bypass circulation on the cardiac muscle cells were studied electron microscopically. Subendocardial hemorrhage was commonly recognized in both ventricles. However, it appeared earlier and more severely in the right ventricle than in the left ventricle. Clear flocculent areas containing numerous filamentous structures were originated from degradation of myofibrils. Degradation of myofibrils occurred within 1 hour after bypass circulation. Degenerative changes of myocardial cells such as zonal lesions, contraction bands and swelling or condensation of mitochondria appeared in both ventricles. Damages of myocardial cells in right ventricular wall were more severer as compared with those in the left throughout the period of observation.

A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Subcellular localization of the protease in porcine skeletal muscle.

A study was done to determine whether the Ca2+-activated muscle protease (CAF) that removes Z disks from myofibrils in the presence of Ca2+ is located in a sedimentable subcellular organelle. Porcine skeletal muscle cells were diced finely with a scalpel and were suspended in 0.25 M sucrose, 4 mM EDTA with a VIRTIS homogenizer. Filtration of the suspended muscle through four layers of cheesecloth removed most of the myofibrils and stromal protein. Nuclear (1,000 gavg for 15 min), mitochondrial-microsomal (50,000 gavg for 60 min), and supernatant fractions were assayed for succinic dehydrogenase, acid ribonuclease, cathepsin D, and CAF activities. Approximately 96% of total succinic dehydrogenase activity, 81% of cathepsin D activity, and 45% of acid ribonuclease activity, but only 14% of total CAF activity, were found in the nuclear and mitochondrial-microsomal fractions. Cathepsin D activity in the nuclear and mitochondrial-microsomal fractions was decreased if assays were done without prior treatment to rupture membranous structures; hence, our cell rupture and homogenization procedures preserved some intact lysosomal organelles. The results indicate that the small amount of CAF activity in the nuclear and mitochondrial-microsomal fractions was due to contamination by supernate and that CAF is not located in a membrane-bounded subcellular particle. Because CAF is active at the intracellular pH and temperature of living skeletal muscle cells and is in direct contact with the cytoplasm of muscle cells, its activity must be regulated by intracellular cellular Ca2+ concentration to prevent continuous and indiscriminate degradation of myofibrils.

Proteins released from myofibrils by CASF (Ca2+-activated sarcoplasmic factor) and trypsin.

The nature of the soluble proteins and peptides released from myofibrils by treatment with CASF (Ca2+-activated sarcoplasmic factor) was investigated by using Polyacrylamide gel electrophoresis in both a nondenaturing and a denaturing (sodium dodecyl sulfate=SDS) solvent and by using gel permeation chromatography on Sepharose 6B. Both CASF and trypsin treatment cause removal of Z-disks before causing other ultrastructurally detectable degradation of myofibrils. CASF treatment of myofibrils releases a protein that is identical to α-actinin, one of the known components of the Z-disk, on the basis of mobility in Polyacrylamide gel electrophoresis in a nondenaturing solvent and in SDS and on the basis of elution from gel permeation columns. Trypsin treatment of myofibrils releases a number of smaller molecular weight products that cannot be identified with any of the known myofibrillar proteins. Hence, CASF seems to remove Z-disks from myofibrils by means of a very specific proteolytic activity that releases ...