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PubMed1971

Long-term organ culture of the salamander heart.

Beating salamander hearts were maintained in tissue culture for periods ranging from 1 to 6 months. After 1, 3, or 6 months of culture, six hearts, along with six control hearts, were fixed for electron microscopy. In control tissue, the sarcoplasmic reticulum usually demonstrated the normal pattern of paired, linearly arranged membranes, although in some cases, the reticulum showed a variation from these membranes to a series of small vesicles. There was no evidence of a T-system of tubules in any of the material examined. Desmosome-Z band complexes were observed in almost all sections of both control and experimental material. A possible role of these complexes in the excitation-contraction mechanism is discussed. In 3 month cultured material, alterations in normal myofibrillar pattern occurred. Small segments of myofibrils branched from one Z band to join the Z band of an adjacent myofibril, or appeared to be fraying out into the sarcoplasm. In 6 month cultured material, myofibrils were fragmented into short segments from which myofilaments frayed out into the sarcoplasm. This filamentous material may be remnants of myofilaments. Despite the morphological changes in myofibrils, the heart pulsation rate, established at the beginning, was maintained throughout the culture period. It is suggested that the alterations, observed in the experimental material, occurred in elements not essential for heart beat maintenance, or that these alterations have not yet progressed to a critical point of affecting the heart beat.

Animals↗
PubMed1983

Switching of filamin polypeptides during myogenesis in vitro.

During chicken skeletal myogenesis in vitro, the actin-binding protein filamin is present at first in association with actin filament bundles both in myoblasts and in myotubes early after fusion. Later in mature myotubes it is found in association with myofibril Z disks. These two associations of filamin are separated by a period of several days, during which the protein is absent from the cytoplasm of differentiating myotubes (Gomer, R., and E. Lazarides, 1981, Cell, 23:524-532). To characterize the two classes of filamin polypeptides we have compared, by two-dimensional peptide mapping, 125I-labeled filamin immunoprecipitated from myoblasts and fibroblasts to filamin immunoprecipitated from mature myotubes and adult skeletal myofibrils. Myoblast filamin is highly homologous to fibroblast and purified chicken gizzard filamins. Mature myotube and adult myofibril filamins are highly homologous but exhibit extensive peptide differences with respect to the other three classes of filamin. Comparison of peptide maps from immunoprecipitated 35S-methionine-labeled filamins also shows that fibroblast and myoblast filamins are highly homologous but show substantial peptide differences with respect to mature myotube filamin. Filamins from both mature myotubes and skeletal myofibrils exhibit a slightly higher electrophoretic mobility than gizzard, fibroblast, and myoblast filamins. Short pulse-labeling studies show that mature myotube filamin is synthesized as a lower molecular weight variant and is not derived from a higher molecular weight precursor. These results suggest that myoblast and mature myotube filamins are distinct gene products and that during skeletal myogenesis in vitro one class of filamin polypeptides is replaced by a new class of filamin polypeptides, and that the latter is maintained into adulthood.

Animals↗
PubMed1984

Connectin filaments in stretched skinned fibers of frog skeletal muscle.

Indirect immunofluorescence microscopy of highly stretched skinned frog semi-tendinous muscle fibers revealed that connectin, an elastic protein of muscle, is located in the gap between actin and myosin filaments and also in the region of myosin filaments except in their centers. Electron microscopic observations showed that there were easily recognizable filaments extending from the myosin filaments to the I band region and to Z lines in the myofibrils treated with antiserum against connectin. In thin sections prepared with tannic acid, very thin filaments connected myosin filaments to actin filaments. These filaments were also observed in myofibrils extracted with a modified Hasselbach-Schneider solution (0.6 M KCl, 0.1 M phosphate buffer, pH 6.5, 2 mM ATP, 2 mM MgCl2, and 1 mM EGTA) and with 0.6 M Kl. SDS PAGE revealed that connectin (also called titin) remained in extracted myofibrils. We suggest that connectin filaments play an important role in the generation of tension upon passive stretch. A scheme of the cytoskeletal structure of myofibrils of vertebrate skeletal muscle is presented on the basis of our present information of connectin and intermediate filaments.

Animals↗
PubMed1994

C2C12 cells: biophysical, biochemical, and immunocytochemical properties.

We examined the myofibril biochemical, structural, and biophysical properties of C2C12, a mouse skeletal muscle cell line (American Type Culture Collection), to assess whether force development and the sensitivity of the myofilaments to calcium could be measured in C2C12 myotubes and whether a cardiac contractile protein, troponin T, is expressed and incorporated into C2C12 myofibrils. When myoblasts fused and differentiated into myotubes, expression of myofilament proteins was initiated. Multiple cardiac and skeletal muscle troponin T isoforms were coexpressed. Cardiac troponin T expression increased and then decreased with time. Fluorescence immunocytochemistry demonstrated incorporation of cardiac troponin T isoforms into the myofibrils. At the time of the biophysical studies, mean myotube diameter was 12 microns (range 5-25 microns), and mean length was 290 microns (range 130-520 microns). The estimated maximum force developed by chemically skinned myotubes at 6-7 days poststarvation, 0.88 +/- 0.12 microN (mean +/- 95% confidence interval, n = 5), was significantly less (P < 0.05) than that at 10-13 days poststarvation, 1.12 +/- 0.12 microN (n = 7). The force-pCa relation yielded a Hill coefficient of 2.9 +/- 0.6 (n = 7) and half-maximal activation at pCa of 5.77 +/- 0.20. The demonstration that the biophysical properties of C2C12 cells can be measured and that cardiac and skeletal muscle troponin T isoforms are incorporated and colocalized into myofibrils suggest that these cells could be a useful model to assess the effects of exogenous native and mutated cardiac and skeletal contractile protein isoforms on myofilament function.

Actin Cytoskeleton↗
PubMed1990

Morphological development of the rat heart growing in oculo in the absence of hemodynamic work load.

We evaluated cardiac muscle development in the absence of hemodynamic work load but in the presence of host factors including blood vessels, nerves, and circulating neurohumoral agents by transplanting 12-day fetal rat ventricle into the anterior eye chamber of adult host rats. Implants were studied by electron microscopy at intervals from 1 to 14 weeks in oculo. For comparison with myocardium developing in oculo, 12-day fetal tissue and 3-, 8-, and 28-day-old normally growing rats were also studied. At 1 week in oculo, myofibrils were laterally located and more frequent than in the 12-day fetus. Fibrils had clear Z bands and H bands, but no M bands. At 10 days in oculo (comparable to birth in normally growing animals), myocyte mitoses were present and tritiated thymidine autoradiography revealed many labeled myocyte nuclei. By 5 weeks in oculo, cells were filled with mature myofibrils with clear M bands and lateral connections between adjacent Z bands. However, myofibril bundles sometimes coursed at sharp angles to each other within single cells. Except for the relative lack of fibrillar polarization and small cell size, ultrastructure of myocytes developing in oculo for 5 or more weeks appeared very similar to myocytes developing in normally growing rats. By 10 weeks in oculo, when in situ growing hearts are clearly in a hypertrophic phase of growth, no mitoses or tritiated thymidine-labeled nuclei were present in myocytes, although labeled nonmyocyte nuclei were present. Morphometric evaluation revealed no change in myocyte diameter or nuclear-to-cytoplasmic ratio from 1 to 3 weeks in oculo, consistent with continued hyperplastic growth. Binucleated cells were present by 3 weeks in oculo and later, and the cytoplasm per nucleus increased fourfold between 3 and 5 weeks in oculo, suggesting conversion to hypertrophic cell growth. We concluded that cells proliferated and differentiated in the absence of a hemodynamic load, but that polarized alignment of myocytes and myofibrils was incomplete.

Age Factors↗
PubMed2004

Myofilament anchoring of protein kinase C-epsilon in cardiac myocytes.

Regulatory proteins on muscle filaments are substrates for protein kinase C (PKC) but mechanisms underlying activation and translocation of PKC to this non-membrane compartment are poorly understood. Here we demonstrate that the epsilon isoform of PKC (epsilon-PKC) activated by arachidonic acid (AA) binds reversibly to cardiac myofibrils with an EC(50) of 86 nM. Binding occurred near the Z-lines giving rise to a striated staining pattern. The delta isoform of PKC (delta-PKC) did not bind to cardiac myofibrils regardless of the activator used, and the alpha isoform (alpha-PKC) bound only under strong activating conditions. Three established PKC anchoring proteins, filamentous actin (F-actin), the LIM domain protein Cypher-1, and the coatamer protein beta'-COP were each tested for their involvement in cytoskeletal anchoring. F-actin bound epsilon-PKC selectively over delta-PKC and alpha-PKC, but this interaction was readily distinguishable from cardiac myofilament binding in two ways. First, the F-actin/epsilon-PKC interaction was independent of PKC activation, and second, the synthetic hexapeptide LKKQET derived from the C1 region of epsilon-PKC effectively blocked epsilon-PKC binding to F-actin, but was without effect on its binding to cardiac myofilaments. Involvement of Cypher-1 was ruled out on the basis of its absence from detergent-skinned myofibrils that bound epsilon-PKC, despite its presence in intact cardiac myocytes. The epsilon-PKC translocation inhibitor peptide EAVSLKPT reduced activated epsilon-PKC binding to cardiac myofibrils in a concentration dependent manner, suggesting that a RACK2 or a similar protein plays a role in epsilon-PKC anchoring in cardiac myofilaments.

Actin Cytoskeleton↗
PubMed1993

Titin aggregates associated with intermediate filaments align along stress fiber-like structures during human skeletal muscle cell differentiation.

Differentiating human skeletal muscle cell cultures were used to study the association of titin with other sarcomeric and cytoskeletal proteins during myofibrillogenesis. Several developmental stages of these cultures were double stained with antibodies to titin in combination with antibodies to alpha-actin, alpha-actinin, myosin heavy chain (MHC), nebulin, desmin, and beta-tubulin. The first indications of titin expression were found in postmitotic mononuclear myoblasts where it is located in a random, punctate fashion. At the light microscope level no evidence was found for an association of these titin spots with any of the other proteins studied, with the exception of MHC, which colocalized with titin in a small minority of the titin expressing cells. Subsequently the titin spots were found to be linked to longitudinally oriented stress fiber-like structures (SFLS), containing alpha-actinin and sarcomeric alpha-actin, but not MHC, nebulin or desmin. Upon further maturation titin antibodies seemed to stain SFLS in a rather homogeneous fashion together with MHC, alpha-actin and alpha-actinin. Thereafter a more periodic localization of titin, MHC, alpha-actin and alpha-actinin on SFLS became obvious. From these structures myofibrils developed as a result of further differentiation. Initially only short stretches with a striated titin, MHC, F-actin and alpha-actinin organization were found. Nebulin was integrated in these young myofibrils at a later developmental stage. Desmin was not found to be incorporated in these myofibrils until complete alignment of the sarcomeres in mature myotubes had occurred. At the ultrastructural level titin antibodies recognized aggregates that were associated with intermediate filaments (IF) in postmitotic mononuclear myoblasts. At a later maturational stage, prior to the development of cross-striated myofibrils, the IF-associated titin aggregates were found in close association with subsarcolemmally located SFLS. We conclude that IF and SFLS play an important role in the very early stages of in vitro human myofibrillogenesis. On the basis of our results we assume that titin aggregates are targeted to SFLS through IF. The association of titin with SFLS might be crucial for the unwinding of titin necessary for the assembly of sarcomeres and the first association of titin with other sarcomeric proteins.

Cell Differentiation↗
PubMed1998

Characterizing titin's I-band Ig domain region as an entropic spring.

The poly-immunoglobulin domain region of titin, located within the elastic section of this giant muscle protein, determines the extensibility of relaxed myofibrils mainly at shorter physiological lengths. To elucidate this region's contribution to titin elasticity, we measured the elastic properties of the N-terminal I-band Ig region by using immunofluorescence/immunoelectron microscopy and myofibril mechanics and tried to simulate the results with a model of entropic polymer elasticity. Rat psoas myofibrils were stained with titin-specific antibodies flanking the Ig region at the N terminus and C terminus, respectively, to record the extension behaviour of that titin segment. The segment's end-to-end length increased mainly at small stretch, reaching approximately 90% of the native contour length of the Ig region at a sarcomere length of 2.8 microm. At this extension, the average force per single titin molecule, deduced from the steady-state passive length-tension relation of myofibrils, was approximately 5 or 2.5 pN, depending on whether we assumed a number of 3 or 6 titins per half thick filament. When the force-extension curve constructed for the Ig region was simulated by the wormlike chain model, best fits were obtained for a persistence length, a measure of the chain's bending rigidity, of 21 or 42 nm (for 3 or 6 titins/half thick filament), which correctly reproduced the curve for sarcomere lengths up to 3.4 microm. Systematic deviations between data and fits above that length indicated that forces of >30 pN per titin strand may induce unfolding of Ig modules. We conclude that stretches of at least 5-6 Ig domains, perhaps coinciding with known super repeat patterns of these titin modules in the I-band, may represent the unitary lengths of the wormlike chain. The poly-Ig regions might thus act as compliant entropic springs that determine the minute levels of passive tension at low extensions of a muscle fiber.

Animals↗
PubMed1997

Assembly of titin, myomesin and M-protein into the sarcomeric M band in differentiating human skeletal muscle cells in vitro.

Immunochemical experiments and in vitro binding studies have revealed that titin/connectin, the elastic protein that spans the whole distance of a half-sarcomere, associates with several myosin-binding proteins of the sarcomeric A and M band. Two of these proteins, M-protein and myomesin, anchor titin in the region of the M band. A detailed molecular map describing the arrangement of titin, M-protein and myomesin in this part of the sarcomere was recently proposed. Furthermore, specific binding sites between the molecules were identified. How these polypeptides function in the assembly of the sarcomeric M band region has, however, remained unclear. Here we describe the distribution patterns of different epitopes recognized by newly developed antibodies against the extreme carboxyterminal portion of titin that is anchored in the M band, during the in vitro differentiation of human skeletal muscle cells. In contrast to a set of antibodies directed against Z band, I band and A band titin epitopes, anti-M band titin did not stain titin aggregates or titin in non-striated myofibrils (NSMF). The M band epitopes were only revealed in their characteristic sarcomeric locations, and were obviously not accessible in the non-striated part of nascent myofibrils, or during earlier developmental stages. We speculate that this phenomenon is associated with "immature" tertiary/quarternary structures of M band titin that avoid preliminary binding of M band proteins. In contrast to earlier observations on myofibrillogenesis in the mouse embryo, all the titin epitopes studied were simultaneously detected in their specific positions. Thus, sarcomere assembly in the widely used in vitro model systems seems to proceed at a much higher speed than in vivo. Similarly, myomesin and M-protein were only perceptible in striated myofibrils. While myomesin antibodies stained myofibrils at the time-point of appearance of the first titin striations, the incorporation of M-protein was found to be slightly delayed. In several myotubes no expression of M-protein was observed even during mature stages. These observations indicate its less important general role in the process of myofibrillogenesis. Furthermore, the relative number of M-protein negative myotubes varied in cultures derived from different muscles. This confirms the observation that cultured satellite cells are predestined to form a certain type of myofibers.

Animals↗
PubMed1981

Electron microscopic study of cardiac lesions induced in rats by isoproterenol and by repeated stress. With suggestion that idiopathic cardiomyopathy may be a "disease of adaptation".

An electron microscopic study of cardiac lesions induced in rats by isoproterenol and by repeated stress is described, and a possible pathogenetic mechanism of idiopathic cardiomyopathy is proposed. Three types of cardiac cells could be roughly differentiated according to the structure and arrangement of myofibrils: 1) normo-type with normal structure and normal arrangement of myofibrils, 2) nondestroyed-degenerated-type in which myofibrils show normal structure and abnormal arrangement, and 3) destroyed-degenerated-type with destroyed myofibrils. Various degrees and frequency of mitochondrial changes (accumulation, variation of size, swelling, loss of cristae and myelin figure), increase of glycogen granules, dilation of sarcoplasmic reticulum or T-tubules and nuclear changes were seen in every type. These 3 types seemed to correlate well with the degree of myocardial beta-adrenergic stimulation by isoproterenol or repeated stress. Normo-types were extensively observed in hearts exposed to mild myocardial beta-adrenergic stimulation. Nondestroyed-degenerated-types and destroyed-degenerated-types were frequently observed in hearts exposed to excessive myocardial beta-adrenergic stimulation. These results show a similarity between cardiac lesions induced in rats by isoproterenol or repeated stress and those seen in human idiopathic cardiomyopathy, and suggest the possibility that idiopathic cardiomyopathy is a "Disease of Adaptation."

Adaptation, Physiological↗
PubMed1998

The cytoskeleton in skeletal, cardiac and smooth muscle cells.

The muscle cell cytoskeleton consists of proteins or structures whose primary function is to link, anchor or tether structural components inside the cell. Two important attributes of the cytoskeleton are strength of the various attachments and flexibility to accommodate the changes in cell geometry that occur during contraction. In striated muscle cells, extramyofibrillar and intramyofibrillar domains of the cytoskeleton have been identified. Evidence of the extramyofibrillar cytoskeleton is seen at the cytoplasmic face of the sarcolemma in striated muscle where vinculin- and dystrophin-rich costameres adjacent to sarcomeric Z lines anchor intermediate filaments that span from peripheral myofibrils to the sarcolemma. Intermediate filaments also link Z lines of adjacent myofibrils and may, in some muscles, link successive Z lines within a myofibril at the surface of the myofibril. The intramyofibrillar cytoskeletal domain includes elastic titin filaments from adjacent sarcomeres that are anchored in the Z line and continue through the M line at the center of the sarcomere; inelastic nebulin filaments also anchored in the Z line and co-extensible with thin filaments; the Z line, which also anchors thin filaments from adjacent sarcomeres; and the M line, which forms bridges between the centers of adjacent thick filaments. In smooth muscle, the cytoskeleton includes adherens junctions at the cytoplasmic face of the sarcolemma, which anchor beta-actin filaments and intermediate filaments of the cytoskeleton, and dense bodies in the cytoplasm, which also anchor actin filaments and intermediate filaments and which may be the interface between cytoskeletal and contractile elements.

Animals↗
PubMed1993

Scanning electron microscopy of tenotomized soleus muscles of the rat.

The effects of tenotomy on the surface morphology of muscle fibers including myotendinous junctions in the rat soleus muscle were studied by scanning electron microscopy (SEM). Using potassium hydroxide (KOH) and collagenase, the extracellular materials were successfully removed to expose the surface of muscle fibers. When the soleus muscle was tenotomized at both proximal and distal ends, virtually all muscle fibers showed marked alterations of the fiber surface characterized by the formation of numerous transverse grooves and folds along their length. Narrow longitudinal grooves and folds of the sarcolemma were also observed. At myotendinous junctions, the fiber ends showed an over-all rounded shape with several short sarcoplasmic processes, indicating that the processes were significantly retracted. These changes were clearly recognizable at 5 days after tenotomy, and most apparent at one week. Thin-section electron microscopy of the same SEM samples demonstrated that such folding of the sarcolemma was not directly related to the sarcomere pattern of the underlying myofibrils, suggesting that, once formed, the folds and grooves were retained for a certain period of time. At 2 and 3 weeks the surface morphology of the fibers underwent a recovery process of restoring the smooth surface on which the cross-striations of the underlying myofibrils were seen. At the fiber ends, sarcoplasmic processes regrew into slender, wavy and short forms. Such sarcoplasmic processes were greater in number and more elaborate than those in the control muscle. At 5 and 6 weeks the fiber surface resumed an almost normal morphology, except that the sarcoplasmic processes at the fiber end were still shorter and more numerous than those in the control. These observations support our previous results obtained by thin-section electron microscopy that the myotendinous junction undergoes a series of morphological changes of collapse and regrowth of the sarcoplasmic processes, reflecting changes in the underlying myofibrils. In conclusion, the changes in the surface morphology of tenotomized muscle fibers were well correlated chronologically to those of myofibrils such as the central core lesion.

Animals↗
PubMed2000

The ultrastructure of skeletal and smooth muscle in experimental protein malnutrition in rats fed a low protein diet.

Light microscopy of the pectoralis muscle of rats on a low protein diet did not show such morphological alterations as atrophy, degeneration, or sarcoplasmic edema, but electron microscopy occasionally demonstrated ultrastructural changes only in the sarcomeres of myofibrils. In the affected sarcomeres, the Z-line was disrupted and often showed a jagged structure. The Z-substance with electron opacity was frequently present flowing along the long axis of myofibrils, here referred to as the streaming of Z-lines. In addition, regular striations formed by the reciprocal arrangement of thick and thin filaments disappeared from the affected sarcomeres, though these filaments were still discernible. Two or more consecutive sarcomeres in a single myofibril were occasionally involved in these changes. A further two or more neighboring sarcomeres at the same level of myofibrils were affected transversely by these structural alterations. On the other hand, the ultrastructure of the intestinal smooth muscle was not affected by protein deficiency. The study suggests that the ultrastructural damage induced by a low protein diet is attributed to the activation of endogenous protease by the excess leaking of Ca2+ into the cytosol as a result of lipid peroxidation of cell membrane by raised free radicals, owing to the depletion of glutathione production by protein deficiency. It also suggests that the smooth muscle cells differ in their susceptibility to protein deficiency from the skeletal muscle cells.

Animals↗
PubMed2001

Morphological changes and recovery process in the tenotomized soleus muscles of the rat.

Tenotomized soleus muscles of adult rats were analyzed morphologically and biochemically with special reference to the recovery process. Light microscopic observations of semi-thin sections showed that the characteristic central core lesion was most extensive at 1 week after tenotomy and began to diminish in extent at 2 weeks until no trace of lesion could be seen by 6th week, as confirmed by thin-section electron microscopy. Three phases of changes in the cross-sectional area of muscle fibers after tenotomy were demonstrated by morphometry: phase I designated as the initial increase up to the 3rd day, phase II as the progressive decrease until the 4th week, and phase III as the recovery to normal or even hypertrophy. In electron microscopy, the earliest alteration of myofibrils was recognized at 3 days after tenotomy. The Z discs showed a wavy or zigzag profile with frequent longitudinal splitting of myofibrils. From the 2nd week on, muscle fibers underwent a process of recovery, replacing the central core lesion with new myofibrils in which a reassembly of thick filaments into bundles of thin filaments took place, with Z discs being aligned adjacent to the peripheral complete myofibrils. In SDS-polyacrylamide gel electrophoresis, the molar ratio of myosin to actin diminished markedly as the central core lesion developed and gradually returned to normal with time, correlating well with the loss and subsequent reassembly of thick filaments.

Actins↗
PubMed1987

Accumulation of newly synthesized myosin heavy chain during thyroxine-induced myofiber type transition.

Slow-tonic myofiber to fast-twitch myofiber conversion was induced in chickens by feeding thyroxine. Incorporation of newly synthesized myosin heavy chain (MHC) into myofibers and myofibrils was followed by immunofluorescence with antibodies specific for fast-twitch MHC and slow-tonic MHC. Presence of more than one type of myosin heavy chain was detected in thyroxine-induced transitional myofibers of chicken pectoralis. Myofibers undergoing a transition were histochemically identical to immunologically cross-reacting, transitional myofibers of normal anterior latissimus dorsi. Newly synthesized MHC appeared to be incorporated uniformly across the cross sectional area of transitional myofibers and incorporated homogeneously into each sarcomere of transitional myofibrils. These observations are consistent with a theory of continuous protein exchange between myofibrillar protein and a non-myofibrillar protein pool, such that every sarcomere of a myofibril, and every myofibril of a myofiber, would be turned over simultaneously.

Animals↗
PubMed1998

Ultrastructure of the atrioventricular junctional area in the heart of Molossus molossus Pallas 1766 (Chiroptera: Molossidae).

The atrioventricular junctional area (AVJA) consists of a group of structures that connects the atrial and ventricular myocardium. Five hearts of an insect-eating bat were studied in light and transmission electron microscopy. In M. molossus, the AVJA consists in a mass of muscle fibers intermingled with variable amount of connective tissue and blood vessels surrounded by the adjacent myocardium and the attachment of the right atrioventricular and aortic valves in the fibrous skeleton. In light microscopy, conducting cells of the AV node and bundle can be distinguished from working cells: smaller size, paler staining reaction and the presence of e sheath of connective tissue surrounding each cell (largely composition by type I collagen fibers). Three cell types are observed in the AVJA. Nodal cells are irregular with few cytoplasmic organelles and several slender sarcolemmal modifications. Myofibrils are sparse and not clearly observable. Transitional cells are spindle-shaped and grouped together into bundles. The cytoplasm, poor in glycogen, has scarce electron-density and myofibrils organized into sarcomeres. Caveolae is observed randomly distributed at the periphery of the cell. The AV bundle cells are elongated with clusters of myofibrils organized in the periphery and a glycogen free area around the nucleus. Ventricular cells are bigger than the atrial ones and show well-developed myofibrils in alternated rows with mitochondria. Lipid droplets are seen near mitochondria and glycogen granules. Intercalated discs and T-tubules are found in working cells but not in conducting ones. The fibrous skeleton has collagen fibers intercalated with fibroblasts.

Animals↗
PubMed1975

Morphometric and autoradiographic study of developing ventricular and atrial myocardium in fetal rats.

The ventricular and atrial myocardia of 19- to 21-day-old rat fetuses have been studied by electron microscopic morphometry and quantitative autoradiography following the injection of 3H-leucine. The myocytes in both these regions contain numerous myofibrils located predominantly near the sarcolemma and oriented parallel to each other and to the long axis of the cell. The width of myofibrils at this stage of development is 0.428 +/- 0.240 mum. in the ventricle and 0.365 +/- 0.185 mum. in the atrium. The volume fractions of myofibrils, mitochondria, smooth endoplasmic reticulum, sarcoplasmic matrix, and sarcolemma in ventricular myocytes are nearly equal to the corresponding fractions in atrial cells. A similarity between ventricular and atrial myocytes was also found in the distribution of newly synthesized proteins as indicated autoradiographically by the concentration of grains over various sarcoplasmic structures. In both tissues the concentration of grains was consistently above average over the sarcolemma, smooth endoplasmic reticulum, and myofibrils; average over the mitochondria; and below average in association with the sarcoplasmic matrix. The specificity of this autoradiographic procedure for labeled amino acids actually incorporated into protein was tested biochemically, and it was found that glutaraldehyde-paraformaldehyde fixation leads to a negligible contamination with non-specifically bound leucine. It is suggested that the high concentration of protein labeling near the sarcolemma is related to the imminent development of the T-system and its associated channels of sarcoplasmic reticulum.

Animals↗
PubMed1992

Proteolytic and physicochemical mechanisms involved in meat texture development.

Development in meat texture is a complex process originating very likely from a softening of the structural elements, especially myofibrils. This process probably involves two sets of mechanisms: 1) an enzymatic mechanism involving at least two of the three proteolytic systems so far identified and present in this tissue, namely lysosomal (cathepsins) and calcium dependent (calpains) proteinases; 2) a physicochemical mechanism based on the important post mortem rise in muscle osmotic pressure which could be twice as high as in live animals. Despite the large progress in muscle enzymology, the nature of the proteinases responsible for the post mortem proteolysis associated with the development in meat texture is still not clearly established. In the present review, data obtained from two different approaches attempting to answer this question were analysed. The first one was based on the identification of a set of structural and biochemical changes associated with meat texture development and to examine which proteolytic system or proteinase would be able to reproduce them when incubated with either myofibrils or muscle fibres as substrate. The second tentatively relates the rate and the extent of the changes in meat texture to the proteolytic equipment of the tissue. The first approach led to the conclusion that changes in muscle proteins and structure can be only explained by considering a synergistic action of both lysosomal and calcium-dependent proteinases. From the second, it was concluded that the process of meat texture development did not depend on the proteinase levels but was related to their initial potential efficiency assessed by measurement of the enzyme/inhibitor ratio. With respect to the physicochemical mechanisms, the post mortem rise in muscle osmotic pressure was shown to be responsible for some biochemical changes occurring in myofibrils. This was further substantiated by the fact that the greatest osmotic pressure values were observed in muscles exhibiting highest tenderising rate. On the other hand we provide evidence suggesting that the substrate, namely myofibrils, might constitute an important limiting step of the efficiency of both types of mechanism. Taken together, the findings presented emphasize that improvement of our knowledge in this field will greatly depend on the development of basic research on these different topics notably: 1) the mechanisms by which proteinases activities are regulated in living and post mortem muscles; and 2) the myofibrillar structure, especially in slow-twitch or type I muscles.

Adenosine Triphosphatases↗