Biomedical subjects

K Trombitás

Publications and source records attributed to K Trombitás.

At least 37 records · Page 2Linked to original sources

PubMed1997

Interaction between titin and thin filaments in intact cardiac muscle.

A 'freeze break' technique and immunoelectron microscopy were used to study the elastic properties of cardiac titin filaments. Small bundles consisting of a few fibres from freshly prepared dog papillary muscle were quickly frozen and broken under liquid nitrogen to fracture sarcomeres in planes perpendicular to the filament axes. Breaks occurred at each of several regions along the sarcomeres. The still-frozen specimens were thawed during fixation to allow elastic filaments to retract. The broken muscle segments were then treated with monoclonal titin antibody 9D10 which labelled a unique epitope in the I-band. In sarcomeres broken at the A-I junction, the titin filaments reacted toward the Z-line, independently of the thin filaments. The retracted epitopes did not reach the Z-line; retraction stopped at the N1-line level. In sarcomeres broken near the Z-line, the titin filaments retracted in the opposite direction, to the tip of the thick filaments. When the break occurred in the A-band, by contrast, the titin-epitope position was unaffected. On the basis of these results, and despite the reported interaction of titin and actin in vitro, it appears that cardiac titin molecules form elastic filaments that are functionally independent of the thin filaments. Near the Z-line, however, the titin filaments seem to associate firmly with the thin filaments.

Actins↗

PubMed1997

Valproate treatment induces lipid globule accumulation with ultrastructural abnormalities of mitochondria in skeletal muscle.

In skeletal muscle of seven children treated with valproic acid (VPA) microvesicular (0.05-2 microns) lipid droplets were observed between the myofibrils predominantly adjacent to the mitochondria. Most of the mitochondria showed ultrastructural abnormalities: several mitochondria were deformed and in several mitochondria abnormal configuration of cristae was observed. The size of some organelles reached the 10-20 microns length with normal diameter. The elongated mitochondria were located either in the long axis of the fibers or in the transverse direction near to the Z discs. In rats receiving valproic acid intraperitoneally for 14 days in daily dose of 100 mg/kg, steatosis developed in the liver, and focal lipid droplets could also be found in the skeletal muscle. The mitochondria of the rat liver appeared as normal, but in muscle the structure and organization of the cristae were disoriented. These observations show that treatment with valproate causes a kind of drug-induced mitochondrial cytopathy with microvesicular lipid deposition in the muscle. The lipid deposits are likely a result of the inhibited mitochondrial fatty acid oxidation. The formation of the abnormal mitochondrial structure could serve as background for the visible consequences of affected lipid metabolism.

Animals↗

PubMed1997

Actin removal from cardiac myocytes shows that near Z line titin attaches to actin while under tension.

The I band of cardiac sarcomeres contains both actin and titin/connectin filaments. Earlier work has suggested that titin binds to actin in situ. This interaction must be weak in the region of the I band where titin behaves elastically. On the other hand, titin may bind strongly to actin in the approximately 100-nm-wide region adjoining the Z line, where titin has been found to be inelastic. To study the putative interaction between titin and actin, techniques for selective removal of actin from different regions of the I band are needed. Here we report studies with a gelsolin fragment (FX-45) and extract actin from rat cardiac myocytes. Actin extraction was biphasic: the majority of actin was extracted in approximately 10 min, whereas actin near the Z line (where titin is inelastic) required a approximately 10-fold longer extraction time. Thus, by controlling the extraction time, we could remove either the full actin filament outside the Z line or just the segment of the actin filament that extends beyond the inelastic region of titin that adjoins the Z line. The actin filament-free I band contained titin filaments, typically with one filament extending from each thick filament. In addition, we observed a dark transverse line (junction line), the location of which in the sarcomere varied linearly with sarcomere length. The position in the sarcomere of the junction line coincided with the binding site of the anti-titin antibody 9D10. Actin removal significantly affected the slack sarcomere length. Slack sarcomere length was 1.85 +/- 0.04 microns in control cells and decreased to 1.71 +/- 0.05 microns after actin near the Z line was extracted. This length reduction may be caused by contraction of the titin segment that becomes exposed after actin removal near the Z line, indicating that titin is not only attached to the actin filament but is also under tension.

Actins↗

PubMed1996

Nonuniform elasticity of titin in cardiac myocytes: a study using immunoelectron microscopy and cellular mechanics.

Titin (also known as connectin) is a muscle-specific giant protein found inside the sarcomere, spanning from the Z-line to the M-line. The I-band segment of titin is considered to function as a molecular spring that develops tension when sarcomeres are stretched (passive tension). Recent studies on skeletal muscle indicate that it is not the entire I-band segment of titin that behaves as a spring; some sections are inelastic and do not take part in the development of passive tension. To better understand the mechanism of passive tension development in the heart, where passive tension plays an essential role in the pumping function, we investigated titin's elastic segment in cardiac myocytes using structural and mechanical techniques. Single cardiac myocytes were stretched by various amounts and then immunolabeled and processed for electron microscopy in the stretched state. Monoclonal antibodies that recognize different titin epitopes were used, and the locations of the titin epitopes in the sarcomere were studied as a function of sarcomere length. We found that only a small region of the I-band segment of titin is elastic; its contour length is estimated at approximately 75 nm, which is only approximately 40% of the total I-band segment of titin. Passive tension measurements indicated that the fundamental determinant of how much passive tension the heart develops is the strain of titin's elastic segment. Furthermore, we found evidence that in sarcomeres that are slack (length, approximately 1.85 microns) the elastic titin segment is highly folded on top of itself. Based on the data, we propose a two-stage mechanism of passive tension development in the heart, in which, between sarcomere lengths of approximately 1.85 microns and approximately 2.0 microns, titin's elastic segment straightens and, at lengths longer than approximately 2.0 microns, the molecular domains that make up titin's elastic segment unravel. Sarcomere shortening to lengths below slack (approximately 1.85 microns) also results in straightening of the elastic titin segment, giving rise to a force that opposes shortening and that tends to bring sarcomeres back to their slack length.

Animals↗

PubMed1996

Titin develops restoring force in rat cardiac myocytes.

When relaxed after contraction, isolated cardiac myocytes quickly relengthen back to their slack length. The molecular basis of the force that underlies passive relengthening, known as restoring force, is not well understood. In a previous study of titin's elasticity in cardiac myocytes, we proposed that titin/connectin develops restoring force, in addition to passive force. This study tested whether titin indeed contributes to the restoring force in cardiac myocytes. Skinned rat cardiac myocytes in suspension were shortened by approximately 20%, using Ca(2+)-independent shortening, followed by relaxation. Cells were observed to relengthen until they reached their original slack sarcomere length. However, the ability to relengthen was abolished after cells had been treated for 12 minutes with trypsin (0.25 microgram/mL, 20 degrees C). Gel electrophoresis showed that this treatment had degraded titin without clearly affecting other proteins, and immunoelectron microscopy revealed that the elastic segment of titin in the I band was missing from the sarcomere. Restoring force was also directly measured, before and after trypsin treatment. Restoring force of control cells was -61 +/- 20 micrograms (per cell) at a sarcomere length of 1.70 microns. Comparison of our results with those of activated trabeculae indicated that a large fraction of restoring force of cardiac muscle originates from within the myocyte. Restoring force of myocytes was found to be depressed after titin had been degraded with trypsin. We conclude that cardiac, titin indeed develops restoring force in shortened cardiac myocytes, in addition to passive force in stretched cells, and that titin functions as a bidirectional spring. Our work suggests that at the level of the whole heart, part of the actomyosin-based active force that is developed during systole is harnessed by titin, allowing for elastic diastolic recoil and aiding in ventricular filling.

Animals↗

PubMed1995

[Mitochondrial DNA deletion in hereditary cardio-encephalo-myopathy].

The case of a female patient with cardio-encephalo-myopathy who died of her illness at one year of age, similarly to her three sisters, is reported. In autopsy samples, like muscle, heart, liver and cerebellum activities of several mitochondrial enzymes were determined. In the skeletal muscle serious decrease of carnitine acetyltransferase was observed (from the normal 4.8 U/g to 0.08 U/g wet weight), while in other tissues this activity was normal. In the muscle activities of several other mitochondrial enzymes were also decreased (cytochrome oxidase, NADH cytochrome C oxidoreductase, citrate synthase), while in other tissues there were no similar changes. Serious distortion was observed in the structure of the majority of mitochondria of muscle and heart by electronmicroscopy. The number of the Purkinje-cells in the cerebellum decreased, and the cells were shrunken, their axons were fragmented and disoriented. Also the structure of the mitochondria was abnormal in the Purkinje-cells, while it was normal in other areas of the cerebrum. In te tissues of the patient normal and deleted mitochondrial DNA coexisted as which could explain the genetic background of this disease at molecular level.

Autopsy↗

PubMed1995

Actin filaments in honeybee-flight muscle move collectively.

To investigate the pattern of actin-filament translation in the intact myofibrillar matrix, we carried out electron micrographic experiments on the "rigor-stretch" model of insect-flight muscle. In this model, thin filaments are mechanically severed from their connections to the Z-line and may then slide freely over the myosin filaments when activated. The model is similar to the in vitro motility assay in that untethered actin filaments slide over myosin, but here the natural filament lattice is retained: sliding takes place through the lattice of thick filaments. We find, in this model, that while the extent of thin filament translation is variable from sarcomere to sarcomere, filaments never translate far enough to enter the opposite I-band. Unlike the in vitro motility assay, where the actin filament translates over the entire thick filament even with "incorrectly" polarized cross-bridges as the sole driver, in this intact filament-lattice model, cross-bridges are apparently unable to move filaments in both directions. We also find that the pattern of filament translation is collective. Although the extent of translation may vary among sarcomeres, in any given half-sarcomere all actin filaments translate by the same degree. Further, the extent of translation is the same in both halves of a given sarcomere. In rare instances where the extent of translation exhibited a transverse gradient across the myofibrillar half-sarcomere, the gradient was similar on both sides of the sarcomere. Filament translation within the sarcomere is thus collective. Some mechanism ensures that nearby but distinctly separated actin filaments move together and that cooperative-like behavior therefore extends to the supramolecular level.

Actin Cytoskeleton↗

PubMed1995

Visualization of the transverse cytoskeletal network in insect-flight muscle by scanning-electron microscopy.

Located at the level of the Z-line, the transverse cytoskeletal network of insect-flight muscle interconnects adjacent myofibrils with one another, and interconnects peripheral myofibrils with the cell membrane. This network has been presumed to keep myofibrils in register, or to distribute tension laterally among myofibrils. In this study, we used scanning-electron microscopy to reveal details of the three-dimensional arrangement of this network. The network is seen to interconnect longitudinal elements of the cytoskeletal network which surround each myofibril. The arrangement is not unlike that seen in vertebrate skeletal muscle. Interestingly, the transverse network makes contact with cell components such as dense bodies and mitochondria. Such contacts imply potential roles over and above those noted above. The network may be involved not only in mechanical function, but possibly also in intracellular communication.

Animals↗

PubMed1995

The mechanically active domain of titin in cardiac muscle.

One of the main contributors to passive tension of the myocardium is titin. However, it is not exactly known what portions of this approximately 1 micron-long molecule are anchored in the sarcomere (hence, are rendered inelastic) and what portions are elastic (hence, are mechanically active in developing passive tension). We assessed the length of the elastic domain of cardiac titin by ultrastructural and mechanical methods. Single cardiac myocytes were stretched by various amounts, and while in the stretched state, they were processed for immunoelectron microscopy. Several monoclonal anti-titin antibodies were used, and the locations of the titin epitopes in the sarcomere were studied as a function of sarcomere length. Only a small fraction (5% to 10%) of the approximately 1000-nm-long molecule behaved elastically under physiological conditions. This mechanically active domain is located close to the A/I junction, and its contour length when unstretched is estimated at approximately 50 to 100 nm. In sarcomeres that are slack (length approximately 1.85 microns), the mechanically active domain is folded on top of itself, and the length of the domain reaches an elastic limit of approximately 550 nm in sarcomeres that are approximately 2.9 microns long.

Animals↗

PubMed1993

Elastic properties of titin filaments demonstrated using a "freeze-break" technique.

A "freeze-break" technique (Trombitás, K.: Acta Biochim. Biophys. Hung. 6:419-427, 1971) and immunoelectron microscopy were used to study the elastic properties of titin filaments. Small bundles of freshly prepared rabbit psoas muscle fibers were quickly frozen and broken under liquid nitrogen to fracture sarcomeres in planes perpendicular to the filament axis, in each of various regions along the sarcomere. The still-frozen specimens were thawed during fixation to allow elastic filaments to retract. The broken specimens were then labelled with monoclonal anti-titin antibodies against an unique epitope in the I-band. The titin epitopes were normally positioned symmetrically about the Z-line. However, in sarcomeres broken at the A-I junction, the epitopes no longer remained symmetrical: the titin filaments in the broken half-sarcomere retracted, independently of the thin filaments, forming a dense band just near the Z-line. The retracted density apparently did not reach the Z-line; retraction stopped at the level of the so-called N1-line. In sarcomeres broken at the Z-line level, the titin filaments retracted in the opposite direction. In this case the titin epitope retracted all the way to the ends of the thick filaments. It appears then that titin molecules form elastic filaments that are independent of thin filaments in most of the I-band. Near the Z-line, however, the titin filaments either have an inelastic domain or associate firmly with the thin filaments at the N1-line level.

Actin Cytoskeleton↗

PubMed1993

Shortening-induced tension enhancement: implication for length-tension relations.

Length-tension relations come in two types--the classical type with linearly descending limb, and the "flat," higher type. The classical type, now confirmed in several laboratories, is obtained when sarcomeres are servo-controlled to maintain constant length. The flat type, also confirmed in several laboratories, is obtained in fixed-end contractions, where some sarcomeres have the opportunity to shorten at least slightly. We find that the higher tensions seen in the flat type are indeed the result of very small shortening: when isometric sarcomeres shorten to a slightly shorter (e.g., by 40 nm) length, they go on to produce considerably more isometric tension than if they had remained at the shorter length throughout contraction. We term this phenomenon "shortening-induced tension enhancement." The phenomenon accounts not only for the higher, flatter length-tension relation seen in fixed-end contractions but can explain the creep of tension that occurs in extended tetani. Thus, several issues concerning the interpretation of length-tension relations are resolved by this newly discovered phenomenon.

Animals↗

PubMed1993

Elastic properties of connecting filaments along the sarcomere.

The elasticity of the connecting filament--the filament that anchors the thick filament to the Z-line--has been investigated using rigor release, freeze-break and immunolabelling techniques. When relaxed insect flight muscle was stretched and then allowed to go into rigor, then released, the recoil forces of the connecting filaments caused sarcomeres to shorten. Thin filaments, prevented from sliding by rigor links, were found crumpled against the Z-line. Thus, rigor release experiments demonstrate the spring-like nature of the connecting filaments in insect flight muscle. In vertebrate skeletal muscle, however, the same protocol did not result in sarcomere shortening. Absence of shortening was due to either smaller stiffness of connecting filaments and/or higher stiffness of the thin filaments relative to insect flight muscle. The spring-like nature of the connecting filament was confirmed with the freeze break technique. When the frozen sarcomeres were broken along the A-I junction, the broken connecting filaments retracted to the N1-line level, independently of the thin filaments, demonstrating the basic elastic nature of these filaments. To study the elastic properties of the connecting filaments along the sarcomere, the muscle was labelled with monoclonal antibodies against a titin epitope near the N1-line, and another very near the A-I junction in the I-band. Before labelling, fibers were pre-stretched to varying extents.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗

PubMed1993

Filament lengths in frog semitendinosus and tibialis anterior muscle fibres.

In frog semitendinosus muscle the descending limb of the length-tension curve is shifted rightward relative to that of tibialis anterior. Both the plateau right corner and the zero-force intercept are equally shifted. To investigate the reason for this shift, we compared filament lengths in the two muscles. Single fibres were mechanically skinned, stretched to reveal filaments clearly, incubated in a solution containing one of several antibodies to enhance filament visualization, and examined by electron microscopy. We found no differences of filament length. Thick filament lengths were 1.62 and 1.61 microns, respectively. I-segment lengths were measured by two methods. With the first, filament length was the same for both muscles, 1.95 or 1.98 microns, depending on the value taken for the troponin repeat; with the second it was 1.92 and 1.94 microns, respectively, for the two muscles. These differences are insignificant. Thus, the reported differences of shape of the length-tension curve are not explainable in terms of differences of filament length.

Actin Cytoskeleton↗

PubMed1993

Elastic properties of the titin filament in the Z-line region of vertebrate striated muscle.

The characteristics of the titin filament in the vicinity of the Z-line were investigated using immunoelectron microscopy. We used monoclonal titin antibodies T-11 and T-12 on single fibres of frog skeletal muscle, and on Z-line-extracted fibres. It is well established that the I-band region of titin is elastic. We find, however, that the elastic properties are not uniform. The T-12 epitope, which binds near the Z-line at the N1-line level, hardly changes position relative to the Z-line as the sarcomere is stretched. This demonstrates the functional inextensibility of the N1-Z-line region. After extreme stretch (above 6-microns sarcomere length), this zone finally does elongate; thus, the titin molecule in this region is intrinsically elastic. The functional inextensibility seen at shorter sarcomere lengths may, therefore, be a result of binding of titin to the actin filament in the zone near the Z-line. When the Z-line was extracted, the T-12 epitope remained in the same position as in the unextracted fibres; it did not retract from the Z-line. Failure to retract implies that functional anchoring of titin is not exclusive to the Z-line, but includes some site closer to the A-band. Combined with the results of the above-mentioned stretch experiment, this result implies a likely binding of titin to the thin filament either focally at the N1 line or all along the entire N1-Z region. Thus, this region of titin is functionally stiff, but intrinsically elastic.

Animals↗

PubMed1993

Contraction-induced movements of water in single fibres of frog skeletal muscle.

Although X-ray diffraction measurements imply almost constant filament separation during isometric contraction, such constancy does not hold at the level of the isolated cell; cell cross-section increases substantially during isometric contraction. This expansion could arise from accumulation of water drawn from other fibre regions, or from water drawn into the cell from outside. To distinguish between these hypotheses, we froze single fibres of frog skeletal muscle that were jacketed by a thin layer of water. Frozen fibres were freeze-substituted, sectioned transversely, and examined in the electron microscope. In fibres frozen during contraction, we found large amounts of water just beneath the sarcolemma, less in deeper regions, and almost none in the fibre core. Such gradients were absent or diminished in fibres frozen in the relaxed state. The water was not confined to the myofibril space alone; we found large water spaces between myofibrils, particularly near mitochondria. Accumulation of water between myofibrils and around mitochondria implies that the driving force for water movement probably lies outside the filament lattice, and may therefore be osmotic. The fact that the distribution was nonuniform-highest near the sarcolemma and lowest in the core--implies that the water was likely drawn from the thin jacket surrounding the cell. Thus, the contractile cycle appears to be associated with water entry into and exit from the cell.

Animals↗

PubMed1993

[Questions concerning two-stage reconstruction of injured flexor tendons. III. Ultrastructure of the tenosynovium in the pseudo-tendon sheath created by using a silicone rod].

Authors have investigated the ultrastructure of the pseudo tendon sheath, formed with silicon rod and man. They have observed a superficial structure, resembling the normal tendon sheath in scanning electron microscopic examination. With transmission electron microscopy phagocyte "A" type and secretion "B" type synovial cells were found. Authors state that the newly formed tenosynovium has an important role in the nutrition of the tendon graft and the prevention of adhesions.

Animals↗

PubMed1991

Nature and origin of gap filaments in striated muscle.

Immunoelectron microscopy was used to study the nature and origin of 'gap' filaments in frog semitendinosus muscle. Gap filaments are fine longitudinal filaments observable only in sarcomeres stretched beyond thick/thin filament overlap: they occupy the gap between the tips of thick and thin filaments. To test whether the gap filaments are part of the titin-filament system, we employed monoclonal antibodies to titin (T-11, Sigma) and observed the location of the epitope at a series of sarcomere lengths. At resting sarcomere length, the epitope was positioned in the I-band approximately 50 nm beyond the apparent ends of the thick filament. The location did not change perceptibly with increasing sarcomere length up to 3.6 microns. Above 3.6 microns, the span between the epitope and the end of the A-band abruptly increased, and above 4 microns, the antibodies could be seen to decorate the gap filaments. Between 5 and 6 microns, the epitope remained approximately in the middle of the gap. Even with this high degree of stretch, the label remained more or less aligned across the myofibril. The abrupt increase of span beyond 3.6 microns implies that the A-band domain of titin is pulled free of its anchor points along the thick filament, and moves toward the gap. Although this domain is functionally inextensible at physiological sarcomere length, the epitope movement in extremely stretched muscle shows that it is intrinsically elastic. Thus, the evidence confirms that gap filaments are clearly part of the titin-filament system. They are derived not only from the I-band domain of titin, but also from its A-band domain.

Animals↗

PubMed1991

Results of ultrastructural analysis of the calf muscles in clubfoot.

Open biopsies were carried out on the muscles of 23 clubfoot patients, ages 9 months to 4 years. Electron microscopic analyses of the so-called "clubfoot muscles" and the peroneal muscles were performed. The changes found were not present in every area of the muscles, but were surrounded by fields of normal structure. Fatty degeneration with fibrosis was observed as the consequence of immobilization. More marked loss was found in the contractile elements. The authors believe that neuromuscular atrophy is a primary cause of congenital clubfoot. The most seriously affected muscles in this study were the tibialis posterior and peroneal muscles. The material failed to prove correlation with age. Based on their observations, the authors suggest finishing all types of immobilization before 1 year of age, when children begin to walk.

Actin Cytoskeleton↗