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PubMed2003

Aberrant myofibril assembly in tropomodulin1 null mice leads to aborted heart development and embryonic lethality.

Tropomodulin1 (Tmod1) caps thin filament pointed ends in striated muscle, where it controls filament lengths by regulating actin dynamics. Here, we investigated myofibril assembly and heart development in a Tmod1 knockout mouse. In the absence of Tmod1, embryonic development appeared normal up to embryonic day (E) 8.5. By E9.5, heart defects were evident, including aborted development of the myocardium and inability to pump, leading to embryonic lethality by E10.5. Confocal microscopy of hearts of E8-8.5 Tmod1 null embryos revealed structures resembling nascent myofibrils with continuous F-actin staining and periodic dots of alpha-actinin, indicating that I-Z-I complexes assembled in the absence of Tmod1. Myomesin, a thick filament component, was also assembled normally along these structures, indicating that thick filament assembly is independent of Tmod1. However, myofibrils did not become striated, and gaps in F-actin staining (H zones) were never observed. We conclude that Tmod1 is required for regulation of actin filament lengths and myofibril maturation; this is critical for heart morphogenesis during embryonic development.

Actinin↗
PubMed1999

Assembly of thick filaments and myofibrils occurs in the absence of the myosin head.

We investigated the importance of the myosin head in thick filament formation and myofibrillogenesis by generating transgenic Drosophila lines expressing either an embryonic or an adult isoform of the myosin rod in their indirect flight muscles. The headless myosin molecules retain the regulatory light-chain binding site, the alpha-helical rod and the C-terminal tailpiece. Both isoforms of headless myosin co-assemble with endogenous full-length myosin in wild-type muscle cells. However, rod polypeptides interfere with muscle function and cause a flightless phenotype. Electron microscopy demonstrates that this results from an antimorphic effect upon myofibril assembly. Thick filaments assemble when the myosin rod is expressed in mutant indirect flight muscles where no full-length myosin heavy chain is produced. These filaments show the characteristic hollow cross-section observed in wild type. The headless thick filaments can assemble with thin filaments into hexagonally packed arrays resembling normal myofibrils. However, thick filament length as well as sarcomere length and myofibril shape are abnormal. Therefore, thick filament assembly and many aspects of myofibrillogenesis are independent of the myosin head and these processes are regulated by the myosin rod and tailpiece. However, interaction of the myosin head with other myofibrillar components is necessary for defining filament length and myofibril dimensions.

Animals↗
PubMed2002

Changes in thiol reactivity and extractability of myofibril bound cardiac troponin C in porcine malignant hyperthermia.

We recently showed that a shift ( approximately 44%) in cardiac myosin isozyme from V3 to V1 occurs in the hearts of malignant hyperthermia (MH)-susceptible pigs and may be important in the disease. To follow up this finding, we investigated whether this myosin isozyme shift results in conformational changes in cardiac troponin C (cTnC). The two cysteine residues (Cys-35 and Cys-84) in the regulatory domain of cTnC make it possible to attach conformational probes to this region. Incorporation of a fluorescent probe, 7-diethylamino-3-(4'maleimidylphenyl)-4-methylcoumarin (CPM), into myofibril-bound cTnC was measured by alkaline urea gel electrophoresis, followed by quantification of the protein and the fluorescent label on the gels. The structural stability of cTnC incorporated into cardiac myofibrils was compared between normal and MH-susceptible pigs by selective cTnC extraction and re-incorporation. Changes were detected in both the reactivity of cTnC with CPM in rigor myofibrils and cTnC incorporation into myofibrils from the hearts of MH-susceptible pigs. These changes are very likely to be a consequence of the cardiac myosin isozyme shift in the hearts of MH-susceptible pigs, which may contribute to the changes in the myofilament response to Ca(2+) binding and to the modulation of cardiac contractility seen in this disease.

Actins↗
PubMed1999

Effects of removal and reconstitution of myosin regulatory light chain and troponin C on the Ca(2+)-sensitive ATPase activity of myofibrils from scallop striated muscle.

In order to examine the involvement of troponin-linked Ca(2+)-regulation, in addition to well-known myosin-linked Ca(2+)-regulation, in the contraction of molluscan striated muscle, myofibrils from Ezo-giant scallop striated muscle were desensitized to Ca(2+) by removing both myosin regulatory light chain and troponin C by treatment with a strong divalent cation chelator, CDTA. The ATPase level in the desensitized myofibrils was about half the maximum level in intact myofibrils regardless of the Ca(2+)-concentration at 25 and 15 degrees C. In the absence of Ca(2+), the ATPase of the desensitized myofibrils was suppressed by myosin regulatory light chain but not affected by troponin C at either temperature. The ATPase was activated at higher Ca(2+)-concentrations by both myosin regulatory light chain and troponin C, but the activating effects of these two proteins were affected differently by temperature. The activation of ATPase by myosin regulatory light chain was much greater than that by troponin C at 25 degrees C, whereas the activation by troponin C was much greater than that by myosin regulatory light chain at 15 degrees C. The maximum activation was only obtained in the presence of both myosin regulatory light chain and troponin C at these temperatures. These findings strongly suggest that the contraction of scallop striated muscle is regulated through both myosin-linked and troponin-linked Ca(2+)-regulation, and that the troponin-linked Ca(2+)-regulation is more significant at lower temperature.

Animals↗
PubMed1988

Localization of paratropomyosin in skeletal muscle myofibrils and its translocation during postmortem storage of muscles.

Using polyclonal antibodies against paratropomyosin, which is believed to modify the actin-myosin interaction in postrigor skeletal muscles, we studied the localization of paratropomyosin in chicken breast muscle myofibrils. Intact myofibrils stained with fluorescent antibodies showed that paratropomyosin was exclusively located at the A-I junction region of sarcomeres. In stretched myofibrils (3.7 micron in sarcomere length), the approximate width of the fluorescent stripes and their relation to the A band remained constant. Removal of the A band from myofibrils led to loss of stainability. During postmortem storage of muscles, on the other hand, paratropomyosin was translocated from its original position at the A-I junction region onto thin filaments. The translocation of paratropomyosin was successfully induced with a calcium ion concentration of 10(-4) M in the presence of protease inhibitors. We therefore conclude that in postrigor muscles, paratropomyosin is released from the A-I junction region following the increase in the sarcoplasmic calcium ion concentration to 10(-4) M, and then binds to thin filaments, which results in weakening of rigor linkages formed between actin and myosin.

Animals↗
PubMed1980

Calcium-activated neutral protease. Its localization in the myofibril, especially at the Z-band.

Calcium-activated neutral protease (CANP) was demonstrated in myofibrils, especially at the Z-band in chicken glycerinated myofibrils, by an immunofluorescent method. The amount of CANP bound to myofibrils was approximately 4 percent of that contained in the whole muscle homogenate. No immunological difference was recognized between the myofibril-bound CANP and the soluble one when examined by Ouchterlony's immunodiffusion analysis. A possible role of myofilbril-bound CANP in the physiological turnover of the myofibrillar proteins is discussed.

Animals↗
PubMed2003

Dynamics of individual sarcomeres during and after stretch in activated single myofibrils.

It is generally assumed that sarcomere lengths (SLs) change in isometric fibres following activation and following stretch on the descending limb of the force-length relationship, because of an inherent instability. Although this assumption has never been tested directly, instability and SL non-uniformity have been associated with several mechanical properties, such as 'creep' and force enhancement. The aim of this study was to test directly the hypothesis that sarcomeres are unstable on the descending limb of the force-length relationship. We used single myofibrils, isolated from rabbit psoas, that were attached to glass needles that allowed for controlled stretching of myofibrils. Images of the sarcomere striation pattern were projected onto a linear photodiode array, which was scanned at 20 Hz to produce dark-light patterns corresponding to the A- and I-bands, respectively. Starting from a mean SL of 2.55 +/- 0.07 microm, stretches of 11.2 +/- 1.6% of SL at a speed of 118.9 +/- 5.9 nm s(-1) were applied to the activated myofibrils (pCa(2+) = 4.75). SLs along the myofibril were non-uniform before, during and after the stretch, but with few exceptions, they remained constant during the isometric period before stretch, and during the extended isometric period after stretch. Sarcomeres never lengthened to a point beyond thick and thin filament overlap. We conclude that sarcomeres are non-uniform but generally stable on the descending limb of the force-length relationship.

Animals↗
PubMed1975

Preparation and properties of vertebrate smooth-muscle myofibrils and actomyosin.

A new technique for obtaining a myofibril-like preparation from vertebrate smooth muscle has been developed. An actomyosin can be readily extracted from these myofibrils at low ionic strength and in yields 20 times as high as previously reported. The protein composition of all preparations has been monitored using dodecylsulfate-gel electrophoresis. By this method smooth muscle actomyosin showed primarily only the major proteins, myosin, actin and tropomyosin, while the myofibrils contained, additionally, three new proteins not previously described with polypeptide chain weights of 60000, 110000 and 130000. The ATPase activities of both the myofibrils and actomyosin preparations are considerably higher than previously described for vertebrate smooth muscle. They are sensitive to micromolar Ca2+ ion concentrations to the same degree as comparable skeletal and cardiac muscle preparations, even though troponin-like proteins could not be identified in these smooth muscle preparations. From the latter observation and the presence of Ca2+-sensitivity in tropomyosin-free actomyosin it is suggested that this calcium sensitivity is, as in some invertebrate muscles, a property of the myosin molecule.

Actomyosin↗
PubMed2002

Titin-based contribution to shortening velocity of rabbit skeletal myofibrils.

The shortening velocity of skeletal muscle fibres is determined principally by actomyosin cross-bridges. However, these contractile elements are in parallel with elastic elements, whose main structural basis is thought to be the titin filaments. If titin is stretched, it may contribute to sarcomere shortening simply because it can recoil 'passively'. The titin-based contribution to shortening velocity (V(p)) was quantified in single rabbit psoas myofibrils. Non-activated specimens were rapidly released from different initial sarcomere lengths (SLs) by various step amplitudes sufficient to buckle the myofibrils; V(p) was calculated from the release amplitude and the time to slack reuptake. V(p) increased progressively (upper limit of detection, approximately 60 microm s(-1) sarcomere(-1)) between 2.0 and 3.0 microm SL, albeit more steeply than passive tension. At very low passive tension levels already (< 1-2 mN mm(-2)), V(p) could greatly exceed the unloaded shortening velocity measured in fully Ca(2+)-activated skinned rabbit psoas fibres. Degradation of titin in relaxed myofibrils by low doses of trypsin (5 min) drastically decreased V(p). In intact myofibrils, average V(p) was faster, the smaller the release step applied. Also, V(p) was much higher at 30 degrees C than at 15 degrees C (Q(10): 2.0, 3.04 or 6.15, for release steps of 150, 250 or 450 nm sarcomere(-1), respectively). Viscous forces opposing the shortening are likely to be involved in determining these effects. The results support the idea that the contractile system imposes a braking force onto the passive recoil of elastic structures. However, elastic recoil may aid active shortening during phases of high elastic energy utilization, i.e. immediately after the onset of contraction under low or zero load or during prolonged shortening from greater physiological SLs.

Animals↗
PubMed1997

Basis of passive tension and stiffness in isolated rabbit myofibrils.

By examining the mechanical properties of isolated skeletal and cardiac myofibrils in calcium-free, ATP-containing solution, we attempted to separate the stiffness contribution of titin filaments from that of weakly bound cross bridges. Efforts to enhance weak cross-bridge binding by lowering ionic strength were met by clear contractile responses. Even at low temperature, myofibrils bathed in low-ionic-strength relaxing solution generated increased force and exhibited sarcomere shortening, apparently caused by active contraction. At normal ionic strength, myofibril stiffness, estimated from the force response to rapid sinusoidal oscillations, increased steadily with sarcomere extension up to a strain limit. No obvious stiffness contribution from weak cross bridges was detectable. Instead, the stiffness response, which was frequency dependent at all sarcomere lengths, was apparently generated by the viscoelastic titin filaments. During imposed stretch-hold ramps, both peak force/stiffness and the amount of subsequent stress relaxation increased with higher stretch rates, larger stretch amplitudes, and longer sarcomere lengths. We conclude that, for a truly relaxed myofibril, both passive force and dynamic stiffness principally reflect the intrinsic viscoelastic properties of the titin filaments.

Animals↗
PubMed1987

Insulin and islet cell transplants: effects on diabetic rat cardiac myofibril ATPase.

Streptozotocin-induced diabetes results in depression of growth rate, cardiac myofibril ATPase activity, and elevated plasma glucose levels. Reversibility of these changes with daily insulin injections and pancreatic islet cell transplants was investigated and compared. Cardiac myofibril ATPase activities (mumol Pi X mg-1 X min-1) were depressed in the uncontrolled diabetic (D) group over the complete range of Ca2+ concentrations tested (e.g., 0.057 +/- 0.017 at 10 microM free Ca2+) with respect to the control (C) group (0.113 +/- 0.009). Neither the transplanted (T) group (0.128 +/- 0.017) nor the insulin injected (I) group (0.111 +/- 0.014) was significantly different from the C animals. Normal growth rates were restored in both I and T groups, whereas in the D group weight gains were negligible in comparison. Cardiac myofibril protein yields (in mg/g wet wt) were not significantly different among groups. These findings indicate that both islet cell transplants and daily insulin injections are capable of normalizing plasma glucose levels, cardiac myofibril ATPase activity, and growth rates in STZ-diabetic rats.

Adenosine Triphosphatases↗
PubMed1993

Spontaneous sarcomeric oscillations at intermediate activation levels in single isolated cardiac myofibrils.

Spontaneous oscillations observed in various heart muscle preparations are widely thought to be triggered by spontaneous release of Ca2+ from the sarcoplasmic reticulum (SR). Here, we report undamped propagated oscillations that occur in the absence of SR. In single cardiac myofibrils treated with Triton X-100 to remove SR and held isometrically, partial activation initiated periodic fluctuations of sarcomere length persisting up to 1 hour. Oscillation characteristics could be readily quantitated by virtue of the small size of the preparation. In an individual sarcomere, the oscillation cycle generally consisted of a slow shortening phase, followed by a phase of rapid lengthening. Oscillations usually propagated along the myofibril--frequently along the entire specimen--in a wavelike fashion (average velocity, 12.3 microns/s at 10 degrees C; Q10, approximately 1.3). The oscillation period was 2.30 and 1.72 seconds at 10 degrees and 20 degrees C, respectively, and was insensitive to stretch. The average oscillation amplitude, which was temperature independent, decreased with stretch from more than 20% of the mean sarcomere length at lengths below 2 microns to zero beyond a sarcomere length of 3 microns. Stiffening of the Z line by labeling with anti-alpha-actinin resulted in a dose-dependent decrease of oscillation amplitude, while the period was not affected. Tension oscillations could not be detected in single myofibrils but were frequently detectable in myofibril doublets, where the oscillation magnitude (approximately 1 microgram) was above the noise floor. Addition of 10 mumol/L ryanodine to the activating solution did not alter oscillation characteristics, as expected, since the oscillations are unrelated to SR calcium release. On the basis of our results, we consider a mechanism for the oscillations in which a length dependence of myofibrillar Ca2+ sensitivity and a dynamic Z-line structure are essential.

Actinin↗
PubMed1976

Dry ice fixation of myofibrils for scanning electron microscopy.

A rapid method of fixation of myofibrils using dry ice is reported. A glass slide or coverslip containing a drop of glutaraldehyde-fixed suspension of myofibrils is placed on dry ice causing the myofibrils to adhere to the glass surface. The specimens are then dehydrated through the alcohols, air dried and metal coated. This technique gives the myofibrils a corrugated appearance under the scanning electron microscope corresponding to the sarcomere banding.

Animals↗
PubMed1976

[Functional characterization of the creatine phosphokinase reactions in heart mitochondria and myofibrils].

The kinetic properties of MM-isozyme of creatine phosphokinase (CPK) bound to heart myofibrils have been determined experimentally. It has been shown that CPK isozymes bound to the heart myofibrils and mitochondria are electrophoretically different, but have very similar kinetic properties. For both isozymes the ATP formation reaction is preferable. However, in heart mitochondria the kinetic properties of CPK are compensated for by a tight functional coupling with ATP-ADP translocase. Due to this coupling the ATP formed in the course of oxidative phosphorylation can be used completely for creatine phosphate production in mitochondria. On the other hand, the kinetic properties of myofibrillar CPK isozyme are such that they provide for the effective utilization of creatine phosphate produced in mitochondria for rephosphorylation of AKP formed in the myofibrils during contraction. It is concluded that in the heart cells energy can be transferred from the mitochondria to the myofibrils by creatine phosphate molecules.

Adenosine Diphosphate↗
PubMed2006

[Estimation of postmortem interval using myofibril fragmentation index].

OBJECTIVE: To study the relationship between postmortem interval and the myofibril fragmentation index of skeletal muscle. METHODS: Rabbit skeletal muscle were left in the room temperature for different postmortem intervals, and the protein concentration of each sample was detected by using biuret method. Furthermore, the myofibril fragmentation index of each sample was measured under the protein concentration level of 0.5 mg/mL. RESULTS: The myofibril fragmentation index increased obviously according to the postmortem interval prolongation. CONCLUSION: The myofibril fragmentation index may be used on the estimation of early postmortem interval.

Animals↗
PubMedDate not supplied

[Aggregation of isolated myofibrils stimulated by their contraction. II. Aggregation mechanism].

Aggregation of contracted myofibrils was studied as a function of experimental conditions at myofibril contraction. The aggregation rate increased at higher concentrations of suspensions and at increased ionic strength of the medium to achieve the maximum at 0.1 M KCL in the last case. The aggregate sizes grow with an increase of ionic strength and concentration of MgATP and reduce with addition of F-actin. Aggregation of myofibrils develops only in the case of their complete or significant contraction. It was suggested that aggregation is stimulated by dehydration of myofibril at contraction.

Actins↗
PubMed1986

Unphosphorylated calf thymus and aorta myosins contract ghost myofibrils.

The MgATPase activity of unphosphorylated gizzard myosin is not stimulated by actin, but the MgATPase activities of unphosphorylated calf thymus and calf aorta myosins are stimulated by actin. This suggested that unphosphorylated thymus and aorta myosins, but not unphosphorylated gizzard myosin, should be able to cause movement. The contractile activities of these myosins were examined using "ghost" myofibrils, skeletal muscle myofibrils which have been depleted of myosin. Ghost myofibrils were reconstituted with unphosphorylated and phosphorylated turkey gizzard, calf aorta, and calf thymus myosins. While ghost myofibrils reconstituted with unphosphorylated gizzard myosin did not contract, those reconstituted with unphosphorylated thymus and aorta myosins did contract. All three phosphorylated myosins supported contraction.

Animals↗
PubMed1985

Heart 6-phosphofructo-1-kinase. Subcellular distribution and binding to myofibrils.

6-Phosphofructo-1-kinase (phosphofructokinase) (ATP:D-fructose-6-P 1-phosphotransferase, EC 2.7.1.11) can be identified in sheep heart homogenates in two forms, a soluble form and a form bound to the particulate fraction. Homogenates from immediately-dissected hearts have the enzyme in the soluble form, while those collected after a delay have the enzyme bound to the particulate fraction. Aldolase appears to show the same change in its location. Homogenization in a solution with concentrated macromolecular species (20% albumin) results in a greater association of phosphofructokinase and of aldolase to the particulate fraction in homogenates from immediately dissected hearts. Phosphofructokinase activity can be solubilized by two specific means: by high ionic strength, which is dependent upon specific salts; or by low ionic strength, which is dependent upon the presence of phosphofructokinase substrates or modifier ligands. These two means of solubilization are affected differently upon decreasing the pH below 6.9: the solubilization at low ionic strength is prevented, whereas phosphofructokinase is still solubilized by high ionic strength. Under the latter condition, the enzyme is in the inactive dimeric state, which can be activated at an alkaline pH. Myofibrils present in the particulate fraction can account for the binding of phosphofructokinase in heart homogenates. Purified myofibrils, when added to heart supernatant fluids, can bind phosphofructokinase at a slightly acidic pH. Conditions for phosphofructokinase binding to myofibrils, as well as its dissociation, follow what was observed with the binding of phosphofructokinase to the particulate fraction. At an acidic pH, and in the presence of a high concentration of ATP, phosphofructokinase exhibits low activity. However, if phosphofructokinase is assayed under these conditions while bound to myofibrils, the enzyme is activated.

Adenosine Triphosphate↗