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PubMed1976

Compositional studies of myofibrils from rabbit striated muscle.

The localization of high-molecular-weight (80,000-200,000-daltons) proteins in the sarcomere of striated muscle has been studied by coordinated electron-microscopic and sodium dodecyl sulfate (SDS) gel electrophoretic analysis of native myofilaments and extracted and digested myofibrils. Methods were developed for the isolation of thick and thin filaments and of uncontracted myofibrils which are devoid of endoproteases and membrane fragments. Treatment of crude myofibrils with 0.5% Triton X-100 results in the release of a 110,000-dalton component without affecting the myofibrillar structure. Extraction of uncontracted myofibrils with a relaxing solution of high ionic strength results in the complete disappearance of the A band and M line. In this extract, five other protein bands in addition to myosin are resolved on SDS gels: bands M 1 (190,000 daltons) and M 2 (170,000 daltons), which are suggested to be components of the M line; M 3 (150,000 daltons), a degradation product; and a doublet M 4, M 5 (140,000 daltons), thick-filament protein having the same mobility as C protein. Extraction of myofibrils with 0.15% deoxycholate, previously shown to remove Z-line density, releases a doublet Z 1, Z 2 (90,000 daltons) with the same mobility as alpha-actinin, as well as proteins of 60,000 daltons and less, and small amounts of M 1, M 2, M 4, and M 5; these proteins were not extracted with 0.5% Triton X-100. The C, M-line, and Z-line proteins and/or their binding to myofibrils are very sensitive to tryptic digestion, whereas the M 3 (150,000 daltons) component and an additional band at 110,000 daltons are products of proteolysis. Gentle treatment of myofibrils with an ATP relaxing solution results in the release of thick and thin myofilaments which can be pelleted by 100,000-g centrifugation. These myofilaments lack M-and Z-line structure when examined with the electron microscope, and their electrophoretograms are devoid of the M 1, M 2, Z 1, and Z 2 bands. The M 4, M 5 (C-protein doublet), and M 3 bands, however, remain associated with the filaments.

Animals↗
PubMed1984

The relationship between stress fiber-like structures and nascent myofibrils in cultured cardiac myocytes.

The topographical relationship between stress fiber-like structures (SFLS) and nascent myofibrils was examined in cultured chick cardiac myocytes by immunofluorescence microscopy. Antibodies against muscle-specific light meromyosin (anti-LMM) and desmin were used to distinguish cardiac myocytes from fibroblastic cells. By various combinations of staining with rhodamine-labeled phalloidin, anti-LMM, and antibodies against chick brain myosin and smooth muscle alpha-actinin, we observed the following relationships between transitory SFLS and nascent and mature myofibrils: (a) more SFLS were present in immature than mature myocytes; (b) in immature myocytes a single fluorescent fiber would stain as a SFLS distally and as a striated myofibril proximally, towards the center of the cell; (c) in regions of a myocyte not yet penetrated by the elongating myofibrils, SFLS were abundant; and (d) in regions of a myocyte with numerous mature myofibrils, SFLS had totally disappeared. Spontaneously contracting striated myofibrils with definitive Z-band regions were present long before anti-desmin localized in the I-Z-band region and long before morphologically recognizable structures periodically link Z-bands to the sarcolemma. These results suggest a transient one-on-one relationship between individual SFLS and newly emerging individual nascent myofibrils. Based on these and other relevant data, a complex, multistage molecular model is presented for myofibrillar assembly and maturation. Lastly, it is of considerable theoretical interest to note that mature cardiac myocytes, like mature skeletal myotubes, lack readily detectable stress fibers.

Animals↗
PubMed1950

The properties of mammalian striated myofibrils isolated by an enzymatic method.

A new method for the isolation of large numbers of individual myofibrils from fresh mammalian skeletal and cardiac muscle has been described. Purification of isolated myofibrils was accomplished by differential centrifugation of fresh frozen sections of muscle which had been mechanically agitated after exposure for 30 to 45 minutes at 0 degrees C. to the action of a dilute solution of trypsin in a phosphate buffer solution with a pH of 7.0 and an ionic strength of 0.25. Isolated skeletal myofibrils of the rabbit and man have similar constant solubility properties. They dissolve in an aqueous mixture of 0.5 N potassium chloride and 0.03 N sodium bicarbonate, giving viscous solutions which exhibit conspicuous birefringence of flow. They are soluble in buffer solutions (ionic strength 0.15) on the acid side of pH 4 and alkaline side of pH 10. If the ionic strength of potassium phosphate buffer solutions is increased to 0.5 or if the ionic strength of phosphate-borate buffer solutions is increased to a similar value by addition of potassium chloride, the isolated myofibrils become soluble at neutrality. Hence, it is possible, first to isolate the myofibrils and then dissolve them without deviating appreciably from physiologic ranges of pH. The extent to which myofibrils are modified by the conditions imposed by the method of isolation is unknown. There is no significant change in microscopic structure or optical birefringence. Furthermore, there is retention of a form of physiological reactivity, for when the isolated skeletal myofibrils are immersed in solutions of adenosinetriphosphate, they promptly and irreversibly change from elongated fibrils with distinct structural detail into dense spherical masses without recognizable microscopic structure.

Adenosine Triphosphate↗
PubMed1976

Structure and function of the two heads of the myosin molecule. I. Binding of adenosine diphosphate to myofibrils during the adenosinetriphosphatase reaction.

1. The myosin content of myofibrils was found to be 51% by SDS-gel electrophoresis. 2. The initial burst of Pi liberation of the ATPase [EC 3.6.1.3] of a solution of myofibrils in 1 M KCl was measured in 0.5 M KCl, and found to be 0.93 mole/mole of myosin. 3. The amount of ADP bound to myofibrils during the ATPase reaction and the ATPase activity were measured by coupling the myofibrillar ATPase reaction with sufficient amounts of pyruvate kinase [EC 2.7.1.40] and PEP to regenerate ATP. The maximum amount of ADP bound to myofibrils in 0.05M KCl and in the relaxed state was about 1.5 mole/mole of myosin. On the other hand, the ATPase activity exhibited substrate inhibition, and the amount of ATP required for a constant level of ATPase activity was smaller than that required for the maximum binding of ADP to myofibrils. 4. The maximum amount of ADP bound to myofibrils in 0.5 M KCl was about 1.9 mole/mole of myosin. When about one mole of ADP was found to 1 mole of myosin in myofibrils, the myofibrillar ATPase activity reached the saturated level, and with further increase in the concentration of ATP one more mole of ADP was found per mole of myosin.

Adenosine Diphosphate↗
PubMed1979

Studies on the post-mortem fragmentation of myofibrils.

1. There was a close relationship between the fragmentation of myofibrils and the tension developed during post-mortem contraction of muscle. The extent of fragmentation was at its maximum when the sarcomeres attained a length of 2.0 to 2.2 micron. 2. The rate of fragmentation of myofibrils depended upon the calcium ion concentration within a range of 10(-5) to 2 x 10(-2) M, with a minimum at pH 6.5. The fragmentation of myofibrils free from muscle fibers was not affected by 10 mM iodoacetate, an irreversible inhibitor of calcium-activated factor (CAF). 3. Incubation of myofibrils with 10 mM CaCl2 caused the release of about 12% of the total myofibrillar proteins after homogenization. The protein solution contained little alpha-actinin, and considerable amounts of 54,000- and 76,000-dalton components which seem to originate from the Z-line. SDS-polyacrylamide gels of troponin prepared from the incubated myofibrils did not change with time of incubation. These findings are in contrast with the proteolytic degradation of Z-lines by CAF treatment, in which alpha-actinin and 87,000 dalton component are released. 4. These data directly demonstrate that the in vitro fragmentation of post-mortem muscle (i.e. duirng its conversion into myofibrils upon mechanical homogenization) is different from that induced by CAF. The possible role of calcium ions during in vitro fragmentation of myofibrils is discussed.

Animals↗
PubMed1990

Effects of zero gravity on myofibril content and isomyosin distribution in rodent skeletal muscle.

The purpose of this experiment was to investigate the effects of 12.5 days of zero gravity (0 g) exposure (Cosmos 1887 Biosputnik) on the enzymatic properties, protein content, and isomyosin distribution of the myofibril fraction of the slow-twitch vastus intermedius (VI) and the fast-twitch vastus lateralis (VL) muscles of adult male rats. Measurements were obtained on three experimental groups (n = 5 each group) designated as flight group (FG), vivarium control (VC), and synchronous control (SC). Body weight of the FG was significantly lower than that of the two control groups (P less than 0.05). Compared with the two control groups, VI weight was lower by 23% (P less than 0.10), whereas no such pattern was apparent for the VL muscle. Myofibril yields (mg protein/g muscle) in the VI were 35% lower in the FG than in controls (P less than 0.05), whereas no such pattern was apparent for the VL muscle. When myofibril yields were expressed on a muscle basis (mg/g x muscle weight), the loss of myofibril protein was more exaggerated and suggests that myofibril protein degradation is an early event in the muscle atrophy response to 0 g. Analysis of myosin isoforms indicated that slow myosin (Sm) was the primary isoform lost in the calculated degradation of total myosin. No evidence of loss of the fast isomyosins was apparent for either muscle following spaceflight. Myofibril ATPase activity of the VI was increased in the FG compared with controls, which is consistent with the observation of preferential Sm degradation. These data suggest that muscles containing a high percentage of slow-twitch fibers undergo greater degrees of myofibril protein degradation than muscles containing predominantly fast-twitch fibers in response to a relatively short period of 0 g exposure, and the primary target appears to be the Sm molecule.

Adenosine Triphosphatases↗
PubMed1999

Modulation by substrate concentration of maximal shortening velocity and isometric force in single myofibrils from frog and rabbit fast skeletal muscle.

1. The effects of magnesium adenosine triphosphate (MgATP; also referred to as 'substrate') concentration on maximal force and shortening velocity have been studied at 5 C in single and thin bundles of striated muscle myofibrils. The minute diameters of the preparations promote rapid diffusional equilibrium between the bathing medium and lattice space so that during contraction fine control of substrate and product concentrations is achieved. 2. Myofibrils from frog tibialis anterior and rabbit psoas fast skeletal muscles were activated maximally by rapidly (10 ms) exchanging a continuous flux of pCa 8.0 for one at pCa 4.75 at a range of substrate concentrations from 10 microM to 5 mM. At high substrate concentrations maximal isometric tension and shortening velocity of both frog and rabbit myofibrils were very close to those determined in whole fibre preparations from the same muscle types. 3. As in frog and rabbit skinned whole fibres, the maximal isometric force of the myofibril preparations decreases as MgATP concentration is increased. The maximal velocity of unloaded shortening (V0) depends hyperbolically on substrate concentration. V0 extrapolated to infinite MgATP (3.6 +/- 0.2 and 0.8 +/- 0.03 l0 s-1 in frog and rabbit myofibrils, respectively) is very close to that determined directly at high substrate concentration. The Km is 210 +/- 20 microM for frog tibialis anterior and 120 +/- 10 microM for rabbit psoas myofibrils, values about half those found in larger whole fibre preparations of the same muscle types. This implies that measurements in whole skinned fibres are perturbed by diffusional delays, even in the presence of MgATP regenerating systems. 4. In both frog and rabbit myofibrils, the Km for V0 is about one order of magnitude higher than the Km for myofibrillar MgATPase determined biochemically in the same experimental conditions. This confirms that the difference between the Km values for MgATPase and shortening velocity is a basic feature of the mechanism of chemomechanical transduction in muscle contraction.

Adenosine Triphosphate↗
PubMed2003

New N-RAP-binding partners alpha-actinin, filamin and Krp1 detected by yeast two-hybrid screening: implications for myofibril assembly.

N-RAP, a muscle-specific protein concentrated at myotendinous junctions in skeletal muscle and intercalated disks in cardiac muscle, has been implicated in myofibril assembly. To discover more about the role of N-RAP in myofibril assembly, we used the yeast two-hybrid system to screen a mouse skeletal muscle cDNA library for proteins capable of binding N-RAP in a eukaryotic cell. From yeast two-hybrid experiments we were able to identify three new N-RAP binding partners: alpha-actinin, filamin-2, and Krp1 (also called sarcosin). In vitro binding assays were used to verify these interactions and to identify the N-RAP domains involved. Three regions of N-RAP were expressed as His-tagged recombinant proteins, including the nebulin-like super repeat region (N-RAP-SR), the N-terminal LIM domain (N-RAP-LIM), and the region of N-RAP in between the super repeat region and the LIM domain (N-RAP-IB). We detected significant alpha-actinin binding to N-RAP-IB and N-RAP-LIM, filamin binding to N-RAP-SR, and Krp1 binding to N-RAP-SR and N-RAP-IB. During myofibril assembly in cultured chick cardiomyocytes, N-RAP and filamin appear to co-localize with alpha-actinin in the earliest myofibril precursors found near the cell periphery, as well as in the nascent myofibrils that form as these structures fuse laterally. In contrast, Krp1 is not localized until late in the assembly process, when it appears at the periphery of myofibrils that appear to be fusing laterally. The results suggest that sequential recruitment of N-RAP binding partners may serve an important role during myofibril assembly.

Actinin↗
PubMed1995

Phalloidin unzips nebulin from thin filaments in skeletal myofibrils.

Fluorescent phallotoxins such as rhodamine-phalloidin take hours to bind uniformly to thin filaments of skeletal myofibrils, after fast initial binding to both ends of thin filaments. Observation of this process in skeletal and cardiac myofibrils and of the resulting re-distribution of nebulin using anti-nebulin antibody showed that: (1) rhodamine-phalloidin binds uniformly to actin in cardiac myofibrils within minutes, in contrast to skeletal myofibrils; (2) overnight pre-incubation of skeletal myofibrils with phalloidin results in uniform initial binding of rhodamine-phalloidin and a changed nebulin localization; (3) pre-incubation of skeletal myofibrils with Ca(2+)-calmodulin results in uniform initial binding of rhodamine-phalloidin; (4) the binding of rhodamine-phalloidin to actin in skeletal myofibrils is unidirectional, i.e. the fluorescence of incorporated rhodamine-phalloidin moves from the pointed ends where it is bound initially toward the barbed end at the Z-band; (5) the unidirectional binding of rhodamine-phalloidin results in redistribution of nebulin, i.e. the initial fluorescent bands associated with the epitopes of bound nebulin antibody change to a single band located close to Z-line. These results indicate that nebulin inhibits rhodamine-phalloidin binding to actin and suggests that the unidirectional rhodamine-phalloidin binding may be due to cooperative competitive binding, i.e. phalloidin 'unzips' nebulin starting from the pointed ends of the thin filaments.

Actins↗
PubMed2006

Mechanical strength of sarcomere structures of skeletal myofibrils studied by submicromanipulation.

The mechanical strength of sarcomere structures of skeletal muscle was studied by rupturing single myofibrils of rabbit psoas muscle by submicromanipulation techniques. Microbeads coated with alpha-actinin were attached to the surface of myofibrils immobilized to coverslip. By use of either optical tweezers or atomic force microscope, the attached beads were captured and detached from the myofibrils. During the detachment of the beads, the actin filaments bound specifically to the beads were peeled off from the bulk structures of myofibrils, thus rupturing the peripheral components of the myofibrils bound to the actin filaments. By analyzing the ruptures thus produced in various myofibril preparations, it was found that the sarcomere structure of myofibrils is maintained by numerous molecular components having the mechanical strength sufficient to sustain the contractile force produced by the actomyosin system. The present techniques could be applied to study the mechanical strength of cellular organelles containing actin filaments as their component.

Actin Cytoskeleton↗
PubMed1997

Dynamics of actin in cardiac myofibrils and fibroblast stress fibers.

The exchangeability of actin in cardiac myofibrils and fibroblast stress fibers was investigated using fluorescent analogue cytochemistry in combination with fluorescence recovery (FR) after photobleaching. Living embryonic chicken cardiac myocytes and fibroblasts were microinjected with rhodamine (rh)-labeled muscle and nonmuscle actins. After incorporation of the fluorescent actin analogue into cellular structures, small areas of labeled structures were photobleached with a laser pulse. In cardiac myofibrils, FR in their proximal striated portions occurred at a slower rate than that in their proximal nonstriated and distal terminal portions with each rh-isoactin injected. Thus, nascent myofibrils at different developmental stages display different actin exchangeabilities. Further, in all portions of myofibrils, FR of rh-muscle actin was faster than that of rh-nonmuscle actin. This indicates that actin molecules in cardiac myofibrils cannot be readily exchanged by heterotypic nonmuscle actin. In fibroblasts, photobleaching of stress fibers yielded similar results in both their proximal mid-points and distal terminal portions, and the FR rate was consistently faster than that observed in any part of the myofibrils. This result seems to be related to the dynamic properties of actin filaments in stress fibers at all portions. Further, the fact that stress fibers possessed a similar exchange rate with muscle and nonmuscle actins appears to be related to a more primitive nature of stress fibers than myofibrils.

Actins↗
PubMed2000

Contractility of single myofibrils of rabbit skeletal muscle studied at various MgATP concentrations.

A novel experimental method was developed to study the contractility of single myofibrils of skeletal muscle. Single myofibrils (ca. 1 microm in diameter) prepared from glycerinated rabbit psoas muscle were suspended between rigid and flexible microneedles by the entwining method. The length changes of the preparations applied via the rigid microneedle by an actuator and the force produced were measured by photo-electrically detecting the nanometer deflections of the flexible microneedle. Single myofibril preparations maintained uniform sarcomere striations during contraction-relaxation cycles. The isometric force produced, the velocity of unloaded shortening, and the force-velocity relationship of single myofibrils were investigated at various MgATP concentrations. The contractility of single myofibrils thus obtained in the absence of ATP regenerative systems was essentially the same as that of skinned muscle fibers under comparable conditions in the presence of ATP regenerative systems. Thus, it was found that (1) the present experimental method is useful for studying the contractility of single myofibrils, and (2) in single myofibril preparations, the MgATP concentration at actomyosin sites is well equilibrated with that in bathing solutions.

Adenosine Triphosphate↗
PubMed1997

Phosphorylation by protein kinase C and the responsiveness of Mg(2+)-ATPase to Ca2+ of myofibrils isolated from stunned and non-stunned porcine myocardium.

Previously we showed in an in situ porcine model that the thiadiazinone derivative [+]EMD 60263, a Ca2+ sensitizer without phosphodiesterase III inhibitory properties, increased contractility more profoundly in stunned than in non-stunned myocardium. This finding was consistent with the observed leftward shifts of the pCa2+/Mg(2+)-ATPase curves of isolated myofibrils induced by [+]EMD 60263. The aim of the present investigation was to study the possible involvement of protein kinase C in the mechanism of reduced Ca2+ responsiveness of myofilaments during stunning. No differences were observed in the maximal activity of the Ca(2+)-stimulated Mg(2+)-ATPase and in the pCa50 of myofibrils isolated from non-stunned and stunned myocardium. After phosphorylation with [gamma-32P]-ATP and excess of purified rat brain protein kinase C, the myofibrils were separated on sodiumdodecylsulphate-polyacrylamide gelectrophoresis and the 32P incorporation counted by the Molecular Imager. Ca2+/ phosphatidylserine/sn-1,2 diolein-dependent 32P incorporation catalyzed by excess of purified rat brain protein kinase C in C-protein, TnT and TnI subunits did not show any differences between myofibrils from non-stunned and stunned myocardium. However, protein kinase C-induced phosphorylation of myofibrils isolated from ventricular myocardium of sham-operated pigs resulted in a marked leftward shift of the pCa50 from 6.03 +/- 0.04 to 6.44 +/- 0.06 (p < 0.05), while porcine heart cyclic AMP-dependent protein kinase-induced phosphorylation resulted in an expected small rightward shift to 5.97, although statistical significance was not reached. Protein kinase C-induced phosphorylation also stimulated (80%) the maximal myofibrillar Mg(2+)-ATPase activity. [+]EMD 60263 (3 microM) produced a leftward shift of the myofibrillar pCa2+/Mg(2+)-ATPase curve which was unaffected by prior protein kinase C-induced phosphorylation. In conclusion, the findings with isolated myofibrils from myocardium of anaesthetized open-chest pigs indicate that protein kinase C might be involved in the mechanism of reduced Ca2+ responsiveness of myofilaments in stunned myocardium. However, at this stage no differences could be found between the maximal activity of the Ca(2+)-stimulated Mg(2+)-ATPase, the pCa50 and the degree of phosphorylation of myofibrils isolated from stunned and non-stunned myocardium.

Actin Cytoskeleton↗
PubMed1997

Breakdown and rebuilding of myofibrils in cultured adult cardiac muscle cells.

The myofibrils of adult rat cardiac muscle cells in culture break down and later reorganize into mature myofibrils. The myofibrillar breakdown and reorganization processes have been investigated with electron microscopical and immunocytochemical studies. The immunocytochemical studies included antibodies to actin, myosin, titin, and alpha-actinin. In addition, rhodamine-labeled phalloidin has been used. These studies revealed that the myofibrils were disorganized into amorphous and/or other forms during breakdown process. Some of these myofibrils undergo degradation and finally extrusion through exocytosis. The reorganization of myofibrils takes place mainly with the participation of the existing myofibrillar proteins in myocytes. This remyofibrillogenesis showed the emergence of punctate alpha-actinin from the existing amorphous alpha-actinin along with the differentiation of titin periodicities, which remained attached to the alpha-actinin structures. The punctate alpha-actinin later differentiated into periodicities, forming Z-lines. The periodicities of actin were differentiated from the amorphous actin and associated with the Z-lines, giving rise to titin, alpha-actinin, and actin complexes. Later, myosin filaments became associated with these complexes, forming sarcomeres where other myofibrillar proteins participated in the formation of mature myofibrils. The temporal sequence of differentiation of periodicities of certain myofibrillar proteins varied among different myocytes and within a single myocyte. The dynamic role of adult cardiac myocytes in the reconstruction of myofibrils is a remarkable phenomenon, which stabilizes adult cardiac muscle cells in long-term culture.

Animals↗
PubMed1993

A structural and kinetic study on myofibrils prevented from shortening by chemical cross-linking.

In previous work, we studied the early steps of the Mg(2+)-ATPase activity of Ca(2+)-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T. (1992) Biochemistry 31, 1564-1569]. The myofibrils were free to contract, and the results obtained refer to the ATPase cycle of myofibrils contracting with no external load. Here we studied the ATPase of myofibrils contracting isometrically. To prevent shortening, we cross-linked them with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). SDS-PAGE and Western blot analyses showed that the myosin rods were extensively cross-linked and that 8% of the myosin heads were cross-linked to the thin filament. The transient kinetics of the cross-linked myofibrils were studied in 0.1 M potassium acetate, pH 7.4 and 4 degrees C, by the rapid-flow quench method. The ATP binding steps were studied by the cold ATP chase and the cleavage and release of products steps by the Pi burst method. In Pi burst experiments, the sizes of the bursts were equal within experimental error to the ATPase site concentrations (as determined by the cold ATP chase methods) for both cross-linked (isometric) and un-cross-linked (isotonic) myofibrils. This shows that in both cases the rate-limiting step is after the cleavage of ATP. When cross-linked, the kcat of Ca(2+)-activated myofibrils was reduced from 1.7 to 0.8 s-1. This is consistent with the observation that fibers shortening at moderate velocity have a higher ATPase activity than isometric fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1993

Development of the excitation-contraction coupling apparatus in skeletal muscle: association of sarcoplasmic reticulum and transverse tubules with myofibrils.

The formation and maintenance of the highly regular organization of membrane systems and proteins in striated muscle require specific membrane-membrane and membrane-cytoskeleton interactions. The development of T-tubules and sarcoplasmic reticulum (SR) was followed in gastrocnemius muscle fibers from chicken embryos between 12 days (E12) and 21 days (E21) of incubation, with particular attention to their relationship with one another and with the myofibrils. The fluorescent lipid analog DiIC16[3] was used to label either the external membranes (plasmalemma and transverse (T)-tubules) or the internal SR in living and fixed muscle. Short membrane invaginations can first be seen in fibers at E14, and at E15 longitudinal T-tubules appear in the periphery of the fibers. A complex network of T-tubules filling the whole fiber diameter develops suddenly at E16. In contrast, SR is abundant at the earliest observed stage (E12) and forms regularly spaced cross striations located at the I-Z-I bands. These correspond to a specific accumulation of smooth membranes around the Z-discs seen in electron micrographs. While SR is specifically associated with the newly formed myofibrils in the periphery of the fibers, the disposition of early T-tubules shows little specific relationship to either SR or the myofibrils. However, electron microscopy shows that junctions between T-tubules and SR are formed during this period (Takekura and Franzini-Armstrong, submitted for publication). Junctions do not acquire a specific relation to the myofibrils until around hatching when triads begin to reorganize into their mature location, the A-I junction. These findings indicate three key events in the organization of T-tubules and SR in the sarcomeres: (1) early SR/Z-line interactions independent of T-tubules; (2) SR/T-tubule interactions to form the triad junctions, independent from the myofibrils; and (3) the late association of the junctional complexes with the myofibrils at the A-I border.

Animals↗
PubMed1994

Effect of phalloidin on the ATPase activity of striated muscle myofibrils.

Phalloidin was shown to increase the ATPase activity and Ca2+ sensitivity of both bovine cardiac and rabbit psoas myofibrils when assayed in a solution containing 50 mM KCl, 100 mM MOPS (pH 7.0), 2 mM MgCl2, 1 mM ATP, 2 mM EGTA, and varying concentrations of Ca2+ (temperature 21-22 degrees C). The phalloidin effect in cardiac myofibrils developed over a time course of several minutes in the presence of 50 microM phalloidin. Relative increase of ATPase activity was maximal at pCa 8 and decreased with decrease in pCa. In cardiac myofibrils the increase was about 70% at pCa 8 and 20% at pCa 4 following 20-30 min pre-incubation with 2 microM or 50 microM phalloidin. The effect persisted after excess phalloidin was washed out. The increase in Ca2+ sensitivity was approximately 0.15 pCa units. For skeletal myofibrils treated with 2 microM phalloidin all changes were considerably less than those seen with cardiac myofibrils and the changes were even less when the myofibrils were exposed to 50 microM phalloidin. These results show that when specifically bound to actin, phalloidin can change the kinetic parameters of the cross-bridge cycle and may also alter the Ca2+ sensitivity of the contractile system. The effects of phalloidin seem to vary with muscle type.

Actins↗
PubMed1991

Differential activation of myofibrils during fatigue in phasic skeletal muscle cells.

In fatigued muscles the T-system is swollen; thus the action potential may fail to travel along the T-system or the T-tubule terminal cisternae signal may fail to bring about TC Ca2+ release. This would lead to a decrease in the number of myofibrils activated and in force development, but if fatigue is the result of a generalized process, all the myofibrils would be affected equally leading to a lower activation of all of them. We have investigated this possibility in isolated twitch muscle fibres by giving them repetitive tetanic stimulations until fatigue developed. The behaviour of myofibrils was followed with cinemicrophotography. Before fatigue, no lack of shortening of myofibrils could be found. During fatigue groups of myofibrils became wavy. When exposed to caffeine, the wavy myofibrils disappeared and tension similar to the control developed. The tension-caffeine concentration relationship was shifted to the left after development of fatigue. In low Na+ solution fatigue developed faster and after reintroducing normal Ringer, tension recovered substantially. K-contractures were smaller during fatigue. These results indicate that in this type of fatigue, a step in the EC coupling chain of events is involved in its development.

Action Potentials↗