The Experts below are selected from a list of 276 Experts worldwide ranked by ideXlab platform
Dilson E. Rassier - One of the best experts on this subject based on the ideXlab platform.
-
Sarcomere Length Nonuniformity and Force Regulation in Myofibrils and Sarcomeres
Biophysical journal, 2020Co-Authors: Felipe De Souza Leite, Dilson E. RassierAbstract:The smallest contractile unit in striated muscles is the sarcomere. Although some of the classic features of contraction assume a uniform behavior of sarcomeres within Myofibrils, the occurrence of sarcomere length nonuniformities has been well recognized for years, but it is yet not well understood. In the past years, there has been a great advance in experiments using isolated Myofibrils and sarcomeres that has allowed scientists to directly evaluate sarcomere length nonuniformity. This review will focus on studies conducted with these preparations to develop the hypotheses that 1) force production in Myofibrils is largely altered and regulated by intersarcomere dynamics and that 2) the mechanical work of one sarcomere in a myofibril is transmitted to other sarcomeres in series. We evaluated studies looking into myofibril activation, relaxation, and force changes produced during activation. We conclude that force production in Myofibrils is largely regulated by intersarcomere dynamics, which arises from the cooperative work of the contractile and elastic elements within a myofibril.
-
isolating Myofibrils from skeletal muscle biopsies and determining contractile function with a nano newton resolution force transducer
Journal of Visualized Experiments, 2020Co-Authors: Martijn Van De Locht, Dilson E. Rassier, Josine M De Winter, Michiel Helmes, Coen A C OttenheijmAbstract:Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of Myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent Myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of Myofibrils might contribute to impaired contractility. If structural damage is present in the Myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in Myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in Myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes Myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-Perot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.
-
extraction of thick filaments in individual sarcomeres affects force production by single Myofibrils
Biophysical Journal, 2020Co-Authors: Andrea C Mendoza, Dilson E. RassierAbstract:Abstract It has been accepted that the force produced by a skeletal muscle myofibril depends on its cross-sectional area, but not on the number of active sarcomeres since they are arranged in series. However, a previous study performed by our group showed that blocking actomyosin interactions within an activated myofibril and depleting the thick filaments in one sarcomere unexpectedly reduced force production. In this study we examined in detail how consecutive depletion of thick filaments in individual sarcomeres within a myofibril affects force production. Myofibrils isolated from rabbit psoas were activated/relaxed using a perfusion system. An extra micro-perfusion needle filled with high ionic strength solution was used to erase thick filaments in individual sarcomeres in real time before myofibril activation. The isometric forces were measured upon activation. The force produced by Myofibrils with intact sarcomeres was significantly higher than the force produced by Myofibrils with one or more sarcomeres lacking thick filaments (p i 0.0001) irrespective of the number of contractions imposed on the Myofibrils and their initial sarcomere length. Our results suggest that the myofibril force is affected by intersarcomere dynamics and the number of active sarcomeres in series.
-
Sarcomere Stiffness during Stretching and Shortening of Rigor Skeletal Myofibrils.
Biophysical journal, 2017Co-Authors: Nabil Shalabi, Malin Persson, Alf Månsson, Srikar Vengallatore, Dilson E. RassierAbstract:Abstract In this study, we measured the stiffness of skeletal muscle Myofibrils in rigor. Using a custom-built atomic force microscope, Myofibrils were first placed in a rigor state then stretched and shortened at different displacements (0.1–0.3 μ m per sarcomere) and nominal speeds (0.4 and 0.8 μ m/s). During stretching, the myofibril stiffness was independent of both displacement and speed (average of 987 nN/ μ m). During shortening, the myofibril stiffness was independent of displacement, but dependent on speed (1234 nN/ μ m at 0.4 μ m/s; 1106 nN/ μ m at 0.8 μ m/s). Furthermore, the myofibril stiffness during shortening was greater than that during stretching and the difference depended on speed (31% at 0.4 μ m/s; 8% at 0.8 μ m/s). The results suggest that the Myofibrils exhibit nonlinear viscoelastic properties that may be derived from myofibril filaments, similar to what has been observed in muscle fibers.
-
microfluidic perfusion shows intersarcomere dynamics within single skeletal muscle Myofibrils
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Felipe De Souza Leite, Fabio C Minozzo, David Altman, Dilson E. RassierAbstract:The sarcomere is the smallest functional unit of Myofibrils in striated muscles. Sarcomeres are connected in series through a network of elastic and structural proteins. During myofibril activation, sarcomeres develop forces that are regulated through complex dynamics among their structures. The mechanisms that regulate intersarcomere dynamics are unclear, which limits our understanding of fundamental muscle features. Such dynamics are associated with the loss in forces caused by mechanical instability encountered in muscle diseases and cardiomyopathy and may underlie potential target treatments for such conditions. In this study, we developed a microfluidic perfusion system to control one sarcomere within a myofibril, while measuring the individual behavior of all sarcomeres. We found that the force from one sarcomere leads to adjustments of adjacent sarcomeres in a mechanism that is dependent on the sarcomere length and the myofibril stiffness. We concluded that the cooperative work of the contractile and the elastic elements within a myofibril rules the intersarcomere dynamics, with important consequences for muscle contraction.
Walter Herzog - One of the best experts on this subject based on the ideXlab platform.
-
activated skeletal muscle Myofibrils have different peak stresses at similar sarcomere lengths when lengthened beyond myofilament overlap
Biophysical Journal, 2017Co-Authors: T R Leonard, Walter HerzogAbstract:We suggested previously that the free-spring region of titin in the I-band can be length-modulated; giving rise to dramatically elevated forces in actively compared to passively stretched Myofibrils at sarcomere length (SL) beyond actin-myosin filament overlap (>4µm).[1]If peak stresses were to vary at very long SL (> 4µm) where titin is the sole contributor (in Myofibrils) to force, then that would suggest that the free-spring length of titin can be length-modulated somehow. The result being that a shorter free-spring titin would generate higher stress at matched SL compared to a myofibril where titin length was not shortened. We stretched calcium activated (pCa+2 3.5 in all experiments) rabbit psoas Myofibrils beyond myofilament overlap (>4µm) while measuring forces and mean SL. Samples were either fully activated (control) or were activated with 2mM BDM. All samples were lengthened (0.1µm/sarcomere/second) following activation. Initial activation stress at SL=2.2µm for BDM was 35.5 ±10.5nN/um2 compared to control 89.3nN/um2. The BDM activated samples had less peak stress (278±32nN/um2) compared to the control (407nN/um2) at similar mean SL 4.7±0.05µm. Previous data [1] for Myofibrils lengthened passively showed less peak stress compared to all experiments here; for mean SL 4.6µm, the mean stress was 135±47nN/um2.Based on these results, we suggest that titin is a molecular spring whose stiffness may be regulated by changes in effective length and that regulation is not an “all-or-none” model. We suggest that titin is a molecular spring whose stiffness is regulated by changes in effective length controlled by force-dependent actin-titin interactions.
-
Residual Force Enhancement in Cardiac Myofibrils
Biophysical Journal, 2015Co-Authors: Kevin Boldt, Venus Joumaa, Walter HerzogAbstract:Residual force enhancement (RFE) is a property of muscle where an activated muscle is stretched from a short to a long length resulting in greater force than is produced isometrically at the long length. This history-dependent property has been identified across skeletal muscle hierarchy including whole muscle, fascicles, fibres, and Myofibrils. However, RFE has not been investigated in cardiac muscle. Therefore, the purpose of this study was to determine if RFE was present in cardiac Myofibrils.Rabbit hearts were dissected and strips of left ventricle were skinned overnight with 1% Triton skinning solution and stored at −20°C. On the day of experiments, the cardiac tissue was blended and a myofibril with a good striation pattern was identified and suspended between a glass needle and a nanolever allowing for length changes and force measurement. The myofibril was set at a sarcomere length (SL) of 2.4 µm and activated to establish a reference contraction before being passively stretched to a SL of 3.2 µm. After a rest period, the myofibril was activated at a SL of 2.4 µm, actively stretched to a SL of 3.2µm, held for one minute and then relaxed. RFE was calculated as the difference between the steady-state force obtained after active stretch and the corresponding predicted isometric force at 3.2 µm based on the reference force, calculated according to the force-length relationship scaled to the filament lengths in rabbit muscles, and accounting for passive force.All Myofibrils (n=5) produced more force when stretched actively compared to the calculated reference isometric force, indicating the presence of RFE in cardiac Myofibrils. The average RFE was 54.8±10.8%.The presence of RFE in cardiac Myofibrils supports that RFE is a universal muscle property. Investigation in cardiac muscle may shed new light into the mechanisms underlying RFE.
-
TITIN HYSTERESIS AND ELASTIICTY IN ACTIVELY STRETCHED MUSCLE Myofibrils
Journal of Undergraduate Research in Alberta, 2015Co-Authors: Craig Martis, Azim Jinha, Tim Leonard, Kaleena Johnston, Walter HerzogAbstract:INTRODUCTION Titin is a protein that spans the length of a half sarcomere in skeletal muscle Myofibrils. It behaves like a molecular spring within the myofibril, playing a role in stabilizing sarcomeres and regulating passive force [1, 2]. Isolated titin has been shown to be essentially elastic if immunoglobulin (Ig) domain unfolding/refolding is prevented [3]. In its native, sarcomeric environment, it has been suggested that stretching and holding a myofibril at very long lengths produces a time-dependent unfolding of all Ig domains, thus, allowing titin’s elastic behavior to be exhibited [4]. Experiments on active Myofibrils showed a decrease in force and a persistent hysteresis throughout a stretch-shortening (SS) protocol, suggesting a time-dependent unfolding of Ig domains [5]. Holding active Myofibrils at long lengths prior to stretch-shortening cycles should allow most (all) of the Ig domains to unfold thus reducing (eliminating) force loss and hysteresis. The goal of this study was to test the hypothesis that holding Myofibrils at long lengths prior to small stretch-shortening cycles would result in essentially elastic properties of Myofibrils, compared to the highly visco-elastic properties for conditions without holding. METHODS Rabbit psoas muscle Myofibrils (n = 5) with clear striation patterns were tested. Single Myofibrils were attached at one end to a glass needle (to control length) and at the other end to a nanolever (to quantify force). Myofibrils were activated at an average sarcomere length of 2.7 µm, and then stretched to a length of 5.2 µm/sarcomere, where they were held for 2 minutes to allow for Ig domain unfolding to occur. The myofibril then underwent a SS protocol with amplitude of ± 0.25 µm (10 cycles) before being shortened to its original length. Myofibril length, diameter, and force were quantified. Diameter was used to calculate cross-sectional area, which accommodated the calculation of myofibril stress from force. Hystereses were calculated as the difference in area under the loading and unloading curves for each SS cycle of the force-length plots. RESULTS Peak stress throughout the 10 cycles remained approximately constant, averaging 102 % relative to the first cycle (Fig 1a). Hysteresis did not follow a specific trend throughout the 10 SS cycles (Fig 1b). DISCUSSION AND CONCLUSIONS The “constant” peak forces are indicative of elastic recoil of Myofibrils during the SS cycles. However, the persistent and random hystereses are indicative of viscous properties. If Ig domains were still unfolding during the SS cycles, peak stresses should also decrease. Since this is not observed, we suggest that all Ig domains are unfolded in this experiment, and that the viscous behaviour producing the hystereses must come from a source other than titin. At this point, any proposition as to the origin of the remnant hystereses is highly speculative but might be associated with titin binding-unbinding to another structural (titin) or contractile (actin) protein that is forming and breaking continuously during the SS cycles.
-
Hysteresis and Efficiency in Passive Skeletal Muscle Myofibrils
Biophysical Journal, 2012Co-Authors: Jens Herzog, Azim Jinha, Tim Leonard, Walter HerzogAbstract:Determining mechanical properties of titin is difficult but the passive properties of single Myofibrils are mostly attributed to titin. We investigated whether myofibril behaviour mirrors that of single titin molecules. Single Myofibrils were subjected to three passive stretch-shortening cycles of up to 3.5μm/sarcomere at a speed of 0.1 μm/sarcomere/second.Stretched-shortened Myofibrils (Figure 1) show reduced force during stretch for a given sarcomere length (SL) and reduced peak force for cycles 2 and 3 compared to cycle 1. Force-SL curves for all 3 cycles during the shortening are similar. We see increased efficiency (shortening energy/lengthening energy) with repeated cycles (37%, 48% and 53%). These properties are in general agreement with results observed in single titin preparations (Kellermayer et al., 1997). Titin properties can be studied using single Myofibrils. In the future, we would like to study titin properties in calcium activated Myofibrils in which active (actin-myosin based cross-bridges forces) are eliminated either by chemical inhibition or by deletion of regulatory proteins on actin.Figure 1. Force and mean sarcomere length for a myofibril subjected to 3 sequential stretch-shortening cycles. Insert shows hysteresis behaviour of a single titin molecule (Kellermayer et al., 1997; Science:276-1112-1116).View Large Image | View Hi-Res Image | Download PowerPoint Slide
-
Role of Sarcomere Disruption in Stretch-Induced Force Loss of Myofibrils
Biophysical Journal, 2010Co-Authors: Appaji Panchangam, Walter HerzogAbstract:Stretching of activated skeletal muscle results in loss of force. In the absence of direct evidence, it is often assumed that sarcomere disruption is the cause of stretch-induced force loss. We stretched mechanically isolated rabbits psoas Myofibrils on the descending limb of the length-tension relationship and asked the specific questions: does sarcomere disruption occur and does it affect the magnitude of stretch-induced force loss? Myofibrils were mounted on an inverted microscope with one end attached to a glass micro needle and the other to a silicon nitrate force lever. Myofibrils (n=11) were maximally activated at an average resting sarcomere length of 2.8 ± 0.2 μm. At peak isometric stress (234 ± 92 kN m−2), Myofibrils were stretched by 34.3 ± 5.2 % at a speed of 3.3 % s−1 and immediately returned to the reference lengths at the same speed. Myofibrils were subsequently relaxed and re-activated after 3-5 minutes of rest to reassess post-stretch stress. Post-stretch isometric stress was reduced by 34 ± 9.6 % compared with pre-stretch stress. Eight out of 11 Myofibrils had no sarcomere disruption after stretching while the remaining 3 Myofibrils had a small percentage of sarcomeres pulled beyond filament overlap suggesting sarcomere disruption. The average stress reduction in the disrupted and non-disrupted Myofibrils was the same (27 ± 13 % vs 36 ± 8%; p = 0.83). We conclude from these results that stretch-induced loss of force in Myofibrils can occur in the absence of sarcomere disruption, and that sarcomere disruption does not increase force loss following myofibril stretch.
Jean M. Sanger - One of the best experts on this subject based on the ideXlab platform.
-
Myofibril assembly and the roles of the ubiquitin proteasome system
Cytoskeleton (Hoboken N.J.), 2020Co-Authors: Jushuo Wang, Syamalima Dube, Dipak K. Dube, Jean M. Sanger, Yingli Fan, Nicodeme Wanko Agassy, Joseph W. SangerAbstract:De novo assembly of Myofibrils in vertebrate cross-striated muscles progresses in three distinct steps, first from a minisarcomeric alignment of several nonmuscle and muscle proteins in preMyofibrils, followed by insertions of additional proteins and increased organization in nascent Myofibrils, ending with mature contractile Myofibrils. In a search for controls of the process of myofibril assembly, we discovered that the transition from nascent to mature Myofibrils could be halted by inhibitors of three distinct functions of the ubiquitin proteasome system (UPS). First, inhibition of pathway to E3 Cullin ligases that ubiquitinate proteins led to an arrest of myofibrillogenesis at the nascent myofibril stage. Second, inhibition of p97 protein extractions of ubiquitinated proteins led to a similar arrest of myofibrillogenesis at the nascent myofibril stage. Third, inhibitors of proteolytic action by proteasomes also blocked nascent Myofibrils from transitioning to mature Myofibrils. In contrast, inhibitors of autophagy or lysosomes did not affect myofibrillogenesis. To probe for differences in the effects of UPS inhibitors during myofibrillogenesis, we analyzed by fluorescence recovery after photobleaching the exchange rates of two selected sarcomeric proteins (muscle myosin II heavy chains and light chains). In the presence of p97 and proteasomal inhibitors, the dynamics of each of these two myosin proteins decreased in the nascent myofibril stage, but were unaffected in the mature myofibril stage. The increased stability of Myofibrils occurring in the transition from nascent to mature myofibril assembly indicates the importance of dynamics and selective destruction in the muscle myosin II proteins for the remodeling of nascent to mature Myofibrils.
-
Myofibril Assembly in Cultured Mouse Neonatal Cardiomyocytes.
Anatomical Record-advances in Integrative Anatomy and Evolutionary Biology, 2018Co-Authors: Jennifer White, Dipak K. Dube, Jushuo Wang, Joseph W. Sanger, Jean M. SangerAbstract:: The formation of Myofibrils was analyzed in neonatal mouse cardiomyocytes grown in culture and stained with fluorescent antibodies directed against myofibrillar proteins. The cardiomyocyte cultures also were exposed to siRNA probes to test the role of nonmuscle myosin IIB expression in the formation of Myofibrils. In culture, new Myofibrils formed in the spreading cell margins surrounding contractile Myofibrils previously assembled in utero. Observations indicated that assembly of mature Myofibrils occurred in three-stages, as previously reported in cultured mouse skeletal muscle. PreMyofibrils, characterized by minisarcomeres with nonmuscle myosin IIB and muscle-specific alpha-actinin bound to actin filaments, formed in the first stage; followed by nascent Myofibrils, the second stage when muscle myosin II and titin were first detected. In the mature myofibril stage muscle myosin II filaments aligned in periodic A-Bands; late assembling proteins, including myomesin and telethonin, were integrated in the sarcomeres, and nonmuscle IIB was absent from the sarcomeres. Treatment of the cultured neonatal cardiomyocytes with gene-specific siRNAs for nonmuscle myosin IIB, led to a marked decrease in the formation of preMyofibrils, and subsequently of mature Myofibrils. Anat Rec, 301:2067-2079, 2018. © 2018 Wiley Periodicals, Inc.
-
Assembly and Maintenance of Myofibrils in Striated Muscle.
Handbook of experimental pharmacology, 2016Co-Authors: Joseph W. Sanger, Dipak K. Dube, Jushuo Wang, Jean M. Sanger, Jennifer White, David PruyneAbstract:In this chapter, we present the current knowledge on de novo assembly, growth, and dynamics of striated Myofibrils, the functional architectural elements developed in skeletal and cardiac muscle. The data were obtained in studies of Myofibrils formed in cultures of mouse skeletal and quail myotubes, in the somites of living zebrafish embryos, and in mouse neonatal and quail embryonic cardiac cells. The comparative view obtained revealed that the assembly of striated Myofibrils is a three-step process progressing from preMyofibrils to nascent Myofibrils to mature Myofibrils. This process is specified by the addition of new structural proteins, the arrangement of myofibrillar components like actin and myosin filaments with their companions into so-called sarcomeres, and in their precise alignment. Accompanying the formation of mature Myofibrils is a decrease in the dynamic behavior of the assembling proteins. Proteins are most dynamic in the preMyofibrils during the early phase and least dynamic in mature Myofibrils in the final stage of myofibrillogenesis. This is probably due to increased interactions between proteins during the maturation process. The dynamic properties of myofibrillar proteins provide a mechanism for the exchange of older proteins or a change in isoforms to take place without disassembling the structural integrity needed for myofibril function. An important aspect of myofibril assembly is the role of actin-nucleating proteins in the formation, maintenance, and sarcomeric arrangement of the myofibrillar actin filaments. This is a very active field of research. We also report on several actin mutations that result in human muscle diseases.
-
Ectopic expression and dynamics of TPM1alpha and TPM1kappa in Myofibrils of avian myotubes.
Cell motility and the cytoskeleton, 2007Co-Authors: Jushuo Wang, Dipak K. Dube, Songman Kang, Jean M. Sanger, Harold L. Thurston, Lynn Abbott, Joseph W. SangerAbstract:From the four known vertebrate tropomyosin genes (designated TPM1, TPM2, TPM3, and TPM4) over 20 isoforms can be generated. The predominant TPM1 isoform, TPM1alpha, is specifically expressed in both skeletal and cardiac muscles. A newly discovered alternatively spliced isoform, TPM1kappa, containing exon 2a instead of exon 2b contained in TPM1alpha, was found to be cardiac specific and developmentally regulated. In this work, we transfected quail skeletal muscle cells with green fluorescent proteins (GFP) coupled to chicken TPM1alpha and chicken TPM1kappa and compared their localizations in preMyofibrils and mature Myofibrils. We used the technique of fluorescence recovery after photobleaching (FRAP) to compare the dynamics of TPM1alpha and TPM1kappa in myotubes. TPM1alpha and TPM1kappa incorporated into preMyofibrils, nascent Myofibrils, and mature Myofibrils of quail myotubes in identical patterns. The two tropomyosin isoforms have a higher exchange rate in preMyofibrils than in mature Myofibrils. F-actin and muscle tropomyosin are present in the same fibers at all three stages of myofibrillogenesis (preMyofibrils, nascent Myofibrils, mature Myofibrils). In contrast, the tropomyosin-binding molecule nebulin is not present in the initial preMyofibrils. Nebulin is gradually added during myofibrillogenesis, becoming fully localized in striated patterns by the mature myofibril stage. A model of thin filament formation is proposed to explain the increased stability of tropomyosin in mature Myofibrils. These experiments are supportive of a maturing thin filament and stepwise model of myofibrillogenesis (preMyofibrils to nascent Myofibrils to mature Myofibrils), and are inconsistent with models that postulate the immediate appearance of fully formed thin filaments or Myofibrils.
-
how to build a myofibril
Journal of Muscle Research and Cell Motility, 2006Co-Authors: Joseph W. Sanger, Jushuo Wang, Songman Kang, Cornelia C Siebrands, Nancy L Freeman, Aiping Du, Andrea L Stout, Jean M. SangerAbstract:Building a myofibril from its component proteins requires the interactions of many different proteins in a process whose details are not understood. Several models have been proposed to provide a framework for understanding the increasing data on new myofibrillar proteins and their localizations during muscle development. In this article we discuss four current models that seek to explain how the assembly occurs in vertebrate cross-striated muscles. The models hypothesize: (a) stress fiber-like structures as templates for the assembly of Myofibrils, (b) assembly in which the actin filaments and Z-bands form subunits independently from A-band subunits, with the two subsequently joined together to form a myofibril, (c) preMyofibrils as precursors of Myofibrils, or (d) assembly occurring without any intermediary structures. The premyofibril model, proposed by the authors, is discussed in more detail as it could explain myofibrillogenesis under a variety of different conditions: in ovo, in explants, and in tissue culture studies on cardiac and skeletal muscles.
Henry Shuman - One of the best experts on this subject based on the ideXlab platform.
-
Distribution and orientation of rhodamine‐phalloidin bound to thin filaments in skeletal and cardiac Myofibrils
Cytoskeleton, 1997Co-Authors: Vladimir Zhukarev, Yale E. Goldman, Jean M. Sanger, Henry ShumanAbstract:Phalloidin staining of muscle does not reflect the known disposition of sarcomeric thin filaments. Quantitative image analysis and steady-state fluorescence polarization microscopy are used to measure the local intensity and orientation of tetramethyl rhodamine-labeled phalloidin (TR-phalloidin) in skinned Myofibrils. TR-phalloidin staining of isolated skeletal Myofibrils labeled while in rigor reveals fluorescence that is brighter at the pointed ends of the thin filaments and Z lines than it is in the middle of the filaments. In cardiac Myofibrils, phalloidin staining is uniform along the lengths of the thin filaments in both relaxed and rigor Myofibrils, except in 0.2-μm dark areas on either side of the Z line. Extraction of myosin or tropomyosin-troponin molecules does not change the nonuniform staining. To test whether long-term storage in glycerol changes the binding of phalloidin to thin filaments in Myofibrils, minimally permeabilized (briefly skinned) Myofibrils, or Myofibrils stored in glycerol for at least 7 days (glycerol extraction) were compared. TR-phalloidin was well ordered throughout the sarcomere in briefly skinned skeletal and cardiac Myofibrils, but TR-phalloidin bound to the Z line and pointed ends of thin filaments was randomly oriented in glycerol-extracted Myofibrils, suggesting that the ends of the thin filaments become disordered after glycerol extraction. In relaxed skeletal Myofibrils with sarcomere lengths greater than 3.0 μm, staining was nearly uniform all along the actin filaments. Exogenous bare actin filaments polymerized from the Z line (Sanger et al., 1984: J. Cell Biol. 98:825–833) in and along the myofibril bind rhodamine phalloidin uniformly. Our results support the hypothesis that nebulin can block the binding of phalloidin to actin in skeletal Myofibrils and nebulette can block phalloidin binding to cardiac thin filaments. Cell Motil. Cytoskeleton 37:363–377, 1997. © 1997 Wiley-Liss, Inc.
-
Distribution and orientation of rhodamine-phalloidin bound to thin filaments in skeletal and cardiac Myofibrils.
Cell motility and the cytoskeleton, 1997Co-Authors: Vladimir Zhukarev, Yale E. Goldman, Jean M. Sanger, Joseph W. Sanger, Henry ShumanAbstract:Phalloidin staining of muscle does not reflect the known disposition of sarcomeric thin filaments. Quantitative image analysis and steady-state fluorescence polarization microscopy are used to measure the local intensity and orientation of tetramethyl rhodamine-labeled phalloidin (TR-phalloidin) in skinned Myofibrils. TR-phalloidin staining of isolated skeletal Myofibrils labeled while in rigor reveals fluorescence that is brighter at the pointed ends of the thin filaments and Z lines than it is in the middle of the filaments. In cardiac Myofibrils, phalloidin staining is uniform along the lengths of the thin filaments in both relaxed and rigor Myofibrils, except in 0.2-μm dark areas on either side of the Z line. Extraction of myosin or tropomyosin-troponin molecules does not change the nonuniform staining. To test whether long-term storage in glycerol changes the binding of phalloidin to thin filaments in Myofibrils, minimally permeabilized (briefly skinned) Myofibrils, or Myofibrils stored in glycerol for at least 7 days (glycerol extraction) were compared. TR-phalloidin was well ordered throughout the sarcomere in briefly skinned skeletal and cardiac Myofibrils, but TR-phalloidin bound to the Z line and pointed ends of thin filaments was randomly oriented in glycerol-extracted Myofibrils, suggesting that the ends of the thin filaments become disordered after glycerol extraction. In relaxed skeletal Myofibrils with sarcomere lengths greater than 3.0 μm, staining was nearly uniform all along the actin filaments. Exogenous bare actin filaments polymerized from the Z line (Sanger et al., 1984: J. Cell Biol. 98:825–833) in and along the myofibril bind rhodamine phalloidin uniformly. Our results support the hypothesis that nebulin can block the binding of phalloidin to actin in skeletal Myofibrils and nebulette can block phalloidin binding to cardiac thin filaments. Cell Motil. Cytoskeleton 37:363–377, 1997. © 1997 Wiley-Liss, Inc.
Wolfgang A Linke - One of the best experts on this subject based on the ideXlab platform.
-
actin titin interaction in cardiac Myofibrils probing a physiological role
Biophysical Journal, 1997Co-Authors: Wolfgang A Linke, Marc Ivemeyer, Horst Hinssen, J. Caspar Rüegg, Siegfried Labeit, Mathias GautelAbstract:The high stiffness of relaxed cardiac Myofibrils is explainable mainly by the expression of a short-length titin (connectin), the giant elastic protein of the vertebrate myofibrillar cytoskeleton. However, additional molecular features could account for this high stiffness, such as interaction between titin and actin, which has previously been reported in vitro. To probe this finding for a possible physiological significance, isolated Myofibrils from rat heart were subjected to selective removal of actin filaments by a calcium-independent gelsolin fragment, and the "passive" stiffness of the specimens was recorded. Upon actin extraction, stiffness decreased by nearly 60%, and to a similar degree after high-salt extraction of thick filaments. Thus actin-titin association indeed contributes to the stiffness of resting cardiac muscle. To identify possible sites of association, we employed a combination of different techniques. Immunofluorescence microscopy revealed that actin extraction increased the extensibility of the previously stiff Z-disc-flanking titin region. Actin-titin interaction within this region was confirmed in in vitro cosedimentation assays, in which multimodule recombinant titin fragments were tested for their ability to interact with F-actin. By contrast, such assays showed no actin-titin-binding propensity for sarcomeric regions outside the Z-disc comb. Accordingly, the results of mechanical measurements demonstrated that competition with native titin by recombinant titin fragments from Z-disc-remote, I-band or A-band regions did not affect passive myofibril stiffness. These results indicate that it is actin-titin association near the Z-disc, but not along the remainder of the sarcomere, that helps to anchor the titin molecule at its N-terminus and maintain a high stiffness of the relaxed cardiac myofibril.
-
basis of passive tension and stiffness in isolated rabbit Myofibrils
American Journal of Physiology-cell Physiology, 1997Co-Authors: Marc L Bartoo, Wolfgang A Linke, Gerald H. PollackAbstract: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.
-
Passive and active tension in single cardiac Myofibrils.
Biophysical Journal, 1994Co-Authors: Wolfgang A Linke, V I Popov, Gerald H. PollackAbstract:Single Myofibrils were isolated from chemically skinned rabbit heart and mounted in an apparatus described previously (Fearn et al., 1993; Linke et al., 1993). We measured the passive length-tension relation and active isometric force, both normalized to cross sectional area. Myofibrillar cross sectional area was calculated based on measurements of myofibril diameter from both phase-contrast images and electron micrographs. Passive tension values up to sarcomere lengths of approximately 2.2 microns were similar to those reported in larger cardiac muscle specimens. Thus, the element responsible for most, if not all, passive force of cardiac muscle at physiological sarcomere lengths appears to reside within the Myofibrils. Above 2.2 microns, passive tension continued to rise, but not as steeply as reported in multicellular preparations. Apparently, structures other than the Myofibrils become increasingly important in determining the magnitude of passive tension at these stretched lengths. Knowing the myofibrillar component of passive tension allowed us to infer the stress-strain relation of titin, the polypeptide thought to support passive force in the sarcomere. The elastic modulus of titin is 3.5 x 10(6) dyn cm-2, a value similar to that reported for elastin. Maximum active isometric tension in the single myofibril at sarcomere lengths of 2.1–2.3 microns was 145 +/- 35 mN/mm2 (mean +/- SD; n=15). This value is comparable with that measured in fixed-end contractions of larger cardiac specimens, when the amount of nonmyofibrillar space in those preparations is considered. However, it is about 4 times lower than the maximum active tension previously measured in single skeletal Myofibrils under similar conditions (Bartoo et al., 1993).
