Intact Sarcomeres Research Articles

Carbon dioxide can inhibit the spoilage potential of Shewanella putrefaciens target at protein in large yellow croakers

In this research, the effect of CO2 (0%, 20%, 60%, and 100%) on the spoilage potential of Shewanella putrefaciens (S. putrefaciens) during 4 °C cold storage was evaluated. Results revealed that the growth curves of S. putrefaciens were effectively affected by CO2 with prolonged lag phases and delayed logarithmic phases. Protein oxidation of inoculated large yellow croakers was noticeably delayed with the treatment of CO2 by keeping higher myofibrillar protein, total sulfhydryl groups and Ca2+-ATPase activity, and lower carbonyl groups. Spatial structures of samples treated with 20%–100% CO2 were better in organized secondary structure and better-stabilized tertiary structure. Meanwhile, the myofibrillar microstructure detected by scanning electron microscope (SEM) and transmission electron microscopy (TEM) was also much well-organized than the control, with relative tightly attached myofibrils and intact sarcomere structure (clear A-band, I-band and M-lines). Furthermore, CO2 treated samples have better texture and stronger moisture holding capacity. These results synergistically demonstrated that the spoilage ability of S. putrefaciens target at proteins was effectively suppressed by CO2.

Structures from intact myofibrils reveal mechanism of thin filament regulation through nebulin.

In skeletal muscle, nebulin stabilizes and regulates the length of thin filaments, but the underlying mechanism remains nebulous. In this work, we used cryo-electron tomography and subtomogram averaging to reveal structures of native nebulin bound to thin filaments within intact sarcomeres. This in situ reconstruction provided high-resolution details of the interaction between nebulin and actin, demonstrating the stabilizing role of nebulin. Myosin bound to the thin filaments exhibited different conformations of the neck domain, highlighting its inherent structural variability in muscle. Unexpectedly, nebulin did not interact with myosin or tropomyosin, but it did interact with a troponin T linker through two potential binding motifs on nebulin, explaining its regulatory role. Our structures support the role of nebulin as a thin filament "molecular ruler" and provide a molecular basis for studying nemaline myopathies.

Abstract 273: Utilization of Proximity-labeling to Identify Sarcomere Assembly Dependent Interactions

The cardiac sarcomere is a multiprotein complex that generates pulsatile contractile force, enabling the heart to circulate vital nutrients, but its molecular components and dynamic interactions are incompletely understood. Cardiac alpha-actinin-2 (encoded by ACTN2 ; referred to as actinin) functions as the structural backbone of the Z-disc and is prone to mutations that result in human heart failure. We hypothesize that through identifying actinin PPIs at the Z-disc, we can identify new sarcomere functions and gain insights into heart failure pathogenesis. Until now, studying sarcomere PPIs has been challenging due to limitations in purifying intact sarcomere complexes and the inability to detect dynamic molecular interactions. To overcome this, we adapted BioID proximity proteomics to comprehensively study actinin PPIs in human induced pluripotent stem cells (hiPSCs) differentiated into cardiomyocytes (iCMs).We engineered an isogenic hiPSC line carrying an in-frame knock-in of BirA* , which encodes a promiscuous biotin ligase, at the ACTN2 locus. Upon differentiation into iCMs, the Actinin-BirA* fusion construct localizes to the Z-disc and, upon biotin supplementation, biotinylates vicinal proteins. We immunoprecipitated and analyzed Z-disc PPIs via quantitative tandem mass spectrometry. Among the >100 PPIs detected by this method, we identified known interactions including sarcomere and focal adhesion proteins, but also new interactions including proteins involved in translation and metabolism. Furthermore, we wanted to better understand which of these protein interactions are dependent on sarcomere assembly and function, potentially elucidating interactions involved in normal cardiomyocyte biology and disease states. Through disruption of sarcomere assembly in our Actinin-BirA* model, we were also able to identify sarcomere-dependent actinin interactions including specific proteins involved in translation, the unfolded protein response, and glycolysis. Further investigation will allow us to better understand the proteomic network around actinin and provide novel insights into human sarcomere biology.

Extraction of Thick Filaments in Individual Sarcomeres Affects Force Production by Single Myofibrils

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 because 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 and relaxed using a perfusion system. An extra microperfusion needle filled with a 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 < 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.

The exon junction complex senses energetic stress and regulates contractility and cell architecture in cardiac myocytes.

The exon junction complex (EJC) is the main mechanism by which cells select specific mRNAs for translation into protein. We hypothesized that the EJC is involved in the regulation of gene expression during the stress response in cardiac myocytes, with implications for the failing heart. In cultured rat neonatal myocytes, we examined the cellular distribution of two EJC components eukaryotic translation initiation factor 4A isoform 3 (eIF4A3) and mago nashi homologue (Mago) in response to metabolic stress. There was significant relocalization of eIF4A3 and Mago from the nucleus to cytoplasm following 18 h of hypoxia. Treating myocytes with 50 mM NaN3 for 4 h to mimic the metabolic stress induced by hypoxia also resulted in significant relocalization of eIF4A3 and Mago to the cytoplasm. To examine whether the effects of metabolic stress on the EJC proteins were dependent on the metabolic sensor AMP kinase (AMPK), we treated myocytes with 1 μM dorsomorphin (DM) in combination with NaN3. DM augmented the translocation of Mago and eIF4A3 from the nucleus to the cytoplasm. Knockdown of eIF4A3 resulted in cessation of cell contractility 96 h post-treatment and a significant reduction in the number of intact sarcomeres. Cell area was significantly reduced by both hypoxia and eIF4A3 knockdown, whilst eIF4A3 knockdown also significantly reduced nuclear size. The reduction in nuclear size is unlikely to be related to apoptosis as it was reversed in combination with hypoxia. These data suggest for the first time that eIF4A3 and potentially other EJC members play an important role in the myocyte stress response, cell contractility and morphology.

Predicting Effects of Tropomyosin Mutations on Cardiac Muscle Contraction through Myofilament Modeling.

Point mutations to the human gene TPM1 have been implicated in the development of both hypertrophic and dilated cardiomyopathies. Such observations have led to studies investigating the link between single residue changes and the biophysical behavior of the tropomyosin molecule. However, the degree to which these molecular perturbations explain the performance of intact sarcomeres containing mutant tropomyosin remains uncertain. Here, we present a modeling approach that integrates various aspects of tropomyosin's molecular properties into a cohesive paradigm representing their impact on muscle function. In particular, we considered the effects of tropomyosin mutations on (1) persistence length, (2) equilibrium between thin filament blocked and closed regulatory states, and (3) the crossbridge duty cycle. After demonstrating the ability of the new model to capture Ca-dependent myofilament responses during both dynamic and steady-state activation, we used it to capture the effects of hypertrophic cardiomyopathy (HCM) related E180G and D175N mutations on skinned myofiber mechanics. Our analysis indicates that the fiber-level effects of the two mutations can be accurately described by a combination of changes to the three tropomyosin properties represented in the model. Subsequently, we used the model to predict mutation effects on muscle twitch. Both mutations led to increased twitch contractility as a consequence of diminished cooperative inhibition between thin filament regulatory units. Overall, simulations suggest that a common twitch phenotype for HCM-linked tropomyosin mutations includes both increased contractility and elevated diastolic tension.

Computing Average Passive Forces in Sarcomeres in Length-Ramp Simulations.

Passive forces in sarcomeres are mainly related to the giant protein titin. Titin’s extensible region consists of spring-like elements acting in series. In skeletal muscles these elements are the PEVK segment, two distinct immunoglobulin (Ig) domain regions (proximal and distal), and a N2A portion. While distal Ig domains are thought to form inextensible end filaments in intact sarcomeres, proximal Ig domains unfold in a force- and time-dependent manner. In length-ramp experiments of single titin strands, sequential unfolding of Ig domains leads to a typical saw-tooth pattern in force-elongation curves which can be simulated by Monte Carlo simulations. In sarcomeres, where more than a thousand titin strands are arranged in parallel, numerous Monte Carlo simulations are required to estimate the resultant force of all titin filaments based on the non-uniform titin elongations. To simplify calculations, the stochastic model of passive forces is often replaced by linear or non-linear deterministic and phenomenological functions. However, new theories of muscle contraction are based on the hypothesized binding of titin to the actin filament upon activation, and thereby on a prominent role of the structural properties of titin. Therefore, these theories necessitate a detailed analysis of titin forces in length-ramp experiments. In our study we present a simple and efficient alternative to Monte Carlo simulations. Based on a structural titin model, we calculate the exact probability distributions of unfolded Ig domains under length-ramp conditions needed for rigorous analysis of expected forces, distribution of unfolding forces, etc. Due to the generality of our model, the approach is applicable to a wide range of stochastic protein unfolding problems.

The Exon Junction Complex proteins are non‐AMP kinase dependent sensors of energetic stress and regulate contractility and sarcomere structure in cardiac myocytes

The heart is known to undergo significant alterations in gene expression during stress and sometimes this adaptation can lead to heart failure. The mechanism by which cells select specific mRNAs for translation is not fully understood, but the Exon Junction Complex (EJC) plays a major role in this process in eukaryotes. The EJC selects the mRNA isoforms to be exported to the cytoplasm and determines whether they are then translated into protein. In this study we hypothesised that the EJC is involved in the regulation of gene expression during the stress response in cardiac myocytes. Using cultured rat neonatal myocytes and subcellular fractionation we examined the cellular distribution of two major components of the EJC in response to metabolic stress. In cardiac myocytes we found that there was a significant relocalisation of eIF4A3 and mago from the nucleus to cytoplasm following 18 hours of hypoxia. To examine the effect of metabolic stress on the EJC in more detail we treated cardiac myocytes with 50mM NaN3 for 4 hours to mimic the metabolic stress induced by hypoxia. Following NaN3 treatment there was a significant relocalisation of eIF4A3 and mago to the cytoplasm and this was associated with the formation of cytoplasmic stress granules. However, immunocytochemical staining with the stress granule marker PABP1 showed that cytoplasmic mago did not colocalise with stress granules. To examine whether the effects of metabolic stress on the EJC proteins was dependent on the metabolic sensor AMP kinase (AMPK), we treated myocytes with 1μM dorsomorphin in combination with NaN3. Dorsomorphin augmented the translocation of mago and eIF4A3 from the nucleus to the cytoplasm. This indicates that the EJC proteins respond to metabolic stress independently of AMPK activity. Moreover, the inhibition of AMPK was associated with increased metabolic cellular stress resulting in the augmented subcellular translocation of EJC proteins. Constructs of eIF4A3 tagged with green fluorescence protein on the N terminus resulted in a predominantly cytosolic protein distribution that did not relocalise in response to NaN3 treatment. This suggests that the N‐terminus of eIF4A3 is necessary for subcellular localisation. Finally, to determine the function of eIF4A3 in cardiac myocytes we knocked down the protein with siRNA. Protein knockdown of eIF4A3 resulted in cessation of cell contractility 96 hours post treatment. There was also a significant reduction in the number of intact sarcomeres suggesting that the contractile filaments were degrading in response to reduced eIF4A3. Taken together, these data suggest that eIF4A3 and other EJC members play an important role in the myocyte stress response, cell contractility and morphology.Support or Funding InformationThis work was supported by the British Heart Foundation project grant PG/10/64/28520 and PG/10/97/28666

Different actions of salt and pyrophosphate on protein extraction from myofibrils reveal the mechanism controlling myosin dissociation.

Myosin is the major functional protein in muscle foods for water retention, protein binding/gelation and fat holding/emulsification. To maximize its functionality, myosin needs to be released from thick filaments. Understanding of the mechanism controlling myosin extraction will help improve quality traits of meat products. The data obtained show that actomyosin binding is the rate-limiting constraint for myosin release in rigor condition. Magnesium pyrophosphate (MgPPi) increased myosin extraction by weakening actomyosin interaction and maximized myosin extraction at 0.4 mol L(-1) NaCl, which was not attained at 1.0 mol L(-1) NaCl in the absence of PPi. Interaction between myosin rod domains is another critical constraint for myosin extraction, which is, rather than PPi, salt dependent. Further, our data suggest that MyBP-C (myosin binding protein C) and M-line might not be of significance in the process of NaCl-induced myosin extraction, though further study was needed. Our study provides new insight into the mechanism that controls myosin extraction from intact sarcomere, which could be applied to maximize myosin function and to improve meat quality in practice.

Abstract 190: A Mechanotransduction Study In Embryonic Zebrafish Heart

Background: While proper heart contraction requires an intact sarcomere structure, contraction reciprocally regulates sarcomere assembly and cardiomyocyte shape during heart morphogenesis and heart remodeling. However, how contraction modulates heart morphogenesis and the underlying molecular mechanism remains unclear. Here we address this question by manipulating cardiac contractility in a developing zebrafish embryo. Results: Cessation of heart contraction was achieved by treating zebrafish embryos with blebbistatin, a myosin II specific inhibitor, or Niferdipine, a Ca 2+ channel inhibitor. Loss of cardiac contractility reduces ventricular chamber size in 1 dpf embryo, but significantly increases ventricular chamber size in 2 dpf and 3 dpf embryos. In both circumstances, cessation of contraction consistently prevents or disrupts the lateral fusion of myofibril. Cessation of contraction-induced heart enlargement is reversible and also dependent on the time of drug treatment. This heart-remodeling event in 2dpf embryo, which manifests as increased cardiomyocyte surface area accompanied by reduced thickness, can be attenuated by reducing the transmural pressure towards the ventricle wall. This mechanotransductive process occurs in the myocardium without need of endocardium. At the molecular level, by screening several compounds, we found that PI3K signaling confers this contraction-regulated cardiomyocyte shape remodeling process without affecting the lateral fusion of sarcomere. Conclusion: Our data in an in vivo animal model distinguish two different and relatively independent functions of contraction in regulating lateral fusion of sarcomere and cardiomyocytes shape remodeling. The contraction-regulated heart shape remodeling is conveyed by PI3k signaling mediated mechanotransduction independent of endocardium.

Modulation of stretch-induced myocyte remodeling and gene expression by nitric oxide: a novel role for lipoma preferred partner in myofibrillogenesis

Prolonged hemodynamic load as a result of hypertension eventually leads to maladaptive cardiac adaptation and heart failure. The signaling pathways that underlie these changes are still poorly understood. The adaptive response to mechanical load is mediated by mechanosensors that convert the mechanical stimuli into a biological response. We examined the effect of cyclic mechanical stretch on myocyte adaptation using neonatal rat ventricular myocytes with 10% (adaptive) or 20% (maladaptive) maximum strain at 1 Hz for 48 h to mimic in vivo mechanical stress. Cells were also treated with and without nitro-L-arginine methyl ester (L-NAME), a general nitric oxide synthase (NOS) inhibitor to suppress NO production. Maladaptive 20% mechanical stretch led to a significant loss of intact sarcomeres that were rescued by L-NAME (P < 0.05; n ≥ 5 cultures). We hypothesized that the mechanism was through NO-induced alteration of myocyte gene expression. L-NAME upregulated the mechanosensing proteins muscle LIM protein (MLP; by 100%; P < 0.05; n = 5 cultures) and lipoma preferred partner (LPP), a novel cardiac protein (by 80%; P < 0.05; n = 4 cultures). L-NAME also significantly altered the subcellular localization of LPP and MLP in a manner that favored growth and adaptation. These findings suggest that NO participates in stretch-mediated adaptation. The use of isoform selective NOS inhibitors indicated a complex interaction between inducible NOS and neuronal NOS isoforms regulate gene expression. LPP knockdown by small intefering RNA led to formation of α-actinin aggregates and Z bodies showing that myofibrillogenesis was impaired. There was an upregulation of E3 ubiquitin ligase (MUL1) by 75% (P < 0.05; n = 5 cultures). This indicates that NO contributes to stretch-mediated adaptation via the upregulation of proteins associated with mechansensing and myofibrillogenesis, thereby presenting potential therapeutic targets during the progression of heart failure.

Modulating Contraction by Binding of MyBP-C to Actin

Myosin-binding protein C (MyBP-C) is a ∼130 kDa protein of the thick filaments of vertebrate skeletal and cardiac muscle. It consists of a linear array of ten or eleven globular, 10-kDa domains from the immunoglobulin (Ig) and fibronectin type III families, and an additional, MyBP-C-specific motif. The cardiac isoform, cMyBP-C, plays a key role in modulating cardiac function, and mutations in MyBP-C cause heart disease. Despite its discovery 40 years ago, the mechanism of MyBP-C function remains poorly understood. In vitro studies suggest that it could modulate contraction by binding to thin filaments, but there has been no evidence for this in situ. We used electron tomography of exceptionally well-preserved skeletal muscle to study the 3D organization of MyBP-C in the intact sarcomere. The tomogram shows that MyBP-C projects perpendicular to the thick filament surface and reaches neighboring thin filaments. This thick-thin filament bridge suggests a possible physical basis for modulating filament sliding and thus contraction. In vitro, binding to actin has been shown to occur via MyBP-C's N-terminal end. To understand the structural basis of this binding, we used negative stain electron microscopy and 3D reconstruction to study F-actin decorated with bacterially expressed N-terminal cMyBP-C fragments. Clear decoration was obtained under a variety of salt conditions. 3D reconstructions showed MyBP-C density starting over subdomain 1 of actin and extending tangentially towards actin's pointed end. Molecular fitting with an atomic structure of a MyBP-C Ig domain suggested that most of the N-terminal domains may be well ordered on actin. The location of binding was such that it appeared to overlap the relaxed (low Ca2+) position of tropomyosin but not the activated position. This suggests that MyBP-C might help determine the state of thin filament activity by modulating tropomyosin position on actin.

Modulation of striated muscle contraction by binding of myosin binding protein C to actin

Myosin binding protein C (MyBP-C or C-protein) is a protein of the thick (myosin-containing) filaments of striated muscle thought to be involved in the modulation of cardiac contraction in response to β-adrenergic stimulation. The mechanism of this modulation is unknown, but one possibility is through transient binding of the N-terminal end of MyBP-C to the thin (actin-containing) filaments. While such binding has been demonstrated in vitro, it was not known until recently whether such a link between thick and thin filaments also occurred in vivo. Here we review a recent paper in which electron microscopy (EM) is used to directly demonstrate MyBP-C links between myosin and actin filaments in the intact sarcomere, suggesting a possible physical mechanism for modulating filament sliding. Molecular details of MyBP-C binding to actin have recently been elucidated by EM of isolated filaments: the results suggest that MyBP-C might contribute to the modulation of contraction in part by competing with tropomyosin for binding sites on actin. New results on the structure and dynamics of the MyBP-C molecule provide additional insights into the function of this enigmatic molecule.

Effect of pH and Electrostatic Interactions on Myofilament Lattice Volume

Changes interfilament lattice spacing is a major determinant of force production in striated muscle with reductions in force generation being observed with both expansion and compression of the lattice spacing relative to the normal physiological values. Previous studies have concluded that lattice spacing depends complexly on the balance of outward repulsive forces and inward attractive and compressive forces between thick and thin filaments. Since lattice spacing has important implications on force generation, we examined the effects of alterations in filament charge, induced by changes in intracellular pH (by the rapid application and withdrawal of 30 mM NH4Cl), on lattice spacing in intact twitching cardiac trabeculae. Since we observed changes in sarcomere length in response to changes in intracellular pH and since changes in sarcomere length induce changes in lattice spacing as a result of the isovolumic volume behavior of intact sarcomeres, sarcomere length was maintained at a fixed value (∼2.2 μm) during the pH interventions. Lattice spacing increased (p < 0.05) following NH4Cl wash in (measured after 5 minutes) when the pH is estimated to increase to ∼7.8 (Balnave 2000 J. Physiol.) to 36.9 +/-0.2. Following the washout of NH4Cl (which is estimated to decrease pH to ∼6.5 (Swietach 2005 J. Physiol.)) lattice spacing increased to 37.6+/−0.4 elevated above (P < 0.05) the space observed at control pH. Since the isoelectric point for myofilaments is ∼5 (Naylor, Biophys J, 1985), our findings suggest that, in addition to electrostatic, van der Waals' and osmotic forces, other pH-sensitive forces are also critical determinants of the lattice spacing, possibly the M-protein, myomesin or other M-line proteins which are postulated to hold thick filaments together in the M-band of striated muscle.

Calcium‐induced contraction of sarcomeres changes the regulation of mitochondrial respiration in permeabilized cardiac cells

The relationships between cardiac cell structure and the regulation of mitochondrial respiration were studied by applying fluorescent confocal microscopy and analysing the kinetics of mitochondrial ADP-stimulated respiration, during calcium-induced contraction in permeabilized cardiomyocytes and myocardial fibers, and in their 'ghost' preparations (after selective myosin extraction). Up to 3 microm free calcium, in the presence of ATP, induced strong contraction of permeabilized cardiomyocytes with intact sarcomeres, accompanied by alterations in mitochondrial arrangement and a significant decrease in the apparent K(m) for exogenous ADP and ATP in the kinetics of mitochondrial respiration. The V(max) of respiration showed a moderate (50%) increase, with an optimum at 0.4 microm free calcium and a decrease at higher calcium concentrations. At high free-calcium concentrations, the direct flux of ADP from ATPases to mitochondria was diminished compared to that at low calcium levels. All of these effects were unrelated either to mitochondrial calcium overload or to mitochondrial permeability transition and were not observed in 'ghost' preparations after the selective extraction of myosin. Our results suggest that the structural changes transmitted from contractile apparatus to mitochondria modify localized restrictions of the diffusion of adenine nucleotides and thus may actively participate in the regulation of mitochondrial function, in addition to the metabolic signalling via the creatine kinase system.

Measurements of the cross-bridge attachment/detachment process within intact sarcomeres by surface plasmon resonance.

We have developed a surface plasmon resonance (SPR) system to monitor the cross-bridge attachment/detachment process within intact sarcomeres from mouse heart muscle. SPR occurs when laser light energy is transferred to surface plasmons that are resonantly excited in a metal (gold) film. This resonance manifests itself as a minimum in the reflection of the incident laser light and occurs at a characteristic angle. The angle of the SPR occurrence depends on the dielectric permittivity of the sample medium adjacent to the gold film. Purified sarcomeric preparations are immobilized onto the gold film in the presence of a relaxing solution. Replacement of the relaxing solution with increasing Ca(2+) concentration solution activates the cross-bridge interaction and produces an increase in the SPR angle. These results imply that the interaction of myosin heads with actin within an intact sarcomere changes the dielectric permittivity of the sarcomeric structure. In addition, we further verify that SPR measurements can detect the changes in the population of the attached cross-bridges with altered concentrations of phosphate, 2,3-butanedione monoxime, or adenosine triphosphate at a fixed calcium concentration, which have been shown to reduce the force and increase the cross-bridge population in attached state. Thus, our data provide the first evidence that the SPR technique allows the monitoring of the cross-bridge attachment/detachment process within intact sarcomeres.

Sarcomeric visco-elasticity of chemically skinned skeletal muscle fibres of the rabbit at rest.

The giant muscle protein titin (connectin), contained in the gap filament that connect a thick filament to the Z-line in a sarcomere, is generally considered to be responsible for the passive force (tension) and visco-elasticity in resting striated muscle. However, whether it can account for all the features of the resting tension response remains unclear. In this paper, we examine the basic features of the 'sarcomeric visco-elasticity' in a single resting mammalian muscle fibre and attempt to account for various tension components on the basis of known structural features of a sarcomere. At sarcomere length of approximately 2.6 microm, the force response to a ramp stretch of 2-5% is complex but can be resolved into four functionally different components. The behaviour displayed by the components ranges from pure viscous type (directly proportional to stretch velocity, ranging from 0.1 to 30 lengths s(-1)) to predominantly elastic type (insensitive to stretch velocity at 1-2 s time scale); simulations show two components of visco-elasticity with characteristically different relaxation times. The velocity-sensitive components (only) are enhanced by filament lattice compression (dextran - 500 kD) and by increased medium viscosity (dextran - 12 kD); also, the relaxation time of visco-elasticity is longer with increased medium viscosity. Amplitude of all the components and the relaxation time of visco-elasticity are increased at longer sarcomere length (range approximately 2.5 - 3.0 microm). The study, and quantitative analyses, extend our previous work on intact muscle fibres and suggest that the velocity-sensitive tension components in intact sarcomere arise from interactions between sarcomeric filaments, filament segments and inter-filamentary medium; the two components of visco-elasticity arise from distinct regions of titin (connectin) molecules.

β-Tropomyosin Overexpression Induces Severe Cardiac Abnormalities

Tropomyosin, a coiled-coil dimer, stabilizes actin filaments and is central to the control of calcium-regulated striated muscle contraction. Striated muscle-specificα-tropomyosin is the predominant isoform in cardiac muscle, with low levels ofβ-tropomyosin restricted to fetal development in the mouse. To understand the functional role of various tropomyosin isoforms during myofilament activation and regulation in the intact sarcomere, we generated transgenic mice that overexpress striated muscle-specificβ-tropomyosin in the adult heart. Our earlier results succinctly demonstrate that overexpression ofβ-tropomyosin in the hearts of transgenic mice decreases endogeneousα-tropomyosin levels while altering diastolic function of the myocardium. To explore further the significance of altering theα- toβ-tropomyosin isoform ratio in developing murine myocardium, we generated transgenic mice which expressβ-tropomyosin at high levels in the heart. The data show that higher levels ofβ-tropomyosin expression are lethal with death ensuing between 10–14 days postnatally. A detailed histological analysis demonstrates that the hearts of these mice exhibit several pathological abnormalities, including thrombus formation in the lumen of both atria and in the subendocardium of the left ventricle. Other changes include atrial enlargement and fibrosis, and diffuse myocytolysis, Physiological analyses using ventricular muscle strip preparations from these mice reveal that both myocardial contraction and relaxation parameters are severely impaired. Thus, these results firmly demonstrate an essential difference in tropomyosin isoform function in physiologically regulating cardiac performance.

Implications of quantal motor action in biological systems.

We demonstrate in this paper that quantal behavior is a central feature of biological motile and contractile systems. Step-like behavior has been demonstrated in the interaction between single molecules and filaments both in the kinesin-microtubule system and in the myosin-actin filament system. We show here that the step-like molecular features appear also in the single intact sarcomere. We studied single sarcomeres of single bumble-bee myofibrils, both in the unactivated and activated states. Myofibril-length changes induced by a motor-imposed ramp were accompanied by corresponding sarcomere-length changes. However, the sarcomere-length changes were stepwise. Computer analysis of the stepwise shortening patterns revealed a step-size distribution containing multiple peaks. In the activated state, the peaks were separated by 2.7 nm per half-sarcomere which is the linear actin-subunit spacing. Thus, translation steps are an integer multiple of the actin-subunit spacing. This result parallels the one observed in the kinesin-tubulin spacing, where step size is a multiple of the tubulin-subunit spacing. In the muscle system, however, the steps are preserved on a macroscopic scale, implying high synchrony. The quantal steps are easily explained by a model in which the actin filament propels itself over stationary cross-bridges: if actin binds to the cross-bridges between steps, then the observed quantal result is inevitable. As probes of contractile phenomena approach the molecular level, the discrete unitary events underlying contraction begin to emerge. Thus, step-like behavior is observed as the single kinesin molecule translates along the microtubule, as the single myosin molecule translates over the actin filament, and as the single isolated titin molecule is stretched.

Quantal Motor Action in Muscle Contraction

AbstractIt is becoming clear that quantal behavior is a central feature of contractile systems. Steplike behavior has been demonstrated in the kinesin - microtubule system and in the myosin - actin filament system-both on molecular scale. We show here that step-like features appear also in the single intact sarcomere. We studied single sarcomeres of single bumblebee myofibrils. Motorimposed ramp length changes on activated myofibrils resulted in sarcomere-length changes that were stepwise. Computer analysis of the stepwise shortening patterns revealed a step-size distribution containing multiple peaks. The peaks were separated by 2.7 nm per half-sarcomere which is the linear actin-subunit spacing. Thus, translation steps are an integer multiple of the actin-subunit spacing. This result parallels the one observed in the kinesin-tubulin spacing where step size is a multiple of the tubulin-subunit spacing. In the muscle system, however, the steps are preserved on a macroscopic scale, implying high synchrony. The quantal steps are easily explained by a model in which the actin filament propels itself over stationary cross-bridges: if actin binds to the cross-bridges between steps, then the observed quantal result is inevitable.