Skeletal Muscle Sarcomere Research Articles

Functional role of myosin-binding protein H in thick filaments of developing vertebrate fast-twitch skeletal muscle.

Myosin-binding protein H (MyBP-H) is a component of the vertebrate skeletal muscle sarcomere with sequence and domain homology to myosin-binding protein C (MyBP-C). Whereas skeletal muscle isoforms of MyBP-C (fMyBP-C, sMyBP-C) modulate muscle contractility via interactions with actin thin filaments and myosin motors within the muscle sarcomere "C-zone," MyBP-H has no known function. This is in part due to MyBP-H having limited expression in adult fast-twitch muscle and no known involvement in muscle disease. Quantitative proteomics reported here reveal that MyBP-H is highly expressed in prenatal rat fast-twitch muscles and larval zebrafish, suggesting a conserved role in muscle development and prompting studies to define its function. We take advantage of the genetic control of the zebrafish model and a combination of structural, functional, and biophysical techniques to interrogate the role of MyBP-H. Transgenic, FLAG-tagged MyBP-H or fMyBP-C both localize to the C-zones in larval myofibers, whereas genetic depletion of endogenous MyBP-H or fMyBP-C leads to increased accumulation of the other, suggesting competition for C-zone binding sites. Does MyBP-H modulate contractility in the C-zone? Globular domains critical to MyBP-C's modulatory functions are absent from MyBP-H, suggesting that MyBP-H may be functionally silent. However, our results suggest an active role. In vitro motility experiments indicate MyBP-H shares MyBP-C's capacity as a molecular "brake." These results provide new insights and raise questions about the role of the C-zone during muscle development.

The Utility of Urinary Titin to Diagnose and Predict the Prognosis of Acute Myocardial Infarction.

Early detection and management are crucial for better prognosis in acute myocardial infarction (AMI). Serum titin, a component of the sarcomere in cardiac and skeletal muscle, was associated with AMI. Thus, we hypothesized that urinary N-fragment titin may be a biomarker for its diagnosis and prognosis. Between January 2021 and November 2021, we prospectively enrolled 83 patients with suspected AMI. Their urinary N-fragment titin, serum high-sensitivity troponin I (hsTnI), creatine kinase (CK), and creatine kinase-MB (CK-MB) were measured on admission. Then, urinary titin was assessed as diagnostic and prognostic biomarker in AMI. Among 83 enrolled patients, 51 patients were diagnosed as AMI. In AMI patients who were admitted as early as 3 h or longer after symptom onset, their urinary titin levels were significantly higher than non-AMI patients who are also admitted 3 h or longer after symptom onset (12.76 [IQR 5.87-16.68] pmol/mgCr (creatinine) and 5.13 [IQR 3.93-11.25] pmol/mgCr, p = 0.045, respectively). Moreover, the urinary titin levels in patients who died during hospitalization were incredibly higher than in those who were discharged (15.90 [IQR 13.46-22.61] pmol/mgCr and 4.90 [IQR 3.55-11.95] pmol/mgCr, p = 0.023). Urinary N-fragment titin can be used as non-invasive early diagnostic biomarker in AMI. Furthermore, it associates with hospital discharge disposition, providing prognostic utility.

Impaired Cardiac and Skeletal Muscle Energetics Following Anthracycline Therapy for Breast Cancer.

Anthracycline-related cardiac toxicity is a recognized consequence of cancer therapies. We assess resting cardiac and skeletal muscle energetics and myocyte, sarcomere, and mitochondrial integrity in patients with breast cancer receiving epirubicin. In a prospective, mechanistic, observational, longitudinal study, we investigated chemotherapy-naive patients with breast cancer receiving epirubicin versus sex- and age-matched healthy controls. Resting energetic status of cardiac and skeletal muscle (phosphocreatine/gamma ATP and inorganic phosphate [Pi]/phosphocreatine, respectively) was assessed with 31P-magnetic resonance spectroscopy. Cardiac function and tissue characterization (magnetic resonance imaging and 2D-echocardiography), cardiac biomarkers (serum NT-pro-BNP and high-sensitivity troponin I), and structural assessments of skeletal muscle biopsies were obtained. All study assessments were performed before and after chemotherapy. Twenty-five female patients with breast cancer (median age, 53 years) received a mean epirubicin dose of 304 mg/m2, and 25 age/sex-matched controls were recruited. Despite comparable baseline cardiac and skeletal muscle energetics with the healthy controls, after chemotherapy, patients with breast cancer showed a reduction in cardiac phosphocreatine/gamma ATP ratio (2.0±0.7 versus 1.1±0.5; P=0.001) and an increase in skeletal muscle Pi/phosphocreatine ratio (0.1±0.1 versus 0.2±0.1; P=0.022). This occurred in the context of increases in left ventricular end-systolic and end-diastolic volumes (P=0.009 and P=0.008, respectively), T1 and T2 mapping (P=0.001 and P=0.028, respectively) but with preserved left ventricular ejection fraction, mass and global longitudinal strain, and no change in cardiac biomarkers. There was preservation of the mitochondrial copy number in skeletal muscle biopsies but a significant increase in areas of skeletal muscle degradation (P=0.001) in patients with breast cancer following chemotherapy. Patients with breast cancer demonstrated a reduction in skeletal muscle sarcomere number from the prechemotherapy stage compared with healthy controls (P=0.013). Contemporary doses of epirubicin for breast cancer treatment result in a significant reduction of cardiac and skeletal muscle high-energy 31P-metabolism alongside structural skeletal muscle changes. URL: https://www. gov; Unique identifier: NCT04467411.

Skeletal muscle fiber hypercontraction induced by Bothrops asper myotoxic phospholipases A2ex vivo does not involve a direct action on the contractile apparatus

Myonecrosis is a frequent clinical manifestation of envenomings by Viperidae snakes, mainly caused by the toxic actions of secreted phospholipase A2 (sPLA2) enzymes and sPLA2-like homologs on skeletal muscle fibers. A hallmark of the necrotic process induced by these myotoxins is the rapid appearance of hypercontracted muscle fibers, attributed to the massive influx of Ca2+ resulting from cell membrane damage. However, the possibility of myotoxins having, in addition, a direct effect on the contractile machinery of skeletal muscle fibers when internalized has not been investigated. This question is here addressed by using an ex vivo model of single-skinned muscle fibers, which lack membranes but retain an intact contractile apparatus. Rabbit psoas skinned fibers were exposed to two types of myotoxins of Bothrops asper venom: Mt-I, a catalytically active Asp49 sPLA2 enzyme, and Mt-II, a Lys49 sPLA2-like protein devoid of phospholipolytic activity. Neither of these myotoxins affected the main parameters of force development in striated muscle sarcomeres of the skinned fibers. Moreover, no microscopical alterations were evidenced after their exposure to Mt-I or Mt-II. In contrast to the lack of effects on skinned muscle fibers, both myotoxins induced a strong hypercontraction in myotubes differentiated from murine C2C12 myoblasts, with drastic morphological alterations that reproduce those described in myonecrotic tissue in vivo. As neither Mt-I nor Mt-II showed direct effects upon the contractile apparatus of skinned fibers, it is concluded that the mechanism of hypercontraction triggered by both myotoxins in patients involves indirect effects, i.e., the large cytosolic Ca2+ increase after sarcolemma permeabilization.

Super relaxed myosins loosen up to different cues in cardiac and skeletal muscle sarcomeres.

Recent papers by Nelson et al. and Pilagov et al. provide important new information on the ever-expanding role of myosin heads in the regulation of contraction.

Variants in ACTC1 underlie distal arthrogryposis accompanied by congenital heart defects.

Contraction of the human sarcomere is the result of interactions between myosin cross-bridges and actin filaments. Pathogenic variants in genes such as MYH7, TPM1, and TNNI3 that encode parts of the cardiac sarcomere cause muscle diseases that affect the heart, such as dilated cardiomyopathy and hypertrophic cardiomyopathy. In contrast, pathogenic variants in homologous genes such as MYH2, TPM2, and TNNI2 that encode parts of the skeletal muscle sarcomere cause muscle diseases affecting skeletal muscle, such as distal arthrogryposis (DA) syndromes and skeletal myopathies. To date, there have been few reports of genes (e.g., MYH7) encoding sarcomeric proteins in which the same pathogenic variant affects skeletal and cardiac muscle. Moreover, none of the known genes underlying DA have been found to contain pathogenic variants that also cause cardiac abnormalities. We report five families with DA because of heterozygous missense variants in the gene actin, alpha, cardiac muscle 1 (ACTC1). ACTC1 encodes a highly conserved actin that binds to myosin in cardiac and skeletal muscle. Pathogenic variants in ACTC1 have been found previously to underlie atrial septal defect, dilated cardiomyopathy, hypertrophic cardiomyopathy, and left ventricular noncompaction. Our discovery delineates a new DA condition because of variants in ACTC1 and suggests that some functions of ACTC1 are shared in cardiac and skeletal muscle.

Two-Molecule Force Spectroscopy on Proteins.

Many elastomeric proteins, which play important roles in a wide range of biological processes, exist as parallel/antiparallelly arranged dimers or multimers to perform their mechanobiological functions. For example, in striated muscle sarcomeres, the giant muscle protein titin exists as hexameric bundles to mediate the passive elasticity of muscles. However, it has not been possible to directly probe the mechanical properties of such parallelly arranged elastomeric proteins. And it remains unknown if the knowledge obtained from single-molecule force spectroscopy studies can be directly extrapolated to such parallelly/antiparallelly arranged systems. Here, we report the development of atomic force microscopy (AFM)-based two-molecule force spectroscopy to directly probe the mechanical properties of two elastomeric proteins that are arranged in parallel. We developed a twin-molecule approach to allow two parallelly arranged elastomeric proteins to be picked up and stretched simultaneously in an AFM experiment. Our results clearly revealed the mechanical features of such parallelly arranged elastomeric proteins during force-extension measurements and allowed for the determination of mechanical unfolding forces of proteins in such an experimental setting. Our study provides a general and robust experimental strategy to closely mimic the physiological condition of such parallel elastomeric protein multimers.

Fluid‐Driven High‐Performance Bionic Artificial Muscle with Adjustable Muscle Architecture

High‐performance artificial muscle is always the pursuit of researchers for robotics. Herein, a bionic artificial muscle is reported called “ExoMuscle” mimicking the sarcomere in skeletal muscle with a bio‐inspired structure to contract “myofilaments” enabling the artificial muscle to mimic the architecture of muscle such as parallel, fusiform, convergent, and pennation and beyond the performance of skeletal muscle. The reported actuators excel in various aspects compared with skeletal muscle including actuation stress (0.41–0.9 MPa), strain (50%), optimal length, velocity‐independence output, power density (10.94 kW kg−1), and efficiency (69.11%). With its own adjustable pennation architecture, it achieves variable actuation stress up to 0.9 MPa meanwhile maintaining high efficiency. Furthermore, ExoMuscle highly conforms to the anatomical complexity of the human body to cooperate with skeletal muscles closely opening the door for bio‐robotics, especially wearable robots.

The organisation of titin at the centre of the vertebrate striated muscle thick filament.

Striated muscle sarcomeres operate by sliding of the regular arrays of interdigitating thin (actin) and thick (myosin) filaments. Thick filaments are held together at their centre by M-band bridges, maintaining sarcomere structure during contraction and relaxation. The giant sarcomeric protein titin is instrumental in maintaining order, since C-terminal truncations of titin lead to loss of A-band alignment and sarcomere failure. The C-terminal sequence of titin encodes a kinase domain, 10 Ig domains and linker sequences, and is associated with the bare zone at the centre of the sarcomere. Unlike the cross-bridge associated sequence, it does not show any correlation with the organisation of the thick filament bare zone and associated M-band bridges. The precise disposition of titin remains unclear, e.g. whether anti-parallel molecules overlap at centre of the filament. We used superresolution light microscopy with new titin antibodies to establish the position of several titin epitopes in the bare zone, together with key markers for the M-band proteins myomesin and M-protein. We show that the titin kinase domain is at the edge of the bare zone, about 70 nm from the M-band centre at the first myosin motor domains and may therefore account for the first of the accessory protein stripes in the A-band. SAXS data of two titin constructs covering several domains of the bare-zone associated sequence, one from the beginning including the kinase and the other from the end, suggest that they are extended structures but with compact linkers. Conversely, the long insert sequence between M-band Ig-domains 3 and 4 (Ig-162 and Ig-163) appears to be more extensive. Our combined data suggests that in the bare zone, titin molecules from each side of the thick filament overlap each other by the width of the M-band.

Computational modeling of cooperative regulation of both strands in thin filaments.

Striated muscle contraction is regulated by thin filaments in response to changes of cytoplasmic Ca2+ concentration. Thin filaments are comprised of ∼27 pairs of regulatory units (RUs), each RU having 7 actins, 1 tropomyosin and 1 troponin. Cooperative interactions between adjacent RUs along thin filaments have been extensively considered, but RUs across thin filaments have received little attention. We used FiberSim—a spatially explicit computational model of the striated muscle sarcomere that includes myofilament compliance (Kosta et al. 2022 Biophs J)—modified to include interactions among neighboring RUs along and across thin filaments. We first simulated Ca2+-dependent changes in thin filaments in the absence of cross-bridges at systolic Ca2+ levels (∼50% activation) with cooperative parameters based on cryo-EM of porcine cardiac thin filaments (Risi et al. 2021 PNAS). Statistical distribution of RU states was analyzed in R as described for cryo-EM data, which allowed comparison between probability matrices obtained from FiberSim simulation and cryo-EM. It shows that FiberSim mimics the probability distribution pattern of RU states obtained from cryo-EM under three conditions: (1) RU on the upper strand is activated; (2) RU on lower strand is activated; (3) RU on lower strand is inactivated. However, the probability distribution exhibits opposite trend when RU on the upper strand is inactivated, which could be explained by the unspecified strand position in the model. Second, isometric force-pCa relationships were simulated using the fitted cooperative parameters, and compared with absence of cooperativity. Results show slightly lower cooperativity because of negative cooperativity across thin filaments. This initial computational modeling of cooperativity that considers RUs both along and across thin filaments aims to bridge structural and functional analyses.

Myosin binding protein h dominates the "c-zone" in ultrafast swimming muscles of developing zebrafish.

The ‘C-zone’ of mature vertebrate skeletal muscle sarcomeres is characterized by the presence of Myosin-Binding Protein C (MyBP-C), which ‘tunes’ contractility via its N-terminal domain interactions with the myosin head region and/or the thin filament. The ultrafast swimming muscles of zebrafish larvae are a perfect model system to define MyBP-C isoform function, due to tractable genetics and easily characterized muscle mechanics. However, liquid chromatography mass spectrometry shows 5-day-old larval muscle expresses MyBP-C at only ∼5% of the ∼1:10 molar ratio with myosin heavy chain (MYH) typically associated with full C-zone occupancy, while a little-studied MyBP-C paralog, Myosin-Binding Protein H (MyBP-H) comprises the remaining ∼95%. MyBP-H is homologous to MyBP-C's C terminus, which binds to the myosin thick filament backbone, but it lacks analagous N-terminal regulatory domains, which are critical to MyBP-C's slowing of muscle shortening. Therefore, the presence of MyBP-H may underlie the extreme contractile speed of larval muscle (>30 lengths/s), and/or indicate its involvement in muscle development. To define MyBP-H's potential roles, we first imaged its sarcomeric localization in larvae expressing transgenic, FLAG-tagged MyBP-H. Immunofluorescence revealed characteristic fluorescence doublets at the sarcomere center, indicating MyBP-H binds throughout the C-zone. Interestingly, MyBP-H-null mutants engineered using CRISPR/Cas9 appear to develop normally, suggesting the developmental impact of MyBP-H may be subtle. Preliminary mechanical studies of MyBP-H-null larval muscles showed no change in twitch kinetics, tetanic force, or the Force:Velocity relationship, which might be expected, given MyBP-C content increases only minimally (From ∼5% to ∼10% of full C-zone occupancy). Although the role of MyBP-H in development remains an enigma, physiologically stressing the larvae may reveal MyBP-H's function. At a minimum, the MyBP-H null larvae provide a clean slate to repopulate the C-zone using transgenically expressed human or disease-causing MyBP-C isoforms.

RNA-Sequencing Reveals Upregulation and a Beneficial Role of Autophagy in Myoblast Differentiation and Fusion.

Myoblast differentiation is a complex process whereby the mononuclear muscle precursor cells myoblasts express skeletal-muscle-specific genes and fuse with each other to form multinucleated myotubes. The objective of this study was to identify potentially novel mechanisms that mediate myoblast differentiation. We first compared transcriptomes in C2C12 myoblasts before and 6 days after induction of myogenic differentiation by RNA-seq. This analysis identified 11,046 differentially expressed genes, of which 5615 and 5431 genes were upregulated and downregulated, respectively, from before differentiation to differentiation. Functional enrichment analyses revealed that the upregulated genes were associated with skeletal muscle contraction, autophagy, and sarcomeres while the downregulated genes were associated with ribonucleoprotein complex biogenesis, mRNA processing, ribosomes, and other biological processes or cellular components. Western blot analyses showed an increased conversion of LC3-I to LC3-II protein during myoblast differentiation, further demonstrating the upregulation of autophagy during myoblast differentiation. Blocking the autophagic flux in C2C12 cells with chloroquine inhibited the expression of skeletal-muscle-specific genes and the formation of myotubes, confirming a positive role for autophagy in myoblast differentiation and fusion.

Sarcomere dynamics revealed by a myofilament integrated FRET-based biosensor in live skeletal muscle fibers

The sarcomere is the functional unit of skeletal muscle, essential for proper contraction. Numerous acquired and inherited myopathies impact sarcomere function causing clinically significant disease. Mechanistic investigations of sarcomere activation have been challenging to undertake in the context of intact, live skeletal muscle fibers during real time physiological twitch contractions. Here, a skeletal muscle specific, intramolecular FRET-based biosensor was designed and engineered into fast skeletal muscle troponin C (TnC) to investigate the dynamics of sarcomere activation. In transgenic animals, the TnC biosensor incorporated into the skeletal muscle fiber sarcomeres by stoichiometric replacement of endogenous TnC and did not alter normal skeletal muscle contractile form or function. In intact single adult skeletal muscle fibers, real time twitch contractile data showed the TnC biosensor transient preceding the peak amplitude of contraction. Importantly, under physiological temperatures, inactivation of the TnC biosensor transient decayed significantly more slowly than the Ca2+ transient and contraction. The uncoupling of the TnC biosensor transient from the Ca2+ transient indicates the biosensor is not functioning as a Ca2+ transient reporter, but rather reports dynamic sarcomere activation/ inactivation that, in turn, is due to the ensemble effects of multiple activating ligands within the myofilaments. Together, these findings provide the foundation for implementing this new biosensor in future physiological studies investigating the mechanism of activation of the skeletal muscle sarcomere in health and disease.

Transcriptome Response of Differentiating Muscle Satellite Cells to Thermal Challenge in Commercial Turkey.

Early muscle development involves the proliferation and differentiation of stem cells (satellite cells, SCs) in the mesoderm to form multinucleated myotubes that mature into muscle fibers and fiber bundles. Proliferation of SCs increases the number of cells available for muscle formation while simultaneously maintaining a population of cells for future response. Differentiation dramatically changes properties of the SCs and environmental stressors can have long lasting effects on muscle growth and physiology. This study was designed to characterize transcriptional changes induced in turkey SCs undergoing differentiation under thermal challenge. Satellite cells from the pectoralis major (p. major) muscle of 1-wk old commercial fast-growing birds (Nicholas turkey, NCT) and from a slower-growing research line (Randombred Control Line 2, RBC2) were proliferated for 72 h at 38 °C and then differentiated for 48 h at 33 °C (cold), 43 °C (hot) or 38 °C (control). Gene expression among thermal treatments and between turkey lines was examined by RNAseq to detect significant differentially expressed genes (DEGs). Cold treatment resulted in significant gene expression changes in the SCs from both turkey lines, with the primary effect being down regulation of the DEGs with overrepresentation of genes involved in regulation of skeletal muscle tissue regeneration and sarcomere organization. Heat stress increased expression of genes reported to regulate myoblast differentiation and survival and to promote cell adhesion particularly in the NCT line. Results suggest that growth selection in turkeys has altered the developmental potential of SCs in commercial birds to increase hypertrophic potential of the p. major muscle and sarcomere assembly. The biology of SCs may account for the distinctly different outcomes in response to thermal challenge on breast muscle growth, development, and structure of the turkey.

Interplay between the Chd4/NuRD Complex and the Transcription Factor Znf219 Controls Cardiac Cell Identity

The sarcomere regulates striated muscle contraction. This structure is composed of several myofibril proteins, isoforms of which are encoded by genes specific to either the heart or skeletal muscle. The chromatin remodeler complex Chd4/NuRD regulates the transcriptional expression of these specific sarcomeric programs by repressing genes of the skeletal muscle sarcomere in the heart. Aberrant expression of skeletal muscle genes induced by the loss of Chd4 in the heart leads to sudden death due to defects in cardiomyocyte contraction that progress to arrhythmia and fibrosis. Identifying the transcription factors (TFs) that recruit Chd4/NuRD to repress skeletal muscle genes in the myocardium will provide important information for understanding numerous cardiac pathologies and, ultimately, pinpointing new therapeutic targets for arrhythmias and cardiomyopathies. Here, we sought to find Chd4 interactors and their function in cardiac homeostasis. We therefore describe a physical interaction between Chd4 and the TF Znf219 in cardiac tissue. Znf219 represses the skeletal-muscle sarcomeric program in cardiomyocytes in vitro and in vivo, similarly to Chd4. Aberrant expression of skeletal-muscle sarcomere proteins in mouse hearts with knocked down Znf219 translates into arrhythmias, accompanied by an increase in PR interval. These data strongly suggest that the physical and genetic interaction of Znf219 and Chd4 in the mammalian heart regulates cardiomyocyte identity and myocardial contraction.

Abstract P1059: Establishing The Mechanistic Basis Of Dilated Cardiomyopathy Associated With The Skeletal Muscle Actin Mutation R256H

Actin, which is expressed in all eukaryotic cells, is an essential component of the cardiac and skeletal muscle sarcomere thin filament. Mutations in actin have been associated with a wide range of diseases, including skeletal myopathies and cardiomyopathies. Humans express six actin isoforms with two that are thought to be highly enriched in sarcomeres and differ by only four amino acids: skeletal muscle actin (ACTA1) and cardiac actin (ACTC1). While ACTC1 is exclusively expressed in the adult heart, ACTA1 is predominantly expressed in skeletal muscle with lower levels detected in cardiac muscle. As such, ACTA1 mutations are well known to cause skeletal myopathy with minimal effects on cardiac function. However, I previously reported a family carrying a novel heterozygous mutation in ACTA1 (ACTA1 R256H) with dilated cardiomyopathy (DCM) and no clinical evidence of skeletal myopathy. The objective of this study is to elucidate the molecular mechanism(s) by which ACTA1 R256H may cause heart failure. I generated ACTA1 R256H/+ human pluripotent stem cells (hPSCs) using Cas9/CRISPR gene editing. Using traction force microscopy, I observed that ACTA1 R256H/+ hPSC-derived cardiomyocytes display reduced contractility. To define relevant mechanisms, I devised a novel approach to purify milligram quantities of recombinant human ACTA1, which has served as a remarkable challenge for the field. Importantly, this purification technique circumvents contamination from endogenous actins and produces functional protein capable of polymerization. Moreover, in vitro motility assays demonstrated that reconstituted human thin filaments containing recombinant ACTA1 are functional and activated in a calcium-regulated manner. Intriguingly, purified ACTA1 R256H appears to have a polymerization defect, suggesting a possible mechanism by which this mutation leads to a defect in contractility. Together, these studies suggest for the first time that a mutation in skeletal muscle actin, ACTA1 R256H, causes cardiomyocyte hypocontractility while also suggesting a potential molecular mechanism. Finally, this study also introduces a new purification method essential for biochemical and biophysical analysis of human ACTA1 mutations.

Ca2+ attenuates nucleation activity of leiomodin.

A transient increase in Ca2+ concentration in sarcomeres is essential for their proper function. Ca2+ drives striated muscle contraction via binding to the troponin complex of the thin filament to activate its interaction with the myosin thick filament. In addition to the troponin complex, the myosin essential light chain and myosin‐binding protein C were also found to be Ca2+ sensitive. However, the effects of Ca2+ on the function of the tropomodulin family proteins involved in regulating thin filament formation have not yet been studied. Leiomodin, a member of the tropomodulin family, is an actin nucleator and thin filament elongator. Using pyrene‐actin polymerization assay and transmission electron microscopy, we show that the actin nucleation activity of leiomodin is attenuated by Ca2+. Using circular dichroism and nuclear magnetic resonance spectroscopy, we demonstrate that the mostly disordered, negatively charged region of leiomodin located between its first two actin‐binding sites binds Ca2+. We propose that Ca2+ binding to leiomodin results in the attenuation of its nucleation activity. Our data provide further evidence regarding the role of Ca2+ as an ultimate regulator of the ensemble of sarcomeric proteins essential for muscle function.Summary StatementCa2+ fluctuations in striated muscle sarcomeres modulate contractile activity via binding to several distinct families of sarcomeric proteins. The effects of Ca2+ on the activity of leiomodin—an actin nucleator and thin filament length regulator—have remained unknown. In this study, we demonstrate that Ca2+ binds directly to leiomodin and attenuates its actin nucleating activity. Our data emphasizes the ultimate role of Ca2+ in the regulation of the sarcomeric protein interactions.

Muscle active force-length curve explained by an electrophysical model of interfilament spacing

The active isometric force-length relation (FLR) of striated muscle sarcomeres is central to understanding and modeling muscle function. The mechanistic basis of the descending arm of the FLR is well explained by the decreasing thin:thick filament overlap that occurs at long sarcomere lengths. The mechanistic basis of the ascending arm of the FLR (the decrease in force that occurs at short sarcomere lengths), alternatively, has never been well explained. Because muscle is a constant-volume system, interfilament lattice distances must increase as sarcomere length shortens. This increase would decrease thin and thick-filament electrostatic interactions independently of thin:thick filament overlap. To examine this effect, we present here a fundamental, physics-based model of the sarcomere that includes filament molecular properties, calcium binding, sarcomere geometry including both thin:thick filament overlap and interfilament radial distance, and electrostatics. The model gives extremely good fits to existing FLR data from a large number of different muscles across their entire range of measured activity levels, with the optimized parameter values in all cases lying within anatomically and physically reasonable ranges. A local first-order sensitivity analysis (varying individual parameters while holding the values of all others constant) shows that model output is most sensitive to a subset of model parameters, most of which are related to sarcomere geometry, with model output being most sensitive to interfilament radial distance. This conclusion is supported by re-running the fits with only this parameter subset being allowed to vary, which increases fit errors only moderately. These results show that the model well reproduces existing experimental data, and indicate that changes in interfilament spacing play as central a role as changes in filament overlap in determining the FLR, particularly on its ascending arm.

A nemaline myopathy-linked point mutation destabilizes the second actin binding site of tropomodulin family proteins

Proper function of striated muscle sarcomeres depends on strict regulation of thin filament lengths. Leiomodin (Lmod) and tropomodulin (Tmod), homologous proteins both with an affinity for actin and tropomyosin, bind to the pointed end of thin filaments, altering actin polymerization dynamics and controlling thin filament lengths. Several mutations in the skeletal isoform of Lmod (Lmod3) have been linked to nemaline myopathy, a disease characterized by aberrant sarcomere assembly, hypotonia, and severe muscle weakness ultimately leading to death.

Abstract 11400: Circulating Glutathionylated Cardiac Myosin Binding Protein C as a Diagnostic Biomarker for Diastolic Dysfunction

Introduction: Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated protein localized to the crossbridge-containing C zones of striated muscle sarcomeres, contributing to regulate cardiac contraction and relaxation. Phosphorylation status of cMyBP-C determines cardiac function, and S-glutathionylation of cMyBP-C is increased in human heart failure (HF). In animal models, we demonstrated that elevated S-glutathionylation of cMyBP-C but not phosphorylation correlates with diastolic dysfunction (DD). Hypothesis: S-glutathionylated cMyBP-C would be a biomarker for DD. Methods: Humans, African Green monkeys, and mice with DD determined by echocardiography (E/e’ ratio > X) were matched by age and gender to controls. Blood samples were acquired and analyzed for S-glutathionylated cMyBP-C by immunoblotting. Results: Circulating S-glutathionylated cMyBP-C in DD patients (N=28) was elevated (fold, 1.57± 0.12, P<0.01, t-test) when compared to controls (N=16). We confirmed these results in mice and monkeys with DD demonstrated by echocardiography. In DD monkeys (N=6), the circulated S-glutathionylated cMyBP-C levels were upregulated (fold, 2.7 ± 0.57, P<0.05, t-test) as compared to those in control (CTL) monkeys (N=6). Similarly, S-glutathionylated cMyBP-C was elevated (fold, 1.61 ± 0.23, P<0.05, t-test) in mice with DD (N=5) as compared to controls (N=5). Conclusions: Glutathionylated cMyBP-C has been associated with DD in animal models, and circulating glutathionylated cMyBP-C was elevated in humans, monkeys, and mice with DD. Glutathionylated cMyBP-C may represent a disease-specific biomarker for the presence of DD.