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15 pages, 3515 KiB

Open AccessArticle

Localization of the Elastic Proteins in the Flight Muscle of Manduca sexta

by Henry Gong, Weikang Ma, Shaoshuai Chen, Geng Wang, Ramzi Khairallah and Thomas Irving

Int. J. Mol. Sci. 2020, 21(15), 5504; https://doi.org/10.3390/ijms21155504 - 31 Jul 2020

Cited by 2 | Viewed by 2375

Abstract

The flight muscle of Manduca sexta (DLM₁) is an emerging model system for biophysical studies of muscle contraction. Unlike the well-studied indirect flight muscle of Lethocerus and Drosophila, the DLM₁ of Manduca is a synchronous muscle, as are the [...] Read more.

The flight muscle of Manduca sexta (DLM₁) is an emerging model system for biophysical studies of muscle contraction. Unlike the well-studied indirect flight muscle of Lethocerus and Drosophila, the DLM₁ of Manduca is a synchronous muscle, as are the vertebrate cardiac and skeletal muscles. Very little has been published regarding the ultrastructure and protein composition of this muscle. Previous studies have demonstrated that DLM₁ express two projectin isoform, two kettin isoforms, and two large Salimus (Sls) isoforms. Such large Sls isoforms have not been observed in the asynchronous flight muscles of Lethocerus and Drosophila. The spatial localization of these proteins was unknown. Here, immuno-localization was used to show that the N-termini of projectin and Salimus are inserted into the Z-band. Projectin spans across the I-band, and the C-terminus is attached to the thick filament in the A-band. The C-terminus of Sls was also located in the A-band. Using confocal microscopy and experimental force-length curves, thin filament lengths were estimated as ~1.5 µm and thick filament lengths were measured as ~2.5 µm. This structural information may help provide an interpretive framework for future studies using this muscle system. Full article

(This article belongs to the Special Issue Sarcomeric Proteins in Health and Disease)

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11 pages, 3116 KiB

Open AccessArticle

Crossbridge Recruitment Capacity of Wild-Type and Hypertrophic Cardiomyopathy-Related Mutant Troponin-T Evaluated by X-ray Diffraction and Mechanical Study of Cardiac Skinned Fibers

by Maki Yamaguchi, Masako Kimura, Tetsuo Ohno, Naoya Nakahara, Nobutake Akiyama, Shigeru Takemori and Naoto Yagi

Int. J. Mol. Sci. 2020, 21(10), 3520; https://doi.org/10.3390/ijms21103520 - 15 May 2020

Cited by 2 | Viewed by 2046

Abstract

X-ray diffraction and tension measurement experiments were conducted on rat left ventricular skinned fibers with or without “troponin-T treatment,” which exchanges the endogenous troponin T/I/C complex with exogenous troponin-T. These experiments were performed to observe the structural changes in troponin-T within a fiber [...] Read more.

X-ray diffraction and tension measurement experiments were conducted on rat left ventricular skinned fibers with or without “troponin-T treatment,” which exchanges the endogenous troponin T/I/C complex with exogenous troponin-T. These experiments were performed to observe the structural changes in troponin-T within a fiber elicited by contractile crossbridge formation and investigate the abnormality of hypertrophic cardiomyopathy-related troponin-T mutants. The intensity of the troponin reflection at 1/38.5 nm⁻¹ was decreased significantly by ATP addition after treatment with wild-type or mutant troponin-T, indicating that crossbridge formation affected the conformation of troponin-T. In experiments on cardiac fibers treated with the hypertrophic cardiomyopathy-related mutants E244D- and K247R-troponin-T, treatment with K247R-troponin-T did not recruit contracting actomyosin to a greater extent than wild-type-troponin-T, although a similar drop in the intensity of the troponin reflection occurred. Therefore, the conformational change in K247R-troponin-T was suggested to be unable to fully recruit actomyosin interaction, which may be the cause of cardiomyopathy. Full article

(This article belongs to the Special Issue Sarcomeric Proteins in Health and Disease)

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19 pages, 4590 KiB

Open AccessArticle

Roles of Dkk2 in the Linkage from Muscle to Bone during Mechanical Unloading in Mice

by Naoyuki Kawao, Hironobu Morita, Shunki Iemura, Masayoshi Ishida and Hiroshi Kaji

Int. J. Mol. Sci. 2020, 21(7), 2547; https://doi.org/10.3390/ijms21072547 - 06 Apr 2020

Cited by 18 | Viewed by 3038

Abstract

Mechanical unloading simultaneously induces muscle and bone loss, but its mechanisms are not fully understood. The interactions between skeletal muscle and bone have been recently noted. Although canonical wingless-related integration site (Wnt)/β-catenin signaling is crucial for bone metabolism, its roles in the muscle [...] Read more.

Mechanical unloading simultaneously induces muscle and bone loss, but its mechanisms are not fully understood. The interactions between skeletal muscle and bone have been recently noted. Although canonical wingless-related integration site (Wnt)/β-catenin signaling is crucial for bone metabolism, its roles in the muscle and bone interactions have remained unknown. Here, we performed comprehensive DNA microarray analyses to clarify humoral factors linking muscle to bone in response to mechanical unloading and hypergravity with 3 g in mice. We identified Dickkopf (Dkk) 2, a Wnt/β-catenin signaling inhibitor, as a gene whose expression was increased by hindlimb unloading (HU) and reduced by hypergravity in the soleus muscle of mice. HU significantly elevated serum Dkk2 levels and Dkk2 mRNA levels in the soleus muscle of mice whereas hypergravity significantly decreased those Dkk2 levels. In the simple regression analyses, serum Dkk2 levels were negatively and positively related to trabecular bone mineral density and mRNA levels of receptor activator of nuclear factor-kappa B ligand (RANKL) in the tibia of mice, respectively. Moreover, shear stress significantly suppressed Dkk2 mRNA levels in C2C12 cells, and cyclooxygenase inhibitors significantly antagonized the effects of shear stress on Dkk2 expression. On the other hand, Dkk2 suppressed the mRNA levels of osteogenic genes, alkaline phosphatase activity and mineralization, and it increased RANKL mRNA levels in mouse osteoblasts. In conclusion, we showed that muscle and serum Dkk2 levels are positively and negatively regulated during mechanical unloading and hypergravity in mice, respectively. An increase in Dkk2 expression in the skeletal muscle might contribute to disuse- and microgravity-induced bone and muscle loss. Full article

(This article belongs to the Special Issue Sarcomeric Proteins in Health and Disease)

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16 pages, 3468 KiB

Open AccessArticle

Estimation of Forces on Actin Filaments in Living Muscle from X-ray Diffraction Patterns and Mechanical Data

by Srboljub M. Mijailovich, Momcilo Prodanovic and Thomas C. Irving

Int. J. Mol. Sci. 2019, 20(23), 6044; https://doi.org/10.3390/ijms20236044 - 30 Nov 2019

Cited by 5 | Viewed by 3034

Abstract

Many biological processes are triggered or driven by mechanical forces in the cytoskeletal network, but these transducing forces have rarely been assessed. Striated muscle, with its well-organized structure provides an opportunity to assess intracellular forces using small-angle X-ray fiber diffraction. We present a [...] Read more.

Many biological processes are triggered or driven by mechanical forces in the cytoskeletal network, but these transducing forces have rarely been assessed. Striated muscle, with its well-organized structure provides an opportunity to assess intracellular forces using small-angle X-ray fiber diffraction. We present a new methodology using Monte Carlo simulations of muscle contraction in an explicit 3D sarcomere lattice to predict the fiber deformations and length changes along thin filaments during contraction. Comparison of predicted diffraction patterns to experimental meridional X-ray reflection profiles allows assessment of the stepwise changes in intermonomer spacings and forces in the myofilaments within living muscle cells. These changes along the filament length reflect the effect of forces from randomly attached crossbridges. This approach enables correlation of the molecular events, such as the current number of attached crossbridges and the distributions of crossbridge forces to macroscopic measurements of force and length changes during muscle contraction. In addition, assessments of fluctuations in local forces in the myofilaments may reveal how variations in the filament forces acting on signaling proteins in the sarcomere M-bands and Z-discs modulate gene expression, protein synthesis and degradation, and as well to mechanisms of adaptation of muscle in response to changes in mechanical loading. Full article

(This article belongs to the Special Issue Sarcomeric Proteins in Health and Disease)

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15 pages, 1894 KiB

Open AccessArticle

Functional Characterization of the Intact Diaphragm in a Nebulin-Based Nemaline Myopathy (NM) Model-Effects of the Fast Skeletal Muscle Troponin Activator tirasemtiv

by Eun-Jeong Lee, Justin Kolb, Darren T. Hwee, Fady I. Malik and Henk L. Granzier

Int. J. Mol. Sci. 2019, 20(20), 5008; https://doi.org/10.3390/ijms20205008 - 10 Oct 2019

Cited by 5 | Viewed by 2868

Abstract

Respiratory failure due to diaphragm dysfunction is considered a main cause of death in nemaline myopathy (NM) and we studied both isometric force and isotonic shortening of diaphragm muscle in a mouse model of nebulin-based NM (Neb cKO). A large contractile deficit was [...] Read more.

Respiratory failure due to diaphragm dysfunction is considered a main cause of death in nemaline myopathy (NM) and we studied both isometric force and isotonic shortening of diaphragm muscle in a mouse model of nebulin-based NM (Neb cKO). A large contractile deficit was found in nebulin-deficient intact muscle that is frequency dependent, with the largest deficits at low–intermediate stimulation frequencies (e.g., a deficit of 72% at a stimulation frequency of 20 Hz). The effect of the fast skeletal muscle troponin activator (FSTA) tirasemtiv on force was examined. Tirasemtiv had a negligible effect at maximal stimulation frequencies, but greatly reduced the force deficit of the diaphragm at sub-maximal stimulation levels with an effect that was largest in Neb cKO diaphragm. As a result, the force deficit of Neb cKO diaphragm fell (from 72% to 29% at 20 Hz). Similar effects were found in in vivo experiments on the nerve-stimulated gastrocnemius muscle complex. Load-clamp experiments on diaphragm muscle showed that tirasemtiv increased the shortening velocity, and reduced the deficit in mechanical power by 33%. Thus, tirasemtiv significantly improves muscle function in a mouse model of nebulin-based nemaline myopathy. Full article

(This article belongs to the Special Issue Sarcomeric Proteins in Health and Disease)

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Review

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26 pages, 2626 KiB

Open AccessReview

Genetic Restrictive Cardiomyopathy: Causes and Consequences—An Integrative Approach

by Diana Cimiotti, Heidi Budde, Roua Hassoun and Kornelia Jaquet

Int. J. Mol. Sci. 2021, 22(2), 558; https://doi.org/10.3390/ijms22020558 - 08 Jan 2021

Cited by 29 | Viewed by 7273

Abstract

The sarcomere as the smallest contractile unit is prone to alterations in its functional, structural and associated proteins. Sarcomeric dysfunction leads to heart failure or cardiomyopathies like hypertrophic (HCM) or restrictive cardiomyopathy (RCM) etc. Genetic based RCM, a very rare but severe disease [...] Read more.

The sarcomere as the smallest contractile unit is prone to alterations in its functional, structural and associated proteins. Sarcomeric dysfunction leads to heart failure or cardiomyopathies like hypertrophic (HCM) or restrictive cardiomyopathy (RCM) etc. Genetic based RCM, a very rare but severe disease with a high mortality rate, might be induced by mutations in genes of non-sarcomeric, sarcomeric and sarcomere associated proteins. In this review, we discuss the functional effects in correlation to the phenotype and present an integrated model for the development of genetic RCM. Full article

(This article belongs to the Special Issue Sarcomeric Proteins in Health and Disease)

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18 pages, 3134 KiB

Open AccessReview

N2A Titin: Signaling Hub and Mechanical Switch in Skeletal Muscle

by Kiisa Nishikawa, Stan L. Lindstedt, Anthony Hessel and Dhruv Mishra

Int. J. Mol. Sci. 2020, 21(11), 3974; https://doi.org/10.3390/ijms21113974 - 01 Jun 2020

Cited by 23 | Viewed by 6470

Abstract

Since its belated discovery, our understanding of the giant protein titin has grown exponentially from its humble beginning as a sarcomeric scaffold to recent recognition of its critical mechanical and signaling functions in active muscle. One uniquely useful model to unravel titin’s functions, [...] Read more.

Since its belated discovery, our understanding of the giant protein titin has grown exponentially from its humble beginning as a sarcomeric scaffold to recent recognition of its critical mechanical and signaling functions in active muscle. One uniquely useful model to unravel titin’s functions, muscular dystrophy with myositis (mdm), arose spontaneously in mice as a transposon-like LINE repeat insertion that results in a small deletion in the N2A region of titin. This small deletion profoundly affects hypertrophic signaling and muscle mechanics, thereby providing insights into the function of this specific region and the consequences of its dysfunction. The impact of this mutation is profound, affecting diverse aspects of the phenotype including muscle mechanics, developmental hypertrophy, and thermoregulation. In this review, we explore accumulating evidence that points to the N2A region of titin as a dynamic “switch” that is critical for both mechanical and signaling functions in skeletal muscle. Calcium-dependent binding of N2A titin to actin filaments triggers a cascade of changes in titin that affect mechanical properties such as elastic energy storage and return, as well as hypertrophic signaling. The mdm phenotype also points to the existence of as yet unidentified signaling pathways for muscle hypertrophy and thermoregulation, likely involving titin’s PEVK region as well as the N2A signalosome. Full article

(This article belongs to the Special Issue Sarcomeric Proteins in Health and Disease)

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21 pages, 4274 KiB

Open AccessReview

Structure and Function of Filamin C in the Muscle Z-Disc

by Zhenfeng Mao and Fumihiko Nakamura

Int. J. Mol. Sci. 2020, 21(8), 2696; https://doi.org/10.3390/ijms21082696 - 13 Apr 2020

Cited by 59 | Viewed by 6310

Abstract

Filamin C (FLNC) is one of three filamin proteins (Filamin A (FLNA), Filamin B (FLNB), and FLNC) that cross-link actin filaments and interact with numerous binding partners. FLNC consists of a N-terminal actin-binding domain followed by 24 immunoglobulin-like repeats with two intervening calpain-sensitive [...] Read more.

Filamin C (FLNC) is one of three filamin proteins (Filamin A (FLNA), Filamin B (FLNB), and FLNC) that cross-link actin filaments and interact with numerous binding partners. FLNC consists of a N-terminal actin-binding domain followed by 24 immunoglobulin-like repeats with two intervening calpain-sensitive hinges separating R15 and R16 (hinge 1) and R23 and R24 (hinge-2). The FLNC subunit is dimerized through R24 and calpain cleaves off the dimerization domain to regulate mobility of the FLNC subunit. FLNC is localized in the Z-disc due to the unique insertion of 82 amino acid residues in repeat 20 and necessary for normal Z-disc formation that connect sarcomeres. Since phosphorylation of FLNC by PKC diminishes the calpain sensitivity, assembly, and disassembly of the Z-disc may be regulated by phosphorylation of FLNC. Mutations of FLNC result in cardiomyopathy and muscle weakness. Although this review will focus on the current understanding of FLNC structure and functions in muscle, we will also discuss other filamins because they share high sequence similarity and are better characterized. We will also discuss a possible role of FLNC as a mechanosensor during muscle contraction. Full article

(This article belongs to the Special Issue Sarcomeric Proteins in Health and Disease)

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