Loss of Sarcomeric Scaffolding as a Common Baseline Histopathologic Lesion in Titin-Related Myopathies
R. Ávila-PoloEdoardo MalfattiXavière LornageChrystel ChéraudIsabelle NelsonJuliette NectouxJohann BöhmRaphaël SchneiderCarola Hedberg‐OldforsB. EymardSoledad MongesFabiana LubienieckiGuy BrochierMai Thao BuiA. MadelaineC. LabasseMaud BeuvinEmmanuelle LacèneAnne BolandJean‐François DeleuzeJulie ThompsonIsabelle RichardAna Lía TaratutoBjarne UddFrance LeturcqGisèle BonneAnders OldforsJocelyn LaporteNorma Beatriz Romero
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Abstract:
Titin-related myopathies are heterogeneous clinical conditions associated with mutations in TTN. To define their histopathologic boundaries and try to overcome the difficulty in assessing the pathogenic role of TTN variants, we performed a thorough morphological skeletal muscle analysis including light and electron microscopy in 23 patients with different clinical phenotypes presenting pathogenic autosomal dominant or autosomal recessive (AR) mutations located in different TTN domains. We identified a consistent pattern characterized by diverse defects in oxidative staining with prominent nuclear internalization in congenital phenotypes (AR-CM) (n = 10), ± necrotic/regenerative fibers, associated with endomysial fibrosis and rimmed vacuoles (RVs) in AR early-onset Emery-Dreifuss-like (AR-ED) (n = 4) and AR adult-onset distal myopathies (n = 4), and cytoplasmic bodies (CBs) as predominant finding in hereditary myopathy with early respiratory failure (HMERF) patients (n = 5). Ultrastructurally, the most significant abnormalities, particularly in AR-CM, were multiple narrow core lesions and/or clear small areas of disorganizations affecting one or a few sarcomeres with M-band and sometimes A-band disruption and loss of thick filaments. CBs were noted in some AR-CM and associated with RVs in HMERF and some AR-ED cases. As a whole, we described recognizable histopathological patterns and structural alterations that could point toward considering the pathogenicity of TTN mutations.Keywords:
Desmin
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Immunoglobulin domain
Immunoelectron microscopy
Cardiac muscle
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Abstract One of the main contributors to passive tension of the myocardium is titin. However, it is not exactly known what portions of this ≈1 μm-long molecule are anchored in the sarcomere (hence, are rendered inelastic) and what portions are elastic (hence, are mechanically active in developing passive tension). We assessed the length of the elastic domain of cardiac titin by ultrastructural and mechanical methods. Single cardiac myocytes were stretched by various amounts, and while in the stretched state, they were processed for immunoelectron microscopy. Several monoclonal anti-titin antibodies were used, and the locations of the titin epitopes in the sarcomere were studied as a function of sarcomere length. Only a small fraction (5% to 10%) of the ≈1000-nm-long molecule behaved elastically under physiological conditions. This mechanically active domain is located close to the A/I junction, and its contour length when unstretched is estimated at ≈50 to 100 nm. In sarcomeres that are slack (length ≈1.8...
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Cardiac muscle
Immunoglobulin domain
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Abstract One of the main contributors to passive tension of the myocardium is titin. However, it is not exactly known what portions of this ≈1 μm-long molecule are anchored in the sarcomere (hence, are rendered inelastic) and what portions are elastic (hence, are mechanically active in developing passive tension). We assessed the length of the elastic domain of cardiac titin by ultrastructural and mechanical methods. Single cardiac myocytes were stretched by various amounts, and while in the stretched state, they were processed for immunoelectron microscopy. Several monoclonal anti-titin antibodies were used, and the locations of the titin epitopes in the sarcomere were studied as a function of sarcomere length. Only a small fraction (5% to 10%) of the ≈1000-nm-long molecule behaved elastically under physiological conditions. This mechanically active domain is located close to the A/I junction, and its contour length when unstretched is estimated at ≈50 to 100 nm. In sarcomeres that are slack (length ≈1.85 μm), the mechanically active domain is folded on top of itself, and the length of the domain reaches an elastic limit of ≈550 nm in sarcomeres that are ≈2.9 μm long.
Immunoelectron microscopy
Immunoglobulin domain
Cardiac muscle
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Titin is a structural protein in muscle that spans the half sarcomere from z-band to M-line. Although there are selected studies on titin’s mechanical properties from tests on isolated molecules or titin fragments, little is known about its behavior within the structural confines of a sarcomere. Here, we tested the hypothesis that titin properties might be reflected well in single myofibrils. Therefore, the purpose of this study was to measure the passive mechanical properties of isolated single myofibrils and evaluate whether these properties reflect the basic mechanical properties of the titin molecule. Single myofibrils from rabbit psoas were prepared for measurement of passive stretch-shortening cycles at lengths where passive titin forces become important. Three repeat stretch-shortening cycles with magnitudes between 1.0-3.0μm/sarcomere were performed at a speed of 0.1μm/s·sarcomere and repeated after a ten minute rest at zero force. These tests were performed in a relaxation solution (passive) and an activation solution (active) where cross-bridge attachment was inhibited with butanedione monoxime. Myofibrils behaved viscoelastically producing an increased efficiency with repeat stretch-shortening cycles, but a decreased efficiency with increasing stretch magnitudes. Furthermore, we observed a first distinct inflection point in the force-elongation curve at an average sarcomere length of 3.5μm that was associated with an average force of 68±5nN/mm -1 . This inflection point was thought to reflect Ig domain unfolding and was missing after a ten minute rest at zero force, suggesting a lack of spontaneous Ig domain refolding. These passive myofibrillar properties are consistent with those observed in isolated titin molecules, suggesting that the mechanics of titin are well preserved in isolated myofibrils, and thus, can be studied readily in myofibrils, rather than in the extremely difficult and labile single titin preparations.
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Elongation
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The aim of this study was to determine the role of titin in preventing the development of sarcomere length nonuniformities following activation and after active and passive stretch by determining the effect of partial titin degradation on sarcomere length nonuniformities and force in passive and active myofibrils. Selective partial titin degradation was performed using a low dose of trypsin. Myofibrils were set at a sarcomere length of 2.4 µm and then passively stretched to sarcomere lengths of 3.4 and 4.4 µm. In the active condition, myofibrils were set at a sarcomere length of 2.8 µm, activated, and actively stretched by 1 µm/sarcomere. The extent of sarcomere length nonuniformities was calculated for each sarcomere as the absolute difference between sarcomere length and the mean sarcomere length of the myofibril. Our main finding is that partial titin degradation does not increase sarcomere length nonuniformities after passive stretch and activation compared with when titin is intact but increases the extent of sarcomere length nonuniformities after active stretch. Furthermore, when titin was partially degraded, active and passive stresses were substantially reduced. These results suggest that titin plays a crucial role in actively stretched myofibrils and is likely involved in active and passive force production.
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Abstract The striated muscle sarcomere contains, in addition to thin and thick filaments, a third myofilament comprised of titin. The extensible region of titin spans the I‐band region of the sarcomere and develops passive force in stretched sarcomeres. This force positions the A‐bands in the middle of the sarcomere, maintains sarcomere length homogeneity and, importantly, is responsible for myocardial passive tension that determines diastolic filling. Recent work suggests that smooth muscle expresses a truncated titin isoform with a short extensible region that is predicted to develop high passive force levels. Several mechanisms for tuning the titin‐based passive tension have been discovered that involve alternative splicing as well as posttranslational modification, mechanisms that are at play both during normal muscle function as well as during disease. Muscle Nerve, 2007
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Cardiac muscle
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In the cross-bridge theory, contractile force is produced by cross-bridges that form between actin and myosin filaments. However, when a contracting muscle is stretched, its active force vastly exceeds the force that can be attributed to cross-bridges. This unexplained, enhanced force has been thought to originate in the giant protein titin, which becomes stiffer in actively compared to passively stretched sarcomeres by an unknown mechanism. We investigated this mechanism using a genetic mutation (mdm) with a small but crucial deletion in the titin protein. Myofibrils from normal and mdm mice were stretched from sarcomere lengths of 2.5 to 6.0 μm. Actively stretched myofibrils from normal mice were stiffer and generated more force than passive myofibrils at all sarcomere lengths. No increase in stiffness, and just a small increase in force, was observed in actively compared to passively stretched mdm myofibrils. These results are in agreement with the idea that titin force enhancement stiffens and stabilizes the sarcomere during contraction and that this mechanism is lost with the mdm mutation.
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Nebulin
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Inflection point
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Abstract The sarcomere is the fundamental structural and functional unit of striated muscle and is directly responsible for most of its mechanical properties. The sarcomere generates active or contractile forces and determines the passive or elastic properties of striated muscle. In the heart, mutations in sarcomeric proteins are responsible for the majority of genetically inherited cardiomyopathies. Here, we review the major determinants of cardiac sarcomere mechanics including the key structural components that contribute to active and passive tension. We dissect the molecular and structural basis of active force generation, including sarcomere composition, structure, activation, and relaxation. We then explore the giant sarcomere-resident protein titin, the major contributor to cardiac passive tension. We discuss sarcomere dynamics exemplified by the regulation of titin-based stiffness and the titin life cycle. Finally, we provide an overview of therapeutic strategies that target the sarcomere to improve cardiac contraction and filling.
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Muscle relaxation
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