Abstract 466: Myopathy Causing Bag3 P209L Protein Leads to Restrictive Cardiomyopathy Caused by Aggregate Formation and Sarcomere Disruption in Cardiomyocytes
Kathrin Graf-RiesenKenichi KimuraAndreas UngerAchim LotherLutz HeinJan DaerrJulia BrauneAstrid OomsGuang LiSean M. WuJörg HöhfeldWolfgang A. LinkeDieter O. FürstBernd K. FleischmannMichael Hesse
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The co-chaperone BAG3 (Bcl-2 associated athanogene 3) is strongly expressed in cross-striated muscles and plays a key role in the turnover of muscle-proteins as a member of the CASA (chaperone-assisted selected autophagy) complex. An amino acid exchange (P209L) in the human BAG3 gene, caused by a single base mutation, gives rise to a severe dominant childhood muscular dystrophy, restrictive cardiomyopathy, and respiratory insufficiency. To get deeper insights into the pathophysiological mechanisms of the disease, we generated a transgenic mouse model of the human mutation BAG3 P209L , in which a fusion protein consisting of the human BAG3 P209L and the green fluorescent protein eGFP can be conditionally overexpressed. Ubiquitous overexpression of BAG3 P209L -eGFP leads to a severe phenotype between the second and fourth week of life, including decreased body weight, skeletal muscle weakness, and heart failure. Echocardiography revealed that the BAG3 P209L -mice suffer from restrictive cardiomyopathy and Sirius-red-staining of heart tissue showed extensive fibrosis. In cardiomyocytes, isolated from hearts of transgenic mice overexpressing BAG3 wt -eGFP or BAG3 P209L -eGFP, BAG3 wt -eGFP stringently localizes to sarcomeres and intercalated discs, whereas cardiomyocytes from BAG3 P209L -eGFP mice displayed formation of BAG3 containing aggregates and disruption of sarcomeres in vivo . While BAG3 P209L -eGFP binding to á-Hsp70, Filamin C and á-HspB8 was unchanged it was less soluble than BAG3 and had a tendency to aggregate, thereby sequestering BAG3 and its clients. Depletion of the BAG3 pool leads to an impairment of CASA and accumulation of damaged proteins, causing sarcomere disintegration leading to restrictive cardiomyopathy.Keywords:
BAG3
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Abstract Myofibrillar myopathy is a clinically and genetically heterogeneous group of muscle disorders characterized by myofibrillar degeneration. Bcl-2-associated athanogene 3 ( BAG3 )-related myopathy is the rarest form of myofibrillar myopathy. Patients with BAG3 -related myopathy present with early-onset and progressive muscle weakness, rigid spine, respiratory insufficiency, and cardiomyopathy. Notably, the heterozygous mutation (Pro209Leu) in BAG3 is commonly associated with rapidly progressive cardiomyopathy in childhood. We describe a male patient with the BAG3 (Pro209Leu) mutation. The patient presented at age 7 years with muscle weakness predominantly in the proximal lower limbs. Histologic findings revealed a mixture of severe neurogenic and myogenic changes. His motor symptoms progressed rapidly in the next decade, becoming wheelchair-dependent by age 17 years; however, at the age of 19 years, cardiomyopathy was not evident. This study reports a case of BAG3 -related myopathy without cardiac involvement and further confirmed the wide phenotypic spectrum of BAG3 -related myopathy.
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The goal of this study was to evaluate if isolated sarcomeres and half-sarcomeres produce a long-lasting increase in force after a stretch is imposed during activation. Single and half-sarcomeres were isolated from myofibrils using micro-needles, which were also used for force measurements. After full force development, both preparations were stretched by different magnitudes. The sarcomere length (SL) or half-sarcomere length variations (HSL) were extracted by measuring the initial and final distances from the Z-line to the adjacent Z-line or to a region externally adjacent to the M-line of the sarcomere, respectively. Half-sarcomeres generated approximately the same amount of isometric force (29.0 ± SD 15.5 nN·μm(-2)) as single sarcomeres (32.1 ± SD 15.3 nN·μm(-2)) when activated. In both cases, the steady-state forces after stretch were higher than the forces during isometric contractions at similar conditions. The results suggest that stretch-induced force enhancement is partly caused by proteins within the half-sarcomere.
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We studied sarcomere performance in single isolated intact frog atrial cells using techniques that allow direct measurement of sarcomere length and force. The purpose of this investigation was to determine whether length-dependent alterations in contractile activation occur in the single isolated cardiac cell. This was accomplished by determining the effect of initial sarcomere length on the time course of sarcomere shortening and force development during auxotonic twitch contractions. The results presented in this paper demonstrate that the velocity of sarcomere shortening, the rate of force development, and the magnitude of force development during auxotonic twitch contractions all increase as initial sarcomere length increases over the range of about 2 micrometers to greater than 3 micrometers. These results indicate that the level of contractile activation increases as initial sarcomere length increases. Also, results are presented that indicate that the rate of increase of contractile activation during a twitch contraction also increases as initial sarcomere length increases. These length-dependent effects on contractile activation in conjunction with the slow time course of contractile activation cause the force-velocity-length relationship to be time-dependent: i.e., the velocity of sarcomere shortening at a given sarcomere length and load depends on the time during the contraction when the sarcomere reaches that length. The results suggest that length-dependent alterations in contractile activation may play a major role in the improved contractile performance that accompanies an increase in initial sarcomere length in cardiac muscle.
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Abstract Human muscle is a hierarchically organised tissue with its contractile cells called myofibers packed into large myofiber bundles. Each myofiber contains periodic myofibrils built by hundreds of contractile sarcomeres that generate large mechanical forces. To better understand the mechanisms that coordinate human muscle morphogenesis from tissue to molecular scales, we adopted a simple in vitro system using induced pluripotent stem cell-derived human myogenic precursors. When grown on an unrestricted two-dimensional substrate, developing myofibers spontaneously align and self-organise into higher-order myofiber bundles, which grow and consolidate to stable sizes. Following a transcriptional boost of sarcomeric components, myofibrils assemble into chains of periodic sarcomeres that emerge across the entire myofiber. By directly probing tension we found that tension build-up precedes sarcomere assembly and increases within each assembling myofibril. Furthermore, we found that myofiber ends stably attach to other myofibers using integrin-based attachments and thus myofiber bundling coincides with stable myofiber bundle attachment in vitro . A failure in stable myofiber attachment results in a collapse of the myofibrils. Overall, our results strongly suggest that mechanical tension across sarcomeric components as well as between differentiating myofibers is key to coordinate the multi-scale self-organisation of muscle morphogenesis.
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The functional unit of muscle is the half-sarcomere in which crossbridges attach and cycle between interdigitating arrays of thick and thin filaments. Half-sarcomeres shorten during contraction if the force produced by the crossbridges is greater than the external force and are stretched if the force produced by the crossbridges is less than the external force. A typical muscle cell will have many thousands of half-sarcomeres in series so the overall performance of a muscle can be a complex function of the behaviour of individual half-sarcomeres. However, until recently, only whole sarcomere lengths could be measured except in electron micrographs. Sarcomere uniformity has long been a topic of interest and it is known, for instance, that in isolated single fibres the sarcomere lengths tend to be longer near the end of the fibre than at the middle. For this reason, Gordon et al. (1966) in their classic study of the force–length relation of single fibres, developed the length clamp and applied it to a middle region of the fibre where the sarcomere uniformity was greatest. It is also recognized that sarcomere non-uniformity can occur in intact muscles, particularly after they are stretched during contraction, often known as eccentric contractions. Thus, Fridén et al. 1981) persuaded men to run down 100 flights of stairs. This resulted in severe pain in the stretched muscle groups in the following 2–3 days and muscle biopsies showed regions of disrupted sarcomeres in which overstretched and understretched sarcomeres could be observed. Sarcomeres are particularly likely to be unstable at long sarcomere lengths (SLs). In mammalian muscles the plateau of the force–length curve lies between SLs 2.0 and 2.4 μm and force falls at longer SLs reaching zero at 3.9 μm (Edman, 2005). Imagine two sarcomeres in series with SLs > 2.4 μm. If one is slightly weaker, then it will tend to be stretched by its stronger neighbour; but the stretching makes it weaker still. This cycle will tend to lead to increasing variability of SLs on the descending limb but not on the ascending limb or the plateau. This potential instability on the descending limb is minimized by various factors, particularly the passive elasticity provided by titin and the fact that the force–velocity curve has a different slope for stretching rather than shortening. These ideas were greatly expanded by Morgan (1990) who pointed out that the force–velocity relation allows very high velocities once the stretching force exceeds about 1.6 × isometric force. Consequently when muscle are stretched moderately rapidly on the descending limb it is possible for the weakest sarcomeres to stretch very rapidly until stabilized at long (non-overlap, > 3.9 μm) sarcomere lengths by the passive force provided by titin and other cytoskeletal proteins. This ‘popping sarcomere’ theory has provided many insights in the behaviour of muscles when stretched during contraction (for recent review see Proske & Morgan, 2001). A new study in this issue of The Journal of Physiology by Telley et al. (2006) makes an important contribution to this story. In a technical tour de force this group has attached fluorescent antibodies to α-actinin in the Z-line and myomesin in the M-band (the centre of the thick filaments). Thus the length of individual half-sarcomeres could be detected rather than the whole sarcomeres. This is potentially important because the two half-sarcomeres of a sarcomere do not necessarily perform in parallel. The preparation used by Telley et al. (2006) was a single (skinned) myofibril of rabbit skeletal muscle which can be rapidly activated and relaxed by appropriate solution changes. From images of the preparation, which contained 20–60 half-sarcomeres, the length of each half-sarcomere can be determined during development of force, during stretch and during the subsequent relaxation. The behaviour of half-sarcomeres turns out to be complex. For instance during contraction some half-sarcomeres shorten while others extend. Less easily understood is that half-sarcomeres that stretched during isometric contraction (weak half-sarcomeres) were not necessarily the ones that show the greatest increase in length during the subsequent stretch. In addition, pairs of half-sarcomeres were observed in which one was short and the neighbour was long (asymmetric sarcomeres). A key point, however, is that no overextended sarcomeres (popped sarcomeres; SL > 3.9 μm) were observed despite conditions which might be expected to trigger popping. Do these observations invalidate the ‘popping sarcomere’ theory? Not yet. Firstly, the SLs used were only just into the descending limb. Secondly, in a myofibrillar preparation most of the desmin will be lost. The authors argue that this should make the preparation more susceptible to sarcomere popping but in some knock-out studies, muscles lacking desmin appear to be resistant to stretch-induced damage (Sam et al. 2000). Thirdly, in the EM study of Brown & Hill (1991) stretched muscles showed over- and under-stretch sarcomeres in myofilaments within a single myofibril, so it is possible that the averaging across a single myofibril disguises some of the heterogeneity of sarcomere lengths. Nevertheless, the approach used by Telley et al. (2006) represents an important step forward for understanding sarcomere properties, and the ability to observe every half-sarcomere in a functioning myofibril will undoubtedly bring new insights into the complexities of muscle contraction.
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The sarcomere pattern and tension of atrial trabeculae isolated from frog hearts have been monitored. The sarcomere length at zero tension varied with the size of the trabeculae but was never less than 1.88 pm, at which length the ends of the thin filaments are at the centre of the A band. Resting tension became large at sarcomere lengths greater than 2.3 m̈m. It was difficult to stretch the trabeculae to produce sarcomere lengths greater than 2.7 m̈m and doing so generally resulted in irreversible changes. Sarcomeres as long as 3.2 m̈m were seen, however, in cells in series with spontaneously contracting fibres. Broadening of the A band at larger sarcomere lengths was interpreted as indicating misalignment of thick filaments and suggests that thick and thin filaments interact in the resting heart. The entire change in length of the central undamaged half of the trabeculae during stretching could be accounted for by the change in sarcomere length.
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When a stretch is imposed to activated muscles, there is a residual force enhancement that persists after the stretch; the force is higher than that produced during an isometric contraction in the corresponding length. The mechanisms behind the force enhancement remain elusive, and there is disagreement if it represents a sarcomeric property, or if it is associated with length nonuniformities among sarcomeres and half-sarcomeres. The purpose of this study was to investigate the effects of stretch on single sarcomeres and myofibrils with predetermined numbers of sarcomeres ( n = 2, 3. . . , 8) isolated from the rabbit psoas muscle. Sarcomeres were attached between two precalibrated microneedles for force measurements, and images of the preparations were projected onto a linear photodiode array for measurements of half-sarcomere length (SL). Fully activated sarcomeres were subjected to a stretch (5–10% of initial SL, at a speed of 0.3 μm·s −1 ·SL −1 ) after which they were maintained isometric for at least 5 s before deactivation. Single sarcomeres showed two patterns: 31 sarcomeres showed a small level of force enhancement after stretch (10.46 ± 0.78%), and 28 sarcomeres did not show force enhancement (−0.54 ± 0.17%). In these preparations, there was not a strong correlation between the force enhancement and half-sarcomere length nonuniformities. When three or more sarcomeres arranged in series were stretched, force enhancement was always observed, and it increased linearly with the degree of half-sarcomere length nonuniformities. The results show that the residual force enhancement has two mechanisms: 1) stretch-induced changes in sarcomeric structure(s); we suggest that titin is responsible for this component, and 2) stretch-induced nonuniformities of half-sarcomere lengths, which significantly increases the level of force enhancement.
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