| 1. |
- Corpeno, Rebeca, et al.
(författare)
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Time-course analysis of mechanical ventilation-induced diaphragm contractile muscle dysfunction in the rat
- 2014
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Ingår i: Journal of Physiology. - : Wiley. - 0022-3751 .- 1469-7793. ; 592:17, s. 3859-3880
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Tidskriftsartikel (refereegranskat)abstract
- Controlled mechanical ventilation (CMV) plays a key role in triggering the impaired diaphragm muscle function and the concomitant delayed weaning from the respirator in critically ill intensive care unit (ICU) patients. To date, experimental and clinical studies have primarily focused on early effects on the diaphragm by CMV, or at specific time points. To improve our understanding of the mechanisms underlying the impaired diaphragm muscle function in response to mechanical ventilation, we have performed time‐resolved analyses between 6 h and 14 days using an experimental rat ICU model allowing detailed studies of the diaphragm in response to long‐term CMV. A rapid and early decline in maximum muscle fibre force and preceding muscle fibre atrophy was observed in the diaphragm in response to CMV, resulting in an 85% reduction in residual diaphragm fibre function after 9–14 days of CMV. A modest loss of contractile proteins was observed and linked to an early activation of the ubiquitin proteasome pathway, myosin:actin ratios were not affected and the transcriptional regulation of myosin isoforms did not show any dramatic changes during the observation period. Furthermore, small angle X‐ray diffraction analyses demonstrate that myosin can bind to actin in an ATP‐dependent manner even after 9–14 days of exposure to CMV. Thus, quantitative changes in muscle fibre size and contractile proteins are not the dominating factors underlying the dramatic decline in diaphragm muscle function in response to CMV, in contrast to earlier observations in limb muscles. The observed early loss of subsarcolemmal neuronal nitric oxide synthase activity, onset of oxidative stress, intracellular lipid accumulation and post‐translational protein modifications strongly argue for significant qualitative changes in contractile proteins causing the severely impaired residual function in diaphragm fibres after long‐term mechanical ventilation. For the first time, the present study demonstrates novel changes in the diaphragm structure/function and underlying mechanisms at the gene, protein and cellular levels in response to CMV at a high temporal resolution ranging from 6 h to 14 days.
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| 2. |
- Lindqvist, Johan, et al.
(författare)
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A myopathy-related actin mutation increases contractile function
- 2012
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Ingår i: Acta Neuropathologica. - : Springer Science and Business Media LLC. - 0001-6322 .- 1432-0533. ; 123:5, s. 739-746
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Tidskriftsartikel (refereegranskat)abstract
- Nemaline myopathy (NM) is the most common congenital myopathy and is caused by mutations in various genes including NEB (nebulin), TPM2 (beta-tropomyosin), TPM3 (gamma-tropomyosin), and ACTA1 (skeletal alpha-actin). 20-25% of NM cases carry ACTA1 defects and these particular mutations usually induce substitutions of single residues in the actin protein. Despite increasing clinical and scientific interest, the contractile consequences of these subtle amino acid substitutions remain obscure. To decipher them, in the present study, we originally recorded and analysed the mechanics as well as the X-ray diffraction patterns of human membrane-permeabilized single muscle fibres with a particular peptide substitution in actin, i.e. p.Phe352Ser. Results unravelled an unexpected cascade of molecular and cellular events. During contraction, p.Phe352Ser greatly enhances the strain of individual cross-bridges. Paradoxically, p.Phe352Ser also slightly lowers the number of cross-bridges by altering the rate of myosin head attachment to actin monomers. Overall, at the cell level, these divergent mechanisms conduct to an improved steady-state force production. Such results provide new surprising scientific insights and crucial information for future therapeutic strategies.
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| 3. |
- Lindqvist, Johan, et al.
(författare)
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The fraction of strongly bound cross-bridges is increased in mice that carry the myopathy-linked myosin heavy chain mutation MYH4(L342Q)
- 2013
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Ingår i: Disease Models and Mechanisms. - : The Company of Biologists. - 1754-8403 .- 1754-8411. ; 6:3, s. 834-840
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Tidskriftsartikel (refereegranskat)abstract
- Myosinopathies have emerged as a new group of diseases and are caused by mutations in genes encoding myosin heavy chain (MyHC) isoforms. One major hallmark of these diseases is skeletal muscle weakness or paralysis, but the underlying molecular mechanisms remain unclear. Here, we have undertaken a detailed functional study of muscle fibers from Myh4(arl) mice, which carry a mutation that provokes an L342Q change within the catalytic domain of the type IIb skeletal muscle myosin protein MYH4. Because homozygous animals develop rapid muscle-structure disruption and lower-limb paralysis, they must be killed by postnatal day 13, so all experiments were performed using skeletal muscles from adult heterozygous animals (Myh4(arl)/+). Myh4(arl)/+ mice contain MYH4(L342Q) expressed at 7% of the levels of the wild-type (WT) protein, and are overtly and histologically normal. However, mechanical and X-ray diffraction pattern analyses of single membrane-permeabilized fibers revealed, upon maximal Ca2+ activation, higher stiffness as well as altered meridional and equatorial reflections in Myh4(arl)/+ mice when compared with age-matched WT animals. Under rigor conditions, by contrast, no difference was observed between Myh4(arl)/+ and WT mice. Altogether, these findings prove that, in adult MYH4(L342Q) heterozygous mice, the transition from weak to strong myosin cross-bridge binding is facilitated, increasing the number of strongly attached myosin heads, thus enhancing force production. These changes are predictably exacerbated in the type IIb fibers of homozygous mice, in which the embryonic myosin isoform is fully replaced by MYH4(L342Q), leading to a hypercontraction, muscle-structure disruption and lower-limb paralysis. Overall, these findings provide important insights into the molecular pathogenesis of skeletal myosinopathies.
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| 4. |
- Ochala, Julien, et al.
(författare)
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A myopathy-linked tropomyosin mutation severely alters thin filament conformational changes during activation
- 2010
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 107:21, s. 9807-9812
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Tidskriftsartikel (refereegranskat)abstract
- Human point mutations in alpha- and beta-tropomyosin induce contractile deregulation, skeletal muscle weakness, and congenital myopathies. The aim of the present study was to elucidate the hitherto unknown underlying molecular mechanisms. Hence, we recorded and analyzed the X-ray diffraction patterns of human membrane-permeabilized muscle cells expressing a particular beta-tropomyosin mutation (R133W) associated with a loss in cell force production, in vivo muscle weakness, and distal arthrogryposis. Upon addition of calcium, we notably observed less intensified changes, compared with controls, (i) in the second (1/19 nm(-1)), sixth (1/5.9 nm(-1)), and seventh (1/5.1 nm(-1)) actin layer lines of cells set at a sarcomere length, allowing an optimal thin-thick filament overlap; and (ii) in the second actin layer line of overstretched cells. Collectively, these results directly prove that during activation, switching of a positive to a neutral charge at position 133 in the protein partially hinders both calcium- and myosin-induced tropomyosin movement over the thin filament, blocking actin conformational changes and consequently decreasing the number of cross-bridges and subsequent force production.
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| 5. |
- Ochala, Julien, et al.
(författare)
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Disrupted myosin cross-bridge cycling kinetics triggers muscle weakness in nebulin-related myopathy
- 2011
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Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 25:6, s. 1903-1913
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Tidskriftsartikel (refereegranskat)abstract
- Nebulin is a giant protein expressed at high levels in skeletal muscle. Mutations in the nebulin gene (NEB) lead to muscle weakness and various congenital myopathies. Despite increasing clinical and scientific interest, the pathogenesis of weakness remains unknown. The present study, therefore, aims at unraveling the underlying molecular mechanisms. Hence, we recorded and analyzed the mechanics as well as the X-ray diffraction patterns of human membrane-permeabilized single muscle fibers expressing nebulin mutations. Results demonstrated that, during contraction, the cycling rate of myosin heads attaching to actin is dramatically perturbed, causing a reduction in the fraction of myosin-actin interactions in the strong binding state. This phenomenon prevents complete thin-filament activation, more especially proper and full tropomyosin movement, further limiting additional binding of myosin cross-bridges. At the cell level, this reduces the force-generating capacity and, overall, provokes muscle weakness. To reverse such a negative cascade of events, future potential therapeutic interventions should, therefore, focus on the triggering component, the altered myosin cross-bridge cycling kinetics.
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| 6. |
- Ochala, Julien, et al.
(författare)
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Myofilament lattice structure in presence of a skeletal myopathy-related tropomyosin mutation
- 2013
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Ingår i: Journal of Muscle Research and Cell Motility. - : Springer Science and Business Media LLC. - 0142-4319 .- 1573-2657. ; 34:3-4, s. 171-175
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Tidskriftsartikel (refereegranskat)abstract
- Human tropomyosin mutations deregulate skeletal muscle contraction at the cellular level. One key feature is the slowing of the kinetics of force development. The aim of the present study was to characterize the potential underlying molecular mechanisms by recording and analyzing the X-ray diffraction patterns of human membrane-permeabilized muscle cells expressing a particular beta-tropomyosin mutation (E41K). During resting conditions, the d(1,0) lattice spacing, Delta(1,0) and I-1,I-1 to I-1,I-0 ratio were not different from control values. These results suggest that, in presence of the E41K beta-tropomyosin mutation, the myofilament lattice geometry is well maintained and therefore may not have any detrimental influence on the contraction mechanisms and thus, on the rate of force generation.
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