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| 2. |
- Li, Meishan, et al.
(författare)
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There is no slowing of motility speed with increased body size in rat, human, horse and rhinoceros independent on temperature and skeletal muscle myosin isoform
- 2011
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Ingår i: Acta Physiologica. - : Wiley. - 1748-1708 .- 1748-1716. ; 202:4, s. 671-681
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Tidskriftsartikel (refereegranskat)abstract
- Aim: The predictions of scaling of skeletal muscle shortening velocity made by A.V. Hill 60-years ago have proven to be remarkably accurate at the cellular level. The current investigation looks to extend the study of scaling of contractile speed to the level of the molecular motor protein myosin at both physiological and unphysiological low temperatures. Methods: A single muscle cell in vitro motility assay to test myosin function, i.e. myosin extracted from short single muscle fibre segments, was used in four species representing a 5 500-fold difference in body mass (rat, man, horse and rhinoceros) at temperatures ranging from 15 to 35 °C. Results: The in vitro motility speed increased as the temperature of the assay increased, but a more profound effect was observed on the slower isoforms, narrowing the relative differences between fast and slow myosin heavy chain (MyHC) isoforms at physiological temperature in all species. The in vitro motility speed varied according to MyHC isoform within each species: I < IIa < IIx < IIb, but the expected scaling relationship within orthologous myosin isoforms was not observed at any temperature. Conclusion: The scaling effect of body size and limb length on shortening velocity at the muscle fibre level, i.e. the decreasing shortening velocity associated with increasing body weight and limb length, was not confirmed at the motor protein level when including mammals of very large size. Thus, other factors than myosin structure and function appear to cause this scaling effect and thin filament isoform expression or myofilament lattice spacing are forwarded as alternative underlying factors.
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| 3. |
- Cacciani, Nicola, et al.
(författare)
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A prospective clinical study on the mechanisms underlying critical illness myopathy : A time-course approach
- 2022
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Ingår i: Journal of Cachexia, Sarcopenia and Muscle. - : John Wiley & Sons. - 2190-5991 .- 2190-6009. ; 13:6, s. 2669-2682
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Tidskriftsartikel (refereegranskat)abstract
- Background: Critical illness myopathy (CIM) is a consequence of modern critical care resulting in general muscle wasting and paralyses of all limb and trunk muscles, resulting in prolonged weaning from the ventilator, intensive care unit (ICU) treatment and rehabilitation. CIM is associated with severe morbidity/mortality and significant negative socioeconomic consequences, which has become increasingly evident during the current COVID-19 pandemic, but underlying mechanisms remain elusive.Methods: Ten neuro-ICU patients exposed to long-term controlled mechanical ventilation were followed with repeated muscle biopsies, electrophysiology and plasma collection three times per week for up to 12 days. Single muscle fibre contractile recordings were conducted on the first and final biopsy, and a multiomics approach was taken to analyse gene and protein expression in muscle and plasma at all collection time points.Results: (i) A progressive preferential myosin loss, the hallmark of CIM, was observed in all neuro-ICU patients during the observation period (myosin:actin ratio decreased from 2.0 in the first to 0.9 in the final biopsy, P < 0.001). The myosin loss was coupled to a general transcriptional downregulation of myofibrillar proteins (P < 0.05; absolute fold change >2) and activation of protein degradation pathways (false discovery rate [FDR] <0.1), resulting in significant muscle fibre atrophy and loss in force generation capacity, which declined >65% during the 12 day observation period (muscle fibre cross-sectional area [CSA] and maximum single muscle fibre force normalized to CSA [specific force] declined 30% [P < 0.007] and 50% [P < 0.0001], respectively). (ii) Membrane excitability was not affected as indicated by the maintained compound muscle action potential amplitude upon supramaximal stimulation of upper and lower extremity motor nerves. (iii) Analyses of plasma revealed early activation of inflammatory and proinflammatory pathways (FDR < 0.1), as well as a redistribution of zinc ions from plasma.Conclusions: The mechanical ventilation-induced lung injury with release of cytokines/chemokines and the complete mechanical silencing uniquely observed in immobilized ICU patients affecting skeletal muscle gene/protein expression are forwarded as the dominant factors triggering CIM.
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| 4. |
- Cacciani, Nicola, et al.
(författare)
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Chaperone co-inducer BGP-15 mitigates early contractile dysfunction of the soleus muscle in a rat ICU model
- 2020
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Ingår i: Acta Physiologica. - : Wiley. - 1748-1708 .- 1748-1716. ; 229:1
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Tidskriftsartikel (refereegranskat)abstract
- Aim Critical illness myopathy (CIM) represents a common consequence of modern intensive care, negatively impacting patient health and significantly increasing health care costs; however, there is no treatment available apart from symptomatic and supportive interventions. The chaperone co-inducer BGP-15 has previously been shown to have a positive effect on the diaphragm in rats exposed to the intensive care unit (ICU) condition. In this study, we aim to explore the effects of BGP-15 on a limb muscle (soleus muscle) in response to the ICU condition. Methods Sprague-Dawley rats were subjected to the ICU condition for 5, 8 and 10 days and compared with untreated sham-operated controls. Results BGP-15 significantly improved soleus muscle fibre force after 5 days exposure to the ICU condition. This improvement was associated with the protection of myosin from post-translational myosin modifications, improved mitochondrial structure/biogenesis and reduced the expression of MuRF1 and Fbxo31 E3 ligases. At longer durations (8 and 10 days), BGP-15 had no protective effect when the hallmark of CIM had become manifest, that is, preferential loss of myosin. Unrelated to the effects on skeletal muscle, BGP-15 had a strong positive effect on survival compared with untreated animals. Conclusions BGP-15 treatment improved soleus muscle fibre and motor protein function after 5 days exposure to the ICU condition, but not at longer durations (8 and 10 days) when the preferential loss of myosin was manifest. Thus, long-term CIM interventions targeting limb muscle fibre/myosin force generation capacity need to consider both the post-translational modifications and the loss of myosin.
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| 5. |
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| 6. |
- Li, Meishan, et al.
(författare)
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Aberrant post-translational modifications compromise human myosin motor function in old age
- 2015
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Ingår i: Aging Cell. - : Wiley. - 1474-9718 .- 1474-9726. ; 14:2, s. 228-235
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Tidskriftsartikel (refereegranskat)abstract
- Novel experimental methods, including a modified single fiber in vitro motility assay, X-ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging-related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P < 0.001) in old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force-generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X-ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age-specific myosin post-translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age.
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| 7. |
- Li, Meishan, et al.
(författare)
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Force-generating capacity of human myosin isoforms extracted from single muscle fibre segments
- 2010
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Ingår i: Journal of Physiology. - : Wiley. - 0022-3751 .- 1469-7793. ; 588:24, s. 5105-5114
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Tidskriftsartikel (refereegranskat)abstract
- Muscle, motor unit and muscle fibre type-specific differences in force-generating capacity have been investigated for many years, but there is still no consensus regarding specific differences between slow- and fast-twitch muscles, motor units or muscle fibres. This is probably related to a number of different confounding factors disguising the function of the molecular motor protein myosin. We have therefore studied the force-generating capacity of specific myosin isoforms or combination of isoforms extracted from short single human muscle fibre segments in a modified single fibre myosin in vitro motility assay, in which an internal load (actin-binding protein) was added in different concentrations to evaluate the force-generating capacity. The force indices were the x-axis intercept and the slope of the relationship between the fraction of moving filaments and the α-actinin concentration. The force-generating capacity of the β/slow myosin isoform (type I) was weaker (P < 0.05) than the fast myosin isoform (type II), but the force-generating capacity of the different human fast myosin isoforms types IIa and IIx or a combination of both (IIax) were indistinguishable. A single fibre in vitro motility assay for both speed and force of specific myosin isoforms is described and used to measure the difference in force-generating capacity between fast and slow human myosin isoforms. The assay is proposed as a useful tool for clinical studies on the effects on muscle function of specific mutations or post-translational modifications of myosin.Myosin is the molecular motor protein in skeletal muscle that generates force and movement. It is expressed in multiple isoforms that have different enzymatic properties. There are isoform specific differences in contractile speed, but there is no consensus if the force generating capacity differs between isoforms. In this study we have modified a single fibre in vitro motility assay to measure both force and speed generated by specific myosin isoforms extracted from short single human muscle fibre segments. It is shown that human slow myosin is weaker and slower than fast myosin. This assay is put forward as a useful tool for future investigations on myosin function in response to modifications associated with muscle disease or ageing.
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| 8. |
- 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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| 9. |
- Ochala, Julien, et al.
(författare)
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Defective regulation of contractile function in muscle fibres carrying an E41K beta-tropomyosin mutation.
- 2008
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Ingår i: The Journal of physiology. - : Wiley. - 1469-7793 .- 0022-3751. ; 586:Pt 12, s. 2993-3004
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Tidskriftsartikel (refereegranskat)abstract
- A novel E41K beta-tropomyosin (beta-Tm) mutation, associated with congenital myopathy and muscle weakness, was recently identified in a woman and her daughter. In both patients, muscle weakness was coupled with muscle fibre atrophy. It remains unknown, however, whether the E41K beta-Tm mutation directly affects regulation of muscle contraction, contributing to the muscle weakness. To address this question, we studied a broad range of contractile characteristics in skinned muscle fibres from the two patients and eight healthy controls. Results showed decreases (i) in speed of contraction at saturated Ca(2+) concentration (apparent rate constant of force redevelopment (k(tr)) and unloaded shortening speed (V(0))); and (ii) in contraction sensitivity to Ca(2+) concentration, in fibres from patients compared with controls, suggesting that the mutation has a negative effect on contractile function, contributing to the muscle weakness. To investigate whether these negative impacts are reversible, we exposed skinned muscle fibres to the Ca(2+) sensitizer EMD 57033. In fibres from patients, 30 mum of EMD 57033 (i) had no effect on speed of contraction (k(tr) and V(0)) at saturated Ca(2+) concentration but (ii) increased Ca(2+) sensitivity of contraction, suggesting a potential therapeutic approach in patients carrying the E41K beta-Tm mutation.
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| 10. |
- 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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