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Cellular and Molecular Mechanisms Underlying Congenital Myopathy-related Weakness
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Clinical Neurophysiology.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Congenital myopathies are a rare and heterogeneous group of diseases. They are primarily characterised by skeletal muscle weakness and disease-specific pathological features. They harshly limit ordinary life and in severe cases, these myopathies are associated with early death of the affected individuals. The congenital myopathies investigated in this thesis are nemaline myopathy and myofibrillar myopathy. These diseases are usually caused by missense mutations in genes encoding myofibrillar proteins, but the exact mechanisms by which the point mutations in these proteins cause the overall weakness remain mysterious. Hence, in this thesis two different nemaline myopathy-causing actin mutations and one myofibrillar myopathy-causing myosin-mutation found in both human patients and mouse models were used to investigate the cascades of molecular and cellular events leading to weakness.

I performed a broad range of functional and structural experiments including skinned muscle fibre mechanics, small-angle X-ray scattering as well as immunoblotting and histochemical techniques. Interestingly, according to my results, point mutations in myosin and actin differently modify myosin binding to actin, cross-bridge formation and muscle fibre force production revealing divergent mechanisms, that is, gain versus loss of function (papers I, II and IV). In addition, one point mutation in actin appears to have muscle-specific effects.  The presence of that mutant protein in respiratory muscles, i.e. diaphragm, has indeed more damaging consequences on myofibrillar structure than in limb muscles complexifying the pathophysiological mechanisms (paper II).

As numerous atrophic muscle fibres can be seen in congenital myopathies, I also considered this phenomenon as a contributing factor to weakness and characterised the underlying causes in presence of one actin mutation. My results highlighted a direct muscle-specific up-regulation of the ubiquitin-proteasome system (paper III).

All together, my research work demonstrates that mutation- and muscle-specific mechanisms trigger the muscle weakness in congenital myopathies. This gives important insights into the pathophysiology of congenital myopathies and will undoubtedly help in designing future therapies.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 45 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 977
Keyword [en]
skeletal muscle, skeletal muscle contraction, atrophy, nemaline myopathy, myofibrillar myopathy, myosin, actin
National Category
Clinical Medicine
Identifiers
URN: urn:nbn:se:uu:diva-219460ISBN: 978-91-554-8894-9 (print)OAI: oai:DiVA.org:uu-219460DiVA: diva2:699934
Public defence
2014-04-16, Hedstrandssalen, Akademiska sjukhuset, ing 70, b.v., Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-03-24 Created: 2014-03-02 Last updated: 2014-04-29
List of papers
1. A myopathy-related actin mutation increases contractile function
Open this publication in new window or tab >>A myopathy-related actin mutation increases contractile function
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2012 (English)In: Acta Neuropathologica, ISSN 0001-6322, E-ISSN 1432-0533, Vol. 123, no 5, 739-746 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
Nemaline myopathy, ACTA1 mutation, Skeletal muscle, Force, Actin, Myosin cross-bridge
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-173318 (URN)10.1007/s00401-012-0962-z (DOI)000302255000009 ()
Available from: 2012-04-25 Created: 2012-04-23 Last updated: 2017-12-07Bibliographically approved
2. Distinct Underlying Mechanisms of Limb and Respiratory Muscle Fiber Weaknesses in Nemaline Myopathy
Open this publication in new window or tab >>Distinct Underlying Mechanisms of Limb and Respiratory Muscle Fiber Weaknesses in Nemaline Myopathy
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2013 (English)In: Journal of Neuropathology and Experimental Neurology, ISSN 0022-3069, E-ISSN 1554-6578, Vol. 72, no 6, 472-481 p.Article in journal (Refereed) Published
Abstract [en]

Nemaline myopathy is the most common congenital myopathy and is caused by mutations in various genes such as ACTA1 (encoding skeletal alpha-actin). It is associated with limb and respiratory muscle weakness. Despite increasing clinical and scientific interest, the molecular and cellular events leading to such weakness remain unknown, which prevents the development of specific therapeutic interventions. To unravel the potential mechanisms involved, we dissected lower limb and diaphragm muscles from a knock-in mouse model of severe nemaline myopathy expressing the ACTA1 His40Tyr actin mutation found in human patients. We then studied a broad range of structural and functional characteristics assessing single-myofiber contraction, protein expression, and electron microscopy. One of the major findings in the diaphragm was the presence of numerous noncontractile areas (including disrupted sarcomeric structures and nemaline bodies). This greatly reduced the number of functional sarcomeres, decreased the force generation capacity at the muscle fiber level, and likely would contribute to respiratory weakness. In limb muscle, by contrast, there were fewer noncontractile areas and they did not seem to have a major role in the pathogenesis of weakness. These divergent muscle-specific results provide new important insights into the pathophysiology of severe nemaline myopathy and crucial information for future development of therapeutic strategies.

Keyword
Actin, Contractile dysfunction, Limb muscle, Nemaline myopathy, Respiratory muscle, Weakness
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-204291 (URN)10.1097/NEN.0b013e318293b1cc (DOI)000319454400003 ()
Available from: 2013-07-30 Created: 2013-07-29 Last updated: 2017-12-06Bibliographically approved
3. Muscle-specific up-regulation of the ubiquitin-proteasome pathway in a mouse model of nemaline myopathy
Open this publication in new window or tab >>Muscle-specific up-regulation of the ubiquitin-proteasome pathway in a mouse model of nemaline myopathy
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Nemaline myopathy, the most common congenital myopathy, is characterized by muscle fibre atrophy.  This compromises contractile performance and ultimately contributes to muscle weakness.  The pathogenic mechanisms remain obscure but may be related to an aberrant protein turnover rate due to an increased activation of the ubiquitin-proteasome pathway.  To verify, this hypothesis, in the present study, we used skeletal muscles from a transgenic mouse model of nemaline myopathy.  We then evaluated the expression of key proteins such as MuRF1 and atrogin-1.  In the slow-twitch soleus muscle, we observed a trend towards a higher level of atrogin-1 whereas in the fast-twitch tibialis anterior muscle, we revealed a greater expression of MuRF1.  These led to divergent effects on protein content and muscle fibre size.  Indeed, in the soleus, a general protein loss and atrophy was found whilst in tibialis anterior, a preferential myosin loss without any clear reduction in the mean muscle fibre size was noticed.  Overall these findings prove for the first time that in nemaline myopathy, the ubiquitin-proteasome pathway (i) is involved in the process of muscle wasting; (ii) is differentially activated in slow- and fast-twitch muscles; (iii) may be targeted as a future therapy to alleviate muscle wasting.

Keyword
myopathy, muscle wasting, atrophy, Murf1, atrogin-1
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-219403 (URN)
Available from: 2014-02-28 Created: 2014-02-28 Last updated: 2014-04-29
4. The fraction of strongly bound cross-bridges is increased in mice that carry the myopathy-linked myosin heavy chain mutation MYH4(L342Q)
Open this publication in new window or tab >>The fraction of strongly bound cross-bridges is increased in mice that carry the myopathy-linked myosin heavy chain mutation MYH4(L342Q)
2013 (English)In: Disease Models and Mechanisms, ISSN 1754-8403, E-ISSN 1754-8411, Vol. 6, no 3, 834-840 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-202382 (URN)10.1242/dmm.011155 (DOI)000318847400029 ()
Available from: 2013-06-24 Created: 2013-06-24 Last updated: 2017-12-06Bibliographically approved

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