“Vague and nebulous is the beginning of all things, but not their end.”
- Khalil Gibran
This thesis delineated the molecular biology of nebulin, one of the largest proteins known. The aim was to elucidate nebulin expression and function, and provide insight into the pathogenetic mechanisms leading to nemaline myopathy, one of the most common congenital myopathies.
Although the picture remains far from complete, the results acquired expand current knowledge of nebulin biology on several frontiers, and form a solid basis for future research. Such knowledge is needed to understand the sequence of events leading to NM, and to evaluate potential therapies.
Nebulin isoform diversity was previously considered a potential mechanism accounting for the selective muscle weakness observed in NM patients. We found, however, that all nebulin isoforms are ubiquitously expressed at mRNA level, throughout the different skeletal muscles studied, hence indicating another mechanism, or level of regulation, underlying the distribution of weakness. Furthermore, the isoform diversity between different skeletal muscles may be much greater at the protein level, or the relative quantities of different isoforms may vary between muscles or myofibres. As our studies on alternative S21 isoforms indicated, differences in isoform usage may arise on more detailed investigation at the myofibre level. Myofibre composition varies between muscles, hence providing a potential explanation for the variability in weakness. Further studies are needed to more widely investigate the nebulin isoform usage in various myofibre types, and during myogenesis.
Nebulin interactions along the length of the protein have been poorly understood because of the technical challenges set by the enormous size of the protein. Our complete panel of nebulin super repeats, covering most of the protein, enabled us to investigate the binding properties of these smaller nebulin fragments in vitro. Although the interactions in vivo are more complex than those studied in vitro, we have gained valuable results that can be utilised in future studies investigating full-length nebulin. Knowledge of differences in actin-binding intensities between super repeats can also be utilised in assessing the potential pathogenicity of mutations in a location-dependent manner. The super-repeat panel will also provide a useful tool for investigating further protein interactions, and for functional testing of potentially pathogenic variants.
The most widely used model organism of NM is the mouse. By studying the effects of different nebulin defects in mice, and comparing with those in NM patients, we have gained a plethora of data shedding light on nebulin function in health and disease. Our novel mouse models are important additions to the NEB-NM mouse model collection. These mouse models have turned
out to be useful in deciphering the pathogenetic mechanisms of NEB-NM, and will be suitable for the assessment of potential therapeutic approaches.
To date, mutations in twelve different genes have been shown to result in NM. Investigating the mechanisms by which mutations in these different genes lead to the same muscle disorder may provide insight into both the common and the partly separate pathways of pathogenesis. By investigating the differences and similarities between the NEB models, and between the models of NM caused by mutations in other genes, we can achieve a more complete understanding of the pathogenetic mechanisms underlying the disorder.
7 A CKNOWLEDGEMENTS
This study was conducted for the most part at the Folkhälsan Institute of Genetics, at the Folkhälsan Research Center and at the Department of Medical and Clinical Genetics, University of Helsinki, over the years 2010-2019. The beginning of the project (I) was carried out at the Genetics Department of the Faculty of Biological and Environmental Sciences, University of Helsinki, and the mouse work (IV) at the Harry Perkins Institute of Medical Research, in Perth, Western Australia. Professor Anna-Elina Lehesjoki, the current head of the Folkhälsan Research Center, as well as all the past and present heads of these institutes are acknowledged for providing excellent resources and facilities for the research.
I wish to thank the Integrative Life Sciences Doctoral Programme for guidance and support, and the following foundations and organisations for funding this Doctoral Thesis project: the Folkhälsan Research Foundation, the Finska Läkaresällskapet, the Sigrid Jusélius Foundation, the Svenska Kulturfonden, the Medicinska Understödsföreningen Liv och Hälsa r.f., the Endeavour Scholarships and Fellowships of the Australian Government, the Academy of Finland, the Association Française contre les Myopathies and the Foundation Building Strength for Nemaline Myopathy. In addition, I am grateful for the financial support received for participation in international congresses from the University of Helsinki Funds, the Chancellor’s travel grant, the World Muscle Society, the Magnus Ehrnrooth Foundation, the Oscar Öflund Foundation, the Finnish Concordia Fund and the Foundation Building Strength for Nemaline Myopathy.
I would like to thank the Faculty of Biological and Environmental Sciences, University of Helsinki, for the opportunity to carry out my post graduate studies, and Professor Juha Partanen for acting as the Custos at my Thesis Defence. I am grateful to the appointed pre-examiners, Professor Olli Carpén and Docent Minna Ruddock for your thorough examination of my thesis during the summer holiday period. Your comments were extremely valuable and allowed me to improve my thesis.
I wish to express my deepest gratitude to my PhD supervisor Docent Katarina Pelin and co-supervisor Docent Mikaela Grönholm. Your endless enthusiasm towards genetics and molecular mechanisms, and vast knowledge of the field of muscle research have greatly helped and inspired me throughout these years. I have learned so much, and I hope that we can continue to unravel the molecular mysteries of muscle together. Many thanks also to the external members of my Thesis Follow-Up Group, Docent Pekka Heino and Professor Päivi Onkamo, for fruitful discussions and the supportive environment at the meetings. Too bad we did not get to organise one of the meetings in Australia…
I have been fortunate to have an additional supervisor, Dr Kristen Nowak, our animal models expert in Perth, Western Australia. It was an absolute pleasure and honour to work with you. In addition to all the scientific advice, you were always there for us, and made sure that we truly enjoyed our stay in Australia. Thank you for everything. I know that I have made a friend for life.
The group leaders in Finland and Australia, Docent Carina Wallgren-Pettersson and Professor Nigel Laing, are sincerely thanked for welcoming me in your research groups. Your stories from the “early days” in the nemaline myopathy research are inspirational. You two are living proof of what a long-lasting, open and supportive collaboration and friendship can bring to the research community. I have been honoured to work with you, and I am forever grateful for all your advice and for providing a rewarding and educative research environment.
I warmly thank all the past and present members of the NEM Group (research group for nemaline myopathy and related disorders in Helsinki). Especially dorktor Vilma-Lotta Lehtokari, a long-standing colleague and friend is sincerely thanked for our legendary brainstorming get-togethers and so many unforgettable moments in the lab and outside. Thank you for always believing in my abilities, and for cheering me on whenever I had a moment of doubt. I would also like to thank Marilotta Turunen, our multitalented lab technician. Without your magic touch and mad skills we would have been in trouble so many times in the lab. Thanks to Kirsi Kiiski, Johanna Lehtonen and Lydia Sagath for all the adventures around the world, and the rest of our NEM-derived peer support group “Lihikset”, Pauliina Repo and Liina Ahlsten for after work bubbles and all the fun times we have had. Sampo Koivunen, thank you for keeping us ladies in check.
Mubashir Hanif, Minttu Marttila and Kati Donner, thank you for laying the foundations for my projects. Special thanks also to all my hard-working interns and students, especially Pavla Hujová and Roosa Harmo in Helsinki, and Rachel Harries in Perth, for your work related to this thesis, and for providing an extra pair of hands and great company during the long days in the lab.
The Folkhälsan lab has been an amazing place to work in, with all the skilful individuals around.
I would like to thank each and every one of you for all the help you have provided, especially Jaakko Sarparanta, pH Jonsson, Anna Vihola, Saara Tegelberg and Helena Luque, just to name a few. Ann-Liz Träskelin, our amazing lab manager and my dedicated trash-buddy, thanks for always being so kind and cheerful. Professor Bjarne Udd, the leader of the neighbouring muscle group, and all the rest of the Udd Group in Helsinki and Tampere, thank you for all your support and collaboration throughout these years. Peter Hackman, Marco Savarese and Mridul Johari, you deserve a special thank you for your friendship and for being such amazing travel buddies to the yearly World Muscle Society meeting (among so many others).
I wish to thank everyone in the Laing lab in Perth for welcoming me there for a year with open arms. Special thanks are due to Elyshia McNamara and Hayley Goullée, my mousketeer dream team, for guiding me through all the new protocols and mouse work. Gina Ravenscroft, thank you for your friendship and all the invaluable advice during my stay and after it. You truly are an expert when it comes to muscle research (and eating out in Perth). Rhonda Taylor, Sarah Beecroft and Denise Howting, you are amazing. Thank you for all the laughs - I will never forget all the Aussie slang you thought me, or the Happy Little Vegemite song (thanks heaps)!
Royston Ong and Joey Coote, your smiles always light up the lab.
At the Department of Biosciences, I have my wonderful office, lab and teaching buddies to thank for the friendships, discussions and laughs. Thank you Minttu Kansikas, Vasse Stratoulias, Riitta Lindström, Mari Palgi, Jukka Kantelinen, Marjaana Pussila, Arja Ikävalko, and everyone else who helped me in the lab during the first years of my PhD project.
All the co-authors of the original publications outside our groups are sincerely thanked for all the hard work you have put in. Professor Caroline Sewry and Dr Mike Lawlor, I really appreciate your enthusiasm and expert eye when it comes to muscle pathology. Professor Julien Ochala, Catherine Wingate, Dr Tony Bakker and Dr Jacob Ross, thank you for your muscle physiology expertise. To the antibody wizards, Professor Glenn Morris, Dr Le Thanh Lam and Dr Ian Holt; I am grateful for the fruitful collaboration in producing new antibodies – nebulin is not an easy target, and you dived fearlessly right into the project. Professor Anders Paetau and Dr Sanna Huovinen, thank you for providing samples and for your expert interpretations of muscle biopsy findings. Thanks to Sari Hujanen, Joni Keto, Dr Panu Somervuo and Docent Petri Auvinen for your work on the expression array.
Thanks to Marjatta Valkama, Jaana Welin-Haapamäki, Åsa Rehn and Nina Forss for efficiently running the administrative and financial department at the Folkhälsan Institute of Genetics.
Thanks to all my dear friends for providing distractions from the life in the lab. Especially my Girls, Inka, Jenni, Laura, Milla, Sanna, Suski, Seija, Emmi, Sari, Taru among others, thank you for being the best support group a girl can have. Special thanks also to Tude, Matti, WG, Sode, Jouni, Iltsu, Janne, Dardan, Jori, Pete, and everyone else in our beautiful group of friends. There is never a dull moment with you. Liisa, Ellu, Katja, Terhi, Pete, Kattainen, and all the rest of the “syöpöt” (ex. juopot?) group, thank you for all the good times, and for trying to understand what it is that I actually do… Dissecting frogs is actually not too far off!
My family has always supported me in my choices, and I am deeply grateful for everything they have done and been for me. My parents Pipsa and Jore, you are two of the bravest, strongest and kindest people I know. It is clear that I got my love for nature, science and adventure from
you. My brother Olli, you are the true scientist in our family. Your knowledge of physics and passion for studying the world around us is truly inspirational. Thank you for always challenging me to think outside the box, and thank you for being you. My sister Tiina, you are just the wittiest and sharpest powerhouse, whom I admire so much. Thank you for always making me laugh, and for being the beautiful soul that you are. Minna and Tuomas, thanks for being such an important a part of our family, and Saku, thanks for all the cuddles and playtimes with your auntie.
Finally, with all my heart I want to thank Samuli, for always being there for me. Thank you for understanding that life in science is not always ordinary, and that in addition to the long hours, nights and weekends, it may mean packing your bags and moving to the other side of the globe with me. Thank you for your patience in drawing the graphics for many of our papers and this thesis with me – I know my scientific vision and your artistic one do not always go together, but the end-result has always been beautiful. In this thesis, you drew the graphics for Fig.1 A and C from scratch in the very limited time we had, and I am forever grateful for that. It has truly been a team effort, and I love to have you as my teammate. I am already looking forward to all the adventures ahead, and there is no-one else I would rather share them with than you.
Helsinki, September 2019
8 R EFERENCES
Abbott, M., Jain, M., Pferdehirt, R., Chen, Y., Tran, A., Duz, M.B., Seven, M., Gibbs, R.A., Muzny, D., Lee, B., Marom, R., Burrage, L.C., 2017. Neonatal fractures as a presenting feature of LMOD3 -associated congenital myopathy. Am. J. Med. Genet. Part A 173(10), 2789–2794.
Abdalla, E., Ravenscroft, G., Zayed, L., Beecroft, S.J., Laing, N.G., 2017. Lethal multiple pterygium syndrome: A severe phenotype associated with a novel mutation in the nebulin gene. Neuromuscul.
Disord. 27(6), 537–541.
Acakpo-Satchivi, L.J.R., Edelmann, W., Sartorius, C., Lu, B.D., Wahr, P.A., Watkins, S.C., Metzger, J.M., Leinwand, L., Kucherlapati, R., 1997. Growth and muscle defects in mice lacking adult myosin heavy chain genes. J. Cell Biol. 139(5), 1219–1229.
Agrawal, P.B., Greenleaf, R.S., Tomczak, K.K., Lehtokari, V.-L., Wallgren-Pettersson, C., Wallefeld, W., Laing, N.G., Darras, B.T., Maciver, S.K., Dormitzer, P.R., Beggs, A.H., 2007. Nemaline myopathy with minicores caused by mutation of the CFL2 gene encoding the skeletal muscle actin–binding protein, cofilin-2. Am. J. Hum. Genet. 80(1), 162–167.
Agrawal, P.B., Joshi, M., Savic, T., Chen, Z., Beggs, A.H., 2012. Normal myofibrillar development followed by progressive sarcomeric disruption with actin accumulations in a mouse Cfl2 knockout demonstrates requirement of cofilin-2 for muscle maintenance. Hum. Mol. Genet. 21(10), 2341–
2356.
Bang, M.-L., Caremani, M., Brunello, E., Littlefield, R., Lieber, R.L., Chen, J., Lombardi, V., Linari, M., 2009. Nebulin plays a direct role in promoting strong actin-myosin interactions. FASEB J. 23(12), 4117–25.
Bang, M.-L., Gregorio, C., Labeit, S., 2002. Molecular dissection of the interaction of desmin with the C-terminal region of nebulin. J. Struct. Biol. 137(1–2), 119–127.
Bang, M.-L., Mudry, R.E., McElhinny, A.S., Trombitás, K., Geach, A.J., Yamasaki, R., Sorimachi, H., Granzier, H., Gregorio, C.C., Labeit, S., 2001. Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies. J. Cell Biol. 153(2), 413–428.
Bang, M.L., Li, X., Littlefield, R., Bremner, S., Thor, A., Knowlton, K.U., Lieber, R.L., Chen, J., 2006.
Nebulin-deficient mice exhibit shorter thin filament lengths and reduced contractile function in skeletal muscle. J. Cell Biol. 173(6), 905–916.
Bonaldo, P., Braghetta, P., Zanetti, M., Piccolo, S., Volpin, D., Bressan, G.M., 1998. Collagen VI deficiency induces early onset myopathy in the mouse: an animal model for Bethlem myopathy.
Hum. Mol. Genet. 7(13), 2135–40.
Buck, D., Hudson, B.D., Ottenheijm, C.A.C., Labeit, S., Granzier, H., 2010. Differential splicing of the large sarcomeric protein nebulin during skeletal muscle development. J. Struct. Biol. 170(2), 325–333.
Castillo, A., Nowak, R., Littlefield, K.P., Fowler, V.M., Littlefield, R.S., 2009. A nebulin ruler does not dictate thin filament lengths. Biophys. J. 96(5), 1856–1865.
Cenik, B.K., Garg, A., McAnally, J.R., Shelton, J.M., Richardson, J.A., Bassel-Duby, R., Olson, E.N., Liu, N., 2015. Severe myopathy in mice lacking the MEF2/SRF-dependent gene leiomodin-3. J. Clin.
Invest. 125(4), 1569–1578.
Chan, C., Fan, J., Messer, A.E., Marston, S.B., Iwamoto, H., Ochala, J., 2016. Myopathy-inducing mutation H40Y in ACTA1 hampers actin filament structure and function. Biochim. Biophys. Acta 1862(8), 1453–1458.
Chandra, M., Manidi, R., Ford, S., Hidalgo, C., Witt, C., Ottenheijm, C., Labeit, S., Granzier, H., 2009.
Nebulin alters cross-bridge cycling kinetics and increases thin filament activation. A novel mechanism for increasing tension and reducing tension cost. J. Biol. Chem. 284(45), 30889–30896.
Chu, M., Gregorio, C.C., Pappas, C.T., 2016. Nebulin, a multi-functional giant. J. Exp. Biol. 219(2), 146–
152.
Colpan, M., Moroz, N.A., Kostyukova, A.S., 2013. Tropomodulins and tropomyosins: working as a team.
J. Muscle Res. Cell Motil. 34(3–4), 247–260.
Conen, P.E., Murphy, E.G., Donohue, W.L., 1963. Light and electron microscopic studies of
“myogranules” in child with hypotonia and muscle weakness. Can. Med. Assoc. J. 89(10), 983–6.
Corbett, M.A., Robinson, C.S., Dunglison, G.F., Yang, N., Joya, J.E., Stewart, A.W., Schnell, C.,
Gunning, P.W., North, K.N., Hardeman, E.C., 2001. A mutation in alpha-tropomyosin slow affects muscle strength, maturation and hypertrophy in a mouse model for nemaline myopathy. Hum. Mol.
Genet. 10(4), 317–328.
Crawford, K., Flick, R., Close, L., Shelly, D., Paul, R., Bove, K., Kumar, A., Lessard, J., 2002. Mice lacking skeletal muscle actin show reduced muscle strength and growth deficits and die during the neonatal period. Mol. Cell. Biol. 22(16), 5887–5896.
Dangain, J., Vrbova, G., 1984. Muscle development in mdx mutant mice. Muscle Nerve 7(9), 700–704.
De Winter, J.M., Ottenheijm, C.A.C., 2017. Sarcomere dysfunction in nemaline myopathy. J. Neuromuscul.
Dis. 4(2), 99–113.
Donner, K., Nowak, K.J., Aro, M., Pelin, K., Wallgren-Pettersson, C., 2006. Developmental and muscle-type-specific expression of mouse nebulin exons 127 and 128. Genomics 88(4), 489–495.
Donner, K., Ollikainen, M., Ridanpää, M., Christen, H.-J., Goebel, H.H., de Visser, M., Pelin, K., Wallgren-Pettersson, C., 2002. Mutations in the β-tropomyosin (TPM2) gene – a rare cause of nemaline myopathy. Neuromuscul. Disord. 12(2), 151–158.
Donner, K., Sandbacka, M., Lehtokari, V.-L., Wallgren-Pettersson, C., Pelin, K., 2004. Complete genomic structure of the human nebulin gene and identification of alternatively spliced transcripts. Eur. J.
Hum. Genet. 12(9), 744–51.
Duboscq-Bidot, L., Xu, P., Charron, P., Neyroud, N., Dilanian, G., Millaire, A., Bors, V., Komajda, M., Villard, E., 2008. Mutations in the Z-band protein myopalladin gene and idiopathic dilated cardiomyopathy. Cardiovasc. Res. 77(1), 118–125.
Dutta, S., Sengupta, P., 2016. Men and mice: Relating their ages. Life Sci. 152, 244–248.
Eulitz, S., Sauer, F., Pelissier, M.-C., Boisguerin, P., Molt, S., Schuld, J., Orfanos, Z., Kley, R.A., Volkmer, R., Wilmanns, M., Kirfel, G., van der Ven, P.F.M., Furst, D.O., 2013. Identification of Xin-repeat proteins as novel ligands of the SH3 domains of nebulin and nebulette and analysis of their interaction during myofibril formation and remodeling. Mol. Biol. Cell 24(20), 3215–3226.
Fan, J., Chan, C., McNamara, E.L., Nowak, K.J., Iwamoto, H., Ochala, J., 2018. Molecular consequences of the myopathy-related D286G mutation on actin function. Front. Physiol. 9(December), 1–5.
Feng, H.Z., Wei, B., Jin, J.P., 2009. Deletion of a genomic segment containing the cardiac troponin I gene knocks down expression of the slow troponin T gene and impairs fatigue tolerance of diaphragm muscle. J. Biol. Chem. 284(46), 31798–31806.
Frontera, W.R., Ochala, J., 2015. Skeletal muscle: A brief review of structure and function. Calcif. Tissue Int. 96(3), 183–195.
Garg, A., O’Rourke, J., Long, C., Doering, J., Ravenscroft, G., Bezprozvannaya, S., Nelson, B.R., Beetz, N., Li, L., Chen, S., Laing, N.G., Grange, R.W., Bassel-Duby, R., Olson, E.N., 2014. KLHL40 deficiency destabilizes thin filament proteins and promotes Nemaline myopathy. J. Clin. Invest.
124(8), 3529–3539.
Gautel, M., Djinović-Carugo, K., 2016. The sarcomeric cytoskeleton: from molecules to motion. J. Exp.
Biol. 219(2), 135–145.
Gautel, M., Goulding, D., Bullard, B., Weber, K., Fürst, D.O., 1996. The central Z-disk region of titin is assembled from a novel repeat in variable copy numbers. J. Cell Sci. 109, 2747–54.
Gillett, G.T., Fox, M.F., Rowe, P.S.N., Casimir, G.M., Povey, S., 1996. Mapping of human non-muscle type cofilin (CFL1) to chromosome 11q13 and muscle-type cofilin (CFL2) to chromosome 14.
Ann. Hum. Genet. 60(3), 201–211.
Gineste, C., De Winter, J.M., Kohl, C., Witt, C.C., Giannesini, B., Brohm, K., Le Fur, Y., Gretz, N., Vilmen, C., Pecchi, E., Jubeau, M., Cozzone, P.J., Stienen, G.J.M., Granzier, H., Labeit, S., Ottenheijm, C.A.C., Bendahan, D., Gondin, J., 2013. In vivo and in vitro investigations of heterozygous nebulin knock-out mice disclose a mild skeletal muscle phenotype. Neuromuscul.
Disord. 23(4), 357–369.
Gineste, C., Duhamel, G., Le Fur, Y., Vilmen, C., Cozzone, P.J., Nowak, K.J., Bendahan, D., Gondin, J.,
Gineste, C., Duhamel, G., Le Fur, Y., Vilmen, C., Cozzone, P.J., Nowak, K.J., Bendahan, D., Gondin, J.,