Functional genomics in zebrafish to disset the pathogenesis of myofibrillar myopathies
Final Report Abstract
Myofibrillar myopathies (MFM) are progressive diseases of human heart and skeletal muscle with a severe impact on life quality and expectancy of affected patients. Although recently several disease genes for myofibrillar myopathies could be identified, today most genetic causes and particularly the associated mechanisms and signaling events that lead from the mutation to the disease phenotype are still mostly unknown. To assess whether the zebrafish is a suitable model system to validate MFM candidate genes using functional genomics strategies, we specifically inactivated known human MFM disease genes and evaluated the resulting muscular and cardiac phenotypes functionally and structurally. Consistently, targeted ablation of MFM genes such as αB-Crystallin, BAG3, Desmin, DNAJB6, FHL1, Filamin C, Myotilin, Plectin and VCP in zebrafish led to compromised skeletal muscle function mostly due to myofibrillar degeneration as well as severe heart failure. Similar to what was shown in MFM patients, MFM gene-deficient zebrafish showed pronounced gene-specific phenotypic and structural differences. These findings indicate that the zebrafish is a suitable model to functionally and structurally evaluate known but also novel MFM disease genes in vivo. In this context, we found that targeted depletion of putative MFM disease genes identified within this consortium (e.g. Strumpellin, Aciculin, SWIP, Atrogin-1) lead to severe myopathic phenotypes affecting both, heart and skeletal muscle, in zebrafish embryos. Our findings provide important new insights into the pathomechanisms underlying human MFM. For instance, we found that overexpression of human FHL 1 mutations (FHL1-H123Y, FHL1-C132F and FHL1-C224W) in wild-type zebrafish embryos didn´t induce myopathy in a dominant-negative mode. By contrast, overexpression of the FHL1-opathy associated human mutations was unable to rescue skeletal and heart muscle myopathy in FHL1 morphant zebrafish embryos, indicating that these autosomal dominant myopathy causing FHL-1 mutations consistently lead to loss of FHL1 function. Valosin-containing protein (VCP)/p97 is a key regulator of cellular proteostasis thereby orchestrating protein turnover and quality control in vivo, processes fundamental for proper cell function. Mutations in VCP frequently lead to severe human myo- and neurodegenerative disorders such as inclusion body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD) or amyotrophic lateral sclerosis (ALS). Within this consortium, we defined the in vivo role of VCP and its novel interactor SWIP (Strumpellin and WASH-Interacting Protein; WASH complex subunit 7). We found that targeted inactivation of both proteins, VCP or SWIP, led to progressive impairment of cardiac and skeletal muscle function, structure and cytoarchitecture without affecting the differentiation of both organ systems. Notably, loss of VCP resulted in compromised protein degradation via the proteasome and the autophagy machinery, whereas SWIP deficiency did not affect the function of the ubiquitin proteasome system (UPS) but led to ER stress and interfered with autophagy function in vivo. Our findings provide novel insights into the in vivo functions of VCP and its novel interactor SWIP and their particular and distinct roles during proteostasis in striated muscle cells. In summary, numerous of our observations made in the zebrafish model are consistent with molecular and ultrastructural findings in MFM mouse models or human MFM patients and demonstrate that the zebrafish is a suitable vertebrate model (1) to study the molecular mechanisms of MFM pathologies and (2) to screen for novel therapeutically active substances in high-throughput in vivo small compound screens (SCS).
Publications
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Strumpellin is a Novel VCP Binding Protein interlinking Hereditary Spastic Paraplegia with Protein Aggregation Diseases. Brain 2010; 133: 2920-2941
Clemen CS, Tangavelou K, Strucksberg K-H, Just S, Gaertner L, Regus-Leidig H, Stumpf M, Reimann J, Coras R, Morgan RO, Fernandez M-P, Hofmann A, Müller S, Schoser B, Hanisch F-G, Rottbauer W, Blümcke I, von Hörsten S, Eichinger L, Schröder R
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Aciculin interacts with filamin C and Xin and is essential for myofibril assembly, remodeling and maintenance. J Cell Sci 2014. 127:3578-3592
Molt S, Bührdel JB, Yakovlev S, Schein P, Orfanos Z, Kirfel G, Winter L, Wiche G, van der Ven PF, Rottbauer W, Just S, Belkin AM, Fürst DO
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In vivo characterization of human myofibrillar myopathy genes in zebrafish. Biochem Biophys Res Commun 2015; 461:217-223
Bührdel JB, Hirth S, Kessler M, Westphal S, Forster M, Manta L, Wiche G, Schoser B, Schessl J, Schröder R, Clemen CS, Eichinger L, Fürst DO, van der Ven PF, Rottbauer W, Just S
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Loss of dihydrolipoyl succinyltransferase (DLST) leads to reduced resting heart rate in the zebrafish. Basic Res Cardiol 2015; 110: 14
Kessler M, Berger I M, Just S, Rottbauer W
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The toxic effect of R350P mutant desmin in striated muscle of man and mouse. Acta Neuropathol. 2015; 129: 297–315
Clemen CS, Stöckigt F, Strucksberg KH, Chevessier F, Winter L, Schütz J, Bauer R, Thorweihe JM, Wenzel D, Schlötzer-Schrehardt U, Rasche V, Krsmanovic P, Katus HA, Rottbauer W, Just S, Müller OJ, Friedrich O, Meyer R, Herrmann H, Schrickel JW and Schröder R
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Vcp and psmf1: Antagonistic regulators of proteasome activity. Biochem Biophys Res Commun. 2015; 463:1210-1217
Clemen CS, Marko M, Strucksberg KH, Behrens J, Wittig I, Gartner L, Winter L, Chevessier F, Matthias J, Turk M, Tangavelou K, Schutz J, Arhzaouy K, Klopffleisch K, Hanisch FG, Rottbauer W, Blümcke I, Just S, Eichinger L, Hofmann A, Schröder R
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Atrogin-1 Deficiency Leads to Myopathy and Heart Failure in Zebrafish. Int J Mol Sci. 2016; 1. pii: E187
Bühler A, Kustermann M, Bummer T, Rottbauer W, Sandri M, Just S
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Knockout of nexilin in mice leads to dilated cardiomyopathy and endomyocardial fibroelastosis. Basic Res Cardiol. 2016
Aherrahrou Z, Schlossarek S, Stoelting S, Klinger M, Geertz B, Weinberger F, Kessler T, Aherrahrou R, Just S, Rottbauer R, Eschenhagen T, Schunkert H, Carrier L, Erdmann J
