The Muscle Cytoskeleton of Mice and Men Structural Remodelling in Desmin Myopathies

Home , Myofibril

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No 754 ISSN 0346-6612 ------From the Department of Integrative Medical Biology, Section for Anatomy, Umeå University, Umeå, Sweden

The Muscle Cytoskeleton of Mice and Men Structural remodelling in desmin myopathies

Lena Carlsson

Umeå 2001

Cover picture: A longitudinal section of the vastus lateralis muscle from a normal human subject. The red dots represent the localisation of desmin in between the myofibrils.

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Courage is very important. Like a muscle, it is strengthened by use. Ruth Gordon

To my family

TABLE OF CONTENTS

ABSTRACT……………………………………………………………………….. 5 ABBREVIATIONS………………………………………………………………... 6 ORIGINAL PAPERS……………………………………………………………… 7 INTRODUCTION………………………………………………………………… 8 The muscle fibre cytoskeleton………………………………………………… 8 Intermediate filaments………………………………………………………… 8 Desmin and other IF proteins in developing muscles…………………………. 9 Desmin in mature skeletal and heart muscle cells……………………………... 10 IF associated proteins…………………………………………………………. 10 The function of desmin………………………………………………………… 11 Desmin in damaged and pathological muscles…………………………………. 11 The desmin gene and mutant phenotypes in mice…………………………….. 12 AIMS OF THE STUDY…………………………………………………………… 14 MATERIALS AND METHODS…………………………………………………. 15 Animal subjects……………………………………………………………….. 15 Human subjects……………………………………………………………….. 15 Preparation and culture of satellite cells………………………………………. 15 Enzymehistochemistry………………………………………………………… 16 Immunocytochemistry………………………………………………………… 16 Immunoelectron microscopy………………………………………………….. 16 Conventional transmission electron microscopy……………………………… 17 RESULTS………………………………………………………………………….. 18 Morphological characterisation of cardiac lesions in desmin K/O mice………. 18 A muscle dystrophy develops in skeletal muscles of desmin K/O mice……… 21 IF and IFAPs in skeletal muscles of WT and desmin K/O mice……………… 23 IF and IFAPs in normal human skeletal muscles……………………………… 23 Altered cytoskeletal organisation in a desmin myopathy….…………………. 23 DISCUSSION……………………………………………………………………… 25 Desmin knock-out mice……………………………………………………….. 25 Human desmin myopathy…………………………………………………….. 25 Morphological alterations in desmin cardiomyopathies………………………. 26 Skeletal muscle morphology in desmin myopathies…………………………... 28 IF and IFAPs in desmin myopathies………………………………………….. 29 The cytoskeleton of neuromuscular and myotendinous junctions……………. 30 Uncertainties and issues to be further investigated…………………………… 30 CONCLUSIONS…………………………………………………………………... 33 ACKNOWLEDGEMENTS……………………………………………………….. 35 REFERENCES…………………………………………………………………….. 36 PAPER I-V…………………………………………………………………………

ABSTRACT

The muscle fibre cytoskeleton of skeletal and heart muscle cells is composed mainly of intermediate filaments (IFs), that surround the myofibrils and connect the peripheral myofibrils with the sarcolemma and the nuclear membrane. Desmin is the first muscle specific IF protein to be produced in developing muscles and is the main IF protein in mature muscles. In skeletal muscle, desmin is particularly abundant at myotendinous and neuromuscular junctions. In the heart an increased amount of desmin is found at intercalated discs and in Purkinje fibres of the conduction system. Interactions between the IFs themselves, and between IFs and other structures such as Z-discs and the sarcolemma, are mediated by intermediate filament associated proteins (IFAPs). A transgenic mice model, which lacks the desmin gene have been developed to study the function of desmin. In these mice, morphological abnormalities are observed in both heart and skeletal muscles. Similar defects have been observed in human myopathies, caused by different mutations in the desmin gene. In the present thesis, skeletal and heart muscles of both wild type and desmin knock-out (K/O) mice have been investigated. Furthermore the cytoskeletal organisation in skeletal muscles from human controls and from a patient with desmin myopathy was examined.

In the desmin K/O mice, no morphological alterations were observed during embryogenesis. These mice postnatally developed a cardiomyopathy and a muscle dystrophy in highly used skeletal muscles. Ruptures of the sarcolemma appear to be the primary event leading to muscle degeneration and fibrosis both in cardiac and affected skeletal muscles. In the heart the muscle degeneration gave rise to calcifications, whereas in skeletal muscles regeneration of affected muscle was seen.

In mature wild type mice, the IF proteins synemin and paranemin, and the IFAP plectin were present together with desmin at the myofibrillar Z-discs, the sarcolemma, the neuromuscular junctions and the myotendinous junctions. Nestin was only found in these junctional regions. In desmin K/O mice, all four proteins were detected at neuromuscular and myotendinous junctions. The normal network of synemin and paranemin were not observed, whereas the distribution of plectin was preserved.

In normal human muscles, synemin, paranemin, plectin and αB-crystallin were colocalised with desmin in between the myofibrils, at the sarcolemma and at myotendinous and neuromuscular junctions. In the human desmin myopathy, the distribution of desmin varied considerably. A normal pattern was seen in some fibres areas, whereas other regions either contained large subsarcolemmal and intermyofibrillar accumulations of desmin or totally lacked desmin. Nestin, synemin, paranemin, plectin and αB-crystallin also exhibited an abnormal distribution. They were often aggregated in the areas that contained accumulations of desmin.

In cultured satellite cells from the patient, a normal network of desmin was present in early passages, whereas aggragates of desmin occurred upon further culturing. In the latter, also the nestin network was disrupted, whereas vimentin showed a normal pattern. αB-crystallin was only present in cells with a disrupted desmin network. Plectin was present in a subset of cells, irrespective of whether desmin was aggregated or showed a normal network.

From the present study it can be concluded that an intact desmin network is needed to maintain the integrity of muscle fibres. Desmin may be an important component in the assembly of proteins, which connect the extrasarcomeric cytoskeleton with the extracellular matrix.

Keywords: desmin, nestin, synemin, paranemin, plectin, αB-crystallin, skeletal muscle, heart muscle, myotendinous junction, motor endplate, costamere.

ABBREVIATIONS

COX Cytochrome c oxidase DMD Duchenne muscular dystrophy DNA Deoxyribonucleic acid ES cell Embryonic stem cell FITC Fluorescein iso-thiocyanate GA Glutaraldehyde GFAP Glial fibrillary acidic protein IF Intermediate filament IFAP Intermediate filament associated protein K/O Knock-out mATP Myofibrillar adenosine triphosphate MyHC Myosin heavy chain NADH-TR Nicotinamide dinucleotide tetrazolium reductase NF Neurofilament PBS Phosphate buffered saline PCR Polymerase chain reaction PAP Peroxidase-anti-peroxidase PFA Paraformaldehyde SDH Succinate dehydrogenase TRITC Tetraethyl rhodamine iso-thiocyanate

Figures 2, 3, 6, 7 and 9 have been published in Acta Physiologica Scandinavica (2001), 171:341-348, and are reproduced with permission from the Editor of the journal.

ORIGINAL PAPERS

The present thesis is based on the following five papers, which in the text will be referred to by their Roman numbers:

I. Thornell L-E, Carlsson L, Li Z, Merickskay M and Paulin D. Null mutation in the desmin gene gives rise to a cardiomyopathy. Journal of Molecular and Cellular Cardiology 29:2107-2124 (1997)

II. Li Z, Merickskay M, Agbulut O, Butler-Browne G, Carlsson L, Thornell L-E, Babinet C and Paulin D. Desmin is essential for the tensile strength and integrity of myofibrils but not for myogenic commitment, differentiation, and fusion of skeletal muscle. Journal of Cell Biology 139(1):129-144 (1997)

III. Carlsson L, Li Z, Paulin D and Thornell L-E. Nestin is expressed during development and in myotendinous and neuromuscular junctions in wild type and desmin knock-out mice. Experimental Cell Research 251:213-223 (1999)

IV. Carlsson L, Li Z, Paulin D, Price MG, Breckler J, Robson RM, Wiche G and Thornell L-E. Differences in the distribution of synemin, paranemin and plectin in skeletal muscles of wild type and desmin knock-out mice. Histochemistry and Cell Biology 114:39-47 (2000)

V. Carlsson L, Fischer C, Sjöberg G, Robson RM, Thornell L-E and Sejersen T. Cytoskeletal derangements in hereditary myopathy with desmin L345P mutation. (Manuscript)

INTRODUCTION

The muscle fibre cytoskeleton specialised sarcolemmal domains, the The muscle fibre cytoskeleton includes costameres (Pardo et al., 1983). proteins, whose primary function is to link and anchor structural cell components, Intermediate filaments especially the myofibrils, the The first muscle IF protein to be mitochondria, the sarcotubular system and distinguished was obtained from chicken the nuclei (Price, 1991; Stromer, 1998). gizzard (Lazarides and Hubbard, 1976). It Three major filamentous components are had a molecular weight of 50kDa and distinguishable within the muscle fibre when used to produce antibodies, was cytoskeleton; intermediate filaments (IFs); shown to stain the myofibrillar Z-discs and microfilaments (actin); and microtubules. filamentous structures of the sarcolemma The IFs are so named because of their in both chicken skeletal and heart muscles. diameter (8-10nm), which is intermediate In the heart the antibodies was also between that of the thick (myosin, 15nm) targeting the intercalated discs. Based on and thin (actin, 6nm) filaments (Ishikawa its proposed linking role in muscles, this et al., 1968). The actin and myosin protein was called desmin, from the Greek filaments are the contractile components of “desmos” = bond, link. Concurrently the myofibrils. They are arranged in another group isolated and characterised a regular repeating units, i.e. the sarcomeres. synonymous protein from pig stomach The IFs, on the other hand, are localised smooth muscle cells (Small and Sobieszek, between the myofibrils at the level of the 1977). They named the protein skeletin, Z-discs, between peripheral myofibrils to based upon its proposed cytoskeletal the sarcolemma, and between the nuclear function. Skeletin was also the name given membrane to myofibrils and the to the 55KDa protein purified from heart sarcolemma. Purkinje fibres (Thornell et al., 1978). The cytoskeleton may also be Immunostaining with antibodies against subdivided into the extra-sarcomeric, the the purified protein revealed strong intra-sarcomeric and the subsarcolemmal staining in conducting system cells of the cytoskeleton, of which the IFs constitute bovine heart (Eriksson et al., 1978). In the the extra-sarcomeric cytoskeleton (for normal myocardium, skeletin was localised reviews, see Berthier and Blaineau, 1997; over the Z-discs and at the intercalated Price, 1991; Small et al., 1992). The intra- discs (Eriksson and Thornell, 1979). sarcomeric cytoskeleton consists mainly of IFs are ubiquitous structures not only titin and nebulin, two large filamentous present in muscle cells. They are composed proteins that are longitudinally arranged of a heterogeneous group of more than 50 within the sarcomeres. Titin extends from proteins, which have been classified into 6 the Z-disc to the M-band, and nebulin from categories according to their tissue-specific the Z-disc along the length of the actin expression, sequence homology and filaments. The subsarcolemmal evolutionary relationship (Dahlstrand et cytoskeleton includes membrane and al., 1992; Fuchs and Weber, 1994; membrane-associated proteins, such as Lazarides, 1980b). The classification of IF vinculin, spectrin, dystrophin, proteins and their primary tissue transmembrane integrins, ankyrin, α- distribution are shown in table 1. actinin and desmin (for reviews, see The IF proteins form intermediate Berthier and Blaineau, 1997; Price, 1991; filaments, either as homodimers or Small et al., 1992). These proteins heterodimers (Fuchs and Weber, 1994). An indirectly connect the most peripheral IF protein is composed of a conserved myofibrils with the extracellular matrix in central rod domain, which is especially

8 important for the formation of intermediate of the IF proteins. In muscle cells a number filaments, and an amino-terminal head of IF proteins have been identified, i.e. domain and a carboxy-terminal tail desmin, vimentin, nestin, cytokeratins, NFs domain, which are very heterogeneous and lamins (Gard and Lazarides, 1980; both in size and sequence among the Kuruc and Franke, 1988; Lazarides, 1980a; different types of IF proteins. These Rober et al., 1989; Sejersen and Lendahl, variable domains are thought to be 1993) (see further below). responsible for the tissue-specific function

Table 1 Classification of intermediate filament proteins

Class Intermediate filament protein Main tissue of expression

Type I Acidic Keratins Epithelia Type II Basic Keratins Epithelia Type III Desmin Skeletal, heart and smooth muscles Vimentin Cells of mesenchymal origin GFAP Glia cells, astrocytes Peripherins Neuronal cells Type IV Neurofilaments L/M/H Neurons Internexins α/β Neurons Type V Lamins A/B/C Nuclear membrane Type VI Nestin Neuroepithelial stem cells

Unclassified Paranemin Skeletal and heart muscles Synemin Skeletal and heart muscles Syncoilin Skeletal and heart muscles Filensin Lens Phakinin Lens

Desmin and other IF proteins in normal mature skeletal muscle fibres developing muscles (Barbet et al., 1991; Fürst et al., 1989). Various IF proteins are expressed at The IF protein nestin, originally identified different stages during the development of in neuroepithelial cells, is also present in skeletal and cardiac muscle. Vimentin is developing skeletal and heart muscles, the characteristic IF protein for where it is transiently coexpressed with mesenchymal cells, some of which are the desmin and vimentin (Kachinsky et al., precursor cells of muscle (Barbet et al., 1994; Sejersen and Lendahl, 1993; Sjöberg 1991; Fürst et al., 1989; Gard and et al., 1994). Nestin becomes Lazarides, 1980; Granger and Lazarides, downregulated postnatally in the rat and is 1979; Tokuyasu et al., 1985). When these absent in mature human skeletal muscles cells commit to a muscle lineage, they (Sejersen and Lendahl, 1993; Sjöberg et express desmin. During early myogenesis al., 1994). However, nestin is detected in desmin becomes incorporated into the pre- the adult human heart, although its existing vimentin filaments and forms localisation has yet to be investigated in longitudinal strands. Upon maturation of detail (Sjöberg, 1997). myotubes these strands are transformed In addition, both cytokeratins and NFs into transversely organised filaments, are present in developing heart muscle in localised in between the myofibrils at the some mammalian species (Gorza and level of the Z-discs (Gard and Lazarides, Vitadello, 1989; Kuruc and Franke, 1988). 1980; Tokuyasu et al., 1985). During later The cytokeratins are mainly observed as stages of myofibrillar maturation, vimentin punctate aggregates at the intercalated is downregulated and is not expressed in

9 discs, whereas the NFs are most abundant al., 1983). Both in heart and skeletal in the heart conducting cells. muscle cells, desmin seems to be especially abundant at regular intervals Desmin in mature skeletal and heart along the sarcolemma (Tokuyasu et al., muscle cells 1983). In the Purkinje fibres and in the Desmin is the main IF protein to be normal myocardium, the membrane expressed in mature skeletal and heart proteins vinculin and spectrin are also muscles. In mammals, desmin is much abundant at intercalated discs and at more abundant in heart muscle cells (2% of intervals along the sarcolemma (Thornell total protein) than in skeletal muscle cells et al., 1985; Thornell et al., 1984). In (0.35%) (Price, 1984). Desmin forms a skeletal muscles both proteins are three-dimensional scaffold around the concentrated in distinct domains at the myofibrillar Z-discs and interconnects the sarcolemma (Porter et al., 1992). This entire contractile apparatus with the localisation is thought to correspond to the subsarcolemmal cytoskeleton, the nuclei costameres (Pardo et al., 1983). Vinculin and other cytoplasmic organelles. In and spectrin have been suggested to serve chicken and rabbit muscles, desmin also as a link between intracellular structures forms longitudinal connections between and the extra cellular matrix (Danowski et the peripheries of successive Z-discs and al., 1992; Shear and Bloch, 1985). The along the plasma membrane (Lazarides and mechanism by which the desmin filaments Hubbard, 1976; Tokuyasu et al., 1983; are anchored to the sarcolemma is Wang and Ramirez-Mitchell, 1983). In currently unknown. However, in vitro skeletal muscles, desmin is enriched at the experiments have showed interactions myotendinous junctions and at between desmin and the membrane protein neuromuscular junctions (Askanas et al., ankyrin (Georgatos et al., 1987). 1990; Sealock et al., 1989; Tidball, 1992). In frog myotendinous junctions, desmin is IF associated proteins present deep within the junctional folds but Connections between IFs, and between IFs not immediately subjacent to the junctional and other structures seem to be mediated membrane (Tidball, 1992). In rat either by the IFs themselves or by neuromuscular junctions, desmin is intermediate filament associated proteins particularly concentrated among and (IFAPs). The IFAPs are generally around the ends of the folds (Sealock et al., identified on the basis of their 1989). This localisation is in agreement copurification and colocalisation with a with the observations of intermediate sized known IF protein. Synemin, paranemin and filaments between the subneural nuclei and plectin are three proteins, which have been the postsynaptic folds in freeze-etched identified as IFAPs. Synemin and frog, rat and snake neuromuscular paranemin are coexpressed with desmin junctions (Hirokawa and Heuser, 1982; and vimentin in developing chicken Yorifuji and Hirokawa, 1989). skeletal and heart muscles (Breckler and In the heart, desmin is particularly Lazarides, 1982; Granger and Lazarides, abundant in the Purkinje fibres. 50-75% of 1980; Price and Lazarides, 1983). In the cytoplasm of cow Purkinje fibres mature chicken muscles, synemin is consists of IFs (Thornell and Eriksson, present together with desmin in skeletal 1981; Thornell et al., 1985). In normal muscles, and paranemin together with cardiomyocytes desmin is also abundant in desmin in heart muscle cells. Both proteins a double band structure at intercalated are supposed to be involved in the discs, the cell-to-cell contact in which both regulation of IF function. Plectin, on the longitudinal and transverse IFs are inserted other hand is widely expressed in many (Ferrans and Roberts, 1973; Thornell et al., different tissues and cell types (Foisner et 1986; Thornell et al., 1985; Tokuyasu et al., 1988; Foisner and Wiche, 1991;

Wiche, 1989; Wiche et al., 1983). In obvious effect on cardiomyocyte muscles, plectin is codistributed with differentiation was observed (Weitzer et desmin between the myofibrillar Z-discs al., 1995). However, these results are in and at the sarcolemma. Since plectin has contrast to those obtained in myogenic binding sites not only for IF proteins, but cells transfected with truncated desmin, also for membrane components, as well as which were able to assemble and laterally actin and tubulin, it has been suggested to align normal striated myofibrils function as a molecule between intra- (Schultheiss et al., 1991). sarcomeric, extra-sarcomeric and the It has also been proposed that desmin subsarcolemmal cytoskeleton (Foisner and may be involved in signal transduction and Wiche, 1991; Wiche, 1989). transport of myogenic factors between the The small heat-shock protein αB- nucleus and the sarcolemma (Li et al., crystallin typical found in the ocular lens, 1994; Weitzer et al., 1995). has also been identified together with desmin at the myofibrillar Z-discs in Desmin in damaged and pathological skeletal and heart muscles (Bennardini et muscles al., 1992; Dubin et al., 1991; Leach et al., Necrosis of muscle cells is a common 1994). In the heart it is also abundant at finding in several muscle diseases intercalated discs and in heart conducting including muscular dystrophies, myositis fibres (Dubin et al., 1991; Leach et al., and metabolic myopathies. Damaged 1994). muscle cells are repaired through a sequence of events, which leads to The function of desmin regeneration of the muscle fibre. Satellite Desmin has been ascribed different cells, the myoblast precursors localised functions. Originally, desmin was between the basal lamina and the plasma considered to have a primarily cytoskeletal membrane of the mature muscle fibre, are function. By interconnecting the myofibrils activated and start to proliferate to form with each other and to the sarcolemma, the new myotubes or to locally repair the IFs were thought to maintain structural damaged fibre (Schultz and McCormick, integrity (Ferrans and Roberts, 1973; 1994). Protein expression in regenerating Lazarides, 1980b; Lazarides and Hubbard, fibres reciprocates that seen in developing 1976). This possibility is further muscles. Thus, desmin is abundant in strengthened by the fact that Purkinje immature muscle fibres, which also fibres maintain their three-dimensional transiently express vimentin and nestin structure despite selective extraction of (Bornemann and Schmalbruch, 1993; membranes and myofibrillar proteins Gallanti et al., 1992; Sarnat, 1992; Sjöberg (Eriksson and Thornell, 1979). et al., 1994; Thornell et al., 1980; Thornell The expression of desmin at early et al., 1983; Young et al., 1994). Small stages of embryogenesis and its atrophic muscle fibres display slight redistribution during myofibrillogenesis, increase in immunoreactivity for desmin also suggests an important role in muscle (Thornell et al., 1983). differentiation (Granger and Lazarides, Irregularities in the desmin staining 1979; Lazarides, 1982; Lazarides and pattern also occur in ring fibres. In these Capetanaki, 1986). This suggestion was pathological fibres increased amounts of supported by in vitro results, showing that desmin is found in the peripheral bundle of myotube formation was totally blocked in myofibrils, which are perpendicularly C2C12 myoblasts treated with desmin oriented to the main myofibres (for antisense RNA (Li et al., 1994). Similarly, reviews, see Goebel, 1995; Thornell et al., in embryonic stem (ES) cells which do not 1983). express desmin, skeletal and smooth The cytoskeletal organisation of muscle formation is inhibited, whereas no desmin is also affected by intense exercise.

In delayed muscle soreness, in particular the desmin gene (Goldfarb et al., 1998; Li after eccentric training, longitudinal et al., 1999; Muñoz-Mármol et al., 1998; extensions of desmin are frequently Saavedra-Matiz et al., 2000; Sjöberg et al., observed between successive Z-discs 1999). These mutations were either (Fridén, 1984; Waterman-Storer, 1991). familiar or sporadic, and were mainly The reorganisation of the cytoskeleton is localised to the rod domain, which is thought to be caused either by the involved in the formation of filaments. increased load applied to the muscle cells, or by the formation of new sarcomeres to The desmin gene and mutant compensate for the increased load (Fridén, phenotypes in mice 1984). Desmin derives from a single copy gene, In the so-called desmin-related which is highly conserved among different myopathies, desmin aggregates are found species. The full length desmin gene has in skeletal and/or heart muscles (Goebel, been cloned and sequenced in a number of 1995; Goebel and Bornemann, 1993). The animals as well as in man (Herrmann et al., lesions are either seen as inclusion bodies 1989; Li et al., 1993; Li et al., 1989; or as granulofilamentous material scattered Tuggle et al., 1999; van Groningen et al., throughout the cytoplasm (Goebel, 1995). 1994). The desmin gene has been mapped The mechanism leading to these to chromosome 2 (band q35) in human accumulations is unknown, but several (Viegas-Pequignot et al. 1989) and to hypotheses have been proposed. In one chromosome 1 (band C3) in mouse (Li et family with inclusion bodies (late onset al., 1990). distal myopathy), an excessive synthesis or Genetic K/O experiments may be ideal inadequate degradation of desmin was for examing the phenotypic effects of suggested to cause the myopathy (Edström various proteins. These transgenic animals et al., 1980). Furthermore, an abnormal can be used to broaden our knowledge on phosphorylation of desmin was observed in the pathophysiology of human diseases. In one family carrying the granulofilamentous order to determine the function of desmin type of the disease (Fardeau et al., 1978). in vivo, Denise Paulin and her co-workers However, in a subsequent study desmin generated transgenic mice, which carry a was excluded as the primary cause of the deletion within their desmin gene (Li et al., myopathy (Vicart et al., 1996). In several 1996) (Fig. 1). At the same time another other myopathies additional muscle research group presented results obtained proteins, such as dystrophin, actin and αB- from mice with a different deletion in the crystallin were accumulated together with desmin gene (Milner et al., 1996). Both desmin (Bertini et al., 1991; Fidzianska et deletions were localised to the first of nine al., 1995; Goebel et al., 1994). exons and they give rise to similar When this study started the genetic phenotypes (for details of the construction defect causing these desmin-related of the desmin targeting vectors, see Li et myopathies was unknown. However, al., 1996; Milner et al., 1996). The desmin recently it has been shown that a mutation K/O mice develop normal muscles. in the gene coding for αB-crystallin, a However, morphological defects in mediator for a correct assembling of the skeletal, cardiac and smooth muscles were desmin filaments, was a common observed in mature animals (Li et al., denominator for one family of the 1996). In the heart, large areas of fibrosis granulofilamentous type (the autosomal and calcifications were observed (Li et al., dominant form of Fardeau) (Vicart et al., 1996; Milner et al., 1996). The left 1998). In other families of the same type, ventricle was particularly affected different mutations have been identified in (Milner et al. 1996)

Fig. 1. Schematic representation showing the genomic structure of the mouse desmin gene, the targeting vector used to disrupt the desmin gene and the mutated desmin gene. The black boxes represent the nine exons. The localisation of the targeting vector within the exon 1 is indicated. β-gal, β-galactosidase; NEO, Neo cassette; TK, TK cassette. Scale bar, 1kb.

AIMS OF THE PRESENT STUDY

The aim of the present thesis was to investigate the structural organisation and possible function of desmin and desmin related cytoskeletal proteins of the muscle fibre cytoskeleton. To address this issue, in addition to normal muscles, muscles from transgenic desmin K/O mice and from a patient with a mutation within the desmin gene were studied. The specific aims were to answer the following questions:

What is the pathogenesis of the lesions in the hearts of desmin K/O mice? - what is the first sign of a lesion and how do they develop?

How do morphological defects develop in skeletal muscles of desmin K/O mice? - what are the early and late events of muscle defects? - what is the pathogenic mechanism behind the defects? - are all types of muscles affected in a similar way?

Can other cytoskeletal proteins such as nestin, synemin, paranemin and plectin compensate for the lack of desmin? Of special interest is the cytoskeleton of myotendinous and neuromuscular junctions, known to contain high amounts of desmin. To be able to answer this question the cytoskeletal organisation of these proteins in wild type mice had to be established.

How are skeletal muscles affected in patients with a mutation in the desmin gene? - is there a correlation between the morphological modifications observed in the desmin K/O mice? For comparison, the cytoskeletal organisation in normal human skeletal muscles had to be determined.

MATERIALS AND METHODS

Human subjects (V) Animal subjects (I-IV) Three muscle biopsies from a patient with Wild type (des+/+) and homozygous a point mutation (L345P, leu→pro) in the mutant (des-/-) mice of the following ages rod domain of the desmin gene (Sjöberg et were used: foetuses at 16 and 18 days of al., 1999) were investigated. The two first gestation, newborn, 5 and 11 day-old, 2, 3, biopsies were obtained from the vastus 4, 6, 10, 12 and 20-week-old. In the mutant lateralis muscle by percutaneous mice, the desmin gene was disrupted by conchotome method, whereas the last inserting a targeting construct coding for biopsy was taken by open surgical bacterial β-galactosidase in-frame into the technique from the deltoid muscle. Muscle first exon of the desmin gene. The plasmid biopsies of the deltoid and vastus lateralis was transferred to ES CK 35 cells and muscles from healthy volunteers were used successfully targeted clones were as controls. The use of muscle biopsies identified by Southern blot analysis. was approved by the Ethics Committee of Modified ES cells were microinjected into the Faculty of Medicine, Umeå University. 3.5-day-old C57BL/6J blastocysts. Each biopsy was divided into four Transgenic founders and subsequent pieces. One was frozen as described above. progeny were bred by backcrossing to Two pieces were stretched on corkplates, C57BI/6JxCBA F1 hybrids. The phenotype fixed with 2% PFA or 2.5% GA for of the mice was identified by polymerase immunoelectron and conventional electron chain reaction (PCR) and Southern blot microscopy, respectively, and the fourth analysis using tail DNA. was used for cell culture. The mice to be used for conventional electron microscopy were perfusion fixed Preparation and culture of satellite cells with 2.5% glutaraldehyde (GA) or a (V) mixture of 4% paraformaldehyde (PFA) Attaching connective tissue was removed and 0.5% GA in 0.1M phosphate buffered under the dissection microscope (Blau and saline (PBS) pH 7.4 . Muscle tissues for Webster, 1981). Then the muscle fragment immunoelectron microscopy were fixed in was minced in F-10 medium to obtain 2% PFA with 0.01% GA in PBS buffer. pieces smaller than 1mm3, which were The heart and the soleus muscles were washed in PBS and digested in 2-3 dissected and further processed. successive treatments with 0.25% trypsin Some of the mice were not perfused. at 37°C during constant stirring for a total Instead the heart, the diaphragm and the of 60 min. The supernatant of each triceps surae muscle, which is composed of digestion step was pooled and cooled on the soleus and gastrocnemius muscles, ice or directly plated onto cell culture were dissected out. Muscle samples were plates in F-10 medium containing 10% mounted in Tissue Tek OCT Compound fetal calf serum and gentamycin in a (Miles Inc., Elkhart, IL, USA) and frozen concentration of 50 ug/ml. Clones were in propane chilled with liquid nitrogen. obtained either by picking single cell These frozen specimens were used for clones with a pipette, or by selective enzyme- and immunohistochemistry. trypsinising a group of cells via the use of a plastic ring. The clones were tested for the presence of myogenic and non- myogenic cells, predominantly fibroblasts, by the use of an antibody against desmin, a muscle-specific protein.

Enzymehistochemistry (I -V) junctions were identified with FITC- Longitudinal and cross sections were conjugated α-bungarotoxin (Sigma, St. serially cut at -20˚C on a Reichert Jung Louis, MO., USA). cryostat (Leica, Nussloch, Germany), and Cells stained according to standard procedures. Cells grown on glass coverslips were Gomori trichrome and haematoxylin-eosin rinsed in PBS and fixed in 4% PFA in PBS stainings were used to get a general for 10 min. After a subsequent rinsing in overview of the sections. A staining for PBS, cells were permeabilised in 0.5% myofibrillar adenosine trihosphate (mATP) Triton-PBS for 5 min and rinsed in PBS. activity, combined with preincubations at Unspecific binding was blocked with 10% pH 4.3, 4.6, 9.4 and 10.3, was used to fetal calf serum and cells were incubated identify the different muscle fibre types. with a primary antibody, rinsed and Nicotinamide dinucleotide tetrazolium incubated in a secondary antibody (FITC, reductase (NADH-TR), α- Zymed Laboratories Inc., San Francisco, glycerophosphate, succinate CA, USA or TRITC/FITC, Dako, dehydrogenase (SDH) and cytochrome c Glostrup, Denmark). Cells were rinsed and oxidase (COX) were used for evaluation of mounted in fluorescent mounting medium mitochondrial activity. Von Kossa and (Dako, Glostrup, Denmark). Alizarin Red stainings were used for Double staining demonstration of calcium deposits. For double staining experiments, a Neuromuscular and myotendinous sequential staining was performed junctions were identified with a staining according to a standard procedure with two for acetylcholinesterase or with antibodies raised in different species interference microscopy. (Beesley, 1993). These antibodies were conjugated to two different fluorochromes, Immunocytochemistry (I-V) which were recognised as red and green Sections light at a certain wavelength. Immuno stainings were performed on Controls sections from both unfixed and PFA-fixed Control sections were treated as above, muscle samples. After rehydration in PBS, except that the primary antibodies were the sections were pre-treated with non- substituted by non-immune serum. immune serum and incubated with primary antibodies for 60 min at 37˚C or overnight Immunoelectron microscopy (III-IV) at 4˚C, in the latter with a tenfold dilution PFA-fixed muscle tissues were cut into 1- of antibodies. In table 2 all primary mm small cubes, cryoprotected in 2.3 M antibodies used in the different papers are sucrose and frozen on stubs in liquid listed. Detection of bound antibodies was nitrogen. Semi-thin sections were cut at - performed with standard indirect 95˚C on a Reichert Ultracut microtome peroxidase-anti-peroxidase (PAP) or equipped with a FCS cryo attachment fluorescence techniques. Visualisation of (Leica, Nussloch, Germany). The sections the antibodies was revealed in a solution were collected on slides and stained with containing diaminobenzidine and hydrogen indirect immunofluorescence (see above). peroxide in the case of PAP staining, Ultra-thin sections were cut at -110˚C and whereas a fluorochrome (FITC, green transferred to grids with a sucrose drop. fluorescence; TRITC, red fluorescence; Cy Sections on grids were washed and 3, red fluorescence; Alexa 488, green immersed in 5% goat serum to block fluorescence; Alexa 568, red fluorescence) unspecific binding. After incubation in (Dako, Glostrup, Denmark and Molecular primary antibodies for 60 min, the sections Probes Inc., Eugene, OR, USA) conjugated were washed and incubated with gold to the secondary antibody was used for labelled (5 or 10nm) secondary antibodies immunofluorescence. Neuromuscular (British Bio Cell, Cardiff, UK) for 30

16 minutes. Sections were post fixed in 2.5% further postfixed in 2% osmium tetroxide GA and counterstained with 2% uranyl for 2 hours. The specimens were oxalate and 4% uranyl acetate (Merck, dehydrated in acetone and embedded in Darmstadt, Germany). The sections were Polybed 812 or Vestopal W. Ultrathin embedded in 1.15% methyl cellulose sections (70 nm thick) were cut, collected (Fluka, Buchs, Switzerland), air-dried and on grids and were stained with uranyl photographed in a Jeol 1200 EX-II electron acetate and lead citrate on a LKB 2168 microscope (Jeol, Tokyo, Japan). ultrostainer. Sections were observed and photographed in a Jeol 1200 Conventional transmission electron EX-II electron microscope (Jeol, Tokyo, microscopy (I-III) Japan). Muscle samples were washed in PBS and divided into small pieces, which were

Table 2. Overview of the antibodies used Antibody Clone Host Paper Desmin D33 Mouse I-V Desmin A0611 Rabbit I, IV Desmin Rabbit II Desmin 37EH11 Mouse I Vimentin 3B4 Mouse I Vimentin Goat II Vimentin V9 Mouse V Nestin 130 Rabbit III Nestin R-401 Mouse III Nestin 4350 Rabbit V Synemin Rabbit IV Synemin 2856 Rabbit IV Paranemin Rabbit IV Paranemin 2318 Rabbit IV Plectin 46 Rabbit IV, V Plectin 10F6 Mouse IV, V αB-crystallin NCL-ABCrys Rabbit V CD44 2C5 Mouse I Adhalin NCL-50DAG Mouse V Dystrophin 1 NCL-DYS1 Mouse V Dystrophin 2 NCL-DYS2 Mouse V Dystrophin 3 NCL-DYS3 Mouse V Sacoglycan NCL-35DAG Mouse V Spectrin 2 NCL-SPEC2 Mouse V Vinculin FB11 Mouse I nNOS Rabbit V Desmoplakin DP2.15 Mouse I Myomesin B4 Mouse III N2.261 Mouse V Fast MHCs WB-MHCf Mouse II Summary of the antibodies used with data on their clone and host.

RESULTS

Morphological characterisation of appeared to be disorganised when cardiac lesions in desmin K/O mice compared to the surrounding non-dividing (Paper I) myocytes. No macroscopical signs of abnormalities in A few (2-3) weeks after birth, the heart of desmin K/O mice were calcified lesions can be observed observed until two weeks postnatally. macroscopically as yellowish-white lesions Using electron microscopy, however, on the exterior of the heart (Fig. 2). These already in 5-day-old mice single or groups lesions contain calcium deposits, as of cardiomyocytes with lower density were revealed by their positive staining with present among apparently normal Alizarin Red or von Kossa. Even though cardiomyocytes with well-organised the extent of the lesions varied, the free myofibrils. The affected myocytes were wall of the right ventricle and the inter- either undergoing degeneration or were in ventricular septum were the most affected. different stages of mitosis. A disrupted At the ultrastructural level, the calcified plasmalemma was an early sign of areas were seen as round or ovoid bodies myocyte degeneration whereas the basal of low density surrounded by concentric lamina seemed to be intact. In these fibres rings of varying density (Fig. 3). The Z-disc streaming, hyper-contraction and remaining myocytes within the lesion were disorganised myofibrils were apparent. scattered and surrounded by interstitial Large variations in the appearance of cells and fibrotic tissue. The calcified mitochondria were observed. Some were lesions lacked myofibrillar ATPase activity rounded and lacked or had fragmented and were depleted of mitochondrial inner membranes, whereas others enzymes, though lysosomal enzymes were contained inclusion densities, which were abundant. Neither degeneration of not present in normal mitochondria. In the cardiomyocytes nor calcification and mitotic myocytes, the mitochondria were fibrosis were observed in the wild type more condensed and the myofibrils mice.

Fig. 2. A) Macroscopic view of a heart from a 9-week-old desmin K/O mouse. The white spotty areas in the right (RV) and left ventricles (LV) represent calcifications. B and C) Transverse serial sections of a heart from a

4-week-old desmin K/O mouse stained for nicotinamide adenine dinucleotide tetrazolium reductase, a mitochondrial enzyme (B) and Alizarin Red, which stains calcium deposits (C). Irregular areas of the free right ventricular wall (RV; cavity of right ventricle) and the interventricular septum (IV) lack mitochondrial activity (light areas in B) and are strongly stained for calcium (dark areas in C).

Fig. 3. A) Electron micrograph of the border of a calcified lesion and the myocardium in a 3-week-old desmin K/O mouse. Abundant interstitial cells (ic) and fibrosis are seen. Two myocytes contain vacuoles (*), which in higher magnification are shown to contain electron dense bodies in destructed mitochondria (B). C) Electron micrograph of a longitudinally sectioned papillary muscle from a 2-week-old desmin K/O mouse. One myocyte contains supercontracted myofibrils with scattered rounded mitochondria in the cytoplasm. In adjacent myocytes regular myofibrils and abundant lipid droplets are present. D) A cardiomyocyte with both normal and disorganised myofibrils (*) is shown. Mitochondria (m), myofibril (mf). Scale bar is 5 µm in A and C, 1.5 µm in B and 1 µm in D.

The intercalated discs were cells. In myocytes facing the border of a characteristically affected in the desmin myocardial lesion remnants of intercalated K/O mice (Fig. 4). In normal mice the discs, appearing as finger-like processes intercalated discs, which are the contact protruding into the area of a former regions between cardiomyocytes, have a myocyte, were observed. These cells were complex structure. The contact region is often hypercontracted and contained composed of desmosomes and gap inclusions filled with mitochondria, in junctions. In the desmin K/O mice the cell- which crystalline electron dense material to-cell contacts were commonly more was frequently observed. distended and contained fewer In the desmin K/O mice the Purkinje desmosomes. The lacunae, which were fibres showed no major alterations, observed between widened junctions of although those in wild type mice were many cardiomyocytes, were often filled shown to contain high amounts of desmin. with remnants of organelles and interstitial

Fig. 4. A) Intercalated disc of a 10-week-old wild type mouse shows a dense zigzag structure (*). B) Intercalated disc of a 6-week-old desmin K/O mouse with lysis (*) in the area, where myofibrils are attached to the disc. C) Intercalated disc of a 3-week-old desmin K/O mouse. The contact region is distended and contains abnormal slender profiles (*). D) A myocyte with supercontracted myofibrils ends abruptly (arrow) into the border region of a lesion in a 3-week-old desmin K/O mouse. Note the lacuna filled with mitochondria (*). Scale bar is 1µm in A-C and 5 µm in D.

A muscle dystrophy develops in skeletal In the soleus muscle a switch in fibre muscles of desmin K/O mice (Paper II) type composition was observed in sections By replacing the desmin gene with a gene stained for mATPase. In 4-week-old wild coding for lacZ, it was possible to visualise type mice the soleus muscle contained a blue reaction product developed 55% fast fibres, whereas in age matched following in the appropriate substrate for desmin K/O mice, the percentage of fast this enzyme. This method showed the fate fibres was reduced to 40%. In 12 week-old of myogenic cells, their differentiation, mice this difference was even more migration and formation of myotubes, pronounced and was in the K/Os 10% in muscle fibres and muscles. comparison to 45% in normal mice. In No morphological alterations occurred accordance with the fibre type switch, the during early muscle development. Somites myosin heavy chain composition also formed, and migration and differentiation modified in the desmin K/O mice. Gel of myogenic cells occurred on time. At electrophoresis of muscle extracts from the birth no anatomical defects were observed soleus muscle of wild type mice, revealed in the desmin K/O mice. They were, three myosin heavy chain (MyHC) however, slightly smaller than wild type isoforms, i.e. slow type I, fast type IIA and mice. IIX/D. In 4-week-old desmin K/O mice a In adult desmin K/O mice 25-50% decrease in the amount of IIA and modifications were observed in the soleus, IIX MyHCs were seen. the diaphragm and the tongue, whereas As observed in the heart, disruption of other muscles were not affected (Fig. 5). the plasmalemma was the first The muscle fibres of the soleus muscle ultrastructural feature of muscle fibre showed large variations with respect to degeneration in skeletal muscle fibres. In fibre types, myosin composition, fibre affected muscle fibres, irregularities in the diameter and organisation of myofibrils myofibrillar organisation were apparent and mitochondria. and the mitochondria were often disorganised and accumulated at the plasmalemma (Fig. 6). However, well- organised myofibrils intermingled with mitochondria were seen in most muscle cells of the young desmin K/O mice. Surprisingly, even in the absence of desmin, in some muscle fibres strands of filamentous material were seen between adjacent myofibrils, and between peripheral myofibrils and the plasmalemma.

Fig. 5. Transverse cryosections of the tongue from 11-day-old (A) and 5-month-old (B) desmin K/O mice. The sections are stained with toluidin blue. Severe muscle degeneration of the tongue is seen in the 5-month-old desmin K/O mouse (B).

In the soleus muscle of older animals, affected muscles, the number of muscle degeneration, regeneration and fibrosis fibres was reduced and replaced by were prominent findings. The regenerative fibrosis. events were characterised by the presence The diaphragm and the gastrocnemius of activated satellite cells, myotubes and muscles, which contained type I, IIA, newly formed muscle fibres. In these IIX/D and IIB MyHCs, revealed no major muscle fibres correct myofibrillar changes with respect to fibre types or assembly was uncommon. In severely myosin content.

Fig. 6. A and B) Electron micrographs of longitudinal sections from a soleus muscle of a 4-week-old desmin K/O mouse. A) A muscle fibre with light cytoplasm and accumulations of mitochondria runs in parallel with fibres with well-organised myofibrils. B) Higher magnification of the boxed area in A shows abundant mitochondria (m) interspersed between disorganised myofibrils (mf). Z (Z-disc) C) Filamentous strands (arrows) interlink two myofibrils. One strand links the myofibrils in the M-band region and another link extends between the M-band of one myofibril to the Z-disc of another myofibril. Scale bar is 5 µm in A and 1 µm in B-C.

IF and IFAPs in skeletal muscles of WT changes with respect to the amount of and desmin K/O mice (Papers III and IV) postsynaptic folds, and number and size of In wild type mice synemin, paranemin and mitochondria in the nerve terminals were plectin are present together with desmin at observed in neuromuscular junctions. the myofibrillar Z-discs and at the Likewise, the degree of folding at plasmalemma. In addition, they are myotendinous junctions was highly abundant at neuromuscular and variable. However, neither of these myotendinous junctions. As expected the alterations seemed to be specific for the developmental IF proteins vimentin and desmin K/Os, as they were also observed nestin are absent in mature muscles. in wild type mice. However, nestin is selectively found in myotendinous and neuromuscular IFs and IFAPs in normal human skeletal junctions. muscles (Paper V) In the desmin K/O mice neither In normal human skeletal muscle fibres vimentin nor nestin maintain their prenatal staining for desmin is seen as transverse upregulation and do not compensate for the striations with a regular periodicity in lack of desmin. Vimentin was expressed longitudinally sectioned muscle fibres, during early muscle development, but was whereas the staining appears as a absent at later stages of development. meshwork in cross-sectioned muscle fibres Nestin, as in the wild type mice, was (Fig. 7). Staining for synemin, plectin and expressed in both primary and secondary αB-crystallin exhibited the same staining myotubes during embyogenesis and was pattern. Staining for paranemin, when downregulated during the first 2-3 weeks present, showed the same pattern, but the of the postnatal period, except at motor staining was very weak. The two plectin endplates and myotendinous junctions. At antibodies differed in their reactivity. The the motor endplates nestin was localised plectin 46 antibody stained the type I fibres between the subneural nuclei and the stronger than type II fibres, whereas the junctional folds, whereas at myotendinous plectin 10F6 antibody showed the reversed junctions it was selectively expressed at pattern. The αB-crystallin antibody stained the sarcolemma and between myofibrils the type I fibres stronger than the type II close to and at the junction. In adult fibres. The antibody against vimentin did desmin K/O mice nestin was also present not stain mature skeletal muscle fibres. The in regenerating muscle fibres. same was true for the nestin antibodies Immunostaining with anti-plectin, at both except that motor endplates and the light and electron microscopy level, myotendinous junctions were detected. showed that plectin was not affected by the lack of desmin and showed the same Altered cytoskeletal organisation in a localisation as in normal mice. In contrast, desmin myopathy (Paper V) synemin and paranemin were generally The muscle biopsies from the desmin absent in the desmin K/O mice. However, myopathy patient were highly abnormal in some muscle fibres synemin and and varied a lot in appearance. Variations paranemin were detected in the in fibre size, fat infiltration and fibrosis subsarcolemmal region. were typical findings. Most fibres were Although synemin and paranemin type I and contained slow MyHC. In were not expressed in muscles of desmin addition many fibres contained MyHC K/O mice, they were selectively expressed isoforms usually expressed during muscle in the postjunctional region of development. Muscle fibre splitting, neuromuscular junctions. Furthermore, at abnormal accumulations of mitochondria myotendinous junctions both proteins were and central nuclei were frequently seen. present within the interdigitating processes The cytoskeletal organisation was highly extending into the tendon. Ultrastructural affected. The normal staining pattern of

23 desmin around the myofibrils was generally not observed. Instead small spots and subsarcolemmal or cytoplasmic aggregates were typical (Fig. 7). In addition some fibres totally lacked desmin. Interestingly, some muscle fibres showed well-organised myofibrils even in the absence of desmin or in the presence of aggregated desmin. Accumulations of nestin, synemin, plectin and crystallin were often present in areas of desmin accumulations. Some fibres lacked dystophin at the plasmalemma, whereas others in addition contained accumulations of dystrophin within the fibres. No vimentin staining was detected in the muscle fibres. In cultured satellite cells from the patient with an L345P mutation a normal network of desmin filaments was Fig. 7. A and B) Longitudinal semi-thin sections of seen in early passages. However a the vastus lateralis muscle from a healthy human disruption of the network became apparent subject (A) and from a patient, who has a point mutation in the desmin gene (B). The section in A after several month in culture. In such cells is stained with a desmin antibody and the section in a normal vimentin network was seen, B is double-stained with a desmin antibody (green) whereas nestin filaments were disrupted. and phalloidin (red). The latter binds to the thin αB-crystallin, which was lacking in cells actin filaments and serves as a marker for with normal desmin filaments, was present myofibrils. A) In the normal muscle, the staining of desmin is seen as regularly spaced dots between the in cells with a disrupted desmin network. myofibrils and at the sarcolemma. B) In the Plectin was present in a subset of cells, pathological muscle, one small sized muscle fibre irrespective of whether a normal or a (*) totally lacks desmin, whereas in the large fibre disrupted network of desmin was seen. desmin is accumulated in the subsarcolemmal region (arrows) and in between the myofibrils. In focal areas traces of a normal desmin staining, regularly spaced dots, is seen. Scale bar is 8 µm in A and 10 µm in B.

DISCUSSION to a defect in the desmin gene and thus are Desmin knock-out mice true desmin myopathies (Dalakas et al., Desmin K/O mice do not express the IF 2000; Goldfarb et al., 1998; Li et al., 1999; protein desmin and represent an ideal Muñoz-Mármol et al., 1998; Sjöberg et al., animal model for studies on the 1999; Sugawara et al., 2000). Other cytoskeleton of muscle cells. This study myopathies with desmin abnormalities can shows that the early stages of muscle be divided into different sub-groups development, as well as subsequent depending on which protein is mutated. maturation of muscle fibres are not Thus the initial family suggested to be a affected by the absence of desmin. No desmin disorder (Fardeau et al., 1978), is anatomical or behavioural defects were now known to be due to a defect in the αB- apparent at birth, although the K/Os were crystallin gene (Vicart et al., 1998). It slightly smaller in comparison to wild type should therefore be referred as αB- mice. However, soon after birth crystallin myopathy (Goebel and Warlo, ultrastructural analysis revealed muscle 2000). Currently 10 different mutations in fibre degeneration in both cardiac and the desmin gene have been described (Fig. skeletal muscles. Curiously, only certain 8). In the true desmin myopathies, clinical skeletal muscles were affected. In order to symptoms generally become apparent in evaluate the mechanisms, which results in early to middle adulthood with muscle muscle cell death, we have investigated in weakness in the lower extremities and gait detail hearts and soleus muscles of desmin disturbances. The myopathy slowly K/O mice. progresses to involve also proximal, We show that already in 5-day-old respiratory, fascial and heart muscles. mice single cadiomyocytes showed signs Occasionally defects in the heart precede of degeneration. In the soleus muscle the those occurring in skeletal muscle (Dalakas first signs of degeneration was seen 5-11 et al., 2000; Goldfarb et al., 1998; Li et al., days after birth. Thereafter a process of 1999). Alterations in the heart appear as continual degeneration, regeneration and conduction defects, arrhytmias and fibrosis was observed. This study confirms congestive heart failure. that lack of desmin postnatally gives rise to The patient we have investigated a cardiomyopathy and a muscular belongs to a family with a desmin dystrophy. Ventricular dilatation and myopathy, which has been thoroughly impaired systolic function are common investigated (Horowitz and Schmalbruch, findings in aged desmin K/O mice (Milner 1994; Milhorat and Wolff, 1943; Sjöberg et al., 2000). These pathological features et al., 1999). The first case was examined reduce the lifespan of the desmin K/O mice and diagnosed as a progressive muscular and make them less tolerant to exercise (Li dystrophy (atrophic distal type) already et al., 1997; Milner et al., 2000). 1923 (Milhorat and Wolff, 1943). In a subsequent study, the histopathology of the Human desmin myopathy muscles was analysed (Horowitz and In some muscle disorders, abnormal Schmalbruch, 1994). A new observation at deposits of desmin have been observed in that time was deposits of desmin in the muscle fibres (Thornell et al., 1983). These interior of muscle cells and in the disorders, which are both hereditary and subsarcolemmal region. Since the disease sporadic, have been referred to as desmin- seemed to be more severe than other distal related myopathies. Recently a number of myopathies, it was suggested to be a these disorders have been shown to be due unique type of adult onset distal myopathy. A recent study has shown that the affected family members have an L345P mutation

25 in the rod domain of the desmin gene mind that the mice totally lack desmin, (Sjöberg et al., 1999). whereas in the human desmin myopathy As our aim was to compare the effects both normal and affected desmin are of a desmin mutation in humans with those present. in desmin K/O mice, one has to keep in

      

Head 1A 1B 2A 2B Tail

Mutation Type Age of onset Skeletal Heart Reference R173-E1791 Familiar 15 + + Muñoz-Mármol et al. 214-2452 Sporadic 40 + - Dalakas et al. A3373 Familiar 20-38 + +/- Goldfarb et al. N3424 Familiar 24-30 + - Dalakas et al. L345P5 Familiar Early-midadult + + Sjöberg et al. A360P6, N393I7 Familiar 2-10 + + Goldfarb et al. L385P8 Sporadic 21 + + Sugarawa et al. R4069 Sporadic 24 + + Dalakas et al. Ile451Met10 Familiar - + Li et al., Dalakas et al.

Fig. 8. A schematic drawing of the human desmin gene. The conserved rod domain is made up by four helix subdomains (1A, 1B, 2A and 2B). The rod domain is flanked by the amino-terminal head domain and the carboxy-terminal tail domain. 10 different mutations in the human desmin gene are indicated with data on their involvement in skeletal and heart muscles.

Morphological alterations in desmin which is exposed to great strain during cardiomyopathies contraction. Desmosomes are the The earliest sign of cell death in the attachment sites for intermediate filaments desmin K/O mice was ruptures of the at the intercalated discs and are involved in plasmalemma in single cardiomyocytes. maintaining the connection between We propose that the cell membrane cardiomyocytes. Therefore the lack of damage gives rise both to leakage of desmin may be related to the observed cellular components from the cell and to an reduction in the number of desmosomes. inflow of Ca2+ into the cell. An increased This in turn may cause the complete osmolarity inside the cells might, due to disruption of intercalated discs observed in swelling, gives rise to the rounded severely affected animals. appearance of the mitochondria. The dense In some areas mitochondria were often bodies seen in some of the mitochondria in dispersed in lacunae between young animals are probably due to calcium cardiomyocytes. These mitochondria seem loading within the mitochondria and would to undergo a progressive degeneration, be the earliest signs of calcification (see since electron dense bodies of varying size further below). were present similar to those observed in The intercalated discs, the contact cardiomyocytes irreversibly damaged by region between cardiomyocytes in series, ischemia (Thornell et al., 1992). Activated were especially affected, indicating an area macrophages were also observed to engulf of minor resistance in the desmin K/O the disintegrated myocytes. It is likely that mice. In normal mice the intercalated discs the fragments of organelles observed have a regular zigzag pattern and contain within the widened space at intercalated an abundance of desmin. It is also an area, discs are remnants of dead cells, which

26 later become calcified. In other areas the lengthening of cardiomyocytes during mitochondria were accumulated in diastole cause ruptures in the abnormally large numbers between plasmalemma, since the cardimyocytes of myofibrils and at the sarcolemma, desmin K/O mice are lacking the normal suggesting that desmin may be involved in cytoskeletal link between the myofibrils maintaining mitochondrial position. The and the sarcolemma. An alternative ongoing degenerative process in desmin hypothesis has been proposed by Milner et K/O mice eventually leads to fibrosis and al. (2000), who suggest that alterations calcification of cell organelles. However, with respect to the positioning and function calcifications in the hearts are not specific of mitochondria initiate cell degeneration, for the lack of desmin, but are merely a since swelling and degeneration of the secondary phenomenon due to mitochondrial matrix were observed before cardiomyocyte degeneration. Calcifications any other structural defects were noticeable occur in other types of cardiomyopathies in (Milner et al., 2000). However mice mice (Eaton et al., 1978; Ivandic et al., younger than 2 weeks were not included in 1996) and in other species, which all the study. We have also observed express desmin (Bajusz et al., 1969; abnormalities in both the positioning and Reichenbach and Benditt, 1969). the structure of mitochondria (see below), Programmed cell death, apoptosis, has but in order to unravel the consecutive recently been considered to be involved in events leading to the cardiomyopathy cardiac myocyte death (Anversa et al., additional studies are needed. 1996; Chien, 1999; Maisch, 1999). In contrast to our results, Milner et al. Therefore we investigated, using electron (1996) have reported that muscle microscopy and immunohistochemistry, if degeneration was predominantly seen in apoptosis might be a cause of cell death in the left ventricles. However we propose the desmin K/O mice. The typical that they have misinterpreted the anatomy ultrastructural signs for apoptosis, of the heart since their figures showed chromatin aggregation at the nuclear calcifications mainly in the right ventricles membrane and fragmentation of the cells, (Thornell et al., 1997). In subsequent were not observed in the hearts of desmin papers they also report that the lesions K/O mice. The expression level of Bax and were more frequently observed in the right Bcl-2, two proteins which appear to ventricles (Capetanaki and Milner, 1998; regulate cell survival, did not reveal any Milner et al., 1999). differences in desmin K/O mice compared Several members of the family to to controls (data not shown). Bcl-2 is which our patient belongs, have had considered to block apoptosis, whereas cardiac conduction defects requiring Bax is a pro-apoptotic protein, which implantation of a permanent pacemaker. induces apoptosis (Hsu et al., 1997; Wolter Heart failure is also the most common et al., 1997). Even though apoptosis might cause of death within the family. At be involved in single cells, it is likely that present it is not known if a specific part of the main pathway of cell death in the the heart is affected preferentially in this desmin K/O mice is by necrosis. family as no heart has been closely Although large variations were examined. However at autopsy, one patient observed in the degree of muscle damage was shown to have a hypertrophic heart in the hearts, we did find that the right with slight fibrosis of the myocardium ventricle and the interventricular septum (Milhorat and Wolff, 1943). In some other were most affected. Based on the desmin myopathies it has been reported localisation of the defects, we suggest that that the right ventricle is the most affected it is not the work-load and the contraction part of the heart, both with respect to per se which initiate degeneration of the conduction defects and the number of cardiomyocytes. It is more probable that desmin aggregates (Ariza et al., 1995;

Goldfarb et al., 1998; Muñoz-Mármol et the soleus muscle which is used for weight al., 1998). Thus the desmin K/O mice bearing and the tongue for eating and seem to reflect the cardiac defects sucking are the most affected. Magnetic observed in human desmin myopathy. resonans imaging (MRI) of the patient with However, a detailed examination of the a mutated desmin gene revealed that the heart from patients with desmin myopathy muscles of the legs and the pelvis region is needed to be able to distinguish were predominantly affected, whereas the similarities and differences with the pectoralis and some of the arm muscles desmin K/O mice. seemed to be almost intact. Such selectivity in muscle involvement seems to Skeletal muscle morphology in desmin be a general phenomenon for many other myopathies muscle diseases, e.g. limb girdle dystrophy In both desmin K/O mice and in the human and oculopharyngeal muscular dystrophy. desmin myopathy, we observed enlarged This might depend on the fact that different muscle fibres, activated satellite cells, skeletal muscles normally exhibit a large myotubes and small fibres with abnormally diversity in both functional and structural organised myofibrils. These observations properties (Bottinelli and Reggiani, 2000; can be assembled into a series of events, Pette and Staron, 1990; Schiaffino and which are typical for degeneration and Reggiani, 1994; Schiaffino and Reggiani, subsequent regeneration of skeletal muscle 1996). cells. A ruptured plasmalemma was the As in other degenerative first defect seen in the skeletal muscles of neuromuscular diseases, abortive desmin K/O mice, and this was similar to regeneration as demonstrated by branched the first lesions observed in the heart (see muscle fibres, extensive fibrosis and fat above). We therefore conclude that desmin infiltration were present in skeletal muscles has an important function in the of both the patient with desmin myopathy maintenance of cell structure during and in the desmin K/O mice. The contraction in both skeletal and heart disorganised myofibrils seen in muscles. Desmin thus belongs to the group regenerating muscle fibres in contrast to of proteins which if affected give rise to a the well-aligned myofibrils in developing muscle disease characterised by muscle muscles, indicate that desmin is needed to weakness due to muscle fibre degeneration correctly assemble myofibrils during the and abortive regeneration. Other proteins regenerative process. Recent studies in previously described which are related to mice have shown that the absence of loss of membrane integrity when lacking desmin prolongs myoblast proliferation or being defect are dystrophin (Hoffman et and delays fusion in regenerating grafts of al., 1987; Koenig et al., 1988), α-2- skeletal muscle (Smythe et al., 2001) and laminin (Helbling-Leclerc et al., 1995), that injection of cardiotoxin results in a plectin (Chavanas et al., 1996; Gache et delayed and incomplete muscle al., 1996), integrin (Hayashi et al., 1993) regeneration (Agbulut et al., 2001). These and sarcoglycans (Bonnemann et al., 1995; studies further support the hypothesis that Lim et al., 1995). All these proteins, when lack of desmin affects muscle fibre affected, give rise to different types of regeneration. muscular dystrophies. Although an abundance of myonuclei In common with the traditional was seen only a few satellite cells were muscular dystrophies different muscle present in the human desmin myopathy groups demonstrated variable degrees of (unpublished observations). This is pathology in both the desmin K/O mice probably due to many cycles of and in the desmin myopathy patient. Our degeneration and regeneration, and is very results on the desmin K/O mice indicate similar to that observed in biopsies of the that extensively loaded muscles, such as affected muscles of patients with

Duchenne muscular dystrophy (DMD). It The continued expression of plectin at has been suggested that muscles from both the Z-discs and at the plasmalemma in patients with DMD contain a number of the desmin K/O mice indicates that this senescent cells, which upon degeneration molecule can act independently of desmin are unable to regenerate but instead are and it might therefore be a component of replaced by fibrosis (Renault et al., 2000). the filamentous links observed in some of The life span of each satellite cell is the muscle fibres of the desmin K/Os. This limited due to shortening of the telomere at suggestion is supported by the fact that each division (Decary et al., 2000). Since plectin is able to crosslink not only to the telomere length most likely reflects the various cellular components, but also to severity of the disease, it would be itself (Andrä et al., 1998; Reipert et al., interesting in future studies to compare the 1999; Steinböck and Wiche, 1999; telomere length in muscles of patients with Svitkina et al., 1996; Wiche, 1998). desmin myopathy with other human Furthermore, the reduced amount of neuromuscular diseases (Decary et al., vinculin and spectrin observed at the 2000). sarcolemma in plectin deficient mice Ultrastructural analysis of muscle indicates that plectin is able to interact also biopsies from the patient with desmin with the other costameric proteins (Andrä myopathy showed myofibrillar inclusions, et al., 1997). which contained electrondense The lack of synemin and paranemin granulofilamnetous material. Similar between myofibrils clearly showed that findings have also been reported in a they are dependent on desmin for their Z- myopathy where there is a defect in the disc association. This is concurrent with αB-crystallin gene (Fardeau et al., 1978; the fact that they can only coassemble into Vicart et al., 1998). Thus, these heteropolymeric IFs together with either granulofilamentous type of inclusions are desmin or vimentin (Bellin et al., 1999a; not specific for desmin mutations, but are Bellin et al., 1999b; Hemken et al., 1997). rather a general phenomenon, which are Both synemin and paranemin have been observed in desmin-related myopathies. shown to interact with α-actinin, a Z-disc component (Bellin et al., 2001; Seiler et IF and IFAPs in desmin myopathies al., 1998). The presence of synemin and Vimentin did not compensate for the paranemin at the sarcolemma, but not in deficient desmin gene, since it was not the myofibrillar area of desmin K/O mice, found to replace desmin either in the adult indicate that these proteins preferentially desmin K/O mice or in the human patient interact with sarcolemmal proteins. This is with desmin myopathy. supported by their ability to interact with Nestin was expressed in regenerating both vinculin and α-actinin at the fibres, in motor endplates and in the costameres (Bellin et al., 2001; Seiler et myotendinous junctions of the mature al., 1998). desmin K/O mice. Since nestin was also In muscle biopsies of the human present in the junctional areas of wild type desmin myopathy nestin, plectin, synemin, mice, it is unlikely to compensate for the paranemin and αB-crystallin were lacking lack of desmin. In a previous study nestin in some areas, whereas they were abundant was shown to be able to polymerise with in others. Such variability seemed to be desmin and vimentin, but cannot form a related to whether desmin was absent homodimer (Sjöberg et al., 1997). respectively aggregated. It is likely that However, since neither desmin nor this variability depends on the fact that vimentin were present in mature muscles mutated desmin disrupts the pre-existing of desmin K/O mice still another protein filament network, as shown by in vitro must be able to be used as an assembling experiments in the present and a previous partner for nestin. study (Sjöberg et al., 1999). Surprisingly,

αB-crystallin was only present in cultured might therefore gives rise to the abnormal cells with a disrupted desmin network. Our muscle weakness and fatigue observed in results strengthen the hypothesis that an the desmin K/O mice (Agbulut et al., upregulation of αB-crystallin is the cells 2001). Since results on the neuromuscular attempt to protect against stress-induced and myotendinous junctions in the human damage (Banwell and Engel, 2000; desmin myopathy are lacking, further Bennardini et al., 1992; Ray et al., 2001). studies will be needed for comparison. The altered expression of dystrophin and spectrin in some fibres clearly indicated Uncertainties and issues to be further that these costameric proteins are either investigated directly or indirectly interacting with Why are only some muscles affected by desmin. Desmuslin, a recently discovered desmin deficiency, even though they all IF protein, might be involved since it has contain the same type of mutation? This is the capacity to interact with desmin at the a question of general importance for many Z-discs and with dystrobrevin, a human myopathies. Morphological studies dystrophin-associated protein at the on desmin K/O mice showed that muscle sarcolemma (Mizuno et al., 2001). degeneration was observed mainly in the Interestingly, in the human desmin soleus, the diaphragm, the tongue and the myopathy desmuslin seemed to be heart (Li et al., 1996; Li et al., 1997; upregulated at the sarcolemma and was Milner et al., 1996; Thornell et al., 1997). also present in the cytoplasm of some Thus it can be concluded that desmin is fibres (Unpublished observations). These important for the maintenance of muscle fibres coincided with those having fibre integrity. The soleus muscles of increased amount of desmin. desmin K/O mice were also shown to generate less force and to fatigue more The cytoskeleton of neuromuscular and quickly even though they contained an myotendinous junctions increased number of slow fibres (Li et al., The cytoskeletal organisation seems to be 1997). Our finding of fibrosis in the soleus particularly complex at neuromuscular and muscle is in agreement with the increased myotendinous junctions, as synemin, passive stiffness reported in a recent study paranemin, plectin, nestin and desmin were on desmin K/O mice (Anderson et al., abundant in these areas in normal human 2001). On the other hand, other and murine muscles. This complexity is physiological studies have showed that fast likely to depend on the specific function of EDL muscles subjected to eccentric the proteins in junctional areas, in force contractions are less vulnerable to injury transmission at myotendinous junctions and they generate greater relative force in and in muscle fibre exitation at comparison to wild type mice (Sam et al., neuromuscular junctions. However a 2000; Wieneke et al., 2000). However, detailed view of their organisation and further analysis of single fibres of the EDL mutual interactions is still lacking. In the muscle showed that some fibres generated desmin K/O mice synemin, paranemin, no or very low tension and displayed no plectin and nestin were all present in these visible striations (Wieneke et al., 2000). junctional areas although desmin was The contradictory results of early and lacking. They might therefore be able to recent studies might therefore be related to partly compensate for the lack of desmin in the level of muscle fibre degeneration in these structures. The neuromuscular the muscles. junctions of desmin K/O mice contain less Is the degree of involvement related to acetylcholine receptors, Na+ channels and the content of desmin in individual postsynaptic foldings in comparison to muscles? The diaphragm, which shows wild type mice (Agbulut et al., 2001). An severe defects in the desmin K/O mice, impaired neuromuscular transmission contains a greater amount of desmin than

30 the biceps femoris muscle (Boriek et al., unknown. The mitochondria seem also to 2001). In rat, the slow soleus muscle is have different functions in various muscle known to contain higher amounts of groups. In situ investigations using skinned desmin than the fast extensor digitorum fibres have shown that fibres from the longus (EDL) muscle (Chopard et al., heart and soleus muscle of desmin K/O 2001). Since the soleus muscle is mainly mice display an increased affinity for ADP composed of type I fibres, one would and a decreased maximal respiration in expect that the type I fibres should be the comparison to control mice (Kay et al., ones which are primarily affected in the 1997; Milner et al., 2000). On the other desmin K/O mice. By contrast muscle hand these respiratory parameters were degeneration was in our study primarily unaffected in the fast gastrocnemius observed in the type II fibres of the soleus muscle. In vitro experiments on isolated muscle. mitochondria also showed no differences Do the differences in muscle in respiratory activity between K/O and involvement reflect the unique functional WT mice. Further analysis is needed to and structural properties of each muscle? clarify the relationship between Are muscles exposed to high tension mitochondrial function and lack of desmin. primarily affected? The diaphragm is Has the phenotype of the desmin K/O exposed to both transverse and longitudinal mice changed? In initial studies extensive loading during each respiratory cycle. A damage was observed in the heart of recent study has shown that desmin in the desmin K/O mice. In recently published diaphragm, but not in the biceps muscle, papers the mice seem to be less affected connects transverse and longitudinal even though they all lack the desmin gene elements of the cytoskeleton (Boriek et al., (Milner et al., 2000; Milner et al., 1999) 2001). These couplings are needed for the (and unpublished observations). It might regulation of force in transverse and be that continuous inbreeding of the least longitudinal directions (Boriek et al., affected animals gives rise to a genetically 2001). However, the tongue, which is not altered offspring (Müller, 1999; Sigmund, subjected to stretch depending on its non- 2000). osseus insertion, exhibits severe Is desmin involved in degeneration in the desmin K/O mice. Do sacomerogenesis? Our study showed that differences in mitochondrial number and desmin was not needed for initial function affect the degree of muscle formation of sarcomeres during degeneration? The mitochondria are able to development, but might affect interact directly or indirectly with both sarcomerogenesis in regenerating muscle desmin and plectin (Kay et al., 1997; fibres. A recent study indicates that desmin Milner et al., 2000; Reipert et al., 1999). is not essential for the regulation of Undoubtedly lack of either of these sarcomere number during lengthening and proteins causes lysis, swelling and shortening of muscle fibres, but may play a aggregation of the mitochondria. However, role in the optimal arrangement of whether this is a direct or indirect effect is sarcomeres (Shah et al., 2001).

CONCLUSIONS

On basis of the results presented in this thesis, it is concluded that: • Degeneration of individual cardiomyocytes and infiltration of interstitial cells in the early postnatal period, were the first signs of cardiac abnormalities in the desmin K/O mice. • A rupture of the plasmalemma is proposed to be the irreversible damage, which finally results in myocyte necrosis. The intercalated discs, which normally contain high amounts of desmin, seem to be especially prone to injury. • Inflow of calcium and calcium loading of mitochondria are suggested to be major pathogenic events leading to the calcified lesions, a typical end stage of myocardial damage in mice. • Lack of desmin gives rise to skeletal muscle degeneration in mice. Muscle fibre degeneration first appeared in the postnatal period in extensively used skeletal muscles. The muscle fibre damage was due to a rupture of the plasmalemma and was followed by reparative events like phagocytosis, activation of satellite cells and formation of myotubes and new muscle fibres. • The lack of desmin in the cytoskeletal links between the contractile myofibrils and the sarcolemma is suggested to be a common denominator for both heart and skeletal muscle damage. • The IF proteins nestin, synemin and paranemin and the IFAP plectin are constituents of the cytoskeleton in both skeletal muscle fibres and cardiac myocytes of normal mice. Interestingly, these proteins were differently affected in the desmin K/O mice. The proteins showed a preserved localisation at neuromuscular and myotendinous junctions, whereas they were differently reorganised around the myofibrils and at the sarcolemma. • The IF proteins nestin, synemin and paranemin, as well as the IFAPs plectin and αB- crystallin are, together with desmin, involved in the cytoskeleton of mature human muscle fibres. Nestin was only detected in neuromuscular and myotendinous junctions, whereas the other proteins, in addition to being present in junctional areas, form transverse links between adjacent myofibrils and between the peripheral myofibrils and the saroclemma. • The skeletal muscles were affected to various degrees in the patient with a L345P desmin mutation. Fibrosis and fat infiltration were typical for severely affected muscles, whereas less affected muscles showed signs of ongoing muscle fibre degeneration and muscle fibre regeneration. • In skeletal muscle fibres from the patient, desmin was irregularly distributed in the main part of the fibres. Desmin was aggregated in bundles in some areas, whereas it was either lacking or showed a normal pattern in others. Also the other cytoskeletal proteins showed an abnormal expression and organisation, indicating their inability to form an appropriate cytoskeleton. The variable organisation of the proteins is proposed to be due to whether normal or mutated desmin is present. • In early passages of cultured satellite cells from the patient, desmin formed a normal network of filaments, whereas the filaments were disrupted upon further culturing. This indicated that the mutated desmin has a negative feedback on filament formation. Curiously synemin and paranemin were never observed in cultured satellite cells and αB- crystallin was only present in cells with disrupted desmin. Nestin was abnormally distributed in cells with a disrupted desmin network, whereas both vimentin and plectin showed the same staining pattern as in the cells with a normal desmin network. Although we are far away from a complete understanding on the complexity on the muscle fibre cytoskeleton, a more complete picture has evolved (Fig. 9). Results from studies on desmin K/O mice can provide us with ideas for further investigations on structure and function of other cytoskeletal components. In addition, single/double/triple K/Os of the

32 various IF proteins and IFAPs can further broaden our knowledge on human skeletal and cardiac myopathies. However, the use of truncated desmin or a mutated desmin instead of a total absence would be even better, as it would be closer to the situation observed in the human desmin myopathies. Another aspect to remember is that there are species differences between mice and men. Transgenic mice are therefore only models for the human genetic disorders.

Fig. 9. A) Schematic drawing of the skeletal muscle cytoskeleton. Two myofibrils composed of sarcomeres, the smallest contractile unit, are shown. The sarcomere with A, I and M-bands, is bounded at both ends by Z-discs. It is composed of thick myosin and interdigitating thin filaments, and an intra-sarcomeric cytoskeleton, which is made up by titin and nebulin. The extra-sarcomeric cytoskeleton consists of intermediate filaments (IFs), mainly composed of desmin. The IFs interlink myofibrils through plectin and other IF related proteins. The IFs also connect the peripheral myofibrils with the sarcolemma and the nuclear membrane. B) Model of the molecular organisation of the cytoskeleton at a costamere (see boxed area in A). The extracellular laminins are attached to integrins and glycoprotein complexes, which form transmembrane links to dystrophin, spectrin, vinculin, talin and α-actinin, which in turn through actin and plectin are linked to the α-crystallin and desmin. The model is tentative, as the interrelationship between some of the proteins is still unknown.

ACKNOWLEDGEMENTS

This study was carried out at the Department of Medical Biology, section for Anatomy, Umeå University. I would like to express my sincere gratitude to all those who have assisted and supported me during the course of the studies. In particular I would like to mention:

Professor Lars-Eric Thornell, my supervisor, for valuable support, encouragement and guidance throughout the work with my thesis. He has also been a true friend during my years at the department. I wish, however, that he could work up the courage to challenge me to a round of golf, even though I do not blame him for being afraid.

Drs Thomas Sejersen, Urban Lendahl, Gunnar Sjöberg, Kristian Borg and Christine Fischer, Stockholm for gift of antibodies and collaboration on the patient with desmin myopathy.

Professor Denise Paulin, Zhenlin Li, Mathias Mericskay, Onnik Agbulut and Charles Babinet, Paris for providing the desmin K/O mice and for fruitful collaboration.

Professor Gillian Butler-Browne, Paris and Dr Maureen Price, Houston for collaboration and linguistic revisions of the manuscripts.

Professors Richard Robson, Ames, Jennifer Breckler, San Francisco and Gerhard Wiche, Vienna for collaboration and generous gift of antibodies.

The “Muscle group”. I would especially like to mention Margaretha Enerstedt, Mona Lindström and Anna-Karin Olofsson, my laboratory colleagues, with whom I share many laughs and memories both at work and at spare time. Thanks also for skilful technical assistance and fruitful discussions about everything and nothing. I also which to thank my research colleagues Eva Carlsson, Fatima Pedrosa- Domellöf, Bengt Johansson, Fouzi Kadi, Jing-xia Liu, Per Stål and Ji-guo Yu for being nice friends and for giving support and valuable advice.

Ingrid Nilsson for secretarial assistance and for arranging pleasant activities. It was always nice when your door was open, when I arrived at work (even though most often I was very early).

Inga Johansson for excellent technical assistance.

Tomas Karlsson and Ulf Ranggård for solving computer tasks.

Bror Berggren for skilful photographic work.

The staff at “Anatomy”, all of whom has in different ways contributed to many joyful and memorable moments.

My parents Ruth and Paul, my brothers Thomas and Bengt, as well as their families, for being the ones you are.

Finally, I am deeply grateful to Olle, my life companion and true love, and my children Niklas and Malin. Without their encouragement, support and belief in me this work never would have come through. I also would like to thank Ray Tomlinson, the inventor of e-mail, who made it possible to keep contact with Niklas, when he moved to Canada.

This work was supported by the Swedish Medical Research Council (12X-03934), the Magnus Bergvall Foundation and the Medical Faculty of Umeå University.

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