Helsinki University Biomedical Dissertation No. 114

Novel insights on functions of the myotilin/palladin family members

Monica Moza

Program of Molecular Neurology Department of Pathology Faculty of Medicine University of Helsinki Finland

Academic dissertation To be publicly discussed with the permission of the Faculty of Medicine of the University of Helsinki, in the small lecture hall, Haartman Institute, on November 14th, 2008, at 12 noon.

Helsinki 2008 Thesis supervisor: Professor Olli Carpén, M.D., Ph.D. Department of Pathology University of Turku and Turku University Hospital Program of Molecular Neurology, Biomedicum Helsinki Department of Pathology, University of Helsinki Helsinki, Finland

Thesis reviewers: Professor Hannu Kalimo, M.D., Ph.D. Department of Pathology, University of Helsinki Helsinki, Finland

Professor Jari Ylänne, Ph.D. Department of Biological and Environmental Science University of Jyväskylä Jyväskylä, Finland

Thesis opponent: Professor Rolf Schröder, M.D., Ph.D. Department of Neuropathology University of Erlangen Erlangen, Germany

ISBN 978-952-10-5096-1 (paperback) ISBN 978-952-10-5097-8 (PDF) ISSN 1457-8433

2 CONTENTS

LIST OF ORIGINAL PUBLICATIONS 5 ABREVIATIONS 6 ABSTRACT 8 REVIEW OF THE LITERATURE 10

1. 10 1.1 Structural and functional differences between striated muscle, smooth muscle and non-muscle cytoskeleton 10 2. cytoskeleton 11 2.1. Actin 11 2.2. Actin-associated 11 2.3. Immunoglobulin-like domain containing proteins 14 3. Skeletal muscle structure and function 15 3.1 Z-disk. Structure, function, components 18 3.1.1 α-Actinin 18 3.1.2. Myotilin, myopalladin and palladin 19 3.1.3. ZASP/Cypher 24 3.1.4. FATZ 25 3.1.5. Filamin C 26 3.1.6. Telethonin/T-cap 26 3.2. Thin filaments 27 3.2.1. Actin 27 3.2.2. Tropomyosin and troponins 27 3.3.3. Capping proteins, tropomodulin and CapZ 28 3.3.4. Nebulin and nebulette 29 3.3. Thick filaments 30 3.3.1. Myosin 30 3.3.2. Titin 30 3.4. Intermediate filaments 32 3.4.1. Desmin 32 4. Skeletal muscle development 32 5. Regulation of striated muscle growth and adaptation processes 34 6. Muscular dystrophies and myopathies 36 6.1. Limb girdle muscular dystrophies (LGMD) 36 6.2. Myofibrillar myopathies (MFM) 37 6.2.1. Myotilinopathy 38 6.2.2. Desminopathies 38 6.2.3. Zaspopathy 39 7. Model organisms 39 3 AIMS OF THE STUDY 41 MATERIALS AND METHODS 42 RESULTS AND DISCUSSION 43

Palladin is a binding partner for profilin (I) 43 Palladin is a binding partner for α-actinin (II) 44 Absence of myotilin in mice does not disrupt morphology and function of striated muscle (III) 46 Myotilin in myofibrillar remodelling (IV) 47 Myotilin and palladin deficiency leads to structural and functional changes of striated muscle (V) 48 CONCLUSIONS AND FUTURE PROSPECTS 52 ACKNOWLEDGEMENTS 54 REFERENCES 56

4 LIST OF ORIGINAL PUBLICATIONS

I. Boukhelifa M.#, M. Moza#, T. Johansson#, A. Rachlin, M. Parast, S. Hüttelmaier, P. Roy, B. Jocusch, O. Carpén, R. Karlsson, C. Otey: The proline-rich palladin is a binding partner for profilin. (# equal contribution). FEBS J, 273:26-33 (2006).

II. Rönty M.#, A. Taivainen#, M. Moza, C. A. Otey, O. Carpén: Molecular analysis of the interaction between palladin and alpha-actinin. (# equal contribution), FEBS Lett, 566:30-34 (2004).

III. Moza M., L. Mologni, R. Trokovic, J. Partanen, G. Faulkner, O. Carpén: Targeted deletion of the muscular dystrophy myotilin does not perturb muscle structure or function in mice. Mol Cell Biol, 27:244-252 (2007).

IV. Carlsson L., J.-G. Yu, M. Moza, O. Carpén, L-E. Thornell: Myotilin - a prominent marker of myofibrillar remodelling. Neuromusc Disord, 17:61-68 (2007).

V. Moza M. #, H.-V. Wang#, W. Bloch, O. Carpén, M. Moser: Analysis of the cardiac and skeletal muscle phenotype in 200 kDa palladin/myotilin double mutant mice (# equal contribution). Submitted.

5 ABREVIATIONS

ADF actin depolymerizing factor ALP α-actinin-associated LIM protein Arp2/3 actin-related protein 2/3 CaM calmodulin CARP cardiac ankyrin repeat protein CH calponin homology CMD congenital muscular dystrophies DARP diabetes-related ankyrin repeat protein DCM DOMS delayed-onsent muscular soreness ECC excitation-contraction coupling ECM extracellular matrix EGFR epidermal growth factor receptor ER endoplasmic reticulum ES embryonic stem (cells) EVH1 Ena/VASP homology F-actin filamentous monomers FATZ a filamin, actinin, and telethonin binding protein of the Z-disk FTLD-U frontotemporal lobar degeneration with ubiquitin-positive inclusions G-actin globular actin GFAP glial fibrillary acidic protein GSK-3β glycogen synthase kinase HDAC5 histone deacetylase 5 HCM hypertrophic cardiomyopathy bHLH basic helix-loop-helix IF intermediate filaments Ig immunoglobulin IGF-l insulin-like growth factor Igl immunoglobulin-like INLVM isolated non-compaction of the left ventricular myocardium LGMD limb-girdle muscular dystrophy MADS MCM1, agamous, deficiens, serum response factor MARP muscle ankyrin repeat protein MBP-H myosin binding protein H MEF mouse embryonic fibroblasts Mena mammalian enabled (protein)

6 MFM myofibrillar myopathy MLC myosin light chains MLCK myosin light chain kinase MLP muscle LIM protein MRF myogenic regulatory factor MOM mitochondrial outer membrane MyHC myosin heavy chain mTOR Akt/mammalian target of rapamycin MURF muscle-specific RING finger MyBP-C myosin-binding protein C Myf5 myogenic factor 5 MyoD myogenic determination NFAT nuclear factor of activated T-cells N-RAP nebulin-related protein (N)-WASP (neuronal) Wiskott–Aldrich syndrome protein PDGF platelet derived growth factor PI3P phosphatidylinositol (3,4,5)-trisphosphate

PIP2 phosphatidylinositol 4,5-bisphosphate PIP phosphatidylinositol 4-monophosphate PI3K type 1 phosphatidyl inositol 3 kinase PKB protein kinase B PKC protein kinase C SBM spheroid body myopathy SPR surface plasmon resonance SR sarcoplasmic reticulum TGF-β tumor growth factor β TMD tibial muscle dystrophy Tn C troponin C TnI troponin I TnT troponin T VASP vasodilator-stimulated phosphoprotein VCPMD vocal cord and pharyngeal weakness Y2H yeast-two-hybrid ZASP Z-disk alternatively spliced PDZ-motif ZM ZASP-like motif

7 ABSTRACT

In skeletal and cardiac muscle cells, the contractile unit sarcomere consists of actin filaments that have constant length, a strict spatial distribution and are aligned with myosin filaments. Actin filaments from adjacent sarcomeres are cross-linked by actin-associated proteins which form the Z-disk. Sarcomeres provide the muscle cell its shape and they are the structural and functional basis of the muscle contraction. In response to intra- and extracellular stimuli, the actin cytoskeleton is continuously reorganizing in non- muscle or smooth muscle cells in order to determine the cell morphology, the cytokinesis, coordinated cell movements and the intracellular transport of organelles. Myotilin, palladin and myopalladin form a small family of proteins containing immunoglobulin- like (Igl) domains. They are associated with the actin cytoskeleton, and all members of the family are suggested to have roles as a scaffold and as regulators of actin organization. Myotilin and myopalladin are expressed as a single isoform only in the striated muscle. Myotilin expression level is higher in the skeletal muscle than in the heart, while myopalladin expression in higher in the heart than in the skeletal muscle. Their expression levels might be directly correlated with their function. On the contrary, the palladin gene gives rise to several palladin isoforms expressed as a result of alternative splicing events. Palladin is expressed not only in the striated muscle but also in other tissue types, such as the nervous tissue, and different isoforms apparently play cell-specific roles. The three members of the family are localized at the Z-disk in the striated muscle and here they have a common binding partner, the bona-fide Z-disk protein, α-actinin. Point mutations in myotilin gene causes muscle disorders of variable phenotype: limb-girdle muscular dystrophy A (LGMD1A), myofibrillar myopathy (MFM) and spheroid body myopathy (SBM). On the other hand, palladin gene mutation has been reported to be associated with familial and increased risk for myocardial infarction. In this study we found that, as shown for other poly-L-proline-containing proteins, palladin directly binds a key molecule involved in actin dynamics, profilin. Profilin has a dual function: it is a promoter of actin assembly and it can also act as an actin-sequestring protein resulting in actin depolymerisation. Palladin was shown to interact with profilin via its second polyproline region and, further more, we demonstrated that the interaction is highly dynamic and of low affinity. In cells, palladin colocalized with profilin in actin-rich bundles near cell edges. These results indicate that 90-92 kDa palladin associates with profilin in regions in which novel actin filaments form and that palladin may be involved in the coordination of actin filament formation. α-Actinin, an important actin cross-linking molecule, connects the cytoskeleton to transmembrane proteins and serves as a scaffold for signalling molecules. We found that palladin binds to and colocalize with α-actinin. The interaction is mediated via a highly conserved region in both myotilin and palladin which is considered as a new binding domain for α-actinin. Pain, stiffness and tenderness in the muscle, or delayed onset muscle soreness (DOMS) appears in untrained persons who perform eccentric exercise. All aspects of DOMS are not yet fully understood, however it is considered that new sarcomeres are formed. This process includes modifications in the Z-

8 disk architecture and composition, such as temporary loss of α-actinin, titin and nebulin and polymerization of G-actin to form foci of increased F-actin labelling in skeletal muscle fibers after the exercise to finally result in supranumerary sarcomeres. Our studies showed that myotilin follows the widened distribution patterns observed for F-actin, suggesting that myotilin acts as a cross-linker and an F-actin stabilizer. In these structures, myotilin is probably replacing α-actinin and, similarly with α- actinin, might be an anchoring site for signalling or scaffolding molecules that are recruited to drive the formation of the new sarcomeres. We propose myotilin as a guardian of the Z-disk as apparently myotilin contributes to actin organization in both mature and forming Z-disks. To further study the functions of myotilin we generated knockout mice. While loss of all palladin isoforms leads to embryonical lethality in mice, we showed that myotilin knockout mouse survives and has no obvious morphological or functional alteration in striated muscle or other organs. Myotilin absence is apparently compensated by other proteins, possibly by members of myotilin/palladin/myopalladin subfamily. This finding is in concordance with the finding that the transgenic expression of a mutant myotilin leads to morphological alterations observed in myotilinopathies, i.e. mutated myotilin appears to have a toxic effect on the structure of the skeletal muscle, whereas myopathy due to myotilin deficiency has not been reported. Further, we have generated and investigated a mouse that lacks myotilin and expresses low levels of the 200 kDa palladin. While the 200 kDa palladin hypomorph mice shows ultrastructural modifications of the heart architecture, we showed that combined absence of myotilin and low levels of 200 kDa palladin led to morphological and functional alterations in skeletal muscle. This work showed that 200 kDa palladin has an important role in the maintenance of normal architecture of the cardiomyocytes and that myotilin deficiency apparently exacerbated the cardiomyocyte defects. This indicates that in skeletal muscle myotilin and 200 kDa palladin may have a similar function, while in the heart their functions might be different.

9 REVIEW OF THE LITERATURE

1. Cytoskeleton

The cytoplasm of all eukaryotic cells contains a scaffold structure named the cytoskeleton. It is a complex network of proteins, organized as three distinct categories of filaments: actin filaments, intermediate filaments and microtubules. These are highly dynamic and continuously reorganizing structures that determine the cell morphology, the cytokinesis, coordinated cell movements in response to intra- and extracellular stimuli and the intracellular transport of organelles. All three different filament types in the cell are structurally and functionally interconnected.

1.1 Structural and functional differences between striated muscle, smooth muscle and non- muscle cytoskeleton

The vertebrates have many differentiated cell types with specific functions. Among them, the muscle cells are terminally differentiated cells specialized for force production. They are divided into two main categories: striated and smooth muscle cells. The striated muscle cells are divided into two different types of cells: the skeletal muscle and the cardiac muscle cells. The skeletal muscle in most situations contract voluntarily to generate movement, but can contract involuntarily producing reflexes. Athough the cardiac muscle cell architecture markedly differs from that of the smooth muscle cell, both function involuntarily. The heart contracts rhythmically to pump blood into the circulation. In non-muscle cells the actin filaments are assembled and disassembled continuously, while in striated muscles the actin filaments are stable and permanently organized as thin filaments. Once formed, their spatial arrangement is maintained throughout the cell’s life and little is known about the length regulation of thin filaments in striated muscle cell (reviewed in Littlefield and Fowler 1998). In a similar manner, myosin forms in the skeletal muscle cell highly organized structures, the thick filaments. Under the light microscope and by electron microscopic investigations, cellular architecture of the skeletal and cardiac muscle appears striated, as light and dark bands are alternating. The precise alignment of the thin and thick filaments makes up the functional structural unit of the striated muscle cell, named sarcomere. The composition of actin filaments and associated proteins differ drastically in striated muscle cells compared to non-muscle cells. The smooth muscle cells are found mainly in the blood vessel walls, gastrointestinal tract, respiratory tract and uterus. Under the control of autonomic nervous system, systems and organs composed of smooth muscle cells are able to contract involuntarily for extended periods of time. The contractile activity is required e.g. for maintenance of the blood pressure or ensuring the movement of the digestive products by peristaltic movements.

10 2. Actin cytoskeleton 2.1. Actin Changes in cell morphology and cell movement are major functions provided by the actin cytoskeleton. Actin is a major protein component found in all eukaryotic cell types and actin are highly conserved through the evolution and between species. Encoded by separate genes mammals have six expressed in a tissue-specific manner. The actins are classified by their isoelectric points in α, β and γ (Vandekerckhove and Weber 1978). Expression of actin isoforms varies in muscle and non-muscle cells. α-Skeletal, α-smooth muscle and α-cardiac are expressed in muscle cells and β and γ are expressed in non-muscle cells. While skeletal muscle-specific actin isoforms are expressed in skeletal muscles, the cardiac actin isoform is not expressed in the skeletal muscle or in smooth muscle cells. However, in skeletal muscle, smooth-muscle actin isoforms are expressed but apparently they do not participate in the actin filament formation (Vandekerckhove and Weber 1978; McHugh et al. 1991). Actin is a 375 amino acid polypeptide chain that interacts with one molecule of ATP or ADP and contains one high affinity binding site and various low affinity sites for divalent cations, such as Ca2+ (Ampe and Vandekerckhove 2005; Carlier et al 1986). Actin monomers are named globular actin (G- actin), while the polymeric form is filamentous actin (F-actin) which consists of assembled filaments. The 42 kDa actin monomers are assembled into twisted filaments in a left-handed helix with 13 monomers in six turns (Holmes et al. 1990; Squire et al. 1997). In the cell, the actin polymers are polar structures and are associated with a large number of actin-binding proteins to form various intracellular specialized structures. Actin structures include the stress fibers, lamellipodia, filopodia, podosomes and the cortical actin meshwork. These structures participate in the organization of the cytoplasm and are continuously assembled and disassembled in response to intra- and extracellular stimuli or during the cell cycle. In addition, they generate mechanical forces whithin the cell, being essential for many contractile activities, such as contraction or separation of daughter cells by the contractile ring during cytokinesis (Pelham and Chang 2002). They participate to the cell-cell and cell-substrate interaction along with adhesion molecules and are involved in the transmembrane signaling (Woods et al 2007).

2.2. Actin-associated proteins

The architecture and function of the actin cytoskeleton is not generated by the actin filaments alone. A number of actin-associated proteins are required to coordinate the assembly and dissassembly of the filaments in various locations inside the cell. In addition, the actin-binding proteins provide the link of the filaments to one another or to other cell structures, such as plasma membrane. The dynamics of actin structures to specific sites within the cell at a given time is strictly regulated both spatially and temporally by the actin-associated proteins. Based on the type of actin they bind and their mode of action, they are divided into several functional subclasses. To date, over 160 actin-binding proteins are known (dos Remedios et al. 2003). Based on structural characteristics they fall into more than 60 classes (Pollard 1999). The activity of actin binding proteins is often modulated by ions, such as Ca2+, by phosphoinositides or by phosphorylation.

11 Proteins binding to monomeric actin contribute to the amount, localization and dynamics of unpolymerized actin molecules in cells (Paavilainen et al. 2004; dos Remedios et al. 2003). Others can bind actin filaments, regulating the nucleation, assembly, disassembly and crosslinking of actin filaments (Pollard 1986; dos Remedios et al. 2003). During the assembly process actin filaments grow due to addition ATP-actin monomers, rapidly at the plus end (the barbed end) and slowly at the minus end (the point end). The dissasembly process occurs by dissociation of ADP-actin from the minus end of the filament. Together, the assembly and disassembly are known as treadmilling. Under physiological ion conditions treadmilling of actin progresses very slowly in vitro when only actin monomers and filaments are present. In contrast, the treadmilling of actin subunits from the minus end to the plus end in cells is very rapid, indicating the participation of the regulatory proteins to achieve this physiological behavior (Fig. 1). Most important proteins for the actin dynamics are actin depolymerizing factor/cofilin (ADF/cofilin) (Bamburg et al. 1999), actin-related protein 2/3 (Arp2/3) complex (Pollard and Beltzner 2002), activators of Arp2/3 complex, such as cortactin and neuronal Wiskott–Aldrich syndrome protein (N-WASP) (Weaver et al. 2002), and profilin (Schluter et al. 1997).

Figure 1. Actin dynamics at the membrane (modified from Pollard et al. 2001) Profilin is represented by small black dots and the blue dots represent actin. From the pool of profilin:actin complexes, actin is rapidly polymerized at the plus end of the filament that pushes the cell membrane forward. Palladin is represented as yellow squares and it binds both actin and profilin. The nucleation complex Arp2/3 is represented by red oval. On an actin filament, from the nucleation complexes daughter filaments grow at an angle of 70 degrees. At the minus end the actin is dissociated fom the actin filament and the bound ADF/cofilin (represented by white arrowheads) is exchanged by profilin.

12 In the cytoplasm a pool of unpolymerized actin monomers is bound to profilin and sequestring proteins, such as thymosin-β4. Activation of WASP/Scar proteins leads to activation of the actin nucleation complex, Arp2/3 complex. This complex is required for both assembly and disassembly of actin filaments that form the leading edge of a cell. An active Arp2/3 complex at the leading edge initiates the growth and anchors branches of actin filaments on existing mother actin filaments at a 700 angle (reviewed in Pollard et al. 2007). The actin filaments grow rapidly due to addition of actin monomers from the profilin:actin pool. Each filament grows transiently, as capping proteins stop the assembly of ATP-actin at the minus end of the filament. As the actin filament ages, the actin-bound ATP is rapidly hydrolyzed and the γ-phosphate is slowly released, this latter process determining the onset of the disassembly reactions. They consist of debranching and binding of ADF/cofilin, which promotes severing and dissociation of ADP-actin monomers from filament end. To return the actin monomers into subunits that can be polymerized, ADP is exchanged into ATP. This is achieved by the small protein, profilin, that plays the role of a nucleotide exchange factor (reviewed in Pollard et al. 2001). Profilin was identified as a G-actin-binding protein (Carlsson et al. 1977). It is an ubiquitous protein of 12-16 kDa, expressed as many isoforms. To date, five mammal profilin isoforms as products of four profilin genes, profilin I, II, III and IV, are known (Polet et al. 2007). By alternative splicing, profilin II gene gives rise to two isoforms. Various eukaryotic cells express simultaneously multiple profilin isoforms which differ by expression patterns and biochemical properties. Profilins play an important role in growth of actin filaments, a process controlled by various signalling cascades. In response, the actin cytoskeleton rapidly and precisely changes its organization and the equilibrium between actin monomers and actin filaments is modified. In addition to G-actin, further studies showed that profilin binds actin- related proteins (Machesky et al. 1994), phosphoinositide lipids (Lassing and Lindberg 1985; Skare and Karlsson 2002) and poly-L-proline domain-containing proteins (Holt and Koffer 2001). These ligands are components of various signaling cascades that modulate the organization of actin filaments. Profilin binds to G-actin at 1:1 stoichiometry (Carlsson et al. 1977) and it occupies the actin- binding site on the G-actin monomer. When bound to actin, profilin exchanges the actin-bound ADP into ATP. It has a higher affinity for ATP-G-actin (Kd = 0.1 mM) than for ADP-G-actin (Kd = 0.5 mM) and renders the functionality to actin monomers, by generation of ATP-G-actin:profilin. The complex can elongate the plus ends of an actin filament, but cannot bind to the minus end. Profilin inhibits spontaneous nucleation of actin filaments and also inhibits the growth of minus end of actin filaments (Pollard 1986). The growth of actin filaments is implemented by profilin-actin complex rather than only by ATP-G-actin, therefore profilin has the role of lowering the critical concentration for polymerization (Pantaloni et al. 1993). Binding of the profilin-actin complex to the plus ends of filaments results in the release of profilin. Another class of profilin binding partners are the phosphoinositide lipids. On profilin, the binding site for PIP2 (phosphatidylinositol 4,5-bisphosphate) and PIP (phosphatidylinositol 4-monophosphate) lies in the same region as for G-actin. Binding of PIP2 and PIP to profilin induces profilin conformation changes and results in profilin-G-actin dissociation (Raghunathan et al. 1992). Profilin is bound to cellular membranes via the PIP2-binding site and a model of profilin activation proceeds by external signals that activate tyrosine kinases which phosphorylate PLCγ1 (phospholipase C gamma 1). The 13 activated enzyme splits PIP2 and results in releasing of profilin from the membrane. The released profilin can subsequently bind G-actin and the formed complex can be used by a growing actin filament (Goldschmidt-Clermont et al. 1991). The third category of ligands for profilin is composed of the polyproline containing proteins or synthetic peptides. Their binding to profilin does not interfere with the binding of profilin to G-actin or phosphoinositide lipids. Of them, two proteins that contribute to the barbed-end filament elongation are the VASP (vasodilator-stimulated phosphoprotein) (Reinhard et al. 1995) and the VASP-related protein Mena (mammalian enabled) (Gertler et al. 1996). They are substrates of cAMP/cGMP dependent protein kinases. With members in several organisms, the actin-filament-nucleating formins (Evangelista et al. 2003) contain stretches of 5-13 proline residues which bind profilin. The role for formins in the actin dynamics is finely tuned by profilin, that acts in vivo as a cofactor. Formins and formin-related proteins are linked to the small GTPase of Rho family signaling cascade that participates in cell morphology, adhesion, cytokinesis, cell polarity and vertebrate limb formation processes (Tapon et al. 1997). Another binding partner is N-WASP (Suetsugu et al. 1998). Profilin is considered an actin sequestering molecule and a key factor of actin dynamics, especially in higher eukaryotes. Profilin seems to have a direct effect on microfilament organization as well as on integration of various signalling transduction events that result in coordination of spatially and temporally essential processes such as cellular morphology, cellular locomotion, organ development or even immune responses to foreign organisms (for an extensive review see Schluter et al. 1997).

2.3. Immunoglobulin-like domain containing proteins

Immunoglobulin (Ig) and Ig-like (Igl) domains are structural protein domains found in several protein categories. They consist of about 100 amino acids with a characteristic structure known as immunoglobulin fold. The basic structure has 7 β strands organized into two antiparallel β-sheets that create a sandwich shape, conformation maintained by hydrophobic internal residues (Murzin et al. 1995). The Ig domains are classified into four major categories: variable (IgV), constant-1 (C1-set), constant-2 (C2-set) and intermediate (I-set) types (Bork et al 1994, Smith and Xue 1997). The best known proteins that contain Ig domains are antibody molecules and several transmembrane proteins, such as antigen presenting molecules, various receptors on immune cells, adhesion molecules and ligands. By mediating protein-protein interactions or acting as modular spacers, the Ig and Igl-domain containing proteins play a multitude of roles in regulating mainly the function of immune system (Harpaz and Chothia 1994). Among them are Sns, Kirre, Roughest and Hibris proteins that can function as adhesion receptors in fusion of other types of cells and that are implicated in myoblast fusion in Drosophila (Artero et al. 2001; Bour et al. 2000; Ruiz-Gomez et al. 2000; Strunkelnberg et al. 2001). Recently, crystallographic studies showed that the extracellular region of the skeletal muscle membrane protein α-dystroglycan contains an Igl type domain (Bozic et al. 2004). This determines the interaction of α-dystroglycan with the extracellular matrix protein laminin-1 that may play a role in anchorage and signaling events via the sarcolemmal membrane. A subfamily of Igl domain-containing intracellular proteins is composed of titin (Maruyama 14 1997), obscurin (Young et al. 2001), myomesin/M-protein (Steiner et al 1998), MyBP-C (myosin-binding protein C) (Weber et al. 1993), myopalladin, myotilin and palladin (Otey et al. 2005). With the exception of the last mentioned, they are rather exclusively components of the skeletal muscle cytoskeleton (Otey et al. 2005). Palladin is expressed ubiquitously and, some of its several isoforms are expressed in striated and smooth muscle cells. The Igl domains found in these proteins do not mediate immunological recognition as the proteins associated with immune cells. Rather, they serve as protein binding domains or contribute to the passive stiffness of the stretched sarcomere, as showed for titin molecules (Linke et al. 1998). The Igl domains found in titin and the Drosophila homologue of titin, kettin, is proposed to bind actin (Jin 2000; van Straaten et al. 1999). In myotilin, Igl domains are proposed to mediate actin- bundling, and are proposed as a novel actin-binding module (von Nandelstadh et al. 2005). Igl domains appear to mediate protein dimerization in myotilin and filamin C (Himmel et al. 2003; von Nandelstadh et al. 2005). In addition, the three carboxy terminus Igl domains in myopalladin mediate the interaction with the α-actinin (Bang et al. 2001). Recently, the Z1 Z2 Igl domains of titin were shown to contribute to the structural stability of the Z-disk, as two titin molecules from opposed sarcomeres are cross-linked by telethonin (Zou et al. 2003). In MyBP-C, the Igl domains mediate the binding of MyBP-C with myosin (Okagaki et al. 1993).

3. Skeletal muscle structure and function

Striated muscle cells, myofibers and cardiomyocytes, form skeletal and cardiac muscles, respectively. A unique feature of the striated muscle cell providing the cell the “striated” appearance is a characteristic highly organized cytoskeletal architecture that forms the myofibrils. The cytoskeleton is organized in contractile units, named sarcomeres. This structure enables the striated muscle cell to produce force, enabling movement of the body, breathing and blood circulation through the vascular system (reviewed in Craig and Padron 2004). An adult human skeletal muscle cell, or myofiber, is a cell of approximately 10-100 μm diameter that can extend tens of centimeters in length. Several muscle fibers are organized into fascicles sourrounded by perimisium and several fascicles sourrouded by epimisium compose a skeletal muscle. A skeletal muscle fiber is a terminally differentiated cell that has multiple nuclei normally located at the cell periphery, adjacent to the sarcolemma. A skeletal muscle cell contains several myofibrils of 1-3 μm in diameter that run parallel to the long axis within the muscle fiber. In striated mucle the endoplasmic reticulum (ER) is named sarcoplasmic reticulum (SR). Each myofibril is surrounded by SR, a structure implicated in Ca2+ storage and cycling. Under the (light) microscope, myofibrils confer the skeletal muscle the striated aspect, as they are composed of repetitive and higly orderded arrays of thin and thick filaments, named myofilaments (Fig. 2.). Together, they form the contractile units of the muscle fiber, the sarcomere. The myofilaments are classified as thin and thick filaments according to their electron microscopic appearance. Thin filaments, called also actin filaments, are polar filaments of precise length composed mainly of actin. Several proteins binding along the filaments, such as tropomyosin that forms a parallel filament, and proteins capping the filament ends, such as CapZ (Schafer et al. 1993), are considered components of the thin filaments. In longitudinal section, thin filaments appear as electron 15 light structures and form the I-band (approximately isotropic in polarized light). In the middle of I-band, an electron dense structure is observed, the Z-disk. In transversal section, the Z-disk is formed by tetragonally organized thin filaments from one sarcomere that interdigitate with the actin filament of the following sarcomere (reviewed in Craig and Padron 2004). Here, actin filaments from opposite directions are cross-linked mainly by α-actinin dimers (Luther 2000). Z-disk is the structural border of adjacent sarcomeres and functionally provides a mechanical and signalling link for the sarcomere (Frank et al. 2006). The thick filaments are composed of polar myosin polymers that form the central part of A-band (anisotropic in polarized light), called H-zone. They are cross-linked by several myosin-binding proteins at the M-line, located in the centre of H-zone. In transversal section, the myosin filaments in the H-zone are organized in a hexagonal manner. The lateral region of A-band adjacent to I-band is composed of overlapping thin and thick filaments. In transversal section, the lateral region of the A-band is composed by a hexagonal array of myosin filaments and each myosin filament is surrounded by 6 to 11 actin filamens (Nistal et al. 1977). In skeletal muscle, actin and myosin are non-covalently bound protein assemblies and account for 70% of myofibrillar proteins (Hanson and Huxley 1957). The third type of filament found in the striated skeletal muscle is the titin filament that connects both the actin and myosin filaments. Titin is a giant molecule that spans half of the sarcomere, from the Z-disk to the M-line (Fig. 2.). Titin is thought to function as a molecular ruler, governing the assembly of myofibrillar structures during myofibrillogenesis (Sanger et al. 2000). In addition, nebulin is another large molecule that binds actin filaments and is suggested to control the actin filament length (McElhinny et al. 2005). Similar to the skeletal muscle, the cardiac muscle cell has a highly organized cytoskeletal architecture organized as myofibrils and sarcomeres. However, the molecular composition of the filaments and other proteins, characterized by the expression of cardiac-specific components, provide structural and functional differences from the skeletal muscle. Different from their skeletal muscle counterparts, cardiac muscle cells are relatively small cells, averaging 10–20 µm in diameter and 50–100 µm in length, and contain one centrally localized nucleus. The individual cardiomyocytes are chemically, mechanically and electrically interconnected via the intercalated disks to form a functional syncytium. The sarcomere is the functional unit of the skeletal muscle. Muscle contraction is a process by which chemical energy is transformed in mechanical force. The action potentials at the sarcolemmal membrane trigger the release of Ca2+ from SR into sarcoplasm. Here, Ca2+ binds to troponin C (TnC) from the troponin complex, promoting a conformational change of troponin-tropomyosin complex, allowing the direct interaction of actin with myosin. Upon interaction, the phosphate (Pi) is released and the actin-myosin interaction drives the tilting of the myosin globular head while still interacting with actin filament. Subsequently, a sliding force is produced with thin and thick filaments sliding past each other, resulting in the muscle contraction. Simultaneously the release of bound ADP from myosin occurs and myosin heads are released from the actin filament interaction. The cycle starts again by binding of another ATP molecule on the myosin head. In skeletal muscle, several cycles of ATP binding and hydrolysis are necessary to produce a contraction (reviewed in Goody 2003).

16

Figure 2. Ultrastructure of a mature skeletal muscle fiber and schematic protein organization in the striated muscle sarcomere (modified from Ottenheijm et al. 2008). The upper panel represents an electron microscopic photograph of a striated muscle, magnification x 4000. The panel below depicts the organization of a mature sarcomere. The thin filaments are represented by a chain of white round dots (actin) that are capped at the Z-disk by CapZ (yellow). To the actin filaments are associated tropomyosin (black thread) and nebulin (red thread) filaments, and troponin complexes (orage dots). The Z-disk is composed of antiparrallel α-actinin molecules (dark green rods), myotilin (violet dot), FATZ (red rod), myopalladin (pink triangle). The titin filament (violet thread) extends from Z- dsk to M-line. At the Z-disk two titin filaments from opposed sarcomers are cross-linked by telethonin (light green). The thick filaments are composed mainly of myosin (dark green). In the middle of the A-band, the M-line is composed of several proteins, among them M-protein (orange triangle), MURF-1 (light blue rod) and the p94

17 enzyme (blue dot).

3.1 Z-disk. Structure, function, components

Z-disk is located in the middle of the I-band and forms the border of two adjacent sarcomeres. Its structure appears as a dark zig-zag in longitudinal sections or as square and “basket array” in transversal section. The Z-disk width can vary between species and may vary according to the fiber type in the same muscle (reviewed in Vigoreaux 1994). The Z-disk is a three dimensional structure composed of several structural proteins and its components are continuously discovered. The Z-disk is the location where the plus ends of actin filaments from opposite sarcomeres are cross-linked together. Similarly, here, the amino termini of titin filaments from opposite sarcomeres meet and are cross-linked.

3.1.1 α-Actinin

α-Actinin is the shortest member of the spectrin protein superfamily, which includes spectrin, dystrophin and utrophin. In higher vertebrates four different α-actinin genes are present (ACTN1 to ACTN4) (Virel and Backman 2004; Honda et al. 1998). ACTN2 and ACTN3 are expressed to striated muscle, whereas ACTN2 is expressed in the cardiac muscle. ACTN3 is absent in approximately 18% of the human population and in chicken (MacArthur and North 2004; North et al. 1999). Its absence apparently induces a shift in structural, metabolic and contraction properties skeletal muscle fibers towards a slow muscle phenotype, without modification of myosin heavy chain expression and it associates with an increase in endurance performance (MacArthur et al. 2008; MacArthur et al. 2007). α-Actinin is a protein of 97 kDa that contains three different domains: an amino terminal actin-binding domain with two calponin homology (CH) domains, a central rod domain composed of four spectrin domains and a carboxy terminus containing two calmodulin (CaM)-like domains. α-Actinin forms antiparallel homodimers via the rod domain and can cross-link actin in both parallel and anti-parallel fashion (Taylor et al. 2000). The CaM domains form the α-actinin EF-hands and, in non-muscle isoforms, they are capable of Ca2+ binding, while in muscle isoforms they are not. The actin-binding capacity of muscle α-actinin isoforms is Ca2+-independent, whereas non-muscle isoforms bind actin in a Ca2+-dependent manner (Burridge and Feramisco 1981; Honda et al. 1998; Weins et al. 2007). In both non-muscle and skeletal muscle cells, α-actinin can have multiple binding partners and its functions are dependent on these multiple interactions. In striated muscle cells, α-actinin is the key protein of the Z-disk where it cross-links both actin filaments and titin Z-repeats from opposed neighboring sarcomeres (Sorimachi et al. 1997). Myopodin is suggested to play a role in linking actin filaments of the same polarity (i.e. from same sarcomere) as it binds along the filaments. In striated muscle, α-actinin has several proteins of the Z-disk as binding partners, such as ALP (associated LIM protein) (Xia et al. 1997), ZASP/Cypher (Z-band alternatively spliced PDZ motif protein) (Faulkner et al. 1999), FATZ/calsarcin (filamin, actinin, and telethonin binding protein of the Z-disk) (Faulkner et al. 2000; Frey et al. 2001; Takada et al. 2001), myopalladin (Bang et al. 2001), myotilin (Salmikangas et al. 1999), titin (Sorimachi et al. 1997). In addition, α-actinin binds to myopodin, a Z-disk actin bundling

18 protein (Faul et al. 2007). Myopodin is also found in the nucleus, and is thought to be a link between nucleus and the Z-disk during development or in stress situations (Weins et al. 2001). In cardiomyocytes, myopodin forms a Z-disk signaling complex with α-actinin, calcineurin, Ca2+/calmodulin-dependent kinase II (CaMKII), muscle-specific A-kinase anchoring protein, and myomegalin (Faul et al. 2007). In non-muscle cells, α-actinin is located in multiple subcellular regions, such as cell-cell and cell- matrix interfaces, lamellipodia and stress fiber dense regions. α-Actinin’s best known role is to bind, cross-link and spatially organize actin filaments both in muscle and non-muscle cells. In non-muscle cells, α-actinin organizes actin into bundles and contribute to their attachment to the plasma membrane (reviewed in Otey and Carpén 2004). The spectrin repeats in the rod domain of α-actinin provide the molecule elasticity and a strong but tightly regulated docking site for several binding proteins or complexes. The region is important for α-actinin’s roles in cell adhesion via its interaction with adhesion molecules, such as integrins or ICAMs (Otey et al. 1990; Carpén et al. 1992). In dense bodies, the non- muscle cell equivalent of Z-disk, α-actinin binds LIM and PDZ domain-containing proteins, such as the Click1 kinase and, respectively, CLP-36, an Enigma/Cypher protein (Bauer et al. 2000; Vallenius and Mäkelä 2002; Vallenius et al. 2000). α-Actinin associates with proteins that are shuttling between cytoskeleton and nucleus, such as Click1 and with zyxin (Reinhard et al. 1999). (For an extensive review on non-muscle α-actinins see Otey and Carpén 2004). Drosophila gene disruption studies have shown that the single α-actinin gene is not absolutely necessary for proper assembly of the contractile machinery, but it is critical for stabilizing the muscle cytoskeleton once contraction begins (Fyrberg et al. 1998).

3.1.2. Myotilin, myopalladin and palladin

Myotilin, myopalladin and palladin are highly homologous within their carboxy terminus, which consists of two or three Igl domains. Whereas myotilin and myopalladin are expressed as a single major isoform and restricted to the striated muscle, palladin is expressed as several isoforms in several types of cells (Fig. 3; reviewed in Otey et al. 2005). Characterized palladin isoforms contain at least three Igl domains as the carboxy terminus is similar for all 200, 140 and 90-92 kDa isoforms. The number of Igl domains in palladin is five for the 200 kDa isoform (Wang et al. 2008) and four or five for the 140 kDa isoform (Otey et al. 2005; Rönty et al. 2005; Wang et al. 2008). The 90-92 kDa palladin isoform has three Igl domains situated at the carboxy terminus of the protein (Parast and Otey 2000, Mykkänen et al. 2001). Myotilin has two Igl domains, most homologous with the two most carboxy terminal Igl domains of palladin. Myotilin has a unique amino terminus, with a serine and threonine rich region and a short hydrophobic stretch between amino acids 57-79. A short region located before the Ig domains shares low degree of homology with a similar region in 90-92 kDa of palladin (Salmikangas et al. 2001). On the other hand, the amino terminus of palladin shares high degree of homology with myopalladin, but polyproline or serine rich regions are not found in myopalladin (Bang et al. 2001). Based on their structure and the expression pattern myotilin, myopalladin and palladin could play both similar and divergent functions in the skeletal muscle. Several palladin isoforms composed of various numbers of Igl domains and unique

19 regions may play cell and tissue specific roles (reviewed in Otey et al. 2005).

Fig.3. Scheme of myotilin/palladin/myopalladin subfamily of proteins. The Igl domains are represented as loops and the proline-rich regions are represented by the dashed bar.

The myotilin gene is located on 5q31 and the coding sequence is composed of 10 exons. The exon I and the beginning of exon II are transcribed, but not translated, forming the 5’untranslated region of the mRNA (Mologni et al. 2005; Salmikangas et al. 1999). Studies in C2C12 mouse cell line showed that promoter region up to –1287 is most important for myotilin expression (Mologni et al. 2005). Myotilin is expressed in various developing tissues of mice and human embryos. In situ hybridization studies showed that myotilin trascript is found in developing central and peripheral nervous system, lung, liver, kidney, skin, digestive tract and skeleton. High levels of expression are reported in the embryonic mouse heart and somites. In the somites, myotilin expression is detected during embryonic day 10 and 11 (Mologni et al. 2001), later than expression of other sarcomeric proteins (Fürst et al. 1989). Expression of myotilin in adult tissues is restricted to the skeletal muscle and heart. Protein evaluation showed that myotilin is found as a single isoform with an apparent molecular weight of 57 kDa. The human myotilin polypeptide is composed of 498 amino acids and the mouse orthologue of 496 amino acids (Mologni et al. 2005). The protein has a unique amino terminus, two Igl domains at the carboxy terminus, and a short carboxy terminal tail (Salmikangas et al. 1999). Myotilin Igl domains are found at 252-341 and 351-441. They are most homologous with the carboxy-terminal Igl domains of palladin 90-92 kDa isoform, and share low homology with Z1 Z2 Ig domains of titin. The amino terminus of myotilin shares very low degree of homology with the amino terminus of palladin 90-92 kDa isoform, it is rich in serines and threonines and has a short hydrophobic stretch located between amino acids 57-79. In adult skeletal muscle and heart myotilin is localized at the Z-disk, where it is thought to participate to the cross-linking of actin filaments from opposite sarcomeres (Salmikangas et al. 2003). Myotilin induces the formation of thick actin bundles when expressed in cells which do not have a highly

20 organized cytoskeleton. Further, actin bundle formation is enhanced when myotilin cooperates with α- actinin (Salmikangas et al. 2003). Myotilin binds G and F-actin via Igl domains that are proposed to form a novel actin-binding motif (von Nandelstadh et al. 2005). Myotilin has several binding partners, all of which are, at least in part, localized at the Z-disk. Myotilin binds filamin C and FATZ (van der Ven et al. 2000; Gontier et al. 2005). Via filamin C, myotilin is a link between the Z-disk and sarcolemma and, eventually, the extracellular matrix, as filamin C binds the integrins (Gontier et al. 2005; van der Ven et al. 2000). Recently, myotilin was shown to bind MURF-1 and -2 (Witt et al. 2005) (MUscle specific RING Finger) proteins associated wih the microtubules and Z-disks (Spencer et al. 2000). MURF-1, MURF-2 and MURF-3 are a specific class of RING finger proteins that are implicated in many functions such as cytoskeletal adaptors, signaling molecules via the association with the kinase domain of titin (Lange et al. 2005; McElhinny et al. 2004) or other myofibril components as myofibrillar proteins, microtubules (Spencer et al. 2000) and/or nuclear factors that regulate gene expression (Dai and Liew 2001; McElhinny et al. 2002) and E3-like ubiquitin ligases (Kedar et al. 2004; Witt et al. 2005). Whether myotilin is ubiquitinated by MURF-1 and degraded by proteasome-dependent proteolysis (Bodine et al. 2001), as shown for troponin I in cardiomyocytes (Kedar et al. 2004) remains to be established. Recent investigations of myotilinopathy patients showed that myotilin containing filament aggregates are also immunostained for ubiquitin and a mutant form of ubiquitin (UBB+) (Olive et al. 2007). Currently, it is hypothesized that mutated myotilin is misfolded and packed in the skeletal muscle aggregates and that oxidative stress may play a role in the abnormal protein aggregation (Janue et al. 2007). However, the correlation between the UPS (ubiquitin proteasome system) and myotilin remains to be investigated. The multimeric signal protein named polyubiquitin-binding protein p62 is involved in the aggregate formation (Wooten et al. 2005) and is found in myotilin positive aggregates. p62 plays a key role in trafficking, regulation of aggregation and inclusion body formation in neurodegenerative diseases such as in tauo-, synucleinopathies and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) (Kuusisto et al. 2008). The inclusions found in neuronal cells are sites for sequestration of misfolded and ubiquitinated proteins triggered for degradation. On the other hand, direct evidence of myotilin ubiquitination and defective degradation or toxic effects are still waiting clarifications. To date, post-translational modifications of myotilin have not been studied. However, several phosphorylation sites are predicted in silico, within the threonine and serine rich region (Selcen and Engel 2004). Myopalladin is a sarcomeric protein of 145 kDa expressed exclusively in skeletal and cardiac muscle as a single isoform. Myopalladin gene is located on located on chromosome 10q21.1. Myopalladin binds via its carboxy terminal region to the SH3 domain of nebulin and nebulette and to the EF-hands of α-actinin (Bang et al. 2001). Numerous SH3 binding motifs present in titin’s PEVK segments of titin isoforms are binding sites for myopalladin, suggesting that titin PEVK and myopalladin may play signaling roles in targeting and orientating nebulin to the Z-line during sarcomere assembly (Ma and Wang 2002). The amino terminus of myopalladin binds to CARP (cardiac ankyrin repeat protein) (Bang et al. 21 2001). CARP belongs to a group of proteins structurally related that includes Ankrd-2/Arpp (ankyrin repeat domain 2/ankyrin repeat protein with PEST and proline-rich region) and DARP (diabetes- associated ankyrin repeat protein) and together they are called MARPs (muscle ankyrin repeat proteins). They are thought to be involved in muscle stress response pathways, as each of the proteins is upregulated by injury and hypertrophy (CARP), stretch or denervation (Ankrd2/Arpp), and during recovery following starvation. MARPs, myopalladin, and the calpain protease p94 appear to be components of signalling pathways detecting myofibrillar stretching and strain with subsequent response by modification in gene expression (Miller et al. 2003). Recently, myopalladin mutations were reported to cause idiopathic dilated cardiomyopathy (Duboscq-Bidot et al. 2008). Palladin is a cytoskeletal-associated protein expressed as many isoforms from a single gene located on human chromosome 4q32.3. The mouse orthologue is located on chromosome 8, cytoband B3.3. The structure of the gene is complex, both in human and mouse, with mouse gene formed by 400kbp and 24 exons. The gene has three promoters, allowing for expression of several transcripts, among which some are tissue specific (Mykkänen et al. 2001; Parast and Otey 2000). Palladin isoforms result from the activity of three promoters, splicing and termination mechanisms (reviewed in Otey et al. 2005). The first described isoform is transcribed from the most 3’ promoter of the gene and is translated as a doublet with apparent molecular weight of 90-92 kDa. It is considered a major palladin isoform as it is widely expressed in several cell lines, a variety of mouse and human tissues, and is ubiquitously expressed in tissues of developing rodents. It has three Igl domains located at the carboxy terminus, with the second and third Igl domains homologous to myotilin Igl domains. The Igl domains are most homologous to IgI of the myopalladin, as the carboxy terminus region of palladin is 63% identical to that of myopalladin (Bang et al. 2001). The amino terminus of the molecule is less homologous to carboxy terminus of myotilin, rather has high homology with myopalladin amino terminus region. The amino terminus region of palladin has polyproline (FPPPP) rich and serine rich domains. Conserved fragments, such as a 15-residue box, previously shown to bind SH3 domain-containing proteins in myopalladin, are present in palladin molecule (Rönty et al. 2005). At least two additional palladin isoforms have been reported. Rönty et al. (2005) described a 140 kDa palladin with five Igl domains, of which the three carboxy terminal ones are identical to the 90-92 kDa isoform (Rönty et al. 2005). This isoform has two additional Igl domains located at the amino terminal region and exclude the middle polyproline stretches. Two other reports (Rachlin and Otey 2006; Rönty et al. 2006) describe the 140 kDa isoform as composed of only four Igl domains, therefore named also 4Ig/140 kDa isoform, the carboxy terminus three Ig identical to the 90-92 kDa isoform and, in addition, a fourth Ig at the very amino terminus. The latter isoform contains also two polyproline stretches located within the middle region of the molecule. The 4Ig/140 kDa isoform is expressed in embryonic mouse tissues and in mouse adult tissues rich in smooth muscle, such as stomach and intestine. Its expression is induced during myofibroblast differentiation in a TGF-β1(tumor growth factor)-induced manner and is thought to participate to the force generation and transmission in the myofibroblasts (Rönty et al. 2006). 22 The longest palladin isoform is thought to be transcribed from the most 5’ promoter. It is detected in neonatal mouse heart, skeletal muscle and bone, while in adult mouse tissues it appears to be expressed in skeletal muscle and heart, and only weakly in lung. The protein is approximately 200 kDa and thought to contain a total five Igl domains (Rachlin and Otey 2006) Palladin isoforms are tightly associated with the actin cytoskeleton. Palladin 90-92kDa isoform is found associated with stress fibers, at focal adhesion sites and cell-cell junctions. Along stress fibers, it is distributed in a punctate pattern and colocalize with α-actinin and these together form stable complexes. Their composition include ArgBP2, a cytoskeleton associated adaptor protein that binds Arg, Abl and Pyk2 kinases. Palladin 90-92 kDa isoform may provide the ArgBP2 localization to the Z-disks, intercalated disks and dense bodies and it links, at least indirectly, palladin with signalling events in (cardio)myocytes, neuronal and glial cells (Rönty et al. 2005). Palladin 90-92 kDa is phosphorylated (Parast and Otey 2000), and at lest one of the kinases showed to phosphorylate palladin is Src (Rönty et al. 2007). Palladin was shown to be required for Src-induced actin remodelling and together with SPIN90 a multiprotein complex is formed. Palladin interacts via its polyproline rich regions with SH3 domains of SPIN90, Src, ArgBP2 and Lasp 1 (Rönty et al. 2007; Rönty et al. 2005; Rachlin and Otey 2006). Along with 90-92 kDa palladin isoform, transfection and immunodetection studies of 4Ig/140 kDa palladin supports the evidence that it is localized at the Z-disk in both embryonic and adult striated muscle cells (Bang et al. 2006; Parast and Otey. 2000; Rönty et al. 2005). Palladin isoform 90-92 kDa contain two polyproline regions closely located, LPPPP and SPPPP, while the 4Ig/140 kDa and 200 kDa isoforms contain four proline rich regions FPPPP, LPPPP and SPPPP that are homologous to ones found in ActA, zyxin, vinculin and Mena/VASP (Rachlin and Otey 2006). The polyproline motifs in palladin molecules are binding sites for EVH1 (Ena/VASP homology 1) domains found in VASP protein family members. In cells, members of VASP protein family are localized to sites of actin assembly (Bear et al. 2000; Rottner et al. 2001), bind and polymerize in vitro actin (Bachmann et al. 1999; Harbeck et al. 2000) and are major substrates for cAMP and cGMP-dependent protein kinases in platelets (Halbrugge et al. 1990; Reinhard et al. 1992; Reinhard et al. 2001). In addition, they are important regulators of actin assembly and their function is crucial in cell adhesion and migration. Palladin interacts with VASP via the interaction of FPXPP motif in palladin and EVH1 domain in VASP. Palladin has been proposed to be a scaffold protein involved in recruiting VASP along the stress fibers and lamellipodia, where rapid actin polymerization takes place (Boukhelifa et al. 2004). Together with the observation that palladin is localized to growth cones of primary cortical neurons, it is thought that palladin’s interaction with VASP plays an important role in axonal outgrowth (Boukhelifa et al. 2001). In addition, 4Ig/140 kDa and 200 kDa isoforms contain a small polyproline stretch EPPP, that is found also in myopalladin and which serves in pallaidn as binding site for Lasp-1 (Rachlin and Otey 2006). Another size variant of ~85 kDa palladin has been reported in developing mouse embryo brains and cultured neurons. Palladin appears to play an important role in the maturation of neurons and their mophological differentiation (Boukhelifa et al. 2001). In addition, palladin appears important for modeling the actin cytoskeleton and subsequently shape of astrocytes in vivo and in vitro (Boukhelifa et 23 al. 2003). This supports the palladin role in actin cytoskeleton remodelling. In cells, dorsal ruffles induced by PDGF (platelet derived growth factor) treatments and podosomes induced by phorbol ester treatments are transient and highly dynamic actin-based structures in cells. Along with the actin-polymerizing proteins, palladin was found to localize to these structures and interact with one of their components, Eps8. Palladin is thought to be a component of Rac-dependent signaling pathways resulting in the modulation of actin-based membrane cell protrusions (Goicoechea et al. 2006). Further, PDGF treatment of cells that induce Src activation or Src activation alone induce actin cytoskeleton remodelling that requires palladin, as palladin knock-down prevents these changes (Rönty et al. 2007). Along with the observation that palladin binds (Mykkänen et al. 2001), its association with Src is thought to implicate palladin in cell migration events, whether normal or cancer cells. This observation has been supported by knock out studies, as palladin-/- MEF (mouse embryonic fibroblasts) cells display disorganized actin cytoskeleton and weak cell-ECM interaction (Liu et al. 2007; Luo et al. 2005). An additional palladin isoform of 50 kDa that lacks the carboxy terminal Igl domains has been observed in MEFs (Luo et al. 2005). To date, a mutation in palladin gene is associated with human pancreatic cancer. The mutation consists of a single amino acid substitution and is located in the palladin molecule at the binding site of α- actinin (Pogue-Geile et al. 2006). However, studies in European patients did not confirm the association (Zogopoulos et al. 2007).

3.1.3. ZASP/Cypher

ZASP/Cypher is a Z-disk protein known also as Cypher/Oracle. It is expressed as many isoforms from a single gene located on 10q22.2-q23.3, in skeletal and cardiac muscle (Faulkner et al. 1999). In humans, at least two isoforms, of 32 kDa and 78 kDa are found (Faulkner et al. 1999), while six murine isoforms have been characterized (Huang et al. 2003; Zhou et al. 1999). They are classified as skeletal or cardiac specific classes upon the presence or absence of skeletal or cardiac domains. Although the isoforms vary in length, both short and long isoforms contain an amino terminus-located PDZ domain, consisting of 80- 120 amino acid residues (Huang et al. 2003; Zhou et al. 1999). The PDZ domain-containing proteins interact with each other, and via the PDZ motif they appear to be directed to multiprotein complexes involved in targeting and clustering of membrane proteins. ZASP/Cypher contains a conserved region, named ZASP-like motif (ZM), present in addition in the ALP (α-actinin-associated LIM protein), CLP36 proteins (Klaavuniemi et al. 2004). The long isoforms contain three carboxy terminal LIM domains. ZASP/Cypher isoforms are developmentally regulated in both skeletal and cardiac muscle. In zebrafish, ZASP/Cypher is important for somite and heart development (van der Meer et al. 2006). In addition, studies on mice showed that ZASP/Cypher knockout is neonatally lethal, as the mice develop a severe congenital myopathy and dilated cardiomyopathy. Rescue experiments with short or long isoforms suggest that either isoform can partially rescue the lethality associated with the absence of ZASP/Cypher (Huang et al. 2003; Zhou et al. 1999). ZASP/Cypher interacts through its PDZ domain and internal region with the major Z-disk actin

24 cross-linker, α-actinin. Point mutations found in cardiomyopathy patients are located in the internal region of ZASP/Cypher, however, they did not affect the ZASP/Cypher co-localization with α-actinin, or the stability of the ZASP/Cypher protein (Klaavuniemi and Ylänne 2006). ZASP/Cypher interacts with structural Z-disk proteins such as α-actinin, nebulette (Holmes and Moncman 2007) and myotilin (von Nandelstadth et al., submitted), as well as with enzymes, such as protein kinase C (PKC). A novel biochemical mechanism of the pathogenesis of dilated cardiomyopathy is suggested by Arimura et al. (2004). They found a late onset cardiomyopathy-associated D626N mutation of ZASP/Cypher that increased the affinity of the LIM domain for PKC. At least five ZASP/Cypher mutations were identified in familial or sporadic DCM (dilated cardiomyopathy) with or without INLVM (isolated non-compaction of the left ventricular myocardium) (Vatta et al. 2003). In addition to ZASP/Cypher mutations associated to cardiac diseases, ZASP/Cypher point mutations are found also to cause skeletal muscle disorders (Griggs et al. 2007; Selcen and Engel 2005).

3.1.4. FATZ

FATZ/myozenin/calsarcin is a recently described protein family expressed as three homologous members, FATZ-1/calsarcin-2/myozenin-1, FATZ-2/calsarcin-1/myozenin-2, and FATZ-3/calsarcin-3/myozenin-3). They are expressed mainly in the striated muscle with significantly lower levels of expression in other tissues, and localize to the Z-disk in striated muscle (Faulkner et al. 2000; Takada et al. 2001). While FATZ-1/calsarcins-2 and FATZ-2/calsarcin-1 are expressed both in skeletal muscle and heart, FATZ- 3/calsarcins-3 appears restricted to the skeletal muscle. FATZ/calsarcin may play role in the skeletal muscle fiber type specification, as FATZ-2/calsarcins-1 is found primarily in cardiac muscle and type I skeletal muscle fibers, FATZ-1/calsarcin-2 is predominantly in type II fibers and FATZ-3, expressed specifically in skeletal muscle, is enriched in type II muscle fibers. Structurally, FATZ/calsarcins are characterized by highly homologous stretches both at the amino and the carboxy termini that mediate the interaction with binding partners for all the family members. Within the Z-disk, FATZ/calsarcins bind α- actinin (Frey and Olson. 2002; Takada et al. 2001), telethonin and filamins A, B, and C (Faulkner et al. 2000; Frey et al. 2000) and myotilin (Gontier et al. 2005). In addition of binding structural proteins, they bind and target to the sarcomere a calcium/calmodulin-dependent serine/threonine phosphatase, calcineurin (Frey and Olson 2002; Frey et al. 2000; Takada et al. 2001). Targeting of calcineurin to subcellular compartments is important for its activation or inhibition in response to physiological and pathological stimuli and its association with downstream effectors, such as NFAT (nuclear factor of activated T-cells). When activated, calcineurin promotes the development of cardiac hypertrophy and cardiomyopathy and the reactivation of a fetal cardiac gene program, which includes genes whose products control contractility, calcium handling and energy metabolism. In skeletal muscle calcineurin influences the muscle fiber type specification, as overexpression of calcineurin in skeletal muscle leads to the conversion of type II to type I fibers. Studies of FATZ-2/calsarcin-1 knockout mouse showed that FATZ-2/calsarcin-1 plays an important role in modulating skeletal and cardiac muscle signaling through its effect on calcineurin activity. Hearts of FATZ-2/calsarcins-1-deficient mice showed exaggerated cardiomyopathy and super-induction of hypertrophic genes in response to pressure overload (Frey et al. 25 2004). This response to mechanical stress suggests that FATZ-2/calsarcins-1 is a negative regulator of stress-induced signaling in vivo that leads to pathological growth of the heart. Recently, mutations in FATZ-2/calsarcin-1 were shown to be associated with HCM (hypertrophic cardiomyopathy) (Osio et al. 2007) and idiopatic DCM (Arola et al. 2007) supporting the participation of FATZ/calsarcin to the molecular machinery transducing the biomechanical stress.

3.1.5. Filamin C

Filamins are a group of proteins ubiquitously expressed as several isoforms of three different genes, FLNA, FLNB, and FLNC. Several filamins are known, as all cells express at least one filamin at some point in the development and the filamin type may change as cells differentiate (Hartwig and Stossel. 1975; Maestrini et al. 1993; Shizuta et al. 1976; Takafuta et al. 1998). While products of FLNA, FLNB genes, filamin A and B, participate in the cytoskeletal structure of various cell types, filamin C (γ-filamin, filamin 2, ABL-L), is a product of FLNC gene and is characteristic to the cardiac and skeletal muscle. Filamins contain an amino terminal actin-binding domain and 24 E-type Igl domains that are separated by two hinge regions (Fucini et al. 1997; Gorlin et al. 1990). Structurally filamin C has a unique insertion in the middle of the Igl domain 20 and localizes subsarcolemmally and to the periphery of the Z-disk (Xie et al. 1998). At the Z-disk filamin C interacts with FATZ and myotilin, while subsarcolemmally localized filamin interacts with γ- and δ-sarcoglycans but not with α- and β-sarcoglycans (Thompson et al. 2000). Filamin also interacts with caveolin 1, a membrane bound protein (Stahlhut and van Deurs 2000) and with Xin in myotendinous junctions (van der Ven et al. 2006). It also interacts with a PI3P (phosphatidylinositol (3,4,5)-trisphosphate) sensor, LL5beta in a PI3K (type 1A phosphoinositide 3- kinase)-independent fashion (Paranavitane et al. 2003; Paranavitane et al. 2007). Filamin C can be cleaved in vitro by the muscle-specific member of calcium-dependent protease family, calpain 3, which regulates its interaction with the sarcoglycans (Guyon et al. 2003). PKC phosphorylates carboxy terminus of filamin C, resulting in protection of filamin C against proteolysis by calpain 1 in COS cells (Raynaud et al. 2006). Calpain cleavage of filamin C could be involved in calpain-mediated remodelling of cytoskeletal-membrane interactions and adhesion, such as those that occur during myoblast fusion and muscle repair.

3.1.6. Telethonin/T-cap

Telethonin, named also T-cap, is a small protein of 19 kDa expressed as a single isoform in skeletal and cardiac muscle (Gregorio et al. 1998; Valle et al. 1997). It is localized at the Z-disks in the mature sarcomere and has several binding partnetrs: the Z-disk proteins FATZ/calsarcins (Faulkner et al. 2000), Ankrd2/Arpp, a member of the MARP family protein (Kojic et al. 2004), the potassium channel β-subunit minK (Furukawa et al. 2001) and titin (Mues et al. 1998). Its expression is developmentally regulated and, at least in part, regulated by neuronal activity (Mason et al. 1999; Schröder et al. 2001). Its interaction with minK proceeds in a phosphorylation dependent manner (Furukawa et al. 2001). During myofibrillogenesis is it a target subtrate for titin kinase, suggesting it may play roles in

26 myofibrillogenesis. Within the mature Z-disk it interacts with Z1Z2 domains of the giant protein titin. Structural studies show that one telethonin molecule functions as a ligand for two titin molecules to form an antiparallel complex. The structure of telethonin is pseudosymmetric and whithin the complex mediates a unique palindromic arrangement of two titin filaments (Zou et al. 2003). This conformation distributes equally the forces between titin’s Z2 and Z1 domains resulting in high stability of the Z-disk structure (Lee et al. 2006). Telethonin interacts with a secreted growth factor member of TGF-β family which is a key negative regulator of skeletal muscle growth, myostatin (Nicholas et al. 2002). Although telethonin does not interfere with the production and processing of myostatin, it is proposed to have a role in blocking the secretion of mature myostatin. However, the biological implications are still unclear. Telethonin also binds the ubiquitin ligase MURF-1 (Witt et al. 2005), which may couple telethonin to proteasome degradation pathway. Telethonin is proposed to play structural roles in the skeletal muscle and as well implicated in dynamic control of myofibrillogenesis and muscle turnover in human skeletal muscle. Telethonin mutations cause LGMD2G (Moreira et al. 2000), a recessive disease characterized by absence of telethonin protein. Point mutations in telethonin gene are found both in HCM (Bos et al. 2006, Hayashi et al. 2004) and DCM (Hayashi et al. 2004).

3.2. Thin filaments

3.2.1. Actin

The actin filaments in the skeletal muscle have precise organization and a constant length. They are anchored at the Z-disk, span the entire I-band and continue into the A-band up to the H-zone. Within A- band, they interdigitate with the myosin filaments of the respective sarcomere and here, one myosin filament is sourrounded by 6 to 11 actin filaments (Nistal et al. 1977). The mature actin filaments appear as filaments of ~ 1μM in length and 10nm in diameter. They are polar structures cross-linked at the Z- disk mainly by α-actinin. Their length is constant and is thought to be maintained by several mechanisms including interactions with other proteins such as templates, capping proteins and polymerization/depolymerization factors. One such protein is the giant protein nebulin that binds actin filaments and is thought to regulate the actin filament size. In addition, titin isoforms were found to correlate with the length of actin filaments (reviewed in Craig and Padron, 2004).

3.2.2. Tropomyosin and troponins

Tropomyosins are a family of fibrous proteins present in virtually all eukaryotic cells. Several isoforms expressed from four different genes are found in vertebrates. Typically alternative splicing and alternative promoter usage enriches the isoform variety. They can form hetero- or homodimers arranged in an in- parallel, in-register coiled-coil that bind lengthwise to actin filaments to form an actin-associated filament (Lehman et al. 2000). α-, β-, and γ-tropomyosins are specific for striated muscle (Perry 2001). They associate with a complex of three troponins, TnI (troponin I), TnC (troponin C), and TnT (troponin T), the

27 latter binding directly to tropomyosin (Perry 1998; Perry 1999). Tropomyosin and troponins form a Ca2+- sensitive complex that mask the myosin binding site on actin filaments. In the absence or low levels of Ca2+, the tropomyosin-troponin complex blocks the binding of myosin to actin by TnI binding to actin and thereby inhibiting actomyosin ATP-ase activity. Upon activation, Ca2+ ions bind to TnC and the complex position is shifted resulting in TnI dissociation from actin-tropomyosin releasing the inhibition. Subsequently, the interaction of actin and myosin results in the filament sliding and contraction. Tropomyosin and troponins function consists of modulating the actin-myosin interaction by sterically blocking myosin-binding sites on actin in the relaxed state and of stabilizing the actin filament structure (reviewed in Gordon et al. 2000). Mutations in tropomyosin and troponin genes cause various forms of nemaline myopathy (Donner et al. 2002; Penisson-Besnier et al. 2007; Laing et al. 1995). Mutations and deletions in tropomyosins and troponins cause also distal arthrogryposis and familial HCM and DCM (Thierfelder et al. 1994). Troponin (TNNI3) mutations were shown to cause restrictive cardiomyopathy (Mogensen et al. 2003).

3.3.3. Capping proteins, tropomodulin and CapZ

The actin filaments of the skeletal muscle are relatively stable and have a constant length that is maintained by capping proteins. Both ends of a thin filament are associated with actin-capping proteins, CapZ and tropomodulin. They regulate the filament growth and simultaneously stabilize it, by inhibiting the loss of actin subunits. CapZ, is a heterodimer of a 36 kDa α-subunit and a 32 kDa β-subunit that binds the plus ends of actin filaments located at the Z-disk and prevents the addition or loss of actin subunits. CapZ binds α- actinin and together anchor the actin filaments to the Z-disk. Studies supporting a critical role for CapZ to normal muscle development show that CapZ organizes and aligns the actin filaments at the Z-disk. CapZ assembles at Z-bodies before the actin filaments are detected in developing myotubes (Schafer et al. 1993). Disruption of actin-CapZ interaction impairs myofibrillogenesis (Schafer et al. 1995) and produces gross myofibrillar disarray (Hart and Cooper 1999). Expression of mutated or non-sarcomeric isoform of CapZ leads to sarcomere disruption and causes cardiomyopathy in transgenic mice (Hart and Cooper

1999). Filament capping by this protein is inhibited by PIP2, suggesting that CapZ may play a role in actin dynamics (Schafer et al. 1996). In addition to anchoring actin, Z-disks bind PKC. Reduction in the 2+ expression of β1 subunit of CapZ protein increases myofilament Ca sensitivity and disrupts PKC- dependent myofilament regulation, revealing a functional significance of CapZ in the transduction of PKC pathway signals to the myofilament (Pyle et al. 2002). Tropomodulin caps the opposite end of actin filaments, the minus ends. It is found as two isoforms, an erythrocyte and a skeletal tropomodulin. Both are be found in striated muscle and while skeletal tropomodulin is localized at the Z-disk, the erythrocyte isoform is localized at the costameres. The capping activity of tropomodulin is enhanced in the presence of tropomyosin, suggesting that the two proteins function as a complex to stabilize actin filament. Tropomodulin binds amino terminus of nebulin at the minus ends of actin filaments and together they may control thin filament lengths in skeletal muscle 28 (McElhinny et al. 2001). Actin filaments in striated muscle are maintained at a constant length, but recent studies show they are relatively dynamic despite the presence of capping proteins, and that regulated actin assembly at minus ends determines the length of thin filaments (Littlefield et al. 2001).

3.3.4. Nebulin and nebulette

Nebulin is a large muscle protein (600-900 kDa) coded by a single gene, NEB. Nebulin forms the fourth filament system in vertebrate muscles. A large number of nebulin transcripts arise by alternative splicing and they vary with the developmental stage and muscle type (Labeit and Kolmerer 1995). Over 95% of the nebulin protein consists of 30-35 amino acids long repeating modules organized into simple repeats. In the central part of the protein they are arranged into a higher order repeating unit, termed a super repeat, each containing seven modules. At each repeat module, conserved actin-binding motifs are found (Jin and Wang 1991; Pfuhl et al. 1994). Nebulin’s inextensible structural properties and the good correlation between the nebulin isoforms and the length of the actin filaments suggest nebulin role as a molecular ruler. As nebulin appears to assemble early during myofibrillogenesis, it is suggested that nebulin acts as a template of the actin filament size during skeletal muscle development. Some modules promote actin nucleation and filament bundling (Jin and Wang 1991; Pfuhl et al. 1994). Within modules, other types of conserved motifs are likely to bind tropomyosin and troponins (Wang et al. 1996). Additionally, nebulin interacts with the capping protein topomodulin, and is proposed to maintain the length of the thin filament in terminally differentiated muscle cells (Horowits 2006; McElhinny et al. 2001; McElhinny et al. 2005). Nebulin motifs that are located at the junction of A band with I band bind to actin, myosin and calmodulin. They can inhibit actomyosin ATP-ase activity and influence the sliding velocity of actin filaments over myosin in a Ca2+/calmodulin-dependent manner in vitro (Root and Wang 2001). The carboxy terminus of nebulin contains an SH3 domain, via which nebulin interacts with myopalladin and, in addition, modules M160-170 of nebulin located at the periphery of the Z-disk interact with desmin (Bang et al. 2001). Interaction of nebulin with the intermediate filament protein desmin may suggest that nebulin contributes to the lateral linkage of the Z-disks of adjacent myofibrils. Nebulette is a cardiac-specific nebulin homologue. It is a protein of approximately 107 kDa containing 22 nebulin-related modules, a carboxy terminus almost identical with nebulin and a unique amino terminus. Similar with nebulin, nebulette binds actin, myopalladin, tropomyosin and is critical for thin filament assembly, their spatial organization and the contractile activity (Bang et al. 2001; Moncman and Wang 2002). Nebulette interacts with filamin C, tropomyosin I and ZASP (Holmes et al. 2007). In cardiac muscle a nebulin-related protein (N-RAP) has been characterized and localizes to the intercalated disks (Luo et al. 1997). Mutations in ACTA1, coding for α actin, and NEB genes are the most common cause of nemaline myopathy (Ilikovski et al. 2001; Gurgel-Giannetti et al. 2001; Pelin et al. 2002). In addition, nebulin levels are reduced in some forms of muscular dystrophy (Campanaro et al. 2002; Wood et al. 1987; Zhang et al. 2006). 29 3.3. Thick filaments

3.3.1. Myosin

The sarcomere thick filaments are composed of several hundred myosin molecules. They are the molecular motors of the muscle contraction. The myosin superfamily is grouped into 15 classes, the type of myosin found in muscles is myosin II. The skeletal muscle and cardiac myosins are encoded by several genes located on chromosome 17 and 14 (Berg et al. 2001). The smooth muscle myosin gene is located on chromosome 16 (Berg et al. 2001). Myosin is a protein of about 500 kDa consisting of two identical heavy chains (MyHC) and two pairs of light chains (MLC), of which one pair of essential light chains and one pair of regulatory light chains. Each heavy chain is about 200 kDa and consists of a globular head region and a long α-helical tail. The tails of two heavy chains twist around each other to form a stable coiled-coil structure and two different light chains associate with each head region to form the complete myosin II molecule. The globular heads of myosin bind actin (Rayment et al. 1993), forming cross- bridges between the thick and thin filaments. In addition, myosin heads bind and hydrolyze ATP, which produces repeated cycles of interaction between myosin heads and actin. The ATP hydrolysis results in conformational changes of myosin that leads to the movement of myosin heads along actin filaments. The actin filament sliding toward the M-line of the sarcomere is the basis of movement in muscle contraction. Various striated muscles have different speed of contraction, function that is achieved by different combinations of MyHC and MLC. The myofiber types are determined by their myosin heavy chain (MyHC) composition that is associated with myofibrillar adenosine triphosphatase (mATPase) activity which, in turn, correlates with muscle function. At least eight striated muscle MyHC are characterized. Mainly, slow type I fibers express slow MyHC, named MyHC β or MyHC I; fast type II fibers are characterized by expression of fast MyHC, named MyHC IIa and IIb (reviewed in Staron 1997). During development, embryonic and perinatal myosins characterize muscle fibers at certain developmental stages, and in addition, are found in extraocular muscles and in regenerating muscles. Mutations in MyHC cause hereditary myosin myopathies, a group of striated muscle disorders with highly variable clinical features. Within the group, the onset and morphology of affected tissues varies widely. The majority of different mutations in myosin result in skeletal and/or cardiomyopathy. To date, over 200 mutations in MYH7 gene are known to cause HCM and DCM. They sum up to 25% of the hypertrophic cardiomyopathies and, together with mutations in MYBPC3 gene that encodes for MyBP-C and accounting for a 26% (Van Driest et al. 2004), they are considered most frequent cause of HCM (reviewed in Oldfors 2007).

3.3.2. Titin

Titin is the largest protein identified to date (3-4.2 MDa) and follows actin and myosin as the third most abundant protein of the skeletal muscle. Organized as the third filament structure of the sarcomere, titin forms a continuous filament system in the myofibrils. Full-length titin spans the I-bands and A-bands of adjacent sarcomeres. The carboxy terminus ends of titin molecules from adjacent sarcomeres overlap at M-line and their amino terminus at Z-line. The full-length titin is a polypeptide of 38138 amino acids

30 (Bang et al. 2001). Several isoforms arise by alternative splicing from a single gene on chromosome 2q. Structurally, titin is highly modular, as about 90% of the molecule consists of Igl domains and fibronectin III-type domains. Unique structural composition provides titin with many crucial roles, from the myoblast differentiation to the fully functional skeletal muscle cell. Early assembly of titin’s repetitive structure during myofibrillogenesis and its interaction with regulatory and sarcomeric components support titin’s role as a template and stabilizer of the sarcomere. The I-band region of titin has unique elastic properties that determine the stiffness of the myofibrillar structures. One of the regions in the titin molecule which is not falling in the repetitive domain category of Igl or fibronectin domains, is a serine/threonine kinase domain that lies near the M-line (Gautel et al. 1995). The role of titin kinase is not completely understood, but it is suggested to serve as a regulator of contractile functions by calcium handling and protein kinase signalling pathways (Peng et al. 2007). Functional studies indicate that Z-disk titin determines the stability of the sarcomere in cell culture (Gregorio et al. 1998; Peckham et al. 1997; Turnacioglu et al. 1997). Within I-band full-length titin and the titin isoforms localized here, contribute to the production of a passive force that maintains the overlap of the thin and thick filaments in the resting muscle. This role is attributed to elastic domains PEVK and N2A in skeletal and cardiac muscle and to cardiac specific N2B domain. The recruitment of these domains proceeds sequentially. Therefore, precise roles for each elastic modules are suggested in a time-, space- and force-dependent manner. The I-band regions of titin and or part of the I-band located titin isoforms can interact with components of the thin filaments, as PEVK region of N2B cardiac isoform interacts with thin filaments in vitro and the interaction appears to be inhibited by Ca2+-binding protein S100/A1 (Kulke et al. 2001; Yamasaki et al. 2001). Mutations in various regions of titin gene are associated with both skeletal muscle and cardiac diseases. Single amino acid substitutions in Z-disk region of titin are found to modify the interaction affinity of titin with telethonin (Matsumoto et al. 2005), or affect the binding to α-actinin (Satoh et al. 1999) were found in HCM patients. Mutation of I-band titin results in familial DCM (Gerull et al. 2002). A zebrafish model resembling DCM consists in a mutation in N2B region of titin (Xu et al. 2002). Additional clues of titin’s roles originated from the observation that skeletal muscles of patients with Duchenne dystrophy have downregulated titin transcrips, and a mutation of N2A causes muscular dystrophy with myositis in mice (Garvey et al. 2002; Tkatchenko et al. 2001). Recently described, homozygous truncations of M-line titin leads to a congenital myopathy with cardiomyopathy (Carmignac et al. 2007). Separately, mutations in titin gene can lead only to skeletal muscle phenotypes. Phenotypic variants are: an autosomal dominant myopathy with proximal weakness and early respiratory muscle weakness characterized by a mutation in the kinase domain of titin (Lange et al. 2005), the muscle dystrophy TMD (tibial muscle dystrophy) and LGMD2J. The latter two are characterized by a mutation named the Finnish FINmaj that consists of a deletion/insertion of 11bp leading to change of four amino acids in an Igl domain of titin located at the periphery of M-line (Hackman et al. 2002; Haravuori et al. 1998). Clinically, both are characterized by high variability of phenotypic expression (Udd et al. 2005).

31 3.4. Intermediate filaments

3.4.1. Desmin

Intermediate filaments (IF) are composed of highly elongated fibrous proteins organized as tetramers and assembled in antiparallel fashion to form nonpolar filaments. Clasifications of IF is based on the amino acid sequence and structure of the proteins. Vimentin, desmin, GFAP (glial fibrillary acidic protein) and peripherin are classified as type III IF proteins. The intermediate filament proteins expressed in skeletal muscles and heart are desmin, vimentin and nestin. Desmin is a 52 kDa protein almost exclusively expressed in striated muscle and endothelial cells (Capetanaki et al. 1984; Lazarides and Hubbard 1976; Traub. 1985). As all IF proteins, desmin has two conserved central rod domains (H1 and H2), four linker regions (L1A, L1B, L2A, L2B) and two globular end domains. Within a muscle cell, desmin filaments form a lattice surrounding the Z-disks. The desmin structure links several myofibril bundles to each other and anchors them to the plasma membrane by interactions with several membrane adhesion complex proteins such as spectrin and ankyrin (Larsen et al. 1999). Desmin is concentrated in specific areas, such as around the Z-disk and at the costameres in striated muscle cells, in dense bodies and around the nucleus. Desmin can form homopolymers or heteropolymers with the same class IF proteins, vimentin and nestin. The desmin lattice is anchored to the thick filaments, as it interacts with myosin, and, in addition, a direct interaction with nebulin was demonstrated by Y2H (Bang et al. 2002). Desmin binds a small chaperone, αB-crystallin (Horwitz 2003), interacts with calpain and caspase, and is a substrate for proteolysis by a muscle-specific calpain (Goll et al. 2003). Desmin could be implicated in the regulation of gene expression, as desmin was found in the nucleus where it interacts with lamins (Tolstonog et al. 2002). Desmin expression is found in the early stages of skeletal, cardiac and smooth muscle formation, however absence of desmin does not impair the primary muscle formation in the desmin knockout mice (Li et al. 1997). Apparently, desmin does not have a direct role in skeletal muscle contraction, rather it indirectly maintains the structural integrity of mature skeletal muscles. Desmin is important for skeletal muscle regenerations, as demonstrated by aberrant skeletal muscle regeneration observed in des-/- mice (Li et al. 1997).

4. Skeletal muscle development

Formation of terminally differentiated skeletal and cardiac muscle is a complex process, which depends on spatially and temporally coordinated activation of several signaling pathways. Most of the skeletal muscles, such as axial and limb musculature, stem from mesodermal precursor cells situated in the somites which in later stages evolve to develop the myotome and, respectively, the dermamyotome. Initially, precursor cells are committed to myoblast lineage, followed by their proliferation and migration to final location in the body. Finally, myoblasts exit from the cell cycle, fuse into multinucleated myotubes and start expressing muscle-specific genes encoding structural and contractile proteins. Two major transcription factor families master the formation of differentiated muscle cells: the myogenic regulatory factor (MRF) family (reviewed in Edmondson and Olson 1993; Olson and Klein

32 1994) and the myocyte enhancer factor 2 (MEF2) family of MADS (MCM1, agamous, deficiens, serum response factor)-box transcription factors (Black and Olson 1998; Naya and Olson 1999). Four members of MRF family are known: myogenic factor 5 (Myf5), MyoD (myogenic determination), myogenic regulatory factor 4 (MRF4/Myf6/herculin) and myogenin (Davis et al. 1987; Emerson 1990). Structurally, they contain a highly conserved basic helix-loop-helix (bHLH) domain (Edmondson and Olson 1993) that mediates the homo- and heterodimerization with E proteins and determines their binding to E-boxes, the latter found in the control region of most muscle-specific genes. Forced expression of any of the MRF family members in cultured non-myogenic cells converts them into myoblasts (Davis et al. 1987). In mice, peaked expression Myf-5 in trunk somites (Smith et al. 1994) contributes to the early specification of mesodermal progenitor cells, their proliferation and migration (Arnold and Winter 1998). MRF4 expression is biphasic, in mouse embryo it is transiently detected in the somite bud and later becomes the predominant MRF in adult skeletal muscle (Bober et al. 1991; Hinterberger et al. 1991; Summerbell et al. 2002). Knockout studies have shown that myogenin is required for the myoblast terminal differentiation into functional myofibers (Buckingham et al. 2003). In early embryonic stages, MyoD activates independently gene expression and can function as the initiator of progenitor cells differentiation (Cao et al. 2006; Ohkawa et al. 2006; Ohkawa et al. 2007) In later stages, MyoD together with transcriptional coactivators induce the expression of muscle contractile proteins (Berkes and Tapscott 2005; Pownall et al. 2002; Tapscott et al. 1990; de la Serna et al. 2005). The MEF2 family members are Mef2a, Mef2c, and Mef2d (Blais et al. 2005) and they bind A/T rich DNA sequences. Members of the MEF2 family act as cofactors for MRF’s, such as myogenin, resulting in muscle-specific lineage transcription of skeletal muscle genes (Molkentin et al. 1995). MEF2C, myogenin and MRF4 play important activity in expression of muscle-specific genes, such as those encoding the contractile proteins myosin heavy and light chains, desmin, α-actinin, nebulin, titin, tropomyosin and troponins, as well as proteins involved in calcium signaling. Myogenesis is also controlled by Pax and Six homeoproteins (Grifone et al. 2005; Spitz et al. 1998) and SRF (serum response factor) (Li et al. 2005). The dominant-negative inhibitor of the MRFs is a family of bHLH factors called Id (inhibitor of binding) (Benezra et al. 1990). Several transcription factors, such as MEF-2, TEF-1 and MNF, are common to the skeletal and cardiac muscle lineages. In the absence of master regulator genes, the heart development is thought to be driven by the interaction between ubiquitous factors and tissue restricted factors. MEF2C is an essential regulator of cardiac myogenesis and right ventricular development (Lin et al. 1997). Other transcription factors, such as Nkx 2.5 and GATA-4, are expressed predominantly in the heart and play important roles in the heart development (reviewed in Sachinidis et al. 2003). Myofibrillogenesis events are frequently studied in vitro by cell culture methods, the differentiation of myoblasts to mature myofibers takes place in approximately seven days. Upon serum withdrawal from culture media cultured mononucleated myoblasts fuse and generate small multinucleated cells, the myotubes. They grow further by fusing end-to-end with other myotubes or myoblasts resulting in formation of myocytes. In vivo and in vitro models show that proteins appear in a well defined temporal order (Fürst et al. 1989; Gregorio and Antin 2000) and recently, the titin kinase has been 33 proposed to control skeletal muscle gene expression and protein turnover (Lange et al. 2005). Classically, formation of a mature sarcomere implies the formation of three forms of myofibrils, association of various proteins with these myofibrils and finally, their spatial arrangement. In early steps of differentiation, premyofibrils are formed as stress fiber-like structures that are decorated by titin and α- actinin in a punctuated pattern. They are composed of non-muscle myosin that interdigitate bipolar actin filaments. The actin filaments are cross-linked in structures called Z-bodies, a non-muscle equivalent and a primordial Z-disk that contains the amino termini of titin. They align laterally and allow that the carboxy termini of titin unfold towards the next Z-body. The carboxy termini of titin molecules that originate from adjacent Z-bodies meet and are bound by myomesin forming the M-band. Other early expressed proteins, such as telethonin, γ-filamin, nebulin, tropomodulin and nebulin incorporate at this stage into premyofibrils to form the nascent myofibril. Further the growth of the sarcomeres in length and alignment of nascent myofibrils results in mature myofibrils (Dabiri et al. 1997; Ehler et al. 1999; Ojima et al. 1999; Rhee et al. 1994; Sanger et al. 2005).

5. Regulation of striated muscle growth and adaptation processes

Striated muscle cells are postmitotically differentiated cells that continuously maintain their composition by protein turnover. Skeletal muscles and heart are sensitive to mechanical environment (i.e. load, unload) and they subsequently adapt their architecture and metabolism by changes in gene expression, modulation of protein synthesis and mitochondrial biogenesis. Mechanical signals are converted into chemical signals capable to generate specific downstream events, leading to muscle structural and functional modifications that aim to better match the functional challenges. Several pathways involving Ca2+ ions, kinases (reviewed in Chin 2005), phosphatases, transcription factors (reviewed in Tidball 2005) and miRNA (Chen et al. 2006) are known to regulate the skeletal muscle growth and adaptation processes. Recently, it also became clear that Z-disk is not only a mere transmitter of generated force through the sarcomere, but an active participant to sense the muscle stretch (Frank et al. 2006). This mechanism enables the cardiomyocytes to detect mechanical load changes leading ultimately to modulation of gene expression. Skeletal muscle exercise is classified in many categories: concentric, eccentric, isometric and isotonic. Of them, eccentric exercise represents the lenghtening of an activated muscle. Performed in an extensive manner by a person unnaccustomed with exercise, it leads to pain, stiffness and tenderness in the muscle. According to the typical symptoms and their appearance, it is called delayed onset muscle soreness (DOMS). Several human and animal studies show after eccentric exercise modifications in skeletal muscle architecture (reviewed in Fridén 2002). After eccentric exercise, modifications in sarcolemmal integrity are detected by discontinuous or loss of sarcolemmmal dystrophin staining and detection of EC matrix protein fibronectin in the skeletal muscle fibers in rat quadriceps muscles (Komulainen et al. 2000). Additionally, the mdx mouse shows an increased permeability of muscle fibers after eccentric exercise (Brusee et al. 1997). Immunofluorescence and protein quantification studies of skeletal muscles subjected to eccentric exercise revealed changes in the localization and/or the amount of cytoskeletal proteins. Specifically, the Z-disk proteins, actin, and intermediate filaments showed localization shifts, such as increased actin staining and decreased α-actinin staining in focal areas (Yu et 34 al. 2003). Evidence from electron microscopy shows alteration of myofibrillar ultrastructure that consists mainly of Z-disk ultrastructural alterations that consist of Z-disk broadening, streaming and disruption. The alterations are proposed to represent the hallmark of muscle with DOMS and the markers of muscle damage (reviewed in Thompson et al. 1999, Lieber and Fridén 2002). Similar Z-disk alterations are also observed in myofibrillar myopathies (Hauser et al. 2000, Claeys et al 2008). Several theories and animal model studies support the hypothesis that eccentric exercise induces damage and necrosis (reviewed in Lieber and Fridén 2002). On the other hand, several human studies support the hypothesis that eccentric exercise induces a remodelling and lengthening of myofibrils (Li JG, 2003, doctoral dissertation). Moreover, it has been showed that one bout of eccentric exercise protects the skeletal muscle from the damage of a second bout (reviewed in McHugh 2003). Studies of Jones et al (1986), Stupka et al. (2000) and Newham et al. (1988) found inflammatory infiltrates in the skeletal muscles after eccentric exercise, however, the degree of pain did not correlate with the structural alterations. On the other hand, studies of Yu et al. (2003) did not find muscle fibre necrosis in human muscles after eccentric exercise and the correlation of inflammatory cells and the ultrastructural alteration of skeletal muscke after eccentric exercise is not yet fully understood. Locally expressed insulin-like growth factor (IGF-l) functions as an autocrine hormone in the skeletal muscle. IGF-l expression increases in vitro and in vivo by mechanical loads (McKoy et al. 1999; Perrone et al. 1995; Yang et al. 1996). Its increased expression correlates with induction of satellite cell proliferation and differentiation in adult skeletal muscle (Musaro et al. 2001) and several in vivo studies demonstrate that increased IGF-l levels leads to increase in muscle mass (Barton-Davis et al. 1999; Coleman et al. 1995; Lee et al. 2004). In skeletal and heart cells, IGF-l binds to its receptor and stimulates PI3K, which, in turn, phosphorylates Akt. This is a serine-threonine kinase, also called protein kinase B (PKB), known as a potent muscle mass regulator (reviewed in Frost and Lang 2007). It functions in Akt/ mTOR (mammalian target of rapamycin) signaling pathway that regulates cell survival, proliferation and protein synthesis. Akt activation results in activation or inhibition of pathways that result in skeletal muscle growth. An Akt substrate is mTOR and downstream of mTOR activation is p70S6K, a potent protein synthesis activator (Baar and Esser 1999). On the other hand, Akt can function as an inhibitor of GSK-3β, a serine/threonine kinase known as a negative regulator of skeletal muscle protein translation (Rommel et al. 2001; Vyas et al. 2002). Akt, mTOR, p70S6K and IGF-1 activity can be induced by mechanical load. They play important roles in promoting muscle growth during development and muscle cell regeneration in the adult (reviewed in Guttridge 2004). IGF-l stimulation of skeletal muscle fibers can induce fluctuations in cytosolic Ca2+ levels, and in turn, Ca2+ levels control the activity of calcineurin. Calcineurin is a serine/threonine phosphatase shown to be targeted at the Z-disk by FATZ/calsarcins or MLP (muscle LIM protein) (Heineke et al. 2005). In response to hypertrophic agonists and alterations sarcomere function that alter Ca2+ handling, calcineurin is activated resulting in regulation of gene expression and enzyme activity of cardiac and skeletal muscle (Molkentin et al. 1998; Sussman et al. 2000). Calcineurin functions by dephosphorylating transcription factors of the NFAT family that subsequently translocate to nucleus and modulate transcription of target 35 genes. Calcineurin/NFAT pathway activation is shown to change skeletal muscle fiber type toward a type I muscle phenotype. Via NFAT and MEF2 family of proteins, calcineurin is thought to participate in signaling pathways that regulate pathological hypertrophic growth of the myocardium and heart failure (reviewed in Wilkins and Molkentin 2004). Ca2+ has a crucial role in both excitation-contraction coupling (ECC) and as an activator of signaling events. In addition to calcineurin activation Ca2+ activates yet other important effectors, as Ca2+- calmodulin-dependent protein kinase II (CaMKII). In cardiomyocytes, its activation determines the nuclear shuttling of histone deacetylase 5 (HDAC5) that control cardiac hypertrophy and heart failure (reviewed in Molkentin 2006). Named also GDF-8, myostatin is a member of the TGF-β superfamily, and is expressed in the striated muscle as a single isoform (McPherron et al. 1997). Myostatin is expressed as a 52 kDa peptide that is processed intracellularly to produce a mature myostatin of 15 kDa. The mature form is secreted as dimers in the circulation, and has skeletal muscle as its target. The activity of mature myostatin is to supress skeletal muscle growth, as shown by knockout and overexpression studies. Knockout myostatin mice showed increased skeletal muscle mass and cardiac size. However, increased cardiac size did not correlate with increased functional parameters, as indicated by investigation of the ejection fraction. Parallel research showed that ventricle mass is increased in the myostatin knockout mouse, but in turn did not influence the fibrosis levels (Artaza et al. 2007; Cohn et al. 2007). In addition to Myf5, MyoD and myogenin are expressed at low levels in adult skeletal muscle (Eftimie et al. 1991; Hughes et al. 1993; Voytik et al. 1993) and in proliferating satellite cells at injury sites, indicating cell commitment to regeneration (Beauchamp et al. 2000; Cornelison et al. 2000; Eftimie et al. 1991). Postnatally, MRF-4 is the most abundant of the MRFs and is preferentially expressed in slow muscle fibers (Walters et al. 2000). The expression of the transcription factors in adult skeletal muscle is thought to participate to the control of muscle fiber phenotype specification.

6. Muscular dystrophies and myopathies

Muscular dystrophies and myopathies are a heterogeneous group of progressive and debilitating disorders characterized by skeletal muscle weakness and cardiomyopathy. They are hereditary disorders and the clinical spectrum greatly varies upon the molecular genetic defect. Classically, the muscular dystrophies are characterized by the breakdown of skeletal muscle intergrity (Kirschner and Bonnemann 2004) and are caused mutations in sarcolemmal proteins. On the other hand, myopathies are characterized by muscle weakness and atrophy that are caused by mutations in intracellular/sarcomeric components. However, several recent papers showed overlapping boundaries of the two categories of diseases (Kirschner and Bonnemann 2004).

6.1. Limb girdle muscular dystrophies (LGMD)

Limb girdle muscular dystrophies are a heterogeneous group of muscular dystrophies (Guglieri et al. 2005; Laval and Bushby 2004). LGMD is typically an inherited disorder presenting as a dominant, recessive, or X-linked genetic defect, however sporadic forms are also reported (Laval and Bushby 2004).

36 Currently, the classification includes the autosomal dominant LGMDs, LGMD1A-1G and autosomal recessive, LGMD2A-2M. Several mutations in genes encoding proteins of the extracellular matrix, glycosylation pathway, sarcolemmal membrane, intracellular proteins, enzymes, nuclear envelope and sarcomeric proteins are underlying LGMD. The disease is characterized by symmetric proximal skeletal muscle weakness of the pelvic and shoulder muscles that can progress to distal muscle groups. Common symptoms are muscle weakness, myotonia, myoglobinuria, pain, cardiomyopathy, elevated serum creatine kinase and rippling muscles. In general, it is a slowly progressing disease, and within many years from the onset, the patient loses muscle bulk and strength. However, severe course of the disease can be encountered in sarcoglycanopathies (LGMD2C, 2D, 2E, 2F), or in the LGMD2J, caused by mutation in titin. Onset in LGMD can be in late childhood, adolescence or even adult life. When the dystrophy begins in childhood the progression appears to be faster and the disease more disabling. Studies show an overlap between forms of LGMD and CMD (congenital muscular dystrophies) due to alterations in essential components of the molecular bridge between sarcolemma and ECM (extracellular matrix) (Kirschner and Bonnemann 2004). Involvement of other muscles than limb muscles, such as respiratory muscles and cardiac muscle is also reported (http://neuromuscular.wustl.edu/musdist/lg.html). LGMD1A is a rare autosomal dominant disease, characterized by weakness of girdle muscles and a dysartric pattern of speech. Initial linkage studies indicated mapping of LGMD1A and VCPMD (vocal cord and pharyngeal weakness) to the same region, however, mutation in myotilin were not found as a causative molecular genetic defect for VCPMD (Garvey et al. 2006). Currently, the only two large families classified as LGMD1A phenotype were found to have point mutations in myotilin gene, leading to single amino acid substitution Ser55Phe and Thr57Ile (Hauser et al. 2000; Hauser et al. 2002).

6.2. Myofibrillar myopathies (MFM)

Myofibrillar myopathies are a clinically and genetically heterogeneous group of disorders that have a common morphological phenotype. The term myofibrillar myopathy was proposed in 1996 as a broad term for a pathological pattern of myofibrillar dissolution, associated with the excess and ectopic accumulation of multiple proteins (Nakano et al. 1996, deBleecker et al. 1996a), that includes mainly desmin, myotilin, αB-crystallin, ZASP (Selcen et al. 2004), dystrophin, NCAM, and beta APP components (De Bleecker et al. 1996a; De Bleecker et al. 1996b), cyclin-dependent kinases and nuclear proteins (Nakano et al. 1997). Several mutations in desmin (Goldfarb et al. 1998), myotilin (Olive et al. 2005; Penisson-Besnier et al. 2006; Selcen and Engel 2004), αB-crystallin (Vicart et al. 1998), ZASP (Griggs et al. 2007; Selcen and Engel 2005) and filamin C (Kley et al. 2007; Vorgerd et al. 2005) have been shown to cause MFM. The disease is also named as desmin-related myopathy, desmin surplus myopathy disease, protein surplus myopathy or protein aggregate myopathy. However, it is unclear if proteins mutated in myofibrillar myopathy show increased amount in the skeletal or cardiac muscle of MFM patients. Currently, a study of MFM caused by desmin mutations showed that myotilin, desmin, and αB-crystallin genes are not overexpressed in the biopsies of these patients (Raju and Dalakas 2007). The disease is characterized by skeletal muscle weakness and variably associated with cardiomyopathy 37 and peripheral neuropathy. In addition, cataract has been reported in MFM caused by mutations in αB- crystallin (reviewed in Horwitz 2003). The clinical phenotype in both sporadically and familial diseases is heterogeneous. The pattern of inheritance in MFM is mainly autosomal dominant and the evolution is slowly progressive. Pathogenesis of the disease is not yet completly understood. It is suggested that myofibrillar desintegration that start at the Z-disk is the first step of the pathogenesis.

6.2.1. Myotilinopathy

Several reports show myotilin mutations cause MFM. Currently, point mutations are located mainly within largest myotilin exon 2 that lead to single amino acid substitution as follows: Ser39Phe (Foroud et al. 2005), Lys36Glu, Ser55Phe, Thr57Ile, Ser60Cys, Ser60Phe, Gln74Lys, Ser95Ile (Olive et al. 2005; Penisson-Besnier et al. 2006; Selcen and Engel 2004). Age of onset and the phenotype are variable. Typically, myotilinopathy is a late onset autosomal dominant disease with slow progression. Predominantly, distal muscle weakness that progresses to involve all limb muscles characterizes the disease. Decreased tendon reflexes, peripheral neuropathy, cardiomyopathy, and involvement of respiratory, facial and trunk muscles have been reported. The pattern of muscle weakness at onset may present as proximal, partially overlapping with LGMD1A phenotype. Of interest is Ser55Phe myotilin mutation as it is reported in phenotypes of both distal and proximal muscle weakness, and that it is a frequent finding in the cohort studies and in our unpublished observations.

6.2.2. Desminopathies

Point and deletion mutations in desmin gene are the molecular genetic defect of desminopathies. Desminopathy is a familial or sporadic disorder that manifests in second to fourth decade, with recessive forms manifesting earlier in life (Goldfarb et al. 1998). Clinically, it is characterized by initial predominant distal lower limb skeletal muscle weakness that progresses slowly without pain towards skeletal muscle atrophy. During the progression of the disease, the pattern of the weakness extends to proximal compartments, upper limbs, trunk and respiratory muscles. The second hallmark of desminopathy is the cardiac involvement manifesting as multiple forms of arrhythmias or true cardiomyopathy that can occur even in the absence of skeletal muscle manifestations (Goldfarb et al. 2004; Schröder et al. 2003). Serum creatine kinase levels are usually only slightly elevated or even normal and the electromyography show a myopathic pattern. Desminopathies are characterized by myopathic alteration of skeletal muscle, accumulations of desmin positive inclusions and myofibrillar abnormalities in the cardiac and skeletal muscle cells. Z-disk streaming and electron-dense granulofilamentous accumulations can be observed in electron microscopy. The desmin-reactive deposits are described as sarcoplasmic bodies, cytoplasmic bodies or spheroid bodies (reviewed by Goebel 1997) and they can be positive for several proteins, such as Z-disk proteins, components of the ubiquitin system, kinases and amyloid. In vitro studies suggest defects in mutant desmin assembly with subsequent loss of function or a toxic effect by accumulation of misfolded protein properly (Schröder et al. 2003).

38 6.2.3. Zaspopathy

Mutations in ZASP exons 4 and 6 (Ala147Thr and Ala165Val) are located adjacent and, respectively, within the ZM motif (Griggs et al. 2007; Selcen and Engel 2005). This is considered important in linking ZASP to the Z-disk by its interaction with α-actinin (Klaavuniemi et al. 2004). Another mutation found is located in exon 9, Arg268Cys (Selcen and Engel. 2005). The disease presents as an autosomal dominant late-onset distal myopathy, initially described by (Markesbery et al. 1974) Cardiac involvement and signs of peripheral neuropathy are reported in a fraction of patients. The pattern of weakness is found proximally and distally. However, distal weakness is predominantly found and is greater distally than proximally both in the Selcen and Engel 2005 and Griggs et al. 2007 studies. Muscle imaging shows that the distal posterior and anterior compartments are affected at the onset and proximal leg muscles are affected at later stages. ZASP expression was not altered and immunohistochemistry showed moderate accumulations of ZASP together with myotilin, desmin, αB-crystallin and α-actinin (Griggs et al. 2007).

7. Model organisms

Advances in understanding genetic disorders and better knowledge of their molecular basis can improve diagnostic, prognostic and genetic counselling. In addition, information on molecular basis of heterogeneous diseases, such as muscular dystrophies, allowed also their classification. For muscular dystrophies, naturally occuring and genetically engineered mouse models are important tools to study the pathogenesis and to develop novel treatment strategies. Palladin knockout mice (pall-/-) are embryonically lethal, as they show neural tube defects and herniation of liver and intestine. Cranial region of neural tube failure to close and defects in ventral closure are thought to be the direct effect of impaired actin cytoskeleton organization and remodelling. Palladin is earliest expressed at E8.5 only in neuronal tissues (rostral and caudal part of neural plate), and later at E9.5 and E10.5 it is expressed ubiquitously. It is thought that palladin is required for initiation of neurulation and normal embryonic development. In addition, palladin is thought to be the first actin cytoskeleton associated protein important for definitive erythropoiesis and erythroblastic island formation, and involved in achieving a normal function of liver macrophages (Liu et al. 2007). Increased apoptosis of erythropoietic cells in pall-/- embryos and abnormal liver macrophage-erythroblasts contacts were observed. However, the deficient erythropoiesis is thought to arise form inappropriate microenvironment consisting of functionally impaired macrophages (Liu et al. 2007). Isolated MEFs from pall-/- are valuable tools to study palladin roles in cell migration, spreading and adhesion. However, the model does not study separately the roles of each palladin isoform, as the pall-/- mice generated by Luo and coworkers (Luo et al. 2005) disrupt the very 3’ end of the palladin gene encoding the carboxy terminus three tandem IgI domains present in at least three palladin isoforms. While desmin knockout (des-/-) mice show normal primary muscle formation, defects appear after birth in skeletal, smooth, and cardiac muscles (Li et al. 1996; Li et al. 1997). The muscle fibers are more susceptible to damage during contraction followed by an aberrant secondary myofibrillogenesis (Li et al. 1997; Milner et al. 1996). Studies of des-/- mouse hearts supported the hypothesis that the desmin filaments do not contribute the passive elasticity of the ventricle wall but rather contribute to the active 39 force generation by supporting sarcomere alignment and force transmission (Balogh et al. 2002). In addition, studies on the des-/- mice showed that desmin might be involved in the distribution of mitochondria, with subsequent consequences for oxidative metabolism. Des-/- mice provide a model for genetic cardiomyopathy where the main factor contributing to impaired cardiac performance is an altered structure and a loss of functional contractile units. In addition, a transgenic mouse that expresses a desmin (D7-des) mutation linked to desmin-related myopathy show intrasarcoplasmic and electron-dense granular filamentous aggregates. In transgenic desmin hearts, desmin filament network and myofibril alignment was disrupted. Functionally, the heart did not respond properly to β-agonist stimulation (Wang et al. 2001). A transgenic mouse expressing Thr57Ile mutated myotilin develop progressive myofibrillar pathology consisting of Z-disk streaming, excess myofibrillar vacuolization and plaque-like myofibrillar aggregation (Garvey et al. 2006). Skeletal muscle functional impairment correlated with the aggregates that are positive for mutated myotilin. It is suggested that mutated myotilin nucleate myofibrillar aggregation by promoting multiple and tight protein-protein interactions. Apparently, myotilin mutations do not render the muscle fiber sensitive to contraction-induced injury, but rather directly impair force production and transmission (Garvey et al. 2006).

40

AIMS OF THE STUDY

The aim of the study was to understand the functions of related proteins myotilin and palladin. Specific aims were: - To identify further the binding partners of 90-92 kDa palladin isoform and determine its role in modeling of the actin cytoskeleton - To investigate the role of myotilin in vivo by generation of myotilin knockout mouse - To characterize myotilin in a model overstretching, delayed onset of muscle soreness (DOMS) - To investigate in vivo the overlapping roles of myotilin and 200 kDa isoform of palladin by generating a myotilin-palladin double mutant mouse.

41 MATERIALS AND METHODS

The materials and techniques used in this thesis are described in detail in the original articles (I-V) and are listed as follows: Production of monoclonal and polyclonal antibodies II, III, IV, V Tissue processing for immunofluorescence, histology and histochemistry IV, V Histochemistry IV, V Immunofluorescence on cells and tissues I, II, III, IV Immunofluorescence on semithin cryosections III Transmission electron microscopy IV, V Production of recombinant DNA constructs I, II Recombinant protein production and purification I, II Cell culture and transfections I, II Surface plasmon resonance experiments and interpretation I Embryonic stem cells culture IV, V Genomic DNA purification and PCR IV, V RNA isolation and RT-PCR IV, V Western Blotting I, II, III, IV Southern Blotting IV, V Northern Blotting IV, V Grip strength testing of laboratory animals IV, V Evand Blue membrane permeability testing IV, V Eccentric exercise and muscle biopsies III

42 RESULTS AND DISCUSSION Palladin is a binding partner for profilin (I) Palladin gene has a complex structure and is transcribed as multiple palladin transcripts. Most expressed palladin isoforms studied have as common feature three carboxy terminal Igl domains. The first characterized palladin isoform is generated by the most 3’ of the three palladin gene promoters (Mykkänen et al. 2001; Parast and Otey 2000). This generates a protein with the apparent molecular weight of 90-92 kDa that appears as a dublet in Westen blots. In tissues of developing rodents, this isoform is currently considered the most abundant form of palladin. The amino terminus of human palladin contains two conserved polyproline stretches, the first located at amino acids 75-85 and the second at 186-220. These consist of a minimum five consecutive prolines and no bulky side chains, including FPPPP sequence. The polyproline sequences are binding regions for profilins, key molecules regulating actin dynamics. Coprecipitation experiments using GST-profilin I and lysates of Swiss 3T3 fibroblasts, HeLa cells and rat astrocytes identified palladin as a binding partner for profilin. Along with palladin, the GST- profilin pulled down its known binding partners, VASP and actin, suggestive of an interaction, either direct or via a complex (I, Fig. 2A). To verify that binding of palladin to GST-profilin is direct and not mediated via co-pelleted proteins, the interaction was further studied by blot overlay. In the assay, myc-tagged palladin was immunoprecipitated from COS 7 cells, resolved by SDS-PAGE and transferred to nitrocellulose filter. The filter was incubated with purified bovine profilin I. The direct binding of profilin on purified palladin was demonstrated using immunoblotting with anti-profilin antibody (I, Fig. 2D). Further, direct binding of purified profilin I was demonstrated by incubation with purified His- tagged mouse palladin bound to beads. The profilin I was detected by immunoblotting (I, Fig. 2C). Fine mapping of the region in palladin that binds profilin was performed using purified protein fragments. Incubation of GST-sepharose-bound GST-tagged human palladin fragments with human carcinoma cell lysate showed specific interaction of human GST-palladin with profilin. In these experiments, profilin interacted with GST-tagged palladin fragments 8-228 and 101-301, but not with GST alone or human GST-palladin 8-173 and 222-387 (I, Fig 2D), indicating that profilin binds to palladin via a sequence that contains the second polyproline stretch. This profilin-binding region lies between 186-220 in human palladin polypeptide, while the most amino terminal located polyproline stretch within amino acids 75-85 does not participate in the binding. To study the direct interaction and its kinetics, binding of palladin and profilin was investigated using BiaCore SPR technology. Amino terminal GST-tagged recombinant palladin fragments were purified and immobilised to the sensor chips. Subsequently, purified profilin I was run over the surfaces on which GST-palladin was immobilized. In Biacore analysis, profilin I interacted with GST-palladin 101-301, but not GST-palladin 222-387 or GST alone (I, Fig. 3). A minor response was obtained using GST-palladin 8-173 as ligand, however, the significance of this respose remains unclear. When compared with the first polyproline stretch in palladin, it is worth noting that the second polyproline stretch consists

43 of larger number of prolines, apparently of two close polyproline stretches (I, Fig. 1). The dynamics of the interaction were studied using gradually increasing concentrations of analyte. The resulting binding curves showed that GST-palladin 101-301 interacted with profilin I in a concentration-dependent fashion, however the saturation was not reached even with a profilin concentration of 320 μM (I, Fig. 3). This did not allow the calculation of the KD, however the features of the interaction, response units obtained and binding curves, allowed conclusion that the interaction is highly dynamic and of low affinity. The features of the interaction resembled the characteristics of the interaction between penta-proline peptides derived from VASP and profilin I, suggesting that VASP and palladin could bind profilin in a similar fashion. In addition to data obtained in vitro, palladin and profilin colocalization studies were performed in vivo, on astrocytes and on Swiss 3T3 fibroblasts. To determine the cellular localization of palladin and profilin, astrocytes were double labeled with palladin and profilin antibodies and examined by fluorescence microscopy. In addition, Swiss 3T3 fibroblasts were transiently transfected with GFP- profilin and stained for palladin. Immunofluorescence microscopic analysis in the cells demonstrated partial overlapping pattern, especially in the actin-rich bundles near cell edges (I, Fig. 4). Profilin is a key protein that controls actin dynamics that is known to bind G-actin and polyproline containing proteins. As profilin is thought to integrate signals that lead to the modulation of actin microfilament growth, the binding of palladin to profilin shows that palladin is an important protein implicated in this process. Palladin has been shown to bind scaffold molecules, such as F-actin (Dixon et al. 2008) and α-actinin (Rönty et al. 2004). It has also been shown to bind signaling molecules, such as EGFR substrate Eps8 that links growth factor stimulation to actin dynamics (Goichoecea et al. 2006). Another partner is Src tyrosine kinase, a well known cytoskeletal remodelling molecule (Rönty et al. 2007). Our studies show that palladin interacts and colocalize with profilin in cells. This interaction might represent the link between the extracellular stimuli to the growth control of actin filaments in specialized regions of the cells, such as dorsal ruffles, lamellipodia and podosomes. In addition, it serves as a docking site for various signaling molecules that determine the fashion of actin microfilament assembly and, as a result, cells migration in response to extracellular stimuli.

Palladin is a binding partner for α-actinin (II) Myotilin was originally identified in a Y2H screening as an α-actinin binding protein (Salmikangas et al. 1999). In myotilin, the α-actinin binding site consists of a small region within the unique amino terminus of the protein (Taivainen et al. unpublished). Myopalladin also binds α-actinin, but the binding site was mapped to the carboxy terminus of the myopalladin, requiring all three Igl domains (Bang et al. 2001). The original report of mouse palladin also demonstrated an association with α-actinin, but this interaction was not further studied (Parast et al. 2000). To explore the binding properties of palladin to α-actinin, several approaches were used. Several Y2H palladin bait constructs were constructed and cotransformed with α-actinin prey constructs. A short fragment, encompassing palladin amino acids 222-280 showed binding to the α-actinin, while none of the

44 constructs lacking it showed binding (II, Fig. 1A). Using Y2H metod, the palladin binding site on α- actinin was mapped to the EF-hands (II, Fig. 1B). Confirmation of the results was done using affinity precipitation assays. Several GST-sepharose bound GST-α-actinin fragments were incubated with labelled in vitro translated HA- or Myc-tagged palladin fragments. The binding of EF hands 3-4 of α-actinin of palladin fragments 8-772 and 101-280, but not to 389-772 was seen (II, Fig. 2). Protein sequence alignment of the palladin fragment 222-280 and myotilin allowed detection of a highly homologous region of 17 amino acids, with 64% identity and 94% similarity (II, Fig. 3). This small region in myotilin is located in the α-actinin-binding region of myotilin. To study the interaction in vivo, cell transfection analyses were used. When transfected in COS 7 cells, GFP-palladin fragments 8-772, 8-387, 101-387 and N-alt-269-715 induced formation of actin bundles (II, Fig. 4). N-alt-269-715 is a palladin variant consisting of a unique amino terminus of other palladin isoform that is fused to the amino acid 269 of the sequence of the 90-92kDa palladin, that did not bind to α-actinin in Y2H (II, Fig. 1). When transfected in COS 7 cells, N-alt-269-715 induces the formation of actin aggregates (II, Fig. 5). In COS 7 cells transfected with GFP-palladin 8-772, α-actinin was targeted to the actin bundles while the transfection of N-alt-269-715 construct did not result in targeting of α-actinin to the newly formed aggregates (II, Fig. 5). This suggested that interaction with α- actinin plays a role in targeting of palladin. To demonstrate a direct association of palladin that is not due to indirect association to actin binding to α-actinin, transfection studies using a mitochondrial outer membrane (MOM) targeting strategy was used. MOM-tagged palladin 8-387 and 222-280 were cotransfected with GFP-α-actinin lacking the actin-binding domain (R1-R4EF) in CHO cells. The GFP-α-actinin R1-R4EF construct is retained in the cytoplasm and leads to the destruction of actin cytoskeleton when transfected alone. When cotransfected with the palladin constructs, the recruitment of GFP-α-actinin R1-R4EF to the mitochondria was observed (II, Fig. 6). This suggests that palladin can target α-actinin to various sites or structure in the CHO cells. The high homology of the α-actinin binding sequence in myotilin and palladin could suggest that the fashion of interaction is similar. Myotilin and α-actinin binding is a high affinity binding (KD = 6 nM) (Taivainen et al, unpublished). The sequence similarity between α-actinin binding sites of myotilin and palladin and, in addition, the remarkable ability of palladin to target α-actinin in cells could indicate a similar high affinity interaction of palladin with α-actinin. α-Actinin is an important cytoskeletal associated protein and in striated muscle cells is considered to be the bona-fide component of Z-disks. α-Actinin is an ubiquitously expressed protein that cross-links actin filaments and it is overexpressed in invasive tumor cells (Gluck and Ben-Ze’ev 1994). It plays important roles in cell-matrix and cell-cell adhesion (reviewed in Otey and Carpén 2004). In brain, where both palladin and α-actinin are highly expressed in neuronal cells and astrocytes, their interaction could serve as a platform for both physiological and pathological processes and in support of this observation, palladin was upregulated in glial scar formation (Boukhelifa et al. 2003). In addition, interaction of

45 palladin with α-actinin and their partial colocalization support the hypothesis that they function together to achieve normal cellular motility and play important role in adhesion. The interaction of palladin with α-actinin may also play a role in integration of signals from outside the cell and may be implicated in the migration of cancer cells.

Absence of myotilin in mice does not disrupt morphology and function of striated muscle (III) Transgenic and knockout mice models have provided important information on several muscle proteins. They have allowed investigation on the consequences of protein absence, overexpression or mutations that form basis of muscular disorders both in animals and in humans. We addressed the question of myotilin’s role in vivo by generation of a knockout mouse, a myotilin null mouse, myo–/–. This was achieved by deletion of exon 3 of the murine myotilin gene, using a targeted deletion strategy consisting of in vivo Cre-loxP-mediated deletion or "floxing out" of the exon 3. After the targeting of exon 3 by homologous recombination, ES cells were used to generate floxed mice (III, Fig. 1). In vivo expression of the Cre recombinase was done by breeding the floxed mice with the Cre-expressing mice, and further breeding generated different genotypes (myo–/–, myo+/–, and myo+/+) that were obtained at the expected Mendelian frequency. At birth, the myo–/– mice were viable and indistinguishable by appearance, behavior (e.g., milk suckling, spontaneous movements), or average birth weight from their myo+/– or myo+/+ littermates (III, Fig. 5A). Our results suggest that during embryogenesis myotilin does not have a crucial role in spite of widespread expression pattern during mouse development. On the other hand myotilin role could be compensated by other homologous proteins, such as palladin or myopalladin. In the adult wild-type mouse, expression studies showed that myotilin is highly expressed in the skeletal and cardiac muscle (Mologni et al. 2005). Investigation of myotilin transcripts in adult mouse tissues by RT-PCR provided the information that mutant myotilin gene is transcribed, however, the absence of myotilin transcript by Northern Blot analysis suggested its degradation (III, Fig. 2). Protein investigation in the skeletal muscle and heart by Western Blot analysis using anti- myotilin antibodies detected a 57 kDa band in tissues of myo+/– or myo+/+ but not in myo-/- mice. This indicated an efficient Cre-mediated deletion of the exon 3 of murine myotilin gene that results in the absence of endogenous myotilin protein from the tissues where myotilin is expressed, the heart and the skeletal muscle (III, Fig. 3A). Adult mouse organs investigated were those, in which myotilin mRNA was detected, such as skeletal muscle, heart, brain and liver. The morphological analysis showed preserved architecture of both adult myo-/- mouse organs and of mouse embryos. Tissue analysis did not reveal any difference between genotypes (III, Fig. 4). The born myo-/- mice were able to develop and sustain their respiratory function, and their post-natal development proceeded in a similar fashion to that of their myo+/+ or myo+/– littermates. Functional evaluation of skeletal muscle function of adult mice using as an indicator the peak force measurement developed by the forelimbs of mice showed similar strength for myo-/- mice when compared with wild type or heterozygous mice (III, Fig. 5C). In addition the voluntary running of the myo-/- mice was similar with the myo+/+ mice suggesting that normal maximal contractile function is preserved in spite of myotilin loss (III, Fig. 5D). 46 The maintenance of a morphologically normal Z-disk and overall cellular architecture and preserved muscle performance of myo-/- animals confirmed that lack of myotilin does not lead to a dystrophic phenotype. This indicates that myotilin is not required for normal striated muscle structure and gross function. Together with the results of Garvey and coworkers (Garvey et al. 2006) showing that transgenic expression of a mutant myotilin leads to morphological alterations observed in myotilinopathies, mutated myotilin appears to have a toxic effect on the structure of the skeletal muscle, whereas myopathy due to myotilin deficiency has not been reported. In addition to the hypothesis that the subfamily members might compensate for myotilin function, our studies indicate that myotilin might also have other than structural functions. This originates form our finding that in myo-/- mice telethonin is upregulated. The complex formed by telethonin/T-cap together with the Z-disk amino terminus of titin and minK is proposed to be a part of a "mechano-electrical feedback" that links mechanical stretch with membrane potentials. In addition, myotilin also binds FATZ, that in turn binds the protein phosphatase calcineurin. While our studies in basal conditions showed largely preserved skeletal muscle architecture and function, telethonin upregulation indicate that myotilin could be a potential component of a stretch transmission mechanism in the skeletal muscle and that challenging conditions might provide a further insight for myotilin functions.

Myotilin in myofibrillar remodelling (IV) Myofibrillar disorganization appearing as Z-disk streaming, Z-disk smearing and Z-disk disruption describe morphology of Z-disk for a part of muscular disorders, including MFM. In the biopsies of patients with MFM caused by myotilin mutations, are observed myofibrillar destruction, electron-dense material of Z-disk origin and Z-disk streaming. Myotilin positive aggregates are suggested to be the most reliable immunocytochemical marker for MFM diagnosis (Selcen et al. 2004) The Z-disk streaming and disruption, or absence of Z-disk components are also the morphological hallmarks of delayed onset muscle soreness (DOMS), a model of overstretching of skeletal myofibers. DOMS appears after eccentric exercise and provides a good model to study the molecular changes during the skeletal muscle remodelling. It is a dynamic process leading to the formation of new sarcomeres and provides the protection when a second bout of exercise is performed. Studies have shown the distribution of major proteins, such as actin, α-actinin, titin and desmin is modified in the regions of sarcomerogenesis. Actin and desmin are found in greater amounts and distributed differently from a normal muscle. On the other hand, α-actinin, nebulin and titin are initially lacking in focal areas and reappearing subsequently (Yu et al. 2003). To study myotilin modifications/behaviour in this model, we have studied the relationship between staining pattern of myotilin and actin, alpha-actinin, desmin and myosin by a semithin cryosectioning technique and immunofluorescent labelling of proteins (III). By staining normal human skeletal muscle with anti-myotilin 151 polyclonal antibody we observed Z-disk localization of myotilin. As a novel finding, double immunofluorescent staining for myosin and myotilin revealed that myotilin staining of Z-disks was more intense in the type I muscle fibers, while type II fibers were moderately stained. In addition, a very low intensity myotilin staining

47 was detected at the M-line (IV, Fig. 1). In fibers affected by the eccentric exercise, myotilin staining was found to be increased in focal areas, such as dots, broad bands, plaques and masses. The extent of the modification varied from a single Z-disk affected to large lesions composed of several sarcomeres (IV, Fig. 3). Increased myotilin staining did not correlate with that of desmin (IV, Fig. 2) or α-actinin (IV, Fig. 4, 5), suggesting that desmin modifications may occur independently from actin or myotilin redistribution. However, myotilin strongly correlated with actin, detected by phalloidin staining which labels F-actin (IV, Fig. 3). Myotilin was found in broad Z-disks that in several occasions show less staining for α-actinin. The study showed myotilin as a prominent marker for myofibrillar remodelling caused by eccentric exercise, process characterized by sarcomerogenesis and lengthening of the myofibrils. In this process, myotilin may play important role by recruiting G-actin and organizing the actin filaments to the newly formed sarcomeres, or may have yet to be characterized binding partners and thus novel functions.

Myotilin and palladin deficiency leads to structural and functional changes of striated muscle (V) We specifically disrupted the palladin 200 kDa isoform by replacing a part of palladin exon 4 and the fourth intron by an IRES-β-galactosidase neomycin selection cassette. Using common procedures for generation of knockout mouse strains from targeted ES cells, we obtained homozygous (200pallhypo/hypo) mice for disrupted palladin gene. Using the galactosidase casette within the palladin gene, the investigation of 200pallhypo/hypo mouse embryos indicated that 200 kDa palladin is expressed in embryonic heart and somites (V, Fig. 1E). Upon analysis, 200pallhypo/hypo mice were indistinguishable from their wild-type littermates, were fertile, and did not develop any overt phenotype. In Northern Blots of heart and skeletal muscle, the mRNA of the palladin 200 kDa isoform appeared as a very large band in the wild-type and a considerably weaker signal was detected in 200pallhypo/hypo. Interestingly, the very low amount of the 200 kDa palladin isoform did not apparently modify the myopalladin expression in heart or skeletal muscle. A significant upregulation of myotilin transcript was observed in skeletal muscle but not in the heart of 200pallhypo/hypo mice (V, Fig. 2A). Analysis of palladin protein in the striated muscle revealed that homozygous mice for the targeted palladin allele express a little amount of a protein band with an apparent size of 200 kDa, and therefore the mice are hypomorph (V, Fig. 2B). Sequencing RT-PCR products revealed an internal splicing event at the border between exon 4 and the β-galactosidase selection cassette producing at least one in-frame transcript. The splicing event resulted in a 10 amino acids shortened product that was expressed as 10% of the total quantity of 200 kDa palladin in wild-type animals (V, Fig. 2C). In contrast to the 140 kDa palladin, expressed early in skeletal muscle progenitor cells, the 200 kDa isoform appers to be expressed late in the differentiation process (Wang et al. 2008; V). Compared with wild-type mice, E11.5 200pallhypo/hypo embryos consistently showed a stronger MyoD and a weaker myogenin signal (V, Fig. 3A). These results suggest that the mutant mice contained more undifferentiated

48 myoblasts than wild-type embryos. In addition, Western blot analysis of primary fibroblasts from wild- type and 200pallhypo/hypo mice showed a reduction of 40-50% of the140 kDa isoform (V, Fig. 3B). Thus, the targeting strategy also influences the expression of the 140 kDa palladin isoform, which most likely causes the differentiation delay of early myoblasts during embryogenesis. In spite of the delay in myogenesis, 200pallhypo/hypo mice were born in a normal Mendelian ratio and investigation of skeletal muscle and heart morphology at light microscopic level showed no obvious alterations (V, Fig. 4A, 5B, 5C). Western blot analyses of proteins did not show an amount difference of the myotilin and telethonin (V, Fig. 7). Transmission electron microscopy of hearts at 2-5 months of age 200pallhypo/hypo mice showed frequently subsarcolemmal vacuoles, that we consider as absent or dissolved myofibrils, a finding never observed in control samples from wild-type mice at similar age (V, Fig. 4C, 6A). Such finding was never observed in skeletal muscle samples from 200pallhypo/hypo mice of different ages. To correlate our finding with a putative marker for cardiac hypertrophy, investigation of CARP protein was used. In 200pallhypo/hypo mouse hearts, CARP levels were increased, whereas no change was detected in skeletal muscle (V, Fig. 4D). The absence or mild phenotypes of myo-/- and 200pallhypo/hypo mice provided us with the hypothesis that the 200 kDa palladin and myotilin could functionally compensate for each other. This is also based on a high similarity of their carboxy termini and their common ligands, e.g. actin and α- actinin. To test the hypothesis, we generated a strain of mice that lack myotilin and express low levels of a truncated 200 kDa palladin, the myo-/-200pallhypo/hypo mice. Mice were obtained successfully, were born at normal ratio and apparently developed similarly with no obvious defect in postnatal development, posture or motor activity. Investigation of skeletal muscle morphology of the myo-/-200pallhypo/hypo mice showed preserved skeletal muscle cell architecture up to 6-8 months of age. However, around 11 months of age, they develop morphological changes consistent with a myopathic phenotype. Compared with wild-type age- matched control mice, several skeletal muscles of the myo-/-200pallhypo/hypo mice exhibit variation in fiber size and increased type II fiber diameter. The size difference is observed for the type II fiber types as determined by NADH-TR (V, Fig 5C) or ATPase stainings (data not shown). In several occasions, skeletal muscles of myo-/-200pallhypo/hypo mice presented with internalized nuclei, a marker of myopathy. However, Gomori trichrome staining did not show increased connective tissue amount in skeletal muscle or heart, supporting the myopathic phenotype rather than dystrophic one (V, Fig. 5D). Moreover, in vivo testing for increased permeability of skeletal muscle fibers using Evans blue dye was not observed in myo- /-200pallhypo/hypo mice, suggesting that increased membrane permeability or necrosis does not contribute to the phenotype of the mice (data not shown). The most obvious morphological alterations were observed in gastrocnemius and quadriceps muscles and, to a lower extent, in tibialis anterior muscle. Transmission electron microscopy on ultrathin transversal sections of heart specimens at 4 months of age shows moderate ultrastructural modifications in 200pallhypo/hypo mice. Alternatively, in addition to apparently normal ventricular myocytes, in myo-/-200pallhypo/hypo mice we found severely disrupted architecture of myofibrils, particularly in the vicinity of cellular borders, either at the 49 intercalated disks or under the sarcolemma (V, Fig 6A). While obvious morphological alterations in routine histological stainings appeared relatively late, early evidence of skeletal muscle modifications was provided by electron microscopic investigations. Earliest modifications observed in 200pallhypo/hypo mice were at the age of 4 months and consisted of the frequent interruption of the Z-disk architecture by tubular structures (V, Fig. 6B). At the age of 11 months, myo-/-200pallhypo/hypo mice showed marked alteration in the alignment of myofibrils, Z-disk fuzziness and accumulation of granular material within the fibers of quadriceps muscle. In the medial region of gastrocnemius, we observed the frequent replacement of Z- disks either by the tubular structures or by granular structures (V, Fig. 6B). Western blot examination of telethonin protein showed similarly increased telethonin levels in the skeletal muscle and heart of myo-/-200pallhypo/hypo mice as in myo-/-200pall+/+ mice. This suggests that while myotilin and 200 kDa palladin share similarities, they could play different roles regarding the functional link to telethonin. Analysis of sarcomeric α-actinin did not show increased levels in striated muscles of myo-/-200pallhypo/hypo. There was no modification in the protein levels of FATZ-1, while about 2-fold increase in ZASP and Ankrd2 protein levels were found in tibialis anterior muscles of myo-/- and myo-/-200pallhypo/hypo mice. In addition, protein analysis of skeletal muscle confirmed the absence of myotilin and the reduced levels of 200 kDa palladin isoform in myo-/-200pallhypo/hypo mice (V, Fig. 7). The upregulation of the two MARP family members, CARP in heart tissues of 200pallhypo/hypo and myo-/- 200pallhypo/hypo mice and Ankrd2 in skeletal muscles of myo-/-200pallhypo/hypo, indicate that both myotilin and 200 kDa palladin might be components of similar stretch response pathways, although in different cell types. Characterization of the functional consequences of myotilin absence and dramatically reduced 200 kDa palladin was done using strength measurements. Forelimb peak force measurements of the myo-/- 200pallhypo/hypo mice showed significant reduction when compared with 200pallhypo/hypo or wild-type mice (V, Fig. 8). It is intriguing however that a functional deficit was observed before the detection of the skeletal muscle structural alterations. Therefore we suggest that myotilin and 200 kDa palladin play an important role in defining the contraction properties of the skeletal muscle that cannot be compensated at least by the third subfamily member, myopalladin. To date, only one study describes association of palladin with cardiac diseases (Horne et al. 2007), while a link of palladin gene modifications with striated skeletal muscle disorders have not been described. Together, the expression pattern of the 200 kDa palladin in striated muscle cells in early development and the cardiac phenotype observed in adult 200pallhypo/hypo mice, indicates that 200 kDa palladin is important for the maintenance of normal sarcomeric architecture of heart muscle cells. In spite the architectural modifications, mice seem to have a normal heart/body weight ratio (personal unpublished observation) and in this context a challenging scenario might provide additional answers regarding role of this protein in maintenance of functional parameters and/or architecture. While defects in heart architecture seems to originate from the reduced levels of 200 kDa palladin, the ultrastructure of myo-/-200pallhypo/hypo mice hearts appeared slightly more disorganized when compared with 200pallhypo/hypo mice. This indicates that in heart, myotilin might play a secondary role to the maintenance of normal cardiomyocyte architecture, while the 200 kDa palladin seems to have a more important role. When the 50 morphology and function were investigated, we found that concomitant loss of myotilin and reduced 200 kDa palladin in myo-/-200pallhypo/hypo mice lead to significant skeletal muscle alteration. The mice showed not only the ultrastructural alteration of myofibrils in cardiomyocytes, but in addition we found a relative late-onset structural and functional impairment of skeletal muscle. Taken together, our findings suggest that palladin 200 kDa isoform plays a unique role in myofibrillar stability in cardiomyocytes, that cannot be fully compensated by myotilin nor myopalladin. In skeletal muscle, both myotilin and 200 kDa palladin play important and overlapping roles in the maintenance of skeletal muscle myofibrillar architecture and their ablation cannot be compensated by myopalladin. In the future, many open questions regarding the functions of this small subfamily of proteins could be answered by generation of the palladin 200 kDa isoform/myotilin/myopalladin triple knockout mice. It would be interesting to generate the palladin 200 kDa isoform/myotilin/myopalladin triple knockout mice to elucidate the completely role of the newly found protein subfamily on striated muscle biology.

51

CONCLUSIONS AND FUTURE PROSPECTS

This study describes further investigation of myotilin and palladin, that together with myopalladin belong to a small subfamily of proteins. Structurally, the members of the subfamily contain Igl domains, and, all of them contain two or three Igl at the carboxy terminus. In addition, others have a variable number of Igl domains also at their amino terminus. Recently, the Igl domains have been proposed as a binding module for actin, and it has been shown that both myotilin and palladin bind and bundle actin via their carboxy terminal Igl domains (von Nandelstadh et al. 2005; Dixon et al. 2008). In the subfamily, only palladin contains two polyproline rich regions that mediate the interaction with a key protein involved in actin dynamics, profilin. Palladin is known to bind and cross-link F-actin into stable actin bundles in stress fibers or lamellipodia in a similar fashion as myotilin, using its Igl domains. Here, palladin acts as a stabilizer of these actin-rich structures. Its direct interaction with profilin might serve as a docking site for profilin:actin complexes and in this fashion contributing to the addition of new G-actin monomers to the filament resulting in controlled growth of the filament and its structural stabilization. Palladin-profilin interaction is very rapid, suggesting that palladin is implicated in organization of actin filamentns in very dynamic situations and of importance in highly motile cells. As another palladin-binding protein we found α-actinin. The proteins are targeting each other in various sites of the cell, but not always fully colocalize, indicating that they have other binding partners and they may serve independent functions. When they colocalize, it happens also in the context of actin- rich structures. In the light that α-actinin serves as a link between the cytoskeleton to transmembrane proteins and as a scaffold for signalling molecules, and, further, that palladin itself binds both scaffolding and signalling molecules, palladin might serve as a docking site for molecules that modulate the organization of actin structures via the α-actinin. Following eccentric exercise, the ultrastucture and functional parameters of a skeletal muscle fiber changes (Thompson et al. 1999). New sarcomeres are formed and DOMS does not appear when a second bout of exercise is done within several days (McHugh et al.2003). It is thought that DOMS is not a destructive process, but rather a remodelling process, the aspect at which detailed aspects are not yet known. In this process, myotilin seems to play an important role, as it is found in the regions of increased F-actin content. This actin is not rigorously organized, rather it is the site where Z-disk components reorganize to form new Z-disks and further, new sarcomeres. In these regions, the staining of the α- actinin at the Z-disk is decreased in intensity and does not colocalize with myotilin. Our studies showed that myotilin is strongly associated with F-actin and myotilin might provide the stability of these F-actin structures. On the other hand, either α-actinin and myotilin might serve as template for the organization of new sarcomeres by recruiting new proteins at a future Z-disk site. In these structures, myotilin is probably replacing α-actinin and, similarly as α-actinin, might be an anchoring site for signalling or scaffolding molecules that are recruited to form the new sarcomeres. Alternatively, myotilin might primarily organize and temporarily stabilize the actin filaments allowing for Z-disk to be assembled. As

52 the assembly of Z-disk advances, myotilin might contribute to a final process of structurally locking the Z-disk in its mature form. It would be rather interesting to know what are the binding partners of myotilin in these situations and if myotilin serves as a template for the Z-disk by promoting a rather rigid F-actin cross-linking. The myotilin knockout study provided essential information on myotilin. Myotilin, although widely expressed in embryonic stages, is dispensable for the mouse embryogenesis, as myotilin knockout mice were born and developed subsequently without any obvious defect. It is possible that other proteins, especially the proteins from the palladin/myopalladin family might functionally compensate for myotilin absence, in spite of rather unchanged 200 kDa palladin and myopalladin expression. Morphological and functional analyses in non-challenging condition showed that myotilin is dispensable for a normal striated muscle function. Together with the information that transgenic expression of mutant Thr57Ile myotilin in mice leads to skeletal muscle morphologic alteration (Garvey et al. 2006), we conclude that possibly the pathogenesis mechanism is linked to a toxic effect of mutant myotilin on striated muscle cells. We further generated a mouse that does not express myotilin and in addition has very low expression of the 200 kDa palladin, the latter as being the major isoform of palladin expressed in striated muscle and localizing at the Z-disk. While the 200 kDa palladin shows ultrastructural modifications of the heart architecture, we demonstrate that combined deficiency of myotilin and low levels of 200 kDa palladin lead to morphological and functional alterations of skeletal muscle. The 200 kDa palladin isoform is highly expressed in heart and has an important role in the maintenance of normal architecture of the cardiomyocytes and in addition, myotilin deficiency exacerbates the cardiomyocyte defects. In the skeletal muscle, we showed that both architecture and function is affected when myotilin is absent and 200 kDa palladin is expressed at low levels. This indicates that in skeletal muscle myotilin and 200 kDa palladin might have a similar function, while in the heart their functions might be different. While expression of myotilin and 200 kDa palladin and many binding partners are characterized, their common or unique roles both in the maintenance and the function of skeletal muscle need further studies. Myotilin and palladin association with the cytoskeleton still awaits investigation in, for example, challenging conditions. In addition, to investigate the roles of the subfamily of proteins a triple palladin 200 kDa isoform/myotilin/myopalladin knockout might be useful to provide the answer if proteins have similar or divergent roles in the development or in the functional adaptation of skeletal muscle. Alhough knowledge on striated muscle biology and physiology has widened considerably, the pathogenic mechanism and a specific treatment in myofibrillar myopathies are yet open questions that require further research.

53 ACKNOWLEDGEMENTS

The research for this thesis was carried out at the Neuroscience Program and Molecular Neurology, Department of Pathology, University of Helsinki in Biomedicum Helsinki, during years 2002-2008.

I would like to thank my supervisor Professor Olli Carpén for providing the opportunity to work in a high-class laboratory and also for introducing me to the wonderful field of pathology. I have greatly benefited from his guidance, support and continuous trust, all these in an inspiring environment. I would like to also thank Professor Veli-Pekka Lehto, Professor Anu Wartiovaara, and Professor Olli Jänne for excellent working facilities.

I would like to thank Docent Anders Paetau and Professor Hannu Kalimo for interesting discussions on the topic of muscular pathology. Acknowledgements go to my thesis reviewers, Professor Hannu Kalimo and Professor Jari Ylänne, for their careful review and useful comments that added value to my thesis.

I acknowledge my Helsinki Biomedical Graduate School follow-up thesis committee Professor Ismo Virtanen and Docent Päivi Miettinen for their constructive comments and encouraging me to put my work together.

Luca Mologni is greatly acknowledged for his avant-garde myotilin work, for sharing his experience and knowledge and also for his friendship. Special thanks go also to Dr. Mikko Rönty. I warmly acknowledge the fruitful collaborations abroad with Professor Carol Otey, Dr. Malika Boukhelifa and the lab members for palladin work, Professor Roger Karlsson and Dr. Thomas Johansson for collaboration on profilin work, Professor Lars-Eric Thornell, Dr. Lena Carsson and Dr. Ji-Guo Yu on myotilin work and Professor Markus Moser and Dr. Hao-Ven Wang for mutant mouse work. In addition, Dr. Ras Trokovic is also acknowledged.

My research adventures go back to Tampere where I made my first steps into the scientific world, for which I am grateful to Professor Olli Silvennoinen and Dr. Daniela Ungureanu.

I thank the current and former members of the Ollilab Dr. Kaija Alfthan, Angela Flint-Oilinki, Dr. Miku Grönholm, Leena Heiska, Dr. Maciej Lalowski, Minja Laulajainen, Dr. Masha Melikova, Dr. Taru Muranen, Olli-Matti Mykkänen, Nilla von Nandelstadh, Dr. Suvi Natunen, Dr. Paula Salmikangas, Heli Suila, Anu Taivainen, and Dr. Fang Zhao for help in everyday matters and for interesting discussions. Helena Ahola, Anu Ahlroos-Lehmus and Tuula Halmesvaara are thanked for helping with the daily routine work. Warm thanks go to Ana-Maria, Henna, Ilse, Alessandro, Lilja, Usama, Alex, Sofia, Nuno and Kati for sharing their experience and for the rush time coffee break chats.

I have been financially supported by grants from the Academy of Finland, the Sigrid Juselius

54 Foundation, Biomedicum Helsinki Foundation, Association Française contre les Myopathies, the Oskar Öflund Foundation, the Finnish Cultural Fund, the Heart Foundation, the Finnish Medical Association, the Aarne Koskelo Foundation, the Maire Taponen Foundation, the Helsinki Biomedical Graduate School, the Ida Montin Foundation, the Paulo Foundation, the Helsinki University Chancellor´s Fund, the University of Helsinki Funds, the Maud Kuistila Memorial Foundation, Paavo Nurmi Foundation and Helsinki Graduate School which are sincerely acknowledged.

I also thank my mother for her love and unconditional support in whatever choices I make in my life. And last but not least, I thank Mihai for his love, his continuous support during all these years, helping in all matters from science to raw work and for cheering up every one of our life’s mornings.

Helsinki, October 2008 Monica Moza

55

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