The gamma-smooth muscle actin isoform in smooth muscle and smooth muscle-like cells

Thesis

Reference

The gamma-smooth muscle actin isoform in smooth muscle and smooth muscle-like cells

ARNOLDI, Richard Guy Marie

Abstract

L'actine est non seulement la protéine la plus abondante dans la plupart des cellules eucaryotes mais également une des plus conservées dans l'évolution. Il reste toutefois à ce jour de nombreuses zones d'ombre, en particulier en ce qui concerne le rôle de ses différentes isoformes. Nous avons développé un anticorps monoclonal capable de reconnaître spécifiquement l'isoforme gamma-musculaire lisse (g-SMA). Les résultats de nos experiences ont permis de mettre en évidence chez plusieurs espèces une distribution différente au niveau tissulaire et cellulaire par rapport aux autres isoformes d'actine, et en particulier par rapport à l'autre actine musculaire lisse (a-SMA). Nos résultats permettent non seulement d'affirmer que l'isoforme g-SMA est exprimée de manière spécifique mais aussi qu'elle possède une modulation propre lui permettant de jouer un rôle bien distinct (non-contractile) par rapport à l'actine a-SMA. Responsable de la compliance et conférerait à l'organe une plus grande résistance face aux forces de dilatation.

ARNOLDI, Richard Guy Marie. The gamma-smooth muscle actin isoform in smooth muscle and smooth muscle-like cells. Thèse de doctorat : Univ. Genève, 2013, no. Sc.

4672

URN : urn:nbn:ch:unige-393344

DOI : 10.13097/archive-ouverte/unige:39334

Available at:

http://archive-ouverte.unige.ch/unige:39334

Disclaimer: layout of this document may differ from the published version.

1 / 1

UNIVERSITE DE GENEVE

Département de Biologie cellulaire FACULTE DES SCIENCES Professeur Didier PICARD

Département de Pathologie et Immunologie FACULTE DE MEDECINE

Professeur Christine CHAPONNIER

The -smooth muscle actin isoform in smooth muscle and smooth muscle-like cells

THESE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Richard ARNOLDI de

Italie

Thèse n° 4672

Atelier d’impression Repromail Genève

2014

2

3

Table of contents

Acknowledgements ………. 5

List of Abbreviations………. 7

Abstract………... 9

Résumé……… 11

Introduction……… 13

1. The Cytoskeleton………. 14

2. Actin……….. 16

2.1 General description……….. 16

2.2 Functions……….. 19

2.3 Regulation………. 19

2.4 Isoforms……… 22

2.4.1 The -SMA isoform……… 28

2.4.2 Actin isoforms in non-muscle cells……….. 30

2.4.3 Actin isoforms in skeletal muscle………. 31

2.4.4 Actin isoforms in cardiac muscle……….. ……… 32

3. Actin isoforms in Smooth Muscle and SM-like cells……. 34

3.1 The Smooth muscle………..…. 34

3.1.1 Visceral smooth muscle……… 36

3.1.2 Vascular smooth muscle……….. 36

3.2 SM-like cells……… 38

3.2.1 Myofibroblasts……….. 38

3.2.2 Myoepithelial cells………... 38

3.2.3 Myoid cells……… 40

3.3 SM actin isoforms……….. 41

3.3.1 SM actin isoforms expression modulation... 46

Aim of the Study………... 52

Results……… 54

4

1. Introduction Paper 1………...………. 55

1.1 Paper 1………... 57

2. Introduction Paper 2………. 73

2.1 Paper 2……… 75

3. Introduction Paper 3………. 89

3.1 Paper 3……… 92

Discussion………... 119

1. Summary……… 120

2. Isoactins are more function-specific than tissue-specific... 121

3. Actin isoform mutations and diseases………... 122

4. Limitations of two common experimental techniques…... 123

4.1 Actin isoforms gene knock-out………... 123

4.2 Live studies using fluorescent protein labeling……..…… 124

5. SM actin isoforms and ABPs……… 124

6. -SMA: a potential marker in breast carcinoma?... 127

7. -SMA regulation………. 128

7.1 -SMA regulation during pregnancy: an implication of hormones and IGF?... 129

8. Perspectives……… 131

8.1 -SMA in the vascular system……… 131

8.2 -SMA in atherosclerosis………. 132

8.3 -SMA in breast cancer……… 133

8.4 SM actin isoforms and ABPs……….. 133

8.5 -SMA regulation……… 133

8.6 In vitro dilation assays……… 134

9. Conclusion………. 134

Appendices………. 135

References……… 142

5

Acknowledgements

First of all, I am very grateful to the members of the Jury: Prof. Didier Picard and Prof.

Annette Draeger for their availability in the co-direction of this work.

I am sincerely grateful to Prof. Christine Chaponnier for having welcomed me in her team, and for having shared with me part of her skills. I wish in particular to thank her strongly for having provided me with all the scientific advices and all the human support, without which this work could not have been accomplished.

I am particularly grateful to Dr. Marie-Luce Bochaton-Piallat and her team for furnishing part of the vascular materials, for all their precious support and advices, and most of all for their friendship.

I wish to thank Prof. Giulio Gabbiani and Prof. Boris Hinz for their precious advises, Dr. Vera Dugina for her precious help with confocal imaging, her acute advises, and her daily encouragements, as well as Dr. Sophie Clément for the pictures of mice cardiomyocytes, and for her constant friendly encouragements.

I wish to thank also Prof. Annelise Wohlwend, and Drs Jean-Christophe Tille and Jean- François Egger for furnishing the human materials, and for their acute and friendly explanations.

I wish to thank Sandra, Aman, Giuseppe, Philippe, and Anthony for their precious technical help, as well as Kristel and Alexandre for their collaboration.

A special thank to Anne-Laure, Emilie, Gaia, Isabelle, Lucie, and Patricia for their support, in particular in the recent troubled months.

A special thank to my friends Ewan, Julian and Thierry, for the great moments in Geneva and abroad.

6 I am grateful to the Department of Pathology and Immunology for accepting me, and to all its members for their various contributions and for the pleasant times.

Last but not least, I wish to specially thank Anita Hiltbrunner for all her fantastic technical help and for their daily support.

Un grand merci maintenant à mon amie Anita qui a toujours été présente au long de ces années aussi bien au CMU que dans les salles de sport et les randonnées.

Matteo e Sara, come riassumere tutti questi anni. Qualcuno ha parlato di amicizia, ma per me siete stati la mia famiglia. Vi voglio bene.

Perrine, la lumière d’ «Elendil », qui a réussi à éclairer les endroits sombres où toutes les autres étaient éteintes.

Un ringraziamento particolare va ai miei genitori Angela e Vittore per tutto quello che hanno fatto per me, per il meraviglioso esempio di vita che sono sempre stati e sopratutto per avere fatto di me quello che sono oggi. Niente sarebbe stato possibile senza di loro.

7

List of Abbreviations

-SMA : alpha-smooth muscle actin

-CAA : alpha-cardiac muscle actin

-SKA : alpha-skeletal muscle actin

-CYA : beta-cytoplasmic actin

-SMA : gamma-smooth muscle actin

-CYA : gamma-cytoplasmic actin ABP : actin binding protein ADP : adenosine diphosphate ANF : atrial natriuretic factor ATP : adenosine triphosphate

BM : basal membrane

ECM : extracellular matrix EGF : epidermal growth factor ESMC : enteric smooth muscle cell FA : focal adhesion

FGF : fibroblast growth factor GAM : goat-anti-mouse

GAR : goat anti-rabbit

GEF : guanine exchange factor GPCR : G-protein coupled receptor GTP : guanosine triphosphate IGF : insulin-like growth factor IGFBP : IGF-binding protein IF : intermediate filament

IFAP : intermediate filament-associated protein LPA : lysophosphatidic acid

mAb : monoclonal antibody

MAP : microtubule-associated protein MC : myoepithelial cell

mRNA : messenger RNA

MT : microtubule

8 MTOC : microtubule-organizing center

MHC : myosin heavy chain

MRTF : myocardin-related transcription factors mTOR : mammalian target of rapamycin

PDGF-BB : platelet-derived growth factor-BB PI3K : Phosphatidylinositide 3-kinase rRNA : ribosomal RNA

ROCK : Rho-associated kinase S1P : sphingosine-1-phosphate sHSP : small heat shock protein

SM : smooth muscle

SMC : smooth muscle cell

SMMHC : smooth muscle myosin heavy chain SRE : SRF-binding element

SRF : serum response factor

TGF- : transforming growth factor 

UTR : untranslated region

VEGF : vascular endothelial growth factor VSMC : vascular smooth muscle cell

9

Abstract

Actin, which is involved in a plethora of functions, is the most abundant protein in most of the cells and is impressively conserved across species. However, not much is known about its different isoforms and even less about the -smooth muscle isoform (-SMA). Until now, this isoform has always been considered as the Cinderella of actins, mainly because of its apparently limited distribution and because of the lack of appropriate tools for a proper investigation. For this reason, we developed a specific monoclonal antibody (mAb) discriminating this isoform. We performed successively several experiments in order to highlight its localisation in different tissues and different species. Our results confirmed some of the existing data, and spotted new evidence for specific tissue and cellular compartmentalization of -SMA. In particular, a different distribution compared with the other SM isoform (-SMA) has been evidenced in the intestine, in the vascular system, in myoepithelial cells, and in various neoplastic or non-neoplastic fibrocontractive situations.

The current paradigm states that -SMA is responsible for contraction in smooth muscle (SM) and SM-like cells, and that -SMA fulfils the same role in the enteric system. According to our analysis we proposed a new hypothesis, in which -SMA is responsible for contraction, whereas -SMA is responsible for compliance and confers a resistance to the organ when submitted to strong dilative forces.

We performed several in vivo experiments and, in particular, we demonstrated that, contrary to -SMA, -SMA expression is upregulated in the SMCs of the uterus in the course of pregnancy and in mammary myoepithelial cells during lactation. Moreover, we showed that - SMA is mainly expressed in highly compliant veins, whereas its expression is very limited in stiff contractile arteries.

We pursued the investigation on different cell cultures and focused on vascular SMCs (VSMCs), in which we observed a different pattern of spatial and temporal expression distinct of -SMA. For the first time we showed that the intracellular location of the two isoforms is different: -SMA fibres extend in the whole cell and connect to vinculin-positive focal adhesions, while -SMA is limited to a more central region without reaching the focal

10 adhesions. This distinct location sustains our hypothesis of a different (non-contractile) function for -SMA.

By coupling a collagen lattice contraction assay with the specific depletion of the two isoforms, we evidenced that the partial depletion of -SMA diminishes the contractility of the cell, despite an increase of -SMA expression. On the other hand, the partial depletion of - SMA has no impact on the contractile properties of the cell.

The treatment of fibroblasts and SMCs with the whole palette of compounds, known to promote the expression of -SMA, (e.g. TGF-, Heparin) has no effect on the expression level of -SMA. Furthermore by screening a library of 1040 out-patented FDA-approved compounds, we identified other candidates that differently regulate the expression of the two SMA isoforms.

Our results suggest that the specific tissular and subcellular locations of -SMA, as well as its proper modulation, reflect a distinct role for this isoform compared to -SMA.

11

Résumé

L’actine est non seulement la protéine la plus abondante dans la plupart des cellules eucaryotes mais également une des plus conservées dans l’évolution. Si elle a été beaucoup étudiée au travers des nombreuses fonctions qu’elle possède dans la cellule, il reste à ce jour de nombreuses zones d’ombre, et en particulier en ce qui concerne le rôle de ses différentes isoformes. L’isoforme d’actine la moins connue est certainement la -musculaire lisse (- SMA), et ceci dû en grande partie, à l’absence d’outils appropriés. Nous avons, par consequent, développé un anticorps monoclonal capable de reconnaître spécifiquement cette isoforme. Les résultats de nos experiences ont permis de mettre en évidence sa localisation dans de nombreux tissus de plusieurs espèces. Si nos résultats confirment la validité des travaux précédants, ils mettent également en évidence une distribution différente aussi bien au niveau tissulaire que cellulaire par rapport aux autres isoformes d’actine, et en particulier par rapport à l’autre actine musculaire lisse (-SMA). Des différences significatives ont notamment été mises en évidence dans l’intestin, le système vasculaire et les cellules myoépithéliales, ainsi que dans de nombreuses fibroses d’origine tumorale ou non.

Le paradigme actuellement admis prétend que l’actine -SMA est responsable de la contraction dans les cellulaires musculaires lisses et similaires, et que l’actine -SMA remplirait la même fonction dans les muscles lisses entériques. A la suite de nos travaux d’analyse nous avons proposé une nouvelle hypothèse, selon laquelle si l’actine -SMA est bien responsable pour la contraction, l’actine -SMA serait, elle, responsable de la compliance et conférerait donc à l’organe une plus grande résistance face aux forces de dilatation.

Nous avons donc mené plusieurs expériences in vivo et nous avons démontré, entre autres, que contrairement à l’actine -SMA, l’expression de l’actine -SMA était fortement augmentée dans les cellules musculaires lisses de l’utérus lors de la grossesse ainsi que dans les cellules myoépithéliales des glandes mammaires pendant la lactation. En outre, nous avons montré que l’actine -SMA est fortement exprimée dans les veines, vaisseaux faiblement contractiles et très expansibles, alors qu’elle n’est que sporadiquement exprimée dans les artères, connues pour être très contractiles et rigides.

12 Nous avons alors étendu notre recherche in vitro et notamment aux cellules musculaires lisses vasculaires. Nous avons montré pour la première fois une distribution intracellulaire différente des deux isoformes d’actine musculaire lisse : les fibres d’actine -SMA s’étendent dans toute la cellule et se connectent aux points d’adhésion focaux, alors que l’actine -SMA est davantage restreinte à une région centrale de la cellule et n’atteint pas les points d’adhésion focaux. Cette localisation distincte soutien notre hypothèse d’une fonction différente (non-contractile) de l’actine -SMA.

En diminuant artificiellement in vitro les deux isoformes d’actine et en menant simultanément des tests de contraction du collagène, nous avons démontré que la diminution partielle de l’actine -SMA dans la cellule abroge sa contractilité et ce, malgré une augmentation de l’expression de -SMA. A contrario, la diminution de -SMA n’a aucun effet sur les capacités contractiles de la cellule.

Le traitement in vitro de fibroblastes et de cellules musculaires lisses avec des molécules connues pour augmenter l’expression d’actine -SMA (p.ex. TGF-, Héparine) n’a aucun effet sur le niveau d’expression de -SMA. En outre, en ayant recours à une librairie de 1040 molécules, nous avons pu identifier d’autres candidats agissant différemment sur la régulation des deux isoformes.

Nos résultats permettent non seulement d’affirmer que l’isoforme -SMA est exprimée de manière spécifique mais aussi qu’elle possède une modulation propre lui permettant de jouer un rôle bien distinct par rapport à l’actine -SMA.

13

Introduction

14

1. The Cytoskeleton

The idea of the importance of « fibrous structures » in various compartments of the living beings has been proposed for centuries (reviewed in (1)), but the concrete existence of a structural intracellular network of proteins has only been revealed over the course of the 20^th century. The term Cytoskeleton, the Cytosquelette in reality, was coined in 1931 by the French embryologist Paul Wintrebert (2), but the concept remained vague for about a quarter of century, while electronic microscopy made its debut in biological research. Pioneering studies (3-7) evidenced then that eukaryotic cells contain three distinct cytoskeletal filaments which exhibit very different assembly properties, supramolecular architectures, dynamic behavior, and mechanical properties: microtubules (MTs), intermediate filaments (IFs), and microfilaments (MFs) (Fig. 1).

Fig. 1 The eukaryotic cytoskeleton is constituted by three distinct systems of filaments

Although the cytoskeleton has been considered for a long time as a peculiarity of eukaryotes, homologs to all major protein of the eukaryotic cytoskeleton have been described in the last decades in all living organisms from prokaryotes (8, 9) to plants (10, 11). Bacteria express MreB, FtsZ, and Crescentin as actin- , tubulin-, and intermediate filament-like proteins (12).

Moreover, there is an increasing amount of newly identified proteins with no eukaryotic homologs, such as WACA proteins and bactofilins, involved in the bacterial cytoskeletal architecture (13). For a detailed study analyzing the relationships between the main

15 constituents of the bacterial, archaeal, and eukaryotic cytoskeletons, see the comprehensive review from Wickstead and Gull (14).

Microtubules are made of protein tubulin and microtubule-associated proteins (MAPs), which assemble into long and straight hollow cylinders of about 25 nm in diameter, generally nucleating from a microtubule-organizing center (MTOC) or centrosome and spreading throughout the cell. Their main roles include the positioning of organelles, intracellular transport, assembly of the mitotic spindle, and the formation of motile structures such as flagella and cilia (15).

Intermediate filaments (IFs) and intermediate filament-associated proteins (IFAPs), which are ropelike fibers of about 10 nm in diameter, are made of various cell-specific subunits forming a large and heterogeneous family of proteins, such as vimentin, desmin or cytokeratin. They usually extend across the cytoplasm from one cell junction to another, providing mechanical strength and resistance to shear stress to the cell (15).

With a diameter of 7-9 nm, actin microfilaments are the thinnest structures of the cytoskeleton. They are relatively flexible compared to MTs, and are often nucleated at the plasma membrane in the so-called “cortex” region, granting both shape and motility to the cell.

The organization of the cytoskeleton has been revealed to be extremely complex from a structural, mechanistic and regulatory perspective, since three distinct systems interact with each other and with hundreds of associated proteins to confer to the cell a great plasticity and to permit a wide-range of functions.

The cytoskeleton is often altered in disease and there is an emerging evidence of its involvement in molecular and cell mechanisms processes leading to severe pathologies such as cancer, cardiovascular disease, myopathies or skin disorders (16). Several mutations, deletions and alterations in components of the cytoskeleton have been shown to affect cell normal functions and eventually to lead to pathological conditions (17-29).

16

2. Actin

2.1 General description

In most eukaryotic cell, actin is by far the most abundant protein, ranging from 5 to 10% (up to 25%) of the total protein content. The highest concentrations of actin are found in the cytoplasm as stable microfilament systems assembled within myofibrillar contractile ultrastructures in striated muscles. Nevertheless, apart of its specialized role in muscle contraction, actin is present in all muscle and non-muscle cells, where it plays a variety of roles thanks to its ability to assemble and disassemble depending on cell requirements (reviewed in (30, 31)).

Actin was discored by Straub in 1942 (32) and purified twenty-four years later from Plasmodium (Physarum polycephalum) by Hatano and Oosawa (33) and later by Ishikawa from nonmuscle cells (34). However, it is the purification of actin at a larger scale from rabbit striated muscle by Spudich et al. (35) that definitively opened the way to in vitro experiments on this protein. Research on this fundamental protein considerably improved only from the middle of the seventies thanks to (i) immunochemical techniques (36), (ii) “gelation” and contraction analysis of cytoplasmic extracts, which led to the identification of actin-associated proteins (37), and (iii) improvement of electronic microscopy, which allowed to visualize the distinct organization of the filaments and to materialize the concept of cytoskeleton (38).

Although the cocrystallization of actin with bovine pancreatic DNase I was first reported in the late 1970s (39), thirteen years passed before the three-dimensional structure of G-actin was resolved at atomic resolution for -skeletal muscle actin (-SKA) complexed with DNase I (40) or gelsolin S1 (41), and for -cytoplasmic actin (-CYA) complexed with profilin (42). A new model for F-actin was thus proposed by Holmes et al. (43), which paved the way for many studies on conformational changes of both actin monomer and filament (44- 48). In 2001, the structure of actin was finally solved from crystals in absence of cocrystallized actin-binding proteins (ABPs) by Dominguez and co-workers (49), which suppressed the tendency of actin to polymerize by modifying the COOH-terminal Cys-374 with a rhodamine label.

17 Fig. 2 Three-dimensional structure model of monomeric actin (G-actin). (http://www.mpimf- heidelberg.mpeg.de/~holmes/newfigs/actin.gif).

Monomeric G-actin is an acidic (IP = 5.4) globular protein of 374-375 amino acids with a molecular weight of approximately 43 kDa (Fig. 2). It is an asymmetric molecule (5.6 X 3.3 X 4.0 nm) clearly divided into two domains of roughly equivalent size (which for historical reasons, are called the large and small domains). These two domains are connected covalently with only two strands of polypeptide chain that are close together at the base of the molecule, allowing relative movement of the domains. Each domain is divided into two subdomains. By definition, subdomains 1 (residues 1-32, 70-144, and 338-375) and 2 (residues 33-69) constitute the small domain, whereas subdomains 3 (residues 145-180 and 270-337) and 4 (residues 181-269) represent the large domain of actin (50). In the centre of the structure, a deep cleft contains one strong-affinity binding site for a nucleotide (usually ATP or ADP-Pi), which binds as a complex with either Mg²⁺ (Kd = 1.2 nM) or Ca²⁺ (Kd = 0.12 nM). Since the bound nucleotide contacts residues from all four subdomains it functions as the coordinating center of the molecule.

Actin microfilaments present a similar structure from plants to animals. According to the model of Holmes et al. (43) and to successive studies (51, 52), actin filament is a two-start right-handed helix with a pitch of 72 nm (Fig. 3). In this model, each monomer is surrounded by four others, yielding a filament diameter of 7-9 nm. Since the monomer-monomer affinity is probably stronger along the two strands than between those strands, the resulting filament is

18 sufficiently rigid. These filaments have a distinct structural polarity, which has been first noticed by Ishikawa in 1969 through decoration with myosin S1 fragments (34).

Fig. 3 Three-dimensional structure model of actin filament (F-actin). Adapted from Holmes et al. (http://www.mpimf-heidelberg.mpg.de/~holmes/newfigs/3.gif)

In vitro, actin polymerization occurs only above a threshold (critical) concentration. Its assembly is not a linear process but can be split in three distinct phases (53): (i) the lag phase:

monomers assemble rather slowly to form trimers (nuclei), (ii) the elongation phase: once the nuclei are formed, a rapid assembly of monomers causes the filament to elongate (54), and (iii) the steady state: the system reaches an equilibrium when the addition of a new monomer at the barbed end is now counterbalanced by the disassembly of another monomer at the pointed end of the filament. However, in the cell, actin filaments are in a continuous process of assembly/disassembly, and in most nonmuscular cells of vertebrates, 50% of actin is present on its monomeric state despite a concentration well above the critical polymerization threshold. This singularity is due to the presence of ABPs, which also help actin filaments to induce the cell shape and drive its motility under the influence of a multitude of intra- and extra-cellular signals (55).

19

2.2 Functions

Actin plays an important role in maintaining cell shape and structures by conferring mechanical strength (contraction and/or tension) often in association with myosin. Since an important property of actin is its ability to produce movement in the absence of motor proteins, microfilaments are strongly involved in cell motility and cytokinesis (56-61). Actin ensures also intracellular organelles and vesicles transport by providing “rails” and by cooperating with MTs and IFs, via MAPs and IFAPs (62-64). Actin also participates in signal transduction by enabling extracellular stimuli to be transmitted downwards in the cell and by regulating the translocation of transcription factors into the nucleus, as well as in ion transport (65), mRNA transport and translation (66), synaptic transmission (67), and in the regulation of enzyme activities. For a comprehensive understanding of actin multifunctionality, see in particular the recent reviews of Schoenenberger et al. and Dominguez et al. (31, 52).

More recently, actin has also been linked to several nuclear functions (see excellent review from de Lanerolle (68)). It has been shown that inside the nucleus actin is required for transcription by all three classes of RNA polymerases and for the transcription of ribosomal genes. Nuclear actin is also involved in rRNA maturation and in the maintenance of nucleolar architecture and chromatin remodeling (69-76).

Considering these multiple cellular functions of actin, it is easy to forecast that alterations in the organization of microfilaments will result in disorganized cell morphology and orientation, uncontrolled cell growth, and abnormal responses to the environment (77-79).

2.3 Regulation

The polymerization equilibrium of G-actin and F-actin in the cytoplasm depends on the intracellular concentration of ATP-bound G-actin, its regeneration rate from ADP-actin pools, the concentration of internal regulatory factors such as Mg²⁺ or ATP (80), and by the activity of many ABPs. Several physiological and pathological extracellular stimuli may promote however the rearrangement of the actin cytoskeleton. In primis, these stimuli are perceived by different receptors, which in turn activate, via selected GEFs, a variety of Rho GTPases, and in particular several members of the Rho, Rac, and Cdc42 families (81, 82). These Rho GTPases regulate downstream effector proteins that modulate the

20 polymerization/depolymerization dynamics of actin microfilaments (83). This pathway can be activated through (i) Tyr kinase receptors (e.g. for PDGF, EGF, FGF, VEGF, or insulin) (84), (ii) heterotetrameric complexes of Ser/Thr kinase receptors (TGF-) (85), (iii) GPCRs (e.g.

LPA, S1P) (86, 87), (iv) integrins (particularly important for the transmission of mechanical stress) (88, 89), (v) E-cadherin-catenins (90, 91), and (vi) non-cannonical Wnt signaling (92, 93).

Fig. 4 A proposed model for -SMA gene promoter regulation. From Kumar et al. (94)

Besides the reorganization of pre-existing actin, microfilament dynamics also require the de novo synthesis of G-actin. A highly complex palette of interacting regulatory elements has been identified in the promoter region of actin genes (95-97) (Fig. 4). Within these elements some are common to the different isoforms, therefore some signaling molecules exert their positive or negative effects on isoactins genes. Other elements, however, are specific for each actin isoform, and are therefore suspected to differentially modulate the expression of a specific actin isoform, either directly, or more probably, through interactions with other regulatory elements. Actin expression-inducing serum response factor (SRF) represents probably one of the most interesting situations (98, 99). All isoactins promoter regions contain in their sequences several SRF-binding elements (SREs), which may act similarly on the expression of all actin isoforms (100). SREs are nevertheless flanked by complementary

21 regulatory elements (Ets), which have a different affinity for various muscle-type specific regulators such as Mhox, Myogenin, nkx 2.5, or nkx 3.1 (101, 102).

An important discovery was that in the cytoplasm G-actin binds several MRTFs cofactors, and that its polymerization into F-actin fibres liberates these factors, thereby inducing the nuclear transcription factor SRF to modulate the expression of genes encoding actin and actin-related proteins.

As a consequence, in addition of to the direct regulation of actin polymerization in the cytoplasm via effector proteins (e.g. ROCK), the activation of RhoA controls MRTF transcriptional activity by regulating the availability of G-actin (103-105).

Table 1 Specific post-translational modifications of actin. From Terman et al. 2013 (106).

Besides all these regulatory mechanisms, actin is also susceptible to covalent modifications of its amino acids. The importance of the N-terminal post-translational modifications, and in particular the acetylation of the heading amino acids, has already been highlighted in early works (107). It has been evidenced that acetylation confers its specificity to actin isoforms, and that it is mandatory for specific interactions with ABPs, as well as for the proper recognition by monoclonal antibodies. Recent studies are evidencing that these covalent modifications of actin are widely employed and regulate actin organization and dynamics (Table 1). Among the most cited post-translational modifications of actin are

22 phosphorylation, acetylation, ADP-ribosylation, and arginylation (for a comprehensive review see Terman et al. 2013 (106)).

2.4 Isoforms

Although some polymerizing forms of actin-like proteins are already present in bacteria (e.g.

MreB (108), ParM (109), and ActM (110)) and in yeast, recombinant DNA techniques have shown that actin exists as a multigene family in protozoa (8), plants (10), and animals (111).

Each isoactin is encoded by a distinct gene and displays a unique temporal and spatial pattern of gene expression (see excellent and comprehensive reviews (112-115). Whereas most of invertebrates present one single (cytoplasmic-type) actin in both muscle and nonmuscle cells¹, a muscle-specific isoform has emerged in evolution with vertebrates². Since fishes, two specific actins are dedicated to, on one hand striated muscles, and on the other hand SMs. The present distribution in actin isoforms appears in reptiles, with the duplication of the prior isoforms to generate cardiac/skeletal isoforms, and vascular/enteric isoforms respectively (Fig. 5).

Fig. 5 A Proposed evolutionary model of actin isoforms. Adapted from Vanderkerckhove et al. (116).

The four muscle actin isoforms are therefore present in one single copy in the genome.

Although the number of coding genes/pseudogenes and copies throughout the genome is very different among species, ranging from one single gene in yeast (117), to six genes in

1 Drosophila melanogaster, for instance, has both muscle and nonmuscle actin isoforms.

2 As of today, this muscle actin is the only specialized isoform expressed in all muscle cells of lower vertebrates.

Higher Vertebrates (since Reptiles) Fishes Lower Vertebrates Invertebrates 1 cytoplasmic

actin 2 cytoplasmic

actins (- and -)

1 muscle

actin 1 striated muscle

actin 1 cardiac muscle

Actin (-card)

1 skeletal muscle

Actin (-sk)

m

1 smooth muscle

actin 1 vascular muscle

Actin (-sm)

1 enteric muscle

Actin (-sm)

23 drosophila (118), and to thirty genes in mammals (6 genes (119) and more than 20 pseudogenes (120)), increasing genetic complexity seems to be associated with progressive expansion of multigene families and the generation of alternative exons.

Despite a palette of isoforms, actins are highly conserved, differing in sequence identity by less than 40% between fungi and humans (113, 121). Studies from Vandekerckhove et al.

(122) have identified six highly conserved vertebrate actin isoforms, the primary structures of which are completely conserved across species from birds to humans (40). The six human actin genes are located on different chromosomes and no genetic linkage is seen among them (123): -CAA gene is on chromosome 15; -SKA gene is on chromosome 1; -SMA gene is on chromosome 10; -SMA gene is on chromosome 2; -CYA gene is on chromosome 17, and -CYA gene is on chromosome 7. Although encoded by a set of structurally related genes that probably evolved from a common ancestor (111), actin isoforms have highly homologous primary sequences (124).

Table 2 Number of different residues between the amino acid sequences of the different actin isoforms. Differences between muscle specific isoforms, between nonmuscle isoforms, and between muscle and nonmuscle isoforms are highlighted in green, blue, and turquoise respectively.

Although there are several differences in the primary sequence that are scattered throughout the molecule (Table 2), the coding sequence heterogeneity lies predominantly in the N- terminus, which contains a cluster of acidic residues (125-127) (Table 3). This variability contributes therefore to a net charge which can be detected by isoelectric focusing. As a consequence, actins have been classified as -, , and -isoforms in order of increasing basicity (5.40, 5.42, and 5.44 respectively) (128, 129). The N-terminal segments of -actins contain four charged amino acid residues, whereas those of - and -actins contain only three acidic residues.

-cardiac -skeletal -smooth -smooth -cyt. -cyt.

-cardiac 4 6 4 22 23

-skeletal 4 8 6 24 25

-smooth 6 8 3 22 23

-smooth 4 6 3 20 23

-cytoplasmic 22 24 22 20 4

-cytoplasmic 23 25 23 23 4

24

0/1 1/2 2/3 3/4 5/6 7/8 9/10 11/12

-cardiac Ac D D E E T T A L V C D …

-skeletal Ac D E D E T T A L V C D …

-smooth Ac E E E D S T A L V C D …

-smooth Ac E E E T T A L V C D …

-cytoplasmic Ac E E E I A A L V I D …

-cytoplasmic Ac D D D I A A L V V D …

Table 3 N-terminal amino acid sequences of actin isoforms of vertebrates. Note that post- translational modifications did remove the methyl/cysteine residues and acetylate the D/E residue at the N-terminus.

Isoforms, which in vertebrates are usually divided into muscle and nonmuscle actins, are also described as class I and II molecules on the basis of their specific N-terminal processing (112). The amino terminal residue of actins is an N-acetyl-Asp or N-acetyl-Glu, although their genes code for proteins with additional N-terminal amino acids that are removed posttranslationally in a stepwise acetylation-dependent processing reaction (130, 131). Class I molecules, are encoded by genes which specify a Met-Asp (Glu) N-terminus. The N-terminus is acetylated early in translation. Following completion of translation, N-acetyl-Met is removed and the Asp (Glu) is acetylated to yield the mature form of the protein. Class II molecules, are encoded by genes which specify a Met-Cys-Asp (Glu) N-terminus. Here, the initiator Met is removed early in translation, and the Cys is acetylated. Following completion of translation, Ac-Cys is removed and the new N-terminus is acetylated (Fig. 6).

The amino acid sequence (and the structure) of actin is thus largely conserved, suggesting for actin a role in primordial reactions and interactions that have been maintained during the evolution as fundamental items for the survey of the cell. Actins from similar tissues from different species are closer than actins from different tissues from the same species. In other words, actin isoform distribution in vertebrates is tissue-specific, and this specificity is conserved across species. Generally speaking, -SKA and -CAA are restricted to skeletal and cardiac muscle respectively, whereas - and -SMAs are predominantly expressed in vascular and enteric SM respectively. - and -CYA isoforms are ubiquitously expressed.

25 Fig. 6 N-terminal posttranslational modifications of actin isoforms. Adapted from Herman (113).

The actin isoforms are expressed in muscle and nonmuscle cells in temporally and spatially regulated patterns (128, 129, 132-134). Many cell types express nevertheless more than one isotype of actin, and the synthesis of specific isoforms is accompanied by their compartmentalization within the cell within different filamentous assemblies such as bundles or networks which may form various ultrastructures such as myofibrils, stress fibres, lamellipodia or filopodia.

Since an intrinsic property of actin filaments is their capability to propagate conformational changes that occur in actin monomer throughout the filament (cooperativity), their structure can be modulated by low amounts of ligands and ABPs (135). Although the same ABPs may have different functions, namely depending on their concentration, their phosphorylation state and the pH of the cell, they can be conventionally separated in distinct classes on the basis of their main mechanism of action (reviewed in Stossel et al. (136) and dos Remedios et al.

(137)).

Class I molecules

-CYA, -CYA

Met - Asp (Glu) -

Ac - Met - Asp (Glu) -

Asp (Glu) -

Ac - Asp (Glu) -

Met - Cys - Asp (Glu) -

Ac - Met - Cys - Asp (Glu) -

Cys - Asp (Glu) -

Asp (Glu) -

Ac - Asp (Glu) - Class II molecules

-CAA, -SKA,

-SMA, -SMA

26 (i) Stabilizing proteins binding to the sides of actin filaments and preventing depolymerization (e.g. tropomyosin); (ii) end-binding proteins (e.g. CapZ, tropomodulin) which bind the extremity of an actin filament (pointed or barbed end) and promote the assembly, inhibit the elongation or decrease the average length of the filaments. Many of them (e.g. gelsolin, severin) may also have a severing activity; (iii) depolymerizing and G-actin sequestering proteins increasing the cellular concentration of G-actin or preventing actin polymerization.

These proteins can operate either by sequestering G-actin (e.g. profilin (138), thymosin 4, DNase I) and modifying the equilibrium between G- and F-actin, or by increasing the rate of actin disassembly (e.g. cofilin, CapZ); (iv) cross-linking proteins which can be divided into bundling proteins (e.g. fimbrin, -actinin, vilin, vinculin) and gel-forming proteins (e.g.

filamin, spectrin). The former crosslink the actin filaments into parallel structures, whereas the latter crosslink with a larger angle two filaments in order to form a meshwork; (v) motor proteins that use F-actin as a track upon which to move (e.g. the myosin family of motors).

Despite similar three-dimensional structures, a few amino acid replacements, primarily distributed in subdomains 1 and 3, may have a significant effect on actin conformation.

Isoforms may therefore differ in the overall position of the small domain relative to the large domain, as well as in local conformations at the N-terminus and, probably, at the C-terminus (42). The N-terminal domain is likely to influence isoform specific functions, thus substitutions in the primary structure of isoactins can modulate actin-actin, actin-myosin, and actin-ABP interactions. Since this region of the actin molecule participates in interactions with many ABPs (121), different affinities of actin isoforms to various ABPs seems likely.

Nevertheless, as of today, because most in vitro experiments have used skeletal actin, the interaction of actin isoforms with ABPs has not been investigated much. Different affinities of actin isoforms to myosin isoforms (139-142), and tropomyosins isoforms were shown (143).

Preferential interaction of cytoplasmic actins with profilin (144, 145) and thymosin 4 (146) as well as that of -CYA with fimbrin (147), l-plastin (148), and with ezrin (149, 150) has been reported. In a cell where different actin isoforms are simultaneously present, the presence of ABPs may lead to the formation of structures selectively enriched in one actin isoform. Once local translation has ceased, isoactins may be sequestered within specific cytoplasmic domains by isoform-specific ABPs.

27 It is not clear why human cells express six actin isoforms, because they seem to some extent interchangeable and the different actin isoforms confer no distinct functional advantage (123).

Since actins interact with many ligands, the differences among the six isoforms might impact various cell structures via different interactions with other cytoskeletal components.

Fig. 7 Similarities and differences of introns location within isoactin genes reflect their evolutionary pattern. From Miwa et al. (151).

Isoforms arised by gene duplication may exert different functions within cells either at the gene level (via different promoter elements), and/or at the mRNA level (via different UTRs), and/or at the protein level (via different functional properties). Isoforms exist as mRNAs and proteins (152). In many cases, the 5’ and/or 3’UTRs of isoform mRNAs differ in addition to the protein coding region (Fig. 7). The evolutionary conservation of some of these isoform- specific UTRs suggests that they are likely to be of functional significance (153, 154).

The high degree of sequence conservation among actin proteins suggests that the multigene family arose by divergence from a single common ancestral gene. Since an evolutionary clock hypothesis predicts that the accumulation of silent or replacement substitutions is proportional to divergence time, it is likely that early vertebrates had only one muscle actin and that the SMA arose only later in evolution (116, 155).



28

2.4.1 The -SMA actin isoform

-SMA sequence has been determined for the first time in 1979 by Vandekerckhove & Weber using chicken gizzard extracts (156). In the following years, thanks to isotype specific 3’UTRs domains, cDNA fragments specific for the different actin isoforms have been identified (154, 157-162). In 1988, McHugh et al. isolated and characterized, for the first time, a cDNA clone specific for rat -enteric SMA, which contains one single amino acid substitution when compared with the chicken gizzard -SMA sequence determined by Vandekerckhove & Weber (substitution of a proline for a glutamine at position 359) (163). In 1990, Miwa & Kamada reported the nucleotide sequence of the human -SMA cDNA (from human stomach) (164). The deduced amino acid sequence is compatible with that of -SMA, as reported by Vandekerckhove, except for the amino acid 359 Pro/Glu substitution already reported in rat by McHugh & Lessard and confirmed by Kim in mouse (165). As a consequence, although nucleotide sequences in coding regions show only about 92%

similarity, -SMA amino acid sequences are completely identical between human, mouse, and rat.

Table 4 Exon and intron organization of the mouse -SMA gene. From Szucsik et al. (155).

In the human genome, there is one single gene for -SMA located on chromosome 2, and extending for about 27 kb (the largest actin gene)³ (151). -SMA gene is divided into nine exons and eight introns, with coding sequences contained in exons 2 through 9 (Table 4). The total exon size of the mouse -SMA gene is 1280 bp or 5.5% of the gene (the smallest amount

3 Sizes of -CYA, -CYA, -SMA, -CAA, and -SKA are 2.8 kb, 3.4 kb, 17 kb, 4.9 kb, and 2.8 kb respectively.

29 among the vertebrate isoforms). The first intron measures over 8600 bp and is the largest intron ever reported for an actin gene (155).

A primary mRNA transcript of approximately 1.4 kb is translated into a 376 amino acids primary product (mature protein is 374 aa). Although the -SMA coding region has a homology of 82.4 to 85.8% with those of other human actin genes, most of the base changes are silent substitutions and, as a consequence, homologies between -SMA and other actins are significantly higher (93.6 to 99.5%).

According to Szucsik, alignment of the mouse, rat, and human -SMA 3’UTR sequences reveals 94% sequence identity for this region between the mouse and the rat, and a 75% level between the mouse and human (155). Their 3’UTRs do not possess the high degree of cross- species, isotype-specific sequence similarities observed for the other actin isoforms (-SMA rat/-CYA rat: 61%; -SMA rat/-CYA human 77%; -SMA rat/ -CYA chicken 66%!).

There is 75% and 76% sequence conservation respectively between human and mouse or rat cDNAs of 3’UTR (similar to -SMA but lower than those of other actin isoform genes (~85%). Introns are located at the same positions in the - and -SMA genes.

Several multiple regulatory elements such as CArG-boxes (CC(A/T)6GG)⁴, E-boxes (CANNTG), AP2 binding site-like sequences, and dinucleotide repeats (T/G residues), are contained within the proximal upstream sequences in all actin genes (Table 5). Although little is known regarding the control of -SMA gene expression (95-97), since they are located at similar relative positions, these elements may be important in controlling actin genes expression and may cooperate as core promoter elements.

CArG- box

CArG- box

C/EBP E- box

CArG- box

E- box

CArG- box

TATA- box

ATG E-box E-box

-434 -339 -253 -176 -108 -78 -71 -27 +1 +830 +850

Table 5. The 3’UTR of -SMA gene contains various potential regulatory sites.

4 The second CArG-box has proven to be a positive transcriptional regulatory site and a major binding site for nuclear factors in the human -SMA gene.

30

2.4.2 Actin isoforms in nonmuscle cells

Most nonmuscle cells express only - and/or -CYAs, although in various proportions⁵ (166).

Previous experiments on endothelial cells double-stained with a -CYA antibody and phalloidin after paraformaldehyde (PFA)-Triton fixation-permeabilization procedure, suggested that -CYA accumulates at the lamellipodium of moving cytoplasm, whereas stress fibers being stained by phalloidin were assumed to be formed of -CYA (167). But, in order to uncover the N-terminus epitope of actin isoform, particular fixation-permeabilization conditions, i.e. PFA-MeOH, are required. Recent results from our laboratory on fibroblasts and epithelial cells, using PFA-MeOH, specific -CYA and -CYA monoclonal antibodies, and siRNA evidenced that stress fibres are formed of -CYA and that the submembranous actin tridimensional network is formed of -CYA filaments (168). The ratio and localization of - and -CYAs being specific for distinct cells and subcellular compartments, overexpression of cytoplasmic actin genes results in dramatic changes in cell phenotype, emphasizing the role of cytoplasmic actins, in contrast to the muscle isoforms, in maintaining cell morphology (169).

Although the vast majority of nonmuscle cells express only CYAs, fibroblasts represent an interesting case in the context of SMA isoforms expression. Fibroblasts and SMCs share several functions, in the normal adult, though some of these activities remain specifically exerted by either the fibroblast (e.g. collagen synthesis) or the SMC (e.g. contractility).

Nevertheless, this separation of tasks may change during pathological conditions with, SMCs developing a synthetic activity and fibroblasts developing muscle-like features. By evolving into myofibroblasts, fibroblasts develop major stress fibres and cell-matrix interactions, and express SM specific proteins such as -SMA and eventually SMMHC, desmin, SM22, and tropomyosin (170). Other differentiation markers of SM, such as smoothelin and h- caldesmon, are expressed only in SMCs (171). Since all fibrocontractive diseases resulting in fibrosis (e.g. pulmonary, kidney and liver fibrosis, and stromal reaction to epithelial tumours) are characterized by the presence of myofibroblasts (172), these activated cells are considered the most common marker of a stromal reaction in various carcinomas such as breast, liver, lung, colon, stomach, prostate, esophagous or pancreas (173-175). For that reason, epithelial

5 In most cells : is around 2 (from only in human erythrocytes and a ratio of 4:1 in human platelets, to 0.5/0.6 in chicken auditory hair cells and intestinal epithelium brush border).

31 tumours, which produce many of the same growth factors that induce fibroblasts to undergo differentiation into myofibroblasts in wounding or fibrosis (176-178), have been described by Dvorak as “wounds that do not heal” (179). Not surprisingly, the levels of several ABPs (thymosin 4, -actinin, tensin, vinculin, merlin, tropomyosin…) decrease also in neoplastic cells (180, 181). Induction of -SMA synthesis in fibroblasts specifically leads to decreased motility of these cells (182). In endothelial and epithelial cells, exogenous -SKA and - CAA rarely are incorporated into stress fibers, instead remaining scattered within the cytoplasm and frequently forming long crystal-like inclusions (183).

2.4.3 Actin isoforms in skeletal muscle

In skeletal muscle, whether the majority of actin (-SKA) is localized in the centre of the cell in the contracting myofibrils (Fig. 8), both cytoplasmic isoforms are absent from these structures. The latter are namely found at the cell periphery in the cortical array of actin filaments (184, 185), and associated with neuromuscular junctions (186), Z-disk lattice, costameres and mitochondria (187, 188). Using a polyclonal anti--actin (staining -SMA and

-CYA isoforms) Pardo showed that in skeletal muscle, -actin is excluded from the contractile apparatus (188).

Fig. 8 Immunofluorescence labelling of -SKA (red) and -CAA (green) isoforms in skeletal muscle (adult rat soleus). Myofibrillar structures of the contractile apparatus are visible in the cell bodies (189).

Though -SKA is the specific isoform, in early muscle development and in prefused cultured myoblasts, only - and -CYAs are present (132). As development progresses, nonmuscle isoactins synthesis gradually ceases and the relative amount of -actin increases until it

32 becomes predominant (190). Then -SMA is downregulated and -CAA expression increases. Whereas skeletal muscle and cardiac actin genes are then coexpressed at the protein (191, 192) and mRNA (193, 194) levels⁶ at birth, -SKA is upregulated and remains the major isoform in adult skeletal muscle. Small amounts of -CAA and its mRNA persist in adult skeletal muscle of different species (191-193). As a consequence, both -CAA and - SMA can be considered foetal forms during skeletal muscle development.

Although -SKA is the predominant isoform in normal skeletal muscles, other isoactins may be expressed under pathological conditions. -CAA may thus be synthesized in skeletal muscle fibers and satellite cells (undifferentiated myoblasts) of injured muscle, in correlation with muscle regeneration, other muscle pathologies and in muscle-derived tumours (195).

Rhabdomyosarcoma, for instance, is a highly heterogeneous group of tumours of soft tissue characterized by the formation of neoplastic skeletal muscle fibres. Since -SMA is normally not found in normal, adult, striated muscle (196), the actin isoform expression pattern in different types of rhabdomyosarcomas may mimic developmental steps of skeletal muscle.

Rhabdomyosarcomas positive for only -CAA and -SMA would correspond to an early developmental stage of the muscle and -SKA positive would reflect a higher degree of differentiation (197).

More than 200 mutations in the -SKA gene causing skeletal muscle diseases have been reported. -SKA mutants generally suffer of severe myopathies, which often result in major disability and early death. Recent works showed that -SKA-deficient mice can be efficiently rescued by the expression of -CAA in skeletal muscle (198), suggesting that the over- expression of the fetal actin isoform in the adult skeletal muscle could be a potential therapy at least for some -SKA mutations (199).

2.4.4 Actin isoforms in cardiac muscle

In cardiac muscle, both sarcomeric actin isoforms are expressed simultaneously. Whether the majority of cardiomyocytes express their typical -CAA isoform, several studies suggested the presence of significant amounts of -SKA in developing, adult, and pathological hearts

6 In embryonic chicken and mouse muscles, -CAA still accounts for 90% and 60-70% respectively.

33 (200). After having developed a new polyclonal antibody specifically recognizing -SKA isoform, Clement and co-workers, evidenced that in normal rat heart -SKA is diffusely distributed within practically all myocardial fibres of the newborn rat, whereas it is surprisingly restricted to a small proportion of adult rat cardiomyocytes (Fig. 9). However, since these -SKA-expressing cardiomyocytes appear to be randomly distributed throughout the ventricles (perhaps more abundant in the left compared to the right ventricle), their precise function remains unclear, although a possible involvement in the regeneration of the cardiac muscle could be plausible (201).

Fig. 9 Immunohistochemical labelling of -SMA (a), -SKA (b), and -CAA (c) isoforms in two-weeks old mice heart. Note the -SMA and -SKA stainings restricted to vascular SM and to a subpopulation of cardiomyocytes respectively, compared to the broad -CAA staining of all the cardiomyocytes. Scale bar: 100 m. From Clement et al. (202).

The question of whether the small amino acid differences between muscle-specific isoactins influence myofilament function remains unsolved since muscle actin isoforms are interchangeable in vitro (203)⁷, but display significant functional differences in vivo, as evidenced by actin knock-out models (e.g. (204, 205)).

Although in normal cardiac muscle, -CAA is considered as the specific isoform, four other actin genes are expressed in cardiomyocytes during development (133). In embryo, - and - CYAs are synthesized in the precardiac mesoderm, in adjacent ectoderm and in the primitive heart. During development, whereas the synthesis of - and -CYAs is reduced, -SMA is

7 Note that during development, other isoforms such as - and -CYAs and -SMA may accumulate in myofibrillar structures in both skeletal and cardiac muscles.

34 transiently expressed in early myocardiogenesis, before being replaced in the developing heart by -SKA and then by -CAA isoform (206, 207). In normal adult myocardium, -SKA and

-CAA represent therefore the sarcomeric actin isoforms (192), although their relative levels may vary with species and age (200, 208, 209).

-CAA, and to a lesser extent -SKA, predominate in mature cardiomyocytes. However, during cardiac hypertrophy or when cultured in presence of hormones or growth factors, cardiomyocytes reexpress proteins normally occurring during fetal development, such as - SMA, -SKA, -MHC or atrial natriuretic factor (ANF) (203, 210). Interestingly, -SMA, which is not expressed in the myocardium at any developmental stage (190, 211, 212), is not expressed. Whereas in vitro expression of -SKA, -SMA, or -SMA in cardiomyocytes has very little effect on their myofibrillar structure and their ability to contract (213), actin isoforms do not seem to be interchangeable in vivo. During induced cardiac hypertrophy, indeed, the expression of -SKA was increased, causing a hyperdynamism and higher contractility of heart (201). This correlation is confirmed by observation of hearts isolated from BALB/c mice, which naturally express high levels of -SKA (214). Furthermore, in - CAA knock-out experiments, the majority (56%) of mice does not survive to term, and the remainder generally dies within 2 weeks of birth. Although an increased expression of - SMA and -SKA is observed in the hearts of newborn mutants, it is insufficient to maintain the myofibrillar integrity. These -CAA deficient mice have been rescued to a certain extent by ectopic expression of -SMA, but their hearts appear extremely hypodynamic and hypertrophied (205). Their myofilaments demonstrated a decreased sensitivity to Ca²⁺, and might display, according to authors, different interactions with tropomyosin, troponin and myosin (215).

3. Actin isoforms in Smooth muscle and SM-like cells

3.1 The Smooth muscle

Higher vertebrates display three different types of muscle cells respectively dedicated to contraction of skeletal, cardiac, and smooth muscles (Fig. 10). Since -SMA is only expressed

35 in SMCs, we will particularly focus on this type of tissue, either surrounding visceral hollow organs or blood vessels.

Fig. 10 Higher vertebrates display three different types of muscle tissues: cardiac, skeletal and smooth (http://apps.uwhealth.org/health/adam/hie/2/19841.htm).

Smooth muscles (SMs) are formed by non-striated mononucleated fusiform cells of about 3 to 6 m in diameter and 100 to 500 m long (volume in the order of 1200 to 1600 m³). These cells are generally disposed as dense layers in the walls of the blood vessels and of all the hollow organs (with the exception of the heart). Their involuntary contraction and relaxation is therefore critical for the function of the vascular, digestive, respiratory and urogenital systems. SMs are usually classified into visceral, vascular, and occasionally airways SMs. In addition to classic SMCs, other cell types display some SM-like features such as the ability to contract and the expression of typical SM proteins, namely actin isoforms, intermediate filaments and ABPs. These cells, which include myofibroblasts, myoepithelial cells and myoid cells, will therefore also be discussed in this chapter.

The primary function of SM is to produce the contraction phenomena in the circulatory, respiratory, gastrointestinal and urogenital systems. However, in the adult organism, the SMC retains significant proliferative, synthetic, and secretory functions compared to skeletal and cardiac myocytes. The variety of physiological roles played by SMCs in the body explains the heterogeneity of their functional and anatomical features. Although SM heterogeneity has been established for a long time, the definition criteria have been refined only during the last

36 decades. As of today, the expression of SMC-specific proteins, in particular the relative expression of - and -SMAs, is the best criterion to appraise heterogeneity of visceral and vascular SMs, as well as of smooth muscle-like cells such as myofibroblasts, myoepithelial and myoid cells. Investigating the heterogeneity and plasticity characteristics of SMCs is of major importance for understanding the mechanisms involved in their normal and pathological adaptations.

3.1.1 Visceral Smooth Muscle

In the walls of hollow organs, enteric SMs are present in several distinct layers surrounding the lumen. A thin layer just beneath the epithelial layer (muscularis mucosae), responsible for local microcontractions of the epithelium, and two (three in stomach) thick perpendiculary- oriented layers (inner circular and outer longitudinal) forming the muscularis propria, which are responsible for peristaltism (Fig. 11).

Fig. 11 A Schematic model of a typical visceral organ wall.

(http://www.lib.mcg.edu/edu/eshuphysio/program/section6/6ch1/6ch1img/page4.jpg)

3.1.2 Vascular smooth muscle

Vessels are formed by three concentric layers: (1) the tunica intima, made up endothelial cells lining the lumen and SMCs, (2) the tunica media, which contains only SMCs, and (3) the

37 tunica adventitia representing the layer of connective tissue containing elements such as fibroblasts, nerve endings, and vasa vasorum (Fig. 12).

Fig. 12 Schematic model of typical vessels walls (artery and vein).

(http://archimede.bibl.ulaval.ca/archimede/files/e8ebe68d-1ee7-4e8e-b018- 852786824b4d/23201001.jpg)

While arteries can be mechanically qualified as highly contractile and poorly compliant vessels, veins are the opposite, being poorly contractile but highly compliant. Since systemic veins are 8-fold more distensible than arteries and have a 3-fold greater volume, they are about 24-fold more compliant than their corresponding arteries. We show that -SMA is abundant and homogenous in the whole media of arteries, whereas -SMA is restricted to the outer layer (where cells are most stretched). The distribution of -SMA is similar to that of - SMA, in both vein adventitia and media (216).

Increased wall pressure initiates SM growth in arterial and venous vessels (217, 218), similar to other hollow organs such as uterus, intestine, and urinary bladder (219-221). Experimental portal hypertension causes a hypertrophy of portal vein SMCs (222). Although the increase in muscle mass is usually accompanied by an increased force-generating ability, in rat portal vein, hypertrophy is associated with decreased force (223). In a rat model of portal vein hypertrophy induced by partial ligation, the cross-sectional area of wall doubled after 7 days, with a moderate decrease of -SMA and increases of -actin, desmin and vimentin (224).