None of the processes that occur during cardiac contraction can be understood without taking into account the role the extracellular matrix (ECM). This three-dimensional structure serves as a framework for all the cells contained in the myocardium. Far from having only a scaffolding function, the ECM is involved in a wide range of tissue processes. ECM composition and arrangement are dynamic and consist not only on collagens, but also proteoglycans decorated with glycosaminoglycans, glycoproteins and other proteins such as proteases that modify the ECM. During cardiac disease, the ECM undergoes remodelling, and this is particularly patent in cardiac conditions involving a strong fibrotic response, such as myocardial infarction and atrial fibrillation.
Arrangement of the extracellular matrix
By acting as a link between the cellular components of the myocardium the ECM integrates in each excitation the contribution of each individual cell and allows for coordinated contraction of the whole tissue at the macroscopic level. The fluid component that fills the extracellular space provided by the ECM allows for the distribution of a wide variety of soluble proteins and other molecules related to many cardiac processes. In addition, various components of the ECM serve as specific attachment sites for soluble molecules, affecting their availability and distribution. Other ECM components, extremely hydrophilic, play a crucial role accumulating water, which provides excellent resistance to compression. The organization, composition and density of the ECM and the extracellular space are dynamic not only in pathological conditions, but also under normal conditions. These three properties are in a constant change in response to different demands, and they all have an impact on the function of the myocardium (Baudino, 2006).
Although functioning as a whole, the extracellular space can be divided into three structures of lower order. The first two, the basement membrane and the interstitial matrix, are part of the structural scaffold created by the ECM. The extracellular fluid, although undoubtedly contributing to the structure, is primarily way of communication between all the components of the tissue.
The basement membrane. The basement membrane is a specialized structure of the ECM that underlies all cellular monolayers of the organism. Its close relationship with cells is critical as it provides tissues with mechanical stability as well as signals that determine cellular polarity and migration. In the heart, its main constituents at the protein level are laminins, collagens IV, XV and XVIII, perlecan, agrin and nidogens 1 and 2 (van Agtmael, 2010; Kramer, 2005; LeBleu, 2007; Iozzo, 2009).
The interstitial matrix. The interstitial matrix is the part of the ECM that contributes to the mechanical properties of the tissue. It appears physically anchored to the basement membrane but forms a different functional unit that, in addition to providing mechanical resistance and structural support to the myocardium, provides the extracellular compartment through which signals and nutrients are distributed and the fibroblasts migrate. In the heart, its main components are collagens I, III, VI and XII, proteoglycans and the matricellular proteins that appear associated with the previous components (Harvey, 1999).
The extracellular fluid. The extracellular fluid surrounds all the cellular and extracellular structures of a tissue. It contains the elements necessary for cellular functioning and viability such as ions (Na+, K+, Ca2+, Cl- and HCO3-), glucose and other sugars, amino acids, fatty acids, neurotransmitters, hormones and growth factors, cytokines and other proteins. These elements derive from cellular secretion in situ and from blood plasma, which remains in continuous exchange with the extracellular fluid by endothelial filtration of the capillaries. The secretory and waste products of the cells exert important roles in the autocrine and paracrine regulation and are drained into the circulation through the lymphatic system (Mow, 1999).
Composition of the cardiac extracellular matrix
The cardiac ECM is composed of a mixture of components of different nature. Proteins, sugars and lipids are normal components of the different extracellular structures. Proteins are particularly prone to associate with other components in order to build hybrid elements such as glycoproteins, proteoglycans or lipoproteins, and they all definitively contribute to the operation and maintenance of the extracellular space as a whole. For this reason, it is difficult to establish an appropriate classification of the protein components of the cardiac ECM (and the extracellular space). Functional classifications yield heterogeneous groups in phylogenic terms, while organizations based solely on phylogeny are difficult to explain functionally. Additionally, although sequence homologies exist for many proteins, post-translational modifications result in significant differences and there is also variability with regards to tissue distribution. Although there is no appropriate classification satisfying all approaches, we will try to be as fair as possible in order to classify the extracellular components in a comprehensive manner. With this in mind, four groups of extracellular proteins will be considered hereinafter:
4) Non-glycosylated proteins and soluble extracellular components
5) Cell-matrix interaction components
1) Proteoglycans (PGs)
Accepted as major structural components of the cartilage ECM from the early 60's, PGs were for years considered to be specific to this tissue. However, in recent decades its presence nas been demonstrated in all tissues, appearing distributed on the interstitial matrix and basement membrane as well as conforming membrane receptors that are important for cell-matrix interactions.
All proteoglycans have a basic structure consisting of a core protein and a variable number of glycosaminoglycans chains (GAGs) anchored to the amino acid protein core at specific locations. PG diversity is based on the variety of genes encoding for protein cores, the different usage of exons contained in these genes, and the variety in length, type and number of GAG chains (Bandtlow, 2000).
PGs are lost during traditional fixation steps. This has caused that few classical studies on the ECM in healthy and pathological conditions focused their attention on the content of PGs (Fomovsky GM, 2009). Although the total number of PGs in mammals does not exceed fifty, the molecular weight range of different protein cores ranges from the small 20 kDa plasma membrane proteoglycan syndecan-4, to the enormous 466 kDa perlecan, in the basement membrane. Additionally, the number of GAG chains is extremely variable between different proteoglycans, and it can range from a single lateral chain (i.e. decorin) to more than 100 (aggrecan). In addition, GAGs content is likely to be affected quantitatively and qualitatively by age, exercise and various pathologies (Yoon JH, 2005). When using identification techniques in which the molecular weight is an essential characteristic (i.e western blot), the number and type of GAG chains can significantly affect the apparent size because it affects both the molecular weight and the electrophoretic properties (Bandtlow, 2000), further complicating their study.
a) Glycosaminoglycans (GAGs), polysaccharides attached to proteoglycans.
GAGs are long linear heterogeneous and negatively charged polysaccharides (Gandhi, 2008). They are fundamental constituents of the extracellular matrix of all tissues. The basic GAG unit consists on disaccharide repeating units composed of an amino sugar and uronic acid. The amino sugar can be N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc) and the uronic acid is glucuronide acid (GlcUA) or iduronic acid (Idou). From this basic configuration, the GAGs obtain their diversity from different combinations of amino sugars and uronic acids, as well as from the types of links established between them and the degree of acetylation and sulfation. The length can vary between 1 and 25,000 repeat units, with molecular weights ranging over three orders of magnitude and a control mechanism of polymerization that remains elusive. With the exception of hyaluronic acid, which is also the only non-sulfated one, GAGs appear normally covalently attached to a protein core, forming proteoglycans (Souza-Fernandes, 2006). The attachment of the GAGs to the core protein requires a specific trisaccharide composed of two galactose residues (Gal) and a xylose (Xyl), attached through O-glycosidic bond to a serine residue. It gives a configuration GAG -Gal-Gal-Xyl-OCH2-Ser-protein (Gandhi, 2008) but again, this anchor region is also susceptible to changes that will be specified later.
Because of their negative charge, GAGs in aqueous solution are surrounded by a layer of water molecules, making them to occupy a huge hydrodynamic volume. When the hydrated GAGs are under compression, water is evacuated and the GAGs reduce their volume until the pressure disappears, returning to their original volume (Gandhi, 2008). This property allows proteoglycans to act as natural cushion and it is fundamental in tissues undergoing continuous cycles of pressure-relief such as cartilage or cardiac muscle. However, the functions of GAGs extend far beyond a mere structural role. They can bind cytokines or growth factors and regulate their distribution and availability, and their degradation products are involved in different signaling cascades. Thus, the GAGs exert a triple role as reservoirs, sinks and cofactors of a large number of signaling molecules (Ernst S, 1995) that mediate adhesion, cell growth and differentiation, and therefore control events associated with inflammation, fibrosis, angiogenesis, or neuronal development among others (Gotte , 2003; Rose, 2004). In addition, GAGs have pharmaceutical applications as anti-coagulants, anti-thrombotic and anti-lipidemics (Yoon JH, 2005). There are four structural families of GAGs:
(i) Chondroitin sulfate and dermatan sulfate
(ii) Heparan sulfate and heparin
(iii) Keratan sulfates I and II
(iv) Hyaluronic acid
(i) Chondroitin sulfate (CS) and dermatan sulfate (DS). These two GAGs arise from the same precursor, chondroitin, with the repeating disaccharide unit GalNAC (β1,4) GlcUA (β1,3) (Yoon, 2005). This molecular form conforms the structure of CS, which is distributed over a wide variety of tissues including heart and cartilage (Bhagavan, 2011). It is part of proteoglycans of the ECM such as versican and Aggrecan or decorin (Mecham, 2011). DS is considered a modified form of CS, derived from the epimerization of GalNAc residues to IdoA (Trowbridge, 2002). It appears mainly distributed in the skin, but is also associated with the PGs Biglycan (Fisher, 1989), versican (Westergren-Thorsson, 1992) and decorin (Day, 1987), present in a great variety of tissues.
(ii) Heparan sulfate (HS) and heparin. Heparan sulfate PGs are present on the surface of all human cells (Yoon, 2005). HS is a linear polymer of repeating units with a usual GlcAβ(1,4)GlNAcα(1,4) composition. Examples of HS PGs of the ECM are perlecan(Farach-Carson, 2007), agrin (Bezakova, 2003) and collagen XVIII (Iozzo, 2005). Heparin may be considered a modified form of HS, but its presence is much more restricted and it does not bind covalently to proteins to form PGs (Nader, 1984).
(iii) Keratan sulfate (KS). PGs with KS residues appear widely distributed in tissues. The main feature of KS is the presence of galactose instead of uronic acid in its basic repeating unit. The structure of this unit is Galβ(1,4)GlcNAcβ(1,3) (Funderburgh JL, 2000). There are two fundamental classes of keratan sulfate. KSI binds to the protein core at asparagine residues via N-glycosidic linkage. KSII establishes O-glycosidic bonds with serine or threonine residues (Funderburgh JL, 2002). Some authors have suggested a third class of KS in which the O-glycosidic bond is established with serine residues via an intermediate mannose (Krusius, 1986). KS PGs include aggrecan and various members of the SLRP family(Funderburgh JL, 2000), which is discussed in greater detail in a later section.
(iv) Hyaluronic acid (HA). Hyaluronic acid is the most abundant non-sulfated GAG in the ECM. It differs from the other GAGs described so far because it never appears covalently attached to a protein core. Furthermore, disaccharide chains of HA polymerize in the extracellular space rather than in the Golgi apparatus. The flexible spiral structure of HA is composed of up to 10,000 repeats of the disaccharide GlcUAβ (1,3)GlcNAcβ(1,4) (Souza-Fernandes, 2006). It is essential for the assembly of the ECM of connective tissues and the stabilization of non-structural elements, weakly attached to the structure of the ECM (Gerdin, 1997). The length of HA chains, together with the anionic charge, yield attraction of enormous volumes of water and make this GAG a particularly determinant element for tissue hydration (Turino, 2003). In addition, HA is involved in other functions, such as tissue repair (Hou, 2005) and protection against infections and proteolytic enzymes of granulocytes (Yang, 2011).
b) Groups of proteoglycans and their functions.
(i) Hyalecticans. The hyalecticans family is composed of four ECM chondroitin sulfate PGs (CSPGs). Aggrecan (GSPG1) and versican (CSPG2) are relatively widely distributed, whereas neurocan (CSPG3) and brevican (CSPG7) are mainly restricted to the central nervous system(Yamaguchi, 2000; Viapiano, 2006; Aspberg, 1997). All hyalecticans are CSPGs secreted with a structure consisting on a broad globular domain at each end and an unfolded intermediate domain that carries the CS chains (Oohira, 2000). Through their globular domains, hyalecticans interact with hyaluronic acid, constituting the main protein binding elements between this basic component of the ECM and the cell surface (Yamaguchi, 2000). Although they have a high homology regarding globular domains, the intermediate region varies considerably in terms of sequence, length and number of CS chains. Splicing variants, glycoforms and stable truncated products are known as well, and further increase the heterogeneity of this small family of PGs (Jones, 2005). Versican is the most ubiquitous hyalectican, and its expression has been detected at the cardiac level. Via its CS chains, versican is recognized by selectins and the membrane proteoglycan CD44, whereas its protein core is recognized by other cellular receptors such as integrins, allowing versican to intervene in processes that drive adhesion and signal transduction between ECM and the cell signals (Wu, 2005).
(ii) SLRPs. Proteoglycans of the SLRP family (small leucine-rich proteoglycans) are biologically active components of the ECM. They belong to the superfamily of proteins with leucine-rich repeats (LRRs), and are spread within a great variety of tissues. The main characteristic of SLRPs is the presence of LRRs flanked by cysteine clusters in the protein core (Huxley-Jones, 2007; Iozzo, 1998; McEwan, 2006; Schaefer, 2008). After their synthesis, the SLRPs are secreted into the pericellular space where they interact with different molecules of the extracellular space and the plasma membrane, modulating a wide variety of processes (Iozzo, 1998; Schaefer, 2008; Brandan, 2008; Perrimon, 2001; Geng, 2006; Barallobre-Barreiro, 2016). For example, SLRPs participate in the fibrilogenesis of collagen and control the availability of growth factors, directly regulating cell growth (Reed, 2002). SLRPs are organized into five different classes based on their N-terminal cysteine clusters, C-terminal repeats (or ears, which are unique to SLRPs), their chromosomal organization, and amino acid and nucleotide sequence homologies. Studies in knockout mice have demonstrated some degree of functional overlap between different SLRPs (Ameye, 2002). Decorin is perhaps the archetypal SLRP. It consists of three domains: N-terminal region containing the first cysteine flanking cluster and the single GAG side chain (CS/DS), a central region with ten LRRs that constitute the main interaction element with other proteins, and the C-terminal region containing the second cysteine flanking cluster. The decorin binds to collagens I, III and VI (Scott, 1981; Thieszen, 1995; Bidanset, 1992), as well as TGFß and myostatin (Nakajima, 2007; Barallobre-Barreiro, 2016) and other proteins, controlling growth and collagen fibrilogenesis (Danielson, 1997; Michelacci, 2003).
(iii) Proteoglycans of the basement membrane. Two PGs appear as fundamental components of mammalian basement membranes; perlecan and agrin (Kruegel, 2010). The first, with a size of 469kDa, is a fundamental component for the stability of the basal membranes of the myocardium (Costell, 1999), presenting domains of interaction with other components of the basal membrane such as collagen IV and laminin (Durbeej, 2010) and with the cell surface (Peng, 1998). The N-terminal domain contains most of the anchoring sites for HS GAGs. The presence of HS combined with the localisation this PG to the basement membrane and the diversity of domains presented by the protein core, enable perlecan to control the pericellular concentration of a large number of molecules. Interestingly, endorepelinl, an N-terminal peptide of perlecan released by proteolytic action, has important angiostatic properties. From its side, agrin is a protein constituted by four domains that share similarities with the perlecan and laminins. It contains at least six binding sites for HS GAGs and five additional N-glycosylation sites. Among its various functions, agrin is a fundamental component in neuromuscular junctions (Ruegg, 1996). Last, collagens XV and XVIII are also PGs of the basement membrane (Tomono, 2002) (although structurally classified as collagens), and will be treated in the section corresponding to the collagens of the multiplexin family.
(iv) Proteoglycans of the plasma membrane. There are several families of PGs in which the protein nucleus is partly integrated into the plasma membrane, with its GAG binding regions on the extracellular side of the membrane. These PGs are important because they exert anchoring and signaling functions between cell and MEC. Syndecans and dystroglycans and CD44 are part of this group that will be briefly discussed below, in the section corresponding to mediator proteins of the cell-matrix interaction.
Glycosylation is the covalent attachment of carbohydrate molecules to the surface of proteins. It is the most prevalent and structurally complex posttranslational modification (Mann, 2003). From a structural point of view, there are two types of glycoproteins: N-glycosylated, in which sugar binds to the amide nitrogen of asparagine (Medzihradszky, 2005), and O-glycosylated, where the bond is produced through hydroxyl oxygen of Hydroxylysine, hydroxyproline, serine or threonine (Peter-Katalinić, 2005). Numerous studies have shown that natural glycosylation increases the molecular stability of proteins (Solá, 2007). In the ECM, many glycoproteins play crucial roles in the basement membrane structure and adhesion (Adams, 2002), and as regulatory molecules (Fishman, 2001; Tuloup-Minguez, 2011). The following, is a non-exhaustive summary of the groups of glycoproteins that constitute the cardiac ECM. It is noteworthy that many groups of ECM proteins carry glycosylations. Therefore, many proteins here classified as collagens or proteoglycans, can also be considered glycoproteins.
a) Glycoproteins that mediate cell adhesion.
Due to the presence of specialized domains and carbohydrate residues, glycoproteins are fundamental components for cell adhesion, mediating the anchorage of the cells to the basement membrane. Laminins and fibronectin are the glycoproteins best characterized in this function. Their presence extends to a great range of tissues.
(i) Laminins. Laminins are heterotrimeric proteins composed of different combinations of a,b and γ chains. Five different a chains, 4 b and 3 γ are currently known, in addition to different splicing variants of these. A common form of this protein, laminin 332, would consist of the combinationa3,b3 and a γ-2 (Tzu, 2008). The different trimers have a tissue-specific distribution (Durbeej, 2010) and play an important role for the structure of the ECM and the anchoring of cells to the basement membrane. Full cross shaped laminins are capable of polymerizing to give supramolecular structures in the form of a network (Cheng, 1997) which constitutes a basic component of the basement membrane. The incorporation of laminin molecules into the ECM is mediated by interactions with other proteins such as collagen IV, nidogen, fibulin and other laminins (Durbeej, 2010).
(ii) Fibronectin. Fibronectin is present as a dimer in blood plasma at micromolar concentrations. Each fibronectin subunit consists of a mosaic of type I (12), type II (2) and type III (15-17) repeating modules, and a variable region that is not homologous to other parts of fibronectin. Unlike laminin, fibronectin does not polymerize under normal physiological conditions and its presence is reduced in the basement membrane, since there is no significant passive accumulation and this is restricted to specialized areas of the cell surface (Peters, 1996). However, during embryonic development or situations of tissue damage (e.g. myocardial infarction or hypertension), fibronectin expression is increased and the tissue also recruits circulating fibronectin. Fibronectin then intervenes in cell-MEC communication and cell migration through its interaction with integrins and other transmembrane receptors (Magnusson, 1998).
b) Matricellular glycoproteins
Matricellular proteins are a heterogeneous group of proteins that interact with surface cellular receptors, other ECM proteins, growth factors and proteases, but do not function as structural proteins per se (Bornstein, 1995). For example, thrombospondin 1, SPARC or tenascin C are capable of disrupting cell-matrix interactions that play a critical role in processes such as tissue remodeling and angiogenesis (Schultz, 2009; Yan, 1999). Due to the molecular and functional diversity of this group of proteins, it would be not appropriate to choose an archetypal model or to focus on any particular example within this class, and we direct the reader to complete reviews focused on matricellular proteins (Rienks, 2016).
Collagens are a large family of proteins with triple-helix conformation present in many tissues. The presence of different types of collagen is important for a variety of functions ranging from structural support to tissue adhesion and cell migration, angiogenesis and tissue repair. Their forms are the main tensile fibrillar component in many tissues, but there are also network-forming collagens which are a key part of basement membranes. In recent years, new forms of collagen with transmembrane domains have been identified, which have further demonstrated the ubiquity and multifunctional nature of this protein family. Structurally, collagen contains three polypeptide chains that form a right-handed alpha superhelix with one staggered residue between adjacent chains (Brodsky and Persikov, 2005) and linked together by hydrogen bonds. There are several heterotrimeric forms (eg collagens I, III), yet most common structures are homotrimers, with three identical chains (collagen II, XVIII). Canonical composition of each chain is enriched in triplet repeats starting with a glycine residue and the second and third positions occupied by proline and 4-hydroxyproline, respectively (van der Rest, 1991). However, this basic structure is modified in different forms of collagen, leading to structural diversity responsible for functional and spatial diversity of this family (Bella, 2006). The N- and C-terminal chains have domains that do not correspond to the pattern described so far, known as NC or "non-collagen" domains, named by the C-terminus in numerical order.
Collagen I is the archetypal form of collagen. It retains the triple helical structure flawless and assembled into fibers to have a predominantly structural role in a wide variety of tissues. In mammals collagen I is the most abundant protein in the body and it is also the most abundant structural protein of the ECM of the healthy heart. There are at least 28 forms of collagen in vertebrates that differ in varying degrees from prototypical collagen I, but nevertheless, the line between collagens and all those proteins known as collagen-like (eg acetylcholinesterase, adiponectin, C1q , ficolin etc) is diffuse (Myllyharju and Kivirikko, 2001), making it difficult to establish a classification based on a closed number of variants. It is even common for some forms of collagen (XVIII, IX) to appear modified with GAGs, which again reflects the difficulty of establishing strict classifications of collagen and the components of the ECM in general. Based on the above basic structure, proteins which are often referred to as collagens, are those with triple-helix structure that have functions in tissue structure or its maintenance.
The following describes, in summary, the different structural families of collagens, with special emphasis on the archetypal member of each family or those with greater relevance to heart.
i) Fibrilar collagens. This group is represented by collagens I, II, III, V, XI, XXIV and XXVII, which are the main source of tensile strength in animal tissues. The diameter of the fibers varies between 12 and 500 nm, and its length is very variable, depending on the tissue and stage of development (Fleischmajer, 1988). However, the tensile properties of fibrillar collagen matrices does not depend exclusively on the size of the fibers, but intra- and intermolecular crosslinks created by the action of lisyl-oxidases (Eyre, 1984). In mammals there are 11 different genes (the missmatching is explained by the heterotrimers) for fibrillar collagens (Huxley-Jones, 2007). With the exception of collagens XXIV and XXVII, they all contain an unbroken fiber-forming domain of about 1000 residues long. They are synthesized as procollagens containing N and C-terminal telopeptides and propeptides (Prockop and Fertala, 1998) with major anchor sites for fibrillogenesis. The presence of C-terminal domains is essential for fibrillogenesis, and they specific targets for anti-procollagen C proteinases (Greenspan, 2005). Within the heart, collagen I is mainly synthesized by cardiac fibroblasts and is subjected to a slow metabolism. Their average time spent on tissue is about 100 days (Swynghedauw 1999).
ii) FACITs: Fibril Associated Collagens with Interrupted Triple helices. Composed of collagens IX, XII, XIV, XVI, XIX, XX, XXI and XXII. These collagen forms are of relatively small length. They contain one or two collagen domains that are anchored to the surface of fibrillar collagens. The most representative and studied of this group is collagen IX, which contains a GAG chain and is covalently attached to the fibers of collagen II in cartilage (Wu, 1992). Similarly, the collagen types XII and XIV are normally associated with type I collagen fibers (Keene, 1991 - Young, 2000), and interact with various SLRP family of proteoglycans (Font, 1996 - Lonzinguez, 1998).
iii) Layered networks-forming collagens. Part of this family are collagens IV, VIII and X. These collagens associate laterally and linearly to form laminar networks rather than fibers. Their functions are diverse, serving as a support for cells and tissues, molecular filtres and as permeable barriers in the developing embryo (Knup C, 2005). Collagen IV, a major component of all basement membranes is undoubtedly the most extensively characterized member of this family. The three helices that form the basic unit of collagen IV are encoded by six different genes (α1 to α6). However, despit the 56 possible combinations to give trimers, the different chains interact specifically to give only three combinations: α1α1α2, and α5α5α6 α3α4α5. In humans, α1 and α2 chains are encoded on chromosome 13 in opposite directions but under the same promoter region, appearing in all tissues. The other forms are encoded on chromosome 2 (α3 and α4) and X (α5 and α6), and have a distribution restricted to certain tissues during development (Khoshnoodi, 2008). Each chain contains an N-terminal domain called 7S, which is 26KD and cysteine and lysine rich, a 120KD collagenous domain and a C-terminal globular domain NC1 of 25KD (Hudson, 2003). Once secreted, triple helices associate to form networks that provide the scaffolding on which other components of the basement membrane such as laminins, perlecan and other proteoglycans may interact (Khoshnoodi, 2008). Both the 7S domain and the NC1 are essential for the formation of the network of collagen IV (Lebleu, 2007 - Than, 2002). It has been shown that collagen IV is essential for the maintenance of the basement membrane, yet is not essential for its formation (Pöschl 2004). At the cellular level, collagen IV receptor interacts with integrins and DDR1 (discoidin domain receptor 1), a tyrosine kinase receptor that is phosphorylated after recognition of the collagen molecule (Leitinger and Hohenester, 2007).
iv) Anchoring fibers-forming collagens. Collagen VII is the only member of this family and the major component of anchoring fibers. These specialized structures provide an additional connection between the basement membrane and underlying interstitial matrix (Keene DR, 1987). Collagen VII has mostly been studied in the skin, and several studies suggest its involvement in the disease called epidermolysis bullosa or "butterfly skin" (Jaervikallio A, 1997). Structurally it is a homotrimer whose network consists of a central segment of 145KD, which forms the triple helix, a segment NC1 (N-terminal) of 145KD and a C-terminal 34-KD segment NC2 (Lunstrum GP,1987). In the extracellular space, the triple helices are antiparallel dimers stabilized in the C-terminal region by two disulfide bonds from cysteines on the NC2 domain, while the NC1 domain interacts with collagen type IV and laminin 332 (Chen M, 1997 ). In addition, observations have been published about a weak "in vitro" interaction between type VII and I collagens. When the latter form fibers, it offers several possible sites of attachment for collagen VII, which could significantly strengthen the interaction between two proteins (Brittingham R, 2006).
v) Beads-forming collagens. This family comprises type VI, XXVI and XXVIII collagens. Collagen VI is the best characterized and appears on the genetic basis of Belthem myopathy and Ullrich dystrophy. Collagen VI is a key protein of the extracellular matrix of muscle tissue among others, and it forms microfibrilar basement membrane-associated networks. The basic unit consists of three distinct chains: alpha-1 (VI) and alpha-2 (VI), both of 140 KD and alpha-3 (VI), of up to 300 KD. This unit is assembled into tetramers in a multistep process that begins inside the cell and ends in the extracellular space (Lampe, 2005). Microfibrils formed by the tetramers are anchored to different molecules including other ECM components like collagen IV and biglycan, in addition to cell adhesion receptors on the surface of myofibers, such as integrins (Wiberg C, 2003 - Lampe AK , 2005). Thus, one of the main functions of collagen VI is to promote the stability of the sarcolemmal membrane during muscle contraction (Kanagawa M, 2006). This protein has also been involved in other processes including the assembly of the basement membrane and cell signaling events such as cell survival and regulation of myofiber size (AK Lampe, 2005).
vi) Multiplexins. Type XV and XVIII collagens are structurally homologous proteoglycans characterized by the presence of an uninterrupted central triple helix flanked by NC regions. Interruption domains permit the molecule a certain degree of flexibility between triple helix regions and allow the formation of polymers which are structurally different to those formed by fibrillar collagens (Iozzo RV, 2005). Tissue-derived type XVIII collagen is bound to heparan sulphate GAGs, while type XV appears to be chondroitin sulphate-associated. Multiplexins can therefore be considered as hybrid forms of collagen. In the heart, type XV collagen is more abundant than the XVIII, but both are ubiquitous and they are expressed in basement membranes of various tissues (Hurskainen 2005). However, it is not the role as structural molecules that makes this group particularly interesting. Although also found in other collagens, multiplexins’ NC1 domains have activity independent of their membership of the entire molecule of collagen. Endostatin (collagen XVIII) and restin (collagen XV) can be released by proteolytic action and bind various extracellular matrix molecules and cell surface to exert key regulatory effects.
MACITs: Membrane-Associated Collagen with Interrupted Triple helices. Type XIII, XXIII and XXV
collagens are type II transmembrane proteins containing a short N-terminal
cytosolic domain and a long interrupted extracellular C-terminal triple helical
domains, whicha are cleaved by a number of proteases. This collagen family has
adhesive properties and the list of identified members continues to grow. Some
of them, like ectodysplasin or gliomedin, are attributed important roles in the
development of the central nervous system (Maertens, 2007).
4) Non-glycosylated proteins and soluble components of the extracellular space.
The cardiac extracellular space comprises more than 120 proteins that are present at any single time point, as a proteomics profile of cardiac ECM revealed (Barallobre-Barreiro, 2012). This study revealed that proteins other than the aforementioned, which can be classified on broad groups, conform the extracellular environment. Proteins such as dermatopontin or matrilins are not glycosylated and do not belong to previously discussed structural groups. They are, however, important regulators of the extracellular matrix. Some of them can be considered matricellular proteins, although this is as mentioned, a structurally heterogeneous group. Proteases are key regulators of processes involving ECM remodeling and will be discussed below. Growth factors such as TGFß and cytokines such as interleukins are secreted locally or can travel with the circulation, affecting a large variety of processes locally. Last, lipoproteins which are often liver-derived can be deposited (specially in blood vessels), and this deposition is largely regulated by ECM proteins.
5) Cell-matrix interaction components.
The function of extracellular space proteins can not be understood without taking into account their interaction with the cellular component of the tissue. Being not constituent parts of the extracellular space, they are key for the establishment of physical interactions cell-matrix and therefore, central for the homeostasis and normal and pathological remodeling of the extracellular matrix. Several proteins are fundamental in the establishment of this communication. The basic aspects of these will be briefly discussed below.
Integrins. These receptors are transmembrane heterodimers composed of α and β subunits, with a long extracellular domain and a small, highly conserved intracellular domain (Humphries, 2000). The extracellular domain interacts with various components of the ECM, and the intracellular domain does so with both cell signaling molecules and cytoskeletal components (Wylie, 1979). In mammals there are 24 pairs (αβ) of different receptors that can overlap in recognition of ligands (Takada, 2007). Cardiac fibroblasts mainly express the α5β1 pair, which recognizes fibronectin and osteopontin, and the αVβ1, α5β3 and α5β3 pairs, which further bind vitronectin (Maitra, 2000). Adult cardiomyocytes express α7β1 dimer (Bracaccio, 2006). Nevertheless, although each cell type has a particular integrin signature, this is a dynamic conformation and it changes rapidly once the cells are extracted from their normal environment (Barczyk, 2010). In general, integrins can be said to be the main link between the ECM and the cytoskeleton (Geiger, 2009), and integrate both not only mechanically, but also as an active element in cellular signaling (Barczyk, 2010).
Syndecans. Syndecans are a family of proteoglycans with transmembrane protein cores. They have a short cytoplasmic domain and an extracellular domain decorated with HS and CS GAGs, which allow the interaction with a large number of growth factors and proteins of the ECM (Tkachenko, 2005; Okamoto, 2003). In cardiofibroblasts, recognition of fibronectin by syndecan-4 is necessary for proper cell migration (Granés, 2003), and syndecan-1 contributes to angiotensin II-induced cardiac fibrosis (Schellings, 2010).
Dystroglycans. The dystroglycans α and β are the products of the same transcript that after its translation undergoes proteolytic action. The α-dystroglycan (68kDa) is located in the pericellular space non-covalently attached to β-dystroglycan (27 kDa), a small transmembrane protein (Ervasti, 1991). α-dystroglycan is highly glycosylated and, through its binding to laminins, agrin and perlecan contributes significantly to the cell-MEC interaction (Michele, 2003).
CD44 / epican. Epican is a transmembrane glycoprotein encoded by a single gene but expressed in different forms by means of alternative splicing and different prostraductional modifications (Greenfield, 1999). Its long extracellular domain has an interaction domain with hyaluronic acid (Aruffo, 1990), although the interaction of CD44 with other GAGs and different proteins of the ECM (Iczkowski, 2006) and matrix metalloproteinases (MMPs) (Cichy, 2002) has been demonstrated, supporting the notion that this membrane receptor has a important role in cell-ECM signaling (Culty, 1992).
DDRs. DDR (discoidin domain receptors) proteins are important tyrosine kinase receptors in for cell-ECM interaction. DDR2 in particular, is considered a marker for certain populations of cardiac fibroblasts and is a fibrillar collagen receptor that mediates the migration and proliferation of fibroblasts (Vogel, 1999).
Homeostasis of the cardiac ECM: Matrix metalloproteinases
The slow and moderate remodelling of the ECM is characteristic of healthy cardiac tissue: ECM synthesis and degradation are in dynamic equilibrium. As mentioned, the structural basis of cardiac ECM is collagen I, which is extremely resistant to proteolytic action (Jeffrey, 1986). However, many other components are also important components of the ECM. The proper maintenance of the equilibrium in the ECM is subject to the action of several groups of proteolytic enzymes that take part in the degradation of the different components. Among these enzymes are ADAMs (a disintegrin and metalloprotease domain-containing protein), ADAMTSs (a disintegrin and metalloprotease domanin with thrombospondin motifs), serum proteases such as (plasmin, neutrophil-derived elastase, cathepsin G), cysteine proteases (cathepsins B, L and S), aspartyl proteases (cathepsin D) and MMPs (matrix metalloproteinases) (Cleutjens,1996).
MMPs are particularly relevant for the remodelling of the cardiac ECM in healthy and pathological conditions. More than 25 members constitute this family of proteases that use zinc as a cofactor. In general, they are synthesized as inactive pro-MMPs in which the catalytic domain remains inaccessible due to interaction of the N-terminal propeptide with Zn2+. Once the propeptide is released by proteolytic action, the catalytic domain is exposed and the enzyme becomes activated (Suzuki, 1995). After secretion, pro-MMPs bind to various molecules of the ECM and remain latent, constituting a reservoir that can be immediately activated upon demand for proteolytic activity in the tissue (Spinale, 2007).
The MMPs identified so far in the myocardium belong to four families (Spinale, 2007; Spinale 2009) and are synthesized by all major cell types of the myocardium. Together, they can degrade almost all of the proteins present in the ECM (Spinale, 2007) and the range of identified MMP substrates is constantly growing (Somerville, 2003). The following table, modified from Somerville et al., 2003, contains the MMPs identified in cardiac tissue.
Importantly, the importance of MMPs expands beyond the degradation of ECM proteins. Several MMPs have proteolytic targets on cytokines, bioactive peptides and growth factors, in turn affecting various processes in the myocardium (Gearing, 1995; Hwang, 2004; Lee, 2004). Moreover, some MMPs have the ability to recognize pro-MMPs and induce their activation (Woessner, 2003).
Due to the effectiveness of these enzymes degrading their substrates, it is essential to establish a strict control of their activity. TIMPs (tissue inhibitors of matrix metalloproteainases) are low molecular weight proteins that bind avidly to MMPs inhibiting their activity. TIMP-4 is the most highly expressed form in cardiac tissue (Greene, 1996). In addition to MMPs, TIMPs inhibit other proteases with important roles in ECM. TIMP-4 specifically recognizes ADAM proteases ADAM-17, -28 and -33 family, highlighting the importance of TIMPs in the regulation of remodeling processes in the ECM (Brew, 2010). MMPs activity is important for the development of various cardiovascular pathologies. The control of their activation, but also of their transcription, are highly regulated processes that depend on various pathways of activation and repression involving cytokines, growth factors, bioactive peptides and physical stimuli (Deschamps, 2006; Tong, 2011; Kandasamy, 2010). Together, all of them contribute to the maintenance of an extracellular environment that allows for appropriate cardiac function.
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