BIOCHEMISTRY OF MUSCLE, MUSCLE CONTRACTION BIOCHEMISTRY OF CONNECTIVE TISSUE

BIOCHEMISTRY OF CONNECTIVE TISSUE

 

Connective tissue

Connective tissue (CT) is a kind of biological tissue that supports, connects, or separates different types of tissues and organs of the body. It is one of the four general classes of biological tissuesthe others of which are epithelial, muscular, and nervous tissues.

All CT has three main components: cells, fibers, and extracellular matrices, all immersed in the body fluids.

Figure 6 (Picture 413.jpg)

 

Connective tissue can be broadly subdivided into connective tissue proper, special connective tissue, and series of other, less classifiable types of connective tissues. Connective tissue proper consists of loose connective tissue and dense connective tissue (which is further subdivided into dense regular and dense irregular connective tissues.) Special connective tissue consists of reticular connective tissue, adipose tissue, cartilage, bone, and blood. Other kinds of connective tissues include fibrous, elastic, and lymphoid connective tissues.

Fibroblasts are the cells responsible for the production of some CT.

Type-I collagen, is present in many forms of connective tissue, and makes up about 25% of the total protein content of the mammalian body.

 

BIOMEDICAL IMPORTANCE

 

Most mammalian cells are located in tissues where they are surrounded by a complex extracellular matrix (ECM) often referred to as connective tissue. The ECM contains three major classes of biomolecules:

(1)        the structural proteins, collagen, elastin, and fibrillin;

(2) certain specialized proteins such as fibrillin, fibronectin, and laminin; and

(3) proteoglycans, whose chemical natures are described below.

 

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The extracellular matrix

 

The ECM has been found to be involved in many normal and pathologic processeseg, it plays important roles in development, in inflammatory states, and in the spread of cancer cells. Involvement of certain components of the ECM has been documented in both rheumatoid arthritis and osteoarthritis. Several diseases (eg, osteogenesis imperfecta and a number of types of the Ehlers-Danlos syndrome) are due to genetic disturbances of the synthesis of collagen. Specific components of proteoglycans (the glycosaminoglycans; GAGs) are affected in the group of genetic disorders known as the mucopolysaccharidoses.

Changes occur in the ECM during the aging process. This chapter describes the basic biochemistry of the three major classes of biomolecules found in the ECM and illustrates their biomedical significance.

Major biochemical features of two specialized forms of ECMbone and cartilageand of a number of diseases involving them are also briefly considered.

 

Functions of connective tissue

- Storage of energy

- Protection of organs

- Provision of structural framework for the body

- Connection of body tissues

- Connection of epithelial tissues to muscle tissues

 

Characteristics of connective tissue and fiber types

Cells are spread through an extracellular fluid.

Ground substance - A clear, colorless, and viscous fluid containing glycosaminoglycans and proteoglycans to fix the bodywater and the collagen fibers in the intercellular spaces. Ground substance slows the spread of pathogens.

Fibers. Not all types of CT are fibrous. Examples include adipose tissue and blood. Adipose tissue gives "mechanical cushioning" to our body, among other functions. Although there is no dense collagen network in adipose tissue, groups of adipose cells are kept together by collagen fibers and collagen sheets in order to keep fat tissue under compression in place (for example, the sole of the foot). The matrix of blood is plasma.

Both the ground substance and proteins (fibers) create the matrix for CT.

 

Types of fibers:Tissue Purpose Components Location

Collagenous fibers Alpha polypeptide chains → tendon, ligament, skin, cornea, cartilage, bone, blood vessels, gut, and intervertebral disc.

Elastic fibers elastic microfibril & elastin → extracellular matrix

Reticular fibers Type-III collagen → liver, bone marrow, lymphatic organs.

 

Structure and functions of collagen.

Collagen is a group of naturally occurring proteins found in animals, especially in the flesh and connective tissues of vertebrates.

It is the main component of connective tissue, and is the most abundant protein in mammals, making up about 25% to 35% of the whole-body protein content. Collagen, in the form of elongated fibrils, is mostly found in fibrous tissues such as tendon, ligament and skin, and is also abundant in cornea, cartilage, bone, blood vessels, the gut, and intervertebral disc. The fibroblast is the most common cell which creates collagen. In muscle tissue, it serves as a major component of the endomysium.

 

 

 

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Fig.Functions of collagen

 

Collagen constitutes one to two percent of muscle tissue, and accounts for 6% of the weight of strong, tendinous muscles. Gelatin, which is used in food and industry, is collagen that has been irreversibly hydrolyzed. Collagen is composed of a triple helix, which generally consists of two identical chains (α1) and an additional chain that differs slightly in its chemical composition (α2). The amino acid composition of collagen is atypical for proteins, particularly with respect to its high hydroxyproline content. The most common motifs in the amino acid sequence of collagen are glycine-proline-X and glycine-X-hydroxyproline, where X is any amino acid other than glycine, proline or hydroxyproline.

 

http://www.elmhurst.edu/~chm/vchembook/images/567collagen.gif

 

Figure 1. Collagen structures

 

Synthesis

First, a three dimensional stranded structure is assembled, with the amino acids glycine and proline as its principal components. This is not yet collagen but its precursor, procollagen. A recent study shows that vitamin C must have an important role in its synthesis. Prolonged exposure of cultures of human connective-tissue cells to ascorbate induced an eight-fold increase in the synthesis of collagen with no increase in the rate of synthesis of other proteins. Since the production of procollagen must precede the production of collagen, vitamin C must have a role in this step. The conversion involves a reaction that substitutes a hydroxyl group, OH, for a hydrogen atom, H, in the proline residues at certain points in the polypeptide chains, converting those residues to hydroxyproline. This hydroxylation reaction organizes the chains in the conformation necessary for them to form a triple helix. The hydroxylation, next, of the residues of the amino acid lysine, transforming them to hydroxylysine, is then needed to permit the cross-linking of the triple helices into the fibers and networks of the tissues.

Collagen synthesis

Figure Collagen Synthesis

 

These hydroxylation reactions are catalyzed by two different enzymes: prolyl-4-hydroxylase and lysyl-hydroxylase. Vitamin C also serves with them in inducing these reactions. in this service, one molecule of vitamin C is destroyed for each H replaced by OH. The synthesis of collagen occurs inside and outside of the cell. The formation of collagen which results in fibrillary collagen (most common form) is discussed here. Meshwork collagen, which is often involved in the formation of filtration systems is the other form of collagen. It should be noted that all types of collagens are triple helixes, and the differences lie in the make-up of the alpha peptides created in step 2.

1.          Transcription of mRNA: There are approximately 34 genes associated with collagen formation, each coding for a specific mRNA sequence, and typically have the "COL" prefix. The beginning of collagen synthesis begins with turning on genes which are associated with the formation of a particular alpha peptide (typically alpha 1, 2 or 3).

2.          Pre-pro-peptide Formation: Once the final mRNA exits from the cell nucleus and enters into the cytoplasm it links with the ribosomal subunits and the process of translation occurs. The early/first part of the new peptide is known as the signal sequence. The signal sequence on the N-terminal of the peptide is recognized by a signal recognition particle on the endoplasmic reticulum, which will be responsible for directing the pre-pro-peptide into the endoplasmic reticulum. Therefore, once the synthesis of new peptide is finished, it goes directly into the endoplasmic reticulum for post-translational processing. Note that it is now known as pre-pro-collagen.

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Fig. Biosynthesis of collagen from Preprocollagen

 

3.          Alpha Peptide to Procollagen: Three modifications of the pre-pro-peptide occur leading to the formation of the alpha peptide. Secondly, the triple helix known as procollagen is formed before being transported in a transport vesicle to the golgi apparatus. 1) The signal peptide on the N-terminal is dissolved, and the molecule is now known as propeptide (not procollagen). 2) Hydroxylation of lysines and prolines on propeptide by the enzymes prolyl hydroxylase and lysyl hydroxylase (to produce hydroxyproline and hydroxylysine) occurs to aid crosslinking of the alpha peptides. It is this enzymatic step that requires vitamin C as a cofactor. In scurvy, the lack of hydroxylation of prolines and lysines causes a looser triple helix (which is formed by 3 alpha peptides). 3) Glycosylation occurs by adding either glucose or galactose monomers onto the hydroxy groups that were placed onto lysines, but not on prolines. From here the hydroxylated and glycosylated propeptide twists towards the left very tightly and then three propeptides will form a triple helix. It is important to remember that this molecule, now known as procollagen (not propeptide) is composed of a twisted portion (center) and two loose ends on either end. At this point the procollagen is packaged into a transfer vesicle destined for the golgi apparatus.

4.          Golgi Apparatus Modification: In the golgi apparatus, the procollagen goes through one last post-translational modification before being secreted out of the cell. In this step oligosaccharides (not monosaccharides like in step 3) are added, and then the procollagen is packaged into a secretory vesicle destined for the extracellular space.

5.          Formation of Tropocollagen: Once outside the cell, membrane bound enzymes known as collagen peptidases, remove the "loose ends" of the procollagen molecule. What is left is known as tropocollagen. Defect in this step produces one of the many collagenopathies known as Ehlers-Danlos syndrome. This step is absent when synthesizing type III, a type of fibrilar collagen.

6.          Formation of the Collagen Fibril: Lysyl oxidase an extracellular enzyme produces the final step in the collagen synthesis pathway. This enzyme acts on lysines and hydroxylysines producing aldehyde groups, which will eventually undergo covalent bonding between tropocollagen molecules. This polymer of tropocollogen is known as a collagen fibril.

Amino acids

Collagen has an unusual amino acid composition and sequence:

              Glycine is found at almost every third residue

              Proline (Pro) makes up about 17% of collagen

              Collagen contains two uncommon derivative amino acids not directly inserted during translation. These amino acids are found at specific locations relative to glycine and are modified post-translationally by different enzymes, both of which require vitamin C as a cofactor.

o              Hydroxyproline (Hyp), derived from proline.

o              Hydroxylysine (Hyl), derived from lysine (Lys). Depending on the type of collagen, varying numbers of hydroxylysines are glycosylated (mostly having disaccharides attached).

Cortisol stimulates degradation of (skin) collagen into amino acids.

Collagen I formation

Most collagen forms in a similar manner, but the following process is typical for type I:

1.          Inside the cell

1.           Two types of peptide chains are formed during translation on ribosomes along the rough endoplasmic reticulum (RER): alpha-1 and alpha-2 chains. These peptide chains (known as preprocollagen) have registration peptides on each end and a signal peptide.

2.           Polypeptide chains are released into the lumen of the RER.

3.           Signal peptides are cleaved inside the RER and the chains are now known as pro-alpha chains.

4.           Hydroxylation of lysine and proline amino acids occurs inside the lumen. This process is dependent on ascorbic acid (Vitamin C) as a cofactor.

5.           Glycosylation of specific hydroxylysine residues occurs.

6.           Triple ɣ helical structure is formed inside the endoplasmic reticulum from each two alpha-1 chains and one alpha-2 chain.

7.           Procollagen is shipped to the Golgi apparatus, where it is packaged and secreted by exocytosis.

2.          Outside the cell

1.           Registration peptides are cleaved and tropocollagen is formed by procollagen peptidase.

2.           Multiple tropocollagen molecules form collagen fibrils, via covalent cross-linking (aldol reaction) by lysyl oxidase which links hydroxylysine and lysine residues. Multiple collagen fibrils form into collagen fibers.

3.           Collagen may be attached to cell membranes via several types of protein, including fibronectin and integrin.

 

Synthetic pathogenesis

Vitamin C deficiency causes scurvy, a serious and painful disease in which defective collagen prevents the formation of strong connective tissue. Gums deteriorate and bleed, with loss of teeth; skin discolors, and wounds do not heal. Prior to the eighteenth century, this condition was notorious among long duration military, particularly naval, expeditions during which participants were deprived of foods containing Vitamin C.

An autoimmune disease such as lupus erythematosus or rheumatoid arthritis may attack healthy collagen fibers.

Many bacteria and viruses have virulence factors which destroy collagen (such as the enzyme collagenase) or interfere with its production.

Molecular structure

The tropocollagen or collagen molecule is a subunit of larger collagen aggregates such as fibrils. At approximately 300 nm long and 1.5 nm in diameter, it is made up of three polypeptide strands (called alpha peptides, see step 2), each possessing the conformation of a left-handed helix (its name is not to be confused with the commonly occurring alpha helix, a right-handed structure). These three left-handed helices are twisted together into a right-handed coiled coil, a triple helix or "super helix", a cooperative quaternary structure stabilized by numerous hydrogen bonds. With type I collagen and possibly all fibrillar collagens if not all collagens, each triple-helix associates into a right-handed super-super-coil referred to as the collagen microfibril. Each microfibril is interdigitated with its neighboring microfibrils to a degree that might suggest they are individually unstable, although within collagen fibrils, they are so well ordered as to be crystalline.

A distinctive feature of collagen is the regular arrangement of amino acids in each of the three chains of these collagen subunits. The sequence often follows the pattern Gly-Pro-X or Gly-X-Hyp, where X may be any of various other amino acid residues. Proline or hydroxyproline constitute about 1/6 of the total sequence. With glycine accounting for the 1/3 of the sequence, this means approximately half of the collagen sequence is not glycine, proline or hydroxyproline, a fact often missed due to the distraction of the unusual GX1X2 character of collagen alpha-peptides. The high glycine content of collagen is important with respect to stabilization of the collagen helix as this allows the very close association of the collagen fibers within the molecule, facilitating hydrogen bonding and the formation of intermolecular cross-links. This kind of regular repetition and high glycine content is found in only a few other fibrous proteins, such as silk fibroin. About 75-80% of silk is (approximately) -Gly-Ala-Gly-Ala- with 10% serine, and elastin is rich in glycine, proline, and alanine (Ala), whose side group is a small methyl group. Such high glycine and regular repetitions are never found in globular proteins save for very short sections of their sequence. Chemically reactive side groups are not needed in structural proteins, as they are in enzymes and transport proteins; however, collagen is not quite just a structural protein. Due to its key role in the determination of cell phenotype, cell adhesion, tissue regulation and infrastructure, many sections of its nonproline-rich regions have cell or matrix association / regulation roles. The relatively high content of proline and hydroxyproline rings, with their geometrically constrained carboxyl and (secondary) amino groups, along with the rich abundance of glycine, accounts for the tendency of the individual polypeptide strands to form left-handed helices spontaneously, without any intrachain hydrogen bonding.

Because glycine is the smallest amino acid with no side chain, it plays a unique role in fibrous structural proteins. In collagen, Gly is required at every third position because the assembly of the triple helix puts this residue at the interior (axis) of the helix, where there is no space for a larger side group than glycines single hydrogen atom. For the same reason, the rings of the Pro and Hyp must point outward. These two amino acids help stabilize the triple helixHyp even more so than Pro; a lower concentration of them is required in animals such as fish, whose body temperatures are lower than most warm-blooded animals. Lower proline and hydroxyproline contents are characteristic of cold-water, but not warm-water fish; the latter tend to have similar proline and hydroxyproline contents to mammals. The lower proline and hydroxproline contents of cold-water fish and other poikilotherm animals leads to their collagen having a lower thermal stability than mammalian collagen. This lower thermal stability means that gelatin derived from fish collagen is not suitable for many food and industrial applications.

The tropocollagen subunits spontaneously self-assemble, with regularly staggered ends, into even larger arrays in the extracellular spaces of tissues. In the fibrillar collagens, the molecules are staggered from each other by about 67 nm (a unit that is referred to as D and changes depending upon the hydration state of the aggregate). Each D-period contains four plus a fraction collagen molecules, because 300 nm divided by 67 nm does not give an integer (the length of the collagen molecule divided by the stagger distance D). Therefore, in each D-period repeat of the microfibril, there is a part containing five molecules in cross-section, called the overlap, and a part containing only four molecules, called the "gap". The triple-helices are also arranged in a hexagonal or quasihexagonal array in cross-section, in both the gap and overlap regions.

There is some covalent crosslinking within the triple helices, and a variable amount of covalent crosslinking between tropocollagen helices forming well organized aggregates (such as fibrils). Larger fibrillar bundles are formed with the aid of several different classes of proteins (including different collagen types), glycoproteins and proteoglycans to form the different types of mature tissues from alternate combinations of the same key players. Collagen's insolubility was a barrier to the study of monomeric collagen until it was found that tropocollagen from young animals can be extracted because it is not yet fully crosslinked. However, advances in microscopy techniques (i.e. electron microscopy (EM) and atomic force microscopy (AFM)) and X-ray diffraction have enabled researchers to obtain increasingly detailed images of collagen structure in situ. These later advances are particularly important to better understanding the way in which collagen structure affects cell-cell and cell-matrix communication, and how tissues are constructed in growth and repair, and changed in development and disease. For example using AFM based nanoindentation it has been shown that a single collagen fibril is a heterogeneous material along its axial direction with significantly different mechanical properties in its gap and overlap regions, correlating with its different molecular organizations in these two regions.

Collagen fibrils are semicrystalline aggregates of collagen molecules. Collagen fibers are bundles of fibrils.

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Fig. Synthesis Of Collagen

 

Collagen fibrils/aggregates are arranged in different combinations and concentrations in various tissues to provide varying tissue properties. In bone, entire collagen triple helices lie in a parallel, staggered array. 40 nm gaps between the ends of the tropocollagen subunits (approximately equal to the gap region) probably serve as nucleation sites for the deposition of long, hard, fine crystals of the mineral component, which is (approximately) Ca10(OH)2(PO4)6. Type I collagen gives bone its tensile strength.

 

Types and associated disorders

Collagen occurs in many places throughout the body. Over 90% of the collagen in the body, however, is of type I.

So far, 28 types of collagen have been identified and described. The five most common types are:

             Collagen I: skin, tendon, vascular ligature, organs, bone (main component of the organic part of bone)

             Collagen II: cartilage (main component of cartilage)

             Collagen III: reticulate (main component of reticular fibers), commonly found alongside type I.

             Collagen IV: forms bases of cell basement membrane

             Collagen V: cell surfaces, hair and placenta

Collagen-related diseases most commonly arise from genetic defects or nutritional deficiencies that affect the biosynthesis, assembly, postranslational modification, secretion, or other processes involved in normal collagen production.

 

Tabl. Types Of Collagen

 

A Number of Genetic Diseases Result From Abnormalities in the Synthesis of Collagen

About 30 genes encode collagen, and its pathway of biosynthesis is complex, involving at least eight enzyme-catalyzed posttranslational steps. Thus, it is not surprising that a number of diseases are due to mutations in collagen genes or in genes encoding some of the enzymes involved in these posttranslational

modifications. The diseases affecting bone (eg, osteogenesis imperfecta) and cartilage (eg, the chondrodysplasias) will be discussed later in this chapter.

Ehlers-Danlos syndrome comprises a group of inherited disorders whose principal clinical features are hyperextensibility of the skin, abnormal tissue fragility,

and increased joint mobility. The clinical picture is variable, reflecting underlying extensive genetic heterogeneity.

At least 10 types have been recognized, most but not all of which reflect a variety of lesions in the synthesis of collagen. Type IV is the most serious because of its tendency for spontaneous rupture of arteries or the bowel, reflecting abnormalities in type III collagen.

Patients with type VI, due to a deficiency of lysyl hydroxylase, exhibit marked joint hypermobility and a tendency to ocular rupture. A deficiency of procollagen

N-proteinase, causing formation of abnormal thin, irregular collagen fibrils, results in type VIIC, manifested by marked joint hypermobility and soft skin.

Alport syndrome is the designation applied to a number of genetic disorders (both X-linked and autosomal) affecting the structure of type IV collagen fibers, the major collagen found in the basement membranes of the renal glomeruli (see discussion of laminin, below). Mutations in several genes encoding type IV collagen fibers have been demonstrated. The presenting sign is hematuria, and patients may eventually develop end-stage renal disease. Electron microscopy reveals characteristic abnormalities of the structure of the basement membrane and lamina densa.

 

 

In epidermolysis bullosa, the skin breaks and blisters as a result of minor trauma. The dystrophic form is due to mutations in COL7A1, affecting the structure of type VII collagen. This collagen forms delicate fibrils that anchor the basal lamina to collagen fibrils in the dermis. These anchoring fibrils have been shown to be markedly reduced in this form of the disease, probably resulting in the blistering. Epidermolysis bullosa simplex, another variant, is due to mutations in keratin 5.

Scurvy affects the structure of collagen. However, it is due to a deficiency of ascorbic acid and is not a genetic disease. Its major signs are bleeding gums, subcutaneous hemorrhages, and poor wound healing. These signs reflect impaired synthesis of collagen due to deficiencies of prolyl and lysyl hydroxylases, both of which require ascorbic acid as a cofactor.

Osteoporosis - Not inherited genetically, brought on with age, associated with reduced levels of collagen in the skin and bones, growth hormone injections are being researched as a possible treatment to counteract any loss of collagen.

Knobloch syndrome - Caused by a mutation in the collagen XVIII gene, patients present with protrusion of the brain tissue and degeneration of the retina, an individual who has family members suffering from the disorder are at an increased risk of developing it themselves as there is a hereditary link.

 

 

 

Characteristics

Collagen is one of the long, fibrous structural proteins whose functions are quite different from those of globular proteins such as enzymes. Tough bundles of collagen called collagen fibers are a major component of the extracellular matrix that supports most tissues and gives cells structure from the outside, but collagen is also found inside certain cells. Collagen has great tensile strength, and is the main component of fascia, cartilage, ligaments, tendons, bone and skin. Along with soft keratin, it is responsible for skin strength and elasticity, and its degradation leads to wrinkles that accompany aging. It strengthens blood vessels and plays a role in tissue development. It is present in the cornea and lens of the eye in crystalline form.

Uses

Collagen has a wide variety of applications, from food to medical. For instance, it is used in cosmetic surgery and burns surgery. It is widely used in the form of collagen casings for sausages.

If collagen is sufficiently denatured, e.g. by heating, the three tropocollagen strands separate partially or completely into globular domains, containing a different secondary structure to the normal collagen polyproline II (PPII), e.g. random coils. This process describes the formation of gelatin, which is used in many foods, including flavored gelatin desserts. Besides food, gelatin has been used in pharmaceutical, cosmetic, and photography industries. From a nutritional point of view, collagen and gelatin are a poor-quality sole source of protein since they do not contain all the essential amino acids in the proportions that the human body requiresthey are not 'complete proteins' (as defined by food science, not that they are partially structured). Manufacturers of collagen-based dietary supplements claim that their products can improve skin and fingernail quality as well as joint health. However, mainstream scientific research has not shown strong evidence to support these claims. Individuals with problems in these areas are more likely to be suffering from some other underlying condition (such as normal aging, dry skin, arthritis etc.) rather than just a protein deficiency.

From the Greek for glue, kolla, the word collagen means "glue producer" and refers to the early process of boiling the skin and sinews of horses and other animals to obtain glue. Collagen adhesive was used by Egyptians about 4,000 years ago, and Native Americans used it in bows about 1,500 years ago. The oldest glue in the world, carbon-dated as more than 8,000 years old, was found to be collagenused as a protective lining on rope baskets and embroidered fabrics, and to hold utensils together; also in crisscross decorations on human skulls. Collagen normally converts to gelatin, but survived due to the dry conditions. Animal glues are thermoplastic, softening again upon reheating, and so they are still used in making musical instruments such as fine violins and guitars, which may have to be reopened for repairsan application incompatible with tough, synthetic plastic adhesives, which are permanent. Animal sinews and skins, including leather, have been used to make useful articles for millennia.

 

Elastin main protein of elastic fibrils, structure and biological role.

Elastin is a protein in connective tissue that is elastic and allows many tissues in the body to resume their shape after stretching or contracting. Elastin helps skin to return to its original position when it is poked or pinched. Elastin is also an important load-bearing tissue in the bodies of vertebrates and used in places where mechanical energy is required to be stored. In humans, elastin is encoded by the ELN gene.

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File:ECM elastic fibres skin.jpg

Function

This gene encodes a protein that is one of the two components of elastic fibers. The encoded protein is rich in hydrophobic amino acids such as glycine and proline, which form mobile hydrophobic regions bounded by crosslinks between lysine residues. Multiple transcript variants encoding different isoforms have been found for this gene. The other name for elastin is tropoelastin. The characterization of disorder is consistent with an entropy-driven mechanism of elastic recoil. It is concluded that conformational disorder is a constitutive feature of elastin structure and function.

Clinical significance

Deletions and mutations in this gene are associated with supravalvular aortic stenosis (SVAS) and autosomal dominant cutis laxa. Other associated defects in elastin include Marfan's Syndrome and emphysema caused by α1-antitrypsin deficiency.

Composition

Elastic fiber is composed of the protein fibrillin and elastin made of simple amino acids such as glycine, valine, alanine, and proline. The total elastin ranges from 58 to 75% of the weight of the dry defatted artery in normal canine arteries.[6] Comparison between fresh and digested tissues shows that, at 35% strain, a minimum of 48% of the arterial load is carried by elastin, and a minimum of 43% of the change in stiffness of arterial tissue is due to the change in elastin stiffness. Elastin is made by linking many soluble tropoelastin protein molecules, in a reaction catalyzed by lysyl oxidase, to make a massive insoluble, durable cross-linked array. The amino acid responsible for these cross-links is lysine. Tropoelastin is a specialized protein with a molecular weight of 64 to 66 kDa, and an irregular or random coil conformation made up of 830 amino acids.

Desmosine and isodesmosine are types of links for the tropoelastin molecules.

Elastic Fibers synthesis

 

Tissue distribution

Elastin serves an important function in arteries as a medium for pressure wave propagation to help blood flow and is particularly abundant in large elastic blood vessels such as the aorta. Elastin is also very important in the lungs, elastic ligaments, the skin, and the bladder, elastic cartilage. It is present in all vertebrates above the jawless fish.

Table 1 summarizes the main differences between collagen and elastin.

 

Table 1. Major differences between collagen and elastin.

 

 

 

In contrast to collagen, which forms fibers that are tough and have high tensile strength, elastin is a connective tissue protein with rubber-like properties. Elastic fibers composed of elastin and glycoprotein microfibrils are found in the lungs, the walls of large arteries, and elastic ligaments.

They can be stretched to several times their normal length, but recoil to their original shape when the stretching force is relaxed.

A. Structure of elastin

Elastin is an insoluble protein polymer synthesized from a precursor, tropoelastin, which is a linear polypeptide composed of about 700 amino acids that are primarily small and nonpolar (for example, glycine, alanine, and valine). Elastin is also rich in proline and lysine, but contains only a little hydroxyproline and hydroxy lysine.

Tropoelastin is secreted by the cell into the extracellular space. There it interacts with specific glycoprotein microfibrils, such as fibrillin, which function as a scaffold onto which tropoelastin is deposited. Some of the lysyl side chains of the tropoelastin poly peptides are oxidatively deaminated by lysyl oxidase, forming allysine residues. Three of the allysyl side chains plus one unaltered lysyl side chain from the same or neighboring polypeptides form a desmosine cross-link (Figure 4.12). This produces elastinan extensively interconnected, rubbery network that can stretch and bend in any direction when stressed, giving connective tissue elasticity (Figure 4.13). Mutations in the fibrillin-1 protein are responsible for Marfan syndromea connective tissue disorder characterized by impaired structural integrity in the skeleton, the eye, and the cardiovascular system. With this disease, abnormal fibrillin protein is incorporated into microfibrils along with normal fibrillin, inhibiting the formation of functional microfibrils. [Note: Patients with OI, EDS, or Marfan syndrome may have blue sclera due to tissue thinning that allows underlying pigment to show through.]

B. Role of 1-antitrypsin in elastin degradation

1. 1-Antitrypsin: Blood and other body fluids contain a protein, α1-antitrypsin (α1-AT, A1AT, currently also called α1-antiproteinase), that inhibits a number of proteolytic enzymes (also called proteases or proteinases) that hydrolyze and destroy proteins. [Note: The inhibitor was originally named α1-antitrypsin because it inhibits the activity of trypsin (a proteolytic enzyme synthesized as trypsinogen by the pancreas] α1-AT comprises more than 90% of the α1-globulin fraction of normal plasma. α1-AT has the important physiologic role of inhibiting neutrophil elastasea powerful protease that is released into the extracellular space, and degrades elastin of alveolar walls, as well as other structural proteins in a variety of tissues (Figure 4.14). Most of the α1-AT found in plasma is synthesized and secreted by the liver. The remainder is synthesized by several tissues, including monocytes and alveolar macrophages, which may be important in the prevention of local tissue injury by elastase.

 

2. Role of 1-AT in the lungs: In the normal lung, the alveoli are chronically exposed to low levels of neutrophil elastase released from activated and degenerating neutrophils. This proteolytic activity can destroy the elastin in alveolar walls if unopposed by the action of α1-AT, the most important inhibitor of neutrophil elastase (see Figure 4.14). Because lung tissue cannot regenerate, emphysema results from the destruction of the connective tissue of alveolar walls.

3. Emphysema resulting from 1-AT deficiency:

In the United States, approximately 25% of patients with emphysema are predisposed to the disease by inherited defects in α1-AT. A number of different mutations in the gene for α1-AT are known to cause a deficiency of this protein, but one single purine base mutation (GAG → AAG, resulting in the substitution of lysine for glutamic acid at position 342 of the protein) is clinically the most widespread.

The polymerization of the mutated protein in the endoplasmic reticulum of hepatocytes causes decreased secretion of

α1-AT by the liver. The accumulated polymer may result in cirrhosis (scarring of the liver). In the United States, the α1-AT mutation is most common in Caucasians of Northern European ancestry.

An individual must inherit two abnormal α1-AT alleles to be at risk for the development of emphysema. In a heterozygote, with one normal and one defective gene, the levels of α1-AT are sufficient to protect the alveoli from damage. [Note: A specific α1-AT methionine is required for the binding of the inhibitor to its target proteases.

Smoking causes the oxidation and subsequent inactivation of that methionine residue, thereby rendering the inhibitor powerless to neutralize elastase. Smokers with α1-AT deficiency, therefore, have a considerably elevated rate of lung destruction and a poorer survival rate than nonsmokers with the deficiency.] The deficiency of elastase inhibitor can be reversed by augmentation therapyweekly intravenous administration of α1-AT. The α1-AT diffuses from the blood into the lung, where it reaches therapeutic levels in the fluid surrounding the lung epithelial cells.

 

Laminin Structure

             cross-shaped glycoprotein

             3 polypeptides a, b1, b2

             carbohydrate (13% by weight)

             Mr 900K

             separate binding domains

                     collagen IV

                     heparin

                     heparin sulphate

                     cell binding

                     cell specific binding - liver, nerve

                     cell surface receptor

 

 

Laminin Function

             cell adhesion

             migration pathways

             stimulates growth of axons

             development and regeneration

             differentiation

             basal laminae

             most abundant linking glycoprotein

 

PROTEOGLYCANS & GLYCOSAMINOGLYCANS

 

The Glycosaminoglycans Found in Proteoglycans Are Built Up of Repeating Disaccharides

 

Proteoglycans are proteins that contain covalently linked glycosaminoglycans. A number of them have been characterized and given names such as syndecan, betaglycan, serglycin, perlecan, aggrecan, versican, decorin, biglycan, and fibromodulin. They vary in tissue distribution, nature of the core protein, attached glycosaminoglycans, and function. The proteins bound covalently to glycosaminoglycans are called core proteins; they have proved difficult to isolate and characterize, but the use of recombinant DNA technology is beginning to yield important information about their structures. The amount of carbohydrate in a proteoglycan is usually much greater than is found in a glycoprotein and may comprise up to 95% of its weight. Figures 2 and 3 show the general structure of one particular proteoglycan, aggrecan, the major type found in cartilage.

Figure 2. Dark field electron micrograph of a proteoglycan aggregate in which the proteoglycan subunits and filamentous backbone are particularly well extended.

 

It is very large (about 2 × 103 kDa), with its overall structure resembling that of a bottle brush. It contains a long strand of hyaluronic acid (one type of GAG) to which link proteins are attached noncovalently.

Figure 3. Schematic representation of the proteoglycan aggrecan.

 

In turn, these latter interact noncovalently with core protein molecules from which chains of other GAGs (keratan sulfate and chondroitin sulfate in this case) project. More details on this macromolecule are given when cartilage is discussed below.

There are at least seven glycosaminoglycans (GAGs): hyaluronic acid, chondroitin sulfate, keratan sulfates I and II, heparin, heparan sulfate, and dermatan sulfate. A GAG is an unbranched polysaccharide made up of repeating disaccharides, one component of which is always an amino sugar (hence the name GAG), either D-glucosamine or D-galactosamine. The other component of the repeating disaccharide (except in the case of keratan sulfate) is a uronic acid, either L-glucuronic acid (GlcUA) or its 5′-epimer, L-iduronic acid (IdUA). With the exception of hyaluronic acid, all the GAGs contain sulfate groups, either as O-esters or as N-sulfate (in heparin and heparan sulfate).

Hyaluronic acid affords another exception because there is no clear evidence that it is attached covalently to protein, as the definition of a proteoglycan given above specifies. Both GAGs and proteoglycans have proved difficult to work with, partly because of their complexity. However, they are major components of the ground substance; they have a number of important biologic roles; and they are involved in a number of disease processesso that interest in them is increasing rapidly.

Proteoglycans (mucoproteins) are formed of glycosaminoglycans (GAGs) covalently attached to the core proteins.

They are found in all connective tissues, extracellular matrix (ECM) and on the surfaces of many cell types. Proteoglycans are remarkable for their diversity (different cores, different numbers of GAGs with various lenghts and compositions).

Glycosaminoglycans forming the proteoglycans are the most abundant heteropolisaccharides in the body. They are long unbranched molecules containing a repeating disaccharide unit. Usually one sugar is an uronic acid (either D-glucuronic or L-iduronic) and the other is either GlcNAc or GalNAc. One or both sugars contain sulfate groups (the only exception is hyaluronic acid).

GAGs are highly negatively charged what is essential for their function.

THE SPECIFIC GAGs OF PHYSIOLOGICAL SIGNIFICANCE ARE :

Hyaluronic acid (D-glucuronate + GlcNAc)

Occurence : synovial fluid, ECM of loose connective tissue

Hyaluronic acid is unique among the GAGs because it does not contain any sulfate and is not found covalently attached to proteins. It forms non-covalently linked complexes with proteoglycans in the ECM. Hyaluronic acid polymers are very large (100 - 10,000 kD) and can displace a large volume of water.

Dermatan sulfate (L-iduronate + GlcNAc sulfate)

http://www.cryst.bbk.ac.uk/pps97/assignments/projects/emilia/dermatan.gif

Occurence : skin, blood vessels, heart valves

 

Chondroitin sulfate (D-glucuronate + GalNAc sulfate)

 

Occurence : cartilage, bone, heart valves ; It is the most abundant GAG.

Heparin and heparan sulfate (D-glucuronate sulfate + N-sulfo-D-glucosamine)

http://www.cryst.bbk.ac.uk/pps97/assignments/projects/emilia/heparin.gif

Heparans have less sulfate groups than heparins

Occurence :

             Heparin :component of intracellular granules of mast cells lining the arteries of the lungs, liver and skin.

 

Figure. Structure of heparin. The polymer section illustrates structural features typical of heparin; however, the sequence of variously substituted repeating disaccharide units has been arbitrarily selected. In addition, non-O-sulfated or 3-O-sulfated glucosamine residues may also occur.

 

 

 

             Heparan sulfate: basement membranes, component of cell surfaces.

 

 

Keratan sulfate ( Gal + GlcNAc sulfate)

http://www.cryst.bbk.ac.uk/pps97/assignments/projects/emilia/keratan.gif

Occurence : cornea, bone, cartilage;

Keratan sulfates are often aggregated with chondroitin sulfates.

 

Structure of proteoglycans

The GAGs extend perpendicular from the core protein in a bottlebrush- like structure.

The linkage of GAGs such as (heparan sulfates and chondroitin sulfates) to the protein core involves a specific trisaccharide linker :

http://www.cryst.bbk.ac.uk/pps97/assignments/projects/emilia/gag.GIF

Some forms of keratan sulfates are linked to the protein core through an N-asparaginyl bond.

The protein cores of proteoglycans are rich in Ser and Thr residues which allows multiple GAG attachment.

 

Proteoglycans Have Numerous Functions

As indicated above, proteoglycans are remarkably complex molecules and are found in every tissue of the body, mainly in the ECM or ground substance.

There they are associated with each other and also with the other major structural components of the matrix, collagen and elastin, in quite specific manners. Some proteoglycans bind to collagen and others to elastin.

These interactions are important in determining the structural organization of the matrix. Some proteoglycans (eg, decorin) can also bind growth factors such as TGF-â, modulating their effects on cells. In addition, some of them interact with certain adhesive proteins such as fibronectin and laminin (see above), also found in the matrix. The GAGs present in the proteoglycans are polyanions and hence bind polycations and cations such as Na+ and K+. This latter ability attracts water by osmotic pressure into the extracellular matrix and contributes to its turgor. GAGs also gel at relatively low concentrations. Because of the long extended nature of the polysaccharide chains of GAGs and their ability to gel, the proteoglycans can act as sieves, restricting the passage of large macromolecules into the ECM but allowing relatively free diffusion of small molecules.

Again, because of their extended structures and the huge macromolecular aggregates they often form, they occupy a large volume of the matrix relative to proteins.

 

SOME FUNCTIONS OF SPECIFIC GAGS & PROTEOGLYCANS

Hyaluronic acid is especially high in concentration in embryonic tissues and is thought to play an important role in permitting cell migration during morphogenesis and wound repair. Its ability to attract water into the extracellular matrix and thereby loosen it up may be important in this regard. The high concentrations of hyaluronic acid and chondroitin sulfates present in cartilage contribute to its compressibility (see below).

Chondroitin sulfates are located at sites of calcification in endochondral bone and are also found in cartilage.

They are also located inside certain neurons and may provide an endoskeletal structure, helping to maintain their shape.

Both keratan sulfate I and dermatan sulfate are present in the cornea. They lie between collagen fibrils and play a critical role in corneal transparency. Changes in proteoglycan composition found in corneal scars disappear when the cornea heals. The presence of dermatan

sulfate in the sclera may also play a role in maintaining the overall shape of the eye. Keratan sulfate I is also present in cartilage.

Heparin is an important anticoagulant. It binds with factors IX and XI, but its most important interaction is with plasma antithrombin III. Heparin can also bind specifically to lipoprotein lipase present in capillary walls, causing a release of this enzyme into the circulation.

Certain proteoglycans (eg, heparan sulfate) are associated with the plasma membrane of cells, with their core proteins actually spanning that membrane. In it they may act as receptors and may also participate in the mediation of cell growth and cell-cell communication.

The attachment of cells to their substratum in culture is mediated at least in part by heparan sulfate. This proteoglycan is also found in the basement membrane of the kidney along with type IV collagen and laminin (see above), where it plays a major role in determining the charge selectiveness of glomerular filtration.

Proteoglycans are also found in intracellular locations such as the nucleus; their function in this organelle has not been elucidated. They are present in some storage or secretory granules, such as the chromaffin granules of the adrenal medulla. It has been postulated that they play a role in release of the contents of such granules. The various functions of GAGs are summarized in Table 2.

 

Table 2. Some functions of glycosaminoglycans and proteoglycans.

 

 

ASSOCIATIONS WITH MAJOR DISEASES & WITH AGING

Hyaluronic acid may be important in permitting tumor cells to migrate through the ECM. Tumor cells can induce fibroblasts to synthesize greatly increased amounts of this GAG, thereby perhaps facilitating their own spread. Some tumor cells have less heparan sulfate at their surfaces, and this may play a role in the lack of adhesiveness that these cells display.

The intima of the arterial wall contains hyaluronic acid and chondroitin sulfate, dermatan sulfate, and heparan sulfate proteoglycans. Of these proteoglycans, dermatan sulfate binds plasma low-density lipoproteins.

In addition, dermatan sulfate appears to be the major GAG synthesized by arterial smooth muscle cells. Because it is these cells that proliferate in atherosclerotic lesions in arteries, dermatan sulfate may play an important role in development of the atherosclerotic plaque.

In various types of arthritis, proteoglycans may act as autoantigens, thus contributing to the pathologic features of these conditions. The amount of chondroitin sulfate in cartilage diminishes with age, whereas the amounts of keratan sulfate and hyaluronic acid increase.

These changes may contribute to the development of osteoarthritis. Changes in the amounts of cer tain GAGs in the skin are also observed with aging and help to account for the characteristic changes noted in this organ in the elderly.

An exciting new phase in proteoglycan research is opening up with the findings that mutations that affect individual proteoglycans or the enzymes needed for their synthesis alter the regulation of specific signaling pathways in drosophila and Caenorhabditis elegans, thus affecting development; it already seems likely that similar effects exist in mice and humans.

 

 

EXAMPLES OF GAG BINDING PROTEINS:

Secreted proteases and antiproteases

For example antithrombin III (AT III) binds tightly to heparin and certain heparan sulfates (so do its substrates). Thus they control the blood coagulation. In the absence of GAGs AT III inactivates proteases (such as thrombin, factors IXa and XIa) very slowly. In the presence of appropriate GAGs these reactions are accelerated 2000-fold.

GAGs are sufficiently long that both protease and protease inhibitor can bind to the same chain (thus the likelyhood of the two proteins binding to each other is increased enormously). GAGs also affect the protein conformation that contributes to improving AT III binding kinetics.

Polypeptide growth factors

Members of the FGF family, as well as several other growth factors, bind to heparin or heparan sulfate. Binding to endogenous GAGs entraps these molecules in ECM from which they may be later released. GAGs can alter the conformation, proteolytic susceptibility and biological activity of some of these proteins. The bound growth factor is resistant to degradation by extracellular proteases. Active hormone is released by proteolysis of the heparan sulfate chains. It occurs during the tissue growth and remodeling after infection.

ECM proteins

Most of the large, multidomain ECM proteins contain at least one GAG binding site.

For example fibrous collagens (type I, III, V) and fibronectin bind to heparan sulfate chains which are attached to the integral membrane core proteins of cell surface proteoglycans such as syndecan and fibroglycan. Cell surface proteoglycans are thought to anchor cells to matrix fibers.

 

Cell-cell adhesion molecules

              For example NCAM (see cadherins) interacts with cell surface heparan sulfate proteoglycans. This interaction is required for its function. NCAM has a distinct heparan binding domain.

              Hyaluronan is bound to the surface receptors (e.g. CD44) of many migrating cells. It is very important during differentiation (for example myoblasts which are undifferentiated muscle cell precursors bear hyaluronan- rich coat that prevents premature cell fusion). Because its loose, hydrated porous structure, the hyaluronan coat keeps cells apart from each other. They are free to move around and proliferate.

When the level of hyaluronan is lower (e.g. because of digesting by hyaluronidase), there is ceesation of cell movement and initiation of cell- cell attachment.

 

 

Mucopolysaccharidoses and collagenoses, their biochemical diagnostics

Mucopolysaccharidoses are a group of metabolic disorders caused by the absence or malfunctioning of lysosomal enzymes needed to break down molecules called glycosaminoglycans - long chains of sugar carbohydrates in each of our cells that help build bone, cartilage, tendons, corneas, skin and connective tissue. Glycosaminoglycans (formerly called mucopolysaccharides) are also found in the fluid that lubricates our joints.

People with a mucopolysaccharidosis disease either do not produce enough of one of the 11 enzymes required to break down these sugar chains into simpler molecules, or they produce enzymes that do not work properly. Over time, these glycosaminoglycans collect in the cells, blood and connective tissues. The result is permanent, progressive cellular damage which affects appearance, physical abilities, organ and system functioning, and, in most cases, mental development.

The mucopolysaccharidoses are part of the lysosomal storage disease family, a group of more than 40 genetic disorders that result when a specific organelle in our body's cells the lysosome malfunctions. The lysosome is commonly referred to as the cells recycling center because it processes unwanted material into substances that the cell can utilize. Lysosomes break down this unwanted matter via enzymes, highly specialized proteins essential for survival. Lysosomal disorders like mucopolysaccharidosis are triggered when a particular enzyme exists in too small an amount or is missing altogether.

Features

The mucopolysaccharidoses share many clinical features but have varying degrees of severity. These features may not be apparent at birth but progress as storage of glycosaminoglycans affects bone, skeletal structure, connective tissues, and organs. Neurological complications may include damage to neurons (which send and receive signals throughout the body) as well as pain and impaired motor function. This results from compression of nerves or nerve roots in the spinal cord or in the peripheral nervous system, the part of the nervous system that connects the brain and spinal cord to sensory organs such as the eyes and to other organs, muscles, and tissues throughout the body.

Depending on the mucopolysaccharidosis subtype, affected individuals may have normal intellect or have cognitive impairments, may experience developmental delay, or may have severe behavioral problems. Many individuals have hearing loss, either conductive (in which pressure behind the ear drum causes fluid from the lining of the middle ear to build up and eventually congeal), neurosensitive (in which tiny hair cells in the inner ear are damaged), or both. Communicating hydrocephalus in which the normal reabsorption of cerebrospinal fluid is blocked and causes increased pressure inside the head is common in some of the mucopolysaccharidoses. Surgically inserting a shunt into the brain can drain fluid. The eye's cornea often becomes cloudy from intracellular storage, and glaucoma and degeneration of the retina also may affect the patient's vision.

Physical symptoms generally include coarse or rough facial features (including a flat nasal bridge, thick lips, and enlarged mouth and tongue), short stature with disproportionately short trunk (dwarfism), dysplasia (abnormal bone size and/or shape) and other skeletal irregularities, thickened skin, enlarged organs such as liver (hepatomegaly) or spleen (splenomegaly), hernias, and excessive body hair growth. Short and often claw-like hands, progressive joint stiffness, and carpal tunnel syndrome can restrict hand mobility and function. Recurring respiratory infections are common, as are obstructive airway disease and obstructive sleep apnea. Many affected individuals also have heart disease, often involving enlarged or diseased heart valves.

Another lysosomal storage disease often confused with the mucopolysaccharidoses is mucolipidosis. In this disorder, excessive amounts of fatty materials known as lipids (another principal component of living cells) are stored, in addition to sugars. Persons with mucolipidosis may share some of the clinical features associated with the mucopolysaccharidoses (certain facial features, bony structure abnormalities, and damage to the brain), and increased amounts of the enzymes needed to break down the lipids are found in the blood.

Types

Seven distinct clinical types and numerous subtypes of the mucopolysaccharidoses have been identified. Although each mucopolysaccharidosis (MPS) differs clinically, most patients generally experience a period of normal development followed by a decline in physical and/or mental function.

Diagnosis

Diagnosis often can be made through clinical examination and urine tests (excess mucopolysaccharides are excreted in the urine). Enzyme assays (testing a variety of cells or body fluids in culture for enzyme deficiency) are also used to provide definitive diagnosis of one of the mucopolysaccharidoses. Prenatal diagnosis using amniocentesis and chorionic villus sampling can verify if a fetus either carries a copy of the defective gene or is affected with the disorder. Genetic counseling can help parents who have a family history of the mucopolysaccharidoses determine if they are carrying the mutated gene that causes the disorders.

 

Definition of CONNECTIVE TISSUE DISEASE

Any of various diseases or abnormal states (as rheumatoid arthritis, systemic lupus erythematosus, polyarteritis nodosa, rheumatic fever, and dermatomyositis) characterized by inflammatory or degenerative changes in connective tissuecalled also collagen disease, collagenolysis, collagen vascular disease

 

SUMMARY

The major components of the ECM are the structural proteins collagen, elastin, and fibrillin; a number of specialized proteins (eg, fibronectin and laminin); and various proteoglycans.

Collagen is the most abundant protein in the animal kingdom; approximately 19 types have been isolated.

Collagen and elastin are fibrous proteins. Collagen molecules contain an abundance of proline, lysine, and glycine, the latter occurring at every third position in the primary structure. Collagen also contains hydroxyproline, hydroxylysine, and glycosylated hydroxylysine, each formed by posttranslational modification.

Collagen molecules typically form fibrils containing a long, stiff, triple-stranded helical structure, in which three collagen polypeptide chains are wound around one another in a rope-like superhelix (triple helix). Other types of collagen form mesh-like networks.

All collagens contain greater or lesser stretches of triple helix and the repeating structure (Gly-X-Y)n.

The biosynthesis of collagen is complex, featuring many posttranslational events, including hydroxylation of proline and lysine.

Diseases associated with impaired synthesis of collagen include scurvy, osteogenesis imperfecta, Ehlers-Danlos syndrome (many types), and Menkes disease.

Elastin confers extensibility and elastic recoil on tissues.

Elastin lacks hydroxylysine, Gly-X-Y sequences, triple helical structure, and sugars but contains desmosine and isodesmosine cross-links not found in collagen.

Elastin is a connective tissue protein with rubber-like properties in tissues such as the lung. 1-Antitrypsin (α1-AT), produced primarily by the liver but also by tissues such as monocytes and alveolar macrophages, prevents elastin degradation in the alveolar walls. A deficiency of α1-AT can cause emphysema and, in some cases, cirrhosis of the liver.

Fibrillin is located in microfibrils. Mutations in the gene for fibrillin cause Marfan syndrome.

The glycosaminoglycans (GAGs) are made up of repeating disaccharides containing a uronic acid (glucuronic or iduronic) or hexose (galactose) and a hexosamine (galactosamine or glucosamine). Sulfate is also frequently present.

The major GAGs are hyaluronic acid, chondroitin 4- and 6-sulfates, keratan sulfates I and II, heparin, heparan sulfate, and dermatan sulfate.

The GAGs are synthesized by the sequential actions of a battery of specific enzymes (glycosyltransferases, epimerases, sulfotransferases, etc) and are degraded by the sequential action of lysosomal hydrolases. Genetic deficiencies of the latter result in mucopolysaccharidoses (eg, Hurler syndrome).

GAGs occur in tissues bound to various proteins (linker proteins and core proteins), constituting proteoglycans.

These structures are often of very high molecular weight and serve many functions in tissues.

Many components of the ECM bind to proteins of the cell surface named integrins; this constitutes one pathway by which the exteriors of cells can communicate with their interiors.

Bone and cartilage are specialized forms of the ECM.

Collagen I and hydroxyapatite are the major constituents of bone. Collagen II and certain proteoglycans are major constituents of cartilage.

Current understanding of the molecular events underlying muscle contraction is embodied in the sliding filament model of muscle contraction. The model is applicable to smooth, skeletal, cardiac, and other contractile activity, including mechanochemical events such as single cell locomotion and receptor endocytosis. Since the biochemistry of these activities are best understood for skeletal muscle, this discussion focus on skeletal muscle (noting, where appropriate, differences in the other muscle types). The biochemical characteristics that differentiate fast-reacting and slow-reacting cells in muscle tissue and the biochemical basis of some common pathophysiological states of muscle, including tetany, fatigue, and rigor mortis are reviewed as well.

There are three types of muscle:

        Skeletal muscle or "voluntary muscle" is anchored by tendons to bone and is used to affect skeletal movement such as locomotion and in maintaining posture. Though this postural control is generally maintained as a subconscious reflex, the muscles responsible react to conscious control like non-postural muscles. An average adult male is made up of 40-50% of skeletal muscle and an average adult female is made up of 30-40%.

        Smooth muscle or "involuntary muscle" is found within the walls of organs and structures such as the esophagus, stomach, intestines, bronchi, uterus, urethra, bladder, and blood vessels, and unlike skeletal muscle, smooth muscle is not under conscious control.

        Cardiac muscle is also an "involuntary muscle" but is a specialized kind of muscle found only within the heart.

Cardiac muscle

Cardiac and skeletal muscle are "striated" in that they contain sarcomeres and are packed into highly-regular arrangements of bundles; smooth muscle has neither. While skeletal muscles are arranged in regular, parallel bundles, cardiac muscle connects at branching, irregular angles. Striated muscle contracts and relaxes in short, intense bursts, whereas smooth muscle sustains longer or even near-permanent contractions.

http://www.youtube.com/watch?v=InIha7bCTjM&feature=related

http://www.youtube.com/watch?v=-pg09F5V63U&feature=related

 

Skeletal muscles comprise about 40% of the mass of the average human body and are formed of long multinucleate, cylindrical cells called muscle fibers. Skeletal muscle fibers are grossly divided into two type; slow twitch (type I) and fast twitch (type II). Type II fibers are further divided into type IIa and type IIb fibers. Type IIa fibers are intermediate fast twitch fibers and can utilize both aerobic and anaerobic metabolism for ATP production. Type IIb fibers are the classic fast twitch fibers. Slow twitch muscle fibers primarily utilize fatty acid oxidation and contain a high concentration of mitochondria and myoglobin. These two facts are the reason that slow twitch fibers are red in color. Fast twitch fibers primarily utilize glucose oxidation to pyruvate for ATP production, contain less mitochondria and myoglobin than slow twitch fibers and thus, are white muscle fibers. Because slow twitch fibers prefer to oxidize fatty acids they are also referred to as oxidative fibers whereas, fast twitch fibers that utilize glucose are referred to as glycolytic fibers. Slow twitch fibers are capable of continuous extended contractions and therefore, do not fatigue quickly. Fast twitch fibers are used for short rapid bursts of energy and as such fatigue more quickly than slow twitch fibers.

The plasma membrane of muscle fibers is known as the sarcolemma. Each muscle is made up of bundles of these fibers, or cells, embedded in a matrix of connective tissue known as the endomysium. The bundle of fibers with its endomysium is surrounded by a more fibrous connective tissue sheath known as the perimysium. The composite of the perimysium and its contents is known as a fasciculus. A complete muscle consists of numerous fasciculi surrounded by a thick outer layer of connective tissue known as the perimysial septa. The translation of contractile activity of individual muscle fibers to anatomical motion take place through this continuous system of connective tissues and sheaths, which ultimately meld into the tendons.

Within the sarcolemma is the sarcoplasm, containing all the usual subcellular elements plus long prominent myofibrils. Each myofibril is composed of bundles of filamentous contractile proteins, some extending from end to end in the cell. Myofibrils are the most conspicuous elements in skeletal myofibers making up about 60% of myofiber protein. A single myofibril is composed of many short structural units, known as sarcomeres, which are arranged end to end. The proteins at the junctions between sarcomeres form the Z line, and thus a sarcomere extends along a myofibril from one Z line to the next Z line. Sarcomeres are composed mostly of actin thin filaments and myosin thick filaments. Sarcomeres represent the minimal contractile unit of a muscle. It is the coordinated contraction and elongation of millions of sarcomeres in a muscle that gives rise to mechanical skeletal activity. The relationship between muscle proteins and muscles is summarized in the table below:

 

Organization of Contractile Proteins in Muscle

 

Thick Filament

Composed of hundreds of long, contractile myosin molecules arranged in a staggered side by side complex.

Thin Filament

Composed of a linear array of hundreds of globular, actin monomers in a double helical. arrangement.

Sarcomere

The unit of contractile activity composed mainly of actin and myosin and extending from Z line to Z line in a myofibril.

Myofiber

A single multinucleate muscle cell containing all the usual cell organelles plus many myofibrils.

Myofibril

End to end arrays of identical sarcomeres.

Muscle

Organized arrays of muscle fibers.

 

 

Organization of the Sarcomere

 The organization of individual contractile proteins making up a sarcomere is a key feature of the sliding filament model. Each sarcomere is composed of hundreds of filamentous protein aggregates, each known as a myofilament. Two kinds of myofilaments are identifiable on the basis of their diameter and protein composition (see image above). Thick myofilaments are composed of several hundred molecules of a fibrous protein known as myosin. Thin myofilaments are composed of two helically interwound, linear polymers of a globular protein known as actin. Thin and thick filaments also contain accessory proteins, described below. Proteins of the Z line, including α-actinin, serve as an embedding matrix or anchor for one end of the thin filaments, which extend toward the center of sarcomeres on either side of the Z line. The Z line proteins often appear continuous across the width of a muscle fiber and seem to act to keep the myofibrils within a myofiber in register. The distal end of each thin filament is free in the sarcoplasm and is capped with a protein known as β-actinin.

Also depicted in the image above is a second disk-like protein aggregate: the M-line, which is centrally located in sarcomeres. Like Z line protein, the M line protein aggregate acts as an embedding matrix, in this case for the myosin thick filaments. Thick filaments extend from their point of attachment on both sides of the M line toward the two Z lines that define a sarcomere.

Within a sarcomere the thick and thin filaments interdigitate so that in cross section they are seen to form a hexagonal lattice, in which 6 thin filaments are arrayed around each thick filament. The thick filaments are also arranged hexagonally to each other. During contraction and relaxation the distance between the Z lines varies, decreasing with contraction and increasing with relaxation. The M line, with its attached thick filaments, remains centrally located in the sarcomere. The thin and thick filaments retain their extended linear structure except in extreme situations. Changes in sarcomere length are caused by the thin filaments being pulled along the thick filaments in the direction of the M line.

 

Structure and functions of sarcoplasma proteins (Myogene, Myoglobine, Myoalbumine)

 

The sarcoplasm of a muscle fiber is comparable to the cytoplasm of other cells, but it houses unusually large amounts of glycosomes (granules of stored glycogen) and significant amounts of myoglobin, an oxygen binding protein. The calcium concentration in sarcoplasma is also a special element of the muscular fiber by means of which the contractions takes place and regulates.

It contains mostly myofibrils (which are composed of sarcomeres), but its contents are otherwise comparable to those of the cytoplasm of other cells. It has a Golgi apparatus, near the nucleus, mitochondria just on the inside of the cytoplasmic membrane or sarcolemma, as well as a smooth endoplasmic reticulum organized in an extensive network.

 

Myoglobin is an iron- and oxygen-binding protein found in the muscle tissue of vertebrates in general and in almost all mammals. It is related to hemoglobin, which is the iron- and oxygen-binding protein in blood, specifically in the red blood cells. The only time myoglobin is found in the bloodstream is when it is released following muscle injury. It is an abnormal finding, and can be diagnostically relevant when found in blood.

Myoglobin (abbreviated Mb) is a single-chain globular protein of 153 or 154 amino acids, containing a heme (iron-containing porphyrin) prosthetic group in the center around which the remaining apoprotein folds. It has eight alpha helices and a hydrophobic core. It has a molecular weight of 17,699 daltons (with heme), and is the primary oxygen-carrying pigment of muscle tissues. Unlike the blood-borne hemoglobin, to which it is structurally related, this protein does not exhibit cooperative binding of oxygen, since positive cooperativity is a property of multimeric/oligomeric proteins only. High concentrations of myoglobin in muscle cells allow organisms to hold their breaths longer. Diving mammals such as whales and seals have muscles with particularly high myoglobin abundance.

Myoglobin was the first protein to have its three-dimensional structure revealed. In 1958, John Kendrew and associates successfully determined the structure of myoglobin by high-resolution X-ray crystallography. For this discovery, John Kendrew shared the 1962 Nobel Prize in chemistry with Max Perutz. Despite being one of the most studied proteins in biology, its true physiological function is not yet conclusively established: mice genetically engineered to lack myoglobin are viable, but showed a 30% reduction in cardiac systolic output. They adapted to this deficiency through hypoxic genetic mechanisms and increased vasodilation. In humans myoglobin is encoded by the MB gene.

 

Meat color

 

Myoglobin forms pigments responsible for making meat red. The color that meat takes is partly determined by the oxidation states of the iron atom in myoglobin and the oxygen species attached to it. When meat is in its raw state, the iron atom is in the +2 oxidation state, and is bound to a dioxygen molecule (O2). Meat cooked well done is brown because the iron atom is now in the +3 oxidation state, having lost an electron, and is now coordinated by a water molecule. Under some conditions, meat can also remain pink all through cooking, despite being heated to high temperatures. If meat has been exposed to nitrites, it will remain pink because the iron atom is bound to NO, nitric oxide (true of, e.g., corned beef or cured hams). Grilled meats can also take on a pink "smoke ring" that comes from the iron binding to a molecule of carbon monoxide. Raw meat packed in a carbon monoxide atmosphere also shows this same pink "smoke ring" due to the same coordination chemistry. Notably, the surface of this raw meat also displays the pink color, which is usually associated in consumers' minds with fresh meat. This artificially induced pink color can persist in the meat for a very long time, reportedly up to one year. Hormel and Cargill are both reported to use this meat-packing process, and meat treated this way has been in the consumer market since 2003. Myoglobin is found in Type I muscle, Type II A and Type II B, but most texts consider myoglobin not to be found in smooth muscle.

 

Role in disease

 

Myoglobin is released from damaged muscle tissue (rhabdomyolysis), which has very high concentrations of myoglobin. The released myoglobin is filtered by the kidneys but is toxic to the renal tubular epithelium and so may cause acute renal failure. It is not the myoglobin itself that is toxic (it is a protoxin) but the ferrihemate portion that is dissociated from myoglobin in acidic environments (e.g., acidic urine, lysosomes).

Myoglobin is a sensitive marker for muscle injury, making it a potential marker for heart attack in patients with chest pain. However, elevated myoglobin has low specificity for acute myocardial infarction (AMI) and thus CK-MB, cTnT, ECG, and clinical signs should be taken into account to make the diagnosis.

 

Structure and bonding

 

Myoglobin contains a porphyrin ring with an iron center. There is a proximal histidine group attached directly to the iron center, and a distal histidine group on the opposite face, not bonded to the iron.

Many functional models of myoglobin have been studied. One of the most important is that of picket fence porphyrin by James P. Collman. This model was used to show the importance of the distal prosthetic group. It serves three functions:

To form hydrogen bonds with the dioxygen moiety, increasing the O2 binding constant

To prevent the binding of carbon monoxide, whether from within or without the body. Carbon monoxide binds to iron in an end-on fashion, and is hindered by the presence of the distal histidine, which forces it into a bent conformation. CO binds to heme 23,000 times better than O2, but only 200 times better in hemoglobin and myoglobin. Oxygen binds in a bent fashion, which can fit with the distal histidine.

To prevent irreversible dimerization of the oxymyoglobin with another deoxymyoglobin species

 

myogen - proteins extracted from skeletal muscle with cold water, largely the enzymes promoting glycolysis; from the residue, alkaline 0.6 mol L-1 KCl extracts actin and myosin as actomyosin, with myosin further separable into two meromyosins by proteinase treatment.

Synonym(s): myosinogen

 

Proteins of the Myofilaments

The biochemical basis of muscle activity is related to the enzymatic and physical properties of actin, myosin, and the accessory proteins that constitute the thin and thick filaments. The following discussion summarizes the key protein components of the myofilaments and their ATP-dependent interactions, which produce contractile activity.

The proteins of the thin and thick filaments can be separated into actin, myosin, and 6 accessory proteins. The accessory proteins are α-actinin, β-actinin, tropomyosin, troponin, C protein, and M line protein. Solubilized myosin molecules are long thin (fibrous) proteins with a molecular weight of about 500,000 daltons.

Each molecule is made up of 6 subunits, 2 very large, heavy chains (HC), and 4 smaller, light chains (LC). In a given muscle fiber the 2 large subunits are identical, although there are different HC isoforms in different types of muscle fibers. Heavy chains contain a long linear C-terminal α-helical domain (1,300 amino acids) and a prominent globular N-terminal domain of about 800 amino acids. The two HC, α-helical domains are helically interwound, giving the molecules a long, rigid superhelical structure with 2 globular headpieces. A complete myosin molecule also contains 4 relatively small proteins which are associated with the globular headpieces. These small proteins, of molecular weight 16,00024,000 daltons, are known as alkali light chains (LC1 or LC3) and DTNB light chains (LC2). Each myosin molecule contains 2 subunits of LC2, 1 associated with each HC globular domain. Each of the globular domains also contains a subunit of either LC1 or LC3, with the proportions of LC1 and LC3 in the myosin molecules varying in myosins from cardiac, skeletal, embryonic, and smooth muscle. All light chains bind Ca2+ with high affinity, are phosphorylated by myosin light chain kinase (MLCK), and generally serve in the regulation of myosin's ATPase activity and its assembly into thick filaments.

 Organization of myofilaments

 

Several functionally important landmarks exist on the myosin molecule. Near the midpoint of the long linear superhelical region is a site defined by its ready susceptibility to proteolytic trypsin digestion. Trypsin cleaves myosin into 2 portions: 1 containing both globular headpieces and some superhelical region, and the other consisting of the remaining superhelical portion of the carboxy terminus. The portion containing the headpiece is known as heavy meromyosin (HMM; molecular weight 350,000). The C-terminal fragment is known as light meromyosin (LMM; molecular weight 125,000).

The significance of the trypsin site is that its susceptibility to protease action is thought to reflect an interruption in the otherwise rigid superhelix, allowing this site to act as one of a hinge point involved in converting the chemical energy of ATP into the mechanical events of contraction and relaxation. A second proteolytic landmark susceptible to papain has also been considered a hinge point. Papain cleaves a site very close to the globular headpieces; these then separate to form 2 subfragments, each known as an SF-1 (for subfragment 1). The remaining superhelical portion of the molecule is known as SF-2. The ATPase activity of the myosin is associated with the SF-1 units.

A thick filament is composed of approximately 400 myosin molecules, 200 arrayed on either side of the M line. These molecules are maintained in bundles by C protein (clamp protein), M line protein and the hydrophobic interactions of the myosin molecules themselves. The myosin molecules are most tightly packed in the regions represented by the LMM portion of the molecules.

At the trypsin hinge point the heavy meromyosin angles sharply outward from the main axis of the thick filament. This extension of the heavy meromyosin away from the main axis of the thick filament helps bring the headpiece into close proximity to the actin thin filaments lying between the thick filaments. The molecular event underlying muscle contraction is the regulated binding of the myosin headpieces to actin thin filaments, followed by rapid myosin conformational changes about its hinge points with the bound actin being translocated toward the M line.

 

Organization of Actin Thin Filaments

 

Thin filaments are composed of many subunits of the globular protein G-actin (42 kD) and several accessory proteins. In thin filaments, G-Actin is polymerized into long fibrous arrays known as F-actin. A pair of linear F-actin arrays is helically wound to form the backbone structure of 1 complete thin filament.

Each G-actin subunit has 1 ADP/ATP binding site, presumed to be involved in polymerizing the thin filament. Once polymerized, the actin is capped and the thin filament stabilized by a protein known as β-actinin. In addition to its nucleotide binding site, each G-actin molecule contains a high-affinity myosin head-binding site. In skeletal and cardiac muscle, accessory proteins of the thin filament (described below) physically regulate the availability of this site for binding myosin. Thus, the accessory proteins control contractile events.

The main thin filament accessory proteins are tropomyosin and troponin. Tropomyosin is a long, rod-like, αβ helically-interwound heterodimer that spans a length of 7 G-actin residues. A pair of tropomyosin molecules is associated with every 7 pairs of G-actin residues along a thin filament, 1 tropomyosin molecule in each of the grooves of the F-actin helix. In relaxed muscle, each tropomyosin molecule covers the myosin binding sites of 7 G-actin residues, preventing interaction between actin and myosin and thus maintaining the relaxed state. The onset of contractile activity involves activating troponin, the second accessory protein of thin filaments. Troponin is a heterotrimer attached to one end of each tropomyosin molecule and to actin, physically linking tropomyosin to actin.

Conformational changes in the bridging molecule, troponin, are responsible for moving tropomyosin on and off myosin binding sites of actin and thus regulating muscle contraction. One of the troponin subunits, troponin-C (Tn-C), is a calmodulin-like calcium-binding protein. When Tn-C binds calcium, the whole troponin molecule undergoes the conformational change that moves the attached tropomyosin away from the myosin binding sites on actin. This event permits nearby myosin heads to interact with myosin binding sites, and contractile activity ensues.

Events on the thin filament can be summarized as follows: Prior to the appearance of free calcium in the sarcoplasm, tropomyosin covers the myosin binding sites on actin. The appearance of calcium in the sarcoplasm leads to calcium binding on Tn-C. The resulting conformational changes in troponin move the attached tropomyosin molecule more deeply into the helix groove of F-actin, uncovering the myosin binding sites on G-actin subunits. The exposed sites are then available to interact with myosin headpieces. Removing calcium from the sarcoplasm restores the original conformational states of troponin and tropomyosin, preventing interaction between actin and myosin and leading to the relaxed state.

 

Myosin and the Power Stroke of Contraction

In a rested, non-contracting muscle, myosin binding sites on actin are obscured and myosin exists a in high-energy conformational state (M*), poised to carry out a contractile cycle. The energy of ATP hydrolysis is used to drive myosin from a low-energy conformational state (M) to the high-energy state, as depicted in the following equation:

 

(M-ATP) <> (M*-ADP-Pi)

 

When cytosolic calcium increases and myosin binding sites on actin become available, an actomyosin complex is formed, followed by the sequential dissociation of Pi and ADP with conversion of myosin to its low-energy conformational state. These events are accompanied by simultaneous translocation of the attached thin filament toward the M line of the sarcomere. The latter events, summarized in the following 2 equations, comprise the power stroke of the contractile cycle. Note that the energy of the power stroke is derived from ATP, via ATP-driven conversion of a low-energy myosin conformational state to a high-energy conformational state. A useful analogy is that ATP cocks the myosin trigger and the formation of an actomyosin complex pulls the trigger, releasing the energy stored in cocking the trigger.

 

(M*-ADP-Pi) + A <> (M*-ADP-A) + Pi

 

(M*-ADP-A) <> (M-A) + ADP

 

At the end of the power stroke the actomyosin complex is remains intact until ATP becomes available. ATP binding to myosin is a very exergonic reaction, with the result that ATP displaces actin from the myosin head as indicated by the equation below. Thus, it is often said that ATP is required for muscle relaxation. It is important to note that in relaxed muscle, myosin is in its high-energy conformational state. Note that the final product (M-ATP) is also the first reactant shown in the first equation above, completing the reactions of the contractile cycle.

 

(MA) + ATP <> (M-ATP) + A

 Troponin

Troponin is a complex of three regulatory proteins (troponin C, troponin I and troponin T) that is integral to muscle contraction in skeletal and cardiac muscle, but not smooth muscle.

Discussions of troponin often pertain to its functional characteristics and/or to its usefulness as a diagnostic marker for various heart disorders.

 

Function

 Troponin is attached to the protein tropomyosin and lies within the groove between actin filaments in muscle tissue. In a relaxed muscle, tropomyosin blocks the attachment site for the myosin crossbridge, thus preventing contraction. When the muscle cell is stimulated to contract by an action potential, calcium channels open in the sarcoplasmic membrane and release calcium into the sarcoplasm. Some of this calcium attaches to troponin which causes it to change shape, exposing binding sites for myosin (active sites) on the actin filaments. Myosin binding to actin forms cross bridges and contraction (cross bridge cycling) of the muscle begins. Troponin activation. Troponin C (red) binds Ca2+, which stabilizes the activated state, where troponin I (yellow) is no longer bound to actin. Troponin T (blue) anchors the complex on tropomyosin.Troponin is found in both skeletal muscle and cardiac muscle, but the specific versions of troponin differ between types of muscle. The main difference is that the TnC subunit of troponin in skeletal muscle has four calcium ion binding sites, whereas in cardiac muscle there are only three. Views on the actual amount of calcium that binds to troponin vary from expert to expert and source to source.

Both cardiac and skeletal muscles are controlled by changes in the intracellular calcium concentration. When calcium rises, the muscles contract, and when calcium falls, the muscles relax.

 Troponin is a component of thin filaments (along with actin and tropomyosin), and is the protein to which calcium binds to accomplish this regulation. Troponin has three subunits, TnC, TnI, and TnT. When calcium is bound to specific sites on TnC, tropomyosin rolls out of the way of the actin filament active sites, so that myosin (a molecular motor organized in muscle thick filaments) can attach to the thin filament and produce force and/or movement. In the absence of calcium, tropomyosin interferes with this action of myosin, and therefore muscles remain relaxed.

Individual subunits serve different functions:

Troponin C binds to calcium ions to produce a conformational change in TnI

Troponin T binds to tropomyosin, interlocking them to form a troponin-tropomyosin complex

Troponin I binds to actin in thin myofilaments to hold the troponin-tropomyosin complex in place

Smooth muscle does not have troponin.

 

Diagnostic use

The troponin test can be used as a test of several different heart disorders, including myocardial infarction.

 

Cardiac conditions

Certain subtypes of troponin (cardiac troponin I and T) are very sensitive and specific indicators of damage to the heart muscle (myocardium). They are measured in the blood to differentiate between unstable angina and myocardial infarction (heart attack) in patients with chest pain or acute coronary syndrome. A patient who had suffered from a myocardial infarction would have an area of damaged heart muscle and so would have elevated cardiac troponin levels in the blood. This can also occur in patients with coronary vasospasm.

It is important to note that cardiac troponins are a marker of all heart muscle damage, not just myocardial infarction. Other conditions that directly or indirectly lead to heart muscle damage can also increase troponin levels. Severe tachycardia (for example due to supraventricular tachycardia) in an individual with normal coronary arteries can also lead to increased troponins for example, presumably due to increased oxygen demand and inadequate supply to the heart muscle.

Troponins are also increased in patients with heart failure, where they also predict mortality and ventricular rhythm abnormalities. They can rise in inflammatory conditions such as myocarditis and pericarditis with heart muscle involvement (which is then termed myopericarditis). Troponins can also indicate several forms of cardiomyopathy, such as dilated cardiomyopathy, hypertrophic cardiomyopathy or (left) ventricular hypertrophy, peripartum cardiomyopathy, Takotsubo cardiomyopathy or infiltrative disorders such as cardiac amyloidosis.

Heart injury with increased troponins also occurs in cardiac contusion, defibrillation and internal or external cardioversion. Increased troponins are commonly increased in several procedures such as cardiac surgery and heart transplantation, closure of atrial septal defects, percutaneous coronary intervention or radiofrequency ablation.

Non-cardiac conditions

The distinction between cardiac and non-cardiac conditions is somewhat artificial; the conditions listed below are not primary heart diseases, but they exert indirect effects on the heart muscle.

Troponins are increased in around 40% of patients with critical illnesses such as sepsis. There is an increased risk of mortality and length of stay in the intensive care unit in these patients. In severe gastrointestinal bleeding there can also be a mismatch between oxygen demand and supply of the myocardium.

Central nervous system disorders can lead to increased sympathetic tone and/or catecholamine release which lead to cardiac overstimulation. This is seen in subarachnoid hemorrhage, stroke, intracranial hemorrhage and (generalized) seizures (in patients with epilepsy or eclampsia, for example).

Patients with end-stage renal disease can have chronically elevated troponin T levels, which are linked to a poorer prognosis. Troponin I is less likely to be falsely elevated.

Strenuous endurance exercise such as marathons or triathlons can lead to increased troponin levels in up to one third of subjects, but it is not linked to adverse health effects in these competitors. High troponin T levels have also been reported in patients with inflammatory muscle diseases such as polymyositis or dermatomyositis. Troponins are also increased in rhabdomyolysis.

Cardiac troponin T and I can be used to monitor drug and toxin induced cardiomyocyte toxicity.

Tropomyosin

 

Tropomyosin is a two-stranded alpha-helical coiled coil protein found in muscle.

All organisms contain structures which provide physical integrity to their cells. These structures are collectively known as the cytoskeleton and one of the most ancient systems is based on filamentous polymers of the protein actin. During evolution a second polymer of the protein, tropomyosin, arose and became an integral part of most actin filaments in animals.

Tropomyosins are a large family of integral components of actin filaments which play a critical role in regulating the function of actin filaments in both muscle and nonmuscle cells. These proteins consist of rod-shaped coiled-coil hetero- or homo-dimers that lie along the α-helical groove of most actin filaments. Interaction occurs along the length of the actin filament with dimers aligning in a head-to-tail fashion.

Tropomyosins are often categorised into two groups, muscle tropomyosin isoforms and nonmuscle tropomyosin isoforms. Muscle tropomyosin isoforms are involved in regulating interactions between actin and myosin in the muscle sarcomere and play a pivotal role in regulated muscle contraction. Nonmuscle tropomyosin isoforms function in all cells, both muscle and nonmuscle cells, and are involved in a range of cellular pathways that control and regulate the cells cytoskeleton and other key cellular functions.

The actin filament system that is involved in regulating these cellular pathways is more complex than the actin filament systems that regulates muscle contraction. The contractile system relies upon 4 actin filament isoforms and 5 tropomyosin isoforms , whereas the actin filament system of the cytoskeleton uses 2 actin filament isoforms and over 40 tropomyosin isoforms.