BIOCHEMISTRY OF MUSCLE, MUSCLE CONTRACTION. BIOCHEMISTRY OF CONNECTIVE TISSUE
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.
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.
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 16,000–24,000 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.
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.
Regulation of Sarcoplasmic Calcium
Events that stimulate muscle activity by raising sarcoplasmic calcium begin with neural excitation at neuromuscular junctions. Excitation induces local depolarization of the sarcolemma, which spreads to the associated T tubule system and deep into the interior of the myofiber. T tubule depolarization spreads to the sarcoplasmic reticulum (SR), with the effect of opening voltage-gated calcium channels in the SR membranes. This is followed by massive, rapid movement of cisternal calcium into the sarcoplasm close to nearby myofibrils. The appearance of calcium very close to the Tn-C subunit of troponin results in the production of multiple myosin power strokes, as long as the available calcium concentration remains greater than about 1μM to 5μM.
Muscle Relaxation
Normally, cessation of contractile activity and a state of relaxation follow electrical quiescence at the myoneural junction. The sarcoplasmic membrane returns to its resting electrical potential (about 60 mV more positive outside), as does the entire T tubule system and the SR membrane. Subsequently, sarcoplasmic calcium is pumped back into the SR cisternae by an extremely active ATP–driven calcium pump, which comprises one of the main proteins of the SR membrane. For each ATP hydrolyzed, 2 calcium ions are moved out of the sarcoplasm, with sarcoplasmic calcium ultimately falling below 0.1μM, or 50– to 100–fold lower than the KD for calcium binding to Tn-C. The cisternal surface of the SR membrane also contains large quantities of a glycoprotein known as calsequestrin. Calsequestrin avidly binds calcium, decreasing its concentration in the cisternae, and thus favoring calcium accumulation. A final repository of sarcoplasmic calcium is the mitochondrial matrix. Mitochondria have a remarkably active calcium pump, driven by the electron transport-generated chemiosmotic potential. Under aerobic conditions this pump uses the energy of electron transport to sequester calcium in the mitochondrial matrix, in preference to the synthesis of ATP.
Red (Slow) Oxidative and White (Fast) Glycolytic Muscles
In addition to the phosphoryl transfers described by above in the last 3 equations above, muscle ATP is also generated by glycolysis and oxidative phosphorylation. Muscles that depend predominantly on oxidative phosphorylation for ATP require abundant oxygen. To ensure its availability, these muscles store appreciable oxygen as oxymyoglobin. Oxidative, myoglobin-containing muscles are red in color because of their high myoglobin and mitochondrial content. Glycolytic muscles lack appreciable myoglobin and appear white. These muscles generally contain abundant stores of glycogen and generate most of their ATP from glycolytic reactions. A major functional difference between red and white muscle cells is that white fibers generate ATP by a short reaction pathway between substrates (eg, glucose) and the appearance of ATP, whereas in red muscle the pathway from substrate (again, glucose) to ATP is comprised of many more reaction steps (eg, glycolysis plus TCA cycle plus electron transport) and is a correspondingly longer process. Consequently, fast-acting skeletal muscles are composed of dominantly glycolytic white fibers while slow-acting muscles such as those that maintain tone are generally red and oxidative.
Function in Skeletal Muscle Contraction
Skeletal muscle is composed of large, multi-nucleated cells (muscle fibers). Each muscle fiber is packed with longitudinal arrays of myofibrils. Myofibrils are composed of repeating protein structures or sarcomeres, the basic functional unit of skeletal muscle. The sarcomere is a highly structured protein array, consisting of interdigitating thick and thin filaments, where the thin filaments are tethed to a protein structure, the z-line. The dynamic interactions between the thick and thin fialments results in muscle contraction.
Myosin belongs to a family of motor proteins and the muscle isoforms of this family comprise the thick filament. The thin filament is comprised of the skeletal muscle isoforms of actin. Each myosin protein ‘paddles’ along the thin actin filament, repeatedly binding to myosin binding sites along the actin filament, ratcheting and letting go. In effect the thick filament moves or slides along the thin filament, resulting in muscle contraction. This process is known as the sliding filament model.ca+ is necessary for contraction.
The binding of the myosin heads to the muscle actin is a highly regulated process. The thin filament is made of actin, tropomyosin, and troponin. The contraction of skeletal muscle is triggered by nerve impulses which in turn stimulate the release of Ca2+. The release of Ca2+ from the sarcoplasmic reticulum causes an increase in the concentration of Ca2+ in the cytosol. Calcium ions then bind to troponin which is associated with tropomyosin. Binding causes changes in the shape of troponin and subsequently causes the tropomyosin isoform to shift its position on the actin filament. This shifting in position exposes the myosin binding sites on the actin filament, allowing the myosin heads of the thick filament to bind to the thin filament.
Structural and biochemical studies suggest that the position of tropomyosin and troponin on the thin filament regulate the interactions between the myosin heads of the thick filament and the binding sites on the actin of the thin filament. X-ray diffraction and cryoelectron microscopy suggest that tropomyosin sterically blocks the access of myosin to the actin filament.
Although this model is well established it is unclear as to whether the movement of tropomyosin directly causes the myosin head to engage the actin filament. As such an alternative model has emerged, whereby the movement of the tropomyosin in the filament functions as an allosteric switch that is modulated by activating myosin binding but does not function solely by regulating myosin binding.
Regulation of Contraction in Smooth Muscle
Smooth muscle is a type of non-striated muscle and unlike striated muscle, contraction of smooth muscle is not under conscious control. Smooth muscle may contract spontaneously or rhythmically and be induced by a number of physiochemical agents (hormones, drugs, neurotransmitters). Smooth muscle is found within the walls of various organs and tubes in the body such as the esophagus, stomach, intestines, bronchi, urethra, bladder and blood vessels.
Role of calcium and ATP
ATP is the immediate source of energy for muscle contraction. Although a muscle fiber contains only enough ATP to power a few twitches, its ATP “pool” is replenished as needed. There are three sources of high-energy phosphate to keep the ATP pool filled.
· creatine phosphate
· glycogen
· cellular respiration in the mitochondria of the fibers.
Diagnostic significance of determination of creatin, creatinin and creatin phosphokinase’s activity in biological fluids
Creatinine
It is used to find out whether your kidneys are working normally. A combination of blood and urine creatinine levels may be used to calculate a “creatinine clearance”. This measures how effectively your kidneys are filtering small molecules like creatinine out of your blood.
Urine creatinine may also be used with a variety of other urine tests as a correction factor. Since it is produced and removed at a relatively constant rate, the amount of creatinine in urine can be compared to the amount of another substance being measured. Examples of this are when creatinine is measured with protein to calculate a urine protein/creatinine ratio (UP/CR) and when it is measured with microalbumin to calculate microalbumin/creatinine ratio (also known as albumin/creatinine ratio, ACR). These tests are used to evaluate kidney function as well as to detect other urinary tract disorders.
Serum creatinine measurements along with age, weight, and gender are used to calculate the estimated glomerular filtration rate (eGFR), which is used as a screening test to look for evidence of kidney damage.
Creatinine may be part of a routine blood test, widely used when someone has non-specific health complaints, or when your doctor suspects your kidneys are not working properly.
Some signs and symptoms of kidney dysfunction include:
Fatigue, lack of concentration, poor appetite or trouble sleeping
Swelling or puffiness, particularly around the eyes or in the face, wrists, abdomen, thighs or ankles
Urine that is foamy, bloody, or coffee-coloured
A decrease in the amount of urine
Problems urinating, such as a burning feeling or abnormal discharge during urination, or a change in the frequency of urination, especially at night
Mid-back pain (flank), below the ribs, near where the kidneys are located
High blood pressure
The test is also used to monitor treatment of kidney disease or to monitor kidney function while you are on certain drugs.
Diagnostic significance of determination of creatin phosphokinase’s activity(CK)
CK is often determined routinely in a medical laboratory. It is also determined specifically in patients with chest pain or if acute renal failure is suspected. Normal values are usually between 60 and 400 IU/L, where one unit is enzyme activity, more specifically the amount of enzyme that will catalyze 1 μmol of substrate per minute under specified conditions (temperature, pH, substrate concentrations and activators. This test is not specific for the type of CK that is elevated.
Elevation of CK is an indication of damage to muscle. It is therefore indicative of injury, rhabdomyolysis, myocardial infarction, myositis and myocarditis. The use of statin medications, which are commonly used to decrease serum cholesterol levels, may be associated with elevation of the CPK level in about 1% of the patients taking these medications, and with actual muscle damage in a much smaller proportion.
There is an inverse relationship in the serum levels of T3 and CK in thyroid disease. In hypothyroid patients, with decrease in serum T3 there is a significant increase in CK. Therefore, the estimation of serum CK is considered valuable in screening for hypothyroid patients.
Lowered CK can be an indication of alcoholic liver disease and rheumatoid arthritis.
Isoenzyme determination has been used extensively as an indication for myocardial damage in heart attacks. Troponin measurement has largely replaced this in many hospitals, although some centers still rely on CK-MB.
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 tissues—the 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.
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.
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
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.
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 iormal collagen production.
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.
Structure and functions of proteoglycans
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)
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)
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
· Heparan sulfate : basement membranes, component of cell surfaces
Keratan sulfate ( Gal + GlcNAc sulfate)
Occurence : cornea, bone, cartilage ;
Keratan sulfates are often aggregated with chondroitin sulfates.
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.
connective tissue diseases
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 tissue—called also collagen disease, collagenolysis, collagen vascular disease