Biochemistry of muscle, muscle contraction

Biochemistry of muscle, muscle contraction.

Muscle

Muscle (from Latin musculus "little mouse" is contractile tissue of the body and is derived from the mesodermal layer

of embryonic germ cells.

It is classified as:

skeletal, cardiac, or smooth muscle.

Function of muscle is to produce force and cause motion, either locomotion or movement within internal organs. Much of muscle contraction occurs without consciousthought and is necessary for survival, like the contraction of the heart, or peristalsis (which pushes food through the digestive system). Voluntary muscle contraction is used to move the body, and can be finely controlled, like movements of the eye, or gross movements like the quadriceps muscle of the thigh.

Muscular System

There are two broad types of voluntary muscle fibers, slow twitch and fast twitch. Slow twitch fibers contract for long periods of time but with little force while fast twitch fibers contract quickly and powerfully but fatigue very rapidly.

The muscular system includes three types of muscles. They are smooth, which are found on the walls of internal organs, cardiac, which is found only in the heart, and skeletal muscles, which help strenthen the body and connect to bones.

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%.

Skeletal muscle is a type of striated muscle, which usually attaches to tendons. Skeletal muscles are used to create movement, by applying force to bones and joints viacontraction

. They generally contract voluntarily (via somatic nerve stimulation), although they can contract involuntarily through reflexes. The whole muscle is wrapped in a special type of connective tissue, epimysium.

Muscle cells (also called muscle fibers) are cylindrical, and are multinucleated (in vertebrates and insects). The nuclei of these muscles are located in the peripheral aspect of the cell, just under the plasma membrane, which vacates the central part of the muscle fiber for myofibrils. (Conversely, when the nucleus is located in the center it is considered a pathologic condition known as centronuclear myopathy.) Skeletal muscles have one end (the "origin") attached to a bone closer to the centre of the body's axis and the other end (the "insertion") is attached across a joint to another bone further from the body's axis. The bones rotate about the joint and move relative to one another by contraction of the muscle (lifting of the upper arm in the case of the origin and insertion described here).

Skeletal muscle cells are stimulated by acetylcholine, which is released at neuromuscular junctions by motor neurons. Once the cells are "excited", their sarcoplasmic reticulum will release ionic calcium (Ca2+) which interacts with the myofibrils to induce muscular contraction (via the sliding filament mechanism). This process also requires adenosine triphosphate (ATP). The ATP is produced by metabolizing creatine phosphate and glucose (stored as glycogen or absorbed from blood) within the muscle cells by mitochondria, as well as by metabolizing fatty acids obtained from the blood and within the cell. Each motor neuron activates a group of muscle cells, and collectively the neurons and muscle cells are known as motor units. When more strength is required than can be obtained from a single motor unit, more units will be stimulated; this is known as motor unit recruitment. This is spatial summation. If more strength is required than can be obtained from the current number of motor units, the motor neurons continue to recruit more motor units. When all the motor units are recruited, there will be no further increase in contraction strength. To increase the force of contraction, it is necessary to increase the frequency of neuronalT firing. This results in tetanic contraction, which is a smooth contraction. This is temporal summation...

· 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.

Smooth muscle is a type of non-striated muscle, found within the tunica media layer of large and small arteries and veins, the bladder, uterus, male and female reproductive tracts,gastrointestinal tract

, respiratory tract, the ciliary muscle, and iris of the eye. The glomeruli of the kidneys contain a smooth muscle-like cell called the mesangial cell. Smooth muscle is fundamentally different from skeletal muscle and cardiac muscle in terms of structure, function, excitation-contraction coupling, and mechanism of contraction.

To maintain organ dimensions against forces, cells are fastened to one another by adherens junctions. As a consequence, cells are mechanically coupled to one another such that contraction of one cell invokes some degree of contraction in an adjoining cell. Gap junctions couple adjacent cells chemically and electrically, facilitating the spread of chemicals (e.g., calcium) or action potentials between smooth muscle cells. Smooth muscle may contract spontaneously (via ionic channel dynamic or Cajal pacemaker cells) or be induced by a number of physiochemical agents (e.g., hormones, drugs, neurotransmitters - particularly from the autonomic nervous system), and also mechanical stimulation (such as stretch).

Smooth muscles have been divided into "single unit" and "multi-unit" or into "phasic" and "tonic" types based on the characteristics of the contractile patterns and characteristics of the smooth muscle. Multi-unit smooth muscle lines the large airways to the lungs and large blood vessels. The ciliary muscles within the eye and the arrector pili muscle of the skin are also multiunit. This smooth muscle contains few gap junctions and the autonomic nervous system innervates each smooth cell and regulates them like motor units so you can have graded responses. Single unit smooth muscle lines all the hollow organs and is most common. This type smooth muscle tends to contract rhythmically, is coupled by numerous gap junctions, and often exhibits spontaneous action potential. Another nomenclature separates smooth muscle by contractile pattern. It may contract phasically with rapid contraction and relaxation, or tonically with slow and sustained contraction. Smooth muscle in various regions of the vascular tree, the airway and lungs, kidneys, etc. is different in their expression of ionic channels, hormone receptors, cell-signaling pathways, and other proteins that determine function. Smooth muscle-containing tissue often must be stretched, so elasticity is an important attribute of smooth muscle. Smooth muscle cells may secrete a complex extracellular matrix containing collagen (predominantly types I and III), elastin, glycoproteins, and proteoglycans.

These fibers with their extracellular matrices contribute to the viscoelasticity of these tissues. Smooth muscle also has specific elastin and collagen receptors to interact with these proteins.

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

This is a specialized muscle that, while similar in some fundamental ways to smooth muscle and skeletal muscle, has a unique structure and with an ability not possessed by muscle tissue elsewhere in the body. Cardiac muscle, like other muscles, can contract, but it can also carry an action potential (i.e. conduct electricity), like the neurons that constitute nerves. Furthermore, some of the cells have the ability to generate an action potential, known as cardiac muscle automaticity.

As the muscle contracts, it propels blood into the heart and through the blood vessels of the circulatory system. For a human being, the heart beats about once a second for the entire life of the person, without any opportunity to rest (Ward 2001). It can adjust quickly to the body's needs, increasing output from five liters of blood per minute to more than 25 liters per minute (Ward 2001). The muscles that contract the heart can do so without external stimulation from hormones or nerves, and it does not fatigue or stop contracting if supplied with sufficient oxygen and nutrients.

The actions of cardiac muscle reflect on the remarkable harmony within a body and the underlying principle that individual entities in nature provide a larger function. In order for the heart to work properly, and have the necessary waves of contraction to pump blood, the cardiac cells must fire in intricate coordination with each other. In doing so, each cell provides a larger function for the sake of the body, allowing the heart to beat properly, while in turn being provided essential nutrients by the body. The coordination of the cardiac cells is essential. Should the cells fire randomly, the heart would not be able to contract in a synchronized manner and pump blood, and the body (and thus the cell) would die.

Structure and functions of myofibril proteins

(myosine, actine, actomyosine, troponine, tropomyosine).

The individual actin protein is called a "globular" protein ("g-actin") because it is globular, or ball-like, in appearance. It takes many of these globular proteins coming together into a long chain to begin to make a microfilament. In fact, the actin microfilament is two of these chains of g-actin twisted up together, and the filamentous form is then called "f-actin." This is all depicted here in this schematic to the right; the two identical chains of actin are drawn in different colors only so that you can see how they come together.

You should be able to see how the microfilament can get long, so it should make sense that it runs along the long axis of the myofilament. You should also be able to see that the actin microfilament, although a doublet, is actually rather thin for its length (which would extend beyond the edges of your monitor)... that's why the actin microfilament has been nicknamed the thin filament. This actin microfilament will also be associated with some other molecules, called troponin and tropomyosin, but we won't get to that in detail until next week.

Myosin microfilaments, like actin microfilaments, are made up of many individual myosin protein molecules. However, the myosin protein is not globular. Instead, it has a head and a tail (these regions are indicated in the figure to the left). And each complete myosin molecule in muscle is actually composed of two of these head-and-tail molecules twisted around each other.

Note that there are three different polypeptides that contribute to this overall myosin protein... that is not important for you to memorize, but you will see it as you browse through the web, so I have it in this photo; you only need to know about the head and the tail for this class.

Then, to make the myosin filament, you have to take these doublet myosin molecules and put them together into large bundles. This big wad of myosin proteins is then nicknamed the thick filament. A single thick filament typically has over 200 myosin molecules in it! So it is really very thick. This is shown in the figure below (the A with the circle over it in this figure stands for Angstroms, which are a unit of length measurement... 1,000,000 Angstroms fit into one micrometer, and 1,000,000 micrometers fit into a meter; you do not have to memorize the dimensions):

Again, the thick filament runs along the long axis of the myofibril. I found the above two drawings from educational sites, but that was a couple of years ago and I have lost the links. I will attempt to find them again!

Arrangement of thin and thick filaments in a myofibril:

The thin and thick filaments are organized into neat bundles called sarcomeres.

You can read about sarcomeres in your book. You can also check out the from a course at Stanford, and look at the sarcomere link for "other browsers." I'm just going to give you the basics here.

Thin filaments attach at a point called the "Z line" so that they are all lined up with one another. This is shown in this schematic to the right. The Z line is the dark line that runs perpendicular to the actin filaments. Actually, it is simply a lot of sticky proteins that anchor the actin filaments in place.

The thick filaments run in between the thin filaments. I have put them in for you to see, but first I had to change the background color of the image so that both the thick and thin filaments would be readily visible. I also had to shrink the components down a bit more.

Here is the image of the thick and thin filaments together:

In order to fit the thick filaments between the thin, I needed 2 sets of the thin filaments. Also, it looks like the thick filaments are just floating in the middle... however, they are anchored by proteins (of the "M line"); I just didn't show that. As you look at this image, the following items should become easier to understand:

1. A sarcomere runs from Z-line to Z-line.

2. Sarcomeres run along the longitudinal axis of the muscle fiber.

3. One sarcomere connects directly to the next... so I extended the drawing above just a little bit to help you imagine this:

4. If you look at the sarcomere from the side , it would look darker in the area where the myosin runs than in the area where the actin runs. Because of this, we can talk about light and dark bands. The dark band, where the myosin runs, is called the A band. The light band, where the actin runs, is called the I band.

5. How does this relate to the myofibril? A chain of sarcomeres, like the one I drew above in point #3, that runs from one end of the muscle fiber to the other end of the muscle fiber is a myofibril. It is these chains of sarcomeres, or myofibrils, that are going to allow for contraction, since they are made up of cytoskeletal machinery. Therefore, our muscle fiber is going to need many of these myofibrils in order to be good at contraction.

Putting the myofibrils back into the muscle fiber...

First of all, there are hundreds of myofibrils in each muscle fiber. These are images of cross sections through muscle fibers... you'll see many dots on the cut edges; each of those dots is a myofibril.

Adjacent myofibrils line up evenly with each other. That means that the Z-lines of every sarcomere in one myofibril lines up with the Z-lines of every sarcomere in the adjacent myofibrils. Because of that, the I bands and the A bands in all the myofibrils within a muscle fiber are lined up. This is a difficult point to be able to understand with mental imagery. Take a look at the image here If you look up again at the drawing above of the sarcomere, you'll see that the sarcomere runs left and right, but the bands run up and down. Just like in this photo. This causes the muscle fibers to look striped, and this appearance is called striated. We can say that the muscle fibers are striated because they have striations (stripes).

Take a look at your textbook Figure 9.5 to see how adjacent myofibrils line up within a muscle fiber. For the sake of clarity, this figure only shows 9 myofibrils within the muscle fiber-- but there would be hundreds!

Now if you think back to the last web page, you'll remember that there are cell membrane invaginations called t-tubules. These t-tubules run into the muscle fiber at the Z-lines (although your book's Figure 9.5 doesn't really show it like that).

Mechanism of muscle’s contraction and relaxation. Role of calcium and ATP.

When the hypopolarizing stimulus of the spike in the T tubules is over, calcium ceases to be released by the cisternae of the sarcoplasmic reticulum and actively pumped into the longitudinal portion of the reticulum. The Ca pump that pumps Ca from the cytosol back into the sarcoplasmic reticulum is an ATPase that is phosporylated and dephosphorylated during the pumping process. It pumps two Ca ions for each ATP hydrolyzed. In muscle, the Ca ATPase accounts for nearly 90% of the membrane protein and therefore is capable of pumping Ca ions rapidly. Typically, the cytosolic Ca concentration is restored to resting levels within 30 milliseconds. When calcium is removed from the myofibrils, ATP replaces ADP on the myosin complex and the myosin-actin bond is broken. Because the muscle is elastic, it will be restored to its resting length in the absence of a further stimulus to release calcium. Shortening is an active process; lengthening is a passive process.

A single cycle of attachment, swivel, and detachment of the myosin head will produce a linear translation of the myofilaments of about 10 nm. If all cross-bridges in a myofibril cycle once synchronously, a relative movement equal to about 1% of the muscle length will occur, but obviously muscles shorten by more than 1%. The total shortening of a sarcomere during contraction may exceed 1,000 nm; therefore the relative movement of a thin and thick filament would be half this amount or 500 nm. To achieve this magnitude of change in total length when each cross-bridge cycle produces a 10-nm shortening, a minimum of 50 cycles must occur. The flexor muscles of the human upper arm can contract at the rate of 8 m/sec, during which they can shorten by as much as 10 cm. This contraction rate gives a contraction rate for the sarcomere of 160 nm/msec. If a stroke of the cross-bridge is taken to be 10 nm, then at this rate there will be a minimum of 16 strokes/msec. Thus, the swivel time for the cross-bridge must be of the order of 60 sec. Calculations for the frog's sartorius muscle, which can shorten at up to 4 cm/sec, indicate a swivel time of about 1 msec, but this contraction occurs at a lower temperature than those in mammals. In any case, it is clear that the swiveling of the cross-bridge must be a fast mechanical process. At the right is an animation that shows the repeated nature of the process.

The contractile characteristics and the mechanisms that cause contraction of vascular smooth muscle (VSM) are very different from cardiac muscle. VSM undergoes slow, sustained, tonic contractions, whereas cardiac muscle contractions are rapid and of relatively short duration (a few hundred milliseconds). While VSM contains actin and myosin, it does not have the regulatory protein troponin as is found in the heart.

Furthermore, the arrangement of actin and myosin in VSM is not organized into distinct bands as it is in cardiac muscle. This is not to imply that the contractile proteins of VSM are disorganized and not well-developed. They are actually highly organized and well-suited for their role in maintaining tonic contractions and reducing lumen diameter.

Contraction in VSM can be initiated by mechanical, electrical, and chemical stimuli. Passive stretching of VSM can cause contraction that originates from the smooth muscle itself and is therefore termed a myogenic response. Electrical depolarization of the VSM cell membrane also elicits contraction, most likely by opening voltage dependent calcium channels (L-type calcium channels), which causes an increase in the intracellular concentration of calcium. Finally, a number of chemical stimuli such as norepinephrine, angiotensin II, vasopressin, endothelin-1, and thromboxane A₂ can cause contraction. Each of these substances bind to specific receptors on the VSM cell (or to receptors on the endothelium adjacent to the VSM), which then leads to VSM contraction.

Troponin and tropomyosin are regulatory proteins that allow the muscle to shorten in the presence of Ca⁺⁺.

The mechanism of contraction involves different signal transduction pathways, all of which converge to increase intracellular calcium.The mechanism by which an increase in intracellular calcium stimulates VSM contraction is illustrated in the figure to the right. An increase in free intracellular calcium can result from either increased flux of calcium into the cell through calcium channels or by release of calcium from internal stores (e.g., sarcoplasmic reticulum; SR). The free calcium binds to a special calcium binding protein called calmodulin. Calcium-calmodulin activates myosin light chain kinase (MLCK), an enzyme that is capable of phosphorylating myosin light chains (MLC) in the presence of ATP. Myosin light chains are 20-kD regulatory subunits found on the myosin heads. MLC phosphorylation leads to cross-bridge formation between the myosin heads and theactin filaments, and hence, smooth muscle contraction.

Intracellular calcium concentrations, therefore, are very important in regulating smooth muscle contraction. The concentration of intracellular calcium depends upon the balance between the calcium the enters the cells, the calcium that is released by intracellular storage sites (e.g., SR), and removal of calcium either back into storage sites or out of the cell. Calcium is re-sequestered by the SR by a ATP-dependent calcium pump. Calcium is removed from the cell to the external environment by either a ATP-dependent calcium pump or by the sodium-calcium exchanger.

Energetic providing of muscle’s work.

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.

Image:Muscle pathways.svg

Creatine phosphate

The phosphate group in creatine phosphate is attached by a "high-energy" bond like that in ATP. Creatine phosphate derives its high-energy phosphate from ATP and can donate it back to ADP to form ATP.

Creatine phosphate + ADP ↔ creatine + ATP

The pool of creatine phosphate in the fiber is about 10 times larger than that of ATP and thus serves as a modest reservoir of ATP.

Skeletal muscle fibers contain about 1% glycogen. The muscle fiber can degrade this glycogen by glycogenolysis producing glucose-1-phosphate. This enters the glycolytic pathway to yield two molecules of ATP for each pair of lactic acid molecules produced. Not much, but enough to keep the muscle functioning if it fails to receive sufficient oxygen to meet its ATP needs by respiration.

However, this source is limited and eventually the muscle must depend on cellular respiration.

Cellular respiration

Cellular respiration not only is required to meet the ATP needs of a muscle engaged in prolonged activity (thus causing more rapid and deeper breathing), but is also required afterwards to enable the body to resynthesize glycogen from the lactic acid produced earlier (deep breathing continues for a time after exercise is stopped). The body must repay its oxygen debt.

Most skeletal muscles contain some mixture of Type I and Type II fibers, but a single motor unit always contains one type or the other, never both.

Properties of White and Red Muscles

The properties of both red and white muscles are summarized in Table. The properties of slow muscle fibers make them most suited to extended periods of contraction where a minimum force is required, e.g., in maintenance of posture. Fast muscle fibers are better suited to short periods of rapid contraction at higher forces, e.g., in sprint running. In fact, during exercise training there may be a differential effect on the two types of muscles. Strength training leads to hypertrophy of mainly white muscles with conversion of FOG to FG fibers. The number of fibers does not increase, but the size of fibers and the number of myofibrils do increase. This increases both the strength and velocity of contraction. Endurance training apparently affects mainly red muscle fibers, causing an increase in concentration of the enzymes of oxidative phosphorylation, an increase in the vascularization of the muscle and conversion of FG to FOG fibers, but no change in the ratio of fast to slow fibers and no change in muscle size.

Red muscles

The ratio of Type I and Type II fibers can be changed by endurance training (producing more Type I fibers).

· The action potential that triggers the heartbeat is generated within the heart itself. Motor nerves (of the autonomic nervous system) do run to the heart, but their effect is simply to modulate — increase or decrease — the intrinsic rate and the strength of the heartbeat. Even if the nerves are destroyed (as they are in a transplanted heart), the heart continues to beat.

· The action potential that drives contraction of the heart passes from fiber to fiber through gap junctions.

· Significance: All the fibers contract in a synchronous wave that sweeps from the atria down through the ventricles and pumps blood out of the heart. Anything that interferes with this synchronous wave (such as damage to part of the heart muscle from a heart attack) may cause the fibers of the heart to beat at random — called fibrillation. Fibrillation is the ultimate cause of most deaths and its reversal is the function of defibrillators that are part of the equipment in ambulances, hospital emergency rooms, and — recently — even on U.S. air lines.

· The refractory period in heart muscle is longer than the period it takes for the muscle to contract (systole) and relax (diastole).

· Cardiac muscle has a much richer supply of mitochondria than skeletal muscle. This reflects its greater dependence on cellular respiration for ATP.

· Cardiac muscle has little glycogen and gets little benefit from glycolysis when the supply of oxygen is limited.

· Thus anything that interrupts the flow of oxygenated blood to the heart leads quickly to damage — even death — of the affected part. This is what happens in heart attacks.

Below: the human heart, with a schematic view of the pathway of blood through the lungs and internal organs. Oxygenated blood is shown in red; deoxygenated blood in blue. Note that the blood draining the stomach, spleen, and intestines passes through the liver before it is returned to the heart. Here surplus or harmful materials picked up from those organs can be removed before the blood returns to the general circulation.

Peculiarities of metabolism in cardiac muscle.

Three-dimensional reconstruction of cardiac muscle, showing organization of myofibrils, T tubules, and sarcoplasmic reticula.

Structurally, cardiac muscle is similar to skeletal muscle in that it is striated, having both thick and thin filaments. It has a well-developed T tubule system, although the sarcoplasmic reticulum is not as large or as extensive as in skeletal muscle. Unlike those in skeletal muscle, the triads of cardiac muscle of humans are located at the Z line, giving only one per sarcomere. The mechanism of excitation-contraction coupling is the same as for skeletal muscle: The membrane action potential leads to an increase in Ca⁺⁺ around the myofilaments that activates myosin-ATPase and leads to sliding of the thin and thick filaments. The source of the calcium is different in cardiac muscle. Because the sarcoplasmic reticulum is poorly developed, it cannot sequester the large amount of calcium that skeletal muscle can. Therefore, much of the calcium for contraction must come from extracellular sources; it comes in during the action potential.

There are a large number of different kinds of cells in cardiac muscle. These include cells of the sinoatrial node, the atrioventricular node, the atrium, the bundle of His, and the ventricle, each with a differently shaped action potential. The details of these differences are beyond the scope of this treatment. For our purposes, it is convenient to distinguish two kinds of cardiac muscle cells: pacemaker cells, like the Purkinje fibers, and contractile cells. Examples of a Purkinje fiber action potential (A) and a contractile cell action potential (B) Both action potentials are much longer in duration than spikes in nerve cells and skeletal muscle cells, 0.5 sec compared to 0.5 to 5.0 msec. The hypopolarizing phase of the Purkinje fiber's action potential is not different from that in skeletal muscle, and it appears to have the same ionic mechanism, i.e., a dramatic increase in sodium conductance. The contractile cell's action potential has two rising phases, a rapidly rising phase, like that in the Purkinje fiber, and a more slowly rising phase. The fast phase has the same mechanism as the rising phase of Purkinje fiber action potentials, but the slower phase is the result of a slow inward current, carried mostly by Ca⁺⁺. Calcium current activation occurs at a more hypopolarized level of the membrane potential than does sodium activation, and the inactivation of the calcium current is less rapid by about two orders of magnitude.

The long plateau of the action potential in cardiac muscle serves two functions: It provides a more prolonged contraction without resorting to tetanus, and it provides a longer refractory period to prevent the heart from contracting prematurely. This plateau is produced by a number of factors, the most important of which is a decrease in potassium conductance with hypopolarization, followed by a slowly developing increase that brings the potassium conductance to a final value just slightly greater than resting levels in Purkinje fibers and to resting levels in contractile cells in about 300 msec. A change in membrane conductance with changes in membrane potential is called rectification by biophysicists. This change in potassium conductance is called anomalous rectification.

Cardiac muscle behaves much like skeletal muscle, but it exerts a passive tension when stretched at much shorter lengths. In fact, when the muscle is stretched from a length even shorter than resting length, there is a resistance to the stretch. In other words, cardiac muscle experiences elastic tension even at resting length (skeletal muscle does not). In addition, the maximum developed tension in cardiac muscle occurs, not at the resting length, but when it is stretched beyond resting length. The result is that when more blood returns to the heart from the veins, the muscle fibers of the heart will be stretched more, and the blood will automatically be pumped out more forcefully than when the heart is just normally full. This is the basis of the Frank-Starling mechanism in the heart.

Diagnostic significance of determination of creatin, creatinin and creatin phosphokinase’s activity in biological fluids

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.

What does the test result mean?

Increased creatinine levels in the blood suggest diseases that affect kidney function. These can include:

damage to or swelling of blood vessels in the kidneys (glomerulonephritis) caused by, for example, infection or autoimmune diseases bacterial infection of the kidneys (pyelonephritis)

death of cells in the kidneys’ small tubes (acute tubular necrosis) caused, for example, by drugs or toxins

prostate disease, kidney stone, or other causes of urinary tract obstruction; or

reduced blood flow to the kidney due to shock, dehydration, congestive heart failure, atherosclerosis, or complications of diabetes

Creatinine blood levels can also increase temporarily as a result of muscle injury and are generally slightly lower during pregnancy.

Low levels of creatinine are not common and are not usually a cause for concern. As creatinine levels are related to the amount of muscle the person has, low levels may be a consequence of decreased muscle mass (such as in the elderly), but may also be occasionally found in advanced liver disease.

Random urine creatinine levels have no standard reference ranges. They are usually used with other tests to reference levels of other substances measured in the urine. Some examples include the microalbuminuria test and urine protein test.

Since creatinine levels are in proportion to muscle mass, women tend to have lower levels than men.

In general, creatinine levels will stay the same if you eat a normal diet. However, eating large amounts of meat may cause short-lived increases in blood creatinine levels. Taking creatine supplements may also increase creatinine.

There are a few drugs that interfere with the creatinine test, although there are some drugs that can cause some impairment in kidney function. Your creatinine levels may be monitored if you are taking one of these 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.

Biochemistry of connective tissue.

Connective tissues are responsible for the form and shape of the animal body and, in addition, provide protection for vital organs and facilitate locomotion. The term connective tissue is also applied in a more restricted sense to structures such as dermis, tendons, fascia, bone, cartilage, and the capsules of the joint. All cells, however, make contacts with surrounding structures that involve connective tissues as components of the extracellular matrix. The matrix possesses chemical, physical, and mechanical properties uniquely suited to the function of tissues and organs of which the cells are a part. The extracellular matrix may be rigid (e.g., bone), elastic (e.g., blood vessel walls), compressible (e.g., cartilage), or liquid (e.g., synovial fluid). Most connective tissue matrices derive these properties by virtue of the content of fibrillar proteins, nonfibrillar macromolecules, and low molecular weight proteins and electrolytes.

The properties of the matrix are therefore determined predominantly by the function of cells, specific for each tissue, which are responsible for synthesis of the matrix components. Many of the functions of the component cells, in turn, are influenced by the character of the extracellular matrix. The properties of connective tissues are also influenced by their relationships to the vascular system from which critical components are derived, such as water, electrolytes, and proteins. Indeed, the walls of blood vessels may themselves be considered as connective tissues. However, although some connective tissues are highly vascular (e.g., bone), others are essentially avascular (e.g., cartilage).

You will be examining several types of connective tissues. General characteristics of connective tissue include; vascularization, lots of intercellular matrix (space between cells), and fibers.

There are three types of fibers found commonly in connective tissue; collagenous, elastic and reticular.

The intercellular matrix can vary in consistancy, from a solid to a fluid. The number of cells in connective tissue when compared to the number of cells in epithelial tissue is considerably less.

The major functions of connective tissue:

is to protect, to support, to transport, to store, and the obvious one "to connect" one tissue type to another.

The most common cell types are fibroblasts

which produce fibres and other intercellular materials.

Classification of Connective Tissue

I. Connective Tissue Proper - encompasses all organs and body cavities connecting one part with another and, equally important, separating one group of cells from another. This is a very large and diverse group of tissues and includes adipose tissue (fat), areolar (loose) tissue, and dense regular tissue, among others.

adipose tissue

II. Specialized Connective Tissues -- this group includes cartilage, bone, and blood. Cartilage and bone form the skeletal framework of the body while blood is the vascular (transport) tissue of animals.

cartilage blood

Cartilage is a somewhat elastic, pliable, compact type of connective tissue. It is characterized by three traits: lacunae, chondrocytes, and a rigid matrix. The matrix is a firm gel material that contains fibres and other substances. There are three basic types of cartilage in the human body: hyaline cartilage, elastic cartilage and fibrocartilage. In this laboratory, you will examine the most common type of cartilage, the hyaline cartilage. Most of the skeleton of the mammalian fetus is composed of hyaline cartilage.

As the fetus ages, the cartilage is gradually replaced by more supportive bone. In the mammalian adult, hyaline cartilage is mainly restricted to the nose, trachea, bronchi, ends of the ribs, and the articulating surfaces of most joints. The function of the hyaline cartilage is to provide slightly flexible support and reduce friction within joints. It also provides structural reinforcement.

The matrix appears as a smooth, solid, blue or pink-coloured substance. Fine protein fibres, are embedded in the matrix, but they are not visible with the light microscope since they do not stain well. Locate the large cartilage cells called chondrocytes, which are trapped within the matrix in spaces called lacunae (singular, lacuna).

Cartilage is a non-vascular tissue. As such, the chondrocytes rely on blood vessels in the tissue surrounding the cartilage for nutrient supply and waste removal. Considering this structural feature, can you make a general comment as to the potential "thickness" of cartilage?

Schematic representation of hyaline cartilage.

Microscopic view of hyaline cartilage.

I. Connective tissue proper

a) Areolar (Loose) Connective Tissue

Areolar connective tissue is the most widespread connective tissue of the body. It is used to attach the skin to the underlying tissue. It also fills the spaces between various organs and thus holds them in place as well as cushions and protects them. It also surrounds and supports the blood vessels.

areolar (loose) tissue

The fibres of areolar connective tissue are arranged in no particular pattern but run in all directions and form a loose network in the intercellular material. Collagen(collagenous) fibres are predominant. They usually appear as broad pink bands. Some elastic fibres, which appear as thin, dark fibres are also present.

The cellular elements, such as fibroblasts, are difficult to distinguish in the areolar connective tissue. But, one type of cells - the mast cells are usually visible. They have course, dark-staining granules in their cytoplasm. Since the cell membrane is very delicate it frequently ruptures in slide preparation, resulting in a number of granules free in the tissue surrounding the mast cells. The nucleus in these cells is small, oval and light-staining, and may be obscured by the dark granules.

Schematic representation of the areolar connective tissue.

Microscopic view of areolar connective tissue.

Loose or areolar connective tissue. Thick pink bands are the protein collogen, while the thin dark threads are the protein elastin.

Adipose Connective Tissue

The cells of adipose (fat) tissue are characterized by a large internal fat droplet, which distends the cell so that the cytoplasm is reduced to a thin layer and the nucleus is displaced to the edge of the cell. These cells may appear singly but are more often present in groups. When they accumulate in large numbers, they become the predominant cell type and form adipose (fat) tissue.

Adipose tissue, in addition to serving as a storage site for fats (lipids), also pads and protects certain organs and regions of the body. As well, it forms an insulating layer under the skin which helps regulate body temperature.

Schematic representation of the adipose connective tissue.

Dense connective tissue is characterized by an abundance of fibres with fewer cells, as compared to the loose connective tissue. It is also called fibrous or collagenous connective tissue because of the abundance of collagen (collagenous) fibres. Little intercellular substance is present. Furthermore, in this tissue type, the fibres are organized in a regular, parallel pattern. Hence, the name – dense regular (fibrous or collagenous) connective tissue.

In addition to the tendons, this type of tissue is also found in ligaments. Hence, the function of this tissue is to anchor skeletal muscle to bone, to attach bone to bone as well as to stabilize the bones within a joint. On your slide, note that the collagen fibres are parallel to one another. Fibroblasts are the only cells visible, and are arranged in rows between the fibres. These fibroblasts function to lay down or create the fibres of the tissue.

Schematic representation of dense regular connective tissue.

Structure and functions of collagen.

Collagen is a fibrous protein that consists of three α-chains (which are not α-helices, 'helpfully' enough), which form a rope-like triple helix, providing tensile strength to the ECM.

Collagen - click for Jmol version
Collagen.

α chains contain GXY repeats: glycine (G) is small, and is the only amino acid that fits in the crowded interior of the triple helix. X is usually proline, which destabilises the formation of a simple α-helix and Y is usually hydroxyproline; the hydrogen-bonding between the OH groups on this hydroxylated form of proline stabilises the triple helix. Hydroxyproline is formed post-translationally by the action of proline hydroxylase. This enzymes has a vitamin-C cofactor, which explains the symptoms of scurvy: tissues containing collagen (gums, skins, capillaries) are weakened, because the unhydroxylated collagen is destroyed without being secreted.

The synthesis of collagen molecules begins on the RER as with all secreted proteins. The pro-α-chains are made on the RER, and are hydroxylated and glycosylated (on hydroxylysines) in the Golgi. Procollagen forms from three α-chains, and possesses terminal 'propeptides'. This procollagen is then secreted from vesicles, and undergoes proteolysis at its ends in the extracellular space, to form mature 100 nm long collagen molecules.

Collagen molecules are then crosslinked into fibrils: oxidative deamination of hydroxylysine and lysine forms reactive aldehyde groups, which link molecules together (and also linkα-chains together too).

Collagen fibrils then self-assemble into fibres, which form characteristically straited 'ropes' under EM. Collagen fibrils have a service life of 10 years or so: most enzymes turn-over in about an hour.

Collagen comes in many different types. Type I collagen is the most common fibrillar collagen (90%), and is found in skin, bone, tendons, etc. Type II collagen provides similar tensile strength to cartilage.

Other sorts of collagen do not form fibres: type IX collagen (and type XII) are fibril-associated collagens, which link type I (or type II) collagen fibrils together. They are more flexible than fibrillar collagens because the GXY-repeats of their α-chains are interrupted more frequently by other amino acids. Type IV and VII collagens are network-forming collagens; they form a meshwork, particularly in basal lamina.

Collagen provides tensile strength to the ECM, but other proteins provide other properties. Elastin forms elastic fibres, which give the ECM its elasticity (surprise!). Elastin is secreted as the tropoelastin precursor. It is then cross-linked in similar way to collagen to form a stretchy net of elastin. Elastic fibres are coated with fibrillin microfibrils. Defects in the fibrillin-1 gene cause Marfan syndrome, which is characterised by weak elastic tissue, causing long fingers, pigeon chest, and the aorta to be very weak. Abraham Lincoln is thought to have had Marfan syndrome.

Biosynthesis of collagen.

Amino acids

Collagen has an unusual amino acid composition and sequence:

· Glycine (Gly) is found at almost every third residue

· Proline (Pro) makes up about 9% 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, derived from lysine. Depending on the type of collagen, varying numbers of hydroxylysines have disaccharides attached to them.

Collagen I formation

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

1. Inside the cell

1. Three peptide chains are formed (2 alpha-1 and 1 alpha-2 chain) in ribosomes along the Rough Endoplasmic Reticulum (RER). These peptide chains (known aspreprocollagen) have registration peptides on each end; and a signal peptide is also attached to each

2. Peptide chains are sent into the lumen of the RER

3. Signal Peptides are cleaved inside the RER and the chains are now known as procollagen

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 hydroxylated amino acid occurs

6. Triple helical structure is formed inside the RER

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, and multiple collagen fibrils form into collagen fibers

3. Collagen is 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. In the human body, a malfunction of the immune system, called anautoimmune disease, results in an immune response in which healthy collagen fibers are systematically destroyed with inflammation of surrounding tissues. The resulting disease processes are called Lupus erythematosus, and rheumatoid arthritis, or collagen tissue disorders.

Many bacteria and viruses have virulence factors which destroy collagen or interfere with its production.

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

The two most common types of fibres are: collagen (collagenous) and elastic. Collagen fibres are for strength while the elastic ones are for elasticity of the tissue. Both the cells and the fibres are embedded in the intercellular substance. The consistency of this substance is highly variable from gelatin-like to a much more rigid material.

The proportions of the cells, fibres, and intercellular substance vary, depending on a particular nature and function of the connective tissue. For example, a strong connective tissue needs a greater proportion of the collagen fibres and fewer cells. An example would be a dense regular connective tissue, which is found in tendons and ligaments. On the other hand, a connective tissue composed of mostly cells would not be very strong. An example would be an adipose (fat) connective tissue.

Elastic fibers are made of elastin and are "stretchable."

Reticular Fibers

Reticular fibers join connective tissues to other tissues.

Structure and functions of proteoglycans.

Metabolism of proteoglycans.

. The extracellular matrix occupies the space between cells. Many of the components of the extracellular matrix are connected to proteins of the cytoskeleton by transmembrane proteins.

The matrix is a complex network of different combinations of collagens, proteoglycans (PG), hyaluronic acid, laminin, fibronectin, and many other glycoproteins including proteolytic enzymes involved in degradation and remodeling of the extracellular matrix.

The matrix plays an important structural and functional role in multicellular organisms. Fibres within the extracellular matrix are collagen fibres, reticular fibres, and elastic fibres. In the dermis of the skin, bone, tendon, organ capsules and many other areas collagen fibres mainly consist of collagen type 1, which constitutes approximately 90 % of the body collagen. Collagen fibers in cartilage consist of collagen type 2. The basement membrane of epithelila consists of collagen type 4. Reticular fibres are made mainly of collagen type 3 and form a mesh-like structure. In most tissues these fibers are produced by fibroblasts. The reticular fibers in hematopoietic and lymphatic tissue are made by reticular cells. Reticular fibers around peripheral nerves are produced by Schwann cells. Smooth muscle cells also produce reticular fibers. Elastic fibres form a three-dimensional network interwoven with collagen fibres. They consist of elastin and microfibrils and are produced by fibroblasts in most cases. Smooth muscle cells produce these fibers in elastic arteries.

The extracellular matrix is more than a scaffold that fills extracellular spaces. Many of its components are engaged in processes mediating cell-to-cell interactions . In many instances the capacity of a cell to proliferate, differentiate, and to express specialized functions intimately depends on the presence and maintenance of an intact extracellular matrix (see, for example: hematopoiesis). Components of the extracellular matrix are involved also in the process of anoikis, a special form of programmed cell death.

Some of the matrix components, in particular the proteoglycans, function as modulators of the biological activities of growth factors. The organization composition, and physical properties the extracellular environment are also essential for the modulation of functions of endothelial cells during angiogenesis.

Proteoglycans are proteins modified by glycosaminoglycans (abbr. GAG, called also mucopolysaccharides). Glycosaminoglycans are long-chain compounds made up of hundreds or less repeating disaccharide units. One of the sugars in each disaccharide unit is a hexosamine (glycosamine). The four main types consist mainly of sulfated heparan sulfate/heparin, chondroitin sulfate/dermatan, keratan sulfate, and the non-sulfated glycosaminoglycan hyaluronic acid. Hyaluronic acid is an extremely long and rigidglycosaminoglycan containing several thousand sugars but no protein core. Linker molecules join proteoglycans to hyaluronic acid. Many proteoglycans contain a core protein which links them to the cellular membrane.

The combination of a core protein and a specific glycosaminoglycan generates a unique proteoglycan with a precise developmental pattern. In addition, Glycosaminoglycans are negatively charged compounds and therefore bind unspecifically to many other substances, including growth factors. Some proteins are known to interact specifically withglycosaminoglycans. The interactions between glycosaminoglycans and cytokines are one of the important mechanisms underlying communication processes between cells that are mediated by secreted and locally acting factors.

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 cell’s 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.

connective tissue diseases

- 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

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.

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 16,000–24,000 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.