Biochemistry, Myoglobin

Fundamentals

Myoglobin is one of the members of the globin superfamily, which also includes hemoglobin. It often gets compared structurally and functionally to hemoglobin. Hemoglobin has four polypeptide chains and four oxygen binding sites. Myoglobin is a single polypeptide chain with one oxygen binding site, which results in the different binding kinetics of the two proteins to oxygen. Myoglobin does so noncooperatively, unlike hemoglobin which binds to oxygen cooperatively as a result of its tetrameric nature. As a result, myoglobin’s oxygen saturation curve is hyperbolic. Hemoglobin displays a sigmoid-shaped curve due to its cooperative binding. Myoglobin exhibits a higher affinity for oxygen than hemoglobin. Therefore, it is very efficient at extracting oxygen from the blood. Myoglobin is mainly present in the striated muscle of vertebrates. Hemoglobin, on the other hand, is found in the bloodstream as a part of erythrocytes. Myoglobin is also present in much lower concentrations in smooth muscle, endothelial, and even tumor cells.[1][2]

Issues of Concern

Myoglobin’s role as a marker for a disease is limited. It is no longer in specific guidelines for the management of acute coronary syndromes. With regards to rhabdomyolysis, myoglobin is not a prognostic criterion and is not diagnostic.[3]

Cellular Level

Myoglobin occurs in the highest concentration in the striated muscles of vertebrates. Specifically, it is in the cytoplasm of cardiac myocytes and the sarcoplasm of oxidative skeletal muscle fibers; this includes skeletal muscle and heart muscle. Myoglobin is also present in much lower concentrations in smooth muscle and endothelial tumor cells.[2]

The expression of the MB gene has also been reported in various tumor cell lines such as breast carcinoma, colon carcinoma, acute leukemia, desmoplastic small round cell tumors, and non-small cell lung cancer.[4]

Molecular Level

Myoglobin is a single polypeptide chain of 154 amino acids. The chain consists of eight α-helices assigned the letters A–H. The molecule contains a heme prosthetic group, which includes a porphyrin ring iron ion. The heme-bound Fe cation can exist in the 2+ (reduced) or 3+ (oxidized) state. The iron ion itself interacts with six different ligands, one of which serves as the binding site for oxygen. This binding site can also function to bind other potential molecules, including CO and NO.[1]

Function

The primary function of myoglobin is to supply oxygen to the muscle. It does this by releasing its oxygen supply to the mitochondria that make up the respiratory chain, helping the myocytes to meet their high energy demands. Myoglobin serves as a buffer of intracellular oxygen concentrations and as an oxygen reservoir in muscle. This concept has support from the fact that diving mammals can have 10- to 30-fold more myoglobin as compared to animals that do not hold their breath for an extended period.[1]

Myoglobin facilitates oxygen diffusion. Myoglobin desaturates at the onset of muscle activity, which increases oxygen’s diffusion gradient from the capillaries to the cytoplasm.

Myoglobin has also been shown to have enzymatic functions. It is necessary for the decomposition of bioactive nitric oxide to nitrate. The removal of nitric oxide enhances mitochondrial respiration. This enhancement is because nitric oxide reversibly inhibits cytochrome oxidase.

Myoglobin, furthermore, functions to remove reactive oxygen species. It can do this by interacting with fatty acids, which may be metabolically important under oxygenated conditions and high energy demands.[2]

Mechanism

Myoglobin contains a Fe(II) cation bound to a heme group, which gives it the ability to bind to oxygen reversibly. Oxygen can bind to the heme residue of myoglobin due to the iron ion’s interaction with six ligands. Four of these ligands are nitrogens that together make a porphyrin ring and share a common plane with the iron. The fifth ligand is the imidazole side chain of His93 that stabilizes the heme group and slightly displaces the iron ion away from the plane. The sixth ligand position is the binding site for oxygen. When oxygen binds to the iron ion, it partially pulls back to the plane of the porphyrin ring. Myoglobin can also bind alternate ligands such as CO, nitrite, and azide molecules. For example, CO binds strongly to myoglobin just as it does to hemoglobin, which forms carboxymyoglobin.[1]

In its reduced form, Fe(II), myoglobin can either be bound to oxygen (oxymyoglobin) or not (deoxymyoglobin). Additionally, the iron ion can be oxidized to form Fe(III), which is known as metmyoglobin. The binding of oxygen is done noncooperatively since myoglobin is monomeric.[5] This binding is why myoglobin’s oxygen saturation curve is hyperbolic and characteristic of normal Michaelis-Menten enzyme kinetics. The sigmoid shape of the hemoglobin oxygen saturation curve is due to its tetrameric makeup and cooperative binding of oxygen.

Testing

Myoglobin testing can occur in the blood or urine. The main indications for checking this lab are for rhabdomyolysis or myocardial infarction. As stated above, the usefulness of these tests is questionable.

With regards to rhabdomyolysis, myoglobin testing in the urine can be from the combination of a urine dipstick and microscopy. In this disease process, you will get a positive urine dipstick test for blood. However, when one looks at the sample under the microscope, there will be no evidence of red blood cells. The urine dipstick shows a false positive for blood because myoglobin also reacts with the orthotolidine test reagent. Serum myoglobin is not necessary for diagnosing or managing rhabdomyolysis. Urine myoglobin is also not used routinely.[6] A urinalysis can reliably predict the absence of myoglobinuria. This test can be used instead of getting urine myoglobin.[7]

Additionally, myoglobin is a sensitive marker for acute myocardial infarction. However, it lacks specificity. Its usefulness comes in evaluating infarct size and reperfusion. Myoglobin gets rapidly released from the myocardium during the injury, and its blood concentration rises in the 30 minutes immediately after the onset of the event. It is one of the earliest markers to increase as the result of ischemic heart disease. It is then rapidly excreted by the kidneys within 24 hours. In this sense, it is an important marker for early detection or exclusion of cardiac damage. In summary, the use of this biomarker for myocardial infarction should be in combination with other criteria in favor of diagnosing myocardial infarction. In current emergency room settings, myoglobin is not part of the diagnostic criteria. Troponins, Hs-cTnI and hs-cTnT, are the gold standard biomarkers currently.[8]

Pathophysiology

Myoglobin knockout models have been created to try to understand its functions more clearly. Mice models have mutated myoglobin to the point of it being undetectable in cardiac and skeletal muscle. The mutation causes several lethal cardiovascular defects in embryos. However, the mice who survived that stage of development exhibited cellular and molecular adaptive responses to deal with the lack of myoglobin. These included having a higher capillary density in the heart to enhance oxygen supply. Their results, along with other similar studies, suggest that although compensatory responses exist, myoglobin is necessary for normal muscle development and function.[9]

Myoglobin also plays a role in the pathophysiology of rhabdomyolysis. The process begins with injury to the myocyte membrane or altered energy production; this leads to an influx of extracellular calcium from along with a release of calcium from the sarcoplasmic reticulum and mitochondria from inside of the cell.  The accumulation of excess intracellular calcium causes destructive processes leading to lysis of the cell and release of its contents, including myoglobin. This protein is the primary constituent of muscle that contributes to renal damage. The first proposed mechanism of its toxicity is tubular obstruction. Myoglobin can precipitate out of solution in the renal tubules. This is exacerbated by intravascular volume depletion and acidosis. The second mechanism is by oxidant injury. Iron in myoglobin can dissociate and lead to the release of free radicals and oxidative damage to the renal parenchyma. The final mechanism is by myoglobin inducing lipid peroxidation. This process creates molecules that act as vasoconstrictors to the renal arterioles.[10]

Clinical Significance

Myoglobin’s main clinical significance comes with its association with muscle damage. In particular, it correlates with rhabdomyolysis and myocardial infarction. These are not disease processes resulting from defective myoglobin but rather ones that cause myoglobin to leak into the blood and urine, causing damage. Rhabdomyolysis carries associations with myoglobinuria and often acute kidney injury. The first description of rhabdomyolysis was in association with crush injuries and trauma. However, in hospitalized patients, more common causes include over-the-counter, prescription, and illicit drug use and alcohol use. Seizures can also cause rhabdomyolysis.[11] Excessive myoglobin excretion in the urine can cause the color to change to red or brown. Fluid resuscitation is the main intervention to treat the acute kidney injury caused by myoglobin.[10]

Myoglobin is also detectable in the urine as the result of hereditary myopathies. A muscle biopsy can be done to identify these if performed after the episode of rhabdomyolysis triggered by exercise. The diagnostic test of choice, however, is molecular genetics.[12]

Myoglobin is detectable in the serum after acute myocardial infarction. However, as described above, it is not currently in the guidelines for diagnosis. It is one of the earliest cardiac biomarkers to increase in concentration in the blood following myocardial infarction.[8]