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Introduction:
Muscular dystrophies are defined as a group of heterogeneous group of diseases characterized by muscle weakness (Rahimov et al, 2013). There are different kinds of muscular dystrophy like Duchenne, Becker, Myotonic, Congenital, Emery-Dreifuss, Facioscapulohumeral, Limb-girdle, Distal, and Oculopharyngeal (Table 1). The most common form is Duchenne muscular dystrophy (DMD). 1 in every 3500 to 5000 newborn males worldwide are affected by it. Symptoms of muscle weakness are seen typically between 3 to 7 years of age. Boys suffering from DMD show delayed development of motor skills like talking, sitting and walking and around puberty lose ambulation (Gao et al, 2015). DMD results from the loss of function due to a mutation in dystrophin present in the muscle cell. Dystrophin provides a structural link between muscle cytoskeleton and the extracellular matrix to maintain muscle integrity. It is rich in myofibers and concentrated into rib-like structures called costameres (structural components linking myofibrils to sarcolemma). Disruption of this protein causes the plasma membrane to be fragile and making it less stiff with an increase in leakiness of the muscle enzymes (Goldstein et al, 2010). Current research suggests the loss of dystrophin as being the primary reason for muscle weakness. However, a potential secondary reason could be the abnormal Ca2+ levels which play a role in muscle necrosis.
Table 1: Characteristics of the Types of MD
NOTE: Verhaert, D., Richards, K., Rafael-Fortney, J. A., & Raman, S. V. (2011). Cardiac Involvement in Patients With Muscular Dystrophies. Circulation: Cardiovascular Imaging, 4(1), 67-76. doi:10.1161/circimaging.110.960740
Skeletal Muscle Structure and Function:
In order to understand the complexities of muscular dystrophies, first we need to understand the basics of the structure of skeletal muscle and how muscle contraction works.
Structure of skeletal muscle
Muscle in our body is surrounded by a connective tissue called epimysium. The muscle is further divided into numerous bundles called fascicles of individual muscle cells by another connective tissue called perimysium. Hundreds of muscle fibers are contained in these elongated fascicles. Fusion of many cells results in the muscle fibers being multinucleated. These nuclei are found in a region below the plasma membrane of the muscle fiber called as sarcolemma. A semi-fluid cytoplasm called the sarcoplasm is present in each muscle fiber. Rod like elements, myofibrils along with the mitochondria surround the sarcoplasm. Each myofibril is composed of thick filaments called myosin and thin filaments called actin (Stanfield, 2013). The sarcoplasmic reticulum, a saclike membranous network surrounds each of the myofibrils, which is closely associated with other structures called transverse tubules (T tubules). The lateral sacs, enlargements of the sarcoplasmic reticulum are present near the T tubules and serve as a place to store calcium. Each T tubule along with two lateral sacs forms a triad. The sarcoplasmic reticulum and T tubules together help in activating muscle contraction. They send signals to the myofibrils from the sarcolemma further enabling a muscle cell to respond to neural input (Stanfield, 2013).
Figure 1: Structure of a skeletal muscle fiber showing its different components
Muscle contraction:
Skeletal muscle are referred to as striated muscle because of their striped like appearance. The protein fibers (thick and thin filaments) present in the myofibrils are arranged in an orderly way giving them the striations. Thick and thin filaments are present in a 2:1 ratio. These myofibrils are made up of repeating units called the sarcomere that is bordered by the Z lines on each of its sides. These Z lines are perpendicular to the long axis and support one of the ends of the thin filaments. M lines connecting the thick filaments together are also perpendicular to the long axis. On the sides of the A band, dark striations are visible due to the overlapping of the thick and thin filaments. However, the center of the A band is lighter because only thick filaments are present without any overlapping between the thick and thin filaments. This is the H zone. The region where only thin filaments are present without any with thick filaments the I band (Stanfield, 2013). In the center of the I band, there is a Z line connecting the thin filaments together. The thin filaments are made up of actin and the thick filaments are made up of myosin. They are arranged in a repeating fashion.
Each of the thin filaments contain the actin monomer call the G (globular) actin, which have a myosin-binding site. G actin are linked end to end forming the F (fibrous) actin strand. Two F actin strands are arranged in a double helix. The thin filaments also contain two regulatory proteins called the tropomyosin and troponin, which help in activating and deactivating contraction. The long fibrous tropomysin extends over the actin monomers blocking the myosin to bind preventing contraction. Troponin is made up of three proteins. One binds to the actin strand. The second protein binds to tropomyosin. Binding of troponin with tropomysion results in exposing of the myosin binding sites on the actin thin filament. The third protein contains calcium-binding sites (Stanfield, 2013).
Figure 2: (a) A myosin molecule (b) Two actin molecules forming the double-helical structure
Figure 3: Sarcomere structure showing the arrangement of protein filaments within the sarcomere
Note: Stanfield, C. L. (2016). Principles of human physiology. Upper Saddle River: Pearson.
The Mechanism of force generation in a muscle:
Muscle contraction occurs by a repeating cycle where the thick and thin filaments slide past each other. This cycle is called the cross-bridge cycle (Stanfield, 2013).
Each cross-bridge cycle involves the five steps:
- Binding of myosin to actin: Myosin is in its energized form of ADP and Pi with high affinity for actin. It is first bound to the ATPase site of myosin head, which further binds to the actin monomer of the thin filament. This step requires the presence of calcium to take place.
- Power stroke: In this step, the myosin and actin are bound together marking the release of Pi from the ATPase site. This step is called the power stroke because force is generated. In this step, the myosin head swivels towards the center of the sarcomere resulting in shortening of the sarcomere and muscle contracting.
- Rigor: In this step, myosin goes to a lower energy state and releases the ADP from the myosin head. This stage is called as rigor because the actin and myosin are bound tightly together. An excess of calcium due to damaged cell membranes or less ATP production due to no energy production could result in the crossbridge cycle getting stuck at this stage. This could also be called as the rigor mortis, which is characterized by the stiffening of body after death.
- Unbinding of myosin and actin: In this step, a new ATP is introduced to the ATPase site of the myosin head resulting in its conformational change. This conformational change decreases the affinity of myosin for actin. Hence, myosin unbinds from actin.
- Cocking of the myosin head: In this step, energy is released. ATP bound to the ATPase site of myosin head is cleaved into ADP and Pi by ATP hydrolysis. Now myosin is in its high-energy form and is bound to the ATPase site of the myosin head. In presence of calcium, the cycle will repeat again starting from step 1.
This cross-bridge cycle continues endless if there is enough ATP available. Troponin and tropomyosin help in regulating this cycle by interacting with calcium and decreasing the available myosin-binding sites (Stanfield, 2013).
Figure 4: The cross-bridge cycle
Note: Stanfield, C. L. (2016). Principles of human physiology. Upper Saddle River: Pearson.
Excitation-Contraction coupling:
The axon terminal of a motor neuron or the presynaptic cell releases Acetylcholine (ACh). On diffusing into the postsynaptic cell or the muscle cell, the ACh binds to receptors in the motor end plate, which has a high density of acetylcholine receptors. This results in the activation of the many ACh receptors creating an end plate potential (depolarization). This end plate potential is higher in the presynaptic cell than a regular postsynaptic potential. This triggers an action potential in the muscle cell. This action potential generates throughout the sarcolemma and the T tubules. As the action potential propagates through the T tubules, calcium stored in the lateral sacs of the sarcoplasmic reticulum is released. Calcium then binds to the troponin resulting in a conformational change causing tropomyosin to move from its position exposing the myosin-binding sites. This calcium-binding to troponin marks the start of muscle cell contraction. When tropomyosin is not bound to troponin, it blocks the myosin-binding sites resulting in relaxation of the muscle (Stanfield, 2013).
Figure 5: Steps that take place during excitation-contraction coupling
Note: Stanfield, C. L. (2016). Principles of human physiology. Upper Saddle River: Pearson.
What is the role of Dystrophin in Muscular Dystrophy?
We have understood how the muscle works- how does it contract and relax as well as how force generation occurs in a muscle. In order to understand how the absence of dystrophin affects the weakening of muscle, we need to understand the function of dystrophin.
Dystrophin and its Associated Proteins:
DMD is known to be the largest known gene that encodes for the dystrophin (Goldstein et al, 2010). This rod-shaped protein is located primarily on the subsarcolemmal of the skeletal muscle plasma membrane. It is used for movement (skeletal muscles) and in heart (cardiac) muscle. However, small amounts of dystrophin are also found in the nerve cells in the brain. The dystrophin binds to the actin through actin binding domains (Culligan et al, 2002). In skeletal and cardiac muscles, dystrophin is part of a group of proteins, a protein complex. They work together to strengthen muscle fibers and protect them from injury during muscle contraction and relaxation. The dystrophin complex provides a structural link between each muscle cell’s cytoskeleton with the framework of proteins and other molecules outside the cell (extracellular matrix). The dystrophin complex may also play a role in cell signaling by interacting with proteins that send and receive chemical signals.
There is very little information about the function of dystrophin in nerve cells. Current research suggests that this protein is important for the normal structure and function of synapses that are specialized connections between nerve cells where cell-to-cell communication occurs (Culligan et al, 2002).
Figure 6: Muscle fiber showing the presence of dystrophin
Note: Duchenne Muscular Dystrophy (DMD) – Causes/Inheritance. (2018, January 31). Retrieved from https://www.mda.org/disease/duchenne-muscular-dystrophy/causes-inheritance
Membrane fragility caused by Dystrophin deficiency:
Dystrophin along with its associated proteins, are found at the sarcolemma along with the sarcomeric Z disc in structures known as costameres. Costameres are present over the Z disc within the plasma membrane and is perpendicular to the long axis of the myofiber. This intracellular position, dystrophin’s capacity to bind actin, argues for a role in transmitting force from the sarcomeres to the extracellular matrix and neighboring cells. A research paper by Rybakova et al. demonstrated the presence of cytoskeletal actin on the inner cytoplasmic face in a peeled membrane, indicating the tight linkage of dystrophin and actin (Goldstein et al, 2010).
Goldstein et al (2010) used mice as the model organism to study DMD. A peeled membrane from the myofibers of the mdx mouse which has a point mutation in the DMD gene, that lacks dystrophin was not found to retain actin. On the other hand, a peeled membrane from the laminin alpha 2-deficient dy mice retained both dystrophin and actin, hence, a specific role for dystrophin in the actin-binding interface. Recently, it was shown that dystrophin directly binds to tubulin and organizes the microtubule network. Because microtubules not only provide cell structure, but also scaffold the Golgi network and direct intracellular traffic, loss of this dystrophin– microtubule interaction is a plausible mechanism for the altered localization of many proteins in dystrophin- mutant animals.
Figure 7: (A) Normal myofiber (B) Dystrophin deficient mdx myofiber
Note: Goldstein, J. A., & Mcnally, E. M. (2010). Mechanisms of muscle weakness in muscular dystrophy: Figure 1. The Journal of General Physiology, 136(1), 29-34. doi:10.1085/jgp.201010436
Membrane leak and rupture caused by Dystrophin deficiency:
DMD releases muscle enzymes, particularly creatine kinase, into the circulation. Some other proteins like lactate dehydrogenase and aldolase also leak into the serum, and these serum proteins are effective as biomarkers of disease. It is unclear whether the leak of these myoenzymes contributes to muscle dysfunction in muscular dystrophy. The disrupted membrane allows for the entry of small molecules into the myofiber syncytium. Ca2+-sensitive dyes has been used effectively to trace calcium entry into muscle cells. The vital tracer Evans blue dye is a small molecule that can enter into muscle lacking dystrophin whereas normal fibers are typically impermeable (Goldstein et al, 2010).
Impaired calcium handling in muscular dystrophy:
The primary defect in DMD is the loss of the membrane cytoskeletal protein dystrophin resulting from specific mutations in the human DMD gene. However, the secondary molecular mechanisms leading ultimately to muscle degeneration is not clear. Abnormal Ca2+ homeostasis has been known to make the skeletal muscle cells more susceptible to necrosis, cell death. Recently, a study shows how Ca2+ levels are higher in individuals with DMD. A Ca2+ deposit-sensitive histochemical red stain called alizarin was used and an increase in positive-reacting cells in the DMD muscles was seen. When repeated on mdx skeletal muscle, a similar result was observed. Fura-2, a fluorescent Ca2+ indicator has also indicated an increase in Ca2+ levels in the dystrophic muscles. This was further confirmed by studying mdx fibres which showed increase in cytosolic Ca2+ levels particularly in dystrophin deficient muscles. This study was not confirmed universally since many other scientists reported no difference in the calcium levels between dystrophin deficient muscles and normal muscles. (Culligan et al, 2002).
Muscular dystrophy affects other body systems:
A patient suffering from muscular dystrophy has a high chance of experiencing respiratory failures. This results from weakening of breathing muscles which further result in a limited lifespan unless mechanically supported (Gao et al, 2015). In patients suffering from muscular dystrophy, the lung muscles lose their elasticity and become less distensible. The weakened muscle is marked by its inability to generate adequate levels of pressure and volume whereas the fatigued muscle is marked by their inability to produce the pressure in response to the constant load. The cause of this distensibility is unknown but one cause could be breathing at a low lung volume which is common in a MD patients. Respiratory muscle weakness varies from person to person depending on the intensity of the disease. The diagnosis can can be delayed since the symptoms are very subtle in the beginning. However, there is a tension time index (force developed by the inspiratory muscles to the time they are being used) which helps in understanding the respiratory muscle fatigue. Children suffering from MD show elevated values of the tension time index. This is indicative of the fact that inspiratory muscles are prone to fatigue beginning from childhood (Lo Mauro A. et al, 2016). In some forms of muscular dystrophy, heart is also affected further resulting in heart failure and irregular heart rhythms (Gao et al, 2015).
Treatments for muscular dystrophy:
There are not a lot of therapies for Muscular Dystrophy that have been discovered. One of the common successful one is the use of corticosteroids. They have proved to benefit the boys suffering from DMD by stabilizing their muscle strength and function and delaying the advancement of scoliosis and cardiomyopathy. Treatment continued even after the loss of independent ambulation showed some improvement in the patient and hence proved beneficial. Available literature and some clinical experience suggest that corticosteroid treatment should be considered for all patients suffering from DMD starting at an early stage of the disease (Mah, 2016). In addition, supportive care for the muscular dystrophy patients has also helped improve the life expectancy of many young adults suffering from DMD (Mah, 2016 and Leung et al 2013). This involves a lot of nonpharmacologic interventions, like physical therapy, occupational therapy, orthopedic surgery, and genetic counseling (Leung et al 2013).
Works cited:
- Gao, Q. Q., & Mcnally, E. M. (2015). The Dystrophin Complex: Structure, Function, and Implications for Therapy. Comprehensive Physiology, 1223-1239. doi:10.1002/cphy.c140048
- Goldstein, J. A., & Mcnally, E. M. (2010). Mechanisms of muscle weakness in muscular dystrophy: Figure 1. The Journal of General Physiology, 136(1), 29-34. doi:10.1085/jgp.201010436
- Leung, D. G., & Wagner, K. R. (2013). Therapeutic advances in muscular dystrophy. Annals of Neurology, 74(3), 404-411. doi:10.1002/ana.23989
- 4. Mah, J. (2016). Current and emerging treatment strategies for Duchenne muscular dystrophy. Neuropsychiatric Disease and Treatment, Volume 12, 1795-1807. doi:10.2147/ndt.s93873
- Rahimov, F., & Kunkel, L. M. (2013). Cellular and molecular mechanisms underlying muscular dystrophy. The Journal of Cell Biology, 201(4), 499-510. doi:10.1083/jcb.201212142
- Stanfield, C. L. (2016). Principles of human physiology. Upper Saddle River: Pearson.
- Duchenne Muscular Dystrophy (DMD) – Causes/Inheritance. (2018, January 31). Retrieved from https://www.mda.org/disease/duchenne-muscular-dystrophy/causes-inheritance
- Culligan, K. G., & Ohlendieck, K. (2002). Abnormal Calcium Handling in Muscular Dystrophy. Retrieved from http://www.bio.unipd.it/bam/PDF/12-4/02491Culligan.pdf
- Verhaert, D., Richards, K., Rafael-Fortney, J. A., & Raman, S. V. (2011). Cardiac Involvement in Patients With Muscular Dystrophies. Circulation: Cardiovascular Imaging, 4(1), 67-76. doi:10.1161/circimaging.110.960740
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