Longitudinal Changes in Muscle Size and Composition in Duchenne Muscular Dystrophy
Steven A. Svoboda, Rebecca Willcocks, Celine Baligand
University of Florida
Abstract
Duchenne muscular dystrophy (DMD) is a tragic genetically X-linked neuromuscular disease that is characterized by the absence of the structural protein dystrophin, which in turn causes cellular muscle damage, lipid tissue infiltration, and progressive muscle weakness. There are presently no cures or treatments to impede muscle deterioration in DMD and therapeutic interventions and clinical trials have been inadequate and limited. The purposes of the present longitudinal study were to assess alterations in size and fat content of muscles in the lower leg of boys with DMD and to investigate the relationships between muscle size, composition, and function over time using a combination of advanced magnetic resonance imaging (MRI) and spectroscopy (MRS) technologies. Two random subjects with DMD were tested at repeated time points over a two-year period. MRI and MRS were performed to determine muscle area and fat content within the muscles. The subjects were tested on the isokinetic dynamometer for isometric strength and timed functional testing. The subjects exhibited distinct differences in disease progression as proved by MRI, MRS, strength, and functional measures over a period of two years. The combination of these non-invasive tools provides insight into the disease progression and will have implication for future treatment evaluation.
Longitudinal Changes in Muscle Size and Composition in Duchenne Muscular Dystrophy
Duchenne muscular dystrophy (DMD) is a tragic hereditary neuromuscular disease, which affects “1 in 3600-6000 live male births” (Bushby, et al., 2009). This disease transpires by a mutation in the X-linked recessive gene, which is responsible for encoding an essential muscle protein, dystrophin (Mathur, et al., 2011). This protein functions to attach the myofilaments to the muscle cell membrane (Mathur, et al., 2010). The absence of functional dystrophin leads to the degeneration and damage of muscle fibers and tissue, starting first in the lower limbs and extremities (Deconinck & Dan, 2007). This can initially be observed through the enlargement of the calf muscles at which then the enlarged muscle tissue becomes replaced and infiltrated with fat and connective tissue (NHGRI, 2010). This progressively results in muscle function loss and weakness in all voluntary muscles, including the heart and breathing muscles (Medline Plus, 2010). In fact, some boys by three to five years of age already begin experiencing profound muscle weakness and physical limitations (Mathur, et al., 2010). Boys diagnosed with DMD lose the ability to walk early on and face premature death (MDA, 2011). By the time boys with DMD reach their adolescent years, most are confined to a wheelchair. As a result, skeletal deformities develop and muscle strength declines which in most cases leads to breathing complications and cardiomyopathy (enlarged heart). Unfortunately, those affected with DMD rarely survive past their 30s (NHGRI, 2010).
There are presently no cures or treatments to impede the muscle deterioration in DMD (Kinali, et al., 2011). In addition, therapeutic interventions have been inadequate and clinical outcome measures have been confined to measures of muscle function, serum biomarkers of muscle degeneration, and invasive muscle biopsies (Cacchiarelli, et al., 2011). Therefore, in order to facilitate the rapid development of more beneficial interventions, it is necessary to obtain further quantitative outcome measures that are noninvasive and receptive to alteration in muscular structure and composition (Kinali, et al., 2011). Magnetic resonance imaging (MRI) and spectroscopy (MRS) can be used to noninvasively study skeletal muscle and has been recently developed and used to track disease progression in DMD (Akima, et al., in press, Mathur, et al., 2011).
Over the past ten years there has been a major advancement in medical imaging technology and predominantly magnetic resonance has demonstrated to be the most appropriate method for the noninvasive assessment and tracking of skeletal muscle composition and physiology (Kinali, et al., 2011). Through state-of-the-art MR imaging and spectroscopy technologies, loss of muscle contractile area, cellular muscle damage, and intramuscular lipid accumulation in the lower-limb muscles of boys with DMD become discernible (Mathur, et al., 2010). Also, even though MRS and MRI are based on the same principles, they both provide complementary information relevant to the progression of DMD. Essentially, MR spectroscopy can measure the water to lipid ratio in muscles by extracting “information about the chemicals that reside on the frequency scale between water and fat in both a qualitative and quantitative manner” (Imaging Research Center, 2000). Furthermore it generates a plot depicting the chemical composition of a region that must be interpreted, instead of producing a black and white resolution image like an MRI (Imaging Research Center, 2000).
In addition to the MRI and MRS methods, functional tests are essential to determine the condition and severity of Duchenne muscle dystrophy in boys, because these tests relate directly to the boys’ ability to perform daily activities. The two main types of functional tests are kinetic and isometric. Kinetic tests involve a variety of free motion exercises, such as running, walking, and stair climbing, whereas isometric tests measure the strength of the muscle in a fixed position. This method allows for the analysis of muscle strength in boys with DMD. Through these methods of tracking the disease, promising therapeutic interventions have the potential of being assessed and validated in boys affected by DMD (MDA, 2011).
The purposes of this study are to assess alterations in muscle cross-sectional area (CSA) of the lower leg in boys with DMD measured by MRI, to evaluate the changes in lipid to water ratio of muscles in the lower leg measured by MR spectroscopy, and to investigate the relationships between muscle size, composition, and function over a period of two years.
Methods
To evaluate disease progression in boys with DMD, a longitudinal study design was used for MRI/MRS measures of the lower extremity (lower leg) muscles. In order to assess sensitivity of noninvasive MR measures to disease progression, all MRI/MRS measures of muscle composition have been acquired in DMD boys in 6-month intervals within a two-year period. A total of two ambulatory DMD boys have been studied longitudinally.
First, cross sectional images of the lower leg of each of the two DMD boys were taken with MR imaging at three or four time points for two years. At the first imaging time point, subject 1 was 9.9 years of age and subject 2 was 8.8 years of age. MRI has the capability to resolve muscle, fat, connective tissue, and bone using the magnetic properties of the body tissues. When placed in a high magnetic field, the alignment of hydrogen atoms’ nuclei within water molecules in the body changes. This will allow the atoms to absorb and emit radio waves, which then can be recorded and read by the scanner. The resulting images of the lower leg display a contrast that can be used to distinguish the different muscles. Then, utilizing the program OsiriX to view the images on the computer, the maximal CSA of five muscles of the lower leg of each DMD boy were determined by outlining or drawing regions of interests (ROI’s) around the boundaries of the individual muscle of interest. These five muscles include the Medial Gastrocnemius (MG), Lateral Gastrocnemius (LG), Peroneals (Per), Soleus (Sol), and the Tibialis Anterior (TA), as illustrated in figure 1.
Next, using MR spectroscopy, a spectrum was obtained from a voxel within the lateral side of the soleus muscle for subject 1 and subject 2, as shown in figure 2. The spectra shown in figure 3, which display water peak and lipid peak, were then used to determine the area under the water peak and lipid peak through integration. Once the areas of water peak and lipid peak were found, they were expressed as a (lipid:(water+lipid)) ratio. This ratio was studied over time.
Finally, functional tests were given to the two DMD subjects. The two boys first were placed in a isokinetic dynamometer, a computerized robotic chair-like machine used to perform a variety of isometric tests. The two types of isometric tests that were implemented were plantar flexion and dorsiflexion. Plantar flexion, which relies on the soleus and gastrocnemius muscles, was measured with the ankle at a right angle and the knee slightly flexed when at rest but straight during contraction. The subjects were instructed to push forward with their toes with as much force as possible for 5 seconds, as if pushing the gas pedal on a car. This was repeated 5 times, with a 1-minute rest period in between trials. Dorsiflexion, which relies on the tibialis anterior, was performed in the identical position as plantar flexion. The subjects were instructed to pull their toes toward them as hard as they could for 5 seconds. This again was repeated 5 times, with a 1-minute rest period in between trials. The next set of functional tests were timed tests which included standing up from the floor, climbing a set of four stairs, and walking a distance of 30 feet as fast as the subjects could. Three trials were completed for each test and every trial was timed with a stopwatch.
Results
The two subjects in this study exhibited distinct differences in disease progression as measured by MRI, MRS, strength, and functional measures over a period of two years. Subject 1 was measured at 9.9, 10.4, 10.9, and 11.9 years of age. He was ambulatory all throughout the measurement period. He grew from 138 to 144 cm over the entire study, with most of the growth in the final year. His weight changed from 51.5 to 62.3 kg over two years. Subject 2 was measured at 8.8, 9.8, and 10.8 years old. He lost the ability to climb stairs and to stand from the floor between time point 1 and time point 2. He lost ambulation between time point 2 and time point 3. He grew from 121 to 124 cm over the first year, but his height was not measured at time point 3 because he could not stand up to be measured. His weight changed from 22.5 to 23.9 to 25 kg over the period of two years.
In subject 1 shown in figure 4, the maximum cross-sectional area of each of the five muscles in the lower leg increased overall during the period of two years. However in subject 2 shown in figure 5, each of the five muscles either decreased in area or remained relatively the same. The soleus, medial gastrocnemius, and lateral gastrocnemius were the three muscles in subject 2 that decreased in area. The tibialis anterior and the peroneals were the other two muscles in subject 2 that stayed generally constant in size. In subject 1, the total muscle area in the lower leg shown in figure 6 steadily increased over time, where as in subject 2 the total muscle area declined.
MRS revealed the average value for the lipid:(lipid+water) ratio and was expressed as a percent. In both subject 1 and subject 2, the muscle fat content of the soleus increased approximately by 20 percent over two years, as shown in figure 7. The relationship between the size and composition of the soleus in subject 1 and subject 2, shown in figure 15, is unexpected. In subject 1, muscle size increases as the percent fat increases. However, in subject 2, muscle size decreases while percent fat increases.
In subject 1, the time taken to complete the 30-foot walk test shown in figure 8 gradually increased over the two-year period. In subject 2, the time taken to complete the 30-foot walking test increased more rapidly than in subject 1 and by the last time point subject 2 was unable to perform the test due to loss of ambulation. Similarly, in subject 1, the time taken to climb 4 stairs shown in figure 9 increased over the 2-year period. This also occurred in subject 2, however more rapidly because at time points 3 and 4 the subject was already unable to complete the test. In subject 1, the time taken to stand up from the floor progressively increased, as shown in figure 10. In subject 2, the time taken to perform this functional test increased dramatically and by the third time-point the subject was not able to complete it due to loss of ambulation.
The plantar flexor torque shown in figure 11 slightly increased in subject 1 over time. In subject 2, the plantar flexor torque decreased over time. In subject 1, the dorsiflexor torque shown in figure 12 increased overall. In subject 2 the dorsiflexor torque steadily diminished over the time period of two years. The plantar and dorsiflexor torques increased in subject 1, whose total muscle area had also increased over two years. However, the plantar and dorsiflexor torques decreased in subject 2, whose total muscle area had declined over the two years.
The specific torque for dorsiflexion shown in figure 13 (calculated by dividing torque by muscle area) in subject 1 and subject 2 both decreased, however at different rates, over the two-year period. The specific torque for plantar flexion (soleus and gastrocnemii muscles) shown in figure 14 decreased in subject 2 and increased slightly in subject 1.
Discussion
This study examined the progression of Duchenne muscular dystrophy using MRI/MRS, strength, and functional measures in two random subjects with DMD. The two subjects exhibited different progressions of the disease and deterioration of muscles over the period of two years. Subject 1 demonstrated an increase in muscle size, lipid infiltration, and loss of muscle function. Yet, strength in subject 1 showed slight improvement over two years most likely due to muscle growth and maturation as the plantar flexor specific torque shows. Additionally, this plantar specific torque did not increase significantly, but the absolute torque did, signifying that torque is due to muscle size in subject 1. If studied over a longer period of time, however, strength and torque will most likely eventually decline. Subject 2 displayed an increase in lipid infiltration, loss of muscle function, and muscle weakness. Yet, muscle size either decreased or remained the same in subject 2 due to loss of ambulation within the two years. Therefore, it is probable that the lateral and medial gastrocnemii and the soleus, which are responsible for walking, decreased in size as a result of not being in use. It can be expected that subject 2 will have a more rapid progression of the disease because his muscles contain more fat and less muscle area than subject 1 who has fat too but also possesses greater muscle area. This relationship of muscle size to fat content in subjects 1 and 2 indicates the heterogeneity in disease progression as well as suggests that atrophy and lipid infiltration might be driven by different physiological mechanisms.
During the monitoring of disease progression in this study, both subjects demonstrated an increased time in the 30-foot walk, climb up four stairs, and stand up from floor functional tests. The results of these functional timed tests indicate that subject 1 and subject 2 are both progressively getting functionally weaker, however at different rates. In fact, the increase in the amount of time to accomplish these functional tests appears to correlate with the loss of ambulation. For example, subject 2 did in fact become nonambulatory between 12 and 24 months of the study, which was preceded by a large increase in times between 6 and 12 months within all the functional tests.
The plantar flexor specific torque for subject 1 demonstrated a minimal increase in strength, although the size of muscles increased over the time points. The plantar flexor specific torque for subject 2 exhibited a decrease in strength and in size of muscles. This points out that muscle function, although related to muscle size, also depends on muscle composition. In the case of subject 1, the overall muscle size increase was accompanied by an increase in fat content, suggesting that the proportion of healthy and functional muscle decreased. The results validate the progression of the disease because by looking at torque per unit of area and eliminating the variable of muscle size, it allows for composition and fat infiltration to be taken into account. Furthermore, due to subject 1’s slight increase in specific torque for plantar flexion, the results demonstrate that muscles may be able to temporarily adapt to increases in fat. These outcome measures emphasize the considerable heterogeneity in individual responses among the DMD subjects.
Collectively, the results underscore the diversity and variation within the individual progression of DMD. Nevertheless, there remains a close relationship in the outcomes and the progressive nature of this disease. These two subjects highlight the heterogeneity of individuals with Duchenne muscular dystrophy because functional decline was more rapid in subject 2 and was accompanied by a decrease in muscle size, while functional decline in subject 1 was slower and accompanied by an increase in muscle size. Although disease progression is variable across the population with DMD, the use of MRI/MRS tracks objectively and non-invasively the extent of muscle atrophy and biochemical composition of the muscles within a compartment. These outcome measures along with the functional and isometric strength tests help illustrate a more complete picture of this disease. However, the study only focused on the lower leg muscles and not the thigh muscles, which also are responsible for ambulation, balance, and mobility. Further investigation is necessary to expand our understanding of the progression of this disease and the application of MRI/MRS and their ability as outcome measures to document healthy and injured tissue as interventions for this disease come to the forefront.
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