DESCRIPTION OF MUSCULAR DYSTROPHY
Duchenne muscular dystrophy (DMD) is a genetic disorder resulting in the loss of the muscle protein dystrophin (30). Dystrophin is a large protein that has been localized on the inner surface of the muscle fiber sarcolemma (30,69). The precise role of dystrophin is not known but appears to be involved in stabilization of the cell membrane. Muscle membranes from fibers deficient in dystrophin are more susceptible to contraction-induced injury (47,51). Consequently, a cycle of injury and repair ensues and results in progressive muscle weakness and fibrosis. Several studies have indicated that the dystrophic diaphragm muscle is affected to a greater degree than limb skeletal muscle (15,63).
As respiratory muscles in humans with DMD progressively deteriorate, pulmonary function likewise worsens. The lung volumes of patients with DMD are significantly different from normal individuals (57), and the patients’ ability to generate inspiratory or expiratory force is markedly impaired (27). Expiratory muscle strength is severely diminished in all individuals after age 7 and continues to decrease with age (27). Because chronic respiratory insufficiency is a major factor contributing to mortality in muscular dystrophy (35), understanding the mechanisms that lead to respiratory muscle dysfunction are critical for the development of therapeutic interventions aimed at preventing respiratory failure in muscular dystrophy.
USING THE MDX MOUSE TO STUDY PULMONARY DYSFUNCTION IN MUSCULAR DYSTROPHY
Discovered by Bulfield et al. (7), the mdx mouse (a mutant of the C57BL/ScSn strain) is similar to DMD because it too lacks dystrophin due to a point mutation in the gene. Although limb skeletal muscle undergoes fairly successful regeneration, the diaphragm muscle from the mdx mouse exhibits many of the same pathological features observed in muscle from DMD patients.
Associated with membrane fragility, muscle fibers in mdx mice experience difficulty in maintaining intracellular calcium homeostasis (18,68). Loss of intracellular Ca2+ homeostasis is thought to lead to hypercontraction of myofibrillar proteins and segmental necrosis in skeletal muscle from mdx mice (13,67). Elevated levels of intracellular calcium may also activate proteases, e.g., calpains (60) that promote myofibrillar protein degradation. As in DMD, a persistent inflammatory response characterized by elevated levels of inflammatory cells (8,13,49) is also observed in mdx skeletal muscle.
Although limb skeletal muscle from mdx mice undergoes little change in fiber size and type composition during the lifespan, significant differences in diaphragm muscle exist between control and mdx mice even at a young (3–4 months) age (52). The most significant difference at this age is that there is a significant reduction in percent of fibers expressing the 2X or 2B myosin heavy chain (MHC) isoform (∼40% in control mice vs ∼27% in mdx mice) (52). In addition, no fibers express embryonic MHC isoform in normal diaphragm whereas this isoform is expressed in ∼10% of mdx diaphragm fibers (52), indicative of muscle fiber regeneration. With advancing age (22–24 months), there is complete loss of fibers expressing 2X, 2B, and embryonic MHC isoform in mdx diaphragm (52). The fact that embryonic MHC isoform disappears with age suggests a decrease in the regenerative capacity of the mdx diaphragm in the later stages of the disease.
Fibrosis is a distinctive feature in mdx muscle (43,63) just as it is in muscles from DMD patients (4). Stedman et al. (63) noted that by 16 months of age, the collagen level in the mdx diaphragm is ∼7 times greater than that of age-matched controls. The level of collagen cross-linking also significantly increases in muscle from dystrophic animals (17,28). The changes in collagen concentration may in part be due to increased rates of collagen synthesis since the level of type I collagen mRNA is significantly increased in mdx muscle (19).
As in DMD, compositional changes in the mdx diaphragm are accompanied by a severe loss of tissue compliance. Stedman et al. (63) noted that the mdx diaphragm is significantly stiffer (i.e., less compliant) than the diaphragm from normal control mice. The changes in muscle stiffness likely contribute to decreased chest wall compliance and subsequent increase in work of breathing.
Besides changes in the passive viscoelastic properties of the diaphragm, the active contractile characteristics of diaphragm muscle also differ markedly between normal healthy mice and mdx mice. In the diaphragms of young (4–6 months) and old (24 months) mdx mice, there is a significant reduction in optimal muscle length (Lo) compared with age-matched controls (41). Lynch et al. (41) also found that in young mdx mice, diaphragm maximal isometric tetanic force (Po) is reduced by approximately 40%, whereas in the diaphragms of old mdx mice, Po is reduced by ∼52%. In this same study, maximal diaphragmatic power was also decreased in young (∼54%) and old (∼69%) mdx mice compared with age-matched controls. Times to peak-twitch-tension and half-relaxation in diaphragms of old mdx mice were significantly prolonged by ∼33% and 27% respectively, compared with young mdx mice (41) and are likely related to changes in fiber type composition observed by Petrof et al. (52).
Because mdx mice appear to live a normal lifespan, it was unclear to what extent impaired diaphragm function affects pulmonary function. Using the barometric technique (38), we determined that mdx mice indeed had a reduction in ventilatory reserve, i.e., an inability to increase minute ventilation in response to a ventilatory stimulus. Ventilatory pattern in awake, spontaneously breathing 12-wk-old normal C57BL/10J (N = 10) and mdx (N = 8) mice was assessed during room air breathing and in response to hypercapnia (Fig. 1). At rest (room air), mdx mice had a significantly (P < 0.05) lower tidal volume (Vt) with no significant change in respiratory rate such that minute ventilation (E) was significantly lower in mdx mice. During CO2-induced hypercapnia, mdx mice were unable to increase breathing frequency and Vt to the same extent as controls, thereby resulting in a significantly lower E at both 4% and 8% CO2 compared with normal controls. These data indicate that ventilatory pattern in young mdx mice is altered under resting conditions, and when challenged with a respiratory stimulus, a significant ventilatory impairment exists.
IS CHRONIC INFLAMMATION RESPONSIBLE FOR MUSCLE WASTING IN MUSCULAR DYSTROPHY?
Although dystrophin-deficiency is the genetic consequence of DMD, this deficiency alone appears to be insufficient to cause the clinical manifestations of muscle weakness and stiffness. An interesting feature of dystrophin-deficiency across species is the expression of grouped and segmental necrosis (1,10,12,14). This feature does not support the “single gene defect” model because not all dystrophic muscle fibers express similar pathology. Grouped fiber necrosis, which is common to all dystrophinopathies, is more typical of extracellular rather than intracellular events (6). This suggests that the extracellular environment is crucial for maintenance of muscle fiber viability.
The extracellular environment in dystrophic muscle is distinctly different from healthy muscle as a consequence of muscle activity. Normal contractile activity results in the accumulation of transient breaks in the sarcolemma, which allow the release of factors that encourage muscle fiber repair (46). However, given the same recruitment history dystrophin-deficient muscle is damaged to a greater degree than healthy muscle due to its innate membrane fragility (33,36,44,48,51). Therefore, the same factors released transiently by healthy muscle to promote healing may have pathologic consequences in dystrophic muscle because they are present chronically.
One pathologic consequence of continual exposure to factors released by injured muscle is a persistent inflammatory response. One DNA microarray study of 8-wk-old mdx limb muscle found that inflammatory mediators and effectors dominate the expression profile; 30% of the 242 differentially expressed genes in this study are associated with inflammation (54). Increases in genes associated with all aspects of inflammation were found including regulation (proinflammatory chemokine ligands and receptors), extravasation (endothelial cell proteins), mononuclear cell infiltration (lymphoid and myeloid markers), and cytolysis (complement system genes). The increase in proinflammatory chemokine ligands and receptors was confirmed in a subsequent study of transcript and protein expression (55). Importantly, several of the inflammatory genes identified in the muscle from mdx mouse were also found to be upregulated in muscle from DMD patients (11).
DNA microarray data showing that inflammatory mediators and effectors dominate the expression profile of dystrophic muscle is supported by histological evidence showing an increase in the number of mononuclear cells (macrophages, mast cells, dendritic cells, and lymphocytes). Healthy muscle normally has a small number of mononuclear cells residing in the endo- and perimysium, often near neurovascular elements (2,22,49). In dystrophic muscle, there are significantly more mononuclear cells, and they are found in the perimysium, in the endomysium adjacent to necrotic and nonnecrotic fibers, and invading necrotic fibers (2,3). The most abundant mononuclear cell types are T cells and macrophages (16,45,61) although the number of mast cells is also increased (22).
All three of these cell types, T cells, macrophages, and mast cells, could contribute to muscle wasting in dystrophic muscle by killing muscle fibers. Cytolysis is the primary function of a subpopulation of T cells. The frequency of these killing T cells is increased by 17- to 18-fold in muscles from mdx mice compared with control (61). Importantly, depleting dystrophin-deficient mice of T cells reduces the degree of histopathology and reduces the number of apoptotic myonuclei (61). A similar reduction in apoptotic nuclei was observed in dystrophic muscle that was devoid of perforin, an essential cytotoxic compound released by T cells (62). These observations provide strong evidence that dystrophic myofiber death occurs through T cell-mediated processes.
Macrophages may also have a role in killing dystrophic myofibers. Reducing the concentration of circulating macrophage also reduces the number of lethally injured fibers in dystrophic muscle (70). In addition, in vitro work shows that co-culturing healthy myotubes with macrophages isolated from dystrophic muscle, at macrophage concentrations found in dystrophic muscle, results in significant myotube cytolysis (70). Macrophages may contribute to muscle wasting in at least two other ways. First, they may participate indirectly by regulating the recruitment of other cells that kill myofibers (31). Second, macrophages secrete tumor necrosis factor-alpha (TNF-α), which can induce skeletal muscle proteolysis and apoptotic-like DNA fragmentation (9,20,40).
Mast cells are another source of compounds that can cause muscle proteolysis and ultimately kill myofibers. Mast cells produce and secrete a variety of compounds including proteases that have been shown to cause muscle fiber necrosis (21,24). The number of mast cells in dystrophic muscle is 3–5 times greater than control (22,49). Moreover, mast cell number and location is highly correlated with disease progression and myofiber necrosis in dystrophic muscles from humans, mice, and dogs (22). Increasing mast cell activity in mdx mice prolongs the transient period of myofiber necrosis seen in limb muscles (25).
Overall, DNA microarray data and histological evidence from several different species suggest that dystrophic muscle is characterized by a persistent inflammatory response. There is a chronic elevation in inflammatory mediators and effectors as well as an elevation in invading mononuclear cells. The cytolytic abilities of several types of mononuclear cells make it likely that they are at least partially responsible for muscle wasting in muscular dystrophy. Support for this notion comes from studies showing that manipulating the number of invading mononuclear cells can significantly alter disease progression in dystrophic limb muscles. Because the consequences of muscle fiber loss in the diaphragm are ultimately lethal in DMD patients, it is imperative to determine whether global or targeted immunosuppression can improve diaphragm muscle morphology and function.
CAN IMMUNOSUPPRESSION IMPROVE VENTILATORY STATUS IN MDX MICE?
We have begun studies using mdx mice designed to attenuate the pathological progression of diaphragm dysfunction by targeting the immune system in mdx mice. We initially examined the impact of short-term (8 wk) daily treatment of mdx mice with prednisone (mdx-pred) (1 mg·kg−1 I.P.) on diaphragm muscle collagen characteristics and level of TGF-β. TGF-β is produced by macrophages and is known to be a potent stimulator of collagen synthesis in vitro (26,29,34,58). Additionally, TGF-β suppresses the production of matrix-degrading metalloproteinases (50,59) and increases the activity of the inhibitor of matrix-degrading metalloproteinases (50,59). TGF-β may therefore be affecting collagen accretion in two ways: first, by increasing transcription of Types I and III collagen, and second by inhibiting degradation of fibrillar collagen. This study was subsequently followed with a long-term treatment study (6 months) in which we assessed ventilatory function, diaphragm contractility and MHC isoform composition, and extent of diaphragm fibrosis. Prednisone, a drug commonly administered to patients with DMD, has a variety of functions that could influence muscle function. With respect to the immune system, prednisone decreases the circulating and infiltrating levels of T lymphocytes in humans (37) and may affect the level of inflammatory cytokines.
Figure 2 illustrates changes in collagen concentration relative to age-matched controls after both short- (8 wk) (28) and long-term (6 months) treatment of mdx mice with prednisone. Diaphragm muscle collagen concentration was ∼40% higher in untreated 12-wk-old mdx mice (mdx-sham) compared with control, whereas there was no difference between the control group and the mdx-pred group. This reduction in collagen concentration in the mdx-pred group was associated with a significant reduction in level of TGF-β (28), thus supporting the idea that TGF-β is involved in the development of diaphragm muscle fibrosis. In the 7-month-old mice, collagen concentration was ∼242% higher in the mdx-sham group compared with controls. Diaphragm muscle collagen concentration was significantly (P < 0.05) lower in the mdx-pred group compared with the mdx-sham group; however, the concentration was still significantly elevated (∼162%) compared with controls. These findings indicate that with respect to collagen accumulation, the impact of prednisone is attenuated over time, or other mechanisms unresponsive to prednisone are involved.
Figure 3 illustrates the ventilatory response of 7-month-old mice during room air breathing and in response to 7% CO2. As expected, the normal control mice significantly increased E above resting values, whereas there was little response in the mdx-sham group. Interestingly, during 7% CO2 exposure, E in the mdx-pred group did not significantly differ from either the control group or the mdx-sham group. Po (N·cm−2, Fig. 4) was significantly and equally depressed in both the mdx-sham and mdx-pred group. Whether or not the improvement in ventilatory reserve (Fig. 3) in the mdx-pred group was due to an increase in total muscle mass and subsequently total force output is not known. It was not associated with any changes in diaphragm muscle MHC isoform composition as assessed by SDS-PAGE and densitometry (Fig. 5). The diaphragm muscle from the 7-month-old control mice contained on average ∼6% slow, 39% 2A, 48% 2X, and 7% 2B MHC isoform. In contrast, the diaphragm from the mdx-sham group contained significantly less slow MHC (∼2% of total) isoform but more 2A MHC isoform (∼50% of total). The percentages of 2X and 2B MHC isoform did not significantly differ between control and mdx-sham. However, when these two isoforms are combined as one, the percentage of 2X/2B MHC isoform is significantly reduced, a finding previously observed by Petrof et al. (52). As previously stated, long-term prednisone administration had no impact on diaphragm muscle MHC isoform composition.
TUMOR NECROSIS FACTOR-α (TNF-α) DELETION IMPROVES VENTILATORY STATUS IN MDX MICE.
We have recently begun studies examining the role of specific inflammatory cytokines in the pathological cascade of DMD. TNF-α is one such cytokine and its concentration is elevated in muscular dystrophy (53,65). TNF-α has pro-inflammatory actions and is produced by a number of inflammatory cells including activated macrophages and T-cells (32). Besides mediating the inflammatory response (5,42), TNF-α also has direct effects on skeletal muscle that may adversely affect its function (39,40,56) and regeneration potential (64,66). We recently examined the impact of long-term TNF deletion on ventilatory pattern (N = 5/group), diaphragm muscle contractility (N = 8/group), and MHC isoform distribution (N = 9/group) (23). For these studies, 10- to 12-month-old TNF-α deficient (TNF-α−) mdx mice were compared with age-matched background strain controls (TNF-α+mdx).
Figure 6 A, B, and C illustrates, respectively, respiratory rate, VT, and E obtained during quiet room air breathing and in response to 7% CO2 in TNF-α− and TNF-α+mdx mice. With respect to RR, there was no significant main effect for either group or gas mixture.
VT did not significantly differ between the TNF-α− and TNF-α+mdx groups during room air breathing. Within the TNF-α+mdx group, VT did not significantly differ between 7% CO2 and room air. Within the TNF-α−mdx group, however, VT was significantly higher during 7% CO2 breathing compared with room air (P < 0.001). Additionally, 7% CO2 breathing resulted in a significantly higher VT in the TNF-α−mdx group compared with the TNF-α+mdx group.
Minute ventilation did not significantly differ during room air breathing between TNF-α−mdx and TNF-α+mdx groups. Within the TNF-α+mdx group, there was no difference in E between 7% CO2 and room air. Within the TNF-α−mdx group, however, E was significantly higher during 7% CO2 breathing compared with room air (P < 0.001). In addition, E was significantly higher in the TNF-α−mdx group compared with the TNF-α+mdx group (P < 0.001) during 7% CO2 exposure.
TNF-α DELETION AFFECTS DIAPHRAGM CONTRACTILITY AND MHC ISOFORM DISTRIBUTION
In this study, neither Lo, time-to-peak twitch tension, time-to-half relaxation, nor peak twitch tension significantly differed between groups. However, Po was ∼36% higher (P < 0.05) in the TNF-α−mdx group than TNF-α+mdx group.
The relative distribution of the diaphragm muscle MHC isoforms is illustrated in Figure 7. The diaphragm contained on average ∼12.9% slow, ∼52.7% 2A, ∼32.4% 2X, and ∼2.0% 2B MHC isoform in TNF-α+mdx group, whereas in the TNF-α−mdx group, the diaphragm contained ∼7.4% slow, ∼52.8% 2A, ∼36.1% 2X, and ∼3.2% 2B. The percentage of Type I (slow) myosin was significantly higher in the TNF-α+mdx group compared with that of the TNF-α−mdx group. There were no significant differences between groups in the percentages of type 2A, 2X, or 2B MHC isoforms. However, when the fast isoforms were pooled together, the TNF-α−mdx group contained a significantly higher percentage of fast MHC in (93.7 ± 0.8) compared with the TNF-α+mdx group (87.1 ± 2.1).
In summary, our current studies indicate prednisone administration attenuates muscle fibrosis, improves the ventilatory response to a respiratory challenge, and reduces levels of TGF-β1 in mdx diaphragm muscle but has no effect on MHC isoform distribution or diaphragm contractility. Elimination of TNF-α, a pro-inflammatory cytokine with multiple effects on skeletal muscle, in mdx mice resulted in significant improvements in ventilatory function and diaphragm strength, and altered the composition of MHC isoforms in the diaphragm. These findings indicate the immune system plays a role in the progressive deterioration of the diaphragm muscle in muscular dystrophy and provide a basis for developing pharmacological approaches to maintain muscle mass and function in this debilitating disease.
The authors are grateful to Jake Barkley, Jacqueline Williams, and Melissa Deering for their technical assistance. This work was supported by the American Lung Association and the Muscular Dystrophy Association.
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