Muscle Injury Induced By Exercise And Different Types Of Contractions In Dystrophic Mdx Mice

BackgroundDuchenne muscular dystrophy (DMD) is a severe X-linked recessive muscular disorder characterized by progressive weakness that affects1in3500live male births. It is caused by a gene mutation in dystrophin, which located at Xp21, followed by the absence of gene products. The clinical manifestation of DMD is the progressive and symmetric weakness on the proximal limbs. The DMD patients seem to share a similar natural history that the onset age is3to5years old, the age of wheelchair-bounded is9to13years old and the life span is15to25years. The pathological changes include necrotic and regenerating fibers, hypercontracted fibers, splitting fibers, endomysium proliferation and adipose tissue infiltration.The precise mechanism leading from the absence of dystrophin to the muscular injury is still debated, though several mechanisms have been proposed during the past two decades, including direct mechanical injury, impaired calcium homeostasis, increased oxidative stress, recurrent muscle ischemia, aberrant cell signaling and inflammatory injury. It has been proven that the intra-cellular calcium is not increased in isolated DMD muscles without contractions. But the calcium could be markedly increased after contractions. Then researchers suggested that contractions may play an essential role in the disturbance of calcium homeostasis. The neuronal isoform of NO synthase (nNOS) knockout mice do not develop muscle disease, which suggests that nNOS does not play a direct role in DMD. Therefore the calcium hypothesis and vascular hypothesis might reveal some downstream mechanisms in development of DMD. Ozawa proposed that as a component of cytoskeleton the dystrophin protein do not have biochemical activities. We propose that some mechanical factor might play an essential role leading from the absence of dystrophin to the muscle fiber necrosis.Ozawa suggested that there is a dystrophin bolt composed of dystrophin, sarcoglycans and dystroglycans. The C-terminal of dystrophin protein links to β-dystroglycan and the N-terminal to the F-actin. In addition, the a-dystroglycan directly connects with Iaminin-a2. Then the dystrophin seems to be a scaffold between cytoskeleton and sarcolemma, basilar membrane.The dystrophin bolt also links the Z-disk to the basilar membrane according to the desmin-vimentin intermediate filaments (DVIF), which distributes densely around individual myofibrils at each Z-disk. The DVIF radiate away from the myofibrils to the costamere and connect to the dystrophin bolt. This system was named the transverse fixation system (TFS) and it plays an important role in maintaining the integrity of sarcomere and sarcolemma.So far, many researchers have got the consent that the dystrophin play an important role in maintaining the integrity of sarcolemma especially during contractions. We found that the muscle injuries of DMD patients have a deep relationship with contractions itself and varies with different types of contractions.①The serum casein kinase (CK) level increases along with exercises. For DMD patient, the serum CK level begins to increase after birth, then elevates progressively, reaches the peak level at3years old, accounts for about100-fold of the upper limits. Followed the peak level, it gets decreased gradually by20%per year. In a short stage, the CK level fluctuates along with the intensity of exercises. Florence revealed that the CK level could increases by10fold of its former level after visiting a zoo, which suggests that exercise is a key factor influencing the CK level of a DMD patient.②The more exercises, the worse the weakness becomes. Almost all the DMD patients share a similar natural history. The motor development is not delayed markedly. Most patients do not get visible weakness until3years old, then the weakness aggravated slowly, followed by a rapid aggravation after7to9years old. All the patient get wheelchair bounded before13years old. It is obvious that the weakness progressed in proportion to the aggregation of exercises.③Hypercontracted fiber Another pathological feature is hypercontracted fiber, which accounts for4.3to8.3%. It is considered that the contraction-induced breakdown of sarcomere and sarcolemma is essential for hypercontracted fibers. Numerous hypercontracted fibers were observed in mdx extensor digitorum longus (EDL) muscles subjected to electric stimulation in our preliminary experiment.④Type2B fibers are destructed early in DMD patients. Type2B fibers decrease in number after2years old and disappear entirely after5years old. Henning proposed an explanation for this is that their sarcolemma is subject to the highest mechanical stress due to rapid, strong contractions.⑤The extraocular muscle (EOM) and intrinsic laryngeal muscles (ILM) are spared in life time of the DMD patients. Researches have suggested that the EOM and ILM have enhanced ability to maintain calcium homeostasis, scavenge oxygen radicals and regenerating. However, there are not any studies shown these abilities directly facilitate EOM and ILM sparing from pathological changes. We found that both EOM and ILM always perform shortening contractions. In contrast, the iliopsoas and quadriceps, which perform numerous lengthening contractions, are initially and severely affected in the natural course of DMD. We prefer a hypothesis that different types of contractions lead to different extents to which DMD muscle fibers are injured, in another word, shortening contractions produce little injury to dystrophin-deficient muscle fibers, while lengthening ones induce marked damage.So far, there is no research gives a direct insight into the relationship between absence of dystrophin and muscle fiber necrosis. And studies on comparing the effect of lengthening, isometric and shortening contractions on dystrophin-deficient muscles are unavailable.Objectives1. To explore the injury purely caused by exercise to dystrophin-deficient muscles.2. To design a protocol to evaluate injuries induced by different types of contractions in vitro.3. To compare the extent of injury induced by different types of contractions in dystrophin-deficient muscles.Methods1. Injuries induced by exercise to mdx triceps surae muscles (TS). Six5-week-old mdx mice were randomly divided into two groups:normal bred and exercise. Another six age-matched C57BL/6mice were used as control. Mice belonging to exercise group received a voluntary wheel running, a moderate exercise, once a day in a super clean bench. The running last for30min, then mice were returned to the barrier system and bred as normal. On the13th day, all mice were subjected to intraperitoneal injections of1%sterile solution (W/V) of evans blue dye (EBD) at a dosage of100μl per10g body weight. On the14th day, the mice were killed with an overdose of pentobarbitone sodium and bilateral TS muscles were dissected out, blotted and weighted, followed by snap-frozen in isopentane precooled with liquid nitrogen and stored at-80℃for later analysis. Alternate frozen sections (8μm thick) were cut from the middle portion of the TS muscle for Haemotoxylin and Eosin (H&E) and EBD slides. The total number of the muscle fibers, percentages of necrotic/regenerating, centralized nuclear, hypercontracted and EBD positive fibers were determined.2. Different types of contraction-induced injury in vitro. The EDL muscles were dissected from nine12-week-old mdx mice and9age matched C57BL/6mice. Then the mdx and control muscles were randomly assigned to either the lengthening (LC), isometric (IC), shortening (SC) contraction-induced injury protocol, respectively. For each group, a total of6muscles were involved. The Po (maximum tetanic isometric force) measured immediately before contraction-induced injury procedure and Po1min after contractions were recorded. The magnitude of the injury was assessed by the force deficit which was calculated as (Po-P0)/Po×100%. Then the muscle was removed from the facility immediately and incubated while held at Lo in oxygenated0.2%procion orange dye (POD)/Ringer solution for a total duration of90min. The muscle was then washed2×5min in normal Ringer solution, after which it was blotted, weighted and embedded in O.C.T compound, snap-frozen in isopentane precooled with liquid nitrogen and stored at-80℃for later analysis. Alternate frozen sections (8μm thick) were cut from the middle portion of the EDL muscle for H&E and POD slides. The total numbers of the muscle fibers and percentages POD positive fibers were determined.3. Effects of unloaded shortening contractions on mdx TS muscles in vivo. Two groups of21-day-old and30-day-old mdx mice were subjected to an achillotenotomy randomly on one TS muscle and left the contralateral muscle intact as a control. In the next14days, mdx mice were bred in barrier system as usual. A triceps surae muscle of a mouse with excised Achilles tendon should only perform the unloaded shortening contractions during movements. Meanwhile, the contralateral one with intact Achilles tendon performs a mixture of shortening, isometric and lengthening contractions. Another two age-matched C57BL/6mice were subjected to the same procedure as normal control. On the13th day, all mice were placed on intraperitoneal injections of1%sterile solution (W/V) of EBD at a dosage of100μl per10g body weight. On the14th day, the mice were killed with an overdose of pentobarbitone sodium and bilateral TS muscles were dissected out, blotted, weighted and snap-frozen. Alternate frozen sections (8μm thick) were cut from the middle portion of the TS muscle for H&E and EBD slides. The total number of the muscle fibers, percentages of necrotic/regenerating, centralized nuclear, hypercontracted and EBD positive fibers were determined.Results1. Injuries induced by exercise to mdx TS muscle. Compared with normal bred mice, exercise had no effect on the wet weight of mdx and C57BL/6muscles (P=0.226and0.567, respectively, n=6). Exercise had no effect on the total number of TS muscle fibers of mdx and C57BL/6mice (P=0.354and0.377, respectively, n=6). Pathological changes, such as necrosis/regeneration, centrally placed nuclei, hypercontracted fibers, were not observed in samples from C57BL/6mice.Compared with the normal bred mdx mice, the exercised mdx mice showed markedly increased percentages of necrotic/regenerating, centrally nucleated, hypercontracted and EBD positive fibers (all P<0.01, n=6).2. Different types of contraction-induced injury in vitro. The lengthening contractions induced greater force deficit than isometric contractions did to muscles of mdx and control mice (all P<0.01). Isometric contractions resulted in greater (P<0.01) and no more (P=0.55) force deficit than shortening contractions did to muscles of mdx and control mice, respectively. For lengthening and isometric contractions, EDL muscles of mdx mice showed greater force deficit than that of control mice (all P <0.01). However, there is no significant difference in force deficit between mdx and control muscles exposed to isometric contractions (P=0.92).For lengthening and isometric contractions, EDL muscles of mdx mice showed higher percentages of POD positive fibers than that of control mice (all P<0.01). There is no significant difference in percentages of POD positive fibers between mdx and control muscles subjected to isometric contractions (P=0.82). Comparing to the isometric contractions, the lengthening contractions induced higher percentages of POD positive fibers to muscles of mdx and control mice (all P<0.01). Isometric contractions led to greater (P<0.01) and no more(P=0.99) percentages of POD positive fibers than shortening contractions did to muscles of mdx and control mice, respectively.3. Effects of unloaded shortening contractions on mdx TS muscles in vivo. Fourteen days after operation, no differences was observed in the number of myofibers between operated and contralateral triceps surae muscles of35-day-old (P=0.29) and44-day-old (P=0.31) C57BL/6mice. However, there was a significantly decreased muscle mass in muscles with excised Achilles tendon compared to the contralaterals in the two age groups of mice (all P<0.01). No necrotic/regenerating, centrally nucleated, hypercontracted or EBD positive fibers were observed in any C57BL/6mice muscles. Therefore, we assumed that any pathological changes occurring during mdx muscle degeneration-regeneration resulted from the unloaded shortening contractions themselves.Fourteen days after operation, triceps surae muscles with excised Achilles tendon showed significantly decreased percentages of necrotic/regenerating, centrally nucleated, hypercontracted and EBD positive fibers compared to the contralateral ones in35-day-old and44-day-old mdx mice (all P<0.01).Conclusion1. Exercise could induce injury to mdx muscles and its sarcolemma in direct.2. The mdx muscles showed greatly and moderately increased susceptibility to lengthening and isometric contraction-induced injury, respectively. However the unloaded shortening contractions induce little injury to muscles of mdx mice.3. The in vitro protocol employed in our research for different types of contraction-induced injury is sensitive and reliable. These protocols are useful to discriminate the effects of different types of contractions on muscles of both dystrophic and control mice.4. We suggest that avoidance of lengthening and isometric contractions with great afterload, and encouragement of mild-afterload shortening contractions should be added to the exercise prescription for DMD patients.