Muscle injury induced by different types of contractions in dystrophic mdx mice

Studies on comparing the effect of lengthening, isometric and shortening contractions on dystrophin-deficient muscles are unavailable. We hypothesized that different types of contractions lead to different extents to which dystrophin-deficient muscles are injured. For this purpose, we developed protocols for different types of contraction-induced injury to mdx muscles in vitro. Force deficits and percentages of procion orange dye positive fibers were employed to assess the extent of injury to each muscle. Our results revealed that both the lengthening and isometric contractions resulted in significantly greater injury to extensor digitorum longus (EDL) muscles of mdx mice than to that of control (C57BL/6) mice. In contrast, the shortening contractions induced very mild and identical injury to EDL muscles of mdx and C57BL/6 mice. Then another protocol was carried out in vivo to ascertain the effect of shortening contractions on mdx muscles by achillotenotomy. Histological assessment revealed that the triceps surae muscles with excised Achilles tendon (EAT) displayed little and significantly milder injury than the normal ones did. In conclusions, the unloaded shortening contractions induce little injury to mdx muscles. The in vitro protocol for different types of contraction-induced injury is sensitive and reliable.     

成肌细胞移植阻止mdx鼠运动诱导的肌损害

目的:观察过量运动对肌营养不良症模型鼠(mdx鼠)骨骼肌的损害作用,以及成肌细胞移植对运动诱导损害肌纤维的保护作用。方法: 采用分离消化法对C57新生鼠的成肌细胞进行体外培养、纯化鉴定后,肌肉注射到mdx鼠左后肢,右后肢肌注DMEM作对照。成肌细胞移植后1个月,让mdx鼠作运动试验3 d后,静脉注射Evans蓝,次日取骨骼肌作冰冻切片,行dystrophin免疫荧光检测。荧光显微镜下,观察Evans蓝和dystrophin阳性纤维数,图像分析比较。 结果: 未移植肢体有26.82%±14.85%的肌纤维显示Evans蓝染色,而移植侧骨骼肌只有10.37%±2.87%的肌纤维显示Evans蓝染色。两者相比有显著差异(P＜0.05)。移植侧肢体有48.32%±6.54%的肌纤维dystrophin阳性,对照侧几乎没有dystrophin阳性肌纤维。Evans蓝染部分没有dystrophin表达。结论: 成肌细胞移植对运动诱导损害的肌纤维具有防护作用。     

Migration of lacZ positive cells from the tibialis anterior to the extensor digitorum longus muscle of the X-linked muscular dystrophic (mdx) mouse

Summary C2 mouse myogenic cells carrying the lacZ gene coding for -galactosidase (-gal) were injected into the tibialis anterior muscle of dystrophin-deficient mdx mice. Introduced cells were shown to have been incorporated into fibres of the injected muscle by virtue of the colocalization of -gal and dystrophin within them. Synthetic Nuclepore membrane inserted between the injected tibialis anterior and adjacent extensor digitorum longus muscle permitted the visualization of cells migrating between the two muscles through the pores of the membrane. Although the exact nature of the cells passing through the Nuclepore could not be determined by this method, they were thought to include implanted myogenic cells. Evidence for this was gained by the presence of -gal/dystrophin positive fibres within the extensor digitorum longus. Incorporation of cells into the adjacent extensor digitorum longus was greater in animals where this muscle had been autografted by the cutting and resuturing of the distal tendon. Autografted extensor digitorum longi differed from those which had not been subject to this procedure, by undergoing extensive fibre degeneration followed by regeneration, and further by the stripping of their surrounding epimysial covering. Implanted cells substantially participated in extensor digitorum longus fibre formation in these mice, up to 31% of their fibres 3 weeks after implantation coexpressing both the introduced lacZ gene product and the dystrophin gene product, the latter not normally expressed within the fibres of this myopathic recipient.     

Dystrophin is expressed in mdx skeletal muscle fibers after normal myoblast implantation.

In mdx mice, the dystrophin gene of the X chromosome is defective and, as a result, immunoreactive dystrophin is undetectable in all muscle fibers of all animals of this highly inbred strain. This study showed that implantation of suspensions of clonal cultures of normal human myoblasts into different regions of quadriceps muscles of 6-to-10-day-old mdx mice or 60-day-old mdx mice (whose muscles have been crushed 4 days before implantation) results in the appearance of scattered fiber segments containing microscopically demonstrable immunoreactive dystrophin. In the animals that received the normal myoblast implantation in the prenecrotic stage of the disease (6 to 10 days of age), the dystrophin-positive fiber segments (demonstrated at ages 35, 45, and 60 days) escaped necrosis. This was determined by the absence of the characteristic chains of central nuclei, a reliable marker of prior necrosis in mdx muscle fibers. By heavy labeling of the nuclear DNA of the transplantable human myoblasts with H3-thymidine during culturing, and by sequential performance of an immunocytochemical staining for dystrophin and autoradiography on the same sections, some dystrophin-positive fiber segments were shown to contain radiolabeled myonuclei. It was concluded that nondystrophic myoblasts fused with host muscle fibers to form mosaic muscle fibers in which the normal dystrophin gene of the implanted myoblasts was expressed. This approach may be employed for the mitigation of the deleterious consequences of a gene defect in recessively inherited human muscle diseases such as Duchenne dystrophy.     

mdx小鼠骨骼肌组织成肌、成脂、成骨基因的特异性表达

背景：干细胞移植是治疗肌营养不良症的有效方法之一,但移植的干细胞在病理骨骼肌中成肌表达较低。 目的：通过比较mdx小鼠和C57小鼠的骨骼肌形态及成肌、成脂、成骨基因表达的差异,探讨mdx小鼠骨骼肌病理改变的可能机制。 方法：取mdx小鼠与C57小鼠的骨骼肌组织行冰冻切片,苏木精-伊红染色和Vonkossa染色观察两种小鼠肌肉组织的形态特征;提取mdx小鼠和C57小鼠骨骼肌组织总RNA,real-time PCR检测成肌、成脂、成骨相关基因的表达。 结果与结论：mdx小鼠骨骼肌有肌纤维坏死和再生,伴有轻度脂肪、纤维结缔组织增生,Vonkossa染色可见钙结节沉积,而C57小鼠的骨骼肌细胞形态清晰,核位于细胞周边。与C57小鼠比较,mdx小鼠肌肉组织成骨、成脂基因表达有不同程度的上调(P < 0.05),而成肌基因表达下调(P< 0.05)。dystrophin基因缺失及成肌基因表达下调、成骨和成脂基因上调是造成mdx小鼠肌肉组织变性坏死的原因。     

Modification of the phenotypic expression of murine dystrophy: a morphological study

The extensor digitorum longus muscles of 4-6-week-old normal mice (129 ReJ) and dystrophic mice (129 ReJ dy/dy) were orthotopically transplanted. Grafted muscles were examined 1, 3, 7, 14, 20, 50, and 100 days post-transplantation. The myofibers of both types of grafts underwent a similar time course of necrosis and regeneration. Other than during the initial necrotic response, no evidence of necrotic myofibers was found in either type of grafted muscle. At 100 days post-transplantation, the grafted normal and dystrophic muscles were essentially similar, except that the dystrophic graft was of smaller size. Based on a comparison of the number of myofibers found at the 100-day grafts' widest girths [631 +/- 59 SEM, for normal grafts (Bourke and Ontell, 1984); 631 +/- 74 SEM, for dystrophic grafts], it is suggested that the regenerative capability of traumatized 4-6-week-old dystrophic muscle is similar to that of traumatized normal muscle. At 100 days post-transplantation, the grafted dystrophic muscle appeared "healthier" than untraumatized muscle from age-matched dystrophic mice, having less variation in myofiber diameter, better fascicular organization, and less connective tissue. The transplantation system demonstrates the possibility of modifying the expression of genetic programming of myopathic disorders using environmental manipulation.     

Sarcoplasmic reticulum function in slow- and fast-twitch skeletal muscles from mdx mice

The aim of the present study was to establish whether alterations in sarcoplasmic reticulum function are involved in the abnormal Ca(2+) homeostasis of skeletal muscle in mice with muscular dystrophy ( mdx). The properties of the sarcoplasmic reticulum and contractile proteins of fast- and slow-twitch muscles were therefore investigated in chemically skinned fibres isolated from the extensor digitorum longus (EDL) and soleus muscles of normal (C57BL/10) and mdx mice at 4 and 11 weeks of development. Sarcoplasmic reticulum Ca(2+) uptake, estimated by the Ca(2+) release following exposure to caffeine, was significantly slower in mdx mice, while the maximal Ca(2+) quantity did not differ in either type of skeletal muscle at either stage of development. In 4-week-old mice spontaneous sarcoplasmic reticulum Ca(2+) leakage was observed in EDL and soleus fibres and this was more pronounced in mdx mice. In addition, the maximal Ca(2+)-activated tension was smaller in mdx than in normal fibres, while the Ca(2+) sensitivity of the contractile apparatus was not significantly different. These results indicate that mdx hindlimb muscles are affected differently by the disease process and suggest that a reduced ability of the Ca(2+)-ATPase to load Ca(2+) and a leaky sarcoplasmic reticulum membrane may be involved in the altered intracellular Ca(2+) homeostasis.     

Critical period in muscle spindle regeneration in grafts of developing rat muscles

Extensor digitorum longus (EDL) muscles from rats at various intervals after birth were grafted into EDL muscles of adult recipients. Three to twelve months after the operation, host muscles containing the grafts were removed and examined for the presence of muscle spindles in the graft. The aim of the study was to establish when muscle spindles become capable of regeneration during development. Regenerated muscles grafted during the first week after birth were virtually spindleless. Grafts of muscles transplanted 10 and 15 days postnatally contained only 5–8 muscle spindles on average. In contrast, the regenerated grafts originating from muscles of 24- and 28-day-old rats were spindle-rich as in mature muscle grafts; the number of spindles in the transplanted EDL muscles (25.0±2.3; mean ±SE) attained values comparable to free standard autografts of these muscles in adult animals. Thus, the critical period after grafting, which also involves the loss of a vascular supply, is considerably longer than the critical period for muscle-spindle survival after nerve injury. Fifteen days after birth, when muscle spindles still survive denervation, only a few regenerated spindles were present in the individual muscle regenerates. We assume that the low resistance of immature spindle capsules to ischaemia accounts for their massive degeneration and abortive spindle regeneration in grafts from 10- to 15-day-old rats.     

Biomechanics of the sarcolemma and costameres in single skeletal muscle fibers from normal and dystrophin-null mice

We studied the biomechanical properties of the sarcolemma and its links through costameres to the contractile apparatus in single mammalian myofibers of Extensor digitorum longus muscles isolated from wild (WT) and dystrophin-null (mdx) mice. Suction pressures (P) applied through a pipette to the sarcolemma generated a bleb, the height of which increased with increasing P. Larger increases in P broke the connections between the sarcolemma and myofibrils and eventually caused the sarcolemma to burst. We used the values of P at which these changes occurred to estimate the tensions and stiffness of the system and its individual elements. Tensions of the whole system and the sarcolemma, as well as the maximal tension sustained by the costameres, were all significantly lower (1.8-3.3 fold) in muscles of mdx mice compared to WT. Values of P at which separation and bursting occurred, as well as the stiffness of the whole system and of the isolated sarcolemma, were ~2-fold lower in mdx than in WT. Our results indicate that the absence of dystrophin reduces muscle stiffness, increases sarcolemmal deformability, and compromises the mechanical stability of costameres and their connections to nearby myofibrils.     

Modulation of insulin-like growth factor (IGF)-I and IGF-binding protein interactions enhances skeletal muscle regeneration and ameliorates the dystrophic pathology in mdx mice

Administration of recombinant human insulin-like growth factor-I (rhIGF-I) has beneficial effects in animal models of muscle injury and muscular dystrophy. However, the results of these studies may have been confounded by interactions of rhIGF-I with endogenous IGF-binding proteins (IGFBPs). To date, no study has examined whether inhibiting IGFBP interactions with endogenous IGF-I can improve muscle fiber regeneration or muscular pathologies. We tested the hypothesis that reducing IGFBP interactions with endogenous IGF-I would enhance muscle regeneration after myotoxic injury and improve the dystrophic pathology in mdx mice. We administered an IGF-I aptamer (NBI-31772; 6 mg/kg per day, continuous infusion) to C57BL/10 mice undergoing regeneration after myotoxic injury or to mdx dystrophic mice. NBI-31772 binds all six IGFBPs with high affinity and releases "free" endogenous IGF-I. NBI-31772 treatment increased the rate of functional repair in fast-twitch tibialis anterior muscles after notexin-induced injury as evidenced by an increase in maximum force producing capacity (P(o)) at 10 days after injury. In contrast, NBI-31772 administration for 28 days did not alter P(o) of extensor digitorum longus (EDL) and soleus muscles or normalized force of diaphragm muscle strips from mdx mice. Although IGFBP inhibition reduced the susceptibility of the fast-twitch EDL and the diaphragm muscle to contraction-mediated damage, it increased muscle fatigability during repeated maximal contractions. Although the results in the myotoxic injury model suggest IGF-I signaling is important in this model, the results in the mdx model are mixed.     

Mechanical and energetic properties of dystrophic (mdx) mouse muscle

The mechanical and energetic properties of extensor digitorum longus (EDL) and soleus muscles of X chromosome-linked muscular dystrophic mutant (mdx) mice aged 4-6 weeks were studied and compared with those of the muscles of normal mice. Maximum tetanic tension, the speed of contraction of relaxation, and the heat production of mdx soleus muscles were not significantly different from those of the normal muscles. However, in mdx EDL muscles, the tension and heat production were significantly reduced, and relaxation was prolonged. To study the cause of these changes in mdx EDL muscles, tension and heat production were measured at various muscle lengths greater than optimum for tension. Both the amount of twitch heat and the heat rate for a tetanus were linearly related to the tension and had non-zero intercepts at zero tension, the activation heat. The twitch activation heat and the tension-related heat in tetani of mdx EDL muscles were not different from those in normal muscles. On the other hand, the tetanus activation heat of mdx EDL muscles was significantly smaller than that of normal muscles. Assuming that the degenerated fibers do not contribute to the active force produced, these results suggest that the amount of Ca2+ released in a contraction is not significantly different between normal and mdx muscles, but the Ca-ATPase activity of the salcoplasmic reticulum is reduced in mdx EDL, which could cause the slowing of relaxation.     

过量运动对肌营养不良模型鼠骨骼肌的损害作用

目的:观察过量运动对Duchenne型肌营养不良症模型鼠(mdx鼠)骨骼肌组织的影响。方法: 让成年和老年mdx小鼠、成年和老年C57小鼠连续进行游泳和提尾倒立运动试验(对照mdx不作运动), 每天1次, 每次13min, 连续3d后, 鼠尾静脉注谢Evans蓝, 次日杀小鼠, 剥去皮肤, 观察mdx鼠、C57鼠全身骨骼肌颜色改变, 对比照相后, 取腓肠肌、膈肌作冰冻切片, 荧光显微镜下, 观察Evans蓝的染色范围, 再作常规HE染色, 光学显微镜下, 观察骨骼肌细胞的组织学改变。结果:肉眼可见运动后的mdx鼠骨骼肌许多部位呈纵形条状蓝色, 而对照mdx鼠和C57鼠几乎无Evans蓝染色;荧光显微镜下, 运动后的mdx鼠骨骼肌可见Evans蓝荧光, 对照mdx鼠偶见此荧光, 而C57鼠无此荧光。光学显微镜下, 可见mdx鼠骨骼肌细胞变大、变圆, 伊红浓染, 胞核致密粗大, 坏死的肌细胞胞内结构降解, 呈无定型结构。膈肌中可见变性坏死的肌纤维居多, 而C57鼠无此改变。结论:过量运动造成了mdx鼠骨骼肌的损害;Evans蓝染色有助于鉴别变性坏死的肌纤维和研究dystrophin缺乏的肌肉的变性过程。     

Power output of fast and slow skeletal muscles of mdx (dystrophic) and control mice after clenbuterol treatment

The mdx mouse is the most commonly used animal model for Duchenne muscular dystrophy. We tested the null hypothesis that 20 weeks of clenbuterol treatment ( approximately 2 mg kg-1 day-1) of mdx and control mice would have no effect on the absolute and specific force (Po, kN m-2) and absolute and normalised power output (W kg-1) of extensor digitorum longus (EDL) and soleus muscles. For mdx and control mice, clenbuterol treatment produced modest increases in the mass of the two muscles but did not increase absolute or specific force or normalised power output. For absolute power output, only the EDL muscles of mdx mice showed a difference following treatment, with the power output of treated mice being 118 % that of the untreated mice. The modest effects of clenbuterol treatment on the dynamic properties of skeletal muscle provide little support for any improvement in muscle function for the dystrophic condition.     

Reduced density of intramembranous particle clusters: freeze-fracture study of mdx mouse muscle plasma membrane

Our previous freeze-fracture study demonstrated the decreased density of intramembranous particles (IMPs) on the protoplasmic (P) face of muscle plasma membranes in mdx mice. However, the molecular mechanism is unknown. In the present freeze-fracture study, we examined whether the reduced P-face IMP density in mdx mice would be caused by depletion of the rosette-like IMP clusters, which are IMP aggregations differing from crystal-like orthogonal arrays (OAs). By comparison with control mice, the P-face plasma membranes of extensor digitorum longus (EDL) and soleus (SOL) muscles of mdx mice demonstrated the following findings: (1) decreased IMP density with subunit particles of OAs and IMP clusters, (2) decreased IMP density without subunit particles of OAs, (3) normal IMP density without subunit particles of OAs and IMP clusters, (4) decreased OA density in EDL muscles and normal OA density in SOL muscles, and (5) decreased IMP cluster densities in both muscles. Thus, the reduced IMP density of P-face muscle plasma membranes in mdx mice may result from the decreased IMP clusters, suggesting the relationship between IMP clusters and the integral membrane proteins is influenced by dystrophin deficiency such as that of dystrophin-associated glycoproteins or other membrane proteins.     

Transplantation of Human Embryonic Myoblasts and Bone Marrow Stromal Cells into Skeletal Muscle of C57BL/10J-mdx Mice

We studied expression of dystrophin in skeletal muscles of C57BL/10J-mdx mice after transplantation of human embryonic and fetal myoblasts and bone marrow stromal cells. Dystrophin-positive areas corresponding to the location of transplanted cell were detected in muscles of all recipient mice after transplantation of different cell cultures, but the distribution of dystrophin characteristic of normal muscle fibers was detected only after transplantation of embryonic myoblasts. Dystrophin distribution in muscle fibers after transplantation of fetal myoblasts and bone marrow stromal cells was atypical.     

Electron microscopic and autoradiographic characterization of hindlimb muscle regeneration in the mdx mouse

The pattern of postnatal growth and development of skeletal muscle in mdx mice was studied by light and transmission electron microscopy and by autoradiography and was compared with that in their normal age-matched controls at 4 and 32 weeks of age. The muscle weights of both the extensor digitorum longus (EDL) and soleus muscles of mdx mice were significantly greater than those in control mice at both ages. Body weights of male and female mdx mice were also increased over controls up to 12 weeks of age. At 4 weeks, both the EDL and soleus muscles exhibited focal areas of degeneration, necrosis, and regeneration of centrally nucleated extrafusal fibers resulting in a wide range of fiber sizes. By 32 weeks, the majority of fibers in both muscles were centrally nucleated, and focal areas of recent regeneration were observed. By electron microscopy, the course of macrophage infiltration into areas of degenerating fibers and the ongoing regeneration of myofibers within redundant cylinders of external lamina were noted. This pattern was frequent in 4-week-old mdx muscles and was present to a lesser degree at 32 weeks. A notable lack of both adipose tissue infiltration and fibrotic change in the endomysium were observed in muscles at both ages. Autoradiograms of muscles from 4-week-old mdx mice injected with tritiated thymidine showed an increased proportion of labeled sublaminal nuclei at 24 and 48 hours after injection compared to controls. At 32 weeks of age, labeling of nuclei in muscles of mdx mice was also greater than in controls, but was reduced compared to muscle labeling in 4-week-old mdx mice. The observed features of mdx muscle tissue suggest that this animal model is more applicable to the study of regeneration dynamics than to Duchenne-type human muscular dystrophy.     

Growth of regenerating skeletal muscle fibers in cats

Extensor digitorum longus (EDL) muscles of cats were grafted heterotopically or orthotopically, and either with or without prior denervation. The autografted muscles were studied at times from 4 to 518 days after grafting. Muscle weight, fiber cross-sectional area, and ultrastructural, histochemical, and biochemical characteristics of regenerating muscle fibers were determined. Prior denervation reduced the mass of muscle at the time of grafting, but had no significant effect on the characteristics of the regenerating muscles. Orthotopic and heterotopic autografts achieved similar recovery of structure. Mean fiber area reached control values. Differentiation into fiber types occurred, but compared to control muscles, autografts had fewer Type I and Type IIA fibers. Electron microscopic analysis of regenerating muscle fibers revealed centrally located nuclei, but otherwise normal ultrastructure. The bimodal distribution of Z-band width was consistent with differentiation into fiber types. Mean data of some morphological variables did not stabilize.     

Reinnervation of fast and slow mammalian muscles by a superfluous number of motor axons

In this study peripheral nerves from flexor digitorum longus, (alien nerve) as well as the deep branch of the muscle's own lateral popliteal nerve were cut and connected to the distal stump of the lateral popliteal nerve. Extensor digitorum longus and tibialis anterior muscles then became reinnervated to a similar extent by either nerve, showing no preference for its own nerve. A significant proportion of the endplates in these muscles remained permanently supplied by more than one axon, and a proportion of the muscle fibres was supplied by both nerves. No ectopic endplates were formed on fast muscle fibres. The same two nerves were also connected to the slow soleus muscle and this muscle became preferentially reinnervated by the nerve to flexor digitorum longus. In contrast to fast muscles, endplates of soleus muscle fibres were only rarely contacted by more than one axon, and ectopic endplates were often found in this muscle. In both types of muscles that had an excess of motor nerves, extensive sprouting persisted for many months. Thus, identical motor nerves induce different patterns of innervation in slow and fast muscles, and muscle fibres do not show a preference for their own nerve.     

Degenerative and regenerative features of myofibers differ among skeletal muscles in a murine model of muscular dystrophy

Skeletal muscle myofibers constantly undergo degeneration and regeneration. Histopathological features of 6 skeletal muscles (cranial tibial [CT], gastrocnemius, quadriceps femoris, triceps brachii [TB], lumbar longissimus muscles, and costal part of the diaphragm [CPD]) were compared using C57BL/10ScSn-Dmd ^mdx (mdx) mice, a model for muscular dystrophy versus control, C57BL/10 mice. Body weight and skeletal muscle mass were lower in mdx mice than the control at 4 weeks of age; these results were similar at 6–30 weeks. Additionally, muscular lesions were observed in all examined skeletal muscles in mdx mice after 4 weeks, but none were noted in the controls. Immunohistochemical staining revealed numerous paired box 7-positive satellite cells surrounding the embryonic myosin heavy chain-positive regenerating myofibers, while the number of the former and staining intensity of the latter decreased as myofiber regeneration progressed. Persistent muscular lesions were observed in skeletal muscles of mdx mice between 4 and 14 weeks of age, and normal myofibers decreased with age. Number of muscular lesions was lowest in CPD at all ages examined, while the ratio of normal myofibers was lowest in TB at 6 weeks. In CT, TB, and CPD, Iba1-positive macrophages, the main inflammatory cells in skeletal muscle lesions, showed a significant positive correlation with the appearance of regenerating myofibers. Additionally, B220-positive B-cells showed positive and negative correlation with regenerating and regenerated myofibers, respectively. Our data suggest that degenerative and regenerative features of myofibers differ among skeletal muscles and that inflammatory cells are strongly associated with regenerative features of myofibers in mdx mice.     

骨髓干细胞移植治疗不同年龄mdx鼠的疗效研究

目的：研究不同年龄的Duchenne型肌营养不良鼠（mdx鼠）与骨髓干细胞移植后缺失蛋白表达的关系。 方法： 获取4-5周C57BL/6小鼠的骨髓干细胞，体外培养3 d，静脉移植到7Gy γ射线预处理的6周龄、8周龄两组各6只mdx鼠。移植12周后，对移植鼠骨骼肌dystrophin蛋白表达情况进行检测。 结果： 6周龄、8周龄两组mdx鼠，静脉移植1.2×107骨髓干细胞，3个月后，分别有16%和7%的骨骼肌纤维表达了dystrophin蛋白。 结论： 静脉移植同种、同系鼠骨髓干细胞的mdx鼠，3个月之后, 不同年龄mdx鼠骨骼肌细胞dystrophin蛋白表达的阳性率不同，幼年鼠骨髓干细胞移植有较高比率的缺失蛋白表达。