Elevated levels of active matrix metalloproteinase-9 cause hypertrophy in skeletal muscle of normal and dystrophin-deficient mdx mice

Matrix metalloproteinases (MMPs) are a group of extracellular proteases involved in tissue remodeling in several physiological and pathophysiological conditions. While increased expression of MMPs (especially MMP-9) has been observed in skeletal muscle in numerous conditions, their physiological significance remains less-well understood. By generating novel skeletal muscle-specific transgenic (Tg) mice expressing constitutively active mutant of MMP-9 (i.e. MMP-9G100L), in this study, we have investigated the effects of elevated levels of MMP-9 on skeletal muscle structure and function in vivo. Tg expression of enzymatically active MMP-9 protein significantly increased skeletal muscle fiber cross-section area, levels of contractile proteins and force production in isometric contractions. MMP-9 stimulated the activation of the Akt signaling pathway in Tg mice. Moreover, expression of active MMP-9 increased the proportion of fast-type fiber in soleus muscle of mice. Overexpression of MMP-9 also considerably reduced the deposition of collagens I and IV in skeletal muscle in vivo. In one-year-old mdx mice (a model for Duchenne muscular dystrophy, DMD), deletion of the Mmp9 gene reduced fiber hypertrophy and phosphorylation of Akt and p38 mitogen-activated protein kinase. Collectively, our study suggests that elevated levels of active MMP-9 protein cause hypertrophy in skeletal muscle and that the modulation of MMP-9 levels may have therapeutic value in various muscular disorders including DMD.     

Protein kinase C activity regulates slow myosin heavy chain 2 gene expression in slow lineage skeletal muscle fibers.

Expression of the slow myosin heavy chain (MyHC) 2 gene defines slow versus fast avian skeletal muscle fiber types. Fetal, or secondary, skeletal muscle fibers express slow MyHC isoform genes in developmentally regulated patterns within the embryo, and this patterning is at least partly dependent on innervation in vivo. We have previously shown that slow MyHC 2 gene expression in vitro is regulated by a combination of innervation and cell lineage. This pattern of gene expression was indistinguishable from the pattern observed in vivo in that it was restricted to innervated muscle fibers of slow muscle origin. We show here that slow MyHC 2 gene expression in the slow muscle fiber lineage is regulated by protein kinase C (PKC) activity. Inhibition of PKC activity induced slow MyHC 2 gene expression, and the capacity to express the slow MyHC 2 gene was restricted to muscle fibers of slow muscle (medial adductor) origin. Fast muscle fibers derived from the pectoralis major did not express significant levels of slow MyHC 2 with or without inhibitors of PKC activity. This differential expression pattern coincided with different inherent PKC activities in fast versus slow muscle fiber types. Furthermore, over-expression of an unregulated PKCalpha mutant suppressed slow MyHC 2 gene expression in muscle fibers of the slow lineage. Lastly, denervation of skeletal muscles caused an increase in PKC activity, particularly in the slow medial adductor muscle. This increase in PKC activity was associated with lack of slow MyHC 2 gene expression in vivo. These results provide a mechanistic link between innervation, an intracellular signaling pathway mediated by PKC, and expression of a muscle fiber type-specific contractile protein gene. Dev Dyn 1999;216:177-189.     

Dihydropyridine receptor isoform expression in adult rat skeletal muscle

 The expression of isoform-specific dihydropyridine receptor Ca²⁺ channel (DHPR) α1-subunit genes in rat diaphragm, soleus and extensor digitorum longus muscles was investigated using RNase protection assays. As expected, mRNA expression levels for the DHPR skeletal muscle isoform were highest in extensor digitorum longus. Unexpectedly, both diaphragm and soleus expressed mRNA for the cardiac isoform at a significant level. Moreover, immunohistochemical experiments provided evidence of the cardiac DHPR isoform at the protein level in muscle fibres. The presence of the cardiac DHPR in the soleus and diaphragm is consistent with a degree of reported cardiac-like excitation-contraction coupling in these muscles, and may be an explanation for some of the therapeutic effects of theophylline in asthmatics, but is likely to serve some other role(s) as well. Received: 18 December 1997 / Received after revision: 5 March 1998 / Accepted: 17 March 1998     

Normal myofibrillar development followed by progressive sarcomeric disruption with actin accumulations in a mouse Cfl2 knockout demonstrates requirement of cofilin-2 for muscle maintenance

Cofilin-2, a small actin-binding protein and member of the AC protein family that includes cofilin-1 and destrin, is predominantly expressed at sarcomeres in skeletal and cardiac muscles. The role of cofilin-2 in muscle development and function is unclear. In humans, recessive cofilin-2 mutations have been associated with nemaline myopathy with minicores. To investigate the functional role of cofilin-2 in vivo, we generated constitutive and muscle-specific cofilin-2-deficient mice using a cre-loxP strategy. Cofilin-2-deficient mice were similar to their wild-type (WT) littermates at birth, but died by day 8. They were significantly smaller, severely weak and had very little milk in their stomachs. The sarcomeric structure was intact at birth, but by Day 7, skeletal muscles showed severe sarcomeric disruptions starting at the Z-line, along with filamentous actin accumulations consistent with a lack of actin depolymerization activity. Cofilin-2-deficient muscles contained elevated numbers of slow fibers and exhibited upregulation of slow fiber-specific genes. Increased amounts of other sarcomeric proteins including α-actinin-2, α-sarcomeric actin and tropomyosin were also present. While destrin was not expressed in either WT or cofilin-2-deficient muscles, cofilin-1 was similarly expressed in developing myofibers of both genotypes. There was no evidence for compensatory changes in expression of either family member in cofilin-2-deficient tissues. The onset of pathology and weakness in cofilin-2-deficient muscles correlated with normal developmental loss of cofilin-1 expression within myofibers, suggesting that cofilin-1 serves as an early developmental sarcomeric isoform. Overall, cofilin-2, although not critical for muscle development, is essential for muscle maintenance.     

Expression of cofilin isoforms during development of mouse striated muscles

Cofilin (CF) is an actin regulatory protein that plays a critical role in actin filament dynamics in a variety of cells. Two cofilin isoforms, muscle-type (M-CF) and nonmuscle-type (NM-CF) encoded by different genes, exist in mammals; in the adult, the former is predominantly expressed in muscle tissues, while the latter is distributed in various non-muscle tissues (Ono et al., 1994). In this study, we examined cofilin isoform expression during skeletal and cardiac muscle development in mice using cDNA probes and antibodies which distinguish the isoforms. We found that the expression of M-CF was initiated in terminally differentiated myogenic cells in both the myotome and limb buds. In myogenic cell cultures, its expression occurred coupled with myotube formation. NM-CF was expressed in developing skeletal and cardiac muscles but disappeared from skeletal muscle during postnatal development, while its expression persisted in the heart, even in adult mice. A similar situation was observed in the heart of other mammals. Thus, it is likely that the both cofilin isoforms are involved in the regulation of actin assembly during myofibrillogenesis. Only M-CF could be involved in actin dynamics in mature skeletal muscle, while both isoforms could be in the mature heart.     

抗阻训练衰老小鼠骨骼肌p53与bax基因的表达

背景：抗阻训练能够缓解肌肉衰减征的发生并能够弱化骨骼肌细胞凋亡。 目的：观察年龄和抗阻训练对SAMP8小鼠骨骼肌p53,bax基因表达的影响。 方法：分别取3月龄(青年组)和6月龄(老年组)的SAMP8小鼠进行8周爬梯运动,每周3次,以不参加运动的同龄小鼠作为对照。 结果与结论：Real-time PCR结果显示,与青年组比较,老年组小鼠骨骼肌纤维p53,bax mRNA的表达量显著上升(P < 0.05),而与相同月龄对照组比较,抗阻训练的小鼠骨骼肌纤维中p53,bax mRNA的表达量明显降低(P < 0.05)。提示抗阻训练能抑制衰老小鼠骨骼肌p53,bax基因的表达。     

Regulation of dihydropyridine receptor and ryanodine receptor gene expression in regenerating skeletal muscle

 One of the the major properties of mature skeletal muscle is its ability to regenerate after injury. The purpose of the present study was to determine whether the expression of genes encoding the dihydropyridine receptor calcium channel (DHPR) and the ryanodine receptor (RyR), which play a critical role in excitation–contraction coupling, is regulated by skeletal muscle regeneration. The process of regeneration was induced by bupivacaine injection in surgically exposed rat extensor digitorum longus (EDL) muscle. After total RNA isolation from the injected and the contralateral control EDL muscles performed 3, 7, 15 and 30 days following injection, Northern blot and RNase protection assays were carried out with four cDNA probes specific for the skeletal and cardiac muscle isoforms of both the DHPR α1-subunit and the RyR. After 3 days, an initial precipitous decrease in the expression of the genes encoding the skeletal muscle isoforms of the DHPR and RyR was observed, followed by an increase. Moreover, regenerating skeletal muscle transiently expressed mRNA for the DHPR cardiac isoform, mainly at the beginning of regeneration. No expression of mRNA for the cardiac RyR was observed. Contraction experiments, performed using EDL muscle at the same times after bupivacaine injection, showed that twitch amplitude was markedly decreased in the absence of external calcium, but only during the early stages of regeneration. Similar findings in relation to expression of skeletal and cardiac muscle DHPR message were previously reported from experiments conducted during early developmental stages using fetal skeletal muscle and muscle cell cultures [Chaudhari N, Beam KG (1993) Dev Biol 155:507–515]. These results suggest that expression of the DHPR cardiac isoform in skeletal muscle could explain certain cardiac-like aspects of excitation–contraction coupling of regenerating skeletal muscle and developing skeletal muscle as well. Received: 2 July 1996 / Received after revision: 18 September 1996 / Accepted: 20 September 1996     

NADH dehydrogenase activity and expression of mRNA of complex I (ND1, 51kDa, and 75kDa) in heart mitochondria of klotho mouse

Mitochondrial enzyme activities and ultrastructure of mitochondria prepared from klotho mutant mice were compared with those in wild-type mice. We also measured the levels of expression of ND1, 51kDa, and 75kDa mRNA associated with the genes encoding NADH dehydrogenase and complex I and that of alpha cardiac myosin heavy chain mRNA in both groups. Mitochondrial NADH oxidoreductase activity was higher in klotho mutant mice during aging than that in wild-type mice. The area of mitochondria per unit area (300 microm2) of cell was almost constant from 4 to 7 weeks of age in both groups. A few large mitochondria were scattered between numerous small mitochondria with compact cristae and myofibrils in klotho mice from 5 weeks of age. The levels of ND1 and 75kDa mRNA were slightly high from 7 weeks of age in klotho mutant mice, whereas they were almost constant in wild-type mice, in spite of reduced expression of alpha cardiac myosin heavy chain mRNA. Our results indicate that klotho protein indirectly plays a role in diminished functional adaptability of enzymes in aged heart muscle, and is required for hypertrophy of cardiac mitochondria.     

Redefinition of Dystrophin Isoform Distribution in Mouse Tissue by RT-PCR Implies Role in Nonmuscle Manifestations of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is caused by a defect in a 427-kDa membrane-associated protein: dystrophin. The DMD gene also encodes several shorter isoforms which are believed to participate in nonmuscle manifestations of DMD, including abnormal retinal electrophysiology, dilated cardiomyopathy, mental retardation, and hearing defects. The purpose of this work was to determine the normal tissue expression of full-length dystrophin (Dp427) and the dystrophin isoforms Dp260, Dp140, Dp116, and Dp71, to aid in understanding what roles these isoforms might play in DMD nonmuscle manifestations. RT-PCR was performed on mRNA isolated from wild-type C57BL/6J mouse tissues, including brain, cardiac muscle, eye, intestine, kidney, liver, lung, skeletal muscle, spleen, stomach, testis, thymus, and uterus. RT-PCR amplification demonstrated that the isoforms were in a number of tissues which had not been revealed by previous Western and Northern blot analyses. Dp427 was expressed at equal levels in all tissues. Dp260 and Dp140 were present in all tissues tested, but the levels of expression varied. Dp116 was expressed in a subset of tissues and levels of expression varied. Dp71 was constitutively expressed in all tissues, suggesting that this isoform plays a basic role in normal tissue function. The expanded tissue distribution supports the hypothesis that dystrophin isoforms serve essential and unique functions, necessitating further investigation into their potential roles in DMD nonmuscle manifestations.     

Comparative expression analysis of the murine palladin isoforms

Palladin fulfils a crucial function as a molecular scaffold in organizing and stabilizing the actin cytoskeleton. At least four major palladin isoforms exist due to different promoter usage and alternative splicing: a 200-kDa isoform, a 140-kDa isoform, and two isoforms with a size of 90-92 kDa. Here, we describe their expression during mouse development and in adult tissues. The 200-kDa isoform is predominantly expressed in developing heart and skeletal muscle. The 140-kDa isoform is expressed in various mesenchymal tissues, and also represents the major isoform of the brain. The 90-92-kDa isoforms are almost ubiquitously expressed with the highest levels in smooth muscle-rich tissues. Immunohistochemical and immunofluorecence staining with an anti-200-kDa isoform-specific antiserum localizes the large isoform to the Z-discs of cardiac and skeletal muscle cells. Interestingly, the expression of this isoform is initiated and increasing during in vitro differentiation and fusion of C2C12 myoblasts, suggesting that the 200-kDa palladin isoform may play a scaffolding role during sarcomeric organization.