Cintia Yuri Matsumura

Autophagy is increased in laminin α2 chain-deficient muscle and its inhibition improves muscle morphology in a mouse model of MDC1A

Congenital muscular dystrophy caused by laminin α2 chain deficiency (also known as MDC1A) is a severe and incapacitating disease, characterized by massive muscle wasting. The ubiquitin-proteasome system plays a major role in muscle wasting and we recently demonstrated that increased proteasomal activity is a feature of MDC1A. The autophagy-lysosome pathway is the other major system involved in degradation of proteins and organelles within the muscle cell. However, it remains to be determined if the autophagy-lysosome pathway is dysregulated in muscular dystrophies, including MDC1A. Using the dy(3K)/dy(3K) mouse model of laminin α2 chain deficiency and MDC1A patient muscle, we show here that expression of autophagy-related genes is upregulated in laminin α2 chain-deficient muscle. Moreover, we found that autophagy inhibition significantly improves the dystrophic dy(3K)/dy(3K) phenotype. In particular, we show that systemic injection of 3-methyladenine (3-MA) reduces muscle fibrosis, atrophy, apoptosis and increases muscle regeneration and muscle mass. Importantly, lifespan and locomotive behavior were also greatly improved. These findings indicate that enhanced autophagic activity is pathogenic and that autophagy inhibition holds a promising therapeutic potential in the treatment of MDC1A.

Prevention of muscle fibrosis and myonecrosis in mdx mice by suramin, a TGF-β1 blocker.

Fibrosis is a pathological feature observed in patients with Duchenne muscular dystrophy (DMD) and in mdx mice, the experimental model of DMD. We evaluated the effect of suramin, a transforming growth factor‐beta 1 (TGF‐β1) blocker, on fibrosis in mdx mice. mdx mice (6 months old) received suramin for 7 weeks. Suramin‐ and saline‐treated (control) mdx mice performed exercise on a treadmill to worsen disease progression. Immunoblotting showed an increase of TGF‐β1 in mdx diaphragm, limb, and cardiac muscles. Suramin decreased creatine kinase in mdx mice and attenuated fibrosis in all muscles studied, except for cardiac muscle. Suramin protected limb muscles against damage and reduced the exercise‐induced loss of strength over time. These findings support a role for TGF‐β1 in fibrinogenesis and myonecrosis during the later stages of disease in mdx mice. Suramin might be a useful therapeutic alternative for the treatment of dystrophinopathies. Muscle Nerve, 2011

Mechanical, biochemical and morphometric alterations in the femur of mdx mice.

The bone tissue abnormalities observed in patients with Duchenne muscular dystrophy are frequently attributed to muscle weakness. In this condition, bones receive fewer mechanical stimuli, compromising the process of bone modeling. In the present study we hypothesize that other factors inherent to the disease might be associated with bone tissue impairment, irrespective of the presence of muscle impairment. Mdx mice lack dystrophin and present cycles of muscle degeneration/regeneration that become more intense in the third week of life. As observed in humans with muscular dystrophy, bone tissue abnormalities were found in mdx mice during more intense muscle degeneration due to age. Under these circumstances, muscle deficit is probably one of the factors promoting these changes. To test our hypothesis, we investigated the changes that occur in the femur of mdx mice at 21 days of age when muscle damage is still not significant. The mechanical (structural and material) and biochemical properties and morphometric characteristics of the femur of mdx and control animals were evaluated. The results demonstrated a lower strength, stiffness and energy absorption capacity in mdx femurs. Higher values for structural (load and stiffness) and material (stress, elastic modulus and toughness) properties were observed in the control group. Mdx femurs were shorter and were characterized by a smaller cortical area and thickness and a smaller area of epiphyseal trabecular bone. The hydroxyproline content was similar in the two groups, but there was a significant difference in the Ca/P ratios. Thermogravimetry showed a higher mineral matrix content in cortical bone of control animals. In conclusion, femurs of mdx mice presented impaired mechanical and biochemical properties as well as changes in collagen organization in the extracellular matrix. Thus, mdx mice developed femoral osteopenia even in the absence of significant muscle fiber degeneration. This weakness of the mdx femur is probably due to genetic factors that are directly or indirectly related to dystrophin deficiency.

Diltiazem and verapamil protect dystrophin-deficient muscle fibers of MDX mice from degeneration: A potential role in calcium buffering and sarcolemmal stability

The lack of dystrophin in mdx mice and in Duchenne muscular dystrophy causes sarcolemmal breakdown and increased calcium influx followed by myonecrosis. We examined whether the calcium channel blockers diltiazem and verapamil protect dystrophic muscles from degeneration. Mdx mice received daily intraperitoneal injections of diltiazem or verapamil for 18 days, followed by removal of the sternomastoid, diaphragm, tibialis anterior, and cardiac muscles. Control mdx mice were injected with saline. Both drugs significantly decreased blood creatine kinase levels. Total calcium content was significantly higher in mdx muscles than in control C57Bl/10. Verapamil and diltiazem reduced total calcium content only in diaphragm and cardiac muscle. Histological analysis showed that diltiazem significantly attenuated myonecrosis in diaphragm. Immunoblots showed a significant increase of calsequestrin and β‐dystroglycan levels in some diltiazem‐ and verapamil‐treated muscles. Possible interactions of these drugs with the sarcoplasmic reticulum and sarcolemma may also contribute to the improvement of the dystrophic phenotype. Muscle Nerve 39: 167–176, 2009

Stretch-activated calcium channel protein TRPC1 is correlated with the different degrees of the dystrophic phenotype in mdx mice

In Duchenne muscular dystrophy (DMD) and in the mdx mouse model of DMD, the lack of dystrophin is related to enhanced calcium influx and muscle degeneration. Stretch-activated channels (SACs) might be directly involved in the pathology of DMD, and transient receptor potential cation channels have been proposed as likely candidates of SACs. We investigated the levels of transient receptor potential canonical channel 1 (TRPC1) and the effects of streptomycin, a SAC blocker, in muscles showing different degrees of the dystrophic phenotype. Mdx mice (18 days old, n = 16) received daily intraperitoneal injections of streptomycin (182 mg/kg body wt) for 18 days, followed by removal of the diaphragm, sternomastoid (STN), biceps brachii, and tibialis anterior muscles. Control mdx mice (n = 37) were injected with saline. Western blot analysis showed higher levels of TRPC1 in diaphragm muscle compared with STN and limb muscles. Streptomycin reduced creatine kinase and prevented exercise-induced increases of total calcium and Evans blue dye uptake in diaphragm and in STN muscles. It is suggested that different levels of the stretch-activated calcium channel protein TRPC1 may contribute to the different degrees of the dystrophic phenotype seen in mdx mice. Early treatment designed to regulate the activity of these channels may ameliorate the progression of dystrophy in the most affected muscle, the diaphragm.

Isobaric Tagging-Based Quantification for Proteomic Analysis: A Comparative Study of Spared and Affected Muscles from mdx Mice at the Early Phase of Dystrophy

Duchenne muscular dystrophy (DMD) is the most common childhood myopathy, characterized by muscle loss and cardiorespiratory failure. While the genetic basis of DMD is well established, secondary mechanisms associated with dystrophic pathophysiology are not fully clarified yet. In order to obtain new insights into the molecular mechanisms of muscle dystrophy during earlier stages of the disease, we performed a comparative proteomic profile of the spared extraocular muscles (EOM) vs. affected diaphragm from the mdx mice, using a label based shotgun proteomic approach. Out of the 857 identified proteins, 42 to 62 proteins had differential abundance of peptide ions. The calcium-handling proteins sarcalumenin and calsequestrin-1 were increased in control EOM compared with control DIA, reinforcing the view that constitutional properties of EOM are important for their protection against myonecrosis. The finding that galectin-1 (muscle regeneration), annexin A1 (anti-inflammatory) and HSP 47 (fibrosis) were increased in dystrophic diaphragm provides novel insights into the mechanisms through which mdx affected muscles are able to counteract dystrophy, during the early stage of the disease. Overall, the shotgun technique proved to be suitable to perform quantitative comparisons between distinct dystrophic muscles and allowed the suggestion of new potential biomarkers and drug targets for dystrophinopaties.

Quantitative Proteomic Analysis Reveals Metabolic Alterations, Calcium Dysregulation, and Increased Expression of Extracellular Matrix Proteins in Laminin α2 Chain–deficient Muscle

Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain–deficient dy3K/dy3K mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain–deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978).

Calcium-binding proteins in skeletal muscles of the mdx mice: potential role in the pathogenesis of Duchenne muscular dystrophy

Duchenne muscular dystrophy is one of the most common hereditary diseases. Abnormal ion handling renders dystrophic muscle fibers more susceptible to necrosis and a rise in intracellular calcium is an important initiating event in dystrophic muscle pathogenesis. In the mdx mice, muscles are affected with different intensities and some muscles are spared. We investigated the levels of the calcium‐binding proteins calsequestrin and calmodulin in the non‐spared axial (sternomastoid and diaphragm), limb (tibialis anterior and soleus), cardiac and in the spared extraocular muscles (EOM) of control and mdx mice. Immunoblotting analysis showed a significant increase of the proteins in the spared mdx EOM and a significant decrease in the most affected diaphragm. Both proteins were comparable to the cardiac muscle controls. In limb and sternomastoid muscles, calmodulin and calsequestrin were affected differently. These results suggest that differential levels of the calcium‐handling proteins may be involved in the pathogenesis of myonecrosis in mdx muscles. Understanding the signaling mechanisms involving Ca2+‐calmodulin activation and calsequestrin expression may be a valuable way to develop new therapeutic approaches to the dystrophinopaties.

Suramin affects metalloproteinase-9 activity and increases beta-dystroglycan levels in the diaphragm of the dystrophin-deficient mdx MOUSE

Introduction: In Duchenne muscular dystrophy and in the mdx mouse, muscle fiber degeneration and subsequent fibrosis lead to cardiorespiratory failure. Previously, we demonstrated that the anti‐fibrotic agent suramin was effective in decreasing fibrosis in mdx muscles. In this study, we were interested to see whether suramin could affect metalloproteinases (MMP) and improve the functional activity of the mdx diaphragm muscle. Methods: Zymography was performed to evaluate MMP‐2 and MMP‐9 activity. Western blotting was used to analyze the levels of beta‐dystroglycan. Muscle function was assessed in hemidiaphragm in vitro preparations. Results: We found that suramin affects metalloproteinase‐9 activity and increases beta‐dystroglycan. Furthermore, suramin also protects against diaphragm muscle fatigue over time. Conclusions: These results show the potential benefits of suramin in maintaining the structure of the dystrophin‐glycoprotein complex. Muscle Nerve, 46:810–813, 2012

Doxycycline ameliorates the dystrophic phenotype of skeletal and cardiac muscles in mdx mice

Introduction: We examined whether doxycycline, an antibiotic member of the tetracycline family, improves the histopathology and muscle function in mdx mice, the experimental model of DMD. Methods: Doxycycline was administered for 36 days (starting on postnatal day 0) and for 9 months (starting at 8 months of age) in drinking water. Histopathological, biochemical (creatine kinase), and functional (forelimb muscle grip strength) parameters were evaluated in limb, diaphragm, and cardiac muscle. Results: Doxycycline significantly minimized the dystrophic phenotype of skeletal and cardiac muscles and improved forelimb muscle strength. The drug protected muscle fibers against myonecrosis and reduced inflammation. Furthermore, it slowed the progression of myocardial fibrosis. Conclusions: This study provides evidence that doxycycline may be a potential therapeutic agent for DMD. Muscle Nerve 46: 400–406, 2012