Enhanced Na+/H+ Exchange Activity Contributes to the Pathogenesis of Muscular Dystrophy via Involvement of P2 Receptors
2007; Elsevier BV; Volume: 171; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2007.070452
ISSN1525-2191
AutoresYuko Iwata, Yuki Katanosaka, Takashi Hisamitsu, Shigeo Wakabayashi,
Tópico(s)Adipose Tissue and Metabolism
ResumoA subset of muscular dystrophy is caused by genetic defects in dystrophin-associated glycoprotein complex. Using two animal models (BIO14.6 hamsters and mdx mice), we found that Na+/H+ exchanger (NHE) inhibitors prevented muscle degeneration. NHE activity was constitutively enhanced in BIO myotubes, as evidenced by the elevated intracellular pH and enhanced 22Na+ influx, with activation of putative upstream kinases ERK42/44. NHE inhibitor significantly reduced the increases in baseline intracellular Ca2+ as well as Na+ concentration and stretch-induced damage, suggesting that Na+i-dependent Ca2+overload via the Na+/Ca2+ exchanger may cause muscle damage. Furthermore, ATP was found to be released continuously from BIO myotubes in a manner further stimulated by stretching and that the P2 receptor antagonists reduce the enhanced NHE activity and dystrophic muscle damage. These observations suggest that autocrine ATP release may be primarily involved in genesis of abnormal ionic homeostasis in dystrophic muscles and that Na+-dependent ion exchangers play a critical pathological role in muscular dystrophy. A subset of muscular dystrophy is caused by genetic defects in dystrophin-associated glycoprotein complex. Using two animal models (BIO14.6 hamsters and mdx mice), we found that Na+/H+ exchanger (NHE) inhibitors prevented muscle degeneration. NHE activity was constitutively enhanced in BIO myotubes, as evidenced by the elevated intracellular pH and enhanced 22Na+ influx, with activation of putative upstream kinases ERK42/44. NHE inhibitor significantly reduced the increases in baseline intracellular Ca2+ as well as Na+ concentration and stretch-induced damage, suggesting that Na+i-dependent Ca2+overload via the Na+/Ca2+ exchanger may cause muscle damage. Furthermore, ATP was found to be released continuously from BIO myotubes in a manner further stimulated by stretching and that the P2 receptor antagonists reduce the enhanced NHE activity and dystrophic muscle damage. These observations suggest that autocrine ATP release may be primarily involved in genesis of abnormal ionic homeostasis in dystrophic muscles and that Na+-dependent ion exchangers play a critical pathological role in muscular dystrophy. Muscular dystrophy is a heterogeneous genetic disease that causes severe skeletal muscle degeneration, characterized by fiber weakness and muscle fibrosis. The genetic defects associated with muscular dystrophy often include mutations in one of the components of the dystrophin-glycoprotein complex, such as dystrophin or sarcoglycans (α-, β-, γ-, and δ-SG).1Duclos F Straub V Moore SA Venzke DP Hrstka RF Crosbie RH Durbeej M Lebakken CS Ettinger AJ van der Meulen J Holt KH Lim LE Sanes JR Davidson BL Faulkner JA Williamson R Campbell KP Progressive muscular dystrophy in α-sarcoglycan-deficient mice.J Cell Biol. 1998; 142: 1461-1471Crossref PubMed Scopus (312) Google Scholar, 2Campbell KP Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage.Cell. 1995; 80: 675-679Abstract Full Text PDF PubMed Scopus (763) Google Scholar, 3Nigro V Okazaki Y Belsito A Piluso G Matsuda Y Politano L Nigro G Ventura C Abbondanza C Molinari AM Acampora D Nishimura M Hayashizaki Y Puca GA Identification of the Syrian hamster cardiomyopathy gene.Hum Mol Genet. 1997; 6: 601-607Crossref PubMed Scopus (250) Google Scholar The dystrophin-glycoprotein complex is a multisubunit complex2Campbell KP Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage.Cell. 1995; 80: 675-679Abstract Full Text PDF PubMed Scopus (763) Google Scholar, 4Campbell KP Kahl SD Association of dystrophin and an integral membrane glycoprotein.Nature. 1989; 338: 259-262Crossref PubMed Scopus (614) Google Scholar, 5Tinsley JM Blake DJ Zuellig RA Davies KE Increasing complexity of the dystrophin-associated protein complex.Proc Natl Acad Sci USA. 1994; 91: 8307-8313Crossref PubMed Scopus (187) Google Scholar that spans the sarcolemma to form a structural link between the extracellular matrix and the actin cytoskeleton.6Ervasti JM Campbell KP A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin.J Cell Biol. 1993; 122: 809-823Crossref PubMed Scopus (1194) Google Scholar Disruption of dystrophin-glycoprotein complex significantly impairs membrane integrity or stability during muscle contraction/relaxation and prevents myocyte survival. This enhanced susceptibility to exercise-induced damage of muscle fibers is observed in dystrophic animals, such as δ-SG-deficient BIO14.6 hamsters and dystrophin-deficient mdx mice, genetic homologues of human limb-girdle and Duchenne muscular dystrophy, respectively. Despite identification of many genes responsible for muscular dystrophy, the pathways through which genetic defects lead to muscle dysgenesis are still poorly understood. Myocyte degeneration has long been attributed to membrane defects, such as increased fragility to mechanical stress. Enhanced membrane stretching results in increased permeability to Ca2+, and the resultant abnormal Ca2+ handling has been suggested to be a prerequisite for muscle dysgenesis. A number of studies have indicated chronic elevation in the cytosolic Ca2+ concentration ([Ca2+]i), beneath the sarcolemma, or within other cell compartments in skeletal muscle fibers or in cultured myotubes from dystrophin-deficient (Duchenne muscular dystrophy) patients and mdx mice.7Mallouk N Jacquemond V Allard B Elevated subsarcolemmal Ca2+ in mdx mouse skeletal muscle fibers detected with Ca2+-activated K+ channels.Proc Natl Acad Sci USA. 2000; 97: 4950-4955Crossref PubMed Scopus (139) Google Scholar, 8Robert V Massimino ML Tosello V Marsault R Cantini M Sorrentino V Pozzan T Alteration in calcium handling at the subcellular level in mdx myotubes.J Biol Chem. 2001; 276: 4647-4651Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 9Fong PY Turner PR Denetclaw WF Steinhardt RA Increased activity of calcium leak channels in myotubes of Duchenne human and mdx mouse origin.Science. 1990; 250: 673-676Crossref PubMed Scopus (285) Google Scholar Recently, we identified one of the stretch-activated channels, the growth factor responsive channel (GRC, TRPV2), which may be involved in the pathogenesis of myocyte degeneration caused by dystrophin-glycoprotein complex disruption.10Iwata Y Katanosaka Y Arai Y Komamura K Miyatake K Shigekawa M A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor-regulated channel.J Cell Biol. 2003; 161: 957-967Crossref PubMed Scopus (225) Google Scholar More recently, we found that Ca2+-handling drugs, such as tranilast and diltiazem, exert protective effects against muscle degeneration in both mdx mice and BIO14.6 hamsters,11Iwata Y Katanosaka Y Shijun Z Kobayashi Y Hanada H Shigekawa M Wakabayashi S Protective effects of Ca2+ handling drugs against abnormal Ca2+ homeostasis and cell damage in myopathic skeletal muscle cells.Biochem Pharmacol. 2005; 70: 740-751Crossref PubMed Scopus (44) Google Scholar suggesting that Ca2+-permeable channels primarily contribute to abnormal Ca2+-homeostasis in dystrophic animals. In addition to the Ca2+-entry pathway across the plasma membrane, it is also plausible that modifications of other ion-transport proteins contribute to genesis of the abnormal Ca2+ homeostasis in muscular dystrophy. We discovered that plasma membrane Na+/H+ exchanger (NHE) inhibitors are highly protective against muscle damage in dystrophic animals. NHE is an important transporter regulating the intracellular pH (pHi), Na+ concentration ([Na+]i), and cell volume, and catalyzing the electroneutral countertransport of Na+ and H+ through the plasma membrane or organelle membranes.12Wakabayashi S Shigekawa M Pouyssegur J Molecular physiology of vertebrate Na+/H+ exchangers.Physiol Rev. 1997; 77: 51-74Crossref PubMed Scopus (565) Google Scholar, 13Orlowski J Grinstein S Diversity of the mammalian sodium/proton exchanger SLC9 gene family.Pflugers Arch. 2004; 447: 549-565Crossref PubMed Scopus (558) Google Scholar, 14Counillon L Pouyssegur J The expanding family of eucaryotic Na+/H+ exchangers.J Biol Chem. 2000; 275: 1-4Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar The housekeeping isoform, NHE1, is activated rapidly in response to various extracellular stimuli, such as hormones, growth factors, and mechanical stressors.12Wakabayashi S Shigekawa M Pouyssegur J Molecular physiology of vertebrate Na+/H+ exchangers.Physiol Rev. 1997; 77: 51-74Crossref PubMed Scopus (565) Google Scholar Enhanced NHE activity would cause elevation of [Na+]i and may produce intracellular Ca2+ overload via reduced Ca2+ extrusion by the plasma membrane Na+/Ca2+ exchanger (NCX). Although Ca2+ overload caused by Na+-dependent ion exchangers has been studied extensively in ischemic hearts,15Karmazyn M Gan XT Humphreys RA Yoshida H Kusumoto K The myocardial Na(+)-H(+) exchange: structure, regulation, and its role in heart disease.Circ Res. 1999; 85: 777-786Crossref PubMed Scopus (353) Google Scholar, 16Xiao XH Allen DG Role of Na+/H+ exchanger during ischemia and preconditioning in the isolated rat heart.Circ Res. 1999; 85: 723-730Crossref PubMed Scopus (90) Google Scholar, 17Imahashi K Kusuoka H Hashimoto K Yoshioka J Yamaguchi H Nishimura T Intracellular sodium accumulation during ischemia as the substrate for reperfusion injury.Circ Res. 1999; 84: 1401-1406Crossref PubMed Scopus (144) Google Scholar such phenomena have not been reported in dystrophic skeletal muscles. The protective effects of NHE inhibitors suggest that in addition to the Ca2+-permeable channel(s), Na+-dependent ion exchangers may be involved in the pathogenesis of muscular dystrophy, presumably through the sustained increase in [Ca2+]i. Here, we first show that the NHE inhibitors, cariporide and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), have protective effects against muscle degeneration in dystrophic BIO14.6 hamsters and mdx mice. We also show that the NHE activity is constitutively enhanced in dystrophic myotubes and that cariporide significantly reduces both the elevated [Na+]i and [Ca2+]i. Furthermore, we show that P2 receptor stimulation with ATP released by stretching may be the mechanism underlying the constitutive activation of NHE. To our knowledge, this is the first report indicating the pathological importance of Na+-dependent ion exchangers in muscular dystrophy. Cariporide was a gift from Aventis Pharma Chem. Ltd. (Frankfurt, Germany), and EIPA and KB-R7943(KBR) were from the New Drug Research Laboratories of Kanebo, Ltd. (Osaka, Japan). Rabbit polyclonal antibodies against NHE1 and NCX1 were described previously.18Tawada-Iwata Y Imagawa T Yoshida A Takahashi M Nakamura H Shigekawa M Increased mechanical extraction of T-tubule/junctional SR from cardiomyopathic hamster heart.Am J Physiol. 1993; 264: H1447-H1453PubMed Google Scholar, 19Bertrand B Wakabayashi S Ikeda T Pouyssegur J Shigekawa M The Na+/H+ exchanger isoform 1 (NHE1) is a novel member of the calmodulin-binding proteins. Identification and characterization of calmodulin-binding sites.J Biol Chem. 1994; 269: 13703-13709Abstract Full Text PDF PubMed Google Scholar, 20Pang T Su X Wakabayashi S Shigekawa M Calcineurin homologous protein as an essential cofactor for Na+/H+ exchangers.J Biol Chem. 2001; 276: 17367-17372Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar Rabbit polyclonal antibody against p44/42 MAP kinase and mouse monoclonal antibody against phospho-p44/42 MAP kinase (T202/Y204) were purchased from Cell Signaling (Beverly, MA). Gadolinium chloride (GdCl3) hexahydrate, ouabain, apyrase, 6-azaophenyl-2′,4′-disulfonic acid (PPADS), suramin, and monensin were purchased from Sigma Chemical (St. Louis, MO). Thapsigargin was from Calbiochem (La Jolla, CA). 22NaCl was purchased from NEN Life Science Products (Boston, MA). Fura-2/acetoxymethylester (AM) and fluo4-AM were from Dojindo Laboratories (Tokyo, Japan) and Molecular Probes (Eugene, OR), respectively. Our study followed institutional guidelines of National Cardiovascular Center for animal experimentation and was performed under the approved protocol. For examination of drug effects, EIPA and cariporide were administered orally in either the drinking water at a drug/body weight ratio of 3 mg/kg per day to 60-day-old BIO14.6 hamsters or 50-day-old mdx mice or age-matched normal controls as described.21Kusumoto K Haist JV Karmazyn M Na+/H+ exchange inhibition reduces hypertrophy and heart failure after myocardial infarction in rats.Am J Physiol. 2001; 280: H738-H745Crossref PubMed Google Scholar Suramin was administered by intraperitoneal injection at 25 mg/kg per day.22Kharlamov A Jones SC Kim DK Suramin reduces infarct volume in a model of focal brain ischemia in rats.Exp Brain Res. 2002; 147: 353-359Crossref PubMed Scopus (73) Google Scholar After continuous administration for periods indicated in legends to each figure, animals were subjected to measurement of creatine phosphokinase (CK) level in serum, histochemical analysis of muscles, and grip test. For the grip test for mdx mice, forelimb grip strength of mdx mice was assessed by timing how long they could support their body weight by holding onto a fine wire net. Each group consisted of more than five mice, all of which were analyzed twice on 2 different days. Skeletal muscles were fixed in phosphate-buffered saline (PBS) containing 10% formalin and embedded in paraffin. Serial 5-μm sections were stained with hematoxylin and eosin (H&E) or Masson's trichrome. The extent of experienced damage occurring in muscles was determined by comparing the number of centrally located nuclei between samples using a light microscopy. Variability of fiber size was obtained by averaging the standard deviations from three to four cross-sectional views of myofibers from three to four animals per group. The extent of fibrosis (blue-staining area) was measured on photographs of Masson's trichrome-stained sections. Myotubes in culture were prepared as described previously.10Iwata Y Katanosaka Y Arai Y Komamura K Miyatake K Shigekawa M A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor-regulated channel.J Cell Biol. 2003; 161: 957-967Crossref PubMed Scopus (225) Google Scholar, 11Iwata Y Katanosaka Y Shijun Z Kobayashi Y Hanada H Shigekawa M Wakabayashi S Protective effects of Ca2+ handling drugs against abnormal Ca2+ homeostasis and cell damage in myopathic skeletal muscle cells.Biochem Pharmacol. 2005; 70: 740-751Crossref PubMed Scopus (44) Google Scholar In brief, myoblast cells were isolated from the gastrocnemius muscles of normal or BIO14.6 hamsters by enzymatic dissociation. Minced muscles (0.3 g) were incubated for 45 minutes at 37°C in 1 ml of Ham's F-12 medium containing 2 U/ml dispase and 1% collagenase. After filtration through a fine mesh nylon filter and preplating to remove fibroblasts, cells were plated with ∼80% confluence onto collagen I-coated culture dishes in growth medium consisting of Ham's F-12 medium supplemented with 20% fetal calf serum and 2.5 ng/ml basic fibroblast growth factor (Promega BRL, Madison, WI) and 1% chick embryo extract (Life Technologies, Inc., Grand Island, NY). One or 2 days after plating, medium was changed to Dulbecco's modified Eagle's medium containing 2% horse serum (Hyclone Laboratories, Logan, UT) to initiate differentiation. Myoblasts begin to fuse and form myotubes in culture within 24 hours. We used the myotubes 2 to 5 days after the switch to differentiation medium. Normal and BIO14.6 myotubes cultured on collagen I-coated silicon membranes or in 24-well dishes were incubated at 37°C for 30 minutes in uptake solution containing 50 mmol/L NaCl, 96 mmol/L choline chloride, 1 mmol/L MgCl2, 0.1 mmol/L CaCl2, 10 mmol/L glucose, 0.1% bovine serum albumin, 10 mmol/L HEPES/Tris, pH 7.4, 37 kBq/ml 22NaCl, and 1 mmol/L ouabain. In some wells, the uptake solution contained 0.1 mmol/L EIPA or/and 0.25 mmol/L GaCl3. After 30 minutes, cells were rapidly washed four times with ice-cold PBS to terminate 22Na+ uptake. Cells were lysed in 0.1 N NaOH, and aliquots were taken for determination of protein and radioactivity. Myoblasts from skeletal muscles were seeded onto 25-mm glass coverslips coated with collagen I (Becton, Dickinson and Company, Franklin Lakes, NJ) and differentiated into myotubes. Myotubes were loaded with 3 μmol/L 2′,7′-bis-(bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) in balanced salt solution (BSS) (146 mmol/L NaCl, 4 mmol/L KCl, 2 mmol/L MgCl2, 1 mmol/L CaCl2, 10 mmol/L glucose, 0.1% bovine serum albumin, and 10 mmol/L HEPES/Tris, pH 7.4) for 10 minutes at room temperature. The coverslip was mounted on a flow chamber and continuously perfused with solutions at 0.6 ml/minute with a Perista pump. Changes in intracellular pH (pHi) were estimated by ratiometric scanning of changes in BCECF fluorescence. Fluorescence was monitored by alternatively exciting at 440 and 490 nm through a 505-nm dichroic reflector and 510- to 530-nm band-path emission filter. Fluorescence images were collected every 10 seconds using a cooled charge-coupled device camera (ORCA-ER; Hamamatsu Photonics, Hamamatsu, Japan) mounted onto an inverted microscope (IX 71; Olympus, Tokyo, Japan) with a ×20 objective (UApo/340; Olympus) and were then processed with AQUACOSMOS software (Hamamatsu Photonics). The pHi value was calibrated with high K+ solution containing 5 μmol/L nigericin adjusted to various pH values. For measurement of [Na+]i, myotubes were incubated with 10 μmol/L sodium-binding benzofuran isophthalate acetoxymethyl ester (SBFI-AM) and Pluronic F-127 (0.05% w/v) in BSS for 120 minutes at room temperature. After washout, SBFI-AM was de-esterified for 20 minutes. SBFI fluorescence was monitored by alternatively exciting at 340 and 380 nm at 1 Hz through a 505-nm dichroic reflector and 510- to 530-nm band-path emission filter. In some experiments we used BSS buffered with 10 mmol/L NaHCO3 (pH 7.4), saturated with 5% CO2 and 95% O2 gas. Fluorescence images were collected as described above for pHi measurement. The fluorescence ratio at 340:380 was calculated with AQUACOSMOS software and [Na+]i was calibrated at the end of each experiment in solutions containing 0, 10, or 20 mmol/L extracellular NaCl in the presence of 10 μmol/L gramicidin, 1 mmol/L ouabain, and 2 μmol/L monensin. For Ca2+ imaging, cells were plated on glass and cultured and loaded with fluo-4 by incubation for 30 minutes at 37°C in 4 μmol/L acetoxymethyl ester (Molecular Probes) in BSS as described previously.10Iwata Y Katanosaka Y Arai Y Komamura K Miyatake K Shigekawa M A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor-regulated channel.J Cell Biol. 2003; 161: 957-967Crossref PubMed Scopus (225) Google Scholar In brief, fluorescence signals in cells were detected by confocal laser-scanning microscopy using a MRC-1024ES system (Bio-Rad, Richmond, CA) mounted on an Olympus BX50WI microscope with a ×60 water immersion lens. The frequency of image acquisition was selected as one image per <1 second. Analysis of single-frame or single-cell integrated signal density was performed with LaserSharp software (Bio-Rad, Hertfordshire, UK). The Ca2+ level was represented as ΔF/Fo, where Fo is the resting fluo-4 fluorescence and ΔF is the difference between peak steady-state fluorescence within 1 to 2 minutes after stimulation and resting fluorescence. In some experiments, we also loaded cells with 4 μmol/L fura-2 acetoxymethyl ester as described above and measured [Ca2+]i by a ratiometric fluorescence method using a fluorescence image processor (Aquacosmos; Hamamatsu Photonics). The excitation wavelength was alternated at 340 and 380 (1 Hz), and we measured the fluorescence light emitted at 510 nm. The fluorescence ratio at 340:380 was calculated. Mechanical stretching was applied to myotubes using a silicon chamber as described previously.10Iwata Y Katanosaka Y Arai Y Komamura K Miyatake K Shigekawa M A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor-regulated channel.J Cell Biol. 2003; 161: 957-967Crossref PubMed Scopus (225) Google Scholar After cells were allowed to attach to the chamber bottom, uniaxial sinusoidal stretching was applied to the chamber at a constant strength from 5 to 20% elongation at 1 Hz for indicated periods. The relative elongation of the silicone membrane was uniform across the whole membrane area. After stretching of myotubes, CK activity in the medium was determined using an in vitro colorimetric assay kit (CK test kit; Wako Pure Chem. Co., Osaka, Japan) according to the protocol provided by the manufacturer. For ATP measurement, myotubes were washed twice with 0.5 ml of BSS 1 hour before stretching. BSS (0.5 ml) was added to the chamber, and uniaxial sinusoidal stretching was applied as above. Aliquots (100 μl) of the BSS solution were taken at selected times to measure the ATP level. The concentration of ATP released from the myotubes was measured using the luciferin-luciferase reaction (ENLITEN; Promega). Quantitative immunoblotting analysis and immunocytochemistry were performed as described previously.10Iwata Y Katanosaka Y Arai Y Komamura K Miyatake K Shigekawa M A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor-regulated channel.J Cell Biol. 2003; 161: 957-967Crossref PubMed Scopus (225) Google Scholar, 11Iwata Y Katanosaka Y Shijun Z Kobayashi Y Hanada H Shigekawa M Wakabayashi S Protective effects of Ca2+ handling drugs against abnormal Ca2+ homeostasis and cell damage in myopathic skeletal muscle cells.Biochem Pharmacol. 2005; 70: 740-751Crossref PubMed Scopus (44) Google Scholar, 23Iwata Y Nakamura H Mizuno Y Yoshida M Ozawa E Shigekawa M Defective association of dystrophin with sarcolemmal glycoproteins in the cardiomyopathic hamster heart.FEBS Lett. 1993; 329: 227-231Crossref PubMed Scopus (57) Google Scholar, 24Iwata Y Sampaolesi M Shigekawa M Wakabayashi S Syntrophin is an actin-binding protein the cellular localization of which is regulated through cytoskeletal reorganization in skeletal muscle cells.Eur J Cell Biol. 2004; 83: 555-565Crossref PubMed Scopus (26) Google Scholar Protein concentration was measured using a bicinchoninic acid assay system (Pierce Chemical Co., Rockford, IL) with bovine serum albumin as a standard. Unless otherwise stated, experiments were performed at 25 ± 1°C and data are represented as means ± SD of at least three determinations. We used unpaired t-test, one-way analysis of variance followed by Dunnett's test, or two-way analysis of variance for statistical analyses. Values of P < 0.05 were considered statistically significant. Oral EIPA protected against muscle degeneration, as shown in sections stained with H&E (Figure 1A). We measured the number of fibers with central nuclei, which was often used as an index for regeneration to compensate for the fiber breakdown. The number of centrally localized nuclei was markedly reduced by treatment with EIPA (Figure 1Ba) or cariporide (see Figure 8, B and C). Among several other abnormal morphological features, dystrophic muscle fibers are known to display greater variations in their cross-sectional area because muscles contain fibers with different sizes, such as necrotic, splitting, and regenerating fibers. EIPA markedly reduced this fiber size variability as determined by the SD of the cross-sectional areas of myofibers (Figure 1Bb). In addition, NHE inhibitor considerably reduced the area of fibrosis stained with Masson's trichrome (see Figure 8Cb). These results suggest that NHE inhibitor prevented muscle degeneration and blocked the resultant regeneration as evidenced by the reduced centrally located nuclei. Furthermore, EIPA markedly reduced CK level in the serum of BIO14.6 hamsters, which is also a marker for muscle degeneration (Figure 1C). In mdx mice, the extent of muscle degeneration reaches the first peak in ∼21 to 28 days and then declines because of regeneration and reaches the second peak in ∼72 days, when muscle degeneration was checked by serum CK level. Therefore, we started the treatment with cariporide in 50-day-old mice to see whether muscle damage during the second period is reduced. As shown in Figure 1D, treatment with cariporide for 22 days (Figure 1Db) markedly prevented muscle damage. Cariporide reduced inflammatory infiltrate (Figure 1D), fibrosis (data not shown, but see Figure 8), and the number of myofibers with central nuclei particularly in mice treated for 60 days (Figure 1E). In control mdx mice, serum CK levels reached a peak in 72 days of age (22 days after start of experiment) and remained at relatively high level until 145 days of age (95 days after start of experiment, Figure 1F). Treatment with cariporide considerably reduced serum CK level in all investigated ages in mdx mice (Figure 1F). Together, these results suggest that the degenerative and accompanying regenerative episodes become rare on treatment with NHE inhibitor. Furthermore, we also evaluated the muscle performance of mice by timing how long they could support their body weight holding onto a fine wire net. Cariporide significantly improved the results of this grip test in mdx mice (Figure 1G). These observations collectively suggest that inhibition of NHE activity confers a significant protective effect against skeletal muscle dysfunction in dystrophic animals.Figure 8Protective effect of suramin against muscle degeneration. A: Effect of suramin and/or cariporide on CK level in serum from BIO14.6 hamsters. Suramin (sur) and/or cariporide (car) were administered by intraperitoneal injection or by oral intake into 60-day-old BIO14.6 hamsters, respectively, and 14 days after drug administration (74-day-old hamsters), serum CK level was measured. Data are means ± SD (n = 4 to 5). *P < 0.05, whereas **P < 0.05 versus either cariporide or suramin alone. B: Masson's trichrome staining of the quadriceps muscle sections from BIO14.6 hamsters. C: Quantitation of fibers containing central nuclei in muscles of BIO14.6 hamsters. Centrally located nuclei (a), fibrosis area (blue region) (b), and variability (c) were measured as described in Materials and Methods. Data are means ± SD (n = 3 to 4). *P < 0.05. D: Effect of suramin on serum CK level in mdx mice. Suramin or PBS (for control) was injected into 14-day-old mdx mice and serum CK level was measured 7 or 14 days after the start of drug injection. E: Effect of suramin on muscle performance measured by the grip test. Data are means ± SD (n = 3 to 5). *†P < 0.05 versus mdx/control or normal/control, respectively. Scale bar = 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our in vivo data prompted us to study the mechanism of the involvement of NHE in skeletal muscle dysgenesis. Immunoblotting analysis revealed that skeletal muscles expressed NHE1, and its expression level was not very different between skeletal muscles from normal and BIO14.6 hamsters (Figure 2A); the normalized relative amount of NHE1 in BIO14.6 was 0.92 ± 0.07 (n = 3) versus normal muscles. NHE1 is distributed mainly in the sarcolemma of the skeletal muscles from normal and dystrophic hamsters (Supplemental Figure 1; see http://ajp.amjpathol.org). Moreover, we did not detect a large difference in the expression level (Figure 2A; 0.95 ± 0.08 versus normal myotubes, n = 3) of NHE1 between cultured myotubes from normal and BIO14.6 hamsters. We next measured the NHE activity after NH4+ prepulse by ratiometric fluorescence measurement with BCECF. In normal myotubes after NH4+ prepulse, the addition of external Na+ induced rapid pHi recovery, reaching only pHi ∼7.0 (Figure 2B). This pHi recovery was attributable to the NHE activity because it was blocked completely by cariporide (Figure 3A). Because half-maximal inhibition occurred at relatively low cariporide concentration ( 7.2) (Figure 2C). Myotubes from BIO14.6 hamsters exhibited significantly higher resting pHi compared with normal animals (Figure 2D). Interestingly, although the PKC activator PMA markedly accelerated the pHi recovery in normal myotubes, PMA accelerated only the initial pHi recovery phase in BIO14.6 myotubes (Figure 3A). Figure 3B shows the pHi dependence of pHi recovery rate measured in myotubes. The pHi dependence was shifted to the alkaline side in BIO14.6 as compared with normal myotubes. In normal myotubes, PMA greatly shifted the pHi dependence to the alkaline side. In contrast in BIO14.6 myotubes, PMA did not induce a large alkaline shift of pHi dependence although it elevated the recovery rate at each pHi. These observations may reflect the high levels of activated NHE in BIO14.6 myotubes, inducing an alkaline shift of pHi dependence resulting in a marginal effect of PMA.Figure 3High Na+/H+ exchange activity in BIO14.6 myotubes. A: Time courses of Na+-induced pHi recovery in normal and BIO14.6 myotubes. Myotubes were subjected to NH4+ prepulse, and then pHi recovery was induced by exposing myotubes to Na+-containing solution. In some experiments, myotubes were exposed to 1 μmol/L PMA or 10 μmol/L cariporide throughout the NH4+ prepulse and pHi recovery phases. B: The pHi dependence of the pHi recovery rates. The pHi recovery rate was calculated from the increment in pHi every 10 seconds and plotted against pHi. Data are means ± SD of five independent experiments.View Large
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