exercise and duchenne muscular dystrophy: …...invited review exercise and duchenne muscular...

INVITED REVIEW

EXERCISE AND DUCHENNE MUSCULAR DYSTROPHY: TOWARDEVIDENCE-BASED EXERCISE PRESCRIPTIONCHAD D. MARKERT, PhD,1 FABRISIA AMBROSIO, PT, PhD,2 JARROD A. CALL, MS,3 and ROBERT W. GRANGE, PhD4

1 Wake Forest Institute for Regenerative Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157, USA2 Department of Physical Medicine and Rehabilitation, University of Pittsburgh, Pittsburgh, Pennsylvania, USA3 Program in Physical Therapy and Rehabilitation, University of Minnesota School of Medicine, Minneapolis, Minnesota, USA4 Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA

Accepted 30 November 2010

ABSTRACT: To develop a rational framework for answeringquestions about the role of exercise in Duchenne muscular dys-trophy (DMD), we focused on five pathophysiological mecha-nisms and offer brief hypotheses regarding how exercise maybeneficially modulate pertinent cellular and molecular pathways.We aimed to provide an integrative overview of mechanisms ofDMD pathology that may improve or worsen as a result of exer-cise. We also sought to stimulate discussion of what outcomes/dependent variables most appropriately measure these mecha-nisms, with the purpose of defining criteria for well-designed,controlled studies of exercise in DMD. The five mechanismsinclude pathways that are both intrinsic and extrinsic to the dis-eased muscle cells.

Muscle Nerve 43: 464–478, 2011

Effective supportive therapy for Duchenne musculardystrophy (DMD), let alone a cure using gene ther-apy, has been a challenge despite identification of themissing protein, dystrophin, over 20 years ago.1 Exer-cise, depending on several poorly researched parame-ters (including frequency, intensity, time, and type),may be either beneficial or detrimental to dystrophicskeletal and cardiac muscle. A recent mini-review ofthe literature2 recommended that systematic researchstudies of animal models of DMD are warranted tobetter define exercise prescription for boys afflictedwith DMD. However, the most commonly used ani-mal model, the mdx mouse, does not fully recapitulatethe disease. Although similar biochemically, mdxmouse muscle and muscle from a DMD patient differ,for example, in terms of regenerative ability3 andcompensatory protein expression (utrophin).4 Thus,some researchers5–9 have used exercise in mdx miceto worsen the dystrophic phenotype. The fact that

exercise in this instance is detrimental does not neces-sarily translate categorically to boys with the diseasefor three reasons: (1) differences exist between thedystrophic mouse and dystrophic human; (2) there isa dearth of data regarding the very wide spectrum ofvariables to consider prior to exercise prescription;and (3) the adaptability to exercise in animal modelsof the disease and in DMD boys is unknown.2,10,11

Although a positive role for exercise is well acceptedin non-diseased populations,12 scientific studies havenot yet resolved whether exercise is therapeutic in adystrophic population.13,14 Furthermore, althoughsome practical recommendations are available,15,16

specific guidelines regarding exercise prescription(the type, frequency, and intensity of exercise) do notexist.17–22 Thus, families of diseased patients are leftwith some fundamental questions. Does exercise helpor hurt my child? What are the specific guidelines forensuring that the exercise is safe and beneficial and,importantly, not detrimental?

To begin development of a rational frameworkfor answering questions regarding the role of exer-cise in DMD patients, this review focuses on five pro-posed pathophysiological mechanisms of the diseaseand offers brief hypotheses regarding how exercisemay modulate the pertinent cellular and molecularpathways of these mechanisms in a beneficial man-ner. The goals of this review are twofold. First, wesought to provide an integrative overview of mecha-nisms of DMD pathology that may improve or wor-sen as a result of exercise training. Second, wesought to expand upon a recent review,2 stimulatingdiscussion of what outcomes/dependent variablesmost appropriately measure these mechanisms, withthe purpose of defining criteria for well-designed,controlled studies of exercise training in DMD. Thefive mechanisms, as defined and discussed byPetrof,23 include: (1) mechanical weakening of thesarcolemma; (2) inappropriate calcium influx; (3)aberrant cell signaling; (4) increased oxidative stress;and (5) recurrent muscle ischemia.

MECHANICAL WEAKENING OF THE SARCOLEMMA

An appropriate starting point for discussion of theissues that affect dystrophin-deficient animals and

Abbreviations: AChR, acetylcholine receptor; ATP, adenosine triphos-phate; Ca2þ, calcium; cGMP, guanosine cyclic monophosphate; DAMPs,damage-associated molecular patterns; DGC, dystrophin–glycoproteincomplex; DHE, dihydroethidium; DMD, Duchenne muscular dystrophy;ECM, extracellular matrix; EGF, epidermal growth factor; FGF, fibroblastgrowth factor; GFP, green fluorescent protein; GI, gastrointestinal; HGF,hepatocyte growth factor; IGF-1, insulin-like growth factor-1; MHC, myosinheavy chain; MPC, muscle precursor cell; MSC, mesenchymal stem cell;NF-jB nuclear factor–kappaB; NIR, near-infrared; NO, nitric oxide; nNOS,neural nitric oxide synthase; PGC-1a, peroxisome proliferator–activated re-ceptor-gamma coactivator 1alpha; PMN, polymorphonuclear neutrophils;RONS, reactive oxygen and nitrogen species; TGF-b transforming growthfactor-beta; VEGF, vascular endothelial growth factor; Wnt, Wingless intprotein

Correspondence to: C. D. Markert; e-mail: [email protected]

VC 2010 Wiley Periodicals, Inc.Published online 00 Month 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mus.21987

Key words: DMD; dystrophic; mdx; mechanisms; muscle physical activity

J_ID: ZA3 Customer A_ID: MUS21987 Cadmus Art: MUS21987 Ed. Ref. No.: 10-0571.R2 Date: 28-February-11 Stage: Page: 464

ID: senthilk I Black Lining: [ON] I Time: 16:18 I Path: //xinchnasjn/01Journals/Wiley/3b2/MUS#/Vol04304/100316/APPFile/JW-MUS#100316

464 April 2011 MUSCLE & NERVE Exercise and DMD

humans with DMD is with the mdx mouse, themost widely studied animal model of the disease,and with mechanical weakening of the sarco-lemma, widely considered a key mechanism under-lying the pathogenesis of the disease. The mdxmouse is accepted as a biochemical and geneticmodel of DMD and has been studied since the1980s.24 The reader is referred to a recent review25

and the TREAT-NMD website (http://www.treat-nmd.eu/research/preclinical/preclinical/) for in-depth discussion of the mdx mouse and other pre-clinical animal models of DMD. Although useful asa biochemical and genetic model of the disease, aweakness of the mdx mouse model is that it doesnot fully recapitulate the dystrophic phenotypeseen in human patients. Indeed, because the mdxmouse is an insufficient model in this regard, vari-ous researchers have intentionally attempted toworsen the mdx phenotype using, for example, agene knockout of utrophin4,26,27 or, more simply,enforced intense exercise. Because exercise was,and often continues to be, intentionally used in ex-perimental designs in this context to exacerbatedamage,5,7,28 it remains unclear whether exerciseprescriptions, designed with other motivations inmind, could have the opposite effect.29 Further-more, the thesis that exercise exacerbates mechani-cal weakening of the sarcolemma has become sowidely accepted in the literature that few research-ers have questioned its origin. Thus, a key require-ment of future research should be to provide the

evidence that defines the effects of exercise on themechanical properties of the sarcolemma.

The 427-kDa dystrophin protein can be isolatedfrom an oligomeric complex of proteins oftenreferred to as the dystrophin–glycoprotein com-plex (DGC). The literature that has hinted towardre-envisioning the role of exercise in DMD hasbeen reviewed.2,29,30 In this section, particularfocus is placed on the underlying mechanism ofmuscle membrane weakening and on studies31–34

that provide counterpoints to the assertion that‘‘…Both sarcolemmal permeability and necrosis ofdystrophin-deficient muscle are exacerbated byphysical exercise and improved by muscle immobi-lization’’35 (Table T11). Important to this discussionis the awareness that enforced exercise and volun-tary exercise are different exercise paradigms.

Components of the DGC include the dystrogly-cans, sarcoglycans, sarcospan, dystrobrevins, syntro-phins, and neural nitric oxide (NO) synthase,which regulates NO signaling.36 Especially whenconsidered as part of the DGC, dystrophin appearsto have a role in cellular signaling (see AberrantCell Signaling section). However, many studies(e.g., see refs. 20, 30, 35, and 37) have focused ondystrophin’s structural contribution, and thesestudies have suggested that it performs a mechani-cal role in stabilizing the sarcolemma during short-ening, lengthening, or isometric muscle actions.38

In each of these contraction types, the goal istransmission of force. In both skeletal and cardiac

Table 1. Studies that suggest exercise is contraindicated in DMD.35

Study reference Study titleMethodological considerations

that potentially confound this interpretation

Karpati et al. (1986)182 Small-caliber skeletal muscle fibersdo not suffer deleterious consequencesof dystrophic gene expression

a,d

Weller et al. (1990)183 Dystrophin-deficient mdx muscle fibersare preferentially vulnerable to necrosisinduced by experimental lengthening contractions

a–f

Mizuno (1992)184 Prevention of myonecrosis in mdx mice:effect of immobilization by the local tetanus method

g

Clarke et al. (1993)73 Loss of cytoplasmic basic fibroblastgrowth factor from physiologicallywounded myofibers…

a–f

Vilquin et al. (1998)185 Evidence of mdx mouse skeletal musclefragility in vivo by eccentric running exercise

a–e

Mokhtarian et al. (1999)186 Hindlimb immobilization appliedto 21-day-old mdx mice…

g

Bansal et al. (2003)187 Defective membrane repair indysferlin-deficient muscular dystrophy

h

aExercise was studied in mouse not human, or was not studied.bExercise was not voluntary.cExercise was biased toward pliometric muscle action38 (‘‘eccentric’’ or ‘‘lengthening‘‘).dFrequency, intensity, time, and type of exercise were not systematically or fully evaluated.eDose response to the exercise was not established.fLongitudinal effects of exercise intervention were not measured.gMechanical or chemical immobilization unlikely to be adopted clinically.hDuchenne muscular dystrophy was not specifically studied



Exercise and DMD MUSCLE & NERVE April 2011 465

muscle, costameres transduce mechanical force37

as it is relayed from muscle to external load. Posi-tive work is the product of muscle force and short-ening, whereas negative work is the product ofmuscle force and muscle lengthening (i.e., alengthening action38). Costameres are proteinassemblies that interact intimately with the sarco-lemma and are believed first to couple the forcegeneration of sarcomeres to the sarcolemma andto other fibers, and second to convert a mechani-cal stimulus to cellular signaling or gene expres-sion response.37 In muscles that lack dystrophin,costameres are disrupted, and sarcolemmal fragility(commonly measured by release of creatine kinaseand pyruvate kinase) ensues.39,40 Of note,increased membrane permeability and membranemechanical weakness are not necessarily the same.A certain threshold of work (most often negativework—i.e., a lengthening action38) is capable ofproducing contraction-induced injury41,42; thisthreshold is diminished in muscle that lacks dystro-phin.43,44 The work-overload theory45 proposedthat a muscle, such as diaphragm, which is contin-uously activated throughout life, would deterioratefaster than a less frequently activated muscle. Inthe context of limited exercise studies available atthat time, the work-overload theory made senseand provided an explanation of cardiorespiratoryfailure in dystrophin deficiency. The theory waschallenged in 1994 by observations in mdx micethat voluntary wheel-running exercise improved,rather than worsened, the contractile function ofthe diaphragm.29

Since that time several more murine studieshave concluded that exercise may be benefi-cial.2,31–34 There is a conspicuous similaritybetween studies, because the exercise was volun-tary. Human studies focused on inspiratory muscletraining in DMD patients46–51 have shown that thefunction of respiratory muscles improves withtraining. Importantly, respiratory muscle training ismost effective when initiated in the early stages ofDMD,20,49,51,52 suggesting an age-dependent effect.Thus, it is reasonable to presume that, based ontheir own volition, dystrophic animals (and boys)may self-regulate the amount of force transmittedto their costameres by limiting their external work.Stated more simply, dystrophic boys might self-impose limits to their physical activity. Althoughfew studies have investigated sensation of intramus-cular fatigue and central fatigue in patients withDMD, it has been observed that cage activity ofmdx mice is diminished relative to controls andthat DMD boys cannot keep up with their peers.Whether this outcome is volitionally or pathophy-siologically imposed, work is performed at the sub-ject’s discretion. We pose the question: Do DMD

patients develop an awareness and memory of agiven workload/physical activity if significant mus-cle pain or myoglobinuria follows the activity53? Ifso, does this affect their choices regarding partici-pation in physical activities? Do they self-regulate?In addition, we reiterate the call54 for increasedpartnership between DMD families and patientregistries and/or clinical trials. Data collectedthrough this partnership might improve ourunderstanding of the amount and type of dailyself-selected physical activity performed by thepatients in relation to the progression of the pa-thology that could guide future studies to quantita-tively define appropriate exercise.

How can we best determine appropriate exer-cise to minimize negative effects on the dystrophicmembrane and maximize positive adaptations toimprove muscle performance? A recent literaturereview of the biology of the DGC included severalreferences that demonstrated exercise was contra-indicated in DMD (Table 1).35 What several ofthese studies reported was that enforced exerciseor lengthening contractions exacerbated the mus-cle pathophysiology, whereas immobilization didnot. A reasonable conclusion on the basis of thesestudies is that inappropriate exercise can indeedbe detrimental. However, the benefits or detri-ments of exercise would be defined more clearly iffuture studies focused on dose–response relation-ships in specific exercise protocols. Of interest isthe question of whether appropriate exercise canimprove the structural and signaling characteristicsof the myofibers that compose dystrophic muscledespite the absence of the DGC. Future studiesshould address this issue by varying the key ele-ments of exercise prescription, such as type, inten-sity, frequency, and duration, to determinewhether there are ideal values (the right ‘‘prescrip-tion’’) by which dystrophic muscles can positivelyadapt to improve function. Such studies couldstimulate new hypotheses regarding the role of dys-trophin as a determinant of the mechanical prop-erties of the sarcolemma.

INAPPROPRIATE CALCIUM INFLUX

Maintenance of calcium homeostasis within skele-tal muscle fibers is important both to confer func-tionality and to prevent excessive calcium-activatedprotease (calpain) activity.55 Other ions, such asNaþ and Cl�, clearly contribute to function andhealth/disease of skeletal muscle fibers.6,56,57

Although the importance of other ionic changes isnot to be downplayed, this section focuses onmechanisms whereby exercise may modify intracel-lular Ca2þ changes in dystrophic muscle.

There exist different schools of thought on thehierarchy of calcium-dependent events leading to



466 Exercise and DMD MUSCLE & NERVE April 2011

myofiber degradation in muscular dystrophy. Onecontends that the mechanism of cell death in dam-aged dystrophic myofibers is mediated by calpains,which exert well-documented calcium-activatedproteolytic activity in dystrophic muscle.58,59 Thisactivity is increased following disruption of the sar-colemma, mechanical or otherwise. Skeletal musclecontains both of the ubiquitous forms of calpain,termed m-calpain and l-calpain, to indicate,respectively, the millimolar and micromolar Ca2þ

concentrations required for their activation. Skele-tal muscle also contains a third muscle-specific cal-pain, called calpain-3. In healthy muscle, calpainactivity is antagonized by calpastatin. Because cal-pastatin concentration is generally in excess, cal-pain activity is effectively regulated. Little informa-tion is available regarding regulation of calpainactivity in dystrophic human muscle. Muscles ofmdx mice have increased calpain concentrationand activity as compared with wild-type counter-parts60,61; dystrophic symptoms are diminished inmdx mice that overexpress calpastatin.62 Symptomsin mdx mice are also ameliorated after treatmentwith BN 82270, a calpain inhibitor63; however, it isunclear whether calpain-inhibitory or antioxidantproperties of this compound were responsible.

A second school of thought contends that themechanism of cell death in damaged dystrophicmyofibers is mediated by the mitochondria, whichare sensitive to the abnormally high levels of intra-cellular Ca2þ that follow the mechanical insult tothe sarcolemma. Recent evidence suggests thatincreased intracellular Ca2þ stimulates the mito-chondria to amplify production of reactive oxygenspecies, resulting in a vicious cycle.64 The mito-chondrial protein cyclophilin D regulates Ca2þ-de-pendent cellular necrosis. Treatment of mdx micewith an inhibitor of cyclophilin D, Debio-25, pro-vided mitochondrial protection.65 Thus, interven-tions that either reduce dysregulation of intracellu-lar Ca2þ or offer mitochondrial protection mightbe expected to ameliorate dystrophic pathology.66

Both of the above neglect the evidence that sug-gests sarcolemmal tearing is not necessarily a pre-requisite for inappropriate Ca2þ influx.67 Forexample, one investigation found differencesbetween dystrophic and control muscle Ca2þ chan-nels or intracellular calcium stores.68 These mecha-nisms appear to foreshadow pathology withoutovert membrane damage. Further reviews areavailable.69–71

In studies of dystrophic mdx muscle fibers, ithas been shown that, without the membrane stabi-lization and mechanical coupling function nor-mally mediated by the dystrophin protein, extra-cellular Ca2þ influx occurs with catastrophicconsequences.72 This influx may occur either as a

result of sarcolemmal tearing73 and/or as dysfunc-tion of stretch-activated cation channels.74 Mislocal-ization or loss of Ca2þ channels may occur in dys-trophic muscle cells,75 reminiscent of the loss ofthe proteins of the DGC. Furthermore, membraneruptures lead to increased calcium leak channel ac-tivity.67 Thus, inappropriate Ca2þ influx may havea structural basis related only indirectly to mechan-ical weakening of the sarcolemma. The questionarises whether exercise may modulate the expres-sion or leakiness of Ca2þ channels, but few stud-ies76,77 have directly considered this. Of note, twostudies28,78 have permitted a clear comparison ofcalcium influx in response to the same exercise re-gime in healthy and dystrophic animals. Thesestudies showed that a 4–6-week exercise protocol,both in wild-type and mdx mice, significantlyincreases the calcium influx via specific channels.It was also shown that the basal and exercise-induced increases in calcium permeability are sig-nificantly higher in mdx than in wild-type mice.Further study of mechanosensitive ion channelshas been recommended.56,79

Again, exercise, appropriately tailored to theDMD patient, may constitute a useful intervention.For example, it is logical that pliometric38 (length-ening) muscle actions expose susceptible regionsof titin, resulting in their increased vulnerability tocalpain proteolysis.68 This molecular mechanism ofcontractile protein degeneration is complementedby organismal observations such as the widelyaccepted notion that lengthening muscle actionsare excessively degenerative, and therefore contra-indicated, for DMD patients. Thus, at the organis-mal level, exercise must be carefully prescribedbased on our knowledge of deleterious molecularmechanisms of degeneration. Once a tailored exer-cise prescription is considered, then questionsregarding its efficacy at both the organismal andmolecular levels may be posed; for example, doesthe exercise intervention improve muscle functionvia increased calpastatin? Finally, exercise may alsomediate a fast-to-slow myosin heavy chain (MHC)shift and concomitant alterations in Ca2þ han-dling80; however, the intensity sufficient to inducea functionally significant MHC shift28 is likely inap-propriate for DMD patients.

ABERRANT CELL SIGNALING

Aberrant cell signaling is a key mechanism under-lying DMD pathophysiology. For the purposes ofthis review, we discuss aberrant cell signalinginvolving two cell types: differentiated cells andstem/progenitor cells.

Differentiated Cells. The interaction of differenti-ated dystrophic muscle cells with differentiatedcells composing microvessels, nerves, muscle cell




neighbors, and the extracellular matrix are consid-ered in what follows.

Muscle–Microvessel Interactions. Vascular endo-thelial growth factor (VEGF) is a key mediator ofangiogenesis, and it has been shown that peroxi-some proliferator-activated receptor-gamma coacti-vator 1alpha (PGC-1a) orchestrates not only VEGFexpression but also oxidative metabolism,81 as ispredicted by its transactivator status.82 Evidence ofconcerted cellular cues was provided by angiomyo-genic studies using green fluorescent protein(GFP)-positive bone marrow–derived cells trans-planted into mice with satellite cells that had beendepleted by irradiation. Transplanted cells took upresidence in both perivascular and satellite cellniches, and the investigators concluded that theendothelial cells of the microvasculature secretedVEGF and a variety of other growth factors essen-tial for trophic support of the transplanted (andhost) cells.83 The capillary-to-fiber ratio is anotherexample of vessel–muscle interaction, and this ra-tio can be manipulated with exercise.84,85 In addi-tion, well-documented changes in fiber type—con-ferring, for example, a switch from oxidative toglycolytic metabolism, that is, slow- to fast-twitchfiber transformation—are correlated with vascularchanges.86–89 Recent evidence has shown that thefrequency of mesenchymal stem cells (MSCs) cor-relates with blood vessel density.90 This suggeststhat an angiogenic intervention, perhaps by exer-cise, may increase the availability of MSCs for tissueregeneration in patients with DMD, although theMSCs would carry the same genetic mutations asthe patients’ somatic cells.

Muscle–Nerve Interactions. Functional muscledenervation is another consequence of aberrantcell–cell signaling; in this case muscle–nerve inter-actions are impaired. Although a case can be madefor anatomic impairments to neuromuscular regen-eration in dystrophic muscle, such as fibroblastgrowth factor (FGF)-induced73 or transforminggrowth factor (TGF)-b–induced fibrosis,91 we limitour focus here to signaling studies. Expressionprofiling of muscle biopsies from patients withDMD show overexpression of developmentallyregulated genes such as acetylcholine receptor(AChR) and embryonic myosin heavy chain, sug-gesting a chronic state of poorly innervated,incompletely differentiated muscle fibers.92 Immu-nostaining confirmed these profiling results. Thesedata were complemented by analysis of the neuro-muscular junction in normal and dystroglycancomplex–null mice,93 demonstrating that thedystroglycan complex is a requirement for nonpa-thologic condensation of AChR and synapse forma-tion. Thus, expression profiling and immuno-histochemistry provide means for further study of

exercise-induced modifications to neuromuscularbiological cascades.

Muscle–Muscle Interactions. Satellite cells arethe myogenic progenitors responsible for postnatalmuscle growth, repair, and regeneration. Severalexperiments have shown that physical associationof muscle satellite cells with mature myofibers hasan antiproliferative effect on the satellite cells.94

However, it is unclear whether the ability of satel-lite cells to proliferate on basal lamina void ofmyofibers (decellularized extracellular matrix)occurs because antiproliferative cues are not pres-ent (e.g., cell–cell inhibition is absent), or whetherproliferation is actually a function of factorssecreted by the basal lamina. Nonetheless, it isclear that the muscle progenitor cell niche hasprofound effects on the potential forregeneration.95

In addition, it has been suggested that muscleprogenitor cells in an aged environment, charac-terized by oxidant stress, are more likely to differ-entiate into adipocytes.96 Some similarities betweenDMD and aging97 suggest further exploration ofcommon pathological mechanisms, such as the fiveconsidered here, and how exercise interventionsmight ameliorate the pathology.

Muscle–Extracellular Matrix Interactions. Theelasticity of the extracellular matrix (ECM) is avariable that directs stem cell fate.98,99 Softer sub-strates promoted adipogenic differentiation ofmesenchymal stem cells, whereas increasinglystiffer substrates promoted myogenic and osteo-genic lineage specification. Sensing the elasticity ofthe microenvironment was ascribed to non-musclemyosin98; primary cilia may also play a role.100 Thebasal lamina is the ECM of muscle cells. Besidesthe elasticity of the matrix, the composition is ofimportance. The basal lamina is formed from col-lagen type IV, laminin, fibronectin, and assortedglycoproteins and proteoglycans.94 Evidence indi-cates that these components serve dual structuraland regulatory roles. For example, the basal lam-ina serves as a scaffold for regeneration of musclefibers, but its components also bind growth factorsand growth inhibitors, such as hepatocyte growthfactor (HGF) and TGF-b, respectively.91,101,102

Muscle–Immune System Interactions. The nuclearfactor–kappaB (NF-jB) pathway sits at the centerof immune-mediated and inflammatory-mediatedcell signaling networks. Accordingly, NF-jB regu-lates the expression of many pro-inflammatorygenes, in cells as diverse as lymphocytes, macro-phages, and muscle fibers.103–105 As a result of NF-jB upregulation, a given lymphocyte or dystrophicmuscle cell is stimulated to secrete cytokines andchemokines, the molecules that are the front-linemediators of inflammation. These molecules




appear to amplify the inflammatory response byattracting additional immune cells to the dystro-phic area106 and also control the time course ofdegeneration–regeneration of the affected mus-cle.107,108 A further review has been published.103

Muscle (Mitochondria)–Immune System Inter-actions. An additional layer of complexity can beadded to the previous discussion of calcium-signal-ing aberrations in the mitochondria by studies thatdescribed mitochondrial damage–associated molec-ular patterns (DAMPs) as attractants of polymor-phonuclear neutrophils (PMNs).109 The phenom-enon may explain, in part, the excessiveinflammation and fibrosis seen in dystrophic,injured muscles. Briefly, if the sarcolemma of agiven dystrophic fiber is damaged and the mito-chondrial DAMPs contained within are no longer‘‘protected’’ from surveillance by the immune sys-tem, then an inflammatory response follows.Although previous studies have briefly addressedmitochondrial–immune system interactions,110–112

considering the inflammation in DMD as a mito-chondria-mediated process deserves further study.Interaction of histones113 and adenosine triphos-phate (ATP)114,115 with neutrophils may alsoexplain the hyperinflammation observed in dystro-phic muscle. Further details on inflammation arepresented in the Increased Oxidative Stresssection.

Involving Stem Cells. The previous subsections dis-cussed the weakening of the sarcolemma and thesubsequent myofiber degradation that occurs inresponse to muscle loading/external work. Wenow focus on events after myofiber degradation,with an emphasis on the regenerative processes.

Following myofiber degradation, myofiber ne-crosis and initiation of the inflammatory responseresults in the secretion of cytokines and growthfactors, such as insulin-like growth factor-1 (IGF-1),HGF, epidermal growth factor (EGF), and VEGF,to name a few.116 These cytokines and growth fac-tors are required for the activation and recruit-ment of muscle satellite cells. Satellite cells, locatedbetween the sarcolemma and the basal lamina,fuse together, or with damaged myofibers, to formregenerated myofibers.117 In the absence of satel-lite cell activation, muscle regeneration cannotoccur. A further review has been published.118

Due to the repeated and continuous rounds ofmyofiber degeneration–regeneration of dystrophicmuscle, the number of satellite cells significantlydecreases over time.119 Moreover, satellite cellsfrom dystrophic muscle demonstrate a markedlydiminished doubling time120 and a decreasedcapacity for myogenic differentiation (unpublishedresults). Unpublished findings from our laboratory

have demonstrated that muscle-derived precursorcells isolated from the hindlimb skeletal muscle ofmdx mice demonstrate significantly reduced myo-tube formation, in vitro, when compared with pre-cursor cells isolated from wild-type controlmuscles, a phenomenon that also characterizesaged skeletal muscle. In addition, accelerated telo-mere shortening occurs in boys with DMD.3,121

As in skeletal muscle models of aging, in theabsence of effective myofiber regeneration, degen-erating dystrophic myofibers become replaced byconnective or fatty tissues, severely limiting force-producing capacity. In aged muscle models, thisshift from effective myogenic regeneration to fibro-sis after skeletal muscle injury is partly a result of alineage conversion of muscle precursor cells.97

With time, muscle precursor cells shift in theirability to restore the original structure of theinjured muscle to a default quick-fix in which theinjured muscle is rapidly patched with a fibroticframework.73 This is due to fibrogenic differentia-tion of muscle stem cells, perhaps mediated byincreased matrix tension and/or cell shape.98,122

This myogenic-to-fibrogenic conversion of muscleprecursor cells has been recently shown to beunder control of the canonical Wingless int (Wnt)signaling pathway.97 With increasing age, there isincreased Wnt signaling activation, which impairsthe myogenic regeneration of muscle stem cells.This raises the question as to whether decreasedinhibition of the Wnt signaling pathway may simi-larly play a role in the increased fibrosis formationthat characterizes dystrophic muscle. A delayedinflammatory response may be responsible for thedelayed reparative regeneration in aged muscle,123

and future studies should aim to address this indystrophic muscle. Testing whether differentialshifts in the macrophage phenotype in healthy vs.DMD samples occurs124 and whether exercise playsa regulatory role is another promising avenue forresearch. The TGF-b pathway has also been impli-cated.92,125 The fibrotic default program and itsunderlying mechanisms should be the topic offuture studies. In-depth reviews of mechanisms ofmesenchymal stem cell therapy126 and the role forstem cells in DMD therapy have beenpublished.127,128

Benefits of Muscle Loading (Exercise) on MyofiberRegeneration. Methods such as gene therapy129,130

and direct growth factor injection131 have beeninvestigated in the laboratory to enhance the re-generative capacity of dystrophic muscle. However,drawbacks, such as cost, feasibility, and safetyissues, may limit their clinical application. A morepractical approach to manipulation of the micro-environment with mechanical loading, by exerciseor electrical stimulation, for example, provides a




non-invasive method that could be widely used topromote the secretion of myogenically favorablegrowth factors and enhance skeletal muscle regen-erative capacity. In both animals and humans, ithas been shown that mechanical overloading isbeneficial for improving muscle quality in healthyskeletal muscle. This occurs via activation and pro-liferation of satellite cells, skeletal muscle angio-genesis, and release of growth factors such as IGF-1 and VEGF.132,133 In vitro, just 24 hours of cyclicalmechanical stimulation induces a significantincrease in muscle precursor cell (MPC) prolifera-tion134 and increased VEGF secretion by MPCs.135

Not only has mechanical loading been linked tostimulation of critical factors that control myofiberregeneration, but it has also been associated withinhibition of myostatin, a negative regulator ofskeletal muscle growth.136–138 We have previouslydemonstrated that addition of a muscle loading

protocol enhances myofiber regeneration by trans-planted MPCs139 and decreases the formation of fi-brosis after a severe contusion injury.134 This sug-gests that mechanotransductive pathways may playa role in determination of the fate of muscle pre-cursor cells responsible for skeletal muscle regen-eration. Accordingly, administration of a runningprotocol in mice is associated with decreasedgenetic expression of Wnt signaling pathways.140

Using recent studies of exercise-induced satellitecell proliferation and activity in aging skeletal mus-cle141 as a model, future studies should investigatefurther how mechanical stimulation may enhancemyofiber repair and decrease fibrosis in dystrophicmuscle models.

INCREASED OXIDATIVE STRESS

Oxidative stress is the accumulation of destructivereactive oxygen and nitrogen species (RONS) suchas superoxide, the hydroxyl radical, and peroxyni-trite (i.e., O2��, OH��, and ONOO��, respectively;Fig. F11A). When oxidative stress is severe and/orprolonged, antioxidants, the body’s naturaldefense system, can be overwhelmed, which canlead to subsequent oxidative damage of lipids, pro-teins, and DNA.

Lipids that comprise the sarcolemma, such aspolyunsaturated fatty acids, are preferentiallyattacked by RONS142 in a process commonlyreferred to as lipid peroxidation. An oxidized poly-unsaturated fatty acid (i.e., peroxydienyl) has thepotential to act similarly to RONS on nearby lipids,proteins (protein carbonylation), and DNA (Fig.1B). This chain reaction, if uninterrupted by anantioxidant, can continue and lead to the destruc-tion of key cellular components (e.g., contractileproteins actin and myosin). Although RONS areelectromolecularly unstable compounds (i.e., elec-tron-seeking), antioxidants (i.e., compounds capa-ble of sustaining an oxidized state long enough tobe destroyed or dimerize) are capable of enervat-ing the oxidative pathway and include the endoge-nous proteins superoxide dismutase, glutathioneperoxidase, and catalase, and exogenous com-pounds such as vitamins C and E.

Physiological processes that contribute to theproduction of RONS include muscle contrac-tion,143 inflammation,144,145 and mitochondrialCa2þ overload. These processes are also susceptibleto modification with DMD. For example, mito-chondria are major producers of RONS,146,147

which are still effectively dealt with by antioxidantsin normal skeletal muscle. However, in DMD, in-tracellular Ca2þ homeostasis is perturbed and canremain chronically high (see Aberrant Cell Signal-ing section). Because mitochondria are sensitive toCa2þ signaling, chronic high intracellular Ca2þ

FIGURE 1. (A) Production of RONS in muscle cell mitochondria

and cytosol. SODmit and SODcyt, mitochondrial and cytosolic

superoxide dismutase; Cat, catalase; Gpx, glutathione peroxi-

dase; O2�, superoxide; NO, nitric oxide; H2O2, hydrogen perox-

ide; �OH, hydroxyl radical; H2O, water; ONOO, peroxynitrite;

(adapted from ref. 181). (B) A polyunsaturated fatty acid

(PUFA) is readily oxidized by RONS such as superoxide anion

to form a peroxydienyl radical (�). Peroxydienyl radicals can oxi-

dize nearby DNA, PUFAs, cysteine residues on transmembrane

proteins, and glycogens found on glycoproteins. These proc-

esses, left unchecked, can lead to destruction of biomolecules.




levels may increase the production of RONS64,148

sufficiently to overwhelm endogenous antioxidantenzyme defenses. This overt oxidative stress leadsto protein degradation and altered contractile pro-tein function.149

The contribution of oxidative stress to thepathophysiology and onset of DMD is largely unex-plored.150 The parallel presence of oxidative stressbyproducts and elevated antioxidant levels hasbeen observed in many models of DMD, thus sup-porting the axiom that oxidative stress contributesto DMD pathology (Table T22). Oxidative stress wasinvestigated in the pre-necrotic state, that is, thetime preceding the first wave of fiber degenera-tion/regeneration, in mdx mice (age 10–21days).151 Despite no signs of muscle pathology,investigators found increases in lipid peroxidationalong with increases in the endogenous antioxi-dant enzymes. Similar findings (i.e., increased lipidperoxidation) have been recapitulated in studiesof patients with DMD,152–159 suggesting that oxida-tive stress and damage contribute to the dystrophicprocess (Table 2).

Therapies for DMD ideally would prevent mus-cle wasting and oxidative stress and improve over-all muscle function. Exercise may appear to becontraindicated for treatment of oxidative stressbecause it increases oxygen consumption and canbe followed by a short inflammatory response,both of which can lead to increased RONS produc-tion. However, a key benefit of exercise arises fromthe overshoot in RNA transcription and subse-quent mRNA translation of antioxidant enzymesthat can protect against oxidative damage even af-ter the exercise has ceased.160,161

The protective effects of exercise against oxida-tive stress have been best documented in healthymuscle. Exhaustive and even moderate exercise ac-tivity in an untrained individual can increase mito-chondrial lipid peroxidation and protein carbony-lation, but these negative effects are bluntedsubsequent to appropriate exercise training.162,163

Indeed, protection from oxidative stress–induceddamage after exercise has been observed afterboth short- and long-term endurance exercisetraining regimens,164,165 as indicated by decreasedlipid peroxidation and increased antioxidant activ-ities that can remain elevated up to 72 hours post-exercise.161 The sustained activity of antioxidantsbeyond the initial oxidative stress induced by theexercise activity may represent the greatest poten-tial for the longer lasting benefits of exercise train-ing. Hence, one of the benefits of regular exerciseis to counter oxidative stresses in the rest periodsbetween exercise activities.

Unfortunately, evidence for similar responsesto exercise in DMD patients is limited. No studies

Tab

le2.

Musc

ula

rd

ystr

op

hy

and

oxi

dativ

est

ress

.

Mod

el

Mark

er

of

oxi

dativ

est

ress

Antio

xid

ants

measu

red

Conse

nsu

sR

efe

rences:

first

auth

or

(year)

Dys

trop

hic

myo

bla

sts

Cell

death

,p

rote

incarb

onyl

atio

n:

cell

death

;:

pro

tein

carb

onyl

atio

nR

and

o(1

998)1

88;

Dis

atn

ik(2

000)1

89

Dys

trop

hic

chic

kens

Lip

idp

ero

xid

atio

n,

pro

tein

carb

onyl

atio

nG

PX

,G

R:

lipid

pero

xid

atio

n;

:p

rote

incarb

onyl

atio

n;

:G

PX

and

GR

Om

aye

(1974)1

90;

Miz

uno

(1984)1

91;

Ohta

(1988)1

92;

Murp

hy

(1989)1

93

Dys

trop

hic

mic

eLip

idp

ero

xid

atio

nS

OD

,G

PX

,G

R,

CA

T:

lipid

pero

xid

atio

n;

:p

rote

incarb

onyl

atio

n;

:S

OD

,C

AT,

GR

,G

PX

Om

aye

(1974)1

90;

Ragusa

(1997)1

94;

Dis

atn

ik(1

998)1

51;

Dud

ley

2006)1

95;

Sp

ass

ov

(2010)1

96

DM

Dp

atie

nt

Lip

idp

ero

xid

atio

n,

chem

ilum

inesc

ence,

*pro

tein

carb

onyl

atio

n

SO

D,

GP

X,

GR

:lip

idp

ero

xid

atio

n;

:chem

ilum

inesc

ence;

:p

rote

incarb

onyl

atio

n;

:G

PX

,G

R

Kar

(1979)1

57;

Matk

ovi

cs

(1982)1

58;

Mechle

r(1

984)1

59;

Jacks

on

(1984)1

56;

Grin

io(1

984)1

54;

Hunte

r(1

986)1

97;

Burr

(1987)1

98;

Aust

in(1

992)1

99;

Reye

s(1

994)2

00;

Hayc

ock

(1996)2

01;

Rod

riguez

(2003)2

02;

Naka

e(2

004)2

03;

Ab

del2007

152;

Gro

sso

(2008)1

55;

Chahb

ouni(2

010)1

53

SO

D,

sup

ero

xid

ed

ism

uta

se;

CA

T,

cata

lase

;G

PX

,glu

tath

ione

pero

xid

ase

;G

R,

glu

tath

ione

red

ucta

se.

*Chem

ilum

inescence

of

urinary

mark

ers

of

oxi

dativ

ed

am

age.2

00




have directly assessed the oxidative stress and anti-oxidant response to exercise in DMD patients,although there have been a few studies using mdxmice. Voluntary wheel running has been shown toelicit beneficial adaptations in dystrophic muscle,such as increased resistance to fatigue, a shift to-ward smaller type IIa fibers, and increased musclestrength,31–33,209 but its is still unclear whetherexercise training can elicit adaptations to improveoxidative stress and antioxidant capacity. Wereported a 22% increase in total antioxidantcapacity in male mdx mice after 3 weeks of volun-tary wheel running.166 Similarly, a 38% decrease inlipid peroxidation and a 44% decrease in proteincarbonylation in white gastrocnemius muscle frommdx mice were reported after an 8-week, low-inten-sity treadmill training regimen. Contradicting thesefindings, a 6-week treadmill training regimenincreased lipid peroxidation in mdx mice, and a4–8-week treadmill training regimen yielded no sig-nificant changes in antioxidant activity.167,168

Indeed, dihydroethidium (DHE) staining of sec-tions of tibialis anterior muscle, used as a markerof exercise-induced oxidative stress, increased.167

These differences could reflect voluntary runningon a wheel vs. enforced running on a treadmill,suggesting this possibility be tested. Studies thatinvestigated exercise-induced attenuation of oxida-tive stress in mdx mice are highlighted in Table T33.

Potential exercise-based therapies for DMDpatients must be rigorously tested and havedefined parameters to assure maximal benefits andavoid exacerbation of the disease.2 Beneficial out-comes of exercise training on oxidative stress inhealthy individuals are easily achievable becauseexercise prescription and training responses arewell defined. Establishing an exercise prescriptionto limit oxidative stress for patients with DMD willrequire well-designed studies that address the fol-lowing criteria:

1. Establish the degree to which dystrophic muscleadapts to exercise.

2. Demonstrate that antioxidant signaling pathwaysare/are not perturbed in dystrophic muscle.

3. Confirm antioxidant activity levels remain ele-vated after physical activity in patients withDMD.

RECURRENT MUSCLE ISCHEMIA

Throughout this review we have discussed the evi-dence that the five pathophysiological mechanismsoutlined by Petrof are not mutually exclusive. Stud-ies in 1998 and 2000169,170 were among the first toprovide mechanistic evidence for recurrent muscleischemia in dystrophic muscle. These studies

Tab

le3.

Musc

ula

rd

ystr

op

hy,

oxi

dativ

est

ress

,and

exe

rcis

e.

Exe

rcis

ep

roto

col

Mark

er

of

oxi

dativ

est

ress

Antio

xid

ants

measu

red

Exe

rcis

ep

roto

col–

ind

uced

outc

om

es

Refe

rence

30-m

inute

horiz

onta

ltr

ead

mill

runnin

g(1

2m

/min

)tw

ice

weekl

y4–8

weeks

tota

l,m

ale

s,age

4–5

weeks

RO

Sle

vels

inp

lasm

a,

skele

talm

usc

lesu

pero

xid

ep

rod

uctio

n

Blo

od

AO

Xactiv

ityR

unnin

gin

cre

ase

dR

OS

leve

lsin

pla

sma

and

skele

talm

usc

lep

rod

uctio

nof

sup

ero

xid

ein

md

x

mic

ecom

pare

dw

ithse

denta

rycontr

ols

;no

effect

on

blo

od

AO

Xactiv

ity

Burd

iet

al.

(2009)1

67

Wheelru

nnin

g(v

olu

nta

ry),

3w

eeks

tota

l,m

ale

s,age

3w

eeks

Card

iac

and

skele

talm

usc

lelip

idp

ero

xid

atio

nB

lood

AO

Xcap

acity

Runnin

gin

cre

ase

db

lood

AO

Xcap

acity

inm

dx

mic

ecom

pare

dto

sed

enta

rycontr

ols

;no

effect

on

card

iac

or

skele

talm

usc

lelip

idp

ero

xid

atio

n

Call

et

al.

(2008)1

66

30-m

inute

horiz

onta

ltr

ead

mill

runnin

g(9

m/m

in)

twic

ew

eekl

y,8

weeks

tota

l,age

4w

eeks

Ske

leta

lm

usc

lelip

idp

ero

xid

atio

n,

skele

tal

musc

lep

rote

incarb

onyl

conte

nt

Ske

leta

lm

usc

leS

OD

,C

AT,

and

GP

Xactiv

ityR

unnin

gd

ecre

ase

dsk

ele

talm

usc

lelip

idp

ero

xid

atio

nand

pro

tein

carb

onyl

conte

nt

inm

dx

mic

ecom

pare

dw

ithse

denta

rycontr

ols

;no

effect

on

skele

talm

usc

leA

OX

activ

ities

Kaczo

ret

al.

(2007)2

04

30-m

inute

horiz

onta

ltr

ead

mill

runnin

g(8

.3m

/min

)tw

ice

daily

6w

eeks

tota

l,m

ale

s,age

4and

16

weeks

Mito

chond

riallip

idp

ero

xid

atio

n,

skele

talm

usc

lelip

ofu

scin

accum

ula

tion

Ske

leta

lm

usc

leS

OD

and

GP

Xactiv

ity,

skele

tal

musc

levi

tam

inE

conte

nt

Runnin

gin

cre

ase

dlip

idp

ero

xid

atio

nand

lipofu

scin

accum

ula

tion,

and

decre

ase

dG

PX

inm

dx

mic

eaged

4w

eeks

com

pare

dw

ithse

denta

rycontr

ols

;no

effects

of

runnin

gon

mark

ers

of

oxi

dativ

est

ress

or

antio

xid

ant

activ

ities

were

ob

serv

ed

inm

dx

mic

eaged

16

weeks

com

pare

dw

ithse

denta

rycontr

ols

Fais

tet

al.

(2001)1

68

Stu

die

ste

stin

gexe

rcis

eon

oxi

dativ

est

ress

inm

dx

mic

eth

at

rep

ort

ed

mark

ers

of

oxi

dativ

est

ress

were

consi

dere

d.

RO

S,

reactiv

eoxy

gen

specie

s;A

OX

,antio

xid

ant;

SO

D,

sup

ero

xid

ed

ism

uta

se;

GP

X,

glu

tath

ione

pero

xi-

dase

;C

AT,

cata

lase

.




Tab

le4.

Ove

rvie

wof

meth

od

s,outc

om

em

easu

res,

and

the

pote

ntia

leffect

of

ap

pro

pria

teexe

rcis

eon

the

five

mechanis

ms

of

Duchenne

musc

ula

rd

ystr

op

hy

path

op

hys

iolo

gy.

Path

op

hys

iolo

gy

Meth

od

suse

dto

eva

luate

Outc

om

em

easu

res

Pote

ntia

leffect

of

ap

pro

pria

teexe

rcis

eR

efe

rences

Mechanic

alw

eake

nin

gof

the

sarc

ole

mm

aE

vans

blu

ed

yein

jectio

n,

Pro

cio

nora

nge

inje

ctio

n,

CK

ass

ay,

curr

ent

cla

mp

,vo

ltage

cla

mp

,his

tolo

gy,

IHC

,fo

rce

pro

ductio

nm

easu

res,

intr

acellu

lar

Ca2þ

ind

icato

r

Perc

enta

ge

of

degenera

ting

fibers

,m

em

bra

ne

dam

age,

mem

bra

ne

ele

ctr

icalp

rop

ert

ies,

fiber

size

,d

egen-r

egen

gro

up

s,am

ount

of

connectiv

etis

sue,

musc

lefo

rce,

intr

acellu

lar

Ca

2þ

Mem

bra

ne

pro

tectiv

eeffect

5–7

,62,

72,

77

Inap

pro

pria

teC

a2þ

influ

xC

urr

ent

cla

mp

,vo

ltage

cla

mp

,C

a2þ

-dep

end

ent

pro

teoly

sis

ass

ay

and

calp

ain

inhib

ition

ass

ay,

imm

unob

lot,

zym

ogra

m,

his

tolo

gy,

IHC

Mem

bra

ne

ele

ctr

icalp

rop

ert

ies,

free

Ca

2þ

concentr

atio

n,

am

ount

of

calp

ain

sand

calp

ast

atin

,si

zeand

num

ber

of

lesi

ons,

am

ount

of

regenera

tion

Imp

rove

dC

a2þ

hand

ling

28,

60–6

2,

67,

68,

70,

71,7

4,

78,

79,

205,

206

Ab

err

ant

cell

signalin

gS

tain

ing

cells

for

pre

sence

of

gro

wth

facto

rs,

measu

rem

ent

of

resp

onse

togro

wth

facto

rs,

lab

elin

g/

mark

ing

cells

and

their

nic

hes,

cyt

oki

ne

exp

ress

ion,

changes

inp

rop

ort

ions

of

infil

tratin

gm

acro

phage

pop

ula

tions,

Mt

DA

MP

leve

ls

Am

ount

of

gro

wth

facto

rs,

cyt

oki

nes,

macro

phages,

and

infla

mm

atio

nA

ctiv

atio

nof

com

pensa

tory

or

anta

gonis

ticsi

gnalin

gp

ath

ways

73,

83,

97,

104,

105,

106,

107,

109,

125,

138,

175

Incre

ase

doxi

dativ

est

ress

Zym

ogra

phy,

ferr

icya

nid

est

ain

ing,

imm

unoflu

ore

scence

stain

ing

Mt

cata

lase

exp

ress

ion,

glu

tath

ione

pero

xid

ase

,m

anganese

SO

Dexp

ress

ion/

activ

ity,

intr

acellu

lar

free

rad

icalaccum

ula

tion

Incre

ase

dM

tantio

xid

ant

cap

acity

67,

173

Recurr

ent

musc

leis

chem

iaFore

arm

blo

od

flow

,near

infr

are

dsp

ectr

osc

op

y,hin

dlim

bp

erf

usi

on,

lase

rD

op

ple

rp

erf

usi

on

imagin

gM

usc

leoxy

genatio

n,

exp

ress

ion

of

NO

Sis

ofo

rms

Angio

genesi

s,im

pro

ved

ab

ility

tob

lunt

alp

ha-

ad

renerg

icva

soconst

rictio

n,

incre

ase

diN

OS

activ

ity

169,

170,

172,

179,

207,

208

Ca

2þ

,calc

ium

;C

K,

cre

atin

eki

nase

;D

AM

P,

dam

age-a

ssocia

ted

mole

cula

rp

att

ern

;d

egen-r

egen,

degenera

tion–re

genera

tion;

IHC

,im

munohis

tochem

istr

y;iN

OS

,in

ducib

lenitr

icoxi

de

synth

ase

;M

t,m

itochond

rial;

SO

D,

sup

ero

xid

ed

ism

uta

se.




showed that myofibers without pathology are sup-ported by increased blood flow during exercise de-spite exercise-dependent adrenergic signals for va-soconstriction. They do this by increased neuralnitric oxide synthase (nNOS) expression and sub-sequent NO release. However, DMD muscle doesnot display increased nNOS expression. Thus, thereflex sympathetic vasoconstriction that accompa-nies contraction of dystrophic muscles is unop-posed by NO-mediated vasodilation, and functionalmuscle ischemia follows. Of note, these studies inDMD patients were performed using near-infrared(NIR) spectroscopy of the flexor digitorum profun-dus, a muscle used preferentially in handgrip exer-cise and in pushing a wheelchair. Since these stud-ies, it has become increasingly clear that deficientoxygenation of DMD tissue is only one of manypathophysiological consequences of impaired vas-cular control. Due to either absence or mislocaliza-tion of skeletal muscle nNOS, fatigue experiencedby DMD patients after mild exercise is explainedby the failure of normal contraction-inducedcGMP-dependent attenuation of local vasoconstric-tion, and this failure causes vascular narrowing inmuscles after exercise.171 Improved anchoring ofnNOS to the sarcolemma and subsequentimproved muscle perfusion during exercise to-gether provide further evidence of a role fornNOS in the prevention of recurrent muscle ische-mia.172 Importantly, utrophin appears to be unableto recruit nNOS to the sarcomlema.173

NO pathway involvement also affects gastricsmooth muscle174 and thus may partially explaingastrointestinal (GI) issues in DMD patients, whohave decreased serum concentrations of NO.175

Mechanical loading regulates NOS expression, butfurther study in dystrophic muscle is needed.176

These proposed focused studies may find targetson the NO pathway, such as phosphodiesteraseinhibitors,167,171 to ameliorate both GI and skeletalmuscle pathology, and may contribute to an inter-disciplinary management plan.54 Clearly, polyther-apy is indicated because, in the absence of anappropriate contraction-induced vasodilation, ex-cessive exercise may exacerbate the ischemia.

Capillary–myofiber contacts are increased inexercised muscles in healthy individuals, particu-larly the young.177 Because an improved pheno-type of mdx mice is observed by developmentallyincreasing the vasculature,178 future studies ofphysical activity in DMD patients might help todetermine whether the response of exercisedmuscles in dystrophic individuals is similar. Ofnote, the microvasculature is dynamic, andchanges might be expected in DMD boys as theyage, and as recruitment of fibers from various mus-cle groups changes. The interaction of these

changes with the muscle progenitor cell nichemight be studied in arm muscles of DMD boysbefore and after use of a wheelchair.

Observations of skeletal muscle ischemia inDMD are difficult to reconcile with observations ofenhanced arteriogenesis and wound repair in mdxmice.179 This difficulty may be due to general spe-cies differences, or there may be differences in thespecific dystrophic pathology (such as sarcolemmalnNOS anchoring172,173) between the species. Or,myofibroblast precursor/progenitor cells mayreceive signals inductive of differentiation intoblood vessels and epithelial tissues. Differentiationinto these types of tissues may occur preferentially,because these tissues require little if any dystro-phin expression.

CONCLUSIONS

A sense of urgency permeates research into thepathophysiological mechanisms underlying DMD.Improved understanding of the pathophysiology iscritical, and the incorporation of exercise into ex-perimental designs could help to mechanisticallydefine the pathophysiology. At present, informedexercise prescription for DMD patients is challeng-ing due to lack of inquiry and lack of evidence.The cumulative effect of further inquiry—informed exercise prescription—might improvethe quality of life of DMD patients by improvingtheir mobility. Table T44 summarizes methods andoutcome measures that might be used in studyingthe role of exercise with respect to its effects onthe five underlying mechanisms of DMD. In thevast majority of preclinical studies in mdx mice,exercise has been used as a means to increase theseverity of the damage, to better understand thepathological cascade, or to better evaluate theeffects of various therapeutics. For this reason andothers discussed in this review, we consider theability of exercise to improve quality of life forDMD patients to be an issue that requires furtherstudy. However, recently published standard oper-ating procedures25 and clinical trial protocols180

are clear steps in the right direction. Defining cri-teria for well-designed, controlled studies of exer-cise training in DMD may lead not only toinformed exercise prescription, but also toenhanced understanding of the pathophysiology ofthe disease.

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