Nitric Oxide 24 (2011) 43–49

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Nitric Oxide

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Nitric oxide regulates the repair of injured skeletal muscle

Lidiane Isabel Filippin a, María José Cuevas b, Elena Lima b, Norma Possa Marroni c, Javier Gonzalez-Gallego b,Ricardo Machado Xavier d,⇑a Serviço de Reumatologia do Hospital de Clínicas de Porto Alegre (HCPA) – e Universidade Federal do Rio Grande do Sul (UFRGS), Brazilb Institute of Biomedicine (IBIOMED), University of Leon, Leon, Spainc Experimental Hepatology and Physiology Laboratory of HCPA, Brazild Serviço de Reumatologia HCPA/UFRGS, Coordinator of the Laboratory of Molecular Biology of Autoimmune and Infectious Disease, HCPA, Brazil

a r t i c l e i n f o

Article history:Received 11 February 2010Revised 1 November 2010Available online 19 November 2010

Keywords:Satellite cellMuscle repairL-NAME

1089-8603/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.niox.2010.11.003

⇑ Corresponding author. Address: Serviço de ReClínicas de Porto Alegre, Rua Ramiro Barcelos 2350-Porto Alegre, RS, Brazil.

E-mail address: rmaxavier@hcpa.ufrgs.br (R.M. Xa

a b s t r a c t

Skeletal muscle repair can be understood as a balance between fibrosis and regeneration, the result ofwhich may lead to complete recovery or loss of muscle function. To study the involvement of nitric oxidein post-trauma muscle repair, we used an experimental murine model of crush injury muscle. Theanimals were divided into four groups, (i) control (CO), (ii) sham trauma, (iii) trauma and (iv) trau-ma + L-NAME. The animals received a single dose of 100 mg/kg of the L-NAME, an inhibitor of nitric oxidesynthase, 2 h after lesion, and the muscle tissue was analyzed in two time-points: 24 h and 7 days.Twenty-four hours after injury, the crushed muscle was characterized by an intense inflammatory cellinfiltrate and edema demonstrated by histological analysis. These changes were accompanied byincreased iNOS, MMP-2 and HGF mRNA transcription and protein expression of the iNOS and MMP-2in the gastrocnemius muscle. Crushing injury also promoted cell proliferation and increase number satel-lite cell, responsible for the regeneration of the muscle fiber. Treatment with L-NAME blocking local NOproduction, greatly attenuated these histological and molecular findings at 24 h. On the 7th day themolecular findings of both groups were comparable to the control (sham trauma) group. However, theL-NAME group showed increase deposition of collagen and decrease of SC expression. These findingsdemonstrate that activation of NO during muscle crush is critical in the early phases of the skeletal mus-cle repair process and indicate its possible role as a regulator of the balance between fibrosis and muscleregeneration.

� 2010 Elsevier Inc. All rights reserved.

Introduction fibrotic scar tissue formation, resulting in better muscle functional

Skeletal muscle satellite cells (SC) are resident myogenic stemcells normally found in a quiescent state in adult skeletal muscles.These cells are mononuclear progenitor cells found in mature mus-cle between the basal lamina and sarcolemma [1–3]. On activation,they enter the cell cycle, divide, differentiate and fuse with musclefibers to repair damaged regions after injuries [4]. The controlmechanisms of this sequential process are not well defined,although it has been reported that mechanical changes in musclecan initiate events that lead to SC activation, such as a disruptionof the integrity of the sarcolemma and basal lamina [5]. Severalcytokines and inflammatory mediators, such as nitric oxide (NO)released by local or infiltrating cells may also play a role [6]. Prob-ably there is a complex combination of these multiple events. Itwould be clinically important to be able to manipulate theseprocesses in order to improve muscle regeneration and minimize

ll rights reserved.

umatologia do Hospital de6� andar/sala645-90035-003

vier).

recovery after injury.During a crushing injury of the muscle fibers, macrophages

invade the area of injury, phagocytose the necrotic tissue andproduce some growth factors that are mitogenic for muscle precur-sor cells, such as fibroblast growth factor (FGF), transforminggrowth factor (TGF-b, interleukin-6), and hepatocyte growth factor(HGF) [2,7,8]. HGF has been recently well studied as an activator ofSC. It is a a–b heterodimer produced by proteolytic cleavage of asingle-chain inactive precursor of 728 amino acids, localized inthe extracellular domain of uninjured skeletal muscle fibers, at-tached to the extracellular matrix [9–11].

The role of NO in the release of HGF from the extracellular ma-trix has been investigated. Tatsumi et al. [12] studied the effects ofadministration of L-NAME, an inhibitor of NOS function, orD-NAME, an inactive stereoisomer, before a stretching stimulus tothe muscle. In vivo activation of SC and HGF release in stretchedmuscle was inhibited by L-NAME, but not by D-NAME, indicatingthat these processes are dependent of local NO production.

The NO-dependent HGF-matrix release could be mediated bymatrix metalloproteinases (MMP), a large family of zinc-dependent endopeptidases that are capable of degrading one or

44 L.I. Filippin et al. / Nitric Oxide 24 (2011) 43–49

several extracellular matrix proteins [13]. Numerous MMPs,including MMP-2, -3, -7, and -9, are found in skeletal muscle[4,14], possibly playing fundamental roles in muscle fiber growthand repair by regulating the integrity and composition of extracel-lular matrix [15]. Yamada et al. [4] demonstrated that MMP-2mediates stretch-induced activation of skeletal muscle SC in aNO-dependent manner. Results from this study also provide evi-dence that NO-activated MMP2 may cause release of HGF fromthe extracellular matrix and contribute to SC activation in vitro.However, in vitro model using single cell cultures may only permitthe study of the second stage of satellite cell activation and thereare no studies in vivo confirming these observations.

This study explored the role of NO during repair process in anexperimental murine model of skeletal muscle injury using anNO synthase inhibitor (nitro-L-arginine methyl ester: L-NAME),with attention to MMP, HGF, SC and total cell proliferation.

Materials and methods

Animals and experimental groups

Male Wistar rats weighing 250–300 g were used. The animalswere caged at 22 �C, with 12-h light–dark cycles and free accessto food and water until the experiments were performed. Allexperiments were performed according to the Guiding Principlesfor Research Involving Animals (NAS) and the Committee of Re-search and Ethics in Health of the Research and PostgraduateGroup of the Hospital de Clínicas de Porto Alegre.

Experimental animals were randomly divided into four groupsof twenty animals each: (i) C (control), (ii) ST (sham trauma +L-NAME), (iii) T (trauma) and (iv) L-NAME (trauma + L-NAME). Aright gastrocnemius injury was induced by a single impact blunttrauma with a press developed by the Centro Industrial de Equipa-mentos de Ensino e Pesquisa Ltda (CIDEP/RS, Brazil), according tothe procedure of Lech [16]. Briefly, injury was produced by a metalmass (0.459 kg) falling through a metal guide from a height of18 cm on the middle third of the gastrocnemius muscle belly.The impact kinetic energy delivered was 0.811 J [17].

Experimental procedures

During the procedure, rats were anesthetized with a cocktail ofketamine chlorhydrate (Ketalar, Parke Davis, 100 mg/kg) and 2%xylazine (Rompun, Bayer, 50 mg/kg), administered i.p. Sham trau-ma rats were also anesthetized to ensure standardization but didnot receive muscle trauma. Animals of the ST and L-NAME groupsreceived a 100 mg/kg dose of L-NAME i.p. 2 h post-trauma. Ratswere killed 24 h (10 animals each group) or 7 days (10 animalseach group) later for molecular and biochemical evaluation andmuscle histological analysis. The animals were anesthetized witha cocktail of ketamine chlorhydrate and 2% xylazine i.p. The gas-trocnemius muscle was rapidly removed from both legs, snap-frozen in liquid nitrogen and stored at �80 �C until analysis. Theentire surgical procedure took less than 10 min. The gastrocnemiusleft paw was used as an internal control.

Histology

For histological examination, a section of muscle from each ani-mal was trimmed and fixed by immersion in 10% buffered formalinfor 24 h. The blocks were dehydrated in a graded series of ethanoland embedded in paraffin wax. Serial 4-mm sections were stainedwith hematoxylin and eosin or picrosirius. Five sections from eachsample were analyzed by two independent pathologists with noprior knowledge of the animal groups.

Morphometric image analysis

The collagen quantification was realized by morphometric im-age analysis through the specific software. Briefly, from each sam-ple, 10 images of the lesion area were captured from randomlyselected high-power fields (magnification, 100�) measured bythe Adobe Photoshop CS3 Extended 10.0 (Adobe Systems Inc, SanJose, CA) computer program and averaged to obtain the sample fi-nal value. The percentage of fibrosis in each image was then calcu-lated using the ratio of the fibrosis area to the total amount ofpixels per image [18].

Real-time RT-PCR

Real-time PCR products were amplified on an Applied Biosys-tems (Applied Biosystems, Weiterstadt, Germany) on an ABI PRISM7000 sequence detection system using TaqMan-Gene ExpressionAssays for HGF (GenBank accession No. D90102.1 andRn00566673_m1), iNOS (GenBank accession No. D12520.1 andRn00561646_m1), MMP-2 (GenBank accession No. U65656.1 andRn01538170_m1), and the housekeeping gene hypoxanthine phos-phoribosyl-transferase (HPRT) (GenBank accession No. M63983.1and Rn01527840_m1) were used. Relative changes in gene expres-sion levels were determined using the 2�DDCT method, asdescribed previously [19]. The cycle number at which the tran-scripts were detectable (CT) was normalized to the cycle numberof HPRT detection, referred to as DCT.

Western blot analysis

For Western blot analysis of satellite cell (monoclonal anti-PAX7; SIGMA–ALDRICH, St. Louis, MO), muscle homogenates wereprepared, after which protein concentration was measured usingthe Bradford assay [19].

Immunohistochemical analysis

The slides were drained and incubated with anti-iNOS (SIGMA–ALDRICH, St. Louis, MO), anti-MMP-2 (SIGMA–ALDRICH, St. Louis,MO – 1:250), and anti-PCNA (Zymed, San Francisco, California,USA – 1:1000), for 1 h at room temperature. Known positive andnegative tissue biopsies were used as observation controls.

Statistical analysis

Results are expressed as mean values with 95% confidenceintervals (95%IC) for symmetric variables and as median and per-centiles (25% and 75%) for asymmetric variables. The data werecompared by analysis of variance (ANOVA); when the analysisindicated the presence of a significant difference, the means werecompared using the Tukey test. Significance was accepted atp < 0.05. Values were analyzed using the statistical package SPSS13.0 (SPSS, Inc., Chicago, IL).

Results

Histological findings and morphometric image analysis

The sham group (untraumatized + L-NAME) animals showednormal tissue architecture (Fig. 1A). At 24 h after trauma, therewas a loss of normal architecture, with clear inflammatory reactionand edema (Fig. 1B). A single administration of the nitric oxide syn-thase inhibitor L-NAME (100 mg/kg i.p. 2 h after the trauma) mark-edly attenuated all the inflammatory histological abnormalities at24 h (Fig. 1C). On the 7th day post-trauma there was distinct

Fig. 1. Histological analysis in muscle section in coloration of the hematoxylin–eosin and sirius red staining. (A) The sham trauma group (untraumatized + L-NAME) animalsshowed normal architecture (HE – 100�) (B) The trauma group (HE – 100�) showed modifications of normal architecture, with edema and inflammatory reaction withimportant infiltrate of neutrophils. (C) (HE – 100�) the treatment with L-NAME decreased the inflammatory infiltrate. (D) The sham trauma group trauma group (sirius red –100�) in 7 days demonstrated normal architecture. (E) 7 days after muscle injury with fewer fibrosis confirmed in (H) in siruis red staining under polarized light (100�). (F)The L-NAME group (HE – 100�) was associated with more intense and focal formation of fibrotic tissue. (I) Important increase of the deposition of collagen (sirius red – 100�)under polarized light.

L.I. Filippin et al. / Nitric Oxide 24 (2011) 43–49 45

diffuse and poorly organized fibrosis and increased collagen con-centration in the trauma group (Fig. 1E). The administration ofL-NAME was associated with more intense and focal formation offibrotic tissue (Fig. 1F). The collagen is refractile under polarizedlight. Sirius red sections examined under polarized light demon-strated higher collagen concentration in L-NAME group (Fig. 1H)when compared with the trauma group (Fig. 1). The collagen quan-tification by morphometric image analysis demonstrated increasedcollagen concentration in both trauma groups, as compared to thecontrol group, however, it was significantly higher in L-NAMEgroup (801.3 ± 219 pixels) than in the trauma group (456.7 ± 88pixels) (p < 0.05) with increased about 75% (Fig. 2).

Fig. 2. Collagen quantification by morphometric image analysis. The collagenquantification demonstrated increased collagen concentration in trauma group ascompared to the control group; however, it was significantly higher in L-NAMEgroup.

iNOS transcription and expression

Since L-NAME exposure appears to decrease the intensity ofacute inflammation, we evaluated its impact on iNOS tissue tran-scription and expression. The trauma group demonstrated an in-crease in the iNOS transcription (Table 1) and expression at 24 hin the muscle fiber (Fig. 3B). On the order hand, L-NAME inhibitedthe increase in the iNOS mRNA transcription by about 34% (Table 1)and expression at 24 h (Fig. 3C). However, at 7 days both the trau-ma and L-NAME treated groups did not present any detectableiNOS transcription or expression (Table 2).

Growth factors transcription and expression

We also studied the influence of L-NAME exposure in MMP-2transcription and expression. This protein is involved in the

Table 1Transcription mRNA of iNOS, MMP-2, and HGF markers in muscle injury rats in 24 h after lesion.

CO (n = 10) ST (n = 10) T (n = 10) L-NAME (n = 10)

iNOSa 100 (99.5–100.5) 174 (196–189) 1365.3 (1245.3–1485.3)* 366.5 (347.1–385.7)MMP-2b 100 (99.4–100.6) 99 (92–100) 202.8 (181.8–223.8)* 167.4 (166.3–168.6)HGFc 100 (98.6–101.4) 87 (75–100) 142.1 (124.3–159.8)* 69.04 (58.9–79.2)

Data were shown by mean ± 95%IC values.CO = control group (n = 10).ST = sham group (n = 10).T = trauma group (n = 10).L-NAME = L-NAME group (n = 10).

a Levels of iNOS mRNA normalized with HPRT as an endogenous reference.b Levels of MMP-2 mRNA normalized with HPRT as an endogenous reference.c Levels of HGF mRNA normalized with HPRT as an endogenous reference.* p < 0.05 higher versus control and L-NAME groups.

Fig. 3. Immunohistochemistry of iNOS at 24 h after trauma. In photomicrography(A) the sham trauma group (untraumatized + L-NAME) showed no expression(immunohistochemistry – 200�). (B) Trauma group (immunohistochemistry –200�) showed increase of iNOS expression in the cytoplasm of the muscle fiber. Inphotomicrography (C) (immunohistochemistry – 200�) the treatment with L-NAMEdecrease iNOS expression.

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breakdown of extracellular matrix in normal physiological remod-eling processes. There was no expression or transcription detectedin the sham trauma (untraumatized + L-NAME) animals (Table 1;Fig. 4A). The trauma group presented moderate expression(Fig. 4B) and an amplified transcription (Table 1) at 24 h. L-NAMEtreated group had decreased transcription (Table 1) and similarexpression levels as the trauma group, but with a different expres-sion pattern that was more restricted (Fig. 4C), with less positivecells as compared to untreated mice. After 7 days, the transcriptiondecreased significantly in both groups (Table 2), however, the cel-lular expression was persistent in the trauma group and absent inthe L-NAME-treated group (Fig. 4D and E).

We analyzed the influence of L-NAME exposure in HGF tran-scription. The trauma group presented an increase of the transcrip-

tion at 24 h. Nevertheless, the L-NAME group showed decreasedtranscription at 24 h (Table 1). There was no difference betweenthese groups after 7 days (Table 2).

Total cell proliferation

We investigated the influence of L-NAME exposure on the localcell proliferation of the injured muscle. The PCNA is a markerwidely used for cell proliferation, including satellite cell prolifera-tion. In our study, the sham trauma group (untraumatized + L-NAME) showed no expression of PCNA (Fig. 5A). The trauma groupdemonstrated an increase in proliferation activity at the injury siteat 24 h after trauma (Fig. 5B). The administration of L-NAME de-creased proliferative activity by about 34% at 24 h (Fig. 5C). After7 days, both groups demonstrated similar cellular proliferation atthe injury site, which was still increased in relation to the controlgroup (Fig. 5D and E).

Satellite cell expression

Finally, we studied the influence of L-NAME under the markermolecule specific for satellite cells, the PAX-7. It is a member ofthe paired box family of transcription factors, which is expressedin cells residing beneath the basal lamina of muscle fibers. Thetrauma group demonstrated a higher positivity at 24 h after trau-ma, while the administration of L-NAME significantly inhibited thisincrease in SC number. After 7 days, both groups demonstratedsimilar levels of the PAX-7 expression (Fig. 6).

Discussion

The importance of satellite cell activity in skeletal muscleregeneration has been considered for some time. It is known thatmuscle repair is initiated by mechanical or chemical stimuli, whichlead to the activation of quiescent satellite cells. On activation, theyenter the cell cycle, divide, differentiate and fuse with muscle fi-bers to repair damaged regions of muscle fibers [4]. In our study,the SC and total cell proliferation was analyzed by PAX-7 expres-sion and anti-PCNA immunohistochemistry, respectively. Com-pared to control, the trauma group exhibited increased SCnumber and total cell proliferation at 24 h post-trauma. However,a single administration of L-NAME, a NOS inhibitor, inhibited thiseffect. These data, in conjunction with the findings of the histolog-ical study (sirius red), where the L-NAME-treated showed greatercollagen concentration (Figs. 1 and 2) indicate that blocking NOproduction in the early phases of muscle injury may tilt thebalance toward development of repair with fibrotic scar tissue pro-duction rather than muscle regeneration. Therefore, it is important

Table 2Transcription mRNA of iNOS, MMP-2, and HGF markers in muscle injury rats in 7 days after lesion.

CO (n = 10) ST (n = 10) T (n = 10) L-NAME (n = 10)

iNOSa 100 (99.5–100.5) 74.4 (70.6–78.2) 61 (57–65) 73.1 (71.5–74.6)MMP-2b 100 (99.3–100.6) 62.4 (45.2–79.6) 123 (107–139)* 137 (122–152)*

HGFc 100 (98.6–101.4) 83.7 (80–87.4) 111.9 (95.5–128.3) 89.5 (78.7–100.3)

Data were shown by mean ± 95%IC values.CO = control group (n = 10).ST = sham group (n = 10).T = trauma group (n = 10).L-NAME = L-NAME group (n = 10).

a Levels of iNOS mRNA normalized with HPRT as an endogenous reference.b Levels of MMP-2 mRNA normalized with HPRT as an endogenous reference.c Levels of HGF mRNA normalized with HPRT as an endogenous reference.* p < 0.05 higher versus control.

Fig. 4. Immunohistochemistry of MMP-2 at 24 h and 7 days after trauma. In photomicrography (A) the sham trauma group (immunohistochemistry – 200�) showed noexpression. In photomicrography (B) (immunohistochemistry – 200�) the trauma group demonstrated moderate expression in 24 h. In (C) the treatment with L-NAME hadsimilar expression levels as the trauma group, but with a different expression pattern that was more restricted in 24 h after lesion. After 7 days the cellular expression waspersistent in the trauma group (photomicrography (D)) and absent in the group L-NAME-treated (photomicrography (E)).

L.I. Filippin et al. / Nitric Oxide 24 (2011) 43–49 47

to identify the mechanisms by which NO might influence SC acti-vation in trauma skeletal muscle.

Allen et al. [9] demonstrated that HGF could act as an activatorof quiescent SC in vitro. Regulation of HGF function in muscle canoccur at the transcriptional level and by its high-affinity bindingto heparin sulfate proteoglycans of the tissue matrix.

Our experiments demonstrated in vivo that local transcriptionof HGF was correlated to the NO synthesis (Table 1), with markedinhibition when NO synthesis was blocked with L-NAME. Tatsumiet al. [20], based in an in vitro experiment, has proposed thatmechanical stretch stimuli trigger an intracellular cascade ofevents in the muscle fibers, which is pH-dependent and involvesNOS activation, eventually leading to HGF release from the extra-cellular compartment and subsequent SC activation. However,there are controversial results on this topic. Wozniak and Anderson[10] using the single-fiber culture model (normal and mdx) demon-strated that both SC quiescence and activation are NO-dependent.

A possible candidate as the NO-dependent factor for HGF-matrix release is the matrix metalloproteinase (MMP) [15].

In the present study, we demonstrated that MMP-2 is beingtranscripted and expressed in skeletal muscle 24 h after trauma(Table 1 and Fig. 4) and that inhibition of NO production byL-NAME was able to decrease this transcription and expression.In parallel there was an increase in the deposition of collagen (sir-ius red staining). Gosselin et al. [21] demonstrated intrinsic dissim-ilarities in collagen metabolism between functionally differentskeletal muscles. Lewis et al. [22] hypothesized that differentialexpression/activity of the gelatinases (MMP-2 and MMP-9) wouldbe observed during different phases of myoblasts to myotube for-mation and that such expression had functional significance.Therefore, NO is a key signal responsible for SC activation andHGF transcription and release after skeletal muscle injury [23].

As a limitation in our study, we have to acknowledge that theinformation concerning iNOS, MMP-2 and PCNA is only semiquan-titative, since no quantitative formal protein expression measure-ment was performed. However, the immunohistochemicalanalysis was done by two blinded pathologists, and the observa-tions were clear-cut and highly correlated.

Fig. 5. Immunohistochemistry of PCNA at 24 h and 7 days after trauma. In photomicrography (A) the sham trauma group (immunohistochemistry – 400�) showed noexpression. (B) the trauma group (immunohistochemistry – 400�) showed moderate total cell proliferation at 24 h after trauma. In photomicrography (C)(immunohistochemistry – 400�) the treatment with L-NAME decreased the expression levels of total cell proliferation. After 7 days the cell proliferation was persistentin both groups (photomicrography (D) and (E)).

Fig. 6. PAX-7 expression by Western blot analysis. (A) The image representativeblot of PAX-7 and the loading control of the b-actin. In graph (B) the PAX-7expression demonstrated no expression in control and sham trauma groups at 24 hand 7 days. The trauma group showed a higher positivity at 24 h after lesion; L-NAME group inhibited this increase in SC number at 24 h. After 7 days, both groupsdemonstrated similar levels of the PAX-7 expression.

48 L.I. Filippin et al. / Nitric Oxide 24 (2011) 43–49

At the cellular level, shear stress generated by damaged fiberswithin the basal lamina is thought to stimulate synthesis of NOby neuronal NOS located in association with the dystrophin–glycoprotein complex beneath the sarcolemma; and endothelialNOS located in muscle smooth [24]. Additionally, to the releaseof nNOS and eNOS, there is production of iNOS by the local inflam-matory process. In our experiments we demonstrated the overex-pression of iNOS by RT-PCR and immunohistochemistry at 24 h

after trauma. Numerous studies have demonstrated increase iNOSin inflammatory process [17,25]. Recently Tatsumi et al. [26] sug-gested that calcium-calmodulin pathway may be involved in theincreased NO synthesis and resulting SC activation.

In summary, we presented in vivo data that support the notionthat the muscle regeneration process depends on initial NO pro-duction, with subsequent matrix metalloproteinase activation, re-lease of hepatocyte growth factor from the extracellular matrix,increased total cell proliferation and increased of SC number.Blocking early NO production, besides decreasing early cell prolif-eration and SC activation, induced increased collagen deposition,indicating that this molecule has an important role in the balanceof regeneration/fibrosis during muscle repair process. Since regen-eration is important for full functional recovery after muscle injury,better comprehension of the diverse factors regulating this balancemay lead to innovative therapeutic approaches to muscle trauma.

Article origin

Serviço de Reumatologia do Hospital de Clínicas de Porto Alegre– (HCPA) e Universidade Federal do Rio Grande do Sul (UFRGS).

Supporting agency

Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior(CAPES), Conselho Nacional de Desenvolvimento Científico e Tec-nológico (CNPq), Fundo de Incentivo a Pesquisa e Eventos (FIPE).

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