Targeted expression of heme oxygenase-1 in satellite cells improves skeletal muscle pathology in dystrophic mice

Background Adult muscle-resident myogenic stem cells, satellite cells (SCs), that play non-redundant role in muscle regeneration, are intrinsically impaired in Duchenne muscular dystrophy (DMD). Previously we revealed that dystrophic SCs express low level of anti-inflammatory and anti-oxidative heme oxygenase-1 (HO-1, HMOX1). Here we assess whether targeted induction of HMOX1 affect SC function and alleviates hallmark symptoms of DMD. Methods We generated double-transgenic mouse model (mdx;HMOX1Pax7Ind) that allows tamoxifen (TX)-inducible HMOX1 expression in Pax7 positive cells of dystrophic muscles. Mdx;HMOX1Pax7Ind and control mdx mice were subjected to 5-day TX injections (75 mg/kg b.w.) followed by acute exercise protocol with high-speed treadmill (12 m/min, 45 min) and downhill running to worsen skeletal muscle phenotype and reveal immediate effects of HO-1 on muscle pathology and SC function. Results HMOX1 induction caused a drop in SC pool in mdx;HMOX1Pax7Ind mice (vs. mdx counterparts), while not exaggerating the effect of physical exercise. Upon physical exercise, the proliferation of SCs and activated CD34− SC subpopulation, was impaired in mdx mice, an effect that was reversed in mdx;HMOX1Pax7Ind mice, however, both in vehicle- and TX-treated animals. This corresponded to the pattern of HO-1 expression in skeletal muscles. At the tissue level, necrotic events of selective skeletal muscles of mdx mice and associated increase in circulating levels of muscle damage markers were blunted in HO-1 transgenic animals which showed also anti-inflammatory cytokine profile (vs. mdx). Conclusions Targeted expression of HMOX1 plays protective role in DMD and alleviates dystrophic muscle pathology. Supplementary Information The online version contains supplementary material available at 10.1186/s13395-024-00346-2.


Introduction
Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular genetic disorder of poor prognosis with 1 per 5000 male births prevalence.Clinically DMD manifests as progressive skeletal muscle weakness and deterioration in motor performance leading to difficulties in walking and eventually wheelchair requirement around the age of 12 [1].
DMD originates from the mutation in dystrophin gene (DMD) and the lack of functional protein scaffolding submembranous components in striated muscle cells.Unmet mechanical demands result in the fragility of cortical cytoskeleton, disruption of myocyte integrity upon contraction, and myofiber degeneration triggering inflammatory reaction, one of the major hallmarks of DMD.At 3-8 weeks of age skeletal muscles of dystrophic mdx mice show massive necrosis followed by vigorous regeneration.Such cycles of muscle damage and regeneration repeat during this intense growth period and from ⁓12 weeks of age onwards, the pathology stabilizes with continuous degeneration (akin to DMD patients) seen solely in diaphragm, which recapitulates the most the human condition.Persistent pathological progression is observed after the age of 12 months, however, adult muscles typically present only mild necrosis while modestly increased fibrosis [1][2][3].
Adult muscle-resident myogenic stem cells, satellite cells (SCs), under trigger-free conditions remain quiescent in a niche on the surface of the muscle fiber, beneath the ensheathing basal lamina.In response to muscle deterioration SCs are activated to play an essential role in regeneration of ruptured fibers generating committed myogenic progenitor cells (myoblasts) [4].In parallel, a subset of activated SCs self-renew and reinstate quiescence to replenish the stem cell reservoir [4][5][6].The high intensity physical exercise that induces muscle insult is associated with SC activation and proliferation during the acute phases of injury [7][8][9].
Both, direct DMD mutation-related, and indirect, dystrophic environment-caused changes in SC biology are involved in DMD progression [10,11].SCs express high levels of dystrophin that mediates cell polarity, critical for asymmetric divisions [11].Increased number of SCs in DMD may be related to the loss of dystrophin-dependent polarity resulting in inefficient generation of committed myogenic progenitors [11].
Heme oxygenase-1 (HO-1, encoded by HMOX1), an enzyme of anti-inflammatory and anti-oxidative potential due to the products of heme degradation [12] is involved in the response to skeletal muscle injury, being expressed mainly in leukocytes [13,14].Enhanced level of HO-1 is detected also in limb skeletal muscles and diaphragm of mdx mice, both in myofibers and inflammatory cells, and in myoblasts differentiated from DMD patients-derived human-induced pluripotent stem cells (hiPSCs) [15].More recently, HO-1 has been linked with postnatal differentiation of stem and progenitor cells [14,16,17], but there is a gap to be filled about HO-1 involvement in SC proliferation and differentiation potential as so far condition-dependent effect of HO-1 on myogenic precursors were reported [14,15,17].
Our previous data showed that the genetic ablation of Hmox1 in dystrophic mice (Hmox1 −/− mdx) aggravated muscle damage, increased inflammation and was associated with impaired exercise capacity [15].In parallel, no significant changes of SC function were detected, likely due to already reduced expression of Hmox1 in dystrophic SCs (in contrast to muscle fibers) [15].In this study, we sought to determine the effect of HO-1 in dystrophic SCs and its contribution to the pathogenesis of DMD.Towards this end, we generated double-transgenic mouse model (mdx;HMOX1 Pax7Ind ) that allows tamoxifen (TX)-inducible HMOX1 expression in SCs of dystrophic muscles.The high intensity physical exercise regimen was applied to reveal immediate effects of HO-1 on muscle pathology and SC function.

Mice
ROSA26LSL-HO1 (B6-Gt(ROSA)26Sor tm(HMOX1) ) mice with human HO-1 cDNA cloned downstream of a floxed stop codon in the endogenous ROSA26 locus were a kind gift from prof.Anupam Agarwal [18].Pax7-CreER T2 (B6.Cg-Pax7tm1(cre/ERT2)Gaka/J) mice with an internal ribosome entry site (IRES)-CreER T2 fusion protein with Cre recombinase fused to a triple mutant form of the human estrogen receptor, inserted downstream of the stop codon of Pax7 gene were purchased form the Jackson laboratory.Cre-ER T2 fusion gene activity is inducible by TX administration.
Animal experiments were performed after approval (73/2019, 26/2020, 315/2020) by the 2nd Institutional Animal Care and Use Committee (IACUC) in Krakow, Poland, according to Polish and European legislation and according to the ARRIVE guidelines.Mice were housed under specific pathogen free (SPF) conditions in individually ventilated cages with a 14 h/10 h light/dark cycle and were kept on a normal, chow diet with water and food available ad libitum.

Tamoxifen administration and treadmill running
Mice were given intraperitoneal injection of TX (Sigma, 75 mg/kg b.w. in ~ 100 µl of corn oil) once a day for 5 consecutive days or with corn oil (~ 100 µl) as a control.TX stock (20 mg/ml) was incubated in 37 •C with shaking until completely dissolved before use.
Upon 7-day waiting period mice were subjected to physical exercise on a treadmill (Exer 3/6, Columbus Instruments).After familiarization trainings (8 m/ min, 15 min) on three consecutive days, the next day mice were run according to the following program: 0-6 m/min-2 min, 6-8 m/min-2 min, 8-10 m/ min-2 min, 10-12 m/min-45 min with downhill inclination of 15° (acute exercise protocol).At day 3 upon exercise regimen (2 weeks after the last TX injection) mice were euthanized with carbon dioxide and the blood was collected from the heart of unconscious animals.Skeletal muscles from one limb were collected for FACS analysis.Caput gastrocnemius and tibialis anterior from the second limb as well as diaphragmae were dissected for histological procedures and gene expression analysis.

Serum LDH and CK activity
The heart blood collected to 1.5 ml Eppendorf tube was left for 10-30 min at room temperature (rt) for clotting.Upon centrifugation (1000 × g, 10 min, 4 •C) serum was collected and stored at -80 •C.
Serum samples were diluted 10 × in water.The activity of lactate dehydrogenase (LDH) and creatine kinase (CK) was measured using the diagnostic Liquick Cor-LDH and Liquick Cor-CK kit (Cormay), respectively, according to manufacturer's protocol.

RT-qPCR
Skeletal muscles: caput gastrocnemius, tibialis anterior and diaphragmae were collected into RNAlater tissue storage reagent (Sigma-Aldrich), frozen in liquid nitrogen and stored at -80 •C.Tissue samples were homogenized in phenol/guanidine-based QIAzol Lysis Reagent (Qiagen).Total RNA was isolated by organic extraction upon addition of chloroform followed by isopropanol precipitation.Ethanol-washed RNA pellets were redissolved in RNase-free water.RNA concentration was determined by NanoDrop Spectrophotometer (Ther-moFisher Scientific).
Reverse transcription (RT) of mRNA was performed using 500 ng of total RNA and RevertAid reverse transcriptase (ThermoFisher Scientific) according to vendor's instructions and carried out in ProFlex PCR System (Applied Biosystems).cDNA (40 ng per sample) was used for quantitative PCR (qPCR) with Sybr ® Green JumpStart ™ Taq ReadyMix ™ (Sigma-Aldrich) and specific primers (Table 1).qPCR was carried out in StepOne Plus Real-Time PCR System (Applied Biosystems) and analyzed with StepOne Software v2.3.Eef2 was used as a housekeeping gene for relative quantification of gene expression based on 2 -ΔCt method.
Immunofluorescent staining of Pax7/HO-1 was performed on caput gastrocnemius frozen sections.Fixation was performed with 4% paraformaldehyde (25 min, rt) and with methanol (5 min, -20 °C).Citrate buffer (10 mM citric acid in water, pH6) was used for antigen retrieval following 30 min cooling down from boiling buffer.Sections were then blocked with 2.5% BSA containing M.O.M. ® (Mouse on Mouse) blocking reagent (Vector Laboratories) for 30 min at rt.Primary antibodies: murine anti-PAX7 (1:100, DSHB) and rabbit anti-HO-1 (1:300, Enzo) were diluted in 0.1% BSA in PBS and added to the sections for overnight incubation at 4 °C in high humidity chamber.Secondary antibodies: anti-mouse IgG Alexa Flour 488 and anti-goat IgG Alexa Fluor 594 (1:500 each, ThermoFisher Scientific) were diluted in 0.1% BSA in PBS and added for 90 min at rt.Finally, the sections were counterstained with DAPI for 10 min, mounted with fluorescent mounting medium (Dako) and covered with a cover glass.Pictures were taken with LSM-880 meta laser scanning confocal microscope (Zeiss).
For immunofluorescent staining of IgG/IgM frozen sections were air-dried for 15-30 min (rt) and blocked with 10% goat serum for 1 h.Rabbit anti-laminin primary antibody (1:500, Sigma-Aldrich) diluted in 1% goat serum in PBS was applied for 1 h at 37 °C, following incubation with a mixture of anti-rabbit IgG Alexa Fluor 568 (1:500, ThermoFisher Scientific) and goat anti-mouse IgG/IgM (H + L) Alexa Fluor 488 (1:400, ThermoFisher Scientific) with Hoechst33342 (1 μg/ml, Sigma-Aldrich) for 30 min at 37 °C.Sections were mounted with fluorescent mounting medium (Dako) and covered with a cover glass.Scans of the whole tissue sections and representative pictures were done with Leica DMi8 microscope with CMOS Leica MC170 HD camera.

Statistics
Numerical data is presented as mean ± standard error (SEM).The outliers were excluded based on the Grubb's test.The n number refers to the number of mice used for experiment and is indicated in the figures legends.Two-way ANOVA (for multiple group comparisons), followed by Tukey's post hoc test was used to determine the statistical significance (p < 0.05).Student's t-test was used additionally for direct comparison of two groups that were not significantly different using ANOVA tests.GraphPad 8 ™ software was used for all statistical analysis.p values 0.05 -0.1 were considered as a statistical tendency.

Generation of transgenic dystrophic mice with SC-specific HMOX1 expression
To obtain inducible HO-1 expression in SCs of dystrophic mice, we generated double-transgenic mice on mdx background (Fig. 1A).In brief, ROSA26LSL-HO1 mice with human HMOX1 cDNA downstream of a floxed stop codon cloned into the endogenous ROSA26 locus were generated previously [18].To excise the stop codon and enable HMOX1 transgene expression, we introduced Cre recombinase in Pax7-CreER T2 mice with an internal ribosome entry site (IRES)-CreER T2 fusion construct inserted downstream of the stop codon of Pax7 gene.Pax7, a SC marker, is expressed and functional, while Cre-ER T2 activity is inducible by tamoxifen (TX), a selective estrogen receptor regulator, that allows Cre nuclear localization.Dystrophic background was introduced by mdx mice breeding with Rosa26LSL-HO1 and Pax7-CreER T2 mice (Fig. 1A).
Obtained double-transgenic mouse model (mdx; HMOX1 Pax7Ind ) allows TX-inducible, Cre-mediated recombination at the LoxP sites, and HMOX1 expression in Pax7 positive cells.In parallel, TX-treated mdx mice served as control of potential side effects of TX.
The body weight of untreated animals (before experiment) was higher in mdx;HMOX1 Pax7Ind than in mdx mice (Fig. 1C).However, in both genotypes neither TX-nor exercise-dependent effect on body weight was detected as measured before and two weeks after the last TX injection (Suppl.Figure 1A).In addition the total weight of hind limb skeletal muscles (caput gastrocnemius, soleus, tibialis anterior, EBL, quadriceps, adductor) collected from one limb measured at the end of experiment was comparable between groups of both genotypes (Suppl.Figure 1B).

TX induces HMOX1 expression in skeletal muscles of transgenic mdx;HMOX1 Pax7Ind mice
Transgenic and control mice aged 13-16 weeks were subjected to the acute physical exercise protocol with high-speed treadmill (12 m/min, 45 min) and downhill running [19] to worsen skeletal muscle phenotype and reveal immediate effects of HO-1 on muscle pathology and SC activation.Mdx;HMOX1 Pax7Ind and mdx mice were given 5-day TX injections and upon 7-day waiting subjected to exercise protocol (Fig. 1B).
Comparable mRNA level of murine Hmox1 was detected in mdx mice and transgenic mdx;HMOX1 Pax7Ind mice both in caput gastrocnemius (GAS) (Fig. 1D) and tibialis anterior (TA) (Fig. 1E).TX administration induced the expression of human HMOX1 (over oil group) specifically in skeletal muscles of transgenic mice, GAS (Fig. 1F) and TA (Fig. 1G).Nonetheless, HMOX1 expression in oil group of both transgenic muscles was higher than in oil-treated mdx counterparts suggesting either the leakage of the Cre-LoxP system and TXindependent action of Cre, or HMOX1 DNA detection (Fig. 1F,G).
In mdx mice, murine Hmox1 was diminished in skeletal muscles on day 3 upon treadmill exercise, with TX exacerbating this decline (Fig. 1H) and opposite trends seen in mdx;HMOX1 Pax7Ind mice (Fig. 1I).One may consider the regulation of Hmox1 by human HO-1 activity in transgenic mice or strain-dependent pattern of Hmox1 regulation.
No HMOX1 expression was detected in mdx mice (Fig. 1J).The induction of human HMOX1 expression after TX treatment was detected in skeletal muscles of untreated transgenic mice and further increased upon exercise (Fig. 1K).This might be related to enhanced activity of ROSA promoter after exercise but requires more in-depth analysis.

HO-1 induction decreases SC pool in mdx;HMOX1 Pax7Ind mice
The high-speed treadmill exercise regimen decreased SC pool in control, oil-treated mice of both HO-1 transgenic and control mice (Fig. 3A).HO-1 induction caused a drop in SC pool, decreasing the percentage of SCs in mdx;HMOX1 Pax7Ind mice (Fig. 3A) to the comparable extent as the physical training (Fig. 3A).
Studies have shown that high intensity physical exercise that induce muscle insult is associated with SC activation and increase in numbers during the acute phases of injury [7][8][9].In HO-1 transgenic (mdx;HMOX1 Pax7Ind ) and control mdx mice, SC activation associated with the loss of CD34, a marker of quiescent SCs [22], increased in response to physical exercise in a comparable manner (Fig. 3B).CD34 − SCs were also more frequent in both genotypes upon TX administration suggesting HO-1-independent stimulation of SC activity (Fig. 3B).
Upon physical exercise, the percentage of proliferating SCs (Fig. 3C) and CD34 − SC subpopulation (Fig. 3D) decreased in control mdx mice, an effect that was reversed in mdx;HMOX1 Pax7Ind mice.Similar pattern was detected in oil-treated and TX-treated mdx;HMOX1 Pax7Ind mice that could be related to the leakage of the Cre-LoxP system (Fig. 3C,D, compare with Figs.1F,G, 2D).
In skeletal muscles of control mdx mice, we observed exercise-dependent decrease in murine Hmox1 expression that correlated with a drop in SC proliferation (Figs.1H,  3C,D).In contrast, exercise induced HO-1 mRNA level in transgenic mice and also increased SC proliferation (Figs.1I, K, 3C, D).On the other hand, no respective changes in HO-1 level was visible in SCs (Fig. 2D).

SC-targeted HMOX1 expression modulates skeletal muscle inflammation in dystrophic mice
Muscle-derived circulating CK and LDH serve as DMD biomarkers reflecting membrane damage, necrosis and extracellular leakage [23,24].Mdx;HMOX1 Pax7Ind mice showed lower serum level of muscle damage markers upon physical exercise in comparison to mdx mice (Fig. 4A,B).
To further outline the effect of HO-1 in SCs we characterized a dystrophic phenotype and the extent of skeletal muscle damage in mdx;HMOX1 Pax7Ind mice (vs.mdx counterparts).The percentage of WBCs (Suppl.Figure 3B) as well as ECs (Suppl.Figure 3C) in hind limb muscles assessed by flow cytometry was not changed by SC-specific HO-1 induction neither in untrained nor in exercise groups and was comparable to mdx counterparts (Suppl.Figure 3B,C).The histological analysis of TA, GAS and DIA muscles of mdx and mdx;HMOX1 Pax7Ind mice did not reveal significant effects of HO-1 on inflammatory response (Fig. 5A-D).However, there was an increase in inflammatory cell infiltration after exercise in TA of mdx mice, but not in HO-1 transgenic mice (Fig. 5B).Also, the basal inflammation extent was moderately higher in GAS of mdx mice than in HO-1 transgenic mice (Fig. 5A,C).In addition, HO-1 transgenic mice revealed anti-inflammatory cytokine profile.The level of antinflammatory interleukin 10 (IL-10) known to limit T cell activation and production of proinflammatory cytokines [25] increased in mdx;HMOX1 Pax7Ind mice (vs.mdx counterparts) both in response to exercise and TX administration (Fig. 5E).In parallel, TX increased IL-10 level in untrained mdx;HMOX1 Pax7Ind mice, while decreased run-induced IL-10 expression suggesting more complex inflammatory effect (Fig. 5E).

SC-specific HMOX1 induction attenuates skeletal muscle necrotic events upon physical exercise
The analysis of skeletal muscles revealed HO-1 dependent anti-necrotic action upon physical exercise in tibialis of mdx;HMOX1 Pax7Ind and opposite pattern seen in TX-treated mdx mice, as evidenced by assessment of IgM and IgG accumulation (Fig. 6A-E).In untreated mdx mice necrotic events decreased in TA after TX treatment while rose back to control values upon physical exercise, in contrast to HO-1 transgenic mice, in which the IgG/IgM accumulation decreased significantly after exercise (Fig. 6A-C).The reverse effect of TX in mdx;HMOX1 Pax7Ind and mdx mice in TA (Fig. 6A-C) resembled the pattern of inflammation extent observed in TA stained with H&E (Fig. 5A,B).No changes were detected between experimental groups in GAS muscle, likely due to already high basal inflammation extent in this muscle in both strains (Fig. 6B,D).HO-1-dependent anti-necrotic action was detected also in diaphragm of mdx;HMOX1 Pax7Ind both in untrained and exercised transgenic mice in comparison to mdx counterparts (Fig. 6B,E).

Discussion
This study points at the role of HO-1 in dystrophic SCs and beneficial effect against skeletal muscle pathology in dystrophic mice.
In the hind limb skeletal muscles of mdx;HMOX1 Pax7Ind mice, the induction of SC-specific expression of HMOX1 decreased SC pool suggesting its role in stimulation of muscle differentiation, without affecting SC activation, that was increased by TX in HO-1 independent manner.In mdx;HMOX1 Pax7Ind mice we detected physical exercise-induced SC proliferation and opposite pattern in control mdx mice.As similar effect was detected in vehicle and TX-treated transgenic mice, direct involvement of HO-1 is uncertain.The changes in SC proliferation corresponded to the pattern of HO-1 expression, that was enhanced by physical exercise in skeletal muscles of HO-1 transgenic mice, while downregulated in mdx counterparts.
At the tissue level, physical exercise-induced necrosis of selective skeletal muscle and associated increase in circulating levels of muscle damage markers was blunted in the animals that expressed HMOX1.SC-targeted HMOX1 expression modulated muscle phenotype in transgenic mice as shown by 1) a decrease of serum level of muscle damage markers upon physical exercise, 2) decreased necrotic events in diaphragm, 3) in TA of trained mice (vs.mdx): decreased necrotic events, while increased (over mdx control) level of anti-inflammatory IL-10.In addition, anti-inflammatory effect of TX was detected in TA in untreated mdx mice as it decreased the necrotic events, although this was not seen in HO-1 transgenic mice.
We previously showed that HO-1 expression is induced in wild type mice in response to exercise or upon skeletal muscle injury as detected mainly in the infiltrating leukocytes [13,14].In mdx mice, enhanced level of HO-1 was detected in inflammatory cells and myofibers of limb skeletal muscles and diaphragm [15].The genetic ablation of Hmox1 (Hmox1 −/− mdx) or pharmacological inhibition of HO activity aggravated muscle damage and inflammation in dystrophic mice and impaired exercise capacity [15].Here, we confirmed HO-1 expression (detected by antibody recognizing murine and human antigen) in WBCs in both mdx;HMOX1 Pax7Ind and mdx mice, that was potentiated by physical exercise (significantly in mdx mice).Strikingly, nearly 90% of CD45 − CD31 − Sca-1 + Muscle biopsies from DMD patients [26] as well as myofibers [10] and hind limb skeletal muscles [27] isolated from mdx mice show elevated SC numbers relative to age-matched healthy controls.Such data contradicts the previously accepted theory of the SC exhaustion in the course of DMD, related to repetitive cycles of muscle degeneration and regeneration [11].Here we show that TX-inducible increase of low basal level of HO-1 in SCs decreased SC number.It suggests the involvement of HO-1 in SC maintenance in DMD but requires mechanistical investigation.
As shown by us recently, although the global Hmox1 knockout aggravated DMD pathology, it did not affect SC phenotype in mdx mice [15].The lack of HO-1 did  decreased proliferation [17].In contrast, cardiotoxininduced muscle injury increased the rate of proliferation of SCs derived from skeletal muscles of Hmox1 −/− mice over Hmox1 +/+ counterparts [14].Thus, the HO-1 involvement in SC function may be dependent on the condition of the muscle and the injury model.
Studies of other groups have provided evidence that TX may act as a regulator of calcium homeostasis [28] and antioxidant, being efficient inhibitor of lipid peroxidation and intramembraneous scavenger of peroxyl radicals [29].Data from murine models have revealed a role for TX in counteracting DMD pathology, attenuating muscle fibrosis and improving the muscle structure [30].Our data show the anti-necrotic effect of TX mainly in TA of untreated mdx mice, but not in mdx;HMOX1 Pax7Ind mice.Such findings require more in-depth analysis.Nonetheless, the recent findings from the TX clinical trial show that although TX was safe and well tolerated, no difference between groups was concluded for the primary efficacy endpoint [31].Due to insufficient clinical evidence, TX is currently not recommended to use in daily clinical practice [31].

Conclusions
Here we showed that targeted expression of HMOX1 plays protective role in DMD and alleviates dystrophic muscle pathology.HO-1 induction in SCs resulted in a drop of SC pool, the effect not visible in mdx controls, without affecting SC activation, that was increased by TX in HO-1 independent manner.HO-1 decreased the percentage of SCs in mdx;HMOX1 Pax7Ind mice to the comparable extent as the physical training.Upon physical exercise, the proliferation of SCs and activated CD34 − SC subpopulation was impaired in mdx mice, an effect that was reversed in mdx;HMOX1 Pax7Ind mice, however, both in vehicle-and TX-treated animals.This suggests the enhanced leakage of the Cre-LoxP system in exercise conditions resulting in enhanced HMOX1 expression.
At the tissue level, targeted induction of HO-1 in SCs of mdx;HMOX1 Pax7Ind mice attenuated dystrophic muscle pathology.HO-1 transgenic mice showed lower serum level of exercise-induced muscle damage markers, blunted necrotic events of selective skeletal muscles and showed anti-inflammatory cytokine profile (vs.mdx).

Fig. 4
Fig. 4 mdx;HMOX1 Pax7Ind mice show lower serum level of muscle damage markers upon physical exercise.A LDH and (B) CK activity in serum of mdx and mdx; Pax7Ind mice.Colorimetric evaluation (n = 5-9).Data is presented as mean ± SEM. # p < 0.05-Two-way ANOVA variation; * p < 0.05 by Two-way ANOVA with Tukey's post hoc test