NRF2 regulates viability, proliferation, resistance to oxidative stress, and differentiation of murine myoblasts and muscle satellite cells

Increased oxidative stress can slow down the regeneration of skeletal muscle and affect the activity of muscle satellite cells (mSCs). Therefore, we evaluated the role of the NRF2 transcription factor (encoded by the Nfe2l2 gene), the main regulator of the antioxidant response, in muscle cell biology. We used (i) an immortalized murine myoblast cell line (C2C12) with stable overexpression of NRF2 and (ii) primary mSCs isolated from wild-type and Nfe2l2 (transcriptionally)-deficient mice (Nfe2l2$^{tKO}$). NRF2 promoted myoblast proliferation and viability under oxidative stress conditions and decreased the production of reactive oxygen species. Furthermore, NRF2 overexpression inhibited C2C12 cell differentiation by down-regulating the expression of myogenic regulatory factors (MRFs) and muscle-specific microRNAs. We also showed that NRF2 is indispensable for the viability of mSCs since the lack of its transcriptional activity caused high mortality of cells cultured in vitro under normoxic conditions. Concomitantly, Nfe2l2$^{tKO}$ mSCs grown and differentiated under hypoxic conditions were viable and much more differentiated compared to cells isolated from wild-type mice. Taken together, NRF2 significantly influences the properties of myoblasts and muscle satellite cells. This effect might be modulated by the muscle microenvironment

Effect of heme oxygenase-1 on the differentiation of human myoblasts and the regeneration of murine skeletal muscles after acute and chronic injury

Background Impaired muscle regeneration is a hallmark of Duchenne muscular dystrophy (DMD), a neuromuscular disorder caused by mutations in the DMD gene encoding dystrophin. The lack of heme oxygenase-1 (HO-1, Hmox1), a known anti-inflammatory and cytoprotective enzyme, was shown to aggravate DMD pathology. Methods We evaluated the role of HO-1 overexpression in human induced pluripotent stem cell (hiPSC)-derived skeletal muscle cells (hiPSC-SkM) in vitro and in the regeneration process in vivo in wild-type mice. Furthermore, the effect of cobalt protoporphyrin IX (CoPP), a pharmacological inducer of HO-1 expression, on regeneration markers during myogenic hiPSC differentiation and progression of the dystrophic phenotype was analysed in the mdx mouse DMD model. Results HO-1 has an impact on hiPSC-SkM generation by decreasing cell fusion capacity and the expression of myogenic regulatory factors and muscle-specific microRNAs (myomiRs). Also, strong induction of HO-1 by CoPP totally abolished hiPSC-SkM differentiation. Injection of HO-1-overexpressing hiPSC-SkM into the cardiotoxin (CTX)-injured muscle of immunodeficient wild-type mice was associated with decreased expression of miR-206 and Myh3 and lower number of regenerating fibers, suggesting some advanced regeneration. However, the very potent induction of HO-1 by CoPP did not exert any protective effect on necrosis, leukocyte infiltration, fibrosis, myofiber regeneration biomarkers, and exercise capacity of mdx mice. Conclusions In summary, HO-1 inhibits the expression of differentiation markers in human iPSC-derived myoblasts. Although moderate overexpression of HO-1 in the injected myoblast was associated with partially advanced muscle regeneration, the high systemic induction of HO-1 did not improve muscle regeneration. The appropriate threshold of HO-1 expression must be established for the therapeutic effect of HO-1 on muscle regeneration

Rola czynnika transkrypcyjnego Nrf2 w regeneracji mięśni i dystrofii mięśniowej Duchenne'a

Dystrofia mięśniowa Duchenne'a (DMD - Duchenne muscular dystrophy) jest najczęściej występującą formą dystrofii mięśniowej, szacuje się, że cierpi na nią 1 na 5000-6000 chłopców. DMD jest recesywną chorobą genetyczną sprzężoną z chromosomem X, powstaje wskutek mutacji w genie kodującym dystrofinę - ważne białko cytoszkieletowe stanowiące główny element kompleksu dystrofina-glikoproteiny. Kompleks ten odpowiada za utrzymanie integralności komórki, zapewniając mechaniczne połączenie pomiędzy cytoszkieletem aktynowym, a macierzą zewnątrzkomórkowa. Utrata dystrofiny zakłóca funkcjonowanie tego kompleksu, prowadząc do niestabilności sarkolemmy, co czyni ją bardziej podatną na uszkodzenia. W konsekwencji wywołuje to nekrozę włókien mięśniowych, chroniczny stan zapalny, zaburzenia procesu regeneracji, które prowadzą do wyczerpania puli mięśniowych komórek satelitarnych (mSCs), a w końcu do zastąpienia tkanki mięśniowej tkanką włóknistą i tłuszczową. Procesy te są wynikiem braku dystrofiny i dodatkowo przyczyniają się do pogłębienia uszkodzeń, prowadząc do atrofii mięśni, utraty ich funkcji, a ostatecznie do przedwczesnej śmierci na skutek niewydolności krążeniowo-oddechowej. Mimo, iż wiele terapii genowych mających na celu przywrócenie ekspresji dystrofiny jest obecnie opracowywanych, farmakologiczne leczenie wykorzystujące kortykosteroidy, leki o właściwościach przeciwzapalnych, jest nadal złotym standardem w opiece nad pacjentami z DMD. Jednak, pomimo korzystnego wpływu na funkcjonowanie mięśni, poprawiającego jakość i długość życia pacjentów, powodują wiele skutków ubocznych. Dlatego określenie nowych celów terapii przeciwzapalnej może przyczynić się do opracowania skuteczniejszych strategii terapeutycznych. Wcześniejsze badania naszej grupy wykazały, że oksygenaza hemowa 1 (HO-1), białko rozkładające hem, hamuje różnicowanie mioblastów i może odgrywać ochronną rolę w DMD poprzez wpływ na stan zapalny oraz regulując różnicowanie mSCs. Czynnik transkrypcyjny Nrf2 (nuclear factor erythroid 2-related factor) odgrywa w komórce kluczową rolę cytoprotekcyjną, poprzez interakcję z sekwencją ARE (Antioxidant Response Element) zlokalizowaną w regionach promotorowych, reguluje ekspresję genów o właściwościach przeciwutleniających i przeciwzapalnych. Celem prezentowanej pracy było zatem określenie wpływu Nrf2 induktora ekspresji HO-1, na właściwości mioblastów, a także jego roli w ostrym i chronicznym uszkodzeniu mięśni w DMD oraz ostrym uszkodzeniu po nastrzyknięciu kardiotoksyną (CTX). W pierwszym etapie badań przeprowadzono doświadczenia wykorzystując i) unieśmiertelnioną linię mysich mioblastów (C2C12), poddanych transdukcji wektorami retrowirusowymi, tak by uzyskać stabilną ekspresję Nrf2 oraz ii) pierwotne komórki satelitarne wyizolowane z myszy o różnym genotypie. W doświadczeniach in vitro wykazano, że nadekspresja Nrf2 promuje proliferację i przeżywalność mioblastów w warunkach stresu oksydacyjnego oraz zmniejsza produkcję reaktywnych form tlenu. Ponadto zaobserwowano, że Nrf2 silne hamuje różnicowanie mioblastów, ekspresję miogenicznych czynników regulatorowych (MRFs) oraz mięśniowo-specyficznych mikroRNA. Co ciekawe, wykazaliśmy, że komórki satelitarne wyizolowane z myszy pozbawionych aktywnego transkrypcyjnie Nrf2 (Nrf2tKO) i hodowane in vitro w atmosferycznym stężeniu tlenu wykazują wysoką śmiertelność. Natomiast, komórki poddane różnicowaniu in vitro w warunkach hipoksji były żywe i dużo bardziej zróżnicowane niż komórki wyizolowane z myszy typu dzikiego (WT). W kolejnym etapie badań sprawdzono wpływ braku aktywności transkrypcyjnej Nrf2 w ostrym i przewlekłym uszkodzeniu mięśni. W tym celu mięsień brzuchaty łydki myszy typu dzikiego oraz myszy pozbawionych aktywnego transkrypcyjnie Nrf2 został nastrzyknięty uszkadzającą mięśnie CTX. W wyniku zastosowania CTX stwierdzono nieznacznie zwiększone uszkodzenie mięśni u myszy Nrf2tKO w porównaniu do myszy WT. Kolejne etapy badań dotyczyły określenia roli Nrf2 w progresji DMD. W tym celu w eksperymentach wykorzystano cztery genotypy myszy: typu dzikiego, pozbawione aktywnego transkrypcyjnie Nrf2 (Nrf2tKO), dystroficzne myszy mdx, które stanowią mysi model DMD oraz unikatowe tzw. podwójne nokauty, pozbawione zarówno dystrofiny, jak i aktywnego transkrypcyjnie Nrf2 (Nrf2tKOmdx). Przeprowadzono liczne analizy w kontekście najbardziej charakterystycznych procesów patologicznych zachodzących w przebiegu DMD, jak: uszkodzenie, zapalenie, zwłóknienie oraz regeneracja mięśni. W badaniach wykazano, że brak aktywności transkrypcyjnej Nrf2 nie wpływa na pogorszenie fenotypu dystroficznego. Ponieważ jednak przebieg dystrofii u myszy mdx jest znacznie łagodniejszy od tego, który jest obserwowany u ludzi, dlatego w kolejnym etapie badań zwierzęta poddano długotrwałym ćwiczeniom wysiłkowym na bieżni w celu zaostrzenia choroby. Zaobserwowano wówczas zwiększone uszkodzenie oraz stan zapalny w mięśniu brzuchatym łydki myszy Nrf2tKOmdx w porównaniu do myszy mdx, co nie było natomiast widoczne w przeponie. Podsumowując, eksperymenty in vitro wskazują na znaczącą rolę czynnika transkrypcyjnego Nrf2 w mioblastach i pierwotnych komórkach satelitarnych. Jednakże wykazano, że brak aktywności transkrypcyjnej Nrf2 ma umiarkowany wpływ na procesy patologiczne zachodzące w mięśniach w różnych modelach uszkodzenia.Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophies that affects 1 in 5000-6000 boys. DMD is a genetic, X-chromosome linked disease caused by the mutations in the DMD gene encoding dystrophin, important cytoskeleton protein, which is the major element of the dystrophin-glycoprotein complex (DGC). DGC is responsible for maintaining cellular integrity, providing the mechanical link of the intracellular cytoskeleton with the extracellular matrix. The loss of dystrophin disrupts the complex, leading to sarcolemmal instability and inducing muscle damages. Consequently it results in muscle necrosis, extensive inflammation, disturbance of regeneration process, related to the depletion of progenitor cells (muscle satellite cells - mSCs) and finally to replacement of skeletal muscles by fibrotic or adipose tissue. Those processes contribute significantly to disease progression aggravating final outcome and leading to muscle atrophy, loss of muscle function, and ultimately to patients' premature death due to cardio-respiratory failures. Although many gene therapies aiming the restoration of dystrophin are being developed, treatment with corticosteroids, anti-inflammatory agents, is still the gold standard of care for DMD patients. Despite the fact that corticosteroids improve the length and quality of patients' life, their prolonged use can cause serious side effects. Therefore, identifying a new target for anti-inflammatory treatment might contribute to the development of novel therapeutic strategies. In previous studies, we have shown that heme oxygenase 1 (HO-1), a heme-degrading protein, inhibits myoblasts differentiation and might play a protective role in DMD modulating inflammation and regulating differentiation of mSCs. Nrf2 transcription factor, that is the major regulator of other anti-oxidant and anti-inflammatory genes interacting with antioxidant response element (ARE) located in their promoter region, including HO-1. Therefore, in the current research, we aimed to study its role in myoblast biology, chronic injury in DMD as well as in acute myodamage. In the first stage of research, we used i) an immortalized cell line of mice myoblasts (C2C12) which were transduced using retroviral vectors to obtain stable expression of Nrf2, and ii) primary mSC isolated from mice of different Nrf2 genotypes. In vitro experiments revealed that Nrf2 promoted myoblasts proliferation and cell viability under oxidative stress condition, as well as decreased production of reactive oxygen species (ROS). Moreover, Nrf2 was found to inhibit differentiation of C2C12 cells decreasing expression of myogenic regulatory factors (MRFs), and muscle-specific microRNAs. Interestingly, we showed also that Nrf2 is indispensable for the viability of mSCs, as lack of its transcriptional activity caused high mortality of cells cultured in vitro in normoxic conditions, whereas mSCs cultured and differentiated in hypoxic condition were viable and much more differentiated in comparison to cells isolated from wild-type mice. In the next step of research, we checked the effect of Nrf2 transcriptional deficiency during acute and chronic muscle injury. Firstly, the gastrocnemius muscle of wild-type (WT) animals and mice lacking transcriptionally active Nrf2 (Nrf2tKO) were injected with cardiotoxin (CTX). We found slightly increased muscle damage in Nrf2tKO mice in comparison to WT counterparts. To investigate the role of Nrf2 in the progression of DMD, experiments were performed on mice of four genotypes: wild type, lacking transcriptionally active Nrf2, mdx mice lacking dystrophin, which are a mouse model of DMD and double knockouts - mice lacking both dystrophin and lacking transcriptionally active Nrf2 (Nrf2tKOmdx). We performed an extensive analysis of muscle functionality and the main pathological features of DMD such as muscle degeneration, inflammation, regeneration, and fibrosis. However, transcriptional ablation of Nrf2 in mdx mice did not aggravate the dystrophic phenotype. Moreover, as mdx mice exhibit quite mild phenotype in comparison to human patients, animals were subjected to long-term treadmill exercises to exacerbate dystrophic condition. Then we observed increased degeneration and inflammation in gastrocnemius muscle, but not in diaphragm in Nrf2tKOmdx in comparison to mdx mice. In conclusion, in vitro experiments showed that Nrf2 significantly influences the properties of myoblast and muscle satellite cells. However, the lack of transcriptionally active Nrf2 only moderately affects muscle pathology in various models of muscle injury

The role of Nrf2 in acute and chronic muscle injury

The nuclear factor erythroid 2-related factor 2 (Nrf2) is considered as a master cytoprotective factor regulating the expression of genes encoding anti-oxidant, anti-inflammatory, and detoxifying proteins. The role of Nrf2 in the pathophysiology of skeletal muscles has been evaluated in different experimental models, however, due to inconsistent data, we aimed to investigate how Nrf2 transcriptional deficiency (Nrf2(tKO)) affects muscle functions both in an acute and chronic injury. The acute muscle damage was induced in mice of two genotypes-WT and Nrf2(tKO) mice by cardiotoxin (CTX) injection. To investigate the role of Nrf2 in chronic muscle pathology, mdx mice that share genetic, biochemical, and histopathological features with Duchenne muscular dystrophy (DMD) were crossed with mice lacking transcriptionally active Nrf2 and double knockouts (mdx/Nrf2(tKO)) were generated. To worsen the dystrophic phenotype, the analysis of disease pathology was also performed in aggravated conditions, by applying a long-term treadmill test. We have observed slightly increased muscle damage in Nrf2(tKO) mice after CTX injection. Nevertheless, transcriptional ablation of Nrf2 in mdx mice did not significantly aggravate the most deleterious, pathological hallmarks of DMD related to degeneration, inflammation, fibrotic scar formation, angiogenesis, and the number and proliferation of satellite cells in non-exercised conditions. On the other hand, upon chronic exercises, the degeneration and inflammatory infiltration of the gastrocnemius muscle, but not the diaphragm, turned to be increased in Nrf2(tKO)mdx in comparison to mdx mice. In conclusion, the lack of transcriptionally active Nrf2 influences moderately muscle pathology in acute CTX-induced muscle injury and chronic DMD mouse model, without affecting muscle functionality. Hence, in general, we demonstrated that the deficiency of Nrf2 transcriptional activity has no profound impact on muscle pathology in various models of muscle injury

Age-dependent dysregulation of muscle vasculature and blood flow recovery after hindlimb ischemia in the mdx model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD), caused by a lack of functional dystrophin, is characterized by progressive muscle degeneration. Interestingly, dystrophin is also expressed in endothelial cells (ECs), and insufficient angiogenesis has already been hypothesized to contribute to DMD pathology, however, its status in mdx mice, a model of DMD, is still not fully clear. Our study aimed to reveal angiogenesis-related alterations in skeletal muscles of mdx mice compared to wild-type (WT) counterparts. By investigating 6- and 12-week-old mice, we sought to verify if those changes are age-dependent. We utilized a broad spectrum of methods ranging from gene expression analysis, flow cytometry, and immunofluorescence imaging to determine the level of angiogenic markers and to assess muscle blood vessel abundance. Finally, we implemented the hindlimb ischemia (HLI) model, more biologically relevant in the context of functional studies evaluating angiogenesis/arteriogenesis processes. We demonstrated that both 6- and 12-week-old dystrophic mice exhibited dysregulation of several angiogenic factors, including decreased vascular endothelial growth factor A (VEGF) in different muscle types. Nonetheless, in younger, 6-week-old mdx animals, neither the abundance of CD31+α-SMA+ double-positive blood vessels nor basal blood flow and its restoration after HLI was affected. In 12-week-old mdx mice, although a higher number of CD31+α-SMA+ double-positive blood vessels and an increased percentage of skeletal muscle ECs were found, the abundance of pericytes was diminished, and blood flow was reduced. Moreover, impeded perfusion recovery after HLI associated with a blunted inflammatory and regenerative response was evident in 12-week-old dystrophic mice. Hence, our results reinforce the hypothesis of age-dependent angiogenic dysfunction in dystrophic mice. In conclusion, we suggest that older mdx mice constitute an appropriate model for preclinical studies evaluating the effectiveness of vascular-based therapies aimed at the restoration of functional angiogenesis to mitigate DMD severity

Heme oxygenase-1 has a greater effect on melanoma stem cell properties than the expression of melanoma-initiating cell markers

Melanoma-initiating cells (MICs) contribute to the tumorigenicity and heterogeneity of melanoma. MICs are identified by surface and functional markers and have been shown to display cancer stem cell (CSC) properties. However, the existence of MICs that follow the hierarchical CSC model has been questioned by studies showing that single unselected melanoma cells are highly tumorigenic in xenotransplantation assays. Herein, we characterize cells expressing MIC markers (CD20, CD24, CD133, Sca-1, ABCB1, ABCB5, ALDHhigh) in the B16-F10 murine melanoma cell line. We use flow cytometric phenotyping, single-cell sorting followed by in vitro clonogenic assays, and syngeneic in vivo serial transplantation assays to demonstrate that the expression of MIC markers does not select CSC-like cells in this cell line. Previously, our group showed that heme-degrading enzyme heme oxygenase-1 (HO-1) can be upregulated in melanoma and increase its aggressiveness. Here, we show that HO-1 activity is important for non-adherent growth of melanoma and HO-1 overexpression enhances the vasculogenic mimicry potential, which can be considered protumorigenic activity. However, HO-1 overexpression decreases clone formation in vitro and serial tumor initiation in vivo. Thus, HO-1 plays a dual role in melanoma, improving the progression of growing tumors but reducing the risk of melanoma initiation

Slow-cycling murine melanoma cells display plasticity and enhanced tumorigenicity in syngeneic transplantation assay

Slow-cycling cancer cells (SCC) contribute to the aggressiveness of many cancers, and their invasiveness and chemoresistance pose a great therapeutic challenge. However, in melanoma, their tumor-initiating abilities are not fully understood. In this study, we used the syngeneic transplantation assay to investigate the tumor-initiating properties of melanoma SCC in the physiologically relevant in vivo settings. For this we used B16-F10 murine melanoma cell line where we identified a small fraction of SCC. We found that, unlike human melanoma, the murine melanoma SCC were not marked by the high expression of the epigenetic enzyme Jarid1b. At the same time, their slow-cycling phenotype was a temporary state, similar to what was described in human melanoma. Progeny of SCC had slightly increased doxorubicin resistance and altered expression of melanogenesis genes, independent of the expression of cancer stem cell markers. Single-cell expansion of SCC revealed delayed growth and reduced clone formation when compared to non-SCC, which was further confirmed by an in vitro limiting dilution assay. Finally, syngeneic transplantation of 10 cells in vivo established that SCC were able to initiate growth in primary recipients and continue growth in the serial transplantation assay, suggesting their self-renewal nature. Together, our study highlights the high plasticity and tumorigenicity of murine melanoma SCC and suggests their role in melanoma aggressiveness

Duchenne muscular dystrophy trajectory in R-DMDdel52 preclinical rat model identifies COMP as biomarker of fibrosis

International audienceAbstractDuchenne muscular dystrophy (DMD) is a fatal muscle-wasting disorder caused by mutations in the Dystrophin gene and for which there is currently no cure. To bridge the gap between preclinical and therapeutic evaluation studies, we have generated a rat model for DMD that carries an exon 52 deletion (R-DMDdel52) causing a complete lack of dystrophin protein. Here we show that R-DMDdel52 animals recapitulated human DMD pathophysiological trajectory more faithfully than the mdx mouse model. We report that R-DMDdel52 rats displayed progressive and severe skeletal muscle loss associated with fibrotic deposition, fat infiltration and fibre type switch. Early fibrosis was also apparent in the cardiac muscle. These histological modifications led to severe muscle, respiratory and cardiac functional impairments leading to premature death around 1 year. Moreover, DMD muscle exhibited systemic inflammation with a mixed M1/M2 phenotype. A comparative single cell RNAseq analysis of the diaphragm muscle was performed, revealing cellular populations alteration and molecular modifications in all muscle cell types. We show that DMD fibroadipogenic progenitors produced elevated levels of cartilage oligomeric matrix protein, a glycoprotein responsible for modulating homeostasis of extracellular matrix, and whose increased concentration correlated with muscle fibrosis both in R-DMDdel52 rats and human patients. Fibrosis is a component of tissue remodelling impacting the whole musculature of DMD patients, at the tissue level but most importantly at the functional level. We therefore propose that this specific biomarker can optimize the prognostic monitoring of functional improvement of patients included in clinical trials.</jats:p