Cardioprotective Effects of Hydrogen Sulfide and Its Potential Therapeutic Implications in the Amelioration of Duchenne Muscular Dystrophy Cardiomyopathy.

Hydrogen sulfide (H2S) belongs to the family of gasotransmitters and can modulate a myriad of biological signaling pathways. Among others, its cardioprotective effects, through antioxidant, anti-inflammatory, anti-fibrotic, and proangiogenic activities, are well-documented in experimental studies. Cardiorespiratory failure, predominantly cardiomyopathy, is a life-threatening complication that is the number one cause of death in patients with Duchenne muscular dystrophy (DMD). Although recent data suggest the role of H2S in ameliorating muscle wasting in murine and Caenorhabditis elegans models of DMD, possible cardioprotective effects have not yet been addressed. In this review, we summarize the current understanding of the role of H2S in animal models of cardiac dysfunctions and cardiac cells. We highlight that DMD may be amenable to H2S supplementation, and we suggest H2S as a possible factor regulating DMD-associated cardiomyopathy.

Dysregulated iron homeostasis in dystrophin-deficient cardiomyocytes: correction by gene editing and pharmacological treatment.

AIMS: Duchenne muscular dystrophy (DMD)-associated cardiomyopathy is a serious life-threatening complication, the mechanisms of which have not been fully established, and therefore no effective treatment is currently available. The purpose of the study was to identify new molecular signatures of the cardiomyopathy development in DMD. METHODS AND RESULTS: For modelling of DMD-associated cardiomyopathy, we prepared three pairs of isogenic control and dystrophin-deficient human induced pluripotent stem cell (hiPSC) lines. Two isogenic hiPSC lines were obtained by CRISPR/Cas9-mediated deletion of DMD exon 50 in unaffected cells generated from healthy donor and then differentiated into cardiomyocytes (hiPSC-CM). The latter were subjected to global transcriptomic and proteomic analyses followed by more in-depth investigation of selected pathway and pharmacological modulation of observed defects. Proteomic analysis indicated a decrease in the level of mitoNEET protein in dystrophin-deficient hiPSC-CM, suggesting alteration in iron metabolism. Further experiments demonstrated increased labile iron pool both in the cytoplasm and mitochondria, a decrease in ferroportin level and an increase in both ferritin and transferrin receptor in DMD hiPSC-CM. Importantly, CRISPR/Cas9-mediated correction of the mutation in the patient-derived hiPSC reversed the observed changes in iron metabolism and restored normal iron levels in cardiomyocytes. Moreover, treatment of DMD hiPSC-CM with deferoxamine (DFO, iron chelator) or pioglitazone (mitoNEET stabilizing compound) decreased the level of reactive oxygen species in DMD hiPSC-CM. CONCLUSION: To our knowledge, this study demonstrated for the first time impaired iron metabolism in human DMD cardiomyocytes, and potential reversal of this effect by correction of DMD mutation or pharmacological treatment. This implies that iron overload-regulating compounds may serve as novel therapeutic agents in DMD-associated cardiomyopathy.

Proteome Profiling of the Dystrophic mdx Mice Diaphragm.

Mdx mice with a spontaneous mutation in exon 23 of the Dmd gene represent the most common model to investigate the pathophysiology of Duchenne muscular dystrophy (DMD). The disease, caused by the lack of functional dystrophin, is characterized by irreversible impairment of muscle functions, with the diaphragm affected earlier and more severely than other skeletal muscles. We applied a label-free (LF) method and the more thorough tandem mass tag (TMT)-based method to analyze differentially expressed proteins in the diaphragm of 6-week-old mdx mice. The comparison of both methods revealed 88 commonly changed proteins. A more in-depth analysis of the TMT-based method showed 953 significantly changed proteins, with 867 increased and 86 decreased in dystrophic animals (q-value < 0.05, fold-change threshold: 1.5). Consequently, several dysregulated processes were demonstrated, including the immune response, fibrosis, translation, and programmed cell death. Interestingly, in the dystrophic diaphragm, we found a significant decrease in the expression of enzymes generating hydrogen sulfide (H2S), suggesting that alterations in the metabolism of this gaseous mediator could modulate DMD progression, which could be a potential target for pharmacological intervention.

miR-378 influences muscle satellite cells and enhances adipogenic potential of fibro-adipogenic progenitors but does not affect muscle regeneration in the glycerol-induced injury model.

Skeletal muscle regeneration relies on the reciprocal interaction between many types of cells. Regenerative capacity may be altered in different disorders. In our study, we investigated whether the deletion of miR-378a (miR-378) affects muscle regeneration. We subjected 6-week-old wild-type (WT) and miR-378 knockout (miR-378-/-) animals to the glycerol-induced muscle injury and performed analyses in various time-points. In miR-378-/- animals, an elevated abundance of muscle satellite cells (mSCs) on day 3 was found. Furthermore, fibro-adipogenic progenitors (FAPs) isolated from the muscle of miR-378-/- mice exhibited enhanced adipogenic potential. At the same time, lack of miR-378 did not affect inflammation, fibrosis, adipose tissue deposition, centrally nucleated fiber count, muscle fiber size, FAP abundance, and muscle contractility at any time point analyzed. To conclude, our study revealed that miR-378 deletion influences the abundance of mSCs and the adipogenic potential of FAPs, but does not affect overall regeneration upon acute, glycerol-induced muscle injury.

Sodium hydrosulfide moderately alleviates the hallmark symptoms of Duchenne muscular dystrophy in mdx mice.

Duchenne muscular dystrophy (DMD) is an incurable disease caused by mutations in the X-linked DMD gene that encodes a structural muscle protein, dystrophin. This, in turn, leads to progressive degeneration of the skeletal muscles and the heart. Hydrogen sulfide (H2S), the pleiotropic agent with antioxidant, anti-inflammatory, and pro-angiogenic activities, could be considered a promising therapeutic factor for DMD. In this work, we studied the effect of daily intraperitoneal administration of the H2S donor, sodium hydrosulfide (NaHS, 100 µmol/kg/day for 5 weeks) on skeletal muscle (gastrocnemius, diaphragm and tibialis anterior) pathology in dystrophin-deficient mdx mice, characterized by decreased expression of H2S-generating enzymes. NaHS reduced the level of muscle damage markers in plasma (creatine kinase, lactate dehydrogenase and osteopontin). It lowered oxidative stress by affecting the GSH/GSSG ratio, up-regulating the level of cytoprotective heme oxygenase-1 (HO-1) and down-regulating the NF-κB pathway. In the gastrocnemius muscle, it also increased angiogenic vascular endothelial growth factor (Vegf) and its receptor (Kdr) expression, accompanied by the elevated number of α-SMA/CD31/lectin-positive blood vessels. The expression of fibrotic regulators, like Tgfß, Col1a1 and Fn1 was decreased by NaHS in the tibialis anterior, while the level of autophagy markers (AMPKα signalling and Atg genes), was mostly affected in the gastrocnemius. Histological and molecular analysis showed no effect of H2S donor on regeneration and the muscle fiber type composition. Overall, the H2S donor modified the gene expression and protein level of molecules associated with the pathophysiology of DMD, contributing to the regulation of oxidative stress, inflammation, autophagy, and angiogenesis.

Co-administration of angiotensin II and simvastatin triggers kidney injury upon heme oxygenase-1 deficiency.

Kidneys are pivotal organ in iron redistribution and can be severely damaged in the course of hemolysis. In our previous studies, we observed that induction of hypertension with angiotensin II (Ang II) combined with simvastatin administration results in a high mortality rate or the appearance of signs of kidney failure in heme oxygenase-1 knockout (HO-1 KO) mice. Here, we aimed to address the mechanisms underlying this effect, focusing on heme and iron metabolism. We show that HO-1 deficiency leads to iron accumulation in the renal cortex. Higher mortality of Ang II and simvastatin-treated HO-1 KO mice coincides with increased iron accumulation and the upregulation of mucin-1 in the proximal convoluted tubules. In vitro studies showed that mucin-1 hampers heme- and iron-related oxidative stress through the sialic acid residues. In parallel, knock-down of HO-1 induces the glutathione pathway in an NRF2-depedent manner, which likely protects against heme-induced toxicity. To sum up, we showed that heme degradation during heme overload is not solely dependent on HO-1 enzymatic activity, but can be modulated by the glutathione pathway. We also identified mucin-1 as a novel redox regulator. The results suggest that hypertensive patients with less active HMOX1 alleles may be at higher risk of kidney injury after statin treatment.

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.

Cytotoxic and Antioxidant Activity of Hypericum perforatum L. Extracts against Human Melanoma Cells from Different Stages of Cancer Progression, Cultured under Normoxia and Hypoxia.

Oxidative stress and the hypoxic microenvironment play a key role in the progression of human melanoma, one of the most aggressive skin cancers. The aim of our study was to evaluate the effect of Hypericum perforatum extracts of different origins (both commercially available (HpEx2) and laboratory-prepared from wild grown (HpEx12) and in vitro cultured (HpEx13) plants) and hyperforin salt on WM115 primary and WM266-4 lymph node metastatic human melanoma cells cultured under normoxic and hypoxic conditions. The polyphenol content, radical scavenging activity, and hyperforin concentration were determined in the extracts, while cell viability, apoptosis, ROS production, and expression of NRF2 and HO-1, important oxidative stress-related factors, were analyzed after 24 h of cell stimulation with HpExs and hyperforin salt. We found that cytotoxic, pro-apoptotic and antioxidant effects depend on the extract composition, the stage of melanoma progression, and the oxygen level. Hyperforin salt showed lower activity than H. perforatum extracts. Our study for the first time showed that the anticancer activity of H. perforatum extracts differs in normoxia and hypoxia. Importantly, the composition of extracts of various origins, including in vitro cultured, resulting in their unique properties, may be important in the selection of plants for therapeutic application.

Nuclear Factor Erythroid 2-Related Factor 2 and Its Targets in Skeletal Muscle Repair and Regeneration.

Significance: Skeletal muscles have a robust regenerative capacity in response to acute and chronic injuries. Muscle repair and redox homeostasis are intimately linked; increased generation of reactive oxygen species leads to cellular dysfunction and contributes to muscle wasting and progression of muscle diseases. In exemplary muscle disease, Duchenne muscular dystrophy (DMD), caused by mutations in the DMD gene that encodes the muscle structural protein dystrophin, the regeneration machinery is severely compromised, while oxidative stress contributes to the progression of the disease. The nuclear factor erythroid 2-related factor 2 (NRF2) and its target genes, including heme oxygenase-1 (HO-1), provide protective mechanisms against oxidative insults. Recent Advances: Relevant advances have been evolving in recent years in understanding the mechanisms by which NRF2 regulates processes that contribute to effective muscle regeneration. To this end, pathways related to muscle satellite cell differentiation, oxidative stress, mitochondrial metabolism, inflammation, fibrosis, and angiogenesis have been studied. The regulatory role of NRF2 in skeletal muscle ferroptosis has been also suggested. Animal studies have shown that NRF2 pathway activation can stop or reverse skeletal muscle pathology, especially when endogenous stress defence mechanisms are imbalanced. Critical Issues: Despite the growing recognition of NRF2 as a factor that regulates various aspects of muscle regeneration, the mechanistic impact on muscle pathology in various models of muscle injury remains imprecise. Future Directions: Further studies are necessary to fully uncover the role of NRF2 in muscle regeneration, both in physiological and pathological conditions, and to investigate the possibilities for development of new therapeutic modalities. Antioxid. Redox Signal. 38, 619-642.

Hydrogen sulfide as a therapeutic option for the treatment of Duchenne muscular dystrophy and other muscle-related diseases.

Hydrogen sulfide (H2S) has been known for years as a poisoning gas and until recently evoked mostly negative associations. However, the discovery of its gasotransmitter functions suggested its contribution to various physiological and pathological processes. Although H2S has been found to exert cytoprotective effects through modulation of antioxidant, anti-inflammatory, anti-apoptotic, and pro-angiogenic responses in a variety of conditions, its role in the pathophysiology of skeletal muscles has not been broadly elucidated so far. The classical example of muscle-related disorders is Duchenne muscular dystrophy (DMD), the most common and severe type of muscular dystrophy. Mutations in the DMD gene that encodes dystrophin, a cytoskeletal protein that protects muscle fibers from contraction-induced damage, lead to prominent dysfunctions in the structure and functions of the skeletal muscle. However, the main cause of death is associated with cardiorespiratory failure, and DMD remains an incurable disease. Taking into account a wide range of physiological functions of H2S and recent literature data on its possible protective role in DMD, we focused on the description of the 'old' and 'new' functions of H2S, especially in muscle pathophysiology. Although the number of studies showing its essential regulatory action in dystrophic muscles is still limited, we propose that H2S-based therapy has the potential to attenuate the progression of DMD and other muscle-related disorders.

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 (Nfe2l2tKO). 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, Nfe2l2tKO 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.

Molecular mechanisms of Duchenne muscular dystrophy and new therapeutic strategies / Molekularne mechanizmy dystrofii miesniowej Duchenne'a i nowe mozliwosci terapeutyczne.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease affecting approximately 1 in 5,000 born boys. It is caused by mutations in the DMD gene encoding dystrophin, which protects muscle fibers upon contraction. Its absence leads to muscle weakening and premature death mostly due to cardio-respiratory failure. Many experimental therapies have been developed to restore functional dystrophin or counteract processes contributing to disease progression. Nonetheless, DMD remains an incurable disease, and glucocorticoids, exerting many side effects, still serve as the "gold standard" of treatment. Hence, there is a need to develop innovative therapeutic options that will at least alleviate the symptoms of DMD. Among them, targeting specific microRNAs (miRs), e.g. miR-378a, restoring normal angiogenesis and the use of cytoprotective factors such as heme oxygenase-1 (HO-1) or hydrogen sulfide (H2S) might be of special interest. In this review, we describe both the pathology of the disease and the aforementioned new therapeutic options in DMD.

miR-378 affects metabolic disturbances in the mdx model of Duchenne muscular dystrophy.

Although Duchenne muscular dystrophy (DMD) primarily affects muscle tissues, the alterations to systemic metabolism manifested in DMD patients contribute to the severe phenotype of this fatal disorder. We propose that microRNA-378a (miR-378) alters carbohydrate and lipid metabolism in dystrophic mdx mice. In our study, we utilized double knockout animals which lacked both dystrophin and miR-378 (mdx/miR-378-/-). RNA sequencing of the liver identified 561 and 194 differentially expressed genes that distinguished mdx versus wild-type (WT) and mdx/miR-378-/- versus mdx counterparts, respectively. Bioinformatics analysis predicted, among others, carbohydrate metabolism disorder in dystrophic mice, as functionally proven by impaired glucose tolerance and insulin sensitivity. The lack of miR-378 in mdx animals mitigated those effects with a faster glucose clearance in a glucose tolerance test (GTT) and normalization of liver glycogen levels. The absence of miR-378 also restored the expression of genes regulating lipid homeostasis, such as Acly, Fasn, Gpam, Pnpla3, and Scd1. In conclusion, we report for the first time that miR-378 loss results in increased systemic metabolism of mdx mice. Together with our previous finding, demonstrating alleviation of the muscle-related symptoms of DMD, we propose that the inhibition of miR-378 may represent a new strategy to attenuate the multifaceted symptoms of DMD.

Simvastatin does not alleviate muscle pathology in a mouse model of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an incurable disease, caused by the mutations in the DMD gene, encoding dystrophin, an actin-binding cytoskeletal protein. Lack of functional dystrophin results in muscle weakness, degeneration, and as an outcome cardiac and respiratory failure. As there is still no cure for affected individuals, the pharmacological compounds with the potential to treat or at least attenuate the symptoms of the disease are under constant evaluation. The pleiotropic agents, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, known as statins, have been suggested to exert beneficial effects in the mouse model of DMD. On the other hand, they were also reported to induce skeletal-muscle myopathy. Therefore, we decided to verify the hypothesis that simvastatin may be considered a potential therapeutic agent in DMD. METHODS: Several methods including functional assessment of muscle function via grip strength measurement, treadmill test, and single-muscle force estimation, enzymatic assays, histological analysis of muscle damage, gene expression evaluation, and immunofluorescence staining were conducted to study simvastatin-related alterations in the mdx mouse model of DMD. RESULTS: In our study, simvastatin treatment of mdx mice did not result in improved running performance, grip strength, or specific force of the single muscle. Creatine kinase and lactate dehydrogenase activity, markers of muscle injury, were also unaffected by simvastatin delivery in mdx mice. Furthermore, no significant changes in inflammation, fibrosis, and angiogenesis were noted. Despite the decreased percentage of centrally nucleated myofibers in gastrocnemius muscle after simvastatin delivery, no changes were noticed in other regeneration-related parameters. Of note, even an increased rate of necrosis was found in simvastatin-treated mdx mice. CONCLUSION: In conclusion, our study revealed that simvastatin does not ameliorate DMD pathology.

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.

Potential of enhancer of zeste homolog 2 inhibitors for the treatment of SWI/SNF mutant cancers and tumor microenvironment modulation.

Enhancer of zeste homolog 2 (EZH2), a catalytic component of polycomb repressive complex 2 (PRC2), is commonly overexpressed or mutated in many cancer types, both of hematological and solid nature. Till now, plenty of EZH2 small molecule inhibitors have been developed and some of them have already been tested in clinical trials. Most of these inhibitors, however, are effective only in limited cases in the context of EZH2 gain-of-function mutated tumors such as lymphomas. Other cancer types with aberrant EZH2 expression and function require alternative approaches for successful treatment. One possibility is to exploit synthetic lethal strategy, which is based on the phenomenon that concurrent loss of two genes is detrimental but the deletion of either of them leaves cell viable. In the context of EZH2/PRC2, the most promising synthetic lethal target seems to be SWItch/Sucrose Non-Fermentable chromatin remodeling complex (SWI/SNF), which is known to counteract PRC2 functions. SWI/SNF is heavily involved in carcinogenesis and its subunits have been found mutated in approximately 20% of tumors of different kinds. In the current review, we summarize the existing knowledge of synthetic lethal relationships between EZH2/PRC2 and components of the SWI/SNF complex and discuss in detail the potential application of existing EZH2 inhibitors in cancer patients harboring mutations in SWI/SNF proteins. We also highlight recent discoveries of EZH2 involvement in tumor microenvironment regulation and consequences for future therapies. Although clinical studies are limited, the fundamental research might help to understand which patients are most likely to benefit from therapies using EZH2 inhibitors.

Dysregulated Autophagy and Mitophagy in a Mouse Model of Duchenne Muscular Dystrophy Remain Unchanged Following Heme Oxygenase-1 Knockout.

Dysregulation of autophagy may contribute to the progression of various muscle diseases, including Duchenne muscular dystrophy (DMD). Heme oxygenase-1 (HO-1, encoded by Hmox1), a heme-degrading enzyme, may alleviate symptoms of DMD, inter alia, through anti-inflammatory properties. In the present study, we determined the role of HO-1 in the regulation of autophagy and mitophagy in mdx animals, a commonly used mouse model of the disease. In the gastrocnemius of 6-week-old DMD mice, the mRNA level of mitophagy markers: Bnip3 and Pink1, as well as autophagy regulators, e.g., Becn1, Map1lc3b, Sqstm1, and Atg7, was decreased. In the dystrophic diaphragm, changes in the latter were less prominent. In older, 12-week-old dystrophic mice, diminished expressions of Pink1 and Sqstm1 with upregulation of Atg5, Atg7, and Lamp1 was depicted. Interestingly, we demonstrated higher protein levels of autophagy regulator, LC3, in dystrophic muscles. Although the lack of Hmox1 in mdx mice influenced blood cell count and the abundance of profibrotic proteins, no striking differences in mRNA and protein levels of autophagy and mitophagy markers were found. In conclusion, we demonstrated complex, tissue, and age-dependent dysregulation of mitophagic and autophagic markers in DMD mice, which are not affected by the additional lack of Hmox1.

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 (Nrf2tKO) affects muscle functions both in an acute and chronic injury. The acute muscle damage was induced in mice of two genotypes-WT and Nrf2tKO 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/Nrf2tKO) 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 Nrf2tKO 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 Nrf2tKOmdx 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.

Hypoxia as a Driving Force of Pluripotent Stem Cell Reprogramming and Differentiation to Endothelial Cells.

Inadequate supply of oxygen (O2) is a hallmark of many diseases, in particular those related to the cardiovascular system. On the other hand, tissue hypoxia is an important factor regulating (normal) embryogenesis and differentiation of stem cells at the early stages of embryonic development. In culture, hypoxic conditions may facilitate the derivation of embryonic stem cells (ESCs) and the generation of induced pluripotent stem cells (iPSCs), which may serve as a valuable tool for disease modeling. Endothelial cells (ECs), multifunctional components of vascular structures, may be obtained from iPSCs and subsequently used in various (hypoxia-related) disease models to investigate vascular dysfunctions. Although iPSC-ECs demonstrated functionality in vitro and in vivo, ongoing studies are conducted to increase the efficiency of differentiation and to establish the most productive protocols for the application of patient-derived cells in clinics. In this review, we highlight recent discoveries on the role of hypoxia in the derivation of ESCs and the generation of iPSCs. We also summarize the existing protocols of hypoxia-driven differentiation of iPSCs toward ECs and discuss their possible applications in disease modeling and treatment of hypoxia-related disorders.

Muscle and cardiac therapeutic strategies for Duchenne muscular dystrophy: past, present, and future.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular childhood disorder that causes progressive muscle weakness and degeneration and results in functional decline, loss of ambulation and early death of young men due to cardiac or respiratory failure. Although the major cause of the disease has been known for many years-namely mutation in the DMD gene encoding dystrophin, one of the largest human genes-DMD is still incurable, and its treatment is challenging. METHODS: A comprehensive and systematic review of literature on the gene, cell, and pharmacological experimental therapies aimed at restoring functional dystrophin or to counteract the associated processes contributing to disease progression like inflammation, fibrosis, calcium signaling or angiogenesis was carried out. RESULTS: Although some therapies lead to satisfying effects in skeletal muscle, they are highly ineffective in the heart; therefore, targeting defective cardiac and respiratory systems is vital in DMD patients. Unfortunately, most of the pharmacological compounds treat only the symptoms of the disease. Some drugs addressing the underlying cause, like eteplirsen, golodirsen, and ataluren, have recently been conditionally approved; however, they can correct only specific mutations in the DMD gene and are therefore suitable for small sub-populations of affected individuals. CONCLUSION: In this review, we summarize the possible therapeutic options and describe the current status of various, still imperfect, strategies used for attenuating the disease progression.