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Hydrogen sulfide (H 2 S) 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 H 2 S 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 H 2 S in animal models of cardiac dysfunctions and cardiac cells. We highlight that DMD may be amenable to H 2 S supplementation, and we suggest H 2 S as a possible factor regulating DMD-associated cardiomyopathy.

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 (H 2 S), suggesting that alterations in the metabolism of this gaseous mediator could modulate DMD progression, which could be a potential target for pharmacological intervention.

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.

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 rising prevalence of diabetes is threatening global health. It is known not only for the occurrence of severe complications but also for the SARS-Cov-2 pandemic, which shows that it exacerbates susceptibility to infections. Current therapies focus on artificially maintaining insulin homeostasis, and a durable cure has not yet been achieved. We demonstrate that our set of small molecule inhibitors of DYRK1A kinase potently promotes β-cell proliferation, enhances long-term insulin secretion, and balances glucagon level in the organoid model of the human islets. Comparable activity is seen in INS-1E and MIN6 cells, in isolated mice islets, and human iPSC-derived β-cells. Our compounds exert a significantly more pronounced effect compared to harmine, the best-documented molecule enhancing β-cell proliferation. Using a body-like environment of the organoid, we provide a proof-of-concept that small-molecule-induced human β-cell proliferation via DYRK1A inhibition is achievable, which lends a considerable promise for regenerative medicine in T1DM and T2DM treatment.

Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous group of monogenic endocrine disorders that is characterised by autosomal dominant inheritance and pancreatic β-cell dysfunction. These patients are commonly misdiagnosed with type 1 or type 2 diabetes, as the clinical symptoms largely overlap. Even though several biomarkers have been tested none of which could be used as single clinical discriminator. The correct diagnosis for individuals with MODY is of utmost importance, as the applied treatment depends on the gene mutation or is subtype-specific. Moreover, in patients with HNF1A-MODY, additional clinical monitoring can be included due to the high incidence of vascular complications observed in these patients. Finally, stratification of MODY patients will enable better and newer treatment options for MODY patients, once the disease pathology for each patient group is better understood. In the current review the clinical characteristics and the known disease-related abnormalities of the most common MODY subtypes are discussed, together with the up-to-date applied diagnostic criteria and treatment options. Additionally, the usage of pluripotent stem cells together with CRISPR/Cas9 gene editing for disease modelling with the possibility to reveal new pathophysiological mechanisms in MODY is discussed.

Doxorubicin (DOX) is a widely used anticancer drug. However, its clinical use is severely limited due to drug-induced cumulative cardiotoxicity, which leads to progressive cardiomyocyte dysfunction and heart failure. Enormous efforts have been made to identify potential strategies to alleviate DOX-induced cardiotoxicity; however, to date, no universal and highly effective therapy has been introduced. Here we reported that cinnamic acid (CA) derivatives exert a multitarget protective effect against DOX-induced cardiotoxicity. The experiments were performed on rat cardiomyocytes (H9c2) and human induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) as a well-established model for cardiac toxicity assessment. CA derivatives protected cardiomyocytes by ameliorating DOX-induced oxidative stress and viability reduction. Our data indicated that they attenuated the chemotherapeutic's toxicity by downregulating levels of caspase-3 and -7. Pre-incubation of cardiomyocytes with CA derivatives prevented DOX-induced motility inhibition in a wound-healing assay and limited cytoskeleton rearrangement. Detailed safety analyses-including hepatotoxicity, mutagenic potential, and interaction with the hERG channel-were performed for the most promising compounds. We concluded that CA derivatives show a multidirectional protective effect against DOX-induced cardiotoxicity. The results should encourage further research to elucidate the exact molecular mechanism of the compounds' activity. The lead structure of the analyzed CA derivatives may serve as a starting point for the development of novel therapeutics to support patients undergoing DOX therapy.

Heme oxygenase-1 (HO-1, encoded by HMOX1 ) is a cytoprotective enzyme degrading heme into CO, Fe 2+ , and biliverdin. HO-1 was demonstrated to affect cardiac differentiation of murine pluripotent stem cells (PSCs), regulate the metabolism of murine adult cardiomyocytes, and influence regeneration of infarcted myocardium in mice. However, the enzyme's effect on human cardiogenesis and human cardiomyocytes' electromechanical properties has not been described so far. Thus, this study aimed to investigate the role of HO-1 in the differentiation of human induced pluripotent stem cells (hiPSCs) into hiPSC-derived cardiomyocytes (hiPSC-CMs). hiPSCs were generated from human fibroblasts and peripheral blood mononuclear cells using Sendai vectors and subjected to CRISPR/Cas9-mediated HMOX1 knock-out. After confirming lack of HO-1 expression on the protein level, isogenic control and HO-1-deficient hiPSCs were differentiated into hiPSC-CMs. No differences in differentiation efficiency and hiPSC-CMs metabolism were observed in both cell types. The global transcriptomic analysis revealed, on the other hand, alterations in electrophysiological pathways in hiPSC-CMs devoid of HO-1, which also demonstrated increased size. Functional consequences in changes in expression of ion channels genes were then confirmed by patch-clamp analysis. To the best of our knowledge, this is the first report demonstrating the link between HO-1 and electrophysiology in human cardiomyocytes.

Inadequate supply of oxygen (O 2 ) 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.

Burkitt lymphoma (BL) is a rapidly growing tumor, characterized by high anabolic requirements. The MYC oncogene plays a central role in the pathogenesis of this malignancy, controlling genes involved in apoptosis, proliferation, and cellular metabolism. Serine biosynthesis pathway (SBP) couples glycolysis to folate and methionine cycles, supporting biosynthesis of certain amino acids, nucleotides, glutathione, and a methyl group donor, S-adenosylmethionine (SAM). We report that BLs overexpress SBP enzymes, phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT1). Both genes are controlled by the MYC-dependent ATF4 transcription factor. Genetic ablation of PHGDH/PSAT1 or chemical PHGDH inhibition with NCT-503 decreased BL cell lines proliferation and clonogenicity. NCT-503 reduced glutathione level, increased reactive oxygen species abundance, and induced apoptosis. Consistent with the role of SAM as a methyl donor, NCT-503 decreased DNA and histone methylation, and led to the re-expression of ID4 , KLF4 , CDKN2B and TXNIP tumor suppressors. High H3K27me3 level is known to repress the MYC negative regulator miR-494. NCT-503 decreased H3K27me3 abundance, increased the miR-494 level, and reduced the expression of MYC and MYC-dependent histone methyltransferase, EZH2. Surprisingly, chemical/genetic disruption of SBP did not delay BL and breast cancer xenografts growth, suggesting the existence of mechanisms compensating the PHGDH/PSAT1 absence in vivo., Competing Interests: P. Juszczynski is a member of the Scientific Advisory Board at Selvita S.A. and served as a consultant for Selvita S.A. Dr. T. Rzymski, Dr. K. Brzozka and P. Kowalczyk are Selvita S.A. employees.

Elevated expression of heme oxygenase-1 (HO-1, encoded by HMOX1 ) is observed in various types of tumors. Hence, it is suggested that HO-1 may serve as a potential target in anticancer therapies. A novel approach to inhibit HO-1 is related to the synthetic lethality of this enzyme and fumarate hydratase (FH). In the current study, we aimed to validate the effect of genetic and pharmacological inhibition of HO-1 in cells isolated from patients suffering from hereditary leiomyomatosis and renal cell carcinoma (HLRCC)-an inherited cancer syndrome, caused by FH deficiency. Initially, we confirmed that UOK 262, UOK 268, and NCCFH1 cell lines are characterized by non-active FH enzyme, high expression of Nrf2 transcription factor-regulated genes, including HMOX1 and attenuated oxidative phosphorylation. Later, we demonstrated that shRNA-mediated genetic inhibition of HMOX1 resulted in diminished viability and proliferation of cancer cells. Chemical inhibition of HO activity using commercially available inhibitors, zinc and tin metalloporphyrins as well as recently described new imidazole-based compounds, especially SLV-11199, led to decreased cancer cell viability and clonogenic potential. In conclusion, the current study points out the possible relevance of HO-1 inhibition as a potential anti-cancer treatment in HLRCC. However, further studies revealing the molecular mechanisms are still needed.

Patients with HNF1A -maturity-onset diabetes of the young (MODY) often develop endothelial dysfunction and related microvascular complications, like retinopathy. As the clinical phenotype of HNF1A -MODY diabetes varies considerably, we used human induced pluripotent stem cells (hiPSCs) from two healthy individuals (control) to generate isogenic lines with mutation in HNF1A gene. Subsequently, control hiPSCs and their respective HNF1A clones were differentiated toward endothelial cells (hiPSC-ECs) and different markers/functions were compared. Human iPSC-ECs from all cell lines showed similar expression of CD31 and Tie-2. VE-cadherin expression was lower in HNF1A -mutated isogenic lines, but only in clones derived from one control hiPSCs. In the other isogenic set and cells derived from HNF1A -MODY patients, no difference in VE-cadherin expression was observed, suggesting the impact of the genetic background on this endothelial marker. All tested hiPSC-ECs showed an expected angiogenic response regardless of the mutation introduced. Isogenic hiPSC-ECs responded similarly to stimulation with pro-inflammatory cytokine TNF- with the increase in ICAM-1 and permeability, however, HNF1A mutated hiPSC-ECs showed higher permeability in comparison to the control cells. Summarizing, both mono- and biallelic mutations of HNF1A in hiPSC-ECs lead to increased permeability in response to TNF- in normal glycemic conditions, which may have relevance to HNF1A- MODY microvascular complications., Competing Interests: The authors declare no conflict of interest.

The daily expression of genes and the changes in gene expression after silencing the heme oxygenase ( ho ) gene were examined in the retina of Drosophila using microarray and SybrGreen qPCR (quantitative polymerase chain reaction) methods. The HO decrease in the morning upregulated 83 genes and downregulated 57 genes. At night, 80 genes were upregulated and 22 were downregulated. The top 20 genes downregulated after ho silencing in the morning modulate phototransduction, immune responses, autophagy, phagocytosis, apoptosis, the carbon monoxide (CO) response, the oxidative stress/UV response, and translation. In turn, the genes that upregulated at night were involved in translation-the response to oxidative stress, DNA damage, and phototransduction. Among the top 20 genes downregulated at night were genes involved in phototransduction, immune responses, and autophagy. For some genes, a low level of HO had an opposite effect in the morning compared to those at night. Silencing ho also changed the expression of circadian clock genes, while the HO decrease during the night enhanced the expression of immune system genes. The results showed that the cyclic expression of HO is important for controlling several processes in the retina, including neuroprotection and those involved in the innate immune system., Competing Interests: The Authors declare no conflicts of interest.

Therapeutic options for amyotrophic lateral sclerosis (ALS) are still limited. Great hopes, however, are placed in growth factors that show neuroprotective abilities (e.g., nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factor (VEGF)) and in the immune modulating features, in particular, the anti-inflammatory effects. In our study we aimed to investigate whether a bone marrow-derived lineage-negative (Lin-) cells population, after autologous application into cerebrospinal fluid (CSF), is able to produce noticeable concentrations of trophic factors and inflammatory-related proteins and thus influence the clinical course of ALS. To our knowledge, the evaluation of Lin- cells transplantation for ALS treatment has not been previously reported. Early hematopoietic Lin- cells were isolated from twelve ALS patients&rsquo; bone marrow, and later, the suspension of cells was administered into the subarachnoid space by lumbar puncture. Concentrations of selected proteins in the CSF and plasma were quantified by multiplex fluorescent bead-based immunoassays at different timepoints post-transplantation. We also chose microRNAs (miRNAs) related to muscle biology (miRNA-1, miRNA-133a, and miRNA-206) and angiogenesis and inflammation (miRNA-155 and miRNA-378) and tested, for the first time, their expression profiles in the CSF and plasma of ALS patients after Lin- cells transplantation. The injection of bone marrow cells resulted in decreased concentration of selected inflammatory proteins (C3) after Lin- cells injection, particularly in patients who had a better clinical outcome. Moreover, several analyzed miRNAs have changed expression levels in the CSF and plasma of ALS patients subsequent to Lin- cells administration. Interestingly, the expression of miR-206 increased in ALS patients, while miR-378 decreased both in the CSF and plasma one month after the cells&rsquo; injection. We propose that autologous lineage-negative early hematopoietic cells injected intrathecally may be a safe and feasible source of material for transplantations to the central nervous system (CNS) environment aimed at anti-inflammatory support provision for ALS adjuvant treatment strategies. Further research is needed to evaluate whether the observed effects could significantly influence the ALS progression.