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

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. 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. 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. 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.

Głowniak-Kwitek U ，Caballero AL ，Bronisz-Budzyńska I ，Kozakowska M ，Andrysiak K ，Stępniewski J ，Łoboda A ，Dulak J ... -  《-》

Heme Oxygenase-1 Influences Satellite Cells and Progression of Duchenne Muscular Dystrophy in Mice.

Pietraszek-Gremplewicz K ，Kozakowska M ，Bronisz-Budzynska I ，Ciesla M ，Mucha O ，Podkalicka P ，Madej M ，Glowniak U ，Szade K ，Stepniewski J ，Jez M ，Andrysiak K ，Bukowska-Strakova K ，Kaminska A ，Kostera-Pruszczyk A ，Jozkowicz A ，Loboda A ，Dulak J ... -  《-》

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

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. 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. 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). Targeted expression of HMOX1 plays protective role in DMD and alleviates dystrophic muscle pathology.

Florczyk-Soluch U ，Polak K ，Jelinkova S ，Bronisz-Budzyńska I ，Sabo R ，Bolisetty S ，Agarwal A ，Werner E ，Józkowicz A ，Stępniewski J ，Szade K ，Dulak J ... -  《Skeletal Muscle》

p-TAK1 acts as a switch between myoblast proliferation phase and differentiation phase in mdx mice via regulating HO-1 expression.

Skeletal muscle transforming growth factor-β-activated kinase 1 (TAK1) continuous excessive phosphorylation was observed in Duchenne muscular dystrophy (DMD) patients and mdx mice. Inhibiting TAK1 phosphorylation ameliorated fibrosis and muscular atrophy, while TAK1 knockout also impaired muscle regeneration. The definite effect and mechanism of p-TAK1 in muscle regeneration disorder is still obscure. In this study, BaCl2-induced acute muscle injury model was used to investigate the role of p-TAK1 in myoblast proliferation and differentiation phase. The results showed that TAK1 phosphorylation was significantly up-regulated in proliferation phase along with Keap1/Nrf2/HO-1 signaling pathway activation, which was down-regulated in differentiation phase yet. In C2C12 cells, inhibiting TAK1 phosphorylation markedly suppressed the expression of heme oxygenase-1 (HO-1), and both myoblast proliferation and differentiation were inhibited. As for activation, p-TAK1 promoted myoblast proliferation via up-regulating HO-1 level. However, excessive TAK1 phosphorylation (induced by 20 ng·mL-1 TGF-β1) notably up-regulated HO-1 expression, inhibiting myogenic differentiation antigen (MyOD) and myogenic differentiation. A mild p-TAK1 level (induced by 5 or 10 ng·mL-1 TGF-β1) was beneficial for myoblast differentiation. In mdx mice, robust myoblast proliferation and differentiation arrest were observed with high p-TAK1 level in skeletal muscle. HO-1 expression was significantly up-regulated. TAK1 phosphorylation inhibitor NG25 (N-[4-[(4-ethylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]-4-methyl-3-(1H-pyrrolo[2,3-b]pyridin-4-yloxy)benzamide) significantly inhibited HO-1 expression, relieved excessive myoblast proliferation and differentiation arrest, promoted new myofiber formation, and eventually improved muscle function. In conclusion, p-TAK1 acted as "a switch" between proliferation and differentiation phase. Mitigating p-TAK1 level transformed myoblast excessive proliferation phase into differentiation phase in mdx mouse via regulating HO-1 expression.

Fan S ，Huang X ，Tong H ，Hong H ，Lai Z ，Hu W ，Liu X ，Zhang L ，Jiang Z ，Yu Q ... -  《-》

Long-Term Protective Effect of Human Dystrophin Expressing Chimeric (DEC) Cell Therapy on Amelioration of Function of Cardiac, Respiratory and Skeletal Muscles in Duchenne Muscular Dystrophy.

Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in dystrophin encoding gene, causing progressive degeneration of cardiac, respiratory, and skeletal muscles leading to premature death due to cardiac and respiratory failure. Currently, there is no cure for DMD. Therefore, novel therapeutic approaches are needed for DMD patients.We have previously reported functional improvements which correlated with increased dystrophin expression following administration of dystrophin expressing chimeric (DEC) cells of myoblast origin to the mdx mouse models of DMD.In the current study, we confirmed dose-dependent protective effect of human DEC therapy created from myoblasts of normal and DMD-affected donors, on restoration of dystrophin expression and amelioration of cardiac, respiratory, and skeletal muscle function at 180 days after systemic-intraosseous DEC administration to mdx/scid mouse model of DMD. Functional improvements included maintenance of ejection fraction and fractional shortening levels on echocardiography, reduced enhanced pause and expiration time on plethysmography and improved grip strength and maximum stretch induced contraction of skeletal muscles. Improved function was associated with amelioration of mdx muscle pathology revealed by reduced muscle fibrosis, reduced inflammation and improved muscle morphology confirmed by reduced number of centrally nucleated fibers and normalization of muscle fiber diameters. Our findings confirm the long-term systemic effect of DEC therapy in the most severely affected by DMD organs including heart, diaphragm, and long skeletal muscles.These encouraging preclinical data introduces human DEC as a novel therapeutic modality of Advanced Therapy Medicinal Product (ATMP) with the potential to improve or halt the progression of DMD and enhance quality of life of DMD patients. Human DEC as a novel therapeutic modality with the potential to improve or halt progression of the DMD disease and enhance quality of life of DMD patients. Graphical abstract represents manufacturing process of the human DEC therapy for the future clinical applications. 1. We report the long-term efficacy of human DEC therapy resulting in increased dystrophin expression and reduced mdx muscle pathology after systemic-intraosseous administration of human Dystrophin Expressing Chimeric (DEC) Cells to the mdx/scid mouse model of DMD. 2. Systemic administration of human DEC therapy resulted in amelioration of cardiac, respiratory and skeletal muscle function as confirmed by echocardiography, plethysmography and standard muscle strength tests respectively. 3. We introduce human DEC as a novel Advanced Therapy Medicinal Product (ATMP) for future clinical application in DMD patients.

Siemionow M ，Langa P ，Brodowska S ，Kozlowska K ，Zalants K ，Budzynska K ，Heydemann A ... -  《-》