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.

Generation of human induced pluripotent stem cell line derived from Becker muscular dystrophy patient with CRISPR/Cas9-mediated correction of DMD gene mutation.

Becker muscular dystrophy (BMD) is an X-linked recessive disorder caused by in-frame deletions in the dystrophin gene (DMD), leading to progressive muscle degeneration and weakness. We generated a human induced pluripotent stem cell (hiPSC) line from a BMD patient. BMD hiPSCs were then engineered by CRISPR/Cas9-mediated knock-in of missing exons 3-9 of DMD gene. Obtained hiPSC line may be a valuable tool for investigating the mechanisms underlying BMD pathogenesis.

Compromised diabetic heart function is not affected by miR-378a upregulation upon hyperglycemia.

BACKGROUND: Cardiac-abundant microRNA-378a (miR-378a) is associated with postnatal repression of insulin-like growth factor 1 receptor (IGF-1R) controlling physiological hypertrophy and survival pathways. IGF-1/IGF-1R axis has been proposed as a therapeutic candidate against the pathophysiological progress of diabetic cardiomyopathy (DCM). We ask whether hyperglycemia-driven changes in miR-378a expression could mediate DCM progression. METHODS: Diabetes mellitus was induced by streptozotocin (STZ) (55 mg/kg i.p. for 5 days) in male C57BL/6 wild type (miR-378a+/+) and miR-378a knockout (miR-378a-/-) mice. As a parallel human model, we harnessed human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM miR378a+/+ vs. hiPSC-CM miR378a-/-) subjected to high glucose (HG) treatment. RESULTS: We reported miR-378a upregulation in cardiac diabetic milieu arising upon STZ administration to wild-type mice and in HG-treated hiPSC-CMs. Pro-hypertrophic IGF-1R/ERK1/2 pathway and hypertrophic marker expression were activated in miR-378a deficiency and upon STZ/HG treatment of miR-378a+/+ specimens in vivo and in vitro suggesting miR-378a-independent hyperglycemia-promoted hypertrophy. A synergistic upregulation of IGF-1R signaling in diabetic conditions was detected in miR-378a-/- hiPSC-CMs, but not in miR-378a-/- hearts that showed attenuation of this pathway, pointing to the involvement of compensatory mechanisms in the absence of miR-378a. Although STZ administration did not cause pro-inflammatory or pro-fibrotic effects that were detected in miR-378a-/- mice, the compromised diabetic heart function observed in vivo by high-resolution ultrasound imaging upon STZ treatment was not affected by miR-378a presence. CONCLUSIONS: Overall, data underline the role of miR-378a in maintaining basal cardiac structural integrity while pointing to miR-378a-independent hyperglycemia-driven cardiac hypertrophy and associated dysfunction.

Generation of human induced pluripotent stem cell lines with HMOX1 promoter polymorphism and CRISPR/Cas9-mediated deletion of exon 50 of DMD gene.

Duchenne muscular dystrophy (DMD), originating from the lack of functional dystrophin, clinically manifests as devastating disease of skeletal muscles with progressive cardiac involvement. HMOX1 promoter polymorphism may reflect different activity of heme oxygenase-1 (HO-1) that may be critical for DMD progression. Here we generated human induced pluripotent stem cell (hiPSC) lines from healthy donors-derived peripheral blood mononuclear cells with different variants of HMOX1 promoter (GT repeats), and engineered by CRISPR/Cas9-mediated deletion of exon 50 of DMD gene. Such in vitro model could add to molecular understanding of DMD and verify the prognostic value of HMOX1 promoter polymorphism.

The multifaceted view of heart problem in Duchenne muscular dystrophy.

Dystrophin is a large protein serving as local scaffolding repetitively bridging cytoskeleton and the outside of striated muscle cell. As such dystrophin is a critical brick primarily in dystrophin-associated protein complex (DAGC) and in a larger submembranous unit, costamere. Accordingly, the lack of functional dystrophin laying at the root of Duchenne muscular dystrophy (DMD) drives sarcolemma instability. From this point on, the cascade inevitably leading to the death of myocyte begins. In cardiomyocytes, intracellular calcium overload and related mitochondrial-mediated cell death mainly contribute to myocardial dysfunction and dilation while other protein dysregulation and/or mislocalization may affect electrical conduction system and favor arrhythmogenesis. Although clinically DMD manifests as progressive muscle weakness and skeletal muscle symptoms define characteristic of DMD, it is the heart problem the biggest challenge that most often develop in the form of dilated cardiomyopathy (DCM). Current standards of treatment and recent progress in respiratory care, introduced in most settings in the 1990s, have improved quality of life and median life expectancy to 4th decade of patient's age. At the same time, cardiac causes of death related to DMD increases. Despite preventive and palliative cardiac treatments available, the prognoses remain poor. Direct therapeutic targeting of dystrophin deficiency is critical, however, hindered by the large size of the dystrophin cDNA and/or stochastic, often extensive genetic changes in DMD gene. The correlation between cardiac involvement and mutations affecting specific dystrophin isoforms, may provide a mutation-specific cardiac management and novel therapeutic approaches for patients with CM. Nonetheless, the successful cardiac treatment poses a big challenge and may require combined therapy to combat dystrophin deficiency and its after-effects (critical in DMD pathogenesis). This review locates the multifaceted heart problem in the course of DMD, balancing the insights into basic science, translational efforts and clinical manifestation of dystrophic heart disease.

Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes, in Contrast to Adipose Tissue-Derived Stromal Cells, Efficiently Improve Heart Function in Murine Model of Myocardial Infarction.

Cell therapies are extensively tested to restore heart function after myocardial infarction (MI). Survival of any cell type after intracardiac administration, however, may be limited due to unfavorable conditions of damaged tissue. Therefore, the aim of this study was to evaluate the therapeutic effect of adipose-derived stromal cells (ADSCs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) overexpressing either the proangiogenic SDF-1α or anti-inflammatory heme oxygenase-1 (HO-1) in a murine model of MI. ADSCs and hiPSCs were transduced with lentiviral vectors encoding luciferase (Luc), GFP and either HO-1 or SDF-1α. hiPSCs were then differentiated to hiPSC-CMs using small molecules modulating the WNT pathway. Genetically modified ADSCs were firstly administered via intracardiac injection after MI induction in Nude mice. Next, ADSCs-Luc-GFP and genetically modified hiPSC-CMs were injected into the hearts of the more receptive NOD/SCID strain to compare the therapeutic effect of both cell types. Ultrasonography, performed on days 7, 14, 28 and 42, revealed a significant decrease of left ventricular ejection fraction (LVEF) in all MI-induced groups. No improvement of LVEF was observed in ADSC-treated Nude and NOD/SCID mice. In contrast, administration of hiPSC-CMs resulted in a substantial increase of LVEF, occurring between 28 and 42 days after MI, and decreased fibrosis, regardless of genetic modification. Importantly, bioluminescence analysis, as well as immunofluorescent staining, confirmed the presence of hiPSC-CMs in murine tissue. Interestingly, the luminescence signal was strongest in hearts treated with hiPSC-CMs overexpressing HO-1. Performed experiments demonstrate that hiPSC-CMs, unlike ADSCs, are effective in improving heart function after MI. Additionally, long-term evaluation of heart function seems to be crucial for proper assessment of the effect of cell administration.

miR-378a influences vascularization in skeletal muscles.

AIMS: MicroRNA-378a, highly expressed in skeletal muscles, was demonstrated to affect myoblasts differentiation and to promote tumour angiogenesis. We hypothesized that miR-378a could play a pro-angiogenic role in skeletal muscle and may be involved in regeneration after ischaemic injury in mice. METHODS AND RESULTS: Silencing of miR-378a in murine C2C12 myoblasts did not affect differentiation but impaired their secretory angiogenic potential towards endothelial cells. miR-378a knockout (miR-378a-/-) in mice resulted in a decreased number of CD31-positive blood vessels and arterioles in gastrocnemius muscle. In addition, diminished endothelial sprouting from miR-378a-/- aortic rings was shown. Interestingly, although fibroblast growth factor 1 (Fgf1) expression was decreased in miR-378a-/- muscles, this growth factor did not mediate the angiogenic effects exerted by miR-378a. In vivo, miR-378a knockout did not affect the revascularization of the ischaemic muscles in both normo- and hyperglycaemic mice subjected to femoral artery ligation (FAL). No difference in regenerating muscle fibres was detected between miR-378a-/- and miR-378+/+ mice. miR-378a expression temporarily declined in ischaemic skeletal muscles of miR-378+/+ mice already on Day 3 after FAL. At the same time, in the plasma, the level of miR-378a-3p was enhanced. Similar elevation of miR-378a-3p was reported in the plasma of patients with intermittent claudication in comparison to healthy donors. Local adeno-associated viral vectors-based miR-378a overexpression was enough to improve the revascularization of the ischaemic limb of wild-type mice on Day 7 after FAL, what was not reported after systemic delivery of vectors. In addition, the number of infiltrating CD45+ cells and macrophages (CD45+ CD11b+ F4/80+ Ly6G-) was higher in the ischaemic muscles of miR-378a-/- mice, suggesting an anti-inflammatory action of miR-378a. CONCLUSIONS: Data indicate miR-378a role in the pro-angiogenic effect of myoblasts and vascularization of skeletal muscle. After the ischaemic insult, the anti-angiogenic effect of miR-378a deficiency might be compensated by enhanced inflammation.

Atorvastatin and Conditioned Media from Atorvastatin-Treated Human Hematopoietic Stem/Progenitor-Derived Cells Show Proangiogenic Activity In Vitro but Not In Vivo.

Myeloid angiogenic cells (MAC) derive from hematopoietic stem/progenitor cells (HSPCs) that are mobilized from the bone marrow. They home to sites of neovascularization and contribute to angiogenesis by production of paracrine factors. The number and function of proangiogenic cells are impaired in patients with diabetes or cardiovascular diseases. Both conditions can be accompanied by decreased levels of heme oxygenase-1 (HMOX1), cytoprotective, heme-degrading enzyme. Our study is aimed at investigating whether precursors of myeloid angiogenic cells (PACs) treated with known pharmaceuticals would produce media with better proangiogenic activity in vitro and if such media can be used to stimulate blood vessel growth in vivo. We used G-CSF-mobilized CD34+ HSPCs, FACS-sorted from healthy donor peripheral blood mononuclear cells (PBMCs). Sorted cells were predominantly CD133+. CD34+ cells after six days in culture were stimulated with atorvastatin (AT), acetylsalicylic acid (ASA), sulforaphane (SR), resveratrol (RV), or metformin (Met) for 48 h. Conditioned media from such cells were then used to stimulate human aortic endothelial cells (HAoECs) to enhance tube-like structure formation in a Matrigel assay. The only stimulant that enhanced PAC paracrine angiogenic activity was atorvastatin, which also had ability to stabilize endothelial tubes in vitro. On the other hand, the only one that induced heme oxygenase-1 expression was sulforaphane, a known activator of a HMOX1 inducer-NRF2. None of the stimulants changed significantly the levels of 30 cytokines and growth factors tested with the multiplex test. Then, we used atorvastatin-stimulated cells or conditioned media from them in the Matrigel plug in vivo angiogenic assay. Neither AT alone in control media nor conditioned media nor AT-stimulated cells affected numbers of endothelial cells in the plug or plug's vascularization. Concluding, high concentrations of atorvastatin stabilize tubes and enhance the paracrine angiogenic activity of human PAC cells in vitro. However, the effect was not observed in vivo. Therefore, the use of conditioned media from atorvastatin-treated PAC is not a promising therapeutic strategy to enhance angiogenesis.

Transcriptomes of human mesenchymal cells isolated from the right ventricle and epicardial fat differ strikingly both directly after isolation and long-term culture.

AIMS: Mesenchymal stromal cells isolated from different tissues are claimed to demonstrate similar therapeutic potential and are often incorrectly named mesenchymal stem cells. However, through comparison of such cells is lacking. This study aimed to compare the transcriptome of mesenchymal cells of the same phenotype isolated from the heart muscle and epicardial fat of the same patient, before and after culture. METHODS AND RESULTS: Cells were isolated from biopsies of the right ventricle and epicardial fat collected from five patients (three men and two women, mean age 59.4 ± 2.6) who underwent heart transplantation due to ischaemic cardiomyopathy. In both tissues, immunophenotyping revealed three distinct populations: (i)CD31- CD45- CD90+ CD34+ CD146- , (ii) CD31- CD45- CD90+ CD34- CD146+ , and (iii) CD31- CD45- CD90- CD34- CD146+ , of which only the first one could be grown after sorting. Material for RNA-seq was collected from these cells before culture (250 cells) and at passage 6 (5000 cells). Transcriptomic analysis revealed that cells of the same phenotype (CD31- CD45- CD90+ CD34+ CD146- ) upon isolation preferentially clustered according to the tissue of origin, not to the patient from whom they were isolated. Genes up-regulated in the right ventricle-derived cells were related to muscle physiology while down-regulated genes included those encoding proteins with transmembrane signalling receptor activity. After six passages, heart-derived and fat-derived cells did not acquire similar transcriptome. Cells isolated from the right ventricle in comparison with their epicardial fat-derived counterparts demonstrated higher level of transcripts related, among others, to RNA processing and muscle development. The down-regulated genes were involved in the nucleosome assembly, DNA packaging and replication, and interleukin-7-mediated signalling pathway. Cells from epicardial fat demonstrated higher heterogeneity both before and after culture. Cell culture significantly changed gene expression profile within both tissues. CONCLUSIONS: This study is an essential indication that mesenchymal cells isolated from different tissues do not demonstrate similar properties. Phenotypic identification and ease of isolation cannot be considered as a criterion in any therapeutic utilization of such cells.

Various roles of heme oxygenase-1 in response of bone marrow macrophages to RANKL and in the early stage of osteoclastogenesis.

Heme oxygenase-1 (HO-1; encoded by Hmox1), a downstream target of the Nrf2 transcription factor, has been postulated to be a negative regulator of osteoclasts (OCLs) differentiation. Here, we further explored such a hypothesis by examining HO-1 effects in different stages of osteoclastogenesis. We confirmed the inhibition of the expression of OCLs markers by Nrf2. In contrast, both the lack of the active Hmox1 gene or HO-1 silencing in OCLs precursor cells, bone marrow macrophages (BMMs), decreased their differentiation towards OCLs, as indicated by the analysis of OCLs markers such as TRAP. However, no effect of HO-1 deficiency was observed when HO-1 expression was silenced in BMMs or RAW264.7 macrophage cell line pre-stimulated with RANKL (considered as early-stage OCLs). Moreover, cobalt protoporphyrin IX (CoPPIX) or hemin, the known HO-1 inducers, inhibited OCLs markers both in RANKL-stimulated RAW264.7 cells and BMMs. Strikingly, a similar effect occurred in HO-1-/- cells, indicating HO-1-independent activity of CoPPIX and hemin. Interestingly, plasma of HO-1-/- mice contained higher TRAP levels, which suggests an increased number of bone-resorbing OCLs in the absence of HO-1 in vivo. In conclusion, our data indicate that HO-1 is involved in the response of bone marrow macrophages to RANKL and the induction of OCLs markers, but it is dispensable in early-stage OCLs. However, in vivo HO-1 appears to inhibit OCLs formation.

Critical View on Mesenchymal Stromal Cells in Regenerative Medicine.

SIGNIFICANCE: The belief in the potency of stem cells has resulted in the medical applications of numerous cell types for organ repair, often with the low adherence to methodological stringency. Such uncritical enthusiasm is mainly presented in the approaches employing so-called mesenchymal stem cells (MSC), for the treatment of numerous, unrelated conditions. However, it should be stressed that such broad clinical applications of MSC are mostly based on the belief that MSC can efficiently differentiate into multiple cell types, not only osteoblasts, chondrocytes and adipose cells. Recent Advances: Studies employing lineage tracing established more promising markers to characterize MSC identity and localization in vivo and confirmed the differences between MSC isolated from various organs. Furthermore, preclinical and clinical experiments proved that transdifferentiation of MSC is unlikely to contribute to repair of numerous tissues, including the heart. Therefore, the salvage hypotheses, like MSC fusion with cells in target organs or the paracrine mechanisms, were proposed to justify the widespread application of MSC and to explain transient, if any, effects. CRITICAL ISSUES: The lack of standardization concerning the cells markers, their origin and particularly the absence of stringent functional characterization of MSC, leads to propagation of the worrying hype despite the lack of convincing therapeutic efficiency of MSC. FUTURE DIRECTIONS: The adherence to rigorous methodological rules is necessary to prevent the application of procedures which can be dangerous for patients and scientific research on the medical application of stem cells. Antioxid. Redox Signal. 00, 000-000.