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.

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Heart-specific CD4+ T cells have been implicated in development and progression of myocarditis in mice and in humans. Here, using mouse models of experimental autoimmune myocarditis (EAM) we investigated the role of heart non-specific CD4+ T cells in the progression of the disease. Heart non-specific CD4+ T cells were obtained from DO11.10 mice expressing transgenic T cell receptor recognizing chicken ovalbumin. We found that heart infiltrating CD4+ T cells expressed exclusively effector (Teff) phenotype in the EAM model and in hearts of patients with lymphocytic myocarditis. Adoptive transfer experiments showed that while heart-specific Teff infiltrated the heart shortly after injection, heart non-specific Teff effectively accumulated during myocarditis and became the major heart-infiltrating CD4+ T cell subset at later stage. Restimulation of co-cultured heart-specific and heart non-specific CD4+ T cells with alpha-myosin heavy chain antigen showed mainly Th1/Th17 response for heart-specific Teff and up-regulation of a distinct set of extracellular signalling molecules in heart non-specific Teff. Adoptive transfer of heart non-specific Teff in mice with myocarditis did not affect inflammation severity at the peak of disease, but protected the heart from adverse post-inflammatory fibrotic remodelling and cardiac dysfunction at later stages of disease. Furthermore, mouse and human Teff stimulated in vitro with common gamma cytokines suppressed expression of profibrotic genes, reduced amount of α-smooth muscle actin filaments and decreased contraction of cardiac fibroblasts. In this study, we provided a proof-of-concept that heart non-specific Teff cells could effectively contribute to myocarditis and protect the heart from the dilated cardiomyopathy outcome.Electronic supplementary materialThe online version of this article (10.1007/s00395-019-0766-6) contains supplementary material, which is available to authorized users.

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Aims Angiotensin (Ang) II signalling has been suggested to promote cardiac fibrosis in inflammatory heart diseases, however the underlaying mechanisms remain obscure. Using Agtr1a-/- mice with genetic deletion of angiotensin receptor type 1 (ATR1) and the experimental autoimmune myocarditis (EAM) model, we aimed to elucidate the role of Ang II-ATR1 pathway in development of heart-specific autoimmunity and post-inflammatory fibrosis. Methods and Results EAM was induced in wild-type (WT) and Agtr1a-/- mice by subcutaneous injections with alpha myosin heavy chain peptide emulsified in complete Freund’s adjuvant. Agtr1a-/- mice developed myocarditis to a similar extent as WT controls at day 21, but showed reduced fibrosis and better systolic function at day 40. Crisscross bone marrow chimera experiments proved that ATR1 signalling in the bone-marrow compartment was critical for cardiac fibrosis. Heart infiltrating, bone-marrow derived cells produced Ang II, but lack of ATR1 in these cells reduced transforming growth factor beta (TGF-β) mediated fibrotic responses. At the molecular level, Agtr1a-/- heart-inflammatory cells showed impaired TGF-β-mediated phosphorylation of Smad2 and TAK1. In WT cells, TGF-β induced formation of RhoA-GTP and RhoA-A-kinase anchoring protein-Lbc (AKAP-Lbc) complex. In Agtr1a-/- cells stabilization of RhoA-GTP and interaction of RhoA with AKAP-Lbc were largely impaired. Furthermore, in contrast to WT cells, Agtr1a-/- cells stimulated with TGF-β failed to activate canonical Wnt pathway indicated by suppressed activity of glycogen synthase kinase-3 (GSK-3)β and nuclear β-catenin translocation and showed reduced expression of Wnts. In line with these in vitro findings, β-catenin was detected in inflammatory regions of hearts of WT, but not Agtr1a-/- mice and expression of canonical Wnt1 and Wnt10b were lower in Agtr1a-/- hearts. Conclusions Ang II-ATR1 signalling is critical for development of postinflammatory fibrotic remodelling and dilated cardiomyopathy. Our data underpins the importance of Ang II-ATR1 in effective TGF-β downstream signalling response including activation of profibrotic Wnt/β-catenin pathway. Translational Perspective Myocardial fibrosis causes impaired cardiac function in inflammatory heart diseases. It has been believed that targeting angiotensin II receptor 1 (ATR1) could not only lower blood pressure, but also effectively block both, immune and fibrotic processes in the heart. By using an experimental autoimmune myocarditis model and ATR1-deficient mice, we show that the specific ATR-dependent mechanism is rather limited to control profibrotic, but not inflammatory response. On the molecular level, ATR1 plays a central role in activation of the canonical Wnt pathway in profibrotic response. Thus, these data help to understand how the actual angiotensin II-ATR1 signalling contributes to immunofibrotic heart diseases.

Systemically delivered adeno-associated viral vector serotype 9 (AAV9) effectively transduces murine heart, but provides transgene expression also in liver and skeletal muscles. Improvement of the selectivity of transgene expression can be achieved through incorporation of target sites (TSs) for miRNA-122 and miRNA-206 into the 3 0 untranslated region (3 0 UTR) of the expression cassette. Here, we aimed to generate such miRNA-122-and miRNA-206-regulated AAV9 vector for a therapeutic, heart-specific overexpression of heme oxygenase-1 (HO-1). We successfully validated the vector functionality in murine cell lines corresponding to tissues targeted by AAV9. Next, we evaluated biodistribution of transgene expression following systemic vector delivery to HO-1-deficient mice of mixed C57BL/6J Â FVB genetic background. Although AAV genomes were present in the hearts of these animals, HO-1 protein expression was either absent or significantly impaired. We found that miRNA-122, earlier described as liver specific, was present also in the hearts of C57BL/6J Â FVB mice. Various levels of miRNA-122 expression were observed in the hearts of other mouse strains, in heart tissues of patients with cardiomyopathy, and in human induced pluripotent stem cell-derived cardiomyocytes in which we also confirmed such posttranscriptional regulation of transgene expression. Our data clearly indicate that therapeutic utilization of miRNA-based regulation strategy needs to consider inter-individual variability.

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.

Adeno-associated viral (AAV) vectors are characterised by low immunogenicity, although humoral and cellular responses may be triggered upon infection. Following systemic administration high levels of vector particles accumulate within the liver. Kupffer cells (KCs) are liver resident macrophages and an important part of the liver innate immune system. Decreased functional activity of KCs can contribute to exaggerated inflammatory response upon antigen exposure. Heme oxygenase-1 (HO-1) deficiency is associated with considerably reduced numbers of KCs. In this study we aimed to investigate the inflammatory responses in liver and to characterise two populations of hepatic macrophages in adult wild type (WT) and HO-1 knockout (KO) mice following systemic administration of one or two doses (separated by 3 months) of self-complementary (sc)AAV9 vectors. At steady state, the livers of HO-1 KO mice contained significantly higher numbers of monocyte-derived macrophages (MDMs), but significantly less KCs than their WT littermates. Three days after re-administration of scAAV9 we observed increased mRNA level of monocyte chemoattractant protein-1 (Mcp-1) in the livers of both WT and HO-1 KO mice, but the protein level and the macrophage infiltration were not affected. Three days after the 1 st and 3 days after the 2 nd vector dose the numbers of AAV genomes in the liver were comparable between both genotypes indicating similar transduction efficiency, but the percentage of transgeneexpressing MDMs and KCs was higher in WT than in HO-1 KO mice. In the primary culture, KCs were able to internalize AAV9 particles without induction of TLR9-mediated immune responses, but no transgene expression was observed. In conclusion, in vivo and in vitro cultured KCs have different susceptibility to scAAV9 vectors. Regardless of the presence or absence of HO-1 and initial numbers of KCs in the liver, scAAV9 exhibits a low potential to stimulate inflammatory response at the analysed time points.