RESUMO
BACKGROUND: Exercise can protect myocardial infarction (MI) and downregulate cardiac Homeodomain-Interacting Protein Kinase 2 (HIPK2). However, the role of HIPK2 in MI is unclear. METHODS: HIPK2-/- mice and miR-222-/- rats, HIPK2 inhibitor (PKI1H) and adeno-associated virus serotype 9 (AAV9) carrying miR-222 were applied in the study. Animals were subjected to running, swimming, acute MI or post-MI remodeling. HIPK2 inhibition and P53 activator were used in neonatal rat cardiomyocytes (NRCMs) and human embryonic stem cell-derived cardiomyocytes (hESC-CMs) subjected to oxygen glucose deprivation/reperfusion (OGD/R). Serum miR-222 levels were analyzed in healthy people and MI patients that were survival or readmitted to the hospital and/or died. FINDINGS: Cardiac HIPK2 protein levels were reduced by exercise while increased in MI. In vitro, HIPK2 suppression by lentiviral vectors or inhibitor prevented apoptosis induced by OGD/R in NRCMs and hESC-CMs. HIPK2 inhibitor-treated mice and HIPK2-/- mice reduced infarct size after acute MI, and preserved cardiac function in MI remodeling. Mechanistically, protective effect against apoptosis by HIPK2 suppression was reversed by P53 activators. Furthermore, increasing levels of miR-222, targeting HIPK2, protected post-MI cardiac dysfunction, whereas cardiac dysfunction post-MI was aggravated in miR-222-/- rats. Moreover, serum miR-222 levels were significantly reduced in MI patients, as well as in MI patients that were readmitted to the hospital and/or died compared to those not. INTERPRETATION: Exercise-induced HIPK2 suppression attenuates cardiomyocytes apoptosis and protects MI by decreasing P-P53. Inhibition of HIPK2 represents a potential novel therapeutic intervention for MI. FUNDING: This work was supported by the grants from National Key Research and Development Project (2018YFE0113500 to JJ Xiao), National Natural Science Foundation of China (82020108002, 81722008, and 81911540486 to JJ Xiao, 81400647 to MJ Xu, 81800265 to YJ Liang), Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-09-E00042 to JJ Xiao), the grant from Science and Technology Commission of Shanghai Municipality (18410722200 and 17010500100 to JJ Xiao), the "Dawn" Program of Shanghai Education Commission (19SG34 to JJ Xiao), Shanghai Sailing Program (21YF1413200 to QL Zhou). JS is supported by Horizon2020 ERC-2016-COG EVICARE (725229).
Assuntos
Proteínas de Transporte/genética , Regulação para Baixo , Exercício Físico/fisiologia , MicroRNAs/sangue , MicroRNAs/genética , Infarto do Miocárdio/genética , Proteínas Serina-Treonina Quinases/genética , Adulto , Animais , Animais Recém-Nascidos , Proteínas de Transporte/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Dependovirus/genética , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Células-Tronco Embrionárias Humanas/química , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Camundongos , Pessoa de Meia-Idade , Infarto do Miocárdio/induzido quimicamente , Infarto do Miocárdio/terapia , Miócitos Cardíacos/química , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Corrida/fisiologia , Natação/fisiologiaRESUMO
Genetic lineage tracing is accomplished using bi-transgenic mice, where one allele is altered to express Cre recombinase, and another allele encodes a Cre-dependent genetic reporter protein. Once Cre is activated (constitutive or in response to tamoxifen), the marker gene-expressing cells become indelibly labeled by the reporter protein. Therefore, daughter cells derived from labeled cells are permanently labeled even if the marker gene that drove Cre recombinase expression is no longer expressed in these cells. This system is commonly used to label putative progenitor cells and determine the fate of their progeny. Here, we describe the use of c-kit-based genetic lineage-tracing mouse line as an example and discuss caveats for performing these types of experiments.
Assuntos
Linhagem da Célula/genética , Rastreamento de Células/métodos , Células-Tronco/química , Células-Tronco/metabolismo , Animais , Expressão Gênica , Genes Reporter , Ligação Genética , Proteínas de Fluorescência Verde/genética , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Animais , Miócitos Cardíacos/química , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-kit/análise , Proteínas Proto-Oncogênicas c-kit/genética , Células-Tronco/citologia , Tamoxifeno/farmacologiaRESUMO
Neuregulin protein 1 (NRG1) is a large (> 60-amino-acid) natural peptide ligand for the ErbB protein family members HER3 and HER4. We developed an agonistic antibody modality, termed antibody ligand mimetics (ALM), by incorporating complex ligand agonists such as NRG1 into an antibody scaffold. We optimized the linker and ligand length to achieve native ligand activity in HEK293 cells and cardiomyocytes derived from induced pluripotent stem cells (iPSCs) and used a monomeric Fc-ligand fusion platform to steer the ligand specificity toward HER4-dominant agonism. With the help of selectivity engineering, these enhanced ALM molecules can provide an antibody scaffold with increased receptor specificity and the potential to greatly improve the pharmacokinetics, stability, and downstream developability profiles from the natural ligand approach. This ligand mimetic design and optimization approach can be expanded to apply to other cardiovascular disease targets and emerging therapeutic areas, providing differentiated drug molecules with increased specificity and extended half-life.
Assuntos
Anticorpos Monoclonais/química , Neuregulina-1/química , Receptor ErbB-4/agonistas , Anticorpos Monoclonais/metabolismo , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/química , Células-Tronco Pluripotentes Induzidas/metabolismo , Cinética , Ligantes , Miócitos Cardíacos/química , Miócitos Cardíacos/metabolismo , Neuregulina-1/metabolismo , Ligação Proteica , Receptor ErbB-4/metabolismo , Transdução de SinaisRESUMO
Delivery of macromolecular nucleotides into the living cells holds a great promise for the development of new therapeutics. However, its abilities for adoptive immunotherapy, cell reprogramming, and primary cell transfection have been long-term hindered by the lack of a system that can locally deliver engineered therapeutic nucleotides (e.g., plasmids, siRNAs, miRNAs) without causing any side effects. In this chapter, the performance of a novel 3D nanoelectroporation system (3D NEP) is highlighted in three scenarios-adoptive immunotherapy, cell reprogramming, and adult mouse primary cardiomyocyte transfection. Detailed protocols were given to introduce the 3D NEP system assembly, as well as their applications in (1) natural killer (NK) cells transfection by delivery of chimeric antigen receptor (CAR) plasmids; (2) mouse embryonic fibroblasts transfection with OSKM factors; and (3) miR-29b molecular beacon (BMs) delivery into primary cardiomyocytes for interrogating the side effect of miR-29b-assisted treatment.
Assuntos
Eletroporação/instrumentação , Fibroblastos/citologia , Células Matadoras Naturais/citologia , Miócitos Cardíacos/citologia , Nucleotídeos/administração & dosagem , Animais , Células Cultivadas , Técnicas de Reprogramação Celular/instrumentação , Técnicas de Reprogramação Celular/métodos , Fibroblastos/química , Humanos , Imunoterapia Adotiva/instrumentação , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/química , Camundongos , Miócitos Cardíacos/química , Nanotecnologia , Transfecção/instrumentação , Transfecção/métodosRESUMO
Genetics is a significant factor contributing to congenital heart disease (CHD), but our understanding of the genetic players and networks involved in CHD pathogenesis is limited. Here, we searched for de novo copy number variations (CNVs) in a cohort of 167 CHD patients to identify DNA segments containing potential pathogenic genes. Our search focused on new candidate disease genes within 19 deleted de novo CNVs, which did not cover known CHD genes. For this study, we developed an integrated high-throughput phenotypical platform to probe for defects in cardiogenesis and cardiac output in human induced pluripotent stem cell (iPSC)-derived multipotent cardiac progenitor (MCPs) cells and, in parallel, in the Drosophila in vivo heart model. Notably, knockdown (KD) in MCPs of RPL13, a ribosomal gene and SON, an RNA splicing cofactor, reduced proliferation and differentiation of cardiomyocytes, while increasing fibroblasts. In the fly, heart-specific RpL13 KD, predominantly at embryonic stages, resulted in a striking 'no heart' phenotype. KD of Son and Pdss2, among others, caused structural and functional defects, including reduced or abolished contractility, respectively. In summary, using a combination of human genetics and cardiac model systems, we identified new genes as candidates for causing human CHD, with particular emphasis on ribosomal genes, such as RPL13. This powerful, novel approach of combining cardiac phenotyping in human MCPs and in the in vivo Drosophila heart at high throughput will allow for testing large numbers of CHD candidates, based on patient genomic data, and for building upon existing genetic networks involved in heart development and disease.
Assuntos
Variações do Número de Cópias de DNA , Cardiopatias Congênitas/genética , Miocárdio/citologia , Proteínas de Neoplasias/genética , Proteínas Ribossômicas/genética , Animais , Células Cultivadas , Estudos de Coortes , Modelos Animais de Doenças , Drosophila , Feminino , Redes Reguladoras de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/química , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/química , Miócitos Cardíacos/citologia , Miócitos Cardíacos/patologia , Estudos RetrospectivosRESUMO
Recent developments of novel targeted therapies are contributing to the increased long-term survival of cancer patients; however, drug-induced cardiotoxicity induced by cancer drugs remains a serious problem in clinical settings. Nevertheless, there are few in vitro cell-based assays available to predict this toxicity, especially from the aspect of morphology. Here, we developed a simple two-dimensional (2D) morphological assessment system, 2DMA, to predict drug-induced cardiotoxicity in cancer patients using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with image-based high-content analysis in a high-throughput manner. To assess the effects of drugs on cardiomyocytes, we treated iPSC-CMs with 28 marketed pharmaceuticals and measured two key parameters: number of cell nuclei and sarcomere morphology. Drugs that significantly perturbed these two parameters at concentrations ≤30 times the human Cmax value were regarded as positive in the test. Based on these criteria, the sensitivity and specificity of the 2DMA system were 81% and 100%, respectively. Moreover, the translational predictability of 2DMA was comparable with that of a three-dimensional cardiotoxicity assay. RNA sequencing further revealed that the expression levels of several genes related to sarcomere components decreased following treatment with sunitinib, suggesting that inhibition of the synthesis of proteins that comprise the sarcomere contributes to drug-induced sarcomere disruption. Based on these features, the 2DMA system provides mechanistic insight with high predictability of cancer drug-induced cardiotoxicity in humans, and could thus contribute to the reduction of drug attrition rates at early stages of drug development.
Assuntos
Antineoplásicos/toxicidade , Cardiotoxinas/toxicidade , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Microscopia Eletrônica/métodos , Miócitos Cardíacos/efeitos dos fármacos , Cardiotoxicidade/patologia , Técnicas de Cultura de Células/métodos , Células Cultivadas , Corantes Fluorescentes/análise , Previsões , Humanos , Células-Tronco Pluripotentes Induzidas/química , Células-Tronco Pluripotentes Induzidas/patologia , Miócitos Cardíacos/química , Miócitos Cardíacos/patologiaRESUMO
Extracellular vesicles (EVs) generated from redox active anticancer drugs are released into the extracellular environment. These EVs contain oxidized molecules and trigger inflammatory responses by macrophages. Using a mouse model of doxorubicin (DOX)-induced tissue injury, we previously found that the major sources of circulating EVs are from heart and liver, organs that are differentially affected by DOX. Here, we investigated the effects of EVs from cardiomyocytes and those from hepatocytes on macrophage activation. EVs from H9c2 rat cardiomyocytes (H9c2 EVs) and EVs from FL83b mouse hepatocytes (FL83â¯bâ¯EVs) have different levels of protein-bound 4-hydroxynonenal and thus different immunostimulatory effects on mouse RAW264.7 macrophages. H9c2 EVs but not FL83â¯bâ¯EVs induced both pro-inflammatory and anti-inflammatory macrophage activation, mediated by NFκB and Nrf-2 pathways, respectively. DOX enhanced the effects of H9c2 EVs but not FL83â¯bâ¯EVs. While EVs from DOX-treated H9c2 cells (H9c2 DOXEVs) suppressed mitochondrial respiration and increased glycolysis of macrophages, EVs from DOX-treated FL83b cells (FL83b DOXEVs) enhanced mitochondrial reserve capacity. Mechanistically, the different immunostimulatory functions of H9c2 EVs and FL83â¯bâ¯EVs are regulated, in part, by the redox status of the cytoplasmic thioredoxin 1 (Trx1) of macrophages. H9c2 DOXEVs lowered the level of reduced Trx1 in cytoplasm while FL83b DOXEVs did the opposite. Trx1 overexpression alleviated the effect of H9c2 DOXEVs on NFκB and Nrf-2 activation and prevented the upregulation of their target genes. Our findings identify EVs as a novel Trx1-mediated redox mediator of immune response, which greatly enhances our understanding of innate immune responses during cancer therapy.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Vesículas Extracelulares/imunologia , Hepatócitos/química , Miócitos Cardíacos/química , Tiorredoxinas/imunologia , Aldeídos/imunologia , Aldeídos/metabolismo , Aldeídos/farmacologia , Animais , Linhagem Celular , Meios de Cultivo Condicionados/química , Vesículas Extracelulares/química , Regulação da Expressão Gênica , Glicólise/efeitos dos fármacos , Hepatócitos/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Oxirredução , Células RAW 264.7 , Ratos , Tiorredoxinas/genéticaRESUMO
Autologous induced pluripotent stem cells (iPSCs) constitute an unlimited cell source for patient-specific cell-based organ repair strategies. However, their generation and subsequent differentiation into specific cells or tissues entail cell line-specific manufacturing challenges and form a lengthy process that precludes acute treatment modalities. These shortcomings could be overcome by using prefabricated allogeneic cell or tissue products, but the vigorous immune response against histo-incompatible cells has prevented the successful implementation of this approach. Here we show that both mouse and human iPSCs lose their immunogenicity when major histocompatibility complex (MHC) class I and II genes are inactivated and CD47 is over-expressed. These hypoimmunogenic iPSCs retain their pluripotent stem cell potential and differentiation capacity. Endothelial cells, smooth muscle cells, and cardiomyocytes derived from hypoimmunogenic mouse or human iPSCs reliably evade immune rejection in fully MHC-mismatched allogeneic recipients and survive long-term without the use of immunosuppression. These findings suggest that hypoimmunogenic cell grafts can be engineered for universal transplantation.
Assuntos
Diferenciação Celular/imunologia , Rejeição de Enxerto/imunologia , Antígenos HLA/genética , Células-Tronco Pluripotentes Induzidas/transplante , Animais , Diferenciação Celular/genética , Rejeição de Enxerto/genética , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Camundongos , Miócitos Cardíacos/química , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Transplante Homólogo/métodosRESUMO
Previous studies have demonstrated that ovariectomy may lead to a reduction in antioxidative biomarkers in the myocardium, thus suggesting that estrogens may serve a protective role in the suppression of oxidative stress. Lycium barbarum polysaccharides (LBP) are a wellknown antioxidant Chinese traditional medicine, which appear to have a similar function to estrogens with regards to the regulation of cardiac function. In the present study, 30 SpragueDawley rats were randomly divided into the following groups: Sham operation group, ovariectomized (OVX) group, estradiol valerate group, highdose LBP (LBPH) group and lowdose LBP (LBPL) group. All of the rats were provided tap water, estradiol valerate or LBP for 12 weeks. In addition, all rats were ovariectomized, with the exception of rats in the sham operation group, which underwent fat removal only. Reactive oxygen species (ROS), malondialdehyde (MDA), glutathione peroxidase (GSHpx), catalase (CAT) and superoxide dismutase activities were subsequently examined. The protein expression levels of cleaved caspase9, cleaved caspase3 and phosphorylatedprotein kinase B (pAkt) were also assessed. The results demonstrated that highdose LBP decreased the enhanced levels of ROS and MDA in OVX rats, whereas GSHpx and CAT activities were increased in the LBPH group compared with in OVX rats. Furthermore, the expression levels of cleaved caspase9 and cleaved caspase3 were significantly upregulated in the OVX group, whereas highdose LBP exerted protective effects on OVX rats by decreasing the expression of apoptotic proteins. Conversely, pAkt expression was decreased in the OVX group and was increased in the LBPH group. These results indicated that LBP is essentially involved in cardiac protection by inhibiting apoptosis in response to oxidative stress. In addition, improvement of antioxidant status by LBP is associated with the Akt signaling pathway in the myocardium of OVX rats.
Assuntos
Estrogênios/metabolismo , Coração/efeitos dos fármacos , Lycium/química , Polissacarídeos/administração & dosagem , Animais , Antioxidantes/administração & dosagem , Apoptose/efeitos dos fármacos , Humanos , Medicina Tradicional Chinesa , Miocárdio/patologia , Miócitos Cardíacos/química , Ovariectomia , Estresse Oxidativo/efeitos dos fármacos , Polissacarídeos/química , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Heart neoplasms are uncommon and usually benign. Hamartoma of mature cardiac myocytes is an unusual lesion with only a few reported cases. It is a heterogeneous mixture of well-differentiated myocytes, fibroblasts, adipocytes and blood vessels. We present a case of hamartoma of mature cardiac myocytes and a concise review of the pertinent literature. A multi-lobulated polypoid tumour attached to the wall of the right atrium was found during an autopsy of a young woman. Microscopy revealed cardiomyocytes, fibrous connective tissue and well-differentiated adipocytes. The immunohistochemical study had a positive immunoreactivity for desmin, muscle-specific actin (HHF-35) and CD34 markers, showing the different types of mesenchymal cells involved. This combination of markers has not been previously used. Other tumours, such as cardiac rhabdomyoma and cardiac myxoma were ruled out due to the differences in histological characteristics and clinical presentation.
Assuntos
Cardiomiopatias/patologia , Hamartoma/patologia , Miócitos Cardíacos/patologia , Actinas/análise , Antígenos CD34/análise , Autopsia , Biomarcadores/análise , Desmina/análise , Evolução Fatal , Feminino , Hamartoma/química , Humanos , Imuno-Histoquímica , Miócitos Cardíacos/química , Adulto JovemRESUMO
Cardiovascular diseases are the main cause of death worldwide, demanding new treatments and interventions. Recently, extracellular vesicles (EVs) came in focus as important carriers of protective molecules such as miRNAs and proteins which might contribute to e.g. improved cardiac function after myocardial infarction. EVs can be secreted from almost every cell type in the human body and can be transferred via the bloodstream in almost every compartment. To provide an all-encompassing overview of studies investigating these beneficial properties of EVs we performed a systematic review/meta-analysis of studies investigating the cardioprotective characteristics of EVs. Forty-three studies were investigated and catalogued according to the EV source. We provide an in-depth analysis of the purification method, size of the EVs, the conducted experiments to investigate the beneficial properties of EVs as well as the major effector molecule encapsulated in EVs mediating protection. This study provides evidence that EVs from different cell types and body fluids provide cardioprotection in different in vivo and in vitro studies. A meta-analysis was performed to estimate the underlying effect size. In conclusion, we demonstrated that EVs from different sources might serve as a promising tool for treating cardiovascular diseases in the future.
Assuntos
Cardiotônicos/uso terapêutico , Vesículas Extracelulares/fisiologia , Angina Estável/sangue , Animais , Líquidos Corporais , Cardiotônicos/farmacologia , Fracionamento Celular , Linhagem Celular , Avaliação de Medicamentos , Vesículas Extracelulares/química , Fibroblastos/química , Fibroblastos/ultraestrutura , Humanos , Precondicionamento Isquêmico Miocárdico/métodos , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/ultraestrutura , Infarto do Miocárdio/sangue , Infarto do Miocárdio/terapia , Miócitos Cardíacos/química , Miócitos Cardíacos/ultraestrutura , Especificidade de Órgãos , Estresse OxidativoRESUMO
The capability to on-line sense tissue function, provide stimulation to control contractility and efficiently release drugs within an engineered tissue microenvironment may enhance tissue assembly and improve the therapeutic outcome of implanted engineered tissues. To endow cardiac patches with such capabilities we developed elastic, biodegradable, electronic scaffolds. The scaffolds were composed of electrospun albumin fibers that served as both a substrate and a passivation layer for evaporated gold electrodes. Cardiomyocytes seeded onto the electronic scaffolds organized into a functional cardiac tissue and their function was recorded on-line. Furthermore, the electronic scaffolds enabled to actuate the engineered tissue to control its function and trigger the release of drugs. Post implantation, these electronic scaffolds degraded, leading to the dissociation of the inorganic material from within the scaffold. Such technology can be built upon to create a variety of degradable devices for tissue engineering of various tissues, as well as pristine cell-free devices with electronic components for short-term in vivo use.
Assuntos
Miócitos Cardíacos/citologia , Alicerces Teciduais/química , Albuminas/química , Animais , Materiais Biocompatíveis/química , Adesão Celular , Proliferação de Células , Dexametasona/química , Portadores de Fármacos , Liberação Controlada de Fármacos , Eletrodos , Ouro/química , Coração , Masculino , Miócitos Cardíacos/química , Miócitos Cardíacos/metabolismo , Polímeros/química , Pirróis/química , Ratos Sprague-Dawley , Propriedades de Superfície , Engenharia Tecidual/métodosRESUMO
RATIONALE: Autologous bone marrow mesenchymal stem cells (MSCs) and c-kit+ cardiac progenitor cells (CPCs) are 2 promising cell types being evaluated for patients with heart failure (HF) secondary to ischemic cardiomyopathy. No information is available in humans about the relative efficacy of MSCs and CPCs and whether their combination is more efficacious than either cell type alone. OBJECTIVE: CONCERT-HF (Combination of Mesenchymal and c-kit+ Cardiac Stem Cells As Regenerative Therapy for Heart Failure) is a phase II trial aimed at elucidating these issues by assessing the feasibility, safety, and efficacy of transendocardial administration of autologous MSCs and CPCs, alone and in combination, in patients with HF caused by chronic ischemic cardiomyopathy (coronary artery disease and old myocardial infarction). METHODS AND RESULTS: Using a randomized, double-blinded, placebo-controlled, multicenter, multitreatment, and adaptive design, CONCERT-HF examines whether administration of MSCs alone, CPCs alone, or MSCs+CPCs in this population alleviates left ventricular remodeling and dysfunction, reduces scar size, improves quality of life, or augments functional capacity. The 4-arm design enables comparisons of MSCs alone with CPCs alone and with their combination. CONCERT-HF consists of 162 patients, 18 in a safety lead-in phase (stage 1) and 144 in the main trial (stage 2). Stage 1 is complete, and stage 2 is currently randomizing patients from 7 centers across the United States. CONCLUSIONS: CONCERT-HF will provide important insights into the potential therapeutic utility of MSCs and CPCs, given alone and in combination, for patients with HF secondary to ischemic cardiomyopathy. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02501811.
Assuntos
Insuficiência Cardíaca/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Miócitos Cardíacos/citologia , Transplante de Células-Tronco/métodos , Terapia Combinada/métodos , Método Duplo-Cego , Estudos de Viabilidade , Insuficiência Cardíaca/etiologia , Humanos , Isquemia Miocárdica/complicações , Miócitos Cardíacos/química , Proteínas Proto-Oncogênicas c-kit , Projetos de Pesquisa , Transplante Autólogo , Resultado do Tratamento , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/terapia , Remodelação VentricularRESUMO
Cardiovascular diseases are responsible for almost one third of all global deaths yearly, and therefore are largely studied. Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) have emerged as an exciting technology for cardiac disease modelling and personalised therapy. Nevertheless, issues concerning functional and molecular maturation are still faced. In addition to this, differentiation protocols generally yield a heterogeneous mixed population comprised of nodal, atrial and ventricular-like subtypes, being unsuitable for therapeutic purposes. Bradykinin (BK) is a vasoactive peptide which exerts important physiological roles in the cardiovascular system, having been previously described as important for cellular, keratinocyte and skeletal muscle differentiation. This project performed in cooperation with PluriCell Biotech, a startup specialized in the production and differentiation of hiPSC-CM, has sought (1) characterizing gene and protein expression of molecular markers of maturation and of subtype specification throughout of differentiation; (2) Assessing the electrical functionality of hiPSC-CM through the characterization of subtype-specific action potentials (APs) and (3) Investigating whether the progress of hiPSCCM maturation is regulated by BK through kinin-B2 receptors (B2R). Our results have validated the model that proposes a developmental-dependent switch between skeletal (ssTnI) and cardiac (cTnI) isoforms of troponin I as differentiation progresses, at least to some extent. Furthermore, prolonged time in culture has resulted in higher levels of expression of the ventricular marker MLC2v and in increased rates of ventricular-like action APs. Electrophysiological analysis of hiPSC-CM reveals a mixed population with AP morphologies correspondent to nodal, atrial and ventricular subtypes, all showing pronounced automaticity as well as other features of immature cardiomyocytes, such as low amplitude and depolarization velocity. Such findings are coherent with those from other groups who have attempted to differentiate mature native-like cardiac cells from pluripotent stem cells sources, without fully succeeding. After showing that differentiating hiPSC-CM express a functional and responsive B2R, the receptor was subjected to chronic activation with 10µM BK and 1µM BK or inhibition with 5µM Firazyr+BK. Even though B2R modulation has not interfered negatively with differentiation yields nor cell morphology, analysis of gene andprotein expression of ssTnI or cTnI and of the ventricular marker MLC2v, have revealed no significant results in comparison to untreated controls. This suggests that BK does not interfere on hiPSC-CM maturation nor subtype specification, although we cannot rule out that it could be leading to other unexplored effects. We recommend a closer look into which intracellular signalling pathways become active upon B2R stimulation in hiPSC-CM, in order to narrow down cellular processes for further investigation
Doenças cardiovasculares são responsáveis por quase um terço de todas as mortes globais anualmente, e por isto o sistema cardiovascular é amplamente estudado. Cardiomiócitos derivados a partir de células-tronco pluripotentes induzidas humanas (hiPSCCM) emergiram como uma promissora tecnologia para modelagem de doenças cardíacas e terapia personalizada. No entanto, desafios acerca de sua maturação funcional e molecular ainda são enfrentados. Além disso, protocolos de diferenciação geralmente levam à obtenção de populações heterogêneas contendo células com fenótipos similares aos de cardiomiócitos nodais, atriais e ventriculares sendo, portanto, inapropriadas para fins terapêuticos. A bradicinina (BK) é um peptídio vasoativo que exerce importantes papeis fisiológicos no sistema cardiovascular, além de ter sido previamente descrita como importante para a diferenciação neuronal, de queratinócitos e de músculo esquelético. Este projeto foi realizado em colaboração com a empresa PluriCell Biotech, uma startup especializada na produção e diferenciação de hiPSC-CM, e buscou (1) caracterizar a expressão gênica e proteíca de marcadores moleculares de maturação e de especificação de subtipos cardíacos durante a diferenciação; (2) avaliar a funcionalidade elétrica de hiPSC-CM por meio da caracterização de seus potenciais de ação (PAs) e (3) Investigar se o progresso da diferenciação de hiPSCCM é regulado por bradicinina por meio do receptor B2 (B2R). Nossos resultados validaram o modelo que propõe um switch na expressão das isoformas funcionais de troponina I esquelética (ssTnI) e cardíaca (cTnI), durante o desenvolvimento e diferenciação celular, pelo menos parcialmente. Além disso, tempo prolongado em cultura resultou em maiores níveis de expressão do marcador ventricular MLC2v, assim como maiores frequências de PAs com morfologias similares a de cardiomiócitos ventriculares. Análise eletrofisiológica de hiPSCCM revelam a existência de uma população mista contendo PAs correspondentes aos subtipos nodais, atriais e ventriculares, assim como pronunciada automaticidade e outros atributos típicos de cardiomiócitos imaturos, como baixa amplitude e devagar velocidade de despolarização. Estes resultados são coerentes com os de outros grupos que ainda não foram totalmente bem-sucedidos em diferenciar células cardíacas maduras similares acardiomiócitos nativos a partir de células-troncos pluripotentes. Após mostrar que as hiPSCCM expressam receptores B2 funcionais e responsivos, submetemos o receptor a uma ativação crônica com BK 10µM e BK 1µM ou inibição crônica com Firazyr 5µM + BK. Apesar da modulação do B2R não ter interferido de forma negativa no rendimento da diferenciação ou na morfologia celular, análise de expressão gênica e proteica de ssTnI e cTnI e do marcador ventricular MLC2v não revelou resultados significativos em comparação aos controles não-tratados. Isto sugere que a BK não interfere na maturação e especificação de subtipos cardíacos em hiPSC-CM, apesar de não podermos ignorar o fato de que ela poderia estar desencadeando outros efeitos inexplorados. Nós recomendamos um estudo mais aprofundado acerca de quais vias de sinalização se tornam ativas após estimulação do receptor B2 em hiPSC-CM, com o objetivo de afunilar quais processos celulares poderiam ser investigados em uma próxima etapa deste estudo
Assuntos
Miócitos Cardíacos/química , Receptor B2 da Bradicinina/análise , Cininas/efeitos adversos , Bradicinina/fisiologia , Doenças Cardiovasculares/patologia , Sistema Cardiovascular , Eletrofisiologia/instrumentação , Células-Tronco Pluripotentes InduzidasRESUMO
Dilated cardiomyopathy (DCM) leads to disturbed contraction and force transduction, and is associated with substantial mortality in all age groups. Involvement of a disrupted composition of the intercalated disc (ID) has been reported. However, in children, little is established about such subcellular changes during disease, because of the pathological mix-up with the ongoing cardiac maturation. This leaves maladaptive remodeling often undetected. We aimed at illustrating subcellular alterations in children diagnosed with DCM compared to age-matched controls, focusing on ID proteins known to be crucially stable under healthy conditions and destabilized during cardiac injury in adults. Left ventricular or septal pediatric specimens were collected from 7 individuals diagnosed with DCM (age: 23 weeks in utero to 8 weeks postnatal) and age-matched controls that died of non-cardiovascular cause. We determined the amount of fibrosis and localization of ID proteins by immunohistochemistry. In pediatric DCM, most ID proteins follow similar spatiotemporal changes in localization as in controls. However, although no mutations were found, the signal of the desmosomal protein Desmoglein-2 was reduced in all pediatric DCM specimens, but not in controls or adult DCM patients. Endocardial and transmural fibrosis was increased in all pediatric DCM patients compared to age-matched controls. Composition of the ID in pediatric DCM patients is similar to controls, except for the localization of Desmoglein-2 and presence of severe fibrosis. This suggests that the architecture of desmosomes is already disturbed in the early stages of DCM. These findings contribute to the understanding of pediatric DCM.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Desmogleína 2/análise , Desmossomos/química , Miócitos Cardíacos/química , Fatores Etários , Autopsia , Biópsia , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/mortalidade , Estudos de Casos e Controles , Desmossomos/patologia , Regulação para Baixo , Feminino , Fibrose , Imunofluorescência , Predisposição Genética para Doença , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Miócitos Cardíacos/patologia , FenótipoRESUMO
Microscopy in combination with contrast-increasing dyes allows the visualization and analysis of organs, tissues, and various cells. Because of their better resolution, the development of confocal and laser microscopes enables the investigations of cell components, which are labeled with fluorescent dyes. The imaging of living cells on subcellular level (also in vivo) needs a labeling by gene transfection of GFP or similar labeled proteins. We present a method for visualization of cell structure in skeletal and heart muscle by label-free Second Harmonic Generation (SHG) microscopy and describe analytic methods for quantitative measurements of morphology and dynamics in skeletal muscle fibers.
Assuntos
Fibras Musculares Esqueléticas/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Microscopia de Geração do Segundo Harmônico/métodos , Animais , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Imageamento Tridimensional , Microscopia Confocal , Fibras Musculares Esqueléticas/química , Miócitos Cardíacos/química , Miosinas/química , Fótons , Sarcômeros/químicaRESUMO
Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal distribution of cAMP at the subcellular level could be important for developing new strategies for the prevention or treatment of unfavorable responses associated with different disease states.
Assuntos
Simulação por Computador , AMP Cíclico/química , AMP Cíclico/metabolismo , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Biologia Computacional , Camundongos , Miócitos Cardíacos/química , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismoRESUMO
BACKGROUND: Fabry disease is caused by mutations in the α-galactosidase A (GLA) gene, which is located in X-chromosome coding for the lysosomal enzyme of GLA. Among many gene mutations, E66Q mutation is under discussion for its pathogenicity because there is no clinical report showing pathological evidence of Fabry disease with E66Q mutation. CASE PRESENTATION: A 65-year-old Japanese female was referred to our hospital for chest discomfort on effort. Transthoracic echocardiography showed severe left ventricular (LV) hypertrophy with LV outflow obstruction. Maximum LV outflow pressure gradient was 87 mmHg, and Valsalva maneuver increased the pressure gradient up to 98 mmHg. According to medical interview, one of her younger sister and a nephew died suddenly at age 42 and 36, respectively. Another younger sister also presented LV hypertrophy with outflow obstruction. Maximum LV outflow pressure gradient was 100 mmHg, and the E66Q mutation was detected similar to the case. Endomyocardial biopsy specimens presented vacuolation of cardiomyocytes, in which zebra bodies were detected by electron microscopic examination. Although the enzymatic activity of GLA was within normal range, the c. 196G>C nucleotide change, which lead to the E66Q mutation of GLA gene, was detected. We initially diagnosed her as cardiac Fabry disease based on the findings of zebra body. However, immunostaining showed few deposition of globotriaosylceramide in left ventricular myocardium, and gene mutations in the disease genes for hypertrophic cardiomyopathy (HCM), MYBPC3 and MYH6, were detected. Although the pathogenicity of the E66Q mutation cannot be ruled out, hypertrophic obstructive cardiomyopathy (HOCM) was more reasonable to explain the pathophysiology in the case. CONCLUSIONS: This is the confusable case of HOCM with Fabry disease with the GLA E66Q mutation. We have to take into consideration the possibility that some patients with the E66Q mutation may have similar histological findings of Fabry disease, and should be examed the possibility for harboring gene mutations associated with HCM.
Assuntos
Cardiomiopatia Hipertrófica Familiar/genética , Doença de Fabry/genética , Mutação , Miócitos Cardíacos/ultraestrutura , alfa-Galactosidase/genética , Idoso , Biópsia , Cardiomiopatia Hipertrófica Familiar/diagnóstico , Cardiomiopatia Hipertrófica Familiar/enzimologia , Cardiomiopatia Hipertrófica Familiar/fisiopatologia , Análise Mutacional de DNA , Ecocardiografia Doppler em Cores , Doença de Fabry/diagnóstico , Doença de Fabry/enzimologia , Doença de Fabry/fisiopatologia , Feminino , Marcadores Genéticos , Predisposição Genética para Doença , Hereditariedade , Humanos , Microscopia Eletrônica , Miócitos Cardíacos/química , Linhagem , Fenótipo , Valor Preditivo dos Testes , Triexosilceramidas/análiseRESUMO
BACKGROUND: Host autoimmune activity in myocarditis has been proposed to play a role in development of cardiac disease, but evidence of autoimmunity and relationship to outcomes have not been evaluated in pediatric myocarditis. METHODS: We performed a multi-institutional study of children with clinical myocarditis. Newly diagnosed patients were followed for up to 12 months and previously diagnosed patients at a single follow-up for serum levels of autoantibodies to human cardiac myosin, beta-adrenergic receptors 1 and 2, muscarinic-2 receptors, and antibody-mediated protein kinase A (PKA) activation in heart cells in culture. Results were compared with those of healthy control children. RESULTS: Both previously diagnosed patient at follow-up (P = .0061) and newly diagnosed patients at presentation (P = .0127) had elevated cardiac myosin antibodies compared with control subjects. Antibody levels were not associated with recovery status at follow-up in either group. PKA activation was higher at presentation in the newly diagnosed patients who did not recovery normal function (P = .042). CONCLUSIONS: Children with myocarditis have evidence of autoantibodies against human cardiac myosin at diagnosis and follow-up compared with control subjects. Differences in antibody-mediated cell signaling may contribute to differences in patient outcomes, as suggested by elevated antibody-mediated PKA activation in heart cells by the serum from nonrecovered patients.
Assuntos
Autoanticorpos/imunologia , Autoimunidade , Miosinas Cardíacas/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/imunologia , Miocardite/imunologia , Miócitos Cardíacos/imunologia , Autoanticorpos/sangue , Criança , Pré-Escolar , Proteínas Quinases Dependentes de AMP Cíclico/análise , Coração/diagnóstico por imagem , Coração/fisiopatologia , Humanos , Lactente , Miocardite/sangue , Miocardite/diagnóstico por imagem , Miócitos Cardíacos/químicaRESUMO
Heart diseases are one of the main causes of death around the world. The great challenge for scientists is to develop new therapeutic methods for these types of ailments. Stem cells (SCs) therapy could be one of a promising technique used for renewal of cardiac cells and treatment of heart diseases. Conventional in vitro techniques utilized for investigation of heart regeneration do not mimic natural cardiac physiology. Lab-on-a-chip systems may be the solution which could allow the creation of a heart muscle model, enabling the growth of cardiac cells in conditions similar to in vivo conditions. Microsystems can be also used for differentiation of stem cells into heart cells, successfully. It will help better understand of proliferation and regeneration ability of these cells. In this review, we present Heart-on-a-chip systems based on cardiac cell culture and stem cell biology. This review begins with the description of the physiological environment and the functions of the heart. Next, we shortly described conventional techniques of stem cells differentiation into the cardiac cells. This review is mostly focused on describing Lab-on-a-chip systems for cardiac tissue engineering. Therefore, in the next part of this article, the microsystems for both cardiac cell culture and SCs differentiation into cardiac cells are described. The section about SCs differentiation into the heart cells is divided in sections describing biochemical, physical and mechanical stimulations. Finally, we outline present challenges and future research concerning Heart-on-a-chip based on stem cell biology.