RESUMO
Myocardial interstitial fibrosis (MIF) is characterized by excessive extracellular matrix (ECM) deposition, increased myocardial stiffness, functional weakening, and compensatory cardiomyocyte (CM) hypertrophy. Fibroblasts (Fbs) are considered the principal source of ECM, but the contribution of perivascular cells, including pericytes (PCs), has gained attention, since MIF develops primarily around small vessels. The pathogenesis of MIF is difficult to study in humans because of the pleiotropy of mutually influencing pathomechanisms, unpredictable side effects, and the lack of available patient samples. Human pluripotent stem cells (hPSCs) offer the unique opportunity for the de novo formation of bioartificial cardiac tissue (BCT) using a variety of different cardiovascular cell types to model aspects of MIF pathogenesis in vitro. Here, we have optimized a protocol for the derivation of hPSC-derived PC-like cells (iPSC-PCs) and present a BCT in vitro model of MIF that shows their central influence on interstitial collagen deposition and myocardial tissue stiffening. This model was used to study the interplay of different cell types-i.e., hPSC-derived CMs, endothelial cells (ECs), and iPSC-PCs or primary Fbs, respectively. While iPSC-PCs improved the sarcomere structure and supported vascularization in a PC-like fashion, the functional and histological parameters of BCTs revealed EC- and PC-mediated effects on fibrosis-related cardiac tissue remodeling.
Assuntos
Diferenciação Celular/genética , Fibrose/terapia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/metabolismo , Neovascularização Patológica/terapia , Órgãos Bioartificiais , Células Endoteliais/citologia , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibrose/genética , Fibrose/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Pericitos/citologia , Pericitos/metabolismo , Sarcômeros/genética , Sarcômeros/metabolismo , Remodelação Ventricular/genéticaRESUMO
Maladaptive cardiac remodeling after myocardial infarction (MI) is increasingly contributing to the prevalence of chronic heart failure. Women show less severe remodeling, a reduced mortality and a better systolic function after MI compared to men. Although sex hormones are being made responsible for these differences, it remains currently unknown how this could be translated into therapeutic strategies. Because we had recently demonstrated that inhibition of the conversion of testosterone to its highly active metabolite dihydrotestosterone (DHT) by finasteride effectively reduces cardiac hypertrophy and improves heart function during pressure overload, we asked here whether this strategy could be applied to post-MI remodeling. We found increased abundance of DHT and increased expression of androgen responsive genes in the mouse myocardium after experimental MI. Treatment of mice with finasteride for 21â¯days (starting 7â¯days after surgery), reduced myocardial DHT levels and markedly attenuated cardiac dysfunction as well as hypertrophic remodeling after MI. Histological and molecular analyses showed reduced MI triggered interstitial fibrosis, reduced cardiomyocyte hypertrophy and increased capillary density in the myocardium of finasteride treated mice. Mechanistically, this was associated with decreased activation of myocardial growth-signaling pathways, a comprehensive normalization of pathological myocardial gene-expression as revealed by RNA deep-sequencing and with direct effects of finasteride on cardiac fibroblasts and endothelial cells. In conclusion, we demonstrated a beneficial role of anti-androgenic treatment with finasteride in post-MI remodeling of mice. As finasteride is already approved for the treatment of benign prostate disease, it could potentially be evaluated as therapeutic strategy for heart failure after MI.
Assuntos
Antagonistas de Androgênios/uso terapêutico , Finasterida/uso terapêutico , Expressão Gênica/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Análise de Variância , Animais , Cardiomegalia/tratamento farmacológico , Linhagem Celular , Di-Hidrotestosterona/metabolismo , Células Endoteliais/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibrose , Células Endoteliais da Veia Umbilical Humana , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Camundongos , Contração Muscular/efeitos dos fármacos , Miocárdio/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Calcium plays a key role in cardiomyocytes (CMs) for the translation of the electrical impulse of an action potential into contraction forces. A rapid, not-invasive fluorescence imaging technology allows for the monitoring of calcium transients in human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) to investigate the cardiac electrophysiology in vitro and after cell transplantation in vivo. The genetically encoded calcium indicators (GECIs) GCaMP6f or RCaMP1h were successfully transfected in the previously established hiPSC line MHHi001-A, together with a cardiac specific antibiotic selection cassette facilitating the monitoring of the calcium handling in highly pure populations of hiPSC-CMs.
Assuntos
Células-Tronco Pluripotentes Induzidas , Potenciais de Ação , Cálcio/metabolismo , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Therapeutic application of induced pluripotent stem cell (iPSC) derivatives requires comprehensive assessment of the integrity of their nuclear and mitochondrial DNA (mtDNA) to exclude oncogenic potential and functional deficits. It is unknown, to which extent mtDNA variants originate from their parental cells or from de novo mutagenesis, and whether dynamics in heteroplasmy levels are caused by inter- and intracellular selection or genetic drift. Sequencing of mtDNA of 26 iPSC clones did not reveal evidence for de novo mutagenesis, or for any selection processes during reprogramming or differentiation. Culture expansion, however, selected against putatively actionable mtDNA mutations. Altogether, our findings point toward a scenario in which intracellular selection of mtDNA variants during culture expansion shapes the mutational landscape of the mitochondrial genome. Our results suggest that intercellular selection and genetic drift exert minor impact and that the bottleneck effect in context of the mtDNA genetic pool might have been overestimated.