RESUMO
The X-linked lysosomal storage disorder Fabry disease originates from GLA gene mutations causing α-galactosidase A enzyme deficiency. Here we generated the GLA knockout hiPSC line MHHi001-A-15 (GLA-KOhiPSC) as an in vitro Fabry disease model by targeting exon 2 of the GLA gene by CRISPR/Cas9 in the established control hiPSC line MHHi001-A. GLA-KOhiPSCs retained the expression of pluripotency markers, trilineage differentiation potential, as well as normal karyotype and stem cell morphology but lacked α-galactosidase A enzyme activity. The GLA-KOhiPSCs represent a potent resource to not only study the Fabry disease manifestation but also screen for novel treatment options.
Assuntos
Sistemas CRISPR-Cas , Doença de Fabry , Células-Tronco Pluripotentes Induzidas , alfa-Galactosidase , Doença de Fabry/genética , Doença de Fabry/patologia , Doença de Fabry/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo , Feminino , Linhagem Celular , Técnicas de Inativação de Genes , Diferenciação CelularRESUMO
Hypertrophic cardiomyopathy (HCM) constitutes the most common genetic cardiac disorder. However, current pharmacotherapeutics are mainly symptomatic and only partially address underlying molecular mechanisms. Circular RNAs (circRNAs) are a recently discovered class of non-coding RNAs and emerged as specific and powerful regulators of cellular functions. By performing global circRNA-specific next generation sequencing in cardiac tissue of patients with hypertrophic cardiomyopathy compared to healthy donors, we identified circZFPM2 (hsa_circ_0003380). CircZFPM2, which derives from the ZFPM2 gene locus, is a highly conserved regulatory circRNA that is strongly induced in HCM tissue. In vitro loss-of-function experiments were performed in neonatal rat cardiomyocytes, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and HCM-patient-derived hiPSC-CMs. A knockdown of circZFPM2 was found to induce cardiomyocyte hypertrophy and compromise mitochondrial respiration, leading to an increased production of reactive oxygen species and apoptosis. In contrast, delivery of recombinant circZFPM2, packaged in lipid-nanoparticles or using AAV-based overexpression, rescued cardiomyocyte hypertrophic gene expression and promoted cell survival. Additionally, HCM-derived cardiac organoids exhibited improved contractility upon CM-specific overexpression of circZFPM2. Multi-Omics analysis further promoted our hypothesis, showing beneficial effects of circZFPM2 on cardiac contractility and mitochondrial function. Collectively, our data highlight that circZFPM2 serves as a promising target for the treatment of cardiac hypertrophy including HCM.
Assuntos
Apoptose , Cardiomiopatia Hipertrófica , Sobrevivência Celular , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , RNA Circular , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , RNA Circular/metabolismo , RNA Circular/genética , Humanos , Animais , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/patologia , Cardiomiopatia Hipertrófica/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Ratos , Apoptose/genética , Células Cultivadas , Espécies Reativas de Oxigênio/metabolismo , RNA/genética , Animais Recém-Nascidos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Mitocôndrias Cardíacas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Fabry disease (FD) is a rare and inherited monogenetic disease caused by mutations in the X-chromosomal alpha-galactosidase A gene GLA concomitant with accumulation of its substrate globotriaosylceramide (Gb3) and multi-organ symptoms. We derived an induced pluripotent stem cell line, MHHi029-A, from a male FD patient carrying a c.959A > T missense mutation in the GLA gene. The hiPSCs show a normal karyotype, expression of pluripotency markers and trilineage differentiation capacity. Importantly, they present the patient-specific mutation in the GLA gene and are therefore a valuable resource for investigating the FD mechanism and identifying novel therapies.