Project description:QT prolongation represents a dangerous and potentially life-threatening electrical event affecting the heart. Thyroid hormones (THs) are critical for cardiac development and heart function. However, little is known about THs influence on ventricular repolarization and controversial effects on QT prolongation are reported. Aim of this study was to characterize the direct effects of THs on the cardiac repolarization of human Induced Pluripotent Stem Cell derived cardiomyocytes (hiPSC-CM). RNA Seq analysis of hiPSC-CM treated with Thyroid Hormone T3 indicates significant deregulation in genes involved in cardiac pathways, in particular in ion homeostasis.
Project description:Energy metabolism is a key aspect of cardiomyocyte biology. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising tool for biomedical application, but they are immature and have not undergone metabolic shift related to early postnatal development. Cultivation of hiPSC-CM in 3D engineered heart tissue (EHT) format leads to morphological maturation. This study compared the mitochondrial and metabolic state of hiPSC-CM in standard 2D culture and the EHT format and determined the influence of contractile activity. HiPSC-CM in EHTs showed ~2-fold higher number of mitochondria (electron microscopy), mitochondrial mass (mitotracker), DNA (Mt-ND1, Mt-ND2), and protein abundance (proteome) than in 2D culture. While hiPSC-CM exhibited the principal ability to use glucose, lactate and fatty acids as energy substrates irrespective of culture format, hiPSC-CM in 3D performed more oxidation of glucose, lactate and fatty acid, and less anaerobic glycolysis. The increase in mitochondrial mass and DNA in 3D was diminished by pharmacological inhibition of contractile force, suggesting that contractile work participates in mitochondrial development hiPSC-CM. In conclusion, contractile work in the EHT format contributes to metabolic maturation of hiPSC-CM.
Project description:DMD is a genetic disease, which leads to muscle weakness and cardiomyopathy. The latter remains incurable, being the main cause of death in DMD, therefore new therapeutic strategies are being sought to provide effective treatment. One of them considers upregulation of utrophin, a protein structurally and functionally homologous to dystrophin. In this study proteomic analysis of dystrophin-deficient and both dystrophin- and utrophin-deficient hiPSC-CM indicated on considerable differences in terms of contraction-related mechanisms. We thus investigated the role of utrophin in the maintenance of electrophysiological properties of DMD hiPSC-CM using the cells with additional utrophin deficiency and with utrophin upregulation. Obtained results indicated on disturbance of calcium handling in DMD hiPSC-CM, even more pronounced in DMD/UTRN KO hiPSC-CM and increased values of AHP in DMD hiPSC-CM. Utrophin upregulation improved both calcium oscillations and AHP values. Our findings highlight utrophin as important in the maintenance of the electrophysiological properties of DMD hiPSC-CM.
Project description:Extracellular Vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, we isolated EV from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors (CPC-EV), immature (CMi-EV) and mature (CMm-EV) cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV were characterized in terms of expression of specific markers, yield, and size. Bioactivity was assessed in human umbilical vein endothelial cells (HUVEC) and hiPSC-CM. Small RNA-Seq was performed to identify the differentially expressed miRNA in the four EV groups. Bioactivity assays showed increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increased hiPSC-CM proliferation. Global miRNA expression profiles corroborated an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification. A stemness maintenance miRNA cluster upregulated in hiPSC-EV was found to target the PTEN/PI3K/AKT pathway. Moreover, hiPSC-EV treatment mediated PTEN suppression and increased AKT phosphorylation. Overall, our findings validate hiPSC as suitable cell biofactories for EV production for cardiac regenerative applications.
Project description:Thyroid Hormone effects on hiPSC-derived cardiomyocytes (hiPSC-CM). Identification of target genes and pathways by RNA-Seq analysis
Project description:To investigate HCM mutation-associated molecular details at the early stage of disease development, we performed bulk RNA-seq analysis at Day 15 of differentiation using RNA isolated from the isogenic control and mutant hiPSC-CMs. We performed pair-end sequencing with >30 million reads per sample with clean read counts of more than 96% for each sample.
Project description:In order to investigate the changes of hiPSC-CMS transcriptome after alcohol treatment and whether losartan has an effect on the changes of hiPSC-CM transcriptome after alcohol treatment, RNAseq was performed on hiPSC-CMs treated with 100mM alcohol and 100mM alcohol co-treated with1uM losartan.
