Transcriptomic analysis (RNA-seq) of early iPSC-CMs between PA-IVS and healthy control.
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ABSTRACT: Transcriptomic analysis by RNA-seq revealed that pathways associated with cell cycle and mitosis were downregulated in day 13 PA-IVS-1v iPSC derived cardiomyocytes.
Project description:Pulmonary atresia with the intact ventricular septum (PA-IVS) is a rare congenital cardiac disease characterized by atresia of the pulmonary valve, resulting in the absence of a connection between the right ventricular outflow tract and pulmonary arteries. PA-IVS is characterized by varying degrees of right ventricular hypoplasia: from single ventricle palliation (1v) to 1½-ventricle palliation (1.5v) and bi-ventricle repair (2v). To understand how cardiac development is impaired in PA-IVS-1v, we generated PA-IVS-1v patient-specific induced pluripotent stem cells (iPSCs). We then ran single-cell RNA-seq to temporally profile transcriptomic changes during cardiac differentiation in healthy control (Control) and PA-IVS-1v iPSCs. We collected differentiating cells at multiple time points corresponding to different development stages, ie. Day 5 (cardiac mesoderm), Day 10 (cardiac progenitor), Day 14 (early cardiomyocyte), and Day 30 (fetal cardiomyocyte). Single-cell transcriptomic analysis indicates that cell lineage commitment of cardiac progenitors is skewed towards epicardial and first heart field progenitors in the differentiating iPSCs from PA-IVS-1v.
Project description:Dilated cardiomyopathy (DCM), a myocardial disorder that can result in progressive heart failure and arrhythmias, is defined by ventricular chamber enlargement and dilatation, and systolic dysfunction. To decipher the basis for the cardiac pathology in titin-mutated patients, we investigated the hypothesis that induced Pluripotent Stem Cell (iPSC)- derived cardiomyocytes (iPSC-CM) generated from patients, recapitulate the disease phenotype.Our findings show that the mutated cardiomyocytes from DCM patients recapitulate abnormalities of the inherited cardiomyopathies.
Project description:The human iPSC line H19101 was differentiated in vitro into cardiomyocytes using a 20-day differentiation protocol (Burridge et al. 2014 PMID 24930130 and Montefiori et al 2018 PMID 29988018 ). 50,000 cardiomyocytes were used in each ATAC-seq experiment. 8 replicates were pooled to obtain the final peak file.
Project description:We used human iPSC-CMs generated from healthy individuals and performed RNA-sequencing after 7 days of trastuzumab treatment to examine the mechanism associated with contraction dysfunction in iPSC-CMs after trastuzumab treatment. Transcriptome analysis revealed the key role of an altered energy metabolism pathway for cardiomyocytes in the disease pathogenesis.
Project description:The generation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offers an unlimited source of patient-specific human cardiomyocytes. However, the use of iPSC-CM in vitro-models to guide the clinic selection of particular drugs for individual patients remains a vision. A major limitation represents the immature phenotype of iPSC-CMs, which alters their sensitivity towards physiological and pathophysiological stimuli, cardioactive drugs or toxic substances, in comparison to adult cardiomyocytes (CMs). In this study, we aim to generate iPSC-CMs with an advanced maturation state by combining the use of MM with the alignment of the cells on nanopatterned surfaces (NP) and induction of an increased contractile workload by electrical stimulation (ES). Under the combined influence of MM, NP and ES, iPSC-CMs develop a more complex cellular structure, increased mitochondrial content and enhanced electrophysiological properties. Furthermore, the response of iPSC-CMs matured under influence of MM, NP and ES to isoprenaline as well as verapamil differs to less mature iPSC-CMs cultured in B27-medium. Taken together, our results reveal that the combination of MM, NP and ES strongly improves the maturation state of iPSC-CMs and that this advanced maturation state critically affects the cell behavior in functional studies as well as response to cardioactive drugs.
Project description:The immaturity of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a major limitation for their use in drug screening to identify pro-arrhythmogenic or cardiotoxic molecules. Here, we demonstrate an approach that combines lipid-enriched maturation medium with a high concentration of calcium, nanopatterning of culture surfaces and electrostimulation to generate iPSC-CMs with advanced electrophysiological, structural and metabolic phenotypes. Systematic testing reveals that electrostimulation is the key driver of enhanced mitochondrial development and metabolic maturation and improved electrophysiological properties of iPSC-CMs. Increased calcium concentration strongly promotes electrophysiological maturation, while nanopatterning primarily facilitates sarcomere organisation with minor effect on electrophysiological properties. Transcriptome analysis reveals that activation of HMCES and TFAM targets contributes to mitochondrial development, whereas downregulation of MAPK/PI3K and SRF targets is associated with iPSC-CM polyploidy. These findings provide mechanistic insights into iPSC-CM maturation, paving the way for pharmacological responses that more closely resemble those of adult CMs.
Project description:N-terminal-acetyltransferases including NAA10 catalyze N-terminal acetylation (Nt-acetylation), an evolutionarily conserved co- and post-translational modification. However, little is known about the role of Nt-acetylation in cardiac homeostasis. To gain insight into cardiac-dependent NAA10 function, we studied a novel NAA10 variant (p.R4S) segregating with QT-prolongation, cardiomyopathy and developmental delay in a large kindred. Here we show that the NAA10R4S variant reduced enzymatic activity, decreased expression levels of NAA10/NAA15 proteins, and destabilized the enzymatic complex NatA. In NAA10R4S/Y-iPSC-CMs, dysregulation of the late sodium and slow rectifying potassium currents caused severe repolarization abnormalities, consistent with clinical QT prolongation. Engineered heart tissues generated from NAA10R4S/Y-iPSC-CMs had significantly decreased contractile force and sarcomeric disorganization, consistent with the pedigree’s cardiomyopathic phenotype. Proteomic studies revealed dysregulation of metabolic pathways and cardiac structural proteins. We identified small molecule and genetic therapies that normalized the phenotype of NAA10R4S/Y-iPSC-CMs. Our study defines novel roles of Nt-acetylation in cardiac regulation and delineates mechanisms underlying QT prolongation, arrhythmia, and cardiomyopathy caused by NAA10 dysfunction.