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Increased predominance of the matured ventricular subtype in embryonic stem cell-derived cardiomyocytes in vivo.

ABSTRACT: Accumulating evidence suggests that human pluripotent stem cell-derived cardiomyocytes can affect "heart regeneration", replacing injured cardiac scar tissue with concomitant electrical integration. However, electrically coupled graft cardiomyocytes were found to innately induce transient post-transplant ventricular tachycardia in recent large animal model transplantation studies. We hypothesised that these phenomena were derived from alterations in the grafted cardiomyocyte characteristics. In vitro experiments showed that human embryonic stem cell-derived cardiomyocytes (hESC-CMs) contain nodal-like cardiomyocytes that spontaneously contract faster than working-type cardiomyocytes. When transplanted into athymic rat hearts, proliferative capacity was lower for nodal-like than working-type cardiomyocytes with grafted cardiomyocytes eventually comprising only relatively matured ventricular cardiomyocytes. RNA-sequencing of engrafted hESC-CMs confirmed the increased expression of matured ventricular cardiomyocyte-related genes, and simultaneous decreased expression of nodal cardiomyocyte-related genes. Temporal engraftment of electrical excitable nodal-like cardiomyocytes may thus explain the transient incidence of post-transplant ventricular tachycardia, although further large animal model studies will be required to control post-transplant arrhythmia.

SUBMITTER: Ichimura H

PROVIDER: S-EPMC7368005 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Increased predominance of the matured ventricular subtype in embryonic stem cell-derived cardiomyocytes in vivo.

Ichimura Hajime H Kadota Shin S Kashihara Toshihide T Yamada Mitsuhiko M Ito Kuniaki K Kobayashi Hideki H Tanaka Yuki Y Shiba Naoko N Chuma Shinichiro S Tohyama Shugo S Seto Tatsuichiro T Okada Kenji K Kuwahara Koichiro K Shiba Yuji Y

Scientific reports 20200717 1

Accumulating evidence suggests that human pluripotent stem cell-derived cardiomyocytes can affect "heart regeneration", replacing injured cardiac scar tissue with concomitant electrical integration. However, electrically coupled graft cardiomyocytes were found to innately induce transient post-transplant ventricular tachycardia in recent large animal model transplantation studies. We hypothesised that these phenomena were derived from alterations in the grafted cardiomyocyte characteristics. In ...[more]

PMID: 32681032

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Project description:In adult cardiomyocytes (CMs), the Na(+)/Ca(2+) exchanger (NCX) is a well-defined determinant of Ca(2+) homeostasis. Developmentally, global NCX knockout in mice leads to abnormal myofibrillar organization, electrical defects, and early embryonic death. Little is known about the expression and function of NCX in human heart development. Self-renewable, pluripotent human embryonic stem cells (hESCs) can serve as an excellent experimental model. However, hESC-derived CMs are highly heterogeneous. A stably lentivirus-transduced hESC line (MLC2v-dsRed) was generated to express dsRed under the transcriptional control of the ventricular-restricted myosin light chain-2v (MLC2v) promoter. Electrophysiologically, dsRed+ cells differentiated from MLC2vdsRed hESCs displayed ventricular action potentials (AP), exclusively. Neither atrial nor pacemaker APs were observed. While I(Ca-L), I(f), and I(Kr) were robustly expressed, I(Ks) and I(K1) were absent in dsRed+ ventricular hESCCMs. Upon differentiation (7+40 to +90 days), the basal [Ca(2+)](i), Ca(2+) transient amplitude, maximum upstroke, and decay velocities significantly increased (P < 0.05). The I(Ca-L) antagonizer nifedipine (1 microM) decreased the Ca(2+) transient amplitude (to approximately 30%) and slowed the kinetics (by approximately 2-fold), but Ca(2+) transients could still be elicited even after complete ICa-L blockade, suggesting the presence of additional Ca(2+) influx(es). Indeed, Ni(2+)-sensitive INCX could be recorded in 7+40- and +90-day dsRed+ hESC-CMs, and its densities increased from -1.2 +/- 0.6 pA/pF at -120 mV and 3.6 +/- 1.0 pA/pF at 60 mV by 6- and 2-folds, respectively. With higher [Ca(2+)](i), 7+90-day ventricular hESC-CMs spontaneously but irregularly fired transients upon a single stimulus under an external Na(+)-free condition; however, without extracellular Na(+), nifedipine could completely inhibit Ca(2+) transients. We conclude that I(NCX) is functionally expressed in developing ventricular hESC-CMs and contributes to their excitation-contraction coupling.

Project description:Introduction: The potentially unlimited number of cardiomyocyte (CMs) derived from human induced pluripotent stem cells (hiPSCs) in vitro facilitates high throughput applications like cell transplantation for myocardial repair, disease modelling, and cardiotoxicity testing during drug development. Despite promising progress in these areas, a major disadvantage that limits the use of hiPSC derived CMs (hiPSC-CMs) is their immaturity. Methods: Three hiPSC lines (PCBC-hiPSC, DP3-hiPSCs, and MLC2v-mEGFP hiPSC) were differentiated into CMs (PCBC-CMs, DP3-CMs, and MLC2v-CMs, respectively) with or without retinoic acid (RA). hiPSC-CMs were either maintained up to day 30 of contraction (D30C), or D60C, or purified using lactate acid and used for experiments. Purified hiPSC-CMs were cultured in basal maturation medium (BMM) or BMM supplemented with ascorbic acid (AA) for 14 days. The AA treated and non-treated hiPSC-CMs were characterized for sarcomeric proteins (MLC2v, TNNI3, and MYH7), ion channel proteins (Kir2.1, Nav1.5, Cav1.2, SERCA2a, and RyR), mitochondrial membrane potential, metabolomics, and action potential. Bobcat339, a selective and potent inhibitor of DNA demethylation, was used to determine whether AA promoted hiPSC-CM maturation through modulating DNA demethylation. Results: AA significantly increased MLC2v expression in PCBC-CMs, DP3-CMs, MLC2v-CMs, and RA induced atrial-like PCBC-CMs. AA treatment significantly increased mitochondrial mass, membrane potential, and amino acid and fatty acid metabolism in PCBC-CMs. Patch clamp studies showed that AA treatment induced PCBC-CMs and DP3-CMs adaptation to a ventricular-like phenotype. Bobcat339 inhibited MLC2v protein expression in AA treated PCBC-CMs and DP3-CMs. DNA demethylation inhibition was also associated with reduced TET1 and TET2 protein expressions and reduced accumulation of the oxidative product, 5 hmC, in both PCBC-CMs and DP3-CMs, in the presence of AA. Conclusions: Ascorbic acid induced MLC2v protein expression and promoted ventricular-like CM subtype in hiPSC-CMs. The effect of AA on hiPSC-CM was attenuated with inhibition of TET1/TET2 mediated DNA demethylation.

Project description:BACKGROUND: Characterization of gene expression signatures for cardiomyocytes derived from embryonic stem cells will help to define their early biologic processes. RESULTS: A transgenic alpha-myosin heavy chain (MHC) embryonic stem cell lineage was generated, exhibiting puromycin resistance and expressing enhanced green fluorescent protein (EGFP) under the control of the alpha-MHC promoter. A puromycin-resistant, EGFP-positive, alpha-MHC-positive cardiomyocyte population was isolated with over 92% purity. RNA was isolated after electrophysiological characterization of the cardiomyocytes. Comprehensive transcriptome analysis of alpha-MHC-positive cardiomyocytes in comparison with undifferentiated alpha-MHC embryonic stem cells and the control population from 15-day-old embryoid bodies led to identification of 884 upregulated probe sets and 951 downregulated probe sets in alpha-MHC-positive cardiomyocytes. A subset of upregulated genes encodes cytoskeletal and voltage-dependent channel proteins, and proteins that participate in aerobic energy metabolism. Interestingly, mitosis, apoptosis, and Wnt signaling-associated genes were downregulated in the cardiomyocytes. In contrast, annotations for genes upregulated in the alpha-MHC-positive cardiomyocytes are enriched for the following Gene Ontology (GO) categories: enzyme-linked receptor protein signaling pathway (GO:0007167), protein kinase activity (GO:0004672), negative regulation of Wnt receptor signaling pathway (GO:0030178), and regulation of cell size (O:0008361). They were also enriched for the Biocarta p38 mitogen-activated protein kinase signaling pathway and Kyoto Encyclopedia of Genes and Genomes (KEGG) calcium signaling pathway. CONCLUSION: The specific pattern of gene expression in the cardiomyocytes derived from embryonic stem cells reflects the biologic, physiologic, and functional processes that take place in mature cardiomyocytes. Identification of cardiomyocyte-specific gene expression patterns and signaling pathways will contribute toward elucidating their roles in intact cardiac function.

Dataset Information

Increased predominance of the matured ventricular subtype in embryonic stem cell-derived cardiomyocytes in vivo.

Publications

Increased predominance of the matured ventricular subtype in embryonic stem cell-derived cardiomyocytes in vivo.

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