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Ascorbic acid induces MLC2v protein expression and promotes ventricular-like cardiomyocyte subtype in human induced pluripotent stem cells derived cardiomyocytes
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. D...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Ivyspring International Publisher
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10334833/ https://www.ncbi.nlm.nih.gov/pubmed/37441603 http://dx.doi.org/10.7150/thno.80801 |
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author | Gao, Yu Su, Liping Wei, Yuhua Tan, Shihua Hu, Zhenyu Tao, Zhonghao Kovalik, Jean-Paul Soong, Tuck Wah Zhang, Jianyi Pu, Jun Ye, Lei |
author_facet | Gao, Yu Su, Liping Wei, Yuhua Tan, Shihua Hu, Zhenyu Tao, Zhonghao Kovalik, Jean-Paul Soong, Tuck Wah Zhang, Jianyi Pu, Jun Ye, Lei |
author_sort | Gao, Yu |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-10334833 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Ivyspring International Publisher |
record_format | MEDLINE/PubMed |
spelling | pubmed-103348332023-07-12 Ascorbic acid induces MLC2v protein expression and promotes ventricular-like cardiomyocyte subtype in human induced pluripotent stem cells derived cardiomyocytes Gao, Yu Su, Liping Wei, Yuhua Tan, Shihua Hu, Zhenyu Tao, Zhonghao Kovalik, Jean-Paul Soong, Tuck Wah Zhang, Jianyi Pu, Jun Ye, Lei Theranostics Research Paper 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. Ivyspring International Publisher 2023-07-03 /pmc/articles/PMC10334833/ /pubmed/37441603 http://dx.doi.org/10.7150/thno.80801 Text en © The author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. |
spellingShingle | Research Paper Gao, Yu Su, Liping Wei, Yuhua Tan, Shihua Hu, Zhenyu Tao, Zhonghao Kovalik, Jean-Paul Soong, Tuck Wah Zhang, Jianyi Pu, Jun Ye, Lei Ascorbic acid induces MLC2v protein expression and promotes ventricular-like cardiomyocyte subtype in human induced pluripotent stem cells derived cardiomyocytes |
title | Ascorbic acid induces MLC2v protein expression and promotes ventricular-like cardiomyocyte subtype in human induced pluripotent stem cells derived cardiomyocytes |
title_full | Ascorbic acid induces MLC2v protein expression and promotes ventricular-like cardiomyocyte subtype in human induced pluripotent stem cells derived cardiomyocytes |
title_fullStr | Ascorbic acid induces MLC2v protein expression and promotes ventricular-like cardiomyocyte subtype in human induced pluripotent stem cells derived cardiomyocytes |
title_full_unstemmed | Ascorbic acid induces MLC2v protein expression and promotes ventricular-like cardiomyocyte subtype in human induced pluripotent stem cells derived cardiomyocytes |
title_short | Ascorbic acid induces MLC2v protein expression and promotes ventricular-like cardiomyocyte subtype in human induced pluripotent stem cells derived cardiomyocytes |
title_sort | ascorbic acid induces mlc2v protein expression and promotes ventricular-like cardiomyocyte subtype in human induced pluripotent stem cells derived cardiomyocytes |
topic | Research Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10334833/ https://www.ncbi.nlm.nih.gov/pubmed/37441603 http://dx.doi.org/10.7150/thno.80801 |
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