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Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency
Cardiomyocytes differentiated from human induced Pluripotent Stem Cells (hiPSC- CMs) are a unique source for modelling inherited cardiomyopathies. In particular, the possibility of observing maturation processes in a simple culture dish opens novel perspectives in the study of early-disease defects...
Autores principales: | , , , , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9676373/ https://www.ncbi.nlm.nih.gov/pubmed/36419836 http://dx.doi.org/10.3389/fphys.2022.1030920 |
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author | Pioner, Josè Manuel Santini, Lorenzo Palandri, Chiara Langione, Marianna Grandinetti, Bruno Querceto, Silvia Martella, Daniele Mazzantini, Costanza Scellini, Beatrice Giammarino, Lucrezia Lupi, Flavia Mazzarotto, Francesco Gowran, Aoife Rovina, Davide Santoro, Rosaria Pompilio, Giulio Tesi, Chiara Parmeggiani, Camilla Regnier, Michael Cerbai, Elisabetta Mack, David L. Poggesi, Corrado Ferrantini, Cecilia Coppini, Raffaele |
author_facet | Pioner, Josè Manuel Santini, Lorenzo Palandri, Chiara Langione, Marianna Grandinetti, Bruno Querceto, Silvia Martella, Daniele Mazzantini, Costanza Scellini, Beatrice Giammarino, Lucrezia Lupi, Flavia Mazzarotto, Francesco Gowran, Aoife Rovina, Davide Santoro, Rosaria Pompilio, Giulio Tesi, Chiara Parmeggiani, Camilla Regnier, Michael Cerbai, Elisabetta Mack, David L. Poggesi, Corrado Ferrantini, Cecilia Coppini, Raffaele |
author_sort | Pioner, Josè Manuel |
collection | PubMed |
description | Cardiomyocytes differentiated from human induced Pluripotent Stem Cells (hiPSC- CMs) are a unique source for modelling inherited cardiomyopathies. In particular, the possibility of observing maturation processes in a simple culture dish opens novel perspectives in the study of early-disease defects caused by genetic mutations before the onset of clinical manifestations. For instance, calcium handling abnormalities are considered as a leading cause of cardiomyocyte dysfunction in several genetic-based dilated cardiomyopathies, including rare types such as Duchenne Muscular Dystrophy (DMD)-associated cardiomyopathy. To better define the maturation of calcium handling we simultaneously measured action potential and calcium transients (Ca-Ts) using fluorescent indicators at specific time points. We combined micropatterned substrates with long-term cultures to improve maturation of hiPSC-CMs (60, 75 or 90 days post-differentiation). Control-(hiPSC)-CMs displayed increased maturation over time (90 vs 60 days), with longer action potential duration (APD), increased Ca-T amplitude, faster Ca-T rise (time to peak) and Ca-T decay (RT50). The progressively increased contribution of the SR to Ca release (estimated by post-rest potentiation or Caffeine-induced Ca-Ts) appeared as the main determinant of the progressive rise of Ca-T amplitude during maturation. As an example of severe cardiomyopathy with early onset, we compared hiPSC-CMs generated from a DMD patient (DMD-ΔExon50) and a CRISPR-Cas9 genome edited cell line isogenic to the healthy control with deletion of a G base at position 263 of the DMD gene (c.263delG-CMs). In DMD-hiPSC-CMs, changes of Ca-Ts during maturation were less pronounced: indeed, DMD cells at 90 days showed reduced Ca-T amplitude and faster Ca-T rise and RT50, as compared with control hiPSC-CMs. Caffeine-Ca-T was reduced in amplitude and had a slower time course, suggesting lower SR calcium content and NCX function in DMD vs control cells. Nonetheless, the inotropic and lusitropic responses to forskolin were preserved. CRISPR-induced c.263delG-CM line recapitulated the same developmental calcium handling alterations observed in DMD-CMs. We then tested the effects of micropatterned substrates with higher stiffness. In control hiPSC-CMs, higher stiffness leads to higher amplitude of Ca-T with faster decay kinetics. In hiPSC-CMs lacking full-length dystrophin, however, stiffer substrates did not modify Ca-Ts but only led to higher SR Ca content. These findings highlighted the inability of dystrophin-deficient cardiomyocytes to adjust their calcium homeostasis in response to increases of extracellular matrix stiffness, which suggests a mechanism occurring during the physiological and pathological development (i.e. fibrosis). |
format | Online Article Text |
id | pubmed-9676373 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-96763732022-11-22 Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency Pioner, Josè Manuel Santini, Lorenzo Palandri, Chiara Langione, Marianna Grandinetti, Bruno Querceto, Silvia Martella, Daniele Mazzantini, Costanza Scellini, Beatrice Giammarino, Lucrezia Lupi, Flavia Mazzarotto, Francesco Gowran, Aoife Rovina, Davide Santoro, Rosaria Pompilio, Giulio Tesi, Chiara Parmeggiani, Camilla Regnier, Michael Cerbai, Elisabetta Mack, David L. Poggesi, Corrado Ferrantini, Cecilia Coppini, Raffaele Front Physiol Physiology Cardiomyocytes differentiated from human induced Pluripotent Stem Cells (hiPSC- CMs) are a unique source for modelling inherited cardiomyopathies. In particular, the possibility of observing maturation processes in a simple culture dish opens novel perspectives in the study of early-disease defects caused by genetic mutations before the onset of clinical manifestations. For instance, calcium handling abnormalities are considered as a leading cause of cardiomyocyte dysfunction in several genetic-based dilated cardiomyopathies, including rare types such as Duchenne Muscular Dystrophy (DMD)-associated cardiomyopathy. To better define the maturation of calcium handling we simultaneously measured action potential and calcium transients (Ca-Ts) using fluorescent indicators at specific time points. We combined micropatterned substrates with long-term cultures to improve maturation of hiPSC-CMs (60, 75 or 90 days post-differentiation). Control-(hiPSC)-CMs displayed increased maturation over time (90 vs 60 days), with longer action potential duration (APD), increased Ca-T amplitude, faster Ca-T rise (time to peak) and Ca-T decay (RT50). The progressively increased contribution of the SR to Ca release (estimated by post-rest potentiation or Caffeine-induced Ca-Ts) appeared as the main determinant of the progressive rise of Ca-T amplitude during maturation. As an example of severe cardiomyopathy with early onset, we compared hiPSC-CMs generated from a DMD patient (DMD-ΔExon50) and a CRISPR-Cas9 genome edited cell line isogenic to the healthy control with deletion of a G base at position 263 of the DMD gene (c.263delG-CMs). In DMD-hiPSC-CMs, changes of Ca-Ts during maturation were less pronounced: indeed, DMD cells at 90 days showed reduced Ca-T amplitude and faster Ca-T rise and RT50, as compared with control hiPSC-CMs. Caffeine-Ca-T was reduced in amplitude and had a slower time course, suggesting lower SR calcium content and NCX function in DMD vs control cells. Nonetheless, the inotropic and lusitropic responses to forskolin were preserved. CRISPR-induced c.263delG-CM line recapitulated the same developmental calcium handling alterations observed in DMD-CMs. We then tested the effects of micropatterned substrates with higher stiffness. In control hiPSC-CMs, higher stiffness leads to higher amplitude of Ca-T with faster decay kinetics. In hiPSC-CMs lacking full-length dystrophin, however, stiffer substrates did not modify Ca-Ts but only led to higher SR Ca content. These findings highlighted the inability of dystrophin-deficient cardiomyocytes to adjust their calcium homeostasis in response to increases of extracellular matrix stiffness, which suggests a mechanism occurring during the physiological and pathological development (i.e. fibrosis). Frontiers Media S.A. 2022-11-07 /pmc/articles/PMC9676373/ /pubmed/36419836 http://dx.doi.org/10.3389/fphys.2022.1030920 Text en Copyright © 2022 Pioner, Santini, Palandri, Langione, Grandinetti, Querceto, Martella, Mazzantini, Scellini, Giammarino, Lupi, Mazzarotto, Gowran, Rovina, Santoro, Pompilio, Tesi, Parmeggiani, Regnier, Cerbai, Mack, Poggesi, Ferrantini and Coppini. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Physiology Pioner, Josè Manuel Santini, Lorenzo Palandri, Chiara Langione, Marianna Grandinetti, Bruno Querceto, Silvia Martella, Daniele Mazzantini, Costanza Scellini, Beatrice Giammarino, Lucrezia Lupi, Flavia Mazzarotto, Francesco Gowran, Aoife Rovina, Davide Santoro, Rosaria Pompilio, Giulio Tesi, Chiara Parmeggiani, Camilla Regnier, Michael Cerbai, Elisabetta Mack, David L. Poggesi, Corrado Ferrantini, Cecilia Coppini, Raffaele Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency |
title | Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency |
title_full | Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency |
title_fullStr | Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency |
title_full_unstemmed | Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency |
title_short | Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency |
title_sort | calcium handling maturation and adaptation to increased substrate stiffness in human ipsc-derived cardiomyocytes: the impact of full-length dystrophin deficiency |
topic | Physiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9676373/ https://www.ncbi.nlm.nih.gov/pubmed/36419836 http://dx.doi.org/10.3389/fphys.2022.1030920 |
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