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...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-11-01
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| Series: | Frontiers in Physiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fphys.2022.1030920/full |
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| author | Josè Manuel Pioner Lorenzo Santini Chiara Palandri Marianna Langione Bruno Grandinetti Silvia Querceto Daniele Martella Daniele Martella Costanza Mazzantini Beatrice Scellini Lucrezia Giammarino Flavia Lupi Francesco Mazzarotto Francesco Mazzarotto Aoife Gowran Davide Rovina Rosaria Santoro Giulio Pompilio Giulio Pompilio Chiara Tesi Camilla Parmeggiani Camilla Parmeggiani Michael Regnier Elisabetta Cerbai David L. Mack Corrado Poggesi Cecilia Ferrantini Raffaele Coppini |
| author_facet | Josè Manuel Pioner Lorenzo Santini Chiara Palandri Marianna Langione Bruno Grandinetti Silvia Querceto Daniele Martella Daniele Martella Costanza Mazzantini Beatrice Scellini Lucrezia Giammarino Flavia Lupi Francesco Mazzarotto Francesco Mazzarotto Aoife Gowran Davide Rovina Rosaria Santoro Giulio Pompilio Giulio Pompilio Chiara Tesi Camilla Parmeggiani Camilla Parmeggiani Michael Regnier Elisabetta Cerbai David L. Mack Corrado Poggesi Cecilia Ferrantini Raffaele Coppini |
| author_sort | Josè Manuel Pioner |
| collection | DOAJ |
| 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). |
| first_indexed | 2024-03-13T06:01:19Z |
| format | Article |
| id | doaj.art-95e6e0e9e12446fa8397f9dbba59be9f |
| institution | Directory Open Access Journal |
| issn | 1664-042X |
| language | English |
| last_indexed | 2024-03-13T06:01:19Z |
| publishDate | 2022-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Physiology |
| spelling | doaj.art-95e6e0e9e12446fa8397f9dbba59be9f2023-06-12T14:40:50ZengFrontiers Media S.A.Frontiers in Physiology1664-042X2022-11-011310.3389/fphys.2022.10309201030920Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiencyJosè Manuel Pioner0Lorenzo Santini1Chiara Palandri2Marianna Langione3Bruno Grandinetti4Silvia Querceto5Daniele Martella6Daniele Martella7Costanza Mazzantini8Beatrice Scellini9Lucrezia Giammarino10Flavia Lupi11Francesco Mazzarotto12Francesco Mazzarotto13Aoife Gowran14Davide Rovina15Rosaria Santoro16Giulio Pompilio17Giulio Pompilio18Chiara Tesi19Camilla Parmeggiani20Camilla Parmeggiani21Michael Regnier22Elisabetta Cerbai23David L. Mack24Corrado Poggesi25Cecilia Ferrantini26Raffaele Coppini27Department of Biology, University of Florence, Florence, ItalyDepartment of Neurofarba, University of Florence, Florence, ItalyDepartment of Neurofarba, University of Florence, Florence, ItalyDepartment of Experimental and Clinical Medicine, University of Florence, Florence, ItalyEuropean Laboratory for Non-Linear Spectroscopy (LENS), University of Florence, Florence, ItalyEuropean Laboratory for Non-Linear Spectroscopy (LENS), University of Florence, Florence, ItalyEuropean Laboratory for Non-Linear Spectroscopy (LENS), University of Florence, Florence, ItalyIstituto Nazionale di Ricerca Metrologica (INRiM), Turin, ItalyDepartment of Neurofarba, University of Florence, Florence, ItalyDepartment of Experimental and Clinical Medicine, University of Florence, Florence, ItalyDepartment of Neurofarba, University of Florence, Florence, ItalyEuropean Laboratory for Non-Linear Spectroscopy (LENS), University of Florence, Florence, ItalyDepartment of Molecular and Translational Medicine, University of Brescia, Brescia, ItalyNational Heart and Lung Institute, Imperial College London, London, United KingdomUnit of Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino IRCCS, Milan, ItalyUnit of Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino IRCCS, Milan, ItalyUnit of Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino IRCCS, Milan, ItalyUnit of Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino IRCCS, Milan, ItalyDepartment of Biomedical, Surgical and Dental Sciences, University of Milan, Milan, ItalyDepartment of Experimental and Clinical Medicine, University of Florence, Florence, ItalyEuropean Laboratory for Non-Linear Spectroscopy (LENS), University of Florence, Florence, Italy0Department of Chemistry “Ugo Schiff”, University of Florence, Florence, Italy1Department of Bioengineering, University of Washington, Seattle, WA, United StatesDepartment of Neurofarba, University of Florence, Florence, Italy1Department of Bioengineering, University of Washington, Seattle, WA, United StatesDepartment of Experimental and Clinical Medicine, University of Florence, Florence, ItalyDepartment of Experimental and Clinical Medicine, University of Florence, Florence, ItalyDepartment of Neurofarba, University of Florence, Florence, ItalyCardiomyocytes 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).https://www.frontiersin.org/articles/10.3389/fphys.2022.1030920/fullhuman iPSC derived cardiomyocytesdystrophin (DMD)substrate stiffnesscalcium handingduchenne muscular dystrophy (DMD) |
| spellingShingle | Josè Manuel Pioner Lorenzo Santini Chiara Palandri Marianna Langione Bruno Grandinetti Silvia Querceto Daniele Martella Daniele Martella Costanza Mazzantini Beatrice Scellini Lucrezia Giammarino Flavia Lupi Francesco Mazzarotto Francesco Mazzarotto Aoife Gowran Davide Rovina Rosaria Santoro Giulio Pompilio Giulio Pompilio Chiara Tesi Camilla Parmeggiani Camilla Parmeggiani Michael Regnier Elisabetta Cerbai David L. Mack Corrado Poggesi Cecilia Ferrantini Raffaele Coppini Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency Frontiers in Physiology human iPSC derived cardiomyocytes dystrophin (DMD) substrate stiffness calcium handing duchenne muscular dystrophy (DMD) |
| 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 | human iPSC derived cardiomyocytes dystrophin (DMD) substrate stiffness calcium handing duchenne muscular dystrophy (DMD) |
| url | https://www.frontiersin.org/articles/10.3389/fphys.2022.1030920/full |
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