The harder the climb the better the view: The impact of substrate stiffness on cardiomyocyte fate

Querceto, Silvia; Santoro, Rosaria; Gowran, Aoife; Grandinetti, Bruno; Pompilio, Giulio; Regnier, Michael; Tesi, Chiara; Poggesi, Corrado; Pioner, Josè Manuel

doi:10.1016/j.yjmcc.2022.02.001

Cited by 11 publications

(12 citation statements)

References 131 publications

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“…In this work, we have studied the Ca-Ts and APs recorded from DMD hiPSC-CMs obtained from a patient with DMD-related cardiomyopathy, carrying a deletion of exon 50 (Δ exon 50) in DMD gene ( Pioner et al, 2020 ). We compared these with the results obtained in control cell lines and a positive isogenic control created by CRISPR-Cas9 targeting the DMD gene (c.263delG), as previously described ( Querceto et al, 2022 ). Previous reports using these DMD hiPSC-CM lines exhibited membrane fragility ( Guan et al, 2014 ) and diminished structural/functional response to an underlying substrate with nanotopographic grooves compared to normal cardiomyocytes that was associated to a lower level of actin cytoskeleton turnover ( Macadangdang et al, 2015 ).…”

Section: Discussionmentioning

confidence: 99%

“…Among these, cardiomyocytes actively probe the rigidity of their extracellular environment by exerting traction forces via transmembrane proteins named integrins ( Santoro et al, 2019 ). However, it is still poorly understood how cardiac cells sense matrix stiffness and how they transduce the mechanical information into specific cellular responses or functional phenotypes ( Querceto et al, 2022 ). Key proteins in this scenario may include dystrophin and the glycoproteic complex (DGC).…”

Section: Discussionmentioning

confidence: 99%

“…anisotropy and stiffness. Substrates with low (e.g., PDMS, Matrigel, hydrogels) and high (e.g., glass, polyurethane) stiffness can provide different levels of external load, influencing the force of contraction and protein expression ( Querceto et al, 2022 ). The current work used different substrates that are far from recapitulating the physiological range of extracellular tissue stiffness.…”

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…A second issue that is often overlooked in current hiPSC-CM research are the effects of the different culture substrates on cardiomyocyte structure and function ( Querceto et al, 2022 ). Increased substrate stiffness and anisotropic surfaces morphology have both shown to be valuable biomimetic tools for advancing the structural and functional maturation of immature cardiomyocytes in culture ( Querceto et al, 2022 ). Specifically, cardiomyocytes increase their contraction force in response to increased stiffness via adaptation of calcium handling and myofibril structural organization ( Rodriguez et al, 2011 ; Ribeiro et al, 2015 ; Ribeiro et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

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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

et al. 2022

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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).

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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

et al. 2022

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Cellular Alignment and Matrix Stiffening Induced Changes in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

House,

Santillan,

Correa

et al. 2024

Adv Healthcare Materials

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Biological processes are inherently dynamic, necessitating biomaterial platforms capable of spatiotemporal control over cellular organization and matrix stiffness for accurate study of tissue development, wound healing, and disease. However, most in vitro platforms remain static. In this study, a dynamic biomaterial platform comprising a stiffening hydrogel is introduced and achieved through a stepwise approach of addition followed by light‐mediated crosslinking, integrated with an elastomeric substrate featuring strain‐responsive lamellar surface patterns. Employing this platform, the response of human induced pluripotent stem cell‐derived cardiomyocytes (hIPSC‐CMs) is investigated to dynamic stiffening from healthy to fibrotic tissue stiffness. The results demonstrate that culturing hIPSC‐CMs on physiologically relevant healthy stiffness significantly enhances their function, as evidenced by increased sarcomere fraction, wider sarcomere width, significantly higher connexin‐43 content, and elevated cell beating frequency compared to cells cultured on fibrotic matrix. Conversely, dynamic matrix stiffening negatively impacts hIPSC‐CM function, with earlier stiffening events exerting a more pronounced hindering effect. These findings provide valuable insights into material‐based approaches for addressing existing challenges in hIPSC‐CM maturation and have broader implications across various tissue models, including muscle, tendon, nerve, and cornea, where both cellular alignment and matrix stiffening play pivotal roles in tissue development and regeneration.

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Pluripotent Stem Cells in Myocardial Tissue Engineering and Heart Regeneration

Mohd Yusof,

Esa,

Tan

2024

Comprehensive Hematology and Stem Cell Research

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The harder the climb the better the view: The impact of substrate stiffness on cardiomyocyte fate

Cited by 11 publications

References 131 publications

Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency

Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency

Cellular Alignment and Matrix Stiffening Induced Changes in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

Pluripotent Stem Cells in Myocardial Tissue Engineering and Heart Regeneration

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