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Human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) offer numerous advantages as a biological model, yet their inherent immaturity compared to adult cardiomyocytes poses significant limitations. This study addresses hiPSCCM immaturity by introducing a physiologically relevant micropatterned substrate for long-term culture and maturation. An innovative microfabrication methodology combining laser etching and casting creates a micropatterned polydimethylsiloxane (PDMS) substrate with varying stiffness, from 2 to 50 kPa, mimicking healthy and fibrotic cardiac tissue. Platinum electrodes were integrated into the cell culture chamber enable pacing of cells at various frequencies. Subsequently, cells were transferred to the incubator for time-course analysis, ensuring contamination-free conditions. Cell contractility, cytosolic Ca 2+ transient, sarcomere orientation, and nucleus aspect ratio were analyzed in a 2D hiPSCCM monolayer up to 90 days post-replating in relation to substrate micropattern dimensions. Culturing hiPSCCMs for three weeks on a micropatterned PDMS substrate (2.5-5 µm deep, 20 µm center-to-center spacing of grooves, 2-5 kPa stiffness) emerges as optimal for cardiomyocyte alignment, contractility, and cytosolic Ca 2+ transient. The study provides insights into substrate stiffness effects on hiPSCCM contractility and Ca 2+ transient at immature and mature states. Maximum contractility and fastest Ca 2+ transient kinetics occur in mature hiPSCCMs cultured for two to four weeks, with the optimum at three weeks, on a soft micropatterned PDMS substrate. MS proteomic analysis further revealed that hiPSCCMs cultured on soft micropatterned substrates exhibit advanced maturation, marked by significant upregulation of key structural, electrophysiological, and metabolic proteins. This new substrate offers a promising platform for disease modeling and therapeutic interventions. STATEMENT OF SIGNIFICANCE: Human induced pluripotent stem cell derived cardiomyocytes (hiPSCCMs) have been transformative to disease-in-a-dish modeling, drug discovery and testing, and autologous regeneration for human hearts and their role will continue to expand dramatically. However, one of the major limitations of hiPSCCMs is that without intervention, the cells are immature and represent those in the fetal heart. We developed protocols for the fabrication of the PDMS matrices that includes variations in its stiffness and micropatterning. Growing our hiPSCCMs on matrices of comparable stiffness to a healthy heart (5 kPa) and grooves of 20 μm, generate heart cells typical of the healthy adult human heart., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for Frontiers in Cell and Developmental Biology and was not involved in the editorial review or the decision to publish this article., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The advent of human induced pluripotent stem cells (hiPSCs) and their capacity to be differentiated into beating human cardiomyocytes (CMs) in vitro has revolutionized human disease modelling, genotype-phenotype predictions, and therapeutic testing. Hypertrophic cardiomyopathy (HCM) is a common inherited cardiomyopathy and the leading known cause of sudden cardiac arrest in young adults and athletes. On a molecular level, HCM is often driven by single pathogenic genetic variants, usually in sarcomeric proteins, that can alter the mechanical, electrical, signalling, and transcriptional properties of the cell. A deeper knowledge of these alterations is critical to better understanding HCM manifestation, progression, and treatment. Leveraging hiPSC-CMs to investigate the molecular mechanisms driving HCM presents a unique opportunity to dissect the consequences of genetic variants in a sophisticated and controlled manner. In this review, we summarize the molecular underpinnings of HCM and the role of hiPSC-CM studies in advancing our understanding, and we highlight the advances in hiPSC-CM-based modelling of HCM, including maturation, contractility, multiomics, and genome editing, with the notable exception of electrophysiology, which has been previously covered., (Copyright © 2023 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.)

Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease and often results in cardiac remodeling and an increased incidence of sudden cardiac arrest (SCA) and death, especially in youth and young adults. Among thousands of different variants found in HCM patients, variants of TNNT2 (cardiac troponin T-TNNT2) are linked to increased risk of ventricular arrhythmogenesis and sudden death despite causing little to no cardiac hypertrophy. Therefore, studying the effect of TNNT2 variants on cardiac propensity for arrhythmogenesis can pave the way for characterizing HCM in susceptible patients before sudden cardiac arrest occurs. In this study, a TNNT2 variant, I79N, was generated in human cardiac recombinant/reconstituted thin filaments (hcRTF) to investigate the effect of the mutation on myofilament Ca 2+ sensitivity and Ca 2+ dissociation rate using steady-state and stopped-flow fluorescence techniques. The results revealed that the I79N variant significantly increases myofilament Ca 2+ sensitivity and decreases the Ca 2+ off-rate constant ( k off ). To investigate further, a heterozygous I79N +/- TNNT2 variant was introduced into human-induced pluripotent stem cells using CRISPR/Cas9 and subsequently differentiated into ventricular cardiomyocytes (hiPSC-CMs). To study the arrhythmogenic properties, monolayers of I79N +/- hiPSC-CMs were studied in comparison to their isogenic controls. Arrhythmogenesis was investigated by measuring voltage ( V m ) and cytosolic Ca 2+ transients over a range of stimulation frequencies. An increasing stimulation frequency was applied to the cells, from 55 to 75 bpm. The results of this protocol showed that the TnT-I79N cells had reduced intracellular Ca 2+ transients due to the enhanced cytosolic Ca 2+ buffering. These changes in Ca 2+ handling resulted in beat-to-beat instability and triangulation of the cardiac action potential, which are predictors of arrhythmia risk. While wild-type (WT) hiPSC-CMs were accurately entrained to frequencies of at least 150 bpm, the I79N hiPSC-CMs demonstrated clear patterns of alternans for both V m and Ca 2+ transients at frequencies >75 bpm. Lastly, a transcriptomic analysis was conducted on WT vs. I79N +/- TNNT2 hiPSC-CMs using a custom NanoString codeset. The results showed a significant upregulation of NPPA (atrial natriuretic peptide), NPPB (brain natriuretic peptide), Notch signaling pathway components, and other extracellular matrix (ECM) remodeling components in I79N +/- vs. the isogenic control. This significant shift demonstrates that this missense in the TNNT2 transcript likely causes a biophysical trigger, which initiates this significant alteration in the transcriptome. This TnT-I79N hiPSC-CM model not only reproduces key cellular features of HCM-linked mutations but also suggests that this variant causes uncharted pro-arrhythmic changes to the human action potential and gene expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Shafaattalab, Li, Gunawan, Kim, Jayousi, Maaref, Song, Weiss, Solaro, Qu and Tibbits.)