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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.)

The zebrafish has proven to be an excellent organism for manipulation of its genome from a long history of transcript down-regulation using morpholino oligimers to more recent genome editing tools such as CRISPR-Cas9. Early forward and reverse genetic screens significantly benefited from the transparency of zebrafish embryos, allowing cardiac development as a function of genetics to be directly observed. However, gradual loss of transparency with subsequent maturation limited many of these approaches to the first several days post-fertilization. As many genes are developmentally regulated, the immature phenotype is not entirely indicative of that of the mature zebrafish. For accurate phenotyping, subsequent developmental stages including full maturation must also be considered. In adult zebrafish, cardiac function can now be studied in great detail due both to the size of the hearts as well as recent technological improvements. Because of their small size, zebrafish are particularly amenable to high frequency echocardiography for detailed functional recordings. Although relatively small, the hearts are easily excised and contractile parameters can be measured from whole hearts, heart slices, individual cardiomyocytes and even single myofibrils. Similarly, electrical activity can also be measured using a variety of techniques, including in vivo and ex vivo electrocardiograms, optical mapping and traditional microelectrode techniques. In this report, the major advantages and technical considerations of these physiological tools are discussed., (Copyright © 2019. Published by Elsevier B.V.)

Background: Diabetes is associated with an increased risk of heart failure, cardiac arrhythmias and sudden cardiac death. We previously showed that ROCK2 expression is elevated in diabetic rat hearts, and that ROCK inhibition acutely improves their contractile function. In the present study we investigated whether inhibition of ROCK or partial deletion of ROCK2 improves impaired Ca 2+ handling in the diabetic heart., Methods: Contractile properties and Ca 2+ transients were measured before and after treatment with the ROCK inhibitor Y-27632 (1 μM) in fluo-4-loaded cardiomyocytes isolated from streptozotocin (STZ)-diabetic or non-diabetic rats. Cardiac function was determined in vivo, and contractile properties and Ca 2+ transients also measured in cardiomyocytes from non-diabetic and STZ-diabetic wild-type (WT) and ROCK2+/- mice., Results: ROCK inhibition improved some parameters of contractile function and Ca 2+ handling in cardiomyocytes from diabetic rat hearts. In addition, ROCK inhibition attenuated the diabetes-induced delayed aftercontractions (DACs) and associated irregular Ca 2+ transients induced by increased [Ca 2+ ]o. Although no overt cardiac dysfunction was detected in diabetic WT mice, cardiomyocytes from these mice also developed arrhythmic Ca 2+ transients in response to increased [Ca 2+ ]. These were attenuated in cardiomyocytes from diabetic ROCK2+/- mice, in association with decreased diastolic Ca 2+ leak and with reduction of the diabetes-induced increased phosphorylation of both CaMKII and the ryanodine receptor (RyR)., Conclusions: These data suggest that ROCK2 contributes to diabetes-induced impaired cardiac Ca 2+ homeostasis, at least in part by promoting CaMKII-mediated phosphorylation of RyR. This may have important clinical implications for the treatment of the increased incidence of dysrhythmias in diabetes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Postoperative arrhythmias such as junctional ectopic tachycardia and atrioventricular block are serious postoperative complications for children with congenital heart disease. We hypothesize that ischemia-reperfusion (I/R) related changes exacerbate these postoperative arrhythmias in the neonate heart and administration of postoperative inotropes is contributory., Objective: The purpose of this study was to study the effects of I/R and postischemic dopamine application on automaticity and rhythmicity in immature and mature pacemaker cells and whole heart preparations., Methods: Single pacemaker cells and whole heart models of postoperative arrhythmias were generated in a rabbit model encompassing 3 primary risk factors: age, I/R exposure, and dopamine application. Single cells were studied using current clamp and line scan confocal microscopy, whereas whole hearts were studied using optical mapping., Results: Four responses were observed in neonatal atrioventricular nodal cells (AVNCs): slowing of AVNC automaticity (from 62±10 to 36 ± 12 action potentials per minute, P<.05); induction of arrhythmicity or increased beat-to-beat variability (0.08 ± 0.04 to 3.83 ± 1.79, P<.05); altered automaticity (subthreshold electrical fluctuations); and disruption of calcium transients. In contrast, these responses were not observed in mature AVNCs or neonatal sinoatrial cells. In whole heart experiments, neonatal hearts experienced persistent postischemia arrhythmias of varying severity, whereas mature hearts exhibited no arrhythmias or relatively transient ones., Conclusion: Neonatal pacemaker cells and whole hearts demonstrate a susceptibility to I/R insults resulting in alterations in automaticity, which may predispose neonates to postoperative arrhythmias such as junctional ectopic tachycardia and atrioventricular block., (Copyright © 2016 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

The amino-terminal regulatory domain of cardiac troponin C (cNTnC) plays an important role as the calcium sensor for the troponin complex. Calcium binding to cNTnC results in conformational changes that trigger a cascade of events that lead to cardiac muscle contraction. The cardiac N-terminal domain of TnC consists of two EF-hand calcium binding motifs, one of which is dysfunctional in binding calcium. Nevertheless, the defunct EF-hand still maintains a role in cNTnC function. For its structural analysis by X-ray crystallography, human cNTnC with the wild-type primary sequence was crystallized under a novel crystallization condition. The crystal structure was solved by the single-wavelength anomalous dispersion method and refined to 2.2 Å resolution. The structure displays several novel features. Firstly, both EF-hand motifs coordinate cadmium ions derived from the crystallization milieu. Secondly, the ion coordination in the defunct EF-hand motif accompanies unusual changes in the protein conformation. Thirdly, deoxycholic acid, also derived from the crystallization milieu, is bound in the central hydrophobic cavity. This is reminiscent of the interactions observed for cardiac calcium sensitizer drugs that bind to the same core region and maintain the "open" conformational state of calcium-bound cNTnC. The cadmium ion coordination in the defunct EF-hand indicates that this vestigial calcium binding site retains the structural and functional elements that allow it to coordinate a cadmium ion. However, it is a result of, or concomitant with, large and unusual structural changes in cNTnC., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In adult cardiac cells the established mechanism of excitation-contraction coupling is by calcium-induced calcium release (CICR) mediated by L-type Ca(2+) channels. However, in neonate cardiomyocytes, a CICR modality involving reverse mode Na(+)-Ca(2+) exchanger (NCX) activity predominates. This has been hypothesized to be due, in part, to the high expression levels of NCX in the neonate heart which drop several fold during ontogeny. Very little is known about the nature of NCX distribution within the cardiomyocyte and how this might change with development given the significant differences in gene expression. We investigated the spatial arrangements of NCX in developing rabbit ventricular myocytes with traditional as well as novel image processing and analysis techniques. Using image segmentation, colocalization analysis was conducted at the whole cell, compartmental (cell periphery and cell interior) and object levels. Because NCX has been suggested to colocalize with caveolin-3 (cav-3) and perhaps form a signaling unit within caveolae, the spatial relationship of NCX relative to cav-3 was also examined in detail. NCX and cav-3 objects were found to be isolated islands of lit voxels that are present after thresholding. These objects were categorized into non-colocalized (0%), lowly colocalized (<50%) and highly colocalized (>50%) subpopulations in both the interior and peripheral compartments. Our results show that NCX and cav-3 are distributed on the peripheral membrane as discrete objects and are not highly colocalized throughout development. 3D distance analysis revealed that NCX and cav-3 objects are organized with a longitudinal and lateral periodicity of about 1mum and that NCX and cav-3 cluster appear to be mutually exclusive on the cell periphery. We conclude that despite the very significant decrease in NCX expression with maturation, qualitatively there were no differences in NCX surface distribution or in the spatial relationship to caveolin 3.

Background: The effects of inhibition of the Na+/H+ exchanger (NHE) on postischemic recovery of the injured neonatal rabbit heart were examined. The NHE may be an important mechanism for reperfusion injury in the neonate heart. The effects of two NHE inhibitors, HOE 642 (HOE) and 5-(N,N-dimethyl)-amiloride (DMA), given during hypothermic cardioplegic arrest, were evaluated., Methods: Isolated working crystalloid-perfused neonatal rabbit hearts were subjected to 10 min of normothermic ischemia to cause injury before undergoing 4 h of hypothermic (10 degrees C) cardioplegic arrest with a single dose of crystalloid solution (controls, n=21) or with the addition of 0.5 micromol/L HOE (n=24) or 30 micromol/L DMA (n=15)., Results: Hearts subjected to HOE had improved recoveries of aortic flow when compared with controls at 15 min and 30 min of reperfusion (35.7+/-1.3 mL/min versus 26.2+/-1.4 mL/min, respectively, at 15 min, P<0.0001; 36.5+/-1.5 mL/min versus 23.6+/-1.6 mL/min, respectively, at 30 min, P<0.0001) and with DMA at 30 min (36.5+/-1.5 mL/min versus 29.9+/-1.9 mL/min, P=0.0214). Cardiac output and systolic pressure were also improved at 30 min in HOE hearts versus controls (P<0.0001)., Conclusions: NHE inhibition with HOE during cardioplegic arrest resulted in improved functional recovery of injured hearts. Further studies in blood-perfused neonatal preparations are warranted.