The human myocardium is a mechanically active tissue whose anisotropic organization is crucial for proper function. Disruption of this organization after injury contributes to adverse remodeling and heart failure. While mechanobiological phenomena like strain avoidance and contact guidance have been used to promote anisotropy, the role of contraction-induced strain - driven by cardiomyocytes - has been largely overlooked. To investigate this, we engineered cardiac microtissues by embedding cardiac cells in constrained collagen hydrogels, modeling aligned (anisotropic) and disorganized (isotropic) tissues. By varying tissue constraint geometry, we created conditions that either promote or restrict anisotropy and assessed the contribution of cardiomyocyte contraction. Using combined in vitro experiments and in silico modeling, we demonstrate that constraint geometry influences tissue anisotropy and that cardiomyocyte contraction enhances this effect via cell-mediated collagen prestretch. Furthermore, establishing anisotropy in contracting microtissues by manipulating mechanical constraints significantly improves sarcomere development and overall tissue contractility. Our approach thus provides evidence of the functional benefits of beating cardiomyocytes in restoring cardiac tissue anisotropy. Additionally, our study offers insights into the dynamic interplay between tissue contractility, mechanical tension, and organization, presenting potential pathways for cardiac regenerative strategies by actively harnessing contraction-induced matrix remodeling to guide tissue architecture and function.

Anthracyclines, key chemotherapy agents, pose cardiotoxicity risks. In a 3-year study of 89 breast cancer patients treated with doxorubicin or epirubicin, more than 50% showed reduced left ventricular ejection fraction and progressive ventricular dilation. Although troponin-I flagged acute damage, it failed to predict long-term remodeling. Using a human methylome atlas, researchers identified 33 heart-specific methylated CpG sites and validated methylated PIH1D1 (mPIH1D1) as a novel biomarker. Elevated mPIH1D1 levels strongly correlated with ventricular dilation but not left ventricular ejection fraction decline, indicating its sensitivity to early cardiac remodeling. mPIH1D1 may complement troponin-I in risk assessment and cardiotoxicity management for patients undergoing anthracycline-based chemotherapy.

Traditional two-dimensional cell cultures and in vivo animal studies fail to fully recapitulate human cardiac physiology, highlighting the urgent need for more relevant human-based models. Engineered three-dimensional cardiac systems - including organoids, engineered heart tissues, and heart-on-chip platforms offer promising alternatives, providing structural and functional insights into cardiac biology. However, a critical limitation of these models is their inability to perform fluid pumping and relaxation, which together define fundamental heart function. Engineered cardiac chambers have emerged to address this gap, enabling physiologically relevant pressure-volume measurements and capturing both contractile and diastolic dynamics that mimic aspects of native cardiac hemodynamics. This mini-review examines the current state of engineered cardiac chambers and highlights their main design features. We discuss their applications in disease modeling and drug testing, and outline key factors influencing the optimization of these models, including balancing biological fidelity with process efficiency through modular design principles. Overall, engineered cardiac chambers represent a unique, powerful platform to improve mechanistic understanding of cardiac disease, offering significant potential to advance cardiovascular research and therapeutic development.

Hydrogels are widely used materials for biomedical applications. In particular, supramolecular hydrogels bioinspired on guanosine-quadruplexes (GQ) have been utilized in the last decade for drug delivery, biocatalysis and biosensing. However, when applied as scaffolds for cell and bioengineering applications, existing GQ hydrogels suffer from important limitations: i) scarcity of fabrication strategies under mild aqueous conditions, ii) quick disintegration within few hours under physiological conditions, iii) narrow tunability of their properties in relation to native tissues. These shortcomings hamper the broad use of GQ hydrogels in bioengineering. Herein, stable and versatile GQ hydrogels are developed as dynamic biomatrices for cell culture and tissue engineering applications. The GQ matrices are easily fabricated under mild aqueous conditions, present improved physical integrity and tunable mechanical properties within physiological ranges, besides fibrillar, porous microstructure, ion conductivity and cytocompatibility. Detailed characterization of GQ self-assembly process driving hydrogelation is presented through various spectroscopic, structural, rheological and microscopic techniques. For the first time, mechanistic insight on the ion conductivity of GQ hydrogels is revealed by electro-rheology. These matrices support the culture of diverse cell types in 2D and 3D, and showcase injectability and printability, making these scaffolds suitable for manufacturing soft medical devices. The results of this study expand the applicability potential of GQ hydrogel for advancing in vitro tissue models.

The maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) remains a major challenge in developing functional in vitro cardiac models. While three-dimensional (3D) culture systems improve structural and metabolic properties, they do not fully recapitulate adult cardiomyocyte physiology. Exogenous electrical stimulation has emerged as complementary strategy to further drive maturation. This review highlights both the maturation effects of electrical pacing in 3D cardiac tissues, including enhanced sarcomere organization, conduction velocity, calcium handling, and contractile function. It also discusses the technological parameters used to achieve these outcomes, such as electric field voltage (EFV), pulse duration (PD), stimulation waveform, electrode materials, and pacing protocols, and how these factors influence hPSC-CM development. Despite progress, further research is needed to optimize stimulation setups and to integrate electrical pacing with other maturation cues. Advancing high-throughput, miniaturized platforms will be essential for translating these models into biomedical applications like drug discovery and disease modeling.

Stem cell-based models of human heart tissue and cardiac differentiation employ monolayer and 3D organoid cultures with different properties, cell type composition, and maturity. Here we show how cardiac monolayer, embryoid body, and engineered heart tissue trajectories compare in a single-cell roadmap of atrial and ventricular differentiation conditions. Using a multiomic approach and gene-regulatory network inference, we identified regulators of the epicardial, atrial, and ventricular cardiomyocyte lineages. We identified ZNF711 as a regulatory switch and safeguard for cardiomyocyte commitment. We show that ZNF711 ablation prevents cardiomyocyte differentiation in the absence of retinoic acid, causing progenitors to be diverted more prominently to epicardial and other lineages. Retinoic acid rescues this shift in lineage commitment and promotes atrial cardiomyocyte differentiation by regulation of shared and complementary target genes, showing interplay between ZNF711 and retinoic acid in cardiac lineage commitment.

The use of human pluripotent stem cells in cardiac tissue engineering has led to significant advances in the development of in vitro models of the human heart. However, full maturation of human pluripotent stem cell-derived cardiomyocytes has not been achieved. Current maturation strategies aim to replicate the native cardiac environment by incorporating the passive and active mechanical cues of the heart. Cardiac preload and afterload are key active mechanical loads that directly influence cardiomyocyte maturation and overall cardiac function. In this review, we explore the role of mechanical stimuli in cardiac development and cardiomyocyte maturation, with a focus on how preload and afterload dynamics can be replicated in vitro using platforms such as engineered heart tissues, stretchable membranes, bioactuators, engineered cardiac chambers, and microtissues. Additionally, we highlight the role of stimulation parameters used in dynamic preload modelling and how the incorporation of these active mechanical loads is applied in disease modelling.

Aims:P-glycoprotein (P-gp), an efflux transporter with diverse compound effects, is a vital part of cardiac function. To determine if the selective substrate tracer [18F]MC225 also functions in cardiac P-gp, micro-engineered heart tissues (µ-EHTs) utilizing human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes were used. This model offers advantages in potentially reducing animal experiments and allowing direct evaluation on human cells. However, its adoption in nuclear medicine remains very limited. This study aims to evaluate [18F]MC225 as a measurement method for cardiac P-gp function using a heart-on-chip model. Methods and results:µ-EHTs were treated with the P-gp inhibitor Tariquidar (200 nM for 30 min) or the P-gp inducer Doxorubicin (1 µM for 24 h) and incubated with [18F]MC225 (1 MBq/mL for 30 min). First, we identified and confirmed the expression of P-gp in the µ-EHTs using immunofluorescent staining, which showed an increase of P-gp expression after Doxorubicin treatment. According to γ-counter measurements, Tariquidar-treated tissues exhibited a higher uptake (117.5 ± 33.67%, n = 24) (P = 0.035) than the control, compared to Doxorubicin-treated tissues which exhibited a lower uptake (63.97 ± 21.89%, n = 20) (P < 0.001) compared to its controls. Autoradiography visualized radioactive distribution in each µ-EHT and confirmed the γ-counter measurements. Conclusion:[18F]MC225 effectively evaluates and measures cardiac P-gp function in µ-EHTs on the heart-on-chip platform. This research sets the stage for future studies using P-gp function to evaluate the efficacy and safety of novel cardiovascular drugs using µ-EHTs.

Functional stem cell-derived heart models offer new avenues for preclinical, animal-free physiological assessment of drug cardiotoxicity. Yet, comprehensive molecular profiling in these models remains limited, leaving key metabolic drivers of cardiotoxicity unexplored. Here, we leveraged an innovative platform and a topology-guided integration framework to unveil the complex dose- and time-dependent metabolic rewiring of the central carbon metabolism caused by doxorubicin-induced cardiotoxicity (DiC) in human heart tissue. Through cross-modal integration of cardiac functionality and metabolomics in 3D engineered heart tissues, we identified 20 metabolites linked to cardiac contraction and differentially affected by doxorubicin exposure. Nine of them, including carnitine esters and uridine 5'-diphosphate (UDP)-glucuronic acid, were never before implicated in DiC and may represent promising candidates for DiC metabolic rescue. By yielding high-resolution insights into complex biological mechanisms, our platform and mathematical framework enable metabolic and functional assessment of cardiotoxicity in engineered heart models, paving the way for innovative advances in preclinical drug development.

Engineered heart tissues (EHTs) have shown great potential in recapitulating tissue organization, functions, and cell-cell interactions of the human heart in vitro. Currently, multiple EHT platforms are used by both industry and academia for different applications, such as drug discovery, disease modelling, and fundamental research. The tissues' contractile force, one of the main hallmarks of tissue function and maturation level of cardiomyocytes, can be read out from EHT platforms by optically tracking the movement of elastic pillars induced by the contractile tissues. However, existing optical tracking algorithms which focus on calculating the contractile force are customized and platform-specific, often not available to the broad research community, and thus hamper head-to-head comparison of the model output. Therefore, there is the need for robust, standardized and platform-independent software for tissues' force assessment. To meet this need, we developed ForceTracker: a standalone and computationally efficient software for analyzing contractile properties of tissues in different EHT platforms. The software uses a shape-detection algorithm to single out and track the movement of pillars' tips for the most common shapes of EHT platforms. In this way, we can obtain information about tissues' contractile performance. ForceTracker is coded in Python and uses a multi-threading approach for time-efficient analysis of large data sets in multiple formats. The software efficiency to analyze circular and rectangular pillar shapes is successfully tested by analyzing different format videos from two EHT platforms, developed by different research groups. We demonstrate robust and reproducible performance of the software in the analysis of tissues over time and in various conditions. ForceTracker's detection and tracking shows low sensitivity to common incidental defects, such as alteration of tissue shape or air bubbles. Detection accuracy is determined via comparison with manual measurements using the software ImageJ. We developed ForceTracker as a tool for standardized analysis of contractile performance in EHT platforms to facilitate research on disease modeling and drug discovery in academia and industry.

Engineered cardiac tissue models for in vitro physiological studies often fail to replicate the pump function of the heart. Despite promising advancements, the use of engineered cardiac chambers is often hindered by complex fabrication processes and invasive characterization techniques. Here, we engineered a human cardiac chamber, referred to as a ‘mini-heart’, by employing a novel sacrificial molding approach within a customized bioreactor. The mini-heart’s pumping capability was confirmed through optical recording of fluid displacement at the engineered tissue inlet, enabling the non-invasive acquisition of hemodynamic parameters such as stroke volume, stroke work, ejection fraction, and developed pressure. Morphological analysis of the engineered tissues revealed organized sarcomeres and extracellular matrix self-determination, highlighting the advantage of our degradable mold technology. Additionally, we have measured calcium transients during both spontaneous and electrically-paced beating, and observed a positive inotropic response to the β-adrenergic agonist drug isoproterenol. Altogether, our biomimicking engineered tissue platform provides a robust tool for exploring cardiac pressure-volume dynamics, thereby facilitating complex disease modelling and drug screening applications in vitro . ### Competing Interest Statement R.P. is a co-founder of Pluriomics (Ncardia) and River BioMedics BV. M.C.R. is a co-founder of River Biomedics BV. * CVD : Cardiovascular disease hPSC : Human pluripotent stem cell CM : Cardiomyocyte EHT : Engineered heart tissue HCF : Human cardiac fibroblast Cx-43 : Connexin 43 ECM : Extracellular matrix Pt : Total pressure Pd : Dynamic pressure Ph : Hydrostatic pressure EDA : End-diastolic area ESA : End-systolic area CTD : Calcium transient duration RT : Relaxation time TTP : Time to peak PV : Pressure-volume cTnT : Cardiac troponin SW : Stroke work European Research Council European Innovation Council, 101070953 Nederlandse Organisatie voor Wetenschappelijk Onderzoek, NWO/ENW-XL 2019.029

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): This work was supported by the European Research Area Network on Cardiovascular Diseases 807 (2016T092) and Hartstichting (2019T104) Background/Introduction/Purpose Cardiotoxicity continues to be a significant factor in drug withdrawal, partly due to the unpredictable nature of animal models. Moreover, the limitation of cancer treatment arises from the potential risk of cardiotoxicity. Identifying patients at risk of developing heart symptoms after therapy will strongly contribute to improved treatment possibilities. Cardiomyocytes derived from human pluripotent stem cells (hPSCs) represent an inexhaustible cell reservoir and hold the potential to provide personalized solutions to address this issue. However, conventional 2D cultures have several limitations, such as tissue complexity and the difficulty to apply repeated dose treatments. Methods Therefore, we developed an engineered heart tissue (EHT) platform to predict functional aspects of cardiotoxicity. We evaluated the effect of several anti-cancer drugs on cardiomyocyte contractility after repetitive treatment and quantified force of contraction and contraction kinetics. However, these traditional EHTs lack the complexity of diverse cell types. Thus, in a next step, we generated an innovative and user-friendly Heart-on-Chip (HoC) model from hPSC-derived cardiomyocytes, endothelial and smooth muscle cells. Under flow, these miniaturized micro-EHTs (μEHTs) self-organize into a cardiomyocyte core surrounded by an endothelial barrier layer mimicking the cardiomyocyte-endothelial interface as observed in vivo. Results Our versatile drug screening systems permit following individual tissues over time. We therefore treated EHTs with DMSO or 1 µM Doxorubicin for 24 hours and monitored tissue contraction force over a time course of 28 days. Doxorubicin treatment initially caused a drop of force, but demonstrated a recovery of force and kinetics 10 days after treatment. To recapitulate a clinically relevant intermittent treatment regime, a second treatment of 1 µM Doxorubicin was administered 21 days after the first dose and showed a similar decrease in contraction force compared with the first exposure to the drug. μEHTs cultured under flow exhibit improved contractile performance and conduction velocity when compared to static culture conditions. Importantly, the presence of an endothelial barrier delayed the cardiotoxic effect of drugs, indicating that endothelial cells form a barrier mimicking the systemic delivery route of drugs to the heart. Conclusions Our systems are characterized by increased physiological accuracy and enable the study of hPSC-cardiomyocyte recovery and the effects of accumulated dose after multiple dosing, allowing individualized cardiotoxicity evaluation. In the future, we can further increase the complexity by including multi-organ-on-chip approaches, as for example heart-liver-on-chip to revolutionize the screening of therapeutic targets and the prediction of drug efficacy.

The synchronization of the electrical and mechanical coupling assures the physiological pump function of the heart, but life-threatening pathologies may jeopardize this equilibrium. Recently, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a model for personalized investigation because they can recapitulate human diseased traits, such as compromised electrical capacity or mechanical circuit disruption. This research avails the model of hiPSC-CMs and showcases innovative techniques to study the electrical and mechanical properties as well as their modulation due to inherited cardiomyopathies. In this work, hiPSC-CMs carrying either Brugada syndrome (BRU) or dilated cardiomyopathy (DCM), were organized in a bilayer configuration to first validate the experimental methods and second mimic the physiological environment. High-density CMOS-based microelectrode arrays (HD-MEA) have been employed to study the electrical activity. Furthermore, mechanical function was investigated via quantitative video-based evaluation, upon stimulation with a beta-adrenergic agonist. This study introduces two experimental methods. First, high-throughput mechanical measurements in the hiPSC-CM layers (xy-inspection) are obtained using both a recently developed optical tracker (OPT) and confocal reference-free traction force microscopy (cTFM) aimed to quantify cardiac kinematics. Second, atomic force microscopy (AFM) with FluidFM probes, combined with the xy-inspection methods, supplemented a three-dimensional understanding of cell-cell mechanical coupling (xyz-inspection). This particular combination represents a multi-technique approach to detecting electrical and mechanical latency among the cell layers, examining differences and possible implications following inherited cardiomyopathies. It can not only detect disease characteristics in the proposed in vitro model but also quantitatively assess its response to drugs, thereby demonstrating its feasibility as a scalable tool for clinical and pharmacological studies.

Cardiotoxicity remains a major cause of drug withdrawal, partially due to lacking predictability of animal models. Additionally, risk of cardiotoxicity following treatment of cancer patients is treatment limiting. It is unclear which patients will develop heart failure following therapy. Human pluripotent stem cell (hPSC)-derived cardiomyocytes present an unlimited cell source and may offer individualized solutions to this problem. We developed a platform to predict molecular and functional aspects of cardiotoxicity. Our platform can discriminate between the different cardiotoxic mechanisms of existing and novel anthracyclines Doxorubicin, Aclarubicin, and Amrubicin. Doxorubicin and Aclarubicin unlike Amrubicin substantially affected the transcriptome, mitochondrial membrane integrity, contractile force and transcription factor availability. Cardiomyocytes recovered fully within two or three weeks, corresponding to the intermittent clinical treatment regimen. Our system permits the study of hPSC-cardiomyocyte recovery and the effects of accumulated dose after multiple dosing, allowing individualized cardiotoxicity evaluation, which effects millions of cancer patients treated annually.