Generation and characterization of novel human induced pluripotent stem cell (iPSC) lines originating from five asymptomatic individuals carrying the PLN-R14del pathogenic variant and a non-carrier relative.

The rare genetic alteration PLN-c.(40_42delAGA), leading to the deletion of arginine 14 (p.R14del) in phospholamban, is associated with dilated and arrhythmogenic cardiomyopathies occurring in early-adulthood. However, some carriers remain asymptomatic with normal lifespans. Here, we report human induced pluripotent stem cell (iPSC) lines generated from peripheral blood mononuclear cells (PBMCs) of five PLN-R14del carriers, who were asymptomatic at the time of blood collection, and one non-carrier family member. Each line exhibited typical iPSC morphology, pluripotency markers, and tri-lineage differentiation. These cell lines provide a valuable model to investigate the mechanisms underlying the onset, progression, and patient-specific resistance to PLN-R14del-induced cardiomyopathy.

Rianú: Multi-tissue tracking software for increased throughput of engineered cardiac tissue screening.

Background: The field of tissue engineering has provided valuable three-dimensional species-specific models of the human myocardium in the form of human Engineered Cardiac Tissues (hECTs) and similar constructs. However, hECT systems are often bottlenecked by a lack of openly available software that can collect data from multiple tissues at a time, even in multi-tissue bioreactors, which limits throughput in phenotypic and therapeutic screening applications. Methods: We developed Rianú, an open-source web application capable of simultaneously tracking multiple hECTs on flexible end-posts. This software is operating system agnostic and deployable on a remote server, accessible via a web browser with no local hardware or software requirements. The software incorporates object-tracking capabilities for multiple objects simultaneously, an algorithm for twitch tracing analysis and contractile force calculation, and a data compilation system for comparative analysis within and amongst groups. Validation tests were performed using in-silico and in-vitro experiments for comparison with established methods and interventions. Results: Rianú was able to detect the displacement of the flexible end-posts with a sub-pixel sensitivity of 0.555 px/post (minimum increment in post displacement) and a lower limit of 1.665 px/post (minimum post displacement). Compared to our established reference for contractility assessment, Rianú had a high correlation for all parameters analyzed (ranging from R2=0.7514 to R2=0.9695), demonstrating its high accuracy and reliability. Conclusions: Rianú provides simultaneous tracking of multiple hECTs, expediting the recording and analysis processes, and simplifies time-based intervention studies. It also allows data collection from different formats and has scale-up capabilities proportional to the number of tissues per field of view. These capabilities will enhance throughput of hECTs and similar assays for in-vitro analysis in disease modeling and drug screening applications.

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues.

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues. This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process. The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Single-cell RNA sequencing reveals maturation trajectory in human pluripotent stem cell-derived cardiomyocytes in engineered tissues.

Cardiac in vitro models have become increasingly obtainable and affordable with the optimization of human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) differentiation. However, these CMs are immature compared to their in vivo counterparts. Here we study the cellular phenotype of hPSC-CMs by comparing their single-cell gene expression and functional profiles in three engineered cardiac tissue configurations: human ventricular (hv) cardiac anisotropic sheet, cardiac tissue strip, and cardiac organoid chamber (hvCOC), with spontaneously aggregated 3D cardiac spheroids (CS) as control. The CM maturity was found to increase with increasing levels of complexity of the engineered tissues from CS to hvCOC. The contractile components are the first function to mature, followed by electrophysiology and oxidative metabolism. Notably, the 2D tissue constructs show a higher cellular organization whereas metabolic maturity preferentially increases in the 3D constructs. We conclude that the tissue engineering models resembling configurations of native tissues may be reliable for drug screening or disease modeling.

Computational design of custom therapeutic cells to correct failing human cardiomyocytes.

Background: Myocardial delivery of non-excitable cells-namely human mesenchymal stem cells (hMSCs) and c-kit+ cardiac interstitial cells (hCICs)-remains a promising approach for treating the failing heart. Recent empirical studies attempt to improve such therapies by genetically engineering cells to express specific ion channels, or by creating hybrid cells with combined channel expression. This study uses a computational modeling approach to test the hypothesis that custom hypothetical cells can be rationally designed to restore a healthy phenotype when coupled to human heart failure (HF) cardiomyocytes. Methods: Candidate custom cells were simulated with a combination of ion channels from non-excitable cells and healthy human cardiomyocytes (hCMs). Using a genetic algorithm-based optimization approach, candidate cells were accepted if a root mean square error (RMSE) of less than 50% relative to healthy hCM was achieved for both action potential and calcium transient waveforms for the cell-treated HF cardiomyocyte, normalized to the untreated HF cardiomyocyte. Results: Custom cells expressing only non-excitable ion channels were inadequate to restore a healthy cardiac phenotype when coupled to either fibrotic or non-fibrotic HF cardiomyocytes. In contrast, custom cells also expressing cardiac ion channels led to acceptable restoration of a healthy cardiomyocyte phenotype when coupled to fibrotic, but not non-fibrotic, HF cardiomyocytes. Incorporating the cardiomyocyte inward rectifier K+ channel was critical to accomplishing this phenotypic rescue while also improving single-cell action potential metrics associated with arrhythmias, namely resting membrane potential and action potential duration. The computational approach also provided insight into the rescue mechanisms, whereby heterocellular coupling enhanced cardiomyocyte L-type calcium current and promoted calcium-induced calcium release. Finally, as a therapeutically translatable strategy, we simulated delivery of hMSCs and hCICs genetically engineered to express the cardiomyocyte inward rectifier K+ channel, which decreased action potential and calcium transient RMSEs by at least 24% relative to control hMSCs and hCICs, with more favorable single-cell arrhythmia metrics. Conclusion: Computational modeling facilitates exploration of customizable engineered cell therapies. Optimized cells expressing cardiac ion channels restored healthy action potential and calcium handling phenotypes in fibrotic HF cardiomyocytes and improved single-cell arrhythmia metrics, warranting further experimental validation studies of the proposed custom therapeutic cells.

Age and Blood Pressure Contribute to Aortic Cell and Tissue Stiffness Through Distinct Mechanisms.

BACKGROUND: Aortic stiffening is strongly associated with both aging and hypertension, but the underlying mechanisms remain unclear. We hypothesized that aging-induced aortic stiffness is mediated by a mechanism differing from hypertension. METHODS: We conducted comprehensive in vivo and in vitro experiments using multiple rat models to dissect the different mechanisms of aortic stiffening mediated by aging and hypertension. RESULTS: A time-course study in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) normotensive rats showed more pronounced aging-associated aortic stiffening in SHR versus WKY. Angiotensin II-induced hypertension was associated with more significant aortic stiffening in older versus young WKY rats. Hypertension aggravated aging effects on aortic wall thickness and extracellular matrix content, indicating combinational effects of aging and hypertension on aortic stiffening. Intrinsic stiffness of isolated aortic vascular smooth muscle cells (VSMCs) increased with age in WKY rats, although no significant difference between older SHR and older WKY VSMCs was observed in 2-dimensional culture, reconstituted 3-dimensional tissues were stiffer for older SHR versus older WKY. A selective inhibitor that reduced hypertension-mediated aortic stiffening did not decrease age-related stiffening in aortic VSMCs and aortic wall. Integrin ß1 and SM22 (smooth muscle-specific SM22 protein) expression were negligibly changed in WKY VSMCs during aging but were markedly increased by hypertension in older versus young WKY VSMCs. A notable shift of filamin isoforms from B to A was detected in older WKY VSMCs. CONCLUSIONS: Our results indicate distinct mechanisms mediating aging-associated aortic VSMC and vessel stiffness, providing new insights into aortic stiffening and the pathogenesis of hypertension in the elderly.

Generation of human induced pluripotent stem cell (iPSC) lines derived from five patients carrying the pathogenic phospholamban-R14del (PLN-R14del) variant and three non-carrier family members.

The R14del pathogenic variant in the phospholamban (PLN) gene (PLN-R14del), has been identified in families with hereditary cardiomyopathy, including dilated and arrhythmogenic cardiomyopathies. Here we have generated human iPSC lines from five PLN-R14del carriers and three non-carrier family members. Peripheral blood mononuclear cells (PBMC) were obtained from the eight individuals and reprogrammed using Sendai viral vector system carrying the Yamanaka factors. All eight lines show typical iPSC morphology, normal karyotype, high expression of pluripotency markers, and possess the ability to differentiate into all three germ layers. These lines represent valuable resources for studying the pathophysiological mechanisms of PLN-R14del associated cardiomyopathy.

Gene editing reverses arrhythmia susceptibility in humanized PLN-R14del mice: modelling a European cardiomyopathy with global impact.

AIMS: A mutation in the phospholamban (PLN) gene, leading to deletion of Arg14 (R14del), has been associated with malignant arrhythmias and ventricular dilation. Identifying pre-symptomatic carriers with vulnerable myocardium is crucial because arrhythmia can result in sudden cardiac death, especially in young adults with PLN-R14del mutation. This study aimed at assessing the efficiency and efficacy of in vivo genome editing, using CRISPR/Cas9 and a cardiotropic adeno-associated virus-9 (AAV9), in improving cardiac function in young adult mice expressing the human PLN-R14del. METHODS AND RESULTS: Humanized mice were generated expressing human wild-type (hPLN-WT) or mutant (hPLN-R14del) PLN in the heterozygous state, mimicking human carriers. Cardiac magnetic resonance imaging at 12 weeks of age showed bi-ventricular dilation and increased stroke volume in mutant vs. WT mice, with no deficit in ejection fraction or cardiac output. Challenge of ex vivo hearts with isoproterenol and rapid pacing unmasked higher propensity for sustained ventricular tachycardia (VT) in hPLN-R14del relative to hPLN-WT. Specifically, the VT threshold was significantly reduced (20.3 ± 1.2 Hz in hPLN-R14del vs. 25.7 ± 1.3 Hz in WT, P < 0.01) reflecting higher arrhythmia burden. To inactivate the R14del allele, mice were tail-vein-injected with AAV9.CRISPR/Cas9/gRNA or AAV9 empty capsid (controls). CRISPR-Cas9 efficiency was evaluated by droplet digital polymerase chain reaction and NGS-based amplicon sequencing. In vivo gene editing significantly reduced end-diastolic and stroke volumes in hPLN-R14del CRISPR-treated mice compared to controls. Susceptibility to VT was also reduced, as the VT threshold was significantly increased relative to controls (30.9 ± 2.3 Hz vs. 21.3 ± 1.5 Hz; P < 0.01). CONCLUSIONS: This study is the first to show that disruption of hPLN-R14del allele by AAV9-CRISPR/Cas9 improves cardiac function and reduces VT susceptibility in humanized PLN-R14del mice, offering preclinical evidence for translatable approaches to therapeutically suppress the arrhythmogenic phenotype in human patients with PLN-R14del disease.

Adverse effects of hydroxychloroquine and azithromycin on contractility and arrhythmogenicity revealed by human engineered cardiac tissues.

The coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic as declared by World Health Organization (WHO). In the absence of an effective treatment, different drugs with unknown effectiveness, including antimalarial hydroxychloroquine (HCQ), with or without concurrent administration with azithromycin (AZM), have been tested for treating COVID-19 patients with developed pneumonia. However, the efficacy and safety of HCQ and/or AZM have been questioned by recent clinical reports. Direct effects of these drugs on the human heart remain very poorly defined. To better understand the mechanisms of action of HCQ +/- AZM, we employed bioengineered human ventricular cardiac tissue strip (hvCTS) and anisotropic sheet (hvCAS) assays, made with human pluripotent stem cell (hPSC)-derived ventricular cardiomyocytes (hvCMs), which have been designed for measuring cardiac contractility and electrophysiology, respectively. Our hvCTS experiments showed that AZM induced a dose-dependent negative inotropic effect which could be aggravated by HCQ; electrophysiologically, as revealed by the hvCAS platform, AZM prolonged action potentials and induced spiral wave formations. Collectively, our data were consistent with reported clinical risks of HCQ and AZM on QTc prolongation/ventricular arrhythmias and development of heart failure. In conclusion, our study exposed the risks of HCQ/AZM administration while providing mechanistic insights for their toxicity. Our bioengineered human cardiac tissue constructs therefore provide a useful platform for screening cardiac safety and efficacy when developing therapeutics against COVID-19.

In silico Cell Therapy Model Restores Failing Human Myocyte Electrophysiology and Calcium Cycling in Fibrotic Myocardium.

Myocardial delivery of human c-kit+ cardiac interstitial cells (hCICs) and human mesenchymal stem cells (hMSCs), an emerging approach for treating the failing heart, has been limited by an incomplete understanding of the effects on host myocardium. This computational study aims to model hCIC and hMSC effects on electrophysiology and calcium cycling of healthy and diseased human cardiomyocytes (hCM), and reveals a possible cardiotherapeutic benefit independent of putative regeneration processes. First, we developed an original hCIC mathematical model with an electrical profile comprised of distinct experimentally identified ion currents. Next, we verified the model by confirming it is representative of published experiments on hCIC whole-cell electrophysiology and on hCIC co-cultures with rodent cardiomyocytes. We then used our model to compare electrophysiological effects of hCICs to other non-excitable cells, as well as clinically relevant hCIC-hMSC combination therapies and fused hCIC-hMSC CardioChimeras. Simulation of direct coupling of hCICs to healthy or failing hCMs through gap junctions led to greater increases in calcium cycling with lesser reductions in action potential duration (APD) compared with hMSCs. Combined coupling of hCICs and hMSCs to healthy or diseased hCMs led to intermediate effects on electrophysiology and calcium cycling compared to individually coupled hCICs or hMSCs. Fused hCIC-hMSC CardioChimeras decreased healthy and diseased hCM APD and calcium transient amplitude compared to individual or combined cell treatments. Finally, to provide a theoretical basis for optimizing cell-based therapies, we randomized populations of 2,500 models incorporating variable hMSC and hCIC interventions and simulated their effects on restoring diseased cardiomyocyte electrophysiology and calcium handling. The permutation simulation predicted the ability to correct abnormal properties of heart failure hCMs in fibrotic, but not non-fibrotic, myocardium. This permutation experiment also predicted paracrine signaling to be a necessary and sufficient mechanism for this correction, counteracting the fibrotic effects while also restoring arrhythmia-related metrics such as upstroke velocity and resting membrane potential. Altogether, our in silico findings suggest anti-fibrotic effects of paracrine signaling are critical to abrogating pathological cardiomyocyte electrophysiology and calcium cycling in fibrotic heart failure, and support further investigation of delivering an optimized cellular secretome as a potential strategy for improving heart failure therapy.

High-resolution integrated piezoresistive sensors for microfluidic monitoring.

Microfluidic devices are traditionally monitored by bulky and expensive off-chip sensors. We have developed a soft piezoresistive sensor capable of measuring micron-level strains that can be easily integrated into devices via soft lithography. We apply this sensor to achieve fast and localized monitoring of pressure, flow, and valve actuation.

Combinatorial Treatment of Human Cardiac Engineered Tissues With Biomimetic Cues Induces Functional Maturation as Revealed by Optical Mapping of Action Potentials and Calcium Transients.

Although biomimetic stimuli, such as microgroove-induced alignment (µ), triiodothyronine (T3) induction, and electrical conditioning (EC), have been reported to promote maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), a systematic examination of their combinatorial effects on engineered cardiac tissue constructs and the underlying molecular pathways has not been reported. Herein, human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) were used to generate a micro-patterned human ventricular cardiac anisotropic sheets (hvCAS) for studying the physiological effects of combinatorial treatments by a range of functional, calcium (Ca2+)-handling, and molecular analyses. High-resolution optical mapping showed that combined µ-T3-EC treatment of hvCAS increased the conduction velocity, anisotropic ratio, and proportion of mature quiescent-yet-excitable preparations by 2. 3-, 1. 8-, and 5-fold (>70%), respectively. Such electrophysiological changes could be attributed to an increase in inward sodium current density and a decrease in funny current densities, which is consistent with the observed up- and downregulated SCN1B and HCN2/4 transcripts, respectively. Furthermore, Ca2+-handling transcripts encoding for phospholamban (PLN) and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) were upregulated, and this led to faster upstroke and decay kinetics of Ca2+-transients. RNA-sequencing and pathway mapping of T3-EC-treated hvCAS revealed that the TGF-ß signaling was downregulated; the TGF-ß receptor agonist and antagonist TGF-ß1 and SB431542 partially reversed T3-EC induced quiescence and reduced spontaneous contractions, respectively. Taken together, we concluded that topographical cues alone primed cardiac tissue constructs for augmented electrophysiological and calcium handling by T3-EC. Not only do these studies improve our understanding of hPSC-CM biology, but the orchestration of these pro-maturational factors also improves the use of engineered cardiac tissues for in vitro drug screening and disease modeling.

Adult human cardiac stem cell supplementation effectively increases contractile function and maturation in human engineered cardiac tissues.

BACKGROUND: Delivery of stem cells to the failing heart is a promising therapeutic strategy. However, the improvement in cardiac function in animal studies has not fully translated to humans. To help bridge the gap between species, we investigated the effects of adult human cardiac stem cells (hCSCs) on contractile function of human engineered cardiac tissues (hECTs) as a species-specific model of the human myocardium. METHODS: Human induced pluripotent stem cell-derived cardiomyoctes (hCMs) were mixed with Collagen/Matrigel to fabricate control hECTs, with an experimental group of hCSC-supplemented hECT fabricated using a 9:1 ratio of hCM to hCSC. Functional testing was performed starting on culture day 6, under spontaneous conditions and also during electrical pacing from 0.25 to 1.0 Hz, measurements repeated at days 8 and 10. hECTs were then frozen and processed for gene analysis using a Nanostring assay with a cardiac targeted custom panel. RESULTS: The hCSC-supplemented hECTs displayed a twofold higher developed force vs. hCM-only controls by day 6, with approximately threefold higher developed stress and maximum rates of contraction and relaxation during pacing at 0.75 Hz. The spontaneous beat rate characteristics were similar between groups, and hCSC supplementation did not adversely impact beat rate variability. The increased contractility persisted through days 8 and 10, albeit with some decrease in the magnitude of the difference of the force by day 10, but with developed stress still significantly higher in hCSC-supplemented hECT; these findings were confirmed with multiple hCSC and hCM cell lines. The force-frequency relationship, while negative for both, control (- 0.687 Hz- 1; p = 0.013 vs. zero) and hCSC-supplemented (- 0.233 Hz- 1;p = 0.067 vs. zero) hECTs, showed a significant rectification in the regression slope in hCSC-supplemented hECT (p = 0.011 vs. control). Targeted gene exploration (59 genes) identified a total of 14 differentially expressed genes, with increases in the ratios of MYH7/MHY6, MYL2/MYL7, and TNNI3/TNNI1 in hCSC-supplemented hECT versus controls. CONCLUSIONS: For the first time, hCSC supplementation was shown to significantly improve human cardiac tissue contractility in vitro, without evidence of proarrhythmic effects, and was associated with increased expression of markers of cardiac maturation. These findings provide new insights about adult cardiac stem cells as contributors to functional improvement of human myocardium.

Correlation between frataxin expression and contractility revealed by in vitro Friedreich's ataxia cardiac tissue models engineered from human pluripotent stem cells.

BACKGROUND: Friedreich's ataxia (FRDA) is an autosomal recessive disease caused by a non-coding mutation in the first intron of the frataxin (FXN) gene that suppresses its expression. Compensatory hypertrophic cardiomyopathy, dilated cardiomyopathy, and conduction system abnormalities in FRDA lead to cardiomyocyte (CM) death and fibrosis, consequently resulting in heart failure and arrhythmias. Murine models have been developed to study disease pathology in the past two decades; however, differences between human and mouse physiology and metabolism have limited the relevance of animal studies in cardiac disease conditions. To bridge this gap, we aimed to generate species-specific, functional in vitro experimental models of FRDA using 2-dimensional (2D) and 3-dimensional (3D) engineered cardiac tissues from FXN-deficient human pluripotent stem cell-derived ventricular cardiomyocytes (hPSC-hvCMs) and to compare their contractile and electrophysiological properties with healthy tissue constructs. METHODS: Healthy control and FRDA patient-specific hPSC-hvCMs were derived by directed differentiation using a small molecule-based protocol reported previously. We engineered the hvCMs into our established human ventricular cardiac tissue strip (hvCTS) and human ventricular cardiac anisotropic sheet (hvCAS) models, and functional assays were performed on days 7-17 post-tissue fabrication to assess the electrophysiology and contractility of FRDA patient-derived and FXN-knockdown engineered tissues, in comparison with healthy controls. To further validate the disease model, forced expression of FXN was induced in FXN-deficient tissues to test if disease phenotypes could be rescued. RESULTS: Here, we report for the first time the generation of human engineered tissue models of FRDA cardiomyopathy from hPSCs: FXN-deficient hvCTS displayed attenuated developed forces (by 70-80%) compared to healthy controls. High-resolution optical mapping of hvCAS with reduced FXN expression also revealed electrophysiological defects consistent with clinical observations, including action potential duration prolongation and maximum capture frequency reduction. Interestingly, a clear positive correlation between FXN expression and contractility was observed (ρ > 0.9), and restoration of FXN protein levels by lentiviral transduction rescued contractility defects in FXN-deficient hvCTS. CONCLUSIONS: We conclude that human-based in vitro cardiac tissue models of FRDA provide a translational, disease-relevant biomimetic platform for the evaluation of novel therapeutics and to provide insight into FRDA disease progression.

Sarco/endoplasmic reticulum Ca2+-ATPase is a more effective calcium remover than sodium-calcium exchanger in human embryonic stem cell-derived cardiomyocytes.

Human pluripotent stem cell (hPSCs)-derived ventricular (V) cardiomyocytes (CMs) display immature Ca2+-handing properties with smaller transient amplitudes and slower kinetics due to such differences in crucial Ca2+-handling proteins as the poor sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump but robust Na+-Ca2+ exchanger (NCX) activities in human embryonic stem cell (ESC)-derived VCMs compared with adult. Despite their fundamental importance in excitation-contraction coupling, the relative contribution of SERCA and NCX to Ca2+-handling of hPSC-VCMs remains unexplored. We systematically altered the activities of SERCA and NCX in human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) and their engineered microtissues, followed by examining the resultant phenotypic consequences. SERCA overexpression in hESC-VCMs shortened the decay of Ca2+ transient at low frequencies (0.5 Hz) without affecting the amplitude, SR Ca2+ content and Ca2+ baseline. Interestingly, short hairpin RNA-based NCX suppression did not prolong the transient decay, indicating a compensatory response for Ca2+ removal. Although hESC-VCMs and their derived microtissues exhibited negative frequency-transient/force responses, SERCA overexpression rendered them less negative at high frequencies (>2 Hz) by accelerating Ca2+ sequestration. We conclude that for hESC-VCMs and their microtissues, SERCA, rather than NCX, is the main Ca2+ remover during diastole; poor SERCA expression is the leading cause for immature negative-frequency/force responses, which can be partially reverted by forced expression. Combinatorial approach to mature calcium handling in hESC-VCMs may help shed further mechanistic insights.NEW & NOTEWORTHY In this study of human pluripotent stem cell-derived cardiomyocytes, we studied the role of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) in Ca2+ handling. Our data support the notion that SERCA is more effective in cytosolic calcium removal than the NCX.

Modulation of chromatin remodeling proteins SMYD1 and SMARCD1 promotes contractile function of human pluripotent stem cell-derived ventricular cardiomyocyte in 3D-engineered cardiac tissues.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have the ability of differentiating into functional cardiomyocytes (CMs) for cell replacement therapy, tissue engineering, drug discovery and toxicity screening. From a scale-free, co-expression network analysis of transcriptomic data that distinguished gene expression profiles of undifferentiated hESC, hESC-, fetal- and adult-ventricular(V) CM, two candidate chromatin remodeling proteins, SMYD1 and SMARCD1 were found to be differentially expressed. Using lentiviral transduction, SMYD1 and SMARCD1 were over-expressed and suppressed, respectively, in single hESC-VCMs as well as the 3D constructs Cardiac Micro Tissues (CMT) and Tissue Strips (CTS) to mirror the endogenous patterns, followed by dissection of their roles in controlling cardiac gene expression, contractility, Ca2+-handling, electrophysiological functions and in vitro maturation. Interestingly, compared to independent single transductions, simultaneous SMYD1 overexpression and SMARCD1 suppression in hESC-VCMs synergistically interacted to increase the contractile forces of CMTs and CTSs with up-regulated transcripts for cardiac contractile, Ca2+-handing, and ion channel proteins. Certain effects that were not detected at the single-cell level could be unleashed under 3D environments. The two chromatin remodelers SMYD1 and SMARCD1 play distinct roles in cardiac development and maturation, consistent with the notion that epigenetic priming requires triggering signals such as 3D environmental cues for pro-maturation effects.

Human Cardiac Ventricular-Like Organoid Chambers and Tissue Strips From Pluripotent Stem Cells as a Two-Tiered Assay for Inotropic Responses.

Traditional drug discovery is an inefficient process. Human pluripotent stem cell-derived cardiomyocytes can potentially fill the gap between animal and clinical studies, but conventional two-dimensional cultures inadequately recapitulate the human cardiac phenotype. Here, we systematically examined the pharmacological responses of engineered human ventricular-like cardiac tissue strips (hvCTS) and organoid chambers (hvCOC) to 25 cardioactive compounds covering various drug classes. While hvCTS effectively detected negative and null inotropic effects, the sensitivity to positive inotropes was modest. We further quantified the predictive capacity of hvCTS in a blinded screening, with accuracies for negative, positive, and null inotropic effects at 100%, 86%, and 80%, respectively. Interestingly, hvCOC, with a pro-maturation milieu that yields physiologically complex parameters, displayed enhanced positive inotropy. Based on these results, we propose a two-tiered screening system for avoiding false positives and negatives. Such an approach would facilitate drug discovery by leading to better overall success.

An Introduction to Computational Modeling of Cardiac Electrophysiology and Arrhythmogenicity.

Mathematical modeling is a powerful tool to study the complex and orchestrated biological process of cardiac electrical activity. By integrating experimental data from key components of cardiac electrophysiology, systems biology simulations can complement empirical findings, provide quantitative insight into physiological and pathophysiological mechanisms of action, and guide new hypotheses to better understand this complex biological system to develop novel cardiotherapeutic approaches. In this chapter, we briefly introduce in silico methods to describe the dynamics of physiological and pathophysiological single-cell and tissue-level cardiac electrophysiology. Using a "bottom-up" approach, we first describe the basis of ion channel mathematical models. Next, we discuss how the net flux of ions through such channels leads to changes in transmembrane voltage during cardiomyocyte action potentials. By applying these fundamentals, we describe how action potentials propagate in models of cardiac tissue. In addition, we provide case studies simulating single-cell and tissue-level arrhythmogenesis, as well as promising approaches to circumvent or overcome such adverse events. Overall, basic concepts and tools are discussed in this chapter as an accessible introduction to nonmathematicians to foster an understanding of electrophysiological modeling studies and help facilitate communication with dry lab colleagues and collaborators.

Cardiac Tissue Engineering Models of Inherited and Acquired Cardiomyopathies.

The lack of biomimetic in vitro models of the human heart has posed a critical barrier to progress in the field of modeling cardiac disease. Human engineered cardiac tissues (hECTs)-autonomous, beating structures that recapitulate key aspects of native cardiac muscle physiology-offer an attractive alternative to traditional in vitro models. Here we describe the use of hECTs to advance our understanding and modeling of cardiac diseases in order to test therapeutic interventions, with a focus on contractile dysfunction in the setting of inherited and acquired forms of cardiomyopathies. Four major procedures are discussed in this chapter: (1) preparation of hECTs from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on single-tissue and multitissue bioreactors; (2) data acquisition of hECT contractile function on both of these platforms; (3) hECT modeling of hereditary phospholamban-R14 deletion-dilated cardiomyopathy; and (4) cryo-injury and doxorubicin-induced hECT models of acquired cardiomyopathy.

Dehomogenized Elastic Properties of Heterogeneous Layered Materials in AFM Indentation Experiments.

Atomic force microscopy (AFM) is used to study mechanical properties of biological materials at submicron length scales. However, such samples are often structurally heterogeneous even at the local level, with different regions having distinct mechanical properties. Physical or chemical disruption can isolate individual structural elements but may alter the properties being measured. Therefore, to determine the micromechanical properties of intact heterogeneous multilayered samples indented by AFM, we propose the Hybrid Eshelby Decomposition (HED) analysis, which combines a modified homogenization theory and finite element modeling to extract layer-specific elastic moduli of composite structures from single indentations, utilizing knowledge of the component distribution to achieve solution uniqueness. Using finite element model-simulated indentation of layered samples with micron-scale thickness dimensions, biologically relevant elastic properties for incompressible soft tissues, and layer-specific heterogeneity of an order of magnitude or less, HED analysis recovered the prescribed modulus values typically within 10% error. Experimental validation using bilayer spin-coated polydimethylsiloxane samples also yielded self-consistent layer-specific modulus values whether arranged as stiff layer on soft substrate or soft layer on stiff substrate. We further examined a biophysical application by characterizing layer-specific microelastic properties of full-thickness mouse aortic wall tissue, demonstrating that the HED-extracted modulus of the tunica media was more than fivefold stiffer than the intima and not significantly different from direct indentation of exposed media tissue. Our results show that the elastic properties of surface and subsurface layers of microscale synthetic and biological samples can be simultaneously extracted from the composite material response to AFM indentation. HED analysis offers a robust approach to studying regional micromechanics of heterogeneous multilayered samples without destructively separating individual components before testing.