Cardiac Disease Modeling Research Articles

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) for modeling cardiac arrhythmias: strengths, challenges and potential solutions.

Ion channels and cytoskeletal proteins in the cardiac dyad play a critical role in maintaining excitation-contraction (E-C) coupling and provide cardiac homeostasis. Functional changes in these dyad proteins, whether induced by genetic, epigenetic, metabolic, therapeutic, or environmental factors, can disrupt normal cardiac electrophysiology, leading to abnormal E-C coupling and arrhythmias. Animal models and heterologous cell cultures provide platforms to elucidate the pathogenesis of arrhythmias for basic cardiac research; however, these traditional systems do not truly reflect human cardiac electro-pathophysiology. Notably, patients with the same genetic variants of inherited channelopathies (ICC) often exhibit incomplete penetrance and variable expressivity which underscores the need to establish patient-specific disease models to comprehend the mechanistic pathways of arrhythmias and determine personalized therapies. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) inherit the genetic background of the patient and reflect the electrophysiological characteristics of the native cardiomyocytes. Thus, iPSC-CMs provide an innovative and translational pivotal platform in cardiac disease modeling and therapeutic screening. In this review, we will examine how patient-specific iPSC-CMs historically evolved to model arrhythmia syndromes in a dish, and their utility in understanding the role of specific ion channels and their functional characteristics in causing arrhythmias. We will also examine how CRISPR/Cas9 have enabled the establishment of patient-independent and variant-induced iPSC-CMs-based arrhythmia models. Next, we will examine the limitations of using human iPSC-CMs with respect to in vitro arrhythmia modeling that stems from variations in iPSCs or toxicity due to gene editing on iPSC or iPSC-CMs and explore how such hurdles are being addressed. Importantly, we will also discuss how novel 3D iPSC-CM models can better capture in vitro characteristics and how all-optical platforms provide non-invasive and high- throughput electrophysiological data that is useful for stratification of emerging arrhythmogenic variants and drug discovery. Finally, we will examine strategies to improve iPSC-CM maturity, including powerful gene editing and optogenetic tools that can introduce/modify specific ion channels in iPSC-CMs and tailor cellular and functional characteristics. We anticipate that an elegant synergy of iPSCs, novel gene editing, 3D- culture models, and all-optical platforms will offer a high-throughput template to faithfully recapitulate in vitro arrhythmogenic events necessary for personalized arrhythmia monitoring and drug screening process.

Noninvasive Quantification of Contractile Dynamics in Cardiac Cells, Spheroids, and Organs-on-a-Chip Using High-Frequency Ultrasound.

Cell-based models that mimic in vivo heart physiology are poised to make significant advances in cardiac disease modeling and drug discovery. In these systems, cardiomyocyte (CM) contractility is an important functional metric, but current measurement methods are inaccurate and low-throughput or require complex setups. To address this need, we developed a standalone noninvasive, label-free ultrasound technique operating at 40-200 MHz to measure the contractile kinetics of cardiac models, ranging from single adult CMs to 3D microtissue constructs in standard cell culture formats. The high temporal resolution of 1000 fps resolved the beat profile of single mouse CMs paced at up to 9 Hz, revealing limitations of lower speed optical based measurements to resolve beat kinetics or characterize aberrant beats. Coupling of ultrasound with traction force microscopy enabled the measurement of the CM longitudinal modulus and facile estimation of adult mouse CM contractile forces of 2.34 ± 1.40 μN, comparable to more complex measurement techniques. Similarly, the beat rate, rhythm, and drug responses of CM spheroid and microtissue models were measured, including in configurations without optical access. In conclusion, ultrasound can be used for the rapid characterization of CM contractile function in a wide range of commonly studied configurations ranging from single cells to 3D tissue constructs using standard well plates and custom microdevices, with applications in cardiac drug discovery and cardiotoxicity evaluation.

Cardiac and intestinal tissue conduct developmental and reparative processes in response to lymphangiocrine signaling.

Lymphatic vessels conduct a diverse range of activities to sustain the integrity of surrounding tissue. Besides facilitating the movement of lymph and its associated factors, lymphatic vessels are capable of producing tissue-specific responses to changes within their microenvironment. Lymphatic endothelial cells (LECs) secrete paracrine signals that bind to neighboring cell-receptors, commencing an intracellular signaling cascade that preludes modifications to the organ tissue's structure and function. While the lymphangiocrine factors and the molecular and cellular mechanisms themselves are specific to the organ tissue, the crosstalk action between LECs and adjacent cells has been highlighted as a commonality in augmenting tissue regeneration within animal models of cardiac and intestinal disease. Lymphangiocrine secretions have been owed for subsequent improvements in organ function by optimizing the clearance of excess tissue fluid and immune cells and stimulating favorable tissue growth, whereas perturbations in lymphatic performance bring about the opposite. Newly published landmark studies have filled gaps in our understanding of cardiac and intestinal maintenance by revealing key players for lymphangiocrine processes. Here, we will expand upon those findings and review the nature of lymphangiocrine factors in the heart and intestine, emphasizing its involvement within an interconnected network that supports daily homeostasis and self-renewal following injury.

JAK2 as a surface marker for enrichment of human pluripotent stem cells-derived ventricular cardiomyocytes

BackgroundHuman pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) hold great promise for cardiac disease modelling, drug discovery and regenerative medicine. Despite the advancement in various differentiation protocols, the heterogeneity of the generated population composed of diverse cardiac subtypes poses a significant challenge to their practical applications. Mixed populations of cardiac subtypes can compromise disease modelling and drug discovery, while transplanting them may lead to undesired arrhythmias as they may not integrate and synchronize with the host tissue's contractility. It is therefore crucial to identify cell surface markers that could enable high purity of ventricular CMs for subsequent applications.MethodsBy exploiting the fact that immature CMs expressing myosin light chain 2A (MLC2A) will gradually express myosin light chain 2 V (MLC2V) protein as they mature towards ventricular fate, we isolated signal regulatory protein alpha (SIRPA)-positive CMs expressing intracellular MLC2A or MLC2V using MARIS (method for analysing RNA following intracellular sorting). Subsequently, RNA sequencing analysis was performed to examine the gene expression profile of MLC2A + and MLC2V + sorted CMs. We identified genes that were significantly up-regulated in MLC2V + samples to be potential surface marker candidates for ventricular specification. To validate these surface markers, we performed immunostaining and western blot analysis to measure MLC2A and MLC2V protein expressions in SIRPA + CMs that were either positive or negative for the putative surface markers, JAK2 (Janus kinase 2) or CD200. We then characterized the electrophysiological properties of surface marker-sorted CMs, using fluo-4 AM, a green-fluorescent calcium indicator, to measure the cellular calcium transient at the single cell level. For functional validation, we investigated the response of the surface marker-sorted CMs to vernakalant, an atrial-selective anti-arrhythmic agent.ResultsIn this study, while JAK2 and CD200 were identified as potential surface markers for the purification of ventricular-like CMs, the SIRPA+/JAK2+ population showed a higher percentage of MLC2V-expressing cells (~ 90%) compared to SIRPA+/CD200+ population (~ 75%). SIRPA+/JAK2+ sorted CMs exhibited ventricular-like electrophysiological properties, including slower beating rate, slower calcium depolarization and longer calcium repolarization duration. Importantly, vernakalant had limited to no significant effect on the calcium repolarization duration of SIRPA+/JAK2+ population, indicating their enrichment for ventricular-like CMs.ConclusionOur study lays the groundwork for the identification of cardiac subtype surface markers that allow purification of cardiomyocyte sub-populations. Our findings suggest that JAK2 can be employed as a cell surface marker for enrichment of hPSC-derived ventricular-like CMs.

A patterned human primitive heart organoid model generated by pluripotent stem cell self-organization

Pluripotent stem cell-derived organoids can recapitulate significant features of organ development in vitro. We hypothesized that creating human heart organoids by mimicking aspects of in utero gestation (e.g., addition of metabolic and hormonal factors) would lead to higher physiological and anatomical relevance. We find that heart organoids produced using this self-organization-driven developmental induction strategy are remarkably similar transcriptionally and morphologically to age-matched human embryonic hearts. We also show that they recapitulate several aspects of cardiac development, including large atrial and ventricular chambers, proepicardial organ formation, and retinoic acid-mediated anterior-posterior patterning, mimicking the developmental processes found in the post-heart tube stage primitive heart. Moreover, we provide proof-of-concept demonstration of the value of this system for disease modeling by exploring the effects of ondansetron, a drug administered to pregnant women and associated with congenital heart defects. These findings constitute a significant technical advance for synthetic heart development and provide a powerful tool for cardiac disease modeling.

ERG potassium channels and T-type calcium channels contribute to the pacemaker and atrioventricular conduction in zebrafish larvae.

Bradyarrhythmias result from inhibition of automaticity, prolonged repolarization, or slow conduction in the heart. The ERG channels mediate the repolarizing current IKr in the cardiac action potential, whereas T-type calcium channels (TTCC) are involved in the sinoatrial pacemaker and atrioventricular conduction in mammals. Zebrafish have become a valuable research model for human cardiac electrophysiology and disease. Here, we investigate the contribution of ERG channels and TTCCs to the pacemaker and atrioventricular conduction in zebrafish larvae and determine the mechanisms causing atrioventricular block. Zebrafish larvae expressing ratiometric fluorescent Ca2+ biosensors in the heart were used to measure Ca2+ levels and rhythm in beating hearts invivo, concurrently with contraction and hemodynamics. The atrioventricular delay (the time between the start of atrial and ventricular Ca2+ transients) was used to measure impulse conduction velocity and distinguished between slow conduction and prolonged refractoriness as the cause of the conduction block. ERG blockers caused bradycardia and atrioventricular block by prolonging the refractory period in the atrioventricular canal and in working ventricular myocytes. In contrast, inhibition of TTCCs caused bradycardia and second-degree block (Mobitz type I) by slowing atrioventricular conduction. TTCC block did not affect ventricular contractility, despite being highly expressed in cardiomyocytes. Concomitant measurement of Ca2+ levels and ventricular size showed mechano-mechanical coupling: increased preload resulted in a stronger heart contraction invivo. ERG channels and TTCCs influence the heart rate and atrioventricular conduction in zebrafish larvae. The zebrafish lines expressing Ca2+ biosensors in the heart allow us to investigate physiological feedback mechanisms and complex arrhythmias.

Gene editing and cardiac disease modelling for the interpretation of genetic variants of uncertain significance in congenital heart disease

BackgroundGenomic sequencing in congenital heart disease (CHD) patients often discovers novel genetic variants, which are classified as variants of uncertain significance (VUS). Functional analysis of each VUS is required in specialised laboratories, to determine whether the VUS is disease causative or not, leading to lengthy diagnostic delays. We investigated stem cell cardiac disease modelling and transcriptomics for the purpose of genetic variant classification using a GATA4 (p.Arg283Cys) VUS in a patient with CHD.MethodsWe performed high efficiency CRISPR gene editing with homology directed repair in induced pluripotent stem cells (iPSCs), followed by rapid clonal selection with amplicon sequencing. Genetic variant and healthy matched control cells were compared using cardiomyocyte disease modelling and transcriptomics.ResultsGenetic variant and healthy cardiomyocytes similarly expressed Troponin T (cTNNT), and GATA4. Transcriptomics analysis of cardiomyocyte differentiation identified changes consistent with the patient’s clinical human phenotype ontology terms. Further, transcriptomics revealed changes in calcium signalling, and cardiomyocyte adrenergic signalling in the variant cells. Functional testing demonstrated, altered action potentials in GATA4 genetic variant cardiomyocytes were consistent with patient cardiac abnormalities.ConclusionsThis work provides in vivo functional studies supportive of a damaging effect on the gene or gene product. Furthermore, we demonstrate the utility of iPSCs, CRISPR gene editing and cardiac disease modelling for genetic variant interpretation. The method can readily be applied to other genetic variants in GATA4 or other genes in cardiac disease, providing a centralised assessment pathway for patient genetic variant interpretation.

AP-1 signaling modulates cardiac fibroblast stress responses.

Matrix remodeling outcomes largely dictate patient survival post myocardial infarction. Moreover, human-restricted noncoding regulatory elements have been shown to worsen fibrosis, but their mechanism of action remains elusive. Here, we demonstrate, using induced pluripotent stem cell-derived cardiac fibroblasts (iCFs), that inflammatory ligands abundant in the remodeling heart after infarction activate AP-1 transcription factor signaling pathways resulting in fibrotic responses. This observed signaling induces deposition of fibronectin matrix and is further capable of supporting immune cell adhesion; pathway inhibition blocks iCF matrix production and cell adhesion. Polymorphisms in the noncoding regulatory elements within the 9p21 locus (also referred to as ANRIL) redirect stress programs, and in iCFs, they transcriptionally silence the AP-1 inducible transcription factor GATA5. The presence of these polymorphisms modulate iCF matrix production and assembly and reduce cell-cell signaling. These data suggest that this signaling axis is a critical modulator of cardiac disease models and might be influenced by noncoding regulatory elements.

Multifocal optical projection microscopy enables label-free 3D measurement of cardiomyocyte cluster contractility.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) models have become an attractive tool for in vitro cardiac disease modeling and drug studies. These models are moving towards more complex three-dimensional microphysiological organ-on-chip systems. Label-free imaging-based techniques capable of quantifying contractility in 3D are needed, as traditional two-dimensional methods are ill-suited for 3D applications. Here, we developed multifocal (MF) optical projection microscopy (OPM) by integrating an electrically tunable lens to our in-house built optical projection tomography setup for extended depth of field brightfield imaging in CM clusters. We quantified cluster biomechanics by implementing our previously developed optical flow-based CM video analysis for MF-OPM. To demonstrate, we acquired and analyzed multiangle and multifocal projection videos of beating hiPSC-CM clusters in 3D hydrogel. We further quantified cluster contractility response to temperature and adrenaline and observed changes to beating rate and relaxation. Challenges emerge from light penetration and overlaying textures in larger clusters. However, our findings indicate that MF-OPM is suitable for contractility studies of 3D clusters. Thus, for the first time, MF-OPM is used in CM studies and hiPSC-CM 3D cluster contraction is quantified in multiple orientations and imaging planes.

Scalable and stable human pluripotent stem cell-derived microvascular-like endothelial cells for cardiac regeneration and disease modelling applications

Abstract Introduction The microvasculature is a vital constituent of the adult human vasculature 1. Cardiac tissue engineering, in vitro models of cardiac remodelling and regeneration, and disease models of the microvasculature would benefit from the inclusion of well-characterised, phenotypically stable microvascular-like endothelial cells (ECs) that could be generated at scale without xenogeneic reagents. Primary microvascular ECs have been utilised for such applications however, they are susceptible to passaging-induced senescence². Moreover, alternative solutions including current protocols for the derivation of human pluripotent stem cell-derived ECs (hPSC-ECs) generate cells with a heterogenous vascular identity that often transdifferentiate into non-EC lineages³. Purpose The purpose of this study was to create a 3D, xenogeneic-free differentiation protocol that yielded stable hPSC-derived microvascular-like ECs (hPSC-MVECs) at scale. Methods Endothelial differentiation of hPSCs was conducted in 2D adherent monolayers and within stirred 3D bioreactors for 12 days. Thereafter, CD31pos ECs were isolated via fluorescence-activated cell sorting (FACS) and cultured for a further 14 days. Gene expression analysis of hPSCs undergoing 2D and 3D differentiation was evaluated via RT-qPCR. The day 26 hPSC-ECs were treated with VEGFA (0 – 50 ng/ml) for a further 7 days and evaluated for CD31 expression via high-content image analysis. The angiogenic profiles of 2D-, 3D-, and 3D-hPSC-ECs cultured in high concentration VEGFA (herein, 3D+V), relative to primary Human Cardiac Microvascular Endothelial Cells (HMVEC-Cs) was assessed using the Proteome Profiler Human Angiogenesis Array. Transcriptomic analysis of these EC populations was subsequently conducted via single-cell RNA sequencing (scRNA-seq). Results Complex vascular clusters emerged by day 12 of the 3D differentiation protocol (fig. 1). The emergent 3D hPSC-ECs had a comparable expression of PECAM1 and CDH5 relative to 2D-derived hPSC-ECs however, VEGFA expression was significantly reduced (P<0.01, fig. 2A). VEGFA treatment at 50 ng/ml yielded phenotypically stable hPSC-ECs (3D+V, fig. 2B). Principal component analysis of the angiogenic factors assayed for revealed 3D+V hPSC-ECs clustered with HMVEC-Cs (fig. 2C). scRNA-seq identified a subset of 3D-derived hPSC-ECs expressing genes associated with microvascular vessels (GMFG, PLVAP), capillary ECs (RGCC, GLUL), and angiogenic tip cells (ESM1, HSPG2). Conclusion The scalable and xenogeneic-free 3D protocol outlined within allows for the attainment of hPSC-derived microvascular-like ECs with high angiogenic potential. The absence of Matrigel enhances the translational capacity of the hPSC-MVECs arising from this protocol that could now be derived from patient-specific hPSC lines for evaluation in pre-clinical models of post-myocardial infarction cardiac regeneration or for in vivo models of cardiac remodelling and microvasculature disease.

Empagliflozin treatment prevented oxidative stress, macrophage infiltration, pro-inflammatory and pro-fibrotic responses in the left ventricle of isoprenaline-induced Takotsubo-like syndrome in rats

Abstract Introduction Takotsubo Syndrome (TTS) is an acute heart failure (HF) syndrome induced by both emotional and physical stressors involving a surge of catecholamines and apical ballooning. Currently, there is no specific treatment for TTS. Sodium-glucose cotransporter2 inhibitors (SGLT2i) showed major cardiovascular protective effects in patients with HF independent of glycemic control. SGLT2i have also been shown to have anti-inflammatory and anti-oxidant effects, and to improve the endothelial function in experimental models of cardiac disease. However, a possible protective role of SGLT2i in TTS is unclear. Thus, this study evaluated the effect of SGLT2i on isoprenaline-induced TTS-like syndrome in rats, and determined the underlying mechanism. Methods 32 female Sprague-Dawley rats (10 weeks old) were divided into 4 groups: control, empagliflozin (30 mg/kg/day provided in the diet for 2 weeks), isoprenaline (Iso, 100 mg/kg, i.p.) to induce a TTS-like syndrome, and Iso plus empagliflozin (IE group). After 6 h of Iso injection, echocardiography was performed to determine left ventricular (LV) ejection fraction (EF) by area and heart rate (HR). At day 3, rats were sacrificed, main mesenteric artery and microvessels were collected for vascular reactivity studies, and LV was divided into base and apex to study mRNA expression levels using RT-qPCR, macrophage infiltration by in situ CD68 immunofluorescence staining, and oxidative stress using dihydroethidium. Results The injection of Iso to rats caused at 6 h an increased LVEF and HR in the Iso and the IE groups as compared to their corresponding controls. The HR response was significantly smaller in the IE group compared to Iso group whereas LVEF was similar. In addition, in mesenteric microvessels but not the main mesenteric artery, Iso group showed significant decreased relaxations to acetylcholine and increased contractile responses to phenylephrine in intact rings both of which were not observed in the IE group. Hearts showed significantly increased mRNA levels of IL-1β, TNF-α, NOX2, VCAM-1, MMP-9, TGF-β1 and collagen-1, oxidative stress levels and CD68 staining in the apex compared to the base in the ISO group, all of which were significantly reduced in IE group. Conclusions The present findings indicate that ISO injection caused in the LV elevated levels of oxidative stress, macrophage infiltration, pro-inflammatory, pro-remodeling and pro-fibrosis responses that were more pronounced in the apex compared to the base, and also endothelial dysfunction in the systemic microcirculation, all of which were significantly prevented by the empagliflozin treatment. They further suggest that SGLT2 appears as an interesting target in TTS.

Characterization of atrial cardiomyopathy subtypes - A cellular electrophysiological comparison and evaluation of appropriate treatment options

Abstract Background Atrial fibrillation occurs in the context of atrial remodeling processes which are the result of an underlying atrial cardiomyopathy (AC). Insufficient understanding of the pathophysiological mechanisms behind AC contributes significantly to the fact that current clinical strategies for the treatment of atrial fibrillation are often ineffective. A systematic investigation of these cellular remodeling processes in the context of the various cardiac diseases underlying AC is still missing. Purpose The aim of this study was to characterize different subtypes of AC associated with common cardiac disease entities by systematic cellular electrophysiological characterization of the atria in combination with molecular and structural investigations. Methods For a comprehensive analysis of the molecular changes associated with AC, we characterized several mouse models of common heart diseases, including models for dilated cardiomyopathy (DCM), HFpEF, mitral regurgitation and immune checkpoint inhibitor (ICI-)mediated myocarditis. Patch clamp experiments were performed on isolated atrial cardiomyocytes to study atrial action potentials (aAP) and different ion currents contributing to the aAP. The cellular electrophysiological parameters of the different models were correlated to clinical parameters (ecg, echocardiography of the atria) of the mouse models and with data from RNA sequencing of the mice’ atria. Results Patch clamp measurements performed on isolated left and right atrial cardiomyocytes revealed distinct differences among the examined models. In all pathophysiological models, the aAP was significantly shorter than in the corresponding wild type mice. The different ion current components we measured various changes in potassium, sodium and calcium currents. The repolarizing TASK-1 potassium current was significantly increased in all models. The DCM model caused by an R636Q mutation in the RBM20 gene showed the most severe changes in cellular electrophysiology. Considering the detected atrial changes, we investigated the effects of SGLTi on atrial cardiomyocytes of the different models. When elevated concentrations of dapa-, empa- and sotagliflozin were administrated on atrial cardiomyocytes, a class-I-antiarrhythmic effect with reduced aAP inducibility and changes in the aAP duration could be observed. Conclusion Characterization of murine models of cardiac disease with respect to classification of AC subtypes revealed various electrical remodeling processes at the level of ion currents involved in aAP formation. A potent inductor of atrial proarrhythmogenity in the AC subtypes was an aAP shortening caused by an increase in repolarizing potassium currents, mediated especially by TASK-1. The most severe electrical remodeling in atria cardiomyocytes was observed in the RBM20-mutant model (DCM). SGLTi exhibit anti-arrhythmic effects in different murine disease models and may provide future treatment options for patients with AC.

Fluorofenidone enhances cardiac contractility by stimulating CICR and CaV1.2

Fluorofenidone (AKF-PD) is a novel pyridone derivative that inhibits fibrosis and inflammation in many tissues. Accordingly, it has been effective in disease models, such as liver failure, nephropathy, and pulmonary fibrosis. However, its potential role in cardiac physiology and pathology has yet to be elucidated. Thus, this paper investigated a possible functional impact of AKF-PD on adult rat cardiac myocytes. Cells were kept in culture for 1–2 days under either control conditions or the presence of AKF-PD (500 μM). They were next examined concerning cell contractility, intracellular Ca2+ homeostasis, and activity of voltage-gated Ca2+ channels. Remarkably, AKF-PD enhanced the percentage of cell shortening and rates of both contraction and relaxation by nearly 100%. A stimulus in Ca2+-induced Ca2+ release (CICR) most likely accounts for these effects because AKF-PD also increased the magnitude of electrically evoked Ca2+ transients. Of note, the compound did not alter the peak value of caffeine-elicited Ca2+ transients, indicating stimulation of CICR at constant sarcoplasmic reticulum Ca2+ load. Since CICR is triggered by the entry of Ca2+ through CaV1.2 (ICa), a possible effect on these Ca2+ channels was also investigated. AKF-PD increased the magnitude of both ICa and maximal macroscopic Ca2+ conductance (Gmax) by about 50%. However, no differences were found in either voltage dependence of inactivation or the amount of maximal immobilization-resistant charge movement (Qmax). Thus, the effect on ICa could be explained by a higher channel's open probability (Po) rather than a greater abundance of channel proteins. Additional data indicate that AKF-PD reduces the rate of Ca2+ extrusion in the presence of caffeine, suggesting inhibition of the Na/Ca exchanger. Overall, these results indicate that AKF-PD upregulates the Po of CaV1.2 and then sequentially enhances ICa, CICR, and contractility. Therefore, the novel compound is also a candidate to be tested in cardiac disease models.

Using Ribonucleoprotein-based CRISPR/Cas9 to Edit Single Nucleotide on Human Induced Pluripotent Stem Cells to Model Type 3 Long QT Syndrome (SCN5A±)

Human induced pluripotent stem cells (hiPSCs) have been widely used in cardiac disease modelling, drug discovery, and regenerative medicine as they can be differentiated into patient-specific cardiomyocytes. Long QT syndrome type 3 (LQT3) is one of the more malignant congenital long QT syndrome (LQTS) variants with an SCN5A gain-of-function effect on the gated sodium channel. Moreover, the predominant pathogenic variants in LQTS genes are single nucleotide substitutions (missense) and small insertion/deletions (INDEL). CRISPR/Cas9 genome editing has been utilised to create isogenic hiPSCs to control for an identical genetic background and to isolate the pathogenicity of a single nucleotide change. In this study, we described an optimized and rapid protocol to introduce a heterozygous LQT3-specific variant into healthy control hiPSCs using ribonucleoprotein (RNP) and single-stranded oligonucleotide (ssODN). Based on this protocol, we successfully screened hiPSCs carrying a heterozygous LQT3 pathogenic variant (SCN5A±) with high efficiency (6 out of 69) and confirmed no off-target effect, normal karyotype, high alkaline phosphatase activity, unaffected pluripotency, and in vitro embryonic body formation capacity within 2 weeks. In addition, we also provide protocols to robustly differentiate hiPSCs into cardiomyocytes and evaluate the electrophysiological characteristics using Multi-electrode Array. This protocol is also applicable to introduce and/or correct other disease-specific variants into hiPSCs for future pharmacological screening and gene therapeutic development.Graphical

Single-Cell Analysis of Contractile Forces in iPSC-Derived Cardiomyocytes: Paving the Way for Precision Medicine in Cardiovascular Disease.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold enormous potential in cardiac disease modeling, drug screening, and regenerative medicine. Furthermore, patient-specific iPSC-CMS can be tested for personalized medicine. To provide a deeper understanding of the contractile force dynamics of iPSC-CMs, we employed Atomic Force Microscopy (AFM) as an advanced detection tool to distinguish the characteristics of force dynamics at a single cell level. We measured normal (vertical) and lateral (axial) force at different pacing frequencies. We found a significant correlation between normal and lateral force. We also observed a significant force-frequency relationship for both types of forces. This work represents the first demonstration of the correlation of normal and lateral force from individual iPSC-CMs. The identification of this correlation is relevant because it validates the comparison across systems and models that can only account for either normal or lateral force. These findings enhance our understanding of iPSC-CM properties, thereby paving the way for the development of therapeutic strategies in cardiovascular medicine.

Biomimetic Approaches in Cardiac Tissue Engineering: Replicating the Native Heart Microenvironment.

Cardiovascular diseases, including heart failure, pose significant challenges in medical practice, necessitating innovative approaches for cardiac repair and regeneration. Cardiac tissue engineering has emerged as a promising solution, aiming to develop functional and physiologically relevant cardiac tissue constructs. Replicating the native heart microenvironment, with its complex and dynamic milieu necessary for cardiac tissue growth and function, is crucial in tissue engineering. Biomimetic strategies that closely mimic the natural heart microenvironment have gained significant interest due to their potential to enhance synthetic cardiac tissue functionality and therapeutic applicability. Biomimetic approaches focus on mimicking biochemical cues, mechanical stimuli, coordinated electrical signaling, and cell-cell/cell-matrix interactions of cardiac tissue. By combining bioactive ligands, controlled delivery systems, appropriate biomaterial characteristics, electrical signals, and strategies to enhance cell interactions, biomimetic approaches provide a more physiologically relevant environment for tissue growth. The replication of the native cardiac microenvironment enables precise regulation of cellular responses, tissue remodeling, and the development of functional cardiac tissue constructs. Challenges and future directions include refining complex biochemical signaling networks, paracrine signaling, synchronized electrical networks, and cell-cell/cell-matrix interactions. Advancements in biomimetic approaches hold great promise for cardiovascular regenerative medicine, offering potential therapeutic strategies and revolutionizing cardiac disease modeling. These approaches contribute to the development of more effective treatments, personalized medicine, and improved patient outcomes. Ongoing research and innovation in biomimetic approaches have the potential to revolutionize regenerative medicine and cardiac disease modeling by replicating the native heart microenvironment, advancing functional cardiac tissue engineering, and improving patient outcomes.

Abstract P2047: Fast Skeletal Myosin Binding Protein-C Expression Exacerbates Dysfunction In Heart Failure

Background: During heart failure, gene and protein expression profiles undergo extensive compensatory and pathological remodeling. We have previously observed that fast skeletal myosin binding protein-C (fMyBP-C) is upregulated in diseased mouse hearts. While fMyBP-C shares significant homology with its cardiac paralog, cardiac myosin binding protein-C (cMyBP-C), there are key differences in the functional differences which may affect cardiac function. Also, it is unknown if the fMyBP-C expression in the heart is a pathological or compensatory response. Objectives: We aim to elucidate the consequence of increased fMyBP-C expression in the heart. Methods: To determine the sufficiency of fMyBP-C to cause cardiac dysfunction, we generated cardiac-specific fMyBP-C over-expression mice. These mice were further crossed into a cMyBP-C null model to assess the effect of fMyBP-C in the heart in the absence of cMyBP-C function. Finally, fMyBP-C knockout mice were subjected to transverse aortic constriction (TAC) to define the requirement of fMyBP-C during heart failure development. Results: We confirmed the upregulation of fMyBP-C in several models of cardiac disease. Genetic over-expression of low levels of fMyBP-C caused mild cardiac remodeling and sarcomere dysfunction. High expression of fMyBP-C in a heart failure model further exacerbated the cardiac disease, hypertrophy and fibrosis. On the other hand, following 8 weeks of TAC, fMyBP-C null mice demonstrated greater protection against heart failure development. Mechanistically, this may be due to the greater instability of the myosin super-relaxed state compared to wild-type littermates. Conclusions: These findings suggest that the elevated expression of fMyBP-C in diseased hearts is a pathological response. Targeted therapies to prevent upregulation of fMyBP-C may prove beneficial in the treatment of heart failure.

Abstract P3161: Engineering Mesenchymal Stem Cell-derived Extracellular Vesicles To Enhance Their Therapeutic Efficacy In Cardiovascular Disease

Background: Extracellular vesicles derived from cardiosphere-derived cells (CDC-EVs) have demonstrated the ability to regulate cardiomyocyte apoptosis and induce cardiomyocyte proliferation in pre-clinical models of acute MI. While CDC-EVs exhibit cardioprotective effects, they require invasive intramyocardial injections to achieve therapeutic efficacy, detracting from their translatability. Mesenchymal stem cells (MSC) also generate cardioprotective EVs (MSC-EVs), which have demonstrated therapeutic efficacy in models of cardiac disease following intravenous administration. However, MSC-EVs lack several of the cardioprotective miRs expressed by CDC-EVs, including miR146a, which has been shown to modulate cardiomyocyte apoptosis. The objective of our current study was to determine the effect of enriching MSC-EVs with cardioprotective CDC-EV miRNA on cardiomyocyte apoptosis. Methods: MSCs were transfected with miR-146a-5p mimics using Trans-IT TKO in serum free growth media. EVs were isolated using a combination of ultrafiltration and size exclusion chromatography (SEC) and characterized by size, morphology and expression of surface markers. RT-PCR was performed on EV samples to quantify miR-146a expression. Primary neonatal mouse cardiomyocytes were treated with MSC-EVs, miR-146a loaded MSC-EVs, CDC-EVs or matched volume vehicle control. Cardiomyocyte apoptosis was assessed by a TUNEL assay. Results: Treatment with miR-146a loaded MSC EVs reduced cardiomyocyte apoptosis compared with cardiomyocytes treated with unmodified MSC exosomes (14±1% vs. 31±3%) (mean ± SEM, p=0.001, one-way ANOVA). miR-146a loaded MSC-EVs and CDC-EVs demonstrated a similar level of reduction in cardiomyocyte apoptosis (14±1% vs. 15±1%). There was no difference in the level of apoptosis between cardiomyocytes treated with unmodified MSC exosomes and cardiomyocytes treated with vehicle control (31±3% vs. 27±1%) (mean ± SEM, p=0.366, one-way ANOVA). Conclusion: Engineering MSC-EVs to express miR-146a enhances their ability to reduce cardiomyocyte apoptosis in vitro . This highlights the potential of cargo engineering as a means to improve the therapeutic efficacy of EVs for cardiovascular disease.

Zebrafish as a model for cardiac disease; Cryo-EM structure of native cardiac thin filaments from Danio Rerio

Actin, tropomyosin and troponin, the proteins that comprise the contractile apparatus of the cardiac thin filament, are highly conserved across species. We have used cryo-EM to study the three-dimensional structure of the zebrafish cardiac thin and actin filaments. With 70% of human genes having an obvious zebrafish orthologue, and conservation of 85% of disease-causing genes, zebrafish are a good animal model for the study of human disease. Our structure of the zebrafish thin filament reveals the molecular interactions between the constituent proteins, showing that the fundamental organisation of the complex is the same as that reported in the human reconstituted thin filament. A reconstruction of zebrafish cardiac F-actin demonstrates no deviations from human cardiac actin over an extended length of 14 actin subunits. Modelling zebrafish homology models into our maps enabled us to compare, in detail, the similarity with human models. The structural similarities of troponin-T in particular, a region known to contain a hypertrophic cardiomyopathy ‘hotspot’, confirm the suitability of zebrafish to study these disease-causing mutations.

APLNR marks a cardiac progenitor derived with human induced pluripotent stem cells

Cardiomyocytes can be readily derived from human induced pluripotent stem cell (hiPSC) lines, yet its efficacy varies across different batches of the same and different hiPSC lines. To unravel the inconsistencies of in vitro cardiac differentiation, we utilized single cell transcriptomics on hiPSCs undergoing cardiac differentiation and identified cardiac and extra-cardiac lineages throughout differentiation. We further identified APLNR as a surface marker for in vitro cardiac progenitors and immunomagnetically isolated them. Differentiation of isolated in vitro APLNR+ cardiac progenitors derived from multiple hiPSC lines resulted in predominantly cardiomyocytes accompanied with cardiac mesenchyme. Transcriptomic analysis of differentiating in vitro APLNR+ cardiac progenitors revealed transient expression of cardiac progenitor markers before further commitment into cardiomyocyte and cardiac mesenchyme. Analysis of in vivo human and mouse embryo single cell transcriptomic datasets have identified APLNR expression in early cardiac progenitors of multiple lineages. This platform enables generation of in vitro cardiac progenitors from multiple hiPSC lines without genetic manipulation, which has potential applications in studying cardiac development, disease modelling and cardiac regeneration.