Human iPSC Cardiomyocytes Research Articles

CRISPRi/a screens in human iPSC-cardiomyocytes identify glycolytic activation as a druggable target for doxorubicin-induced cardiotoxicity.

Doxorubicin is limited in its therapeutic utility due to its life-threatening cardiovascular side effects. Here, we present an integrated drug discovery pipeline combining human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs), CRISPR interference and activation (CRISPRi/a) bidirectional pooled screens, and a small-molecule screening to identify therapeutic targets mitigating doxorubicin-induced cardiotoxicity (DIC) without compromising its oncological effects. The screens revealed several previously unreported candidate genes contributing to DIC, including carbonic anhydrase 12 (CA12). Genetic inhibition of CA12 protected iCMs against DIC by improving cell survival, sarcomere structural integrity, contractile function, and calcium handling. Indisulam, a CA12 antagonist, can effectively attenuate DIC in iCMs, engineered heart tissue, and animal models. Mechanistically, doxorubicin-induced CA12 potentiated a glycolytic activation in cardiomyocytes, contributing to DIC by interfering with cellular metabolism and functions. Collectively, our study provides a roadmap for future drug discovery efforts, potentially leading to more targeted therapies with minimal off-target toxicity.

Abstract 6295: Role of MAPK11 in the protective mechanisms against toxicity of tyrosine kinase inhibitors

Abstract Introduction and purpose of the study: Targeted therapies with Tyrosine Kinase Inhibitors (TKIs) have drastically improved the survival of cancer patients. However, long term exposure to TKIs has been associated with severe adverse events such as heart failure, impairing patient survival and life quality, and limiting treatment options. Cardioprotective effect of MAPK11 has been described against cardiac ischemic disease. In addition, MAPK11 has also been reported as an important mediator in cancer development and progression. Therefore the aim of this study is to evaluate the potential therapeutic and dual anti-cancer and cardio-protective effect of MAPK11 inhibition during TKI treatment. Experimental procedures: For this study we used the TKI ponatinib as a paradigm of cardiac toxicity. Ponatinib has been used as a gold standard therapy for the drug resistant type of Chronic Myeloid Leukemia (CML) due to T315I-ABL1 mutation, and has been associated with high risk of cardiac toxicity. We performed in vitro functional assays to evaluate the cardiotoxicity and cardiac protection using human iPSC-cardiomyocytes (hiPSC-CMs), and anti-cancer effect in T315I mutated KCL-22 CML cells. The cardiotoxicity was evaluated by impairment of cardiomyocytes contractile function during TKI (ponatinib) treatment, the cardiac-protection was evaluated by restoration of contractile function post-inhibition of MAPK11 with RNA silencing (siRNA) technique. The anti-cancer effect was evaluated using a cell viability and proliferation assay during inhibition of MAPK11 by siRNA and ABL1 inhibition as a positive control. Summary of unpublished data: Pretreatment with siRNA MAPK11 inhibition significantly improved the contractile function of the cardiomyocytes during treatment with ponatinib when compared to those that did not receive MAPK11 inhibition. Moreover, when compared to the positive control of siRNA ABL1 inhibition, the MAPK11 inhibition showed similar anti-cancer effect in the T315I mutated KCL-22 CML cells proliferation and survival assay. Conclusions: Our study suggests that the inhibition of MAPK11 is a potential druggable therapeutic target with dual action of anti-cancer effect for the mutant form of CML, as well as a substantial protective effect against the cardiac toxicity associated with the TKI ponatinib that could bring insights in the development of safer oncological drugs. Citation Format: Anna Pavlovna Hnatiuk, David Staudt, Mark Mercola. Role of MAPK11 in the protective mechanisms against toxicity of tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6295.

ERMA (TMEM94) is a P-type ATPase transporter for Mg2+ uptake in the endoplasmic reticulum

Intracellular Mg2+ (iMg2+) is bound with phosphometabolites, nucleic acids, and proteins in eukaryotes. Little is known about the intracellular compartmentalization and molecular details of Mg2+ transport into/from cellular organelles such as the endoplasmic reticulum (ER). We found that the ER is a major iMg2+ compartment refilled by a largely uncharacterized ER-localized protein, TMEM94. Conventional and AlphaFold2 predictions suggest that ERMA (TMEM94) is a multi-pass transmembrane protein with large cytosolic headpiece actuator, nucleotide, and phosphorylation domains, analogous to P-type ATPases. However, ERMA uniquely combines a P-type ATPase domain and a GMN motif for ERMg2+ uptake. Experiments reveal that a tyrosine residue is crucial for Mg2+ binding and activity in a mechanism conserved in both prokaryotic (mgtB and mgtA) and eukaryotic Mg2+ ATPases. Cardiac dysfunction by haploinsufficiency, abnormal Ca2+ cycling in mouse Erma+/- cardiomyocytes, and ERMA mRNA silencing in human iPSC-cardiomyocytes collectively define ERMA as an essential component of ERMg2+ uptake in eukaryotes.

Electrophysiological Changes in the Rabbit Ventricular Wedge and Human-Induced Pluripotent Stem-Cell Derived (IPSC) Cardiomyocytes Translate to Severe Arrhythmia Observed in a Canine Toxicology Study, Not Predicted by Standard In Vitro Ion Channel Assays.

During drug discovery, small molecules are typically assayed in vitro for secondary pharmacology effects, which include ion channels relevant to cardiac electrophysiology. Compound A was an irreversible inhibitor of myeloperoxidase investigated for the treatment of peripheral artery disease. Oral doses in dogs at ≥5mg/kg resulted in cardiac arrhythmias in a dose-dependent manner (at Cmax, free ≥1.53μM) that progressed in severity with time. Nevertheless, a panel of 13 different cardiac ion channel (K, Na, and Ca) assays, including hERG, failed to identify pharmacologic risks of the molecule. Compound A and a related Compound B were evaluated for electrophysiological effects in the isolated rabbit ventricular wedge assay. Compounds A and B prolonged QT and Tp-e intervals at ≥1 and ≥.3μM, respectively, and both prolonged QRS at ≥5μM. Compound A produced early after depolarizations and premature ventricular complexes at ≥5μM. These data indicate both compounds may be modulating hERG (Ikr) and Nav1.5 ion channels. In human IPSC cardiomyocytes, Compounds A and B prolonged field potential duration at ≥3μM and induced cellular dysrhythmia at ≥10 and ≥3μM, respectively. In a rat toxicology study, heart tissue: plasma concentration ratios for Compound A were ≥19X at 24hours post-dose, indicating significant tissue distribution. In conclusion, in vitro ion channel assays may not always identify cardiovascular electrophysiological risks observed in vivo, which can be affected by tissue drug distribution. Risk for arrhythmia may increase with a "trappable" ion channel inhibitor, particularly if cardiac tissue drug levels achieve a critical threshold for pharmacologic effects.

Vascularization of PEGylated fibrin hydrogels increases the proliferation of human iPSC-cardiomyocytes.

Studies have long sought to develop engineered heart tissue for the surgical correction of structural heart defects, as well as other applications and vascularization of this tissue has presented a challenge. Recent studies suggest that vascular cells and a vascular network may have regenerative effects on implanted cardiomyocytes (CM) and nearby heart tissue separate from perfusion of oxygen and nutrients. The goal of this study was to test whether vascular cells or a formed vascular network in a fibrin-based hydrogel would alter the proliferation of human iPSC-derived CM. First, vascular network formation in a slowly degrading PEGylated fibrin hydrogel was optimized by altering the cell ratio of human umbilical vein endothelial cells to human dermal fibroblasts, the inclusion of growth factors, and the total cell concentration. An endothelial to fibroblast ratio of 5:1 and a total cell concentration of 1.1 × 106 cells/mL without additional growth factors generated robust vascular networks while minimizing the number of cells required. Using this optimized system, human iPSC-derived CM were cultured on hydrogels without vascular cells, hydrogels with unorganized encapsulated vascular cells, or hydrogels with encapsulated vascular cells organized into networks for 7 days. CM proliferation and gene expression were assayed following 7 days of culture on the hydrogels. The presence of vascular cells in the hydrogel, whether unorganized or in vascular networks, significantly increased CM proliferation compared to an acellular hydrogel. Hydrogels with unorganized vascular cells resulted in lower CM maturity evidenced by decreased expression of cardiac troponin t (TNNT2), myosin light chain 7, and phospholamban compared to hydrogels without vascular cells and hydrogels with vascular networks. Altogether, this study details a robust method of forming rudimentary vascular networks in a fibrin-based hydrogel and shows that a hydrogel containing endothelial cells and fibroblasts can induce proliferation in adjacent CM, and these cells do not hinder CM gene expression when organized into a vascular network.

CRISPRi gene modulation and all-optical electrophysiology in post-differentiated human iPSC-cardiomyocytes

Uncovering gene-phenotype relationships can be enabled by precise gene modulation in human induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs) and follow up phenotyping using scalable all-optical electrophysiology platforms. Such efforts towards human functional genomics can be aided by recent CRISPR-derived technologies for reversible gene inhibition or activation (CRISPRi/a). We set out to characterize the performance of CRISPRi in post-differentiated iPSC-CMs, targeting key cardiac ion channel genes, KCNH2, KCNJ2, and GJA1, and providing a multiparametric quantification of the effects on cardiac repolarization, stability of the resting membrane potential and conduction properties using all-optical tools. More potent CRISPRi effectors, e.g., Zim3, and optimized viral delivery led to improved performance on par with the use of CRISPRi iPSC lines. Confirmed mild yet specific phenotype changes when CRISPRi is deployed in non-dividing differentiated heart cells is an important step towards more holistic pre-clinical cardiotoxicity testing and for future therapeutic use in vivo.

Comparing the effects of chemical Ca2+ dyes and R-GECO on contractility and Ca2+ transients in adult and human iPSC cardiomyocytes

We compared commonly used BAPTA-derived chemical Ca2+ dyes (fura2, Fluo-4, and Rhod-2) with a newer genetically encoded indicator (R-GECO) in single cell models of the heart. We assessed their performance and effects on cardiomyocyte contractility, determining fluorescent signal-to-noise ratios and sarcomere shortening in primary ventricular myocytes from adult mouse and guinea pig, and in human iPSC-derived cardiomyocytes. Chemical Ca2+ dyes displayed dose-dependent contractile impairment in all cell types, and we observed a negative correlation between contraction and fluorescence signal-to-noise ratio, particularly for fura2 and Fluo-4. R-GECO had no effect on sarcomere shortening. BAPTA-based dyes, but not R-GECO, inhibited in vitro acto-myosin ATPase activity. The presence of fura2 accentuated or diminished changes in contractility and Ca2+ handling caused by small molecule modulators of contractility and intracellular ionic homeostasis (mavacamten, levosimendan, and flecainide), but this was not observed when using R-GECO in adult guinea pig left ventricular cardiomyocytes. Ca2+ handling studies are necessary for cardiotoxicity assessments of small molecules intended for clinical use. Caution should be exercised when interpreting small molecule studies assessing contractile effects and Ca2+ transients derived from BAPTA-like chemical Ca2+ dyes in cellular assays, a common platform for cardiac toxicology testing and mechanistic investigation of cardiac disease physiology and treatment.

Simultaneous Widefield Voltage and Dye-Free Optical Mapping Quantifies Electromechanical Waves in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Coupled electromechanical waves define a heart's function in health and diseases. Optical mapping of electrical waves using fluorescent labels offers mechanistic insights into cardiac conduction abnormalities. Dye-free/label-free mapping of mechanical waves presents an attractive non-invasive alternative. In this study, we developed a simultaneous widefield voltage and interferometric dye-free optical imaging methodology that was used as follows: (1) to validate dye-free optical mapping for quantification of cardiac wave properties in human iPSC-cardiomyocytes (CMs); (2) to demonstrate low-cost optical mapping of electromechanical waves in hiPSC-CMs using recent near-infrared (NIR) voltage sensors and orders of magnitude cheaper miniature industrial CMOS cameras; (3) to uncover previously underexplored frequency- and space-varying parameters of cardiac electromechanical waves in hiPSC-CMs. We find similarity in the frequency-dependent responses of electrical (NIR fluorescence-imaged) and mechanical (dye-free-imaged) waves, with the latter being more sensitive to faster rates and showing steeper restitution and earlier appearance of wavefront tortuosity. During regular pacing, the dye-free-imaged conduction velocity and electrical wave velocity are correlated; both modalities are sensitive to pharmacological uncoupling and dependent on gap-junctional protein (connexins) determinants of wave propagation. We uncover the strong frequency dependence of the electromechanical delay (EMD) locally and globally in hiPSC-CMs on a rigid substrate. The presented framework and results offer new means to track the functional responses of hiPSC-CMs inexpensively and non-invasively for counteracting heart disease and aiding cardiotoxicity testing and drug development.

Leveraging optogenetics for CRISPRi screens in human iPSC-cardiomyocytes.

New gene modulation methods, e.g., interference or activation CRISPR (CRISPRi/a), are deployable in a highly parallel format and allow selective perturbation of genes of interest in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). When combined with high-throughput (HT) methods for quantitative assessment of cellular responses, they provide new means for functional genomics in human heart cells and engineered tissues for translational studies. Here we demonstrate how all-optical electrophysiology, in part fueled by optogenetics for actuation and/or sensing of functional responses, can be used in conjunction with CRISPRi modulation to yield a HT platform. Our recent studies explore the possibility to use CRISPRi in post-differentiated hiPSC-CMs to selectively perturb key genes underlying cardiac electrophysiology. We validated the use of our all-optical technology for HT analysis as part of a comprehensive characterization pipeline to link electromechanical functional outputs to molecular correlates, identified by CRISPRi perturbation in hiPSC-CMs. The approach entails the short-term genetic engineering of already differentiated cells and the validation of a pipeline for mRNA or protein analysis post-functional assessment, along with correlative and dimension-reduction studies. Our results demonstrate proof of principle utility of optical methods as partner technology to CRISPRi perturbations that yield mild but specific functional changes relevant to cardiac electromechanics and remodeling during disease. This technology can be useful for multiparametric assessment of cardiotoxicity and drug testing in human cells.

Cardiotoxicity assessment of VEGFR-tyrosine kinase inhibitors by human iPSC cardiomyocyte and pharmacovigilance analysis

Cardiotoxicity assessment of VEGFR-tyrosine kinase inhibitors by human iPSC cardiomyocyte and pharmacovigilance analysis

Single-cell transcriptomic profiling reveals specific maturation signatures in human cardiomyocytes derived from LMNB2-inactivated induced pluripotent stem cells.

Mammalian cardiomyocyte maturation entails phenotypic and functional optimization during the late fetal and postnatal phases of heart development, both processes driven and coordinated by complex gene regulatory networks. Cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) are heterogenous and immature, barely resembling their adult in vivo counterparts. To characterize relevant developmental programs and maturation states during human iPSC-cardiomyocyte differentiation, we performed single-cell transcriptomic sequencing, which revealed six cardiomyocyte subpopulations, whose heterogeneity was defined by cell cycle and maturation states. Two of those subpopulations were characterized by a mature, non-proliferative transcriptional profile. To further investigate the proliferation-maturation transition in cardiomyocytes, we induced loss-of-function of LMNB2, which represses cell cycle progression in primary cardiomyocytes in vivo. This resulted in increased maturation in LMNB2-inactivated cardiomyocytes, characterized by transcriptional profiles related to myofibril structure and energy metabolism. Furthermore, we identified maturation signatures and maturational trajectories unique for control and LMNB2-inactivated cardiomyocytes. By comparing these datasets with single-cell transcriptomes of human fetal hearts, we were able to define spatiotemporal maturation states in human iPSC-cardiomyocytes. Our results provide an integrated approach for comparing in vitro-differentiated cardiomyocytes with their in vivo counterparts and suggest a strategy to promote cardiomyocyte maturation.

Abstract 15594: Decreasing the High Risk of Ponatinib-Induced Cardiovascular Toxicity in Chronic Myeloid Leukemia

Introduction: Cardiovascular (CV) toxicity of cancer therapeutics is a growing problem. Ponatinib is one of the most cardiotoxic Tyrosine Kinase Inhibitors (TKIs) and is used as a highly effective TKI against ABL T315I mutation in Chronic Myeloid Leukemia (CML). Long-term exposure to ponatinib increases CV events including myocardial infarction, heart failure, stroke, peripheral vascular disease and venous thrombosis. There is need of novel therapeutics to provide treatment for CML while avoiding CV toxicity. Hypothesis: Using in vitro and in vivo models of CV toxicity can make possible to reengineer ponatinib to minimize its toxicity. Methods: We developed new safer analogues of ponatinib using fragment-based approach. Their anti-tumor efficacy was tested in 2 different CML cell lines and CML patient samples. We assayed for myocardial toxicity by measuring contractile function in human iPSC-cardiomyocytes (hiPSC-CMs) using high-throughput functional imaging, for vascular toxicity by measuring vasculogenesis in human microvascular endothelial cells (HMVECs), and for bone marrow toxicity by colony formation assay in human bone marrow cells (HBMCs). Finally, we confirmed the safer CV profile and adequate anti-tumor efficacy in an in vivo xenograft mouse model of CML. Results: The new analogues inhibited T315I BCR-ABL kinase activity similar to ponatinib and suppressed T315I mutant CML tumor growth in vitro and in vivo . Compared to ponatinib, the new compounds showed markedly decreased adverse effects on contractility of hiPSC-CMs, vasculogenesis in HMVECs and bone marrow toxicity in HBMCs in vitro . The therapeutic window was increased in vivo , leading to regression of human T315I mutant CML xenografts comparable to ponatinib but without increased levels of cardiac troponin. Additionally, we identified multiple kinases, including FGFR1, that were inhibited by ponatinib but not the analogues, suggesting that there is a specific set of kinases responsible for ponatinib toxicity. Conclusions: This study demonstrates that chemical reengineering can generate novel, CV-safe TKIs that retain effective therapeutic properties against CML carrying ABL T315I mutation, but that exhibit minimal CV toxicity compared to ponatinib.

Abstract 12531: Electrophysiological Mechanisms of Tachycardia-Induced Cardiomyopathy in Human Myocardium

Introduction: Tachycardia-induced cardiomyopathy (TCM) is a reversible form of heart failure. However, the effects of persistent tachycardia leading to TCM in the human are less understood limiting appropriate management of patients with this underrecognized condition. This study investigated the electrophysiological mechanisms of TCM. Methods and Results: Tachycardic in vitro electrical stimulation (120 bpm versus 60 bpm control) of human specimen was performed. In human twitching ventricular trabeculae from patients with preexistent heart failure, tachycardia for 8h compromised systolic force, while diastolic tension and relaxation time were increased (n=6/8 patients each). Human iPSC-cardiomyocytes (iPSC-CM) of healthy donors were utilized as human model suitable for extended electrical pacing. After 24h of tachycardia, systolic Ca 2+ -transient amplitude of iPSC-CM was reduced, while diastolic Ca 2+ levels and cytosolic Ca 2+ elimination were not changed (Fura-2, n tachycardia/control=49/44 cells). 7d of tachycardia resulted in a progressive decline of Ca 2+ -transient amplitude together with an impaired Ca 2+ elimination (n=73/66) thereby demonstrating the progression of TCM. Sarcoplasmic reticulum (SR) Ca 2+ load (caffeine application) and SERCA activity (K sys -K caff ) were reduced after 7d of tachycardia (n=13/13). Besides an impaired SERCA activity, we could identify an increased diastolic SR Ca 2+ spark frequency after 7d of tachycardia (n=76/79) as another mechanism of reduced SR Ca 2+ load. In voltage clamp studies, I CaL density was reduced after 7d of tachycardia (n=11/12), which may further contribute to impaired systolic Ca 2+ -handling. Conversely, action potential duration was prolonged after 7d of tachycardia compared to control (n=19/21). As a potential explanation for prolonged action potentials however, late sodium current (I NaL ) was enhanced after 7d of tachycardia (n=26/19). Conclusions: This study demonstrates for the first time in human myocardium that persistent tachycardia induces early adverse electrophysiological remodeling that may underlie the progression of TCM. These findings may help to understand the pathophysiology of an underrated but prevalent disease

Senolytics rejuvenate the reparative activity of human cardiomyocytes and endothelial cells

Abstract Background Senescent cells have emerged as bona fide drivers of ageing and age-related cardiovascular disease, with senescent cells accumulating in the aged heart and following damage/injury. We have shown that removal of senescent cells using senolytics can rejuvenate the regenerative capacity of the aged heart. Purpose We investigate the effects of cell senescence and action of the senolytics, Dasatinib (D) and Quercetin (Q), on human cardiomyocyte and endothelial cell viability, survival, proliferation, angiogenesis and senescence in vitro. Methods We developed a transwell insert co-culture stress-induced premature senescence model system in vitro. Doxorubicin was used to induce premature senescence to cardiac stromal progenitor cells or HUVECs, which were seeded on the bottom chamber. In separate well plates, untreated cells (non-senescent) were seeded on the bottom chamber and acted as control. Human iPSC-cardiomyocytes (iPSC-CMs) or HUVECs were seeded on the top chamber insert. Co-cultures were left for 7 days and then iPSC-CMs or HUVECs in the top chamber were analysed for viability, proliferation and markers of senescence, and the conditioned medium analysed for SASP factors. The cultures were then treated with D+Q (0.5μM D with 20μM Q) for 3 days to clear the senescent cells in the bottom chamber. Then 7 days later the iPSC-CMs or HUVECs in the top chamber were analysed for viability, proliferation and the markers of senescence, and conditioned medium analysed for SASP factors (total of 17 days). Results Co-culture of iPSC-CMs with senescent cells led to decreased iPSC-CM viability (64±11% vs. 100±3% control; p<0.05) and proliferation (5±2% vs. 11±1% control; p<0.05). There was no increased senescence in co-cultured iPSC-CMs. Treatment with senolytics D+Q resulted in rescue of viability (79±23% 17d co-culture+D+Q; 64±9% 17d co-culture; 100±4% 17d control) and proliferation (7±2% 17d co-culture+D+Q; 3±0.3% 17d co-culture; 8±0.3% 17d control). Similar effects were observed with HUVECs, which showed decreased viability (83±5% vs. 100±6% control; p<0.05) and proliferation (21±7% vs. 44±7% control; p<0.05) when co-cultured with senescent HUVECs. Treatment with senolytics ameliorated HUVEC viability (61±22% 17d co-culture+D+Q; 21±12% 17d co-culture; 100±5% 17d control), proliferation (30±5% 17d co-culture+D+Q; 22±4% 17d co-culture; 48±4% 17d control) and angiogenesis (Score relative to control: 1.1±0.5 D+Q conditioned media; 0.2±0.1 senescent cell conditioned media; 1.0±0.3 control media). Luminex analysis of the senescent cell conditioned media revealed upregulation of SASP factors, but the level of SASP factors were reduced with application of D+Q. Conclusion We show that D+Q senolytics have therapeutic potential in rejuvenating the reparative activity of cardiomyocytes and endothelial cells. These results open the path to further studies on using senolytic therapy in age-related cardiac deterioration and rejuvenation. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Heart Research UK

A large-scale functional high-throughput screening identifies miR-515 and miR-519e as potent inducers of human iPSC-cardiomyocyte proliferation

Abstract Introduction Ischemic heart failure persists as a global health problem despite optimized medical and adjunctive device therapies. Loss of cardiomyocytes in the absence of a proliferative response comprise a major contributor to pathological remodeling and death in this patient population. Experimental studies have shown that microRNAs (miRNAs) may be used as a therapeutic option to reinduce adult cardiomyocyte proliferation. Purpose This study thought to evaluate proliferative potential in human cardiomyocytes after overexpression and inhibition of 2019 miRNAs. Methods To identify miRNAs that regulate cardiomyocyte proliferation, we performed functional high-throughput screenings in human iPSC-derived cardiomyocytes (hiPSC-CM) after transient hypoxia. Herein, 2019 miRNA-mimics for overexpression and 2019 anti-miRs for inhibition were individually transfected to examine EdU-incorporation in hiPSC-CM. MiR-mimic-515 and miR-mimic-519e that induced the highest EdU-uptake, were further assessed by immunostaining and molecular methods for markers indicative of early and late mitosis. In addition, RNA-Sequencing in hiPSC-CM after overexpression of miR-515 and miR-519e was performed to examine differential gene expression and miRNA-modulated pathways involved in cardiomyocyte proliferation. Results Using a functional high-throughput screening, we assessed differential proliferative potential of 2019 miRNAs after transient hypoxia by transfecting both miR-inhibitor and miR-mimic libraries in human iPSC-derived cardiomyocytes (hiPSC-CM). Overexpression of 28 miRNAs substantially induced proliferative activity in hiPSC-CM, with an overrepresentation of miRNAs belonging to the C19MC-cluster and adjacent miR-371–373 family. Two of these miRNAs, miR-515 and miR-519e increased markers of early and late mitosis, with an additive cardiomyocyte turnover after transient hypoxia and substantially increased Aurora B-kinase activity in midbodies, indicative of cell division. These findings were supported by molecular studies using qRT-PCR, Western blot, and RNA-Sequencing after overexpression of miR-515 and miR-519e showing substantial alterations of signaling pathways relevant for cardiomyocytes proliferation in human iPSC-CM. Conclusion Collectively, these results support a critical role of miR-515 and miR-519e for induction of proliferation in human cardiomyocytes under hypoxic conditions, such as present in patients with ischemia-driven cardiomyopathy. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): This work was supported by the German Centre for Cardiovascular Research (DZHK), Deutsche Stiftung für Herzforschung (DSHF) and OPO Foundation.

Fabrication of human myocardium using multidimensional modelling of engineered tissues

Biofabrication of human tissues has seen a meteoric growth triggered by recent technical advancements such as human induced pluripotent stem cells (hiPSCs) and additive manufacturing. However, generation of cardiac tissue is still hampered by lack of adequate mechanical properties and crucially by the often unpredictable post-fabrication evolution of biological components. In this study we employ melt electrowriting (MEW) and hiPSC-derived cardiac cells to generate fibre-reinforced human cardiac minitissues. These are thoroughly characterized in order to build computational models and simulations able to predict their post-fabrication evolution. Our results show that MEW-based human minitissues display advanced maturation 28 post-generation, with a significant increase in the expression of cardiac genes such as MYL2, GJA5, SCN5A and the MYH7/MYH6 and MYL2/MYL7 ratios. Human iPSC-cardiomyocytes are significantly more aligned within the MEW-based 3D tissues, as compared to conventional 2D controls, and also display greater expression of C ×43. These are also correlated with a more mature functionality in the form of faster conduction velocity. We used these data to develop simulations capable of accurately reproducing the experimental performance. In-depth gauging of the structural disposition (cellular alignment) and intercellular connectivity (C ×43) allowed us to develop an improved computational model able to predict the relationship between cardiac cell alignment and functional performance. This study lays down the path for advancing in the development of in silico tools to predict cardiac biofabricated tissue evolution after generation, and maps the route towards more accurate and biomimetic tissue manufacture.

Abstract P3105: Improving Cardiovascular Toxicity Of Chronic Myeloid Leukemia Therapy.

Introduction: Ponatinib is one of the most cardiotoxic Tyrosine Kinase Inhibitors (TKIs), but continues to be used in clinical practice as it is the only TKI effective against the most common ABL T315I mutation in Chronic Myeloid Leukemia (CML). Long-term exposure to ponatinib increases cardiovascular events including myocardial infarction, heart failure, stroke, peripheral vascular disease and venous thrombosis. Novel therapeutics are needed to provide treatment for this common form of CML while avoiding cardiovascular side effects. Hypothesis: Chemical reengineering can create novel TKIs effective against T315I mutant CML but with reduced cardiovascular toxicity. Methods: Using fragment-based approach, we generated new, safer analogues of ponatinib. The anti-tumor efficacy of these analogues was tested in 2 different CML cell lines (K562 T315I and KCL22 T315I) and in CML patient samples. We assayed for myocardial toxicity by measuring contractile function in human iPSC-cardiomyocytes (hiPSC-CMs) using high-throughput functional imaging, and assayed for vascular toxicity by measuring vasculogenesis in human microvascular endothelial cells (HMVECs). Finally, we confirmed the safer cardiovascular profile and adequate anti-tumor efficacy in an in vivo xenograft mouse model of CML. Results: The new analogues inhibited T315I BCR-ABL kinase activity similar to ponatinib and suppressed T315I mutant CML tumor growth in vitro and in vivo . Compared to ponatinib, the new compounds showed markedly decreased adverse effects on contractility of hiPSC-CMs and vasculogenesis in HMVECs in vitro . The therapeutic window was increased in vivo , leading to regression of human T315I mutant CML xenografts comparable to ponatinib but without increased levels of cardiac troponin. Additionally, we identified multiple kinases, including FGFR1, that were inhibited by ponatinib but not the analogues, suggesting that there is a specific set of kinases responsible for ponatinib toxicity. Conclusions: This study demonstrates that chemical reengineering can generate novel, cardiovascular-safe TKIs that retain effective therapeutic properties against CML carrying ABL T315I mutation, but that exhibit minimal cardiovascular toxicity compared to ponatinib.

Abstract LB077: Reengineering ponatinib to minimize cardiovascular toxicity

Abstract Introduction and Purpose of the Study: The advent of small molecule Tyrosine Kinase Inhibitors (TKIs) has revolutionized cancer treatment and greatly improved patient survival. However, the life-threatening cardiovascular toxicity of many TKIs present major concerns. Ponatinib is the most effective treatment for patients harboring the common T315I mutation of BCR-ABL that drives Chronic Myeloid Leukemia (CML) yet is one of the most cardiotoxic of TKIs. Ponatinib-induced events include myocardial infarction, heart failure, stroke, peripheral vascular disease and venous thrombosis. The aim of this study is to reengineer the chemical structure of ponatinib to minimize its adverse cardiovascular effects by using in vitro and in vivo models of cardiovascular toxicity. Experimental Procedures: We developed in vitro cardiovascular toxicity assays using human iPSC-cardiomyocytes (hiPSC-CMs) and human microvascular endothelial cells (HMVECs), and assessed anti-tumor efficacy in T315I mutated K562 CML cells. The cardiotoxicity was determined by impairment of cardiomyocyte contractility function, the vascular toxicity was defined by inability to form capillary-like networks in vasculogenesis assays. In vitro screening of ponatinib analogues probed chemical moieties responsible for its cardiovascular toxicity that were confirmed using mice xenograft mice models with human T315I mutant K562 cells. Summary of Unpublished Data: Compared to ponatinib, refined analogues showed markedly decreased adverse effects on HMVECs vasculogenesis and hiPSC-CMs contractility in vitro. The therapeutic window was increased in vivo, leading to regression of human T315I mutant CML xenografts comparable to ponatinib but without increasing serum levels of cardiac troponin. The kinase inhibition profiles of the “safe” analogues compared to ponatinib identified candidate mediators of ponatinib-induced toxicity. Validation by selective knockdown revealed likely mediators of cardiotoxicity, including FGFR. Conclusions: We succeeded in engineering a cardiovascular safe TKI that retained effective therapeutic properties against BCR-ABL T351 mutated form of CML, but with substantially decreased cardiovascular toxicity. Citation Format: Anna Pavlovna Hnatiuk, Arne A. Bruyneel, Dhanir Taylor, Mallesh Pandrala, Arpit Dheeraj, Wendy Li, Ricardo Serrano Fernandez, Dries A. Feyen, Michelle M. Wu, Prashila Amatya, Isabel Morgado, Volker Wiebking, Matthew H. Porteus, Sanjay Malhotra, Mark Mercola. Reengineering ponatinib to minimize cardiovascular toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB077.

Identification of the Hub Genes and the Signaling Pathways in Human iPSC-Cardiomyocytes Infected by SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus Type 2 (SARS-CoV-2) is an enveloped single-stranded RNA virus that can lead to respiratory symptoms and damage many organs such as heart, kidney, intestine, brain and liver. It has not been clearly documented whether myocardial injury is caused by direct infection of cardiomyocytes, lung injury, or other unknown mechanisms. The gene expression profile of GSE150392 was obtained from the Gene Expression Omnibus (GEO) database. The processing of high-throughput sequencing data and the screening of differentially expressed genes (DEGs) were implemented by R software. The R software was employed to analyze the Gene Ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The protein–protein interaction (PPI) network of the DEGs was constructed by the STRING website. The Cytoscape software was applied for the visualization of PPI network and the identification of hub genes. The statistical analysis was performed by the GraphPad Prism software to verify the hub genes. A total of 516 up-regulated genes and 191 down-regulated genes were screened out. The top 1 enrichment items of GO in biological process (BP), Cellular Component (CC), and Molecular Function (MF) were type I interferon signaling pathway, sarcomere, and receptor ligand activity, respectively. The top 10 enrichment pathways, including TNF signaling pathway, were identified by KEGG enrichment analysis. A PPI network was established, consisting of 613 nodes and 3,993 edges. The 12 hub genes were confirmed as statistically significant, which was verified by GSE151879 dataset. In conclusion, the hub genes of human iPSC-cardiomyocytes infected with SARS-CoV-2 were identified through bioinformatics analysis, which may be used as biomarkers for further research.

Effects of Atrial Fibrillation on the Human Ventricle.

Rationale:Atrial fibrillation (AF) and heart failure often coexist, but their interaction is poorly understood. Clinical data indicate that the arrhythmic component of AF may contribute to left ventricular (LV) dysfunction.Objective:This study investigates the effects and molecular mechanisms of AF on the human LV.Methods and Results:Ventricular myocardium from patients with aortic stenosis and preserved LV function with sinus rhythm or rate-controlled AF was studied. LV myocardium from patients with sinus rhythm and patients with AF showed no differences in fibrosis. In functional studies, systolic Ca2+ transient amplitude of LV cardiomyocytes was reduced in patients with AF, while diastolic Ca2+ levels and Ca2+ transient kinetics were not statistically different. These results were confirmed in LV cardiomyocytes from nonfailing donors with sinus rhythm or AF. Moreover, normofrequent AF was simulated in vitro using arrhythmic or rhythmic pacing (both at 60 bpm). After 24 hours of AF-simulation, human LV cardiomyocytes from nonfailing donors showed an impaired Ca2+ transient amplitude. For a standardized investigation of AF-simulation, human iPSC-cardiomyocytes were tested. Seven days of AF-simulation caused reduced systolic Ca2+ transient amplitude and sarcoplasmic reticulum Ca2+ load likely because of an increased diastolic sarcoplasmic reticulum Ca2+ leak. Moreover, cytosolic Na+ concentration was elevated and action potential duration was prolonged after AF-simulation. We detected an increased late Na+ current as a potential trigger for the detrimentally altered Ca2+/Na+-interplay. Mechanistically, reactive oxygen species were higher in the LV of patients with AF. CaMKII (Ca2+/calmodulin-dependent protein kinase IIδc) was found to be more oxidized at Met281/282 in the LV of patients with AF leading to an increased CaMKII activity and consequent increased RyR2 phosphorylation. CaMKII inhibition and ROS scavenging ameliorated impaired systolic Ca2+ handling after AF-simulation.Conclusions:AF causes distinct functional and molecular remodeling of the human LV. This translational study provides the first mechanistic characterization and the potential negative impact of AF in the absence of tachycardia on the human ventricle.