Stem Cell-derived Cardiomyocytes Research Articles

Unleashing Potential: First Vietnamese 3D in vitro Model using Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Advancements in studying cardiovascular diseases (CVDs) face challenges related to cell sources and utilizing animal models. To mitigate these limitations, three-dimensional (3D) in vitro modeling using human induced pluripotent stem cell-derived cardiomyocytes (iCMs) has emerged as a promising approach. In this study, human induced pluripotent stem cells (hiPSCs) were successfully differentiated into cardiomyocytes and subsequently used to generate spheroids via the hanging drop method. We comprehensively characterized the formation, fusion, and beating capability of the generated spheroids. The hiPSCs were efficiently differentiated into cardiomyocytes, exhibiting contractile activity and expressing cardiac-specific markers such as α-actinin and cardiac troponin. Subsequent results demonstrated the successful formation of iCM-derived cardiac spheroids within 48 hours, with beating activity observed after two days cultured in an ultra-low attachment 96-well plate. In addition, similar to the iCM monolayer, the established spheroids also positively expressed α-actinin. Notably, the spheroids exhibited rapid assembly and maintained survival and stable beating throughout the observation period. These findings highlight the potential of establishing a robust in vitro iCM-derived 3D spheroids model for drug testing and cardiovascular disease modeling, typically for Vietnamese people.

Quiescent-yet-excitable pluripotent stem cell-derived cardiomyocytes engraft without fatal arrhythmias in minipig and primates

Abstract Background Pluripotent stem cell-derived cardiomyocyte (PSC-CM) transplantation is a promising therapy for subacute myocardial infarction (MI), but transient engraftment-related arrhythmia (EA) is major barrier to clinical development (1, 2). We hypothesize that PSC-CM genetically edited to remove automaticity but otherwise functionally intact will engraft in vivo with greater arrhythmic safety. Methods Combinatorial CRISPR/Cas9 genome editing to screen for electrically quiescent PSC-CM yielded a line deficient in the depolarization-associated genes HCN4, CACNA1H, and SLC8A1, combined with overexpression of the hyperpolarization-associated gene KCNJ2. Initially, we transplanted 150 million (n=3) and 500 million gene-edited (n=2) and 150 million wildtype (n=7) PSC-CMs into a minipig model of EA, and we monitored the animals for up to seven weeks on continuous ECG for arrythmia without anti-arrhythmic therapy. In a second study, we transplanted 750 million gene-edited (n=2) and wildtype (n=1) PSC-CMs into sub-acutely infarcted Macaca nemestrina primates. Sham transplant (n=1) was also performed, and all animals were observed for up to three months with immunosuppression and beta-blocker/amiodarone therapy. Results Gene-edited PSC-CM successfully engrafted and structurally coupled with the host myocardium of minipigs (Figure 1) and nonhuman primates (Figure 2). No significant EA occurred after transplanting edited PSC-CM at the 150 million dose, whereas brief EA (<24 hours) was observed and well tolerated at the higher 500 million dose. All five minipigs survived to prespecified timepoints without complication. Conversely, wildtype PSC-CM controls demonstrated uncontrolled and fatal ventricular tachycardia (VT). Of the seven WT control minipigs, only four survived (43% mortality); three expired or were euthanized due to unstable VT or ventricular fibrillation. Arrythmia burden for both 150 and 500 million doses were significantly reduced compared to the 150 million wildtype dose in the first 30-days post-transplant (p<0.0001 for both). We then tested edited PSC-CM in a clinically relevant nonhuman primate model of sub-acute myocardial infarction using standard medical therapy including modest antiarrhythmic therapy. No arrythmia was noted in both nonhuman primates dosed with 750 million edited PSC-CM whereas significant arrhythmia was observed with wildtype despite beta blocker and amiodarone therapy. All pig and primate grafts were structurally coupled with host myocardium. Conclusion Genetically-edited PSC-CM without pacemaker-like activity significantly reduced arrythmia burden and prevented arrhythmic death in a dose-escalation study in minipig. No arrhythmia was observed in a clinically relevant nonhuman primate model of cardiac remuscularization for sub-acute myocardial infarction. This study supports continued investigation of this novel strategy to improve safety and tolerability of PSC-CM transplantation.Edited PSC-CM transplant in minipigEdited PSC-CM transplant in primates

HIF-1 regulates common bidirectional gene expression changes during hypoxia and iron deficiency in human induced pluripotent stem cell derived cardiomyocytes

Abstract Background Heart failure regardless of cause is characterised by extensive transcriptional reprogramming and a switch to foetal developmental programmes via unknown mechanisms (1-3). An additional factor that adversely impacts heart failure patients is iron deficiency (ID)(4). ID is also associated with worse prognosis in acute coronary syndromes (5). However, the mechanisms by which ID interferes with clinical outcomes in heart disease remain incompletely understood. Purpose The purpose of this study was to investigate gene expression changes in human cardiomyocytes during hypoxia and ID, two important stressors in the context of ischaemia and heart failure. Methods In this study, we use human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) derived through a protocol that promotes left ventricle cardiomyocyte differentiation (6). Cells were harvested at different stages of differentiation (day 6 and day 32) and mature (day 32) hiPSC-CMs were treated with 30 μM deferoxamine to induce ID either under normoxic or hypoxic conditions with 1% O2. hiPSC contraction was assessed using musclemotion software and metabolism through use of a Seahorse extracellular flux analyser. RNA was isolated using standard isolation kits and RNA-seq (n=5) was performed. Data was analysed for gene expression by alignment with the human hg19 reference genome using the software STAR. A log2 fold change of 1 and P value of <0.0001 were considered significant. Results We show that both hypoxic and ID hiPSC-CMs demonstrate impaired contractility and reduced oxygen consumption, features of cardiomyocytes from failing hearts. Importantly, hiPSC-CMs exposed to hypoxia, ID or the combination of the two exhibit bidirectional gene expression changes with significant overlap (Figure 1). Focusing on the commonly affected gene set, it becomes evident that hypoxia & ID work via similar mechanisms involving activation of the HIF-1 pathway, that in turn orchestrates the metabolic switch to glycolysis and glutamine oxidation similar to foetal cells. Overall, hypoxia promotes a shift towards immature gene programmes, with 45% of dysregulated genes in hypoxia mimicking expression patterns of less mature cells. Interestingly changes in long non-coding RNA (lncRNA) profiles in hypoxia and ID mimic gene expression changes in these conditions (figure 2). Although restoring oxygen levels in hypoxic hiPSC-CMs rescues the hypoxic transcriptional signature, hiPSC-CMs previously exposed to hypoxia can use glycolytic pathways more efficiently upon further exposures to hypoxia. Conclusion These results shed light into the mechanisms ID impacts heart disease patients and are suggestive that HIF-1 is a key player in orchestrating the gene switch in ID and hypoxia.

Characterisation of hypertrophic response and the effect of a GATA4-targeting compound on the phenotype of hypertrophic cardiomyopathy patient-derived hiPSC-cardiomyocytes

Abstract Introduction Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. Patient-derived human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an informative platform to study disease pathomechanisms. Transcription factor GATA4 serves a significant role in cardiomyocyte hypertrophy, and GATA4-targeted compounds inhibit hypertrophy [1]. Purpose This study aims at identifying phenotypic differences between patient-derived hiPSC-CMs and control hiPSC-CMs and at investigating the effect of a novel GATA4-targeting compound C-2021 on the patient-derived hiPSC-CMs phenotype. Methods hiPSC-CMs were differentiated from a healthy, control line (iPS(IMR90)-4; here, control hiPSC-CMs) and from HCM patient-derived hiPSCs (HCM hiPSC-CMs) that carry a mutation in the MYBPC3 gene. Two types of hypertrophic stimuli were applied to hiPSC-CMs: mechanical stretch and endothelin-1 (ET-1) treatment. For pharmacological assays, compound C-2021 (at 30 µM) or vehicle (0.1 % DMSO) were introduced to hiPSC-CMs one hour prior to the stimuli. hiPSC-CMs were exposed to cyclic equibiaxial mechanical stretching for 24 h or 72 h using Flexcell FX-5000 Tension System (Flexcell International Corporation) [2]. The expression of hypertrophic markers (such as NPPB, NPPA) and structural proteins (including α-myosin and β-myosin heavy chains; MYH6 and MYH7, respectively) was investigated with qPCR. The relative gene expression was analysed using the 2-ΔΔCt method using 18S and ACTB as reference genes. Furthermore, hiPSC-CMs were subjected to ET-1 at 100 nM for 24 h and proBNP expression was quantified using high-content analysis (HCA). Results HCM hiPSC-CMs displayed increased basal expression of NPPB (Fig. 1A-B), lower expression of MYH6 (Fig. 1C) and higher expression of MYH7 (Fig. 1D) compared to control hiPSC-CMs, which is in line with the hypertrophic disease phenotype. Control and HCM hiPSC-CMs responded to 24 h and 72 h mechanical stretching by increased NPPB expression (Fig. 1A-B). At 72 h, the increase in NPPB expression was more pronounced in the HCM hiPSC-CMs (Fig. 1B). The compound C-2021 inhibited stretch-induced NPPB expression after 72 h stretching. HCA results indicate that ET-1 induced a 17-fold increase in proBNP-positive control hiPSC-CMs, and this effect was attenuated by C-2021 treatment. Interestingly, ET-1 did not enhance proBNP expression in HCM hiPSC-CMs (Fig. 2). Conclusions Our results show that the HCM hiPSC-CMs differ from the control hiPSC-CMs in response to hypertrophic stimuli and in myosin heavy chain isoform expression. Additionally, C-2021 may have anti-hypertrophic effects in both in control and HCM hiPSC-CMs based on the downregulation of NPPB expression, and the decrease in proBNP-positive hiPSC-CMs. Further studies are needed to elucidate the exact mechanisms underlying HCM and the action of C-2021 on the HCM hiPSC-CM phenotype.

Effective cardiac mRNA delivery using lipid nanoparticles

Abstract Background/Introduction Heart failure is a global health burden for which there is no curative treatment available that aims to recover the impaired cardiac function.(1) Messenger RNA (mRNA) has emerged as a promising tool for protein replacement therapy. Endogenous mechanisms that enable repairing the heart can potentially be triggered by cardiac messenger RNA (mRNA) delivery.(2,3) Furthermore, the mRNA molecule can be modified in its secondary structure in order to reduce its immunogenicity and susceptibility to degradation by RNases. Clinical application of modified mRNA (modRNA) is challenged by the lack of effective and safe delivery systems. Lipid nanoparticles (LNPs) represent a well characterized class of mRNA delivery systems, which were recently approved for clinical usage in mRNA-based covid-19 vaccines. Purpose We compared cardiac delivery efficiency between different mRNA-LNPs formulations and naked mRNA in citrate buffer upon intramyocardial administration in mice. Furthermore, we aimed to identify cardiac cell types that were targeted. Finally, we assessed whether LNP administration induces a local immunogenic response. Methods Different LNP formulations were tested in vitro and in vivo for luciferase or GFP – encoding modRNA delivery. In vitro, LNP mediated modRNA delivery was assessed in human endothelial cells (HMEC-1), induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) and induced pluripotent stem cell -derived fibroblasts (iPS-FBs). In vivo, mice undergoing open chest heart surgery under general anaesthesia were intramyocardially injected with 2.5μg modRNA in LNPs or in citrate buffer. In order to quantify tissue distribution of mRNA delivery levels, luciferase expression was measured in various organs 24h after administration. Moreover, to identify cellular uptake specificity, histology was performed on mice treated with GFP modRNA. Results mRNA-LNPs outperformed naked mRNA upon intramyocardial administration in mice (figure 1 a, d-i). Although LNPs were mainly in the injection site in the left ventricular wall (Figure 1 b), the liver showed to be the main mRNA-LNP target organ(figure 1 a,b). mRNA-LNPs showed targeting of epicardial and interstitial cardiac cells (figure 1c). Among the tested C12-200 LNP formulations, no differences in in vivo delivery efficiency were observed (Figure 1 a, d). Most importantly, variations on the ionizable lipid LNP component determined the LNP local immune response. Conclusion(s) We present a proof of concept of a promising strategy for delivering mRNA to the heart by using LNPs as delivery systems, which proved to be more efficient than the current state of the art (mRNA in citrate buffer). LNP optimization to better target the heart could introduce a novel approach to achieve cardiac tissue repair.

Massive expansion of functional human iPSC-derived cardiomyocytes by concomitant glycogen synthase kinase-3 beta inhibition and removal of cell-cell contact

Abstract Background Adult mammalian heart has limited capacity for regeneration. Cardiac injuries such as myocardial infarction lead to significant cardiomyocyte (CM) loss and subsequent heart failure with significant morbidity and mortality. Cell-based therapeutic approaches such as injection of stem cell-derived cells or transplantation of engineered cardiac tissue patches have shown promise to re-muscularize the damaged myocardium and enhance cardiac functions. Rationale: Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (< 5-fold). Results Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e. 100 to 250-fold) by glycogen synthase kinase-3β (GSK-3β) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show GSK-3β inhibition suppresses CM maturation while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10- to 25-times greater expansion beyond GSK-3β inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from Yes associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. Conclusion In summary, we uncovered a molecular interplay that enables massive hiPSC-CMs expansion for large-scale drug screening and tissue engineering applications.

Enhanced myofilament calcium sensitivity aggravates abnormal calcium handling and diastolic dysfunction in patient-specific induced pluripotent stem cell-derived cardiomyocytes with MYH7 mutation

Hypertrophic cardiomyopathy (HCM), the most common inherited heart disease, is frequently caused by mutations in the β-cardiac myosin heavy chain gene (MYH7). Abnormal calcium handling and diastolic dysfunction are archetypical features of HCM caused by MYH7 gene mutations. However, the mechanism of how MYH7 mutations leads to these features remains unclear, which inhibits the development of effective therapies. Initially, cardiomyocytes were generated from induced pluripotent stem cells from an eight-year-old girl diagnosed with HCM carrying a MYH7(C.1063 G>A) heterozygous mutation(mutant-iPSC-CMs) and mutation-corrected isogenic iPSCs(control-iPSC-CMs) in the present study. Next, we compared phenotype of mutant-iPSC-CMs to that of control-iPSC-CMs, by assessing their morphology, hypertrophy-related genes expression, calcium handling, diastolic function and myofilament calcium sensitivity at days 15 and 40 respectively. Finally, to better understand increased myofilament Ca2+ sensitivity as a central mechanism of central pathogenicity in HCM, inhibition of calcium sensitivity with mavacamten can improveed cardiomyocyte hypertrophy. Mutant-iPSC-CMs exhibited enlarged areas, increased sarcomere disarray, enhanced expression of hypertrophy-related genes proteins, abnormal calcium handling, diastolic dysfunction and increased myofilament calcium sensitivity at day 40, but only significant increase in calcium sensitivity and mild diastolic dysfunction at day 15. Increased calcium sensitivity by levosimendan aggravates cardiomyocyte hypertrophy phenotypes such as expression of hypertrophy-related genes, abnormal calcium handling and diastolic dysfunction, while inhibition of calcium sensitivity significantly improves cardiomyocyte hypertrophy phenotypes in mutant-iPSC-CMs, suggesting increased myofilament calcium sensitivity is the primary mechanisms for MYH7 mutations pathogenesis. Our studies have uncovered a pathogenic mechanism of HCM caused by MYH7 gene mutations through which enhanced myofilament calcium sensitivity aggravates abnormal calcium handling and diastolic dysfunction. Correction of the myofilament calcium sensitivity was found to be an effective method for treating the development of HCM phenotype in vitro.

Abstract 18022: Multiplexed Generation and Functional Annotation of Hypertrophic Cardiomyopathy-Associated MYH7 Missense Variants in Isogenic Gene-Edited Stem Cell-Derived Cardiomyocytes

Hypertrophic cardiomyopathy (HCM) is an inheritable cardiac condition affecting 1:200-500 people and is characterized by left ventricular hypertrophy that can cause heart failure. Autosomal-dominant, missense mutations in MYH7 (encoding MHC-β) cause 33% of genotype + HCM cases. Despite this, 75% of MYH7 missense variants are of unknown clinical significance (VUS). Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can elucidate the effect(s) of VUS, however, new variant identification by genetic testing outpaces methods to gene edit hiPSCs. Thus, we developed a method called CR ISPR a On- T arget E diting R etrieval (CRaTER) which increased successful gene-editing 25-fold over current methods. We leveraged CRaTER to enrich for edited hiPSCs, generating a library of 113 MYH7 missense variants. As mutations can reduce protein stability and abundance, we flow sorted MYH7 variant hiPSC-CMs into bins based on differing MHC-β protein abundance followed by next generation sequencing to calculate variant abundance scores. This multiplexed assay identified 31 residue substitutions as functionally abnormal, including all tested pathogenic variants and VUS. MHC-β depletion was validated in clonal hiPSC-CMs and by Western blotting of patient myocardium with a MYH7 mutation. Additionally, we found that severe MHC-β depletion is associated with increased cell size, a hallmark pathological phenotype of HCM. Finally, single-cell contractility analyses of hiPSC-CMs with a pathogenic MYH7 variant revealed hyperdynamic physiology relative to isogenic controls, suggesting an association between MHC-β loss and systolic dysfunction. Together, CRaTER enables the generation of isogenic hiPSC variant libraries at unprecedented scale for differentiation to disease-relevant cell types. Functional analyses of MYH7 variant hiPSC-CMs have revealed a novel correlation between MHC-β protein depletion and pathogenicity that accurately predicts MYH7 variant effect and has implications for future MYH7 variant effect interpretations.

Abstract 15544: Manipulating Oxygen-Releasing Levels and Oxidative Stress to Enhance Viability and Functionality of Human Pluripotent Stem Cell-Derived Cardiomyocytes for Myocardial Infarction Therapy

Introduction: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold great promise for myocardial infarction (MI) treatment. Although O 2 -releasing engineering approaches have been developed to enhance stem cell engraftment, a significant gap persists in knowledge regarding the oxidative stress controlling viability and functionality of hPSC-CMs. Hypothesis: Preconditioning hPSC-CMs with appropriate oxidative levels will enhance their adaptation and engraftment in the infarcted heart. Aims: To develop a novel system that produces a controlled release of O 2 to supply engineered heart tissue for regenerative therapies. Methods: An O 2 -releasing hydrogel system (ORHS) was engineered using peroxides (sodium percarbonate) and antioxidants (β-carotene) encapsulated in PLGA-based particles. The effect of ORHS on hPSC-CMs was evaluated through oxidative stress assays, cell viability assessments, and contractility measurements. RNA-seq profiling was performed to analyze the gene expression of hPSC-CMs in response to ORHS. After implantation of ORHS-hPSC-CMs in infarcted hearts of immunodeficient mice, heart functions were monitored with echocardiography. Implanted cell engraftment was assessed using in vivo imaging systems and immunostaining. Results: The dissolved O 2 levels were increased in ORHS for 7 days, while the oxidative stress was minimized by β-carotene. The optimal ORHS significantly enhanced the viability and mitigated cell death of hPSC-CMs under hypoxic stress conditions. ORHS also enhanced cell contractility and improved calcium signaling in hPSC-CMs. Genes related to maturation and functionality (such as TNNT2, TNNI3, GJA1, and RYR2) were upregulated by ORHS in hPSC-CMs. Implantation of ORHS-hPSC-CMs for 4 weeks resulted in improved ejection fraction, reduced ventricular remodeling, alleviated infarct size, and scar formation. Furthermore, the engraftment and survival of hPSC-CMs were significantly increased by ORHS compared to control hydrogels, as demonstrated by luciferase reporter assays and troponin immunostaining with TUNEL. Conclusion: The development of ORHS offers a promising approach for inducing oxidative preconditioning and achieving enhanced regenerative outcomes of hPSC-CMs.

Abstract 15579: Stem Cell Modeling of a Candidate Genetic Variant in a Family With Complex Inherited Cardiomyopathy

Introduction: Inherited cardiomyopathies, such as arrhythmogenic right (or left) ventricular cardiomyopathy (ARVC; ALVC) and dilated cardiomyopathy (DCM), can lead to sudden cardiac death. Mechanisms of arrhythmogenesis in these cardiomyopathies are not fully understood, with ~ 20% of patients carrying a pathogenic variant in a validated cardiomyopathy-associated gene. Aims: Here, we investigate a family with highly variable expression of arrhythmogenic cardiomyopathy. The two affected siblings have differential ventricle involved and arrhythmia burden: with one fulfilling criteria for ARVC, and the other for ALVC. Their father has a non-ischemic cardiomyopathy with ventricular arrhythmias and a family history of sudden death. The maternal side is unaffected. The sibling with ALVC harbors a paternally inherited rare missense variant of uncertain significance (VUS) in plakoglobin ( JUP) (c.356C>T; p.Pro119Leu), an ARVC-associated gene that does not segregate in the sibling with ARVC. Hypothesis: Study of patient-specific inducible pluripotent stem cell-derived cardiomyocytes (iPSC-CM) can define an elusive genetic etiology as well molecular pathways associated with cardiomyopathy in the affected family members. Methods: We reprogrammed iPSC lines from 3 affected family members and 2 unaffected maternal controls and differentiated them into cardiomyocytes. Results: Differentiation to iPSC-CMs resulted in transcriptionally upregulated markers of cardiac stress and contractile function in affected family members ( NPPA, NPPB, MYH7 ). iPSC-CM harboring JUP c.356C>T did not differ in plakoglobin RNA or protein expression. Hypertrophy was observed in the iPSC-CMs from affected family members after isoproterenol stimulation. Whole exome sequencing results revealed rare candidate variants (MAF<5x10 -5 , REVEL>0.7) that segregated in affected family members. Conclusions: iPSC-CMs from affected family members recapitulate cardiomyopathy phenotypes. Affected family members harbor novel candidate variants not previously associated with cardiomyopathies. Future work will utilize base editing to evaluate for compound contributions of several novel VUS on cardiomyopathy phenotypes in iPSC-CM from family members.

Abstract 16714: An Injectable Thermo-Responsive Hydrogel as a Tool for Cardiac Tissue Engineering Efforts: In vitro and in vivo Biocompatibility Assessment

Background: Heart failure (HF) is a major contributor to the global burden of cardiovascular disease. Current treatments for HF do not regenerate muscle nor function and so cardiac transplantation remains the only definitive treatment for HF. Thus, novel therapies for HF are still needed. To overcome this limitation, we engineered an injectable thermo-responsive hydrogel that transitions from a liquid-solution to a gel-based matrix upon reaching body temperature allowing for a liquid injection rapidly followed by gel localization. Previously, we demonstrated the potential of our hydrogel for cardiac tissue applications using primary cardiomyocytes. Goal: The goal of this project is to assess the biocompatibility of the thermo-responsive hydrogel with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and its effect on mice heart tissue. Methods: 3D cell culture: 9x104 hiPSC-CM were encapsulated for 21 days using 150μl of hydrogel solution (1.5% w/w) and cell function was assessed afterwards. Mouse intracardial injection: a left thoracotomy was performed via the fourth intercostal space in an adult C57BL/6 mouse model. Then, three injections of 10 μl of either (i) lipofectamine, (ii) hydrogel (1% w/w) or (ii) saline were injected into the left ventricle anterior wall. After injections, the thoracotomy site was closed, and the animals recovered. Four weeks post inject the tissues were harvested and prepared for studies. Results: We found that our hydrogel promotes hiPSC-CMs alignment, elongation with increased Cx43 localization and improved contraction function when compared with traditional 2D fibronectin controls. Furthermore, the in-vivo biocompatibility in mice (intracardial injection) proved that our hydrogel is well tolerated by cardiac tissue. Conclusion: Our results indicated that the injectable thermos-responsive hydrogel is a biocompatible material with potential to be used in cardiac tissue engineering efforts.

Abstract 15426: Stress-Induced Transcriptomic Shift in Human iPSC-Derived Cardiomyocytes Carrying Hypertrophic Cardiomyopathy Associated MYH7 G256E Mutation

Introduction: Hypertrophic cardiomyopathy (HCM) is a monogenic disorder caused by heterozygous (HET) mutations in sarcomere genes such as MYH7. Recently, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying HET HCM mutations have shed new insights into the molecular mechanisms of HCM. However, it remains unclear whether the cellular phenotypes of low penetrant mutations such as MYH7 G256E and others, can be adequately elucidated. Hypothesis: We hypothesize that the incorporation of cellular stressors can enhance the manifestation of pathogenic HCM phenotypes in low penetrant MYH7 mutations. Methods: We generated HET and homozygous (HOM) hiPSC-lines for mutation G256E in the MYH7 locus, which is associated with HCM. We performed single-cell RNA sequencing (scRNAseq) with CellPlex technology on wildtype (WT), HET, and HOM iPSC-CMs at day 30 and 60 of cardiac differentiation to establish baseline cellular transcriptional phenotype. We then treated day 30 hiPSC-CMs with 1μM isoproterenol treatment for 5 days or glucose starvation for 10 days and performed scRNAseq to assess their transcriptional shift. Additional Digital Droplet PCR studies were performed to investigate the MYH7 mutant allele expression. Results: In day 30 iPSC-CMs we detected a large clone-to-clone and batch-to-batch variability transcriptionally that obscured the identification of a consistent mutant phenotype in HET MYH G256E vs WT hiPSC lines. These phenotypes correlated with a 1:1 ratio of mutant vs WT allele expression. Implementation of long-term culture or treatment with isoproterenol or glucose starvation led to enhanced hypertrophic and oxidative phosphorylation gene expression in HET hiPSC-CMs. Intriguingly, the expression of MYH6 increased in MYH7-mutant hiPSC-CMs. Conclusions: The application of cellular stressors can enhance the manifestation of hypertrophic phenotypes in HET MYH G256E hiPSC-CMs and suggests a potential mechanism for the variable penetrance of MYH7 mutations in HCM patients.

Abstract 15609: ParSE-seq: A Calibrated Multiplexed Assay for Clinical Interpretation of Putative Splice-Altering Variants

Background: Up to 10% of disease-associated variants are predicted to affect RNA-splicing. Interpreting the clinical significance of putative splice-altering variants outside the 2-base pair canonical splice sites remains difficult without functional studies. Goal: To develop a calibrated, high-throughput assay to adjudicate variant effects on RNA splicing. Methods: We established Parallel Splice Effect Sequencing (ParSE-seq), a multiplexed minigene assay, to test variant effects on RNA splicing quantified by high-throughput sequencing. We used the tool SpliceAI to prioritize exonic and intronic candidate splice variants, and ClinVar to select curated benign and pathogenic controls in SCN5A , which encodes the major voltage-gated cardiac sodium channel. We transfected 271 barcoded minigene constructs into HEK cells or induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and calibrated to the American College of Medical Genetics and Genomics scheme. Variants were interpreted using the calibrated functional data, and experimental data correlated to SpliceAI predictions. We studied selected variants by automated patch clamp (APC) in HEK cells and by assessing splicing and channel function directly in CRISPR-edited iPSC-CMs. Results: ParSE-seq revealed the splicing effect of 245 SCN5A variants in iPSC-CMs. The assay had concordant scores for 43/45 benign and pathogenic control variants, achieving both PS3- and BS3-strong levels of evidence after calibration. 41/114 exonic variants and 30/66 intronic variants with determinate scores disrupted splicing. 9 of 42 Variants of Uncertain Significance were reclassified, and 30 of 34 variants with conflicting interpretations were reinterpreted using the functional data. Predictions from SpliceAI performed well on experimental data (AUC = 0.96). We identified 3 clinically relevant splice-altering missense variants that had normal function when tested with a cDNA-based APC assay. A splice-altering intronic variant, c.1891-5C>G also disrupted splicing and sodium channel electrophysiology when introduced into iPSC-CMs by CRISPR-editing. Conclusions: ParSE-seq is an efficient, calibrated, high-throughput assay to facilitate splice-altering variant interpretation.

Abstract 12784: Ischemic Cardiac Autophagy/Mitophagy is Regulated by TRPML1 and Mitochondrial Reactive Oxygen Species

Introduction: In myocardial infarction (MI), activated autophagy/mitophagy helps degrade cytoplasmic macromolecules/proteins/ organelles (especially damaged mitochondria), recycle breakdown products, and prevent oxidative damage and cell death. It had reported that reactive oxygen species (ROS) activates the transcription factor EB (TFEB) via a lysosomal transient receptor potential mucolipin 1 (TRPML1) Ca 2+ efflux. TFEB is thought to be the master regulator of lysosome biogenesis. Hypothesis: We investigated whether lysosomal TRPML1 was activated by ROS and contributed to ischemic cardiac autophagy/mitophagy. Methods: MI was induced in wild type C57BL/6J mice by ligated left anterior descending coronary artery. Mice were studied at three weeks after MI surgery. Results: The lysosomal-restricted TRPML1 Ca 2+ release channel was significantly upregulated by 84.8% in cardiac border zone (BZ) and 55.4% in a remote zone after MI. As expected, dephosphorylated TFEB (i.e., active TFEB), mitophagy marker Parkin, autophagy marker LC3II, and lysosome marker LAMP1 increased substantially after MI. Autophagy degradation was upregulated as measured by SQSTM1/p62. TRPML1 protein expression was reduced in a dose-dependent manner by TRPML1 shRNA AAV9 (AAV9 was injected 1 week after MI for two weeks). The protein levels of LC3II and LAMP1 significantly decreased in MI BZ after AAV9 transfection. Mitochondrial ROS scavenger (mitoTEMPO, 1 mg/kg/day, i.p.) treatment for two weeks (starting at 1 week after MI) significantly inhibited TRPML1, LAMP1, LC3II and Parkin expression in the MI BZ. Conversely, when TRPML1 was overexpressed in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), the protein levels of LC3II/Parkin were increased. Speaking to human relevance, TRPML1 was significantly increased in human patients with ischemic cardiomyopathy. Conclusion: Autophagy and mitophagy after MI are regulated by TRPML1 and mitochondrial ROS.

Abstract 13336: Myocardial Protection of Chitosan Cardiac Muscle Patch in Myocardial Infarcted Mice

Background: Poor grafted rate of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) via direct injection into the myocardium limits the efficacy of cardiac repair after myocardial infarction (MI). Chitosan-based scaffolds has regarded as functional biomaterial for designing delivery systems in cardiac therapies. This study aimed to enhancing the potency of cardiac repair with the hiPSC-CMs loaded chitosan cardiac patch (hCCMP). Methods: The patches were fabricated with chitosan. Characterization of the chitosan patches was evaluated through the detection of swelling rate and porosity. Biocompatibility of the hiPSC-CMs loaded patches was analysed by scanning electron microscope (SEM) and CCK8 assay. The mice were randomly divided into 4 groups (n=10 for each). Animals underwent MI-inductive surgery followed by epicardium implantation of hCCMP or Empty-patch (without hiPSC-CMs); Sham and MI mice without further intervention were set as control. 4 weeks after MI, heart function was measured via echocardiography. Engraftment was evaluated with human cardiac troponin T staining. Infarction size, left ventricular anterior wall thickness, cardiac remodeling, angiogenesis, arterialization and apoptosis was detected via immunofluorescence in infarcted boder area. Results: SEM showed that the chitosan patch had regular pore size and good 3D structure; porosity was about (96.7±0.37)%. On day3, the patch swelling rate was about (715±64.55)%. SEM, CCK8 assay showed a normal cells growth in the chitosan patch. The hiPSC-CMs grafted well with chitosan patch delivery systems and was associated with significantly improvement in LV heart function, infarction size, LV anterior wall thickness, cardiac remodeling, angiogenesis and cardiomyocyte apoptosis 4 weeks after MI. Conclusions: The 3D chitosan cardiac patch with hiPSC-CMs loaded can enhance the potency of cardiac repair after MI and present a novel and promising therapic strategy.

Abstract 15451: Regulations of Nrf2 by Nf-κB in Ischemic Heart Failure

Introduction: Oxidative stress is an important pathophysiological factor in the development and progression of ischemic heart failure (IHF). Nuclear factor E2-related factor 2 (Nrf2) is a major regulator of the protective antioxidant response and has been found to decrease by 50% in IHF. However, the mechanism of Nrf2 downregulation remains incompletely understood. NF-kB is a critical transcription factor that influences many cellular processes and is activated in IHF with increased reactive oxygen species (ROS). We hypothesize that activation of NF-kB may underlie the decreased Nrf2 activity in IHF. Methods and Results: To induce IHF, we performed left anterior descending coronary artery (LAD) ligation surgery in Sprague Dawley rats and FVB/NJ mice. We found a significant reduction in cardiac function after LAD surgery in ischemia/reperfusion (I/R) mice relative to sham-operated mice using echocardiography. Tissue sample analyses reveal a crucial role of NF-kB activation in the regulation of Nrf2 in IHF. Additionally, after 2 weeks of I/R, we found that rats treated with Nrf2 overexpressed lentivirus demonstrated greater pressure development than rats with vehicle treatment. Using rat cardiac myoblast cells (H9C2) and western blot analysis, we tested the expression of cytosolic and nuclear Nrf2 and p65 (a component of NF-kB) following time- and dose-dependent treatments of H2O2. We found that p65 activation inhibited Nrf2 activity, while inhibitions of p65 nuclear translocation prevented the decrease in Nrf2 nuclear activities. We then generated a knockout of p65 in H9C2 cells and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using CRISPR-Cas9 technology to test NF-κB regulation on the Nrf2 activities, which further strengthened our previous findings. Conclusions: Taken together, our data support the critical role of NF-kB activation in the regulation of Nrf2 in IHF, providing new insights into possible molecular targets for the treatment of IHF through the regulation of the protective antioxidant response.

Abstract 16452: Targeting Notch3 Pathway and Using Extracellular Vesicles From Induced Pluripotent Stem Cell-Derived Cardiomyocytes is Effective in Porcine Chronic Myocardial Injury Model

Introduction: Heart failure is the leading cause of hospital admission in the modern U.S. We previously demonstrated that exosomes (Ex) from iPSC-derived cardiomyocytes (iCMs) significantly restored murine peri-infarct region. We found that miR20/92/363 (CORE miRNA) activated iCM proliferation and myocardial restoration by repressing the Notch3 pathway. However, the optimal pre-clinical method to target the Notch3 pathway through Ex, miRNA, and siRNA remains to be assessed. Hypothesis: Exosomes are the most effective biologic for chronic porcine heart failure model. Methods: iPSCs were generated from four heart failure patients (Pt 1-4) and differentiated into iCMs. Ex were isolated from the iCM culture media and used to treat iCMs subjected to hypoxia-injury. The in vitro findings were then confirmed in vivo in Yorkshire pigs subjected to myocardial ischemia-reperfusion injury for one hour via balloon occlusion of the left descending artery. One month later, chronic heart failure model was developed, and percutaneous catheter system (Biocardia, Inc) delivered 10 11 Ex, 4200 μg of CORE miRNA, or 4200 μg of siNotch3 into porcine peri-infarct region. MRI and mRNA seq analyzed the porcine myocardium. Results: Viability of hypoxia-injured iCMs treated by siNotch3 (132.50% viability ± 23.54% SD)*, CORE miRNA (127.79% ± 10.06%)*, Pt 1 Ex (74.88% ± 3.11%)*, Pt 2 Ex (72.90% ± 4.50%)*, Pt 3 Ex (79.11% ± 7.23%)*, and Pt 4 Ex (77.81% ± 3.31%)* all significantly improved iCM viability compared to non-treated hypoxia-injured iCMs (26.09% ± 10.75%, *p<0.05). In porcine chronic heart failure model (n=4 - 6/treatment group), the mean absolute change in LVEF of PBS (control), siNotch3, CORE miRNA, and Pt 1 Ex treatments were -3.55 ± 3.27, 1.19 ± 3.17*, 2.34 ± 1.53*, and 3.53 ± 3.59*, respectively (*p<0.05). mRNA seq, gene ontology, and KEGG analysis of porcine myocardium suggested that myocardial repair occurred through the TGFβ pathway and cell cycle re-entry of cardiomyocytes. Conclusion: siRNA, miRNA, and exosomes are all effective methods to repair the porcine myocardium after chronic myocardial injury. Exosome showed the highest functional restoration. Determination of mechanism and optimal biologic and dose are underway.

Abstract 15334: Laminin Alpha 2 Increases the Protective Effect of Exosomes in Human IPSC-Derived Cardiomyocytes During Ischemia-Reperfusion

Acute myocardial infarction (AMI) is a leading cause of death and disability worldwide. With early treatment, restoration of blood flow to ischemic myocardium can be achieved and therefore reduce mortality risk. Evidence suggests that mesenchymal stromal cell (MSC)-derived exosomes (EXO) promote myocardial repair after AMI due to paracrine activity. Laminin alpha 2 (LAMA2) is a major cardiac extracellular matrix (ECM) component and can be used to produce cardiac-specific EXO. Here we coated MSC-derived EXOs in LAMA2 to enhance their recognition by human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We hypothesized that LAMA2-coated MSC-EXO would confer protective effects on CMs during ischemia-reperfusion. The LAMA2-EXO had a similar yield of particles, with a size range of 130nm as the control (gelatin-EXO). No cytotoxic effect was observed in CMs after 3-day exposure to LAMA2-EXO. CMs were then treated with LAMA2-EXO two hours prior to 4h ischemia (1% O 2 , 5% CO 2 , and glucose-free media). LAMA2-EXO had a 2-fold protective effect compared to non-treatment, and after 1.5h of reperfusion (5% CO 2 , CM-enriched media) CMs treated with LAMA2-EXO recovered faster than the control group. Conversely, due to the laminin 511/411 ECM component of endothelial cells (ECs), we did not see a significant protective effect nor a difference between LAMA2-EXO vs. the control in the EC ischemia-reperfusion group. This is the first report of suggesting the specificity of LAMA2-EXO to treat CM post-exposure to ischemia-reperfusion injury. Overall, these results indicate that LAMA2-EXO can be a promising treatment to target CM survival in ischemic cardiovascular disease.

Prioritizing cardiovascular disease-associated variants altering NKX2-5 and TBX5 binding through an integrative computational approach

Cardiovascular diseases (CVDs) are the leading cause of death worldwide and are heavily influenced by genetic factors. Genome-wide association studies (GWAS) have mapped > 90% of CVD-associated variants within the non-coding genome, which can alter the function of regulatory proteins, like transcription factors (TFs). However, due to the overwhelming number of GWAS single nucleotide polymorphisms (SNPs) (>500,000), prioritizing variants for in vitro analysis remains challenging. In this work, we implemented a computational approach that considers support vector machine (SVM)-based TF binding site classification and cardiac expression quantitative trait loci (eQTL) analysis to identify and prioritize potential CVD-causing SNPs. We identified 1,535 CVD-associated SNPs that occur within TF footprints and putative enhancers in the human heart and 14,218 variants in linkage disequilibrium (LD) with genotype-dependent gene expression in cardiac tissue. Using ChIP-seq data from two essential cardiac TFs (NKX2-5 and TBX5) in human induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CM), we trained a large-scale gapped k-mer SVM (LS-GKM-SVM) model to identify CVD-associated SNPs that altered NKX2-5 and TBX5 binding sites. The model was tested by scoring human heart TF genomic footprints within putative enhancers and measuring in vitro binding through electrophoretic mobility shift assay (EMSA). Five variants predicted to alter NKX2-5 (rs59310144, rs6715570, and rs61872084) and TBX5 (rs7612445 and rs7790964) binding were prioritized for in vitro validation based on the magnitude of the predicted change in binding and because they are eQTLs in cardiac tissue. All five variants altered NKX2-5 and TBX5 DNA binding. In summary, we present a bioinformatic approach that considers tissue-specific eQTL analysis and SVM-based TF binding site classification to prioritize CVD-associated variants for in vitro experimental analysis.

Current status and emerging trends of cardiac metabolism from the past 20 years: A bibliometric study

BackgroundAbnormal cardiac metabolism is a key factor in the development of cardiovascular diseases. Consequently, there has been considerable emphasis on researching and developing drugs that regulate metabolism. This study employed bibliometric methods to comprehensively and objectively analyze the relevant literature, offering insights into the knowledge dynamics in this field. MethodsThe data source for this study was the Web of Science Core Collection (WoSCC), from which the collected data were imported into bibliometric software for analysis. ResultsThe United States was the leading contributor, accounting for 38.33 % of publications. The University of Washington and Damian J. Tyler were the most active institution and author, respectively. The American Journal of Physiology-Heart and Circulatory Physiology, Journal of Molecular and Cellular Cardiology, Cardiovascular Research, Circulation Research, and American Journal of Physiology-Endocrinology and Metabolism were highly influential journals that published numerous high-quality articles on cardiac metabolism. Common keywords in this research area included heart failure, insulin resistance, skeletal muscle, mitochondria, as well as topic words such as cardiac metabolism, fatty acid oxidation, glucose metabolism, and myocardial metabolism. Co-citation analysis has shown that research on heart failure and in vitro modeling of cardiovascular disease has gained prominence in recent years and making it a research hotspot. ConclusionResearch on cardiac metabolism is steadily growing, with a specific focus on heart failure and the interplay between mitochondrial dysfunction, insulin resistance, and cardiac metabolism. An emerging trend in this field involves the enhancement of maturation in human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) through the manipulation of cardiac metabolism.