Dilated Cardiomyopathy Research Articles

Abstract Tu062: FLNC Deficiency Triggers Unfolded Protein Response and Leads to Dilated Cardiomyopathy

Background: The FLNC gene encodes filamin C, which is highly expressed in cardiomyocytes. Dysfunction of filamin C, due to different types of FLNC mutations, has been reported to be associated with various types of cardiomyopathy. Truncating mutations in FLNC often result in insufficient expression of filamin C, resulting in dilated cardiomyopathy (DCM). However, the specific processes via which the deficiency of filamin C causes DCM remain incompletely comprehended. Aims: To investigate the pathogenic mechanisms of DCM caused by the deficiency of filamin C in cardiomyocytes using a mouse model with cardiac-specific knockout of Flnc and to explore potential therapeutic approaches. Methods and Results: We used tamoxifen to induce cardiac-specific Flnc gene knockout in cardiomyocytes and found that mice with cardiac-specific Flnc knockout exhibited left ventricular dilation, heart failure, increased heart weight, cardiomyocyte enlargement and fibrosis, suggesting that Flnc deficiency in cardiomyocytes causes DCM. Transcriptional profiling of primary cardiomyocytes isolated on the seventh day after tamoxifen injections showed the activation of ER stress and the unfolded protein response (UPR) upon Flnc deletion. Then we measured the expression levels of proteins in UPR pathway in cardiac tissue and found that PDI and pIRE1a proteins were significantly upregulated, while there were no changes in the other two branches of UPR, including the PERK and ATF6 branches. Intraperitoneal injection of a PDI inhibitor E64FC26 significantly improved heart function and reduced fibrosis in cardiac-specific Flnc deletion mice. Conclusion: Cardiac-specific knockout of Flnc leads to DCM in mice, during which the UPR pathway is activated. Treatment with the PDI inhibitor E64FC26 improves the heart function and the fibrosis in Flnc -deficient DCM mice, offering novel perspectives on the pathological mechanisms of DCM and providing new insights into therapeutic strategies in treating DCM caused by FLNC dysfunction.

Abstract We147: The Utility of Cardiopulmonary Exercise Testing (CPET) Amongst Male and Female athletes to Differentiate Between Physiological Left Ventricular Enlargement and Mildly Depressed Resting Systolic Function and Athletic Individuals with Dilated Cardiomyopathy.

Background: Athletes frequently reveal a dilated left ventricle (LV),some also demonstrate a co-existent mildly depressed LV ejection fraction, raising the possibility of mild dilated cardiomyopathy (DCM). The maximal oxygen consumption(VO2max) is a valuable tool for assessing fitness, largely determined by cardiac output. References ranges derived from the general population, may have limitations when assessing athletes with primary myocardial disease. This study investigated the role of VO2max and VO2max predicted (V02pred) to differentiate between physiological LV dilatation and athletic individuals with DCM in male and female athletes. Methods: 147 male and 35 female athletes with dilated LV(mean age 35.4years, 34.5 years respectively) underwent a 25watt cycle ergometer continuous ramp protocol. 75% males and 67% females participated in endurance sports, remaining participating in mixed sports. 52 had mild DCM, 62 defined as healthy control athletes(dilated LV) and 68 were Grey zone (dilated LV and mildly depressed LVEF) Results: DCM males (N=39) revealed a lower mean VO2max than male control (N=46), 34.9ml/kg/min and 48ml/kg/min respectively (p value <0.001). DCM females(N=13) showed lower VO2max of 29.9ml/min/kg than female control (N=16) who revealed similar values to male controls (48 ml/kg/min)(p value <0.001). 62 male Grey zones and 6 female Grey zone revealed lower VO2max than controls 43.1ml/min/kg and 34.1ml/min/kg respectively. DCM females had lower VO2pred than control females 119% and 161% respectively but significantly higher than their male counterparts, means of 106% and 123% respectively (p value <0.001). Male and female grey zones had lower mean VO2pred than controls, 121% and 106% respectively. Higher VO2max of ≥38ml/kg/min and VO2pred of ≥120% gave a sensitivity, specificity of 84.7% and 77%respectively to differentiate healthy athletes and athletic DCMs in males and females. Only41.1% Grey zone athletes were able to achieve above these thresholds. Conclusion: Female athletes with dilated LV may reveal supranormal VO2max which can equal their male counterparts. Furthermore, athletic females with physiological or pathological LV dilatation can reveal higher VO2pred than male athletes. Most athletic DCM individuals show lower CPET parameters than controls athletes but can still achieve greater than normal measurements seen in healthy general population. VO2 may have a role in assisting in differentiating healthy athletes from athletic DCM.

Abstract We077: Mechanisms of T-lymphocyte Dysfunction in Dilated Cardiomyopathy Patients

Preclinical studies have shown that T-cells infiltrate the heart during heart failure (HF), promoting remodeling. However, it remains unclear whether activated T-cells infiltrate the human failing hearts also. Importantly, cellular mechanisms that are involved in T-cell activation during human HF are unknown. To address this question, we analyzed published single-cell RNA sequencing data from patients with dilated cardiomyopathy (DCM) and compared it with control hearts. Our analysis focused on characterizing the gene expression of cardiac T-cells to uncover potential mechanisms underlying their role in human HF. We found that patients with DCM had significantly more cardiac CD3+ T-cells (%CD45+ cells; 35.5±5.9 vs 22.0±0.8 p < 0.01), with gene signatures indicative of higher TCR dependent antigenic activation, proliferation, and exhaustion. Although there were no differences in the proportion of CD4+ helper and CD8+ cytotoxic T-cells between groups, patients with DCM had a higher proportion of activated (%CD45+ cells, 6.6±0.8 vs 3.1±0.3 p = 0.03) and proliferative CD4+ T-cells (%CD45+ cells, 8.0±0.8 vs 3.1±0.3 p < 0.01). Additionally, genes involved in TNF and TGFβ signaling were significantly enriched in cardiac T-cells derived from patients with DCM (normalized enrichment scores = 2.35, p adj < 0.01, and 1.72, p adj < 0.01, respectively),consistent with preclinical studies published by us and others. Recently, we also showed that dysfunctional T-cells exhibit increased estrogenic signaling with activation of Estrogen Receptor (ER)α in mice. Thus, we also looked at estrogen signaling in T-cells infiltrated into the DCM hearts and found that, indeed, cardiac T-cells in DCM patients exhibit significant upregulation of genes involved in estrogen signaling (normalized enrichment score = 1.69, p adj < 0.01). This comprehensive analysis, for the first time, shows that T-cells are antigenically activated, actively infiltrate the failing human hearts and exhibit increased estrogenic signaling during DCM, providing novel insights and potential avenues for targeted immunotherapies.

Abstract We103: Integration of deep learning assisted high-content screening and deep tissue-phenotyping to identify cardioprotective compounds in dilated cardiomyopathy

With a prevalence of 4:10,000, dilated cardiomyopathy (DCM) is the most common hereditary heart disease, characterized by dilatation of one or both ventricles and impaired systolic and diastolic function. Numerous mutations have been identified to cause DCM. TITIN-truncating variants (TTNtv) are commonly observed in 20% of the genetic DCM cases. Progress in drug discovery for DCM has been hampered by the lack of robust high-throughput/high-content analysis pipelines of disease-specific cell and tissue platforms for deep structural and functional phenotyping. To address this challenge, we have developed a two-tiered human induced pluripotent stem cell (iPSC)-based screening approach, comprised of high-content cardiomyocyte imaging with artificial intelligence (AI)-driven image and data analyses as well as deep tissue phenotyping in engineered heart muscle (EHM) models. As a DCM use case, we have developed several isogenic human iPSC-lines with and without a previously reported and pathologically relevant A-band-TTNtv mutation (c.70692_70693insAT; p.T23565SfsX5; Gerull et al. 11788824, Gramlich et al. 25759365, Fomin et al. 34731013) Using fixed weights from a convolutional deep neural network trained on ImageNet, we generated unbiased deep embeddings from each 2D cell model and applied these to train machine learning (ML) models to detect morphological disease phenotypes. Our ML model is able to confidently separate healthy controls from TTNtv cells with high accuracy (>93%), specificity (WT >79%) across different batches/plate layouts and generate concentration-response curves, demonstrating platform robustness and sensitivity. Building upon this work, we will present our latest efforts applying the platform technology for a chemogenomic screening campaign to identify novel cardioprotective drugs using a library of 7,100 bioactive compounds and validation assays, including measuring functional changes in EHMs. The phenotypic profiling platform, as presented here, can be readily adapted to other disease-relevant cell types and pathologies. Our results demonstrate the power of combining iPSC-CMs with cytological profiling and deep learning as well as tissue-level phenotyping to accelerate drug discovery for patients with DCM.

Abstract Mo093: Rescue of Desmin Insufficiency Restores Contractile Function in Cardiomyocytes with MYH7 E848G Dilated Cardiomyopathy Variant

Dilated cardiomyopathy (DCM), characterized by systolic dysfunction, is a significant cause of heart failure. Myosin heavy chain 7 ( MYH7 ) pathogenic variants are common causes of DCM; however, no specific disease-modifying therapy for MYH7 DCM exists. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing the pathogenic MYH7 E848G DCM variant, we confirmed hypocontractility at the single cell level as assessed with traction force microscopy. hiPSC-CMs were cultured on polyacrylamide hydrogels with physiological stiffness in 15x100 µm patterned Matrigel-coated rectangles, each containing one cell. Upon 1 Hz electrical stimulation, contracting cells displaced fluorescent beads embedded within the gel, which were measured to estimate twitch force. MYH7 E848G/+ hiPSC-CMs had reduced maximum twitch force (97 ± 11 nN, n=46 cells) compared to MYH7 +/+ hiPSC-CMs (179 ± 18 nN, n=49 cells), recapitulating the disease phenotype. RNAseq analysis of MYH7 E848G/+ hiPSC-CMs compared to isogenic control MYH7 +/+ hiPSC-CMs found significant downregulation of desmin ( DES) (76.4 ± 8.2% reduction, n=4 bio reps), which encodes a muscle-specific type III intermediate filament. Desmin protein expression in MYH7 E848G/+ hiPSC-CMs was significantly attenuated relative to MYH7 +/+ hiPSC-CMs (90.9 ± 1.8% reduction, n=4 bio reps), matching gene expression data. Gene expression of the transcription factor MEF2C , a known transactivator of DES, was also reduced (40.4 ± 7.0% reduction, n=4 bio reps), suggesting MYH7 E848G/+ suppresses the MEF2C - DES signaling axis. We hypothesized restoring desmin protein levels may improve contractility in MYH7 E848G/+ hiPSC-CMs. To test this, we created an adeno-associated virus (AAV9) packaged with full-length desmin cDNA fused with a T2A self-cleaving peptide and mApple fluorescent reporter. hiPSC-CMs were transfected with this mApple-T2A-DES AAV at 1 MOI and contractility was assessed via traction force microscopy. mApple + MYH7 E848G/+ hiPSC-CMs had increased maximum twitch force (141 ± 11 nN, n=75 cells) relative to mApple - MYH7 E848G/+ hiPSC-CMs (65 ± 20 nN, n=21 cells), approaching the maximum twitch force of mApple + MYH7 +/+ hiPSC-CMs (165 ± 10 nN, n=87 cells). In conclusion, suppression of the MEF2C - DES signaling axis is a potential novel mechanism by which pathogenic MYH7 variants cause DCM. By leveraging this mechanistic insight, we found that rescuing desmin insufficiency can partially restore contractile function.

Abstract Or116: One Carbon Metabolism Defect In Failing Hearts

Aberrant cardiac metabolism has long been hypothesized to contribute to heart failure (HF). However, understanding of cardiac metabolism in HF remains incomplete, especially in humans. To fill this gap, we performed metabolomic and RNA sequencing analysis of 48 non-failing (NF) and 39 dilated cardiomyopathy (DCM) hearts. Among many differences, metabolites from one-carbon metabolism were significantly reduced, especially in methionine cycle; methionine (Met) and S-adenosylmethionine (SAM), a universal methyl donor of methylation. We next studied the mechanism leading to decreased Met/SAM in DCM hearts. We first quantitatively defined methionine cycle in the heart: studies in neonatal rat ventricular myocytes (NRVMs), and in vivo steady-state infusions with isotope tracing, showed that Met was mostly imported or degraded from protein rather than recycled from homocysteine (~10%), suggesting that a defect in recycling is unlikely to explain low Met/SAM in DCM hearts. We next explored the impact of low SAM on cardiac function by generating mice with cardiac specific deletion of SAM synthase. Constitutive KO mice were largely embryonic lethal, and the few survivors showed dramatic systolic dysfunction and died within 12 weeks. Inducing loss of SAM synthase in adults led to 50% mortality after 2 weeks, again with marked cardiac dysfunction. Finally, we explored potential mechanisms by which low SAM can drive heart failure. In human DCM, numerous methylation products were low, including most notably creatine, a SAM methylation product and essential metabolite for cardiac energetics. Creatine is not thought to be synthesized in the heart, but we found that, to our surprise, NRVMs synthesized ~50% of their creatine pool when external supply was low, and synthesis was dependent on SAM availability. Moreover, in vivo steady state infusions revealed that in intact animals, even in the absence of stressors, ~10% of creatine in the heart is synthesized locally. In summary, we find that: (1) the majority of Met in cardiomyocyte is imported or derived from protein degradation, with rapid turnover; (2) Met and its product SAM are reduced in human DCM; (3) loss of SAM in murine heart leads to profound heart failure; (4) creatine is surprisingly synthesized from SAM in cardiomyocytes, and its levels are reduced in human DCM. Together, these findings suggest that a defective Met cycle may contribute to low creatine and ultimately HF.

Abstract Or104: Targeting Tropomyosin Overlap Flexibility via Small Molecule Intervention in a Murine Dilated Cardiomyopathy Model

Familial dilated cardiomyopathy (DCM) is a genetic heart disorder characterized by enlargement of one or both ventricles, rendering the heart unable to pump blood efficiently. Mutations in cardiac thin filament (CTF) proteins have been associated with characterized clinical manifestations of DCM. Genetic segregation identified a mutation in α-Tropomyosin (Tm) at residue 230 (D230N-Tm) in two unrelated, multigenerational families linked to severe, early onset DCM. To meaningfully link genotype to phenotype our group sought to investigate the effects of D230N-Tm on the biophysical structure of Tm filament. We showed that D230N-Tm caused a compaction of the cardiac Troponin T (cTnT)–Tm overlap region accompanied by a decreased in flexibility of the Tm filament. Given the altered biophysical structure we investigated whether we could target this decrease in flexibility via small molecule intervention and improve function. Computational modeling and biophysical experiments identified small molecule 4-Hydroxy Duloxetine β-D-Glucuronide (Z06) shown to improve compaction of the cTnT-Tm overlap and restore flexibility of the Tm filament. We hypothesized that the decrease in flexibility of Tm impairs its ability to shift through its different conformational states, leading to impaired crossbridge formation. To investigate whether Z06 improves myofilament function, we performed an NADH-coupled ATPase assay using isolated myofibrils from our D230N-Tm transgenic mice and compared them to Non-Transgenic (NTg) littermates. ATPase activity was measured at saturating calcium (100uM) in the presence (250uM) and absence (0uM) of Z06. In the absence of Z06, D230N-Tm exhibited a decrease in ATPase activity (1.793±0.203 umol/min) compared to NTg littermates (5.420±0.562 umol/min), a common trend in DCM models. In the presence of 250uM Z06, D230N-Tm ATPase rates improve to levels comparable to NTg mice (4.816±0.544 umol/min). Moving forward, we will perform calcium titrations, allowing us to investigate if Z06 can restore cooperativity of myofilament activation. Additionally, we will test if the restorative effects of Z06 are present at the myocellular level, and if so, we will optimize delivery to test in vivo effects of Z06 in our D230N-Tm mouse model.

Abstract We146: CHD8 is Essential for Postnatal Heart Function through Preserving Mitochondrial Integrity

Objective: Epigenetic regulation plays a crucial role in heart development and disease, although its mechanisms remain elusive. Known mutations in epigenetic regulators have been linked to cardiac disease. For instance, the mutation in the chromodomain helicase DNA-binding (CHD) protein 7 gene, which leads to CHARGE syndrome, is known to results in heart defects. However, the role of CHD8, an interacting partner of CHD7, in the heart has not been investigated. This study aims to explore the function of CHD8 in the heart. Methods and Results: We generated a cardiac-specific Chd8 conditional knockout mouse line (Chd8 cKO) by breeding Chd8 fl/fl mice with aMHC Cre mice. Over 60% of Chd8 cKO mice died between 7 and 9 months of age, with increased heart weight to bodyweight ratio and reduced cardiac function, increased fibrosis, and signs of dilated cardiomyopathy. Electron microscopy analysis revealed disordered sarcomeres and escalated mitophagy in CHD8-deficient hearts. Using Genome-wide transcriptome analysis though RNA sequencing, we discovered that genes involved in mitochondrial oxidative phosphorylation and muscle contraction were down-regulated in Chd8 cKO hearts. Proteins in the electron transport chain complexes were down regulated and mitophagy marker LC3 increased in the Chd8 cKO hearts compared with the control mice. Upregulated pathways include inflammatory response suggesting that the innate immune response may contribute to dilated cardiomyopathy. Conclusions: Our findings underscore CHD8 as a key regulator of cardiac function and regeneration by influencing chromatin accessibility of genes involved in mitochondrial oxidative phosphorylation and contractility. Overall, our study provides new insights into the epigenetic regulation of cardiac function and the CHD8’s role in heart development and disease. It implies that activating CHD8 may be a promising therapeutic strategy for treating dilated cardiomyopathy and other heart-related diseases.

Abstract Mo096: High-Throughput Functional Determination of TNNI3 Variant Pathogenicity

Introduction: Mutations in the thin filament protein TNNI3 (cardiac troponin I) can perturb calcium cycling and cause hypertrophic, restrictive, and dilated cardiomyopathies. However, the majority of TNNI3 variants observed in patients have unknown functional significance and uncertain contribution to disease. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful system for studying the effect of genetic variants on human cardiomyocyte function. TNNI3 has been challenging to study in these cells as they predominantly express TNNI1. In this study, we combined a novel scalable in vitro gene replacement method with simultaneous measurement of contractility and calcium cycling to determine the functional effect of TNNI3 variants. Hypothesis: Using our gene replacement platform, we expect that TNNI3 variants causing Hypertrophic Cardiomyopathy (HCM) will increase force generation and calcium sensitivity relative to wild-type, whereas TNNI3 variants causing Dilated Cardiomyopathy (DCM) will decrease force generation and calcium sensitivity relative to wild-type. Methods: We generated double knockout TNNI1 -/- TNNI3 -/- hiPSCs. Cardiomyocytes differentiated from these hiPSCs were transduced with lentivirus carrying either wild-type or mutant TNNI3 during replating onto polyacrylamide gels with fluorescent beads. After a week of incubation, the cells were stained with a fluorescent calcium probe, and contractility, diastolic tension, and calcium sensitivity were measured by our high-throughput physiologic imaging and analysis platform. Results: hiPSC-CMs expressing the HCM-associated variant R192H show increased peak force, diastolic tension, and calcium sensitivity relative to wild-type TNNI3, whereas hiPSC-CMs expressing the DCM-associated variant K36Q show decreased peak force and calcium sensitivity relative to wild-type TNNI3. Conclusion: We validated our TNNI3 in vitro gene replacement and high-throughput physiologic imaging and analysis platform by comparing reference HCM and DCM variants to wild-type TNNI3. Ongoing scaled studies will evaluate the naturally occurring variants in ClinVar to characterize their functional effect on cardiac physiology.

Abstract Mo098: LMNA R190W Mutation Associated With Dilated Cardiomyopathy Causes Hypercontractility in iPSC-derived Cardiomyocytes and Engineered Heart Muscles

Familial dilated cardiomyopathy (DCM) caused by mutations in sarcomeric proteins have been extensively studied in vitro , however less is known about DCM caused by alterations in non-sarcomeric genes like Lamin A (LMNA). LMNA is a ubiquitously expressed nuclear intermediate filament protein that regulates nuclear structure, chromatin access and gene expression. LMNA mutations account for 6% of DCM, and affected patients develop drug-therapy resistant severe heart failure and lethal arrythmias that are not amenable to currently available therapies. Most identified LMNA-cardiomyopathy mutations have been associated with severely depressed cardiac systolic function in patients and cardiomyocyte hypocontractility in vitro . However, the underlying mechanisms are incompletely understood. This project examines the mechanisms by which a patient-derived pathogenic mutation, LMNA R190W, leads to cardiac dysfunction at the cellular and tissue levels. We hypothesize that LMNA R190W mutation causes dysregulated expression of calcium handling genes, eventually leading to altered contractility. First, we investigated early disease pathogenesis using iPSC-derived cardiomyocytes. Intriguingly, single cardiomyocyte traction force microscopy studies showed cardiomyocyte hypercontractility, and calcium flux assays revealed prolonged calcium transients. Second, in human engineered heart muscle tissues, LMNA CMs were also consistently hypercontractile. Through gene expression studies, we identified upregulation of a cardiac-specific sarcomeric calcium regulator, calsequestrin 2 (CASQ2), which modulates intracellular calcium availability and localization, and thereby indirectly impacts calcium-dependent contraction and electrophysiology. siRNA-mediated knockdown of CASQ2 in iPSC-CMs decreased cellular calcium content, suggesting Casq2 is necessary for increased calcium and contractility in LMNA R190W cells. These findings suggest that in the initial stages of LMNA R190W-associated cardiomyopathy, cardiomyocyte contractility is not diminished, but actually augmented, and this may be related to aberrant calcium handling. We posit that chronic inappropriate calcium overload may eventually lead to cell death, and predispose to cardiomyopathy. Understanding the initial processes driving the development of LMNA cardiomyopathy will inform more accurate mechanism-based subclassification of mutations, and the rational development of precision therapies.

Abstract Tu059: Single-Nucleus Transcriptomics Demonstrates Endothelial Cell Expansion in Failing Human Right Ventricles

Background: Right ventricular failure (RVF) is most commonly observed as a sequela of left ventricular failure (LVF)—for which it more than doubles the risk of mortality. Moreover, isolated RVF is the most common cause of death in pulmonary hypertension and is a leading cause of major adverse events in systemic right ventricle congenital heart diseases. Therapies which effectively treat LVF show poor efficacy for RVF. For example, beta blockade, a cornerstone of LVF therapy, has no efficacy in RVF. Therefore, there is a need for broader exploratory research to identify pathways which are dysregulated in RVF and which could serve as targets for future therapies. Methods/Results: To this end, we performed single-nucleus RNA sequencing (N=11) on RV myocardium from nonfailing or dilated cardiomyopathy (DCM) hearts subgrouped by preserved systolic and diastolic RV function (pRV) or RVF. After cell-type assignment and differential expression analysis, we find a progressive upregulation of pro-angiogenic signaling in vascular endothelial cells (ECs) from nonfailing to pRV and RVF. There is a concomitant expansion of the EC pool from nonfailing to pRV and RVF (EC percentage 5%, 9%,15% for NF, pRV, and RVF; p < 0.05 for one-way ANOVA). We find increased HIF-mediated and VEGF-mediated signaling in these ECs, which may be mediating this expansion. Surprisingly, there is also increased expression of genes regulated by type 1 interferon signaling, which is thought to be anti-angiogenic. Discussion: In summary, we have conducted a single-nuclei transcriptomic study of human RVF and found evidence for increased angiogenic signaling and EC expansion. These findings are significant, as the balance of pro-angiogenic and anti-angiogenic factors in RVF is still unclear, with studies in animal models showing conflicting results in terms of changes in macro/microvasculature in RVF. Here, we provide evidence that in human RVF the balance tends to lead toward a pro-angiogenic phenotype, even in dysfunctional RVs. Orthogonal studies to validate the causal nature of these observed changes in pathogenesis of RVF are currently ongoing.

Abstract We110: Novel allosteric ligand designed to prevent Autoantibody targeting the Angiotensin Type 1 Receptor (AT1R) in cardiovascular diseases (CVD)

Background: The clinical data suggest an association of autoantibodies (AAb) targeting angiotensin II type1 receptor (AT1R) and outcomes in heart failure (HF) and preeclampsia. Notably, patient’s risk increases 2 to 4-fold for heart diseases and hypertension. Although AT1R blockers (ARBs) have emerged as a key component in the therapeutic arsenal against CVD, we believe that rather than completely blocking AT1R signaling, maintaining a balanced signaling is crucial for cardiovascular homeostasis. Therefore, we posit that reducing AT1R overactivation and blocking AAb-mediated activation could benefit cardiac health and attenuate the progression of CVD. In our previous study (PMID:36440576), we identified small molecules capable of inhibiting AAb-induced AT1R signaling. Leveraging these observations, we have developed a novel AT1R allosteric ligand that inhibits autoantibody binding to AT1R while still retaining normal AT1R signaling. Methods: Using computational and medicinal chemistry approaches, we designed a series of novel AT1R ligands and obtained compounds with >98% purity through chemical synthesis. Monoclonal antibodies mimicking AT1R-AAb have been generated. Serum samples from dilated cardiomyopathy (DCM) and preeclampsia (PE) patient cohorts were collected and presence of AT1R-AAb was determined using ELISA and epitope-peptide competition studies. Pharmacological characterization of compounds was performed using calcium mobilization, 125I -AngII binding, ex vivo vasoconstriction, and ELISA assays. Results: We identified an AT1R allosteric compound that reduced AngII-mediated calcium signaling with a 5-fold shift in EC 50 . The IC 50 was determined to be 18 µM. It reduces the AngII-mediated vasoconstriction while blocking both monoclonal and patient-derived autoantibody binding with an IC 50 of 5 µM. Conclusion: We have identified a novel AT1R allosteric ligand with negative allosteric modulator (NAM) properties, which has the potential to block autoantibodies and could serve as a new generation of ARBs. Uniquely, it retains beneficial AT1R signaling without competing with AngII, and its allosteric ligand property can be tailored to achieve optimal AT1R signaling.

Abstract Tu050: Deficiency Of Mitochondrial Disulfide Relay Carrier Leads To Cardiac Hypertrophy

Background: Innate immune activation is critical for cardiac hypertrophy; however, its upstream regulation is less well understood. Coiled-coil-helix-coiled-coil-helix domain 4 (CHCHD4) is an essential carrier for the import of various mitochondrial proteins including TP53 -regulated inhibitor of apoptosis 1 (TRIAP1). CHCHD4 was recently identified as a dysregulated gene in patients with dilated cardiomyopathy and was separately found to mediate innate immune signaling in skeletal muscle. Therefore, we hypothesized that CHCHD4 may play a role in the development of cardiac hypertrophy by regulating innate immunity. Methods: Genetically modified C57BL/6 mice overexpressing CHCHD4 (CHCHD4 Tg) or haploinsufficient in CHCHD4 ( CHCHD4 +/- ) were utilized. Mice were injected either with low dose (2 mg/kg) or high dose (30 mg/kg) isoproterenol (ISO) daily for up to 3 wk to induce hypertrophy. Results: CHCHD4 +/- mice developed cardiac hypertrophy (HW/BW (mg/g): 3.62 ± 0.15 CHCHD4 +/- vs 2.70 ± 0.13 wild-type (WT) or 2.55 ± 0.16 CHCHD4 Tg, p<0.0001) when allowed to age to 28 wk and showed reduced left ventricular ejection fraction (EF) at 1 year when compared with wild-type or CHCHD4 Tg mice (EF (%): 50.94 ± 0.94 CHCHD4 +/- vs 58.33 ± 0.78 wild-type or 57.88 ± 1.32 CHCHD4 Tg, p<0.0001). After low dose ISO treatment, CHCHD4 +/- mice had reduced EF at 2 wk compared to WT or CHCHD4 Tg mice (EF (%): 44.60 ± 1.74 CHCHD4 +/- vs 54.28 ± 0.86 wild-type, p<0.0001; or vs 52.76 ± 1.33 CHCHD4 Tg, p=0.0006). After high dose ISO treatment, WT mice developed cardiac hypertrophy by 1 wk and impaired EF by 3 wk, while CHCHD4 Tg mice remained normal. ISO treatment caused reduction of CHCHD4 levels in both the 24 hr and 1 wk states. This reduction was associated with decreased TRIAP1 and activation of the cGAS-STING-NFkB pathway in wild-type, but not CHCHD4 Tg mice. Further, plasma mitochondrial DNA (mtDNA) was 2.3 times higher in WT mice treated with high dose ISO for 1 wk compared to WT control (n=8-11/group, p=0.011), while that of CHCHD4 Tg mice was not significantly changed by ISO treatment. Conclusions: Our findings suggest that CHCHD4 may be a key upstream regulator of innate immune activation mediating cardiac hypertrophy and that cell-free mtDNA in blood could be a potential biomarker of pathogenesis.

Abstract We013: Modeling Sex and Stress using Induced Pluripotent Stem Cell Derived Cardiomyocytes

Despite causing 1 in 3 deaths worldwide, cardiovascular disease and the heart have only recently been studied in women. Contrary to previous belief, the female heart is not just a smaller replica of a male heart. Almost all heart diseases show sexual differences in risk, treatment, and symptoms. Even with these differences there is a lack of female models in animal and cellular studies. Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are a powerful human model to test baseline and stressed sex differences at the cellular level. We hypothesized that using stressors that mimic disease conditions (drugs, substrate stiffness) in multiple male and female hiPSC-CMs will result in differential sex-based responses. We ran Traction Force Microscopy on 3 male (n = 424 cells, 3 batches) and 3 female (n = 318 cells, 3 batches) cell lines on 10 kPa polyacrylamide hydrogels. Male cells had 42% higher twitch force (p = 8.8E-6) with 25% higher contraction (p = 1.77E-5) and relaxation (p = 0.0006) velocities. However, female cells had a larger spread area (823 +/- 17.32 µm 2 vs. 756 +/- 21.64 µm 2 , p = 0.0018). To validate hiPSC-CMs as a model we compared preliminary RNA-seq to publicly available Heart Cell Atlas sc/snRNA-seq data sets of primary ventricular cardiomyocytes. They had similar differentially expressed genes (DEGs) as our hiPSC-CMs. Furthermore, DEGs had significantly enriched gene sets including a 25-fold enrichment in hypertrophic cardiomyopathy (FDR = 1.6E-7), 23-fold in dilated cardiomyopathy (FDR = 1.6E-7), and 16-fold in cardiac muscle contraction (FDR = 6.3E-4). We analyzed stiffness by differentiating and culturing hiPSC-CMs on 10 kPa and 100 kPa stiffness hydrogels. Our RNA-seq analysis at day 10 and 30 after differentiation found 7000 DEGs at 10 kPa while only 556 DEGs at 100 kPa. We looked hypercontractility caused stres and performed RNA-seq on day 60 wild type and hypercontractile mutant hiPSC-CMs. The wild type was dosed with positive inotrope levosimendan to induce hypercontractility while the hypercontractile mutant was dosed with negative inotrope verapamil to lower contractility. Hypercontractility had significant upregulation of the cellular response to stress including ER stress, stress-activated MAPK cascades, stress fibers, and cardiac muscle hypertrophy in response to stress. These differences can identify possible gene targets for preventing cardiovascular disease and inform personalized medicine.

Abstract Or107: MyBP-HL nonsense mutations fail in sarcomere incorporation leads to modifications in myocyte contraction trough calcium modifications.

Introduction: Heart function depends on the cardiomyocyte contractile apparatus and proper sarcomere protein expression. Mutations in sarcomere genes cause inherited forms of cardiomyopathy and arrhythmias, including atrial fibrillation. AF is the most common cardiac arrhythmia and is associated with ischemic stroke, heart failure, and substantial morbidity and mortality. Recently, our group described a novel sarcomere component, myosin binding protein-H like (MyBP-HL), that is mainly expressed in the cardiac atria. MyBP-HL is composed of two immunoglobulin domains and one fibronectin domain, with homology to last three C-terminal domains of cardiac myosin binding protein-C (cMyBP-C). cMyBP-C nonsense mutations prevent his incorporation into the sarcomere. C-MyBP-C truncated peptides are identified by cells as poisoning peptides, and they are rapidly degraded. cMyBP-C nonsense mutations are causative of hypertrophic cardiomyopathy (HCM). The MyBP-HL Arg255stop variant has been described in humans and has been linked to ventricular conduction system abnormalities, atrial enlargement, dilated cardiomyopathy, and atrial and ventricular arrhythmias. The gnomAD database reports 9 nonsense mutations for MYBPHL in humans (Gln29, Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250, Arg255, and Tyr307). Hypothesis: MYBPHL nonsense mutations fail to incorporate into the sarcomere will lead to contractile disfunction. Results: We demonstrated that MyBP-HL overexpression showed to be able to incorporate in a similar pattern to cMyBP-C C-zone doublets. Nevertheless, nonsense mutations disrupt normal sarcomere localization. MyBP-HL nonsense mutations promote an increase in contraction amplitude and slower relaxation kinetics. Also, we observed an increase in sarcomere length. Similarly, MyBP-HL nonsense mutations lead to increase in amplitude peak of calcium transients, and reduction in velocity to maximal calcium peak. In accordance with calcium modifications, we observed through mass spectrometry an increase in calpain 6 abundance promoted by MyBP-HL nonsense mutations. Conclusion: Based on these data, we demonstrated that MYBPHL nonsense mutations prevent MyBP-HL incorporation into the thick filament. The failure of MyBP-HL truncating peptides to incorporate into the sarcomere leads to a deficiencies in myocytes contraction patterns, changes in calcium patterns and increase in degradation proteins abundance, which are related to AF prevalence.

Abstract Mo059: Leucine-Rich Repeat-Containing Protein 10 Modulates Human Cardiac L-Type Ca 2+ Channels but not T-Type Ca 2+ Channels

Background: Leucine-rich repeat-containing protein 10 (LRRC10) is a novel regulator of the cardiac L-type Ca 2+ channel/current (LTCC/I Ca,L ). LRRC10 is crucial for cardiac regeneration in multiple species, and LRRC10 variants have been associated with dilated cardiomyopathy in humans. LRRC10 increases I Ca,L in HEK-293 cells expressing rabbit Ca V 1.2 subunit, and knockout of LRRC10 in zebrafish and mouse cardiomyocytes (CM) reduces I Ca,L . However, LRRC10-mediated regulation of human cardiac LTCC has not been demonstrated, and the mechanism by which LRRC10 modulates LTCC is not known. Moreover, the effect of LRRC10 on the T-type Ca 2+ channel/current (TTCC/I Ca,T ), which has been implicated in CM cell cycle regulation, has never been tested. Methods: LRRC10 knockout ( LRRC10 –/– ) human induced-pluripotent stem cells (hiPSC) were generated from wild-type (WT) DF19-9-11T hiPSCs using CRISPR-Cas9 techniques. LRRC10 –/– and WT hiPSCs were differentiated to CMs using the standard GiWi protocol. Whole-cell I Ca,L and I Ca,T were examined in LRRC10 –/– and WT hiPSC-CMs. Whole-cell I Ca,L was also assessed in HEK-293 cells expressing human cardiac LTCC subunits (Ca V 1.2, β 2 , and α 2 δ) with or without human LRRC10. To identify the LRRC10-interacting site of Ca V 1.2, GST-pulldown assays were performed using purified GST-fusion constructs of the intracellular domains of rabbit Ca V 1.2 and lysates from HEK-293 cells expressing human LRRC10. Ca V protein sequences were analyzed using the Clustal Omega program. Results: Genetic ablation of LRRC10 in hiPSC-CMs decreased peak I Ca,L (WT -29.4±2.2 pA/pF, LRRC10 –/– -15.0±1.8 pA/pF, p<0.01) but did not significantly affect I Ca,T magnitude (WT -3.9±0.5 pA/pF, LRRC10 –/– -4.0±0.7 pA/pF, p>0.05 [NS]). LRRC10 increased peak I Ca,L in HEK-293 cells expressing human LTCC (with LRRC10 -156.0±19.8 pA/pF, without LRRC10 -60.2±12.0 pA/pF, p<0.05). LRRC10 was pulled down by the GST-Ca V 1.2-N-terminus but not by other GST-Ca V 1.2 constructs. Sequence analyses revealed an invariant exon 2-encoded segment of the N-terminus (NT) that was present in both human and rabbit LTCC Ca V 1.2 but not in TTCC Ca V 3.1 and Ca V 3.2. Conclusions: The Ca 2+ influx-potentiating effect of LRRC10 is conserved in human LTCC and is specific to regulate I Ca,L , not I Ca,T . The invariant Ca V 1.2 NT segment is a potential LRRC10-interacting site. The LRRC10 –/– hiPSC line is a promising tool for future investigations into the roles of LRRC10-LTCC interaction in human cardiac biology and disease.

Abstract Mo053: Acacetin's Antiarrhythmic Potential in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Harboring Hypertrophic and Dilated Cardiomyopathy-Related Mutations

Background: Hypertrophic cardiomyopathy (HCM) is often caused by mutations such as p.R943X in the Myosin Binding Protein C3 (MYBPC3), leading to early and delayed afterdepolarizations, myofibrillar disarray, and calcium dysfunction, which may induce lethal arrhythmias. Dilated cardiomyopathy (DCM) can also result from mutations, such as the p.R14del mutation in the phospholamban (PLN) protein, which is crucial for calcium signaling regulation, leading to arrhythmogenesis when provoked. This study explores the arrhythmogenic potential in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the HCM MYBPC3 p.R943X mutation and the DCM PLN p.R14del mutation. Methods: We utilized a 384-well optical physiological system to record action potentials (APs) in hiPSC-CMs carrying the MYBPC3 p.R943X mutation and the DCM PLN p.R14del mutation, including their isogenic controls. Action potential metrics were measured under spontaneous and electrically paced conditions. The effects of Acacetin, NS-5806, nifedipine, and GS-967 on the recorded AP types were analyzed to understand the underlying channelopathies and arrhythmogenic potential. Special attention was given to the variability between the HCM MYBPC3 p.R943X and DCM PLN p.R14del cell lines' susceptibility to arrhythmogenesis and their isogenic controls. Results: The HCM MYBPC3 p.R943X hiPSC-CMs exhibited a significant increase in early afterdepolarizations, non-sustained and sustained ventricular tachycardia, and AP notches compared to their isogenic control. AP notches and arrhythmogenic events were notably provoked by NS-5806 in both the HCM MYBPC3 p.R943X and DCM PLN p.R14del hiPSC-CMs. However, these were significantly ameliorated (p<0.05) by Acacetin administration (5 µM), highlighting its potential as an effective antiarrhythmic in managing both HCM- and DCM-induced arrhythmias. GS-967 also showed efficacy in reducing early afterdepolarizations (EADs) in the HCM model. Conclusions: This study provides novel insights into the arrhythmogenic potential of hiPSC-CMs carrying MYBPC3 p.R943X and PLN p.R14del mutations, underscoring the significant role of Acacetin in attenuating these effects. It paves the way for developing targeted therapies that address the complex molecular mechanisms underlying arrhythmogenesis in cardiomyopathies. The findings suggest that Acacetin could be a promising candidate for the pharmacologic treatment of cardiomyopathies characterized by arrhythmogenic risks.

Analysis of pediatric heart transplantation supported by extracorporeal membrane oxygenation

Objective: To summarize the clinical characteristics of patients with end-stage heart failure who receive heart transplant under extracorporeal membrane oxygenation (ECMO) support. Methods: The clinical data of 12 pediatric patients who received heart transplant with ECMO support in the Seventh Medical Center of Chinese People's Liberation Army General Hospital and Guangdong Provincial People's Hospital, from January 2019 to December 2023 was collected. The data included sex, age, weight, diagnosis, pre-ECMO lactate level, left ventricular ejection fraction (LVEF), vasoactive-inotropic score (VIS), and preoperative ECMO running time. Surgical data included cold ischemia time of the donor heart, cardiopulmonary bypass time, intraoperative use of immunosuppressant, postoperative use of ECMO, duration of postoperative ECMO, rate of successful weaning from ECMO, and survival discharge rate. The paired t-test was performed to compare cardiac function indices before and after left ventricular decompression. Results: The 12 patients ranged in age from 1.1 to 15.8 years, and weighted from 8 to 63 kg. Ten children were diagnosed with dilated cardiomyopathy, one with myocardial underdensification, and one with a novel heterozygous mutation of the SCN5A gene causing overlap syndrome complicated by fatal arrhythmia. Before ECMO, the lactate ranged from 0.6 to>15.0 mmol/L, the LVEF from 6.5% to 43%, and VIS from 3 to 108. Four patients underwent left ventricular decompression supported by preoperative ECMO, and their pulse pressure was significantly increased after decompression ((17.8±2.1) vs. (9.8±1.5) mmHg, 1 mmHg=0.133 kPa, t=11.31, P=0.001), while there was no apparent change in LVEF ((26.8±4.4)% vs. (24.9±4.9)%, t=1.75, P=0.178). A total of 7 children received a second run of ECMO after surgery and 3 of them successfully weaned off ECMO and survived to discharge. In the entire cohort, 10 were successfully weaned from ECMO and 8 survived to discharge. Conclusions: For children with end-stage heart failure supported by ECMO, left ventricular decompression can significantly improve pulse pressure. These patients will eventually require heart transplantation.

Abstract Mo116: Evaluating MYBPC3 and MYBPHL missense mutations on sarcomere incorporation

Hypertrophic cardiomyopathy (HCM) is one of the common genetic heart diseases and causes hypercontractility and pathological thickening of the myocardium. Cardiac myosin binding protein-C (cMyBP-C) is an important regulator of cardiac contractility and MYBPC3 mutations accounts for 40% of HCM cases, resulting in the truncation and loss of cMyBP-C. While cMyBP-C is expressed in both atria and ventricle, its recently identified homologous partner, myosin binding protein-HL (MyBP-HL) is solely expressed in the atria. Loss of function mutations in MYBPHL cause dilated cardiomyopathy in humans and mice. MyBP-HL and cMyBP-C bind to specific positions on the thick filament within the C-zone of sarcomere. In the atria, there is a 1:1 ratio between MyBP-HL and cMyBP-C, whereas cMyBP-C is solely expressed in the ventricle at double the cMyBP-C content of the the atria, establishing a stoichiometric relationship between these two proteins in their ability to bind to the thick filament within the sarcomere. Within the ventricle, heterozygous missense mutations within the necessary myosin binding domains of cMyBP-C may prevent proper myosin binding and sarcomere incorporation, but function may still be preserved due to the other unaffected allele. Since the atria expresses MyBP-HL and cMyBP-C, we hypothesize that MYBPC3 and MYBPHL missense mutations are more severe due to the competition for binding sites within the sarcomere between these proteins. We have identified MYBPC3 and MYBPHL missense mutations and variants to evaluate their pathogenicity and sarcomere incorporation via immunofluorescence microscopy within neonatal rat ventricular cardiomyocytes. To test these mutations, we created a “Mini-C” construct consisting of the thick filament binding domains of cMyBP-C. We validated our Mini-C construct via transfection and co-localization with endogenous cMyBP-C in neonatal rat ventricular cardiomyocytes (NRVMs). Our data demonstrate that known pathogenic MYBPC3 missense mutations have improper sarcomere incorporation within NRVMs. Disease associated MYBPHL missense mutations also show improper sarcomere incorporation. To test the effects of missense mutations on myosin binding protein stoichiometry within the sarcomere, we have created and validated a T2A/P2A construct, expressing our Mini-C construct, a fluorescent reporter, Td-Tomato, and our hMyBP-HL construct at consistent and reproducible expression levels via western blot and mass spectrometry.

Abstract Tu114: Prostaglandin E2 Alters Mitochondrial Energy Metabolism in the Murine Heart

Prostaglandin E 2 (PGE2) is an autacoid that acts via 4 receptors (EP1-EP4), with EP3 and EP4 being the most prominent in the heart. We previously reported that cardiomyocyte specific knockout of EP4 (EP4 KO) results in dilated cardiomyopathy. Additionally, transgenic mice that overexpress EP3 in the cardiomyocyte (EP3 TG) develop heart failure. Recently published data from our laboratory suggests EP4 KO results in mitochondrial dysfunction, likely via EP3 receptor signaling. We therefore hypothesized that PGE2 alters energy metabolism in the heart via EP3. To test our hypothesis, we performed gene array analysis on left ventricles from adult EP4 KO and wild type (WT) mice. Significant reductions (2.1-fold) in carnitine palmitoyltransferase 2 ( Cpt2 ) were observed in EP4 KO vs. EP4 WT. CPT2 is a key enzyme in fatty acid oxidation and deficiencies in humans have been shown to cause cardiomyopathy. Substantial reductions in CPT2 were also observed in C57 mice after 2 weeks of myocardial infarction (0.22 ± 0.04 a.u. in sham controls vs. 0.039 ± 0.06 a.u. after MI. p<0.01) by western blot analysis. Moreover, treatment with the EP3 receptor agonist, sulprostone, reduced CPT2 protein expression significantly in cardiomyocytes isolated from C57 mice (1.0 ± 0.0 a.u. in vehicle control vs. 0.67 ± 0.07 a.u. in sulprostone treated. p<0.005). These data suggest that PGE2 via its EP3 receptor reduces CPT2 expression. In fact, RNAseq analysis on left ventricles from EP3 TG and EP3 WT mice showed significant reductions in Cpt 2 expression as early as 5 wks of age (log 2 FC = -1.17. p<0.01). RNAseq also showed reductions in other key fatty acid oxidation/transport genes in EP3 TG mice ( Acsl1 ; log 2 FC= -0.73, Acads , log 2 FC= -0.59, Cd36 , log 2 FC= -0.64. p<0.005). Lastly, to further investigate the role of the EP receptors on energy metabolism in the heart, we isolated cardiomyocytes from EP4 KO and EP4 WT mice and performed metabolomics. As anticipated, we observed significant reductions in L-carnitine (7.6 ± 1.13 fmol/cell in EP4 WT vs. 2.1 ± 0.32 fmol/cell in EP4 KO. p<0.005) and several acyl carnitine products. Altogether our data supports our hypothesis that PGE2 signaling via its EP3 receptor impairs mitochondrial energy metabolism in the heart by reducing fatty acid oxidation.