bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract Mo024: Integrated analysis of
ex vivo
biomechanical ventricular remodeling and imaging-based
in vivo
strain changes in a pre-clinical murine model of radiation-induced cardiotoxicity
Authors: Mukherjee Tanmay , Elliott Sarah , Higgins Taylor-Jade , Wansapura Janaka , Alluri Prasanna , Avazmohammadi Reza
Published: October 2024

Introduction:
Thoracic radiation therapy (RT) is commonly used for the treatment of many common malignancies, such as lung and breast cancer, but carries a high risk of radiation-induced cardiotoxicity (RIC). Existing diagnosis of RIC is challenged by subclinical ventricular dysfunction, not captured by global indices of cardiac function. Understanding RIC-driven biomechanical remodeling of the myocardium will provide novel insights into developing high-fidelity imaging approaches for early detection of RIC.

Methods:
A pre-clinical model of RIC was developed by administering whole heart irradiation under image guidance in mice (8 Gy x 5) over five consecutive days. A longitudinal study was carried out to investigate the biomechanical response of the left ventricle (LV). Cardiac magnetic resonance imaging was performed to estimate end-systolic strains. Mechanical testing was performed on the harvested LV free wall (LVFW) specimens to measure passive stiffness, calculated as the tangent to the ensemble stress-strain curve at 30% strain.

Results:
A significant increase in passive LVFW stiffness was observed at 3M post-RT (Figs. 1A and B). Significant declines in both global circumferential strain (GCS) and global radial strain (GRS) at 3M post-RT, compared to respective measurements at pre-RT, were observed (Figs. 1C and D). Suppressed contractile strains suggested biomechanical remodeling of the LV, including impaired contractility and passive stiffening of the LVFW, consistent with ex-vivo measurements.

Conclusion:
We have established a direct correlation between ex-vivo mechanical testing (elevated stiffness) and reduced contractile strains in in-vivo analysis in a mouse model of RIC. Such integrated analysis will pave the way towards developing highly sensitive imaging approaches that non-invasively recapitulate the ventricular remodeling events associated with RIC, thereby enabling early detection of RIC in patients receiving thoracic RT.
Abstract Mo026: ATP-dependent citrate lyase Drives Left Ventricular Dysfunction by Metabolic Remodeling of the Heart
Authors: Liu Shijie , Gao Yaqi , Kim Kyoungmin , Gammon Seth , Tan Lin , Pham Janet , Khanna Radhika , Gould Benjamin , Salazar Rebecca , Lien Evan , Hanson Blake , Vander Heiden Matthew , Lorenzi Philip , Taegtmeyer Heinrich , Piwnica-Worms David , Martin James , Karlstaedt Anja
Published: October 2024

Background:
Metabolic remodeling is a hallmark of the failing heart. Oncometabolic stress during cancer increases the activity and abundance of the ATP-dependent citrate lyase (ACL,
Acly
), which promotes histone acetylation and cardiac adaptation. ACL is critical for the de novo synthesis of lipids, but how these metabolic alterations contribute to cardiac structural and functional changes remains unclear.

Methods:
We utilized MyH6-Cas9 mice (male and female) and CRISPR/Cas9 gene editing to generate a cardiac-specific Acly knockdown (AclyKD) mouse model. To assess how the loss of
Acly
impacts cardiac energy substrate metabolism, we conducted positron emission tomography in vivo and ex vivo stable isotope tracer labelling using working heart preparation. We conducted a multi-omics analysis using RNA-sequencing and mass spectrometry-based metabolomics. Experimental data were integrated into computational modelling using the metabolic network CardioNet to identify significantly dysregulated metabolic processes at a systems level.

Results:
Loss of ACL expression causes a left-ventricular cardiomyopathy by histology and echocardiography. FDG-PET/CT imaging in male and female mice demonstrated an increased glucose uptake and utilization in AclyKD mice, suggesting a substrate switch from fatty acid oxidation towards glucose. These findings were corroborated by stable isotope tracing, demonstrating that AclyKD suppresses fatty acid oxidation while enhancing lipid remodelling. AclyKD also reduced the efflux of glucose-derived citrate from the mitochondria into the cytosol, confirming that citrate is required for reductive metabolism in the heart. Computational flux analysis and integrative multi-omics analysis indicate that ACL activity supports lipid biosynthesis in the heart. Blocking mitochondrial isocitrate dehydrogenase or alpha-ketoglutarate dehydrogenase sensitized cardiomyocytes to ACL inhibition, and overexpressing cytosolic isocitrate dehydrogenase was sufficient to prevent citrate accumulation.

Conclusions:
Our work demonstrates a previously undescribed function of ACL as a regulator of cardiac energy substrate metabolism and lipid modulation.
Abstract Mo042: Poison Peptides May Contribute to Dysregulation of SERCA in Heart Failure
Authors: Phillips Taylor , Cunningham Jacob , Seflova Jaroslava , Cho Ellen , Edassery Seby , Bovo Elisa , Zima Aleksey , Kirk Jonathan , Robia Seth
Published: October 2024

Proteolysis increases in heart disease, resulting in an accumulation of degraded membrane protein fragments in heart cells. Previous studies have shown that diverse hydrophobic alpha helices can bind and non-specifically inhibit the cardiac calcium pump SERCA. Thus, we hypothesized that partially degraded fragments of transmembrane proteins may directly inhibit SERCA or compete with native peptide regulators of the pump, like such as phospholamban. The resulting dysregulation of SERCA may contribute to calcium mishandling in heart failure. To test these hypotheses, purified cell membranes from non-failing or dilated cardiomyopathy human hearts were subjected to SDS-PAGE and mass spectrometry to identify protein fragments smaller than 26 kD. 1800 proteins were detected, of which 92 were upregulated >3 fold in the failing heart samples, consistent with increased production of small fragments from these membrane proteins. 9 of the upregulated fragments containing transmembrane segments were selected for further analysis based on sequence hydrophobicity and structural similarity to endogenous micropeptide regulators of SERCA. Of these 9 fragments, 6 showed ER localization, suggesting potential for a toxic interaction with SERCA. The relative binding of these candidates to SERCA was quantified with FRET microscopy. Fragments of three membrane proteins (SEC61B, VAMP3, VAMP8) showed an avid interaction with SERCA. To measure SERCA inhibition, we utilized a calcium activity assay was performed usingin which HEK293T cells were transfected with SERCA, ryanodine receptor, candidate peptides and rCepia as an ER calcium indicator. ER-calcium level was normalized to minimum fluorescence after caffeine addition and an ionomycin induced maximum fluorescence after addition of ionomycin and calcium and caffeine induced minimum. Unregulated Candidate poison peptides were compared to SERCA alone and SERCA-PLB phospholamban controls were also performed. . SEC61B demonstrated the a strongest inhibition, most (similar to phospholamban, ) while VAMP3 and VAMP8’s effect was more modest. This type of non-nativeThus, we propose accumulation of toxic transmembrane peptide fragments may inhibition could be contributinginhibit SERCA and contribute to the calcium mishandling seen in heart failure. Future experiments will be performed to quantify the relative abundance of the fragments compared to endogenous regulatory micropeptides.
Abstract Mo044: Minimally Invasive Delivery of Mesenchymal Stem Cell-derived Exosomes Improves Heart Repair in Pigs
Authors: Li Yuan , Tasoudis Panagiotis , Huang Ke , Cheng Ke
Published: October 2024

Myocardial infarction (MI) is one of the primary manifestations of cardiovascular events. Human mesenchymal stem cell-derived exosomes (hMSC-Exo) were shown to promote cardiac repair after MI. To harness the therapeutic effect, prolong the therapeutic window, and achieve minimally invasive delivery, hMSC-Exo was embedded in hyaluronic acid (HA) hydrogel. The intrapericardial cavity (iPC) is a natural “pocket” surrounding the myocardium. Clinically, iPC procedures were used to treat heart diseases, which could make iPC injection a viable route for drug delivery. In addition, porcine models were highly relevant for pre-clinical studies due to their physiological and anatomical similarities to human patients. Therefore, we hypothesized that minimally invasive delivery of MSC-Exo in HA gel via iPC injection in a porcine MI model could improve pig cardiac function and cardiomyocyte (CM) proliferation. Here, a pig MI model was created using percutaneous coronary balloon catheter occlusion with C-arm fluoroscopy. Yorkshire pigs underwent video-assisted thoracoscopic surgery for iPC injection of MSC-Exo. Cardiac function studies were conducted using echocardiography (echo) to measure left ventricle ejection fraction (LVEF) and left ventricle fractional shortening (LVFS). Histology and immunohistochemistry (IHC) analyses were performed to investigate CM proliferation. Single nucleus RNA-sequencing (snRNA-sq) analysis was performed to identify key gene features and phenotype changes that may be affected by the MSC-Exo treatment. As a result, the iPC injection of MSC-Exo improved cardiac function, enhanced cardiomyocyte proliferation, and induced phenotype alteration in the pig MI model. Echo depicted significantly enhanced LVEF and LVFS on Day 28 in the MSC-Exo group. Histology showed reduced fibrotic percentage and improved infarct wall thickness. IHC analysis showed a significant increase in CM proliferation. SnRNA-seq gene and pathway enrichment elucidated upregulated CM gene features involved in cell proliferation and ECM interaction. In conclusion, these results demonstrated significant improvements in pig cardiac function and CM proliferation. Interestingly, immature cardiomyocytes of a different phenotype emerged in the MSC-Exo group. These findings provide an unprecedented dataset to facilitate the clinical translation of minimally invasive cardiac repair therapeutics.
Abstract Mo046: GJA1-20k Restores Connexin-43 Trafficking and β-Catenin Signaling in Myocytes Lacking Desmoplakin
Authors: Maalouf Mario , Nguyen Vu , HUNTER JENNIFER , Shimura Daisuke , Shaw Robin
Published: October 2024

Background:
Arrhythmogenic Cardiomyopathy (ACM) is an insidious hereditary heart disease that results in structural and electrical cardiac abnormalities leading to arrhythmogenesis and heart failure. ACM is characterized by cardiomyocyte loss, decreased cellular coupling, and fibrofatty replacement. The disease arises from mutations in desmosomal genes, such as plakophilin (PKP), desmoglein (DSG), and desmoplakin (DSP). The pathogenesis of this disease is linked to dysfunction at the level of several cellular processes and pathways, including Wnt/β-catenin signaling. Recent evidence has shown that GJA1-20k, a truncated isoform of Connexin-43 (Cx43) generated by internal translation, has a therapeutic role in preserving Cx43 trafficking and cell-cell coupling in ACM of DSG origin. This study aims to investigate the role of GJA1-20k in protecting against ACM of DSP origin by evaluating its effect in cell models on Cx43 trafficking and Wnt/β-catenin signaling.

Methods:
DSP knockdown was performed on HEK cells and mouse neonatal cardiomyocytes. The role of GJA1-20k was determined using imaging, biochemical, and molecular biology techniques.

Results:
Protein levels of Cx43 and β-catenin were decreased on Western blot (WB) upon DSP knockdown. These findings were corroborated through imaging wherein the signal intensity for Cx43 at cellular junctions and β-catenin at the membrane was significantly decreased compared to the control group. β-catenin signal intensity was also decreased within the nucleus. Upon administration of GJA1-20k, protein levels of Cx43 were normalized on WB. Moreover, the ratio of phosphorylated β-catenin (inactive) to total β-catenin was decreased. Imaging studies identified a significant increase in Cx43 and β-catenin at the membrane in ACM models treated with GJA1-20k. This was also accompanied by an increase in β-catenin signal intensity within the nucleus. A TOP-flash luciferase assay was performed and revealed a significant increase in β-catenin transcriptional activity in the presence of GJA1-20k despite DSP knockdown.

Conclusions:
These results suggest that GJA1-20k is able to rescue Cx43 trafficking and recover Wnt/β-catenin signaling in cells and cardiomyocytes lacking DSP. The ability of GJA1-20k to restore dysfunctional genetic pathways involved in arrhythmogenesis and fibro-adipogenesis make it an attractive potential candidate for targeted therapy against ACM.
Abstract Mo049: Loss of Y chromosome and cardiovascular events in chronic kidney disease
Authors: Zeiher Andreas
Published: October 2024

Background:
Chronic kidney disease represents one of the strongest risk factors for cardiovascular diseases, and particularly for heart failure. Despite improved pharmaceutical treatments mortality remains high. Recently, experimental studies demonstrated that mosaic loss of Y chromosome (LOY) associates with cardiac fibrosis in mice. Since diffuse cardiac fibrosis is the common denominator for progression of all forms of heart failure, we determined the association of LOY on mortality and cardiovascular disease outcomes in patients with chronic kidney disease.

Methods:
LOY was quantified in patients with stable CKD (CARE for HOMe study, derivation cohort, N=279) and dialysis patients (4D study, validation cohort, N=544). The association between LOY and mortality, combined cardiovascular and heart failure-specific endpoints, and echocardiographic measures was assessed.

Results:
In CARE for HOMe, mortality was higher in men as compared to women across the entire range of impaired renal function. The frequency of LOY increased with age. LOY was associated with increased mortality (HR 2.58, 95%CI 1.33-5.03) and risk for cardiac decompensation or death (HR 2.30, 95%CI 1.23-4.27). Patients with LOY >17 % showed a significant decline of ejection fraction and an increase of E/E’ within five years. Consistently, validation in the 4D study revealed that LOY was associated with increased mortality (HR 3.13, 95%CI 1.97-4.99), higher risk of death due to heart failure and sudden cardiac death (HR 4.14, 95%CI 2.00-8.57), but not atherosclerotic events (see figure). Patients with LOY >17 % showed significantly higher plasma levels of soluble ST2, a biomarker for myocardial fibrosis. Mechanistically, intermediate monocytes from patients with LOY >17 % showed significantly higher CCR2 expression and higher plasma levels of the CCR2 ligand CCL2, which may have contributed to increased heart failure events.

Conclusions:
LOY identifies male patients with chronic kidney disease at high risk for mortality and heart failure events.
Abstract Mo051: Phototherapy to stimulate carnosine activity and enhance global heart function: A promising adjunctive therapy for congestive heart failure (CHF)
Authors: Kneller James
Published: October 2024

Background:
The emerging field of cardiac optogenetics uses pulses of light to stimulate optically-sensitive proteins in the heart, influencing cellular physiology with unprecedented spatiotemporal resolution. Effects include increased glutathione and carnosine activity, and promotion of ATP synthesis. Clinical benefits include improved functional capacity, heart rhythm stability, and relief form congestive heart failure (CHF). Transcutaneous phototherapy devices are now available (LifeWave Inc, San Diego, USA), which may provide these benefits without the requirement for an external power source.

Objective:
To investigate whether upregulation of carnosine activity using a wearable cutaneous phototherapy device may improve echocardiographic measures of global heart function, cardiac contractility, and hemodynamics.

Methods:
The Carnosine
®
adhesive skin patch (34 mm outside diameter) contain an organic crystalline material that reflects back specific frequencies of infra-red light known to activate carnosine dipeptides throughout the body. These function as a high-energy phosphate system known to enhance muscle function. Study participants (n=12 healthy volunteers (7 male, 5 female), 40-66 years of age) underwent a focused baseline echocardiogram, which included atrial (A) and early (E) mitral valve inflow, peak tissue velocity (E’) at both the lateral and septal mitral annulus, and left ventricular ejection fraction (LVEF). 10-14 patches were then applied along the sternum and across the left thorax (3
rd
-5
th
interspace), incorporating the anterior axilla. The echocardiogram was repeated 20-30 minutes after patch application.

Results:
LVEF was improved following application of the phototherapy device, increasing from 59.8±6.3% to 66.65±5.8% (Δ 6.3%, p<0.05), indicating an improvement in overall cardiac function. Septal E’ also increased, from 7.3±0.7 cm/s to 8.2±0.6 cm/s (Δ 0.89 cm/s, p<0.05), indicating a direct effect on contractility. E and A wave velocities increased from 63.7 cm/s to 66.1 cm/s (Δ 2.5±0.4 cm/s, p<0.08) and from 56.8 cm/s to 62.6 cm/s (Δ 5.7±0.7 cm/s, p<0.1), indicating augmented hemodynamics trending towards significance.

Conclusion:
Self-powered wearable phototherapy devices may enhance myocardial contractility and overall cardiac function. Such technologies represent a promising therapy for CHF.
Abstract Mo057: Computational Modeling Reveals Enhanced Ca
2+
-Driven Arrhythmias in Female Patients with Atrial Fibrillation
Authors: Zhang Xianwei , Wu Yixuan , Smith Charlotte , Louch William , Morotti Stefano , Dobrev Dobromir , Grandi Eleonora , Ni Haibo
Published: October 2024

Background and Aim:
Substantial sex-based differences have been reported in atrial fibrillation (AF), with female patients experiencing worse symptoms, more complications from treatment, and greater risk of stroke and mortality. Recent studies revealed sex-specific alterations in AF-associated Ca
2+
dysregulation, whereby female myocytes more frequently exhibit proarrhythmic Ca
2+
-driven instabilities compared to male myocytes. Here, we aim to obtain a mechanistic understanding of the Ca
2+
-handling disturbances and Ca
2+
-driven arrhythmogenic events in males vs females and establish their responses to Ca
2+
-targeted interventions.

Methods and Results:
We incorporated known sex differences and AF-associated changes in the expression and phosphorylation of key Ca
2+
-handling proteins and in ultrastructural properties and dimensions of atrial myocytes into our recently developed 3D atrial myocyte model that couples electrophysiology with spatially detailed Ca
2+
-handling processes. Simulations show increased incidence of Ca
2+
sparks in female vs male myocytes in AF, in agreement with previous experimental reports. Additionally, our female model exhibited elevated propensity to developing pacing-induced spontaneous Ca
2+
releases (SCRs,
Figure 1A,B
) and augmented beat-to-beat variability in action potential (AP)-elicited Ca
2+
transients compared with the male model (
Figure 1C
). Parameter sensitivity analysis uncovered precise arrhythmogenic contributions of each component that was implicated in sex and/or AF alterations (
Figure 2
). Specifically, increased ryanodine receptor phosphorylation in female AF myocytes emerged as the major SCR contributor, while reduced L-type Ca
2+
current was protective against SCRs for male AF myocytes. Furthermore, simulations of Ca
2+
-targeted interventions identified potential strategies to attenuate Ca
2+
-driven arrhythmogenic events in female atria (e.g., t-tubule restoration, and inhibition of ryanodine receptor and sarcoplasmic/endoplasmic reticulum Ca
2+
-ATPase), and revealed enhanced efficacy when applied in combination.

Conclusions:
Our sex-specific computational models of human atrial myocytes uncover the increased female propensity to Ca
2+
-driven arrhythmogenic events vs male in AF, and identify combined Ca
2+
-targeted interventions as promising approaches to treat AF in female patients. Our study establishes that AF treatment may benefit from sex-dependent strategies informed by sex-specific mechanisms.
Abstract Mo064: Na
+
leak channel NALCN plays an essential role in mineralocorticoid-induced hypertension through nonrenal mechanisms.
Authors: LEE HYE RYEONG , park solah , SUNG DONG JUN , AN JIN RYEOL , SEO MI SEON , NOH HYUN JU , KIM SUNG HEA , KIM BO KYUNG , PARK SANG WOONG , Bae Young Min
Published: October 2024

In human and animal hyperaldosteronism-related hypertension, the increased blood volume has been regarded as a primary contributor to hypertension generation. However, increases in arterial contractility (and thus peripheral resistance) are also frequently reported in various hypertension models, including the mineralocorticoid-induced hypertensions such as DOCA-salt hypertension. Moreover, non-renal mechanisms (i.e., direct regulation on the cardiovascular ion channels) of the mineralocorticoid-induced hypertension are reportedly important. Na
+
-leak channel (NALCN) was first cloned as Na
+
/Ca
2+
channel family in 1999. However, its role in providing background cation conductance and contributing to normal neuronal excitability has been uncovered relatively recently and little is known about the roles of NALCN in cardiovascular system and pathogenesis of hypertension. Here, we show that aldosterone directly upregulates NALCN expression in arterial smooth muscle independent of kidney and contribute to the increases in arterial contractility, thereby contributing to the development of hypertension. Remarkably, mere the transient knockdown of arterial NALCN greatly ameliorated the development of hypertension along with arterial hypercontractility. Moreover, it is very interesting that our findings suggest that NALCN is activated by oxidative stimuli and this activation can be prevented by -gliflozin series SGLT2 inhibitors. From these, we propose that NALCN is a very promising and potent target for the treatment of various hypertensions including mineralocorticoid-related one.
Abstract Mo084: Stem Cell-Based Functional Genomics Unravel A Novel Vascular Smooth Muscle Cell State Induced By The 9p21 Coronary Artery Disease Risk Locus
Authors: Salido Elsa , de Medeiros Vieira Carolina , Zade Rohan , Lo Sardo Valentina
Published: October 2024

Background:
Genome-wide association studies have identified common genetic variants (Single Nucleotide Polymorphisms – SNPs) at ~300 human genomic loci linked to coronary artery disease (CAD) susceptibility. Among these genomic regions, the most impactful is the 9p21.3 CAD risk locus, which spans a 60 kb gene desert and encompasses ~80 SNPs in high linkage disequilibrium. We have previously generated isogenic induced Pluripotent Stem Cells (iPSCs) lines from risk and non-risk donors at 9p21.3 and performed a complete deletion of the locus. By using iPSC-derived vascular smooth muscle cells (iPSC-VSMCs) we demonstrated that the risk haplotype at 9p21.3 causes altered expression of numerous genes essential for muscle function, including contraction and adhesion. Notably, deletion of the risk haplotype restores the non-risk phenotype, suggesting a gain of function effect. Here, we aimed to identify the transcriptomic signature and state trajectories of VSMCs induced by the 9p21.3 risk haplotype and validate them through functional assays and ex-vivo analysis of human coronary arteries.

Methods:
We have used iPSCs from individuals carrying the risk and non-risk haplotype at 9p21.3 and isogenic knockout lines. After VSMCs differentiation induction, mature VSMCs were used for single cell RNA sequencing using 10X Chromium platform and functional assays.

Results:
Our analysis revealed that the 9p21.3 risk haplotype prompts VSMCs to acquire a novel cellular state showing reduced plasticity and divergent from other VSMC states previously linked to CAD. We found markers of alternative lineages, including osteogenic and neuronal, coupled with altered expression of genes within other CAD loci, and functional defects. We identified a set of new molecular markers crucial to define risk-associated VSMCs and uncovered their upstream regulators.

Conclusions:
Our study provides insights into CAD pathogenesis driven by the 9p21.3 risk locus and identified new gene regulatory networks essential for maintaining the normal functionality of the muscle layer of the arteries. Leveraging the power of iPSCs we present novel concepts about the biology of VSMCs and shed light on the impact of the strongest CAD genetic risk factor in preserving a healthy cellular state within the vasculature.
Abstract Mo088:
RBFOX2
haploinsufficiency impairs cardiomyocyte adhesion and contractility through faulty RNA metabolism
Authors: Huang Mengmeng , Gao Xinlei , Trembley Michael , Prondzynski Maksymilian , Tharani Yashasvi , Nawar Farina , Zheng Rongbin , Aigner Stefan , Yeo Gene , Pu William , Chen Kaifu , Burns C. Geoffrey , BURNS Caroline
Published: October 2024

Background:
Hypoplastic left heart syndrome (HLHS) is a severe form of congenital heart disease that is uniformly lethal if left untreated. Protein damaging mutations in one allele of
RBFOX2,
a highly conserved RNA binding protein, are enriched in HLHS patients, suggesting they are causal. However, it remains unclear how
RBFOX2
haploinsufficiency contributes to disease pathogenesis as heterozygous animal models appear healthy.

Methods:
We generated and characterized
RBFOX2
heterozygous and null human induced pluripotent stem cells (hiPSCs) and assessed their phenotypes in differentiated cardiomyocytes (iPSC-CMs) in 2D culture. To learn if altered function might be revealed in a more native microenvironment, we also generated 3D engineered heart tissues (EHTs) from
RBFOX2
heterozygous hiPSC-CMs. To delineate the underlying molecular mechanisms, we performed bulk RNA-seq to reveal differential gene expression (DGE) and alternative splicing events (ASEs) between the different genetic cohorts and eCLIP-seq to detect RNAs directly bound by RBFOX2 in the CM lineage.

Results:
We observed dose-dependent reductions in cell adhesion, size, maturation, respiration, calcium handling, and action potential duration with no significant effect on proliferation in 2D culture. Although myofibril alignment and contractility were completely abolished in null hiPSC-CMs, these features were surprisingly preserved in heterozygous cells. However, in EHTs, we found that RBFOX2 is haploinsufficient for tissue maturation and contractility, suggesting that myocardial-intrinsic defects contribute to
RBFOX2
-mediated HLHS. Integration of DGE, ASEs and eCLIP-seq datasets uncovered significant enrichment of DGE and/or ASEs of RBFOX2-bound and unbound transcripts involved in cell adhesion between the extracellular matrix (ECM) and the actin cytoskeleton/sarcomere network. Included in the direct targets with altered expression and alternative splicing was the ECM ligand
FIBRONECTIN 1
(
FN1
), which when downregulated was previously associated with HLHS.

Conclusions:
Our data suggest that RBFOX2 sits atop an expression and splicing hierarchy that promotes optimal CM cell growth, adhesion, and contractility that protects the heart from HLHS pathogenesis.
Abstract Mo090: Altered Cardiac Cell Populations in Hypoplastic Left Heart Syndrome
Authors: Morton Sarah , Brown Kemar , Wei Eric , Gorham Joshua , MCDONOUGH BARBARA , Beyer Martin , Neyazi Meraj , Layton Olivia , Seidman Jonathan , Seidman Christine
Published: October 2024

Background:
Congenital heart disease (CHD) affects ~1% of infants. Hypoplastic left heart syndrome (HLHS), a severe form of CHD in which the left ventricle is underdeveloped, is associated by a 15% incidence of heart failure by 6 years of age. Only 6% of HLHS patients have a genetic cause identified on exome sequencing, limiting the ability of patients to receive a diagnosis and potentially benefit from targeted treatments.

Hypothesis:
Novel HLHS genes are differentially expressed in HLHS cardiac tissues, or in cells with differential abundance in HLHS hearts.

Goals:
To use single nucleus RNA sequencing of HLHS patient cardiac tissues to identify candidate HLHS genes.

Methods:
Single nucleus RNA sequencing (nucSeq) was performed on paired left and right ventricular tissues (LV, RV) from 9 participants with HLHS. Left ventricular cardiac tissues from children (4) and adults (12) without CHD were used for comparison. Filtering with CellBender and Solo were used to remove low-quality nuclei. Analysis was performed in R using the
Seurat
package.

Results:
HLHS LV tissues had a higher proportion of cardiomyocytes than pediatric or adult control tissues, (78.7% vs 64.1%, p=0.005; 78.7 % vs 47.0%, p=1.8E-08, respectively), and a lower proportion of mural cells (3.1% vs 9.3%, p=3.9E-05; 3.1 % vs 23.5%, p=7.3E-15, respectively). By contrast, there were similar proportions of endothelial cells in the HLHS, pediatric and adult tissues (7.1%, 8.8% and 7.9%, respectively; p=0.56). Within HLHS tissues, the LV had a higher proportion of cardiomyocytes than the RV (78.7% vs 51.2%, p=4.8E-04) while the proportion of endothelial cells were similar in the LV and RV (7.3% and 9.1%, respectively; p=0.33). LV tissues from HLHS participants 2-18 years of age had fewer capillary endothelial cells than pediatric controls (31.2% vs 57.8%, p=0.001), and gene markers of HLHS endothelial cells included
PBX1
, which is required for Hox D-3 mediated angiogenesis, as well as
ARHGAP26
and
RORA,
inhibitors of VEGF signaling.

Conclusion:
HLHS cardiac tissues have a higher proportion of cardiomyocytes in the LV despite a smaller chamber size. Differences in gene expression that accompany the differences in cell proportion could identify novel HLHS genes as well as potential therapeutic targets.
Abstract Mo102: Swine Model of Heart Failure with Preserved Ejection Fraction Driven by Lipoprotein Lipase Inhibition and Enhanced Cardiac Low-Density Lipoprotein Receptor Expression
Authors: Condor Jose Manuel , Robleto Emely , Bagno Luiza , Ionescu Simona , Clavellina Diana , Rosado Marcos , Rodriguez Jose , Saad Ali , Takeuchi Lauro , Balkan Wayne , WEBSTER KEITH , Hare Joshua , Shehadeh Lina
Published: October 2024

Background:
Heart failure with preserved ejection fraction (HFpEF) poses an escalating public health threat, marked by growing incidence and high mortality. In the cardiometabolic phenogroup of HFpEF that includes diabetes and obesity, intramyocardial lipid content is a prognostic indicator of diastolic dysfunction and adverse outcome. Our group recently reported a mouse model of cardiometabolic HFpEF wherein myocardial lipotoxicity was induced by systemic inhibition of lipoprotein lipase (LPL) with Poloxamer 407 (P407) and cardiac overexpression of the LDL receptor (LDLR), conditions that have been documented in clinical HFpEF. The model demonstrates myocardial lipid accumulation, fibrosis, arrhythmia, and diastolic dysfunction. To reproduce this model in large animals we implemented the same protocol in pigs.

Methods:
Female Yorkshire swine (~25 Kg) were subjected to one of two protocols including: 1) single intracoronary (i.c.) injection of 10
13
AAV9-cTnT-LDLR particles and biweekly intraperitoneal (i.p.) P407 at 1g/Kg, (high dose, n=2), or 2) Double i.c. injections of 10
13
AAV9-cTnT-LDLR particles and biweekly i.p. P407 at 0.25g/Kg) (low dose, n=2). Cardiac structure and function were evaluated by MRI, and hemodynamics measured by PV-Loop at baseline, 4, 8, and 12 weeks. Blood was collected biweekly for complete blood count (CBC), basic biochemistry, and liver and lipid panels. Tissues were collected for protein and histopathological analysis.

Results:
In both strategies, the animals developed high LDL-cholesterol and cardiac hypertrophy with preserved EF. High dose P407 led to higher triglycerides, VLDL, HDL, liver injury (ALT > 200U/L), and steatosis at 4 weeks. One of the 2 pigs died at 3 weeks. Low dose P407 and high dose viral particles induced HFpEF at ~12 weeks with increased LV mass, relative wall thickness, end-diastolic pressure, and tau. Serum LDL-C accumulated from an initial value of 161 to 344.5mg/dL at 12 weeks. No significant liver injury was present until week 12 (ALT<88 U/L, AST<59 U/L). Both strategies did not exhibit abnormalities in CBC or other biochemical parameters.

Conclusions:
Low dose inhibition of LPL combined with cardiac overexpression of LDLR confers HFpEF in swine.
Abstract Mo106: Inhibition of
microRNA-200c
promotes regeneration of the ischemic injured myocardium through activation of developmental pathways
Authors: Leonard Riley , Sweat Mason , Eliason Steve , Zimmerman Kathy , Batz Ariana , Kutschke William , Weiss Robert , Amendt Brad
Published: October 2024

Ischemic injury and adverse post-infarction myocardial remodeling are major causes of heart failure worldwide. Strategies designed to offset cell death with cardiomyocyte regeneration have, to date, not translated to routine clinical practice.
Mir-200c
modulates the transcripts of cardiogenic transcription factors (TFs)
Tbx5
,
Gata4
,
Mef2c
to promote myocyte maturation. We hypothesized that inhibition of
mir-200c
activity would result in postnatal myocyte de-differentiation and replication sufficient to ameliorate post-infarction decompensation after ischemic injury. To test this hypothesis, we preformed left coronary ligation on Wild-Type (WT) and transgenic mice expressing an RNA inhibitor against
miR-200c
(
PMIS-C
). Echocardiographic left ventricular (LV) ejection fraction (EF) fell from 88.41 ± 1.08% (WT) and 90% ± 1.42% (
PMIS-C
) (p=NS) to 28% ± 11.55% (WT) and 36% ± 9.35% at 1 day post injury (DPI) (p =NS). At 21DPI, LVEF was 27% ± 4.31% (WT) and 64% ± 4.25% (
PMIS-C
) [p ≤ 0.0001]. Post-infarction LV chamber dilation was reversed in
PMIS-C
mice (1.136 ± 0.19 v 0.67 ± 0.06 p ≤ 0.004 LV Vol/mass). By 9 WPI,
PMIS-C
heart function within non-significantly levels of sham and function prior to injury. These results indicated that inhibiting
miR-200c
following ischemic injury can recover cardiac function and prevent LV dilation. Moreover, trichrome stain showed a decrease in fibrosis 3 WPI (WT: 57% ± 17.46 v
PMIS-C
: 12% ± 3.01 p ≤ 0.001) and 9 WPI. To understand the mechanism causing this phenomenon, we analyzed expression of cardiac progenitor markers. Fold change in expression of TFs Tbx5, Gata4, Mef2c, and Isl1 were increased at 1 and 3 DPI in
PMIS-C
. At 1 WPI,
PMIS-C
border zone CMs express markers of a “progenitor-like” cell state seen during cardiogenesis. Conclusions: Inhibiting
miR-200c
abrogates post-infarction LV remodeling and significantly restores LV systolic function. Ongoing studies are investigating the therapeutic use of
PMIS-C
through delivery via AAV and nanoparticles.
Abstract Mo112: ARA290, a small peptide non-erythropoietic Erythropoietin derivative, ameliorates heart functional deterioration in a chronic cardiac stress model
Authors: Gime Suri , Winicki Nolan , Ahmet Ismayil , Morrell Christopher , Lakatta Edward
Published: October 2024

Objective:
To determine whether ARA290 can prevent or delay age-related declines in cardiac function and structure in the TG
AC8
heart failure model.

Methods:
The study consists of 9 TG
AC8 +
mice that were randomly assigned to treatment group (n=5) or non-treatment saline group (n=4). Both groups received intraperitoneal injections of ARA290 (100 μg/kg) or saline tri-weekly. Monthly echocardiographic assessment of heart systolic function prior to, and monthly during 7 months of treatment was performed starting at 10 months of age. Two Way ANOVA was used to analyze the data.

Results:
At baseline, 10 months of age, prospective treatment and non-treatment group did not exhibit any statistically significant difference in their measure of ejection fraction (EF), fractional shortening (FS), and stroke volume (SV) (
P
> 0.05).

ARA290 treatment preserved EF, FS, and SV, (compared to a progressive, time dependent deterioration of these systolic functions in the saline treated group). Significant interaction terms for EF (
P
< 0.04), FS (
P
< 0.03), and SV (
P
< 0.02), and time indicated that the ARA290 effect depended upon treatment time.

Conclusion:
The prevention of the decline in heart systolic functional by ARA290 suggests that this small peptide derivative of EPO has therapeutic potential as a novel intervention to retard declines in cardiac function that accompany chronic heart diseases.
Abstract Mo118: Myofilament proteolysis may underlie contractile remodeling in atrial fibrillation
Authors: Cizauskas Hannah , Burnham Hope , Panni Azaria , Pena Alexandra , Alvarez-Arce Alejandro , Davis Therese , Araujo Kelly , Delligatti Christine , Edassery Seby , Kirk Jonathan , Arora Rishi , Barefield David
Published: October 2024

Atrial fibrillation (AFib) is the most common cardiac rhythm disturbance. Treatment of AFib involves restoration of the atrial electrical rhythm. Following rhythm restoration, a period of depressed mechanical function, including decreased blood flow velocity and reduced atrial contractility, known as atrial stunning, occurs. This suggests that defects in contractility occur in AFib and are revealed upon restoration of rhythm. To assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared to sinus rhythm atria. In atrial cardiomyocytes that were useable for skinned single cardiomyocyte force-calcium studies, we found reduced force of contraction. There were no significant differences in myosin heavy chain isoform expression. Resting tension is decreased in the AFib samples correlating with reduced full-length titin in the sarcomere. We measured degradation of other myofilament proteins including cMyBP-C, actinin, MHC, and cTnI, showing significant degradation in the AFib samples compared to sinus rhythm atria. Many of the protein degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. Skinned cardiomyocytes from AFib atria showed increased calcium sensitivity that was consistent with increased cTnI degradation and decreased TnI phosphorylation. These results provide an understanding of the contractile remodeling that occurs in AFib.
Abstract Mo125: Translating ECG responses to drugs across sexes: model development and validation in the clinic
Authors: Shetty Roshni , Morotti Stefano , Sobota Vladimir , Bayer Jason , Ni Haibo , Grandi Eleonora
Published: October 2024

Background:
Sex-based differences in cardiac electrophysiology and their potential to impact susceptibility to cardiac arrhythmia and response to treatment are widely recognized. For example, female sex is an independent risk factor for drug-induced Long QT syndrome, Torsade de Pointes, and sudden cardiac death. However, arrhythmia studies have been predominantly performed in males, thus limiting our understanding of sex-specific arrhythmia mechanisms and the development of sex-specific metrics for arrhythmia risk. Computational tools are a powerful approach for integrating sex differences across scales and studying such multi-level phenomena, accelerating challenging and costly human and animal experiments.

Aim:
To develop a quantitative tool that predicts drug-induced responses in female electrocardiogram (ECG) features based on ECG data collected in males (and vice versa).

Methods:
Male and female 1D cable models of ventricular cardiomyocytes were constructed by incorporating transmural heterogeneity and sex-specific parameterizations. We constructed populations of male and female models by randomly perturbing baseline parameters, and derived pseudo-ECGs from computed extracellular potentials. We then utilized lasso regression to map ECG features (QRS duration, QT interval, T-peak-end duration, and T-wave amplitude) extracted from the male pseudo-ECG onto the female ECG features (and vice versa) (
Fig 1
).

Results:
Our simulated data-driven translator accurately predicted female pseudo-ECG features from male data in an independent simulated test set (R
2
>0.95). This predictive capability was further assessed in an independent dataset simulating the effects of 95 drugs at different concentrations (
Fig 2A
), exhibiting an average translation error <1% for the QT interval (
Fig 2B
). Proof-of-concept application to ECG data sourced from publicly available FDA-approved datasets, in patients of both sexes under various anti-arrhythmic drugs and drug combinations, demonstrates high accuracy (
Fig 3
) and validates the translator's applicability to the clinic.

Conclusions:
Our findings underscore the translator's potential in conducting sex-specific cardiac safety and efficacy assessments, enhancing the precision of evaluations in females. We plan to expand our translator capability to predict sex-specific effects of perturbations, including physiological and pathophysiological stressors.
Abstract Mo127: Nitrate Supplementation Plus Voluntary Activity in C57Bl/6 Mice Improves Fitness and Impairs Cardiac Calcium Handling In a Sex Specific Fashion
Authors: Bisset Elise , Banga Shubham , Howlett Susan
Published: October 2024

Nitrate supplements are commonly combined with aerobic exercise to potentially improve performance and cardiac function. Whether this combination benefits cardiac function at the organ and cellular levels is unclear. We will determine if nitrates affect fitness or cardiac function in both sexes. C57Bl/6 mice (9-mos) were given 1mM sodium nitrate (drinking water) and/or access to a running wheel for 3 mos. Cardiac function (echocardiography), blood pressure (tail cuff) and fitness (treadmill) were measured. Calcium transients (Fura-2) and contractions (2Hz) were measured in ventricular myocytes and calcium handling proteins were measured by qPCR. In both sexes, nitrates did not change blood pressure or voluntary wheel running although females ran more than males (3.7±0.3km vs 2.2±0.3km, p=0.002). In exercised female mice, nitrates increased fitness from baseline while control did not (top speed at exhaustion: 24.2±1.4 to 28.0±1.5 cm/s, p=0.07 vs 23.3±1.0 to 30.7±2.0 cm/s, p=0.002) but nitrate supplemented males showed no fitness benefit. However, while exercise improved cardiac function and contractility compared to sedentary mice, nitrates plus exercise impaired cardiac function in a sex-specific manner. Nitrate-treated males who exercised had lower E/A ratios (1.8±0.3 vs 1.3±0.1, p=0.01) and worse diastolic function than control exercised males. By contrast, nitrate-treated female mice who exercised had smaller global longitudinal strain (-19.5±1.5 vs -11.8±3.0%, p=0.01) than controls, indicative of worse systolic function. Corresponding changes were seen at the cellular level. Exercised males given nitrates had slower a calcium transient decay than controls (44.8±4 vs 73.8±6 ms, p=0.01), while females given nitrates had slower velocity to peak calcium (0.031±0.004 vs 0.018±0.003 µm/ms, p=0.005) and smaller cellular contractions (2.9±0.5 µm vs 1.4±0.5 µm: p=0.03). Cardiac calcium handling proteins were also modified in females. Exercise plus nitrates caused a decrease in SERCA2a and ryanodine receptor 2 expression (mRNA) compared to the control exercise group. Studies in males are ongoing. Exercise plus nitrates increased fitness levels in only females and reduced cardiac function in a sex specific way when compared to exercise alone. This suggests a potential balance between benefits to fitness and potential cardiac complications that could affect athletes taking nitrate supplementation.
Abstract Mo130: Epigenetic Mechanisms Regulate Sex Differences in Cardiac Reparative Functions of Bone Marrow Progenitor Cells
Authors: Gurrala Charan , Roy Rajika , Cheng Zhongjian , Garikipati Venkata , TRUONGCAO MAY , Joladarashi Darukeshwara , Mallaredy Vandana , Cimini Maria , Gonzalez Carolina , Magadum Ajit , Ghosh Jayashri , Benedict Cindy , Koch Walter , Kishore Raj
Published: October 2024

Historically, lower incidence of CVD and related deaths in women as compared with men of the same age has been attributed to female sex hormones, particularly estrogen and its receptors. Autologous Bone marrow stem cell (BMSC) clinical trials for cardiac cell therapy overwhelmingly included male patients; however, meta-analysis data on these trials suggest a better functional outcome in women as compared with men. A direct comparison of the mechanisms governing sex-specific cardiac reparative activity in bone marrow-derived mesenchymal stem cells (BMSCs), with and without the influence of sex hormones, remains unexplored. This study was designed to identify mechanisms that regulate superior reparative properties of female endothelial progenitor cells (EPCs) post-MI, particularly epigenetic mechanisms. Male (M), female (F), and ovariectomized female (OVX) mice-derived EPCs were subjected to a series of molecular and epigenetic analyses followed by
in vivo
functional assessments of cardiac repair. RNA sequencing and other quantitative assays showed a similar genetic profile between F-EPCs and OVX-EPCs, distinct from M-EPCs that displayed significant up-regulation of inflammation-related genes. F-EPCs and OVX EPCs secrete higher levels of proangiogenic factors, lower levels of proinflammatory cytokines and show better cardiac reparative activity after intra-cardiac injections in a male mouse model of myocardial infarction (MI). Epigenetic sequencing revealed a marked difference in the occupancy of the gene repressive H3K9me3 mark, particularly at transcription start sites of key angiogenic and proinflammatory genes in male EPCs compared with female and ovariectomized EPCs. Our study unveiled that functional sex differences in EPCs is, in part, mediated by differential epigenetic regulation of the proinflammatory and anti-angiogenic gene CCL3, orchestrated by the control of H3K9me3 by histone methyltransferase, G9a/Ehmt2. Our research highlights the importance of considering sex of donor cells for progenitor-based tissue repair.