bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract Mo075: Cigarette smoking causes somatic mutations in cardiac endothelial cells
Authors: Zhou Zinan , Huang August Yue , Mao Shulin , Hilal Nazia , Chhouk Brian , Walsh Christopher , Lee Eunjung Alice , Chen Ming-Hui , Choudhury Sangita
Published: October 2024

Cigarette smoking (CS) is a major contributor to cardiovascular diseases (CVDs), responsible for one in every four CVD-related deaths. Despite this, the precise mechanisms through which smoking induces CVDs remain elusive. The chemicals in cigarette smoke, particularly oxidants, are known to instigate oxidative stress and activate the vessel wall. Alongside the dysregulation of cellular functions, oxidative stress can lead to DNA damage. Accumulation of DNA damage and somatic mutations is a recognized hallmark of aging, a significant risk factor for various age-related diseases, including CVDs, neurodegenerative diseases, and cancer. Recent studies have indicated a substantial increase in somatic mutations, with mutational signatures reflective of oxidative stress, in bronchial epithelial cells from smokers. Given their proximity in the blood circulation, these pathophysiological findings could naturally extend to the coronary system. Therefore, we hypothesized that CS induces somatic mutations and injuries in cardiac endothelial cells (ECs), laying the mechanisms for cigarette smoking-induced CVDs. To test this hypothesis, we conducted single-cell whole-genome sequencing (scWGS) on 105 cardiac endothelial cells isolated from postmortem hearts of 6 normal individuals, 3 smokers, and 4 smokers with atherosclerotic cardiovascular disease (ASCVD). Our results revealed a significant increase in somatic single nucleotide variants (SNVs) and insertions and deletions (indels) in cardiac ECs from smokers and those with ASCVD. These excess somatic mutations exhibited Catalogue Of Somatic Mutations In Cancer (COSMIC) mutational signatures, including SBS4, SBS29, SBS40, SBS92, and ID3, indicative of DNA damage and mutagenesis associated with cigarette smoking, oxidative stress, and formaldehyde exposure. Further analysis of single-nucleus RNA sequencing (snRNA-seq) data from postmortem hearts of 3 normal individuals, 4 smokers, and 1 smoker with ASCVD demonstrated upregulated pathways involved in apoptosis, EC migration, and vascular development. In conclusion, our study demonstrates that chemicals in cigarette smoke exert direct genotoxic effects on cardiac ECs, mirroring their impact on bronchial epithelial cells. Cigarette smoking significantly accelerates the accumulation of somatic mutations in cardiac ECs, resulting in apoptosis and vascular damage. Consequently, this damage leads to a secondary activation of angiogenesis for vascular repair.
Abstract Mo078: Identifying Genetic Determinants of Phytosterol Levels: A Genome-Wide Association and Meta-Analysis Study Unveils a New Locus Influencing Campesterol Serum Concentrations
Authors: Alenbawi Jamil , Al-Sarraj Yasser , Ismail Umlai Umm-Kulthum , Kadhi Ayat , Hendi Nagham , Nemer Georges , Albagha Omar
Published: October 2024

Background:
Sitosterolemia, a rare inherited disorder, stems from mutations in the ABCG5/ABCG8 genes, which play a critical role in plant sterol transport. These genetic anomalies result in reduced phytosterol excretion, causing an accumulation in the body that can lead to serious health issues, such as premature coronary artery disease.

Hypothesis:
The study aimed to uncover the genetic determinants influencing plant sterol levels within the Middle East/North Africa (MENA) population, focusing on individuals from Qatar. It hypothesized that unique genetic variations might influence these levels, contributing to the development of sitosterolemia.

Aims:
To conduct the first genome-wide association study (GWAS) in the MENA region to identify genetic variants affecting plant sterol levels in the Qatari population, and to compare these findings with those from populations of European ancestry.

Methods:
The study utilized the Metabolon platform for analyzing serum levels of β-sitosterol and campesterol, combined with genome sequence data from the Qatar Genome Program. A trans-ancestry meta-analysis compared our findings with those from a large European cohort, while conditional analysis helped identify specific single nucleotide polymorphisms (SNPs) associated with these sterols.

Results:
The research identified two independent SNPs related to β-sitosterol ((rs145164937 and rs4299376) and two to campesterol (rs7598542 and rs75901165) in the Qatari population, alongside previously reported variants (
P
<5.0 ×10
-8
) .Notably, one variant (rs75901165) was located in the Intraflagellar Transport 43 (IFT43) gene, diverging from the common ABCG5/8 locus. The study also supported the association of variants in the SCARB1 and ABO genes with sitosterolemia and assessed a polygenic risk score's efficacy based on European GWAS data in the Qatari cohort.

Conclusion(s):
The findings underscore the significance of conducting population-specific genetic research to understand better and manage conditions like sitosterolemia. This research highlights the importance of personalized treatment approaches for hyperlipidemia and the value of establishing comprehensive multi-omics databases tailored to specific populations.
Abstract Mo079: Analyzing the Predicted Impact of Published SCN5A Variants in Juvenile-Onset Sick Sinus Syndrome
Authors: Rajasekaran Snekha , Foksinska Aleksandra , Crowder Camerron , Might Matthew
Published: October 2024

Background:
Sick sinus syndrome (SSS) is a cardiac condition caused by sinoatrial node dysfunction, leading to arrhythmias due to the inability to transmit signals properly to the upper chambers of the heart. While age-related degeneration of the sinoatrial node is the most common contributing factor to SSS, congenital disorders involving genetic variants can also lead to its development, even in pediatric patients. To explore the genetic basis of SSS in the pediatric population (age < 19 years), we conducted a comprehensive review of case reports to identify associated genetic variants. Our focus centered on variants reported in the sodium channel protein type 5 subunit alpha (
SCN5A
) gene, which has been reported to contribute to various ventricular arrhythmias, including SSS.

Methods:
A comprehensive literature review of case reports of juvenile-onset SSS was conducted using PubTator/PubMed and the Online Mendelian Inheritance in Man (OMIM) database to identify
SCN5A
variants. Case reports were selected if they met the following criteria: at least one patient diagnosed with SSS at or under the age of 19 with a detected variant in the
SCN5A
gene. The structural effects of identified variants were assessed using the HOPE web service.

Results:
Data compiled from the literature review provided a total of 53 cases related to juvenile-onset SSS and the
SCN5A
gene. Among the reported patients, many exhibited multiple arrhythmogenic phenotypes alongside SSS, including progressive cardiac conduction disease, idiopathic ventricular tachycardia, and Brugada syndrome. The majority of identified variants were missense, although truncating and insertion/deletion variants were also documented. All three inheritance variations - heterozygous, homozygous, and compound heterozygous - were observed in this set of patients. Although many of the variants had some functional studies performed to determine their pathogenicity, some of the variants did not have a predicted impact. For these variants, we characterized the location and biochemical properties of the variants, comparing them to the variants with known functional impact.

Conclusion:
Future studies will explore the functional impact associated with the predicted variants through modeling specific mutations in
SCN5A
, using cardiomyocytes
in vitro
. Additionally, using further published studies and surveying the protein structure analysis of
SCN5A
variants will aid in characterizing those that are specific to SSS.
Abstract Mo085: Direct effect of sodium-glucose cotransporter 2 (SGLT2) inhibitors on cardiomyocyte function as a potential therapy for Phospholamban cardiomyopathy
Authors: Hnatiuk Anna , Li Alexander , Staudt David , Serrano Ricardo , Mercola Mark
Published: October 2024

Introduction:
Phospholamban (PLN) cardiomyopathy is a dilated and arrhythmogenic cardiomyopathy caused by a single deletion of arginine in PLN gene (PLN-R14del). Clinically, It presents with heart failure, ventricular arrhythmias and sudden cardiac death, and there is still no effective and/or specific treatment besides the heart transplant.

Hypothesis:
Sodium-glucose cotransporter 2 (SGLT2) inhibitors have shown cardiovascular outcomes improvement in general heart failure patients in multiple clinical trials. We hypothesize that SGLT2 inhibitors could ameliorate the contractile dysfunction in PLN cardiomyopathy.

Goals:
Identify whether there is a direct cardiac effect of SGLT2 inhibitors on PLN cardiomyopathy.

Approach:
We used a human iPSC-derived cardiomyocyte (hiPSC-CMs) model of PLN cardiomyopathy that mimics the impaired contractility of the disease to evaluate the contractile function after 4 days of Empagliflozin treatment by using functional cellular imaging assays. Additionally, we evaluated the effect of Empagliflozin treatment on CaMKII phosphorylation and activation of apoptosis mediators by measuring protein expression. All the experiments were compared to results using a CRISPR-engineered isogenic (healthy) line control hiPSC-CMs.

Results:
4 days of Empagliflozin treatment significantly restored contractile function, decreased levels of CaMKII phosphorylation, and decreased apoptotic activity in hiPSC-CMs with PLN cardiomyopathy when compared to their healthy (isogenic) control.

Conclusions:
Our study provided evidence that SGLT2 inhibitors might serve as a therapeutic strategy to ameliorate contractile dysfunction and suppress arrhythmia in PLN cardiomyopathy.
Abstract Mo086: Functional impact of phosphorylation-site mutations on PKA response of Kir2.1
Authors: Munawar Saba , Anderson Corey , Reilly Louise , Melnick Molly , Brown Kyle , Ge Ying , Eckhardt Lee
Published: October 2024

Background:
Kir2.1 channels maintain the cardiac resting membrane potential and assist in phase 3 repolarization. PKA-mediated phosphorylation modulates Kir2.1 function, impacting heart excitability and rhythm. Using as mass-spetometry based integrated top-down and bottom up proteomic approach, we identified five novel and confirmed a 6
th
phosphorylation site. However, which of these residues impact channel function has not been previously identified.

Methods:
HEK293 cells were engineered to stably express either WT-Kir2.1. Ablation of the phosphorylation site was achieved with an alanine substitution of the PKA phosphorylation residue (S13A, S14A, S313A,Y326A, T347A, and S425A). Whole-cell patch clamp experiments of WT-Kir2.1 or the phosphorylation mutants were performed using a standard protocol. Following the initial recording, cells were perfused with PKA-stimulating cocktail (20µM Forskolin and 10µ IBMX) for 4 minutes and the protocol was repeated. Clampfit 10.7, GraphPad Prism, and Origin 20 were used for Data Analysis.

Results:
HEK293 cells stabling expressing Kir2.1 demonstrate typical inward rectification with a maximum outward current at -50mV and responds to PKA-cocktail with 2.0 pA/pF increase in maximum outward current. Both S13A and S313A had a normal response PKA stimulation. In contrast, ablation of S425 and S14 resulted in no PKA response. T347A showed little response to PKA; however, the current density is very low at baseline, indicating that this residue is necessary for normal Kir2.1 current density. The Y326 is known to be critical for normal protein folding, and ablation of the site resulted in a complete loss of function.

Discussion:
Of the 6 PKA sites identified by a proteomic approach, only S425 and S14 functionally influence Kir2.1 response to PKA stimulation. Further molecular dynamics simulation investigation will provide deeper insight into the conformational changes of Kir2.1 in response to phosphorylation at these sites and the relationship of channel phosphorylation with clinical channel mutation residues.
Abstract Mo087: A shared gene regulatory network underlies atrial pathophysiology in atrial fibrillation and heart failure mouse models
Authors: Lazarevic Sonja , Perez-Cerventes Carlos , Wang Zhezhen , Shen Kaitlyn , Gadek Margaret , Chapski Douglas , McKinsey Timothy , Vondriska Thomas , Ruthenburg Alexander , Park David , Moskowitz Ivan
Published: October 2024

Atrial fibrillation and heart failure have a strong epidemiologic link, however the mechanisms underlying their mutual risk remain unresolved. We revealed a remarkable correlation of both gene expression and genomic features in the atria of atrial fibrillation (AF,
Tbx5
deletion) and heart failure (HF, TAC banding) mouse models.
Tbx5
- and TAC-dependent gene expression in the left atria demonstrated a correlation coefficient of 0.8. Remarkably, 118 transcription factors were dysregulated in both models and 109 shared directionality in both cases, suggesting a shared transcriptional response. Differential non-coding transcriptional profiling to identify altered
cis
-regulatory regions demonstrated a correlation coefficient of 0.81 between the models, including ~1,800 shared differentially expressed ncRNAs, of which ~400 emanated from accessible candidate regulatory elements. Integration with chromatin architecture allowed identification of lineage-specific shared AF and HF gene regulatory networks. A TBX5-driven ncRNA-defined regulatory element drives expression of
Klf15,
an inhibitor of cardiac hypertrophy, in the wild-type cardiomyocytes that is downregulated in both mouse and human heart failure. Whereas in mouse HF and AF a disease-specific ncRNA-defined regulatory element is upregulated with
Sox9
, an important transcriptional regulator of fibroblast activation. This work indicates that AF and HF initiate a shared atrial pathological genomic response, including downregulation of a wild-type GRN that normally prevents injury-based disease signaling and upregulation of a disease GRN that promotes atrial remodeling, uncovering a common genomic underpinning of atrial disease.
Abstract Mo089: Fontan-Associated Liver Disease On A Chip
Authors: Rezapourdamanab Sarah , Singh Yamini , Norton Sophia , Hwang Boeun , Jin Linqi , Salar Amoli Mehdi , Karnik Shweta , Avazmohammadi Reza , Neelakantan Sunder , Dasi Lakshmi , Romero Rene , Bauser Heaton Holly , Serpooshan Vahid
Published: October 2024

Introduction:
The Fontan procedure is performed in children with single ventricle physiology, and has led to excellent short-term palliation and survival rates but at the cost of long-term complications. Fontan-associated liver disease (FALD) results from hemodynamic changes following the Fontan procedure, leading to elevated central venous pressures, chronic alterations in hepatic perfusion, and resultant hepatic fibrosis. Improved understanding of FALD pathophysiology, which may provide insights for prevention and treatment is challenged by the lack of experimental models. Current
in vitro
liver models, mostly 2D or suspension hepatic cell cultures, lack biomimicry. This study presents a 3D bioprinted perfusable human liver model for
in vitro
study of hepatic fibrosis in response to altered flow conditions in FALD patients.

Methods:
Vascular 3D model of hepatic sinusoid was biofabricated using hybrid bioinks in two distinct peripheral layers and a central lumen. The outer layer was cast with hepatic (HepG2) cells encapsulated in a mixture of gelatin methacrylate (GelMA) and collagen type I. The inner layer consisted of hepatic stellate cells (HSCs) in GelMA. Human endothelial cells (ECs) were seeded onto the central lumen. 3D cellular constructs were cultured
in vitro
for three weeks in static or dynamic conditions, by circulating the culture media at varying flow conditions to simulate sinusoid pressure characteristic of healthy
vs.
disease state. Using Volcano system in cath lab, we invasively measured the pressure in mid-channel. Cell viability, proliferation, maturation into functional liver tissue, and tissue stiffness were evaluated via microindentation, flow hemodynamics assays, metabolomic assays, and immunohistochemistry analyses.

Results:
Bioengineered liver constructs cultured under static and dynamic conditions demonstrated high cell viability and growth and full endothelialization of the sinusoid channel. HepG2 cells exhibited long-term function and maturation, forming clusters. Under FALD flow conditions, there were notable changes in EC-HepG2 cell morphology compared to dynamic flow in the healthy range, accompanied by reduced levels of functional markers.

Conclusion:
This proof-of-principle study introduces an innovative and highly biomimetic 3D model of hepatic sinusoid, which can serve as a robust platform for
in vitro
investigations of complex cell-microenvironment interactions in the context of FALD as well as other hepatic disorders.
Abstract Mo094: Cardiomyocytes Differentiated from Human Induced Pluripotent Stem Cells Show Robust Phenotypes to Test Gene Therapy Strategies for Duchenne Muscular Dystrophy
Authors: Keegan Abiageal , Gonzalez Adriana Fernanda , Torres-Bigio Sofia , Mamuyac Leahlyn , Doan Hung , Zhang Rongruo , Lau Fiona , Eguchi Asuka
Published: October 2024

Introduction:
Duchenne muscular dystrophy is a severe muscle wasting disease caused by the lack of dystrophin expression. Patients die of heart failure in their second or third decade of life. Delivery of full-length dystrophin is not practical due to the large size of the gene; however, smaller variants of dystrophin, called microdystrophins, have shown promise in restoring function to the skeletal muscle. These microdystrophins are currently being dosed to patients in clinical trials; however, therapeutic efficacy in the heart is unknown due to challenges in modeling DMD-associated heart failure in animal models.

Hypothesis:
We hypothesized that microdystrophin gene therapy can ameliorate disease phenotypes in DMD iPSC-cardiomyocytes.

Methods:
This study uses human induced pluripotent stem cells (iPSCs) to model the functional deficits of cardiomyocytes. By comparing DMD cells with isogenic controls where the dystrophin mutations are corrected, we are able to focus on disease phenotypes attributed to the disease in vitro. We performed viability assays to assess cell survival, ratiometric calcium imaging to evaluate calcium handling, and traction force microscopy to measure the force of contraction.

Results:
We found that iPSC-cardiomyocytes exhibit poor viability, aberrant calcium handling, and reduced force of contraction. When we delivered three microdystrophin variants to DMD iPSC-cardiomyocytes, we found the microdystrophins improved cell viability and partially rescued functional deficits.

Conclusions:
These findings demonstrate that this platform can be used to test the therapeutic efficacy of dystrophin variants in the molecular context of human cells.
Abstract Mo096: High-Throughput Functional Determination of TNNI3 Variant Pathogenicity
Authors: Staudt David , Tran Peter , Han Dongju , Serrano Ricardo , Mercola Mark
Published: October 2024

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
Authors: Hector-Greene Melissa , Kapustin Jacob , Greenberg Lina , Greenberg Michael , Lavine Kory
Published: October 2024

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 Mo100: Predictive Accuracy of Rightward Extrapolation of the Left Ventricular End-Diastolic Pressure-Volume Relationship in Healthy Swine
Authors: Konecny Filip , Mackey Ryan , Hudson Emily , Graser Luke , Weil Brian
Published: October 2024

Objective:
Myocardial stiffness is a primary determinant of left ventricular (LV) diastolic function and can be quantitatively described by the end-diastolic pressure-volume relationship (EDPVR). The EDPVR is traditionally derived by measuring LV end-diastolic pressure (EDP) and volume (EDV) during transient reductions in venous return (preload). However, the accuracy of this approach in predicting changes in LVEDP in response to a rise in LVEDV via rightward extrapolation has not been directly studied. Accordingly, we sought to assess the predictive accuracy of preload reduction-derived EDPVRs in estimating LV diastolic stiffness (ΔLVEDP/ΔLVEDV) in healthy swine.

Methods:
LVEDP and LVEDV were measured in swine (n=6) at rest and during transient inferior vena cava (IVC) occlusion (reduced preload), transient aortic occlusion (increased afterload), and intravenous phenylephrine (PE; 0.5 mg/kg/hr; increased afterload and preload). Pressure-volume data collected during IVC occlusion were used to generate the EDPVR, the accuracy of which was determined via comparison to measurements of ΔLVEDP/ΔLVEDV during aortic occlusion and PE.

Results:
The LV diastolic stiffness coefficient was greater when derived via aortic occlusion vs. IVC occlusion (0.057±0.013 vs. 0.022±0.004; p=0.03). As a result, IVC occlusion-derived EDPVRs underestimated the rise in LVEDP during aortic occlusion (7.6±2.2 vs. 13.5±2.1 mmHg; p=0.08). Similarly, ΔLVEDP/ΔLVEDV in response to aortic occlusion (0.96±0.28 mmHg/mL) tended to be higher than that predicted from the IVC occlusion-derived EDPVR (0.44±0.09 mmHg/mL; p=0.12). Compared with aortic occlusion, PE elicited a greater rise in LVEDV (31.5±5.4 vs.18.6±4.2 mL; p=0.08) and LVEDP (20.8±1.3 vs. 13.5±2.1 mmHg; p=0.01) but ΔLVEDP/ΔLVEDV was comparable between each condition (0.78±0.16 mmHg/mL vs. 0.96±0.28 mmHg/mL; p=0.60). However, ΔLVEDP/ΔLVEDV derived from PE tended to be higher than that from IVC occlusion (0.78±0.16 mmHg/mL vs. 0.44±0.09 mmHg/mL; p=0.10).

Conclusions:
Rightward extrapolation of the EDPVR derived via IVC occlusion underestimates the rise in LVEDP when LVEDV is increased in the normal heart. This was observed with increased afterload as well as when afterload and preload were simultaneously increased via PE. These findings reinforce the importance of assessing the hemodynamic response to increased LV loading directly, rather than extrapolating from EDPVRs created via transient preload reduction.
Abstract Mo103: Aberrant Trans- and De- Nitrosylation Underpins Nitrosative Stress in Cardiometabolic HFpEF
Authors: Li Zhen , Gehred Natalie , Gromova Tatiana , LaPenna Kyle , Sharp Thomas , Chen Jingshu , Shambhu Smitha , YU Xiaoman , Goodchild Traci , Borch Jensen Martin , Vondriska Thomas , Lefer David
Published: October 2024

Background:
Heart failure with preserved ejection fraction (HFpEF) is among the leading causes of cardiovascular mortality and morbidity. Recent reports suggest excessive myocardial protein S-nitrosylation (-SNO), a hallmark of nitrosative stress, contributes to HFpEF pathophysiology. However, the role of transnitrosylases, enzymes that induce protein-SNO, or denitrosylases, enzymes that eliminate protein-SNO, have not been investigated in the context of HFpEF.

Research Questions and Goals:
We sought to investigate the regulation of protein nitrosylation dynamics in a rat model of cardiometabolic HFpEF.

Methods:
Echocardiographic assessment, invasive hemodynamic measurements, and exercise testing were conducted in WKY or ZSF1 obese (Ob) rats to evaluate HFpEF severity. Nitric oxide (NO) bioavailability and protein-SNO levels were determined in the heart tissue. Endothelial-dependent vascular reactivity was assayed to further evaluate NO signaling. In parallel, single-cell (sc) RNA sequencing was performed to determine the transcriptional changes in trans- and denitrosylases, as well as NO synthases.

Results:
We observed progressive deterioration in LV diastolic function (significantly elevated E/e’ and LV end-diastolic pressure) and exercise performance in ZSF1 Ob when compared to WKY controls. Notably, ZSF1 Ob rats exhibited an age-dependent elevation in protein-SNO levels, despite significant reduction in NO bioavailability and signaling. Targeted sc transcriptomic analysis revealed significantly elevated expression of transnitrosylases such as hemoglobin subunit α and β in cardiomyocytes, endothelial cells, and cardiac fibroblasts from ZSF1 Ob hearts. In contrast, significantly reduced expression of denitrosylases such as thioredoxin 2 was found in the ZSF1 Ob cardiomyocytes. No change was observed in expression of NO synthases (nNOS, iNOS, eNOS).

Conclusion:
Herein, we demonstrate a profound disconnect between insufficient NO bioavailability and nitrosative stress in ZSF1 Ob rat model of HFpEF. Our data suggests the pathological accumulation of nitroso - proteins, may be attributed in part to the derangement of transnitrosylases and denitrosylases expression. Restoration of physiological protein nitrosylation dynamics may represent a novel therapeutic approach for HFpEF, and warrants further investigation.
Abstract Mo108: Sex-specific Impact of Glutathione Precursor-supplemented Diet on the Aging Mouse Heart
Authors: Angelini Aude , Garcia Marquez Grecia , Malovannaya Anna , Fiorotto Marta , Salzman Alexander , Jain Antrix , Trial JoAnn , Taffet George , Cieslik Katarzyna
Published: October 2024

Common features of the aging heart include dysregulated metabolism, inflammation, and fibrosis, which can all contribute to diastolic dysfunction. Imbalanced oxidative stress can exacerbate each of these conditions, worsening the age-related cardiac defects.

Therefore, we hypothesized that increasing natural antioxidant defenses (glutathione) may favor a healthier cardiac aging phenotype. Twenty-one month-old mice were fed for 12 weeks with a diet supplemented in two glutathione precursors, Glycine and N-Acetyl Cysteine (GlyNAC) or with a control diet. Heart function was measured noninvasively and longitudinally at baseline and then every 6 weeks.

We found that GlyNAC reduced the age-associated increase in left atrial volume in old male, but not in old female mice ( 12% ± 3% in GlyNAC vs 20% ± 2% in control males (*); 11% ± 3% in GlyNAC vs 16% ± 3% in control females; * P=0.03). It also improved exercise performance in males. Subsequent unbiased cardiac proteome analysis identified that GlyNAC modestly changed global protein expression in males and pointed to the significant changes in two pathways: mitochondria-metabolism and extracellular matrix. Further analysis performed on mitochondrial extracts determined that GlyNAC diet led to an increase in Ndufb8, a subunit of the mitochondrial respiratory chain complex (1.76±0.3 in GlyNAC males vs 0.7±0.1 in control males, P=0.02). In the GlyNAC old male mitochondria, we found an improved catalytic activity for CPT1b (7.51±0.79 vs 3.74±0.32 in control males; P=0.002) and an increased CrAT activity (1.99±0.23 vs 1.06±0.08 in control males; P=0.005). Both enzymes are involved in fatty acid metabolism. Hearts from GlyNAC-fed old males exhibited enrichment in Fmod, a protein that can inhibit collagen fibril formation (1.08±0.03 vs 0.89±0.05, P=0.01), possibly reducing extracellular matrix stiffness. In females, the expression or enzymatic activities of these proteins were not affected by the GlyNAC-supplemented diet.

In summary, our study supports the concept that aged male and female hearts exhibit basic phenotypical differences that may affect the response to interventions. These observations may have direct relevance in cardiac gerontology.
Abstract Mo114: Assessing Diastolic Performance in Working Myocardial Slices from ZSF1 Rats
Authors: Hancock Emmaleigh , Price Alexa , Kabir Raihan , Weingart Shaina , Palmer Brad , Caporizzo Matthew
Published: October 2024

Diastolic dysfunction is a prevalent and therapeutically intractable feature of heart failure which arises from increased ventricular compliance and impaired relaxation. Properly assessing diastolic performance is challenging because it requires simultaneous in-vivo measurement of ventricular pressure and volume. Furthermore, identifying diastolic dysfunction may require the simultaneous application of a stress such as physical exertion. Herein, we demonstrate a preparation that recapitulates the complexity of diastole in working myocardial slices: a force-feedback system providing precision control of pacing frequency, preload, afterload, and a tunable stretch at the end of diastole that mimics late filling driven by atrial systole. We assess the diastolic performance (isovolumic relaxation, early and late filling characteristics) in working myocardial slices from a known model of diastolic dysfunction, ZSF1 Obese rats and their Lean control. We find that parameters relating to myocardial performance such as slice work, stroke length, and twitch force are the same between Lean and Obese animals but parameters of diastolic performance are altered in the Obese rats. Specifically, we observe a decreased separation between peak early and late filling with a reduced diastolic filling interval at the same pacing frequency while we do not observe a difference in isovolumic relaxation time. Taken together, these results indicate that while the work output of myocardium is preserved in ZSF1 Obese rats, a reduction in available filling time and early filling efficiency may indicate a reduced capacity maintain output at higher heart rates.
Abstract Mo116: Evaluating
MYBPC3
and
MYBPHL
missense mutations on sarcomere incorporation
Authors: Araujo Kelly , Pena Alexandra , Alvarez-Arce Alejandro , Wittenkeller Lucas , Barefield David
Published: October 2024

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 Mo117: Investigation of Mechanisms to Modulate Contractility in 13-Lined Ground Squirrels
Authors: Patterson Maighdlin , Chiang Po-Jen , Carey Hannah , Ball Karen , Chung Charles
Published: October 2024

Introduction:
During hibernation, 13-lined ground squirrels cycle between torpor and intermittent bouts of arousal (IBA). Bouts of IBA last 12-48 hours, during which heart rates rise from 2-4 bpm in torpor to 300 bpm during IBA before returning to torpor. Thus, rates of contractility and relaxation and their mechanisms must rapidly change. Phosphorylation of cardiac troponin I (cTnI) at serine 23/24 (pSer23/24) has been shown to decrease calcium sensitivity and increase the rate of myocardial relaxation, enabling faster heart rates. Heat shock protein 27 (HSP27) has been shown to protect contractility by stabilizing troponin.

Hypothesis:
We hypothesized that cTnI Ser23/24 phosphorylation would be higher in summer and IBA and that HSP27 would be found in higher quantities during torpor and IBA.

Methods and Results:
Left ventricular tissue (summer, n=8; torpor, n=8; IBA, n=8) was solubilized, separated by SDS-PAGE, and blotted with antibodies for quantification. The pSer23/24 to total troponin ratios were higher in summer compared to torpor (1.22±0.75 vs 0.32±0.19, p=0.003) and IBA (0.57±0.19, p=0.03), but not different between IBA and torpor. No significant season-dependence was observed for the serine 43 cTnI phosphorylation to total troponin ratio (torpor 1.45±0.69, summer 1.05±0.34, IBA 1.15±0.84). No significant season-dependence was observed in HSP27 (normalized to GAPDH) (summer 0.09±0.02, IBA 0.14±0.10, torpor 0.16±0.08). Nor was there a significant season-dependence observed in HSP90 (normalized to GAPDH) (summer 1.57±0.66, IBA 1.13±0.19, torpor 1.22±0.21).

Conclusions:
None of these mechanisms fully explain the rapid change in contractility observed during the hibernating periods of 13-lined ground squirrels. Other post-translational modifications of troponin or modifiers of HSP activity may modify the reported results. Such regulation may identify new treatments for humans where rapid changes in heart rate or contractility occur.
Abstract Mo119: PI3Kgamma regulates CamKII mediated phosphorylation of phospholamban and SR calcium cycling
Authors: Mohan Maradumane , Witherow Conner , Papay Robert , Sun Yu , Stenson Kate , Ashok Anushruti , Naga Prasad Sathyamangla
Published: October 2024

Although phosphoinositide 3-kinase (PI3Kγ) null mice (PI3Kγ
-/-
) show increased ventricular rate/contraction, it is unknown whether PI3Kγ regulates calcium recycling machinery underlying this phenotype. Primary adult cardiomyocytes from PI3Kγ
-/-
mice show altered sarcoendoplasmic reticulum (SR) calcium cycling following caffeine. Unexpectedly, PI3Kγ
-/-
cardiomyocytes showed significant reduction in phosphorylation of phospholamban (PLN) at Thr17, a key regulator of SR calcium re-uptake without changes in phosphorylation at Ser16. Furthermore, loss in PLN phosphorylation in PI3Kγ
-/-
cardiomyocytes was associated with augmented interaction with SR calcium ATPase (SERCA). Surprisingly, cardiomyocyte-specific overexpression of kinase-dead PI3Kγ (PI3Kγ
inact
) in global PI3Kγ
-/-
mice (PI3Kγ
inact
/PI3Kγ
-/-
) normalized caffeine-induced calcium re-uptake, PLN phosphorylation at Thr17 and decreased PLN-SERCA interaction. These data suggested kinase-independent function of PI3Kγ in regulation of SR calcium load and PLN phosphorylation. Since phosphorylation of Thr17 of PLN is carried out by Ca
2+
/Calmodulin dependent protein kinase (CamKII), we probed for the role of PI3Kγ in regulation of CamKII in PLN phosphorylation. Mechanistically, PI3Kγ exhibits scaffolding function in recruitment of CamKII to PLN at SR to mediate PLN phosphorylation. Furthermore, we showed that PI3Kγ directly interacts with CamKII. The study unravels a yet to be recognized kinase-independent role of PI3Kγ in regulating PLN with implications in cardiac function.
Abstract Mo122: Investigating the Role of the Lysine Methyltransferase SMYD1 in Striated Muscle Motor Domain Folding
Authors: Hunt Dakota , Martin Thomas , Leinwand Leslie
Published: October 2024

Properly coordinated folding and assembly of myosin into the sarcomere thick filament is critical for cardiac and skeletal muscle development, function, growth, and maintenance. The motor domain of myosin heavy chain (MHC) requires muscle-specific chaperones for proper folding, making it currently impossible to fold in non-muscle cells. Previous studies found that HSP90 and UNC45B are necessary, but not sufficient, for folding the motor domain, indicating other factors are involved. Defining the minimal set of factors for motor domain folding would allow for development of an improved expression system and accelerate the study of myosin mutations and myosin-targeted drug development. We hypothesize that the muscle-specific lysine methyltransferase SMYD1 contributes to MHC folding and stability. We found SMYD1 associates with UNC45B, HSP90, and MHC in the adult human heart. To study motor domain folding we generated a human MYH7 motor domain fused to GFP (MD::GFP). Transfection into COS-7 cells resulted in unfolded aggregates while adenoviral infection of C
2
C
12
myotubes leads to folded and functional MD::GFP. Interestingly, the folded MD::GFP contains lysine trimethylation, which may arise from the methyltransferase activity of SMYD1. When COS-7 cells were co-transfected with UNC45B and SMYD1, we observed increased total protein levels of non-functional MD::GFP without affecting RNA levels as assessed by RT-qPCR. This effect was not observed when MD::GFP was co-transfected with UNC45B or SMYD1 alone and is dependent on HSP90. We found that UNC45B and SMYD1 stabilized MD::GFP as determined by cycloheximide chase assays. We also found SMYD1 associates with MD::GFP, suggesting this stabilizing effect is due to a direct interaction. Interestingly, a catalytically inactive SMYD1 also increased MD::GFP protein levels in COS-7 cells, consistent with our observation that no significant changes in global protein methylation were observed in the presence of WT SMYD1. Our data suggests that SMYD1 stabilizes the motor domain of striated muscle myosin in conjunction with UNC45B and HSP90, in a manner that is independent of its methyltransferase activity. However, UNC45B and SMYD1 are insufficient for motor domain folding in a non-muscle cell line, suggesting that additional folding factors or proper stoichiometric ratios of chaperones/co-chaperones may be required.
Abstract Mo123: Post-transcriptional Mechanisms of BAG3 Regulation in Ischemia-Reperfusion Injury
Authors: Sherer Laura , Zied Ahmed , Martin Thomas , Door Michaela , Delligatti Christine , Kirk Jonathan
Published: October 2024

The co-chaperone BAG3 is critical for protein quality control at the cardiac sarcomere. BAG3 binds to Hsp70 and coordinates the assembly of the CASA (chaperone-assisted selective autophagy) complex, thus supporting protein homeostasis and cardiomyocyte contractility. Decreased BAG3 levels are associated with heart disease, whereas BAG3 overexpression rescues ventricular function in animal models of heart failure (HF). Despite BAG3’s potential as a therapeutic target, the mechanisms underlying BAG3 regulation are largely unresolved. Here, we investigate the mechanisms of BAG3 downregulation after stress. We found that BAG3 protein is reduced in human cardiomyopathies compared to non-failing hearts, yet there is an increase in
bag3
mRNA transcript, suggesting BAG3’s downregulation in heart disease may be controlled post-transcriptionally. To identify these post-transcriptional pathways, we subjected neonatal rat ventricular myocytes (NRVMs) to prolonged hypoxia-reoxygenation (H/R) stress, which recapitulated the decrease in BAG3 levels observed in human heart disease. Notably, disrupting Hsp70 binding to BAG3 in NRVMs via the drug JG-98 decreased BAG3’s half-life by ~90%, suggesting that Hsp70 protects BAG3 from degradation. Loss of Hsp70-mediated protection could contribute to declining BAG3 levels, so we quantified Hsp70 abundance after H/R stress in NRVMs, finding no significant change. We also found that overexpressing inducible Hsp70 did not rescue BAG3 levels. This data suggests that BAG3 downregulation in H/R stress is not caused by loss of Hsp70 binding/protection. To examine BAG3 regulation
in vivo,
we subjected wildtype mice to ischemia-reperfusion injury. After 24 hours, we observed a decrease in BAG3 levels in the left ventricle and no significant change in Hsp70 abundance. Interestingly, the decline in full-length BAG3 (85 kDa) was accompanied by an increase in a BAG3 cleavage product at 74 kDa. We analyzed this product via mass spectrometry, discovering that it lacks a third of the WW domain, which is involved in autophagy. In future experiments, BAG3 cleavage will be explored as a potential mechanism of BAG3 loss. Such mechanisms will provide insight into how to maintain BAG3 levels, and thus cardiac function, during stress.
Abstract Mo134: Restoring NaV1.5 Mutant Functionality in Arrhythmia with ManNAc Supplementation
Authors: Tarantino Adriana , Melgari Dario , Lavota Ivana , Rota Paola , Frosio Anthony , Rivolta Ilaria , Pappone Carlo , Anastasia Luigi
Published: October 2024

The SCN5A gene encodes the human cardiac sodium channel NaV1.5, essential for cardiac excitability and action potential propagation. Mutations in SCN5A are linked to various cardiac disorders, including Brugada Syndrome (BrS), typically leading to channel loss-of-function. Understanding alterations in NaV1.5 expression and sodium current density is vital for comprehending arrhythmogenesis and formulating therapeutic strategies.

Post-translational modifications, especially sialylation, significantly impact channel functionality. We previously demonstrated that BrS patients show decreased protein sialylation, correlating with reduced sodium channel activity. This study aimed to investigate sialic acid's role in regulating NaV1.5 activity, testing if sialylation induction via mannosamine (ManNAc) supplementation could restore channel activity in-vitro. Preliminary tests with exogenous sialidases and ManNAc supplementation affirmed our hypothesis.

We examined ManNAc's effect on NaV1.5 mutants common in BrS, which typically exhibit partial functional loss. We overexpressed mutations p.A344S, p.K1479A, and p.G1406R in HEK293A cells and measured sodium currents before and after ManNAc supplementation to stimulate sialic acid synthesis. ManNAc effectively restored sodium currents in mutants with intracellular and extracellular loop mutations. However, the p.1406G>A mutant, affecting the pore structure, showed no improvement, suggesting that sialic acid mainly influences channel membrane trafficking rather than direct glycosylation. This underscores the complexity of sialic acid's role and points to trafficking pathways as potential therapeutic targets. Notably, the treatment neither increased nor decreased sodium currents in wild-type NaV1.5 channels or in the CaV3.2 calcium channel, underscoring its specificity.

In summary, our findings demonstrate that ManNAc supplementation can selectively restore the function of certain NaV1.5 mutants without affecting wild-type channels, highlighting the potential of targeting sialylation pathways for SCN5A-related arrhythmia treatments. Future research should aim to unravel these mechanisms further, providing insights into novel treatments for SCN5A-related arrhythmias.