bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract Mo093: Rescue of Desmin Insufficiency Restores Contractile Function in Cardiomyocytes with
MYH7
E848G Dilated Cardiomyopathy Variant
Authors: Loiben Alexander , Chien Wei-Ming , Mckinstry Ashley , Ahmed Dania , Yang Kai-chun
Published: October 2024

Dilated cardiomyopathy (DCM), characterized by systolic dysfunction, is a significant cause of heart failure. Myosin heavy chain 7 (
MYH7
) pathogenic variants are common causes of DCM; however, no specific disease-modifying therapy for
MYH7
DCM exists. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing the pathogenic
MYH7
E848G DCM variant, we confirmed hypocontractility at the single cell level as assessed with traction force microscopy. hiPSC-CMs were cultured on polyacrylamide hydrogels with physiological stiffness in 15x100 µm patterned Matrigel-coated rectangles, each containing one cell. Upon 1 Hz electrical stimulation, contracting cells displaced fluorescent beads embedded within the gel, which were measured to estimate twitch force.
MYH7
E848G/+

hiPSC-CMs had reduced maximum twitch force (97 ± 11 nN, n=46 cells) compared to
MYH7
+/+

hiPSC-CMs (179 ± 18 nN, n=49 cells), recapitulating the disease phenotype. RNAseq analysis of
MYH7
E848G/+
hiPSC-CMs compared to isogenic control
MYH7
+/+
hiPSC-CMs found significant downregulation of desmin (
DES)
(76.4 ± 8.2% reduction, n=4 bio reps), which encodes a muscle-specific type III intermediate filament. Desmin protein expression in
MYH7
E848G/+
hiPSC-CMs was significantly attenuated relative to
MYH7
+/+
hiPSC-CMs (90.9 ± 1.8% reduction, n=4 bio reps), matching gene expression data. Gene expression of the transcription factor
MEF2C
, a known transactivator of DES, was also reduced (40.4 ± 7.0% reduction, n=4 bio reps), suggesting
MYH7
E848G/+
suppresses the
MEF2C
-
DES
signaling axis. We hypothesized restoring desmin protein levels may improve contractility in
MYH7
E848G/+
hiPSC-CMs. To test this, we created an adeno-associated virus (AAV9) packaged with full-length desmin cDNA fused with a T2A self-cleaving peptide and mApple fluorescent reporter. hiPSC-CMs were transfected with this mApple-T2A-DES AAV at 1 MOI and contractility was assessed via traction force microscopy. mApple
+
MYH7
E848G/+
hiPSC-CMs had increased maximum twitch force (141 ± 11 nN, n=75 cells) relative to mApple
-
MYH7
E848G/+
hiPSC-CMs (65 ± 20 nN, n=21 cells), approaching the maximum twitch force of mApple
+
MYH7
+/+
hiPSC-CMs (165 ± 10 nN, n=87 cells). In conclusion, suppression of the
MEF2C
-
DES
signaling axis is a potential novel mechanism by which pathogenic
MYH7
variants cause DCM. By leveraging this mechanistic insight, we found that rescuing desmin insufficiency can partially restore contractile function.
Abstract Mo095: Variant Effect Mapping by Combining Base-editing Mutagenesis and Multiplexed Functional Assays Using Human Cardiomyocytes
Authors: Yamamoto Yuta , Parikh Victoria , Ashley Euan
Published: October 2024

Introduction:
MYBPC3
is the most prevalent gene in hypertrophic cardiomyopathy (HCM). However, the pathogenicity of most
MYBPC3
variants remains unknown, and it is still challenging to interpret variants identified in patients. Although multiplexed functional assays using cancer cell lines are powerful approaches to test large number of variants in a single experiment and facilitate the interpretation of variants, there is a limitation of the assessment of genes expressing in specific cell types, such as sarcomeric genes including
MYBPC3.

Hypothesis:
The approach integrating base-editing mutagenesis and multiplexed functional assays using iPSC-derived cardiomyocytes (iPSC-CMs) will enable us to prospectively evaluate
MYBPC3
variants in a high-throughput manner and illuminate the pathophysiological mechanism of HCM.

Methods:
We focused on the intron-exon boundary including intron 11 and exon 12 in
MYBPC3
and introduced variants into iPSCs by CRISPR-X, which can target multiple genomic locations and introduce diverse editing, and adenine base editor. After cardiac differentiation, we performed multiplexed functional assays. iPSC-CMs were immunostained for cMyBP-C and BNP expressions and sorted based on those expressions using fluorescence-activated cell sorting. The ubiquitin-proteasome activity associated with cellular proteostasis was analyzed by ubiquitin fusion degradation reporter system. The variant scores were calculated by comparing the frequency of each variant in each population sorted based on phenotype using next-generation sequencing.

Results:
About 200 variants located at the mutagenesis target region were evaluated via in situ mutagenesis and each functional assay. The pathogenic/likely pathogenic variants from ClinVar exhibited higher variant scores compared to benign/likely benign variants in each assay. Even in variants that have not yet been reported clinically, nonsense variants and several missense and intronic variants including splice site variants showed higher variant scores compared to synonymous variants.

Conclusion:
This study demonstrates that our novel approach can prospectively evaluate
MYBPC3
variant effect at scale and provide clues to elucidate pathophysiological mechanism of HCM.
Abstract Mo104: Cardiolipin-Metabolite Crosstalk in HFpEF
Authors: Kamiar Ali , Williams Monique , Condor Jose Manuel , Robleto Emely , Kovilakath Anna , Kanashiro-Takeuchi Rosemeire , Takeuchi Lauro , Cardoso Alisson , Alborghetti Marcos , de Carvalho Pereira Jose Geraldo , Shinzato Mariane , Franchini Kleber , Webster Keith , Lopaschuk Gary , Hare Joshua , Cowart Lauren , Shehadeh Lina
Published: October 2024

Background:
Transition from fatty acid oxidation to glycolysis is linked to oncometabolism and cardiac hypertrophy. We hypothesize that such a metabolic shift is pivotal in driving HFpEF in 3 murine models. The citric acid cycle (CAC) enzyme αKGDH is dysregulated in HFpEF patient biopsies and Alport hearts. Therefore, we also hypothesize more generalized CAC dysregulation at αKGDH associated with HFpEF.

Methods & Results:
Alport and LDLR/P407 mice were generated on 129J background, and HFD/L-NAME HFpEF on C57Bl6N, (n= 5M,5F/group). Two groups of Alport and LDLR/P407 mice were fed a 2% αKG diet, and a 3rd subgroup of Alport mice injected with AAV9-CMV-αKGDH. PET-CT of
18
F-FDG showed significant increased cardiac glucose uptake in Alport and LDLR/P407 mice up to 8 wk of treatment. αKG diet conferred significantly increased cardiac glucose uptake and worse diastolic dysfunction in LDLR/P407 mice. αKGDH protein expression was reduced by ~40% (p<.05) in the HFD/L-NAME hearts. Targeted metabolomic analysis by LC-MS/MS indicated normalization of intermediate metabolites in LDLR/P407 hearts by αKG diet and significantly shortening life spans (p<.001). The αKG diet exacerbated adverse remodeling of cardiolipin (CL) in LDLR/P407 hearts. Expression of CL remodeling enzymes, CL Synthase (p<.05) and Tafazzin (p<.0001), were altered in the LDLR/P407 hearts. NADH increased in Alport and LDLR/P407 hearts suggesting decreased ATP availability and impaired oxidative phosphorylation. HFD/L-NAME HFpEF mice showed a possible block in CAC flux at αKGDH, with increased αKG and reduced succinyl-CoA. Acetyl-CoA and αKG were increased in Alport hearts. Serum metabolic signatures in 273 patients by
1
H NMR stratified into non-HF, pre-HFpEF, and HFpEF by the H2FPEF algorithm, indicated elevated serum αKG levels associated with patients with higher H2FPEF scores. Leucine is increased in LDLR/P407 and HFD/L-NAME hearts consistent with published findings in patients with HFpEF. These 2 models also have increased levels of purines. Increased G-6P in LDLR/P407 hearts supports increased glucose uptake. Glutamine is increased in HFD/L-NAME, consistent with published findings in patients with HFpEF.

Conclusions:
Dysregulated αKG and CL remodeling modulate oncometabolism and energy transduction that exacerbate HF in Alport, LDLR/P407, and HFD/L-NAME mice. Circulating αKG may constitute a marker of CAC dysfunction in HFpEF patients and αKGHD activation as a potential therapeutic goal.
Abstract Mo111: Comparison of Pre-Clinical Models of Heart Failure with Preserved Ejection Fraction Using Multimodal Approaches
Authors: Wassenaar Jean , Sangowdar Pranoy , Smart Charles , Fehrenbach Daniel , Zhong Lin , Colvin Daniel , Miller Francis , Dendy Jeffrey , Doran Amanda , Madhur Meena
Published: October 2024

There are various pre-clinical models of heart failure with preserved ejection fraction (HFpEF) designed to model different clinical phenotypes of HFpEF. However, detailed assessment and head to head comparison of diastolic function by invasive and non-invasive techniques in these models is lacking. Our goal was to comprehensively evaluate cardiac structure and function in three HFpEF mouse models – deoxycortisterone acetate (DOCA)-salt, Nω-nitro-l-arginine methyl ester high fat diet (LNAME+HFD), and aged female mice fed HFD with angiotensin infusion (Ang+HFD) – using both non-invasive and invasive methods. We employed pulse wave and tissue Doppler echocardiography, cardiac magnetic resonance imaging (CMR), and left ventricular (LV) catheterization in the commonly used C57BL/6J mouse, enabling direct comparisons between models. Cardiac hypertrophy was assessed by heart weight (HW) to tibia length (TL) ratio. The Mann-Whitney test was used for statistical testing within each model. We found that on echocardiography, all models demonstrated diastolic dysfunction with significant differences (p < 0.05) or trends for significance (p < 0.1) on tissue doppler peak mitral annular velocity during early filling (e’), ratio of peak early mitral flow velocity (E) to e’ (E/e’), ratio of peak early mitral flow velocity (E) to e’ (E/e’), isovolumetric relaxation time (IVRT), and E pressure half time (E PHT). CMR was notable for findings of reduced right ventricular ejection function in the LNAME+HFD group (36.1±2.7% vs 49.8±1.4% in chow controls, p<0.01). On invasive hemodynamics, only DOCA-salt had a higher LV diastolic time constant (t, 11.7±1.4 vs 6.9±0.8 ms in sham controls, p=0.02) and only Ang+HFD had a higher minimum rate of pressure change in the ventricle (dP/dt
min
, -3506±342 vs -5268±227 in chow fed, vehicle infusion controls, p<0.01). No model showed a difference in LV end diastolic pressure. For cardiac hypertrophy, we found that both DOCA-salt (8.1±0.2 vs 10.3±0.5, p<0.01) and Ang+HFD (9.2±0.3 vs 11.5±0.4, p<0.01) models demonstrated increased HW to TL ratio, while LNAME+HFD did not. These results highlight differences in cardiac structure and function in HFpEF mouse models and the need for multi-modal assessment in pre-clinical studies.
Abstract Mo113: An Ovary-Intact Postmenopausal HFpEF Animal Model.
Authors: Strom Joshua , Farman Gerrie , Gohlke Jochen , Van Der Pijl Robbert , Granzier Hendrikus (Henk) , Methawasin Mei
Published: October 2024

Background:
The incidence of HFpEF in women significantly escalates following the age of menopause. This trend cannot solely be attributed to chronological aging, as evidenced by the more gradual increase in prevalence among men with HFpEF. This pattern suggests that menopause is a provocative event for HFpEF. However, the underlying mechanisms remain elusive and challenging to investigate in human subjects; moreover, the attempt to create HFpEF in ovariectomized (OVX) mice was unsuccessful. We aim to create an animal model of postmenopausal HFpEF that resembles the characteristics of HFpEF in women with natural menopause.

Methods:
We use the 4-vinyl cyclohexene dioxide (VCD) to induce “ovary-intact” menopause, combined with the 2hit regimen (HFpEF inducing regimen elicited by high-fat diet and nitric oxide synthase inhibitor) to model postmenopausal HFpEF. VCD gradually causes ovarian failure, providing the perimenopausal transition and preserving residual ovarian stroma, mimicking natural menopause. The HFpEF phenotypes were studied at the in vivo LV by pressure-volume (PV) analysis and at the single cell level by intact cell force measurement.

Results:
The Female-VCD-2hit model demonstrates diastolic dysfunction at both the LV and the cellular levels. At the LV level, the increase in the end-diastolic pressure-volume relation (EDPVR) stiffness coefficient and LV end-diastolic pressure (LVEDP) indicates a heightened stiffness of the LV chamber and an elevation in LV filling pressure, an HFpEF-like condition. At the cardiomyocyte level, the female-VCD-2hit model exhibits an increase in cellular diastolic stiffness, suggesting that the observed LV diastolic stiffness is derived from the stiffness of the cardiomyocytes. We compared the female-VCD-2hit mice with the OVX mice subjected to the 2hit regimen. The 2hit-OVX model showed a milder form of diastolic dysfunction, observed as a smaller increase in LV diastolic stiffness (EDPVR) and normal LV filling pressure (LVEDP).

Conclusions:
Female mice, either premenopausal or ovariectomized, are more resistant to the development of HFpEF, unlike their ovary-intact menopausal (VCD-treated) counterparts, which exhibit a vulnerability to HFpEF, making the VCD-2hit model a suitable model to study postmenopausal HFpEF. The LV diastolic dysfunction observed in the 2hit model is attributed to mechanical dysfunction at the cardiomyocyte level.
Abstract Mo121: Sarcomere activation biosensor reveals key functional differences in live cell active states between cardiac and skeletal muscle
Authors: Martin Ashley , Metzger Joseph
Published: October 2024

The sarcomere is the functional unit of striated muscle contraction. Previous studies have provided valuable information about the sarcomeric proteins and modulators that are necessary for activation of contraction through inter-myofilament signaling. This work has revealed key differences in contraction kinetics and paradigms between cardiac, slow skeletal, and fast skeletal muscle. We seek here to expand these studies through the use of myofilament incorporated Förster Resonance Energy Transfer (FRET) fluorescent biosensor in live, intact muscle. This biosensor fuses fluorescent donor and acceptor proteins to the N- and C-terminals of fast skeletal and cardiac (slow) troponin C, respectively, and allows for the monitoring in real-time of the stepwise physiological mechanism of muscle sarcomere activation in an intact system which preserves significant features of the muscle, including excitation-contraction coupling and load. This biosensor has been designed and validated in both cardiac and skeletal muscle and using intact muscles from transgenic animals we have demonstrated FRET fluorescence in cardiac papillaries, extensor digitorum longus, and soleus muscles. This biosensor has shown unique sarcomere activation properties between cardiac and skeletal muscle, notably the emergence of a prolonged return to baseline of the FRET signal in loaded fast and slow skeletal muscle, which is evidence of a “primed state” of myofilament activation unique to skeletal muscles under load. Elucidation of the “primed state” of sarcomere activation is evidence of a transient memory of recent activity enabling the enhanced contraction properties of skeletal muscle. This finding advances previous understanding of skeletal muscle activation, including summation and tetanic contraction, which is not possible in cardiac muscle. The implementation of this biosensor provides a new approach to interrogate the essential physiological features of muscle sarcomere activation and contraction and adds a valuable new tool for small molecule and gene-based discoveries for muscle disease remediation.
Abstract Mo137: Modulation of the Inflammatory and Fibrotic Effects of Angiotensin on Cardiomyocytes through YAP-mediated Transcription.
Authors: Ninh Van , Brand Cameron , Barlow Melissa , Aydin Sidar , Francisco Jamie , Del Re Dominic , Heller Brown Joan
Published: October 2024

Introduction:
Signaling through the transcriptional co-activator Yes-associated protein (YAP) has been shown to protect the myocardium against ischemic or mechanical stress.

Research Question:
In light of the critical role of inflammation on adverse cardiac responses we examined the possibility that YAP regulates inflammatory responses to Angiotensin II (AngII)-mediated stress.

Approach:
Cardiomyocyte specific YAP-KO mice were generated, implanted with AngII minipumps, infused with AngII (1.5 mg/kg/min) or vehicle for periods of 3 hrs to 3 days and hearts collected for analysis.

Results:
YAP deletion markedly enhanced AngII-induced mRNA expression of pro-inflammatory cytokines and chemokines (e.g. CCL2, CXCL2, IL-6) at 3 or 24 hrs in the cardiomyocyte, but not non-myocyte cell fraction. Expression of mRNA for the hypertrophic genes ANP and MHC-β, in contrast, was decreased in YAP KO mice. After 3 days of AngII infusion there was enhanced mRNA for pro-fibrotic genes (Col1a1, Col3a1 and Periostin) and increased staining for COL1A1 and F4/80, indicative of increased fibrosis and inflammatory cell recruitment in the absence of YAP signaling. Single cell RNA sequencing revealed specific subsets of genes that were upregulated in the cardiomyocyte subcluster from YAP KO mice. These included pro-inflammatory cytokines, cJun family genes, TLR4 and, surprisingly, CaMKIId. Elevated activation of CaMKIIδ in response to AngII in YAP KO hearts was confirmed by increased phosphorylation of phospholamban. Cardiomyocytes expressing siYAP also showed enhanced expression of TLR4 and CaMKIIδ.

Conclusion:
Upregulated expression of cJun, TLR4 and CaMKII occurs in the absence of YAP and this conspires to create a greatly enhanced inflammatory milieu which fuels adverse responses to external insults. YAP activation serves to suppress these responses and thus temper development of maladaptive inflammation.
Abstract Or104: Targeting Tropomyosin Overlap Flexibility via Small Molecule Intervention in a Murine Dilated Cardiomyopathy Model
Authors: Vasquez Catherine , Lynn Melissa , Tardiff Jil
Published: October 2024

Familial dilated cardiomyopathy (DCM) is a genetic heart disorder characterized by enlargement of one or both ventricles, rendering the heart unable to pump blood efficiently. Mutations in cardiac thin filament (CTF) proteins have been associated with characterized clinical manifestations of DCM. Genetic segregation identified a mutation in α-Tropomyosin (Tm) at residue 230 (D230N-Tm) in two unrelated, multigenerational families linked to severe, early onset DCM. To meaningfully link genotype to phenotype our group sought to investigate the effects of D230N-Tm on the biophysical structure of Tm filament. We showed that D230N-Tm caused a compaction of the cardiac Troponin T (cTnT)–Tm overlap region accompanied by a decreased in flexibility of the Tm filament. Given the altered biophysical structure we investigated whether we could target this decrease in flexibility via small molecule intervention and improve function. Computational modeling and biophysical experiments identified small molecule 4-Hydroxy Duloxetine β-D-Glucuronide (Z06) shown to improve compaction of the cTnT-Tm overlap and restore flexibility of the Tm filament. We hypothesized that the decrease in flexibility of Tm impairs its ability to shift through its different conformational states, leading to impaired crossbridge formation. To investigate whether Z06 improves myofilament function, we performed an NADH-coupled ATPase assay using isolated myofibrils from our D230N-Tm transgenic mice and compared them to Non-Transgenic (NTg) littermates. ATPase activity was measured at saturating calcium (100uM) in the presence (250uM) and absence (0uM) of Z06. In the absence of Z06, D230N-Tm exhibited a decrease in ATPase activity (1.793±0.203 umol/min) compared to NTg littermates (5.420±0.562 umol/min), a common trend in DCM models. In the presence of 250uM Z06, D230N-Tm ATPase rates improve to levels comparable to NTg mice (4.816±0.544 umol/min). Moving forward, we will perform calcium titrations, allowing us to investigate if Z06 can restore cooperativity of myofilament activation. Additionally, we will test if the restorative effects of Z06 are present at the myocellular level, and if so, we will optimize delivery to test in vivo effects of Z06 in our D230N-Tm mouse model.
Abstract Or105: Constitutive Deletion of the Obscurin Ig58/59 Domains Induces Structural and Electrophysiological Remodeling in Atria
Authors: Brong Annie , Grogan Alyssa , Cavalcante Joca Humberto , Boyman Liron , Kaplan Aaron , Ward Christopher , Greiser Maura , Kontrogianni-Konstantopoulos Aikaterini
Published: October 2024

Introduction:
Obscurin is a giant cytoskeletal protein that supports muscle development, tethers intracellular compartments and regulates contraction. In the
ObscnΔIg58/59
mouse model, expressing obscurin lacking Immunoglobulin (Ig) domains 58 and 59, aging males exhibit irregular heart rhythm with prominent atrial fibrillation, atrial enlargement, and progressive remodeling of the ventricles. A mechanistic basis for the emergence of arrhythmia early on could not be identified in
ObscnΔIg58/59
ventricles, suggesting that the atria are preferentially impacted by deletion of the obscurin Ig58/59 module prior to the ventricles.

Hypothesis:
We hypothesize that Ig58/59 deletion elicits unique structural, electrical, and functional consequences in the atria preceding ventricular maladaptation.

Aims:
I. Examine the structural organization of
ObscnΔIg58/59
atria

II. Assess Ca
2+
handling in isolated atrial cardiomyocytes and determine molecular alterations.

Methods:
Electron microscopy and super resolution microscopy were utilized to visualize sarcomeric ultrastructure and the transverse-axial tubule (TAT) network, respectively. Ca
2+
handling activity and synchrony were also resolved, while Phos-Tag gels were used to quantify T-cap phospho-species.

Results:
ObscnΔIg58/59
atria exhibited misalignment of Z-disks. Spontaneous and stimulated Ca
2+
cycling behavior were [AB1] differentially disrupted in atrial cardiomyocytes in 6- and 12-month
ObscnΔIg58/59
males. Relatedly, atrial cells showed an age-dependent deterioration in TAT architecture. Finally, as a function of aging,
ObscnΔIg58/59
atria displayed alterations in the expression and phosphorylation of T-cap, a Z-disk protein implicated in the integration of t-tubules with the sarcomeric cytoskeleton.

Conclusions:
The structural and electrical alterations that arise in
ObscnΔIg58/59
atria precede ventricular dysfunction and coincide with the emergence of atrial fibrillation. Collectively, our work indicates that the atria are principally affected by Ig58/59 elimination, instigating arrhythmias and morphological alterations with age. The
ObscnΔIg58/59
mouse model has thus emerged as a proxy for atrial cardiomyopathy.
Abstract Or111: Titin cleavage disrupts sarcomere-adhesion tensional homeostasis triggering cardiomyocyte arrhythmia
in vitro
Authors: Pricolo Maria Rosaria , López-Unzu Miguel , Vicente Natalia , Morales-Lopez Cristina , Perez Franco Wendy , Dumitru Andra C. , Espinosa Barea Francisco Miguel , Sanchez Mateo I. , Oria-Muriel Manuel , Silva-Rojas Roberto , Herrero-Galan Elias , Garcia-Garcia Ricardo , Alegre-Cebollada Jorge
Published: October 2024

The cardiac tissue experiences several forms of mechanical stress, including contraction of cardiomyocytes and forces derived from the stiffness of extracellular matrix (ECM). Preserving homeostatic mechanical forces is crucial for a healthy heart, yet the mechanisms involved are not well-explored. Our study delves into cardiomyocyte responses following the sudden release of mechanical tension across titin, a giant sarcomere protein crucial for setting cardiomyocyte stiffness. Through the expression of virally transduced TEV protease (TEVp) in neonatal mouse cardiomyocytes (NMCMs), we induced the cleavage of mutant titin including the specific TEV protease recognition site (TEVs) within the mechanically active I-band region of the protein. Titin cleavage was validated by 1.8% polyacrylamide SDS-PAGE electrophoresis, revealing a primary band at ~2.2 MDa in TEV-positive NMCMs, corresponding to digested titin fragment A-M. This cleavage, confirmed at 2 days post-infection (dpi) with ~80% efficiency, reached 100% by 5 dpi. Consequently, the Young’s modulus of affected cardiomyocytes dropped by 50%, as determined by Atomic Force Spectroscopy. Sarcomere disarray increased over time, as evidenced by immunofluorescence, yet this effect did not induce cell death or proliferation. Cell viability was confirmed by the fact that affected cardiomyocytes are able to contract spontaneously, although titin cleavage resulted in reduced beating amplitude and induced arrhythmic behavior. Considering this arrhythmogenic situation, we tested cell-cell adhesion through dissociation assays, which revealed significantly reduced intercellular adhesion in affected cardiomyocytes. RNAseq confirmed involvement of pathways related to heart contraction, anchoring junctions, and cell junctions. Specifically, dysregulated genes associated with gap-junction and costamere complexes were observed at 5 dpi. Alterations of adhesion were further confirmed by reductions in connexin 43 and focal adhesion length in immunofluorescence experiments, which also showed mislocalization of important adhesive proteins. In conclusion, titin cleavage in neonatal cardiomyocytes leads to sarcomere disarray, perturbed cell-cell adhesion and arrhythmia. These findings highlight the importance of full mechanical integrity of the titin filament in living cardiomyocytes.
Abstract Or113: Identification of Newly Synthesized Protein in Cardiomyocytes after Myocardial Infarction
Authors: Zhang Feng , Liang Jie , Cusick Jenna , Wang Fangfei , Li Ainyu , Liu Shijie
Published: October 2024

Compared to mRNA, proteins perform ultimate functions in the heart and can be rapidly upregulated or downregulated in response to environmental inputs. Identifying how the proteome is altered in cardiomyocytes (CMs) after myocardial infarction (MI) will improve our understanding of the stress response signaling after MI and contribute to developing new therapeutic strategies. Here, we developed a method for determining which proteins in CMs are newly synthesized after MI by using mice expressing a mutant methionyl-tRNA synthetase that incorporates the non-canonical amino acid azidonorleucine into the newly synthesized peptides of CMs. We found that the proteins synthesized at the highest levels in homeostasis are involved in sarcomere structure and metabolism, which aligns with immunofluorescence staining results that showed enrichment of newly synthesized proteins localized at the sarcomere and intercalated disc. We showed that α-actin, a critical component of the sarcomere, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme for glucose metabolism, are highly dynamic and require constant protein synthesis for maintenance. After MI, protein synthesis in CMs within the infarct area is halted, and the border zone CMs showed reduced protein synthesis. Most notably, proteins associated with mitochondrial oxidative phosphorylation are reduced compared with sham. By integrating with spatial transcriptomics, we also discovered that the MI-induced genes are enriched in CMs within the infarcted areas. These CMs exhibit decreased general protein synthesis, indicating a decoupling of transcriptional and translational regulation following MI. Collectively, our findings revealed that CMs rapidly respond to injuries by controlling protein synthesis, which is independent of the expression of mRNAs.
Abstract Or114: Inhibiting IL-1 Signaling to Cardiac Fibroblasts Protects Against Acute Viral Myocarditis
Authors: Elias David , Jo Wonyoung , Talor Monica , Cihakova Daniela
Published: October 2024

Background:
Understanding what factors perpetuate cardiac inflammation in patients with unremitting inflammatory dilated cardiomyopathy (iDCM) is critical for developing curative therapies. We have previously demonstrated that inflammatory fibroblasts (IFs), a subset of cardiac fibroblasts (CFs) that promote inflammation, depend on IL-17A signaling to drive iDCM progression in experimental autoimmune myocarditis. Here, we demonstrate that IL-1 signaling more potently activates IFs than IL-17A. IL-1 signaling has been implicated in the pathogenesis of viral myocarditis, the most common etiology of myocarditis, though the mechanism is unclear.

Hypothesis:
We hypothesize that IL-1 signaling to CFs induces their differentiation into IFs, which drive pathogenic inflammation during viral myocarditis.

Methods:
Primary murine CF cultures were used to characterize IF activation by various cytokines. Next, we both performed qPCR on sorted CFs and used a CCL2-mCherry reporter mouse line to phenotype IF activation during Coxsackievirus B3 (CVB3) myocarditis. Finally,
in-vivo
fibroblast-specific IL-1 signaling was genetically ablated using PDGFRα
cre
IL1r1
fl/fl
mice. We infected PDGFRα
cre
and PDGFRα
cre
IL1r1
fl/fl
mice with CVB3 and assessed myocarditis severity during the peak of inflammation using histological scoring and flow cytometry.

Results:
Innate, Th1, Th2, and Th17 related cytokines such as IL-1β, IFNγ, IL-13, and IL-17A induced unique IF gene expression signatures. IL-1β most potently activated IFs with a pro-myeloid cytokine inflammatory profile. During CVB3 myocarditis, IF activation peaked on day 3 post infection and was sustained until day 10. Ablating IL-1 signaling to CFs reduced the magnitude of cardiac inflammation by 45% ± 14% (p=0.002) during the peak of CVB3 myocarditis with a reduction in monocyte, CD4+ T cells, CD8+ T cells, and NK cell recruitment. Gene expression analysis of sorted CFs from mutant and control mice revealed reduced expression of pro-myeloid chemokines such as CXCL1 and CCL2 on day 3 of CVB3 myocarditis.

Conclusions:
Cardiac fibroblasts exhibit remarkable plasticity allowing them to respond to a changing micro-environment. Blocking IL-1 signaling to IFs during CVB3 myocarditis inhibited production of pro-inflammatory chemokines resulting in attenuation of myocardial inflammation. Therefore, early IF activity acts as a rheostat to modulate the magnitude of cardiac inflammation during myocarditis.
Abstract Or120: CD40 is an immune checkpoint regulator that potentiates myocardial inflammation through activation and expansion of CCR2
+
macrophages and CD8 T-cells
Authors: Jimenez Jesus , Amrute Junedh , Ma Pan , Wang Xiaoran , Dai Raymond , Lavine Kory
Published: October 2024

Novel immune checkpoint therapeutics including CD40 agonists have tremendous promise to elicit antitumor responses in patients resistant to current therapies. Conventional immune checkpoint inhibitors (PD-1/PD-L1, CTLA-4 antagonists) are associated with serious adverse cardiac events including life-threatening myocarditis. However, little is known regarding the potential for CD40 agonists to trigger myocardial inflammation or myocarditis. Here, we leveraged genetic mouse models, single cell sequencing, and cell depletion studies to demonstrate that an anti-CD40 agonist antibody reshapes the cardiac immune landscape through activation of CCR2
+
macrophages and subsequent recruitment of effector memory CD8 T-cells. We identify a positive feedback loop between CCR2
+
macrophages and CD8 T-cells driven by IL12b, TNF, and IFN-γ signaling that promotes myocardial inflammation and show that prior exposure to CD40 agonists sensitizes the heart to secondary insults and accelerates LV remodeling. Collectively, these findings highlight the potential for CD40 agonists to promote myocardial inflammation and potentiate heart failure pathogenesis.
Abstract Tu019: Single-Cell RNA Sequencing Identifies IFNICs as a Cellular Target for Mitigating the Progression of Abdominal Aortic Aneurysm and Rupture Risk
Authors: Le Sheng , Wu Jia , Liu Hao , Du Yifan , Wang Dashuai , Luo Jingjing , Yang Peiwen , Ran Shuan , Hu Poyi , Chen Manhua , Ye Ping , Xia Jiahong
Published: October 2024

Introduction:
Abdominal aortic aneurysm (AAA) represents a life-threatening condition characterized by medial layer degeneration of the abdominal aorta. Nevertheless, knowledge regarding changes in regulators associated with aortic status remains incomplete. A thorough understanding of cell types and signaling pathways involved in the development and progression of AAAs is essential for the development of medical therapy.

Methods and Results:
We harvested specimens of the abdominal aorta with different pathological features in Angiotensin II (AngII)-infused
ApoE
-/-
mice, conducted scRNA-seq, identified a unique population of interferon-inducible monocytes/macrophages (IFNICs), which were amply found in the abdominal aortic aneurysms (AAAs). Gene set variation analysis (GSVA) revealed that activation of the cytosolic DNA sensing cGAS-STING and JAK-STAT pathways promoted the secretion of type I interferons in monocytes/macrophages and differentiated them into IFNICs. We generated myeloid cell-specific deletion of Sting1 (
Lyz2
-Cre
+/-
;
Sting1
flox/flox
) mice and performed bone marrow transplantation and found that myeloid cell-specific deletion of
Sting1
or
Ifnar1
significantly reduced the incidence of AAA, aortic rupture rate and diameter of the abdominal aorta. Mechanistically, the activated pyroptosis- and inflammation-related signaling pathways, regulated by IRF7 in IFNICs, play critical roles in the developing AAAs.

Conclusion:
IFNICs is a unique monocyte/macrophage subset implicated in the development of AAAs and aortic rupture.
Abstract Tu025: Exploring Metabolomic Profiles in Vitamin D Deficient and Obese Individuals: Relevance to Arterial Stiffness
Authors: Elmoselhi Adel , Bouzid Amal , Seif Allah Mohamed , ibrahim zeinab , Siddiqui Ruqaiyyah , Khan Naveed Ahmed , Al-Hroub Hamza M. , Harb Mohammad , Soares Nelson Da Cruz , Hamoudi Rifat A
Published: October 2024

Introduction:
Cardiovascular diseases (CVDs) continue to pose a significant global health challenge, with vitamin D deficiency and obesity emerging as prominent risk factors. Arterial stiffness is recognized as a pivotal predictor and an independent risk element for CVDs. This study explores the metabolomic profiling of blood samples obtained from individuals afflicted with early arterial stiffness due to vitamin D deficiency and obesity, compared with a control group.

Methods:
The study comprised nine participants with vitamin D deficiency (vitamin D level of 20 ng/ml or lower), and obese participants (BMI of 30 or higher), along with eleven control participants (with vitamin D levels above 20 ng/ml and normal BMI). Pulse Wave Velocity (PWV) measurements were conducted using the SphygmoCor device. Whole blood was collected from each participant in EDTA tubes, followed by centrifugation at 4200 RPM for 10 minutes to obtain plasma samples, which were then stored in a -80°C freezer. Metabolomic analysis was carried out using Liquid Chromatography coupled with tandem mass spectrometry (LC-MS/MS). A two-tailed p-value < 0.05 was considered statistically significant for all analyses.

Results:
The average PWV value relative to the participant's age was 19.4 ± 4.7 m/s in the group with both vitamin D deficiency and obesity, compared to 14.7 ± 2.1 m/s in the control group (p < 0.05), indicating increased arterial stiffness. Furthermore, eleven metabolites exhibited statistically significant differences in patients with vitamin D deficiency and obesity. Of these, eight metabolites demonstrated increased expression levels, including Nutriacholic acid, N-Acetylputrescine, Succinylacetone, Adenosine monophosphate, Elaidic acid, Niacinamide, DUMP, and 2-Pyrrolidinone, while three metabolites exhibited decreased expression: PC (18:1(9Z)/18:1(9Z)), Trimethylamine, and Imidazole. Additionally, top enrichment analysis revealed metabolic pathways predicted to be altered in the context of dysfunctional enzymes, encompassing processes such as nicotinamide acid uptake, fatty-acyl-CoA synthase (n-C18:0CoA), and extracellular NADP nucleosidase, among others.

Conclusion:
The metabolomic alterations identified in our study offer insights into potential biomarkers for evaluating arterial stiffness and present novel targets for therapeutic interventions in vitamin D deficient and obese individuals.
Abstract Tu028: NADPH Oxidase Inhibition Antagonizes Endothelial Pro-inflammatory and Pro-oxidant Signaling Resulting in Enhanced Coronary Vasorelaxation in Diabetic Models
Authors: Banerjee Debolina , Xing Hang , Nho Ju-Woo , Li Janelle , Iddrisu Hanisah , Kanuparthy Meghamsh , sellke frank , Feng Jun
Published: October 2024

Introduction:
NADPH oxidase (NOX) overactivation and reactive oxygen species amplification may underlie worse cardiac outcomes in patients with diabetes. Our group has shown impaired endothelium-dependent coronary vasodilation in diabetes, with restored endothelial function following NOX inhibition. Persistently high levels of oxidative stress may be driving this impaired coronary microvascular reactivity by altering small-conductance potassium channel activity. We hypothesized that NADPH oxidase inhibition would be vasoprotective through reductions in pro-oxidant and pro-inflammatory signaling.

Methods:
Human coronary arterial endothelial cells (HCAECs) were obtained from diabetic (D) and nondiabetic (C) patients (N = 4 per group). HCAECs were cultured in normo- or hyperglycemic conditions, with one hyperglycemic group receiving NADPH oxidase inhibitor apocynin (DT). Molecular signaling was assessed using immunoblotting.

Results:
Diabetes resulted in significantly increased pro-oxidant markers, including NOX 4 (p = 0.005), phosphorylated endothelial nitric oxide synthase (p-eNOS) (p = 0.007), and eNOS (p = 0.003). Pro-inflammatory Nrf2 was also increased (p = 0.0003), with a trend toward increased NFkB (p = 0.09) in D compared to C. Antioxidant peroxiredoxin (PRDX) 1 was significantly reduced in D compared to C (p = 0.02). NOX inhibition rescued these effects, with reversed trends in NOX 4 (p = 0.008), p-eNOS (p = 0.03), and PRDX 1 (p = 0.02) following apocynin treatment. NOX inhibition resulted in
de novo
decrease in NOX 1 in DT when compared to D (p = 0.008) and C (p = 0.02), and decrease in phosphorylated NFkB (p-NFkB) when compared to D (p = 0.02). There was a trend toward decreased total NFkB (p = 0.07) and p-NFkB:NFkB ratio (p = 0.06) and increased antioxidant thioredoxin (TXN) 2 (p = 0.05) in diabetic cells following treatment. Antioxidant superoxide dismutase (SOD) 2 (p = 0.01) and TXN 1 (p = 0.02) were increased in D when compared to C, while apocynin treatment reversed these changes (SOD 2, p = 0.0006; TXN 1, p = 0.04). There were no significant differences in NOX 2, SOD 1, catalase, PRDX 2, or PRDX 3 between the groups.

Conclusions:
Apocynin overall suppressed pro-oxidant and pro-inflammatory signaling in human diabetic coronary endothelial cells, resulting in improved coronary endothelium-dependent vasorelaxation. Cardioprotective strategies that incorporate NADPH oxidase inhibition in the setting of diabetes warrant further investigation.
Abstract Tu034: Endothelial cell regeneration by secretome obtained from P53 silenced bone marrow derived mesenchymal stromal cells (BM-MSCs)
Authors: Nandula Seshagiri Rao , Sen Sabyasachi
Published: October 2024

Introduction:
Diabetes is associated with impaired endothelial function. Studies have demonstrated that endothelial progenitor cells (EPCs) and mature endothelial cells (EC) are susceptible to apoptosis in hyperglycemic environment. Hyperglycemia leads to p53 gene over-expression and p53 gene silencing prior to hyperglycemia exposure prevents cellular senescence, improves mitochondrial function and helps vascular regeneration.. However, it is not possible to obtain sufficient quantities of secretome for therapeutic purposes from hematopoietic CD34+ve stem cells (HSCs). Our goal in this project was to test whether conditioned media (CM) obtained from p53 silenced human stem cells (other than CD34+ve cells) can also improve endothelial regeneration exposed to hyperglycemia. We compared CM obtained from adipose and bone marrow derived mesenchymal stem cells on endothelial regeneration

Methods:
Ad-human-P53 (TP53)-shRNA was used to silence P53 and Ad-scrambled-null-shRNA was used as control in bone marrow derived mesenchymal stromal cells. To collect conditioned media BM-MSCs were cultured in exosome free FBS media for 5 days, following transduction with Ad-p53sh or Ad-null, as the case may be. After transduction, cells were grown in media containing 2% exosome free FBS for only 48h and the collected supernatant was concentrated 10-fold to obtain the CM. Endothelial regeneration was tested using
endothelial wound healing assay kit
( from Cell Biolabs # CBA-120-T). We compared CM obtained from null and p53sh in adipose and bone marrow derived MSCs on human endothelial cells in normal and high glucose conditions

Results:
We observed that CM from bone marrow derived MSCs increased the proliferation of endothelial cells in both normal and high glucose conditions, more so than adipose derived MSCs. We are currently studying the role of exosomes from this CM on cell proliferation and we are also analyzing the secretome (exosomal and non- exosomal fraction) by mass spectroscopy.

Conclusion:
P53 silenced BMMSC-CM appears to improve endothelial regeneration in hyperglycemia similar to the effect of p53-silenced HSCs. This maybe secondary to the fact that both these cells are derived from bone marrow and p53 silencing improves mitochondrial function. The CM obtained from BM-MSCs are in sufficient quantities and may have a prominent therapeutic role in treating diabetes vascular complications such as diabetic wound healing.
Abstract Tu041: Gut microbiome
Bifidobacterium
is associated with improvement in severity of heart failure with reduced ejection fraction
Authors: Mamic Petra , Shi Handuo , Tang Wai Hong , Sonnenburg Justin , Khush Kiran , Heidenreich Paul , Huang Kerwyn , Snyder Michael
Published: October 2024

Background:
Heart failure (HF) has been associated with dysregulated gut microbiome. However, less is understood about how gut microbiome alterations relate to changes in HF severity. We sought to understand how gut microbial shifts and associated host signatures related to changes in clinical status of patients with HF with reduced ejection fraction (HFrEF).

Methods:
Twenty-six adults with HFrEF were recruited and completed 2 study visits approximately 6 months apart. At each visit they provided stool sample for microbiome profiling (metagenomic sequencing), and fasting blood samples for untargeted metabolomics (liquid chromatography–mass spectrometry) and cytokine assay (Luminex antibody-conjugated bead capture). Subjects also completed dietary surveys. Clinical improvement was measured as between-visit change in the New York Heart Association (NYHA) class.

Results:
Between-visit NYHA class improved in 6 individuals; 20 patients remained stable or worsened. After accounting for demographics, body mass index, and diet, genus
Bifidobacterium
was significantly enriched in the improved group and increased over time (Figure 1).
Bifidobacterium
was directly associated with circulating malonic acid, which is implicated in cardio-protection and cardiomyocyte regeneration, and with indole-3-propionate (IPA), anti-inflammatory gut microbial metabolite of dietary tryptophan (Figure 2). There was also a positive correlation between
Bifidobacterium
and IL10, a major anti-inflammatory cytokine that plays a role in cardiac remodeling.

Conclusion:
Bifidobacterium
was independently associated with longitudinal improvement in functional status in patients with HFrEF. Additionally,
Bifidobacterium
correlated with circulating IPA, malonic acid, and IL10. Our findings support (1) gut
Bifidobacterium
as a potential biomarker of clinical improvement in HFrEF and a possible new therapeutic target, and (2) potential downstream mediators through which
Bifidobacterium
may regulate HF pathophysiology.
Abstract Tu056: A Two-Hit HFpEF-like Mouse Model with Accelerated Disease Onset
Authors: Nehra Sarita , Dokania Manoj , Gupta Ankit , Garcia Ricardo , Dudhgaonkar Shailesh , Mazumder Tagore Debarati , CK Neethu , Wagh Somnath , Kale Prajakta , Selvam Sabariya , Anand Amit , Luettgen Joseph , Bennett Nikki , Gulia Jyoti
Published: October 2024

Introduction:
Heart failure with preserved ejection fraction (HFpEF) is a heterogenous syndrome with multiple co-morbidities and has limited therapeutic options. To better understand the underlying mechanisms and develop novel therapies, it is important to focus on development of pre-clinical models that mimic the multi-factorial nature of HFpEF.

Goal & Methodology:
We aimed to develop a mouse HFpEF model by incorporating unfavourable metabolic axis, in db/db mice carrying mutation in lectin receptor, with Ang-II induced impaired hemodynamic conditions. Briefly, we infused Angiotensin II (Ang II, 1500 ng/kg/min) subcutaneously in 12-week-old wild type (WT) and db/db mice for 4 weeks (Fig 1). SGLT2 inhibitor (SGLT2i) was used as a pharmacological intervention.

Results:
The db/db + Ang II mice showed impaired diastolic function reflected by increase in E/A, E/e’ and global longitudinal strain. This increase was not observed in the db/db + Ang II mice upon treatment with SGLT2i. The ejection fraction was preserved across all groups. Interestingly, db/db + Ang II group did not develop cardiac hypertrophy & fibrosis as indicated by unchanged LV mass, picosirius red staining and unaltered expression of Nppa, Nppb & Acta2 transcripts. Investigation of lipid metabolism showed reduction of fatty acid synthase in db/db (+/-Ang II) groups in comparison to WT animals. Also, phosphorylation of ATP citrate lyase in db/db + Ang II group was diminished compared to db/db animals. Among the glucose metabolism markers, lactate dehydrogenase and pyruvate dehydrogenase showed decreasing trend in all groups compared to WT group.

Conclusion:
The present mouse model recapitulates metabolic and hemodynamic aspects of HFpEF pathophysiology. Our model is useful for basic research as well as quick evaluation of targets and assets focused on cardiac metabolism with the advantage of faster disease onset and clinically relevant echocardiographic endpoints.
Abstract Tu069: Development of a Novel Biallelic, Haploinsufficient Mouse Model of
MYBPC3
Related Hypertrophic Cardiomyopathy
Authors: Meisner Joshua , Bullard Abbey , Friedline Sabrina , Rechsteiner Olivia , Previs Michael , Helms Adam
Published: October 2024

Background:
No current animal models re-capitulate MyBP-c haploinsufficiency and hypertrophic cardiomyopathy (HCM) observed in patients with heterozygous
MYBPC3
truncating variants, the most common genetic cause of HCM. We recently identified a hypomorphic (partial) loss of function
MYBPC3
variant that increases disease severity in HCM patients when combined with an
MYBPC3
truncating variant.

Hypothesis:
Additive effects from a hypomorphic loss of function
MYBPC3
variant observed in human HCM will result in MyBP-c protein haploinsufficiency and HCM in a biallelic murine model.

Methods:
To test
MYBPC3
dose effects, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were first derived from an HCM patient with biallelic hypomorphic and complete loss of function variants in
MYBPC3
. We then generated
Mybpc3
knock out (Mybpc3
KO
) and hypomorphic knock in (Mybpc3
KI
) alleles in mice. Control (Mybpc3
+/+
), monoallelic, and biallelic mice and iPSC-CMs were characterized with morphometric measurements, echocardiography, mass spectroscopy, and RNA sequencing.

Results:
Biallelic iPSC-CMs demonstrated haploinsufficiency not present in heterozygous models through dose reduction in
MYBPC3
protein (Fig 1A). Similarly, biallelic Mybpc3
KI/KO
mice had a reduction in
Mybpc3
transcripts (62±3%, p<0.001) with 43±11%% reduction in MyBP-c protein (Fig 1B) and cardiac hypertrophy (10±5% increase in heart mass, p=0.002, Fig 1C).

Conclusions:
Graded reduction in
Mybpc3
transcripts in a biallelic mouse model results in MyBP-c haploinsufficiency similar to that observed in HCM patients. Corresponding hypertrophy establishes this approach as a clinically relevant, novel model of
MYBPC3
HCM.