bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P432: Alpha-actinin 2 Missense Variant And Its Role In Cardiac Muscle Force Production And Diastolic Dysfunction
Authors: Rodriguez Garcia Michelle C , Landim-Vieira Maicon , Schmeckpeper Jeffrey , Wang Lili , Shoemaker Ben B , Chase Prescott B , Knollmann Bjorn C , Pinto Jose R
Published: November 2021

ACTN2
gene encodes for the alpha-actinin 2 protein, which has an actin-binding role. In cardiac muscle, alpha-actinin 2 is located in the Z-disc of the sarcomere, where it anchors myofibrillar actin filaments. A 48-years old female patient presented with ventricular fibrillation arrest was successfully resuscitated. Coronary angiography exhibited normal coronary arteries. Cardiac MRI did not show myocardial scar tissue or arrhythmogenic right ventricular dysplasia (ARVD). Echocardiogram showed normal left ventricular ejection fraction, stage II diastolic dysfunction and elevated right ventricular systolic pressure. Genetic testing identified a missense variant of unknown significance in
ACTN2
(a gene previously associated with dilated cardiomyopathy/ARVD), and in
SCNB2
(encodes sodium voltage-gated channel beta subunit 2, a gene previously associated with long QT syndrome). We investigated the effects of the
ACTN2
variant on cardiac muscle contraction, providing an inside view of the importance of
ACTN2
in sarcomere function. Left ventricular free wall samples were obtained from the patient’s explanted heart at the time of cardiac transplantation. Mechanics of contraction were measured in permeabilized cardiac muscle preparations (CMPs) in the presence of 3% dextran T-500 (to restore the myofilament lattice to physiological dimensions) at 30°C. The experiments were performed at sarcomere lengths 2.1 μm and 2.3 μm achieved by HeNe laser diffraction. CMPs containing
ACTN2
mutant protein displayed increased myofilament Ca
2+
sensitivity of isometric force and lower maximal isometric force compared to human donor samples (control). Sinusoidal stiffness was decreased at all levels of Ca
2+
activation and isometric force in
ACTN2
variant CMPs. The rate of tension redevelopment was faster at all levels of Ca
2+
activation and isometric force. Alpha-actinin 2 is an important sarcomeric protein that regulates the kinetics of cardiac muscle contraction. This work establishes the importance of alpha-actinin 2 in the Z-disc mechanotransduction to thin and thick filament interactions.
Abstract P435: Probiotic Supplements Could Prevent Diet-induced Mitochondrial Dysfunction In The Liver Hepatocytes Of Apolipoprotein-e Knockout Mice
Authors: St-Amant Antoine J , Cougnaud Lise , Vuckovic Dajana , Bergdahl Andreas
Published: November 2021

A poor diet can modify the intestinal microbiota, known as gut dysbiosis, which causes hepatocyte mitochondrial dysfunction through the release of endotoxins in the portal vein. Mitochondrial dysfunction in the liver leads to non-alcoholic fatty liver disease (NAFLD). Epidemiological evidence shows that most deaths from NAFLD are related to cardiovascular diseases (CVD) demonstrated by impaired vasodilatation as well as increased arterial stiffness, calcification, inflammation, and intimal-medial thickness. Therefore, the objective of our study was to investigate the potential of probiotic supplements to prevent a diet-induced mitochondrial dysfunction in hepatocytes. Apolipoprotein-E knockout mice—genetically modified animals that develop dyslipidemia—were placed in 3 diet groups including a control, western style (high in fats and carbohydrates), as well as a low-carbohydrate high-protein diet. Each group was then further divided in 3 subgroups based on the dose of probiotics administered, including a control, low (0.5 B/dose) and high (5 B/dose) dose of probiotics, for a total of 9 groups (n= 8-12 animals/group). The probiotic supplements contained a 1:1 ratio of
Lactobacillus Helveticus
and
Bifidobacterium Bifidum
. The bodyweight and water intake were monitored weekly. After 6 weeks, the liver mitochondria were analyzed by high resolution respirometry using a sequential substrate addition protocol (malate, octanoylcarnitine, ADP, glutamate, pyruvate, cytochrome C, succinate and FCCP) to investigate mitochondrial maximal respiration, leak, membrane damage and uncoupling. Our preliminary results show that the groups that received a high dose of probiotics gained less weight, had a higher respiration following the addition of ADP (a biomarker of complex V efficiency) and a lower respiration following FCCP (a biomarker of mitochondrial uncoupling), as compared to control. Supplements of probiotics could promote weight loss and prevent a diet-induced mitochondrial dysfunction in liver hepatocytes, an important risk factor for NAFLD and therefore, CVD. This project could translate into future research directions in preventative medicine, for example by investigating the potential of probiotics in the prevention of mitochondrial dysfunction in other organs, including the heart and the vascular system.
Abstract P443: Loss Of Pkm2 Alters Myocardial Metabolism And Increases Oxidative Stress After Infarction
Authors: Lee Katie C , Williams Allison L , Shohet Ralph V
Published: November 2021

Introduction:
Pyruvate kinase (Pkm1) directs pyruvate to the TCA cycle for oxidative metabolism in the healthy heart. Our lab described a hypoxia-mediated switch to the alternatively spliced isoform Pkm2, enhancing pyruvate to lactate conversion. Recently, we have also found that Pkm2 knockout (KO) mice had profound depletion of basal glucose in the heart compared to control mice. Pkm2 has also been shown to reduce oxidative damage and promote cardiomyocyte cell proliferation after myocardial infarction (MI). We hypothesize that the upregulation of Pkm2 alters metabolic pathways after injury to promote glycolysis and preserve ATP production in hypoxia, which protects the heart from the stresses of hypoxia and injury.

Methods:
Global Pkm2 KO mice were subjected to permanent ligation of the left anterior descending coronary artery to mimic an MI. RNA-seq analysis of left ventricles from control (n=8) and Pkm2 KO mice (n=8) before and 3 days after sham or MI surgery was performed. Semiquantitative real-time PCR (qPCR) was used to confirm changes in selected genes of interest.

Results:
Loss of Pkm2 did not alter gene expression substantially at baseline. 68 genes were differentially expressed in Pkm2 KO hearts after MI (q<0.05, FDR<0.05) not observed in control MI hearts. MI in Pkm2 KO hearts resulted in considerable reduction of transcripts of enzymes in the insulin signaling pathway, mitochondrial oxidative phosphorylation, fatty acid metabolism, and increase in transcripts encoding enzymes in the pentose phosphate pathway, response to oxidative stress, and apoptotic signaling. qPCR of selected genes involved in glucose metabolism confirmed RNA-seq results.

Conclusions:
RNA-seq analysis of Pkm2 KO hearts demonstrated that loss of Pkm2 altered gene expression of metabolic and mitochondrial enzymes. Conversely, Pkm2 KO hearts showed increased abundance of pro-apoptotic markers which may be a result of increased oxidative stress.
Abstract P449: Connexin43 As A Therapeutic For Arrhythmogenic Right Ventricular Cardiomyopathy
Authors: Zhang Jing , Zanella Fabian , Bradford William H , Fujita Kyohei , Karakikes Ioannis , Lyon Robert C , Mezzano Valeria , Roberts Jason D , Carromeu Cassiano , Gu Yusu , Martin Jody L , Muotri Alysson R , Scheinman Melvin , Peterson Kirk L , Sheikh Farah
Published: November 2021

Limited efforts have been focused on the interventions which could therapeutically alter arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), a fatal cardiac disease of the desmosomal (mechanical) cell-cell junction. The desmosome is a critical target for intervention as mutations in desmosomal genes underlie 40-50% of ARVD/C populations and its dysregulation is associated with severe cardiac electrical and structural alterations, which facilitate myocardial failure, arrhythmias and premature death in these populations. Cardiomyocyte reduction of the predominant ventricular gap junction protein connexin43 is a molecular alteration that underlies desmosomal deficits and arrhythmias in ARVD/C. However, the role of connexin43 in structural alterations associated with ARVD/C remains unclear. We intervened with connexin43 reduction in human and mouse models of ARVD/C via connexin43 restoration strategies, which revealed beneficial effects in both ARVD/C models. We show ARVD/C human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes that recapitulate desmosomal structural defects and reveal connexin43 diminution alterations that are reflective of disease found in donor ARVD/C hearts. Connexin43 restoration was sufficient to rescue cardiac physiological deficits and increase desmosomal gene expressions in ARVD/C hiPSC derived cardiomyocytes, encompassing structural alterations.
In vivo
studies exploiting a mouse model of ARVD/C harboring severe desmosomal structural alterations revealed that cardiac connexin43 restoration was sufficient to prolong lifespan and restore cardiac desmosomal proteins. Herein, we provide evidence for non-canonical functions for connexin43, classically associated with electrical function, in the mechanical modulation of junctions. Our findings have broad implications in exploiting connexin43 as a therapeutic in advanced diseases associated with cardiac structural defects.
Abstract P454: The Interaction Of P300 With Brg1 Acetylated The Histone H3 Globular Domain In Heart Failure
Authors: uehara sho , Morimoto Tatsuya
Published: November 2021

Background:
Epigenetic regulatory mechanisms such as histone post-translational modifications are involved in the development of heart failure. Although the acetylation of tail domains, such as H3K9, has been extensively studied, that of H3K122, the globular domain, has received much less attention. Acetylation of the globular domain directly activates transcription by destabilizing histone-DNA binding. However, the acetylation of these domains during the transition from left ventricular hypertrophy (LVH) to heart failure (HF) remains unknown.

Methods and Results:
Primary cultured cardiomyocytes prepared from neonatal rats were treated with phenylephrine (PE). PE increased the acetylation of H3K9 and H3K122. The acetylation of H3K9 and H3K122 on the promoters of ANF and BNP, which are hypertrophic reaction genes, was increased in cardiomyocyte hypertrophy. To investigate whether the transcriptional coactivator p300 is involved in the acetylation of H3K9 and H3K122, p300 knockdown was used. p300 knockdown suppressed PE-induced cardiomyocyte hypertrophy and the acetylation of H3K9 and H3K122. In Dahl-salt sensitive rats,
in vivo
chromatin-immunoprecipitation assays revealed that the acetylation of H3K9 on the promoter of the hypertrophic response genes was significantly increased in LVH, but the acetylation of H3K122 was not increased in LVH. However, H3K122 acetylation was significantly increased in HF. On the other hand, there was no difference in the amount of recruitment of p300 in LVH and HF. Interestingly, immunoprecipitation-WB showed that binding of p300 with BRG1, a key component of the SWI/SNF complex, was enhanced in HF. The recruitment of BRG1 increased significantly in HF compared to LVH. Moreover, PFI-3, a BRG1 inhibitor, significantly suppressed a PE-induced increase in cardiomyocyte surface area, the mRNA levels of ANF and BNP, and the acetylation of H3K9 and H3K122 in cultured cardiomyocytes.

Conclusion:
This study demonstrates that the acetylation of H3K122 is enhanced via the interaction of p300 and BRG1 in heart failure, providing novel insights into the epigenetic regulatory mechanism governing transcriptional activity in these processes.
Abstract P467: Cardiac-specific Deletion Of Voltage Dependent Anion Channel 2 Leads To Dilated Cardiomyopathy By Altering Calcium Homeostasis
Authors: Shankar Thirupura S , Anandamurugan Ramadurai Dinesh Kumar , Steinhorst Kira , Sommakia Salah , Badolia Rachit , THODOU KROKIDI ASPASIA SPYRIDOULA , Calder Dallen , Navankasattusas Sutip , Ling Jing , Kwon Ohyun , Cheng Emily , Richardson Russel , Sachse Frank B , Schredelseker Johann , Spitzer Kenneth , Chaudhuri Dipayan , Drakos Stavros G
Published: November 2021

Voltage dependent anion channel 2 (VDAC2) is a mitochondrial outer membrane porin known to play a significant role in apoptosis and calcium signaling. Abnormalities in cellular calcium homeostasis often leads to electrical and contractile dysfunction and can cause dilated cardiomyopathy and heart failure. Previous literature suggests that improving mitochondrial calcium uptake via VDAC2 rescues arrhythmia phenotypes in genetic models of impaired cellular calcium signaling. However, the direct role of VDAC2 in intracellular calcium signaling and cardiac function is not well understood. To elucidate the role of VDAC2 in calcium homeostasis, we generated a cardiac-specific deletion of
Vdac2
in mice. Our results indicate that loss of VDAC2 in the myocardium during development causes severe impairment in excitation-contraction coupling by reducing mitochondrial calcium uptake (n=3, p<0.05) and thereby impairing intracellular calcium signaling. VDAC2 knock-out mice showed a significant reduction in RYR-mediated calcium release (F/F
0
) and rate of calcium uptake by SERCA2a [tau(msec)] compared to control mice (N=3, WT=54, KO=38, p<0.0001 (F/F
0
) and p<0.05 (tau)). We also observed adverse cardiac remodeling which progressed to severe dilated cardiomyopathy and death (N=6, p<0.0001). Reintroducing VDAC2 in 6-week-old knock-out mice partially rescued the cardiomyopathy phenotype evident from improvement in ejection fraction and fractional shortening (n=3, p<0.05). Improving mitochondrial calcium uptake via VDAC2 using a VDAC2 agonist efsevin, increased cardiac contractile force in a mouse model of pressure-overload induced heart failure (N=8, n=22, p<0.05). In conclusion, our findings demonstrate that VDAC2 plays a crucial role in cardiac function by influencing mitochondrial and cellular calcium signaling. Through this role in cellular calcium dynamics and excitation-contraction coupling VDAC2 emerges as a plausible therapeutic target for heart failure.
Abstract P470: Disrupted HRAS/GRB2 Signaling In Induced-cardiomyocytes Derived From Patients With Noonan Syndrome Carrying SOS1 Gene Mutation
Authors: SIGAMANI VINOTH , Gurusamy Narasimman , RAJASINGH SHEEJA , Johnson Rajasingh
Published: November 2021

Background:
Noonan syndrome (NS), a dominant autosomal genetic disorder that prevents normal development, and exhibits cardiac defects, which is estimated to appear in 50% to 90% of patients. Son of sevenless homolog 1 (SOS1) gene mutation has been identified as a major gene causing NS and attributes to the development of cardiomyopathy and congenital heart defects. SOS1 is a guanine nucleotide exchange factor for RAS and is known to interact with growth factor receptor-bound protein 2 (GRB2). Recently, we have generated induced pluripotent stem cells (iPSCs)-derived cardiomyocytes (iCMCs) from cardiac fibroblasts obtained from a NS patient carrying SOS1 gene variant 1654A>G.

Hypothesis:
Since NS is known to have aberrant RAS-MAPK signaling, we hypothesize that iCMCs derived from NS patient (NS-iCMCs) may have atypical RAS signaling leading to the development of cardiomyopathy.

Methods and Results:
We have compared the normal skin fibroblast-derived iPSCs (N-iPSCs) and N-iCMCs with NS patient-derived induced NS-iPSCs and NS-iCMCs. Our qRT-PCR results showed that the mRNA expressions of signaling molecules HRAS, GRB2 and SOS1 were significantly decreased in NS-iCMCs compared with N-iCMCs (Figure A), and further confirmed through the protein expression by Western immunoblotting (Figure B). These results were in association with a significantly decreased mRNA and protein expressions of cardiac transcription factor GATA4, and structural proteins alpha sarcomeric actinin-2 (ACTN2), cardiac troponin T (TNNT2) and tropomyosin alpha-1 (TPM1) in NS-iCMCs compared with N-iCMCs. Further studies are underway to explore the difference in the guanine nucleotide exchange factor (GEF) activity and ERK activation between NS-iCMCs and N-iCMCs.

Conclusion:
Our current findings clearly indicate that the SOS1-associated signaling molecules HRAS and GRB2 were disrupted in NS-iCMCs, which may result in the development of cardiomyopathy in NS patients.
Abstract P471: Mechanisms Controlling Regression Of Cardiac Fibrosis By Removal Of Pressure Overload
Authors: Neff Lily , Van Laer An , Baicu Catalin F , Zile Michael R , Bradshaw Amy
Published: November 2021

Background:
Antecedent conditions, like aortic stenosis, can induce left ventricular pressure overload (LVPO), that can lead to Heart Failure with Preserved Ejection Fraction (HFpEF). Myocardial fibrosis and stiffness are key characteristics of HFpEF. Cardiac fibroblasts are the primary cell type regulating ECM production and deposition. In previous studies, biopsies isolated at the time of SAVR surgery, to correct stenosis, and then at 1-year and 5-years post-SAVR showed reductions in hypertrophy and fibrosis demonstrating these processes can regress. However, cellular mechanisms, including fibroblast activity, are poorly defined.

Objective:
Define mechanisms that contribute to remodeling of ECM before and after LVPO.

Methods:
LVPO was induced using transverse aortic constriction (TAC). LVPO was relieved by removal of the band (unTAC) at 4 wks. Cardiomyocyte cross-sectional area (CSA), collagen volume fraction (CVF), and protein production was measured by histology and immunoblot for five time points: nonTAC, 2wk TAC, 4wk TAC, 4wk TAC+2wk unTAC, and 4wk TAC+4wk unTAC.

Results:
In response to LVPO, myocyte CSA increased by 23% at 2wk TAC and by 47% at 4wk. CVF increased by 64% and 204% at 2wk and 4wk TAC, respectively, versus nonTAC. In 2wk TAC hearts, SMA, a marker of fibroblast activation was increased as was production of two collagen cross-linking enzymes, lysyl oxidase (LOX) and LOXL2, in the absence of significant increases in markers of ECM degradation. After unloading, myocyte CSA decreased by 20% in 2wk unTAC versus 4wk TAC and CVF decreased by 38% in 4wk unTAC versus 4wk TAC. Coincident with decreases in CVF, levels of pro-MMP2 increased at 2wk unTAC as did levels of degraded collagen measured by collagen hybridizing peptide reactivity. Whereas markers of ECM deposition, LOX and LOXL2, were not increased in unTAC myocardium, a resurgence of SMA production occurred in 2wk unTAC.

Conclusions:
In LVPO hearts, hypertrophy was characterized by increases in myocyte CSA, greater CVF, and fibroblast activation with increased production of pro-fibrotic ECM. After unloading, hypertrophy and fibrosis significantly decreased accompanied by increases in ECM degrading activity and reductions in proteins that contribute to collagen assembly.
Abstract P477: A Novel
SMYD1
Variant (c.302A>G) Leads To Dilated Cardiomyopathy And Biventricular Heart Failure.
Authors: Szulik Marta , Reyes-Mugica Miguel , Marker Daniel F , Ghaloul-Gonzalez Lina , Franklin Sarah
Published: November 2021

The lysine methyltransferase SMYD1 was first identified in mice and shown to be important for embryonic cardiac development. Subsequently, we reported the first analysis of SMYD1 in adult myocardium and demonstrated that cardiomyocyte-specific loss of SMYD1 lead to progressive cardiac hypertrophy and heart failure, and showed that this enzyme is necessary to maintain metabolic homeostasis through transcriptional regulation of mitochondrial energetics in adult mice. While SMYD1 has been the subject of several additional studies in zebrafish and mice, since it was first identified, only in the last few years have human patients been identified with variants in the
SMYD1
gene thought to be responsible for their cardiomyopathies. Specifically, two patients have been identified to date, the first patient displaying hypertrophic cardiomyopathy had a
de novo
heterozygous variant (c.814T>C) and the second patient with left ventricular non-compaction cardiomyopathy and arrhythmias had a truncating heterozygous variant (c.675delA). Here we report a third patient with biventricular heart failure containing a homozygous variant (c.302A>G; p.Asn101S) in the
SMYD1
gene which was identified by a whole exome sequencing. Our histopathological analysis of cardiac tissue and skeletal muscle from the proband showed abnormalities in myofibrillar organization in both cardiac and skeletal muscle suggesting that SMYD1 is necessary for sarcomere assembly and organization. In addition, we observe markedly abnormal myocardium with extensive fibrosis and multifocal calcification, and our ultrastructural (EM) analysis revealed presence of abnormal mitochondria with reduced and irregular or lost cristae. Lastly, we have performed structural modeling of SMYD1 containing the p.Asn101Ser variant (N101S) and report how this variant may affect the enzymatic activity of SMYD1 due to its proximity to the substrate binding site. The identification of this novel variant constitutes the third patient with a SMYD1 variant displaying cardiomyopathy and provides insights into the molecular functionality of this protein. In addition, this is the first analysis of tissue from a patient expressing a SMYD1 variant which provides critical insights into the role of SMYD1 in the heart and how loss of function mutations can effect cardiac physiology.
Abstract P482: Elucidating And Characterizing The Molecular Mechanistic Role Of Phospholamban L39 Stop In The Pathophysiology Of Cardiomyopathy Using Patient-derived Human Induced Pluripotent Stem Cells And Humanized Knock-in Mouse Model Systems
Authors: Devendran Anichavezhi , Bailey Rasheed , Kar Sumanta , Stillitano Francesca , Turnbull Irene , Fish Kenneth , Dubois Nicole , Wickramasinghe Nadeera , Hajjar Roger , Costa Kevin , Kranias Evangelia , Trivieri Maria
Published: November 2021

Background:
Heart failure (HF) is a complex clinical condition associated with substantial morbidity and mortality worldwide. The contractile dysfunction and arrhythmogenesis related to HF has been linked to the remodelling of calcium (Ca
++
) handling. Phospholamban (PLN) has emerged as a key regulator of intracellular Ca
++
concentration. Of the PLN mutations, L39X is intriguing as it has not been fully characterized. This mutation is believed to be functionally equivalent to PLN null (KO) but contrary to PLN KO mice, L39X carriers develop a lethal cardiomyopathy (CMP). Our study aims at using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from homozygous L39X carriers to elucidate the role of L39X in human pathophysiology. Our plan also involves the characterization of humanized L39X knock-in mice (KM), which we hypothesize will develop a CMP from mis-localization of PLN and disruption of Ca
++
signalling.

Methodology and Results:
Mononuclear cells from Hom L39X carriers were obtained to generate 11 integration-free patient-specific iPSC clones. The iPSC-CMs were derived using established protocols. Compared to the WT iPSC-CMs, the Hom L39X derived-CMs PLN had an abnormal cytoplasmic distribution and formed intracellular aggregates, with the loss of perinuclear localization. There was also a 70% and 50% reduction of mRNA and protein expression of PLN respectively in L39X compared to WT iPSC-CMs. These findings indicated that L39X PLN is both under-expressed and mis-localized within the cell. To validate this observation
in-vivo,
we genetically modified FVB mice to harbour the human L39X. Following electroporation, positively transfected mouse embryonic stem cells were injected into host blastocysts to make humanized KM that were subsequently used to generate either a protamine-Cre (endogenous PLN driven expression) or a cardiac TNT mouse (i.e., CMP specific).

Conclusion:
Our data confirm an abnormal intracellular distribution of PLN, with the loss of perinuclear accumulation and mis-localization, suggestive of ineffective targeting to or retention of L39X. The mouse model will be critically important to validate the
in-vitro
observations and provides an ideal platform for future studies centred on the development of novel therapeutic strategies including virally delivered CRISPR/Cas9 for
in-vivo
gene editing and testing of biochemical signalling pathways.
Abstract P484: Defining Disease Specific Cardiac Regulatory Networks
Authors: Lazarevic Sonja , Wang Zhezhen , Shen Kaitlyn , Gadek Margaret , Perez-Cervantes Carlos , Park David , Moskowitz Ivan
Published: November 2021

The transcriptional basis of homeostasis and disease is implied by the non-coding nature of genetic variation identified by GWAS. Functional genomic approaches to unveil the gene regulatory networks (GRNs) relevant to disease are therefore a high priority. Most models for transcriptional dysregulation presume that perturbation of a wild-type gene regulatory network causes disease risk. For example, the T-box transcription factor (TF) TBX5, is essential for atrial rhythm homeostasis and directly drives a physiologically relevant GRN composed of cardiac channel genes. Alternatively, the expression of many genes pertinent to cardiac pathology are upregulated after the removal of
Tbx5,
implicating a disease-specific GRN absent from the wild-type atrium. We applied TF-dependent noncoding RNA (ncRNA) profiling, using differential deep ncRNA sequencing from atria of wild-type and
Tbx5
mutant mice, to identify TBX5-dependent enhancers and ncRNAs that were only activated following TBX5 removal. We hypothesized that these regulatory elements would reveal disease-response enhancers, essential for coping with atrial dysfunction. To identify the cell-specific regulatory elements, we generated cell-type specific Assay from Transposase-Accessible Chromatin (ATAC) datasets for left atrial tissue, cardiac fibroblasts, and cardiomyocytes. Overlap with the
Tbx5
-repressed ncRNAs defined candidate cell-type specific regulatory elements. Candidate regulatory elements were identified upstream of Sox9, a known modulator of cardiac fibrosis, along with other cardiac stress-response pathways, including mediators of TGF-β signaling. Activation of the enhancer at Sox9 was confirmed in isolated cardiac fibroblasts treated with TGF-β. We hypothesized that the disease-acquired GRN in TBX5 mutant atria may be generalizable to other cardiac insults. We therefore examined the transcriptional and genomic changes in the left atria of the heart failure Transverse Aortic Constriction (TAC) mouse model. This analysis revealed remarkable correlation between differentially expressed genes and ncRNAs between TAC and TBX5 mutant disease models. The conservation of the coding and non-coding transcriptional response between arrhythmia and heart failure models supports a paradigm of a common disease-specific GRN that mediates the physiologic consequences of distinct cardiac diseases.
Abstract P486: Decellularized Extracellular Matrix Microparticles Promote Heart Regeneration And Neovascularization In Post-mi Mice
Authors: Wang Xinming , Ansari Ali , Pierre Valinteshley , Senyo Samuel
Published: November 2021

Hypothesis and objective:
We hypothesize that solid hydrogel particles will improve therapeutic efficacy of hydrogels derived from decellularized fetal hearts (dECM) on myocardial infarction (MI). In this study we developed dECM microparticles (MPs) to compare with liquid hydrogel. The objectives are 1) develop dECM MPs for increased stability at site of injection; 2) establish reduced degradation
in vivo
; and 3) evaluate potential regenerative benefits from dispersed particles of dECM.

Methods:
Microparticles were generated by electrospray of solubilized dECM. Microparticles were injected immediately after coronary artery ligation in adolescent mice. Echocardiography and histology were conducted 21days post-MI.

Results:
Electrospray with emulsification produced solid dECM microparticles ranging in size from nanometers to microns depending on parameters. The dECM MPs showed prolonged release of proteins and decreased degradation rate
in vitro
compared to dECM hydrogel. The dECM MPs were observed to protect cardiac function (Fig. 1), lower fibrosis, stimulate cardiogenesis, and promote vascularization compared to MI control. Cardiogenesis and neovascularization were observed with dECM microparticles treatment compared to hydrogel treatment (Fig. 2). This study suggests the solid dECM particles provides unique advantages as an injectable therapy directed at the ischemic heart.
Abstract P487: Impact Of Oxidative Dna Damage On Heart Health
Authors: Lowran Kaitlin , Wu Colin , Petersen Ingrid
Published: November 2021

The accumulation of DNA damage in human cardiomyocytes causes apoptosis which can lead to heart failure or other cardiovascular diseases. Although the effects of oxidative stress on heart health and on the DNA Damage Response network are well-known, the two fields have evolved as separate areas of research. The precise impact of oxidative DNA damage on cardiomyocyte contractile function still remains poorly understood. The human FANCJ helicase participates in multiple DNA repair pathways, including interstrand crosslink repair and double-stranded break repair. We have shown previously that FANCJ targets and unfolds 8-oxoguanine modified DNA secondary structures that arise from oxidative damage. We predict that human cardiomyocytes expressing mutations of FANCJ would be more susceptible to oxidative DNA damage and will negatively influence their contractile motion. To test this, hiPSC-CMs were treated with hydrogen peroxide, camptothecin, or bleomycin to induce different forms of DNA damage. The relative abundance of single-stranded DNA breaks and double-stranded DNA breaks were determined by modified comet assays, while contractile function was monitored using video-based detection methods. Cells that overexpress FANCJ protein were able to overcome the chemical stress from hydrogen peroxide. On the contrary, cells that produce a FANCJ K141/K142AA variant, which was previously characterized in the lab, resulted in a hypersensitivity to double-stranded DNA breaks. Based on this evidence, FANCJ plays a vital role in alleviating the effects of oxidative stress. Our long-term goal is to use the established methods to develop functional assays that characterize the cardiovascular risks of other FANCJ variants. These assays can be used to develop screening methods to identify patients who may be predisposed to FANCJ-associated cardiovascular diseases.
Abstract P491: Myosin-7 E848G Mutation Induces P53-associated Cell Death That Leads To Impaired Tissue Contractility In Patient-derived Induced Pluripotent Stem Cell Model Of Hypertrophic Cardiomyopathy
Authors: Loiben Alexander , Friedman Clayton , Chen Wei-Ming , Chung Benjamin , Yang Kai-chun
Published: November 2021

Introduction:
Familial hypertrophic cardiomyopathy (HCM), affecting 1 in 500 adults, is characterized by idiopathic thickening of the heart and occasional impaired systolic function. Mechanisms through which cardiac sarcomeric mutations manifest in HCM are poorly understood.

Hypothesis:
We previously identified a novel MYH7 E848G mutation associated with HCM. We hypothesize E848G induces cell death that results in impaired tissue contractility in a dose-dependent manner.

Methods:
We created MYH7 expressing CMs with WT/WT, E848G/WT, or E848G/E848G alleles by CRISPR/Cas9 gene-editing patient-specific induced pluripotent stem cells (hiPSCs). hiPSC-derived cardiomyocytes were metabolically purified and cocultured with stromal cells on PDMS posts to create 3D engineered heart tissues (EHTs) or cultured as a monolayer.

Results:
Day 65 monolayer E848G/E848G CMs had 48.5% effective cell number relative to WT/WT. p53 (2.80 ± .11-fold), p21 (7.24 ± .18-fold), and BAX (1.64 ± 0.14-fold) mRNA transcripts were upregulated in day 60 monolayer E848G/E848G relative to WT/WT. E848G/E848G EHTs (n = 12) exhibited lower maximum active twitch force (104.6 ± 18.2 μN) and smaller 2D projected area (5.31 ± 0.22 mm
2
) at day 14 relative to WT/WT (n = 15; 238.0 ± 20.4 μN; 6.87 ± 0.26 mm
2
). E848G/WT EHTs (n = 7) had intermediate twitch force (168.7 ± 12.2 μN) and 2D area (6.18 ± 0.36 mm
2
).

Conclusion:
These results suggest the MYH7 E848G mutation induces p53-associated cell death that leads to reduced tissue contractility. Ongoing studies will elucidate the molecular mechanism through which E848G activates cell death pathways.

Figure:
Representative EHTs. L-R: WT/WT, E848G/WT, E848G/E848G.
Abstract P494: Truncated Titin Protein In Dilated Cardiomyopathy
Authors: McAfee Quentin , Chen Christina Yingxian , Caporizzo Matthew , Morley Michael , Babu Apoorva , Jeong Sunhye , Brandimarto Jeff , Bedi Kenneth , Flam Emily , Cesare Joseph , Cappola Tom , Margulies Kenneth B , Prosser Benjamin , Arany Zoltan
Published: November 2021

Truncating variations in the gene coding for titin (TTNtv) have been known to cause dilated cardiomyopathy for nearly 20 years. Efforts to detect direct evidence of either haploinsufficiency or dominant negative mechanisms have thus far failed, leaving the mechanism open to controversy. By analyzing a collection of 184 post-transplant human hearts, 22 of which bear TTNtv’s, we show evidence supporting both haploinsufficient (lack of sufficient full length titin to maintain normal cardiomyocyte contractility) and dominant-negative (toxic gain of function due to truncated titin) mechanisms. Using allele specific proteomics as well as epitope specific agarose gel immunoblotting we show that TTNtv are present in human myocardium at the expected molecular weight and bear only the epitopes expected to be present in TTNtv protein. TTNtv associate with the sarcomere bearing insoluble fraction of human myocardium but are more weakly attached to the sarcomere than full length titin, consistent with their lack of thick filament and M-line attachment sites. We further show that DCM hearts bearing TTNtv have less full length titin than non-TTNtv bearing DCM hearts, by both total protein and in ratio to sarcomeric proteins, indicating TTN haploinsufficiency is also present in TTNtv hearts. This unambiguous detection of TTNtv protein in the myocardium of DCM combined with a reduction in full length titin supports a combined dominant negative and haploinsufficient mechanism of pathogenesis of TTNtv induced DCM.
Abstract P496: Exosomes From Brown Adipose Tissue Contain Cardiac Gene-regulatory Elements
Authors: Shettigar Vikram , Sturgill Sarah , Hu Benjamin , Baer Lisa , Peres Valgas Da Silva Carmem , Stanford Kriistin , Ziolo Mark T
Published: November 2021

Exosomes, extracellular vesicles <150 nm, are vehicles for transporting information (i.e., cargo) allowing tissue to tissue communication. Depending on the cargo, exosomes can have beneficial or detrimental effects. Brown Adipose Tissue (BAT) is a thermogenic organ that modulates metabolism. BAT is also an endocrine organ affecting function of various distant tissue. We have recently shown that BAT is an important modulator of the healthy and diseased heart. Adipose tissue is a large source of circulating exosomes, but the effects of BAT and changing BAT activity on circulating exosome number and cargo are unknown. Identifying the role of BAT in modulating exosome number and cargo is important since the myocardium is highly responsive to exosomes. We used various known approaches that increase BAT activity (cold exposure, BATcold) or decrease BAT activity (BAT removal (BATless), obesity (HFD), aging (old)) and examined the number and content of circulating exosomes. Upon BAT activation via cold exposure, there was a large increase in circulating exosome numbers (see figure). All approaches that results in decreased BAT activity resulted in a decrease in circulating exosome numbers (see figure). We further examined the role of changing BAT activity on the content (i.e., cargo) of the exosomes, specifically focusing on miRNA. Interestingly, changing BAT activity resulted in large changes to the content of the exosomes, with some miRNA increasing levels and other miRNA decreasing levels. Some of these identified miRNA have been shown to exert beneficial effects on the heart and many miRNA having no defined effect on cardiac function. We believe that these BAT activated exosomes have the combination and proportion of circulatory miRNA necessary to enhance and maintain heart function. There is a great need for new strategies and approaches for treatment of cardiovascular disease (CVD). Our data suggest that a novel treatment strategy for CVD can be derived from BAT exosomes.
Abstract P497: Myocardial Infarct Outcome And Weight Gain In Menopause Mice Are Mitigated During Probiotic Oligofructose Treatment
Authors: Hoyer-Kimura Christina H , Solomon Alice E , Fricks Joshua P , Kathuria Brij , Leber Christian , Salcedo Victoria , Duca Frank , Konhilas John P
Published: November 2021

The average woman spends over 40% of their life in menopause, during which estrogen’s protective roles are lost and women suffer an increased risk of developing inflammatory pathologies including obesity and worsened cardiovascular disease outcomes. Yet, safe, and effective menopause therapies are still unavailable.

The gut microbiome impacts both metabolic and circulating estrogen homeostasis. A dysbiotic (unhealthy) gut can contribute to pathologies that are also observed during menopause including increased obesity and worsened myocardial infarct (MI) outcomes. We have previously demonstrated that gut manipulation through prebiotics (Oligofructose [OFS]), attenuates myocardial infarct injury, improved glucose tolerance, and reduced weight gain in mice.

We hypothesize that loss of estrogen during menopause results in intestine morphological and microbial changes, worsens MI outcomes and increases weight gain, which can be mitigated with OFS treatment. Menopause was induced in female mice fed a high-fat-diet (HFD, 45% fat) via 4-vinylcycohexane diepoxide intraperitoneal injections (controls were injected with sesame oil), menopause (14+days diestrus) was determined by cytology. Following menopause, mice were treated for 6wks with 10% OFS supplemented into the diet (provided ad libitum; controls were fed HFD food only). Mice underwent 3day permanent ligation prior to sacrifice. We observed that menopause results in increased villi density in the duodenum and ileum, and that menopausal mice had a significant increase in both duodenum villi length and wall thickness. Menopause was associated with a shift in the gut microbiota composition. Relative abundance of Alistipes was reduced in menopausal mice, compared to premenopausal mice, and is restored with OFS treatment. Furthermore, menopausal mice had increased weight gain and infarct size compared to premenopausal mice, and OFS attenuated the differences between menopause and perimenopause weight gain over time and infarct size. Together these data demonstrate menopause is associated with intestinal morphological changes and shifts in the gut microbiome, and that in the presence of OFS infarct size and weight gain in menopausal mice are mitigated.
Abstract P506: Sirt6 Overexpression Inhibits Senescence And Inflammation In Human Mesenchymal Stem Cells
Authors: Neupane Rahul , Joladarashi Darukeshwara , Youker Keith , Krishnamoorthi Muthu Kumar , Bhimaraj Arvind , Guha Ashrith , Amirthalingam Thandavarayan Rajarajan
Published: November 2021

Background:
Mesenchymal Stem Cells (MSCs) offer regenerative and therapeutic potential in an injured tissue in diabetic conditions. But the functional efficiency of MSCs has been shown to decrease with diabetes and aging. This reduces the potential of cell-based therapy in diabetic patients. Recent studies have established the role of sirtuin family proteins in metabolic disease, inflammation, longevity, and DNA repair. However, their potential to be used as a therapeutic target in cardiomyopathy and heart failure remain unexplored.

Objective:
To investigate the role of Sirtuin 6 (SIRT6) in mesenchymal stem cells senescence and cardiac regeneration.

Methods and Results:
We performed genomics and proteomics in the human control and diabetic heart tissues collected from the heart transplant. Human MSCs were treated with high glucose and mouse MSCs were derived from the bone marrow of diabetic
db/db
mice for this study. We found that SIRT6 expression is reduced in the myocardium of diabetic patients compared to non-diabetic controls. The SIRT6 expression decreased in high glucose-treated human MSCs compared to mannitol-treated control MSCs as well as in
db/db
mice MSCs compared to control mice MSCs. These high glucose-treated human MSCs and
db/db
mice MSCs showed increased expression of senescence and inflammation-related markers like that in diabetic human myocardium. Furthermore, we used small interfering RNA (SIRT6-siRNA) in human MSCs to knock down the SIRT6 gene and validated our findings. Indeed, the knockdown of SIRT6 promoted senescence and inflammation in those MSCs. Finally, we used adeno-associated viruses (AAV-SIRT6) to overexpress SIRT6 in MSCs treated with high glucose and performed proteomic analysis. SIRT6 overexpression in high glucose-treated MSCs reduced the expression of senescence and inflammation related genes and proteins.

Conclusion:
Our results highlight the importance of SIRT6 in diabetic myocardium and its role in balancing senescence and inflammation in MSCs. The validation of such
in vitro
studies in a diabetic mouse model (
db/db
) along with transplantation of SIRT6 overexpressed
db/db
MSCs into the myocardium of diabetic mice could open doors to successfully use MSC therapy in diabetic patients in the future.
Abstract P508: Hyperglycemia Exacerbated Sars-cov-2 Spike Protein-induced Electrophysiological Dysfunctions In Hipsc-derived Cardiomyocytes
Authors: Wong Chloe , Nguyen Thong B , Tong Michael , Matsui Takashi , Zhang Yiqiang
Published: November 2021

Background:
Cardiac arrhythmias, heart failure, and myocarditis are common symptoms associated with coronavirus 2 (SARS-CoV-2) infection. The cytopathic effect of SARS-CoV-2 is initiated by spike protein (S-protein) binding to the ACE2 receptor. However, cellular mechanisms of SARS-CoV-2 causing cardiac complications, particularly in a subset of the population such as diabetic patients, remain unclear.

Aims:
To determine electrophysiological cytopathic effects of SARS-CoV-2 S-protein in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).

Methods and Results:
CMs were differentiated from WTC-11, then re-plated on 48-well microelectrode array (MEA) plates and cultured under normal or hyperglycemic conditions (Glucose 5 mM (NG), and Glucose 20 mM (HG)) for three days. These HiPSC-CMs were treated with recombinant S-protein with or without insulin supplementation. Cardiac modules in AxIS MEA program were used to acquire and analyze electric and contractile functions. Beat period and field potential duration were significantly prolonged, while conductance velocity and spike slope were accelerated in HG without insulin compared to that in HG with insulin (p<0.05). S-protein-induced perturbations in local extracellular action potential (LEAP) matrices (e.g., LPD30, 50, 90) and Contractility matrices in HG were mitigated by insulin treatment. Higher concentration S-protein induced perturbations in normal hiPSC-CMs (NG+insulin). One day after S-protein treatment, CM functions appeared comparable to the pretreatment state, although changes in some electro-contractile matrices persisted.

Conclusion:
These results demonstrate SARS-CoV-2 viral structural protein can induce acute electrophysiological dysfunctions in hiPSC-CMs, which is influenced by glucose level. This establishes a new model for dissecting the mechanism of cardiac complications in viral infection.
Abstract P511: Sirtuin 5 Overexpression Protects Against Pressure Overload-Induced Ventricular Dysfunction
Authors: Guo Hanjia , Baliirra Rachael , Skinner Mary , Kumar Surinder , Michan Shaday , Day Sharlene , Sinclair David , Lyssiotis Costas , Stein Adam , Lombard David B
Published: November 2021

Heart failure (HF) is defined as an inability of the heart to pump blood sufficiently to meet the metabolic demands of the body. HF with systolic dysfunction is caused by a progressive decline in contractile function and chronic hemodynamic overload, and characterized by ventricular hypertrophy and remodeling, neurohormonal compensation mechanisms, and myocardial damage. Transverse aortic constriction (TAC) is a well-established model for inducing hypertrophy and HF in rodents. Mice globally deficient in sirtuin 5 (SIRT5), a NAD
+
-dependent deacylase, are hypersensitive to cardiac stress and display increased mortality after TAC. Prior studies assessing SIRT5 functions in the heart have all employed loss-of-function approaches. In this study, we generated SIRT5 overexpressing (SIRT5OE) mice, and evaluated their response to chronic pressure overload using TAC. Compared to littermate controls, elevated SIRT5 levels promoted maintenance of cardiac contractile function after 4 weeks of pressure overload, at which point control mice had developed systolic dysfunction, characterized by decreased EF, coupled with ventricular dilation, remodeling and fibrosis. Transcriptomic analysis revealed that SIRT5 suppresses key HF sequelae, including metabolic switch from fatty acid oxidation to glycolysis and immune activation (
i.e.,
TGFβ, IL6, Renin-Angiotensin, and NFAT, and fibrotic signaling pathways). We conclude that SIRT5 is a limiting factor in the preservation of cardiac function in response to experimental pressure overload.
Abstract P514: The Role Of Sweet And Umami Taste Receptors In The Heart
Authors: Papadaki Maria , Osorio- Valencia Sara , Kirk Jonathan A
Published: November 2021

The tongue can distinguish between five different tastes via the taste receptors, which are G-protein coupled receptors (GPCRs). There are two classes of taste receptors, the TAS1 (T1) and TAS2 (T2) families, and the T1R1-T1R3 dimer senses the umami taste and T1R2-T1R3 senses the sweet taste. Recently, the taste receptors have also been found in the brain, lungs, intestine and pancreas, where they sense changes in the nutrient environment and respond through GPCR signalling. Given the importance of glucose and amino acid metabolism in the heart, we hypothesized that the sweet and umami taste receptors have an important function in the heart. Using a variety of technologies and disease states, we have identified that T1R1, T1R2 and T1R3 are expressed in the heart. More specifically, mass spectrometry of a dog model of dyssynchrony has shown the presence of T1R1, T1R3 and T1R2. RNA seq of human patients who received a Left Ventricular Assist device and those who did not also revealed the presence of T1R1 and T1R3. The expression of these proteins was also confirmed using Western blot. We further showed T1R2 and T1R3 protein is localized in the plasma membrane of the cardiomyocytes by immunofluorescence (colocalized with Na/K ATPase) and PM enrichment. When we compared the taste receptor protein levels in dilated cardiomyopathy (DCM) compared to donor heart tissue, we found that T1R2 was overexpressed in DCM, showing that taste receptors may be important in nutrient sensing in disease. Furthermore, when neonatal rat ventricular myocytes were treated with sweet and umami agonists (aspartame for the sweet taste receptor and monosodium glutamate for the umami receptor), they had increased calcium transients as shown by an increase in peak calcium. Cardiomyocytes treated with aspartame also showed a decrease in time to relax. We hypothesize that in the heart, sweet and umami receptors induce positive inotropy upon a change in nutrient environment.