bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P340: Histone Methyltransferase Smyd1a Protects Heart From Ischemic Injury By Regulating Mitochondrial Respiration
Authors: Szulik Marta , Valdez Steven , Walsh Maureen , Bia Ryan , Davis Kathryn , Horiuchi Emilee , O'Very Sean , Laxman Anil , Miller Mickey R , Wang Li , June Garcia-Llano , Tracy Christopher , Drakos Stavros G , Funai Katsuhiko , Chaudhuri Dipayan , Boudina Sihem , Franklin Sarah
Published: November 2021

SMYD1a, a myosin-specific histone lysine methyltransferase, plays a major role in regulating disease-induced remodeling in the adult heart. Previously, we demonstrated that the inducible loss of this chromatin-bound enzyme is sufficient to induce cardiac hypertrophy and failure
in vivo
, which is preceded by downregulation of mitochondrial proteins involved in oxidative phosphorylation (OXPHOS), and reduction of mitochondrial respiration capacity. However, our most recent data in transgenic mice (TG) displaying inducible, cardiomyocyte-specific overexpression of SMYD1a show that these mice are protected from ischemic injury after permanent occlusion (PO) of the LAD manifested by reduced infarct size and cardiac dysfunction compared to littermate controls (WT), suggesting that SMYD1 plays a protective role in the heart and mitigates disease-induced remodeling. Additionally, global proteomic evaluation of cardiac tissue from TG mice showed unique expression of metabolic enzymes, including proteins from the electron transport chain, and our high-resolution mitochondrial respirometry analysis showed that overexpression of SMYD1a leads to increased oxygen consumption rates through Complex I and II. To further asses OXPHOS efficiency in TG mice we subjected them to permanent occlusion of the LAD and evaluated ATP production rates in isolated mitochondria from TG and WT mice, by measuring the molar amount of ATP produced per mole of atomic oxygen consumed (known as ATP:O ratio). Interestingly, we observed a significant increase in ATP:O ratio in TG mice 24h after PO suggesting that they are much more efficient at producing ATP. Finally, we show that the global regulation of mitochondrial respiration in TG mice occurs through transcriptional control of
Ppargc1α
. Our results confirm that cardiac expression of
Ppargc1α
was significantly reduced in WT mice (48h after PO) but maintained at basal levels in TG mice, which also corroborated with our ChIP-qPCR data showing SMYD1a binding to the
Ppargc1α
promoter and regulating its expression. Overall, these results show that SMYD1a can mitigate ischemic injury and adverse remodeling in the adult myocardium, which occurs through
Ppargc1α
expression and regulation of cardiac energetics and metabolism.
Abstract P341: Juvenile Exposure To Doxorubicin Differentially Alters The Cardiac Response To Angiotensin II And Isoproterenol In Adult Mice
Authors: Grant Marianne K , Zordoky Beshay N
Published: November 2021

Hypertension is the most significant risk factor for heart failure in doxorubicin (DOX)-treated childhood cancer survivors. We previously developed a two-hit mouse model of juvenile DOX-induced latent cardiotoxicity that is exacerbated by adult-onset angiotensin II (ANGII)-induced hypertension. Nevertheless, it is still not known how juvenile exposure to DOX would predispose the heart to other cardiovascular pathologic stimuli that do not cause hypertension. The objective of this work was to compare the effects of ANGII to those of isoproterenol (ISO) in adult C57BL6/N mice pre-exposed to DOX as juveniles. Five-week-old male mice were administered a low dose of DOX (4 mg/kg/week) or saline for 3 weeks and then allowed to recover for 5 weeks. Thereafter, mice were either infused with ANGII (1.4 mg/kg/day) or injected with ISO (10 mg/kg/day) for 14 days. Juvenile exposure to DOX abrogated the hypertrophic response to both ANGII and ISO, while it failed to correct ANGII- and ISO-induced upregulation in the hypertrophic markers ANP and BNP. ANGII, but not ISO, worsened cardiac function in DOX-exposed mice as measured by echocardiography. Cardiac fibrosis was also exacerbated by ANGII, but not ISO, in DOX-exposed mice as evident by Masson’s trichrome staining and upregulation of the inflammatory and fibrotic markers,
Cox-2, Col1a1, Col3a1
, and
galectin-3
. In conclusion, the current work demonstrates that ANGII causes more severe deterioration in cardiac function and adverse cardiac remodeling in DOX-exposed mice when compared to ISO. The comparison between DOX/ANGII and DOX/ISO models is critical to understanding why hypertension is the most significant risk factor for heart failure in DOX-treated childhood cancer survivors and thereby devising effective therapeutic strategies against this significant clinical problem.
Abstract P343: Physiologic Mechanical Stress In An Induced Pluripotent Stem Cell Derived Cardiomyocyte Model Of Duchene Muscular Dystrophy-related Cardiomyopathy Treated With A Membrane Resealant
Authors: Fullenkamp Dominic E , Curtin Jodi L , Amaral Ansel P , Harris Sloane I , Burridge Paul W , Demonbreun Alexis R , McNally Elizabeth M
Published: November 2021

Heart failure is leading cause of morbidity and mortality in the X-linked disease Duchenne muscular dystrophy (DMD). DMD is due to mutations the gene encoding dystrophin. Dystrophin localizes to the costamere in skeletal and cardiac muscle and is part of the larger dystrophin complex, which forms a critical connection linking the sarcomere to extracellular matrix. Disruptions in this complex lead to membrane fragility and multiple forms of muscular dystrophy, most of which have significant cardiac involvement. Therapeutic strategies for DMD include FDA-approved agents for exon skipping, as well as micro-dystrophins gene therapy, which is currently in clinical trials. Despite this progress, there is inadequate information as to how these and other novel agents will affect the DMD heart. Given the critical importance of cardiac muscle efficacy for any therapeutic for DMD and importance of membrane fragility in the disease phenotype, we assessed the susceptibility of patient-derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) harboring an out-of-frame exon 46-47
DMD
deletion. iPSC-CMs were reprogrammed via a standard approach and differentiated, expanded, and purified by published methods. Troponin T flow cytometry was performed to and a minimum troponin T positivity >85% positivity was set for inclusion. DMD and control cells were plated onto flexible silicone membranes and subjected to equibiaxial strain as physiologic mechanical stressor, consistent with the inciting pathologic insult in DMD. Troponin and LDH release were assessed as clinically-relevant biomarkers of injury. Physiologic stress parameters were defined using troponin and LDH release relative to unstressed conditions. A membrane resealant being developed for treating DMD was shown to reduce troponin and LDH release in DMD iPSC-CMs, and also showed benefit in control cells. This work provides a ready platform for assessing therapeutics that target not only DMD-related cardiomyopathy, but other forms of cardiomyopathy and myocardial injury.
Abstract P345: A MRI Displacement-based Deep-learning Semantic Segmentation Tool For Left-ventricular Longitudinal Strain Analysis In Cardiotoxicity
Authors: Kar Julia , Cohen Michael V , Poorsala Teja , McQuiston Samuel A , Revere Cheri , Judice-Yates Angela , Malozzi Christopher M
Published: November 2021

Global longitudinal strain (GLS) computed in the left-ventricle (LV) is an established metric for detecting cardiotoxicity in breast cancer patients treated with antineoplastic agents. The purpose of this study was to develop a novel, MRI-based, deep-learning semantic segmentation tool that automates the phase-unwrapping for LV displacement computation in GLS. Strain analysis via phase-unwrapping was conducted on 30 breast cancer patients and 30 healthy females acquired with the Displacement Encoding with Stimulated Echoes (DENSE) sequence. A ResNet-50 deep convolutional neural network (DCNN) architecture for automated phase-unwrapping, a previously validated ResNet-50 DCNN for chamber quantification and the Radial Point Interpolation Method were used for GLS computation (Figure 1). The DCNN's performance was measured with F1 and Dice scores, and validated in comparison to the robust transport of intensity equation (RTIE) and quality guided phase-unwrapping (QGPU) conventional algorithms. The three techniques were compared by intraclass correlation coefficient with Cronbach’s alpha (C-alpha) index. Classification accuracy with the DCNN was F1 score of 0.92 and Dice score of 0.89. The GLS results from RTIE, QGPU and DCNN were -16.0 ± 2%, -16.1 ± 3% and -15.9 ± 3% (C-alpha = 0.89) for patients and -18.9 ± 3%, -19.0 ± 4% and -18.9 ± 3% (C-alpha = 0.92) for healthy subjects. Comparable validation results from the three techniques show the feasibility of a DCNN-based approach to LV displacement and GLS analysis. The dissimilarities between patients and healthy subjects demonstrate that DCNN-based GLS computation may detect LV abnormalities related to cardiotoxicity.
Abstract P347: Transcriptional Features Of Biological Age Maintained In Cultured Cardiac Interstitial Cells
Authors: Echeagaray Oscar E , Kim Taeyong , Casillas Alex , Monsanto Megan M , Sussman Mark A
Published: November 2021

Introduction:
Ex vivo expansion of cells is necessary in regenerative medicine to generate large populations for therapeutic use. Adaptation to culture conditions prompt an increase in transcriptome diversity and decreased population heterogeneity in cKit+ cardiac interstitial cells (cCICs). The “transcriptional memory” influenced by cellular origin remains unexplored and is likely to differ between neonatal versus senescent input cells undergoing culture expansion.

Approach:
cCICs isolated from neonatal and adult cardiac tissue (Left Ventricular Assist Device; LVAD). Single cell libraries from
in vitro
expanded cells were prepared following five passages as previously demonstrated by our group to promote transcriptional homogeneity. “Transcriptional memory” was surveyed via bioinformatic analysis including unsupervised clustering, differential expression analysis, gene ontology and pathway analysis. Transitional states were assessed through pseudotime analysis.

Results:
Cell cycle imprint associated with biological age after culture was observed in Neonatal cCICs via upregulation of G2M genes. LVAD derived cCICs retained a widespread senescent profile, in particular high expression of interleukins and elements of the senescence associated secretory phenotype (SASP). Nuclear pore complex TPR and UBC9 and translation initiation factors, displayed age-associated downregulation of elements in the RNA transport and processing pathway. Pathway and co-expression analysis of fibroblast markers Ddr2, Tcf21, Vimentin, Periostin and Collagen deposition markers indicated a primed fibrotic phenotype in senescent cells. A small subset of cCICs exist in a transcriptional continuum between “youthful” phenotype and the damaged microenvironment of adult tissue in which they were embedded.

Conclusion:
The influence of age, pathology and the cellular stress associated to the
in vivo
tissue microenvironment persist after culture adaptation, influencing targets of 1) cell cycle, 2) senescence associated secretory phenotype (SASP), 3) RNA transport, and 4) ECM-receptor/fibrosis. The connate transcriptional phenotypes offer fundamental biological insight and highlights cellular input as a consideration in culture expansion and adoptive transfer protocols.
Abstract P348: Pathophysiological Basis Of A Compound Variant In Calcium And Sarcomere Regulation Causing Cardiac Arrhythmias And Hypertrophic Cardiomyopathy
Authors: Kumar Mohit , Bazrafshan Sholeh , Dhandapany Perundurai , Haghighi Kobra , Singh Rohit , Knoell Ralph , Kranias Evangelia , Sadayappan Sakthivel
Published: November 2021

Rationale:
Hypertrophic cardiomyopathy (HCM) is common inheritable heart disease. HCM is highly associated with arrhythmias and/or sudden death. Studies show that molecular defects in calcium handling impairing the cardiomyocyte contractility is a predominant cause. However, the pathophysiology underlying HCM with arrhythmias is not well understood, hindering the identification of novel therapies.

Objective:
To investigate the pathophysiological consequences of compound variants, consisting of Histidine Rich Calcium Binding Protein gene (
HRC
S96A
) and an intronic 25bp deletion in cardiac myosin binding protein-C (
MYBPC3
Δ25bp
).

Methods and Results:
Clinical data revealed that co-segregation of HRC
S96A
and MYBPC3
ΔInt32
results in cardiac arrhythmia, heart failure, and sudden cardiac death in South Asians. To determine the cellular/molecular trigger underlying the pathophysiology of this dual variant, we used humanized, knock-in, heterozygous mouse models, including
HRC
S81A
(equivalent to
HRC
S96A
)
MYBPC3
Δ25bp
,
HRC
S81A
/
MYBPC3
Δ25bp
(double variant, DV), and wild-type controls. Echocardiography revealed a significant decrease in the percentage of ejection fraction and fractional shortening in DV mice, as well as the presence of diastolic dysfunction, at 12 weeks of age, compared to single-variant and wild-type mice. Electrocardiogram tracing of DV mice showed the presence of stress-induced arrhythmias, such as ventricular tachycardia after caffeine and epinephrine administration. Using isolated cardiomyocytes
in vitro
, Calcium transient experiments indicated a significant decrease in fractional shortening, Ca
2+
transient amplitude, and a higher number of after-contractions in cardiomyocytes from DV mice. DV mouse hearts showed increased phosphorylation of CaMKII and SR Ca
2+
leak by cardiomyocytes. Inclusion of the CaMKII inhibitor KN-93 rescued the increases in SR Ca
2+
leak and in aftercontractions.

Conclusion:
Impaired Ca
2+
-handling, owing to the
HRC
S96A
variant, aggravates SR Ca
2+
leak and aftercontractions in
MYBPC3
Δ25bp
cardiomyocytes, subsequently triggering cardiac arrhythmias and sudden death
in vivo
.
Abstract P351: The Post-transcriptional Regulations Of Centrosomal
Plp
Mrna In
Drosophila
Authors: Fang Junnan
Published: November 2021

Centrosomes, functioning as microtubule organizing centers, are composed of a proteinaceous matrix of pericentriolar material (PCM) that surrounds a pair of centrioles.
Drosophila
Pericentrin (Pcnt)-like protein (PLP) is a key component of the centrosome that serves as a scaffold for PCM assembly. The disruption of
plp
in
Drosophila
results in embryonic lethality, while the deregulation of Pcnt in humans is associated with MOPD II and Trisomy 21.We recently found
plp
mRNA localizes to
Drosophila
embryonic centrosomes. While RNA is known to associate with centrosomes in diverse cell types, the elements required for
plp
mRNA localization to centrosomes remains completely unknown. Additionally, how
plp
translation is regulated to accommodate rapid cell divisions during early embryogenesis is unclear. RNA localization coupled with translational control is a conserved mechanism that functions in diverse cellular processes. Control of mRNA localization and translation is mediated by RNA-binding proteins (RBPs). We find PLP protein expression is specifically promoted by an RNA-binding protein, Orb, during embryogenesis; moreover,
plp
mRNA interacts with Orb. Importantly, we find overexpression of full-length PLP can rescue cell division defects and embryonic lethality caused by
orb
depletion. We aim to uncover the mechanisms underlying embryonic
plp
mRNA localization and function and how Orb regulates
plp
translation.
Abstract P355: Comparable Impairment Of Vascular Endothelial Function By A Wide Range Of Electronic Nicotine Delivery Devices
Authors: Rao Poonam , Tan Kelly , Han Daniel D , Derakhshandeh Ronak , Springer Matthew L
Published: November 2021

Introduction:
Electronic nicotine delivery systems (ENDS; i.e., vaping devices) such as e-cigarettes, heated tobacco products, and newer coil-less ultrasonic vaping devices are promoted as less harmful alternatives to combustible cigarettes. However, the cardiovascular health effects of these devices are understudied. We investigated whether exposure to aerosol from a wide range of ENDS devices with and without nicotine, including a new ultrasonic vaping device, impairs endothelial function.

Hypothesis:
ENDS aerosols irrespective of nicotine, flavors, and heating coil impair endothelial function comparably to smoke from cigarettes.

Methods:
We exposed 11 groups (n=8), of anaesthetized rats to aerosol from propylene glycol (PG), vegetable glycerin (VG), PG+VG without nicotine, a USONICIG Zip ultrasonic vaping device, previous generation e-cigarettes (tank style with freebase nicotine), 5% nicotine JUUL pods of three flavors (Virginia tobacco, Mango, and Menthol), and an IQOS heated tobacco product; with Marlboro Red cigarette smoke and clean air as controls, to a single session of 10 cycles of pulsatile 5s exposure over 5 minutes. Endothelial function was quantified as arterial flow-mediated dilation (FMD) using micro-ultrasound.

Results:
Aerosol/smoke from all conditions except air significantly impaired FMD (Figure: each colored line denotes one rat, black horizontal bars denote means). The extent of impairment ranged from 40%-67% although there were no significant differences between groups. Nicotine absorption into the blood varied widely from these undiluted aerosols (those containing nicotine), with IQOS being the highest, and USONICIG and previous generation e-cig being the lowest.

Conclusions:
A single session of exposure to aerosol from a wide range of ENDS, including multiple types of e-cigarettes, a heated tobacco product, and an ultrasonic vaping device, all impair endothelial vascular function comparably to combusted cigarettes.

Key words:
JUUL, IQOS, ultrasonic vaping device, e-liquids, flow-mediated dilation
Abstract P356: Dual Dysfunction Of Kir2.1 Underlies Conduction And Excitation-contraction Coupling Defects Promoting Arrhythmias In A Mouse Model Of Andersen-tawil Syndrome Type 1
Authors: Macias Alvaro , González-Guerra Andrés , Moreno-Manuel Ana I. , Cruz Francisco M. , García-Quintáns Nieves , Gutiérrez Lilian K. , Roche-Molina Marta , Bermúdez-Jiménez Francisco J. , Andrés Vicente , Vera-Pedrosa Maria L. , Martínez-Carrascoso Isabel , Bernal Juan A. , Jalife José
Published: November 2021

Background:
Andersen-Tawil syndrome type 1 (ATS1), caused by trafficking deficient mutations in the gene
KCNJ2
coding the inward rectifier K
+
channel Kir2.1, is associated with life-threatening arrhythmias, which in some patients resemble catecholaminergic polymorphic ventricular tachycardia (CPVT), but the mechanisms are poorly understood. We tested the hypothesis that dysfunction of two different populations of mutant Kir2.1 channels, one at the sarcolemma, and the other at the sarcoplasmic reticulum (SR) membrane, directly alters conduction and intracellular calcium dynamics, respectively, to promote the ATS1 phenotype and arrhythmias that resemble CPVT.

Methods:
We generated a new mouse model of ATS1 by a single i.v. injection of cardiac specific adeno-associated viral (AAV) transduction with Kir2.1
Δ314-315
.
In-vivo
and cellular, structural and functional analyses of the model were carried out by electrocardiogram (ECG), magnetic resonance imaging (MRI), intracardiac stimulation, patch-clamping, membrane fractionation, western blot, immunolocalization and live calcium imaging.

Results:
Our mouse model carrying mutant Kir2.1
Δ314-315
recapitulated the ATS1 phenotype without modifying ventricular function. On ECG, Kir2.1
Δ314-315
mice had prolonged PR, QRS and QT intervals and occasional U waves. They showed significantly slower conduction velocities than wildtype mice in response to flecaidine-induced Na
+
-channel blockade, additional QT prolongation in response to isoproterenol, and increased vulnerability to cardiac fibrillation. Cardiomyocytes from Kir2.1
Δ314-315
mice had significantly reduced inward rectifier K
+
and Na
+
inward currents, depolarized resting membrane potential and prolonged action potential duration. Immunolocalization in wildtype cardiomyocytes and skeletal muscle cells revealed a novel SR microdomain of functional Kir2.1 channels contributing to intracellular Ca
2+
homeostasis. Kir2.1
Δ314-315
cardiomyocytes showed defects in SR Kir2.1 localization and function, which contributed to abnormal spontaneous Ca
2+
release events.

Conclusions:
Cardiac-specific AAV transduction with Kir2.1
Δ314-315
in mice recapitulates the ATS1 phenotype by disrupting localization and function of Kir2.1 channels at the SR, and the Kir2.1-Na
V
1.5 channelosome at the sarcolemma. These results reveal a novel dual mechanism of arrhythmogenesis in ATS1 involving defects in Kir2.1 channel trafficking and function at two different microdomains. They also provide the first demonstration at the molecular level of the mechanism underlying the overlap between ATS1 and CPVT associated with defects in intracellular calcium homeostasis.
Abstract P362: Gata4 Dimerization May Be A Target For Heart Failure Therapy
Authors: Shimizu Satoshi , Hara Kodai , Hishiki Asami , Sunagawa Yoichi , Katanasaka Yasufumi , Miyazaki Yusuke , Funamoto Masafumi , sari nurmila , Shimizu Kana , Hasegawa Koji , Hashimoto Hiroshi , Morimoto Tatsuya
Published: November 2021

Introduction:
Cardiac hypertrophy is regulated by activation of GATA4. Although GATA4 post-translational modification such as acetylation by p300 is well examined, the details of the activation mechanism of GATA4 are still unclear. The purpose of this study is to investigate whether GATA4 dimerization involved in transcriptional activation and cardiomyocyte hypertrophic responses.

Methods and Results:
A GST pull-down assay using GST fusion GATA4 full-length and deletion mutants demonstrated that GATA4 308-326, including the acetylation site, was required for the dimerization of GATA4. A DNA pull down assay showed that the C-zinc finger motif (256-295) and the acetylation site were required for the DNA binding capacity of GATA4. IP-WB using nuclear extract from HEK293T cells expressing FLAG- or HA-tagged GATA4 showed that co-expression of p300 increased the formation of the homo-dimer as well as acetylation of GATA4. The GATA4 homo-dimer was disrupted by both acetyl-deficient GATA4 and HAT-deficient p300. This result indicates that acetylation of GATA4 is important for dimerization of GATA4. Overexpression of the deletion mutant containing a GATA4 308-326 (G4D) prevented p300-induced GATA4 dimerization but not the p300 binding nor acetylation of GATA4. ChIP assay and DNA pulldown assay showed that G4D did not inhibit the p300-induced DNA binding of GATA4. In cardiomyocytes, the G4D inhibited phenylephrine-induced ANF and ET-1 promoter activities and cardiomyocyte hypertrophy. To perform the X-ray crystal structure analysis, recombinant GATA4 fragment including GATA4 308-326 was highly purified. The X-ray diffraction data of obtained crystals was collected. Resolution of the crystal was 3.1Å, which was insufficient for phase determine. To obtain a high-quality crystal, GATA4 fragment was crystallized in international space station, in collaboration with JAXA. Resolution of the crystal was 3.16Å, which was similar to the best data before obtained.

Conclusions:
These results suggest that GATA4 dimerization may play an important role in hypertrophy-response gene transcription. It is expected to elucidate the GATA4 dimerization mechanism and targeted this dimerization will lead to the development of a noble heart failure therapy.
Abstract P363: Annexin-A1 Deficiency Exaggerates Cardiac Remodeling In Angiotensin II-induced Hypertension
Authors: Jackson Kristy , Singh Jaideep , Ng Yen Zhi , Peng Cheng , Velagic Anida , Scott Tara , Woodman Owen L , Ritchie Rebecca H , Head Geoff , Qin Chengxue H
Published: November 2021

Introduction:
We have previously demonstrated that the naturally-occurring anti-inflammatory and pro-resolving protein Annexin-A1 (Anx-A1) limits the acute inflammatory response post myocardial infarction, but its impact on chronic inflammation, such as hypertension, has not been explored. This study aims to investigate the role of Anx-A1 in a preclinical model of hypertension, induced by angiotensin-II (Ang-II).

Methods:
15-week-old male C57BL/6 or ANXA1
-/-
were anesthetized (isoflurane, 2-4% v/v) and implanted with an osmotic minipump randomly assigned to receive Ang-II (0.7mg/kg/day) or vehicle (saline). Radiotelemetry recordings of blood pressure were taken at 10 intermittent timepoints from baseline to the end of the 29-day infusion period. Animals were euthanized with pentobarbitone (100mg/kg; i.p.) at endpoint and organ weights recorded and normalized to bodyweight. Left ventricle (LV) samples were stained with picrosirius red to assess total LV collagen deposition.

Results:
Ang II-induced mice at the end of the study had elevated mean arterial pressure (MAP), cardiac hypertrophy and fibrosis compared to normotensive mice (Table). Anx-A1 deficient mice given Ang II had an even greater increase in MAP and cardiac remodeling compared to WT. Interestingly, MAP of Anx-A1 deficient mice at baseline is significantly higher compare to C57BL/6 counterparts (Table).

Conclusion:
This is the first study to demonstrate that deficiency of Anx-A1 exaggerates cardiac remodeling in AngII-induced hypertension, suggesting that endogenous Anx-A1 might play previously unappreciated physiological role in regulating blood pressure. This supports the development of Anx-A1 based pharmacotherapy against hypertension-induced cardiac damage.
Abstract P375: Gender-based Cardio-protective Functional Dimorphism Of Bone Marrow Endothelial Progenitor Cells And Their Exosomes: Estrogen-independent Epigenetic Mechanisms
Authors: Gurrala Charan T , Garikipati Venkata , Cheng Zhongjian , Mallaredy Vandana , Cimini Maria , Joladarashi Darukeshwara , TRUONGCAO MAY , Wang Chunlin , Huang Grace , Gonzalez Carolina , Magadum Ajit , Roy Rajika , Benedict Cindy , Koch Walter J , Kishore Raj
Published: November 2021

Introduction:
Estrogen or estrogen receptor-dependent mechanisms in enhancing the cardioprotective efficacy of bone marrow endothelial progenitor cells (BM-EPC) is well-established in preclinical studies. However, the efficacy of estrogen does not reflect in the data from randomized cardiovascular clinical trials, suggesting an estrogen-independent role of female BM-EPC in eliciting enhanced cardiac protection compared to males.

Hypothesis:
Epigenetic mechanisms may contribute to the sex-specific dimorphism of Sca-1
+
/CD31
+
BM-EPC in regulating cell-homing, pro-angiogenic and anti-inflammatory functions in the ischemic myocardium leading to enhanced reparative function of female progenitor cells.

Methods & Results:
Transplantation of GFP-BM-mononuclear cells from male and female GFP transgenic mice into the BM of lethally irradiated recipient male C57BL/6 mice resulted in the enhanced mobilization of female Sca-1
+
CD31
+
/GFP
+
BM-EPC into circulation post-MI. A higher number of female BM-EPC homed to the ischemic myocardium and significantly improved LV functions and capillary density post-MI compared to male BM-EPC. Female BM-EPC showed increased expression of bFGF, VEGFR2, SDF-1α, and IL-10 genes, thereby efficiently promoted endothelial tube formation
in vitro
compared to male BM-EPC. Transplantation of female BM-EPC and their exosomes into post-MI male mice improved LV cardiac function, reduced scar size, and improved capillary density compared to male BM-EPC and exosomes. Male BM-EPC showed an increased expression of G9a/Ehmt2, an H3K9me3 methyltransferase, and Dnmt3a DNA methyltransferase compared to female BM-EPC. In contrast, Kdm6b/JMJD3, H3K27me3 demethylase was highly expressed in female BM-EPC compared to males. Treatment of BM-EPC of both sexes with 17-β-estradiol did not alter the expression of Kdm6b/JMJD3. Male BM-EPC highly expressed repressive gene marks, H3K9me3, and H3K27me3 compared to females. Compared to the male, BM-EPC from female and ovariectomized (OXV) female mice showed equally high expression of angiogenic genes ANGPT-1, MDK, PLAU, Tie-2, and VEGFR2 and lower levels of inflammatory cytokines, TNFα, IFNγ, IL-1β, and CCL3. Conditioned medium from female and OVX BM-EPC equally promoted enhanced migration and tube formation of HUVEC
in vitro,
compared to male BM-EPC.

Conclusions:
An estrogen-independent epigenetic mechanism likely governs the enhanced cardiac reparative properties of female BM-EPC.
Abstract P381:
Ecklonia Stolonifera
Okamura Extract Suppresses Hypertrophic Responses In Cardiomyocytes And Development Of Heart Failure By Inhibiting P300-HAT Activity
Authors: Katagiri Takahiro , Sunagawa Yoichi , Funamoto Masafumi , Katanasaka Yasufumi , Miyazaki Yusuke , Shimizu Kana , Shimizu Satoshi , Hasegawa Koji , Morimoto Tatsuya
Published: November 2021

Introduction:
Heart failure is the leading cause of death in the world. Cardiomyocyte hypertrophy is observed during the development of heart failure, suggesting that its inhibition is a potential target for the prevention and treatment of heart failure. In this study, we screened a natural compound library using cultured cardiomyocytes and found that
Ecklonia stolonifera
Okamura extract (ESE) suppressed cardiomyocyte hypertrophy. ESE, a perennial brown alga, has been reported to have various bioactive effects, such as antioxidant and anti-inflammatory activity, but its effect on heart failure is still unclear. Therefore, we investigated whether ESE has an inhibitory effect on cardiomyocyte hypertrophic response and on the progression of heart failure in post-myocardial infarction (MI) rats.

Methods and Results:
First, primary cultured cardiomyocytes from neonatal rats were treated with ESE and then stimulated with phenylephrine (PE) for 48 hours. ESE (1000 μg/mL) significantly suppressed PE-induced increases in cardiomyocyte surface area, hypertrophic response gene transcription, and acetylation of histone H3K9. An
in vitro
p300-HAT assay indicated that ESE directly inhibited p300-HAT activity (IC50: 505 μg/mL). Next, one week after the ligation of the left anterior descending artery, rats with moderate MI (left ventricular fractioning shorting (LVFS) <40%) were randomly assigned to three groups: vehicle (saline) (n=9), ESE (0.3 g/kg) (n=10), or ESE (1 g/kg) (n=10). Daily oral administration was repeated for 8 weeks. After treatment, LVFS was significantly higher in the ESE (1 g/kg) group (23.3 ± 0.7%, p<0.05) than in the vehicle group (16.6 ± 1.3%). Next, the hearts were isolated and histological analysis, evaluation of gene transcription, and measurement of histone H3K9 acetylation. were performed. ESE treatment significantly suppressed MI-induced increases both in myocardial cell diameter and in the mRNA levels of hypertrophic response genes. ESE also inhibited MI-induced perivascular fibrosis and the acetylation of histone H3K9.

Conclusion:
These results suggest that ESE suppresses both hypertrophic responses in cardiomyocytes and the development of heart failure by inhibiting p300-HAT activity. Further studies are needed to clarify the effectiveness of ESE for heart failure therapy.
Abstract P382: Transcriptomic Analysis Of Human Left Atrial Tissue Reveals Mitochondrial Gene Co-expression
Authors: Rennison Julie H , Wass Sojin Y , Lin Cheryl R , Sun Han , Hsu Jeffrey , Cantlay Catherine , Castel Laurie , Lovano Beth , McHale Meghan , Gillinov A M , Moravec Christine S , Smith Jonathan D , Chung Mina K , Barnard John , Van Wagoner David R
Published: November 2021

Atrial fibrillation (AF) risk is heritable. High rate electrical activity in AF requires increased energy. Atrial mitochondrial structure and function are altered in AF patients in an effort to generate adequate ATP through oxidative phosphorylation. Genomic studies have identified putative AF risk genes, but the association of AF risk genes with expression of mitochondrial genes is unclear. We tested the hypothesis that putative AF risk genes are co-expressed with mitochondrial genes that play a role in atrial energy production. RNA-seq was performed on left atrial appendage (LAA) tissues obtained from 251 cardiac surgery patients. RNA coexpression profiles were evaluated for 222 putative AF risk genes. Genes encoding proteins that localize to the mitochondria were identified using MitoCarta 2.0. Changes in metabolic pathways were detected using Ingenuity Pathway Analysis (IPA). Our analysis identified 128 AF risk genes that coexpressed with at least one mitochondrial gene. The highest level of mitochondrial gene coexpression was evident with PCCB, in which 30% (253 of 848) of coexpressed genes were mitochondrial. CASQ2 (24%, 104 of 431) and ASAH1 (20%, 37 of 182) also showed high levels of mitochondrial gene coexpression. The IPA Oxidative Phosphorylation Pathway was significantly altered (p<0.05) for 26 AF risk genes, with CASQ2 (9.42E-79), MYH6 (4.73E-78), YWHAE (4.96E-75), and TTN (4.30E-71) the most strongly associated (Table). Additionally, 12 AF risk genes coexpressed with genes encoded by mitochondrial DNA (mtDNA) (Table). ASAH1, CASQ2, MYH6, NACA, NUCKS1, PLN, TTN, and YWHAE coexpressed with all 13 mtDNA encoded components of the electron transport chain. Many AF risk genes show significant coexpression with mitochondrial genes. We propose that genetic risk scores based on these AF risk genes may identify a subset of AF patients that would benefit from AF therapies that enhance metabolic activity.
Abstract P383: 3D-collagen Matrix Enhanced Integrins And Matrix Metalloproteinases Expression In Cardiac Mesenchymal Cells
Authors: Muthusamy Senthilkumar , Nath Asha V , Ajit Shilpa , PR Anil K
Published: November 2021

Introduction:
Use of cardiac mesenchymal cells (CMCs) has been shown to improve cardiac function following myocardial infarction. Main drawback in cardiac cell therapy is the major loss of injected cells within few hours. Increase the retention of these injected cells could increase their efficacy, where cardiac patches with various cell types showed better outcome. Among, collagen patch plays lead role as a cell-laden matrix in cardiac tissue engineering. Creating a detailed understanding of how collagen matrix changes the cellular phenotype could provide seminal insights to regeneration therapy.

Hypothesis:
Growing CMCs in three dimensional (3D) collagen matrix could alter the expression of extracellular matrix (ECM) and adhesion molecules, which may enhance their efficacy.

Methods:
The bovine type I collagen was chemically modified and solubilized in culture medium with photo-initiator. The mouse CMCs were isolated and resuspended in collagen solution, printed using 3D bioprinter and UV-crosslinked to form 3D-CMC construct. The 3D-CMC construct was submerged in growth medium and cultured for 48h and analyzed for the expression of ECM and adhesion molecules (n=5/group). CMCs cultured in regular plastic tissue culture dish was used as control.

Results:
RT profiler array showed changes in the ECM and adhesion molecules expression, specifically certain integrins and matrix metalloproteinases (MMPs) in CMCs cultured 3D collagen construct compared to 2D monolayer. Subsequent qRT-PCR analysis revealed significant (p<0.01) upregulation of integrins such as Itga2 (2.96±0.13), Itgb1 (3.18±0.2) and Itgb3 (2.4±0.27) and MMPs such as MMP13 (37.2±3.36), MMP9 (5.23±1.06) and MMP3 (7.14±2.07). Western blot analysis further confirmed significant elevation of these integrins and matrix metalloproteinases at protein level. Collagen encapsulation did not alter the expression of N-cadherin in CMCs, which is a potential mesenchymal cadherin adhesion molecule.

Conclusion:
Integrin αβ heterodimers transduce signals that facilitate cell homing, migration, survival and differentiation. Similarly, MMPs plays vital role in cell migration and proliferation. Our results demonstrate that the 3D-collagen Niche enhances the expression of certain integrins and MMPs in CMCs. This suggest that the efficacy of CMCs could be magnified by providing 3D architecture with collagen matrix and further in vivo experiments would reveal functional benefits from CMCs for clinical use.
Abstract P385: Sarcolipin Reduction Prevents Mitochondrial Dysfunction In Dystrophic Myocardium
Authors: Mareedu Satvik , Fefelova Nadezhda , Xie Lai-hua , Babu Gopal J
Published: November 2021

Rationale & Hypothesis:
Sarcolipin (SLN), an inhibitor of sarco/endoplasmic reticulum Ca
2+
ATPase (SERCA) is abnormally high and enriched in the mitochondrial associated membrane (MAM) fractions of dystrophic hearts. We, therefore, hypothesized that in the dystrophic cardiomyocytes, SLN upregulation causes abnormal elevation of intracellular Ca
2+
(Ca
2+
i
) including Ca
2+
mishandling in the MAM region, resulting in sustained elevation of mitochondrial Ca
2+
(Ca
2+
m
)
,
which in turn affects mitochondrial structure and function.

Approach:

We chose dystrophin mutant and utrophin deficient, mdx:utr
-/-
mice for our studies and generated SLN haploinsufficient mdx:utr
-/-
(mdx:utr
-/-
:sln
+/-
) mice. Single-cell Ca
2+
transients and

Ca
2+
m
efflux were measured in isolated cardiomyocytes to determine

Ca
2+
i
handling and

Ca
2+
m
content, respectively. Mitochondrial membrane potential was assessed with membrane-permeant dyes in isolated cardiomyocytes followed by confocal imaging. Mitochondrial respiration was measured using Complex activity assays and Seahorse metabolic profiling in ventricular tissue lysates and isolated mitochondria respectively. Mitochondrial structure, number, and area were evaluated using electron micrographs.

Results:
Heterozygous deletion of the SLN gene normalized SLN expression and improved Ca
2+
i
cycling and prevented
Ca
2+
m
overload in dystrophic cardiac myocytes. Furthermore, reducing SLN expression prevents loss of membrane potential and improves mitochondrial respiration in the dystrophic cardiac myocytes. Transmission electron microscopic analyses of the dystrophic heart revealed significant cristae loss in the mitochondria globally as well as in the MAM-associated mitochondria. On the other hand, the reduction in SLN expression prevents these changes.

Conclusions:
In conclusion, reduction in SLN expression preserves the SR-mitochondrial interface and restores the mitochondrial function by mitigating the

Ca
2+

overload, membrane potential loss, and structural damage. These changes improve cardiac function and prevent the development of cardiomyopathy in

mdx:utr
-/-

mice. Our findings suggest that SLN reduction could be a potential therapeutic strategy for the treatment of Duchenne muscular dystrophy and associated cardiomyopathy.
Abstract P394: Angiotensin-(1-7) Attenuates Diastolic Dysfunction And Cardiac Remodeling In Murine Model Of Heart Failure With Preserved Ejection Fraction
Authors: Edalat Pariya , Gomes Karina , Ballasy Noura N , Jadli Anshul S , Belke Darrell D , Patel Vaibhav B
Published: November 2021

Background:
Heart failure with preserved ejection fraction (HFpEF) is a global public health epidemic that accounts for half of the heart failure cases. Various therapeutic approaches have been tested to block the activation of the Renin-Angiotensin System (RAS), including AT1R blockers (ARBs), Angiotensin-Converting Enzyme (ACE) inhibitors (ACEi), and direct renin inhibitors (DRIs) with modest to negligible benefits. The discovery of ACE2, a novel homolog of ACE, has advanced our understanding of the RAS. ACE2 is a monocarboxypeptidase that degrades Ang II into Ang-(1-7), which works via the activation of the Mas receptor. It has been well understood that the actions of Ang-(1-7) attenuate cardiac remodeling, production of ROS, and cardiac fibrosis.

Objective:
To determine the therapeutic role of Ang-(1-7) in HFpEF and identify the molecular mechanism related to its action.

Methods and Results:
To generate a murine model of HFpEF, male WT mice (n=24) were subjected to HFD in addition to eNOS inhibition with L-NAME (0.5 g l-1 in drinking water), as previously described. The control group (n=12) received chow diet and normal tap water. The murine model of HFpEF was validated using the non-invasive transthoracic echocardiography and invasive pressure-volume (PV) loop analyses, which exhibited diastolic dysfunction as well as cardiac hypertrophy. To evaluate the effects of Ang-(1-7) on HFpEF, animals were administered with either saline (n=12) or Ang-(1-7) (n=12) (24 μg/kg/day) for four weeks. Ang-(1-7) treatment improved diastolic function by reducing LVEDP (Ctrl: 8.267±1.254; HFD+L-NAME: 17.64±1.925; Ang-(1-7): 9.100±1.578) and Tau value (Ctrl: 7.365±0.5752; HFD+L-NAME: 9.224±0.3569; Ang-(1-7): 7.381±0.3041). Furthermore, Ang-(1-7) reduced cardiac hypertrophy by reducing the phosphorylation level of MAPK ERK 1/2 (Ctrl: 0.9074±0.1088; HFD+L-NAME: 1.212±0.1369; Ang-(1-7): 0.5615±0.1502) and increasing the phosphorylation level of AMPK (Ctrl: 0.1502±0.1502; HFD+L-NAME: 0.6127±0.06414; Ang-(1-7): 0.7852±0.1006). Ang-(1-7) treatment also reduces cardiomyocytes’ size and decreases interstitial fibrosis, as indicated by WGA and PSR staining.

Conclusion:
Ang-(1-7) treatment attenuated the development of HFD+L-NAME-induced HFpEF, reduced cardiac hypertrophy, and improved metabolic function.
Abstract P400: Treatment With The AMPK Agonist AICAR Alleviates Age-associated Cardiac Defects In The Mouse By Distinct Sex-specific Mechanisms
Authors: Angelini Aude , Ortiz-Urbina Jesus , Trial JoAnn , Entman Mark L , Taffet George E , Cieslik Katarzyna A
Published: November 2021

Heart failure is a major cause of mortality in the elderly. Features of cardiac aging include diastolic dysfunction and interstitial fibrosis with sex-specific differences.

We treated old male and female mice (21 months-old) 3 times a week for 3 months with the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR, 0.166 mg/g BW). We previously reported that AICAR normalizes the aged fibroblast phenotype.

In a longitudinal study, we found that AICAR attenuates the age-associated increase in left atrial volume, an indirect indicator of diastolic dysfunction, in female AICAR-treated mice (-29% ±5%) but not in males (-7% ±6%). Cardiac fibroblasts from AICAR-treated mice expressed decreased pro-collagen levels by 42% (mean fluorescence intensity, P=0.0003 for females, and P=0.05 for males). Cardiac fibroblasts cultured on decellularized cardiac matrices also had a reduced expression of pro-collagen when treated with AICAR (0.5mM, 7 days). Myocardial hydroxyproline level (an indicator of total collagen content) was reduced in female hearts (from 0.83 ± 0.07 in controls to 0.48 ± 0.05 in AICAR-treated, P=0.006). Female cells also exhibited a reduced expression of periostin after treatment (by 65%, P=0.02). By contrast, age-matched control males had a lower cardiac level of hydroxyproline (-65%, P=0.0002) and periostin (-45%, P=0.0004) than females, and were not affected by the treatment.

Accumulation of defective mitochondria is a hallmark of aging. Since AICAR can favor mitophagy, we isolated mitochondrial fractions from the hearts of old mice undergoing treatment. We found that AICAR decreased Parkin level in the small mitochondria fraction (0.75±0.09, P=0.028) in males, but no significant change was found between the groups of females. The reduction of Parkin may suggest improved clearance of defective mitochondria.For all experiments, we used 4-10 animals per group. One-way Anova or student’s T-test evaluated statistical significance.

In conclusion, age-associated cardiac remodeling leads to distinct patterns between male and female mice. AICAR treatment can have sex-specific effects: it reduces fibrosis in females but may promote mitophagy in males, translating into an improvement of heart function via distinct mechanisms.
Abstract P404: Membrane Composition Of Cardiac-derived Extracellular Vesicles Changes With Vesicle Origin And Determines Uptake
Authors: Bheri Sruti , Hoffman Jessica R , Park Hyun-Ji , Davis Michael E
Published: November 2021

Introduction:
Myocardial infarction (MI) is a leading cause of mortality worldwide. The potency of cell-based therapies for MI is increasingly attributed to the release of extracellular vesicles (EVs) which consist of a lipid/protein membrane and encapsulate RNA cargo. Specifically, EVs from ckit+ progenitor cells (CPCs) and mesenchymal stromal cells (MSCs) are shown to be pro-reparative, with clinical trials ongoing. Despite copious research into EV cargo, the role of donor cell type on EV membrane composition and its effects on EV uptake mechanism by recipient cells remain unclear. This is crucial for designing EV-based therapeutics as uptake mechanism dictates the functionality of the cargo. Thus, we hypothesized that (1) EV membrane composition varies by donor cell type and (2) this variation covaries with the mechanism of uptake.

Methods:
EVs were isolated using differential ultracentrifugation from four cardiac cell types: CPCs, MSCs, cardiac endothelial cells (CECs) and rat cardiac fibroblasts (RCFs) grown in normoxia (18% O
2
) or hypoxia (1% O
2
) to mimic ischemic conditions. EVs were characterized for size and concentration. EV lipid membrane profile was assessed through LC/MS/MS. Donor cell’s role on EV uptake mechanism was determined by inhibiting known uptake pathways (clathrin, dynamin, macropinocytosis and caveolae/lipid raft) with small molecules and quantifying CEC/RCF endocytosis of EVs with flow cytometry. Finally, partial least squares regression was used to determine the most important lipids involved in EV uptake mechanism.

Results:
EVs were successfully isolated and characterized. The EV membrane lipid profiles clustered by donor cell type. Uptake mechanism of EVs varied based on both donor and recipient cell type with dynamin mediated endocytosis being the most common. Further, the uptake mechanism was independent of normoxic/hypoxic conditioning. Finally, supervised learning methods revealed specific lipid classes (sphingolipids and glycerophospholipids) covaried with EV uptake mechanism.

Conclusion:
This work highlights the importance of the understudied EV membrane and its role in delivering therapeutic cargo. Active donor cell selection for efficient EV uptake will allow for more potent EV-based MI therapies.