bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 264: Upstream and Downstream Regulators of Lysophosphatidic Acid Receptor 4 are Critical for Cardiac Differentiation and Repair
Authors: Cho Hyun-jai , Lee Jin-Woo , Lee Choon-Soo , Kim Hyo-Soo
Published: October 2020

The therapeutic application of cell therapy to repair the damaged heart requires a comprehensive understanding of the process of cardiac differentiation of stem cells. We discovered a cardiac-specific marker, lysophosphatidic acid receptor 4 (LPAR4), which is G protein-coupled receptor (GPCR) and demonstrated its functional significance during cardiac differentiation. We screened GPCR expressing on mouse cardiac progenitor cells at differentiation day 3 compared to mouse undifferentiated pluripotent stem cells (PSCs). Among candidates, we identified LPAR4. We have found that in both mouse and human PSCs, LPAR4 has a transient expression pattern during cardiac differentiation. During in vitro differentiation of mouse and human PSCs toward cardiomyocytes, LPAR4 expression peaked for 3-5 days and then and declined immediately. Treatment with ODP (LPAR4 specific agonist) followed by p38MAPK blocker (SB203580) in the cardiac differentiation protocol significantly increased cardiac differentiation efficiency. Then we performed RNA sequencing to find transcription factors that regulate LPAR4. We screened 2-fold up-regulated transcription factors that related to cardiac differentiation during human iPSCs toward cardiomyocytes. The expression of LPAR4 is regulated by SOX17 expression, demonstrating that SOX17 is an upstream regulator of LPAR4. During cardiac differentiation, when knocking down SOX17 by siRNA, the expression of LPAR4 decreased as the expression of SOX17 mRNA decreased, resulting in no progression of cardiac differentiation. Besides, cardiac differentiation did not progress when SOX17 was overexpressed. SOX17 plays a role early in cardiac differentiation and that the expression should be reduced. However, it is assumed that sox17 lentivirus was gene-integrated and continued expression without decreasing expression. SOX17 overexpression needs further study. In conclusion, we demonstrated that LPAR4 is a novel cardiac progenitor cell marker and modulation of the upstream and downstream regulators shown functional significance during cardiac differentiation. Furthermore, our findings provide new insight into cell-free cardiac repair by the modulation of LPAR4 positive cells in the heart.

Funding Source: This study was supported by "Strategic Center of Cell and Bio Therapy for Heart, Diabetes & Cancer" (HI17C2085) through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea.
Abstract 265: Systemic Inflammation and Cardiac Macrophage Infiltration Accompany Stretch-induced Myocardial Injury in Swine Subjected to Transient Pressure Overload
Authors: Weil Brian R , Kosmerl Kristen , Nsumbu Dorcas , Young Rebeccah , Allen Shannon , Canty John M
Published: October 2020

Objective:
Transient pressure overload (TPO) elicits stretch-induced myocyte injury in the absence of ischemia or infarction, but the extent to which this is associated with inflammation is unclear. The present study was designed to assess immune activation after TPO in swine and compare the inflammatory cytokine profile to that observed after myocardial infarction (MI).

Methods:
Swine received a brief infusion of phenylephrine (18 mg/hr iv; 30-60 min) to induce TPO and were studied for 1 hr (n=5), 3 hr (n=6), or 24 hr (n=6) before analysis of myocardial macrophage gene expression (CD68) via qPCR. Serial blood sampling was performed to assess neutrophil and monocyte counts as well as circulating cardiac troponin I (cTnI), IL-6, TNF-α, and CRP. Cytokine levels were compared to those from a separate group (n=5) subjected to a 60 min LAD occlusion to produce MI.

Results:
TPO increased circulating cTnI (from 20±5 ng/L to 340±58 ng/L at 3 hr and 1520±616 ng/L at 24 hr; p<0.01) without evidence of infarction. Compared with controls (n=6), swine subjected to TPO exhibited increased CD68 gene expression at 1, 3, and 24 hr (
A
). This was accompanied by a rise in peripheral blood neutrophils and monocytes (
B
) as well as an elevation in circulating IL-6, TNF-α, and CRP that was comparable to that observed after MI (
C
).

Conclusion:
Myocyte injury following TPO elicits mobilization of neutrophils and monocytes and a rise in circulating inflammatory cytokines comparable to that observed after MI. This is accompanied by an increase in cardiac macrophages and may be an important mechanism by which repetitive episodes of TPO lead to interstitial fibrosis and diastolic dysfunction without anatomic hypertrophy.
Abstract 271: Mitochondrial DNA Copy Number in Peripheral Blood Cells is Associated With All -Cause Mortality in Older Cardiovascular Patients
Authors: Galenko Oxana , Juan James , Horne Benjamin D , Knight Stacey , Carlquist John F
Published: October 2020

Background:
Mitochondrial DNA copy number (Mt CN) is a surrogate marker of mitochondrial function. Variations in Mt CN have been associated with several age-related diseases. Lower Mt CN may indicate impaired cellular energy production whereas higher Mt CN may compensate for energy disbalance but also may lead to cellular damage through oxidative stress. This study evaluated the association of Mt CN and all-cause mortality in older cardiovascular patients (Pt).

Methods:
The study was approved by the Intermountain Healthcare Institutional Review Board. Consenting subjects (n=2,253) were participants in the INSPIRE registry undergoing cardiac catheterization at the Intermountain Heart Institute. Total DNA was extracted from EDTA stabilized blood with either Puregene (Qiagen) or Reliaprep (Promega) reagents. Relative Mt CN measurements were performed with multiplexed real-time polymerase chain reaction coamplifying a stable site of Mt D- Loop and a region of a single copy nuclear β-2-microglobulin gene(β2M) and calculated as a ratio of Mt to β2M DNA. Cox regression was used to evaluate the association of Mt CN with all-cause mortality, with adjustment considering 32 covariables. Further adjustment entered the Intermountain Risk Score (IMRS), a validated mortality risk predictor.

Results:
Mean Pt age was age 62.3±13.8 yrs.; 65.2% were male; 1,122 (50%) died during 25.4 years of follow-up. Mt CN was lower for males (539±449 vs 654
±
1053 for females; p=0.004). Mt CN was 616±917 in decedents and 542±438 in survivors (p=0.014). In univariable Cox regression, Quartile 1 versus Quartile 4 of Mt CN was associated with the highest mortality risk (hazard ratio [HR]=1.45, CI=1.22, 1.71, p<0.001). This association remained significant after multivariable adjustment (HR=1.30, CI=1.10, 1.54, p=0.003). There was minimal correlation between IMRS and Mt CN continuous values (r= -0.09 for males, r= -0.13 for females). In Cox regression, adjustment for IMRS and covariables showed Mt CN remained associated with mortality (HR=1.30, CI=1.08, 1.57, p=0.006).

Conclusions:
Low Mt CN is independently associated with higher risk of all-cause mortality. Further studies validating this finding and examining potential underlying physiologic protective mechanisms may prove to be of therapeutic and prognostic value.
Abstract 274: Activation of CaMKII Signaling Pathway Contributes to the Pathogenesis of Genetic Hypertrophic Cardiomyopathy
Authors: Perea Gil Isaac , Bellbachir Nadjet , Gavidia Alexandra A , Arthur Jennifer , Zhang Yuan , Vadgama Nirmal , Oikonomopoulos Angelos , Roura Santiago , Wu Joseph C , Bayes-Genis Antoni , Karakikes Ioannis
Published: October 2020

Background:
Hypertrophic cardiomyopathy (HCM) is the most prevalent form of genetic cardiomyopathy associated with ventricular arrhythmia and sudden cardiac death. To date, several pathogenic variants of sarcomeric protein genes have been identified in genetic HCM. However, the mechanisms by which specific mutations cause the hallmark phenotypes of HCM remain largely unresolved. There is a major unmet need for a better understanding of disease mechanisms in order to predict individuals at risk for sudden cardiac death and design mechanism-based therapeutics.

Methods:
We modeled HCM
in vitro
using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from HCM patients with a history of ventricular arrhythmias and sudden cardiac death harboring a heterozygous mutation in the Tropomyosin 1 gene (TPM1, c.107 A>G). We studied the functional and molecular consequences of the TPM1 mutation in iPSC-CMs to determine the molecular mechanism of HCM pathogenesis.

Results:
In mutant cardiomyocytes we observed impaired mechanical relaxation, aberrant intracellular calcium cycling and increased incidence of delayed after-depolarizations when compared to isogenic controls, recapitulating the HCM phenotype
in vitro
. Genome wide transcriptional profiling revealed a significant enrichment of genes associated with calcium dysregulation and the activation of the Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) pathway in mutant cardiomyocytes. Conversely, pharmacological inhibition of CaMKII pathway rescued the arrhythmic phenotype of mutant cardiomyocytes
in vitro
.

Conclusions:
Taken together, our data suggest a mechanistic link between the activation of CaMKII signaling pathway and the pathogenesis of sarcomeric HCM. These results provide a better understanding of the arrhythmogenic signaling pathway associated with the HCM phenotype and could potentially lead to mechanism-based targeted therapeutics for genetic HCM patients.
Abstract 278: Metabolic Reprogramming is Essential for Cell Cycle Progression in Cardiomyocytes
Authors: Abouleisa Riham , McNally Lindsey , Ou Qinghui , Choudhary Krishna , Thomas Reuben , Hill Bradford G , Mohamed Tamer m
Published: October 2020

Myocardial infarction causes irreversible loss of cardiomyocytes (CMs) and often leads to heart failure. To replace the lost cells, we identified a combination of cell-cycle regulators that induces stable cytokinesis in adult post-mitotic cells. Specifically, adenoviral overexpression of cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 (collectively known as ‘four factors’, or simply 4F) induced cell division in ~15% of post-mitotic mouse, rat, and human CMs. Identifying the major roadblocks during the process of CM proliferation is a key for advancing this field. This was not possible before due to the lack of efficient methods to induce CM proliferation. The goal of the current study is to understand why a subpopulation of CMs divide while most CMs, despite expressing 4F, resist cell cycle reentry. To investigate transcriptional changes during cell cycle progression at the single cell level, we conducted temporal single cell RNAseq on 60-day-old matured hiPS-CMs infected with either LacZ (control) or 4F for 24, 48 and 72 h. We found a unique cell population that appears 48 h after infection with the 4F; this population was identified as the proliferating population and expressed high levels of cytokinesis genes (Ki67, Aurora Kinase A and B, E2F1, CDC20, ANLN, TK1, CCNA2, PLK1 and PCNA). Consistent with our published data, this population represents ~15% of the total CMs population expressing 4F. Compared with the quiescent population from the same sample, this unique population of proliferating CMs shows significant upregulation of the cell cycle program and major downregulation of mitochondrial electron transport chain genes. In line with these transcriptomic changes, hiPS-CMs had 50% lower rates of oxidative phosphorylation 48 h after 4F infection. Furthermore, in 4F-overexpressing iPS-CMs, stable isotope tracing demonstrated significantly higher enrichment of glucose-derived
13
C in NAD and UDP-HexNAc, suggesting activation of NAD synthesis and the hexosamine biosynthetic pathway. We conclude that proliferating CMs diminish catabolic pathway activity and augment biosynthetic pathway activity. The capacity of CM subpopulation to reprogram their metabolism is likely to facilitate their ability to complete cell division.
Abstract 281: Human Atrial Extracellular Matrix Drives Differentiation of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes Toward an Atrial Phenotype
Authors: Mesquita Fernanda , Morrissey Jacquelynn , Xi Yutao , Monnerat Gustavo , Nogueira Fabio , Domont Gilberto , Sampaio Luiz , Hochman-mendez Camila , Taylor Doris A
Published: October 2020

Extracellular matrix (ECM) can directly modulate cell proliferation, migration and differentiation by mediating diverse growth factors and signaling interactions. Protocols for cardiomyocyte differentiation of induced pluripotent stem cells (iPSCs) that recapitulate cardiac development frequently result in a mixed cardiac cell population dominated overwhelmingly by ventricular-like cells. Utilizing the inherent biological capabilities of decellularized ECM (dECM) from human myocardium, we developed a method for committing human iPSCs to an atrial-like cell phenotype. We employed a modified decellularization method to generate small particles (125-500 μm) of human atrial and ventricular dECM. The particles presented a fractal dimension (1.63 and 1.71) that suggested self-similarity across particle sizes of both atrial and ventricular dECM. Quantifications of DNA (3.37±0.50 and 2.77±0.62% of cadaveric), GAG (0.44±0.08 and 0.59±0.13 μg/mg), and SDS (2.46±1.20 and 2.91±2.53 μg/mg) validated the absence of difference of atrial and ventricular dECM. Proteomic profiling revealed dECM chamber-specific clustered populations. Ventricular and atrial dECM segregated into ventricular and atrial parts based on component 1 (19.5%) and component 2 (13.9%). A total of 14% of atrial proteins were matrisome atrial-related and 13% of ventricle proteins were matrisome ventricular-related. Myocytes differentiated in the presence of atrial dECM showed similar differentiation efficiency (66.6±10.2 vs 65.5±12.7% of cTNT) and, importantly, increased atrial markers, as confirmed by qPCR (SLP and COUPF-I) and flow cytometry (43.5%±12.7% vs 23.9%±10.8% of MLC2a) in comparison to control. We observed an increase in atrial cells (38.4% vs 14.8%) by action potential duration (APD), with statistical differences in cAPD10 (57.1±20.2 vs 104.4±48.7 ms) and cAPD20 (76.2±22 vs 126±47.4 ms). Altogether, we demonstrate that human atrial ECM retains cues to drive cardiac differentiation to an atrial fate, doubling the number of atrial cells with a functional atrial phenotype. These findings are a critical step toward generating sufficient quantities of atrial cells, which can be used for chamber-specific cardiac disease modeling and drug development.
Abstract 283: Western Diet Induces Diastolic Dysfunction Linked to Impaired Cardiac Lipid Storage Dynamics That Predate Systolic Dysfunction
Authors: Lewandowski E Douglas K , Carley Andrew N , Lewandowski E Douglas D
Published: October 2020

Western diet (WD), containing high fat and high refined sugar, is associated with increased risk of cardiovascular disease. However, mechanisms linking WD to cardiomyopathy are far from understood. We hypothesize that WD induces cardiac dysfunction by dysregulation of cardiac lipid dynamics. Adult C57BL/6J mice were fed with either standard chow (CON, fat 4.5 %, sucrose 0%) or western diet (WD, fat 45%, sucrose 21%). At 20 weeks, WD mice exhibited higher body mass, reduced glucose tolerance, and increased isovolumetric relaxation time (IVRT), with no systolic dysfunction vs. CON. However, at 24 weeks, WD caused both systolic and diastolic dysfunction with decreases in left ventricular ejection fraction (66±2% CON vs. 54±2 WD, P <0.05) and fractional shortening (35±2% CON vs. 27±1 WD), with elevated IVRT and E/E’ ratio vs CON. Real time, dynamic-mode
13
C NMR and endpoint LC/MS of isolated perfused hearts supplied
13
C palmitate and
13
C oleate, with unlabeled glucose and lactate, revealed elevated TG content (28% increase vs. CON) with reduced TG turnover in WD hearts (35% decrease vs. CON), concurrent with early diastolic dysfunction at 20 weeks and preceding systolic dysfunction at 24 weeks. WD induced lower content of the TG lipase, ATGL (48% vs. CON), with no change in the TG synthase, DGAT1, suggesting elevated cardiac TG and lower TG turnover are due to reduced TG hydrolysis and not TAG synthesis. Impaired lipid storage dynamics can cause increased generation of potentially lipotoxic acyl intermediates, leading to cardiac dysfunction. The mitochondrial long chain fatty acid transporter, CPT1b was reduced (50%), with no change in the lesser, co-expressed CPT1a isoform in WD hearts. Despite reduced CPT1b in WD hearts, contributions from LCFAs to acetyl CoA production in the citric acid cycle were unchanged, suggesting that the WD provided sufficient LCFAs for mitochondrial oxidation and that the reduced CPT1b was not limiting. In conclusion, western diet expands the TG pool but impairs fat storage dynamics via low ATGL, leading to early diastolic dysfunction and eventual systolic dysfunction. Nutrient overload from WD leads to impaired cardiac lipid dynamics with early diastolic dysfunction that is distinguished from HFpEF due to later systolic dysfunction.
Abstract 288: Distribution of Pathological Features and Inflammatory Cells in Patients With Giant Cell Myocarditis
Authors: SHANGYU LIU , Yan Yao
Published: October 2020

Background:
Giant-cell myocarditis (GCM) is a rare disease with a poor prognosis. The typical pathological features of GCM include an infiltration of multinucleated giant cells accompanied by numerous inflammatory immune cells. However, the etiology and pathophysiology of GCM remain largely unclear.

Methods:
Eight patients with pathological diagnoses with GCM underwent heart transplantation at our center. Hematoxylin- eosin (H-E) and Masson’s tri-chrome staining were performed on biopsies of the free walls of the right and left ventricles and interventricular septa of the original hearts to determine the characteristic distribution of cardiac lesions and the composition of infiltrating immune cells. A multiplex immunohistochemistry and multispectral imaging analysis were applied to further classify the specific types of inflammatory immune cells.

Results:
Inflammation found in a descending frequency gradually from the epicardium to the endocardium in the free wall of the left ventricle, but concentrated on the surface of right ventricular septum. Typical inflammatory infiltration and pathological changes were observed in the right-sided ventricular septum samples from all 8 patients. Numerous inflammatory immune cells, particularly CD4
+
T cells, were detected in the lesion, which surrounded the emerging multinucleated giant cells. CD8
+
T cells and a small number of regulatory T cells were scattered in the periphery.

Conclusions:
In GCM, cardiac lesions appear to concentrate particularly beneath the epicardium of the left ventricular free wall and the right side of the ventricular septum. These findings provide a rationale for the diagnostic use of conventional endocardial biopsy. The findings further suggest that myocardial injury is mediated by a variety of lymphocytes, especially CD4
+
T cells.
Abstract 289: Perm1 is a Novel Regulator of Cardiac Energetics and Function
Authors: Oka Shinichi , Sabry Amira , Horiuchi Amanda , Cawley Keiko , O'Very Sean , Zaitsev Maria , Shankar Thirupura S , Byun Jaemin , Huang Chun-yang , Xu Xioayong , Torres Natalia S , Bakhtina Anna , Stubben Chris , Franklin Sarah , Saijoh Yukio , Drakos Stavros G , Sadoshima Junichi , Warren Junco S
Published: October 2020

Regulation of mitochondrial energetics is key to maintain cardiac function. Perm1 (
P
GC-1 and
E
RR-
r
egulator,
m
uscle
1
) was previously shown to enhance exercise endurance in skeletal muscle through increasing mitochondrial bioenergetics. Despite the significant expression levels of Perm1 in the heart, its role in the healthy and diseased hearts has never been investigated. We found that cardiac Perm1 was downregulated in the mouse failing heart subjected to pressure overload for 4 weeks (24.4 ± 5.9 % of sham operated, p<0.05) and in patients with advanced heart failure (55.2 ± 13.1 % of donors, p<0.05) suggesting a role of Perm1 in cardiac pathology. Phenylephrine (PE)-induced hypertrophy in cardiomyocytes was accompanied by downregulation of Perm1 (55.7 ± 5.7 % of control, p <0.05), and adenovirus-mediated overexpression of Perm1 rescued PE-induced downregulation of estrogen-related receptor alpha (ERRα), a key transcriptional regulator of mitochondrial energetics, and its target gene, Ndufv1 (Complex I), suggesting that downregulation of Perm1 contributes to the development of mitochondrial dysfunction in response to hypertrophic stimuli. Pathway enrichment analysis in cardiomyocytes where Perm1 was knocked-down by siRNA (siPerm1) revealed that the most downregulated pathway was metabolism, while upregulated pathways were mostly related to synthesis and assembly of collagen fibrils (i.e. Col3a1, Col5a1, Col11a1, Lox). Cell stress test using Seahorse XF analyzer showed that basal respiration and ATP production were significantly reduced in siPerm1 cardiomyocytes (40.7 % and 23.6 % of scrambled-siRNA, respectively, both p <0.05). Luciferase reporter gene assay further revealed that Perm1 dose-dependently increased the promoter activity of the ERRα gene and known targets of ERRα, Ndufv1 and Ndufs1 (Complex I). Furthermore, systemic Perm1-knockout mice developed heart failure (3 months old ejection fraction 38.8 %), concurrent with downregulation of proteins involved in oxidative phosphorylation, such as Ndufaf4 and Uqcr11 (-6.14 and -2.39 in fold change, respectively), and increased fibrosis. These results suggest that Perm1 is a novel regulator of cardiac metabolism and function, that enhances energetics and suppresses fibrosis.
Abstract 291:
In Vitro
Matured Human Embryonic Stem Cell-derived Cardiomyocytes Form Grafts With Enhanced Structure and Improved Electromechanical Integration in Injured Hearts
Authors: Dhahri Wahiba , Sadikov Valdman Tamilla , Qiang Beiping , Masoudpour Hassan , Ceylan Eylul , Wulkan Fanny , Laflamme Michael A
Published: October 2020

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) have tremendous promise for application in cardiac repair, but their immature phenotype greatly limits their translational potential. The present study was designed to two hypotheses: 1) that previously reported methods to promote the maturation of hESC-CMs by culture on soft polydimethylsiloxane (PDMS) substrates can be upscaled to the quantities required for transplantation studies; and 2) that PDMS-matured hESC-CMs will stably engraft in injured hearts and form graft myocardium with enhanced structural and functional properties. First, we cultured hESC-CMs on either PDMS or tissue culture plastic (TCP) for 20 and 40 days, then phenotyped the resultant populations. All hESC-CMs were engineered to express the fluorescent voltage-sensitive protein ASAP1 to facilitate in vitro and in vivo electrophysiological studies. Relative to their counterparts on TCP, hESC-CMs on PDMS at both time-points exhibited increased cardiac gene expression as well as a more mature structural and electrophysiological phenotype in vitro. Single-cell transcriptomics confirmed enrichment of cardiac maturation markers including gene pathways involved in cardiac contraction, extracellular matrix organization, sarcomerogenesis, and adult heart development in PDMS versus TCP cultures. Next, we transplanted day 20 or 40 TCP vs PDMS ASAP1+ hESC-CMs into injured guinea pig hearts. Recipient hearts were later analyzed by ex vivo optical voltage mapping studies and histology. While CMs from both substrates showed similar capacity for engraftment, grafts formed with PDMS-matured myocytes had more mature structural properties including enhanced alignment, sarcomere lengths and maturation marker expression. Most importantly, graft formed with PDMS-matured myocytes showed improved electrophysiological properties including better host-graft electromechanical integration and more rapid and uniform propagation. We conclude that large quantities of matured hESC-CMs can indeed be economically produced by these methods. Moreover, PDMS-matured myocytes form large intramyocardial grafts with enhanced cardiac structure and electrical function, thereby establishing that maturation prior to transplantation meaningfully improves outcomes in vivo.
Abstract 296: The MicroRNA
-134-5p
Induces Calcium Deposition by Inhibiting Hdac5 in Vascular Smooth Muscle Cells
Authors: Choe Nakwon , SHIN SERA , Joung Hosouk , Ryu Juhee , Kim Young-Kook , Ahn Youngkeun , Kwon Duk-hwa , Kook Hyun
Published: October 2020

Calcium deposition in vascular smooth muscle cells (VSMCs) is a form of ectopic ossification in blood vessels. It can result in rigidity of the vasculature and an increase in cardiac events. Here, we report that the microRNA-134-5p (
miR-134-5p)
potentiates inorganic phosphate (Pi)-induced calcium deposition in VSMCs by inhibiting histone deacetylase 5 (HDAC5). Using miRNA microarray analysis of Pi-treated rat VSMCs, we first selected
miR-134-5p
for further evaluation. Quantitative RT-PCR confirmed that
miR-134-5p
was increased in Pi-treated A10 cells, a rat VSMC line. Transfection of
miR-134-5p
mimic potentiated the Pi-induced increase in calcium contents.
miR-134-5p
increased the amounts of bone runt-related transcription factor 2 (RUNX2) protein and bone morphogenic protein (BMP2) mRNA in the presence of Pi but decreased the expression of osteoprotegerin (OPG). Bioinformatic analysis showed that the HDAC5 5’UTR was one of the targets of
miR-134-5p
. Hdac5 expression was downregulated by
miR-134-5p
mimic in an
Hdac5
5’UTR-dependent manner. Overexpression of
Hdac5
mitigated the calcium deposition induced by
miR-134-5p
. Our results suggest that a Pi-induced increase of
miR-134-5p
may cause vascular calcification through repression of HDAC5.
Abstract 298: Flavin Containing Monooxygenase 2 in Cardiac Fibrosis
Authors: Hu Xinyang , Ni Cheng
Published: October 2020

Cardiac fibrosis is associated with clinical outcome in patients with heart failing. There is no information concerning whether flavin containing monooxygenase 2 (FMO2) is involved in the fibrotic response. Here, we demonstrate that FMO2 is consistently down-regulated after myocardial infarction (MI) in rats, non-human primates and patients with chronic MI. Furthermore, FMO2 knockdown or knockout results in significantly increased cardiac fibrosis accompanied with impaired heart function in rats. Compensation of non-myocyte FMO2 using the strategy of miR133a/1a TS aided lentivirus inhibits the cardiac fibrosis and restores the deteriorated cardiac function following MI in rats.
In vitro
studies showed FMO2 expression in neonatal rat cardiac fibroblasts (NRCFs) is dramatically decreased after treatment of TGF-β. FMO2 deficiency in CFs isolated from FMO2-/- rats induces augmented collagen deposition, fibroblast-myofibroblast transdifferentiation and increased phosphorylated SMAD2/3. However, overexpression of FMO2 in NRCFs or CFs from MI patients suppresses this exact process. Mechanistically, we demonstrate using mass spectrometry that FMO2 combines with cytochrome p450 superfamily 2J3 (CYP2J3) at the ubiquitination site of the latter one after TGF-β stimulation, and then blocks the CYP2J3 ubiquitination. The accumulated CYP2J3 expression induces the up-regulation and nuclear translocation of SMURF2, the E3 ubiquitin-protein ligase specifically degrades phosphorylated SMAD2/3, through a negative feedback as another substrate of SMURF2, in addition, FMO2 can competitively exploit CYP2J3 from SMURF2, thus in turn promotes nuclear translocation of SMURF2 to degrade phosphorylated SMAD2/3. Furthermore, in non-human primate MI model, delivery of non-myocyte FMO2 over-expression lentivirus significantly decreases the cardiac fibrosis and improves heart function. In summary, our study demonstrates for the first time that FMO2 is a critical determinant of cardiac fibrosis by interfering TGF-β/SMAD2/3 and provides a novel potential target for treating cardiac fibrosis.
Abstract 306: Loss of a Disintegrin and Metalloproteinase-15 Impairs Fibroblast Activation Following Myocardial Infarction Resulting in Ventricular Rupture
Authors: Chute Michael J , Shen Mengcheng , Jana Sayantan , Wang Wang , Kassiri Zamaneh
Published: October 2020

Background:
Myocardial infarction (MI) occurs when blood flow to a region of the myocardium is interrupted. Over time, the damaged myocardium is replaced with scar tissue through activation of an inflammatory phase, followed by a reparative and a maturation phase. A disintegrin and metalloproteinase-15 (ADAM15) is a membrane-bound enzyme that is expressed in inflammatory cells and the heart, and its expression in the heart is increased following myocardial infarction. However, its function in heart disease has not yet been explored. We hypothesized that ADAM15 deficiency will impede the inflammatory response post-MI, which could reduce inflammation but also hinder scar formation causing adverse remodeling of the surviving myocardium.

Methods:
MI was induced in adult male wildtype (WT) and ADAM15-deficient (
Adam15
-/-
) mice by permanent ligation of the left anterior descending artery. LV structure, systolic and diastolic functions were assessed by echocardiography. Hearts were excised at 3-days or 1 week post-MI, processed for histological and molecular analyses. Fibrillar collagen organization was assessed by Second Harmonic Generation. Cardiac fibroblasts (cFBs) were isolated from WT or Adam15
-/-
hearts, and subjected to ischemia (hypoxia + nutrient deletion)
in vitro
.

Results:
Adam15
-/-
mice exhibited significantly compromised survival post-MI, mainly due to LV rupture. Cardiac contractility was reduced in
Adam15
-/-
compared to WT-MI mice. Collagen fibers in the scar tissue were distorted and scarce in
Adam15
-/-
MI hearts, associated with reduced levels of a major cross-linking enzyme, lysyl oxidase.
In vitro, Adam15
-/-
cFBs showed impaired myofibroblast transformation under ischemic conditions, suggesting attenuated fibroblast activation.

Conclusion:
Adam15
-deficiency impairs the wound healing process post-MI, leading to deleterious remodelling and LV rupture.
Abstract 308: Reductive Carboxylation Contributes to Cardiac Adaptation in Response to the Oncometabolite D2-hydroxyglutarate
Authors: Karlstaedt Anja , Faubert Brandon , Vitrac Heidi M , Salazar Rebecca L , Gould Benjamin D , DeBerardinis Ralph , Taegtmeyer Heinrich
Published: October 2020

Cancer cells rewire metabolism to support tumor growth and proliferation. In isocitrate dehydrogenase (IDH) 1 and 2 mutant tumors, increased plasma levels of the oncometabolite D-2-hydroxyglutarate (D2-HG) are associated with systemic effects, including myopathy. D2-HG causes inhibition of alpha-ketoglutarate dehydrogenase (AKGDH), which is associated with reduced cardiac contractile function. How tumor cells influence the metabolism of cardiomyocytes remains mostly unknown. Specific cancer cells use glutamine-dependent reductive carboxylation to circumvent defective mitochondrial metabolism by producing citrate and acetyl-CoA for lipid synthesis, which tumors require for growth. Here, we explore the hypothesis that inhibition of AKGDH by the oncometabolite D2-HG increases glutamine-dependent reductive carboxylation in the heart. We combined ex vivo rat heart perfusions with mass-spectrometry-based stable isotope tracer studies and in silico metabolic flux analysis. In response to D2-HG-mediated inhibition of AKGDH, we observed an increased reductive carboxylation of alpha-ketoglutarate to citrate rather than oxidative decarboxylation. This pathway increases glutamine uptake and glutamine-derived citrate formation in both working rat heart perfusions and cultured adult mouse ventricular cardiomyocytes. When we perfused rat hearts with 13C-labelled D2-HG, we observed a similarly increased formation of citrate. To identify which IDH isoform is responsible for redirecting carbon flux, we modulated IDH1, 2, and 3 in adult mouse ventricular cardiomyocytes using siRNAs. Reduced expression of IDH1 impaired reductive formation of citrate. Importantly, we observed a significant correlation between reductive citrate formation and epigenetic modifications of histones, including increased histone 3 lysine 9 acetylation and di-methylation. To explore these observations, we conducted ChIP-sequencing and identified distinct transcriptional remodeling. Taken together, we demonstrate how oncometabolic stress in the heart causes redirection of central carbon metabolism via reductive carboxylation, and provide evidence of how reductive-citrate formation may induce epigenetic modifications in the heart.
Abstract 312: The Tricuspid Valve Also Maladapts: Evidence From Sheep With Functional Tricuspid Regurgitation
Authors: Rausch Manuel K , Meador William , Malinowski Marcin , Jazwiec Tomasz , Mathur Mrudang , Timek Tomasz
Published: October 2020

The objective of this work was to determine whether tricuspid valve leaflets maladapt to functional regurgitation as they do in the mitral valve. If they did, pharmacological control of the underlying mechanisms could open opportunities to support tricuspid valve surgery and improve currently poor outcomes. To this end, we used a biventricular heart failure model to induce functional tricuspid valve regurgitation in sheep. We sacrificed animals once they showed signs of heart failure (with in 2-3 weeks). In those animals (and controls), we measured tricuspid anterior leaflet area and thickness, collagen content via ELISAs, tissue stiffness via mechanical testing, and cellular changes via immunohistochemistry. We found a statistically significant increase with disease in leaflet area (p=0.004), thickness (p=0.037), collagen content (p=0.001), circumferential leaflet stiffness (p=0.001), and SMA (p=0.030), Figure 1. Our findings imply that in approximately 2-3 weeks of biventricular heart failure and associated functional tricuspid regurgitation, tricuspid valve leaflets underwent substantial remodeling. Specifically, it appears that activation of valvular interstitial cells increased collagen syntheses resulting in leaflet growth, thickening, and stiffening. No other previous study has demonstrated tissue maladaptation on all functional scales in the tricuspid valve. Our work is relevant as it implies that pharmacological control of this maladaptive response could allow for the promotion of the positive effects (e.g., area increase) while suppressing negative effects (e.g., fibrosis).
Abstract 322: Chamber-specific Mitochondrial Remodeling in Atrial Fibrillation
Authors: Emelyanova Larisa , Warner Catherine , Sharp Ryan , Kurian Justin , Recchia Fabio A , David Kress , Rizvi Farhan , Khan Mohsin , Arshad Jahangir
Published: October 2020

Introduction:
Studies have shown that right (RA) and left (LA) have different susceptibilities toward developing atrial fibrillation (AF). However, the molecular basis of these differences are not well characterized. Given these, we hypothesize that AF is associated with dissimilar energetic remodeling in the RA and LA and presence of interatrial differences might be a potential reason for different susceptibility of the atria to AF.

Objective:
The aim was to compare differences in mitochondrial oxidative phosphorylation system (OXPHOS) between the RA and LA in patients with and without AF (non-AF) and determine whether the same differences in chamber-specific sensitivity toward AF exist in the animal model of AF.

Methods:
The RA and LA tissue were collected from non-AF and AF dogs and consented patients undergoing elective open-heart surgery. Gene expression profiling of 72 genes involved in OXPHOS was performed using the Mitochondrial Energy Metabolism RT2 Profiler PCR Array. Comparison between the AF and non-AF groups were determined by using a pairwise differential expression analysis. A Venn diagram tool was applied to visualize expressed genes that overlap.

Results:
Expression of 14 and 25 genes was significantly reduced in the human and dog RA, correspondingly. Unlike, expression of only 2 and 9 genes was significantly reduced in human and dogs LA. The Venn diagram demonstrates the overlaps (common genes) and differences between the genes in patients and dogs with AF (Fig. A, B).

Conclusion:
AF is associated with different chamber-specific energetic remodeling suggesting that dissimilar mechanisms may contribute to the development and progression of AF.
Abstract 331: Impact of Acetylated Cyclophilin D on the Mitochondrial Permeability Transition Pore
Authors: Beutner Gisela , Perkins Jacob , Sardari Ronak A , Porter George A
Published: October 2020

Background:
The mitochondrial matrix protein cyclophilin D (CypD) is a key regulator of mitochondrial function. CypD controls electron transport chain activity and ATP synthesis by regulating the permeability transition pore (PTP). The activity of CypD is regulated by several post-translational modifications including acetylation of lysine 166 in the mouse.

Objective:
To investigate how acetylation at lysine 166 of CypD specifically in the heart modifies its ability to regulate the PTP and the ATP synthase.

Results:
We generated a conditional cardiac knock-in mouse model where lysine 166 has been mutated into glutamine (CypD
K166Q
) to mimic permanent acetylation of CypD. The mice were either +/+, +/- or -/- for the expression of native CypD. Results show that mitochondrial oxygen consumption was not affected by the expression of CypD
K166Q
. The calcium retention capacity (CRC) was measured with Arsenazo III and decreased significantly when CypD
K166Q
was expressed. The CypD inhibitor cyclosporine A significantly increased the CRC in WT mice. However, cyclosporine A was did not inhibit CypD in the hearts of mice expressing only CypD
K166Q
or in addition to wild-type CypD. The ability of the ATP synthase to create dimers or oligomers was assessed by western blotting and the hydrolysis of ATP in in-gel assays and shows that expression of CypD
K166Q
decreased the assembly of the ATP synthase into dimers or oligomers.

Conclusions:
Our data show that the expression of CypD
K166Q
increases the sensitivity of PTP opening to calcium and limits the assembly of ATP synthase into oligomers.
Abstract 333: Cardiomyocyte Death and Fibrotic Scarring in the Infarcted Neonatal Mouse Heart
Authors: Mohr Mary , Tao Ge , Li Shuang , Roddy Patrick
Published: October 2020

As one the leading causes of death in the United States, myocardial infarction (MI) occurs every 40 seconds, causing severe public health burden. Following MI, the loss of healthy cardiomyocytes leads to decreased contractility and eventually heart failure. Mature mammalian cardiomyocytes have a low turnover rate at only 0.5-2% per year, insufficient for repopulating damaged myocardium after MI. However, a contradictory discovery was made showing that the neonatal mammalian heart is regenerative, although this reparative ability is lost within days after birth. A great amount of effort is needed to understand the mechanisms underlying neonatal cardiomyocyte regeneration. In the current project, we attempt to profile different types of cell death in regenerating and non-regenerating mouse models following MI, in order to gain insights into a favorable type of cardiomyocyte death during regeneration. We induced MI in postnatal day 1 (P1, regenerative), and postnatal day 7 (P7, non-regenerative) mouse hearts by left anterior descending artery occlusion (LAD-O). The progressive scar formation was assessed using Masson’s Trichrome staining at multiple timepoints up to 14 days after MI. At each time point, we profile three major types of regulated cell death, apoptosis, necroptosis, and ferroptosis, using immunofluorescence staining. We also used AC16, a human cardiomyocyte cell line, to investigate the role of cell density in the regulation of ferroptosis. We found that the scar formation was most dynamic between 2 and 3 days after MI and that the course of scar formation varied greatly between P1 and P7 hearts. Immunofluorescence of different cell death markers reveal differentially progressed cell death between P1 and P7 hearts after MI. Our results indicate a different pattern of cardiomyocyte death in the regenerative P1 heart compared to the non-regenerative P7 heart, that could be more favorable for myocardial regeneration.
Abstract 335: Distinct Effects of Eicosapentaenoic Acid and Docosahexaenoic Acid Linked Phospholipids on Membrane Structure
Authors: Mason Preston , Sherratt Samuel , Bhatt Deepak L
Published: October 2020

In REDUCE-IT, prescription eicosapentaenoic acid (EPA), an omega-3 fatty acid (O3FA), significantly reduced ischemic events in patients with diabetes or cardiovascular disease. Treatments containing docosahexaenoic acid (DHA) have not shown similar benefits in other trials. EPA and DHA differentially influence lipid oxidation, signal transduction, fluidity, and cholesterol domain formation, potentially due to distinct membrane interactions. Small angle x-ray diffraction approaches compared the separate and combined effects of 1-palmitoyl-2-eicosapentaenoyl-sn-glycero-3-phosphocholine (PL-EPA) and 1-palmitoyl-2-docosahexaenoic-sn-glycero-3-phosphocholine (PL-DHA) at a 1:20 ratio in membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PL-OA) and cholesterol (C) at a 0.3:1 C:PL ratio. As a control, we also evaluated membranes consisting of PL-OA and C only. Electron density profiles (electrons/Å
3
vs Å) generated from the diffraction data served to determine membrane structure, including its width or
d
-space, at 1 Å resolution (Fig. 1A). Addition of PL-EPA increased membrane hydrocarbon core electron density over a broad area ± 0-10 Å from the membrane center, indicating an energetically favorable extended membrane orientation for EPA, stabilized by van der Waals interactions (Fig. 1B). By contrast, PL-DHA increased electron density in the phospholipid head group region concomitant with disordering in the hydrocarbon core ± 0-9 Å (Fig. 1C). Addition of the PL-O3FAs did not alter the overall membrane width (57 ± 1 Å). The combination of PL-EPA with PL-DHA highly attenuated their separate effects on the hydrocarbon core and headgroup (Fig. 1D). The contrasting EPA and DHA effects on membrane structure signify distinct molecular orientations that may contribute to observed differences in biological activity.
Abstract 339: Runx1 Knock-down by Adeno Associated Viral Mediated-delivery of Short Hairpin RNA Preserves Cardiac Contractility Following Myocardial Infarction
Authors: Martin Tamara P , Cameron Ewan , Nicklin Stuart , Loughrey Christopher M
Published: October 2020

Myocardial infarction (MI) is a leading cause of heart failure (HF) and mortality worldwide. Preservation of contractile function and prevention of adverse cardiac remodelling following MI are essential to limit progression to HF. Expression of the Runx1 transcription factor in border zone cardiomyocytes is increased within 24 hours after MI. Genetically modified mice with Runx1 deficiency demonstrate an absence of cardiac dilation and preservation of left ventricular (LV) contractility following MI without a change of infarct size. Here, we investigate if adeno-associated viral (AAV)-mediated gene transfer to knock-down Runx1 expression post-MI attenuates adverse cardiac remodelling in mice. Male C57BL/6J mice underwent LAD ligation followed by intravenous injection of AAV-Runx1-shRNA or control AAV-scramble-shRNA (1x10
11
viral genomes). LV contractility (% fractional shortening, FS) was assessed by M-mode echocardiography. RNAscope assessed Runx1 expression in border zone cardiomyocytes and infarct size was histologically quantified using Picro sirius red stain.
Runx1
expression within border zone cardiomyocytes was reduced by 30% in AAV-Runx1-shRNA treated mice (37.8±4.5 vs. 53.9±6.4% of cardiomyocytes; AAV-Runx1-shRNA vs. control). LV contractile function was significantly preserved as early as 1 day post-MI (%FS, day 1; 37.7±1.4
vs
. 27.6±1.4, day 7; 37.2 ±1.2
vs
.28.2±1.4; LV internal dimension during systole day 1; 2.9±0.2
vs
. 2.4±0.1, day 7; 3.1±0.2
vs
. 2.5±0.1mm; AAV-Runx1-shRNA vs. control,
P
<0.05). Interestingly, infarct size was reduced with AAV-Runx1-shRNA (31.7±2.0
vs
. 40.4±1.4% of LV; AAV-Runx1-shRNA
vs
. control,
P
<0.05). In conclusion, a therapeutic approach utilising AAV-mediated knock-down of Runx1 preserved LV contractility and reduced infarct size following MI. These data further highlight Runx1 as a key driver of adverse cardiac remodelling and a therapeutic target with translational potential to prevent adverse cardiac remodelling following MI.
Abstract 347: Comparison of Six Minute Walk Test and Cardiopulmonary Exercise Test Results in Postural Orthostatic Tachycardia Syndrome Patients
Authors: Yousphi Adeel , Bakhtiar Ayesha , Sheikh Adnaan , Yang Lan , Khan Shahkar A , Mahmood Esha , Mistry Hemanshi , Suleman Amer
Published: October 2020

Introduction:
Postural orthostatic tachycardia syndrome (POTS) is a form of dysautonomia that can present as orthostatic tachycardia, dizziness, nausea, and anxiety, and many other symptoms that can be debilitating enough to cause functional impairment. The cardiopulmonary exercise test (CPET) has become a reliable test for evaluating the functional capacity (fc) in POTS patients. The test requires utilization of a bicycle ergometer, blood pressure apparatus, ECG, pulse oximeter, and a face mask that measures the oxygen uptake and carbon dioxide release. The 6-minute walk test (6MWT) is another test that can also be utilized to assess fc in patients.

Objective:
The aim of this study is to differentiate whether the 6MWT and CPET give similar results with regards to fc in POTS patients.

Methods:
A total of 27 POTS patients visiting the clinic consented to participate in the voluntary study. There were 2 males and 25 females, with ages between 16 to 58 years. For the 6MWT, we used wrist worn pulse oximeter, electronic tablet device, sphygmomanometer and a lap counter. Patients walked in a straight flat corridor which was marked at 3 meter intervals, for six minutes. At the end of 6 minutes, patients were asked to stop and the six minute walk distance (6MWD) was recorded. The predicted 6MWD, the distance the patient was expected to walk based on patient’s sex, age, height and weight was calculated by the application installed on the tablet. The percentage of the predicted 6MWD that was actually walked by the patients was calculated (6MWD % predicted), and considered as their fc. Using the electronic medical records, we obtained their CPET fc results, where fc was calculated as a percentage of the predicted maximal oxygen uptake per unit body weight per unit time (VO
2max
) that was actually consumed. The VO
2max
measurement depends on patient’s sex, age, weight and height. Pearson correlation coefficient was used to compare the results of the 6MWT (6MWD % predicted) and CPET (fc).

Results:
Pearson correlation coefficient shows that there is no linear association between the CPET (fc) results and 6MWT (6MWD % predicted) results in POTS patients, with p=0.3368.

Conclusion:
The results convey no statistically significant association between the CPET (fc) results and the 6MWT (6MWD % predicted) results in POTS patients; the results of the 6MWT were not similar to that of the CPET in terms of fc testing in POTS patients. This suggests that the 6MWT has limited utility in POTS patients.