bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 319: Mirna-125b Reduces Ischemia / Reperfusion (i/r)-induced Apoptosis Through Targeting Multiple Targets in Intrinsic and Extrinsic Apoptosis Pathways
Authors: Wang Peipei , Chen Qiying , Zhou Yue , Richards Arthur M
Published: July 2021

Apoptosis is mediated through extrinsic and intrinsic pathways, both play a role in ischemia/reperfusion (I/R) injury. Predicted targets for miRNA-125b include extrinsic pathway mediators Traf6 and Tnfrsf1b, and intrinsic mitochondria regulators Bcl-2 family pro-apoptotic effectors Bak1 and BH3-only facilitators Bim, Bmf, Puma. We hypothesized that miRNA-125b directly targets multiple genes to reduce I/R-induced apoptosis. Myoblast H9c2 cells underwent 16 hours 0.2% O
2
hypoxia followed by 2 hours re-oxygenation (H-R, simulating I/R) and were transfected with miRNA-125b mimic vs. scrambled mimic control (25 nmol, miR-125b-M vs. MC) and miR-125b inhibitor vs. inhibitor control (miR-125b-I vs. IC). Cell count/viability, WST assay, cell injury-induced LDH release and apoptotic marker Casp3/7 were measured. Cells were trypsinized for assessment of apoptosis (7-AAD and annexin V double staining) and lysed for RT-qPCR and western blot (WB) analyses. pCMV-Myc-Bak1 plasmids were cloned and transfected into H9c2 for recovery studies. The effects were verified in neonatal rat ventricular myocytes (NRVM). miRNA-125b-M significantly reduced H-R injury as indicated by higher cell count/viability and WST activity, and reduced LDH (miR-125b-M vs. MC p<0.05). qPCR confirmed that (1) miR-125b expression was reduced in H-R; (2) RISC-loaded (immunoprecipitation pull-down Ago-2) miR-125b increased by ~35 fold and reduced to ~3% following mimic and inhibitor transfection respectively; (3) multiple apoptosis-related genes were reduced by miR-125b-M, Bak1, Bmf, Bim, Puma, Traf6 and Tnfrsf1b. All changes were confirmed by WB. Luciferase reporter assays indicated miR-125b bound to the 3’-UTR of all genes tested except Traf6. Total apoptotic cell numbers and Casp3/7 release were significantly reduced by miR-125b-M. The protective effect of miRNA-125b was partially abolished by Bak1 overexpression (pCMV-Myc-Bak1 and miR-125b co-transfection). Protective effects of miRNA-125b were further verified in NRVM. MiRNA-125b inhibitor reversed protective effects and target changes at mRNA and protein level. miR-125b is powerfully cardioprotective in I/R injury due to directly targeting multiple genes in the extrinsic and intrinsic apoptotic pathways.
Abstract 330: Serum Mir-129 and Mir-21 Predict 100-day Mortality in Acute Heart Failure Patients
Authors: Tao Lichan , Bei Yihua , Shen Shutong , Li Jin , Gao Rongrong , Wu Xiaoting , Xiao Junjie , Li Xinli
Published: July 2021

Background:
Heart failure is a common disease worldwide and it could be divided as chronic heart failure (CHF) and acute heart failure (AHF). Circulating microRNAs (miRNAs, miRs) have been reported to be novel biomarkers of diagnostic, prognostic and predictive values in cardiovascular diseases. However, little is know about using circulating miRNAs as biomarkers for mortality in AHF patients.

Methods and results:
A total of 151 AHF patients were enrolled in this study. Ten miRNAs involved in the regulation of AHF including miR-129, miR-675, miR-622, miR-146a, miR-155, miR-21, miR-18b, miR-92b, miR-126 and miR-22 were determined by reverse transcription polymerase chain reactions using total RNA isolated from serum of those 151 patients with AHF enrolled in our center. After a follow-up period of 100 days, 16 patients died and based on that, we found that expression levels of serum miR-129 (p=0.032) and miR-21-5p (p=0.001) were significantly lower in those patients died within 100 days. The kaplan cumulative survival analysis confirmed that patients with higher levels of miR-129 (p=0.036) and miR-21 (p=0.001) had significantly higher survival rate.

Conclusion:
Serum low levels of miR-129 and miR-21 predict 100-day mortality in AHF patients.
Abstract 333: AAV9 Serca2a Gene Transfer Reverses Some but Not All Electrophysiological Deficits in a Chronic Model of Congestive Heart Failure
Authors: Xie Chaoqin , Chen Jiqiu , Hu Jun , Chaanine Antoine H , Jeong Dongtak , Kohlbrenner Erik , Hajjar Roger J , Akar Fadi G
Published: July 2021

SERCA2a gene transfer (GT) to the failing heart improves it mechanical function. The electrophysiological (EP) consequences of SERCA2a GT are less clear. We investigated the EP substrate, total (t) and phosphorylated (p) Cx43 expression in a chronic model of heart failure (HF) with & without SERCA2a GT.

Methods:
After 2 mo of aortic banding rats underwent 30min LAD occlusion & reperfusion for another 2 mo. Rats developed widespread proliferation of reactive fibrosis in ischemic & remote zones and were randomized to receive AAV9 GFP (HF) or AAV9 SERCA2a (HF treatment) at the time of LAD occlusion/reperfusion. Hearts from normal (Sham, N=6), HF (AAV9.GFP, N=7), and HF treatment (AAV9.SERCA2a, N=9) rats were studied using optical mapping. Cx43 levels (t & p) were measured.

Results:
HF rats exhibited significant APD prolongation (by 50%) & CV slowing (by 30%). More importantly APD heterogeneity was increased 2.5 fold (p=0.006). The ratio of S368 p-to-t Cx43 was reduced in the ischemic zone of HF rats where SERCA2a expression was decreased (Fig). AAV9 SERCA2a GT increased SERCA2a levels in the ischemic and remote zones. Surprisingly, APD & CV were comparable (p=NS each) in HF and HF treatment groups. In contrast SERCA2a GT reduced APD heterogeneity by 30% relative to untreated rats (p=0.03), fully restored pCx43 expression to sham levels in the ischemic zone and abolished regional differences in p-to-t Cx43 (Fig).

Conclusions:
AAV9 SERCA2a GT reverses key EP and molecular deficits that are causally related to arrhythmias in HF. This therapy, however, fails to reverse other hallmark features of HF, which are likely dependent on widespread ion channel and structural remodeling, namely fibrosis.
Abstract 335: Regulation of Muscle Contractility by a Family of SERCA-Inhibitory Micropeptides
Authors: Anderson Douglas M , Anderson Kelly M , Chang Chi-Lun , Makarewich Catherine A , Nelson Benjamin R , McAnally John R , Shelton John M , Liou Jen , Bassel-Duby Rhonda , Olson Eric N
Published: July 2021

Functional micropeptides can be concealed within RNA transcripts that have been putatively annotated as non-coding. We recently discovered a muscle-specific micropeptide, named myoregulin (MLN), that inhibits the activity of SERCA, the membrane pump that controls muscle relaxation by regulating Ca2+ uptake into the sarcoplasmic reticulum (SR). Genetic deletion of MLN in mice enhances Ca2+ handling in skeletal muscle and improves exercise performance. MLN shares structural and functional similarity with phospholamban (PLN) and sarcolipin (SLN), two well-studied micropeptides that regulate cardiac contractility and disease. Here we identify an additional member of this micropeptide family, named endoregulin (ELN), that specifically overlaps with the expression of SERCA3, the dominant Ca2+ ATPase in endothelial cells that controls the contractility of vascular and visceral smooth muscles. ELN encodes a single transmembrane alpha helix that localizes to the endoplasmic reticulum (ER), where it forms a stable complex with SERCA3. In cell based assays, ELN inhibits SERCA-dependent Ca2+ uptake into the ER and controls ER calcium levels. Due to the essential role of SERCA3 in regulating vascular smooth muscle contractility, ELN represents a potential regulator of vascular tone and novel therapeutic target for the treatment of cardiovascular disease.
Abstract 343: Bayesian Selection of Modifier Genes in Hypertrophic Cardiomyopathy Through Whole Genome Sequencing
Authors: Wheeler Matthew , Waggott Daryl , Grove Megan , Dewey Frederick , Pan Cuiping , Pavlovic Aleks , Goldfeder Rachel , Puckelwartz Megan , Day Sharlene , McNally Elizabeth , Dorn Gerald W , Ashley Euan
Published: July 2021

Background:
Technological advances have greatly reduced the cost of whole genome sequencing. For single individuals clinical application is apparent, while exome sequencing in tens of thousands of people has allowed a more global view of genetic variation that can inform interpretation of specific variants in individuals. We hypothesized that genome sequencing of patients with monogenic cardiomyopathy would facilitate discovery of genetic modifiers of phenotype.

Methods and Results:
We identified 48 individuals diagnosed with cardiomyopathy and with putative mutations in MYH7, the gene encoding beta myosin heavy chain. We carried out whole genome sequencing and applied a newly developed analytical pipeline optimized for discovery of genes modifying severity of clinical presentation and outcomes. Using a combination of external priors and rare variant burden tests we scored genes as potential modifiers. There were 96 genes that reached a modifier score of 6 out of 12 or better (9=2, 8=8, 7=17, 6=69). We identified NCKAP1, a gene that regulates actin filament dynamics, and CAMSAP1, a calmodulin regulate gene that regulates microtubule dynamics, as top scoring modifiers of hypertrophic cardiomyopathy phenotypes (score=9) while LDB2, RYR2, FBN1 and ATP1A2 had modifier scores of 8. Of the top scoring genes, 21 out of 96 were identified as candidates a priori. Our candidate prioritization scheme identified the previously described modifiers of cardiomyopathy phenotype, FHOD3 and MYBPC3, as top scoring genes. We identified structural variants in 21 clinically sequenced cardiomyopathy associated genes, 13 of which were at less than 10% frequency. Copy number variants in ILK and CSRP3 were nominally associated with ejection fraction (p=0.03), while 8 genes showed copy gains (GLA, FKTN, SGCD, TTN, SOS1, ANKRD1, VCL and NEBL). Structural variants were found in CSRP3, MYL3 and TNNC1, all of which have been implicated as causative for HCM.

Conclusion:
Evaluation of the whole genome sequence, even in the case of putatively monogenic disease, leads to important diagnostic and scientific insights not revealed by panel-based sequencing.
Abstract 345: Dramatic ATP Depletion Without Concomitant Declines in ATP Synthesis Rates Suggests Impaired Energy Metabolism Mechanisms in Adrenergic-Deficient Mouse Embryos
Authors: Peoples Jessica N , Baker Candice , Ebert Steven
Published: July 2021

High cardiac energy demands increase during embryonic development, requiring oxidative phosphorylation converting ADP to ATP in mitochondria to meet these demands. We have recently shown that adrenergic hormones are required to maintain sufficient cardiac energy metabolism during embryonic development, but the specific mechanism(s) underlying this regulation are not known. Mouse embryos lacking adrenergic hormones, norepinephrine (NE) and epinephrine (EPI), due to targeted loss of the essential
dopamine β-hydroxylase
(
Dbh
) gene, have remarkably decreased steady-state ATP/ADP ratios. To determine if ATP synthesis was affected, we examined the rate of ATP formation in adrenergic-deficient and control embryonic hearts. Our rate data have shown that despite > 50-fold decrease of steady-state ATP concentrations in

Dbh
-/-

embryos, the rate of ATP synthesis was not significantly different in adrenergic-competent and deficient embryos. This indicates that respiratory complexes in mitochondria are capable of producing ATP in adrenergic-deficient embryos, and suggest that ATP is either consumed at a faster rate than it is produced or its production is limited
in vivo
due to limited access to metabolic substrates (“starvation”). These findings reveal new mechanistic insights about how adrenergic hormones regulate energy metabolism during embryonic development.
Abstract 354: Sirt1 Negatively Regulates Cardiac Inflammation Induced by Metabolic Stress
Authors: Schesing Kevin , Sadoshima Junichi , Oka Shinichi
Published: July 2021

Obesity is a major risk factor for heart disease. Metaflammation is a metabolically driven, chronic, low-grade inflammatory reaction that is seen in the heart under obese conditions. Cardiac myocytes are known to produce inflammatory cytokines such as interleukin-6 (IL-6). However, whether and how cardiac myocytes mediate metaflammation is unknown. Silent information regulator 1 (Sirt1), a histone deacetylase involved in numerous metabolic pathways, facilitates an anti-inflammatory function through inhibition and deacetylation of the NFkB p65 subunit. This project aims to examine whether Sirt1 plays a role in cardiac local metaflammation. Mice with cardiac specific Sirt1 gene deletion (c-Sirt1 KO), Sirt1 overexpression (Tg-Sirt1), and relevant controls (Flox/Flox and WT respectively) were each placed on high fat and regular chow diets. The mice were euthanized after 3 months and their hearts were extracted for genetic and molecular analyses. Sirt1 mRNA and protein levels were significantly decreased under HFD feeding conditions (50% reduction in HFD feeding compared to normal diet, P<0.05). Acetylation of p65 was significantly induced by HFD feeding, and was further enhanced in c-Sirt1 KO mice, but inhibited in Tg-Sirt1 mice. Cardiac inflammation was enhanced in the c-Sirt1 KO group as evidenced by a significant increase in mRNA levels of IL-6 in mice fed regular chow (3-fold increase in c-Sirt1 KO vs. Sirt1 Flox/Flox, p<0.05) and HFD (5-fold increase in c-Sirt1 KO vs. Sirt1 Flox/Flox, p<0.05). Intracellular IL-6 signaling steps, such as phosphorylation of STAT3, were enhanced in c-Sirt1 KO, but inhibited in Tg-Sirt1 mice under HFD feeding conditions. These results suggest that cardiac myocytes mediate metaflammation partly through downregulation of Sirt1.
Abstract 356: Angiotensin II as a Paracrine Factor in Heart Environment
Authors: Pereira Isabela C , Rodrigues Maria Laura P , Teodoro Lívia G , Vieira Fabiano F , Gomes Roseli A
Published: July 2021

Besides its potent vasoconstrictor action, the Angiotensin II (Ang II) has other important biological roles as autocrine and paracrine actions, being fundamental in cardiovascular homeostasis. Changes in the levels of this substance in the pericardial fluid (PF) are associated with the development of cardiovascular diseases. The aim of this study was to evaluate the presence of members of the classical and alternative pathways of the Renin Angiotensin System (RAS), as well as one of its main effectors peptides, Ang II, in PF of patients with coronary artery disease and assess the role of Ang II as a factor able to promote the process of epithelial-mesenchymal transition epicardial mesothelial cells in genetically modified mice. Male and female patients with coronary artery disease and controls patients were involved in this study. The ELISA’s results showed significant amounts of Ang II, Angiotensin Converting Enzyme (ACE) and chymase in PF coronary artery disease male patients. Chymase positive mast cells were identified by immunohistochemical and quantitative analysis demonstrated a greater density of mast cells in this group. Expression of AT1 and AT2 was detected by immunohistochemistry in patients of male and female groups with coronary artery disease and controls. The scaling of the parietal mesothelium is a process seen in all groups, although when quantified showed no significant differences between them. Ang II was not able to increase the epithelial-mesenchymal transition of the epicardial cells in infarcted mice genetically modified. The presence of constituents of the classical and alternative pathways of RAS in the PF suggest an important role for this fluid as a modulator of cardiac functions. Specifically in relation to Ang II, their biological roles in the cardiovascular system still need to be further investigated. The identification in the cardiovascular microenvironment of paracrine factors and your action mechanisms, which stimulate and promote cardiac repair, may allow a better understanding of heart disease and the development of new therapies.
Abstract 357: Manganese Superoxide Dismutase: a Novel Mediator of Heart Failure Development and Progression
Authors: Miriyala Sumitra , Chandra Mini , Maxey Benjamin , St. Clair Daret K , Panchatcharam Manikandan
Published: July 2021

Manganese Superoxide Dismutase (MnSOD), an antioxidant enzyme that catalyzes the conversion of superoxide radicals (O
2
•-) in mitochondria. Constitutive activation mitochondrial reactive oxygen species (ROS) has been implicated in both the pathogenesis and the progression of cardiovascular disease. Absence of
SOD2
(gene that encodes MnSOD) is found to be embryonic lethal in animal models due to impairment of mitochondrial function, most noticeably in the heart. In our earlier investigation, we have shown that the MnSOD mimetic, MnTnBuOE-2-PyP
5+
distributes 3-fold more in mitochondria than in cytosol. The exceptional ability of MnTnBuOE-2-PyP
5+
to dismute O
2
•- parallels its ability to reduce ONOO– and CO3–. Based on our earlier reports, we have generated mice that specifically lack MnSOD in cardiomyocytes (Mhy6-SOD2
Δ
). These mice showed early mortality ~4 months due to cardiac mitochondrial dysfunction. Oxidative phosphorylation (OXPHOS) in mitochondria is the predominant mode for O
2
consumption in cells, and the mitochondria are the primary source of ROS in cells due to leaked electrons. FACS analyses using Mito-Tracker Green indicated that the mass of mitochondria per cell was slightly decreased in the Mhy6-SOD2
Δ
to the wild type. We then examined OXPHOS levels in Mhy6-SOD2
Δ
v.s. wild type using a Seahorse XF analyzer. The rate of oxygen consumption per cells was signi[[Unable to Display Character: ﬁ]]cantly lower in Mhy6-SOD2
Δ
cardiomyocytes than that in wild type. The most noticeable difference in the O
2
consumption was found in the presence of FCCP (H+ ionophore / uncoupler). FCCP is an inner membrane pore opener which resets the proton gradient between the mitochondrial matrix and the interspace, resulting in continuous transport of protons and consuming O
2
at the maximum potential. Remarkably, while the FCCP treatment increased O
2
consumption in wild type, the treatment showed no effect on the O
2
consumption in the Mhy6-SOD2
Δ
cardiomyocytes. The result indicated that the low basal OXPHOS activity in Mhy6-SOD2
Δ
was due to unusually low OXPHOS potential. We examined glycolysis in these cells by measuring extracellular acidi[[Unable to Display Character: ﬁ]]cation (ECAR) and the pattern exactly opposite to that of oxygen consumption rate (OCR) was observed for glycolysis rates between Mhy6-SOD2
Δ
and wild type.
Abstract 363: Recovery of Exercise Intolerance and Ventricular Dysfunction of Infarcted Spontaneously Hypertensive Rats After Oral Treatment With an Agonist of Adenosine Receptor
Authors: da Silva Jaqueline S , Sudo Roberto T , Debom Roberto , Barreiro Eliezer J , Fraga Carlos A , Zapata-Sudo Gisele
Published: July 2021

Severe ventricular dysfunction is observed in spontaneously hypertensive rats (SHR) submitted to myocardial infarction (MI). The present work evaluates the cardioprotective effects of oral administration of a novel agonist of adenosine A2A receptor, LASSBio-294, in infarcted SHR. Methods: Male SHR (12-14 wks old) were randomly divided into groups: sham and infarcted (MI) which were either treated orally with vehicle or LASSBio-294 (10 mg/kg) for 28 days. Before and after the animals were treated with LASSBio-294, cardiac function and exercise capacity were evaluated through the echocardiography and treadmill test. Mean blood pressure (MBP), left ventricular end diastolic pressure (LVEDP) and negative dP/dt were also determined. Fibrosis in heart sections were detected using H&E staining. Immunohistochemical staining for TNF-alpha and SERCA2a in LV tissues were observed. Results: MI in SHR reduced the running distance from 257.9 ± 13.2 to 39.0 ± 4.4 m which normalized to 296.0 ± 26.4 m after treatment with LASSBio-294. Reduced anterior wall thickness was observed after MI (0.51 ± 0.14 mm) which was prevented with treatment (1.65 ± 0.21 mm). Ratio of early and late transmitral filling velocity was reduced from 1.48 ± 0.09 to 0.99 ± 0.04 and recovered to 1.35 ± 0.07 after treatment. MBP was reduced from 169.0 ± 5.6 to 120.4 ± 7.4 mmHg in SHR-IM treated with LASSBio-294. Increased LVEDP of 25.6 ± 3.2 observed in SHR-IM was reduced to 7.3 ± 1.0 mmHg after treatment. The -dP/dt was reduced in SHR-MI to -5698 ± 408.1 mmHg/s and returned to -7894 ± 631.6 mmHg/s after LASSBio-294 treatment. There was an increase in collagen deposition after MI (from 14.5 ± 3.5 to 59.8 ± 5.4 %) which was prevented with LASSBio-294 treatment (29.5 ± 2.2 %). Increase of positive staining for TNF-α was observed in SHR-MI (from 9.5 ± 1.0 to 32.3 ± 2.1%) which recovered in SHR-MI treated group (14.4 ± 1.3%). Also, the expression of SERCA2a was reduced in ventricular muscle from SHR-IM (from 68.7 ± 5.1 to 21.4 ± 2.3 %) which partially recovered to 40.6 ± 1.19% with LASSBio-294 treatment.

Conclusion:
LASSBio-294 reduced exercise intolerance, prevented cardiac remodeling and diastolic dysfunction in infarcted SHR.
Abstract 364: Pathological Effects of Trimethylamine N-oxide (TMAO) on Pressure Overload-induced Heart Failure
Authors: Organ Chelsea L , Otsuka Hiroyuki , Bradley Jessica , Bhushan Shashi , Trivedi Rishi , Tang W. H. Wilson , Li Xinmin , Hazen Stanley , Lefer David J
Published: July 2021

Rationale:
Trimethylamine N-oxide (TMAO), a metabolite formed in the metabolism of dietary phosphatidylcholine, is elevated in the circulation of patients at increased risk for heart attack and adverse prognosis during heart failure.

Objective:
We investigated the effects of dietary choline and TMAO on the severity of heart failure following transverse aortic constriction (TAC).

Methods and Results:
Male C57Blk/6J mice were fed either control diet, a diet containing choline (1.2%) or a diet containing TMAO (0.12%) at 3 weeks prior to surgical TAC and were studied for 12 weeks. Left ventricular (LV) structure and function were monitored at 3 week intervals and myocardial tissue was collected at 12 weeks. Plasma TMAO levels were significantly (p < 0.01) increased in the choline (28.64 ± 2.30 μM) and TMAO (28.18 ± 4.27 μM) compared to the control group (1.87 ± 0.26 μM). Left ventricular ejection fraction (LVEF) was significantly (p < 0.05) worse in mice fed TMAO compared to control diet. LV end-diastolic and end-systolic diameters were significantly (p < 0.05) increased in the TMAO group compared to control diet. Myocardial fibrosis as measured with Picrosirius Red staining was also significantly greater (p < 0.01) in the TMAO and choline groups. Circulating BNP levels were significantly (p < 0.05) increased in the TMAO and choline groups.

Conclusions:
These data demonstrate that heart failure severity is significantly enhanced in mice fed diets containing either TMAO or choline. Our results suggest that consumption of food high in dietary nutrients that increase TMAO levels such as phosphatidylcholine may increase heart failure severity.
Abstract 366: Disease Phenotype Assessment Across a Library of iPSC-Derived Cardiomyocytes From Patient Cohorts Carrying Distinct Mutations for Familial Hypertrophic Cardiomyopathy
Authors: Tsai Jason , Lam Jason , Sanchez-Freire Veronica , Gadkari Rishali , Agarwal Maya , Bian Jing , Huang Guanyi , Magal Ashita , Lan Feng , Lee Andrew S
Published: July 2021

Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in the young, and is the most common inherited heart defect affecting 1 in 500 individuals worldwide. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have been demonstrated to model aspects of HCM, but only one iPSC model has been reported for a single HCM mutation in one gene. Here we compare disease phenotypes across a library of patient-specific HCM iPSC-CMs carrying distinct mutations to assess the range of phenotypes that may present in iPSC-CMs derived from different patient cohorts. iPSCs were generated from three patient cohorts carrying known hereditary mutations for HCM in TNNI3, TNNT2, and MYH7 and family-matched controls. Disease phenotypes in patient-specific iPSC-CMs were modeled using immunostaining, Ca2+ imaging, multielectrode array, and video analysis of contractile motion. HCM iPSC-CMs displayed a range of disease phenotypes as assessed by cell size, Ca2+ homeostasis, electrophysiology, and contractile arrhythmia. Different HCM mutations resulted in distinct disease phenotype presentation. Importantly, identical mutations demonstrated similar readouts across multiple lines and clones whereas distinct mutations exhibited differential disease phenotypes. These findings indicate disease-specific iPSC-CMs present with a range of phenotypes for HCM that vary by specific mutation and that iPSC libraries are important for cellular characterization of diseases such as HCM.

Figure 1. Derivation and disease phenotype modeling of iPSC-CMs generated from patients carrying distinct familial HCM mutations and family-matched controls.
Abstract 371: Down Regulation of ECM Genes Laminin and Collagen IV Lead to Preserved Cardiac Function With Age and Increased Lifespan in Drosophila
Authors: Sessions Ayla O
Published: July 2021

Increased deposition of extracellular matrix (ECM) is observed in all advanced age heart failure patients, but current model systems are complex and slow to age. To investigate the effect of extracellular remodeling on mechanical function in genetically tractable, rapidly aging, and simple model organisms, we employed Drosophila melanogaster, which has a simple trilayered heart tube.

We found that two common wildtype strains of Drosophila, i.e. yellow-white (yw) and white-1118 (w1118), exhibit different cytoskeletal and ECM remodeling with age. Using a recently developed nanoindentation method to measure cardiomyocyte stiffness and high speed optical imaging to assess contractility of intact Drosophila hearts, we found that yw flies had stiffer intercalated discs (ICD) and exhibited diastolic dysfunction with age. On the other hand, w1118 flies had a shorter lifespan compared to yw, did not exhibit ICD stiffening, had a less severe diastolic dysfunction, and showed an increase in ECM layer thickness between ventral muscle (VM) and cardiomyocyte (CM) layers of the heart tube. To modulate ECM and assess its effect in the aged w1118 flies, we knocked-down ECM genes LamininA and Viking (homologous to Collagen IV). Both ECM KD genotypes exhibited diastolic dilation with increased fractional shortening at adult (1wk) and aged (5wk) time points. The LamininA KD resulted in decreased cardiomyocyte stiffness correlating with increased relaxation velocities in adult flies and preservation of shortening and relaxation velocities in aged flies over controls. However, both the LamininA and Collagen IV KD flies experienced a basal increase in the decoupling of their cardiomyocytes as determined by heart period variance and % fibrillar heart-beats. These conductance issues were not enough to counteract the increased cardiac output and performance with age, and the Collagen IV KD outlived controls by 1.5 weeks median survival and the LamininA KD by 3 weeks. This suggests that the cell-ECM contacts in the basement membrane are intimately tied not only to the coupling of the cardiomyocytes of the Drosophila heart tube but also to cytoskeletal remodeling, but perhaps different ECM proteins have different mechanisms for interacting with the cardiomyocyte cytoskeleton.
Abstract 374: Cardiac Fibroblasts are Essential During Pathological Stress
Authors: Kuwabara Jill T
Published: July 2021

Cardiac fibroblasts reportedly play a role in normal heart function and are responsible for adverse remodeling after heart injury. Although fibroblast activities can be detrimental during disease, a basal level of cardiac fibroblast activity is required to maintain structural integrity and prevent rupture. We use transgenic mouse models to demonstrate that reduced fibroblasts numbers have serious consequences during pathological stress. At baseline, we observe a dramatic loss of the cardiac fibroblast lineage, which results in vasculature alterations, such as capillary dilation and decreased capillary density. Baseline changes in basement membrane (laminin) and cardiomyocyte structure have also been identified in fibroblast deficient hearts. These phenotypic changes become exacerbated after surgery indicating that fibroblasts are necessary for crosstalk between other cell types in the heart. In addition, we predict that the loss of fibroblasts will cause enhanced deterioration in cardiac function after injury due to reduced structural integrity of the heart. We will use transverse aortic constriction (TAC) as a pressure-overload model. After 5 weeks of TAC, we observe a ~50% (Baseline 71.99±6.06, TAC5wks 22.09±1.89) decrease in ejection fraction in fibroblast deficient hearts compared to a ~16% (Baseline 76.21±7.08, TAC5wks 59.59) decrease in control hearts. Our data suggest that a specific level of cardiac fibroblast activity is required to maintain normal heart function. Our goal is to identify both deleterious and beneficial roles of fibroblasts in the response of the heart to the types of pathological stress commonly encountered in patients.
Abstract 378: Endothelial Cell Hypoxia-inducible Factor (HIF) Activity is Necessary for Right Ventricular Remodeling in a Murine Model of Chronic-hypoxic Pulmonary Hypertension
Authors: Kolb Todd M , Singer Ben , D’Alessio Franco , Damarla Mahendra , Damico Rachel L , Hassoun Paul M , Semenza Gregg L , Shimoda Larissa A
Published: July 2021

Introduction:
Pulmonary hypertension induced myocardial remodeling is required to minimize RV wall stress as pulmonary arterial pressures increase. Myocardial angiogenesis is presumed to play an integral role in early RV remodeling, though molecular regulators remain undefined. Endothelial cell (EC) expression of HIF-1 is necessary for adaptive LV remodeling during chronic pressure overload, with loss of EC-specific HIF-1 activity associated with reduced LV capillary density. We hypothesized that EC HIF activity may also be necessary for adaptive RV remodeling and angiogenesis in a model of chronic RV pressure overload.

Methods:
We used a murine model of chronic hypoxic pulmonary hypertension (CH-PH) to induce RV remodeling. Conditional transgenic mice lacking expression of the obligate HIF-1α/HIF-2α co-factor ARNT (aryl hydrocarbon receptor nuclear translocator) in Tie2+ lineage cells (

ARNT
fl/fl

;

Tie2-Cre
+/-

) and

ARNT
fl/fl
;Tie2-Cre
-/-

controls were exposed to normobaric hypoxia (10% FiO
2
) for 1-5 weeks. We measured RV hypertrophy (RVH), RV systolic pressure (RVSP), and myocardial EC proliferation (flow cytometry) as a marker for sprouting angiogenesis.

Results:
After three weeks of CH-PH, RVH was observed in

ARNT
fl/fl
;Tie2-Cre
-/-

control mice, as anticipated (0.332±0.02 vs. 0.231±0.02;
P
< 0.05). CH-PH induced RVH in

ARNT
fl/fl
;Tie2-Cre
-/-

mice was associated with an early (1 week) increase in RV EC proliferation (vs. LV, 4.5±1.3% vs.1.8±0.5%;
P
< 0.05). Conversely,

ARNT
fl/fl

;
Tie2
-

Cre
+/-

mice did not develop RVH after 3 weeks of CH ( 0.222±0.01 vs. 0.241±0.02;
P
= NS), and early RV EC proliferation was attenuated (vs. LV, 2.9±1.2% vs. 1.5±0.3%;
P
= NS). Despite similar increases in CH-PH induced RVSP after 3 weeks, after 5 weeks RVSP normalized in

ARNT
fl/fl

;
Tie2
-

Cre
+/-

mice, but remained elevated in

ARNT
fl/fl
;Tie2-Cre
-/-

controls.

Conclusions:
These preliminary findings confirm that EC-specific HIF activity is necessary for CH-PH induced RVH, and suggest that reduced EC proliferation (and potentially angiogenesis) may contribute to abrogated RV remodeling in

ARNT
fl/fl

;
Tie2
-

Cre
+/-

mice. We hypothesize that late normalization of RVSP in these mice may be indicative of premature RV failure.
Abstract 382: Reduced Mitochondrial Function and Metabolic Dysregulation in Aneurysmal Fibulin-4 Mice.
Authors: van der Pluijm Ingrid , van Heijningen P.M. , IJpma A. , van Vliet N. , Sluiter W , Davis E.C. , Ringuette L.J. , Dekkers D.H.W. , Ghazi S. , Que I. , Kaijzel E.L. , te Riet L. , Gabriels S.I.C. , Mastroberardino P.G. , van der Linden R. , Vermeij M. , Demmers J.A. , Das D. , Yanagisawa H. , Kanaar R. , Essers J.
Published: July 2021

Thoracic aortic aneurysms are a life-threatening condition often diagnosed too late. The underlying mechanism is largely unknown. An accurate and early predictive biomarker for aneurysm formation has not yet been identified, although some molecular processes, such as disturbed TGF-β signalling, have been implicated. To discover novel robust biomarkers, we aimed to better understand the molecular mechanisms involved in aneurysm initiation and progression.

In Fibulin-4
R/R
mice, the extracellular matrix protein Fibulin-4 is 4-fold reduced, resulting in progressive ascending aneurysm formation and early death around 3 months of age. We performed LC-MS/MS proteomics and transcriptomics analyses on the aortas of Fibulin-4
R/R
and Fibulin-4
+/+
mice. Protein and gene data sets were separately analysed for genotype specific differences with Ingenuity Pathway analysis tools. Intriguingly, we observed alterations in mitochondrial composition in aortas from Fibulin-4
R/R
mice. Consistently, functional studies in Fibulin-4
R/R
vascular smooth muscle cells (VSMCs) revealed lower oxygen consumption rates, but increased acidification rates compared to Fibulin-4
+/+
. The mitochondria in VSMCs of Fibulin-4
R/R
mice were reduced in size and had increased complex I-IV levels. Furthermore, aortas of aneurysmal Fibulin-4
R/R
mice displayed increased levels of ROS. Consistent with these findings, gene expression analyses revealed the dysregulation of metabolic pathways. In accordance, ketone levels in the blood of Fibulin-4
R/R
mice were reduced and liver fatty acids were decreased, while liver glycogen was increased. As predicted by these findings, activity of PGC1α, a key regulator between mitochondrial function and organismal metabolism, was downregulated in Fibulin-4
R/R
VSMCs.

In conclusion, our data indicate altered mitochondrial function and metabolic dysregulation, leading to increased ROS levels and altered energy production, as a novel mechanism, which may contribute to thoracic aortic aneurysm formation. This discovery will not only provide new biomarkers that can be validated in human aortas, but they will also provide the rational for new interventions such as alterations in diet to prevent aneurysm formation.
Abstract 383: 17 beta-Estradiol Enhances Mitochondrial Function of Cerebral Arteries From Ovariectomized Female Rats
Authors: Rutkai Ibolya , Dutta Somhrita , Walter Korey A , Katakam Prasad V , Busija David W
Published: July 2021

Previous studies have provided indirect evidence that circulating sex hormones alter the function of the cerebral circulation perhaps via effects on mitochondrial dynamics. However, effects of estrogen on mitochondrial respiration have never been directly examined. We have previously observed a difference in the mitochondrial function of cerebral arteries from male and female rats but the exact mechanisms are not clear.

We tested the hypothesis that mitochondrial respiration in isolated cerebral arteries from ovariectomized (OVX) Sprague Dawley rats treated with a 21 d release, 0.5 mg of 17 β-estradiol pellet (OVX+E) was enhanced compared with arteries from placebo treated OVX rats. The Seahorse Bioscience XFe24 system was used to measure mitochondrial oxygen consumption rate (pM/min/μg protein) in cerebral arteries. Western blot was used to investigate the arterial expression of proteins. Radioimmunoassay was used to measure serum estradiol level.

Treatment with 17 β-estradiol resulted in a higher serum estradiol level (146.9±18.16 pg/ml) and uterus weight (0.15±0.0058 g) in the OVX+E compared with the OVX (14.7±1.2 pg/ml, 0.07±0.003, respectively; p<0.05). The components of mitochondrial respiration in pM/min/μg protein including basal respiration (147±9), ATP production (44±4), proton leak (102±7), and maximal respiration (212±13) were elevated in OVX+E compared with OVX (105±13, 21±4, 50±6, 138±10, respectively; p<0.05). Expression of the mitochondrial DNA encoded Complexes I and III, the nuclear DNA encoded Complexes II, IV, V, and the voltage-dependent anion channel protein were similar in all groups. However, the ratios of phosphorylated endothelial and neuronal nitric oxide (NO) synthase normalized to total protein were significantly (p<0.05) elevated in the OVX+E (1.43±0.06, 1.15±0.2, respectively) compared with OVX group (0.92±0.06, 0.53±0.19, respectively).

Our findings provide direct evidence for sex-specific differences in mitochondrial function on freshly isolated cerebral arteries. Thus, estradiol replacement enhances the efficacy of the oxidative phosphorylation resulting in an increased mitochondrial respiration which is not due to increased mitochondrial protein expression but may be due to enhanced NO.
Abstract 387: Role of Glycolysis in Cardiac Adaptation to Exercise
Authors: Gibb Andrew , Bhatnagar Aruni , Hill Bradford
Published: July 2021

Cardiac stressors affect the function and structure of the heart with prominent effects on metabolism. While the heart typically derives most (~80%) of its energy from fatty acids, the exercised heart is associated with an enhanced capacity to utilize a variety of carbon sources to sustain energy demands that would be advantageous during increased workloads and ischemic events. Due to the central role of glycolysis in glucose metabolism, it is likely that changes in the glycolytic rate regulate cell growth, homeostasis and stress responses. In this study, we assessed how exercise affects glucose metabolism in the myocardium and whether genetically decreasing glycolysis in the heart - using a validated, cardiac-specific, dominant negative form of phosphofructokinase 2 (kd-PFK2) - affects cardiac adaptation to exercise. Adult male FVB/NJ mice were subjected to forced treadmill running for 30 days. Exercise performance was measured and echocardiographic analysis was used to assess changes in cardiac function. Immunoblotting was performed to quantify the abundance of key enzymes involved in glucose metabolism. Exercise training increased exercise capacity by 70% (p < 0.01) and work performed by 69% (p < 0.01). Exercise training also induced cardiac hypertrophy (15%; p < 0.01) associated with a 1.4 fold increase in mitochondrial content (p < 0.05). Cardiac function remained largely unchanged with a significant reduction of 5% in ejection fraction (p < 0.01) and an increase in the left ventricle chamber dimensions (p < 0.05). Relative myocardial abundance of thirteen glycolytic and glucose metabolism enzymes remained unchanged. The kd-PFK2 mice showed that low levels of glycolysis in the heart promote cardiac hypertrophy (15%; p < 0.01) and prevent physiological hypertrophy due to exercise. In comparison with WT mice, the kd-PFK2 mice had a 20% lower exercise capacity (p < 0.05). These findings suggest that the glycolytic rate regulates cardiac adaptations to exercise and may mediate some of the beneficial effects of exercise training on exercise capacity.
Abstract 39: CD47 Regulates Aberrant Ca2+ Channel Current in Cardiac Myocytes
Authors: Sharifi-Sanjani Maryam , Gabris Beth , Li Qiao , Salama Guy , Isenberg Jeffrey S
Published: July 2021

Background:
Left ventricular (LV) heart failure (HF) affects over 5 million Americans with 50% of HF patients dying within 5 years of diagnosis. Therapeutics, while only relieving symptoms and have multiple side effects, have not completely resolved this process. HF is associated with cardiac myocyte cytosolic Ca2+ overload due to impaired sarcoplasmic reticulum (SR) and/or membrane Ca2+ handling. We recently found that a widely expressed cell surface receptor CD47 and its high affinity ligand, thrombospondin-1 (TSP1), are expressed on cardiac myocytes. We further showed that activated CD47 increases isolated neonatal cardiac myocyte cytosolic Ca2+ levels in part through SR Ca2+ channels. We postulated that TSP1-CD47 signaling may in part play a role in LV HF secondary to dysregulation of Ca2+, though the specific Ca2+ dynamics through which TSP1-CD47 signaling controls cardiac Ca2+ remains to be determined.

Methods:
Isolated hearts from mutated mice lacking CD47 (CD47-/-) and wild type (WT) mice with and without 4 weeks of transverse aortic constriction (TAC) were studied in ex vivo perfused heart Langendorff using dual optical mapping of Cai transients and action potential or voltage measurements. Isolated cardiac myocyte experiments were performed using whole cell voltage-clamp analysis.

Results:
In TAC-stressed WT murine hearts studied in the Landendorff system, tetracaine (an inhibitor of SR ryanodine receptor2) blunted arrhythmia induced by electrical stimulation. Isoproterenol-induced Ca2+ transient irregularities in hearts from WT mice were ameliorated in CD47-/- mice. Further, hearts from WT mice treated with peptide 7N3, a TSP1 mimetic, showed induced Ca2+ transient irregularities and elicited early after depolarization which was also attenuated with K2O1 (ryanodine receptor2 stabilizer) treatment. Voltage-clamp studies of isolated adult mouse cardiac myocytes showed that 7N3 peptide increased the L-type Ca2+ channel current (ICa,L) whereas the scrambled control peptide did not alter ICa,L.

Conclusion:
These data demonstrate that CD47 activation may be a trigger for aberrant myocyte Ca2+ handling typically associated with HF. Targeting of TSP1-CD47 signaling may open a new therapeutic avenue to be further studied in HF.
Abstract 40: Discovery and Characterization of the Novel Muscle-Specific Membrane Protein Smco1
Authors: Papizan James B , McAnally John R , Bassel-Duby Rhonda , Olson Eric N
Published: July 2021

Mutations in numerous membrane proteins cause debilitating myopathies. The discovery of novel muscle-specific, membrane proteins would likely provide insight into mechanisms of disease and potentially yield new therapeutic targets. Through bioinformatics screening for muscle-specific membrane proteins with unknown function, we identified C3orf43 or single-pass membrane protein with coiled-coil domains 1 (Smco1). Consistent with bioinformatics predictions, Smco1 is expressed exclusively in cardiac and skeletal muscle. We demonstrate with chromatin immunoprecipitation and luciferase promoter assays that Smco1 is a Mef2-regulated gene with robust expression occurring shortly after birth. Immunofluorescent analysis demonstrates Scmo1 localizes to the cardiomyocyte sarcolemma and intercalated disks. Talen-mediated disruption of Smco1 in mice results in stunted postnatal growth, cardiac hypoplasia and skeletal muscle myopathy as early as postnatal day 15. While studies are on going to determine the function of Smco1, our findings reveal an essential role of Smco1 in striated muscle structure and function. The identification of heart- and muscle-specific membrane proteins will likely illuminate the mechanisms of muscular membrane diseases.
Abstract 402: Selective Protein Secretion Upon Endoplasmic Reticulum Stress Protects Cardiac Myocytes
Authors: Doroudgar Shirin , Thuerauf Donna J , Stastna Mirka , Voelkers Mirko , Van Eyk Jennifer E , Glembotski Christopher C
Published: July 2021

Protein secretion is important for proper cardiac myocyte function. Many secreted proteins are synthesized and folded in the sarco- endo-plasmic reticulum (SR/ER). A number of diseases, including heart disease, alter the ER in ways that impair ER protein folding, causing ER stress, which can result in cardiac myocyte dysfunction and decreased viability. In studies aimed at assessing the effects of ER stress on cardiac myocyte viability, heart disease-related ER stress was mimicked by treating neonatal rat ventricular myocytes (NRVM) with either tunicamycin (TM) or thapsigargin (TG), which inhibit SR/ER protein glycosylation or decrease SR/ER calcium, respectively. When treated in high culture media volumes, both TM and TG caused cardiac myocyte death; however, in low culture media volumes, while TM still caused death, remarkably, TG was protective, suggesting that potentially protective factors were secreted in response to TG but not TM. To characterize these factors, the identities of proteins in control-, TM-, and TG-conditioned medium from NRVM were determined by proteomic approaches using high performance liquid chromatography and mass spectrometry. Twenty-four different proteins, known to be synthesized in the ER, were identified in control-conditioned medium. The levels of eighteen of these proteins, including extracellular matrix proteins, hormones, and growth factors were decreased in TM- and TG-conditioned medium. However, the levels of three SR/ER-resident, calcium-binding chaperones, glucose regulated protein 78 (GRP78), glucose regulated protein 94 (GRP94), and calreticulin were increased in TG-conditioned medium but not in TM-conditioned medium. Furthermore, we found that ischemia/reperfusion, which decreases SR/ER calcium, upregulated secretion of the proteins selectively secreted in response to TG. Thus, while ER stress mediated by TM or TG decreases the movement of most proteins through the secretory pathway, TG, which mimics the effects of heart disease on SR/ER calcium in cardiac myocytes, selectively enhances the secretion of a subset of proteins, which confer protection. Therefore, proteins once thought to be permanent residents of the SR/ER may have novel, extracellular, protective roles in the diseased heart.
Abstract 41: Different MicroRNA Expression Profiles in Murine Aorta and Carotid Arteries
Authors: Curty-Costa Ernesto , Hoffmann Luísa , Silva Rosane , Ürményi Turán P , Faffe Debora S
Published: July 2021

Atherosclerosis, a major cause of death, affects vascular walls diffusely. Some vascular beds are, however, preferentially involved. Small noncoding RNAs, microRNAs, have emerged as key regulators of gene expression in either physiological or pathophysiological processes. Here, we investigated whether different vascular beds, commonly affected by atherosclerosis, present specific microRNA expression profiles. To this end, aorta (Ao) and carotid (Ca) arteries from two male Wistar rats (200 g) were dissected, total RNA was extracted with Trizol, and the small RNA fraction was enriched using magnetic beads. Sequencing was performed using RNA-Seq on Ion Torrent PGM platform and data were analyzed using CLC Genomics Workbench software. We identified 372 and 305 microRNAs in Ca and Ao, respectively - the arteries shared 280 microRNAs. The majority of the 20 most expressed microRNAs were similar between the arteries. Of these top 20, 90% in the Ao were also among the most expressed in Ca, and in Ca 65% were also among the most expressed in Ao. Both mechano and platelet-microRNAs were identified in both arteries. Note that overexpression of mechano-miR-21 is reported to attenuate lipid accumulation and to reduce inflammation, preventing atherosclerosis. We detected mechano-miR-21, however, only in the Ao. We also found that murine aorta and carotid arteries express differently 29 microRNAs (p<0.05). Among the 29 microRNAs, 8 (28%) have been related to atherosclerosis. Of these, 6 were more expressed in Ca (miR-181, miR-9a-1, miR-9a-2, miR-511, miR-146, miR-27), and 2 were more expressed in Ao (miR-144 and miR-322). Note that 3 of the 6 more expressed microRNAs in Ca are related to atherosclerosis, that is: miR-181 regulates cell stress and proliferation; miR-511 regulates cholesterol synthesis, and miR-27 is a known marker of the disease. Taken together, our results suggest that specific microRNA expression profiles may play a role in the vascular behavior during the atherosclerotic process.