bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P358: Cardiac Innervation Remodeling And Impaired Brain Derived Neurotrophic Factor (bdnf) Levels In Physiological Aging Vivo Model
Authors: Elia Andrea , Cannavo Alessandro , Gambino Giuseppina , Cimini Maria , Ferrara Nicola , Kishore Raj , Paolocci Nazareno , RENGO Giuseppe
Published: November 2021

Aging is a multifactorial process associated with gradual loss of function and decay involving several neurohormonal systems, such as the autonomic nervous system (ANS). Progressive remodeling of ANS, induces a circulating catecholamines spillover and cardiac autonomic fibers depletion with raising both morbidities and mortality risk. Neurotrophic factors (NF) play a pivotal role in modulating neuronal function and are impaired in cardiovascular disorders. Whether and how physiological aging impacts these neurobiomarkers and cardiac innervation remains still unclear. Therefore, we investigated the impact of aging on neurotrophins (such as BDNF and NGF) production and secretion and its consequences, on cardiac nervous system homeostasis. In vivo, we used young (age: 3 months; n=10) and old (age: 24 months; n=11) male Fisher rats. In vitro, human neuroblastoma cells (SH-SY5Y) were stimulated with serum withdrawn from both experimental groups. Old rats showed a significant reduction in overall ANS fiber density, sympathetic (marked by dopamine β-hydroxylase, dβh) and cholinergic compartment (evidenced by vesicular acetylcholine transporter, VaChT) compared to the young group, assessed by immunohistochemical staining. In addition, we observed a marked downregulation of GAP-43 and BDNF protein levels in left ventricle total lysates via immunoblot analysis, in aged hearts as opposed to young ones. Conversely, no changes were observed in NGF protein expression. To further investigate the autocrine effect of aging on autonomic nerve fibers, we treated SH-SY5Y cells in vitro, with blood serum obtained by young or old rats. Both stimuli induced a remarkable increase in neuronal sprouting, as evidenced via crystal violet assay. Nevertheless, we found a bulky drop in the neuronal function of cells stimulated with old rat serum. Interestingly, this effect was accompanied by a sizeable blunt in GAP-43 and BDNF protein levels, compared to cells treated with young rat serum. Taken together, our data suggest that neuronal function impairment aging-induced associated with significant BDNF impoverishment, might favor maladaptive remodeling of cardiac ANS.
Abstract P361: Reduced Cardiac Tmem65 In Mouse Hearts Results In Intercalated Disc Defects And Eventual Dilated Cardiomyopathy With Cardiac Fibrosis
Authors: Teng Allen C , Gu Liyang , Di Paola Michelle , Williams Zachary , Au Aaron , Callaghan Neal , Hakem Zadeh Farigol , Zhao Yu-Qing , Fatah Meena , Chatterjee Diptendu , Jourdan J. J , Simmons Craig A , Kislinger Thomas , Yip Christopher , Gourdie Robert G , Hamilton Robert M , Gramolini Anthony
Published: November 2021

The intercalated disc (ICD) is unique membrane structure that is indispensable to normal heart function. However, its structural organization is not well understood. Previously, we showed that the ICD-bound transmembrane protein 65 (Tmem65) was required for connexin 43 (Cx43) localization in cultured mouse neonatal cardiomyocytes. Here, we investigated the role of Tmem65 in ICD organization
in vivo
. A mouse model was established by injecting CD1 mouse pups (3-7 days after birth) with recombinant adeno-associated virus 9 (rAAV9) harboring Tmem65 (or scrambled) shRNA. Quantitative polymerase chain reaction (qPCR) and immunoblots confirmed greater than 85% reduction in Tmem65 expression (7.1±0.7% remained for Tmem65 proteins; 14.4±2.5% remained for Tmem65 transcripts,
n
=4) in mouse ventricles compared to control hearts. Tmem65 knockdown (KD) mice exhibited heart failure-like symptoms as early as 3 weeks post viral administration. Specifically, Tmem65 KD mice developed eccentric hypertrophic cardiomyopathy in 3 weeks and dilated cardiomyopathy with severe cardiac fibrosis in 7 weeks, as confirmed by H&E and Masson’s Trichrome staining. Echocardiography and electrocardiography, respectively, showed depressed hemodynamics (19.27±1.46ml/min for cardiac output in control hearts vs. 6.63±0.52ml/min for Tmem65 KD hearts,
n
=6) and impaired conduction, including prolonged PR (22.7±1.85ms in control hearts vs. 28.89±3.85ms in Tmem65 KD hearts, n≥8), QRS intervals (10.47±0.42ms in control hearts vs. 16.35±0.36ms in Tmem65 KD hearts, n≥8), and slowed heart rate (415±10bpm in control hearts vs. 347±16bpm in Tmem65 KD hearts, n≥8) in Tmem65 KD mouse hearts.

Immunoprecipitation and super-resolution microscopy confirmed the physical interaction and localization between Tmem65 and voltage-gated sodium channel β subunit (β1) at the ICD and this interaction was evidently required for the establishment of perinexal nanodomains and voltage-gated sodium channel 1.5 (NaV1.5) localization to the ICD. Disrupting Tmem65 function, thus, impaired perinexal structure, reduced conduction velocity, and ultimately resulted in cardiomyopathy
in vivo
.
Abstract P364: Liraglutide Protects Against Angiotensin-induced Diastolic Dysfunction And Is Associated With Altered Acetyl-coa Metabolism
Authors: Rutledge Cody A , Lopez Mabel , Mullett Steven , Wendell Stacy , Goetzman Eric , Kaufman Brett A
Published: November 2021

Background:
The mechanisms underlying diastolic dysfunction remain complicated and poorly understood. Previously implicated mechanisms include increased interstitial fibrosis and oxidative damage, altered calcium handling, and mitochondrial dysfunction. In this study, we investigate changes in the cardiac metabolic profile from mice with Angiotensin-II (Ang)-induced diastolic dysfunction. Because treatment with the GLP-1 agonist liraglutide (Lira) relieves Ang-induced diastolic dysfunction, we examined altered metabolites in Ang and Ang+Lira mice to identify metabolic pathways involved.

Methods:
8-wk mice were implanted with Ang pumps (1000 ng/kg/min) +/- Lira (0.2 mg/kg/day) therapy for 4 wks and compared to sham mice. Baseline and 4-wk echocardiography was performed. Hearts were collected for RNA-sequencing, Western blot, histology, radiolabeled palmitate assay, and targeted metabolomics by liquid chromatography-mass spectrometry to assess metabolic changes.

Results:
After Ang treatment, mice had significant diastolic dysfunction but only mild hypertrophy based on echo and histologic measures and no evidence of systolic change. Compared to sham mice, Ang mice had reduced E/A (Sham, 2.67±0.40; Ang, 1.71±0.11; p<0.05) and peak reverse longitudinal strain rate (rLSR;Sham, 8.48±0.65/s; Ang, 6.081±0.33/s; p<0.05, consistent with diastolic dysfunction. Ang+Lira mice had improved E/A (3.57±0.50, p<0.01 vs. Ang) and rLSR (8.65±0.68/s, p<0.01 vs Ang). Targeted metabolomic analysis of hearts found significantly increased accumulation of Acetyl-CoA in Ang mice (peak area 0.14±0.02) compared to sham (0.04±0.02 ,p<0.05) that is restored by Lira therapy (0.02±0.01 ,p<0.05 vs sham and Ang). A radiolabeled palmitate oxidation assay found decreased palmitate oxidation in both Ang and Ang+Lira mice compared to sham, but interestingly, also demonstrated significantly lower acid soluble metabolite oxidation in the Ang+Lira mice vs. Ang alone.

Conclusions:
We found that Ang-treated mice accumulate myocardial Acetyl-CoA, suggesting a defect in TCA flux. Lira resolves the increase in Acetyl-CoA and improved measures of diastolic dysfunction. This data may implicate a novel metabolic pathway in the development of diastolic disease.
Abstract P365: Neutrophils Contribute To Doxorubicin-induced Cardiotoxicity
Authors: Bhagat Anchit , Kleinerman Eugenie
Published: November 2021

Doxorubicin is one for the most effective chemotherapy agents for the treatment of childhoodcancer. Unfortunately Dox treatment causes damage to the heart and childhood cancer survivorsare at higher risk of developing cardiovascular disease at an earlier age than community controls.Understanding the mechanisms by which Dox induces cardiotoxicity is crucial to identifyingpreventive interventions. Innate immune cells, in particular neutrophils, have been shown to playa role in other forms of cardiac disease such as atherosclerosis and myocardial infarction.However, the role of these immune cells in Dox-induced cardiotoxicity is poorly understood.We hypothesized that neutrophils contribute to the cardiac damage caused by Dox treatment andinvestigated this hypothesis using our juvenile mouse cardiotoxicity model. Mice were treated withDoxorubicin for 2 weeks. Echocardiograms were performed before and 24 hours after therapy.Hearts were then harvested, and the heart tissue evaluated for neutrophil infiltration using flowcytometry. There was a decrease in heart function as quantified by echocardiography (ejection fraction [EF] and fractional shortening [FS]) 24 hours after therapy which persisted for 12 weeks. There was also an elevation in neutrophils in heart tissue of Dox-treated mice as compared to controls. To determine whether neutrophils contributed to the functional decline seen in the Dox-treated mice, neutrophils were depleted using an anti-Ly6G antibody prior to Dox treatment. Neutrophil depletion was confirmed by flow cytometry in the blood and hearts of Dox-treated mice. Neutrophil depletion prevented the Dox-induced decrease in EF and FS seen at 24 hours and 12weeks after therapy. Additionally, we also analyzed heart sections for fibrosis and found that neutrophil depletion protected against excessive collagen deposition 12 weeks after therapy. Together these findings suggest that neutrophils contribute Dox-induced cardiotoxicity and that targeting neutrophils may be a potential strategy to prevent Dox-induced cardiotoxicity.
Abstract P371: Fusion Of C-type Natriuretic Peptide With Elastin-like Polypeptide For Enhanced Pharmacological Activity
Authors: Jowett James D , Arnold Susan , Umsted Bret , Sendecki Dave , Dickerhoff Dave , Lee John
Published: November 2021

C-type natriuretic peptide (CNP) is a pluripotent molecule produced by the endothelium and myocardium with therapeutic potential in pulmonary hypertension, myocardial infarction, and heart failure. However, the native peptide’s short elimination half-life of under 3 minutes limits its use clinically. To improve the pharmacokinetic profile of CNP we recombinantly expressed the CNP peptide as a fusion with an elastin-like polypeptide (ELP). ELPs are biopolymers that undergo reversible temperature-dependent phase separation and form a subcutaneous depot upon injection. CNP-ELP fusion proteins were expressed in
E. coli
, purified, and showed
in vitro
potency 2-4 fold less than native CNP. When administered subcutaneously in mice CNP-ELP showed a significantly enhanced PK profile due to the slow release of the fusion into the circulation with detectable fusion protein 24 hours after a single injection. In addition, the CNP-ELP increased growth velocity in mice significantly greater than that of native peptide indicating an
in vivo
activation of the CNP receptor, NPR-B. These findings represent a new method of administering a long acting CNP peptide for treatment of different cardiovascular diseases.
Abstract P378: Metabolic Dysregulation Of Endothelial Sk Channels Via Protein Kinase C
Authors: Xing Hang , Shi Guangbin , Liu Yuhong , Sellke Frank W , Feng Jun
Published: November 2021

Introduction:
Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease, which predisposes to ischemic cardiovascular events. Small conductance calcium-activated-potassium (SK) channels are largely responsible for coronary arteriolar relaxation mediated by endothelium-dependent hyperpolarizing factors. Diabetic inactivation/inhibition of endothelial SK channels contributes to endothelial dysfunction. Endothelial dysfunction during diabetes (DM) is also associated with increases in metabolites NADH, and PKC. The pyridine nucleotide NADH has been recently found to inactivate endothelial SK channels. The overexpressed and activated PKC has been shown to play an important role in diabetes-induced endothelial dysfunction. However, it is undefined if PKC is involved in the metabolite NADH dysregulation of endothelial SK channels.

Hypothesis:
We hypothesized that PKC in a signaling cascade whereby NADH dysregulates endothelial SK channels.

Methods:
SK channels currents of human coronary artery endothelial cells were measured by whole cell patch clamp method in the presence or absence of NADH, and/or PKC activator PMA, PKC inhibitors or endothelial PKC
α
/PKCβ knock-down by using short interfering RNA.

Results:
NADH (30-300μM, n=7-9) or PKC activator PMA (30-300μM, n=6-12) reduced endothelial SK current density (p<0.05 vs. control (n=15), Fig. A-D), whereas the selective PKC
α
inhibitor LY333531 (50nM, n=12) significantly reversed the NADH-induced SK channel inhibition (p>0.05 vs. control (n=15), Fig. E). PKC
α
knock-down failed to affect NADH (n=13) and PMA (n=10) inhibition of endothelial SK currents (Fig. F). In contrast, PKCβ knock-down significantly prevented NADH (n=12) and PMA (n=6)-induced SK inhibition (p>0.05, vs. control, Fig. G).

Conclusions:
The metabolite NADH dysregulation of endothelial SK channels was via PKCβ activation.
Abstract P387: Deceasing Tissue Stiffness Improves The Efficacy Of Extracellular Matrix-based Therapy For Myocardial Infarction
Authors: Wang Xinming , Senyo Samuel
Published: November 2021

Hypothesis and objective:
We hypothesize that lowering heart stiffness improves heart regeneration induced by extracellular matrix (ECM) proteins. In this study we investigated heart post-injury responses in non-regenerative mice hearts with modulated tissue stiffness and ECM hydrogel. The objectives are 1) determining if heart stiffness affects heart regeneration in response to extracellular biomolecules; and 2) identifying specific signaling pathways that regulate fibroblasts and cardiomyocytes mechanosensitivity to extracellular proteins.

Methods:
P5 mouse heart stiffness was modulated by BAPN and genipin injections. Solubilized porcine fetal ECM was injected immediately after ligating coronary artery. Heart function and histology were conducted on day 3 and week 3 post-myocardial infarction (MI). A mice ventricle explant model was used to investigate the molecular mechanisms.

Results:
Heart stiffness was lowered from 50kPa to 9kPa by BAPN and increased to 142kPa by genipin. We observed that fetal ECM treatment preserved cardiac output, reduced fibrosis, and promoted cardiomyocyte cell cycle activity. Decreasing tissue stiffness further improved the therapeutic efficacy of fetal ECM on heart post-MI response (Fig.1). Decreasing tissue stiffness lowered fibrosis in non-ECM treated MI control hearts, but did not affect the other aspects. Using a ventricle explant model of various stiffness, molecular evidence demonstrated that agrin-YAP signaling pathway is involved in the mechano-regulation of fetal ECM-induced heart regeneration. Agrin expression was elevated and cardiomyocyte YAP activation was not affected by changing tissue stiffness in control (no-ECM) mechano-modulated explants. However, YAP activation in fetal-ECM treated explants was increased by softening tissue. We also demonstrated that ECM hydrogel inhibits fibroblast to myofibroblast differentiation through CAPG protein.
Abstract P388: Extra-cardiac BCAA Catabolism Lowers Blood Pressure And Protects From Heart Failure
Authors: Murashige Danielle , Jang Cholsoon , Neinast Michael , Levin Michael , Jung Jae Woo , Lambert Jonathan P , Garbincius Joanne , Kim Boa , Marti-Pamies Ingrid , Flam Emily , Hoshino Atsushi , Yang Steven , Roberts Emilia , Li Li , Assoian Richard K , Elrod John W , Rabinowitz Joshua , Arany Zoltan
Published: November 2021

Pharmacologic activation of branched chain amino acid (BCAA) catabolism is protective in numerous models of heart failure (HF). How this protection occurs has remained unclear, although a causative block in cardiac BCAA oxidation has been proposed. We use here in vivo heavy isotope infusion studies to show that cardiac preference for BCAA oxidation increases, rather than decreases, in multiple models of HF. We use various genetic models to show that cardiac-specific activation of BCAA oxidation does not protect from HF, even though systemic activation of BCAA oxidation does. Lowering plasma and cardiac BCAAs by genetic means is also not sufficient to confer protection comparable to that conferred by pharmacologic activation of BCAA oxidation, suggesting alternative mechanisms of protection. Surprisingly, telemetry and invasive hemodynamic studies showed that pharmacological activation of BCAA catabolism lowers blood pressure, a well-established cardioprotective mechanism. The effects on blood pressure occurred independently of nitric oxide (NO), and reflected a vascular resistance to adrenergic constriction. Finally, mendelian randomization studies revealed that elevations in plasma BCAAs portend higher blood pressure in large human cohorts. Together, these data indicate that activation of BCAA oxidation lowers blood pressure and protects from heart failure independently of any direct effects on the heart itself.
Abstract P391: Inhalation Of Airborne Particulate Matter Alters Cardiovascular Function Of Alzheimer-prone Mice
Authors: O'Piela Devin R , Saldana Ty A , Aslaner David M , Gorr Matthew W , Mackos Amy R , Wold Loren E
Published: November 2021

Air pollution has detrimental effects on cardiovascular and lung function, and the extent of its pathological consequences continues to be uncovered. Recently, air pollution has been implicated in the development of Alzheimer Disease (AD) progression. AD and heart failure are common co-morbidities, giving reason to believe that cardiovascular dysfunction may contribute to AD. A known contributor to cardiovascular dysfunction-particulate matter (PM
2.5
, < 2.5 μm diameter)—is a critical component of air pollution and is considered a risk factor for heart failure and AD development. This co-morbidity pattern and shared environmental risk factor prompted the hypothesis that PM
2.5
contributes to cardiovascular dysfunction in a transgenic mouse model of AD. We tested our hypothesis by subjecting 6-month-old transgenic (APP) and non-carrier wildtype (WT) male mice to filtered air (FA) or PM
2.5
for 5 days/week, 6 hours/day for 3 months (n = 34). Following exposure, echocardiography, pressure-volume (PV) loops, and respiratory mechanics were performed to detect cardiac and pulmonary changes associated with genotype and exposure conditions among the 3-month group. Echocardiography revealed left ventricular anterior wall thickness in systole was significantly elevated among PM-exposed APP mice compared to FA-exposed APP controls. PV data demonstrated significant reduced end-systolic elastance in PM-exposed mice compared to FA-exposed mice in both WT and APP mouse models, demonstrating impaired contractility. PV loops also showed that the time constant of isovolumetric relaxation was increased in PM-exposed compared to FA-exposed WT mice. APP mice experienced higher lung resistance and central airway resistance with an increasing dose of methacholine. Taken together, these findings indicate airborne particulate matter exposure causes cardiac and pulmonary dysfunction in a transgenic mouse model of AD.
Abstract P392: The Effect Of Cell Sex On Magnetic Nanoparticle Uptake Of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes
Authors: Mahmoudi Morteza , Serpooshan Vahid , Yang Phillip C , Heydarpour Mahyar
Published: November 2021

Introduction:
It is well understood that the occurrence, progress, and treatment of heart failure, which is a leading cause of death worldwide, is sex-specific. Over the past decade, the majority of efforts in myocardial regeneration have been centered on cell-based cardiac repair. A promising cell source for these efforts is patient-specific human cardiomyocytes (CMs) differentiated from human inducible pluripotent stem cells (hiPSCs). However, successful use of hiPSC-CMs faces a major limitation, the poor engraftment and electromechanical coupling of transplanted cells with the host myocardial tissue. Magnetic nanoparticles (NPs) demonstrate great potential to address this challenge for treating heart failure via cell therapies. In particular, superparamagnetic iron oxide NPs (SPIONs) have been used to label hiPSC-CMs and, with the aid of external magnetic field, improve their engraftment and electromechanical coupling in the heart tissue. However, the critical role of cell sex in the uptake and labeling efficacy of NPs has not been evaluated.

Hypothesis:
Significant differences in the molecular and structural (e.g., actin structures and distribution) characteristics of male and female hiPSC-CMs affect their labeling efficacy with SPIONs.

Methods and Results:
To test our hypothesis, we first performed RNA-Seq analysis on three male and three female (healthy) hiPSC-CM lines. The normalized outcomes were analyzed by edgeR package. We next calculated gene-expression differential between male and female CMs. The results revealed 58 genes with significant differences between the male and female cells (p-value < 0.01). The highest observed sex-specific variation in genes was related to tophit gene (MEG3: logFC = 7.32, P-value = 5.63e
-06
), which is the maternally expressed imprinted gene with a great role in cardiac angiogenesis. Among the identified genes, a number of those were related to the cellular cytoskeletal structures including actin. We probed possible structural differences between actin filaments organization and distribution of male and female hiPSC-CMs using the stochastic optical reconstruction microscopy (STORM) technique. The results demonstrated substantial differences in organization, distribution, and morphology of actin filaments between male and female CMs. Incubation of SPIONs with male and female hiPSC-CMs revealed higher uptake of NPs (~ 3 folds) in female cells as compared to the male cells. The significant differences in the uptake of SPIONs by male
vs.
female cells could be attributed to the distinct organization, distribution, and morphology of actin in male
vs.
female cells.

Conclusions:
Our results indicate that male and female hiPSCs-CMs respond differently to the labeling SPIONs.
Abstract P397:
MTCH2
As A Modifier Of Cardiomyopathy
Authors: Fischer Julie A , Puckelwartz Megan , Wolf Matthew , Quattrocelli Mattia , Pesce Lorenzo , Bauer Rosemary , Kearns Samuel , McNally Elizabeth M
Published: November 2021

Background:
Cardiomyopathy is a highly heritable disorder that carries a significant risk for heart failure and arrhythmias. Most inherited cardiomyopathies are characterized by variable penetrance and expressivity, which in part arises from additional genetic variation, known as genetic modifiers.

Methods and Results:
Genomic profiling of human cardiomyopathy cases identified enriched genetic variation in the gene
MTCH2.
Specifically, a truncating variant was found to be overrepresented in patients with cardiomyopathy compared to controls.
MTCH2
encodes a mitochondrial carrier protein that has a role in regulating oxidative phosphorylation. To investigate fundamental mechanisms by which MTCH2 contributes to cardiac and metabolic phenotypes, we generated a knockdown model of the
Drosophila MTCH2
ortholog,
Mtch.
We found that cardiac-specific
Mtch
reduction in flies produced heart tube dilation and reduced function as well as a shortened life span, documenting a clear role Mtch in the myocardium. Metabolomic profiling demonstrated cardiac deficiency of
Mtch
lowered the flux of glucose-derived metabolites to the citric acid cycle associated with reduced downstream oxygen consumption and ATP synthesis, causing an energy deficit. We generated a deletion of
MTCH2
using gene editing in HEK293 cells. Similar to the fly model, these cells demonstrated reduced oxygen consumption in the presence of glucose, but not fatty acids, and had a higher level of inhibitory phosphorylation of pyruvate dehydrogenase, a critical regulator of glucose metabolism. These data suggest MTCH2 influences the efficiency of glucose oxidation and substrate usage, an important mode of cardiac energy generation, especially in the setting of heart failure.

Conclusions:
We identified
MTCH2
as a modifier of the cardiomyopathy phenotype in humans. Reduction of
MTCH2
resulted in impaired cardiac function with reduced oxygen consumption and increased glycolysis in a substrate dependent manner. Since failed hearts are more dependent on glycolysis, these data support that reduction of MTCH2 promotes heart failure and provides a mechanism by which MTCH2 acts as a deleterious genetic modifier in heart failure.
Abstract P402: Caveolin-1 Upregulation Leads To Decreased Erg And Stress-induced Ventricular Tachycardia In Cystic Fibrosis Hearts
Authors: Dai Dao-Fu
Published: November 2021

Background:
Cardiac manifestations of cystic fibrosis are classically accepted as cor pulmonale, yet emerging evidence supports that cardiac abnormalities can develop early when respiratory function is preserved. Myocardial cystic fibrosis transmembrane regulator (CFTR) channels, inactive in normal conditions, are activated in response to catecholamine stress.

Methods and Results:
In multiple mouse models of homozygous CFTR mutations (S489X or F508d), we demonstrate increased susceptibility to ventricular tachycardia (VT) in response to epinephrine, and this is associated with prolongation of QTc intervals (repolarization). Despite having normal left ventricular ejection fraction and histology, CF S489X
mut/mut
hearts showed 460 proteins significantly altered compared with WT hearts by tandem mass tag (TMT) proteomics. The Ingenuity Pathway Analysis of proteomics data revealed mitochondrial dysfunction and increased caveolin-1 signaling among the top pathways affected in CF S489X
mut/mut
hearts. In mouse hearts and human induced pluripotent stem cells derived cardiomyocytes (iPS-CM) with CFTR mutations, we observed increased ER stress and caveolin-1 (cav-1). These changes are concomitant with reduced levels of the ERG channel, a K+ channel contributing to phase 3 repolarization, mutations of which cause long QT syndrome II and VT. Patch clamp analysis showed decreased phase 2-3 repolarization current (including hERG current) in CF F508d iPS-CM compared with control iPS-CM, consistent with the findings of decreased membranous hERG and elevated Cav-1. Suppression of Cav-1 successfully restores membranous hERG in two separate lines of mutant iPS-CM generated from CF patients with F508d mutation. In F508d
mut/mut
mice, suppression of cardiac Cav-1 upregulation by reducing ER stress using TUDCA or crossing with heterozygous Cav-1
+/-
abolished the stress-induced VT. Our analysis of a large-scale real-world clinical database from TriNetX, including 4520 CF men, 3510 CF women and the same number of well-matched controls demonstrated novel findings of increased risk for QT prolongation and VT in CF patients. Retrospective analysis of 126 CF patients with available EKG in our hospital revealed significant QTc prolongation in CF patients compared with matched controls, for both males (CF 447.8±4.5 vs cont. 429.2±2.5) and females (CF 454.0±3.5 vs cont.437.5±2.4). The QTc interval is not affected by lung function (%predicted FEV1) and is an independent predictor of survival in CF patients.

Conclusions:
In mice and iPS-CM models of CF, we demonstrated impaired repolarization and increased susceptibility to stress-induced VT. This is mediated by ER-stress-induced upregulation of Cav-1, which leads to internalization of ERG and subsequent decrease in repolarization current.
Abstract P403: Three-dimensional Bioprinting Of Adhesive Cardiac Patch Systems
Authors: Chen Shuai , Tomov Martin , Ning Liqun , Gil Carmen , Hwang Boeun , Bauser-Heaton Holly , Serpooshan Vahid
Published: November 2021

Rapid advancements in 3D bioprinting have enabled the design of functionalized bioinks to create scaffolds with robust adhesive properties. These constructs are of great interest in cardiac tissue engineering, where the integration of grafted patch with the host myocardium, through sutures, staples, or adhesives, faces risks such as bleeding, cytotoxicity, and infection. We introduce the first generation of functional adhesive bioinks that can be bioprinted through various modalities to fabricate patch structures with intrinsic adhesive properties. A dopamine-modified hyaluronic acid methacrylate (HAMA-Dopa) and gelatin methacrylate (gelMA) composite bioink is used to create the patch via air and embedded bioprinting. Constructs are crosslinked onto a collagen sheet substrate simulating the host cardiac tissue (
Figure 1A-C
).

We developed novel
in vitro
methods to assess adhesion properties of printed patch under shear, tension, or dynamic loading (
Figure 1C
). Our approach allowed for steady and precise application of stress to the adhesion interface. Embedded-printed HAMA-Dopa/gelMA scaffold showed significantly enhanced adhesion strength (1025 Pa) compared to gelMA (495 Pa) and HAMA (477 Pa) control groups under tension, and under shear (548 Pa
vs.
234 and 239 Pa). The adhesion strength of air-printed HAMA-Dopa/gelMA constructs was markedly higher (10,128 Pa) than the embedded ones, possibly due to the more effective,
in-situ
crosslinking. We also investigated the dynamic adhesive properties in aqueous environment using an
ex vivo
beating heart model. Air-printed HAMA-Dopa/gelMA showed the greatest adhesion under wet conditions, tolerating 345,600 cycles. We further characterized printing fidelity, mechanical properties, swelling, and biocompatibility of HAMA-Dopa/gelMA constructs, demonstrating adequate functionality of this bioprinted adhesive scaffold for cardiac tissue engineering applications.

Figure 1
. Summary of workflow to develop bioprinted adhesive scaffolds.
A:
Embedded (top) or air (bottom) bioprinting was used to create 3D patch geometries.
B:
Printing fidelity assessment and optimization.
C:
Different adhesion mechanisms were assessed using novel customized tools and approaches to apply tensile (
C-i
), shear (
C-ii
), and dynamic (
C-iii
) loading.
Abstract P409: GSK-3β Deficiency In Cardiomyocyte Induces Cardiac Progenitor Cell Proliferation In The Ischemic Heart
Authors: Yusuf Ayesha , Qaisar Rizwan , Woodgett James R , Ahmad Firdos
Published: November 2021

The cardiomyocytes are terminally differentiated cells and ischemia-induced cardiomyopathy is an irreparable loss. Novel strategies are needed to induce resident cardiac progenitor cells (CPCs) proliferation
in situ
to enhance the possibility of cardiac regeneration. Here we sought to identify a potential role for glycogen synthase kinase-3β (GSK-3β), a critical regulator of cell proliferation and differentiation, in CPCs proliferation in the ischemic heart. Cardiomyocyte-specific conditional GSK-3β knockout (cKO) and littermate control mice were recruited and challenged with myocardial infarction (MI). The cardiac function was assessed using trans-thoracic M-mode echocardiography. The level of CPC proliferation in the ischemic cKO hearts was determined at 2, 4 and 8 weeks post-MI through immunofluorescence labeling of stem cell marker c-Kit. To confirm the lineage of identified c-Kit -positive cells (KPCs) in the heart, a hematopoietic lineage marker was stained along with c-Kit. The cardiac left ventricular chamber dimension (LVID) and contractile functions were comparable until 2 weeks post-MI. The cKO mice displayed significantly preserved LV chamber [LVIDd(mm); 5.01±0.67 vs.6.09±0.65,
p
=0.01] and contractile function [LVEF(%); 31.98±8.52 vs. 18.06±7.11.,
p
=0.01] in comparison to control mice at 4 week post-MI. Consistent with protective phenotypes, an increased percentage of KPC was observed in the cKO hearts at 4 and 6-weeks post-MI which was accompanied by an increased level of cardiomyocyte proliferation. Further analysis revealed that the observed increased number of KPCs in the ischemic cKO hearts were mostly from a cardiac lineage as the majority of identified KPCs were negative for the hematopoietic marker, CD45. In conclusion, our findings strongly suggest that GSK-3β inhibits CPCc proliferation post-ischemia, and loss of GSK-3β in cardiomyocytes promotes resident CPCc proliferation potentially through paracrine mechanisms.
Abstract P412: Mitochondrial Metabolic Transcriptome Profiles During Cardiomyocyte Differentiation From Mouse And Human Pluripotent Stem Cells
Authors: Cho Sung Woo , Kim Hyoung Kyu , Han Jin , Sung Ji-Hee
Published: November 2021

Simultaneous increase of myofibrils and mitochondria is a key process of cardiomyocyte differentiation from pluripotent stem cells (PSCs). Specifically, development of mitochondrial oxidative energy metabolism in cardiomyocytes is essential to providing the beating function. Although previous studies reported that mitochondrial oxidative metabolism have some correlation with the differentiation of cardiomyocytes, the mechanism by which mitochondrial oxidative metabolism is regulated and the link between cardiomyogenesis and mitochondrial function are still poorly understood. In the present study, we performed transcriptome analysis on cells at specific stages of cardiomyocyte differentiation from mouse embryonic stem cells (mESCs) and human induced PSCs (hiPSCs). We selected highly upregulated mitochondrial metabolic genes at cardiac lineage commitment and time-dependent manner during cardiomyocyte differentiation and identified the protein-protein interaction network connecting between mitochondrial metabolic and cardiac developmental genes. We found several mitochondrial metabolic regulatory genes at cardiac lineage commitment (Cck, Bdnf, Fabp4, Cebpa, Cdkn2a in mESC-derived cells and CCK, NOS3 in hiPSC-derived cells) and time-dependent manner during cardiomyocyte differentiation (Eno3, Pgam2, Cox6a2, Fabp3 in mESC-derived cells and PGAM2, SLC25A4 in hiPSC-derived cells). Notably, mitochondrial metabolic proteins are highly interacted with cardiac developmental proteins time-dependent manner during cardiomyocyte differentiation rather than cardiac lineage commitment. Furthermore, mitochondrial metabolic proteins are mainly interacted with cardiac muscle contractile proteins rather than cardiac transcription factors in cardiomyocyte. Therefore, mitochondrial metabolism is critical at cardiac maturation rather than cardiac lineage commitment.
Abstract P419: Revealing The Dynamics Of Vesicle Formation During Clathrin-mediated Endocytosis In Living Endothelial Cells
Authors: Nawara Tomasz , Rao Tejeshwar , Mattheyses Alexa
Published: November 2021

Clathrin-mediated endocytosis (CME) is an essential cellular process for internalizing nutrients and therapeutics at endothelial cell barriers. Studying the formation of cargo containing endocytic vesicles in living cells is challenging due to the limited resolution of fluorescence microscopy and the highly dynamic nature of CME. Moreover, it is currently unknown how the physiological conditions present in vasculature affect CME in endothelial cells. To address this challenge we used a novel microscopy approach, Simultaneous Two-wavelength Axial Ratiometry (STAR), to image vesicle formation dynamics with nanometer axial resolution in living cells. High-throughput analysis revealed that 80% of
de novo
clathrin accumulations contributed to endocytosis while 20% remained flat, consistent in both human umbilical vein endothelial cells (HUVECs) and our test-bed model green monkey kidney fibroblast-like (Cos-7) cells. We next investigated the interplay between coat curvature and clathrin accumulation in vesicle initiation to identify the mechanism of vesicle formation. Our results support the flexible model of vesicle formation with curvature and clathrin accumulation initiating together at shorter-lived vesicles (<20s) and through a flat-to-curved transition of clathrin lattices at longer-lived vesicles (>20s). Finally, we addressed if physiological conditions present in vasculature alter the dynamics of vesicle formation. We show that increasing osmotic pressure decreased the total number of internalizations but had no impact on the number of flat clathrin accumulations or the mechanism of vesicle formation in Cos-7 cells. In future research, we will test the hypothesis that HUVECs have distinct mechanisms to retain vesicle formation under osmotic pressure or shear stress conditions similar to their native environment. Additionally targeted drug delivery to vascular endothelial cells, for example nanocarriers binding to flat lattices or CCVs leading to different therapeutic outcomes or bioavailability, can potentially be informed by identifying clathrin morphology and dynamics and the mechanisms of endocytosis using STAR microscopy.
Abstract P420: Nano-Triciribine Reduces SARS2-CoV-2 Infection By Sequestering ACE2 And The Novel Host Factor LDLR
Authors: Condor Capcha Jose Manuel , Iansen Irion Camila , Lambert Guerline , Chahdi Ahmed , Buchwald Peter , Dykxhoorn Derek , Farzaneh Hamidreza , deo Sapna , Daunert Sylvia , Young Karen C , Shehadeh Lina A
Published: November 2021

Background:
People with previous CVD hospitalized for COVID-19 have elevated death rate. We reported that patients with diabetes and HF higher protein levels of the low density lipoprotein receptor (LDLR). We hypothesized that LDLR is a novel host factor for the SARS-CoV-2-Spike (S2S) protein that may be regulated by the Akt inhibitor Triciribine (TCN), a drug being tested in Phase III studies for breast cancer. We also hypothesized that nano-formulation of Triciribine (NanoTriciribine; NTCN) would enhance its efficacy and allow for intranasal delivery.

Methods:
Interactions between the recombinant proteins Spike-RBD (receptor binding domain), ACE2, LDLR and its ectodomains (EGFA-EFFB, C2-C5 and C2) were analyzed by binding assays and co-IP in HepG2, HK2, and 293T cells. Viral entry assays were performed with 2 S2S pseudoviruses using 293T cells + hACE2 and TMPRSS2 or Furin protease. The effect of NTCN or the LXR agonist GW-3965 on viral uptake (pseudotyped VSVΔG-GFP*S2S or chimera VSV-S2S-eGFP virus) was assessed. Akt, pAkt, ACE2, and LDLR levels were determined in 293T+hACE2 by flow cytometry. Assays were done in triplicates and 1-way-ANOVA with Tukey’s correction was used for statistics.

Results:
RBD protein binds modestly to the human LDLR (EC
50
:10μM) and its C2-C5 ectodomain (EC
50
:13.8μM). Co-IP revealed a novel and strong LDLR-ACE2 interaction in several human cell lines. LDLR overexpression in human cells increased the uptake of VSVΔG-GFP*S2S (FC=2.32;p<0.001) and chimera virus (FC=.33; p<.0001). NTCN and TCN each reduced pAkt/Akt ratio. 1μM TCN or NTCN reduced LDLR (7.2%;p<.01 & 15.6%;p<0.0001) and ACE2 (32%;p<0.05 & 44.7%;p<.01) cell surface expression, respectively. 1μM NTCN or GW-3965 reduced S2S viral entry by 64.2% (p<.0001) and 40.7% (p<.01), respectively, confirming a role for LDLR in S2S infection. In hACE2tg mice, chimera VSV-S2S caused significant lung infection as measured by qPCR, GFP expression in proximal and distal lung airway epithelial cells, and electron microscopy. Intranasal delivery of NTCN was well tolerated.

Conclusions:
LDLR enhanced S2S viral entry supporting the elevated COVID-19 susceptibility seen in patients with heart disease. NTCN is a promising candidate for prophylactic treatment against COVID-19.
Abstract P421: Analysis Of The Functional Relevance Of Human Beta-myosin Heavy Chain Post-translational Modifications
Authors: Landim-Vieira Maicon , Childers Matthew C , Wacker Amanda , Rodriguez Garcia Michelle C , Singh Rakesh , Whitson Bryan , Brundage Elizabeth , Janssen Paul M , Chase P B , Biesiadecki Brandon , Regnier Michael , Pinto J Renato , Parvatiyar Michelle S
Published: November 2021

Sarcomeric proteins have been shown to be a target of post-translational modifications (PTMs). Phosphorylation and acetylation of several sarcomeric proteins have been reported to be important for fine-tuning of myocardial contractility. Given the emerging importance of understanding the potential role of PTMs on cardiac muscle performance in healthy and diseased states, we sought to identify novel PTMs on human cardiac beta-myosin heavy chain (beta-MHC). We found several high confidence beta-MHC peptides modified by K-acetylation and S- and T-phosphorylation found in non-diseased, ischemic, and non-ischemic human heart samples. Using bottom-up proteomics and label-free quantification, we identified seven high-confidence peptides (K34, K58, S210, T215, K429, K951, K1195) with K951 displaying significant reduction in acetylation levels in both ischemic and non-ischemic failing hearts compared to donor hearts. Molecular dynamics simulations were performed to better understand the functional significance of the beta-MHC PTMs. Focus was placed on modifications in the regions with greatest potential functional significance as well as modified residues with significantly altered abundance in diseased states (K951-Ac at the myosin tail nearby a binding site for myosin heads in the super-relaxed state). K951 is located in the myosin tail (S2) at the C-terminal end of simulated structure. In both unmodified and modified simulations, the tail fragment showed significant flexibility and partial unfolding at the C-terminus. In the unmodified simulations, the inter- and intra-helical contacts were maintained. However, when beta-MHC is acetylated at residue 951, these helical contacts were altered as the uncharged acetylated residue no longer formed strong hydrogen bonds with a residue of the opposite chain. This facilitated changes increase in inter-helical contacts, an increase in inter-helical distance, and disruption of the coiled-coil tail domain structure. Our study suggests that there are distinct differences in beta-MHC acetylation levels that appear to be influenced more by location of the modified residues than the type of heart disease (ischemic- and non-ischemic heart failure). Additionally, we speculate that these PTMs have the potential to modulate the interactions between beta-MHC and other regulatory sarcomeric proteins, as well as ADP-release rate of myosin, flexibility of S2 fragment, and cardiac myofilament contractility under normal and heart failure condition.
Abstract P428: A New Mouse Model Of Myocardial Lipids, Fibrosis, Arrhythmia And Diastolic Dysfunction Induced By Hyperlipidemia And Cardiac LDLR
Authors: Williams Monique , Iansen Irion Camila , Condor Capcha Jose Manuel , Lambert Guerline , Seo Grace , Goldberger Jeffrey J , Shehadeh Lina A
Published: November 2021

Background:
Hyperlipidemia is a major risk factor for CVD. Patients with HF with preserved ejection fraction (HFpEF) have more myocardial lipid accumulation than patients with reduced EF (HFrEF). RNASeq data from cardiac biopsies showed downregulation of the gene for lipoprotein lipase (LPL) that degrades triglycerides, in HFpEF patients compared to healthy and HFrEF controls. Poloxamer-407 (p407) induces hyperlipidemia by blocking LPL and subsequent increase in plasma triglycerides and low-density lipoprotein (LDL) cholesterol. We hypothesized that mice treated with p407 and cardiac LDL-Receptor (LDLR) over-expression (OE) develop hyperlipidemia, myocardial lipid accumulation, and diastolic dysfunction resulting in HFpEF and arrhythmias.

Methods:
Baseline cardiac function was assessed by echo for male and female C57Bl6 mice (n=9) for 2 groups: 4wk biweekly i.p. p407-injections with (n=4) or without (n=3) single i.v. injection with AAV9-cTnT-LDLR. Cardiac function was assessed by echocardiography at 3 and 4 wks. Blood Pressure (BP) and Whole Body Plethysmography (WBP) were assessed during wk4. Ttest was used for statistics. PR and ORO staining and telemetry were performed at wk4.

Results:
At wk3, P407 and LDLR OE led to alterations in diastolic function (increased IVCT, IVRT, MV E/E’, MPI, and NFT) and increased LV wall thickness, p<0.05. At wk4, there was pulmonary hypertension (increased mean pulmonary arterial pressure, decreased pulmonary acceleration time
p
<.05).Histology showed excessive myocardial lipids and fibrosis, and telemetry showed incidents of second-degree and higher-degree AV block. The group injected solely with p407 show
e
d alterations in diastolic function (increased IVCT, IVRT, NFT, LVMPI, LVMPI NFT
p<.05
) and decreased EDV, ESV, EDLVM, ESLVM,
p<.05
at wk4. All groups had preserved %EF and no abnormalities in BP or WBP.

Conclusions:
P407 and cardiac LDLR OE induce a drastic decline in cardiac diastolic function over a shorter period of time compared to p407 alone. Diastolic dysfunction was observed in wk3 followed by pulmonary hypertension, arrhythmia, myocardial lipid accumulation and fibrosis in wk4. This new model may allow for more rapid investigations of cardiac abnormalities seen in HFpEF patients.
Abstract P430: VSMCs Increase Glutamine Utilization For Energy Metabolism During Obesity
Authors: Roggerson Krystal M , Francis Sharon
Published: November 2021

Obesity increases the risk of developing cardiovascular disease through vascular remodeling though the underlying mechanisms are not entirely understood. However, metabolic fuel partitioning and mitochondrial flexibility during energy metabolism may play a critical role. We demonstrated serum and glucocorticoid-inducible kinase 1 (SGK-1) is up-regulated in the vasculature of diet-induced obese mice and that SGK-1 deletion is protective against obesity-induced vascular remodeling by metabolically reprogramming vascular smooth muscle cell (VSMC) energy metabolism towards oxidative phosphorylation (OXPHOS) and away from glycolysis. Mitochondrial substrate availability and utilization of the primary metabolic fuels glucose, long chain fatty acids (LCFAs) and glutamine can drive metabolic reprogramming. Therefore, alterations in fuel utilization may contribute to vascular remodeling during obesity. The purpose of this study was to examine SGK-1’s role in 1) fuel dependency: a cell’s reliance for a specific fuel and 2) fuel capacity: a cell’s ability to oxidize a specific fuel to meet cellular energy demand under low-fat and high-fat diet-induced obesity. Using the MitoXpress Oxygen Consumption assay which measures OXPHOS, primary VSMCs isolated from wildtype (WT) and SMC-specific SGK-1 knockout (smSGK-1 KO) mice fed a 10% kcal low-fat or 45% kcal high-fat diet for eight weeks were seeded in a 96-well plate at a density of 6x10
4
cells/well in culture medium. To assess fuel dependency, cells were treated with fuel pathway inhibitors UK5099, Etomoxir or BPTES to block glucose, LCFA or glutamine oxidation, respectively. To measure fuel capacity, VSMCs were treated with a combination of two pathway inhibitors simultaneously. Next, samples were overlaid with a fluorescent extracellular oxygen consumption reagent, sealed with high-sensitivity mineral oil, then signals were read at 1.5-minute intervals for 2 hours at Ex/Em= 380/650 nm. Our results show WT VSMCs are exclusively glucose-dependent for OXPHOS regardless of dietary conditions. However, SGK-1 deletion induces a dependency for all three fuels for OXPHOS in VSMCs under low- and high-fat conditions. Even though WT and smSGK-1 KO VSMCs preferentially oxidized glucose for OXPHOS under low-fat conditions; SGK-1 deletion resulted in a 2.2-fold increase in glutamine capacity. Alternatively, WT VSMCs exposed to obesogenic conditions preferentially oxidized glutamine whereas SGK-1 deletion induced a nearly equal partitioning of all three fuels during obesity suggesting elevated mitochondrial flexibility. Overall, this study suggests SGK-1 increases glucose dependency for energy metabolism under physiological and obesogenic conditions. Also, increased glutamine utilization for OXPHOS during obesity may be an underlying cause of VSMC dysfunction and subsequent vascular impairment.