bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 113: Recombinant Tafazzin Enzyme Replacement Therapy Rescues Metabolic and Functional Defects in a Mouse Model of Barth Syndrome
Authors: Thomas Corinne J , Awata Junya , Dinca Ana A , Chien Wei-Ming , Blanton Robert , Aronovitz Mark , Martin Gregory L , Richey Lauren , Tam Kelly , Strathdee Douglas , Chin Michael T
Published: October 2019

Barth syndrome (BTHS) is an X-linked recessive disease where patients most commonly die from cardiomyopathy-induced heart failure before middle age. BTHS is caused by mutations in the tafazzin (TAZ) gene, resulting in defective TAZ protein. TAZ is an enzyme that generates mature cardiolipin (CL) from monolysocardiolipin (MLCL) in the mitochondrial membrane, a reaction essential for normal mitochondrial function. Current therapies can only treat the symptoms of BTHS. In this study, we propose an enzyme replacement therapy for BTHS which utilizes recombinant human TAZ fused to a cell penetrating peptide (hTAZ-CTP) to facilitate tissue uptake. The efficacy of this protein was tested
in vitro
on C2C12 TAZ-knockout (TAZ-KO) skeletal myoblasts and
in vivo
on a myocardial-specific TAZ conditional knockout mouse, modelling the cardiomyopathy associated with BTHS.

In vitro
tests of TAZ-KO cells, using oxygen consumption rate as a measure of mitochondrial activity, showed treatment of the cells with hTAZ-CTP effected a partial rescue of the fatty acid oxidation capabilities of the TAZ-KO cells.
In vivo
tests showed that BTHS mice display increasing septal wall thickness over time, an effect halted upon treatment with hTAZ-CTP. Pressure-volume (PV) loop analysis indicated that heart function, impaired in the vehicle-treated BTHS mouse, was similar between treated mice and normal mice. The ratio of MLCL/CL, a direct measure of TAZ enzymatic activity, was measured in heart mitochondria isolated from BTHS and control mice after treatment. The vehicle treated BTHS mouse showed the high MLCL/CL ratio typical of BTHS patients, whereas the MLCL/CL ratios in protein-treated mice matched the much lower ratio of the control mice. Similarly, oxygen consumption rate measurements of these isolated heart mitochondria demonstrated partial rescue by hTAZ-CTP treatment. Coupled with the lack of toxicity observed in the liver, spleen, kidney, and heart due to hTAZ-CTP injection, these results indicate that TAZ enzyme replacement therapy has great potential as a future treatment for BTHS.
Abstract 115: Surgical Bilateral Stellate Ganglionectomy Reduces Mitochondrial Reactive Oxygen Species (mROS) and Prevents Sudden Cardiac Death (SCD) in Non-ischemic Heart Failure (HF)
Authors: Dey Swati , O'Rourke Brian , DeMazumder Deeptankar
Published: October 2019

Introduction:
Current therapies for SCD are limited. Bilateral sympathetic stellate ganglionectomy (
BSG
) is a promising new adjunctive therapy in patients with ischemic HF who have incessant ventricular tachyarrhythmias (
VT/VF
) despite optimal medical management. The underlying mechanisms are unknown. The effect of BSG in non-ischemic HF is unclear.

Hypothesis:
BSG prevents VT/VF and SCD in non-ischemic HF by restoring sympathovagal balance and reducing mROS
. METHODS:
We used a unique pressure-overload guinea pig model that closely mimics human non-ischemic HF, including a high incidence of spontaneous VT/VF/SCD. We randomized the animals to non-failing controls, HF+Sham, or HF+BSG surgery. We used intention to treat analysis to evaluate VT/VF-free survival. We analyzed continuous ECG (VT/VF burden, heart rate variability, QT variability) and echo (cardiac function). In isolated left ventricular (LV) myocytes, we measured Ca
2+
transients and mitochondrial reactive oxygen species (mROS) using targeted ratiometric probes.

Results:
Half of the HF+Sham animals experienced SCD in 4 weeks. In contrast, BSG abolished SCD, reduced VT/VF, and increased parasympathetic tone (beyond corresponding reductions in sympathetic tone). Fractional shortening of the LV was markedly reduced in HF+Sham (0.28±0.02) vs. control (0.50±0.01), but preserved by BSG (0.45±0.02). Whereas HF+Sham increased mROS levels in LV myocytes (fractional oxidation, F
ox
: 0.53±0.015) compared to control (0.29±0.020), BSG reduced mROS (0.38±0.035). Mitochondrial antioxidant capacity, as indexed by H
2
O
2
challenge of LV myocytes, was compromised in HF+Sham (F
ox
: 0.81±0.040) compared to control (0.44±0.061), but preserved by BSG (0.48±0.023).

Conclusions:
Surgical BSG prevents SCD in non-ischemic HF by reducing mROS, decreasing sympathetic tone, and by increasing parasympathetic tone (possibly by facilitating cholinergic transdifferentiation). This promising new minimally-invasive surgical therapy may be particularly useful in patients with non-ischemic HF on optimal medical therapy who require recurrent ICD shocks to prevent SCD. Patients in the early stages of HF who are not eligible for ICDs but remain at higher SCD risk may also benefit from BSG surgery.
Abstract 119: Multi-Omics Investigation of Cardiomyocyte-to-Fibroblast Crosstalk in Human iPSC Models
Authors: Lau Edward , Chandy Mark J , Williams Damon R , Shrestha Rajani , Rhee June-Wha , Wu Joseph C
Published: October 2019

Introduction:
Cardiac cells communicate with each other in part through secreted proteins known as cardiokines. The total repertoire of proteins secreted by cardiac cells is unknown. We aim to apply human iPSC models and large-scale multi-omics to identify secreted proteins and the crosstalk signals they mediate.

Method:
We optimized an experimental protocol to recover and identify cell-specific secreted proteins from human iPSC-cardiac cells. Human iPSCs were differentiated into cardiomyocyte (CM) and endothelial cells (EC) using established protocols, after which secreted proteins were extracted from the conditioned medium for analysis. We combined multiplexed aptamer-based large-scale protein quantification platform and high-resolution mass spectrometry to identify secreted proteins from iPSC-CM, iPSC-ECs, and primary ventricular fibroblasts. An in-silico filter was implemented to prioritize bona fide cardiokines over proteins externalized by passive lysis.

Result:
We identified 146 candidate cardiokines at 1% false discovery rate, including cell-specific cardiokines as well as a common core secretome of three cardiac cells. We analyzed the data to identify potential signals secreted by cardiomyocytes to mediate fibroblast function, using a ligand-receptor model based on proteomics and single-cell transcriptomics data to prioritize cardiokines secreted by iPSC-CMs and which bind to fibroblast-expressed receptors. To examine their roles in fibrosis regulation, we selected three candidate cardiokines (PLAU, FGF7, and CXCL12) for verification using immunodetection, and exposed their recombinant proteins at multiple concentrations to human ventricular fibroblasts. The results nominated cardiokine-specific effects on recipient cell gene expression including evidence of modulation of fibroblast transcription and translation pathways.

Conclusion:
We demonstrate a multi-omics approach in iPSC models to explore the large-scale human secretome of multiple cardiac cell types. The approach holds promise for identifying disease markers and understanding intercellular communication in cardiac development and diseases.
Abstract 120: Methamphetamine-induced Cardiomyopathy Associated With Mitochondrial Dysfunction, Cardiac Fibrosis and Hypertrophy
Authors: Abdullah Chowdhury S , Aishwarya Richa , Alam Shafiul , Morshed Mahboob , Kolluru Gopi K , Traylor James , Miriyala Sumitra , Panchatcharam Manikandan , Woolard Matthew D , Goeders Nicholas E , Lu Xioa-Hong , Dominic Paari S , Kevil Christopher G , Orr A. Wayne , Harris Norman R , Gavins Felicity N. E , Bhuiyan Md. Shenuarin
Published: October 2019

Introduction:
Methamphetamine (METH) is one of the most commonly abused illicit drugs in the United States, exerting a range of adverse effects upon multiple organ systems. Cardiovascular complications are among the major causes of death in METH users. METH-induced cardiomyopathy is a poorly characterized disease entity as METH-induced molecular perturbations, and histopathological changes in the heart remain under-explored.

Objectives:
We studied histopathology in the hearts of human METH users. We also observed the histological alteration and changes in mitochondrial function in mice that received ‘binge’ administration of METH.

Methods and Results:
We obtained 32 autopsy heart samples from humans with positive toxicology for chronic METH use and performed Sirius Red and Masson’s Trichrome (MT) staining on left ventricular (LV) sections. Notably, chronic METH user hearts showed intense perivascular and interstitial fibrosis in LVs. ‘Binge’ METH administration in mice for 4 weeks showed an increase in heart weight-to-tibia length and increase in myocyte cross-sectional area in WGA stained LVs compared to saline-treated mice. Sirius red and MT staining also showed an increase in perivascular and interstitial fibrosis in METH mice heart. Isolated mitochondria from METH-treated mice heart showed suppressed mitochondrial bioenergetics measured by Seahorse Analyzer. Immunoblotting in heart lysates and mitochondrial fractions showed altered mitochondrial dynamics regulatory proteins expression in METH mice compared to control saline group. METH-treated cultured neonatal rat ventricular cardiomyocytes also showed suppression of mitochondrial respiration and mitochondrial network disorganization indicating a direct effect of METH on cardiomyocytes.

Conclusions:
We report that maladaptive cardiac fibrotic remodeling is typical in a human and pre-clinical mouse model of METH abuse. ‘Binge’ METH exposure in mice induces cardiac hypertrophy, cardiac fibrosis, and suppression of mitochondrial respiration. Thus, chronic METH use induces maladaptive cardiac remodeling associated with mitochondrial dysfunction.
Abstract 122: Reliability Analysis for Image-based Non-invasive Pressure Quantification in Aortorenal Artery Systems
Authors: Wu Hao , Khan Monsurul , Du Xiaoping , Sawchuk Alan P , YU Huidan W
Published: October 2019

Non-invasive quantification of fractional flow reserve (FFR) based on CT angiography has great potential to replace invasive catheterization for quantifying the amount of ischemia caused by a specific arterial stenosis (AS), promising for lower medical cost and better patient care. In clinical practice, the reliability of quantifying the 4-D approximate pressure (p
a
) and distal pressure (p
d
) across an AS is critical since FFR=p
d
/p
a
by definition. The objective of this work is to assess the effects of uncertainties (empiricalness) in the 3-element Windkessel model (WK3) used as exit boundary conditions (BC) on the quantification of p
d
and p
a
in aortorenal artery systems. A developed and validated in-house software (
InVascular
, see Fig. 1(a)), featured with revolutionarily fast speed for image-based computational hemodynamics, is used for the uncertainty quantification analysis. Fifteen total aortic and renal arteries were studies in five male patients aged 64 to 83. In each case, the effects of the uncertainty on the 4-D pressure quantification are assessed using the First Order Second Moment method. We conduct analyses where the input variables are normally distributed with their coefficients of variation being 3%. Figure 1(b) shows the effects of uncertainty on the systolic pressure (SP) with their means, standard deviations, and 95% confident intervals. The results indicate that the uncertainty (measured by standard deviation) in SP in the aorta is higher than that in the renal arteries. The uncertainty is minimal for the severe stenosis (in red), implying more reliable noninvasive pressure quantification in stenosed arteries.
Abstract 139: Systemic Analysis and Discovery of Embryonic Stem Cell-derived Exosomal Long Non-coding RNAs as Potential Therapeutic Modulators of Myocardial Repair
Authors: Huang Grace
Published: October 2019

Cardiovascular disease is the leading cause of death in the U.S. Exogenous stem cell therapy has emerged as a novel therapeutic approach and various stem cell types, including embryonic stem cells (ESCs), bone marrow stem cells and mesenchymal stem cells, have been studied for cardiac regenerative cell therapy. Even though there was great enthusiasm with promising initial results in pre-clinical disease models, stem cell-based therapy demonstrated modest benefits in clinical trials. Emerging evidence suggests that stem cells can secrete a large number of soluble factors to evoke a resident cell response for tissue regeneration. In this perspective, stem cells-derived exosome therapy has been recently put forward as an alternative cell-free modality for cardiac repair after myocardial infarction (MI). Our previous study indicated that ESC-derived exosomes (ESC-exo) promote endogenous cardiac repair and enhanced cardiac function following MI. However, the mechanisms underlying ESC-exo mediated cardiac repair and regeneration still need to be fully elucidated. RNA-sequencing data on ESC-exo revealed that non-coding RNAs, including long non-coding RNAs (lncRNA), are selectively sorted into exosomes and highly enriched compared to control fibroblasts. Unlike exosomal miRNA, exosomal lncRNAs have not been well-studied in the context of tissue repair. To investigate the reparative role of lncRNA in protecting against MI, we first systemically analyzed and discovered ESC-derived exosomal lncRNAs
in silico.
We analyzed novel lncRNAs from ESC- and MEF-exo based on differential expression by microarray analysis, lncRNA classification, human homology and non-coding potential prediction. For lncRNAs enriched in the ESC-exo population, we selected 2 potential lncRNAs of interest for further study: Gm4890 and XLOC 01990, which are confirmed for expression by qRT-PCR results. Gm4890 and XLOC are mainly located in nucleus rather than cytoplasm implicating their function on transcriptional regulation. Ongoing studies are evaluating the functional roles for these ESC-exo enriched lncRNAs.
Abstract 155: Lin28 Enhances Cardiac Progenitor Cell Ability to Repair the Heart by Reprogramming Cellular Metabolism
Authors: Yuko Antonia , Kurian Justin , Kasatkin Nicole , Busch Kelsey , AlHawaj Fares , Ma Lena , Khan Mohsin , Mohsin Sadia , Houser Steven , Wang Hong
Published: October 2019

Rationale:
The adult Heart is largely a dormant organ supporting limited cellular turnover. In contrast, neonatal cardiac tissue proliferates and is capable of regeneration while operating under a specialized metabolic state. During transition to adulthood, cardiac metabolism undergoes a rapid shift that coincides with termination of regenerative processes. Whether altering cardiac metabolism recapitulates regenerative potential remains untested. Recently, introduction of Lin28, a metabolic regulator of pluripotency, enhances tissue repair after injury. Nevertheless, there are no studies characterizing the effect of Lin28 on cardiac repair.

Objective:
Determine the effect of Lin28 on cardiac progenitor cell function and cardiac repair after injury.

Methods and Results:
Lin28 expression coincides during heart development with c-kit and declines postnatal with complete abrogation in 3-week-old adult heart as measured by qRT-PCR and immunohistochemistry. CPCs were engineered with Lin28-GFP (CPCLin) lentivirus GFP expressing CPCs were used as controls (CPC-G). CPCLin demonstrated increased proliferation measured by CyQuant compared to CPC-G, concurrent with decreased apoptosis. Interestingly, CPCLin demonstrated a significant increase in lactate production, pyruvate kinase activity, glucose uptake together with enhancement of glycolysis and expression of glycolytic enzymes compared to CPC-G. Oxidative metabolism was also upregulated together with increased intracellular ATP in CPCLin compared to controls. Additionally, CPCLin demonstrated significantly reduced ROS generation as measured by CM-H
2
TMROS based FACS analysis compared to CPC-G. To determine in vivo efficacy, CPCLin and CPC-G were transplanted in the heart after myocardial infarction. CPC-Lin hearts showed significant increase in cardiac structure and function 8 weeks after MI. Increased persistence and proliferation of transplanted CPC-Lin cells together with reduced apoptosis were observed in CPCLin hearts compared to control CPC-G hearts 2 days after transplantation.

Conclusions:
Lin28 modification of CPCs reprograms cellular metabolism in CPCs enhancing proliferation and survival including ability to repair the heart after myocardial injury.
Abstract 156: Endogenous Cardiomyocyte Dedifferentiation and Cycling Revealed by Single-Cell Imaging and Single-Nucleus Transcriptomic Analysis
Authors: Zhang Yiqiang , Gago-Lopez Nuria , Li Ning , Zhang Zhenhe , Alver Naima , MacLellan William R
Published: October 2019

Although the mammalian heart had been recognized as a post-mitotic non-regenerative organ, recent evidence demonstrated a measurable cardiomyocyte (CM) renewal at low rates in normal hearts and augmented in post-injury myocardium. However, the resources and cellular processes of such a new CM formation, and the underlying molecular programs remained largely undetermined. While CMs in neonatal hearts or adult CMs in tissue culture can undergo (partial) dedifferentiation and proliferation, the roles of
in vivo
CM dedifferentiation and cycling were unclear. Here, we report a multi-reporter transgenic mouse models featuring efficient adult CM (ACM) genetic cell fate mapping and real-time CM dedifferentiation reporting. In this triple-transgenic model, upon tamoxifen-induced gene recombination, only ACMs switched from expressing red fluorescent protein (RFP) to green fluorescent protein (GFP), and non-CMs remained expressing RFP. The nuclear blue fluorescent protein (BFP) signal was expressed in normal mature ACMs but reduced in neonatal or cultured dedifferentiated ACMs. Hence, all 4 plausible modes of new CM formation can be visualized using this model. Using ImageStream for single-cell imaging analysis, we demonstrated that non-myocytes (including putative cardiac progenitor cells) contributed negligibly to new ACM formation at baseline or after myocardial infarction (MI). In contrast, there was a significant increase in dedifferentiated (e.g. GFP
+
BFP
-
) CMs in post-MI hearts compared to the sham. Cell cycling activity was enhanced within the dedifferentiated CM population compared to normal ACMs (GFP
+
BFP
+
). Furthermore, we applied massive whole-heart single-nucleus RNA-seq to dissect the transcriptomes of dedifferentiated CMs. We found that CMs in post-MI hearts identified with dedifferentiation reporter had significant down-regulations of genes in networks for hypertrophic growth, cardiac contractile functions and metabolism, but up-regulation in signaling pathways for cell survival, active cell cycle and proliferation. Therefore, cellular dedifferentiation may be an important prerequisite for endogenous CM cell cycling and proliferation and explain the measurable cardiac myogenesis seen after MI.
Abstract 163: NFkB Promotes Oxidative Stress-induced Necrosis and Ischemia Reperfusion Injury Through NRF2-ARE Pathway
Authors: Guo Xiaoyun , Chen Yi , Steinmetz Rachel , Hong Siqi , He Hui , Zeng Yachang , Liu Qinghang
Published: October 2019

Objective:
Despite the well-studied pro-survival function of nuclear factor-κB (NFκB), recent studies suggest that NFκB may also play a pathogenic role in myocardial ischemia injury and adverse remodeling. This study aims to define a new pro-cell death role of NFκB in response to oxidative stress and the functional implications in ischemia reperfusion (I/R) injury.

Methods and Results:
We identified an unexpected pro-cell death role of NFκB in oxidative stress-induced necrosis, and provide new mechanistic evidence that NFκB, in cooperation with HDAC3, negatively regulates NRF2-ARE anti-oxidative signaling through transcriptional silencing. Genetic deletion of NFκB-p65 inhibits, whereas activation of NFκB promotes, oxidative stress-induced cell death and HMGB1 release, a biomarker of necrosis. Moreover, simulated ischemia reperfusion (sI/R) and doxorubicin (Dox) treatment both induce NFκB-luciferase activity in cardiomyocytes, and inhibition of NFκB diminishes sI/R- and Dox- induced necrosis. Importantly, NFκB negatively regulates NRF2-ARE activity and the expression of anti-oxidant proteins. Mechanistically, co-immunoprecipitation reveals that p65 is required for the association between NRF2 and HDAC3 and transcriptional silencing of NRF2-ARE activity. Further, the ability of HDAC3 to repress NRF2-ARE activity is lost in p65-/- cells. The HADC inhibitor TSA and NFκB inhibitor BMS-345541 both increase NRF2-ARE activity and promote cell survival following sI/R.
In vivo
, NFκB transcriptional activity in the heart is significantly elevated after I/R injury, which is abolished by cardiac-specific deletion of p65. Moreover, ablation of p65 using
p65
fl/fl-Nkx-Cre mice reduces myocardial infarct size after acute I/R, and prevents chronic remodeling and contractile dysfunction after myocardial infarction.

Conclusions:
Our results identified NFκB as a key regulator of oxidative stress-induced necrosis by suppressing the NRF2-ARE anti-oxidant pathway through an HDAC3-dependent mechanism. Ablation of NFκB-p65 attenuates oxidative stress-induced necrosis and I/R injury, suggesting a new pathogenic role of the NFκB pathway, and thus a therapeutic target, in myocardial ischemic injury and remodeling.
Abstract 164: Hypoxia-Induced Cardiomyocyte Mitophagy and Mitochondrial Permeability Transition are Inhibited by Bnip3 Phosphorylation
Authors: Martens Matthew D , Field Jared T , Mughal Wajihah , Rampitsch Christof , Ivanco Tammy , Diehl-Jones William , Gordon Joseph W
Published: October 2019

Bnip3 is a hypoxia-inducible initiator of cardiomyocyte cell death and has also been implicated as a mitochondrial receptor for the cellular mitophagy machinery. Previous work from our group demonstrates that the prostaglandin E1 analogue, misoprostol, prevents hypoxia-induced mitochondrial dysfunction and is a potent activator of PKA. We hypothesize that misoprostol alters the phosphorylation status of Bnip3, inhibiting its ability to induce cardiomyocyte mitophagy, mitochondrial dysfunction and cell death. Using a rodent model of neonatal hypoxia, in combination with rat primary ventricular neonatal cardiomyocytes (PVNC’s) and H9c2 cells, we assessed the effect of hypoxia and misoprostol drug treatments on mitochondrial function, mitophagy, and cell viability. In postnatal day 5 rats, hypoxia caused a 30% increase in the concentration of serum cardiac troponin-I, a clinically relevant marker for cardiomyocyte cell death, which was absent with the addition of misoprostol drug treatments (n=3). Using PVNC’s we further demonstrated that hypoxia reduced measures of mitochondrial function including membrane potential (47%) and maximal respiration (46%), which were restored back to control levels with the addition of misoprostol (p<0.01). Furthermore, hypoxia induced a 36% increase in mitophagy, concurrent with a 210% increase in mitochondrial permeability transition, both of which were reversed with misoprostol drug treatment (p<0.01). Using a combination of mass spectrometry and mutagenesis, we also show that PKA directly phosphorylates the transmembrane domain of Bnip3 to inhibit its function. Mechanistically, when the PKA phosphorylation site on Bnip3 was neutralized, the protective effect of misoprostol on mitochondrial membrane potential, mitophagy and permeability transition was lost. Taken together, these results demonstrate a foundational role for Bnip3 phosphorylation in the molecular regulation of cardiomyocyte mitochondrial dysfunction. These findings further identify a pharmacological mechanism, through PKA, which may ultimately be able to prevent hypoxia-induced myocardial injury.
Abstract 166: MCL-1 Facilitates the Removal of Damaged Mitochondria via the Mitophagy Receptor BNIP3
Authors: Moyzis Alexandra G , Lally Navraj S , Najor Rita A , Leon Leonardo J , Gustafsson Åsa B
Published: October 2019

Myeloid Cell Leukemia-1 (MCL-1) is an anti-apoptotic BCL-2 family protein that is necessary to maintain cardiac homeostasis in the adult heart. MCL-1 localizes to two distinct mitochondrial locations in myocytes, both on the outer mitochondrial membrane (MCL-1
OM
) and in the mitochondrial matrix (MCL-1
Matrix
). Our lab previously showed that cardiac-specific ablation of MCL-1 at both of these mitochondrial locations in mice led to severe contractile dysfunction and compromised mitochondrial function. Intriguingly, these defects were accompanied by signs of necrotic, rather than apoptotic, cell death. This indicates that MCL-1 has an alternate role in maintaining mitochondrial homeostasis in cardiac myocytes. Unexpectedly, we found that MCL-1 induces mitochondrial clearance in response to treatment with the chemical uncoupler FCCP in a Parkin-independent manner. Hypoxia is also known to induce mitochondrial clearance, and overexpression of MCL-1 further enhances hypoxia-mediated mitophagy. Fluorescence imaging identified MCL-1-positive mitochondria sequestered inside autophagosomes. MCL-1-mediated clearance is abrogated in autophagy deficient Atg5-/- cells, confirming that clearance is occurring via the autophagy pathway. Mutation of MCL-1’s BH3 domain (G198E D199A) does not affect its ability to induce clearance, suggesting that this role may be independent of its anti-apoptotic function. Also, replacing MCL-1’s BH domains with those of BCL-2, does not affect its ability to induce mitophagy. Next, we investigated whether MCL-1 functions as a mitophagy receptor and promotes removal of damaged mitochondria by binding to directly to LC3 through one or more of its three putative LC3-Interacting Region (LIR) motifs. Endogenous MCL-1 and LC3 co-immunoprecipitate in response to stress induced by FCCP. However, mutating each of MCL-1’s individual LIR motifs, as well as generating combined mutations in all three, does not affect MCL-1-mediated mitophagy. Instead, we found that MCL-1 interacts with the known mitophagy receptor BNIP3 both
in vitro
and
in vivo
. Thus, our data suggest that MCL-1 promotes elimination of dysfunctional mitochondria by positively regulating the mitophagy receptor BNIP3.
Abstract 177: Hemodynamic Status From Photoplethysmography, Signs of Successful Pregnancy
Authors: Chang Chi-Wei , Xie Dan ni , Wang Shu jia , Li Xao ran , Li Meng jie , Huang Chun , Wang An ran , Zhou Li , Zhu Xiao feng , Li Yan mei , Huang Zi-Xin , Wang Gin-Chung
Published: October 2019

The key physiological condition for successful pregnancy is a critical issue in reproductive science. Many studies have shown that the pregnancy process requires a lot of subtle cardiovascular adaptation, including a decrease in systemic vascular resistance, an increase in maternal heart rate, stroke volume, and cardiac output. Those adaptation will also lead to changes in the arterial pulse spectrum. Since pregnancy requires the mother's competency to change the hemodynamic state, an interesting question is if the pre-pregnancy hemodynamics of the cardiovascular system affect the clinical pregnancy rate of each embryo transfer. Therefore, we initiated an observational study to validate our conjecture. We recruited 116 women of childbearing age(range 22 to 41 years) who planned to carry out in vitro fertilization(IVF). Each subject underwent photoplethysmography(PPG) before embryo implantation. We calculated the first ten harmonic amplitudes (C1~C10) and phases (P1~P10) from the pulse data of PPG using the Fourier transform method, and tracked these women to 42 days after implantation to confirm the successful pregnancy. The results showed that the subjects with successful IVF had higher C4 and C7(P=0.006; P=0.018), compared with those who failed the IVF. We then classified the enrolled patients into quartile groups of C4 and C7 respectively. Log rank test further proved that the cumulative rates of successful pregnancy were significant higher for those with the higher C4 and C7, taking groups with smaller values as reference(Figure 1). In conclusion, maternal hemodynamic state before implantation have an impact on the successful pregnancy rate during IVF.
Abstract 178: Development of a New Structural Family of Microbial Choline Trimethylamine Lyase Inhibitors for the Treatment and Prevention of Cardiovascular Disease
Authors: Duzan Ashraf S
Published: October 2019

Ashraf Duzan, BPharm., MS
1,2
; Xiaodong Gu, Ph.D.
1,3
; Adam Roberts, Ph.D.
1,3
; Jennifier Buffa, MS
1,3
; James Anderson
1,3
, Valentine Gogonea, Ph.D
1,2,3
Stanley L. Hazen, MD, Ph.D.
1,2,3
.

1
Department of Cellular & Molecular Medicine,
3
Center for Microbiome and Human Health,
4
Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, Ohio 44195;
2
Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115

Recent clinical research points to trimethylamine N-oxide (TMAO), a gut microbiota generated metabolite, as a biomarker associated with acute thrombotic event and cardiovascular disease risks, and a direct causative contributor to these adverse phenotypes. Our goal in this project is to develop novel microbial enzyme inhibitors that alter the biosynthetic pathway of TMAO in vivo through the selective potent inhibition of gut microbial choline trimethylamine (TMA) lyase activity, the rate limiting step in TMA and TMAO generation in vivo. TMA generation by gut microbiota is predominantly catalyzed by the gut microbial enzyme pair CutC/D, members of the microbial choline utilization (
cut
) gene cluster. We are using structure activity relationships (SAR) to predict new classes of chemical structures as potential efficient inhibitors. Thus, we are exploring the synthesis of new chemical compounds, non-lethal to the gut microbial community, with high inhibitory potency in multiple
in vitro
assays employing evolutionarily diverse microbial
cutC/D
(primary and secondary screens), polymicrobial communities (tertiary screens), and for inhibitors that pass the above screening assays, progression to
in vivo
studies. We are preparing and assessing inhibitors that can work either through irreversible non-competitive or competitive mechanisms, possess appropriate physico-chemical pharmaceutical properties and have minimal systemic exposure to the host in effort to minimize possibility of side effects. Our leading candidates have excellent enzyme blocking efficiency and display good pharmacokinetic/pharmacodynamics properties.
Abstract 179: Resistin Regulates ATP-citrate lyase Expression a Key Metabolic Enzyme of Lipogenesis and Histone acetylation in Human Macrophage
Authors: Haider Nezam , Moly Pricila , Lu Weifeng , Zhou Wei
Published: October 2019

BACKGROUND:
Human resistin synthesized by macrophages is associated with insulin resistance, type 2 diabetes, atherosclerosis, and chronic inflammation. We used a comparative global protein expression profiling of human macrophage treated with resistin to identify downstream targets and intracellular signaling pathways involving hyper-resistinemia-associated metabolic disorders by HPLC-ESI-MS/MS. ATP-citrate lyase (ACLY), is an essential cytosolic enzyme for generating acetyl-CoA, a key metabolite for glycolysis, de novo lipogenesis, cholesterol synthesis, and histone acetylation. Its dysregulation is associated with diabetes, hypercholesterolemia, and oncogenesis and is a potent therapeutic target . In this study, we explored the regulation of ACLY and its phosphorylation by resistin.

METHODS:
Mϕ derived from THP1 cells were treated with human resistin for 24-72 hrs. Samples of total protein tryptic digest of untreated control and resistin-treated Mϕ were analyzed by HPLC-ESI-MS/MS. Total protein were also analyzed by western blot for ACLY and its phosphorylation . Gene expression was measured by Quantitative Real-time PCR.

RESULTS:
ACLY showed a significant (ρ < 0.05) differences in expression between control and resistin treated groups by quantitative mass spectrometry. ACLY formed protein-protein interaction (PPI) with 23 out of 162 significantly differentially regulated proteins. Level of ACLY protein decreased 2 fold after 48 hours of resistin treatment. However, after 72 hours of resistin treatment ACLY proteins increase with a 4-5 fold increase in its serine 455 phosphorylation (Figure 1). In human macrophage, ACLY mRNA also increased ~2 fold after 24 hours of resistin treatment (Figure 2).

CONCLUSION:
Resistin significantly changed the gene expression of ACLY in humanMϕ. Although there was an initial decrease in ACLY protein with no change in its phosphorylation. However, a longer 72 hours treatment of resistin induce ACLY protein in human macrophages and a very significant increase in its phosphorylation. ACLY plays a pivotal role in many metabolic and epigenetic processes, this late ACLY activation may mediate the effect of resistin on chronic diseases like diabetes, atherosclerosis, and chronic inflammation.
Abstract 181: Anti-inflammatory Effects of Macitentan on Monocrotaline-induced Pulmonary Hypertension in a Rat Model
Authors: Kim Kyung-hee , Rha Seung-Woon , Park Yoonjee , Byun Jae Kyeong
Published: October 2019

Background:
Inflammation is closely linked to pulmonary arterial hypertension (PAH). Macitentan (MAC) reduces morbidity and mortality among advanced-stage pulmonary arterial hypertension (PAH) patients. However, data regarding anti inflammatory benefits of MAC treatment at an early stage of PAH is lacking.

Methods:
One week after monocrotaline (MCT) injection, rats were randomly assigned to MAC (n=30), normal saline (MCT, n=30). Fourteen sham rats (Sham) were included for comparison. Right ventricular (RV) systolic function was assessed via echocardiography as the RV fractional area change (RV-FAC). An invasive pressure-volume analysis using a Millar conductance catheter was performed 8 weeks after MCT injection. Rats were subsequently euthanized for histopathologic analysis. Levels of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1,6) and nuclear factor-kappa B (NF-κB) activity in serum were determined with enzyme-linked immunosorbent assay.

Results:
RV-right atrial pressure gradient on echocardiography was significantly increased 3 weeks after MCT injection, but was maintained in the Sham. On invasive hemodynamic analyses, RV end-systolic (203±79 μL) and end-diastolic volumes (315±83 μL), pulmonary artery systolic pressure (90±6 mmHg), and end-systolic pressurevolume relationship (–264±23) were significantly worse in the MCT vs. in the MAC (98±45 μL, 225±35 μL, 39±12 mmHg, and –147±42, respectively, all p<0.05). On histopathology, both RV and lung fibrosis were significantly reduced in the MAC. MAC significantly ameliorated PAH by acting against pulmonary vascular remodeling, decreasing macrophage infiltration, and reducing pro-inflammatory cytokine expression and nuclear factor-kappa B (NF-κB) activity in the lungs of the MCT-treated rats.

Conclusions:
MAC treatment improves haemodynamic parameters in established pulmonary hypertension. These results suggest that MAC saline ameliorates the progression of pulmonary hypertension induced by monocrotaline in rats, which may be associated with anti-inflammatory effects.
Abstract 183: Phosphodiesterase 9 Inhibition Improves Cardiometabolic Profile in Female Mice Independent of Estrogen Status
Authors: Mishra Sumita , Zhu Guangshuo , Rodriguez Susana , Wong William , Kass David
Published: October 2019

Approximately 6.5 million Americans are living with heart failure (HF) currently and the number of persons is projected to increase 46% by 2030. One particularly notable syndrome associated with increasingly severe obesity is called - Heart failure with a preserved ejection fraction (HFpEF); which represents half of all HF patients world-wide. Epidemiologic insights have provided incremental evidence that female sex is a stronger risk factor for HFpEF, particularly post-menopausal women. Loss of estrogen after menopause reduces estrogen receptor induced-NO-cGMP-PKG activation and consequently less benefit from Phosphodiesterase 5(PDE5) inhibition which principally hydrolyze NO-derived cGMP that appears compromised in HFpEF patients. PDE9 targets cGMP generated by Natriuretic peptide (NP) signaling and is significantly increased in hearts from HFpEF patients. This supports a role of PDE9 in the heart and its value as a therapeutic target for abnormalities relevant to HFpEF. We created a murine model of postmenopausal obesity and mild cardiac stress induced HFpEF. Genetic ablation or pharmacological inhibition of PDE9 conferred higher contractile performance with increased %EF, %FS and improved diastolic relaxation and filling. Echo-MRI analysis showed an improvement in whole body metabolism with significant decrease in percent body fat associated with enhanced O
2
consumption and increased energy expenditure. The mechanisms underlying the protective effects are increased mitochondrial function and enhanced expression of transcriptional regulators pgc1α and pparα, driving mitochondrial fatty acid oxidation and increased energy dissipation. Our work identifies for the first-time reversal of changes in substrate metabolism, and mitochondrial dynamics as a direct consequence of PDE9 inhibition in heart and fat. Proteomic analysis also revealed a dynamic cross-talk of PDE9 protein with biological networks of mitochondrial membrane proteins and proteins regulating cellular metabolism. This study greatly enhances our understanding of how the cGMP/PKG activation system can be modulated in females with reduced sex hormone- paving the way for gender-selective targeting in patients with obesity and HFpEF.
Abstract 191: Difference in Calcium Sensitivity Between Right and Left Ventricles With Lower Expression of Calcium Binding Proteins in Right Ventricular Myocytes
Authors: Jeon Young Keul , Jang Ji Hyun , Youm Jae Boum , Zhang Yin Hua , Kim Sung Joon
Published: October 2019

Left ventricle (LV) and right ventricle (RV) have distinctive structural and functional characteristics as well as heterogeneous physiological properties. The RV is exposed to a relatively low pulmonary vasculature, which results in less mechanical afterload. Consistent with these physiological differences, the RV has a thinner free wall than the LV, and the movement of its contraction is geometrically different. Despite these definite differences, the studies of basic excitation-contraction coupling and calcium homeostasis of RV has been less studied than in LV. To establish the interventricular difference, we evaluated the basic electrophysiological and calcium-contractile properties of myocyte with or without β-adrenergic stimulation. Analyses of contraction and Ca
2+
signaling and action potential duration (APD) in isolated RV myocytes showed more prominent APD prolongation with less significant changes in sarcomere shortening and calcium transient, implying less efficient E-C coupling RV myocytes. To investigate the prolonged APD of RV, we measured transient outward potassium (Ito) current and L-type calcium channel (LTCC) current of two ventricles. Although the peak current of two ion channel was not different significantly in two ventricle, the half inactivation voltage of RV Ito was smaller than LV. Comparing with LV, RV myocytes showed round peak, slower early relaxation, and faster late relaxation, suggesting the difference of calcium sensitivity between two ventricles. To investigate the difference, we examined the expression level of calcium-binding proteins that regulate myofilament activities. Using immunoblotting from enriched protein of myofilament fraction, we found that the calcium binding proteins such as troponin I were lower in RV. Taken together, our results suggest that calcium binding proteins of RV was differ from that of LV, which induce the modified calcium sensitivity. We confirmed that reduced calcium binding proteins induced the decrease of calcium sensitivity and modified E-C coupling using Bers’ mathematical rat cardiomyocyte model. The calcium sensitivity of RV is a clue to explain the different physiological properties of the RV.
Abstract 201: Roles of PIKfyve in Cardiac Fibroblast Migration
Authors: Luo Guangming , Weisman Lois S
Published: October 2019

Cell migration is fundamental to proper development of the heart as well as other organs. Our characterization of a hypomorphic PIKfyve
β-geo/β-geo
mouse mutant, revealed that has an abnormally small heart, and raised the possibility that this could be due in part to a defect in cell migration. Moreover, a few studies suggested that PIKfyve, the sole lipid kinase that generates phosphatidylinositol 3,5-bisphosphate (PI3,5P2) and provides most of the PI5P, may have a role in migration of cultured cells. However, the localization of PIKfyve and the molecular mechanisms whereby PIKfyve regulates cell movement remain elusive. Here I show that inhibition of PIKfyve significantly impairs cell migration in cultured cell lines, as well as in primary cardiac fibroblasts, which are essential for proper heart development. Moreover, expression of a hyperactive PIKfyve mutant promotes cell migration. In order to detect the localization of endogenous PIKfyve, we used CRISPR-Cas9 genome editing to tag endogenous PIKfyve with an HA peptide at its N-terminus. Interestingly, in addition to PIKfyve localization on endosomes, a pool of PIKfyve was located at the cell leading edge. We observed PIKfyve in both lamellipodia and filopodia. Notably, we found that acute addition of PI3,5P2 or PI5P for 5 min altered filopodia dynamics. Our preliminary results suggest that PI3,5P2 and PI5P alter different properties of filopodia. These findings suggest that cell migration may be controlled in part by PI3,5P2 and PI5P which arise due to PIKfyve localization to the cell leading edge. Together, these studies suggest that mechanistic insights into how PIKfyve regulates cell migration may reveal new tools or concepts that can be utilized to study heart development in mammals.
Abstract 215: Assessing the Efficacy of Novel RYR2 Inhibitor, EL20, in Induced Pluripotent Stem Cell Derived Cardiomyocytes from a Catecholaminergic Polymorphic Ventricular Tachycardia Patient
Authors: Word Tarah A , Landstrom Andrew , Quick Ann , Shak Mayra , Miyake Christina , Allen Hugh D , Wehrens Xander
Published: October 2019

Background:
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac arrhythmia syndrome that leads to sudden cardiac death in children and young adults. The most common form of CPVT is caused by autosomal-dominant mutations in the gene encoding cardiac ryanodine receptor type-2 (
RYR2)
. Mutations in
RYR2
promote excessive calcium (Ca
2+
) leak from the sarcoplasmic reticulum (SR), triggering potentially lethal arrhythmias. Recently, we demonstrated that the tetracaine derivative EL20 specifically inhibits RYR2, normalizes Ca
2+
handling, and suppresses arrhythmias in a CPVT mouse model. We hypothesized that EL20 will also normalize Ca
2+
handling and arrhythmias in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs).

Materials/Methods:
Blood samples from a child carrying RYR2 mutation R176Q/+ (RQ) and a mutation-negative relative (WT) were collected, processed into peripheral blood mononuclear cells (pBMCs), and reprogrammed into iPSCs using Sendai virus system. Functional iPSC-CMs were derived using Stemdiff
TM
kit. Differentiation of CMs was validated using qPCR and immunofluorescent staining. Confocal Ca
2+
imaging was used to monitor RyR2 activity. Axiom Biosystem
TM
microelectrode arrays (MEA) was used to assess arrhythmogenic action potential (AP) and rates.

Results:
Successfully differentiated CMs showed increased levels of Ca
2+
-handling markers and decreased levels of stem cell markers. Baseline Ca
2+
imaging revealed a 4-fold increase in calcium spark frequency (CaSpF) in RQ iPSC-CMs (3.08±0.58 a.u., n=12) vs. WT (0.69±0.22 a.u., n=13, p<0.01). Strikingly, EL20 normalized CaSpF in RQ iPSC-CMs (0.60±0.16 a.u., n=9, p<0.05 vs. baseline). Furthermore, EL20 decreased the erratic beat period rate of spontaneous APs occurring in RQ iPSC-CMs to a rate similar to WT. Importantly, EL20 had no significant effect on the beat period rate or field potential duration of WT iPSC-CMs.

Conclusion:
Our results demonstrate that EL20 normalizes Ca
2+
handling and AP beat rate frequency in patient-derived RYR2-R176Q/+ iPSC-CMs with no observed off-target effects, positioning this novel RyR2-inhibitor as a promising therapeutic candidate for treatment of CPVT.
Abstract 223: Dynamic Multimodality Imaging Monitored Anti-inflammation Therapy for Myocardial Infarction: Exploring the Role of MCC950 in Murine Model
Authors: Li Xiang , Yang Weidong , Wang Jing
Published: October 2019

Objectives:
The aim of this study is applying non-invasive multimodality imaging to investigate the potential therapeutic effects of MCC950, a novel selective NLRP3 inflammasome inhibitor, in myocardial infarction (MI) model.

Methods:
C57BL/6 mice were subjected to the MI or Sham model.
18
F-FDG PET/CT was undertaken for monitoring any post-MI inflammation changes on days 1, 3, 5 and 7.
18
F-FDG PET/CT and echocardiography were both used to quantitate any alterations of infarct size and cardiac function on days 7 and 28 post-MI.

Results:
Dynamic
18
F-FDG PET/CT revealed that MI+MCC950 group had a lower accumulation of
18
F-FDG at infarct region as early as day one, and remained lower until day seven post-MI when compared to the MI group (Infarct/remote SUV
mean
ratios, from day 1 to 7 respectively: 2.37±0.11, 3.10±0.28, 2.74±0.43, 1.89±0.26, P < 0.05.). What’s more, MCC950 inhibited infarct size expanding on day 7 (31.90±1.60%
vs
36.30±3.31%, P < 0.05
vs
MI group). The use of echocardiography suggested that MCC950 treatment increased ejection fraction (EF) and fraction shortening (FS), (EF: 48.72±3.86%
vs
39.64±2.85%, P < 0.05; FS: 22.93±2.58%
vs
17.54±1.91%, P < 0.05), meanwhile decreased end-diastolic volume (EDV) and end-systolic volume (ESV) when compared with that in the MI group on day 28 (EDV: 70.91±3.31
vs
82.40±5.30 ul, P < 0.05; ESV: 38.95±4.38
vs
50.29±3.76 ul, P < 0.05).

Conclusions:
MCC950 reduced inflammatory response, infarct size and preserved cardiac function post-MI. Dynamic multimodality imaging may be potential approaches for basic research and future clinical translation in the use of anti-inflammation therapy for MI.