bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 226: Statin Therapy is Associated With Decreased Neovascularization in Symptomatic Carotid Atherosclerotic Plaque: A Pathological Analysis
Authors: Konishi Takao , Tanaka Shinya
Published: February 2019

Background:
Statin treatment has been shown to be associated with decreased intraplaque neovascularization in human carotid arteries by various imaging modalities or in animal models by pathological analysis. However, whether statin treatment attenuates intraplaque neovascularization in human carotid plaque has not been fully described. The aim of this study was to analyze the possible relationships between statin treatment and intraplaque microvessels (MVs) in patients undergoing carotid endarterectomy (CEA).

Methods:
Among consecutive 79 patients with advanced carotid artery stenosis (>70%) between May 2015 and February 2017, 66 patients without statin therapy (group 1) and 13 patients with statin therapy (group 2) were analyzed. The specimens were stained with hematoxylin/eosin and elastica-Masson. Immunohistochemistry was performed, using an endothelial specific antibody to CD31, CD34 and PDGFRβ. The number of intraplaque MVs was analyzed in each specimen. MV density was defined as number of MVs per mm
2
.

Results:
The mean number of MVs was fewer in group 2 when compared with that in group 1 (25.8±17.6 vs. 50.8±31.8 /section, P<0.001). The mean MV density was lower in group 2 when compared with that in group 1 (1.06±0.84 vs. 2.19±1.43 /mm
2
, P<0.001). There was no significant difference in the mean concentration of low-density lipoprotein cholesterol between group 1 and group 2 (121±32 mg/dl vs. 105±37 mg/dl, P=0.118).

Conclusions:
This pathological analysis of carotid atherosclerotic plaque suggests that statin therapy is associated with decreased intraplaque neovascularization in symptomatic patients undergoing CEA. This plaque stability might be derived from pleiotropic effect beyond lipid lowering of statin therapy.
Abstract 229: Non-invasive, Patient-specific Assessment of LVAD Modeled With Consideration of LV Ejection and Function
Authors: Yu Huidan (Whitney) , Deb Anurag , Khan Monsurul , Chen Rou , Yang Yang , Wang I-Wen
Published: February 2019

The use of left ventricular assist device (LVAD) is a proven therapy for end-stage heart failure either as bridge-to-transplant or destination therapy. The improvement of LVADs with greater reliability, durability, and miniaturization require corresponding innovations in surgical techniques to fully realize the better clinical outcomes, shorter length of stay, and lower cost to the health care system. One key direction in surgical advancement is the minimally invasive implantation techniques. Traditional LVAD implantation is most often performed with full sternotomy and anastomosis of the outflow graft (OG) to ascending aorta. Minimally invasive techniques will require the option of potential OG anastomosis to other arterial sites such as axillary artery or descending aorta. The consideration of a patient’s initial native cardiac function and subsequent evolution on LVAD support becomes more critical. This
research objective
of this work is to fill in a
GAP
between a critical need for clinical guidelines of optimal LVAD implant and a reliable pre-surgical assessment that is patient-specific and noninvasive. We present a parametric study using image-based computational modeling and analysis, Fig 1 (a), to non-invasively assess the blood supply to upper/low body through 15 combinations between LVAD pumping and native LV ejection (b). The flow and kinetic energy distribution from ascending aorta to upper/low body are shown in (c) and (d) respectively. It is found that more LV ejection results in more flow and kinetic energy going to the upper body. The variability of LVAD flow and delivery of kinetic energy has impact on end-organ perfusion and should be addressed in future optimization of LVAD implantation.
Abstract 236: Histone Variant H2A.Z Defines Cell Identity in Smooth Muscle Cells as Revealed by Single-Cell Transcriptomics
Authors: Wang Li , Yao Fang , Yu Peng
Published: February 2019

Background:
Histone variants endow chromatin with specific structures, and play essential roles in development and diseases. However, little is known about their roles in controlling cell identity in vascular diseases.

Methods and Results:
Given the cell heterogeneity in atherosclerotic lesions, we applied single-cell RNA-Seq to analyze human diseased arteries, and identified histone variant H2A.Z as a key histone signature to maintain vascular smooth muscle cell (VSMC) identity. We show that H2A.Z occupies genomic regions near VSMC marker genes and its occupancy is decreased in VSMC undergoing dedifferentiation. Mechanistically, H2A.Z occupancy preferentially promotes nucleosome turnover, facilitates the recruitment of Smad3 and Med1, thereby activating VSMC marker gene expression. In human diseased vascular tissue, H2A.Z expression is dramatically decreased. Notably,
in vivo
overexpression of H2A.Z rescued injury-induced loss of VSMC identity and neointima formation.

Conclusions:
Together, our data introduce dynamic occupancy of a histone variant as a novel regulatory basis contributing to cell fate decisions, and imply H2A.Z as a potential intervention node for vascular diseases.
Abstract 267: Safety and Efficacy of a Combinatorial CCN5/SERCA2a Gene Delivery Approach for Arrhythmia Suppression in a Chronic Model of Angiotensin II (ANG) Induced Cardiac Hypertrophy and Failure
Authors: Raad Nour , Jeong Dongtak , Park Woo Jin , Akar Fadi
Published: February 2019

Background:
Structural & electromechanical remodeling promotes the incidence of ventricular tachycardia (VT) during progression from compensated hypertrophy to heart failure. The multifactorial nature of VT during disease progression presents a major challenge for conventional anti-arrhythmic approaches.

Objective:
To determine the safety & efficacy of the antifibrotic matricellular protein CCN5 and a novel combinatorial vector carrying CCN5 and SERCA2a (CCN5/S2a) for prevention of VT.

Methods:
Adult mice (N=15) underwent chronic ANG infusion (3mg/kg/d) for 2 wk. Mice were subsequently randomized for treatment with AAV9 vectors carrying CCN5, CCN5/S2a, or an empty vector and were maintained for another 2 wk. Age matched mice served as controls (Ctrl). Cardiac function was serially assessed by echocardiography. Optical mapping was performed in perfused hearts before & after challenge with isoproterenol and rapid pacing. VT threshold (VT-t) was defined as the pacing frequency required for induction of sustained VT.

Results:
ANG infusion resulted in a biphasic change in fractional shortening (FS) consistent with an early phase of compensated hypertrophy followed by decompensation. Late decompensation was associated with adverse electrophysiological (EP) remodeling, including action potential prolongation (21.3±2.4ms in ANG vs 17.5±1.9ms in Ctrl), conduction slowing (0.43±0.04m/s in ANG vs 0.57±0.08m/s in Ctrl) and susceptibility for pacing induced VT (VT-t 21.3±1.2Hz in ANG vs 25.8±1.4Hz in Ctrl). At a cut-off frequency of 22Hz, 5/5 ANG vs 0/7 Ctrl heart were prone to VT. Treatment of ANG mice with CCN5 and the combinatorial CCN5/S2a vector suppressed VT susceptibility by increasing VT-t (25.9±2.9Hz for CCN5, 28.4±2.6Hz for CCN5/S2a). Mechanistically, CCN5/S2a delivery reversed EP and structural remodeling, which in turn prevented the formation of the critical lines of block that maintained VT circuits. In addition, CCN5/S2a fully abrogated the late decrease in FS.

Conclusion:
Delivery of a combinatorial AAV9 vector carrying CCN5/S2a during the early phase of compensated hypertrophy prevents the progression of electrical and structural remodeling, thereby suppressing the incidence of VT while preserving mechanical function.
Abstract 280: MCL-1
Matrix
Promotes Mitochondrial Fusion and Protects Against TAC Induced Hypertrophy
Authors: Leon Leonardo J , Moyzis Alexandra G , Gustafsson Asa B
Published: February 2019

The anti-apoptotic BCL-2 protein MCL-1 is highly expressed in the myocardium, and we have previously found that deletion in cardiomyocytes leads to mitochondrial dysfunction and rapid development of heart failure. MCL-1 exists both in the outer mitochondrial membrane (MCL-1
OM
) and in the mitochondrial matrix (MCL-1
Matrix
). Because

Mcl-1
-/-

mice lack both MCL-1
Matrix
and MCL-1
OM
, the functional role of each form remains unknown. While studies have implicated MCL-1
OM
in regulating apoptosis, very little is known about the functional role of MCL-1
Matrix
. Here, we show that mitochondria are responsive to alterations in cellular metabolism, and culturing mouse embryonic fibroblasts (MEFs) in “oxidative” media containing galactose or acetoacetate led to a significant increase in the MCL-1
Matrix
/MCL-1
OM
ratio and elongation of mitochondria. We confirmed that MCL-1
Matrix
overexpression promoted mitochondrial fusion and reduced maximal mitochondrial respiration under baseline conditions. Interestingly, the elongated mitochondria were initially protected against FCCP-induced fission and mitophagy. However, prolonged exposure to FCCP resulted in abrogation of MCL-1
Matrix
import. Accumulation of MCL-1
Matrix
on the outer membrane led to its degradation and subsequent induction of mitophagy. Furthermore, to investigate its functional role in the heart, we generated cardiac specific MCL-1
Matrix
(αMHC-MCL-1
Matrix
) transgenic mice. The mice show no differences in cardiac structure and function compared to wild-type litter mates at 3 months. However, ultrastructural analysis revealed the presence of enlarged mitochondria in myocytes from αMHC-MCL-1
Matrix
mice. Finally, we subjected WT and αMHC-MCL-1
Matrix
mice to transverse aortic constriction (TAC) and we found that αMHC-MCL-1
Matrix
mice had reduced heart weight to body weight (HW/BW) ratio and decreased expression of hypertrophy markers ANF, BNP, and MHC-β at two weeks post-TAC compared to WT. Thus, our studies indicate that cardiac overexpression of MCL-1
Matrix
promotes mitochondrial enlargement in myocytes and alleviates stress placed on the heart during pressure overload.
Abstract 291: The Type I Interferon Receptor Amplifies Innate Immune Responses After Myocardial Infarction
Authors: Ng Richard P , King Kevin R , Aguirre Aaron D , Arlauckas Sean P , Weissleder Ralph
Published: February 2019

Ischemic heart disease (IHD), the leading cause of death in the world, begins with myocardial infarction (MI). Overly exuberant innate immune responses are detrimental to post-MI healing and repair. Efforts to achieve post-MI cardioprotection focused on blocking NFκB-mediated inflammation have been largely unsuccessful. We recently discovered that MI leads to cytosolic DNA sensing in leukocytes and triggers the interferon regulatory factor 3 (IRF3)-dependent expression of interferon stimulated genes (ISGs). We found that genetic inhibition of IRF3 after MI reduces inflammation, limits ventricular dilation and contractile dysfunction, and improves survival. In this work, we test two hypotheses. 1) Type I interferons (IFNs), secreted by a few DNA-sensing/IRF3-activated leukocytes, signal via type I interferon alpha/beta receptors (IFNARs), to amplify ISG expression after MI. 2) Pharmacologic inhibition of type I IFN/IFNAR signaling blocks response amplification and significantly reduces ISG expression.

We established DNA-stimulated bone marrow derived macrophages (BMDMs) as an
in vitro
model of the post-MI type I IFN response and measured the expression of ISGs (
Cxcl10
,
Irf7
,
Ifit1
, and
Ifit3
) by qPCR. We quantified and compared changes in ISG expression caused by direct DNA stimulation, incubation with conditioned media containing secreted factors from DNA-stimulated cells, and treatment with an IFNAR neutralizing antibody. Compared to unstimulated BMDMs, ISG expression was amplified by >35-fold in BMDMs stimulated by either DNA or conditioned media. However, there was no significant amplification when the experiments were performed in the presence of an IFNAR neutralizing antibody. In a mouse model of MI, single cell RNA-Seq revealed that IFNAR-deficient mice have reduced numbers of ISG-expressing leukocytes compared to wild type controls, which indicates that IFNAR-dependent amplification of innate immune responses occurs after MI
in vivo
.

Collectively, these results demonstrate that IFNAR ligands are important amplifiers of DNA-induced innate immune responses after MI and suggest that anti-interferon therapy should be further investigated
in vivo
to improve clinical outcomes after MI.
Abstract 299: Theranostic nanoparticles for Thrombosed Vessels: H
2
O
2
-Activatable Contrast-Enhanced Photoacoustic Imaging and Antithrombotic Therapy
Authors: Lee Dongwon
Published: February 2019

A thrombus (blood clot) is formed in the injured vessels to maintain the integrity of vasculature. A thrombus, composed mainly of activated platelets and fibrin, obstructs arteries or veins, leading to various life-threatening diseases. During thrombus formation, H
2
O
2

(hydrogen peroxide) is overproduced by activated platelets and other vascular sources and serves as a key mediator of platelet activation and aggregation, facilitating inflammation and development of obstructive vascular diseases. In this regard, high level of H
2
O
2

holds great potential as diagnostic and therapeutic markers of obstructive capsular diseases. Inspired by the distinctive physicochemical characteristics of thrombi such as abundant fibrin and the significant of elevated level of H
2
O
2
, we developed H
2
O
2
-activatable novel nanomedicine (T-FBM) that could detect thrombi through the amplification of ultrasound/photoacoustic signal and serve as an antithrombotic nanomedicine. T-FBM nanoparticles were designed to target fibrin-rich thrombi and be activated by H
2
O
2
to generate CO
2
bubbles to amplify the photoacoustic signal. In the phantom studies, T-FBM nanoparticles showed significant amplification of ultrasound/photoacoustic signals in a H
2
O
2
-triggered manner. T-FBM nanoparticles also exerted H
2
O
2
-activatable antioxidant, anti-inflammatory and antiplatelet activities on endothelial cells. In mouse models of carotid arterial injury, T-FBM nanoparticles significantly enhanced the photoacoustic contrast specifically in thrombosed vessels and significantly suppressed thrombus formation. We anticipate that T-FBM nanoparticles hold great translational potential as nanotheranostics for H
2
O
2
-associated cardiovascular diseases.
Abstract 301: An m6A Demethylase, FTO Mediates Post-transcriptional mRNA Modifications to Regulate Cardiac and Cardiomyocyte Function
Authors: Mathiyalagan Prabhu , Adamiak Marta , Mayourian Joshua , Liang Yaxuan , Sassi Yassine , Agarwal Neha , Jha Divya , Ishikawa Kiyotake , Zhang Shihong , Kohlbrenner Erik , Yin Xiaoke , Chepurko Elena , Chen Jiqiu , Trivieri Maria G , Singh Rajvir , Mayr Manuel , Fish Kenneth , Lebeche Djamel , Hajjar Roger J , Sahoo Susmita
Published: February 2019

Background:
Post-transcriptional modifications in the form of m6A (N6-methyladenosine) regulate mRNA fate and translation, miRNA biogenesis, lncRNA function and several cellular processes. However, m6A mechanisms in the mature post-mitotic tissues such as the mammalian heart remain unexplored. Here, we investigated the role of transcriptome-wide m6A in cardiac protein expression as well as lncRNA function.

Methods:
Using biochemical assays, we investigated the spatiotemporal gene expression patterns of m6A regulators in non-failing, failing (ischemic) human hearts, in mouse and pig MI models. In mouse heart, we mapped transcriptome-wide m6A by developing state-of-the-art MeRIP-seq coupled with novel bioinformatics analysis. To investigate the functional relevance of m6A to cardiac proteome, we used SILAC-LC-MS. By silencing (siRNA) m6A-reader proteins, we investigated the role of m6A in cardiomyocyte mRNA stability, decay and nuclear export. For in vivo myocardial gene delivery, we used AAV9 and adenovirus vectors. Finally, we performed immunohistology in cardiomyocytes and mouse heart tissues to study nuclei size, fibrosis and angiogenesis.

Results:
We discovered that the expression of m6A demethylase, FTO is decreased in ischemic myocardium and cardiomyocytes, thus in vivo FTO gene delivery resulted in attenuation of ischemia-induced increase in m6A and decrease in cardiac contractile function post-MI. FTO overexpression in mouse heart and human cardiomyocytes revealed selective demethylation (MeRIP-seq) of cardiac contractile transcripts resulting in induced contractile protein expression (SILAC-LC-MS) thus rescuing heart function post-MI. Mechanistically, we demonstrate that the cardioprotective mechanism of FTO is mediated by selective demethylation of cardiac contractile transcripts under ischemia, which prevents mRNA degradation as well as enhanced nuclear compaction. Finally, we demonstrate that FTO overexpression in mouse models of MI resulted in decreased fibrosis and enhanced angiogenesis.

Conclusion:
Our study provides the first description of a cardiac active m6A demethylase working at post-transcriptional level as a critical regulator of cardiac contractile function, fibrosis and angiogenesis.
Abstract 304: Protein Phosphatase 1 Contributes to Atrial Stunning in Atrial Fibrillation
Authors: Perike Srikanth , Alsina Katherina M , Sridhar Arvind , Wehrens Xander H , Darbar Dawood , McCauley Mark D
Published: February 2019

Background:
Atrial fibrillation (AF) is the most common cardiac arrhythmia. AF increases stroke risk 5-fold, and is partially mediated by reduced atrial contractility (stunning). However, it’s mechanism is relatively unknown. Recent data from our lab indicate protein phosphatase 1 (PP1) dephosphorylation of myosin light chain 2a (MLC2a) as contributor to Atrial stunning in AF

Aim:
To determine role of PP1 regulatory subunit (PPP1R12C) and catalytic subunit (PP1c) in modifying atrial sarcomere phosphorylation in AF

Methods:
We analyzed protein binding and phosphorylation of PPP1R12C, PP1c, and MLC2a in transfected HL1 and HEK cells, and AF prone
Pitx2
null+/–
mice. We also characterized the MRCK site on PPP1R12C that regulates PP1c and MLC2a phosphorylation using BDP5290 (PPP1R12C phosphorylation inhibitor), phosphomimetic (T560D) and phospho-ablated (T560A) versions of PPP1R12C

Results:
In

Pitx2
null

+/–
mice, PPP1R12C increased by 2-fold (
P
<0.01) and had a 40% reduction in S-19-MLC2a phosphorylation (
P
<0.058). BDP5290 increased binding of PPP1R12C-PP1c and MLC2a >3-fold in HL1 and HEK cells (
P
<0.01). Also, the constitutively activated T560A site on PPP1R12C significantly increased binding of PPP1R12C-MLC2a and PP1c

Conclusion:
In

Pitx2
null+/-

mice, increased expression of PPP1R12C is associated with PP1 holoenzyme targeting to sarcomeric MLC2a, and is associated with reduced S19-MLC2a phosphorylation. Additionally, BDP5290 and PPP1R12C (T560A) enhanced PPP1R12C-PP1c interaction and decreased MLC2a phosphorylation, thereby modelling PP1 activity in AF. Future studies will examine effects of altered
Ppp1r12c
expression on atrial contractility
in vivo
Abstract 360: Effective Three-element Windkessel Model based on Doppler Ultrasound Images for Noninvasive Quantification of Trans-stenotic Pressure Gradient in Aortorenal System
Authors: Khan Monsurul , Sawchuk Alan P , Deb Anurag , Chen Rou , Motaganahalli Raghu L , Fang Xin , Yu Huidan(Whitney)
Published: February 2019

The research objective is to develop and validate a noninvasive method to quantify trans-stenotic pressure gradient in aortorenal system through image-based computational hemodynamics (ICH) for supporting clinical decision of stenting therapy. Renal artery stenosis (RAS) causes renovascular hypertension but the true severity of RAS is hard to be determined thus effective stenting therapy cannot be well planned. We hypothesize that the trans-stenotic pressure gradient truly reflects the severity of RAS. Recently, ICH has merged as a unique tool, using clinical CT and Doppler images as well as the 3-element WindKessel model. As schematized in Fig. 1(a), ICH noninvasively quantify the live hemodynamics, e.g. pressure in Fig. 1(b), in human vessels. One challenge in ICH is to introduce the appropriate values of proximal resistance (r), distal resistance (R), and compliance (C). We have developed a comprehensive algorithm to determine these values by comparing the computational flow-rates at the exits of aortic artery (AA), left renal (LR), and right renal (RR) arteries with the corresponding flow-rates from Doppler images. The image data of the study case consist of CT data, Doppler images at the juxtarenal AA, LR and RR arteries, and invasive pressure measurement at the corresponding arteries from pressure wire transducers. The r, R, and C values determined through the Doppler images are shown in Fig. 19(c). The three non-invasively computed pressure waves are in good agreements with the invasively measured pressure waves (Fig. 1(d)). The validated ICH is a practical tool to noninvasively measure pressure gradients so that physicians can make better decision at treatment of RAS.
Abstract 364: Metabolic Syndrome Induced by High Fat/high Fructose Diet in C57bl/6 Mice Impaired Cardiac Remodeling During Pregnancy
Authors: Yang Yijun , Joo Yunichel , Wang Tao , Borghetti Giulia , Harper Shavonn , Bryan Christopher , Mohsin Sadia , Houser Steven R
Published: February 2019

Rationale:
Due to the change of life style in the past decades, the incidence of metabolic syndrome (MetS) and obesity in women of childbearing age has been increased. Evidences have shown that complications during pregnancy, such as obesity and gestational diabetes, strongly link to future heart diseases. During pregnancy, the heart undergoes a physiological remodeling with mild ventricular dilation and hypertrophy. However, the effects of MetS on cardiac phenotype during pregnancy are not well understood. Moreover, animal models for MetS so far are not well established, which are usually species limited, gender limited and lack of translational significance. Thus, it’s important to develop an animal model which resembles the etiology of MetS in human beings.

Objective:
Establish a model of MetS in female C57BL/6J mice and determine the effects of MetS on cardiac remodeling during pregnancy.

Methods and Results:
To establish a mouse model of MetS, high fat (49% kcal)/high fructose (35.6% kcal) diet are given to C57BL/6J mice at the age of 8-week. Mice fed with normal diet (10% kcal fat) served as control. To better characterize the model within different genders, male mice are also subjected to the high fat/ high fructose (HFHF diet) and control diet. After 12 weeks feeding, mice fed with HFHF diet showed significantly higher body weight and gonadal fat weight compared with control mice in both genders. Also, mice fed with HFHF diet showed impaired glucose tolerance and higher serum total cholesterol level. However, the blood pressure showed no difference between groups in both genders. Overall, the symptoms of obesity, impaired glucose tolerance and dyslipidemia suggest the development of MetS. Female mice after 12-week special diet feeding will be subjected to breeding. Cardiac function is then evaluated by echocardiography before and during pregnancy. Female mice that developed MetS showed impaired cardiac function and pathological remodeling during pregnancy.

Conclusion:
We’ve developed a mouse model of MetS in both female and male C57BL/6J mice strain with 12 weeks feeding of HFHF diet, which resemble the western diet that affects human beings nowadays. Females that have MetS showed impaired cardiac function and pathological remodeling during pregnancy.
Abstract 366: The Role of Mitochondrial Calcium Uniporter Complex in Cardiac Contractility and Function
Authors: Tomar Dhanendra , Shanmughapriya Santhanam , Roy Rajika , Zhang Xueqian , Song Jianliang , Schumacher Sarah M , Madesh Muniswamy , Cheung Joseph Y , Koch Walter J , Rajan Sudarsan
Published: February 2019

Cardiac contractility is regulated by the intracellular Ca
2+
concentration fluxes which are maintained by multiple channels and transporters. Mitochondrial Calcium Uniporter (MCU) is the highly selective Ca
2+
ion channel responsible for the electrophoretic Ca
2+
uptake into the mitochondrial matrix. Earlier studies on the cardiac-specific acute MCU knockout and transgenic dominant-negative MCU mice have demonstrated that mitochondrial Ca
2+
(
m
Ca
2+
) signaling is necessary for cardiac ‘‘fight-or-flight’’ contractile response, however, the role of
m
Ca
2+
in the beat-to-beat regulation of cardiac contractility is highly debated. The MCU channel function is regulated by varied molecular regulatory components as well as ROS mediated modification at the Cys-96 residue of the core MCU protein. Our earlier studies have demonstrated that loss of MCU Regulator 1 (MCUR1) in cardiomyocytes results in the impaired
m
Ca
2+
uptake. To dissect the precise role of MCU in regulating cytosolic Ca
2+
transients associated with excitation-contraction (E-C) coupling and cardiac functions, we have employed the loss-of-function, cardiac-specific MCUR1 knockout mouse and a gain-of-function MCU
C96A
knockin mouse generated by CRISPR/Cas9 approach. Using these novel mouse models, both by in vivo analyses of cardiac physiology and in vitro experiments including single-cell cardiac contractility, calcium transients, and electrophysiology measurements, our study demonstrates that
m
Ca
2+
buffering actively participates in E-C coupling. MCUR1/MCU regulated
m
Ca
2+
buffering in cardiomyocytes, although insignificant under basal condition, becomes critical in stress-induced conditions and actively participates in regulating the
c
Ca
2+
transients during adrenergic stimulation. Our data indicate that MCUR1 ablation offers protection against pressure-overload cardiac hypertrophy and MCU
C96A
KI mice exhibit significantly decreased LV ejection fraction at baseline. In summary, our results provide critical insights into the mechanisms by which the MCU channel contributes in regulating the contractile function of the cardiomyocytes and the role of
m
Ca
2+
in the development and progression of heart failure.
Abstract 378: Evaluation of Patient Specific
MTERF4
Variants in Gene Edited Human iPSC-derived Cardiomyocytes
Authors: Marquez Maribel , McDermott-Roe Chris , Grzybowski Mike , Helbling Daniel , Dimmock David P , Verbsky James W , Geurts Aron M
Published: February 2019

Mitochondrial Transcription Termination Factor 4 (
MTERF4
) is a transcription factor involved in mitochondrial ribosomal biogenesis and was identified as a gene of interest via whole exome sequencing in a pediatric patient with hypertrophic cardiomyopathy (HCM) at Children’s Hospital of Wisconsin. The variants of interest identified in
MTERF4
have not been previously reported in the literature or associated with HCM. Here, we combined precision genome editing via CRISPR/Cas9 system with human iPSC-derived cardiomyocytes (hiPSC-derived CMs) to model the HCM patient-specific
MTERF4
variants. We hypothesize that
MTERF4
variants are contributing to cardiomyocyte impairment, leading to the development of the hypertrophic phenotype. To improve our efficiency of CRISPR/Cas9 precision genome editing in hiPSCs, we are using a co-targeting with selection method.
MTERF4
mutant clones and control hiPSCs (obtained from co-targeted but unmodified by CRISPR/Cas9) were identified by Sanger sequencing and assessed for pluripotency using immunostaining and gene expression.
MTERF4
clones and controls were subjected to our modified Palecek matrix-overlay method for CM differentiation and evaluated at 3-4 weeks for cell size, mitochondrial function, and gene expression. The hiPSC-derived CMs cell size analysis by average pixel area of the
MTERF4
mutant indicated that they are significantly (p=0.0012) larger compared to
MTERF4
control. The assessment of mitochondrial function demonstrated that the maximal mitochondrial respiration may be reduced in
MTERF4
mutant hiPSC-derived CMs compared to control (p=0.056). In known cardiovascular disease genes (NPPA, NPPB, GATA4, TNNT, MYL7, MYH7) associated with HCM, gene expression (qRT-PCR) were elevated. Preliminary data support our hypothesis through changes in CMs size, mitochondrial function, and transcriptional expression for one
MTERF4
mutant clone and control. This approach has generated an
in vitro
tool to evaluate aspects diseases such as cardiomyopathy that can be used for diagnostic screening and therapy. Repeating our initial studies, and adding mechanistic studies, with additional
MTERF4
mutant and control clones will further validate our hypothesis.
Abstract 393: Is High Grade Atrioventricular Block Reversible in Hypothyroidism
Authors: Nwafo Nnamdi A
Published: February 2019

Background:
Hypothyroidism is considered a reversible cause of atrioventricular block (AVB) but rigorous studies to support this are limited. An important consideration in clinical practice is whether pacemaker placement should be deferred in patients with hypothyroidism who present with high degree AV block while awaiting reversal of block. This period of waiting places patients at risk of morbidity and mortality, while extending length of stay and cost of care.

Objective:
To evaluated if high degree AVB in hypothyroidism is in fact reversible, and therefore justify delaying pacemaker(PM) placement.

Method:
Retrospective review of all adult admission with high degree (Mobitz type II or type III) AVB admitted to a Regional hospital over a 10 year period. Out of 271 cases identified, 41 had hypothyroidism (either prior history or newly diagnosed), and were matched by age and sex with 41 patients with AVB but without hypothyroidism. Each group of 41 patients had 25 females (61%) and 16 men (39%). Placement of PM is assumed to indicate lack of reversibility of AVB. Statistic analysis for matched pairs was by McNemar’s test with Yates continuity correction.

Results:
In the hypothyroidism group, 31 of 41 patients(76%) received PM, while 34 of 41 patients (83%) in the group without hypothyroidism received PM. The calculated test statistic was 12.02, and when referred to the Chi-squared distribution table with 1 degree of freedom, gives a probability of < 0.01, which is highly significant. 14 of the 41 patients(34%) with hypothyroidism had thyroid dysfunction based on thyroid hormone levels, with 10(24%) having sub-clinical hypothyroidism, and 4 (10%) having overt disease. The mean duration in days from time of admission to time of PM placement in the hypothyroidism group was 4.35, while that in the group without hypothyroidism was 2.26.

Conclusion:
The finding rejects the null hypothesis that there is no difference in need for PM (and thus reversibility of AVB) in both groups, suggesting AVB may in fact be reversible in hypothyroidism. Larger and more rigorous studies are therefore warranted to definitively determine if AVB in hypothyroidism is reversible, and adopt delaying PM placement while awaiting reversal, as standard of care.
Abstract 394: The Role Of Camkii-dependent Late Sodium Current In Ischemia/reperfusion-associated Arrhythmogenesis
Authors: Greer-Short Amara , Howard Taylor , Satroplus Tony , Patel Nehal , Nassal Drew , Mohler Peter , Hund Thomas
Published: February 2019

Ischemic heart disease is the leading cause of death in the world. Importantly, CaMKII activity is upregulated during ischemia/reperfusion (I/R). However, the mechanisms by which CaMKII induces arrhythmias remains incompletely understood. We tested the hypothesis that CaMKII-dependent upregulation of the late Na+ current (INa,L) via direct phosphorylation of Nav1.5 at Ser571 is responsible for increased arrhythmia susceptibility during I/R. The role of CaMKII-dependent regulation of INa,L was explored using mice harboring Nav channel variants Nav1.5-Ser571E (S571E, phosphomimetic) and Nav1.5-Ser571A (S571A, phosphorylation incompetent). Isolated, perfused WT, S571E and S571A hearts were subjected to global ischemia (15 min) followed by reperfusion (15 min). Immunoblots demonstrated a significant increase in phosphorylated Nav1.5 at Ser571 in WT hearts during I/R compared to baseline (2.2±0.3 vs 1.0±0.3, respectively; P<0.05, n=5). Optical mapping using the voltage-sensitive dye di-4-ANEPPs (4 μM) revealed a significant increase in action potential duration in WT and S571E mice compared to S571A during I/R (WT: 55.2±0.6 ms*, S571E: 70.0±2.3 ms*, S571A: 46.0±2.9 ms; *P<0.05 vs. S571A, n=5/genotype). Furthermore, spontaneous arrhythmia incidence was increased in WT and S571E hearts (WT: 0.60±0.06 events/min*, S571E: 0.81±0.14 events/min*, S571A : 0.23±0.03 events/min; *P<0.05 vs. S571A, n=5/genotype). Interestingly, arrhythmia inducibility using an S1-S2 protocol was also increased in WT and S571E hearts (WT: 80%*, S571E: 100%*, S571A: 0%; *P<0.05 vs. S571A, n=5/genotype). The Na+ channel blocker mexiletine (10 μM) normalized APD and decreased arrhythmia inducibility in WT hearts during I/R (0% arrhythmia incidence in WT hearts treated with mexiletine). These data indicate a central role for CaMKII-dependent phosphorylation of Nav1.5 and augmentation of INa,L in the development of arrhythmogenesis during I/R.
Abstract 402: Exclusion of Major Cell Types in the Murine Heart Enriches for Cardiac Pericytes
Authors: Park Shuin , Langerman Justin , Ardehali Reza
Published: February 2019

Cardiac fibrosis is one of the multitude of cardiac pathologies that occur in the event of acute myocardial injury and heart failure. Formation of scar tissue in the myocardium results in disrupted cardiomyocyte gap junction networks and reduced cardiac contractility. Cardiac pericytes are a heterogeneous population of mural cells that have been attributed to having a pro-fibrotic role in injury. However, due to the lack of specific markers for this cell, there has been difficulty in properly characterizing this population. We aimed to conduct single cell RNA-sequencing on this population to discover novel markers for cardiac pericytes and identify pro-fibrotic subpopulations that promote perivascular fibrosis. To avoid using current markers for cardiac pericytes, which lack specificity, we utilized an exclusion-based approach to enrich for the cardiac pericyte population in an unbiased manner. Using a combination of transgenic mice (αMHC
GFP/+
;Col1a1
GFP/+
) and fluorescence-activated cell sorting (FACS), we excluded all major cardiac cell types, including cardiomyocytes, fibroblasts, endothelial cells, hematopoietic cells, smooth muscle cells, and cardiac neurons. We used Drop-sequencing technology to capture single cells and conducted RNA-sequencing. Cardiomyocytes (isolated by Langendorff), fibroblasts (FACS sorted Col1a1
+
cells), endothelial cells (FACS sorted CD31
+
cells), and smooth muscle cells (isolated from murine aortas) were also subjected to single-cell RNA-sequencing for comparison of gene expression profiles. Cells collected by the exclusion-based approach exhibited high expression of known pericyte markers and low expression of markers for other cell types. The t-SNE plots of all the populations revealed that the majority of cardiac pericytes shared similar gene expression profiles with smooth muscle cells while some cells clustered closer with fibroblasts. The use of an unbiased exclusion-based approach, coupled with single cell RNA-sequencing, allows for the identification of unique subpopulations of cardiac pericytes. Further investigation as to the functional roles of these cells will uncover potential therapeutic targets for treating cardiac fibrosis.
Abstract 409: Ablation of Endothelial Sirt3 Exacerbates Pressure Overload-induced Heart Failure
Authors: He Xiaochen , Zeng Heng , Chen Jian Xiong
Published: February 2019

Cardiovascular disease leads to cardiac dysfunction and eventually heart failure which is the leading cause of death in the United States. Our previous data indicates that global ablation of SIRT3 exacerbates acute injury after myocardial ischemia and that deletion of endothelial SIRT3 (SIRT3 ECKO) leads to development of cardiac dysfunction. In the present study, we demonstrates that loss of SIRT3 in endothelial cells results in upregulation of TIGAR, but downregulation of HIF-1α, HIF-2α, glucose transport GLUT-1, glycolytic enzymes PFK-1, and PFKFB3. To further investigate the role of endothelial SIRT3 on cardiac hypertrophy and heart failure during chronic stress, SIRT3 ECKO and their control SIRT3 LoxP mice were subjected to pressure overload-induced by transverse aortic constriction (TAC) for 7 weeks. Our results demonstrate that the ratio of heart weight to tibia length was increased in both strains of mice, in which SIRT3 ECKO + TAC mice exhibited more severe cardiac hypertrophy. In addition, systolic function was significantly decreased in SIRT3 ECKO + TAC mice compared to SIRT3 LoxP + TAC mice, as evidenced by lower EF and FS at week 7. In contrast, diastolic function decreased after TAC surgery in a similar manner in both strains of mice, despite that pre-existing diastolic dysfunction was found in SIRT3 ECKO mice. Similarly, there was no difference in coronary flow reserve between both strains of mice subjected to TAC. Moreover, pressure overload-induced upregulation of Apelin, Angiopoietin-1, GLUT-4, and ERK phosphorylation were decreased in SIRT3 ECKO + TAC mice, whereas the expression of pro-inflammatory molecules, ICAM-1 and VCAM-1, was significantly increased in SIRT3 ECKO + TAC mice. These results implicate a critical role of SIRT3 in regulating glucose uptake and glycolytic function in endothelial cells. Ablation of endothelial SIRT3 exacerbates pressure overload-induced cardiac hypertrophy and heart failure via impaired glucose metabolism and increased pro-inflammatory molecules.
Abstract 410: Prostaglandin E2 Reduces Carnitine Palmitoyltransferase 2 in Adult Mouse Cardiomyocytes
Authors: Harding Pamela , Bryson Timothy D , Datta Indrani , Wang Yun , Levin Albert M
Published: February 2019

Prostaglandin E2 (PGE2) signals differently through its 4 receptor subtypes (EP1-EP4) to elicit diverse physiologic/pathologic effects. We previously reported that PGE2 via its EP3 receptor reduces cardiac contractility and male mice with cardiomyocyte-specific deletion of the EP4 receptor develop dilated cardiomyopathy. To determine pathway(s) responsible for the phenotype, we performed gene array on left ventricles from these mice followed by Ingenuity Pathway Analysis (IPA) which demonstrated that genes differentiating WT mice and EP4 KO mice were significantly overrepresented in mitochondrial (p=2.51x10
-28
) and oxidative phosphorylation (p=3.16 x10
-30
) pathways. Importantly, carnitine palmitoyltransferase 2 (cpt 2), an enzyme that transports fatty acids across the inner mitochondrial membrane was down-regulated in EP4 KO mice. We therefore hypothesized that PGE2 via EP3 decreases cpt 2 to impair mitochondrial function. To test this hypothesis, we used isolated mouse cardiomyocytes (AVM) from 16-18 week old male C57Bl/6 mice and treated them with either 1 μM of the EP3 agonist sulprostone (sulp), or PGE2. Treatment of AVM with sulp for 24 hrs. decreased cpt 2 levels from a control level of 1.0 to 0.57 ± 0.15, p < 0.05 and treatment with PGE2 decreased cpt 2 levels to 0.74 ± 0.27. Since cpt 2 is regulated by the transcription factor NR4A2 in other cell types, we investigated whether NR4A2 was regulated by PGE2 or sulp. NR4A2 levels were reduced after 4 hr. treatment with either PGE2 or by treatment with sulp; from a control value of 1.0 to 0.70 ± 0.16 and to 0.86 ± 0.01 respectively, p< 0.05 for sulp, n = 3. To associate these changes with mitochondrial function, we measured complex I activity with a spectrophotometric assay. Complex I activity was reduced by 50% (p < 0.05) by 4 hr. treatment with PGE2, from 1.32 ± 0.36 to 0.66 ± 0.08 mOD/min, and this was associated with reduced expression of multiple genes from mitochondrial pathways including sub units of mitochondrial NADH dehydrogenase ubiquinone flavoprotein (Nduf), a component of complex I. In conclusion, these results suggest that increased PGE2 and its EP3 receptor in heart disease may contribute to impaired mitochondrial function and provide yet another link between inflammation and cardiac dysfunction.
Abstract 413: NAD
+
-dependent Pathogenic Mechanisms and Metabolic Interventions for Mitochondrial Disease and its Associated Cardiomyopathy
Authors: Lee Chi Fung , Caudal Arianne , Abell Lauren , Gowda G. A. Nagana , Tian Rong
Published: February 2019

Leigh syndrome (LS) is a congenital mitochondrial disease. Patients with LS develop metabolic abnormality and progressive dysfunctions in brain, skeletal and cardiac muscles, eventually leading to early death. A mouse model of LS with deficiency of a Complex I subunit, Ndufs4-null mice (KO), was used in this study. Depleted NAD
+
level and NAD
+
redox imbalance were observed in KO brain and lead to inhibitions of poly (ADP-ribose) polymerase (PARP)-dependent DNA repair and SOD2 activity via protein hyperacetylation. These inhibitions promoted oxidative stress and increased H2Ax phosphorylation, a DNA damage marker, leading to neurological impairments in KO. To counteract the NAD
+
depletion associated with the pathogenesis of KO, we targeted NAD
+
salvage pathway to increase NAD
+
pool size. Supplementation of nicotinamide mononucleotide (NMN), a NAD
+
precursor, doubled lifespan of KO mice from 55 to 110 days, but did not attenuate neurological impairments, owing to the failure to elevate NAD
+
levels in brain. We next determined the underlying mechanisms by which NMN extended lifespan of KO without impacting brain NAD
+
pool. NMN elevated NAD
+
levels in skeletal and cardiac muscles of KO, resulting in blunted hyperacetylation and attenuated systolic dysfunction. NMN lowered lactate levels in serum and muscles of KO, associated with suppression of hypoxic signaling (lowered HIF1α, LDHA levels). The data suggest a NAD
+
-sensitive mechanism that suppresses glycolysis in KO treated by NMN via hypoxic signaling. While acetylation levels of HIF1α protein were unchanged in KO, acetylation of glutamate dehydrogenase (GDH) was upregulated in KO and suppressed by NMN. Deacetylation of GDH after NMN treatment promoted upregulation of α-ketoglutarate (KG) level in KO, a co-substrate needed for HIF1α degradation. The results suggested that KG may be beneficial to KO. To determine the therapeutic potential of KG, dimethyl-ketoglutarate (DMKG), a cell permeable form of KG, was administered to KO. DMKG extended lifespan and improved systolic function in KO, associated with suppressed hypoxic signaling. Collectively, this study identified NAD
+
-dependent pathogenic mechanisms that may lead to translatable interventions for LS and mitochondrial cardiomyopathy.
Abstract 416: Mnsod Cardiomyocyte-specific Knockout Live Up to Four Months: Role in Heart Failure Development and Progression
Authors: Panchatcharam Manikandan , Bhattarai Susmita , Sharma Sudha , Michael LaBrie Timothy , Tupper Hailey D , Bhuiyan Md. Shenuarin , Chandra Mini , Miriyala Sumitra
Published: February 2019

In our earlier investigation, we have shown that the manganese superoxide dismutase (MnSOD) mimetic, MnTnBuOE-2-PyP
5+
distributes 3-fold more in mitochondria than in cytosol. The exceptional ability of MnTnBuOE-2-PyP5+ to quench O2
•-
parallels its ability to reduce ONOO
–
and CO3
–
. The absence of SOD2 (a gene that encodes MnSOD) knocked out globally is found to be embryonic lethal. We have generated mice that specifically lack MnSOD in cardiomyocytes (Mhy6-SOD2
Δ
). Amazingly, we could prolong the lethality for up to four months in these conditional specific knockout mice (Mhy6-SOD2
Δ
) as compared to global knockout mice. FACS analyses using Mito-Tracker Green indicated that the mass of mitochondria per cell was slightly decreased in the Mhy6-SOD2
Δ
to the wild type. We then examined Oxidative phosphorylation (OXPHOS) levels in Mhy6-SOD2
Δ
v.s. Wild-Type using a Seahorse XF analyzer. The rate of oxygen consumption per cells was significantly lower in Mhy6-SOD2
Δ
cardiomyocytes than that in wild-type. The most noticeable difference in the O
2
consumption was found in the presence of FCCP (H
+
ionophore/uncoupler). Remarkably, while the FCCP treatment increased O
2
consumption in wild-type, the treatment showed no effect on the O
2
consumption in the Mhy6-SOD2
Δ
cardiomyocytes. The result indicated that the low basal OXPHOS activity in Mhy6-SOD2
Δ
was due to unusually low OXPHOS potential. We examined glycolysis in these cells by measuring extracellular acidification (ECAR), and the pattern precisely opposite to that of oxygen consumption rate (OCR) was observed for glycolysis rates between Mhy6-SOD2
Δ
and wild-type. Herein we present the first study ever done to show the existence of cardiac-specific MnSOD knockout mice and the importance of antioxidant enzyme MnSOD that is guarding/protecting the myocardium from cardiac failures.
Abstract 422: Recognition of Pharyngeal Pain as an Angina Equivalent
Authors: Grevious Salih N , Fernandes Marcelo , Rivera Manuel
Published: February 2019

A 64-year-old man presented to his physician with a 5-day history of throat pain and hoarseness. An initial diagnosis of pharyngitis was made, and the patient was sent home with a 7-day course of antibiotic therapy. He returned 6 days after his initial visit with persistent symptoms, which he now described as worsening on exertion such as when lifting weights. He was sent for a neck computed tomography to evaluate cervical soft tissues: No masses, enhancing lesions, lymphadenopathies, or imaging findings suggesting vocal cord paresis were noted. An ECG was ordered by the patient’s primary care physician and demonstrated new lateral T-wave inversions. The patient was also referred to ENT, and via direct laryngoscopy normal true vocal cords and questionable evidence of laryngopharyngeal reflux were visualized. He was then recommended to begin lifestyle modifications and start empiric treatment with omeprazole twice daily.

Due to the ECG findings, an exercise stress test was ordered. During the exercise stress test, He developed throat tightness and the study was converted to a Lexiscan nuclear stress test. The stress test revealed inferior wall ischemia of moderate severity. Consequently, a coronary cath demonstrated quadruple vessel disease with 75% occlusion of left main, 70% stenosis of proximal LAD, 60% stenosis of the first diagonal, and total occlusion of the mid RCA. After undergoing quadruple coronary bypass surgery, the patient experienced a total resolution of his throat pain.

Discussion:
When approaching craniofacial pain elicited with exertion, it is essential for the clinician to consider ischemic heart disease as an etiology. Furthermore, the link of pain with physical activity and, additionally, pain relief with rest should drive the physician to consider cardiac ischemia. Isolated craniofacial pain or sole pharyngeal pain of cardiac origin are infrequent indicators of significant coronary artery disease. In a prospective multicenter study, only 6% of patients with confirmed ischemic cardiomyopathy presented with craniofacial pain as the only complaint.

This case illustrates the importance of a thorough history and physical examination as well as a broad differential diagnosis when evaluating patients with throat pain and hoarseness.