bluuebunny/crossref_metadata_2025 huggingface dataset:
The Mediterranean Diet and Cardiovascular Health
Authors: Martínez-González Miguel A. , Gea Alfredo , Ruiz-Canela Miguel
Published: February 2019

The Mediterranean diet (MedDiet), abundant in minimally processed plant-based foods, rich in monounsaturated fat from olive oil, but lower in saturated fat, meats, and dairy products, seems an ideal nutritional model for cardiovascular health. Methodological aspects of Mediterranean intervention trials, limitations in the quality of some meta-analyses, and other issues may have raised recent controversies. It remains unclear whether such limitations are important enough as to attenuate the postulated cardiovascular benefits of the MedDiet. We aimed to critically review current evidence on the role of the MedDiet in cardiovascular health. We systematically searched observational prospective cohorts and randomized controlled trials which explicitly reported to assess the effect of the MedDiet on hard cardiovascular end points. We critically assessed all the original cohorts and randomized controlled trials included in the 5 most comprehensive meta-analyses published between 2014 and 2018 and additional prospective studies not included in these meta-analyses, totaling 45 reports of prospective studies (including 4 randomized controlled trials and 32 independent observational cohorts). We addressed the existing controversies on methodology and other issues. Some departures from individual randomization in a subsample of the landmark Spanish trial (PREDIMED [Prevención con Dieta Mediterránea]) did not represent any clinically meaningful attenuation in the strength of its findings and the results of PREDIMED were robust in a wide range of sensitivity analyses. The criteria for causality were met and potential sources of controversies did not represent any reason to compromise the main findings of the available observational studies and randomized controlled trials. The available evidence is large, strong, and consistent. Better conformity with the traditional MedDiet is associated with better cardiovascular health outcomes, including clinically meaningful reductions in rates of coronary heart disease, ischemic stroke, and total cardiovascular disease.
Influence of Sex on Relationship Between Total Anatomical and Physiologic Disease Burdens and Their Prognostic Implications in Patients With Coronary Artery Disease
Authors: Kim Chee Hae , Koo Bon‐Kwon , Lee Joo Myung , Shin Eun‐Seok , Park Jonghanne , Choi Ki Hong , Hwang Doyeon , Rhee Tae‐Min , Zhang Jinlong , Choi You‐Jung , Lee Seo‐Young , Choi Jin‐Ho , Doh Joon‐Hyung , Nam Chang‐Wook , Wang Jianan , Chen Shaoliang , Kuramitsu Shoichi , Tanaka Nobuhiro , Matsuo Hitoshi , Akasaka Takashi
Published: February 2019

Background

Total atherosclerosis disease burden is associated with clinical outcomes in patients with coronary artery disease. However, the influence of sex on the relationship between total anatomical and physiologic disease burdens and their prognostic implications have not been well defined.

Methods and Results

A total of 1136 patients who underwent fractional flow reserve (FFR) measurement in all 3 major coronary arteries were included in this study. Anatomical and physiologic total disease burden was assessed by SYNTAX (Synergy Between Percutaneous Coronary Intervention With Taxus and Cardiac Surgery) score, residual SYNTAX score, a total sum of FFR in 3 vessels (3‐vessel FFR), and functional SYNTAX score. The primary end point was major adverse cardiac events, a composite of cardiac death, myocardial infarction, and ischemia‐driven revascularization at 2 years. There were no differences in angiographic diameter stenosis, SYNTAX score, or residual SYNTAX score between women and men. However, both per‐vessel FFR (0.89±0.10 versus 0.87±0.11,
P
<0.001) and 3‐vessel FFR (2.72±0.13 versus 2.69±0.15,
P<
0.001) were higher in women. Multivariable Cox regression analyses showed that total anatomical and physiologic disease burdens were significantly associated with 2‐year major adverse cardiac events, and there was no significant interaction between sex and total disease burden for clinical outcomes.

Conclusions

Despite similar angiographic disease severity, both per‐vessel and per‐patient physiologic disease severity was less in women than in men. There was no influence of sex on prognostic implications of total anatomical and physiologic disease burdens in patients with coronary artery disease.

Clinical Trial Registration

URL:
https://www.clinicaltrials.gov
. Unique identifier: NCT01621438.
Abstract 101: Endothelial Arnt Regulates Microvascular Endothelial Barrier Function in Heart Failure Through a Novel Mmp3 Pathway
Authors: Zheng Mei , Knapp Maura , Andrade Jorge , Cheng Anna , Sladojevic Nikola , Birukov Konstantin G , Zhao Qiong , liao James K. , Wu Rongxue (Rosie)
Published: February 2019

Background:
Increased microvascular permeability and cardiac interstitial edema formation are major contributors to progressive myocyte dysfunction during ischemia/reperfusion (IR) injury. Effective clinical therapies for IR injury are lacking, primarily because the mechanisms linking IR to cardiac microvascular permeability and edema are largely unknown. Results from my current studies indicate that vascular permeability is limited by the expression of Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT), a transcription factor that regulates gene expression by homodimerizing or by heterodimerizing with hypoxia-inducible factor (HIF) 1α, HIF2α, or other cofactors. We hypothesize that endothelial ARNT mediates cardiac remodeling following IR.

Methods and Results:
We have recently generated inducible VE-cadherin-Cre
ERT2
endothelial cell (EC)-specific ARNT knockout mice (ecARNT
-/-
), which, after ecARNT
-/-
induction by oral tamoxifen administration, display increased cardiac endothelial permeability with preserved contractile function of the heart. However, after 24 hours of ischemia-reperfusion, ecARNT
-/-
mice exhibit severe cardiac blood vessel leakage and impaired cardiac function. Following one month of IR, ecARNT
-/-
mice have a 50% mortality rate, and impaired cardiac function, as determined by EF% and FS%. Only 10% mortality is observed in the control group (ARNTf/f mice treated with tamoxifen). Furthermore, data from microarray analysis show that matrix metalloproteinases (MMPs) are significantly upregulated in ARNT deficient mouse cardiac microvascular endothelial cells (CMVECs), with a particularly significant increase in MMP3. MMP3 can cleave most extracellular matrix constituents and its inhibition attenuates the increase in cell permeability observed in ecARNT
-/-
CMVECs. Finally, use of an MMP inhibitor in vivo rescues heart failure induced by IR in ecARNT
-/-
mice. Taken together, these results suggest that ecARNT may mediate heart failure during IR through an MMP3 pathway.

Conclusion:
Endothelial ARNT is a critical regulator of endothelial function and could potentially serve as a therapeutic target for heart failure in response to IR injury.
Abstract 106: Identifying Cardiac Conduction System-specific Enhancers That Link Cell-type Specification With Human Conduction Defects
Authors: Bhattacharyya Samadrita , Bhakta Minoti , Wang Lin , Munshi Nikhil V
Published: February 2019

The Cardiac Conduction System (CCS) is composed of a small set of specialized cardiomyocytes (CMs) that generate and propagate the electrical impulse required for contraction of the cardiac chambers. Genome Wide Association Studies (GWAS) have identified nearly fifty cardiac rhythm associated Single Nucleotide Polymorphisms (SNPs), and recent reports have characterized how selected SNPs impact CCS gene regulatory networks to influence cardiac conduction. However, a global view of CCS regulatory networks remains to be elucidated, and the vast majority of cardiac conduction associated SNPs remain uncharacterized. Such in-depth analyses have been hampered by our inability to specifically interrogate genome accessibility and RNA expression in the CCS. In order to identify genome-wide
cis
regulatory elements that correlate with CCS cell-type specification, we generated and rigorously characterized novel knock-in (KI) Cre driver lines that label the Atrio-Ventricular Node (AVN) and ventricular conduction system (VCS) to add to the limited CCS-specific genetic toolkit. In our study, we also used a previously characterized

Shox2
KI-Cre

driver to label the sinoatrial node (SAN), the primary pacemaker of the heart. Given a variety of issues associated with FACS and Laser Capture Microdissection, we optimized the INTACT (Isolation of Nuclei TAgged in specific Cell Types) method for mammalian heart tissue. To isolate cell-type-specific nuclei, we used Rosa26-Sun1-myc-sfGFP-Tag mice in combination with CCS-specific Cre driver lines. Following isolation and enrichment of CCS-specific nuclei, we performed Omni-ATAC-seq to identify open regions of chromatin (e.g. enhancer elements) and differentially enriched transcription factor motifs for individual CCS cellular sub-types without prior knowledge of cell-type specific regulators. Most cardiac transcriptomes in the literature do not distinguish between individual subtypes. Therefore, using bulk nuclear RNA-Seq, we also generated CCS-subtype specific transcriptomes. We anticipate that our datasets will provide an important starting point for establishing mechanistic links between CCS-specific enhancer elements and disease-associated SNPs identified by GWAS.
Abstract 112: Doxorubicin Alters Cardiac Fibroblast Phenotype
Authors: Mancilla Trevi R , Aune Gregory J
Published: February 2019

The oldest cohort of childhood cancer survivors are entering into their sixth decade. The milestone comes with the knowledge that this population is at a 15 times greater risk of cardiovascular events than the general population. One factor in this is doxorubicin, a chemotherapeutic drug commonly used to treat pediatric cancers. Our study investigates the effect of doxorubicin on proliferating cells in the heart, specifically, cardiac fibroblasts.

Doxorubicin inhibits cardiac fibroblast growth and migration. Cardiac fibroblasts also undergo a shift in gene expression when exposed to doxorubicin. Inflammatory cytokines and their receptors are upregulated, along with extracellular matrix regulatory proteins. ECM structural proteins and adhesion molecules are downregulated compared to control cells. Of note, monocyte chemoattractant protein-1, and its receptor C-C motif chemokine receptor-4, are upregulated over 40-fold compared to control cells.

Doxorubicin has multiple mechanisms of actions that target proliferating and non-proliferating cells differently. Cardiac fibroblasts are proliferative cells with a major role in multiple processes of cardiac homeostasis. This study lays the foundation of the phenotypic changes cardiac fibroblasts undergo when exposed to doxorubicin. These changes may create a microenvironment that primes the heart for adverse cardiac remodeling.
Abstract 114: A Role for Brain-derived Neurotrophic Factor in Duchenne Cardiomyopathy
Authors: Galindo Cristi L , Awgulewitsch Cassandra P , Soslow Jonathan H , Zhong Lin , Carrier Erica J , Singh Anand P , Umbakar Prachi , Zhang Qinkun , Raucci Frank , Markham Larry W , Lal Hind , Hatzopoulos Antonis K
Published: February 2019

Brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin receptor kinase B (TrkB) harbor cardioprotective qualities that may attenuate CM. In a small cohort of DMD patients we found that BDNF blood levels positively correlated with preserved ejection fraction (EF) and less fibrosis, and carriers of the common BDNF single nucleotide polymorphism rs6265 (Val66-Met) tended to exhibit earlier age of onset of fibrosis with subsequent progression to LV dysfunction, compared to non-carriers. We thus hypothesized that BDNF/TrkB signaling delays CM progression in DMD. To test this hypothesis, we administered the TrkB agonist 7,8-dihydroxyflavone (DHF) to mdx
4cv
; mTR
KO
mice in their drinking water for 26 weeks, beginning at 8 weeks of age. Based on echocardiography, DHF treatment preserved cardiac output compared to vehicle-treated controls. Conversely, mdx
4cv
; mTR
KO
mice injected intraperitoneally with the TrkB inhibitor (K252a) displayed bradycardia and PR interval prolongation, as measured by EKG, as well as acute (10-20 min) reduction in percent EF and fractional shortening, as measured by echocardiography. K252a also significantly reduced sarcomere shortening in isolated murine cardiomyocytes. BDNF might also contribute to cardiac repair. Using humanized BDNF polymorphic mice, which have the Val66-Met mutation, we found that post-myocardial infarction cardiac dysfunction was significantly exacerbated in Met/Met mice compared to Val/Val littermate controls. Finally, we evaluated the role of BDNF/TrkB in human cardiomyocytes differentiated from induced pluripotent cells obtained from DMD patients (DMD iPSC-CMs) and found that they highly express BDNF protein in lysates and supernatants. DMD iPSC-CMs also expressed a truncated version of TrkB that lacks the tyrosine kinase domain essential for canonical BDNF/TrkB signaling. Nonetheless, DMD iPSC-CMs responded to treatment with recombinant BDNF, including increased phosphorylation of GSK-3α, mTOR, AMPK, and MSK1/2. Considered together, our results indicate that BDNF plays a protective role in the dystrophic heart and might represent a novel therapeutic candidate for DMD cardiomyopathy.
Abstract 203: Short Telomeres Induce p53 and Autophagy and Modulate Age-Associated Changes in Cardiac Progenitor Cell Fate
Authors: Hariharan Nirmala , Matsumoto Collin , Jiang Yan , Sussman Mark
Published: February 2019

Aging severely limits myocardial repair and regeneration. Delineating the impact of age-associated factors such as short telomeres is critical to enhance the regenerative potential of cardiac progenitor cells (CPCs). We hypothesized that short telomeres activate p53 and induce autophagy to elicit the age-associated change in CPC fate. We compared mouse strains with different telomere lengths for phenotypic characteristics of aging and also isolated CPCs from them. Wild mouse strain Mus musculus castaneus (CAST) possessing short telomeres exhibits early cardiac aging evidenced as diastolic dysfunction, hypertrophy, fibrosis, and senescence, as compared with common lab strains FVB and C57 bearing longer telomeres. CAST CPCs with short telomeres demonstrate altered cell fate as characterized by cell cycle arrest, senescence, basal commitment, and loss of quiescence. Consistent findings of altered cell fate are also evident in old CPCs isolated from aged mice and adult human failing heart CPCs with significantly shorter telomeres. Elongation of telomeres using a modified mRNA for telomerase restores youthful properties to CAST CPCs, evidenced as increased proliferation and quiescence as well as reduced senescence and commitment. Short telomeres induce autophagy in CPCs, a catabolic protein degradation process, as evidenced by reduced p62 and increased accumulation of autophagic puncta. Pharmacological inhibition of autophagosome formation reverses the cell fate to a more youthful phenotype. Mechanistically, cell fate changes induced by short telomeres are partially p53 dependent, as p53 inhibition rescues senescence and commitment observed in CAST CPCs, coincident with attenuation of autophagy. In conclusion, short telomeres activate p53 and autophagy to tip the equilibrium away from quiescence and proliferation toward differentiation and senescence, leading to exhaustion of CPCs. This study provides the mechanistic basis underlying age-associated cell fate changes that will enable identification of molecular strategies to prevent senescence of CPCs.
Abstract 210: Assessment of Cardiac Cell Culture Electrical Excitability Using Strength Duration Relationships
Authors: Sayegh Michael N , Fernandez Natasha , Cho Hee Cheol
Published: February 2019

Understanding the factors that influence the electrical excitability is important for successful engineering and therapeutic translation of cardiac tissue. Electric field excitation is widely utilized in vitro and in vivo to assess the response of cardiomyocytes to electrical excitation. The excitability of cardiac tissue is influenced by a variety of factors, including anatomic location in the heart, disease status, and age. We dissect properties that alter the electrical excitability of ventricular myocardial tissue constructs in vitro in 2D and 3D models of neonatal rat ventricular myocytes, using a custom-made integrated live-cell imaging and pacing setup. We find that for both 2D and 3D constructs, the electrical signal strength correlates negatively with the threshold stimulation duration required for pacing and driving the engineered tissue, and the resulting strength-duration curve is well approximated by the Lapicque-Hill model of electrically excitable membranes. By comparing strength-duration relationships, we find that pacing 2D constructs at 2 Hz requires 1.7 to 1.9 times the stimulus strength compared to pacing at 1 Hz (n=3, p<0.05). Similarly, we find that pacing 3D constructs at 3 Hz requires 1.2 to 1.5 times the stimulus duration compared to pacing at 1 Hz (n=3, p<0.05). By comparing strength-duration relationships of 3D spheroids of diameters ranging from 100 to 210 um (n=7), we find that the signal strength required for capture at 10 ms duration correlates negatively with the spheroid diameter (r =-0.87, p=0.02). When cell culture anisotropy is induced by seeding the cells on abraded plastic substrate, we find that a 1.3 to 1.9 times higher threshold duration of stimulus is required to pace and drive cultures aligned perpendicular to the direction of the electric field compared to those aligned parallel to the direction of the electric field (n=3, p<0.05). Finally, when fibroblasts are added in equal density to the NRVMs, we find a significant increase in threshold duration at lower stimulation strength (n≥2, p<0.05). Therefore, we conclude that 2D and 3D primary NRVM cultures are better electrically entrained when driven at slower frequencies, are large in size and aligned with the electric field, and have low fibroblast content.
Abstract 212: Adrenergic Signaling Genes Are Not All Expressed During Myogenesis in Individual Human iPSC-derived Cardiomyocytes
Authors: Torres Barba David , Vazquez Eliseo T , Sharma Janhavi S , Cortez-Toledo Elizabeth , De la Cruz Cabrera Omar , Lopez Javier E
Published: February 2019

Background:
Adrenergic receptors (AR) in an individual cardiomyocyte (CM) are not uniformly expressed at the single-cell level (Myagmar et al., 2017). The timing and cellular distribution of AR signaling (ARS) genes in individual human iPSC-derived CMs (hiPSC-CMs) have not yet been reported.

Objective:
To map out the transcription program of ARS genes during hiPSC-CM myogenesis using single-cell transcriptomics (scRNA Seq.).

Methods:
132 ARS in CM genes curated by the Kyoto Encyclopedia of Genes and Genomes were studied. The CMs were derived from 2 commercially available hiPSC lines using sequential GSK3 and Wnt signaling inhibition. The CMs were assayed by flow cytometry with sarcomeric myosin heavy chain (MYHC6/7) protein, a biomarker of myogenesis. Baseline cells (iPSCs, n=24) and day 14 (n=45), 30 (n=64), and 60 (n=5) post differentiation cells were sampled for scRNA Seq. using the Fluidigm C1 platform. Data from 10 somatic cell preparations and iPSCs defined the signal specificity and biological noise in the system. The expression patterns were categorized as induced, repressed, or neither based on the median transcript per million for individual cells normalized to iPSCs. A
p
-value of <0.05 by ANOVA was used for significance.

Results:
The CMs had spontaneous contractions by D14 and increased from <1% in iPSCs to 21%, 37% and 59% at D14, D30 and D60 post differentiation (adj. p <0.03) respectively. None of the iPSCs had MYH6/7 transcripts, while >90% of differentiating cells did. MYH6/7 median expression increased 15-fold from D14 to D30 (p<0.0001) and 60-fold to D60 (p<0.06). Of 132 ARS genes, 60 (44%) had no detectable expression out to D60. Of the 72 genes with expression, 18 (25%) were induced in parallel to MYH6/7, while 22 (31%) were repressed. The remaining 32 genes (44%) had no distinctive pattern. From 18 induced genes (14% of all), 8 were sarcomeric genes that preceded expression to most ARS genes without induction (86% of all).

Conclusion:
Single-cell transcriptomes in derived hiPSC-CMs revealed a lack of uniformity between upstream ARS genes and early sarcomeric genes. Defining the timing and cell-to-cell variability of the ARS gene program will be critical to understanding its mechanistic relationship to the sarcomeric gene program in humans.
Abstract 214: A Microrna-hippo Pathway Functions in Cardiac Conduction System Homeostasis and Regeneration
Authors: Wang Jun
Published: February 2019

The cardiac conduction system (CCS) is required for initiating and maintaining regular rhythmic heartbeats. CCS defects can give rise to cardiac arrhythmia, a leading cause for morbidity worldwide. Hippo signaling, a known organ size and growth regulator, has recently been shown to inhibit cardiomyocyte proliferation and renewal. However, the role for Hippo signaling in the CCS is unknown. Here, we found that adult mice display cardiac arrhythmias when Hippo signaling is specifically disrupted in the CCS, indicating an important role for Hippo in maintaining CCS homeostasis. We discovered that
miR-17-92
represses Hippo signaling by directly repressing
Lats2
, a core Hippo pathway component and that Hippo activity is increased in
miR-17-92
deleted hearts. Importantly, disruption of
miR-17-92
in the CCS gives rise to cardiac arrhythmias in adult mice. Notably, specific injury of the CCS causes severe lethal cardiac arrhythmias, whereas inactivation of
Lats
efficiently rescues the phenotype. In addition, based on data from the chromatin immunoprecipitation deep sequencing with an antibody against the Hippo signaling effector Yap, we found that Hippo signaling regulates genes involved in the CCS homeostasis and regeneration. Together, our findings indicate that maintenance of CCS homeostasis requires proper levels of Hippo signaling, and that repression of Hippo signaling protects the hearts from CCS damage and subsequent arrhythmogenesis.
Abstract 217:

TBX5
Clover2
/NKX2-5
TagRFP

hiPSCs for Simultaneously Isolating Lineage-Specific Cardiovascular Cells
Authors: Zhang Joe Z , Termglinchan Vittavat , Itzhaki Ilanit , Wang Vicky , Shao Ningyi , Chang Alex , Ma Ning , Liu Chun , Kitani Tomoya , Sayed Nazish , Wu Haodi , Lam Chi , Blau Helen , Wu Joseph
Published: February 2019

In vertebrate, the heart is the first organ to develop, comprises multiple highly specialized cell types arising from different lineages. Early animal studies have identified two cardiac lineages: the first and second heart fields which contribute to the four-chamber heart formation. These cardiac progenitor cells develop into different parts of the heart and any perturbation of them frequently leads to congenital heart disease. However, given the difficulty in studying human embryology, current understanding of the hierarchy of cardiac lineages is primarily based on mouse models. The recent breakthrough in generation of human induced pluripotent stem cells (hiPSCs) from somatic cells and de novo differentiation into cardiomyocytes offers an unprecedented opportunity to investigate the genetic control of the cellular process of the heart development and investigate cardiac lineages under human genetic background. Despite the prominent advantages of hiPSCs in cardiac research, heterogeneity of hiPSC-derived cardiomyocytes (hiPSC-CMs) significantly impede their applications in regenerative medicine, disease modeling, and personalized drug testing. Using CRISPR/Cas9 genome-editing technology, I have generated a dual-color iPSC reporter line by targeting fluorescent protein sequences to two transcription factors (TBX5 and NKX2.5) which play a critical role in cardiac differentiation. Using this novel reporter line, I was able to simultaneously isolate four distinct subpopulations which exhibited different gene expression patterns. Continuous culture after isolation, it was found that TBX5
+
NKX2.5
+
subpopulation primarily differentiated into ventricular myocytes (90% purity), whereas TBX5
-
NKX2.5
+
subpopulation differentiated into atrial myocytes (80% purity). Moreover the TBX5
+
NKX2.5
-
subpopulation exhibited the epicardial lineage characteristics and contributed to nodal myocytes (80% purity). However, TBX5
-
NKX2.5
-
subpopulation developed into endothelial cells. In conclusion, using this reporter line, different lineage subpopulations can be identified and isolated from a heterogeneous pool of cardiac cells. These purified myocyte subtypes hold the potential for precise disease modeling and drug testing.
Abstract 218: Biofunctional Tissue-engineered Vascular Grafts Induce Vascular Regeneration
Authors: Pan Yiwa , Wu Yifan , Qin Kang , Wei Yongzhen , Kong Deling , Zhao Qiang
Published: February 2019

Vascular graft has proven to be an effective strategy for the treatment of cardiovascular diseases. However grafts made solely from synthetic polymers cannot satisfy the clinic requirements in terms of low long-term patency, because they only provide a simple structural or physical replacement without the biological functions. In order to address the issue, we aim to fabricate vascular grafts which could mimic native extracellular matrix (ECM) in terms of both physiological structure and bio-functions. Different types of strategies and techniques have been explored to realize this purpose, including graft structure optimization, surface/interface functionalization, and controlled delivery of vascoactive molecules.Up to now, we have successfully fabricated a kind of vascular grafts with dual surface functions of anti-thrombogenicity and rapid endothelialization. Through structural optimization, the problem of limited cellularization in vascular grafts has been effectively resolved, and thus the vascular regeneration as well as the homeostasis has been improved. Furthermore, incorporation of some specific cytokines (such as Dickkopf-3) into the micro/nano- fibrous vascular grafts could enhance the migration vascular progenitor cells (VPCs) into the grafts and further induce the differentiation of them, contributing to endothelialisation and intima/media formation. In addition, vascular grafts loaded with exosomes derived from mesenchymal stem cells have been successfully prepared, and delivered exosomes exhibited immunomodulation function which is very important for the vascular regeneration and inhibition of calcification.
Abstract 231: Identification of a
Titin
Enhancer using hiPSC-Derived Cardiomyocytes and CRISPR/Cas9 Genome Editing
Authors: Neyazi Meraj , Schmid Manuel , Sharma Arun , Wasson Lauren K , Gorham Joshua M , DePalma Steven , Kim Seong Won , Seidman Jonathan G , Seidman Christine E
Published: February 2019

Dilated cardiomyopathy (DCM), a disorder that occurs in 1:250 individuals, is associated with rates of mortality of 20% within 5 years of diagnosis and is a leading cause for heart failure and cardiac transplantation. Mutations in the massive sarcomere protein titin (encoded by
TTN
) are the most common genetic cause of DCM, occurring in 10-20% of cases. As dominant DCM mutations truncate titin (TTNtv) and result in haploinsufficiency, we predict that strategies to increase the expression of the wild type (WT)
TTN
allele might attenuate the damaging effects of TTNtv. We used bioinformatic analyses to identify a putative
TTN
enhancer within intron 1. To confirm its function, we deleted 658 bp from intron 1 that encompasses the putative
TTN
enhancer in human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 genome editing. We used qPCR and RNA sequencing of RNA harvested from hiPSC-derived cardiomyocytes (hiPSC-CMs) and demonstrated that a homozygous deletion in this region leads to decreased
TTN
gene expression compared to the WT control (0.344 fold change, p < 0.001), and also to decreased expression of other sarcomeric genes such as
TNNT2
(0.074 fold change, p < 0.001),
MYH6
(0.18 fold change, p < 0.001),
MYH7
(0.008 fold change, p < 0.001), and
ACTN2
(0.118 fold change, p < 0.001). The expression of transcription factors (TF) that have binding sites in this region is also affected, such as
MEIS2
(0.4 fold change, p < 0.001) and
KLF6
(0.33 fold change, p < 0.001), both of which are known to be involved in cardiogenesis. These TF may act as a link between the deletion in
TTN
Intron 1 and a decreased transcription of other cardiac-relevant genes. We also utilized Assay for Transposase-Accessible Chromatin Sequencing (ATAC-Seq) on WT hiPSC-CMs to identify open regions of chromatin that are accessible for TF binding. This provided additional evidence that this 658 bp region in
TTN
intron 1 has enhancer activity. Ongoing studies are aiming to refine the
TTN
Intron 1 enhancer by further studies using CRISPR/Cas9, CRISPR droplet sequencing (CROP-Seq), and luciferase-based enhancer activity assays. If confirmed, we expect that increasing the activity of this 658 bp region with small molecules may provide a novel therapeutic target for DCM caused by TTNtv.
Abstract 232: NFkB1 Pathway is a Major Dysregulated Biological Pathway in Cardiac Myocytes in Myocyte-Specific Lamin A/C Deficient Mice
Authors: Auguste Gaelle , Gurha Priyatansh , Lombardi Raffaella , Marian Ali J
Published: February 2019

Lamin A/C (LMNA) is a major component of the nuclear membrane, involved in regulation of gene expression. Mutations in the
LMNA
gene cause a heterogeneous group of diseases, collectively referred to as laminopathies. Cardiac involvement is one of the most prominent feature of the laminopathies, presenting with dilated cardiomyopathy (DCM), conduction defects, and sudden death. Molecular mechanisms involved in the pathogenesis of cardiac phenotypes in laminopathies are not well known.

To gain insights into molecular pathogenesis of DCM caused by LMNA deficiency,
Lmna
gene was deleted in cardiac myocytes (
Myh6-Cre:Lmna
Flox
). Homozygous (
Myh6-Cre:Lmna
F/F
) had a normal cardiac function and histology at 2 weeks of age. However, the mice showed progressive cardiac dilatation and dysfunction, interstitial fibrosis, increased apoptosis, and died by 4 weeks of age. To identify transcriptomic changes that precede cardiac dysfunction, cardiomyocytes were isolated from 2-week old wild type (WT) and
Myh6-Cre:Lmna
F/F
mice and analyzed by RNA-Seq. A total of 1,420 genes were differentially expressed between WT and
Myh6-Cre:Lmna
F/F
. Pathways analysis of differentially expressed genes (DEGs) showed enrichment of inflammatory, epithelial to mesenchymal transition, and cell death pathways, whereas mitochondrial oxidative function, adipogenesis, and fatty acid metabolism pathways were depressed in
Myh6-Cre:Lmna
F/F
. Consistently, DEGs were enriched for the binding motif of RELA, the main transcription factor of the NFκB1 pathway. Activation of the NFκB1 pathway was validated by western blot showing increased nuclear localization of RELA. To determine therapeutic benefits of targeting the NFκB1 pathway, mice are being treated a specific inhibitor of NFκB (IKK-16). Effects on survival, cardiac function, apoptosis, and fibrosis are being analyzed.

In conclusion, LMNA deficiency results in dysregulation of a large number genes, including NFκB1 pathway target genes that precede the onset of cardiac dysfunction and fibrosis. The findings point to the prominent role of inflammation in the development of cardiac dysfunction in myocyte-specific LMNA deficiency and identify the NFκB1 pathway as a therapeutic target in DCM caused by LMNA deficiency.
Abstract 234: Nrf2-Dependent Transcriptional and Post-Transcriptional Regulatory Responses in Reductive Stress Myocardium
Authors: Quiles Justin M , Pepin Mark E , Cinghu Senthilkumar , Challa Anil Kumar , Wende Adam R , Crossman David , Namakkal-Soorappan Rajasekaran
Published: February 2019

Background:
Transient activation of nuclear factor, erythroid 2 like 2 (Nfe2l2/Nrf2), a master regulator of antioxidant transcription, protects from oxidative insult. However, sustained Nrf2 signaling causes proteotoxic reductive stress (RS). Using Nrf2
-
/
-
mice, we recently uncovered several cardiac microRNAs (miRNAs) potentially expressed in an Nrf2-dependent manner. Here, we utilized cardiac-specific constitutively active Nrf2 transgenic (caNrf2-Tg) mice to elucidate necessary and sufficient miRNAs, and hypothesized that miRNA dysregulation underlies RS.

Methods:
Next-generation RNA sequencing (RNAseq) was used to compare mRNA and miRNA transcriptomes of 6-month low (TgL) and high (TgH) transgene-expressing caNrf2-Tg mice to non-transgenic (NTg) controls (n=3-4/group). Differentially expressed genes (DEGs) were run through Ingenuity Pathway Analysis (IPA). Real-time qPCR validated RNAseq results (n=4-6/group).
In silico
target prediction correlated miRNA changes with corresponding mRNA levels.

Results:
Principal component and hierarchal clustering analyses revealed distinct TgL and TgH transcriptomes. Relative to NTg, TgL mice exhibited 246 DEGs, 214 of which were consistent in TgH hearts. Strikingly, TgH mice displayed 1031 DEGs. As expected, IPA of DEGs indicated enhanced free radical scavenging; however, caNrf2 expression also dose-dependently enriched hypertrophic signaling, protein ubiquitination and the unfolded protein response. While 16 miRNAs were significantly altered in TgL hearts, a total of 101 miRNAs were detected TgH mice, several of which were predicted to account for mRNA changes in RS pathways. Notably, miR-671-3p and miR-455-5p, two miRNAs decreased in Nrf2
-
/
-
mice, were reciprocally increased in caNrf2-Tg mice suggesting that Nrf2 is necessary and sufficient for their function in the heart.

Conclusion:
While RS underlies human mutant protein aggregation cardiomyopathy, molecular determinants of pathogenesis downstream of Nrf2 remain unknown. Here, we identified key transcriptional responses and pathways associated with RS. These results link post-transcriptional regulation with redox imbalances in the heart, and highlight novel miRNAs for future mechanistic studies.
Abstract 238: Ube2v1 Promotes Mutant αB-crystallin-induced Protein Aggregation
Authors: Xu Na , McLendon Patrick , Gulick James , Osinska Hanna , Robbins Jeffrey
Published: February 2019

Background:
Compromised protein quality control causes the accumulation of misfolded proteins and intracellular aggregates, contributing to cardiac disease and heart failure. An unbiased genome-wide short hairpin RNA screen was performed to seek novel genes that could prevent or clear proteotoxic aggregates in a model of desmin-related cardiomyopathy, caused by the R120G mutation of αB-crystallin (CryAB
R120G
). Among a total of 236 hits, we validated a candidate gene named ubiquitin-conjugating E2 enzyme variant 1 (ube2v1). Ube2v1 expression is detectable in the heart and the literature states that it must bind to ube2n to form a heterodimer. The heterodimer then catalyzes the synthesis of Lys63-linked poly-ubiquitin chains, participating in DNA repair and the damage response. Ube2v1 protein levels were increased in CryAB
R120G
transgenic hearts. However, the function of ube2v1 in cardiac proteotoxicity is completely unknown. To assess whether and how ube2v1 impacts cardiac protein aggregation catalyzed by cardiomyocyte-specific expression of mutated CryAB.

Methods and Results:
Neonatal rat ventricular cardiomyocytes (NRVMs) were infected with adenoviruses expressing either wild-type CryAB or CryAB
R120G
. Subsequently, loss- and gain-of-function experiments were performed using ube2v1 siRNA or recombinant adenovirus mediated ube2v1 overexpression. Knockdown of ube2v1 decreased aggregate accumulation and attenuated cytotoxicity caused by CryAB
R120G
expression, while overexpressing ube2v1 enhanced aggregate formation. Ubiquitin proteasome system (UPS) function was analyzed using a UPS reporter protein consisting of a short degron, CL1, fused to the COOH-terminus of green fluorescent protein (GFPu). Knockdown of ube2v1 improved proteasomal function and promoted the degradation of insoluble ubiquitinated proteins in CryAB
R120G
cardiomyocytes, but did not alter autophagic flux.

Conclusions:
Inhibition of ube2v1 improves CryAB
R120G
-induced aggregate formation through enhancing proteasome activity. It provides a novel therapeutic target to treat cardiac proteinopathies.
Abstract 242: Drug Repurpose Using Human Stem Cell Models of Cardiac Arrhythmias
Authors: Yazawa Masayuki , Song LouJin , Morikawa Kumi
Published: February 2019

For translational approach toward drug development, we has investigated the molecular mechanisms underlying various inherited cardiac diseases and identified lead compounds to test the derivatives in iPSC and mouse models. In this study, we focus on a lethal disease, Timothy syndrome (TS), which is characterized as a developmental disorder containing multiple organ dysfunctions including autism and cardiac arrhythmia such as long QT syndrome (type 8, LQTS8) and ventricular tachycardia. TS is caused by
de novo
gain-of-function mutations in a voltage-gated calcium channel Ca
V
1.2, which is essential for cardiac excitation-contraction coupling. Previous reports demonstrated that beta blockers and calcium channel blockers were not sufficient to prevent the lethal arrhythmias in TS patients. In fact, some TS infants died shortly after birth. Therefore, new drug development or FDA drug repurposing is really crucial for the patients. We generated and characterized human iPSC and mouse models of TS. Also, we have used familial LQTS8 and LQTS1 iPSC lines to generate more LQTS iPSC models. Interestingly, our chemical tests identified that an FDA-approved drug, fluvoxamine, rescued cardiac phenotypes in TS (de novo LQTS8), familial LQTS8 and LQTS1 iPSC models. Fluvoxamine has been clinically used for depression and anxiety disorder as a selective serotonin reuptake inhibitor (SSRI). Fluvoxamine is also known to activate Sigma 1 receptor (Sigma-1R). To explore the mechanisms underlying the beneficial effects of fluvoxamine, we tested several Sigma-1R agonists (ex. PRE-084 as a non-SSRI) and antagonists (ex. NE-100) and other SSRIs which do not have any effect on Sigma-1R function (ex. peroxetine) on TS iPSC-derived cardiomyocytes. The results demonstrate that Sigma-1R activation using PRE-084 could rescue the phenotypes in TS models. The molecular mechanism in which Sigma-1R activation rescues cardiac arrhythmic phenotypes is novel. Furthermore, we share our next strategy of chemical synthesis for the development of novel Sigma-1R agonists, which are less permeable to the brain through the blood-brain barrier (BBB), as new therapeutic compounds for LQTS.
Abstract 248: β2 Adrenergic Receptor Deletion Alters Leukocyte Subtype Inter Organ Localization and Survival Following Myocardial Infarction
Authors: Bajpai Anamika
Published: February 2019

Leukocytes are rapidly recruited to the heart following myocardial ischemia (MI) where they regulate a wide variety of cardiac remodeling responses including cardiomyocytes survival and infarct stabilization. We recently reported that mice lacking β2AR expression in cells of hematopoietic origin displayed 100% mortality via cardiac rupture associated with decreased post-MI leukocyte recruitment. Beyond a deficiency in recruitment, we hypothesized that β2AR expression in leukocytes is necessary to promote their survival in the milieu of ischemic cardiac tissue. To assess this, we generated chimeric wild-type (WT) mice harboring either WT or β2ARKO bone marrow via transplantation (BMT), subjected them to sham or MI surgery and assessed leukocyte sub-type localization and survival at different time points. At 1d post-MI there was an increase in TUNEL staining of both cardiomyocytes and CD45
+
cells in β2ARKO BMT versus WT BMT hearts. To begin to identify the leukocyte populations that account for this increased death response, we performed flow cytometry analysis of cells isolated from bone marrow, blood, spleen and heart. Congruent with our previous findings, there was less accumulation of CD45
+
and CCR2
+
leukocytes, Ly6G
+
neutrophils and Ly6C
+
monocytes in the heart and peripheral blood 4d post-MI in β2ARKO BMT mice, with reciprocally increased levels of these populations in the spleen. Annexin V staining confirmed that despite fewer CD45
+
cells being recruited to the heart post-MI, a proportionally higher number of these cells were apoptotic. Further, we detected a generalized enhancement in the apoptosis of CD45
+
leukocytes, monocytes and neutrophils in particular, within the bone marrow and spleen. These data suggest that, in addition to decreasing the recruitment of leukocytes to the heart following MI, the absence of β2AR impairs their survival, exacerbating the loss of cardiomyocytes and cardiac repair.
Abstract 260: Adverse Transverse-Tubule Remodeling in a Rat Model of Heart Failure is Attenuated by Triiodothyronine (T3) Treatment
Authors: An Shimin , Kuruvilla Jes , Muncan Adam , Gilani Nimra , Huang Yuan , Zhang Youhua , Gerdes Anthony M , Ojamaa Kaie
Published: February 2019

Pre-clinical animal studies have shown that triiodothyronine (T3) replacement therapy improves cardiac contractile function after myocardial infarction (MI). We hypothesized that T3 treatment could prevent adverse post-MI cardiomyocyte remodeling by maintaining T-tubule (TT) organization, thus improving contractility.

Methods:
Coronary artery ligation (MI) or sham surgeries were performed on female Sprague-Dawley rats (aged 12 wks), followed by treatment with T3 (5ug/kg/d) or vehicle in drinking water for 16 wks (10/gp). After echocardiographic/hemodynamic analyses, ventricular myocytes were isolated by collagenase digestion and plated on laminin-coated glass coverslips for TT analysis or analyzed for contractility using IonOptix’s edge-detection method. Live cell staining with wheat germ agglutinin (WGA)-488 or di-8-ANNEPS labeled sarcolemma and TT, and multiple z-stack images were captured by scanning confocal microscopy and TT organization measured using TTorg program. Ventricular RNA was analyzed by qPCR.

Results:
Echocardiography and
in vivo
hemodynamic measurements showed significantly improved systolic and diastolic function in T3- vs vehicle-treated MI rats. IonOpix analysis of isolated ventricular myocytes showed significant dysfunction in measures of relaxation in MI myocytes compared to sham, and improvements with T3: max relaxation velocity (Vmax), 2.98±1.41 vs 1.59±0.33, p<0.05; time to Vmax (sec), 0.23±0.04 vs 0.31±0.02, p<0.001, MI+T3 vs MI). Similarly, time to peak contraction was shortened by T3 treatment (0.16±0.02 vs 0.20±0.01 sec., MI+T3 vs MI; p<0.01. TT power measured by TTorg analysis of WGA-488 or di-8-ANNEPS labeled myocytes showed significant disorganization of the TT system in MI myocytes that was normalized by T3-treatment (TTpower = 82±7 sham, 74±8 MI and 79±6 MI+T3; sham vs MI p<0.0001; MI vs MI+T3, p<0.0001). qPCR results indicated that reduced expression of Bin1, Jph2, RyR2 and LTCC in the MI myocardium was normalized by T3. In conclusion, T3 treatment prevented TT disorganization in the post-MI cardiomyocyte, normalized contractile and relaxation parameters and improved cardiac output, thus supporting the therapeutic potential of T3 in heart failure.
Abstract 266: Sex Differences in Survival and Pathological Responses of Hearts, Cardiac Myocytes and Fibroblasts
Authors: Peter Angela K , Christensen Tova L , Walker Cierra J , Trexler Christa L , Ozeroff Christopher D , Lugo Kimberly R , Perry Amy R , Anseth Kristi S , Leinwand Leslie A
Published: February 2019

Introduction:
Biological sex is an important modifier of cardiovascular disease (CVD). However, women and female animals have traditionally been excluded from clinical trials and most preclinical research studies, resulting in therapeutics that are not as effective, or with different side effects, in women relative to men.

Methods and Results:
We show here that expression of α- and β-adrenergic receptors, cardiac stiffness, myofibroblast proliferation, and expression of fibrotic markers are sexually dimorphic at baseline. To determine whether pathogenesis was different between the sexes, the β-adrenergic agonist, isoproterenol (ISO), was administered to rats. In response to 7-days of ISO treatment, female animals were more likely to survive than their male counterparts (percent survival: 44% in males versus 77% in females). Both sexes developed significant cardiac hypertrophy, activation of cardiac fetal genes (ANF and β-MHC) compared to vehicle controls. Cardiac myocytes from ISO treated animals took less time to reach peak shortening and displayed increased departure velocity; however, only male ISO treated cells exhibited reduced relaxation times and increased return velocities. Importantly, ISO treated animals developed significant fibrosis prompting us to analyze isolated cardiac fibroblasts at baseline and in response to ISO. We observed significantly increased expression of α-SMA and Col1a, two indicators of myofibroblast activation, only in fibroblasts isolated from ISO treated males. Both ISO treated male and female fibroblasts showed significant increases in periostin and decreases in TCF21, indicative of increases in myofibroblasts and decreases in quiescent fibroblasts, respectively. In addition, both sexes revealed increased fibroblast proliferation
in vivo
, as measured by EdU incorporation, in response to ISO.

Conclusions:
Chronic
in vivo
β-adrenergic receptor stimulation revealed dramatic sex differences in mortality, cardiac hypertrophy, myocyte contractility, and cardiac fetal gene expression. Increases in myofibroblast differentiation and fibroblast proliferation should lead to a robust fibrotic response in both male and female ISO treated animals.
Abstract 273: Autophagy Impairment is Associated With Defects in Mitochondrial Bioenergetics in Doxorubicin Cardiomyopathy
Authors: Abdullah Chowdhury S , Alam Shafiul , Aishwarya Richa , Miriyala Sumitra , Panchatcharam Manikandan , Pattillo Christopher B , Orr Anthony W , Sadoshima Junichi , Hill Joseph A , Bhuiyan Md. Shenuarin
Published: February 2019

Introduction:
Doxorubicin (Dox) associated cardiotoxicity limits its use in clinical practice. Despite extensive studies it is still unclear if Dox-induces autophagy activation or inhibition. Also a role for autophagy dysregulation contributing to mitochondrial dysfunction in Dox-cardiotoxicity is unknown.

Hypothesis:
Dox-mediated impairment of the autophagy degradation process contributes to mitochondrial dysfunction during the development of cardiomyopathy.

Methods and results:
Time course study of acute Dox treatment (20mg/kg, i.p.) showed accumulation of LC3-II in heart lysates at day five. We also confirmed Dox-induced autophagosome accumulation
in vivo
by using GFP-LC3 and mRFP-GFP-LC3 transgenic (Tg) mice. The autophagy flux assay confirmed that Dox-induced accumulation of autophagosome is due to blockade of the lysosomal degradation process. Mitochondria isolated from acute Dox-treated hearts (at day 5) showed significant suppression of the oxygen consumption rate (OCR). Chronic Dox-cardiotoxicity induced by four weekly consecutive i.p. Injections of Dox (5 mg/kg each) also exhibited time-dependent accumulation of LC3 puncta in GFP-LC3 Tg hearts. LC3-II levels in both whole cell and mitochondrial fraction were increased in Dox-treated hearts. The mitochondria in chronic dox-treated hearts (12 weeks) also showed significant suppression of OCR. To test whether autophagy induction/activation can rescue mitochondrial dysfunction in Dox-cardiotoxicity, we subjected cardiac specific αMHC-Atg7 Tg mice to acute Dox treatment. Mitochondria from Dox-treated αMHC-Atg7 Tg mice (at day 5) exhibited significant preservation of mitochondrial respiratory parameters including basal, ATP linked and maximal respiration.

Conclusions:
Our study confirmed that both acute and chronic Dox-cardiotoxicity is associated with inhibition of the autophagic degradation process associated with suppression of mitochondrial respiration. Notably, autophagy activation/induction preserves mitochondrial function in Dox-treated hearts, underscoring the importance of autophagy in maintaining physiologically normal mitochondrial respiration in hearts under genotoxic stress.