bluuebunny/crossref_metadata_2025 huggingface dataset:
Blood Pressure and Heart Rate Variability in Preschool Children Exposed to Smokeless Tobacco in Fetal Life
Authors: Nordenstam Felicia , Norman Mikael , Wickström Ronny
Published: October 2019

Background

Maternal smoking during pregnancy has been associated with higher blood pressure and autonomic imbalance in the offspring. However, it has been difficult to determine the selective prenatal and postnatal contributions as children frequently have been exposed to smoking both before and after birth. The specific role of nicotine is also unclear. We aimed to determine whether exclusive prenatal exposure to nicotine from maternal use of smokeless tobacco (Swedish snus) in pregnancy was associated with blood pressure and autonomic heart rate control in their children.

Methods and Results

We measured oscillometric blood pressures in forty 5‐ to 6‐year‐old children with snus exposure in fetal life (n=21) and in tobacco‐free controls (n=19). Taking the child′s age and height into account, snus‐exposed children had 4.2 (95%
CI
, 0.2–8.1) mm Hg higher systolic blood pressure than controls (
P
=0.038). The corresponding sex‐, age‐, and height‐standardized systolic blood pressure centiles were 61 and 46 (95%
CI
of the difference, 2–28) (
P
=0.029). Heart rate variability was tested in 30 of the children. The spectral heart rate variability variable low‐frequency/high‐frequency ratio was higher (median, 0.69; interquartile range, 0.45–1.21) in snus‐exposed children than in controls (median, 0.21; interquartile range, 0.32–0.57;
P
=0.034).

Conclusions

Prenatal snus exposure was associated with higher systolic blood pressure and altered heart rate variability at 6 years of age. These findings may indicate adverse prenatal programming of nicotine, but implications for cardiovascular health in later life remain to be studied. Meanwhile, women should be recommended to abstain from all types of tobacco and nicotine products during pregnancy.
Novel Screening Method Identifies PI3Kα, mTOR, and IGF1R as Key Kinases Regulating Cardiomyocyte Survival
Authors: Elmadani Manar , Khan Suleiman , Tenhunen Olli , Magga Johanna , Aittokallio Tero , Wennerberg Krister , Kerkelä Risto
Published: October 2019

Background

Small molecule kinase inhibitors (
KI
s) are a class of agents currently used for treatment of various cancers. Unfortunately, treatment of cancer patients with some of the
KI
s is associated with cardiotoxicity, and there is an unmet need for methods to predict their cardiotoxicity. Here, we utilized a novel computational method to identify protein kinases crucial for cardiomyocyte viability.

Methods and Results

One hundred forty
KI
s were screened for their toxicity in cultured neonatal cardiomyocytes. The kinase targets of
KI
s were determined based on integrated data from binding assays. The key kinases mediating the toxicity of
KI
s to cardiomyocytes were identified by using a novel machine learning method for target deconvolution that combines the information from the toxicity screen and from the kinase profiling assays. The top kinases identified by the model were phosphoinositide 3‐kinase catalytic subunit alpha, mammalian target of rapamycin, and insulin‐like growth factor 1 receptor. Knockdown of the individual kinases in cardiomyocytes confirmed their role in regulating cardiomyocyte viability.

Conclusions

Combining the data from analysis of
KI
toxicity on cardiomyocytes and
KI
target profiling provides a novel method to predict cardiomyocyte toxicity of
KI
s.
Abstract 103: Single Cell Transcriptome Analyses Reveal Novel Targets for Therapeutic Neovascularisation by Resident Endothelial Cells in the Heart
Authors: Li Ziwen , Solomonidis Emmanouil G , Duffin Rodger , Dobie Ross , Mahalhaes Marlene S , Henderson Beth E , Louwe Pieter A , D'Amico Gabriela , Hodivala-Dilke Kairbaan M , Shah Ajay M , Mills Nicholas L , Simons Benjamin D , Gray Gillian A , Henderson Neil C , Baker Andrew H , Brittan Mairi
Published: October 2019

Aims:
A better understanding of the pathways that regulate regeneration of the coronary vasculature is important for future strategies to treat patients with heart disease. We investigated (i) the clonal dynamics of endothelial cells (EC) associated with neovascularization in the ischemic border region (ii) transcriptional signatures of regenerative EC in the ischemic heart using single cell RNA-sequencing (iii) the functional relevance of selected targets.

Methods:
MI was induced in ‘EC-Confetti’ mice by coronary artery ligation. EC clonal proliferation was quantified or hearts dissociated for scRNAseq. Immunofluorescence staining for targets identified by scRNAseq was performed on cardiac tissue from patients with ischemic heart disease. EC proliferation was assessed
in vitro
following siRNA gene silencing.

Results:
EC-Confetti mice express YFP, RFP, GFP, or CFP specifically in EC. Fluorophores are inherited by EC progeny following proliferation, allowing quantitative clonal analysis. Clonal proliferation was significantly increased in the infarct border at 7 days post-MI compared to healthy hearts (
P
<0.0001). Ten transcriptionally discrete EC clusters were defined following scRNAseq with 3 clusters predominantly composed of cells from the MI group, indicating their gene expression profiles may be relevant to neovasculogenic pathways. We selected plasmalemma vesicle associated protein (Plvap) for further study and confirmed EC-specific increased Plvap expression in ischemic border regions of human (
P
=0.002) and mouse (
P
=0.002) hearts, compared to healthy myocardium. siRNA gene silencing of Plvap significantly inhibited EC proliferation (
P
= 0.0038), strong evidence that Plvap can directly modulate EC function.

Conclusions:
Generation of new blood vessels following ischemic injury in the mouse heart is predominantly mediated by clonal proliferation of resident EC. We present a gene expression atlas of resident cardiac EC, and the transcriptional hierarchy underpinning endogenous vascular repair following MI. This resource identifies novel targets, including Plvap, that may augment myocardial perfusion post-MI, and inform future design of strategies aimed at promoting vascular perfusion in ischemic heart disease.
Abstract 107: Role of p53 Acetylation in Mediating Myocardial Angiogenesis and Diabetic Cardiomyopathy
Authors: zeng Heng , Chen Jian Xiong
Published: October 2019

The prevalence of diabetic heart failure (HF) is increasing at an alarming rate, it is therefore urgent to identify potential therapeutic targets. Accumulating evidence demonstrates the important role of acetylation of mitochondrial proteins in HF and diabetes. In this study, we investigated the role of p53 acetylation in diabetic impairment of myocardial angiogenesis and determine whether inhibition of p53 acetylation can improve cardiac function in the setting of diabetes. The acetylation of p53 was significantly increased in hearts of diabetic db/db mice, hyperglycemic STZ mice, and high fat-diet-induced obese mice, which was associated with the reduced level of SIRT3. Knockout of SIRT3 increased p53 acetylation, which in turn led to impairment of angiogenesis and cardiac function in mice. Using cultured cardiomyocytes exposed to high glucose (HG) and diabetic db/db mice, we further explored the roles of p53 acetylation on angiogenic growth factor expression, angiogenesis and cardiac function. We found that exposure of cardiomyocytes to HG resulted in upregulation of p53 acetylation. This was accompanied by a reduction of Sirt3 expression. Transfection with Ad-Sirt3 significantly reduced hyperglycemia-induced p53 acetylation. Overexpression of Sirt3 increased the expression of HIF-1α and Ang-1 and VEGF, together with reduction of oxygen consumption and ROS formation. Hyperglycemia-induced caspase-3 expression and apoptosis was also significantly suppressed by Sirt3. Knockdown of p53 in cardiomyocytes by using siRNA significantly increased the expression of HIF-1α and Ang-1 and VEGF. Treatment with p53 siRNA significantly decreased oxygen consumption.
In vivo
, Ad-Sirt3 treatment significantly inhibited p53 acetylation in the heart of db/db mice. Ad-Sirt3 treatment upregulated the expression of HIF-1α, Ang-1 and VEGF. Similarly, mutations at acetylation sites K117/161/162 and K98 of p53 (p53
4KR
) upregulated the expression of HIF-1α and increased angiogenesis in mouse heart. Ad-Sirt3 treatment increased angiogenesis, and reduced ROS formation and apoptosis in the diabetic heart. These led to significantly improved cardiac function. Our study suggests that p53 acetylation may be a novel target for diabetic cardiomyopathy.
Abstract 109: Site-specific Acetylation of Cardiac Troponin I Regulates Myofilament Relaxation and Calcium Sensitivity
Authors: Woulfe Kathleen C , Lin Ying H , Schmidt William , Cammarato Anthony , Foster D B , Biesiadecki Brandon J , McKinsey Timothy A
Published: October 2019

Objective:
Cardiac troponin I (cTnI) is an essential regulator of cardiac contractility and relaxation. Mutations within key regions of this regulator lead to cardiomyopathies. Further, post-translational modification of cTnI through phosphorylation impacts myofibril relaxation and calcium sensitivity. Recent studies have also demonstrated that myofibril proteins are acetylated leading to faster relaxation. These studies highlight the potential significance of myofilament acetylation; however, it is not known if site-specific acetylation of cTnI can lead to mechanical changes in the myofibril. The objective of this study was to determine if acetylation at a single site of cTnI (lysine-132; K132) is sufficient to alter myofibril protein mechanics.

Methods:
Adult rat ventricular cardiomyocytes infected with adenoviral constructs expressing either cTnI K132 replaced with glutamine (K132Q; to mimic acetylation) or K132 replaced with arginine (K132R; to prevent acetylation) were subjected to cell contractility and isolated myofibril mechanic measurements. Additionally, skinned myofibrils were exchanged with troponin containing wildtype (WT) or K132Q cTnI and mechanics assessed. Finally, dynamics of reconstituted thin filaments containing WT or K132Q cTnI were assessed by in vitro motility assay.

Results:
Cardiomyocytes expressing cTnI K132Q relaxed faster and had decreased calcium sensitivity compared to WT cTnI at the whole cell and myofibril level. Relaxation or calcium sensitivity did not differ between cardiomyocytes infected with WT cTnI and cTnI K132R. Myofibrils with cTnI K132Q exchanged ex vivo demonstrate faster relaxation and decreased calcium sensitivity as well as decreased motility.

Conclusions:
Our results indicate for the first time that acetylation at a specific cTnI lysine can alter myofibril relaxation and calcium sensitivity. This work underscores the importance of understanding how acetylation affects specific sarcomeric proteins in the context of cardiac disease, and suggests that modulation of myofilament lysine acetylation may represent a novel therapeutic target to alter cardiac relaxation.
Abstract 111: Wnt Signaling Inhibition Rescues Voltage-Gated Na
+
Current in Brugada Syndrome Patient Cardiomyocytes
Authors: Liang Wenbin , Lu Aizhu , Chu Cencen , Wang Jerry
Published: October 2019

Aims:
Both inherited arrhythmogenic diseases (such as Brugada Syndrome) and heart failure are associated with reduced voltage-gated Na
+
current (I
Na
) which promotes lethal arrhythmias and sudden deaths. We and others have shown that Wnt/β-catenin signaling (Wnt signaling), which is active in heart disease, inhibits I
Na
in rat and mouse cardiomyocytes. But whether Wnt signaling regulates I
Na
in human cardiomyocytes and represents a novel therapeutic target is unknown. This study aims to investigate if Wnt signaling inhibits I
Na
in human cardiomyocytes, to elucidate the underlying mechanisms, and to test if blocking Wnt signaling can rescue I
Na
in Brugada Syndrome patient cardiomyocytes.

Methods and Results:
Cardiomyocytes were differentiated from human induced pluripotent stem cells (hiPSC-CMs) that were derived from healthy volunteers or a Brugada Syndrome patient. Whole-cell patch-clamp technique was used for I
Na
measurement. Activation of Wnt signaling in healthy hiPSC-CMs led to a 69% reduction in I
Na
amplitude (peak current at -20 mV: -10.5±2.8 pA/pF, n=9 cells vs. control -33.6±4.1 pA/pF, n=6 cells, p<0.01) by reducing
SCN5A
mRNA (encoding the pore-forming α subunit of I
Na
, Na
v
1.5). In addition, Wnt signaling also reduced Na
v
1.5 glycosylation causing a depolarizing shift of I
Na
activation curve (a mechanism found in human, but not in rodent, cardiomyocytes). Blocking Wnt signaling in Brugada hiPSC-CMs with shRNA-mediated β-catenin knockdown led to a 13-fold increase in I
Na
amplitude (p<0.01, n=9 cells), offsetting the fundamental genetic defect. Consistent with increased I
Na,
blocking Wnt signaling also upregulated
SCN5A
mRNA and Na
v
1.5 protein, without affecting expression of other cardiac ion channels in Brugada hiPSC-CMs.

Conclusions:
This study demonstrated Wnt-inhibition of human cardiac I
Na
and, using Brugada Syndrome as an example, demonstrated that blocking Wnt signaling is a novel therapeutic strategy to rescue I
Na
in heart disease.
Abstract 112: Endogenous-Antigen-Specific T Cell Receptor Activation of CD4+ T Cells in the Heart is Required for Maladaptive Cardiac Remodeling Due to Pressure Overload
Authors: Ngwenyama Njabulo , Carrillo-Salinas Francisco , Aronovitz Mark , Kirabo Annet , Harrison David G , Alcaide Pilar
Published: October 2019

Cardiac pressure overload is associated with an adaptive immune response that drives maladaptive cardiac remodeling. CD4+ T cells are activated and expanded specifically in the heart-draining mediastinal lymph nodes (mLNs) and infiltrate the left ventricle (LV) in response to transverse aortic constriction (TAC). However, the specific mechanisms triggering T cell activation during the progression of TAC-induced HF remain unknown. We hypothesized that T cell receptor (TCR)-dependent activation of CD4+ T cells by endogenous antigens initiates and sustains maladaptive cardiac remodeling during the progression of TAC induced HF. We evaluated TCR mediated CD4+ T cell activation in the LV of Nur77
GFP
mice, which transiently express GFP exclusively upon TCR stimulation. Strikingly, we found LV-infiltrated GFP
+
CD4+ T cells that increased in number as maladaptive remodeling and cardiac dysfunction progressed in response to TAC. Next generation sequencing of LV-sorted GFP
+
CD4+ T cells after 8 weeks of TAC revealed a limited repertoire of TCR clones relative to the periphery, demonstrating a restricted CD4+ T cell response to endogenous antigens. We further assessed the requirement for endogenous antigens by immunizing OT-II transgenic mice, which exclusively express a TCR specific for exogenous chicken ovalbumin (OVA), with OVA in the onset of TAC. While OVA induced CD4+ T cell activation and infiltration into the LV, this was not sufficient to induce cardiac dysfunction, demonstrating the importance of an endogenous antigen-specific response. Reactive isolevuglandins (IsoLGs) are formed during oxidative stress, and rapidly adduct to self-proteins forming endogenous neoantigens that elicit CD4+ T cell activation. We treated mice with the IsoLG scavenger 2-hydroxybenzylamine (2-HOBA) in the onset of TAC, which significantly reduced accumulation of IsoLG-protein adducts in dendritic cells, prevented CD4+ T cell activation in the mLNs, and prevented cardiac dysfunction. Thus, our results demonstrate that CD4+ T cell recognition of endogenous antigens in the LV, including IsoLG-protein adducts, via the TCR is required for maladaptive cardiac remodeling and cardiac dysfunction in pressure overload-induced HF.
Abstract 123: Osteopontin Regulates Adult Cardiomyocyte Division in a Mouse Model of Pressure Overload Induced Heart Failure
Authors: Irion Camila I , John-Williams Krista , Chahdi Ahmed , Yousefi Keyvan , Fernandez Yanelys R , Hatzistergos Konstantinos E , Hare Joshua M , Webster Keith , Shehadeh Lina A
Published: October 2019

Background:
Our previous work showed that pharmacological blockade of Osteopontin (OPN) signaling can prevent and reverse heart failure induced by pressure overload in a transverse aortic construction (TAC) mouse model. Surprisingly, OPN Knockout (KO) mice subjected to 3 month TAC had worse cardiac function and bigger hearts than wild type (WT) TAC mice, despite lack of cardiomyocyte hypertrophy. We hypothesized that OPN KO increased adult cardiomyocyte proliferation in TAC-induced heart failure.

Methods:
Male C57Bl/6 (n=17) or OPN KO (n=11) mice were subject to TAC for 3 months. The protein levels of the mitosis marker H3P was quantified using immunofluorescence in paraffin-embedded myocardial sections. Myocytes were co-stained with WGA and MLC2 to count the number of myocytes per field. Adult primary cardiomyocytes from WT hearts were isolated and analyzed with co-imunoprecipitation (Co-IP) to study the interation between the regeneration factor YAP1, OPN and OPN-regulated proteins such as LDLR. For validation and mechanistic studies, more Co-IP experiments were performed in proliferative human liver HEPG2 cells. To study the effect of OPN blockade on YAP1 nuclear translocation, HEPG2 cells and human iPSC- derived cardiomyocytes (hiPS-CMs) were treated with an IgG or OPN blocking antibody for 24 hours followed by immunostaining for YAP1 and PITX2.

Results:
Nuclear H3P normalized to myocyte count was significantly increased in OPN KO relative to WT TAC hearts (Fold Change = 1.4; p=0,04). Co-IP results revealed a novel interaction between OPN, LDLR and YAP1. Stimulation of β2 adrenergic receptor increased the formation of this multi-molecular complex in a time-dependent manner. Blockade of OPN by a monocolonal antibody for 24 hours caused nuclear localization of YAP1 and PITX2 in HEPG2 cells and hiPS-CMs.

Conclusion:
OPN regulates the mitotic program in adult cardiomyocytes. Furthermore, the interaction of OPN with LDLR and YAP1 to form a new multi-molecular protein complex is regulated by β2-cAMP signaling pathway. Importantly, OPN regulates nuclear translocation of the regeneration factors YAP1 and PITX2, suggesting that OPN signaling may be important for adult cardiomyocyte division in TAC and potentially myocardial infarction and aging.
Abstract 134: Direct Reprogramming of Fibroblasts into a Cardiovascular Tissue
Authors: Cho Jaeyeaon , Kim Sangsung , Yoon Young-sup
Published: October 2019

Background:
Direct reprogramming of fibroblasts into cardiomyocytes (CMs), endothelial cells (ECs), or smooth muscle cells (SMCs) has emerged as a promising strategy for cell-based therapy. Moreover, tissue engineering has become a crucial means to enhance the effects of the cell therapy. However, simultaneous reprogramming of somatic cells into all three cell types or a tissue-like structure has not been investigated despite its significance. Here, we aimed to directly reprogram fibroblasts into a cardiovascular tissue containing all three cell types and determined their regenerative effects on infarcted hearts.

Methods and results:
Adult mouse fibroblasts were transfected with a pre-selected cardiac microRNA mimic, miR-208b-3p, and treated with ascorbic acid and BMP4 one day after the transfection for 9 days. At day 10, cells were reprogrammed into a tissue-like structure referred to as reprogrammed cardiovascular tissue (rCVT), which contains reprogrammed CM (16.9 ± 8.0% positive for TNNT2)-, EC (28.4 ± 3.5% positive for PECAM1)-, and SMC (12.0 ± 2.9% positive for SMTN)-like cells, and 14 identified mouse proteins of extracellular matrix. Sarcomeric striations, calcium oscillations and action potentials were shown in reprogrammed CMs. Reprogrammed ECs took up Dil-conjugated acetylated low density lipoprotein and reacted with DAF-FM diacetate, suggesting production of nitric oxide by the cells. Therapeutic effects of rCVT were determined on a mouse model of myocardial infarction with echocardiography and histological analysis over 4 months. Implantation of this rCVT onto the infarcted mouse heart reduced regional cardiac strains and improved cardiac function. Histological examination showed migration of reprogrammed cells from rCVT into the infarcted hearts. Migrated rECs and rSMCs contributed to vessel formation in collaboration with host vascular cells. Migrated rCMs initially displayed immature characteristics but became mature over time and formed gap junctions with host CMs.

Conclusions:
This study demonstrates that a combination of miR-208b-3p, AA and BMP4 can directly reprogrammed single adult somatic cells into a cardiac tissue-like structure containing CMs, ECs, and SMCs, and that rCVT is effective for cardiac repair.
Abstract 137: The Role of TNNI3K in Adult Mammalian Heart Regeneration
Authors: Gan Peiheng
Published: October 2019

Adult mammalian heart regenerative capacity is thought to be extremely limited. All fetal and newborn mouse cardiomyocytes are mononuclear and diploid, and most become polyploid during the first postnatal week. Our lab showed that mononucleated diploid cardiomyocytes (MNDCMs) retain regenerative capacity, and that the percentage of this subpopulation in the adult mouse heart is variable across strains and correlated with the regeneration capacity of mouse heart. We reported cardiac troponin I-interacting kinase (TNNI3K), a novel cardiac specific gene, to be a key regulator of MNDCM frequency. Nonetheless, a detailed mechanism of how
Tnni3k
regulates MNDCM frequency is still lacking. TNNI3K is a MAPKKK by sequence, but no change in MAPK signaling pathways (p38, ERK1/2, JNK) occurs in
Tnni3k
knock-out mice. To distinguish whether
Tnni3k
plays a kinase role, we created a mouse line harboring the kinase-dead mutant of
Tnni3k (Tnni3k-k489r)
, which recapitulates the high MNCM frequency of
Tnni3k
knock-out mice. Engineered
Tnni3k
truncations in mice yield no stable protein, and these mice also show a high MNCM frequency. Interestingly, we found that naked mole-rat (
Heterocephalus glaber
) has a
Tnni3k
truncation mutation, doesn't express a stable TNNI3K protein, and has a high level of MNCM frequency and cardiomyocyte polyploidy. These results indicate that
Tnni3k
may influence cardiomyocyte karyokinesis and cytokinesis across mammalian species. We also found that several human
TNNI3K
polymorphisms compromise kinase activity, which may influence variation in MNDCM frequency and heart regenerative capacity in the human population.
Abstract 141: Identification of a Key Regulator for Activating Bmp Signalling and Promoting Mesodermal Differentiation in Induced Pluripotent Stem Cells
Authors: Kishino Yoshikazu , Yuasa Shinsuke , Fukuda Keiichi
Published: October 2019

Background:
Recently, patient’s own induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) are expected to be cell sources for regenerative therapy. In stem cell differentiation into cardiomyocytes, activation of BMP and Activin signallings in early phase and suppression of Wnt signalling in late phase are important. Although small molecule chemical compounds and recombinant proteins are currently used for differentiation, however, it is better that all recombinant proteins are replaced by chemical compounds, which cost cheaper and improve the efficiency of iPSC-CM differentiation. Several chemical compounds inhibiting Wnt signalling have been reported in previous researches. On the other hand, BMP signalling activator has not been developed because there is little knowledge of factors which strongly modify BMP signalling in various cells. To address this issue, we tried to screen factors activating BMP signalling strongly.

Methods and Results:
To develop the screening system, we used luciferase assay including BMP-responsive element (BRE) and confirmed that our screening system promptly responds to BMP-4 stimulation. Then, we focused on the molecular target, which has a strong impact on BMP signalling. We screened the possible BMP signal regulators which were reported in previous papers by RNA interference in HEK293T cells. Among many candidate regulators, we found that knockdown of
Tripartite motif 33 (TRIM33)
, E3 type ubiquitin ligase, significantly and reproducibly enhanced BRE-luciferase assay and induced the expression of
Id-1
gene as innate reporter gene of BMP signalling. Next, we investigate whether the suppression of
TRIM33
promotes the differentiation of iPSCs into mesodermal cells. We confirmed that
TRIM33
knockdown by RNA interference promoted the expression of mesodermal marker
Brachyury T
and epithelial-mesenchymal transition marker
Slug2
in 2-dimensional cardiac differentiation.

Conclusions:
We successfully detected the factor activating BMP signalling.
TRIM33
inhibition enhanced the expression of
Id-1
gene and promoted mesodermal differentiation. In the future, chemical compounds targeting
TRIM33
may be useful for regenerative medicine as BMP signalling activators.
Abstract 145: Isolation & Characterization of Heart-field Specific Cardiomyocytes
Authors: Pezhouman Arash , Engel James L , Nguyen Ngoc B , Skelton Rhys J , Zhao Peng , Gilmore Blake W , Hornstein Nicholas , Ardehali Reza
Published: October 2019

Cardiovascular disease (CVD) is the leading cause of death worldwide. Human embryonic stem cell (hESC)-derived cardiovascular progenitors (CVPs) or cardiomyocytes (CMs) represent a promising candidate for cell-based therapies to treat CVD. Myocardial infarction leads to extensive CM death mainly within the left ventricle, which is predominantly derived from the first heart field (FHF) during embryonic development. We postulate that the generation of chamber-specific CMs may play a key role in the development of safe and efficacious regenerative therapy. As a first step, we generated and characterized a FHF-specific TBX5-TdTomato
+/W
hESC reporter line. We show that TBX5
+
cells represent an enriched population of FHF CVPs that can give rise to CMs, endothelial, and smooth muscle cells
in vitro
. Interestingly, we observed that TBX5
-
cells can also generate contractile CMs. Bulk RNA-sequencing analysis at different stages of development suggested that TBX5
-
cells are enriched for second heart field (SHF) CMs. To enable prospective isolation of FHF and SHF CMs, we generated a double transgenic TBX5-TdTomato
+/W
/NKX2-5
eGFP/W
hESC reporter line. We performed detailed electrophysiological, functional, and transcriptional studies to characterize the heart-field specificity of these hESC-derived CMs. Electrophysiological studies revealed that, despite the presence of atrial and ventricular action potentials (APs) in both FHF and SHF, there are significant differences in their AP duration and cycle length. In addition, both FHF and SHF CMs responded appropriately to adrenergic stimuli. Single-cell RNA sequencing analysis confirmed the absence of nodal genes within these populations and provided evidence of unique molecular signatures for isolating FHF- and SHF-like CMs. Finally, we identified CORIN as a novel cell surface marker for FHF CMs. Our studies provide a platform for investigating
in vitro
cardiovascular development, drug screening, and may facilitate a safe approach for cell therapy in heart disease.
Abstract 151: Organized 3D Neo-Angiogenic and Neo-Lymphangiogenic Vascular Networks for Cardiac Regeneration
Authors: Lou Xi , Thiruvanamalai Valarmathi T , Zhang Jianyi
Published: October 2019

Introduction:
Cardiovascular tissue engineering has been an area of intense investigation. The major challenge to these approaches has been the inability to vascularize and perfuse the
in vitro
engineered tissue constructs. Engineering a tissue of clinically relevant magnitude requires the formation of extensive and stable microvascular networks within the tissue.

Hypothesis:
Functional vascularized cardiac graft can be generated by the interaction of multipotent human mesenchymal stem cells (hMSCs) and human induced pluripotent stem cell-derived embryonic cardiac myocytes (hiPSC-eCMs) in a 3D collagen cell carrier (CCC) scaffold.

Methods and Results:
To achieve the above aim, we have developed an
in vitro
3D functional vascularized cardiac muscle construct using hiPSC-eCMs and hMSCs. Initially, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were co-cultured on CCCs for 7 days under vasculogenic culture conditions. hCMVECs/hMSCs underwent maturation and differentiation characteristic of microvessel morphogenesis and formed extensive plexuses of capillary networks. Next, the hiPSC-eCMs and hMSCs were co-cultured onto these generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. hiPSC-eCMS/hMSCs underwent maturation and differentiation, and demonstrated spontaneous rhythmic beating. The vascular and cardiac phenotypic inductions were analyzed at the morphological, immunological, biochemical, molecular and functional levels. Expression analyses of the differentiated cells revealed neo-angiogenesis and/or neo-lymphangiogenesis as well as neo-cardiomyogenesis.

Conclusions:
Our unique 3D co-culture system provides us the apt
in vitro
functional prevascularized 3D cardiac graft that can be utilized for myocardial repair and/or regeneration.
Abstract 154: Human Highly Proliferative Cells Acquire Endothelial Phenotype and Promote Healing After Experimental Myocardial Infarction
Authors: Yin Haifeng , Ryzhov Sergey , Robich Michael P , Quinn Reed , Kramer Robert S , Vary Calvin P , Sawyer Douglas B
Published: October 2019

Introduction:
The human adult heart contains a subset of mesenchymal cells characterized by high proliferative potential and capability to differentiate into different cell types. We recently demonstrated that a high level of ErbB2 expression is associated with differentiation of human highly proliferative cells (hHiPC) into endothelial cells in vitro. Based on our findings, we hypothesized that ErbB2
high
hHiPC play a critical role in early revascularization and repair of injured heart. To test this hypothesis, we performed xenotransplantation of human ERBB2
high
hHiPC into mouse hearts and analyzed the phenotype of transplanted cells and cardiac function on day 7 after experimental myocardial infarction (MI).

Methods:
The effect of ErbB2
high
hHiPC was tested in immunodeficient NSG mice. MI was induced by permanent ligation of the left coronary artery. Human ERBB2
high
hHiPC (2.5 x 10
5
cells) were injected into the peri-infarct zone immediately after MI. Echocardiography was performed on unsedated mice before and on days 7 after MI. Examination of transplantation efficiency and hHiPC phenotype was performed using flow cytometric analysis of cell suspension obtained from left ventricles (LV).

Results:
We found that up to 25% of cardiac endothelial cells were represented by cells that originated from hHiPC, as identified by specific expression of human CD31, in mouse LV, on day 7 after MI. The total number of cells expressing mouse or human endothelial cell markers, CD31 and CD105, within non-immune cardiac cell population was higher in mice that received injection of hHiPC compared to control mice (PBS injection) (4.7 vs. 2.9 x 10
5
cells, hHiPC vs. PBS,
p= 0.026
). Echocardiographic analysis revealed that mice which received an injection of ERBB2
high
cells demonstrated significantly improved cardiac function compared to control mice injected with PBS (fractional shortening: 21.6% vs. 15.3% for hHiPC vs. PBS,
p=0.023
).

Conclusion:
ERBB2
high
hHiPC survive, undergo endothelial cell differentiation in vivo, and contribute to the pool of cardiac endothelial cells after experimental myocardial infarction. A population of ErbB2
high
hHiPC obtained from adult human hearts can be used to improve the revascularization of injured myocardium or other tissues.
Abstract 162: Liproxstatin-1 Treatment Protects the Myocardium Against I-R Injury By Decreasing VDAC1 Levels and Increasing GPX4 Activity
Authors: Feng Yansheng , Madungwe Ngonidzashe B , Tombo Nathalie , Liu Li , Aliagan Abdulhafiz , Bopassa Jean C
Published: October 2019

Introduction:
Ferroptosis is a non-apoptotic form of cell death characterized by iron-dependent accumulation of lethal lipid reactive oxygen species (ROS) and liproxstatin-1 is a potent inhibitor of ferroptosis. We investigated whether Liproxstatin-1 can protect the heart against ischemia/reperfusion (I/R) injury and determined the mechanism underlying cardioprotection.

Methods:
Isolated hearts from C57BL/6J mice were subjected to 35 min ischemia followed 120 min reperfusion and treated with Liproxstatin-1 (200nM). Myocardial infarct size was assessed by TTC staining at the end of reperfusion. Mitochondrial integrity was measured by analyzing cristae morphology and ROS production was assessed using Amplex red method. The calcium retention capacity (CRC) required to induce mitochondrial permeability transition pore (mPTP) opening was measured using calcium green dye. The levels of mitochondrial proteins cyclophilin D (CypD), Glutathione peroxidase 4 (GPX4), VDAC1, 2 and 3 were assessed by Western blot. And GPX4 activity was measured using a kit.

Results:
We found that liproxstatin-1 treatment decreased myocardial infarction size, ROS production, and VDAC1 expression as well as preserved mitochondrial structural integrity by protecting cristae from damage. These mitochondrial effects of liproxstatin-1 were associated with the increase in GPX4 levels and activity. But, we also found that liproxstatin-1 did not affect the CypD, VDAC2, and VDAC3 expression as well as mitochondrial CRC.

Conclusion:
Together, these results indicate that liproxstatin-1 treatment protects myocardium against I/R injury by increasing GPX4 activity resulting in the decrease in mitochondrial ROS production; preserving mitochondrial structure as well as a decrease in VDAC1 (but not VDAC 2 and 3) expression. Liproxstain-1 cardioprotective action does not involve the regulation of the mPTP opening. These results point to the cardioprotective role of this anti-ferroptosis compound after I/R.
Abstract 170: Exosomal AAV-mediated SERCA2a Gene Transfer Improves Cardiac Function in a Mouse Model of Heart Failure
Authors: Adamiak Marta , Jha Divya , Liang Yaxuan , Mathiyalagan Prabhu , Agarwal Neha , Kohlbrenner Erik , Chepurko Elena , Jeong Dongtak , Ceholski Delaine , Dubois Nicole , Hajjar Roger , Sahoo Susmita
Published: October 2019

Adeno-associated viruses (AAVs) are promising therapeutic tools for gene delivery to the heart. However, pre-existing antibodies (NAbs) to many cardiotropic AAV serotypes naturally present in humans pose a critical challenge for the translation of gene therapies to clinical applications. Here, we describe the use of exosomal AAVs (eAAV) as a robust heart gene delivery system that improves transduction efficiency while protecting from pre-existing immunity to the viral capsid. To obtain eAAV specimens from conditioned medium from AAV-producing HEK-293T cells, we have developed a state-of-the-art multi-step ultracentrifugation strategy. We demonstrated through electron microscopy-based visualization, size distribution measurements and distribution of AAV genomes in post-centrifugation iodixanol gradients, that our purification process enables isolation of eAAVs with high purity and minimal contamination with standard AAVs. Efficiency of heart targeting was then evaluated for eAAV9 or eAAV6 and standard AAV9 or AAV6 in human cardiomyocytes (hCMs)
in vitro
and in passive immunity nude mouse model
in vivo
. Regardless of the presence or absence of NAbs, we demonstrated that eAAVs are more efficient in transduction of cells in the same titer ranges as standard AAVs. To test the therapeutic efficacy, eAAV9-SERCA2a or AAV9-SERCA2a were injected intramyocardially in post-myocardial infarction (MI) mice preinjected with NAbs. Remarkably, eAAV9-SERCA2a outperformed standard AAVs 6 weeks post-MI, significantly improving cardiac function in the presence of NAbs (%EF 55.14 ± 3.50 vs. 27.31 ± 1.63, respectively). Additionally, we demonstrated
in vivo
that eAAV9-mediated gene delivery is more specific to CMs than to other cell types present in the heart, which suggests that eAAVs preserve cardiotropic properties of AAV9 serotype. With examination of colocalization of eAAVs and markers specific for endosomes (Rab5 and Rab7) in hCMs
in vitro
, our preliminary data indicated that eAAV infectious entry potentially involves trafficking via endocytic compartments. In conclusion, these results underline the therapeutic potential of eAAVs to evade NAbs, and to facilitate the clinical translation of AAV-based gene therapies to a larger human population.
Abstract 174: Investigating Subclinical Cardiotoxicity in Long-term Breast Cancer Survivors Following Chemotherapy With DENSE-based 3D Strain Analysis
Authors: Baldwin Bryant , Johnson Shane , Malozzi Christopher , Cohen Michael V , Figarola Maria S , Chouhdry Anam , Rel Eduardo , Yudice Angela Y , McQuiston Samuel A , Kar Julia
Published: October 2019

This study investigated if left ventricular (LV) mechanical contractile parameters in addition to LV ejection fraction (LVEF) indicated cardiac remodeling and fibrosis in patients exposed to cardiotoxic chemotherapy agents (CCA). Cardiac deformation data were obtained using single-scan acquisitions with the Displacement Encoding with Stimulated Echoes (DENSE) MRI sequence. Contractile analysis consisted of automated myocardial boundary detection and unwrapping 3D DENSE phase images for intra-myocardial displacements, and followed by analyzing torsion and 3D strains with the meshfree Radial Point Interpolation Method (RPIM). Data were acquired on 13 CCA-exposed patients who were undergoing chemotherapy and/or care for cardiac complications. DENSE LVEF measurements in patients were validated against steady-state free precession (SSFP) MRI data, all contractility computations were compared to healthy subjects and Bland-Altman agreements established between strains computed by independent observers. A significant difference was not found between DENSE and SSFP LVEF computations (52 ± 11% vs 48 ± 15%, p=0.33). Significant differences were seen with enlarged LV diameters in patients versus healthy subjects (6.0 ± 1.1 cm vs 4.9 ± 0.7 cm, p<0.001) and similarly for torsion and longitudinal strain but not in LVEF results (p > 0.05). Bland-Altman agreements were 0.01 ± 0.06 for longitudinal strain and 0.1 ± 1.9
°
for torsion. Statistical analysis confirm changed LV size and function in patients and indicate remodeling that is otherwise not demonstrated by LVEF measurements, which may precede clinically apparent heart failure development and worsened morbidity.
Abstract 176: The Effects of Age, Menopause, and Body Mass Index on Blood Pressure and Radial Pulse Wave in Women
Authors: Chen Chih-Yu , Chang Chi-Wei , Chang Yi-Ting , Wang Sheng-Hung , Wang Gin-Chung Wang
Published: October 2019

Age, gender, and body mass index (BMI) play an important roles in cardiovascular risk. However, the independent effects of menopause on the ventricular-arterial system in female individuals have not been fully investigated. Thus, this study aimed to assess the effect of age, menopause and body mass index on different hemodynamic parameters including systolic blood pressure (SBP), diastolic blood pressure (DBP) and harmonics of radial pulse wave. This study enrolled 327 female subjects(230 pre-menopausal and 97 post-menopausal), aged 20-87 and without cardiovascular history. SBP and DBP were evaluated by an automatic blood pressure monitor. A 12-second continuous radial pulse data was recorded using medical grade pulse measuring instrument and standard protocol. Spectrum analysis of radial pressure wave was calculated and transformed into first five harmonic amplitudes(C1~C5). We further performed multivariable linear regression models to assess the independent effects of age, menopause, and body mass index on SBP, DBP, and C1 to C5 respectively. The mean age, BMI, SBP, and DBP in enrolled subjects were 43±13 yeas, 23±4 kg/m
2
, 116±16 mmHg, and 69±11 mmHg. Regression analysis demonstrated that age was positively correlated with SBP(P<0.001), whereas effect of age exerted no significant influence on DBP. BMI had positive associations with SBP and DBP(P<0.001), while menopause had no significant effect on neither SBP nor DBP. Multivariable linear regression showed that menopause had significant effects on C1 (P<0.05) and C3 (P<0.05). C1 is associated with atherosclerosis and is an independent risk predictor for adverse cardiac events. We proposed that an increase in cardiovascular risk during menopause may be due to hormonal changes exacerbating the progression of atherosclerosis and reflected in the radial pulse spectrum. Conclusively, this study demonstrates that age and BMI have an independent effect on SBP, whereas menopause has no significant independent effect on SBP. The effects of menopause on C1 and C3, independent of age and BMI, may partially explain the reasons of dramatically increased cardiovascular risk during menopause and provide clues to the underlying mechanisms of the change in cardiovascular system.
Abstract 184: Cardioprotective Effects of Brain-derived Neurotrophic Factor rs6265 Polymorphism in Duchenne Cardiomyopathy
Authors: Raucci Frank J , Singh Anand P , Soslow Jonathan H , Zhong Lin , Aljafar Wejdan , Lessiohadi Natasja , Awgulewitsch Cassandra P , Umbarkar Prachi , Zhang Qinkun , Smith Holly M , Cannon Presley L , Carrier Erica J , Hatzopoulos Antonis K , Lal Hind , Galindo Cristi L
Published: October 2019

We previously identified brain-derived neurotrophic factor (BDNF) as a putative cardiac-specific biomarker for preserved cardiac function in Duchenne Muscular Dystrophy (DMD). BDNF has cardioprotective qualities that may attenuate dilated cardiomyopathy, and a common, functional BDNF single nucleotide polymorphism rs6265 (Val66-Met) may be correlated with increased risk of cardiovascular events. We hypothesized that BDNF is protective in the setting of DMD and that BDNF’s beneficial effects are diminished in rs6265 allelic carriers. We compared circulating BDNF levels with cardiovascular and skeletal muscle functional parameters in DMD patients stratified by carrier status. Positive association of circulating BDNF with left ventricular ejection fraction in DMD patients was restricted to non-carriers of the rs6265 allele, whereas carriers exhibited worse cardiac function with increased BDNF, more fibrosis and worse skeletal muscle function. To more directly assess association of rs6265 with cardiac function, we used mice with knock-in of BDNF polymorphic Val66Met allele. RNASeq showed higher expression of extracellular matrix genes in Val66Met hearts, and there was a negative correlation of cardiac BDNF protein levels with fibrosis. Additionally, there were baseline echocardiographic and ECG differences, and isolated cardiomyocytes had decreased contractility. This overall cardiac phenotype was substantially enhanced when Val66Met mice were crossed with muscular dystrophy mice. Considered together, our results indicate that BDNF plays a protective role in the dystrophic heart and may represent a novel therapeutic candidate for DMD cardiomyopathy.
Abstract 190: Polycystin-1 Assembles With Kv Channels to Govern Cardiomyocyte Repolarization and Contractility
Authors: Altamirano Francisco , Schiattarella Gabriele G , French Kristin M , Kim Soo Young , Engelberger Felipe , Kyrychenko Sergii , Villalobos Elisa , Tong Dan , Schneider Jay W , Ramirez-Sarmiento Cesar A , Lavandero Sergio , Gillette Thomas G , Hill Joseph A
Published: October 2019

Mutations in the gene encoding polycystin-1 (PC1) underlie autosomal dominant polycystic kidney disease (ADPKD). ADPKD patients present with multiple cardiovascular co-morbidities believed to be caused by renal dysfunction. LV hypertrophy and diastolic dysfunction can manifest during childhood or in young adults prior to a formal diagnosis of hypertension, and evidence suggests that LV function is impaired in ADPKD patients with normal or moderately reduced kidney function. These facts suggest that cardiomyocyte-autonomous effects may contribute to the cardiovascular abnormalities seen in ADPKD. Contractile function (systolic and diastolic) measured by echo was significantly reduced in PC1 cKO (
Pkd1
F/F
;αMHC-Cre) mice compared with controls (
Pkd1
F/F
). PC1 cKO cardiomyocytes manifest impaired contractility and smaller and slower Ca
2+
transients. Using a multidimensional approach, we discovered that cardiomyocytes lacking PC1 have shorter action potentials (APD50/90) and decreased SERCA activity. These alterations impair EC-coupling and decrease SR Ca
2+
loading during pacing. Remarkably, square pulses under voltage clamp (-80 to +10 mV) produced Ca
2+
transients with similar amplitude between genotypes, which highlights that alterations in action potential (AP) duration drive most of the EC-coupling changes. PC1-deficient cardiomyocytes manifested an increase in outward K
+
currents (I
to
, I
Kslow1/2
and I
ss
) but not in inward currents (I
K1
). PC1 over-expression in HEK293T cells reduced the currents of heterologously expressed Kv4.3/2.1/1.5 channels. The inhibitory effects of PC1 on Kv4.3 currents were mediated by PC1-CT (C-terminus) through its coiled-coil domain (CCD). Interestingly, a naturally occurring human mutant PC1
R4228X
, located in the CCD, manifested no suppressive effects on Kv4.3 channel. Finally, to begin to test for relevance to human pathology, we found that PC1 ablation reduces AP duration, and PC1-CT over-expression had the opposite effect in human stem cell-derived cardiomyocytes. Our findings uncover a novel role for PC1 controlling action potential duration and SERCA. PC1-deficient cardiomyocytes manifest impaired contractility, likely contributing to contractile dysfunction in ADPKD patients.
Abstract 198: Single-cell Reconstruction of Differentiation Trajectory Reveals a Critical Role of Ets1 in Human Cardiac Lineage Commitment
Authors: Ruan Hang , Liao Yingnan , Wang Li , Han Leng
Published: October 2019

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) is an essential model system, but the dynamic process of this
in vitro
differentiation, especially the pivotal stages of cardiac cell lineage specification is not fully understood. Here, we generated a rich resource of Single-Cell RNA-sequencing of more than 7,500 single cells to capture the differentiation process of
in vitro
hESC to CMs across six key time points. We characterized the inter-subpopulation relationship demonstrating that co-existing side populations could play an essential role in early cardiac development by bestowing a supporting cellular microenvironment. Through analyzing the dynamic regulatory networks, loss-of-function screen, and systematically validation of binding signals through ChIP-sequencing, we pinpointed the essential regulatory role of
ETS1
in mediating early cardiomyocyte development.