bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 226: Recellularization of Xenograft Heart Valves Reduces the Xenoreactive Immune Response in an
In Vivo
Rat Model
Authors: Bozso Sabin J , EL-Andari Ryaan , Zhu Lin Fu , Adam Benjamin , Moon Michael C , Freed Darren H , Nagendran Jayan , Nagendran Jeevan
Published: October 2020

Background:
Current xenograft valve constructs provoke an intense immune response that may lead to valve dysfunction. Our aim was to address the role of autologous mesenchymal stem cell (MSC) recellularization of xenogenic valves on the activation of the xenoreactive immune response in an
in-vivo
rat model.

Methods:
Explanted aortic valve constructs from female Hartley guinea pigs were procured and decellularized, followed by recellularization with syngeneic Sprague-Dawley rat MSCs. The recellularized aortic valve xenografts were then implanted into the infrarenal aorta of recipient female Sprague-Dawley rats. Grafts were implanted as either syngeneic grafts, non-decellularized (fresh), decellularized and recellularized xenografts. Rats were euthanized after 7-days, exsanguinated and the grafts explanted. Total serum immunoglobulin was quantified and histological analysis perfomed to analyze the immune response.

Results:
Overall survival to endpoint was significantly lower in the decellularized xenograft group (67%; 4/6), compared to fresh (100%; 6/6) and recellularized grafts (100%; 6/6). Similarly grafts in the decellularized group were more likely to have completely thrombosed (50%; 2/4), compared to fresh (33%; 2/6) and recellularized grafts (0%; 0/6). Decellularized guinea pig xenografts, when implanted into rats
in-vivo
, result in significantly reduced total serum immunoglobulin production and significantly reduced graft cellular infiltrate when compared to fresh xenografts. Moreover, when decellularized guinea pig xenografts were recellularized with syngeneic rat MSCs there was an additional decrease in total serum immunoglobulin production and graft cellular infiltrate when compared to both fresh and decellularized xenografts. Importantly, recellularized guinea pig xenografts had an equivalent total immunoglobulin production and graft cellular infiltrate when compared to syngeneic rat aortic valve controls.

Conclusions:
Autologous MSC recellularization of xenogenic valves reduces the xenoreactive immune response in an
in-vivo
rat model and may be an effective approach to decrease the progression of xenograft valve dysfunction.
Abstract 234: Identification of a Novel Cardiomyocyte-enriched Long Noncoding Rna Counteracting Against Heart Failure Development
Authors: Sato Michio , Miyata Keishi , Kadomatsu Tsuyoshi , Node Koichi , Oike Yuichi
Published: October 2020

Background:
The number of patients with heart failure (HF) continues to increase as the elderly population grows, and mortality remains high, with about 50% of HF patients dying within 5 years of diagnosis. Therefore, defining molecular mechanisms underlying HF pathologies is urgently needed.

Methods and Results:
Using a gene trapping approach in mouse ES cells, we identified
Caren
(for

car

diomyocyte-

en

riched noncoding transcript), a novel cytoplasmic lncRNA abundantly expressed in cardiomyocytes.
Caren
expression in mice markedly decreased in heart failure (HF) induced by transverse aortic constriction (TAC), AngiotensinII loaded or aging.
Caren
ablation accelerated HF-related phenotypes, whereas
Caren
transgenic (Tg) mice in cardiomyocytes resisted TAC-induced HF. Furthermore,
Caren
overexpression in cardiomyocytes activated mitochondrial biogenesis and attenuated TAC-induced activation of Ataxia Telangiectasia Mutated (ATM) serine/threonine kinase, a regulator of the DNA damage response (DDR). Next, we generated Tg mice expressing
Caren
fragments (fragments A-E, from 5’ to 3’) respectively, by inserting that fragment into the corresponding endogenous
Caren
genomic locus. None of these Tg mice exhibited an anti-HF effect against TAC, suggesting that anti-HF effects of
Caren
may be more dependent on a structural motif displayed by the full-length lncRNA rather than on a specific
Caren
sequence. Consistently, restoration of expression of full-length
Caren
in TAC-induced failing cardiomyocytes slows HF progression in mice injected with AAV6-
Caren
. Mechanistically,
Caren
inhibited translation of mRNA encoding ‘
Caren
Target Protein 1’ (CTP1), which activates the ATM-DDR pathway and reduces mitochondrial respiratory capacity in cardiomyocytes. CTP1
+/-
mice resisted HF development, strongly suggesting that
Caren
protects against HF by suppressing CTP1 expression.

Conclusion:
This is the first study reporting that
Caren
, a novel cytoplasmic lncRNA
,
counteracts HF development and progression by inactivating the ATM-DDR pathway and activating mitochondrial bioenergetics, in part, by suppressing CTP1 translation in cardiomyocytes. Our findings could encourage development of RNA-based strategies to combat HF development.
Abstract 236: Aldehydes in Cigarette Smoke Impair Vascular Endothelial Function
Authors: Rao Poonam , Nabavizadeh Rafsanjani Pooneh , Han Daniel , Schick Suzaynn , Springer Matthew
Published: October 2020

Introduction:
Exposure to tobacco and marijuana smoke impairs vascular endothelial function. While the particulate phase of smoke is heavily implicated, the role of volatile constituents is unclear. Smoke contains aldehydes, which are known to cause endothelial dysfunction. We explored whether two aldehydes found in smoke, acrolein and acetaldehyde, can induce endothelial dysfunction.

Hypothesis:
Aldehydes in smoke impair endothelial function.

Methods:
We exposed 4 groups of anesthetized rats to 3 ppm acrolein and 10-11.5 ppm acetaldehyde gases (concentrations relevant to levels in secondhand smoke), Marlboro Red cigarette sidestream smoke at modest levels (600 μg/m
3
PM2.5) as a positive control, and clean air through the gas generation system as a negative control. Exposure was continuous for 10 minutes. Endothelial function (flow-mediated dilation; FMD) was quantified pre- and post-exposure by measuring femoral artery diameter with ultrasound before and after 5 min of transient ischemia and expressed as % vasodilation.

Results:
Impairment of FMD was observed for acrolein (10.8±1.7(SD)% vs. 5.8±2.9%, p=.001), acetaldehyde (8.8±2.0% vs. 6.0±2.5%, p=.001), and cigarette smoke (9.4±2.9% vs. 5.8±2.0%, p=.002), but not for air (7.9±2.0% vs. 9±3.2%, p=.44) (figure; each colored line denotes a rat pre- and post-exposure; bars denote means).

Conclusions:
Acrolein and acetaldehyde at levels found in secondhand smoke impair endothelial function. Our results suggest that despite a potential role of particles, volatile aldehydes may mediate part of the endothelial dysfunction caused by exposure to smoke.
Abstract 245: Cardiac Mitofusin-1 is Suppressed in Non-responding Patients With Heart Failure
Authors: Hsiao Yung Ting , Shimizu Ippei , Wakasugi Takayuki , Jiao Shuang , Watanabe Tohru , Kashimura Takeshi , Yoshida Yohko , Hanawa Haruo , Ozaki Kazuyuki , Minamino Tohru
Published: October 2020

Background/Introduction:
Mitochondria are dynamic regulators of cellular metabolism and homeostasis. The dysfunction of mitochondria has long been considered a major contributor to aging and age-related diseases. The prognosis of severe heart failure is still unacceptably poor and it is urgent to establish new therapies for this critical condition. Some patients with heart failure do not respond to established multidisciplinary treatment and they are classified as “non-responders”. The outcome is especially poor for non-responders, and underlying mechanisms are largely unknown.

Purpose:
Studies indicate mitochondrial dysfunction has causal roles for metabolic remodeling in the failing heart, but underlying mechanisms remain to be explored. This study tried to elucidate the role of Mitofusin-1 in a failing heart.

Methods:
We examined twenty-two heart failure patients who underwent endomyocardial biopsy of intraventricular septum. Patients were classified as non-responders when their left-ventricular (LV) ejection fraction did not show more than 10% improvement at remote phase after biopsy. Fourteen patients were classified as responders, and eight as non-responders. Electron microscopy, quantitative PCR, and immunofluorescence studies were performed to explore the biological processes or molecules involved in failure to respond. In addition to studies with cardiac tissue specific knockout mice, we also conducted functional
in-vitro
studies with neonatal rat ventricular myocytes.

Results:
Twenty-two patients with IDCM who underwent endomyocardial biopsy were enrolled in this study, including 14 responders and 8 non-responders. Transmission electron microscopy (EM) showed a significant reduction in mitochondrial size in cardiomyocytes of non-responders compared to responders. Quantitative PCR revealed that transcript of mitochondrial fusion protein, Mitofusin-1, was significantly reduced in non-responders. Studies with neonatal rat ventricular myocytes (NRVMs) indicated that the beta-1 adrenergic receptor-mediated signaling pathway induced microRNA-140 3p and 5p, which is negatively regulated Mitofusin-1 expression. Suppression of Mitofusin-1 resulted in a significant reduction in mitochondrial respiration of NRVMs. We generated left ventricular pressure overload model with thoracic aortic constriction (TAC) in cardiac specific Mitofusin-1 knockout model (c-Mfn1 KO). Systolic function was reduced in c-Mfn1 KO mice, and EM study showed an increase in dysfunctional mitochondria in the KO group subjected to TAC.

Conclusions:
Mitofusin-1 becomes a biomarker for non-responders with heart failure. In addition, our results suggest that therapies targeting mitochondrial dynamics and homeostasis would become next generation therapy for severe heart failure patients.
Abstract 249: Plasma Exosomes Impair Angiogenesis in Ischemic Hind Limb of Diabetic Mice- Role of Histone Methylation
Authors: Cheng Zhongjian , Truongcao May M , Wang Chunlin , Garikipati Venkata NS , Tang Yan , Cimini Maria , Benedict Cindy , Huang Grace , Mallaredy Vandana , Goukassian David , Verma Suresh K , Koch Walter J , Kishore Raj
Published: October 2020

Background:
Critical limb ischemia (CLI) is one of most prevenient cardiovascular disease in diabetic patients. Recent evidence suggests that altered cargo and function of plasma exosomes (plasma-Exo) may play an important role in diabetes-induced cardiovascular complications. Here, we tested the hypotheses that inhibition of exosome biosynthesis/release improves ischemic hind limb (IHL) repair in db/db mice.

Methods:
Plasma-Exo from db/+ and db/db mice were isolated by density-gradient ultracentrifugation. Unilateral IHL in mice was conducted by ligation of left femoral artery. Blood perfusion in IHL was measured by Laser Doppler Imager.

Results:
Diabetic plasma-Exo impaired tube formation/migration of human microvascular endothelial cells (HMVECs) and blood perfusion in IHL of C57BL/6J mice. Exosome inhibitor GW4869 improved blood flow, capillary density, cell survival, and rescued necrosis of toe/toenail and fibrosis in IHL muscle of db/db mice. Mechanistically, diabetic plasma-Exo decreased secretion of pro-angiogenic factor Ang I&II, artemin, FGF2 and IGFBP1&2, and increased repressive transcriptional mark H3K27me3 and its methylase enhancer of zest homolog-2 (EZH2) in HMVECs. EZH2 inhibitor GSK343 rescued diabetic plasma-Exo-impaired tube formation and secretion of FGF2/artemin from HMVECs. Moreover, GW4869 reduced EZH2 and H3K27me3 protein expression in lung microvascular ECs of IHL db/db mice. Finally, diabetic plasma-Exo increased H3K27me3 level at promoter of artemin and FGF2.

Conclusions:
Diabetic plasma-Exo impair angiogenesis and IHL injury repair. Diabetic plasma-Exo impair reparative property of ECs via, at least in part, enhancement of EZH2/H3K27me3/artemin and FGF2 cascade. Inhibition of plasma-Exo biosynthesis/secretion improve IHL repair in db/db mice. Plasma-Exo may be a novel target for prevention/treatment of CLI in diabetic patients.
Abstract 250: Acting Instructor
Authors: Zhang Huiliang , Alder Nathan , Wang Wang , Szeto Hazel , Marcinek David , Rabinovitch Peter S
Published: October 2020

Rational:
Aging-associated diseases, including cardiac dysfunction, are increasingly common in the population. However, the mechanisms of physiologic aging in general, and cardiac aging in particular, remain poorly understood. While effective medical interventions are available for some kinds of heart failure, one age-related impairment, diastolic dysfunction in Heart Failure with Preserved Ejection Fraction (HFpEF) is lacking a clinically effective treatment.

Methods and Results:
Using the pH indicator cpYFP in the model of naturally aging mice and rats, we show direct evidence of increased mitochondrial proton leak in aged heart mitochondria following a pH gradient stress. Furthermore, we identified Adenine Nucleotide Translocator 1 (ANT1) as mediating the increased proton permeability of old cardiomyocytes. Most importantly, acute (2 hours)
in vitro
treatment with the tetra-peptide drug SS-31 (elamipretide) reverses age-related excess proton entry, decreases the mitochondrial flash activity and mitochondrial permeability transition pore (mPTP) opening and rejuvenates mitochondrial function. Moreover, we show that SS-31 benefits the old mitochondria by direct association with ANT1 and stabilization of the mitochondrial ATP synthasome, leading to substantial reversal of diastolic dysfunction.

Conclusion:
Our results uncover excessive mitochondrial proton leak as a novel mechanism of age-related cardiac dysfunction and elucidate how SS-31 is able to reverse this clinically important complication of cardiac aging.
Abstract 256: Human
In Vitro
Model for Preclinical Evaluation of Irreversible Electroporation Devices Used for Cardiac Ablation
Authors: Casciola Maura , Feaster Tromondae K , Pakhomov Andrei G , Blinova Ksenia
Published: October 2020

Irreversible Electroporation (IRE) has gained significant interest as an alternative treatment for atrial fibrillation (AF). Typically, to terminate AF, thermal energy is delivered to the arrhythmic tissue causing rare but serious damage to surrounding areas. IRE is a non-thermal approach that has the potential to be faster yet more targeted. We are developing a novel, species relevant, standard laboratory test to evaluate IRE cardiac ablation using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model. hiPSC-CMs were cultured in monolayers. Pulses were delivered by a AVOZ D7B generator through contact electrodes. Electric field (E) distribution maps were obtained with numerical simulations, Sim4Life v3.2. Electroporation (EP) was quantified by florescence with YO-PRO-1 10 minutes after exposure. hiPSC-CMs were exposed to a range of electrical parameters including 120 unipolar pulses of 5 μs duration, at 1 kHz frequency (Fig. 1). We found that the EP threshold was ~1.5 kV/cm in line with previous reports on single adult ventricular mouse cardiomyocytes (Semenov et al. 2018). The treated area increased with the voltage (V) applied. These data suggest that this assay can delineate the extent of EP. In this work we will vary different pulse parameters (durations, amplitude, shape, repetition rate and number) to evaluate their effects on different human cell lines anatomically adjacent to the heart.

Fig.1: (A) Representation of the EP threshold determined by overlaying the E map (B) with a fluorescence image of pulse-treated hiPSC-CMs (C). To define the E threshold the distributions values obtained by modeling 1 V applied was scaled by the experimental V.
Abstract 261: Evaluating
MTCH2
as a Modifier of Cardiomyopathy
Authors: Fischer Julie A , Quattrocelli Mattia , Puckelwartz Megan , Wolf Matthew , McNally Elizabeth M
Published: October 2020

Background:
Cardiomyopathy is a highly heritable disorder that carries a significant risk for heart failure and arrhythmias. The onset, severity, and progression of cardiomyopathy is influenced by genetic variation, with the most common form of inheritance in familial cases being autosomal dominant. All inherited cardiomyopathies are characterized by variable penetrance and expressivity, which in part arises from additional genetic variation, known as genetic modifiers.

Methods and Results:
Broad genomic profiling of human cardiomyopathy cases identified enriched genetic variation in the gene
MTCH2.
Specifically, a loss of function variant was found to be enriched in patients with cardiomyopathy.
MTCH2
single nucleotide polymorphisms have also been linked to obesity, underscoring a critical role for
MTCH2
in metabolic regulation.
MTCH2
encodes a mitochondrial carrier protein that has a role in regulating oxidative phosphorylation. In order to investigate fundamental mechanisms by which MTCH2 contributes to cardiac and metabolic phenotypes, we generated a knockdown model of the
Drosophila MTCH2
ortholog,
Mtch.
This knockdown model mimics what is seen in human carriers with the heterozygous loss of function allele. In the Drosophila model,
Mtch
RNA was reduced by approximately half. We found that cardiac-specific
Mtch
deficiency in flies produced heart tube dilated and reduced function as well as a shortened life span, documenting a clear role for cardiac Mtch. Cardiac deficiency of
Mtch
increased circulating lactate levels in flies. Oxygen consumption was reduced in cardiac
Mtch
deficiency flies in the presence of glucose, but not palmitate. Thus, loss of Mtch2 alters oxygen consumption in a substrate dependent manner.

Conclusions:
We identified MTCH2 as a modifier of the cardiomyopathy phenotype in humans. Reduction of
Mtch
in flies resulted in impaired cardiac function and reduced oxygen consumption under certain metabolic condition. As failed hearts are more dependent on glycolysis, these data support that reduction of MTCH2 promotes heart failure and provides a mechanism by which MTCH2 acts as a deleterious genetic modifier in heart failure.
Abstract 272: Epigenetic Regulation Involved in Diabetes-induced Impairment in Myocardial Reparative Function of Endothelial Progenitor Cell-derived Exosomes
Authors: Huang Grace , Hildebrand Alycia , Benedict Cindy , Cimini Maria , Wang Chunlin , Cheng Zhongjian , Garikipati Venkata N , Mallaredy Vandana , Kishore Raj
Published: October 2020

Myocardial infarction (MI) occurs frequently in patients with diabetes resulting in higher mortality and morbidity than non-diabetic patients. We and others have shown that bone marrow-derived endothelial progenitor cells (EPCs) promote cardiac neovascularization and attenuate ischemic injury in animal models. Moreover, emerging evidence supports that exosomes (Exo) mediate stem cell therapy by carrying cell-specific biological signatures and by inducing signaling via transfer of bioactive molecules to target cells. However, autologous cell-based therapies yielded modest clinical results, suggesting that cellular/Exo reparative function may be compromised on a background of disease such as diabetes. In addition, recent studies suggest epigenetic mechanisms, such as histone methylation for gene silencing, promotes diabetes-induced vascular complication. Therefore, we hypothesized that diabetic EPCs produce exosomes of altered and dysfunctional content which compromise EPC reparative function in ischemic heart disease via epigenetic alterations. We collected EPC-Exo from non-diabetic mice (Lepr
db/+
) and diabetic mice (Lepr
db/db
) and examined their effect on tube formation and cardiomyocyte/endothelial cell survival
in vitro
as well as their reparative effects on permanent and acute ischemia/reperfusion (I/R) myocardial ischemic injuries
in vivo
. Diabetic EPC-Exo promoted neonatal rat cardiomyocyte cell apoptosis under hypoxic stress and repressed endothelial tube formation and cell survival compared to cells treated with WT EPC-Exo.
In vivo
studies revealed diabetic EPC-Exo significantly attenuated cardiac function, reduced capillary density, increased fibrosis and infarct size in permanent LAD ligation and I/R MI models. Mechanistically,H3K9Me3 was increased in mouse cardiac endothelial cells treated with diabetic EPC-exo, suggesting inhibition of angiogenic genes. Our results provide evidence that diabetic EPC-derived exosomes lose their cardiac reparative activities. Specific angiogenic genes will be examined by CHIP analysis of H3K9Me3. Reversing EPC-Exo function by manipulating H3K9Me3 expression will augment autologous therapies in regenerative medicine.
Abstract 275:
In Vitro
Inflammatory Assays for Decellularized Xenogeneic Heart Valves
Authors: Vedepo Mitchell , Flores Kyra , Jacot Jeffrey
Published: October 2020

Intro:
There is great interest in a decellularized xenogeneic heart valve for valve replacement due to the shortage of human tissue available for implant. However, decellularized xenografts have a poor clinical history due to incomplete antigen removal. The inflammatory potential of decellularized heart valves can be evaluated
in vitro
using assays that measure antigen presence and cytokine production by cultured human macrophages. This study tests the inflammatory potential of porcine aortic valves before and after decellularization.

Methods:
Decellularization was confirmed by histology and DNA quantification. Enzyme linked immunosorbent assays (ELISAs) tested for inflammatory potential using pooled human serum as a primary antibody. Additional tissue samples were cultured with THP-1 derived macrophages and the production of monocyte chemoattractant protein-1 (MCP-1) was measured.

Results:
Decellularization resulted in 99% removal of DNA. The decellularized valve conduit was found to be 2.49% as inflammatory as native tissue (Figure 1). Human macrophages cultured with native tissue produced approximately 10-fold more MCP-1 compared to decellularized tissue.

Impact:
The
in vitro
assays described quantify a reduced inflammatory response for decellularized heart valves. Future use of these assays can be used to further decrease the inflammatory potential, increasing the safety of decellularized xenogeneic heart valves.
Abstract 282: Role of a Disintegrin and Metalloproteinase 15 in Cardiac Hypertrophy and Fibrosis Following Pressure Overload
Authors: Aujla Priya , Jana Sayantan , Chute Michael , Kassiri Zamaneh
Published: October 2020

Introduction:
Disintegrin and metalloproteinases (ADAMs) are membrane-bound cell surface enzymes that are capable of both proteolytic functions (via the metalloproteinase domain) and adhesive functions (via the disintegrin domain), whereby they can influence cell function and extracellular matrix (ECM) remodelling in the heart. ADAM15 is unique among the ADAMs, as it is also capable of degrading ECM proteins. ADAM12 and ADAM17 have been reported to regulate cardiac hypertrophy, but the role of ADAM15 in cardiac hypertrophy is not known. This study investigates the role of ADAM15 in cardiac hypertrophy and fibrosis following pressure overload.

Methods & Results:
Genetically modified male ADAM15-deficient (

Adam15
-/-
)

and wildtype (WT) mice were subjected to cardiac pressure overload by transverse aortic constriction (TAC). Cardiac function and structural remodelling were assessed using echocardiography at 2-, and 6-wks post-TAC. Hearts were excised at 2-, or 6-wks post-TAC.

Adam15
-/-

hearts presented greater hypertrophy and decreased cardiac systolic function at 6wks post-TAC, but no difference at 2wks post-TAC compared to WT-TAC mice.

Adam15
-/-

hearts also showed exacerbated fibrosis at 6wks post-TAC, but not at 2wks post-TAC, compared to WT. Mechanical strain (i.e. pressure overload) triggers two temporally activated pathways leading to an initial compensatory hypertrophy, which can culminate to decompensation and dilated cardiomyopathy. Consistent with the greater hypertrophy, phosphorylation of ERK1/2, JNK1/2/3, and GSK3β was increased in

Adam15
-/-

mice. The calcineurin-NFAT pathways can mediate pressure overload-induced hypertrophy, but we found that Adam15-deficiency did not impact this pathway. The mechanism responsible for this function of ADAM15 requires further investigation.

Conclusion:
This study reports a novel cardioprotective function for ADAM15 in pressure overload, where loss of ADAM15 promotes cardiac fibrosis and decompensated cardiac hypertrophy but does not alter the compensated hypertrophic response.
Abstract 286: Targeting Formyl Peptide Receptors as a Novel Approach to Treat Hypertension
Authors: Qin Chengxue X , Jackson Kristy L , Giddy Thomas , Deo Minh , Helen Kiriazis , Woodman Owen L , Ritchie Rebecca H , Head Geoffrey
Published: October 2020

Introduction:
It is estimated that 15-30% of hypertensive patients are resistant to available drugs; and thus identifying novel therapeutic strategies is imperative. We have previously demonstrated that the formyl peptide receptor-1/2 (FPR) small-molecule agonist, Cmpd17b, exhibits anti-inflammatory and vasodilator actions. We investigated the efficacy of Cmpd17b to reduce hypertension and its associated end-organ damage.

Methods:
Hypertensive (BPH/2J) and normotensive (BPN/3J) mice underwent 24h conscious blood pressure recordings via telemetry from baseline until 4 weeks post Cmpd17b treatment (50 mg/kg/day, i.p.) or vehicle (8% tween80 in saline with 10% dimethyl sulfoxide, i.p.). The abdominal aorta dimension was assessed via ultrasound imaging at baseline and study end.

Results:
Hypertensive mice exhibited elevated mean arterial pressure compared to normotensive mice at baseline (Table). Cmpd17b reduced mean arterial pressure, left ventricle fibrosis, angiotensinogen expression and improved vascular distensibility.

Conclusion:
This study is the first to demonstrate that chronic FPR agonist administration reduces blood pressure in hypertensive mice, and further supports development of FPR-based therapies against hypertension-related organ damage.
Abstract 299: Cardiac Fibroblast-derived FGF21 is a Novel Therapeutic Target for Cardiac Pathological Remodeling
Authors: Furukawa Nozomi , Koitabashi Norimichi , Matsui Hiroki , Yokoyama Tomoyuki , KURABAYASHI Masahiko
Published: October 2020

Background:
Fibroblast Growth factor 21(FGF21) is an endocrine factor, secreted mainly by the liver, that exerts metabolic actions. It has been shown that FGF21 has an anti-hypertrophic action for cardiac hypertrophy. We previously reported that murine cardiac FGF21 expression was upregulated by pressure-overload. The objective of this study was to clarify the role of cardiac fibroblast-derived FGF21 in cardiac pathological remodeling.

Methods and Results:
We generated fibroblast-specific/tamoxifen-inducible FGF21 knockout mice crossing by FGF21flox mice and Col1a2-CreERT mice (FB-FGF21KO). FGF21flox control mice and KO mice were induced cardiac hypertrophy by transverse aortic constriction (TAC). After 3-weeks TAC surgery, we evaluated the cardiac function by echocardiography or conductance catheter. TAC-mediated cardiac hypertrophy or impairment of systolic function were exacerbated in FB-FGF21KO mice. Conductance catheter showed that Tau, diastolic relaxation marker, was exacerbated in FB-FGF21KO mice with TAC. FB-FGF21KO heart with TAC showed significant upregulation in profibrotic genes and down-regulation in Ca2+ ATPase in comparison with control TAC heart. Cardiac Sirt1, which plays a central role in energy metabolism or oxidative stress, increased by TAC was significantly attenuated in FB-FGF21KO heart.

Conclusion:
Deletion of FGF21 in cardiac fibroblasts exacerbates cardiac dysfunction in response to pressure overload suggesting that cardiac fibroblasts regulates pathological remodeling via FGF21 regulation.
Abstract 301: Cavin-2 Deletion Attenuates Fibroblast Activation and Cardiac Fibrosis via TGF-β Signaling by Interacting With Hsp90
Authors: Higuchi Yusuke , Ogata Takehiro , Nakanishi Naohiko , Sakamoto Akira , Tsuji Yumika , Tomita Shinya , Matoba Satoaki
Published: October 2020

Introduction:
Heart failure (HF) is a progressive disease associated with high morbidity and mortality. A major cause of HF is the adverse tissue remodeling with interstitial fibrosis. Excessive extracellular matrix (ECM) accumulation is involved in the poor outcome. Transformation to myofibroblasts in resident fibroblasts has an important role for cardiac fibrosis on pressure-overload heart failure. TGF-β signaling is one of the critical pathways in fibroblast activation. Caveolae are major plasma membrane domain defined as 50-100 nm vesicular structures. Caveolins and cavins are known as caveolar-related proteins. TGF-β signaling is tightly connected with caveolae. A previous report shows that induction of Cav-1 expression leads to suppression of TGF-β signaling and an improvement of fibrosis. Although cavin-2/Serum deprivation response protein (SDPR) is also abundant in fibroblasts, the role of cavin-2 in cardiac fibrosis and function remains unknown.

Methods and Results:
To clarify the role of cavin-2 in cardiac fibroblasts in the pressure-overloaded heart, we performed TAC operation on cavin-2
flox/flox
mice and fibroblast-specific cavin-2 knockout (Postin
Cre
; SDPR
flox/flox
) mice (cavin-2 cKO). Four weeks after TAC, left ventricular fractional shortening (LVFS) was preserved with a significant reduction of cardiac fibrosis in cavin-2 cKO mice. Fibrosis-associated mRNA expression (
Col1a1, Ctgf, Col3
) and α1 type I collagen deposition were reduced in the hearts of cavin-2-cKO mice after TAC. Transdifferentiation of fibroblasts into activated myofibroblasts is a defining feature of fibrosis. Myofibroblasts express αSMA and secrete ECM proteins via Smad signaling. αSMA-positive cells without showing vascular structure were reduced in cavin-2 cKO after TAC. In mouse embryonic fibroblasts (MEFs), cavin-2 deficiency reduced TGF-β1-induced αSMA production and fibrosis-associated mRNA expression. On the other hand, adenovirus-mediated cavin-2 overexpression significantly increased αSMA production and the fibrosis-associated mRNA expressions. Furthermore, TGF-β1-induced Smad2/3 phosphorylation was attenuated in cavin-2 KO MEFs compared to wild-type (WT) MEFs. A protein-Protein interaction screening using a promiscuous biotin ligase, called BioID, revealed that cavin-2 interacted with Hsp90ab1 and Hsp90b1 which modulate collagen synthesis through regulation of SMA and SMAD pathway. The protein expression level of Hsp90 in cavin-2 KO MEFs significantly decreased compared with that in WT MEFs.

Conclusions:
Our observations suggest that cavin-2 contributes to the development of cardiac fibrosis through the differentiation from fibroblasts into myofibroblasts via TGF-β/Smad signaling. cavin-2 may be a novel therapeutic target for cardiac fibrosis.
Abstract 321: Exercise Preconditioning Diminishes Solute Carrier Protein Expression and Cardiac Doxorubicin Accumulation
Authors: Smuder Ashley J , Mor Huertas Andres , Morton Aaron B
Published: October 2020

Doxorubicin (DOX) is an anthracycline antibiotic used in cancer treatment. Unfortunately, the clinical use of this highly efficacious anticancer drug is limited due to the development of irreversible and dose-dependent cardiomyopathy. While the mechanisms responsible for DOX-induced cardiotoxicity remain unclear, previous work demonstrates that the incidence of cardiac dysfunction greatly correlates to the concentration of DOX transported into the cardiomyocytes. In this regard, we recently demonstrated that endurance exercise performed prior to DOX administration is sufficient to reduce the accumulation of DOX within the heart and prevent left ventricular dysfunction. While the mechanisms for the exercise-induced reduction in cardiac DOX levels are unknown, we hypothesize that endurance exercise alters the expression of solute carrier proteins (SLCs) required for the influx of DOX into the heart. To determine if exercise training can alter SLC protein expression in the heart, animals underwent two weeks of treadmill running (5 days/week; 60 min/day; 30m/min). Twenty-four hours following completion of the exercise training period, animals received either saline or DOX (20mg/kg i.p.) treatment. DOX is a slightly basic cationic compound that is thought to be transported into cells by organic cation transporters. Analysis of the expression of the high affinity organic cation transporters SLC22A4 and SLC22A5 revealed a significant increase in their expression in DOX treated animals, which was mitigated by exercise preconditioning. In addition, the low affinity organic cation transporter SLC22A1 was also increased in DOX treated animals compared to exercise trained animals, while SLC22A3 expression did not differ between groups. Finally, exercise training also reduced the DOX-induced increase in the expression of the mitochondria-localized SLC transporter SLC25A1. These data demonstrate that DOX enhances the expression of several SLCs in the heart, and that exercise may ameliorate cardiac DOX accumulation by preventing the upregulation of proteins with the potential to promote DOX cellular influx.
Abstract 328: Enhanced Therapeutic Effects of Mesenchymal Stem Cells for Hindlimb Ischemia by a Nitric Oxide Hydrogel Through Mesenchymal-endothelial Transition
Authors: Zhang Kaiyue , Chen Shang , Huang Haoyan , Wang Chen , Li Zongjin
Published: October 2020

Therapeutic angiogenesis with mesenchymal stem cells (MSCs) is promising for the clinical treatment of peripheral artery disease (PAD). However, the heterogeneous proangiogenic nature of MSCs is a key challenge in developing more effective treatments with MSCs for therapeutic angiogenesis purposes. Here, we propose to enhance the therapeutic function of human placenta-derived MSCs (hP-MSCs) in hindlimb ischemia therapy by using nitric oxide (NO)-releasing chitosan hydrogel (CS-NO). Our data showed that the co-transplantation of CS-NO hydrogel with hP-MSCs remarkably improved the grafting of hP-MSCs and ameliorated the functional recovery of ischemic hindlimbs. Moreover, we found that the neovascularization of damaged hindlimbs was significantly increased after co-transplanting CS-NO hydrogel and hP-MSCs, as confirmed by bioluminescence imaging (BLI). Further analysis revealed an endothelial-like status transformation of hP-MSCs in the presence of NO, which was identified as a potential mechanism contributing to the enhanced endothelium-protective and proangiogenic capacities of hP-MSCs that promote angiogenesis in mouse models of hindlimb ischemia. In conclusion, this study provides a promising approach for using NO hydrogel to improve the proangiogenic potency of MSCs in ischemic diseases, and the strategy used here facilitates the development of controlled-release scaffolds for enhancing the therapeutic efficiency of MSCs in angiogenic therapy.
Abstract 336: Interleukin 4 and 13 Signaling in Myeloid Cells Regulates Cardiac Regeneration
Authors: Alvarez Santiago , Paddock Samantha , Omeara Caitlin
Published: October 2020

Introduction:
Heart failure is a very important disease in the United States and worldwide, with a projected prevalence of 8 million by 2030. Current therapy partially decreases progression; however, mortality continues to be high, and progression to terminal HF is still significant, requiring heart transplant and other interventions that further increase morbidity and overall disease burden and costs. Therefore, the need to develop new therapies that target HF progression. Our prior work and published studies demonstrate that IL13 promotes cardiac regeneration, however, the mechanism mediating this response is currently unknown. IL13 and IL4 share a common receptor, and both cytokines are known to polarize macrophages into a pro-reparative phenotype. Here, we hypothesize that IL4/13 signaling in myeloid cells promotes heart regeneration after cardiac injury.

Methods and Results:
We compared cardiac regeneration in IL4Ra null/FLOXed (IL4Rα-/fl) and IL4Rα-/fl lysosome M Cre littermate mice, knocking out IL4Rα in myeloid cells. We performed apical resection on postnatal day 1 (P1) mice and assessed cardiac regeneration by histological analysis. We found that mice lacking IL4Ra in myeloid cells (IL4rafl/-, LysMCre) had significantly impaired cardiac regenerative capacity compared to Cre negative littermate controls. We also performed gene expression analysis by qPCR in whole hearts collected at 2dpi to quantify macrophages markers. Cre positive mice trended higher for expression of arginase 1 and mannose receptor 1 (p=0.055 and 0.057, respectively; student t-test) compared to Cre negative littermates.

Conclusions and Discussion:
Lack of IL4/13 signaling in myeloid cells impairs cardiac regeneration after injury in neonatal mice and modulated expression of immune markers. To confirm these results, flow cytometry to quantify immune cell infiltration can be performed. Other components that limit damage extension can also be assessed, like angiogenesis and cardiomyocyte proliferation. Our data suggest that IL4/IL13 signaling polarizes myeloid cells to a pro-reparative state in the neonatal heart. Additional studies are required to delineate the specific cell population mediating this response.
Abstract 340: Myocyte-borne Bdnf is Essential to Limit Post-ischemic Cardiac Injury and Dysfunction
Authors: Jun Seungho , Agrimi Jacopo , Cannavo Alessandro , Keceli Gizem , Oeing Christian U , Eremiev Alexander , Marcucci Lorenzo , Megighian Aram , RENGO Giuseppe , Koch Walter J , Paolocci Nazareno
Published: October 2020

Brain-derived neurotrophic factor
(BDNF)/
tyrosine receptor kinase B
(TrkB) signaling is essential for normal cardiac contraction/relaxation. Alterations in this pathway, i.e., defective neuronal BDNF, account for post-ischemic cardiac injury. Less clear, however, is whether myocyte-borne BDNF has a role in this setting. We generated myocyte-selective BDNF knock-out (myoBDNF
-/-
) mice, using Myh6-Cre mice crossed with BDNF
floxed
mice, confirming
bdnf
deletion via RT-PCR in isolated myocytes. Hearts from 12-wk old myoBDNF
-/-
mice and WT littermates underwent global ischemia (30 min) and reperfusion (2 h). At this age, the two strains had similar left ventricular (LV) sizes and fractional shortening [63±1.1 (WT)
vs.
60±1.2% (myoBDNF
-/-
)]. At reperfusion, myoBDNF
-/-
hearts displayed larger infarct size compared to WT (38±3
vs.
14±2%, n=9, p<.0001) and worsened LV functional recovery (
Fig. 1
). For example, the rate-pressure product recuperated only by 14±1.5 in myoBDNF
-/-
mice
vs.
36±3% in WT (p<.0001). The two groups had similar heart rates at 2 h reperfusion, however myoBDNF
-/-
mice markedly lost their contractility [dP/dt
max
= 436±58
vs.
1407±142 mmHg/sec (WT), p< .0001], likely due to the exacerbated myocyte loss. Accordingly, post-ischemic troponin I release was significantly higher in myoBDNF
-/-
than in WT mice (0.9±0.04
vs.
1.3±0.04 ng/ml, p<.0002). Thus, deleting
bdnf
in myocytes severely limits recovery after ischemia, directly linking myocyte-borne BDNF to the heart response to injury. Therefore, preserving or enhancing autologous myocyte BDNF generation offers new avenues to counter cardiac ischemic injury and subsequent heart failure progression.
Abstract 344: Circulating MicroRNA Signature as a Predictive Biomarker for Postoperative Heart Failure
Authors: Rizvi Farhan , Kroboth Stacie , Emelyanova Larisa , Ross Gracious R , Singh Maharaj , Kress David , Tajik A. Jamil , Jahangir Arshad
Published: October 2020

Background:
Advancements in cardiac surgical techniques have led to decreasing operative risk. However, postoperative heart failure (PoHF) continues to be a major risk factor for adverse cardiac events in 20-35% of patients after cardiac surgery, with a 10-fold increase in 30-day mortality. Prediction of PoHF is challenging, particularly in patients with preserved ventricular function. Circulating microRNAs (miRNAs) recently were identified to predict HF or AF after surgery, but their role in predicting PoHF is not known. This study aimed to find novel noninvasive circulating biomarkers along with clinical factors that can identify patients at risk of developing PoHF immediately after surgery.

Methods:
Patients undergoing CABG surgery with no previous history of HF, ventricular or supraventricular tachycardia were recruited, and preoperative blood assessed for circulating levels of protein biomarkers using ELISA. Differences in relative plasma levels of 13 miRNAs between the PoHF and no-PoHF groups were assessed by qPCR. Preoperative echocardiography was obtained. SAS was used for statistical analysis and ROC curve.

Results:
Out of 68 patients, 13 developed PoHF (19.1%, mean age 64.1±11.6y, 53.8% males), whereas 55 (mean age 68.3±12.4y) remained free of HF. Patients who developed PoHF had lower LVEF (51.4±13.7 vs 58.2±9.9, P<0.05) with no differences in prevalence of hypertension, diabetes, hyperlipidemia, obesity, previous myocardial infarction, stroke, COPD, sleep apnea, or use of cardiac medications. The correlation matrix of all 13 miRNAs was transformed in a principal component (PC), resulting in 3 main clusters with eigenvalue >1. PC cluster2 consisted of miR-23a, -23b, -25 and -26a2, principally involved in oxidatives stress, fibrosis and contractility, and had the strongest association (AUC=0.797; P<0.01) with PoHF. A model combining PC cluster2 with age and LVEF improved sensitivity and specificity of the model to identify patients at risk of PoHF (AUC=0.880; 95% CL, 0.761-0.991; P<0.001)

Conclusion:
Our study demonstrates that miR-23a, -23b, -25 and -26a2 may be useful predictors of PoHF. Circulating miRNA as biomarkers may have diagnostic potential to preoperatively, noninvasively identify patients at risk of developing PoHF.
Abstract 352: Optimization of a CRISPR Activation Human Pluripotent Stem Cell Line for Screening of Candidate Regulators of Cardiomyocyte Maturation
Authors: Karbassi Elaheh , Bertero Alessandro , Kadota Shin , Fields Paul , Pabon Lil , Reinecke Hans , Hauschka Stephen , Murry Charles E
Published: October 2020

Human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) applications for cell therapy and disease modeling are limited due to the cells’ lack of resemblance structurally and functionally to adult cardiomyocytes. To understand hPSC-CM maturation, we characterized two established approaches to mature cardiomyocytes—long term culture (aging of cells in a dish) and
in vivo
transplantation to an infarcted adult rat heart. RNA sequencing of hPSC-CMs from these systems demonstrated that
in vivo
transplantation is much more effective in maturing hPSC-CMs, inducing a more adult-like cardiac gene program (e.g. upregulation of
TNNI3, MYL2, SCN5A
), compared to cells kept in culture up to one year. Using this dataset, we identified candidate drivers of hPSC-CM maturation, including transcription factors and chromatin regulators, that we hypothesize are necessary to program hPSC-CMs to an adult-like state. To test the relationship between transcription factor regulation and hPSC-CM maturation, we developed a constitutive CRISPR activation (CRISPRa) pluripotent stem cell line to upregulate these transcriptional regulators upon addition of guide RNAs (gRNA). This cell line expresses nuclease-deficient Cas9 fused to the transcriptional activator VPR (dCas9-VPR), driven by the strong CAG promoter and targeted to the
AAVS1
safe harbor site. In pluripotent stem cells, target genes are upregulated up to 150-fold when gRNA is present; however, after differentiation into cardiomyocytes, dCas9-VPR transgene expression is silenced, and dCas9-VPR levels are insufficient to activate gRNA-targeted genes. To optimize CRISPRa for cardiomyocyte applications, we are generating alternative stem cell lines with dCas9-VPR targeted to the human
ROSA26
safe harbor site or driven by a cardiac-specific troponin T promoter, testing the regulation of transgene expression mediated by safe harbor site or promoter respectively. The characterization of these CRISPRa cell lines provides insights into CRISPR expression regulation and genome engineering strategies for applications in stem cells and hPSC-CMs. We will use this system to screen for maturation regulators and identify key combinations that are effective in programming hPSC-CMs towards an adult-like state.
Abstract 356: Rice Bran Extracts and Its Active Compound, γ-oryzanol, Prevent Particulate Matters-induced Endothelium Senescence
Authors: Gong Dal-Seong , Sharma Kushal , KANG KI-WOON , Oak Min-Ho
Published: October 2020

Particulate matters (PMs) derived from are known to be responsible for a wide range of diseases. Recent studies have indicated that long-term exposure PMs is associated with several cardiovascular diseases, at least in part, by increase of inflammation, oxidative stress and nitric oxide (NO) balance disturbance. The present study was to investigate the protective effect of rice bran and its active compound, γ-oryzanol (γ-Orz), on PMs-induced premature endothelial senescence, and if then, to explore the underlying mechanism. Premature senescence was induced in porcine coronary arterial endothelial cells (PCAECs) by incubating 30 μg/mL of PM10. Senescence-associated β-galactosidase (SA-β-gal) activity, oxidative stress, expression level of cell cycle regulatory proteins, nicotinamide adenine dinucleotide phosphate(NADPH) oxidase, and local angiotensin system (LAS) component were evaluated. The result showed that senescence associated-β-gal activity in the PMs-treated PCAECs was attenuated by rice bran extracts (RBEs) and its major constituent, γ-Orz. Pretreatment with γ-Orz inhibited PMs-induced oxidative stress in PCAECs. Similarly, γ-Orz restored the cell proliferation of PCAECs by inhibiting PMs -induced cell cycle arrest. Western blot analyses revealed that cell cycle regulatory protein such as p53, p16, and p21 in senescent PCAECs treated with γ-Orz were reduced from PMs-exposed cells. In addition, γ- Orz prevented senescence associated NADPH oxidase, LAS activity of PM10-exposed cells. Pretreatment of γ-Orz restored PM10-induced endothelial dysfunction by upregulation of endothelial NO synthase expression. The present study demonstrated that γ-Orz protects against PMs induced PCAECs senescence, potentially through abrogating oxidative stress pathway. Taken together, this study can serve as a pharmacological use of rice bran extracts and γ-Orz for the prevention and/or treatment of air pollution-associated coronary artery diseases.