bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 118: Adipose Inflammation Is Critically Involved in the Progression of Heart Failure During Pressure Overload
Authors: Shimizu Ippei , Minamino Tohru , Yoshida Yohko , Katsuno Taro , Komuro Issei , Kobayashi Yoshio
Published: July 2021

Several clinical studies have shown that insulin resistance is prevalent among patients with impaired cardiac function and that systemic insulin resistance is the risk factor for the development of heart failure; however, underlying mechanisms have not been fully elucidated. We have previously reported that increased p53 level in adipose tissue is crucially involved in adipose inflammation and insulin resistance during pressure overload. Here we show that . Pressure overload increased sympathetic activity and promoted lipolysis in adipose tissue. Accelerated lipolysis resulted in increases of reactive oxygen species and DNA damage, leading to up-regulation of adipose p53. This up-regulation activated the NF-kappaB pathway and induced adipose inflammation and insulin resistance. Genetic disruption of adipose p53 markedly attenuated adipose inflammation and metabolic abnormalities associated with heart failure. We also observed that cardiac function and survival in the chronic phase of heart failure were significantly better in adipose tissue p53-deficient mice than control littermates. Pharmacological inhibition of adipose p53 after imposing pressure overload also improved cardiac dysfunction as well as insulin resistance in the chronic phase of heart failure. These results suggest that inhibition of adipose inflammation is a potential target for treating metabolic abnormalities and systolic dysfunction in patients with heart failure.
Abstract 123: SUMO1 Modification Regulates SR Calcium ATPase Pump, SERCA2a in Heart Failure
Authors: Kho Changwon , Lee Ahyoung , Jeong Dongtak , Oh Jae Gyun , Chaanine Antoine , Park Woo Jin , Hajjar Roger
Published: July 2021

Background:
The cardiac calcium ATPase, SERCA2a, is a critical pump responsible for Ca2+ re-uptake during excitation-contraction coupling. Impaired Ca2+ uptake resulting from decreased expression and reduced activity of SERCA2a is a hallmark of heart failure. Accordingly, restoration of SERCA2a expression by gene transfer has proved to be effective in improving cardiac function in heart-failure patients, as well as in animal models. However, the underlying mechanisms of SERCA2a’s dysfunction remain incompletely understood.

Methods and Results:
In this study, we show that SERCA2a is modified by SUMO1 at lysine sites 480 and 585 and that this SUMOylation is essential for preserving SERCA2a ATPase activity and stability in mouse and human cells. SUMO1 and SERCA2a SUMOylation levels were both decreased in mouse and pig models of heart failure and failing human left ventricles. To determine whether reduced SUMO1 levels are responsible for reduced SERCA2a protein levels and reduced cardiac function, we used an adenovirus associated virus-mediated gene delivery approach to up-regulate SUMO1 in trans aortic constriction-induced mouse model of heart failure. We found that increasing SUMO1 levels led to a restoration of SERCA2a levels, improved hemodynamic performance, and reduced mouse mortality. By contrast, down-regulation of SUMO1 using small hairpin RNA accelerated cardiac functional deterioration and was accompanied by decreased SERCA2a function.

Conclusion:
In this study, we study a new mechanism for modulation of SERCA2a activity and beneficial effects of SUMO1 in the setting of heart failure. It suggests that changes in post-translational modifications of SERCA2a could negatively affect cardiac function in heart failure. Our data may provide a new platform for the design of therapeutic strategies for heart failure.
Abstract 153: Creation of Cell Sheet-Based Bioengineered Cardiac Tissue Using Pluripotent Stem Cell-Derived Cells
Authors: Matsuura Katsuhisa , Aoki Shinako , Haraguchi Yuji , Shimizu Tatsuya , Hagiwara Nobuhisa , Okano Teruo
Published: July 2021

Recent evidences have suggested that current cardiac cell therapy contributes to the improved cardiac function through mainly the paracrine effects. Thereby the bioengineered functional heart tissue is expected to function for repairing the broad injured heart. We have developed the cell sheet-based bioengineered vascularized cardiac tissue, however the system to collect the enough amount of cells from ES/iPS cells and the function of ES-derived cardiac tissue remain elusive. Recently we have established the cultivation system with the suitable conditions for expansion and cardiac differentiation of mouse ES cells and human iPS cells via embryoid body formation using three-dimensional bioreactor with the continuous perfusion system. For the cardiac differentiation experiments, we used several mouse ES cells that express EGFP or neomycin resistant gene under the control of αMHC promoter. At 10 days of differentiation, mouse ES cells increased up to 300 times (6.0×10
6
cells/mL). After the further 8 days of cultivation with the purification step, we collected around 5.0×10
8
cells in the 1L bioreactor culture and 99% of cells were positive for myosin heavy chain. The co-culture of ES-derived cardiomyocytes with the appropriate number of primary cultured fibroblasts on the temperature-responsive culture dishes enabled to form the cardiac cell sheets. Furthermore, when ES-derived cardiomyocytes were co-cultured with ES-derived endothelial cells, robust endothelial cell network was observed in the cardiac cell sheets. Mouse ES- or human iPS-derived cardiomyocytes in cell sheets beat spontaneously and synchronously and connexin43 was expressed at the edge of the adjacent cardiomyocytes. Furthermore the action potential propagation was observed between ES/iPS-derived cardiac cell sheets. These findings suggest that pluripotent stem cell-derived cardiomyocytes and endothelial cells might be useful for creating cell-sheet-based functional cardiac tissue and the layered stem cell-derived cardiac tissue might promote not only the cardiac regenerative medicine but also the understanding the molecular mechanisms of heart diseases.
Abstract 156: The Nitroxyl Donor Angeli's Salt Elicits Concomitant Concentration-Dependent Coronary Vasodilatory and Inotropic Actions in the Intact Rat Heart
Authors: Ritchie Rebecca H , Chin Kai Yee , Qin Chengxue , Cao Nga , Kemp-Harper Barbara K , Woodman Owen L
Published: July 2021

Nitroxyl (HNO) is a novel redox sibling of NO• that elicits vasodilator, antihypertrophic and superoxide-suppressing actions. HNO is however resistant to scavenging by superoxide, and has high reactivity with thiols (enabling potent enhancement of LV function). In the present study, hearts from adult male Sprague-Dawley rats were Langendorff-perfused at constant pressure. Dose-response curves to the HNO donor Angeli’s salt were performed following preconstriction with U46619 to ∼50% of baseline coronary flow (CF). Angeli’s salt (10pmol-10µmol, n=6) elicited significant dose-dependent increases in left ventricular systolic pressure (LVSP), LV developed pressure (LVDP), LV end-diastolic pressure (LVEDP), LV±dP/dt, and CF. The effects of the highest dose of Angeli's salt studied on LV function are shown in the Table; these were significantly inhibited by co-administration of the HNO scavenger L-cysteine (4mM, n=6) or the soluble guanylyl cyclase inhibitor ODQ (10µM, n=5). The EC
50
for Angeli's salt-induced vasodilatation was increased from -8.6±0.3mol to -7.5±0.2mol (p<0.01) and -7.6±0.3mol (p<0.05) by L-cysteine and ODQ, respectively. Although CGRP is a putative mediator of HNO actions, both the inotropic and vasodilator actions of Angeli's salt were insensitive to the CGRP antagonist CGRP
8-37
(0.1µM, n=5). The vehicle for Angeli’s salt, 0.01M NaOH, had no effect on any of these parameters (n=3, results not shown). Our results demonstrate that the cardiac effects of Angeli’s salt are thiol-sensitive, and mediated by HNO and cGMP, but not CGRP. HNO donors represent potential therapy for the failing heart, alone or in addition to standard care.
Abstract 167: MicroRNA Profiles of Non--ST-Elevation Myocardial Infarction
Authors: Ward Jeanine , de la Puente Rafael , McManus David
Published: July 2021

Significance:

MicroRNAs (miRNAs) are short, stable, noncoding translational regulators used as early biomarkers for numerous disease processes. Although specific miRNA patterns are associated with STEMI, the profile and timecourse of miRNA release in NSTEMI is currently unknown but of potential clinical significance.Materials and

Methods:

With IRB approval, patients identified with chest pain were identified at the UMass Medical Center. Upon ED arrival (T0), a K2EDTA tube of whole blood was obtained with simultaneous troponin cTnI levels. After a 6 hour (T6) observation period, another additional K2EDTA tube was drawn, again contemporaneous with cTnI levels. Patients were selected based on an elevated cTnI level (greater then or equal to ≥ 0.04 ng/ml) at T6 as compared to a negative cTnI levels (< 0.04 ng/ml) at T0. MiRNA was isolated from patient plasma using a Trizol extraction procedure. Real-time reverse transciptase polymerase chain reaction (QRT-PCR) was performed for 94 a priori miRNAs common to cardiovascular development or disease.

Results:

Of the 94 miRNAs examined, 11 demonstrated greater than 2-fold change as compared to baseline (Figure 1). A subset of this cohort, namely miRNAs 208a, 483-5p, and 601 revealed statistically significant fold difference (p< 0.05). MiRNA 208a is associated with acute MI. MiRNA 601 has not been previously shown correlated with heart disease.

Conclusion:

This investigation reveals a novel 11 member miRNA profile, including miRNA 208a, 483-5p, and 601, which is associated with NSTEMI. Our findings suggest this miRNA profile by be used as a potential biomarker of myocardial injury.
Abstract 172: The Role of Activin A in Maternal Diabetes-Induced Cardiac Malformations
Authors: Zhao Zhiyong
Published: July 2021

Diabetes mellitus in pregnancy causes heart defects, especially atrioventricular septal defects (AVSDs), in infants. Manifestation of AVSDs results from dysmorphogenesis of the endocardial cushions in the embryo, which is regulated by members of the transforming growth factor (TGF) β family. Among the TGFβs, we have previously observed that the
inhibin
βA
gene, which encodes a protein to form homodimers as activin A, is markedly downregulated by maternal diabetes. To further determine the involvement of inhibin βA and its signaling in diabetic embryopathy, the levels of inhibin βA protein and activation of its downstream transcription factors, Smad2 and Smad3, were examined using immunohistochemical, proximity ligation, and immunoblot assays, and shown to be decreased in the embryonic hearts of diabetic mice induced via intravenous injection of streptozotocin. To investigate the role of activin A in hyperglycemia-induced cardiac malformations, mouse embryos at embryonic day 9.5 (E9.5) cultured in high glucose (22 mM) were treated with activin A (50 ng/ml) for 24 hours. The treatment rescued the development of the endocardial cushions and cell proliferation (BrdU-incorporation assay) in the myocardium, similar to those in normal glucose control (8.3 mM). The role of activin A in endocardial cell migration, an important process for cellularization of the cardiac jelly, was investigated by treating E10.5 endocardial cushion explants cultured in high glucose with activin A (50 ng/ml) in a collagen matrix-based assay system. The treatment significantly increased the number of migrating cells, compared with that in high glucose-treated group, to the level in the control group. These effects were associated with restoration of the activation of Smad2/3. The results demonstrate that the activin-Smad2/3 signaling system plays an important role in cardiac malformation in diabetic embryopathy.
Abstract 188: Atrial Tachyarrhythmia in Rgs5-Null Mice
Authors: Qin Mu , Wang Teng , Hu He , Liu Yu , Huang He , Li Hong L , Huang Cong X
Published: July 2021

Abstract
Aims
The aim of this study was to elucidate the effects of regulator of G-protein signaling 5 (Rgs5), a negative regulator of G protein-mediated signaling, on atrial repolarization and tachyarrhythmia (ATA) in mice.

Methods and Results
In present study, the incidence of ATA were increased in isolated mouse Rgs5
-/-
hearts during program electrical stimulation (PES) (46.7%, 7 of 15) and burst pacing (26.7%, 4 of 15) compared with wild-type (WT) mice (PES: 7.1%,1 of 14; burst:7.1%,1 of 14) (P<0.05). And the duration of ATA also shown longer in Rgs5

[[Unable to Display Character:
+
]]/-

heart than that in WT, 2 out of 15 hearts exhibited sustained ATA (>30s) but none of them observed in WT mice. Atrial prolonged repolarization was observed in Rgs5
-/-
hearts including widened P wave in surface ECG recording, increased action potential duration (APD) and atrial effective refractory periods (AERP), all of them showed significant difference with WT mice (P<0.05). At the cellular level, whole-cell patch clamp recorded markedly increased densities of repolarizing K
+
currents including I
Kur
(at + 60mV: 14.0 ± 2.2 pF/pA) and I
to
(at + 60mV: 16.7±1.3 pA/pF) in Rgs5
-/-
atrial cardiomyocytes, compared to those of WT mice (at + 60mV I
to
: 20.4 ± 2.0 pA/pF; I
kur
: 17.9 ± 2.0 pF/pA) (P<0.05).

Conclusion
These results suggest that Rgs5 is an important regulator of arrhythmogenesis in the mouse atrium and that the enhanced susceptibility to atrial tachyarrhythmias in Rgs5
-/-
mice may contribute to abnormalities of atrial repolarization.
Abstract 196: Self-Organized Formation of Cardiac Pacemaker Tissues in Embryoid Bodies
Authors: Hashem Sherin I , Claycomb William C
Published: July 2021

The pacemaker tissues of the heart are a complex set of specialized cells that initiate the rhythmic heart beat. The sinoatrial node (SAN) serves as the primary pacemaker, whereas the atrioventricular node (AVN) serves as a subsidiary pacemaker under conditions of SAN failure. The elucidation of genetic networks regulating the development of these tissues is crucial for understanding the mechanisms underlying arrhythmias and for the design of biological pacemakers. At present, there is no
in vitro
model system in which these specialized cells are defined or targeted. Here we report efficient self-organized formation of the two pacemaker nodes in three-dimensional aggregate cultures of mouse embryonic stem (ES) cells known as embryoid bodies (EBs), as well as the isolation of ES cell-derived AVN cells. A
Shox2-lacZ
knockin ES cell line carrying a
Cx30.2
enhancer-RFP construct was used to generate EBs. The
Shox2
gene, a determinant of the SAN genetic cascade, was used to delineate the SAN in EBs. The
Cx30.2
enhancer was used to direct RFP expression to the AVN in EBs. Using live fluorescent imaging and immunohistochemistry, we demonstrate that these genetic markers reproducibly delineate cell clusters which express nodal proteins. These clusters are functionally connected with, and consistently located adjacent to the contracting region of the EB in an organized manner. We demonstrate that EBs generated using
Shox2
knockout ES cells exhibit a hypoplastic SAN phenotype that includes reduction in spontaneous contraction rates and altered expression of Shox2 downstream targets. We isolated an ES cell-derived AVN cell line using a genetic selection technique and demonstrated that these cells display nodal characteristics such as calcium dependent spontaneous depolarizations and an AVN gene expression profile. When these cells were grown as three-dimensional aggregates they induced synchronous contraction of surrounding cardiac myocytes in co-culture experiments. Using molecular markers, we have generated a reproducible model system and have isolated an AVN cell line that will be invaluable tools for studying the molecular pathways regulating the development of the cardiac pacemaker tissues and molecular composition of these specialized cells.
Abstract 202: Insulin Resistance as an Independent Predictor of Atherosclerosis Burden in Nondiabetic Patients with Acute Myocardial Infarction
Authors: Karrowni Wassef , Li Yan , Jones Philip G , Buchanan Donna M , El Accaoui Ramzi , Cresci Sharon , Abdallah Mouin , Lanfear David E , Maddox Thomas M , McGuire Darren K , Spertus John A , Horwitz Phillip A
Published: July 2021

Introduction:
Experimental evidence suggests that insulin resistance (IR) is a major risk factor for accelerated atherosclerosis through indirect effects on systemic risk factors and direct effects on insulin signaling in vascular endothelium and plaque macrophages.

Hypothesis:
IR in persons without diabetes mellitus is associated with multi-vessel coronary artery disease (CAD) in acute myocardial infarction (AMI) patients.

Methods:
We examined 1111 non-diabetic AMI patients (HbA1c ≤ 6.5) enrolled in the multicenter TRIUMPH (years 2004-2008) registry. IR was estimated using the homeostasis model assessment of IR (HOMA-IR: fasting insulin (mU/l) x fasting blood glucose (mmol/l)]/22.5). The primary outcome was the extent of angiographic atherosclerosis (≤1-vessel vs. multi-vessel disease). Modified Poisson regression models were used to examine the association between IR and multi-vessel CAD.

Results:
Subjects were divided into quartiles based on HOMA-IR values (quartile 1: male <=1.35, female <=1.26; quartile 4: male >4.00, female >3.64). After adjusting for age, sex, hypertension, smoking, family history of CAD, LDL, HDL, and body mass index (BMI), IR was independently associated with multi-vessel CAD (Figure). As compared to the lowest quartile of IR, the highest quartile had more multi-vessel CAD [RR (95% CI)- 1.35 (1.11-1.64)].

Conclusion:
In a large cohort of non-diabetic AMI patients, IR was associated with more advanced, multi-vessel CAD, independent of lipid levels and other components of the metabolic syndrome. This association is consistent with established experimental evidence and supports IR as a potential target for preventing atherosclerosis.
Abstract 215: Systemic Inflammation Exacerbates Electrophysiological Remodeling and Arrhythmia Following Myocardial Infarction
Authors: De Jesus Nicole , Wang Lianguo , Ripplinger Crystal M
Published: July 2021

Introduction: Inflammatory macrophages infiltrate the heart following myocardial infarction (MI) and secrete proteases and cytokines that may contribute to adverse electrophysiological remodeling. Atherosclerosis, the leading cause of MI, is associated with elevated systemic inflammation. Using a novel combination of optical mapping and molecular imaging, we sought to determine if systemic inflammation exacerbates electrophysiological remodeling and arrhythmogenesis.

Methods: A protease activatable fluorescent sensor (Prosense680) was injected I.V. 4 days post MI in mice. Optical mapping of transmembrane potential was performed 5 days post MI on Langendorff-perfused hearts to assess electrophysiology. Fluorescence reflectance imaging of Prosense680 was then performed to visualize and quantify inflammatory protease activity. Lipopolysaccharide (LPS:10 μ g/day, n=6) was injected I.P. to promote systemic inflammation or saline as control (SAL, n=7).

Results: Inflammation levels were greater in MI+LPS vs MI+SAL hearts (5.626±1.5 vs 1.73±0.25 A.U., p<0.02) and negligible in sham-operated hearts (SH, n=4). MI+LPS hearts had prolonged action potential (AP) duration vs MI+SAL vs SH (62±4.5 vs 56±1.8 vs 45±3.7 ms, p<0.05) and increased AP rise time (5.5±0.5 vs 4.2±0.2 vs 2.9±0.3 ms, p<0.05). A single premature pacing stimulus induced ventricular tachycardia in 67%(4 of 6) of MI+LPS vs 15%(1 of 7) MI+SAL vs 0% (0 of 4) SH, p<0.02.

Conclusions: Elevated post-MI inflammation may contribute to electrophysiological remodeling and arrhythmias. These findings have important implications for the etiology of post-MI arrhythmias in humans with systemic inflammation.
Abstract 238: Role of Erythropoietin Signaling in the Biology of Mouse Cardiac Progenitor Cells
Authors: Zafiriou Maria P , Noack Claudia , Didie Michael , Unsoeld Bernhard , El-Armouche Ali , Bergmann Martin W , Zimmermann Wolfram H , Zelarayan Laura C
Published: July 2021

Erythropoietin (Epo) was shown to improve cardiac function following ischemia reperfusion mainly via neo-angiogenesis and anti-apoptotic mechanisms. We found EpoR expression to be particularly high in adult cardiac progenitor cells (CPCs). Thus, we reasoned that Epo may play a role in the biology of these cells.

We isolated CPCs from adult C57BL/6 hearts by enzymatic digestion and filtration (pore size: 30 µm). By means of immunofluorescence microscopy (IF) and flow cytometry (FC) we analyzed EpoR expression in the CPCs. 24±3% of the investigated cardiac cells were positive for EpoR with 3±2% of these being c-kit+ and 28%±2% Sca-1+. 52% of the EpoR+ cells expressed endothelial cell markers (40±2% CD34+, 9±2% FLK1+). 42±4% expressed myocyte markers (αMHC+, cTNT+). IF revealed a progenitor-like population with immature cell morphology and proliferation potential (ki67+). Cell cycle analysis showed an enrichment of αMHC+ EpoR+ cells in S and G2 phase (49±7%, n=3) as compared to the αMHC- EpoR- population (13±3%, n=3). Moreover, we tested the effect of Epo in the biology of these CPCs in vitro. At d14 we observed a two-fold increase of GATA4+ and cTnT+ cardiac cells in the co-cultures treated with Epo (n=3). CPC cycle arrest abrogated the aforementioned effects, suggesting that Epo influences mainly CPC proliferation. Finally, we tested the potential of Epo to protect against ischemia by inducing the proliferation of these αMHC+ CPCs in vivo in a myocardial infarction (MI) model. 4 weeks post MI, echocardiography did not reveal a significant functional improvement of the Epo receiving mice (2x, 2U/g Epo i.p). Nevertheless, FC analysis of the progenitor pool showed a significant augmentation of αMHC+ and cTnT+ cells (Sham: 19±3% vs Epo 35±3%, n=5; MI: 10.6±2.3%, n=6 vs Epo 20.3±1.9%, n=8). These data suggest an activation of myogenic progenitors by Epo, despite the lack of apparent regeneration under the investigated conditions.

In conclusion, we found that EpoR is expressed in a putative cardiomyogenic progenitor cell pool in the adult heart. Epo drives their proliferation in vitro and in vivo even upon acute cardiac injury. We are currently investigating the long-term consequences of the observed progenitor cell activation in models of chronic ischemic injury.
Abstract 270: The Core Function of Calcineurin
Authors: Ritter Oliver , Czolbe Martin , Seidelmayer Lea , Nordbeck Peter , Williams Tatjana
Published: July 2021

Background:
Recent evidence demonstrates that not only NFAT, but also calcineurin is translocated into the nucleus upon hypertrophic stimulation. Previously it was also demonstrated that calpain-mediated degradation caused a constitutive active calcineurin. We hypothesised that nuclear calcineurin is an intranuclear Ca
2+
sensor hypertrophied myocardium and that inhibition of nuclear translocation of calcineurin is a therapeutic strategy to prevent hypertrophy.

Methods:
Employing a transgene mouse model with conditional calpastatin overexpression (”tet-off”, resulting in calpain inhibition), different adenoviral calcineurin mutants and confocal microscopy in isolated adult cardiac myocytes we investigated calcineurin translocation and nuclear Ca
2+
transients. Assessment of cardiac function if transgenic animals was performed by 7T MRI.

Results:
We could demonstrate that chronic Ang II stimulation of mice caused calpain-mediated degradation of calcineurin resulting in a constitutive active calcineurin with nuclear translocation. The constitutive active calcineurin in the nucleus escaped further degradation by the UPS and sustained an ongoing hypertrophic response, even after removal of Ang II. Inhibition of nuclear translocation of activated calcineurin by a small inhibitory peptide prevented myocardial hypertrophy in vivo. Transgenic inhibition of calpain activity by calpastatin overexpression prevented proteolysis of calcineurin and allowed for relocation of calcineurin from the nucleus back to the cytosol and regression hypertrophy after removal of Ang II. We were also able to demonstrate that Ang II increases nuclear Ca
2+
transients via InsP3 receptors and that calcineurin is able to act as nuclear Ca
2+
sensor detecting local Ca
2+
release from the nuclear envelope via InsP3R. Nuclear calcineurin mutants that are defective for Ca
2+
activation failed to activate NFAT dependent transcription.

Conclusion:
This provides an explanation how Ca
2+
and calcineurin can regulate transcription in cardiomyocytes in response to neurohumoral signals apart from Ca
2+
changes in contraction regulation.
Abstract 286: Long Noncoding RNAs Are Differentially Expressed in Heart Failure
Authors: Robinson Emma L , Haider Syed , Hei Hillary , Lee Richard T , Foo Roger S
Published: July 2021

Heart failure comprises of clinically distinct inciting causes but a consistent pattern of change in myocardial gene expression supports the hypothesis that unifying biochemical mechanisms underlie disease progression. The recent RNA-seq revolution has enabled whole transcriptome profiling, using deep-sequencing technologies. Up to 70% of the genome is now known to be transcribed into RNA, a significant proportion of which is long non-coding RNAs (lncRNAs), defined as polyribonucleotides of ≥200 nucleotides. This project aims to discover whether the myocardium expression of lncRNAs changes in the failing heart.

Paired end RNA-seq from a 300-400bp library of ‘stretched’ mouse myocyte total RNA was carried out to generate 76-mer sequence reads. Mechanically stretching myocytes with equibiaxial stretch apparatus mimics pathological hypertrophy in the heart. Transcripts were assembled and aligned to reference genome mm9 (UCSC), abundance determined and differential expression of novel transcripts and alternative splice variants were compared with that of control (non-stretched) mouse myocytes. Five novel transcripts have been identified in our RNA-seq that are differentially expressed in stretched myocytes compared with non-stretched. These are regions of the genome that are currently unannotated and potentially are transcribed into non-coding RNAs. Roles of known lncRNAs include control of gene expression, either by direct interaction with complementary regions of the genome or association with chromatin remodelling complexes which act on the epigenome.Changes in expression of genes which contribute to the deterioration of the failing heart could be due to the actions of these novel lncRNAs, immediately suggesting a target for new pharmaceuticals.

Changes in the expression of these novel transcripts will be validated in a larger sample size of stretched myocytes vs non-stretched myocytes as well as in the hearts of transverse aortic constriction (TAC) mice vs Sham (surgical procedure without the aortic banding).
In vivo
investigations will then be carried out, using siLNA antisense technology to silence novel lncRNAs in mice.
Abstract 30: Endurance Exercise Induces Cardiac Hypertrophy in Aged Nrf2-/- Mice
Authors: Namakkal Soorappan Rajasekaran , Devdoss Dinesh , Kannan Snkaranarayanan , Olsen Curtis , Subbanna Gounder Sellamuthu , Davidson Christopher J , Whitehead Kevin , Abel E. Dale
Published: July 2021

Background:

Radical forms of oxygen and nitrogen species (ROS/RNS) are highly reactive with nucleic acids, proteins and lipids and promote their oxidation. Normally, cellular ROS/RNS concentrations are tightly controlled by the inducible antioxidant system, which is predominantly regulated by the transcription factor Nrf2 (nuclear erythroid-2 like factor-2) and its cytosolic repressor protein, Keap1. We hypothesized that a decrease or an abrogation of Nrf2 impairs cardiac function and induce hypertrophy upon endurance stress in aging heart.

Methods:

Age-matched wild-type (WT) and Nrf2-/- (KO) mice (n=12/gp) at 2 and >20 months were subjected to endurance exercise stress (EES; 20 meter/min, 12% grade) and assessed the activation of Nrf2/ARE-dependent transcriptional mechanisms in the heart. Cardiac hypertrophy was determined by echocardiography, heart/body weight ratio and biochemical/molecular marker analyses.

Results:

Interestingly, both old-WT and Nrf2-/- mice exhibited oxidative stress on EES due to significant decrease or abrogation of Nrf2 nuclear levels, respectively, suggesting that aged-WT is equally susceptible to stress as Nrf2-/- mice. Age-dependent loss of Nrf2 decreased the transcription of Nrf2-dependent antioxidants and thereby elevated ROS levels to cause a more oxidized intracellular environment. Importantly, the loss of Nrf2 induced cardiac hypertrophy upon endurance stress in the aged (>20 mon) mice. At the end of 2-weeks of endurance stress, both the old-WT and Nrf2-/- mice had developed cardiac hypertrophy. Also, qPCR analysis showed significant (p<0.05) upregulation of hypertrophy markers (ANF and BNF) in the old-WT or Nrf2-/- when compared to sedentary WT mice confirmed cardiac hypertrophy due to loss of Nrf2. These results indicate that either a decrease or an abrogation of Nrf2 can increase susceptibility to stress induced hypertrophy in an aging heart.

Conclusions:

We conclude that enhancing protein levels and stability of nuclear Nrf2 could activate the transcription of major antioxidant enzymes and biosynthesis of key antioxidants. Enhancing protective mechanisms against oxidative stress in the elderly is expected to prevent or delay the onset of age-associated cardiac hypertrophy and cardiomyopathy.
Abstract 301: Decreased Right Ventricular Function in a Novel Model of Pulmonary Insufficiency in Rat
Authors: Jiang Rong , Shen Ming , Ding Guoliang , Wagner Mary B , Kirshbom Paul
Published: July 2021

The population of adult patients with right ventricular (RV) dysfunction is increasing due in part to the increasing number of children with corrected congenital heart defects that now survive to adulthood. We have developed a novel model of pulmonary insufficiency (PI) to investigate the time course of right ventricular failure due to volume loading.

Methods:
PI was created in Sprague-Dawley rats by disrupting the pulmonary valve. After a limited thoracotomy, a custom made hook was inserted through the RV apex into the RV-pulmonary artery annulus and moved back and forth several times to disrupt the valve. After 16-18 weeks, function was assessed by echocardiography (Visualsonics 2100) and RV function was measured using a Langendorff isolated heart system.

Results:
Evidence of PI was noted by regurgitant flow using color Doppler. There was no difference in RV weight or left ventricular function by echo between the sham and PI groups. Tricuspid annular plane systolic excursion (TAPSE) was significantly lower in PI compared to sham (2.46±0.19, n=8 vs. 3.16±0.13, n=5, p<0.05) indicating diminished RV function. Furthermore, in isolated hearts, developed pressure (DP) was significantly smaller in PI compared to sham (25.5±2.7, n=9 vs. 36.5±3.6 mmHg, n=5, p<0.05) as was the minimum derivative of pressure (dPmin, -500±46, n=9 vs. -731±81 mmHg/s, n=5, p<0.02). We separated the PI group into mild-PI and moderate-PI based on the amount of regurgitant flow. Isolated hearts were perfused with dobutamine to determine RV functional reserve. For the sham group, there was a dose-dependent increase of DP in response to dobutamine (36.5±3.6 vs. 38.0±1.7 vs. 51.2±4.1 mmHg for baseline, 100 nM, and 1 μ M (n=5), p<0.05 for 1 μ M vs. baseline and 100 nM). Similar results were found for dPmin and dPmax. In contrast, dobutamine did not significantly increase any functional parameters in the mild or moderate-PI groups.

Conclusions:
Disruption of the pulmonary valve in a rat model caused mild to moderate PI. Measures of RV function (TAPSE and DP) were decreased in the PI group. Response to dobutamine was robust in the sham group and diminished in both mild and moderate PI groups. This is a novel model of PI that can be used to investigate the mechanisms of RV failure in response to volume overload.
Abstract 326: TRPV2 Regulates Cardiomyocyte Function via Altering Ca
2+
Cycling
Authors: Gao Xiaoqian , Koch Sheryl , Jiang Min , Robbins Nathan , Cai Wenfeng , Varma Priyanka , Tranter Michael , Jones W Keith , Wang Hong-Sheng , Rubinstein Jack
Published: July 2021

TRPV2 is a member of transient receptor potential vanilloid (TRPV) family. As a Ca
2+
channel, it can detect various stimuli such as noxious heat (>52°C), membrane stretching, as well as a number of exogenous chemicals, including probenecid, 2-aminoethoxydiphenyl borate, and lysophospholipids. TRPV2 has been found in many tissue types, including neuron and kidney, but the function of TRPV2 in the heart is poorly understood. Here we show TRPV2 is involved in the Ca
2+
cycling process and then regulates the function of the cardiomyocyte.

We identified the mRNA expression of TRPV2 in the cardiac tissues of mice using real-time PCR. By performing echocardiography we found that administration of probenecid, a selective TRPV2 agonist, increased cardiac ejection fraction in mice. This positive inotropic effect of probenecid was also shown in Langendorff perfused mice hearts as increased peak +dP/dt. In isolated ventricular myocytes, we found that probenecid significantly increased myocyte fractional shortening in a dose-dependent manner, which was fully blocked by ruthenium red, a non-selective TRPV2 blocker. We also performed fluorescent studies to examine myocyte Ca
2+
cycling. We found that probenecid significantly increased Ca
2+
transient and resting-state Ca
2+
sparks and this effect was eliminated by ruthenium red. When Ca
2+
storage in sarcoplasmic reticulum (SR) was depleted with caffeine, and SR Ca
2+
reuptake was blocked by thapsigargin at the same time, probenecid did not show any effects in either Ca
2+
transient or Ca
2+
sparks. Our patch clamp experiments indicate that probenecid treatment does not trigger any significant transmembrane Ca
2+
influx. These results point to the important role of TRPV2 in regulating SR Ca
2+
release.

In conclusion, TRPV2 activation may contribute to increased SR Ca
2+
release, leading to the enhancement of myocyte contractility. Thus, TRPV2 plays a potentially important role in controlling the cellular function of heart.
Abstract 341: Transplantation of Cardiac Tissue Sheets Including Defined Cardiovascular Cell Populations Differentiated from Human-Induced Pluripotent Stem Cells Ameliorates Cardiac Dysfunction After Subacute Myocardial Infarction
Authors: Masumoto Hidetoshi , Ikeda Tadashi , Shimizu Tatsuya , Okano Teruo , Sakata Ryuzo , Yamashita Jun K
Published: July 2021

BACKGROUNDS:
To realize cardiac regeneration with human induced pluripotent stem cells (hiPSCs), efficient differentiation from hiPSCs to defined cardiac cell populations (cardiomyocytes [CMs]/ endothelial cells [ECs]/ vascular mural cells [MCs]), and transplantation technique for fair engraftment are required. Recently, we reported that mouse ES cell-derived cardiac tissue sheet transplantation to rat myocardial infarction (MI) model ameliorated cardiac function after MI (Stem Cells, in press). Here we tried to extend this technique to hiPSCs.

METHODS & RESULTS:
We have reported an efficient cardiomyocyte differentiation protocol based on a monolayer culture (PLoS One, 2011), in which cardiac troponin-T (cTnT)-positive CMs robustly appeared with 50-80% efficiency. In this study, we further modified the protocol to induce vascular cells (ECs/MCs) together with CMs by vascular endothelial cell growth factor supplementation, resulted in proportional differentiation of cTnT+-CMs (62.7±11.7% of total cells), VE-cadherin+-ECs (7.8±4.9%) and PDGFRb+-MCs (18.2±11.0%) at differentiation day 15 (n=12). Then, these cells were transferred onto temperature-responsive culture dishes (UpCell dishes; CellSeed, Tokyo, Japan) to form cardiac tissue sheets including defined cardiac populations. After 4 days of culture, we successfully collected self-pulsating cardiac tissue sheets with 7.0×10
5
±2.3 (n=12) of cells consisted of CMs (46.9±15.9% of total cells), ECs (4.1±3.7%), and MCs (22.5±15.7%). Three-layered hiPSC-derived cardiac sheets were transplanted to a MI model of athymic rat heart one week after MI. In transplantation group, echocardiogram showed a significant improvement of systolic function of left ventricle (fractional shortening: 22.6±5.0 vs 36.5±8.0%, p<0.001, n=20) and a decrease in akinetic length (20.8±9.7 vs 2.5±7.7%, p<0.001, n=20) (pre-treatment vs 4weeks after transplantation). We also succeeded in generation of larger sheets (1.6 inch diameter) with the same method.

CONCLUTIONS:
Transplantation of hiPSC-derived cardiac tissue sheets significantly ameliorates cardiac dysfunction after MI. Thus, we developed a valuable technological basis for hiPSC-based cardiac cell therapy.
Abstract 342: Increased Mitochondrial Permeability Transition Pore Opening Dominates Ischemia-Reperfusion Injury in the Aged Female Rat Heart
Authors: Machikas Alexandra M , Hunter James C , Lopez Veronica , Korzick Donna H
Published: July 2021

Background:
Cardiovascular disease remains the leading cause of death in older post-menopausal women. Ischemia/Reperfusion (I/R) injury triggers mitochondrial calcium (Ca
2+
) overload inducing mitochondrial permeability transition pore (MPTP) opening, mitochondrial dysfunction, and cell death potentially by necrosis, apoptosis, and/or autophagy.

Purpose:
We sought to determine if age-associated estrogen deficiency increases mitochondrial Ca
2+
sensitivity, providing a possible mechanism for increased vulnerability to I/R injury in older women.

Methods:
Mitochondrial respiration (MR) was assessed in isolated mitochondria from ventricles of adult (6 mo; n=15) and aged (24 mo; n=18) ovary-intact or ovariectomized (OVX) female F344 rats. MR at complexes I and II was compared in the absence (State 2) and presence (State 3) of ADP to calculate respiratory control index (RCI; state3/state 2). Reduced RCI following Ca
2+
addition was used to assess Ca
2+
sensitivity, while mitochondrial Ca
2+
retention capacity was measured to quantify MPTP opening (CRC; n=4-5/group) prior to and following coronary artery ligation (55 min I and 6 hr R). Apoptosis was examined using DNA laddering and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Markers of autophagy were evaluated by western blotting and mitochondrial morphology through electron microscopy (EM).

Results:
Significant age-dependent decreases in RCI for complex I (12%) and complex II (8%) were observed in the absence of Ca
2+
, and correlated with increased necrosis in aged hearts revealed by triphenyltetrazolium chloride (TTC) staining (p < 0.05). Ca
2+
exposure decreased MR (18-30%; p < 0.05) in Complex I of aged and OVX mitochondria vs adults. Furthermore, CRC worsened with age requiring less Ca
2+
to open the MPTP. Reduced DNA laddering and TUNEL staining combined with increased beclin-1 and cathepsinD expression in aged vs. adult further support a dominant role for necrosis over apoptosis underlying cell death in aged females (n=4-5/group). EM revealed morphological alterations with age and OVX.

Conclusion:
Decreased MR and increased MPTP opening with aging are likely causal in necrotic cell death mechanisms associated with I/R injury observed in post-menopausal women.
Abstract 4: The Mitochondrial Ca
2+
Transport and ATP Production Through Sigma-1 Receptor in Heart
Authors: Fukunaga Kohji , Tagashira Hideaki
Published: July 2021

Objective:
Although sigma-1 receptor is originally postulated as an opioid receptor in the central nervous system, we recently defined the higher expression of sigma-1 receptor in cardiac ventricle and kidney as compared to brain tissues (Expert Opin Ther Targets 2010;14:1009-1022). To address the question whether mitochondrial Ca
2+
transport and ATP production in heart are regulated by sigma-1 receptor stimulation, we tested the anti-hypertrophic effects of the specific sigma-1 receptor agonist, SA4503 in transverse aortic constriction (TAC) mice.

Methods:
We treated mice with SA4503
(0.1, 0.3 and 1.0 mg/kg) orally once a day for 4 weeks after TAC. The cardiac constriction was monitored by echocardiography. The mitochondrial
Ca
2+
transport and ATP production with or without SA4503 treatment were measured in cultured neonatal cardiomyocytes.

Results: The sigma-1 receptor
expression in the left ventricle (LV) decreased significantly over the 4 weeks.
SA4503 administration significantly attenuated TAC-induced myocardial hypertrophy concomitant with the recovery of sigma-1 receptor expression in LV.
SA4503
also ameliorated the impaired LV fractional shortening.
We also investigated the role of sigma-1
receptor
for sarcoplasmic reticulum (SR)-mitochondrial Ca
2+
transport in cultured neonatal rat ventricular cardiomyocytes. Exposure to angiotensin II (Ang II) for 72 hr elicited marked cardiomyocyte hypertrophy and declined phenylephrine (PE)-induced Ca
2+
mobilization into cytosol and mitochondria. SA4503 treatment restored significantly the reduced PE-induced Ca
2+
mobilization into mitochondria. Importantly, The Ang II-induced hypertrophy in vitro and transverse aortic constriction-induced cardiac dysfunction in vivo were associated with the reduced ATP concentration, which was completely restored by SA4503 treatment. NE-100, a sigma-1
receptor
selective antagonist, abolished these effects induced by SA4503.

Conclusion:
The specific sigma-1 receptor agonist, SA4503 ameliorates AngII-induced cardiomyocyte hypertrophy and TAC-induced cardiac dysfunction through restoration of SR-mitochondria Ca
2+
transport via sigma-1
receptor
stimulation, thereby promoting mitochondrial ATP production.
Abstract 41: Explant-Derived Cells from Chronic Heart Failure Activated Endothelial-Mesenchymal Transition and Attenuated Pluripotency Genes Expression
Authors: Zakharova Liudmila , Nural Hikmet , Gaballa Mohamed A
Published: July 2021

Cardiac progenitor cells are generated from atria explants; however the cellular origin and the mechanisms of cell outgrowth are unclear. Using transgenic tamoxifen-induced Willms tumor 1 (Wt1)-Cre/ERT and Cre-activated GFP reporter mice, we found approximately 40% of explant-derived cells and 74% of explant-derived c-Kit+ cells originated from the epicardium. In atria from sham hearts, Wt1+ cells were located in a thin epicardial layer, while c-Kit+ cells were primarily found within both the sub-epicardium and the myocardium, albeit at low frequency. No overlap between c-Kit+ and Wt1+ cells was observed, suggesting that epicardial Wt1+ cells do not express c-Kit marker in vivo, but more likely the c-Kit marker was acquired in culture. Compared with 4 days in culture, at day 21 we observed 7 folds increase in Snail gene expression; 32% increase in α-smooth muscle actin (SMA) marker, and 30% decrease in E-cadherin marker, suggesting that the explant-derived cells underwent epithelial to mesenchymal transition (EMT) in vitro. Cell outgrowths released TGF-β (1036.4 ± 1.18 pm/ml) and exhibited active TGF-β signaling, which might triggered the EMT. Compared to shams, CHF cell outgrowths exhibited elevated levels of EMT markers, SMA (49% vs. 34%) and Snail (2 folds), and reduced level of Wt1 (11% vs. 22%). In addition, CHF cell outgrowths had two folds increase in Pai1 gene expression, a direct target of TGF-β signaling. In c-Kit+ cells derived from CHF explants, Nanog gene expression was 4 folds lower and Sox 2 was 2 folds lower compared with cells from shams. Suppression of EMT in cell outgrowth increased the percentage of c-Kit+ and Wt1+ cells by 17%, and 15%, respectively. Also suppression of EMT in c-Kit+ cells resulted in 4 folds increase in Nanog and 3 fold increase in Sox2 gene expressions. Our results showed that CHF may further exuberates EMT while diminishes the re-activation of pluripotency genes. Thus, EMT modulation in CHF is a possible strategy to regulate both the yield and the pluripotency of cardiac-explant-derived progenitor cells.
Abstract 56: Shifting AAV Mediated Expression Profile to Heart by an Integrated Transcriptional Targeting Strategy
Authors: Rabinowitz Joseph E , Wang Jinhui , Gorsky Joshua , Peterson Jonathan
Published: July 2021

Adeno-associated viral (AAV) vectors are promising platforms for cardiac gene transport, but their broad tropism is a drawback after systematic delivery. To divert gene expression of AAV6 or 9 to heart, an integrated transcriptional targeting strategy was explored. By comparing cardiac GFP expression of scAAV6 and 9-CMV-GFP in C57/B6 or Balb/C mice, a heart-bias in C57/B6 and liver bias in Balb/C was seen. This makes the native liver biased tropism of AAV6 or 9 in Balb/C mice more suitable for the transcriptional targeting study. Luciferase (Luc) gene expression driven by cardiac specific promoters; brain natriuretic peptide (BNP), cardiac ankyrin repeat kinase (CARK), myoglobin (Myo), alpha cardiac actin (a-CARD), Troponin I (Tni), and Troponin T (Tnt), were compared to CMV promoter post retro orbital injection of 2.5x1010 viral particles of AAV6 or 9. In contrast to the previously reported heart biased signal seen in C57/B6 mice, signal was observed mainly in the liver of Balb/C mice. In vitro Luc activity, mRNA abundance and viral genomes (VG) assays were performed in liver and heart samples. Surprisingly, ∼10 folds higher Luc activity and RNA abundance in heart were observed for Tnt, Tni and a-CARD promoters than CMV promoter post AAV9 delivery, while a reduction of 100 fold was observed in liver. Copy number assay revealed six fold higher VG in liver than in heart for all promoters used including CMV. This indicates Tnt, Tni and a-CARD promoters have strong heart biased activity, without a tropism change as determined by VG copy numbers. To further develop the transcriptional targeting strategy three copies of mir122 binding site were inserted into the 3'UTR of AAV9-CMV-Luc and Balb/C mice were injected with 5x1010 viral particles. This resulted in ∼30 fold less Luc activity when compared to AAV9 without mir122 binding sites, with no change in VG copy number. In conclusion, in a mouse strain biased for liver expression after systemic delivery, transcriptional targeting to heart can be accomplished by using strong cardiac biased promoter integrated with mir122 binding sites in the 3’UTR.