bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 109: Regulation of TNF-α Signaling in Cardiomyocytes via a Novel Interaction Between TNF-α Receptor and RASSF1A
Authors: Mohamed Tamer M , Zi Min , Maqsood Arfa , Prehar Sukhpal , Neyses Ludwig , Oceandy Delvac
Published: July 2021

Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that plays key roles in the pathogenesis of heart failure. Cardiomyocytes express the TNF-α receptor (TNFR), however, the mechanism of TNF-α signal transmission in cardiomyocytes is not completely understood. Recent studies showed that in cancer cells TNFR is regulated by Ras-association domain family 1 isoformA (RASSF1A). Therefore, we investigated whether RASSF1A modulates TNF-α signalling in cardiomyocytes. We used RASSF1A knockout (KO) mice and wild type (WT) controls and stimulated them with TNF-α (10µg/kg i.v.). In WT mice acute treatment with low dose of TNF-α increased cardiac contractility as indicated by the change in end systolic elastance (Ees) (baseline Ees (mmHg/μl), 3.3±0.5; Ees after TNF-α stimulation, 5.7±0.8, P<0.05) which is consistent with previously published data (Circulation 2004; 109:406–411). However, KO mice showed a blunted contractile response following acute TNF-α treatment (baseline Ees: 3.05±0.4 vs Ees after TNF-α: 2.54±0.3). Consistently, isolated cardiomyocytes from WT mice showed 40% increase in calcium transient amplitude in response to TNF-α (10 ng/ml) stimulation (n=24 cells, p<0.05). However, KO cardiomyocytes showed no significant increase in calcium transient amplitude following the same stimulation (n=24 cells). We also found that RASSF1A formed a molecular complex with TNF-α receptor in cardiomyocytes and this interaction was essential in the recruitment of TRADD and TRAF2, the major downstream effectors of TNF-α signalling. By mapping the interaction domain we found that the C-terminal region of RASSF1A was responsible for the formation of TNF-α receptor complex. Furthermore, using an adenoviral-mediated shRNA construct we found that cardiomyocytes lacking RASSF1A exhibited reduced activation of NFkB, a downstream target of TNF-α. Overall, our data indicate an essential role of RASSF1A in regulating TNF-α signalling in cardiomyocytes, with RASSF1A being key in the formation of TNF receptor complex and in the signal transmission to the downstream targets.
Abstract 113: SUMO1 Gene Transfer Rescues Cardiac Function in a Large Animal Model of Heart Failure
Authors: Tilemann Lisa M , Ishikawa Kiyotake , Kho Changwon , Lee Ahyoung , Aguero Jaime , Rapti Kleopatra , Santos Gallego Carlos , Kohlbrenner Erik , Fish Kenneth M , Hajjar Roger J
Published: July 2021

Recently, small ubiquitin-related modifier 1 (SUMO1) was found to enhance the activity and stability of the cardiac sarcoplasmic reticulum Ca2+ ATPase, SERCA2a. In both, human and rodent models of heart failure (HF), the total amount of myocardial SUMO1 is decreased and its knock down results in severe HF. Adeno-associated vector (AAV) mediated SUMO1 gene transfer significantly improves cardiac function in murine models of HF. As a critical step towards clinical translation, we evaluated the effects of SUMO1 gene transfer in a swine model of ischemic heart failure. One month after balloon occlusion of the proximal LAD, 21 animals were randomized to receive either AAV1.SUMO1 at two doses, AAV1.SERCA2a, AAV1.SUMO1+AAV1.SERCA2a, or saline via antegrade coronary infusion. In addition, three pigs served as controls and underwent sham procedures. The ejection fraction and the maximum dP/dt significantly increased after gene transfer of SUMO1 at both doses, SERCA2a and the combination of SUMO1 and SERCA2a (p=0.034, p=0.028) compared to saline infusion. The increase in maximum dP/dt was most pronounced in the group that received both SUMO1 and SERCA2a. Furthermore, the increase in end-systolic and end-diastolic volumes was normalized in the treatment groups, while they further deteriorated in the saline group (p=0.001, p=0.022). SUMO1 and SERCA2a gene transfer significantly improved cardiac function and concomitant gene delivery of SUMO1 and SERCA2a had a synergistic effect on improving these parameters in the HF animals. These results strongly support the critical role of SUMO1 for SERCA2a function and underline the therapeutic potential in heart failure patients.
Abstract 126: Gene Deletion of Dipeptidyl Peptidase I Reduces Myocyte Loss and Preserves Cardiac Function After Myocardial Ischemia-Reperfusion Injury
Authors: Qi Zhao , Kolpakov Mikhail , Hooshdaran Bahman , Rafiq Khadija , Guo Xinji , Wang Tao , Pham Christine , Sabri Abdelkarim
Published: July 2021

Introduction and Hypothesis:
Chronic inflammation predisposes tissue to adverse cardiac remodeling and contributes to heart failure (HF) progression. The mechanisms whereby inflammation contributes to cardiac remodeling after cardiac injury has largely focused on action of reactive oxygen species and cytokines/chemokines and their role on myocyte growth and extracellular matrix (ECM) remodeling. However, the role of inflammatory proteases on myocyte loss and adverse cardiac remodeling has not been studied and are the focus of this study.

Methods and Results:
We subjected wild type and mice lacking Dipeptidyl peptidase I (DPPI), a lysosomal enzyme involved in the cleavage and activation of major inflammatory serine proteases, to myocardial ischemia for 30 min followed by reperfusion for 24hrs (IRI) and assessed the role of DPPI gene deletion on inflammatory proteases activation, myocyte apoptosis and cardiac remodeling and function. DPPI KO mice show markedly reduced inflammatory serine protease activity in response to myocardial IRI compared to wild type mice. DPPI deletion also markedly reduced myocyte apoptosis, limited infarct size and improved cardiac function in response to IRI. The role of inflammatory serine proteases in cardiac remodeling was further demonstrated after in-vivo cardiac transfer of cathepsin G, a neutrophil derived serine protease that has been shown to induce myocyte death in-vitro. Mice with cathepsin G injection for 5 days show increased interstitial collagen degradation and myocyte apoptosis and presented cardiac dilatation and contractile dysfunction.

Conclusions:
These findings reveal the role of DPPI as a key mediator of postischemic/reperfusion injury and show that inflammatory derived proteases contribute to the pathological cardiac remodeling and dysfunction.
Abstract 134: Electrical Stimulation Improves Calcium Handling in Engineered Heart Tissue
Authors: Godier-Furnémont Amandine , Tiburcy Malte , Vunjak-Novakovic Gordana , Zimmermann Wolfram
Published: July 2021

Purpose:
Despite considerable advances in cardiac tissue engineering, morphology and functionality of engineered heart tissue (EHT) needs to be further improved, likely by making use of more “physiologic” engineering paradigms. We hypothesized that electrical stimulation may improve calcium handling in EHTs through enhanced maturation of the excitation coupling apparatus.

Methods:
EHTs were prepared from neonatal rat ventricular heart cells and after one week, transferred to dynamic stretchers. EHTs were cultured with (2 or 4 Hz) or without (non-stimulated ‘NS’) electrical stimulation from day 7 to 13 (Ionoptix C-Pacer: 2 ms square biphasic pulse, 4 Volts). At day 13, functional (twitch tension at 2 Hz field stimulation) were performed, and EHTs were processed for Western Blot analysis. In addition, EHT-derived single cardiomyocytes loaded with fura-2 (1 µM) were analyzed using an Ionoptix setup to assess alterations in calcium handling.

Results:
Culture under electrical enhanced twitch tension of EHTs (2 Hz vs. NS: 0.83 ± 0.05 vs. 0.62 ± 0.03, p<0.0001, n=8/group), lowered excitation thresholds, and increased upstroke velocity. Assessment of sarcoplasmic reticulum (SR) functionality by post-rest potentiation revealed improved calcium storage capacity in EHTs (NS vs. 2 Hz vs. 4 Hz: 10.2 ± 1.4 vs. 11.7 ± 1.4 vs. 17.8 ± 1.3 % at 30 s rest; p<0.05, n=7/7/5). Ryanodine (1 µM, to block SR-calcium release) reduced EHT twitch tension to 88.3 ± 2.1 % [NS] vs. 87.2 ± 2.4 % [2 Hz] vs. 75.1 ± 4.5 % [4 Hz] (n=8/group, p<0.05) baseline force, indicating greater reliance on the SR for force generation, particularly in the 4 Hz stimulated group. Ryanodine receptor and SERCA protein expression increased with 4 Hz stimulation. In isolated cardiomyocytes, 4 Hz stimulated cells displayed faster upstroke velocity and relaxation times, approaching that of cardiomyocytes isolated from day 13 postnatal rats.

Conclusions:
Electrical stimulation of dynamically loaded EHTs contributes to increased functionality and appears to improve excitation-contraction coupling through enhanced Calcium storage and release capacity. This combined biomimetic stimulation method may overcome the sub-physiologic SR functionality of classical cell culture models.
Abstract 147: The AMP-Activated Protein Kinase (AMPK) -Dependent Regulation of Endothelial Cell Angiotensin-Converting Enzyme (ACE) Expression Involves p53 and MicroRNA-143/145
Authors: Fleming Ingrid , Kohlstedt Karin
Published: July 2021

High ACE-levels are associated with cardiovascular disease development, but little is known about its expressional regulation. ACE is a target of the miR143/145-cluster in murine vascular smooth muscle cells and can also be affected by AMPK activation in monocytes. Since shear stress activates AMPK in endothelial cells, we determined whether flow alters ACE expression and the involvement of AMPK and miR143/145.

Shear stress decreased ACE expression in human endothelial cells, an effect prevented by
downregulating
(siRNA)
the
AMPK
α2
subunit, but not AMPKα1.Pharmacological AMPK activation also suppressed endothelial ACE expression and AMPKα2
-/-
(not AMPK
α

1
-/-
) mice

expressed
significantly higher ACE levels in different
arteries
than
wild-type littermates.
While
basal blood pressure was comparable in AMPKα2
+/+
and AMPKα2
-/-
mice, AMPKα2
-/-
mice were more sensitive to the hypotensive effect of the ACE-inhibitor ramipril and demonstrated an impaired bradykinin-induced hindlimb-vasodilatation. The suppressive effect of the miR-143/145-cluster on human endothelial ACE was confirmed by overexpression of pre-miR143/145 and AMPKα2 downregulation decreased
miR143/145-levels
. Since f
low increased mature and pre-miR-143/145 levels, but not pri-miR143/145 levels, we focused on the post-transcriptional regulation of miR143/145 by AMPK via p53
.

Indeed,
p53 expression and phosphorylation (Ser15) increased in response to shear stress, and were sensitive to treatment with AMPK
α
2 (but not AMPK
α
1) siRNA.
The data indicate that, AMPKα2 suppresses endothelial ACE expression via p53 activation and upregulation of miR143/145. Since AMPK (and p53) dysregulation is associated with several diseases (e.g. diabetes, cancer), their effect on miRNA143/145 and ACE might mediate disease-associated cardiovascular disorders.
Abstract 17: MicroRNA-30c Modulates Diabetes-Induced Cardiac Hypertrophy via PI3K/Akt Signaling Pathway
Authors: Raut Satish K , Kumar Akhilesh , Singh Gurinder B , Sharma Rajni , Saikia Uma , Khullar Madhu
Published: July 2021

Cardiomyocyte hypertrophy leading to cardiac failure is one of the characteristic alterations in Diabetic Cardiomyopathy. Several regulatory mechanisms mediate altered gene expression in DCM. Alterations in several myocardial miRs have been demonstrated in cardiac hypertrophy but the role of miR-30c in the pathophysiology of diabetes induced cardiac hypertrophy is not known.

Objective:
miR-30c may be involved in modulating expression of key genes (Rac1, Cdc42 and Pak1) of PI3K/Akt signalling pathway involved in cardiac hypertrophy in DCM.

Method:
DCM was induced in Wistar rats by STZ-high fat diet combination and animals were sacrificed after 12 weeks of diabetes. The expression of miR-30c, Rac1, Cdc42 and Pak1 genes and markers of hypertrophy (ANP and β-MHC) was studied in cardiac tissues (n=6) and control rats (n=6) by qRT-PCR.

Result:
We observed 4 fold increased expression of ANP and 2.2 fold of β-MHC genes in DCM group as compared to control group (n=6) (p<0.05). Decreased expression of miR-30c (3.3 fold) was seen in DCM model at 12 weeks compared to control group (n=6) (p<0.05). Expression of three specific targets of miR-30c i.e. Cdc42, Pak1, and Rac1, regulating cardiac hypertrophy, was found to be significantly increased by 1.94 fold, 1.8 fold, and 1.2 fold in cardiac tissues of DCM model respectively and was inversely related to miR-30c expression.

Conclusion:
Our results suggest that down regulation of miR-30c in cardiac tissue may play a key role in regulation of diabetes associated cardiac hypertrophy by modulating PI3K/Akt signalling pathway.
Abstract 170: Inflammation Imaging in the Primate Heart Using
111
In-Labeled Anti-Tenascin-C Antibody
Authors: Hiroe Michiaki , Ageyama Naohide , Yasutomi Yasuhiro , Kurosawa Hiroyuki , Fujimoto Ousuke , Arano Yasushi , Imanaka-Yoshida Kyoko
Published: July 2021

Myocardial inflammation after myocardial infarction and myocarditis may cause cardiac remodeling, leading to congestive heart failure and arrhythmias. Tenascin-C (TNC), an extracellular matrix glycoprotein, is not normally expressed but specifically expressed associated with active inflammation. The aim of this study was to explore the myocardial expression of TNC after myocardial infarction in
Macaca fascicularis
(crab-eating monkey) using
111
In-labeled anti-TNC antibody (
111
In-TNC-Fab’). The left coronary artery was permanently ligated in two monkies and 4 days later, we performed dual-isotope single-photon emission computed tomography imaging (SPECT) of
111
In-TNC-Fab’ and
99m
Tc methoxy- isobutyl isonitrile (MIBI), and compared with those of an age-matched control. Then, dual autoradiography was compared with histology and immunostaining for TNC of the heart. Dual-isotope SPECT demonstrated the regional myocardial uptake of
111
In-TNC-Fab’ in the areas of decreased uptake of MIBI. On autoradiography, the radioactivities were observed in the border zone between the infarcted and the noninfarcted area of MI hearts. The hot spots corresponded with the positively immunostained areas. In contrast, no radioactivites of
111
In-TNC-Fab’ were detected in the control. These data clearly indicated that, using
111
In-TNC-Fab’, we can visualize the inflammatory lesions followed by myocardial infarction of the primate.
Abstract 192: Histological and Functional Assessment of Human Placental Stem Cells in an Induced Myocardial Infarction in Rats
Authors: Makhoul Georges , Ma Yu-Ting , Duong Minh , Chiu Ray C J , Cecere Renzo
Published: July 2021

Purpose:
Human bone marrow mesenchymal stem cells (hbmMSCs) have been studied extensively for myocardial regenerative therapy. However, such cells require invasive procreation and suffer from donor age-related declining quality. Recently, a more abundant resource of young MSCs has been isolated from an otherwise discarded organ: the human placenta mesenchymal stem cells (hpMSCs). In this study, we wanted to examine the survival, differentiation, and functionality of xenogeneic hpMSCs when implanted into an induced myocardial rat infarction.

Methods:
To inspect their stemness,hpMSCs underwent an
In Vitro
cardiac cell differentiation in a DMEM medium containing 5 Azacytidine. Additionally, hpMSCs were tested in a myocardial infarction animal model. Female Lewis rats (40 animals) underwent left coronary artery ligation. Animals were divided into 4 groups. Group 1 was injected with hpMSCs in the peri-infarct region. Groups 2 and 3 received hbmMSCs and
In Vitro
differentiated hbmMSCs into cardiomyocytes respectively. Cell free medium was injected in group 4. Echocardiography was performed at baseline, day 4, weeks 3, 6, and 9 after ligation. Myocardial tissues were harvested and studied immunohistochemically for specific muscular and cardiac markers (Actin and Troponin I) on weeks 6 and 10.

Results:
In Vitro
differentiation into cardiomyocyte lineage was achieved with the hpMSCs. HpMSCs were detected within rat myocardium by week 6 after their implantation. These cells stained positively for Actin and Troponin I. Preliminary echocardiographic data show cardiac functional increase in group 1. Whether hpMSCs can provide a superior effect than hbmMSCs or differentiated hbmMSCs is being investigated.

Conclusions:
In Vitro
studies indicated that the hpMSCs can be differentiated into cardiac cells. When implanted into the rat infarcted myocardium, hpMSCs survived and were able to differentiate into cardiomyocytes. These cells appeared to increase cardiac function in an induced myocardial infarction rat model. With the advantages of easy availability and young age, hpMSCs could be more suitable for myocardial regenerative therapy.
Abstract 195: Leukemia Inhibitory Factor Stimulates Cardiac Stem Cell--Derived Cardiomyocyte Renewal After Myocardial Infarction Using a Genetic Fate-Mapping Study
Authors: Kanda Masato , Nagai Toshio , Takahashi Toshinao , Liu Mei Lan , Kondou Naomichi , Naito Atushiko T , Akazawa Hiroshi , Kobayashi Yoshio , Komuro Issei
Published: July 2021

Cardiac stem cells or precursor cells have the potential to regenerate cardiomyocytes, but their role in the efficacy of cardioprotective drugs remains controversial. Therefore, using a genetic fate-mapping model, we tested the hypothesis that leukemia inhibitory factor (LIF) influences cardiac stem cells and stimulates endogenous cardiomyocyte renewal after myocardial infarction (MI). We generated MerCreMer-LacZ mice in which more than 99.9% of the cardiomyocytes in the left ventricular field showed positive 5-bromo-4-chloro-3-indolyl-β-d-galactoside (Xgal) staining just after tamoxifen injection (5mg/kg/d for 2 weeks). Thus, every Xgal-negative cardiomyocyte was derived from a stem or precursor cell after tamoxifen administration. The number of Xgal-negative cardiomyocytes did not change during normal aging spanning 1 year. However, at 3 months after MI, the MI mice had more Xgal-negative cells than the control mice (57.0 ± 12.0 and 3.0 ± 2.6 cells per section, respectively; P < 0.01). The side population (SP) cell fraction contained label-retaining cells, which differentiated into Xgal-negative cardiomyocytes after MI. Among the cytokines secreted after MI, LIF expression increased to 130 fold in MI mice but rapidly decreased within 1 week. Therefore, we injected the LIF plasmid at the time of MI to keep blood LIF concentrations high and examined its effect on regeneration. At 1 month after MI, the MI + LIF group (118.6 ± 51.5 cells per section) had more Xgal-negative cells than the MI + PBS group (37.0 ± 5.5 cells per section; P < 0.05). Echocardiography showed significant recovery of fractional shortening in the LIF-treated group only. Next, we immunohistochemically analyzed the effect of LIF on SP cells. On BrdU administration at 1 week after MI, the percentages of BrdU-positive SP cells in LIF- and PBS-treated mice were 59%, and 35%, respectively, suggesting that LIF influenced SP cell proliferation. The percentages of Ki67- and phosphorylated histone-3-positive SP cells were also higher in LIF-treated mice. Taken together, LIF may stimulate stem cell-derived cardiomyocyte regeneration in part by activating SP cells. We believe our findings will help provide a novel therapeutic strategy for cardiogenesis.
Abstract 199: Whole-Transcript Genomics Reveals Atrial KCNE1 Downregulation in a Model of Postoperative Atrial Fibrillation Susceptibility
Authors: Kant Ritu , Hu Zhaoyang , Kanda Vikram , Christini David J , Heerdt Paul , Abbott Geoffrey W
Published: July 2021

Lone atrial fibrillation (AF) is associated with various ion channel gene sequence variants, notably the common S38G loss-of-function polymorphism in the
KCNE1
K
+
channel ancillary subunit gene. New-onset postoperative AF (PAF) generally occurs 48-72 hours after major surgery, particularly following procedures within the chest, but its molecular bases remain poorly understood. Here, to begin to address this gap in knowledge, we analyzed molecular changes in the left atrium (LA) three days following left upper lung lobectomy in Sinclair swine in relation to simultaneous changes in hemodynamics, LA effective refractory period (ERP), and the capacity to sustain electrically-induced AF. Relative to control pigs (no previous surgery), lobectomy increased mean pulmonary artery pressure (16 ± 3 vs 22 ± 5 mm Hg;
P
= 0.045) and pulmonary vascular resistance (273 ± 93 vs 481 ± 142 dyn¶s/cm
5
;
P
= 0.025), whereas other hemodynamic parameters were unchanged. Left atrial ERP measured from a 300 ms cycle length trended toward reduction in lobectomy animals (187 ± 37 vs 170 ± 44 ms,
P
>0.05). None of the lobectomy pigs developed spontaneous postoperative AF as assessed by telemetric ECG. However, all lobectomy pigs, but none of the controls, were able to sustain AF induced by a 10 s burst of rapid pacing for ≥30 s (
P
=0.0079), independent of atrial ERP; AF was sustained ≥60 s in 3/5 postoperative pigs versus 0/5 controls and correlated with a shorter ERP overall (
P
=0.023). Whole-transcript genomics of left atrial tissue using 25 probes per transcript revealed 13 genes with expression changes ≥2-fold in postoperative pigs. Strikingly, of these, atrial
KCNE1
showed the statistically strongest link to surgery, and was the most downregulated gene in the postoperative group compared to controls (2.2-fold reduced,
P
=0.0027), recapitulated at the protein level with western blotting (
P
=0.039). The data suggest
KCNE1
remodeling as a potential marker or influencing factor in the early progression of, or susceptibility to, PAF [[Unable to Display Character: –]] uncovering a possible common predisposing genetic factor in PAF and lone AF.
Abstract 200: Hypercoagulabilty Panel Testing in Neonates Undergoing Cardiac Surgery
Authors: Emani Sirisha , Zurakowski David , Baird Christopher , Pigula Frank , Cameron Trenor , Emani Sitaram M
Published: July 2021

Thrombosis is a crucial contributor of morbidity and mortality in neonates undergoing cardiac surgery. Although there is published data on several factors of the hemastatic system, there is no data correlating factor expression and/or function with thrombosis in neonates. We tested the hypothesis that hypercoagulability markers are predictive of thrombosis in neonates undergoing cardiac surgery. Sixty neonates undergoing cardiac surgery were tested for thrombin generation assay; coagulation factors; antithrombin III, protein C, protein S, and factor VIII; fibrinolytic inhibitors; thrombin-activatable fibrinolytic inhibitor, plasminogen activator inhibitor; and presence of cardiolipin antibodies by immunoassays. Factor V Leiden mutation was also tested in a few patients utilizing single nucleotide polymorphism assays. In this pilot study, thrombosis occurred in 15% of the neonates undergoing cardiac surgery. Significant risk factors associated with thrombosis were pre-mature birth, use of cardio pulmonary bypass, and single ventricle physiology. Hypercoagulability factors associated with thrombosis determined by univarent analysis were elevated thrombin generation, enhanced expression of thrombin-activatable fibrinolytic inhibitor and plasminogen activator inhibitor as well as presence of cardiolipin antibodies and factor V Leiden mutation. No correlation was observed between thrombosis and expression of coagulation factors antithrombin III, protein C, protein S, and factor VIII. Multivarient analysis has proven to show thrombin generation, thrombin-activatable fibrinolytic inhibitor, and presence of cardiolipin antibodies as multivariable predictors of thrombosis. These significant hypercoagulability markers are independent predictors of thrombosis. Thus thrombosis predictability can help in post-operative management and care for neonates undergoing cardiac surgery by regulating pro- and/or anti-coagulation therapy.
Abstract 201: Protective Effect of Nicotinamide Mononucleotide Administration Against Cardiac Ischemia-Reperfusion Injury
Authors: Yamamoto Takanobu , Zhai Peiyong , Sadoshima Junichi
Published: July 2021

Background:
Nicotinamide adenine dinucleotide (NAD) participates in redox reactions as an electron-transferring molecule, thereby serving as a cofactor for metabolism. In the NAD biosynthetic salvage pathway, nicotinamide phosphoribosyltransferase (Nampt) which forms nicotinamide mononucleotide (NMN) is the rate-limiting enzyme and exhibits the protective effect against ischemia/ reperfusion (I/R) injury in the hearts of mice with cardiac-specific overexpression of Nampt. In addition, administration of NMN is reported to reverse the pancreatic beta-cell dysfunction observed in Nampt heterozygous knockout mice.

Methods:
To elucidate the protective effect of NMN against cardiac I/R injury, NMN was administered to mice subjected to I/R injury either before ischemia or immediately before reperfusion. Area at risk (AAR) and infarct area (IA) were evaluated 24 hours after reperfusion.

Results:
To confirm the distribution of NMN in the heart, NAD and NADH, into which NMN is quickly converted in the heart, were measured. One hour after NMN administration (500mg/ kg body weight, i.p.), the NAD and NADH contents of the NMN group (n=3) were significantly increased, whereas the NAD/NADH ratio was unchanged compared to the vehicle group (n=3) (NAD: NMN=483±42 pmole/mg tissue, vehicle=183±46 pmole/mg tissue, p<0.01; NADH: NMN=262±25 pmole/mg tissue, vehicle=96±17 pmole/mg tissue, p<0.01; NAD/NADH ratio: NMN=1.85±0.09, vehicle=1.82±0.18, n.s.). In the mice administered NMN or vehicle 30 min before ischemia, IA/AAR in the NMN group was smaller than that in the vehicle group (AAR: NMN=26±0.5%, vehicle=27±0.4%, n.s.; IA/AAR: NMN=23±1.8%, vehicle=42±1.5%, p<0.01, n=4). On the other hand, in the mice administered NMN or vehicle immediately before reperfusion, IA/AAR in the NMN group was not different from that in the vehicle group (AAR: NMN=27±0.5%, vehicle=26±1.0%, n.s.; IA/AAR: NMN=37±1.4%, vehicle=34±2.2%, n.s., n=4).

Conclusions:
NMN administration significantly increased the NAD content in the heart and exhibited a protective effect against cardiac I/R injury. Increasing NAD content before ischemia, rather than after reperfusion, may minimize I/R injury in clinical settings, such as in acute coronary syndrome before coronary intervention.
Abstract 210: PPARγ Activation Prevents Cardiac Dysfunction in Sepsis via Stimulation of Energy Production
Authors: Drosatos Konstantinos , Khan Raffay S , Trent Chad M , Schulze P Christian , Homma Shunichi , Goldberg Ira J
Published: July 2021

Septic cardiac dysfunction has been attributed to either inflammation or suppression of fatty acid oxidation (FAO). We hypothesized that energy deprivation accounts for cardiac dysfunction in lipopolysaccharide (LPS)-induced sepsis. LPS compromised cardiac function, increased cardiac inflammatory markers (interleukin (IL)-1α, IL-6 and tumor necrosis factor (TNF)α) mRNA, and reduced FAO, ATP and FAO-related gene expression: Peroxisome proliferator activating receptor (PPAR)α, cluster of differentiation 36, lipoprotein lipase (LpL), fatty acid transport protein, carnitine palmitoyl-transferase (Cpt)1, PPARγ coactivator (PGC-1)α and PGC-1β in C57BL/6 mice. LC-MS/MS analysis of cardiac lipids showed that total ceramide, which might account for defective FAO and ceramide species were not modulated by LPS. Electron microscopy and ATPase6 DNA levels showed that LPS-treated animals had fewer and smaller cardiac mitochondria. PPARα agonist, WY-14643, did not prevent cardiac dysfunction and suppression of FAO-associated gene expression in LPS-treated mice. In contrast, LPS did not affect cardiac function of mice with cardiomyocyte-specific expression of PPARγ (αMHC-PPARg), although cardiac IL-1α, IL-6 and TNFα mRNA levels were increased. Despite a 77% reduction in PPARα, mRNA levels of FAO-associated genes, such as acyl-CoA oxidase, PGC-1α, PPARδ, Cpt1 increased over 10-fold as compared to saline-treated αMHC-PPARg mice. Similarly, cardiac FAO and ATP content was 2.6 and 5-fold greater in LPS-treated αMHC-PPARγ mice compared to LPS-treated C57BL/6 mice. Moreover, treatment of C57BL/6 mice with LPS and the PPARγ agonist, rosiglitazone, prevented cardiac dysfunction, increased FAO and relevant gene expression, while IL-1α, IL-6 and TNFα mRNA levels increased. Rosiglitazone prevented LPS-mediated reduction of PGC-1α and maintained the number and size of mitochondria. Treatment of C57BL/6 mice with a lethal dose of LPS (15 mg/kg) and repeated doses of rosiglitazone (30mg/kg/day for 72 hours) improved survival, mobility and overall appearance of LPS-treated mice. Thus, PPARγ activation stabilized cardiac energy and prevented cardiac dysfunction and mortality in septic animals despite cardiac inflammation and PPARα downregulation.
Abstract 220: Induction of Heme Oxygenase-1 Enhances Matrix Metalloproteinase-9 Secretion and Myocardial Fibrosis
Authors: Okada Muneyoshi , Ikeda Shiro , Yamawaki Hideyuki , Hara Yukio
Published: July 2021

Background:
Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme in heme catabolism into carbon monoxide, biliverdin and free iron. Whereas HO-1 exerts cardioprotective effect, such as anti-hypertrophic and anti-apoptotic effect in cardiomyocytes, the influence of HO-1 on cardiac fibroblasts has not been clarified. We examined the effect of HO-1 induced by cobalt protoporphyrin IX (CoPPIX) on matrix metalloproteinase (MMP)-9 secretion, a biomarker of cardiac remodeling, in cardiac fibroblasts.

Methods and Results:
Cardiac fibroblasts were isolated from male Wistar rats. Secreted MMP-9 protein into culture medium and HO-1 expression in cell lysates were measured by Western blotting. CoPPIX (0.1-1 μ M) increased HO-1 expression and enhanced interleukin (IL)-1β (4 ng/ml, 24 h)-induced MMP-9 secretion (n=4). Mn
2
(CO)
10
(0.3-10 μ M), a carbon monoxide donor, increased IL-1β-induced MMP-9 secretion (n=4), although bilirubin (0.3-10 μ M), a metabolite of biliverdin, and FeSO
4
(0.3-10 μ M), a free iron donor, had no effect. Mn
2
(CO)
10
increased reactive oxygen species (ROS) generation and the enhanced effect of Mn
2
(CO)
10
on IL-1β-induced MMP-9 secretion was abolished by an antioxidant EUK-134 (10 μ M, n=4). We also examined the effect of CoPPIX on isoproterenol (5 mg/kg/day, subcutaneous injection, for 1 or 7 days)-induced cardiac remodeling in male Wistar rats. CoPPIX (1 mg/kg) was administered intraperitoneally every 2 days and this treatment increased HO-1 expression. Hearts were excised at the day 2 or day 8. Myocardial fibrosis was evaluated by Azan staining. At the day 8, CoPPIX worsened isoproterenol-induced myocardial fibrosis significantly (n=6), but had no effect on the increase of heart weight. CoPPIX also enhanced isoproterenol-induced MMP-9 expression at the day 2 (n=10).

Conclusion:
Induction of HO-1 by CoPPIX enhances IL-1β-induced MMP-9 secretion via ROS generation through heme-derived carbon monoxide in cardiac fibroblasts and isoproterenol-induced myocardial fibrosis and MMP-9 expression. These findings suggest that HO-1 induction does not necessarily exert cardioprotective effects.
Abstract 223: A Case for Hypoxia and Long QT Syndrome in Sudden Infant Death Syndrome
Authors: Neary Marianne , Mohun Timothy J , Breckenridge Ross A
Published: July 2021

INTRODUCTION: Long QT syndrome is considered an important factor in the pathogenesis of Sudden Infant Death Syndrome (SIDS). Prolonged QTc intervals (c=corrected for heart rate) in SIDS are sometimes caused by mutations in genes encoding ion channels. Other causes remain largely idiopathic.

HYPOTHESIS: Risk factors for SIDS, including maternal bed sharing, head covering and high altitude, are associated with a reduced oxygen environment. Our studies investigate a link between hypoxia and long QT syndrome in the neonate.

METHODS: We characterised, for the first time, changes in the murine neonatal electrocardiogram (ECG) at: 0, 1, 3, 6, 12, 24 hours (n=12) and 2, 6 and 10 days (n=25) after birth. We investigated whether birth into hypoxia 10% O2 (n=16) and genetically elevating cardiac hypoxic signalling in neonatal mice alters the course of changes in ECG morphology (n=14). We analysed the ECG for heart rate and parameters associated with dysrrhythmia and sudden death, including the QTc interval.

RESULTS: In the hours and days following birth, we observed a steady increase in heart rate (p<0.0001) and decrease in QTc interval (p<0.05). When neonates were raised in hypoxia for 24 hours, the trends in heart rate (p<0.001) and QTc interval (p<0.0001) were abolished and risk of neonatal death was 52% (17 out of 33) over 24 hours. In transgenic neonatal mice with elevated cardiac hypoxic signalling, we observed a significant bradycardia (p<0.0001) and elongated QTc interval (p<0.0001) compared to controls at ten days after birth, with death occurring pre-weaning.

>CONCLUSIONS: Following birth there are significant changes in ECG morphology, including an increase in heart rate and decrease in QTc interval. Hypoxia diminishes these changes resulting in bradycardia and elongated QTc intervals. We hypothesise that the increase in ambient oxygen concentration after birth drives the maturation of cardiac electrical conduction, failure of which predisposes to dysrhythmia and sudden death. This is consistent with known risk factors of SIDS and provides a link between neonatal hypoxia and ECG repolarisation abnormalities.
Abstract 236: Heart Failure Remodeling: Local Endogenous Erythropoietin and Erythropoietin Receptor Expression
Authors: Leszek Przemyslaw , Sochanowicz Barbara , Brzoska Kamil , Piotrowski Walerian , Danko Bozena , Kusmierczyk Mariusz , Rozanski Jacek , Rywik Tomasz , Kruszewski Marcin
Published: July 2021

Erythropoietin (EPO) via erythropoietin receptor (EPOR) maintains myeloid erythropoiesis, however in a failing heart (FH) it seems to exert cytoprotective, antiapoptotic, proangiogenic effects.

Aim:
to assess (myocardial) M-EPOR expression and to find out if erythropoietin (M-EPO) is produced in human heart; secondary to elucidate the mechanisms related to M-EPO/M-EPOR expression.

Methods:
We used 33 explanted FH, compared to 11 non-failing hearts (NFH). Patients characteristics: left ventricle (LV - EF 22±11%), NTproBNP (5464±4825 pg/ml). Serum variables: iron (62±32ug/dl), ferritin (156±122ng/ml), transferrin receptor (3.8±2.6mg/l), EPO (29.5±44.4mIU/ml), HT (39.8±5.3%), Hb (13.2±1.7g/dl), creatinine clearance (CrCl 68±23 ml/min), Na+ (138±5.4 mmol/l).

Myocardial assesment: M-EPO, M-EPOR, iron storage protein - ferritin (M-FR), iron acquisition protein transferrin receptor (M-TfR), Iron Regulatory Protein 1 (M-IRP1) - by ELISA - ng/mg protein - expression; myocardial iron load - by Instrumental Neutron Activation Analysis, µg/g.

Results:
In FH and NFH M-EPO/M-EPOR expression were detected; M-EPO was elevated in failing LV (in FH vs NFH 4.7±1.6 vs 3.7±1.4, p=0.0346; ng/mg protein), no changes in M-EPOR was found. M-EPO correlated with M-EPOR from both ventricles (LV- left r=0.63, p=0.0002; RV - right r=0.42, p=0.0161). With regard to anemia (by WHO), patients were divided into two subgroups: A+ (n=11) and A- (n=22). In A+ vs A- serum EPO (47±23 vs 28±30 mIU/l, p=0.0473) was higher. In myocardium A+ M-TfR was lower than in A- both LV (136±61 vs 218±134; p=0.0446) and RV (130±92 vs 199±143; p=0.0688), however no differences in M-EPO/M-EPOR/M-FR/M-IRP1 and iron load were found. In LV, A+ subgroup, IRP1 correlation with M-EPO (r=0.79, p=0.0107) and M-EPOR (r=0.76, p=0.0184).

Conclusions:
M-EPO/M-EPOR are synthesized locally in human hearts. In failing LV M-EPO synthesis is significantly increased. M-EPO production correlated with M-EPOR expression. M-EPO/EPOR expression is associated with regulatory protein IRP1. The presence of anemia accompanied by high plasma EPO is not connected with modification of myocardial expression: M-EPO/M-EPOR/M-FR/M-IRP1, iron load, except for M-TfR.
Abstract 241: Cardiac-Specific Overexpression of Runx2 Mediates Cardiac Hypertrophy and Dysfunction in Mice
Authors: Nakayama Hiroyuki , Hamatani Tatsuto , Kumagai Shohei , Tonegawa Kota , Yamashita Tomomi , Fujio Yasushi
Published: July 2021

Backgrounds:
Recent studies demonstrated that the osteopontin (OPN), an acid phosphoprotein plays pivotal roles in cardiac hypertrophy and failure. An osteogenic transcription factor Runx2 regulates the expression of OPN in osteoblasts. In the present study, we examined the pathological role of Runx2 in cardiac hypertrophy and failure.

Methods and Results:
Runx2 expression was detected in neonatal cardiomyocytes and upregulated in heart 14 days after myocardial infarction (MI) as well as 7days after transverse aortic constriction (TAC) procedures. To determine the functional role of Runx2 in heart, we generated transgenic mice (TG) with inducible cardiac-specific overexpression of Runx2. Two TG lines (low and high) were obtained and high-expressing TG (HE-TG) showed premature death within 8 weeks of age specifically in male mice. At two months of age, the survived male and female HE-TG displayed significant increases in heart weight/body weight ratio (mg/g) compared to controls (control; 4.95±0.26, n=6 vs HE-TG; 6.63±0.12, n=5, p<.05). Consistent with those results, the expression of hypertrophic marker genes such as atrial natriuretic factor (ANF) and αskeletal actin significantly increased in HE-TG heart assessed by real-time RT-PCR analysis. In addition, HE-TG mice demonstrated decreased fractional shortening assessed by echocardiography (control; 44.1±1.89%, n=9 vs HE-TG; 23.9±3.48%, n=7, p<.05). HE-TG mice demonstrated significantly lower heart rate (control; 630±18 bpm, vs HE-TG; 350±74 bpm, n=3 each, p<.05) and complete atrioventricular block by telemetry analysis. In response to pressure overload, low expressing TG (LE-TG) demonstrated higher mortality and enhanced cardiac hypertrophic response after TAC (control; 6.20±0.23, n=6 vs LE-TG; 6.90±0.26, n=4, p<.05).

Conclusions:
Targeted expression of Runx2 in heart mediates cardiac dysfunction and hypertrophy in mice. Thus, Runx2 could be a novel therapeutic target for heart failure.
Abstract 243: The Role of Reactive Oxygen Species in Hyperglycemia-Induced Attenuation of Anesthetic Preconditioning in Induced Pluripotent Stem Cell-Derived Cardiomyocytes
Authors: Canfield Scott , Twaroski Danielle , Bai Xiaowen , Kikuchi Chika , Bosnjak Zeljko J
Published: July 2021

Anesthetic Preconditioning (APC) protects the myocardium from ischemia/reperfusion injury. The cardioprotective effects of APC is diminished or even eliminated in individuals with diabetes mellitus and/or hyperglycemia. The development of patient-specific induced pluripotent stem cells and their differentiation capability has provided us with an
in vitro
model

to study the inefficiency of APC in these individuals.To investigate the underlying mechanisms involved in the attenuation of APC in both diabetic individuals and in hyperglycemia we utilized cardiomyocytes derived from Type 2 diabetic patient and healthy individual iPSCs, (T2DM-iPSCs and N-iPSCs, respectively).

Contracting cardiomyocytes were dissociated and selected by the expression of green fluorescent protein under the transcriptional control of myosin light chain-2v. Cardiomyocytes were exposed to varying glucose concentrations (5, 11, and 25 mM). Lactate dehydrogenase (LDH) release was measured using a colorimetric cytotoxicity assay kit and read spectrophotometrically. Mitochondrial membrane potential and reactive oxygen species (ROS) generation were measured with confocal microscopy.

APC reduced oxidative stress-induced lactate dehydrogenase (LDH) release in cardiomyocytes derived from both N-iPSCs- and T2DM-iPSCs in 5 and 11 mM glucose concentrations, but not in 25 mM glucose. Baseline membrane potential was similar between non-diabetic- and Type 2 diabetic-derived cardiomyocytes; however 25 mM glucose hyperpolarized the mitochondrial membrane potential. T2DM-iPSC-derived cardiomyocytes had an increase in ROS baseline levels compared to N-iPSC-derived cardiomyocytes. Additionally, high glucose concentrations increased oxidative stress-induced ROS production compared to lower glucose conditions in both cell lines.

Our preliminary data shows that high glucose generates excessive ROS and hyperpolarizes the mitochondrial membrane and may contribute to the inefficiency of diabetic and/or hyperglycemic individuals to be anesthetically preconditioned. By utilizing human iPSC-derived cardiomyocytes we can begin to understand the inability of hyperglycemic and diabetic individuals to be anesthetically preconditioned.
Abstract 244: Regenerative Metabolome and Energetic Signaling Mechanisms Governing Heart Renewal Capacity
Authors: Zhang Song , Nemutlu Emirhan , Terzic Andre , Dzeja Petras
Published: July 2021

The mammalian heart loses its regenerative potential within a week after birth. Although under certain circumstances adult cardiomyocytes can re-enter the cell cycle, initiate DNA synthesis and progress towards mitosis, however, they do not complete a conventional mitosis cycle and proceed towards endomitosis resulting in polyploidy. This suggests that high-energy dependent steps in cell cycle and myofibrillar disassembly are malfunctioning in adult cardiomyocytes. It is known that nucleotide-based energetic signaling and metabolic environment are critical for initiation of tissue regeneration after injury. The regulatory mechanisms and energetic-metabolic signaling circuits that govern cardiomyocyte cell cycle withdrawal and binucleation are poorly understood. Using mouse neonatal (2 and 10 days) hearts, with high and low regenerative capacity, respectively, we have determined metabolomic profiles and dynamics of phosphotransfer circuits and AMP-signaling using 18O-phosphoryl labeling mass-spectrometric and 18O-assisted 31P NMR techniques. The results demonstrate that loss of heart regenerative capacity 10 days after birth is associated with marked changes in heart metabolome and adenylate kinase (AK)-catalyzed phosphotransfer flux. Among most altered metabolites were putrescine and myo-inositol, involved in tissue regeneration, Krebs cycle and amino acid metabolites, creatinine, glucose-6-phosphate and lactate indicating rearrangements in energy and substrate metabolic systems. AK flux was the only phosphotransfer flux significantly upregulated in hearts at 10 days of age along with increased in mitochondrial and glycolytic capacities and mitochondrial AK2 expression. Increased AK2 expression was associated with augmented AMP phosphorylation, measured by generation of β-ATP[18O], and reduction of AMPK activity. Thus, increased channeling of AMP into mitochondria and reduction of AK-AMP-AMPK signaling axis could contribute to the withdrawal from completion of cell cycle in terminally differentiated adult cardiomyocytes. The uncovered metabolic signaling mechanism opens new avenues for targeted regulation of heart regenerative potential critical for repair of injured hearts.
Abstract 246: Transendocardial Injections of Allogeneic Mesenchymal Stem Cells Demonstrate Reversal of Left Ventricular Remodeling to Baseline
Authors: McCall Frederic C , Williams Adam R , Suncion Viky Y , Karantalis Vasileios , Zambrano Juan P , Heldman Alan W , Hare Joshua M
Published: July 2021

Background:
Mesenchymal stem cells (MSCs) improve ventricular function post MI

Hypothesis:
Transendocardial stem cell injections (TESI) reverse remodeling in a swine model of chronic ischemic cardiomyopathy (CIC).

Methods:
Gottingen swine (n=12), underwent a closed-chest, LAD occlusion-reperfusion model to create chronic ischemic cardiomyopathy. Three months post-MI, transendocardial injections of allogeneic MSCs (n=6) or sham injection (control group, n=6) were administered to the infarct and border zones. Cardiac MRI and pressure volume loops were obtained at baseline, during injection, and 6 months post injection. The three dimensional sphericity index (SI) was determined using the formula, SI= (π x LVLA3)/6, where LVLA is the left ventricle long axis length as measured in a 4-chamber long axis cardiac MRI end diastolic frame perpendicular to the line from the annulus of the mitral valve to the apex.

Results:
The sphericity index increased in both groups 3 months after MI, from 0.268 ± 0.054 to 0.348 ± 0.050 in the sham injection group and 0.277 ± 0.051 to 0.380 ± 0.059 in the TESI group. In the sham injection group, SI remained stable 9 months post sham injections indicative of irreversible remodeling post MI. In contrast, the injection group SI continued to decrease from 0.380 ± 0.059 to 0.346 ± 0.064 and 0.280 ± 0.50 at 3 and 6 months post TESI, respectively.

Conclusions:
Measurements of SI show that transendocardial stem cell injections of allogeneic MSCs demonstrate the potential to reverse left ventricular remodeling in CIC patients to geometric configurations comparable to their pre-MI shapes.
Abstract 247: Expression and Activity of K
ATP
Channel in Human Cardiomyocytes Derived from Induced Pluripotent Stem Cells of Nondiabetic and Type II Diabetic Patients
Authors: Canfield Scott , Bosnjak Zeljko J , Stadnicka Anna
Published: July 2021

Pharmacological preconditioning with volatile anesthetics (APC) protects the myocardium against ischemia/reperfusion injury via multiple pathways that include the cardiac sarcolemmal (sarc) KATP channel. However, APC is altered or absent in metabolic diseases such as diabetes. To investigate the underlying mechanisms we evaluated the expression and activity of cardiac sarcKATP channel in cardiomyocytes (CMs) differentiated from induced pluripotent stem cells of non-diabetic (N-iPSCs) and type II diabetic (DM-iPSCs) patients.

Differentiation of human iPSCs into CMs was confirmed by positive immunostaining for cardiac-specific markers and expression of green fluorescent protein under transcriptional control of cardiac promoter myosin light chain-2v that was delivered by lentiviral vector. Immunocytochemical analysis of cardiac sarcKATP channel subunits was carried out with Anti-Kir6.2 and Anti-SUR2A antibodies. Images were captured using a laser-scanning confocal microscope. Single KATP channel activity was monitored from excised inside-out patches at membrane potentials +80 mV to -80 mV, in symmetrical 140 mM K+. The channel was closed by 1 µM glibenclamide and 2 mM ATP.

Immunocytochemical analysis showed the presence and colocalization of Kir6.2 and SUR2A proteins in CMs derived from both N-iPSCs and DM-iPSCs. Interestingly, sarcolemmal expression of these subunits appeared lower in DM-iPSCs. Electrophysiological recordings demonstrated that at 5 µM ATP internal single sarcKATP channels were open more frequently in N-iPSCs (7 of 16 cultures) than in DM-iPSCs (3 of 16 cultures). When measured at +40 mV, the single channel current amplitude (2.2 pA) and conductance (55 pS) were typical for the cardiac sarcKATP channel in N-iPSCs. By contrast, the DM-iPSC channels flickered between the open and closed state, resulting in higher noise level and decreased current amplitude (1.6 pA).

This is the first report of sarcKATP channel expression and activity in CMs differentiated from human N-iPSCs and DM-iPSCs. The study suggests that compromised APC in human diabetic heart may in part result from altered expression of sarcKATP channel subunits and altered channel kinetics.