bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 316: Role of an Inflammasome Adaptor Molecule ASC (Apoptosis-associated Speck-like Protein Containing a Caspase Recruitment Domain) in Myocardial Ischemia-Reperfusion Injury
Authors: Kawaguchi Masanori , Takahashi Masafumi , Hata Takeki , Takahashi Yasuko , Morimoto Hajime , Ise Hirohiko , Sagara Junji , Hongo Minoru , Taniguchi Shun’ichiro , Ikeda Uichi
Published: July 2021

Inflammatory responses play a key role in the pathophysiology of myocardial ischemia-reperfusion (I/R) injury. ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) is an adaptor protein that forms “inflammasome” whose activation leads to caspase-1-dependent interleukin (IL)-1β generation and subsequent inflammatory responses; however, the role of ASC in myocardial I/R injury remains unclear. Baseline left ventricular (LV) function was unaltered in ASC-deficient (ASC
−/−
) mice. ASC
−/−
(n=42) and wild-type control (WT) (n=42) mice were subjected to 30 min LAD occlusion, followed by reperfusion. ASC
−/−
mice showed reduced infarct area (infarct area/area at risk: 18.7% vs. 28.6% at 48 h, p< 0.01) and scar formation (scar/LV area: 9.7% vs. 14.6% at 14 days, p< 0.01) after myocardial I/R. Echocardiography showed improved LV dysfunction (%FS: 35.2 vs. 28.4 at 7 days, p< 0.01; 34.0 vs. 25.7 at 14 days, p< 0.01) and dimensions (LVEDD [mm]: 3.88 vs. 4.21 at 7 days, p< 0.01; 3.99 vs. 4.43 at 14 days, p< 0.01) in the ASC
−/−
mice after myocardial I/R. Immunohistochemistry revealed that infiltration of macrophages (Mac3) and neutrophils (Gr-1) was markedly decreased in the injured myocardium of the ASC
−/−
mice (48 hr [/mm
2
]: 1226 vs. 884, p< 0.01; 782 vs. 554, p< 0.01, respectively); however, there was no difference of neovascularization (CD31) in the ischemic area. Double immunofluorescent staining showed that ASC expression was clearly observed in the infiltrated macrophages and neutrophils in the injured myocardium. Real-time RT-PCR analysis demonstrated that the myocardial expression of inflammatory cytokines, such as IL-1β, IL-6, and MCP-1, after I/R were significantly decreased in the ASC
−/−
mice, compared to that in the WT mice. Further,
in vitro
experiments showed that LPS-induced production of these inflammatory cytokines in the ASC
−/−
bone marrow cells was significantly decreased. These findings demonstrate that ASC deficiency prevents inflammatory cell infiltration and cytokine expression, thereby resulting in the improvement of LV dysfunction and remodeling after myocardial I/R injury, and suggest that ASC is a novel therapeutic target for myocardial I/R injury.
Abstract 323: Overexpression of Coupling Factor 6 Causes Salt-Induced Congestive Heart Failure by Tissue Acidosis-Mediated Reactive Oxygen Species
Authors: Ashitate Toshihiro
Published: July 2021

Reactive oxygen species (ROS) is associated with the development of congestive heart failure (CHF) in animals and humans. We have showed that coupling factor (CF6), a component of ATP synthase, suppresses prostacyclin and nitric oxide generation by intracellular acidosis, which is closely related to the generation of ROS and CHF. On the other hand, high salt intake, one of the most important factors to initiate and exacerbate CHF, increases ROS generation. We thus investigated the role of CF6 in the genesis of CHF by using transgenic mice (TG) overexpressing CF6 and exposing them to high-salt diet. In TG, the introduced gene of human CF6 was expressed in overall tissues including the heart, and upregulated by 2 fold. Baseline echocardiographic data, ROS generation, protein expression of sarcoplasmic/endoplasmic reticulum of Ca
2+
-ATPase (SERCA)-2, associated with cardiac muscle relaxation, and phospholamban, and the gene expression related to ATP synthesis and glycolysis, and the genes α- and β-MHC in the heart were similar between 7-week-old TG and wild type mice (WT). When the mice were fed with high-salt diet (8% salt) for 20–24 weeks, fractional shortening of the left ventricle was decreased in TG compared to WT (29±3% vs 39±2%, p< 0.05). The protein expression of SERCA-2 was decreased by 90±29% in TG compared with WT, whereas that of phospholamban, an inhibitor of SERCA-2, was increased by 141±31% in TG (both p< 0.05). In cDNA microarray analysis of the heart, the genes related to ATP synthesis, such as ATP synthase (0.29±0.10 fold) and cytochrome C (0.30±0.04), and those related to glycolysis were decreased in TG mice (all p< 0.05). The gene expression of β-MHC as the fetal isoform of MHC was increased in TG heart, whereas that of α-MHC as the adult isoform was unchanged. The myocardial level of 8-iso-prostaglandin F
2α
, a marker of ROS generation, was increased by 83±31% in TG compared with WT (p< 0.05). The mortality rate at the period of 35 weeks was greater in TG (7/14 (50%)) than in WT (1/13 (8%)) (p< 0.05 by log rank test), although neither TG nor WT were dead under normal salt diet. These suggest that CF6 induces the development of mitochondrial dysfunction and systolic and diastolic dysfunction, and eventually causes CHF by enhancing of ROS generation.
Abstract 337: Activation of Myocardial AMP-activated Protein Kinase by Metformin Prevents Progression of Heart Failure in Dogs; Involvement of eNOS Activation
Authors: Sasaki Hideyuki , Asanuma Hiroshi , Fujita Masashi , Takahama Hiroyuki , Asakura Masanori , Kim Jiyoong , Minamino Tetsuo , Takashima Seiji , Sanada Shoji , Ito Shin , Min Kyungduk , Komamura Kazuo , Mochizuki Naoki , Kitakaze Masafumi
Published: July 2021

Background;
Several studies have shown that metformin activates AMP-activated protein kinase (AMPK), which mediates potent cardioprotection against ischemia-reperfusion injury. AMPK is also activated in experimental failing myocardium, suggesting that activation of AMPK is beneficial for the pathophysiology of heart failure. We investigated whether metformin prevents oxidative stress-induced cell death in rat cardiomyocytes and attenuates the progression of heart failure in dogs.

Methods and Results;
The treatment with metformin (10 μmol/L) protected the rat cultured cardiomyocytes against cell death due to H
2
O
2
exposure (50 μmol/L) as indicated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), TUNEL staining, and flow cytometry. These effects were blunted by an AMPK inhibitor, compound-C (20 μmol/L), suggesting that the activation of AMPK decreased the extent of apoptosis-induced cell death due to H
2
O
2
exposure. Continuous rapid ventricular pacing (230/min for 4 weeks) in dogs caused heart failure and the treatment with metformin (100 mg/kg/day PO, n=8) decreased left ventricular (LV) end-diastolic dimension (32.8±0.4 vs. 36.5±1.0 mm, p< 0.01) and pressure (11.8±1.1 vs. 22±0.9 mmHg, p< 0.01), and increased LV fractional shortening (18.6±1.8 vs. 9.6±0.7 %, p< 0.01) along with enhanced phosphorylation of AMPK and the decreased the number of TUNEL-positive cells of the LV myocardium compared with the vehicle group (n=8). Interestingly, metformin increased the protein and mRNA levels of endothelial nitric oxide synthase of the LV myocardium and plasma nitric oxide levels. Metformin improved the plasma insulin resistance without increased myocardial GLUT-4 translocation. Furthermore, the subcutaneous administration of AICAR (50 mg/kg/every other day), another AMPK activator mediated the equivalent effects to metformin, strengthening the pivotal role of AMPK in reduction of apoptosis and prevention of heart failure.

Conclusions;
Activation of myocardial AMPK attenuated the oxidative stress-induced cardiomyocyte apoptosis and prevented the progression of heart failure in dogs, along with eNOS activation. Thus, metformin or AICAR may be applicable as a novel therapy for heart failure.
Abstract 338: Cardiac-type Calcium Release Mechanism in Cardiac Precursor Cells
Authors: Altomare Claudia , Barile Lucio , Marangoni Stefano , Rocchetti Marcella , Mostacciuolo Gaspare , Giacomello Alessandro , Messina Elisa , Zaza Antonio
Published: July 2021

Cardiac progenitor cells (CPCs) form three-dimensional structure named cardiospheres (CSs). While CSs are seen as partially committed cardiac precursors, it is unknown whether they already display cardiac-type molecular functions. Whereas IP3-mediated Ca
2+
release is a shared property of many cell types, Ca
+
release through RyR channels is muscle specific.

Aims:
To test if cardiac-type Ca
2+
release mechanism is present in CPCs and whether it may develop during the CSs stage.

Methods:
Cells arose from murine cardiac explants in culture were studied prior to CSs formation (pre-CSs) and after expansion of CSs (post-CSs). Ca
2+
transients were detected in wide optical confocal fields by Fluo4-AM fluorescence. RyR- and IP3-R-mediated Ca
2+
release from intracellular stores was tested by exposures to caffeine (CAF, 10 mM) or ATP (200 μM) respectively in Ca
2+
-free conditions. The expression of the cardiac-specific RyR isoform (RyR2) was tested by immunolabeling and western-blot analysis.

Results:
In isolated cells CAF-induced response was almost exclusive of post-CSs cells (22.4 % vs. 3.62 %; p<0.05); ATP-induced response was already present in pre-CSs cells and showed only a small increase in post-CSs ones (94.3 % vs 86.1 % p<0.05). The ratio between post-CSs and pre-CSs responses was 6.2 for CAF and 1.1 for ATP. Both CAF and ATP responses were suppressed by the SERCA inhibitor CPA (50 μM), thus confirming intracellular stores as the Ca
2+
source. ATP response only was suppressed by the IP3-R blocker 2APB (10 μM). Ryanodine (20 μM) prevented the response to CAF, but not to ATP. In post-CSs RyR2 protein levels were higher than in pre-CSs and similar to those of adult myocytes. Immunoistochemistry analysis of post-CSs cells results in a distribution of RyR2 expression consistent with immature neonatal cardiomyocytes previously described.

Conclusions:
At variance with IP3-mediated signaling, RyR mediated Ca
2+
release develops during maturation within the CSs environment, along with expression of the cardiac RyR isoform. Detection of caffeine-induced Ca
2+
responses may be useful in identifying cardio-specific functional maturation in progenitor cell populations.
Abstract 350: Toll-like Receptor 2 Activation, Especially In Circulating Leukocytes, Mediates Myocardial Ischemia/Reperfusion Injury In Mice
Authors: Arslan Fatih , Pasterkamp Gerard , Timmers Leo , van Middelaar Ben , Doevendans Pieter A , de Kleijn Dominique P
Published: July 2021

OBJECTIVES. Myocardial ischemia/reperfusion (MI/R) injury is characterized by an inflammatory response through NF-κB, increase of infarct size and worsening of cardiac function. Toll-like receptors (TLRs) are part of innate immunity and initiate the same inflammatory reaction. Here we studied in vivo to what extent TLR2 is involved in myocardial damage and what the relative contribution is of TLR2 expression in parenchymal cells and leukocytes to myocardial damage during MI/R in mice.

METHODS. C57Bl6J mice underwent 30 minutes ischemia - 24 hours of reperfusion. Four experimental groups were studied: TLR2 knock-out (TLR2 KO) mice (n=10), saline treated wild-type (WT) mice (n=10), generated chimeric WT mice with TLR2 KO bone marrow (BLOOD KO; n=7) and chimeric TLR2 KO mice with WT hematopoietic cells (ORGAN KO; n=7). Saline was administered via the tail vein 5 minutes prior to reperfusion. After 24 hours, the LCA was ligated again at the level marked by the suture left in place. Mice were terminated and infarct size (IS) was measured as a percentage of the area at risk (AAR) using 4% Evans’ blue dye injection in the aortic root and triphenyltetrazolium chloride (TTC) staining (fig. 1). Data are presented as Mean±SEM.

RESULTS. The AAR as percentage of the left ventricle was similar between groups: TLR2 KO 41%, saline 41%, Blood KO 41%, Organ KO 42%. Saline treatment resulted in 34.5%±3.3 of infarction, whereas in TLR2 KO mice infarct size decreased to 23.0%±2.9 (p=0.029 vs. saline). Infarct size in BLOOD KO mice was 22.9%±2.7 (p=0.024 vs. saline), while ORGAN KO mice had 33.9%±3.2 (p=0.998 vs. saline) of infarction within the area at risk (fig. 2).

CONCLUSION. TLR2 deficiency significantly reduces infarct size with ~33% compared to saline treatment in mice after 30 minutes of ischemia and 24 hours of reperfusion. We show for the first time that TLR2 expression in circulating leukocytes plays an important role in infarction after MI/R injury. Systemic inhibition of TLR2 may be a potential therapeutic target in the treatment of patients with acute myocardial infarction.
Abstract 355: Snf1-related kinase is Upregulated in the Ischemic Heart and Regulates Cellular Metabolism and Death
Authors: Burke Michael A , Ochenjele George , Fernandez Richard P , Ardehali Hossein
Published: July 2021

Sucrose non-fermenting (Snf1)-related kinase (SNRK) is a novel kinase homologous to the Snf1 kinase of yeast and the AMP-activated protein kinases (AMPK) of mammals. SNRK is expressed in a variety of tissues; however, its role in the cardiovascular system is unclear. While AMPK and Snf1 regulate metabolism, the primary function of SNRK is not known. We hypothesized that like AMPK, SNRK regulates cellular metabolism and influences cell survival. Western blots of protein extracts from several cell types showed that SNRK is widely expressed and exists in different forms in the nucleus and cytoplasm. We next assessed the effects of ischemia, hypoglycemia and increased AMP on SNRK levels. Dog hearts subjected to ischemia displayed a 41% increase in SNRK levels (
P
<0.01), while explanted hearts from patients with ischemic cardiomyopathy showed a 220% increase compared to controls (
P
<0.05). SNRK mRNA levels and subcellular location were altered by hypoglycemia and increased AMP levels. The role of SNRK on cell death and metabolism was then assessed by transfecting HEK293 cells with GFP, a SNRKGFP fusion construct, or SNRKGFP containing a mutation in the nuclear localization sequence (SNRKnls). SNRKGFP overexpression increased cell death by 10% (
P
=0.007) compared to controls both in the absence and presence of oxidant stress. Nuclear respiratory factors (NRF)2α and -2β (transcription factors that induce mitochondrial biogenesis) were upregulated with SNRK overexpression by 30% and 90%, respectively (
P
<0.05). SNRKnls did not induce a significant change. SNRK overexpression, but not SNRKnls, resulted in a 15% reduction in cellular ATP content compared to controls (
P
=0.0001). A gene array performed on cells overexpressing either SNRKGFP or GFP showed that SNRK overexpression strongly correlates with numerous nuclear and metabolic functions. These data show that SNRK is upregulated in ischemic heart disease and suggest that it induces cell death. SNRK also appears to be a component of the cellular metabolic machinery, likely exerting its actions via entry into the nucleus. Thus, by altering cellular metabolism, SNRK may play an important role in the response of the heart to ischemia, and may provide a novel target for cardiovascular therapeutic interventions.
Abstract 356: Global but not Liver-specific Inactivation of CarcinoEmbryonic Antigen-related Cell Adhesion Molecule 1 Results in Cardiac Hypertrophy and Lipid Accumulation
Authors: Morgan Eric E , Ledford Kelly J , Khouri Samer , Purohit Anish , Bowman Thomas , Najjar Sonia M , Pierre Sandrine V
Published: July 2021

Background:
Liver fatty acid synthase (FAS) activity is acutely inhibited by transient increases in insulin via endocytosis of the CarcinoEmbryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1) and its subsequent binding to FAS. Mice with liver-specific inactivation of Ceacam1 (LSACC1) and mice with null mutation of Ceacam1 (Cc1
−/−
) exhibit hyperinsulinemia, insulin resistance, visceral obesity and increased circulating triglycerides (TG) and free fatty acids (FFA).

Hypothesis:
Cc1
−/−
and LSACC1 hearts exhibit elevated cardiac lipid content and hypertrophy.

Results:
Using 2D echocardiography, hypertrophy as evidenced by increased relative wall thickness (RWT; [calculated as: (septal wall + posterior wall thickness)/left ventricular end diastolic dimension]) in Cc1
−/−
mice compared to wild-type (WT) littermates (0.59 ± 0.06 vs 0.42 ± 0.03; n=14/group; p<0.05). RWT was not different between LSACC1 and WT (0.59 ± 0.04 vs. 0.64 ± 0.05; n=7/group). Histological sections stained with H&E demonstrated a significant increase in myocyte thickness in Cc1
−/−
compared to WT(11.0 ± 0.3 μm vs 6.9 ±0.2μm; p<0.001; Figure

, A), and sections stained with Oil Red O revealed accumulations of neutral lipid in Cc1
−/−
hearts (Figure

, B). Myocardial triglyceride content and lipoperoxidation were significantly increased in Cc1
−/−
hearts compared to WT (Figure

, C).

Conclusions:
Cc1
−/−
mice but not LSACC1 mice exhibit myocardial lipid accumulation and hypertrophy. These data suggest that cardiac CEACAM1 plays a key role in regulating cardiac fatty acid metabolism and that cardiac lipid accumulation and hypertrophy in the Cc1−/− mice result, in part, from the loss of cardiac CEACAM1.

Figure.

Representative photomicrographs (×400) of cardiac tissue from
Cc1
+/+
and
Cc1
−/−
mice demonstrating increased cardiac myocyte thickening (Panel A, H&E staining) and netural lipid accumulation (Panel B oil red O staining. Myocardial triglyceride content and lipid peroxidation as determined by the level of thiobarbituric acid reactive substances (TBARS) were significantly elevated in hearts from
Cc1
−/− mice (Panel C).
Abstract 378: Gene Delivery of H11 Kinase/Hsp22 Provides Cardioprotection by A Nitric Oxide-dependent Mechanism
Authors: Chen Li , Shen You-Tang , Hong Chull , Vatner Stephen F , Depre Christophe
Published: July 2021

H11 kinase/Hsp22 (H11K) is a small heat shock protein (hsp) which is upregulated in ischemic heart disease, both in animal models and in patients. Cardiac-specific overexpression of H11K in a transgenic (TG) mouse provides protection against ischemic damage that is equivalent to ischemic preconditioning (IPC). Because studies in mice can not always be translated to patients, we examined the role of H11K in mediating cardiomyopathy in a swine model. An adenovirus harboring the H11K sequence, or a LacZ control, was injected in the potential area-at-risk (AAR) perfused by the left circumflex coronary artery (CA) in chronically instrumented swine (n=5/group). Three days after injection, CA occlusion (O) for 60 min, followed by 3 days of reperfusion (R), induced myocardial infarction. Although the AAR was similar between groups, the MI/AAR was reduced significantly (P<0.05) in the H11K-injected group (32±5%) compared to the LacZ group (50±5%). Three days following reperfusion, regional wall thickening in the ischemic region was reduced by 58±2% of baseline in H11K group vs. 82±7% in LacZ (P<0.05). Compared to LacZ, H11K-injected myocardium showed a significant (P<0.05) 4-fold increase in H11K, and a 2- to 4-fold increase in the expression of the inducible form of nitric oxide synthase (NOS), a key mediator of IPC. To substantiate a role of NOS, the effects of CAO/CAR were examined in H11K-injected pigs (n=4) after pretreatment with 35 mg/kg of the NOS inhibitor L-NNA. In these pigs, the cardioprotection of H11K was abolished, i.e., MI/AAR was 67±6% (NS vs. LacZ control). These results were further substantiated in the TG mouse, pretreated or not with L-NNA for 2 days. After 45 min no-flow ischemia followed by 6h reperfusion, the MI/AAR was 5.0±0.4% in non-treated TG and 36±6% in L-NNA-treated TG mice (P<0.01). Therefore, overexpression of H11K in a large mammalian model of ischemic heart disease induces significant cardioprotection. The mechanism involves upregulation of iNOS, since cardioprotection is no longer evident in the presence of NOS inhibition with L-NNA.
Abstract 382: The Bone Morphogenetic Protein Pathway Mediates the Effects of H11 Kinase/Hsp22 on Cardiac Cell Growth and Survival
Authors: Sui Xiangzhen , Li Dan , Hedhli Nadia , Qiu Hongyu , Gaussin Vinciane , Depre Christophe
Published: July 2021

The bone morphogenetic protein (BMP) pathway is a major signaling mechanism during cardiac development but it has no clear function in the post-natal heart. Here, we tested the hypothesis that BMP mediates the physiological effect of the cardiac chaperone H11Kinase/Hsp22 (H11K).

Expression of H11K increases during both cardiac ischemia and overload, and its cardiac-specific over-expression in a transgenic (TG) mouse is sufficient to provide major protection against ischemia and to promote cardiac cell growth, which involves the activation of phosphatidylinositol-3-kinase (PI3K) and of its effector Akt. We tested whether H11K-induced activation of PI3K is mediated by BMP. Microarray comparison between hearts from TG and wild type (WT) mice showed an up-regulation of the BMP receptor subunits Alk3 and BMPR-II, as well as of the BMP receptor ligand BMP4, which was confirmed at the protein level (P<0.01 vs WT). Activation of the BMP pathway in TG mice was confirmed by increased phosphorylation of the canonical BMP effectors Smad 1/5/8 (P<0.01 vs WT). The mechanism was further studied in isolated cardiac myocytes. Adeno-mediated over-expression of H11K was accompanied by significant 2–3-fold increase in PI3K activity, phospho-Akt, Smad 1/5/8 phosphorylation and cell growth as measured by [3H]phenylalanine incorporation, and by a 70% reduction in H2O2-mediated apoptosis (all values, P<0.01 vs control). All these changes mediated by H11K in myocytes were abolished upon addition of the BMP antagonist noggin. In pull-down experiments, H11K co-precipitated with both Alk3 and BMPR-II, and increased the association of these two subunits into a functional receptor. Accordingly, Smad 1/5/8 phosphorylation in presence of BMP4 was enhanced by 5-fold upon H11K over-expression, whereas it was decreased by 3-fold upon H11K knockdown (both, P<0.01 vs control), which shows that H11K potentiates the BMP receptor signaling pathway. Therefore, potentiation of the BMP receptor pathway by H11K promotes the activation of the PI3K/Akt pathway and dictates the physiological effects of H11K on cardiac cell growth and survival, which shows a novel role for BMP signaling in post-natal heart.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 389: Human Cardiac Sympathetic Nerves Switch the Neurotransmitter Property from Catecholaminergic to Cholinergic in Patients with Severe Heart Failure
Authors: Kanazawa Hideaki , Ieda Masaki , Kimura Kensuke , Arai Takahide , Manabe Haruko , Kimura Tokuhiro , Okada Yasunori , Ueda Hatsue , Fukuda Keiichi
Published: July 2021

[Background]
Congestive heart failure (CHF) is characterized by activation of the sympathetic nervous system (SNS) with depletion of norepinephrine (NE) stores, which was initially considered to be the result of excess NE secretion and the loss of noradrenergic nerve terminals. Recent studies however have revealed that it is caused by down regulation of NE synthesis and re-uptake, although the molecular mechanism of down regulation of the sympathetic neuronal function remains unknown. We recently found in an animal model of CHF that the cardiac SNS switches the neurotransmitter property from catecholaminergic to cholinergic, mediated by cytokines LIF and CT-1 secreted from failing myocardium. This study was designed to investigate whether or not this cholinergic transdifferentiation of cardiac SNS occurs in patients with CHF.

[Methods & Results]
(1) We analyzed 8 samples from patients who died of non-cardiac causes obtained at autopsy (control group), and 8 samples from patients with CHF (CHF group). Five of them died of CHF, and 3 were obtained from native hearts of transplant recipients. (2) The heart weight was significantly higher in the CHF group. (3) The gross morphology of the cardiac SNS did not differ between the two groups. HE and Masson trichrome staining showed disorganized cardiomyocytes and interstitial fibrosis in CHF. (4) Immunostaining for tyrosine hydroxylase (TH, sympathetic nerve marker) revealed that the epicardial nerve bundles and stellate ganglia of the control group had a predominance of TH
+
nerves, whereas those of CHF group were significantly decreased. (5) Immunostaining for choline transporter (CHT, cholinergic neuron marker) revealed that CHT
+
neurons were markedly increased in the epicardial nerve bundles of CHF hearts compared with the control group. Some nerves co-expressed both TH and CHT markers. (6) Immunostaining for choline acetyl transferase (ChAT, a cholinergic neuron marker) revealed that stellate ganglia had a lot of ChAT
+
neurons compared with the control. (7) Nissl staining showed that there was no difference between the two groups in neuron number in the stellate ganglia.

[Conclusions]
These results indicated that in patients with CHF the cardiac sympathetic nerves also had cholinergic nerve properties.
Abstract 390: Antioxidative and Anti-inflammatory Effects of Statin Modulating Autonomic Nerve Function in Hypercholesterolemic Rabbits
Authors: Liu Yen-Bin , Wu Chau-Chung , Lin Lian-Yu
Published: July 2021

Background:
We previous showed statins could prevent or reverse cardiac nerve sprouting and sympathetic hyperinnervation induced by hypercholesterolemia (HC) in rabbit models. The significance and mechanisms are unclear.

Methods and Results:
We studied heart rate variability (HRV) and baroreflex sensitivity (BRS) in 23 rabbits with HC chow, standard chow (Control), or HC chow and atorvastatin. Myocardial content of nitrotyrosine, foam cell formation (RAM 11-positive cell) and serum high-sensitivity C reactive protein (hs-CRP) were measured in 56 rabbits fed with standard chow, HC chow or HC chow and atorvastatin. The status of Rac1 was also examined. The HRV and BRS were significantly reduced in HC rabbits (Control vs. HC; SDNN: 11.7±3.8 vs. 6.0±4.1 ms, p <0.01; RMSSD: 14.6±7.5 vs. 2.8±1.2 ms, p<0.001; LF/HF: 0.29±0.18 vs. 2.0±2.15, p <0.04; BRS: 8.5±4.6 vs. 2.2±2.2 ms/mmHg, p <0.01). Atorvastatin treatment prevented the decreases in HRV and BRS of HC rabbits (Statin, vs. HC; SDNN: 12.0±8.4 ms, p <0.1; RMSSD: 18.2±17.3 ms, p<0.02; LF/HF: 0.39±0.36, p=0.09; BRS: 11.2±4.8 ms/mmHg, p <0.01). Myocardial levels of nitrotyrosine were progressively increased while increasing the dietary cholesterol (Control: 0.34±0.01 nM; 0.25% HC: 0.68±0.04 nM; 0.5% HC: 1.15±0.25 nM; p<0.001). Following statin treatment, levels of nitrotyrosine were significantly reduced (p<0.001), and there was a dose-response relationship between the dosage of atorvastatin and myocardial nitrotyrosine levels. Myocardial RAM 11-positive cells and serum hs-CRP levels were increased in HC (Control vs. HC; RAM 11: 2.3±1.7 vs. 13.1±6.7 /microscopic field, p<0.01; hs-CRP: 1.6±1.0 vs. 7.7±5.2 mg/dl, p<0.01). Atorvastatin significantly reduced the levels of these inflammatory markers (p<0.05). In addition, atorvastatin administration significantly decreased the accentuated myocardial Rac1 expression in HC rabbits.

Conclusion:
HC rabbits have increased cardiac inflammation and heightened sympathetic tone. Atorvastatin significantly decreased the myocardial inflammation and reduced the cardiac sympathetic tone in HC rabbits. A reduction in myocardial Rac1 by atorvastatin may serve as a molecular mechanism for this reversing cardiac neural remodeling.
Abstract 419: Human ApoB100/CETP Transgenic Mouse is a Useful Animal Model for Evaluation of HDL-C Elevation and Suppression of Atherosclerosis by Peroxisome Proliferator-Activated Receptor Delta Agonist
Authors: Naya Noriyuki , Fukao Keita , Nakamura Akemi , Nomura Kohji , Hara Seijiro , Hanasaki Kohji
Published: July 2021

PPARδ is one of the transcription factors that regulate lipid metabolism. Recently, PPARδ agonists have been reported to induce the elevation of plasma HDL-C levels in obese mice, rhesus monkeys as well as humans, indicating their potentials as a new class of HDL-C raising agent. In addition, anti-atherosclerotic effects of PPARδ agonists were reported in LDLR-KO and ApoE-KO mice. In these mice, however, the lipoprotein profiles are greatly different from those in humans in terms of the deficiency in CETP and ApoB100, and thus the anti-inflammatory effects were regarded as a main anti-atherosclerotic mechanism of PPARδ agonists. It has been reported that human ApoB100/CETP transgenic (hApoB100/CETP-Tg) mice have similar lipoprotein profiles with humans. In this study, we used hApoB100/CETP-Tg mice placed under a western diet for evaluation of GW501516, one of the most potent and selective PPARδ agonists, in order to investigate the linkage between HDL-C elevation and anti-atherosclerotic potency. For evaluation of plasma HDL-C levels, the hApoB100/CETP-Tg mice were orally treated with GW501516 for 1 week and its potency was compared with fenofibrate, a PPARα agonist used in the clinic. For evaluation of the anti-atherosclerotic effect, the mice were orally treated with GW501516 for 18 weeks and atherosclerosis at the aortic valves was determined by cross-sectional lesion analysis. Serum lipoprotein parameters were periodically monitored and lipoprotein profiles were analyzed by FPLC method. Treatment with GW501516 resulted in significant elevation of plasma HDL-C levels with more potency compared to fenofibrate (24% and 15% elevation at 10mg/kg, respectively). Serum apoA-I was also increased by the similar ratio as HDL-C, and the particle size of HDL was not changed, suggesting that the numbers of HDL particle are increased by GW501516. Long term treatment of GW501516 (3 and 10 mg/kg) resulted in dose-dependent suppression of atherosclerosis (42 and 57% inhibition, respectively) with a strong correlation with HDL-C elevation (p<0.001). By using hApoB100/CETP-Tg mice, PPARδ was confirmed as a promising target of anti-atherosclerotic therapy as a new class of HDL-C raising agent.
Abstract 421: Aldose Reductase Protects Against the Early Atherosclerotic Lesion Formation InApoe-null Mice
Authors: Srivastava Sanjay , Barski Oleg , Bhatnagar Aruni
Published: July 2021

Atherosclerotic lesion formation is associated with extensive oxidation of unsaturated lipids and the accumulation of lipid oxidation products. Products of lipid oxidation, particularly aldehydes, stimulate cytokine production and enhance monocyte adhesion. Aldehydes generated by oxidized lipids are metabolized by several biochemical pathways, of which aldose reductase (AR)-catalyzed reduction represents a metabolic fate common to both free and phospholipid esterified aldehydes. Herein, we tested the hypothesis that inhibition of AR could aggravate atherosclerotic lesion formation by preventing the removal and the detoxification of aldehydes generated by oxidized lipids. In atherosclerotic lesions of apoE-null mice, AR protein was associated with macrophage-rich regions and its abundance increased with lesion progression. Treatment of 8 week old apoE-null mice with AR inhibitors sorbinil or tolrestat for 4 weeks increased lesion formation in the aortic arch (P<0.01) and the aortic sinus (P<0.01). No change in lesion formation was observed when 24 week old mice were fed AR inhibitors for 12 weeks. To probe the role of AR in atherogenesis further, we generated AR
−/−
/apoE
−/−
mice. Lesions of 8 week old AR
−/−
/apoE
−/−
mice maintained on high fat diet for 4 or 12 weeks were significantly larger throughout the aortic tree (P<0.01 for both the groups) when compared with age-matched AR
+/+
/apoE
−/−
mice. Lesions in AR
−/−
/apoE
−/−
mice exhibited increased collagen (P<0.01) and macrophage content (P<0.01) and a decrease in smooth muscle cells (P<0.01). GC-MS analysis showed that the concentration of AR substrates HNE and hexanal was increased by 2.5–3 fold (P<0.01) in the plasma of AR
−/−
/apoE
−/−
mice as compared with AR
+/+
/apoE
−/−
mice. Immunohistochemical analysis showed greater accumulation of protein-HNE adducts in arterial lesions of AR
−/−
/apoE
−/−
mice. These observations suggest that AR is up regulated during atherosclerosis and that this protein protects against early stages of atherosclerotic lesion formation by removing aldehydes generated by lipid oxidation.
Abstract 485: Follistatin-Like 1 is a Novel Secreted Muscle Protein that Confers Cardiovascular Protection
Authors: Ouchi Noriyuki , Oshima Yuichi , Walsh Kenneth
Published: July 2021

Increasing evidence indicates that skeletal muscle secretes factors, referred to as myokines, that affect the behavior of neighboring or remote organs. Myogenic Akt signaling confers vascular protection during normal tissue growth. We focused on the identification of novel factors secreted from Akt1-activated skeletal muscle that have protective effects on the cardiovascular systems through paracrine mechanisms. Transgenic Akt1 overexpression in skeletal muscle led to myofiber growth that was coupled to an increase in muscle capillary density. Follistatin-like 1 (Fstl1) was selected as a potential myokine after a screening of upregulated transcripts in skeletal muscle of inducible Akt1 transgenic mice by microarray analysis. Myogenic Akt activation led to an induction of Fstl1 by muscle and increased circulating levels of Fstl1. Ischemic hindlimb surgery also induced Fstl1 expression by muscle and increased Fstl1 levels in serum. Intramuscular administration of an adenoviral vector expressing Fstl1 (Ad-Fstl1) accelerated flow recovery and increased capillary density in the ischemic hindlimbs of wild-type mice. These stimulatory effects were dependent on the upregulation of Akt-endothelial nitric oxide synthase (eNOS) signaling axes within endothelial cells. In cultured human umbilical vein endothelium cells, Ad-Fstl1 stimulated migration and differentiation into network structures, and inhibited apoptosis under conditions of serum deprivation. The pro-angiogenic actions of Fstl1 were mediated through its ability to activate Akt-eNOS signaling pathways. Furthermore, Ad-Fstl1 protected neonatal rat ventricular myocytes from hypoxia/reoxygenation-induced apoptosis and this protective effect was dependent on Akt activation. The intravenous administration of Ad-Fstl1 to wild-type mice resulted in a marked reduction in myocardial infarct size following ischemia-reperfusion injury that was accompanied by a significant reduction in apoptosis in the heart. These data indicate that Fstl1 is a novel Akt1-regulated myokine that can function to promote the new blood vessel growth in response to ischemia and protect against myocardial ischemia-reperfusion injury through its ability to activate Akt signaling in remote tissues.
Abstract 1367: The Nad(p)h Oxidase Component Nox4 Is Partially Localized In Mitochondria In Cardiac Myocytes
Authors: Ago Tetsuro , Kuroda Junya , Pain Jayashree , Sadoshima Junichi
Published: July 2021

Reactive oxygen species (ROS) regulate cell growth, death, and differentiation in cells. NAD(P)H oxidases are major sources of ROS in the cardiovascular system. Among the NAD(P)H oxidase protein family (Nox1–5), Nox4 is expressed in both the heart and cultured cardiac myocytes. However, the function of Nox4 in myocytes remains unknown. In order to examine expression of Nox4 in myocytes in detail, we made mouse monoclonal Nox4 antibodies, using the C-terminal cytoplasmic region of Nox4 (residues 307–578) as the antigen. After screening more than 1,000 clones, we chose a clone (3D2) that detected recombinant and endogenous Nox4 in mouse hearts and in neonatal rat cardiac myocytes without crossreacting with other Nox isoforms. Endogenous Nox4 was detected as a 55 kD protein in cardiac myocytes in immunoblot analyses. Immunocytochemistry showed that endogenous Nox4 is localized in the peri-nuclear region in myocytes. When HA-tagged Nox4 was expressed in myocytes, staining with HA antibody and that with the Nox4 antibody exhibited an overlapping pattern in the peri-nuclear region. Interestingly, staining of endogenous and exogenous Nox4 was co-localized with that with Mitotracker, a mitochondrial marker. Fluorescent intensity of Mitosox, an indicator of superoxide production in mitochondria, was enhanced in myocytes overexpressing Nox4. Consistently, in mice with cardiac-specific overexpression of Nox4 (Tg-Nox4), expression of Nox4 was increased 2.5 fold in the mitochondrial fraction. Superoxide dismutase-inhibitable NAD(P)H-dependent superoxide production in the mitochondrial fraction was greater in Tg-Nox4 than in non-transgenic mice (NTg) (1.7 fold). Proteomic analysis demonstrated that mitochondrial proteins, related to the TCA cycle and the electron transport, such as mitochondrial aconitase and NADH dehydrogenase, are strongly oxidized in Tg-Nox4, compared to NTg. These results indicate that Nox4 may be partially localized in mitochondria. Although leakage from electron transport chain accounts for the majority of superoxide production in mitochondria, Nox4 may participate in the superoxide production in mitochondria thereby modulating mitochondrial oxidative stress in cardiac myocytes.
Abstract 1394: Sublethal Levels of Aldehydes Augmented Cardiac Anti-Oxidant Defense through Activation of eIF2
α
-ATF4 Pathway via GCN2 Kinase
Authors: Katayama Takaharu , Sano Motoaki , Endo Jin , Hayashida Kentaro , Matsuhashi Tomohiro , Tokudome Satori , Kageyama Toshimi , Yuasa Shinsuke , Adachi Takeshi , Suematsu Makoto , Nishimaki Kiyomi , Ohsawa Ikuroh , Ohta Shigeo , Ogawa Satoshi , Fukuda Keiichi
Published: July 2021

[Introduction] Despite an increase in the levels of aldehydes, the heart from aldehyde dehydrogenase (
ALDH
)
2*2
-transgenic (Tg) mice, loss of function model of ALDH, exhibited a greater tolerance to oxidative stress via activation of amino acid metabolism leading to glutathione biosynthesis. This study was designed to identify the signaling cascades responsible for the activation of amino acid metabolism by aldehydes. [Methods & Results] (1) Phosphorylation of
α
-subunit of eukaryotic translation initiation factor 2 (eIF2
α
) and subsequent translational activation of ATF4 have been shown to induce amino acid metabolism as a common response to a wide variety of stressors. Consistent with this, phosphorylation levels of eIF2
α
and protein expression of ATF4 were increased in
ALDH2*2
-Tg hearts. (2) Among four eIF2
α
kinases, general control non-depressible (GCN)2 kinase, a sensor for amino acid insufficiency, was activated in
ALDH2*2
-Tg heart. (3) Quantification of intracellular amino acid demonstrated that free histidine concentration in
ALDH2*2
-Tg heart was selectively reduced by 50% compared to that in non-Tg littermates. (4) To clarify the functional significance of observed reduction in histidine,
ALDH2*2
-Tg mice were fed a high histidine diet. The phosphorylation levels of eIF2
α
and the protein levels of ATF4 were diminished by 50% in
ALDH2*2
-Tg mice fed the high histidine diet, in agreement with the normalization of histidine concentration. Accordingly, both enhanced tolerance to ischemia-reperfusion injury and elevated levels of glutathione were partially diminished in the heart from
ALDH2*2
-Tg mice fed the high histidine diet compared to
ALDH2*2
-Tg mice fed normal chow. (5) In culture, exposure to 4-hydroxy-2-nonenal (4-HNE) phosphorylated GCN2 and eIF2
α
and increased protein levels of ATF4 in a time-dependent manner. (6) siRNA-mediated knockdown of GCN2 abrogated 4-HNE-induced induction of amino acid metabolic genes. [Conclusions] Activation of eIF2
α
-ATF4 pathway via GCN2 kinase might be of special importance in the transcriptional control that coordinately promotes amino acid metabolism in response to aldehydes. Intracellular depletion of free histidine is at least partly involved in the activation of GCN2 kinase by aldehydes.
Abstract 1416: IGF-1 Gene Transfer Promotes Connexin-43 Expression and Enhances Integration of Bone Marrow Stem Cells in the Infarcted Heart
Authors: Jiang Shujia , Haider Khawaja , Idris Niagara M , Ashraf Muhammad
Published: July 2021

For functional benefits, bone marrow stem cells should engraft and develop functional syncitium with the host myocytes in the infarcted heart. We report that IGF-1 overexpression significantly enhances connexin-43 (Cx-43) in mesenchymal stem cells (MSCs) which promotes their survival and functional integration with host myocardium post-engraftment. IGF-1 gene transduced MSCs (
IGF-1
MSCs) expressed higher IGF-1 (
p
<
0.001
) as compared with GFP gene transduced cells (
GFP
MSCs). After 8h anoxia, MSCs, H2C9 and endothelial cells treated with
IGF-1
MSCs conditioned medium (
IGF-1
CM) showed higher survival (
p
<
0.001 vs
GFP
MSCs conditioned medium (
GFP
CM) supported by higher Akt activation which was abolished by wortmannin. Immunodetection showed Cx-43 plaques in the peripheral areas besides punctate distribution in the cytoplasm around the nucleus in
IGF-1
MSCs and
IGF-1
CM treated MSCs whereas
GFP
MSCs and
GFP
CM treated MSCs showed Cx-43 expression in regions of intimate cell to cell contact only. IGF-1 overexpression or
IGF-1
CM treatment of MSCs also induced higher level Akt and p27 activation, and Cx-43 expression as compared with
GFP
MSCs (
p
=
0.02
) which was abolished by wortmannin. For in vivo studies, 70
μ
l DMEM without cells (group-1) or containing 1x10
6 GFP
MSC (group-2) or
IGF-1
MSC (group-3) were injected intramyocardially in female rat model of coronary artery ligation (n=16/group). The animals were harvested on 7 days and 6 weeks after cell engraftment. FISH and
sry
-gene analysis on 7 day showed extensive survival of the male donor cells in the heart. Real-time PCR and Western blot on the heart tissue showed higher level of Cx-43 and activation of p27 in group-3 (
p
<
0.05
vs group-2). The number of TUNEL
+
cells was lower in group-3 (
p
=
0.01
vs group-2). Immunostaining for
α
-sarcomeric actin and Cx-43 showed extensive myogenic differentiation of
IGF-1
MSCs and gap junction formation between adjacent neofibers. Indices of heart function including ejection fraction (63.05±1.37%) and fractional shortening (28.54±0.92%) were improved in group-3 (
p
<
0.001
vs group-2
)
. IGF-1 enhances Cx-43 expression in MSCs and promotes their engraftment and functional integration in the heart and improves heart function.
Abstract 1452: Mutation of a SWI/SNF-associated Protein Leads to Massive Cardiac Hyperplasia in the Zebrafish Mutant
heart of stone
Authors: Dahme Tillman , Just Steffen , Hassel David , Meder Benjamin , Katus Hugo A , Rottbauer Wolfgang
Published: July 2021

Mature cardiac myocytes are considered postmitotic. The process, how cardiac precursors lose the ability to proliferate is unknown. The identification of factors that control the proliferative potential of cardiac myocytes would be an important step towards unraveling the process and ultimately to overcome it for therapeutic purposes. The zebrafish is an intriguing model organism for this objective, because zebrafish cardiac myocytes retain the ability to proliferate and therefore to regenerate cardiac injuries. In a forward genetics screen in zebrafisch, we have isolated the recessive, embryonically lethal mutant
heart of stone
(
hst

HJ163

). Hearts of
hst
-mutant zebrafish are characterized by wall-thickening of the cardiac ventricles up to almost complete obliteration of the ventricular lumen. Histology revealed massive hyperplasia of ventricular cardiac myocytes. Hyperplasia is caused by an increase in cardiomyocyte proliferation. By positional cloning we narrowed down the
heart of stone
locus to one gene, which encodes an evolutionary highly conserved SWI/SNF-associated protein, known to be involved in transcriptional regulation. Sequencing of cDNA and genomic DNA identified a splice donor-site mutation leading to intron-integration with premature termination of protein translation in
hst
-mutant zebrafish. Injection of wildtype mRNA into
hst
-mutant embryos fully rescues the mutant phenotype, while injection of morpholino-modified antisense oligonucleotides directed against
hst
-transcripts copies the
hst
phenotype, demonstrating the causality of the identified mutation. In order to define how the mutation of a SWI/SNF-associated protein causes hyperplasia of cardiac myocytes we conducted yeast-two-hybrid screens with a cardiac cDNA-library and the human homologue of
hst
as a bait. Surprisingly, we identified several sarcomeric proteins such as troponin T and α-actinin as robust interactors. By immunofluorescence staining we indeed found distinct nuclear and sarcomeric pools of the
hst
homologue in rat heart sections. It appears, that this SWI/SNF-associated protein shuttles between the sarcomere and the nucleus in order to transmit proliferative signals from the contractile apparatus to the transcriptional machinery.
Abstract 1463: Neuregulin-1 Promotes Embryonic Stem Cell Differentiation into the Cardiac Lineage in vitro
Authors: Sun Maoyun , Bian Yun , Caggiano Anthony O. , Yan Xinhua , Morgan James P.
Published: July 2021

Our previous studies demonstrated that transplantation of embryonic stem cell (ESCs) derived cardiomyocytes improved the cardiac function in myocardial infarction injured mice. However, significant challenges remain to be solved, such as how to produce sufficient amount and mature differentiated cardiomyocytes for the therapy. Neuregulin-1 (NRG-1) is essential for the development of the heart. Here, we tested whether NRG-1 could promote the differentiation of ESCs into cardiomyocytes. Differentiation of murine ESCs was induced by the hanging-drop method. NRG-1 was added at different points of ESC differentiation. The expression of cardiac specific genes, as well as NRG-1 and its erbB receptors were measured by quantitative RT-PCR and Western blot analysis. Our results showed that during the course of hanging-drop induced ESC differentiation, the mRNA level of cardiac-specific structure proteins cardiac troponin T (cTNT), cardiac troponin I (cTNI), myosin light chain 2a (MLC2a) and alpha-sarcomeric actin was elevated. The mRNA level of cardiac transcription factors NKX2.5 and GATA4 was also increased. In addition, beating EBs first appeared on day7 and about 90% of beating EBs was observed on day12. Further, the mRNA level of NRG-1α and NRG-1β, and both mRNA and protein levels of erbB receptors were elevated during the differentiation of ESCs. When NRG-1 was added during a certain developmental window, the mRNA level of cTNT, MLC2a and MEF2c, as well as the protein level of cTNT, CXN40, a marker for cardiac conduction system, and transcriptional factors NKX2.5, GATA4 were increased in NRG-1 treated ESCs compared to untreated cells. These results demonstrated that NRG-erbB signaling was activated during the course of hang-drop induced ESC differentiation; NRG-1 treatment further promoted ESC differentiation into the cardiac lineage
in vitro.
Our results suggest that NRG-1 might be used to produce more and/or more mature ESC derived cardiomyocytes for the treatment of cardiovascular diseases in the clinical setting.