bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P086: Serca1a Expression Exacerbates Cardiac Arrhythmia and Leads to Heart Failure and Early Mortality in a Mouse Model of Exercise-Induced Sudden Cardiac Death
Authors: Kalyanasundaram Anuradha , Lacombe Veronique , Belevych Andriy , Knollmann Bjorn C , Periasamy Muthu , Gyorke Sandor
Published: March 2022

Abnormal calcium (Ca
2+
) handling has been implicated in a range of cardiac diseases, including heart failure, a disease state characterized by compromised systolic and/or diastolic myocardial function and pathological remodeling. Excessive diastolic SR calcium release via the Ryanodine receptor (RyR2) has been suggested to deplete the SR Ca
2+
store thereby reducing cardiac contractility. Although abnormal RyR2 activity and elevated SR Ca
2+
leak are characteristic features of advanced heart failure (HF) in both humans and animal models, clear evidence is lacking whether altered RyR2 function plays a causal role in HF development. Indeed, while nearly 80 mutations in RyR2 have been identified and associated with SR Ca
2+
leak, none of these mutations have been reliably linked to HF. One possible explanation for this is that the SR Ca
2+
leak caused by the mutations is too small and/or compensated by other processes involved in Ca
2+
homeostasis. In the present study we sought to generate a genetic model with chronically increased diastolic release by combining overexpression of the fast skeletal muscle isoform of SERCA, SERCA1a, with ablation of CASQ2. Strikingly, the new hybrid strain (CASQ2 null/1a) exhibit early mortality (avg dying age 39.30days) due to heart failure preceded by pathological remodeling including eccentric hypertrophy, significant dilation of the atrial and ventricular chambers, necrosis induced cell death and widespread fibrosis. Animals are susceptible to ventricular arrhythmias at baseline in the absence of adrenergic triggers and show significant systolic dysfunction. Similarly, cardiomyocytes display increased SR Ca
2+
load along with significant disturbances in RyR2 mediated Ca
2+
release at baseline and are increasingly prone to spontaneous calcium waves. Our results demonstrate that excessive Ca
2+
leak via the RyR2 can lead to severe contractile dysfunction and heart failure. Since abnormal RyR2 activity has been implicated in human HF, our results suggest that up-regulation of SERCA activity as a therapy for HF must be advocated with extreme caution.
Abstract P096: Eya4, a Transcription Cofactor Crucial in Acquired Heart Disease
Authors: Williams Tatjana , Schoenberger Jost , Hundertmark Moritz , Czolbe Martin , Panther Franziska , Ritter Oliver
Published: March 2022

Introduction:
We previously identified a mutation in the human transcriptional cofactor Eya4 (E193) as cause of familial dilated cardiomyopathy (DCM) and heart failure. Upon interaction with real transcription factors such as Six family members, Eya4 is recruited to and interacts with target genes. One of the few known Eya-Six targets expressed in the heart is the cyclin-dependent kinase inhibitor p27kip1, which has been shown to inhibit hypertrophic growth in adult cardiomyocytes, is. We therefore hypothesize that Eya4/Six1 regulates targets relevant in normal cardiac function.

Methods and results:
We examined p27 expression in response to Eya4 in permanent mammalian cell lines. Western blot analysis demonstrated that an overexpression of Eya4 led to a significant downregulation of p27, whereas the overexpression of E193 had no effect on p27 levels. Studies using a p27 promoter fragment including Six1 consensus sites cloned in front of a Luciferase reporter gene proved Eya4 acts as a suppressor of p27 already at the transcriptional level, whereas E193 could not sufficiently inhibit p27 expression. Further transfection and knockdown experiments revealed that under basal conditions an Eya4 overexpression decreased protein synthesis in primary cardiac myocytes. We constructed transgenic mouse models with a constitutive myocardial overexpression of E193 and Eya4 to study the precise role of Eya4 in the heart. First analysis of these animals using magnetic resonance imaging to visualize cardiac structures in detail show that an overexpression of E193 leads to an age related onset of DCM as seen in an increase in LVEDV, whereas Eya4 overexpressing mice show no signs of heart disease.

Conclusion:
In summary, we identified a mutation in Eya4 to cause DCM. We now provide evidence that the Eya4/Six1 signalling cascade is also relevant in more common forms of acquired heart disease. Eya4/Six1 seems to regulate the expression of p27kip1, an important inhibitor of the development of hypertrophy in postmitotic cardiomyocytes. Studies using E193 overexpressing mice support our hypothesis whereas tempering the Eya4/Six1 signalling cascade disturbs cardiac physiology.
Abstract P097: Endothelial Overexpression of
LOX-1
Decreases Arterial Thrombosis and TF Expression in Vivo
:
Role of SIRT-1 and NFκB
Authors: Akhmedov Alexander , Camici Giovani G , Stivala Simona , Holy Erik W , Breitenstein Alexander , Lohmann Christine , Beer Juerg-Hans , Matter Christian M , Luescher Thomas F
Published: March 2022

Background

-The hallmark of the initiation of atherosclerotic lesion is foam cell formation, and oxidized LDL (OxLDL) is believed to play a key role in the initiation of the atherosclerotic process. OxLDL is internalized by several receptors, such as SR-AI/II, SR-BI, CD36, and CD68. OxLDL is also internalized by endothelial cells, but this uptake depends on receptors other than the classic scavenger receptors. In 1997, a lectin-like oxidized LDL receptor-1 (LOX-1, OLR1) was identified in bovine aortic endothelial cells. LOX- 1 is a type II membrane glycoprotein with an apparent molecular weight of 50 kDa. It has a C-terminal extracellular C-type lectin-like domain. This lectin-like domain is essential for binding to OxLDL. Binding of OxLDL to LOX-1 induces several cellular events in endothelial cells, such as activation of transcription factor NF-kB, upregulation of MCP-1, and reduction in intracellular NO, which may trigger the onset of cardiovascular events or accelerate the development of atherosclerosis.

Methods and Results

- We generated endothelial-specific
LOX-1
transgenic mice using the Tie2 promoter (
LOX-1TG
). 12-week-old male
LOX-1TG
and wild-type (WT) mice were applied for carotid artery thrombosis model.
LOX-1TG
mice developed carotid artery thrombosis within a mean occlusion time of 36.96±4.83 min, while WT control mice occluded within a mean time period of 22.75±3.87 min (n=10, P < 0.05). Initial blood flow in carotid artery did not differ between both groups of mice. Decreased occlusion time was in LOX-1TG mice vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression, macrophage accumulation and aortic fatty streaks were increased, while eNOS phosphorylation and endothelial function were reduced. In endothelial cells of
LOX-1TG
mice, reactive oxygen species were increased and the transcription factors NF-κB and Oct-1 activated. In atherosclerotic

LOX-1TG/ApoE
−/−

mice,high cholesterol diet increased VCAM-1 expression, number of macrophages, T-cells as well as plaque size.

Conclusions

-Thus, our data suggest that
LOX-1
plays a protective role in the arterial thrombosis when expressed at unphysiological levels. Therefore,
LOX-1
might represent a novel therapeutic target for atherosclerosis.
Abstract P102: Apelin Regulates the Function of Angiotensin II Type 1 Receptor Through APJ
Authors: Sun Xiao , Iida Shinichiro , Senbonmatsu Rina , Maruyama Kei , Inagami Tadashi , Nishimura Shigeyuki , Senbonmatsu Takaaki
Published: March 2022

Apelin and its G protein-coupled receptor (GPCR) APJ, which is most closely related to the angiotensin II (Ang II) type 1 receptor (AT
1
) but Ang II does not bind to APJ, are potent regulators of the cardiovascular system. Although recent studies have suggested that apelin-APJ reverses the function of Ang II-AT
1
, the mechanism remains unclear because the accumulating evidences indicates that apelin-APJ may contribute to both cardioprotection and pathological progression. In APJ and AT
1
co-expression human embryonic kidney (HEK) 293 cells, we found that APJ and AT
1
created receptor heterodimers. Co-expression with APJ significantly suppressed the phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2) induced by Ang II-AT
1
, whereas apelin eliminated this ligand-independent function of APJ out of relation to its heterodimerization. The pharmacologically non-activated APJ upon apelin stimulation elicited by the Gi/o-specific inhibitor pertussis toxin (PTX) restituted the pERK1/2 level similar to that of AT
1
and APJ co-expression without apelin stimulation. However, the co-expression of the beta-2-adrenergic receptor (β
2
AR) or the pharmacologically non-activated Ang II type 2 receptor (AT
2
) induced by the AT
2
-specific antagonist, PD123319, did not suppress pERK1/2 through Ang II-AT
1
. Pretreatment with 30 nM of the AT
1
blocker (ARB) TA-606A suppressed 50% of the AT
1
-mediated pERK1/2, whereas this suppression raised to 75% when the non-activated APJ was co-expressed. In contrast, 120 nM of TA-606A only reached 34% suppression when it was co-expressed with activated APJ with apelin. Taken together, we demonstrated that apelin may regulate the function of AT
1.
Non-activated APJ may suppress Ang II-AT
1
signaling, whereas this ligand-independent function was diminished with apelin activation. These results may contribute to ARB treatment in the clinical setting.
Abstract P115: Selective Vasoconstriction of the Ductus Arteriosus in the Rat by Stimulation of Thromboxane A
2
Receptor
Authors: Yokota Tomohiro , Aida Takashi , Yokoyama Utako , Minamisawa Susumu
Published: March 2022

Objective: Patent ductus arteriosus (PDA) is a common life-threatening complication of premature infants. Cyclooxygenase inhibitors are frequently used for pharmacologic therapy of PDA to inhibit the synthesis of prostaglandin E
2
(PGE
2
) that is the most potent vasodilator in the DA. However, they often cause severe side-effects. Thromboxane A
2
(TXA
2
), a vasoconstrictive lipid mediator, induces vascular contraction via TXA
2
receptor (TP). TXA
2
is also known to counteract prostacycline, a vasodilative lipid mediator. Therefore, we hypothesized that TXA
2
-TP stimulation can be an alternative strategy to induce DA closure by counteracting PGE
2
.

Methods and Results: Quantitative RT-PCR analysis revealed that the expression of TP mRNA was higher in the DA than in the aorta during fetal periods. When the selective TP agonist U46619 was intraperitoneally injected into Wister rat fetuses at embryonic day 19th (e19) and e21, we found that U46619 selectively constricted the fetal DA in a dose-dependent manner even though the level of circulating PGE
2
was supposed to be high. The vasoconstrictive effect of U46619 was weaker at e19 than at e21 in the DA, suggesting that immature DA is less sensitive to TP stimulation. Importantly, U46619 also showed vasoconstrictive effect on two different types of postnatal PDA models: immature PDA and hypoxia-induced PDA. These effects of U46619 were stronger than the effect of indomethacin that is the most common cyclooxygenase inhibitor. In addition, we found that U46619 exhibited little vasoconstrictive effect on other vessels such as the aorta, pulmonary arteirs, carotid arteries, renal arteries, and portal veins. Furthermore, U46619 was not likely to induce microthrombosis in the pulmonary capillary arteries. These results suggest that TP agonists could selectively promote the DA constriction without severe complications and be an alternative potent vasoconstrictor for patients with PDA.
Abstract P116: The Physiological Ventricular Growth Signal Can Be Determined Using Infants with Congenital Heart Disease as Models
Authors: Foker John E , Berry James M , Harvey Brian A , Pyles Lee A
Published: March 2022

Objectives
The signal for ventricular growth has not been defined. This basic developmental question was studied using infants with congenital heart lesions as models. Our first hypothesis was that clinical ventricular hypoplasia is a developmental rather than a primarily genetic defect and, therefore, catch-up growth can be induced. Our clinical observations also led to the hypothesis that forward flow across the atrioventricular (AV) valve (mitral or tricuspid) is what generates the growth signal. To test these hypotheses we analyzed clinical data from infants with a variety of congenital defects including three groups of patients with a hypoplastic ventricle in whom a procedure was carried out to increase flow across the AV valve.

Methods
Infants with one of several congenital heart problems had right and left ventricular volumes (RV, LV) assessed by biplane echo and indexed to body surface area (m
2
). The degree of hypoplasia was calculated using nomograms to determine the number of standard errors of the mean (SEM) below the expected volume (Table 1). Hypoplasia was considered significant when the SEM < −2.0. The three groups were studied before and after (3–6 months) procedures which increased AV flow.

Results
Other possible growth mechanisms were assessed. (1) High wall stress with systemic or supra-systemic pressures produced no net cavitary growth unless AV valve flow was increased. (2) Significant retrograde flow from semilunar valve regurgitation did not increase ventricular size until failure developed. Therefore, no evidence was found for other growth mechanisms.

Conclusions
1) Patients with congenital heart disease have a variety of defects, some of which can serve as models to answer basic developmental questions. 2) Increased AV valve flow provides the signal which induces ventricular growth. 3) Operations which increased AV valve flow induced catch-up growth of hypoplastic ventricles and allowed beneficial two-ventricle repairs in these patients.
Abstract P127: Carnitine Palmitoyltransferase 1b Deficiency Leads to Exacerbated Cardiac Dysfunction and Hypertrophy in Mice Subjected to Pressure Overload
Authors: He Lan , Kim Teayoun , Liu Jian , Wang Peiyong , Wood Philip A , Yang Qinglin
Published: March 2022

Carnitine palmitoyltransferase 1 (CPT1) is a primary rate-limiting enzyme that controls the entry of long-chain fatty acids into the mitochondrial matrix. CPT1b is the predominant isoform in the heart essential for myocardial fatty acid oxidation (FAO). Inhibition of myocardial FAO by specific CPT1 inhibitors has been proposed to be cardioprotective, but with mixed results from animal and human studies. To gain more specific insights, the present study investigates the effect of CPT1b deficiency in mice subjected to transverse aorta constriction (TAC)-induced pressure-overload. Because homozygous knockout of CPT1b causes embryonic lethality, we used the overtly normal heterozygous CPT1b knockout (CPT1b+/−) mice and their wild type (WT) littermates for the study. Under a severe pressure-overload condition, CPT1b+/− hearts showed substantially increased mortality compared with WT hearts. Under a milder pressure-overload condition, CPT1b+/− mice showed more pronounced cardiac hypertrophy than WT mice. Echocardiographic measurement revealed greater increases of posterior wall thickness at diastole, left ventricular (LV) internal dimension at systole and LV mass in CPT1b+/− than in WT mice. Stroke volume, ejection fraction and fraction shorting were also further decreased in CPT1b+/− mice. Based on assessments of heart-weight to body-weight ratio, molecular markers of cardiac hypertrophy, the cross-sectional area of cardiomyocytes, fibrosis and cardiomyocyte apoptosis, cardiac pathological hypertrophy was more pronounce in CPT1b+/− than in WT mice. Transmission electron microscope assessment revealed a reduced mitochondrial volume in CPT1b+/− compared with WT hearts after TAC. CPT1b+/− heart sections exhibited dramatic myocardial lipid accumulation with numerous lipid droplets. Moreover, CPT1b+/− hearts exhibited substantially elevated triglycerides and ceramide contents compared with WT hearts. Therefore, we conclude that CPT1b deficiency is detrimental to the heart under the pressure-overload condition with exacerbated cardiac dysfunction and progressive development of pathological hypertrophy due to lipotoxicity, thus cautious should be taken in evaluating CPT1b as a therapeutic target for heart disease.
Abstract P137: Metabolomic Analysis of the Early and Late Hypertrophic Heart
Authors: Brooks Alan C , Sansbury Brian E , Xie Zhengzhi , Brainard Robert E , Watson Lewis J , Brittian Kenneth R , Prabhu Sumanth D , Jones Steven P , Bhatnagar Aruni , Hill Bradford G
Published: March 2022

The metabolic adaptations to acute myocardial pressure overload are characterized by alterations in metabolism that drive the hypertrophic response and that balance workload with energy demand. Under conditions of chronic pressure overload, it is known that substrate utilization becomes less flexible and that the heart shifts energy preference from fatty acids to glucose. Nevertheless, the metabolic changes that underlie the progression of compensated hypertrophy to heart failure are incompletely understood and attempts to correct the known metabolic defects to delay decompensation have been largely unsuccessful. To identify key changes in metabolic phenotype that could underlie progression to heart failure, we measured metabolites in a transverse aortic constriction (TAC) mouse model using an unbiased metabolomic approach. Hearts were harvested 1 d, 1 wk and 8 wks after sham or TAC operation, and metabolites were extracted from the hearts and analyzed via GC/MS and LC/MS/MS. The signal intensities of 288 named metabolites were re-scaled to median values. Welch’s t-test and two-way ANOVA were used to identify metabolites that changed significantly with pressure overload and progression to heart failure. Echocardiographic measurements showed a significant decrease in ejection fraction after 1 d (65±2% vs. 49±5%) and 8 wks (61±2% vs. 34±8%) of TAC; 1 wk of TAC showed a compensated phenotype characterized by a largely preserved ejection fraction. One day after TAC, only 1.7% of the metabolites changed significantly; however, nearly all amino acids measured were increased by 1 wk. By 8 wks, amino acids returned to near sham levels and a significant and robust decrease in phospholipid, carnitine, inositol, sterol, and fatty acid metabolites occurred. These findings demonstrate that the temporal changes in metabolic phenotype are more complex than previously thought. The preservation of pathways involved in lipid and amino acid metabolism may be important for maintaining myocardial energetics and preventing pump failure under conditions of chronic pressure overload.
Abstract P139: Heart Failure Is Initiated By and Progresses Because of Normal Responses of Energy Metabolism to Stress
Authors: Foker John E , Berry James M , Harvey Brian A , Befera Nicholas T , Tveter Kevin J , Bianco Richard W
Published: March 2022

Objectives
The mechanism underlying heart failure (HF) after an index cardiac event is unknown but should have several characteristics. HF is a modern problem, so it will likely be a general response and be able to encompass the variety of initiating cardiac lesions as well as significant stress (e.g. exertion). The consequences should lead to the many alterations found in HF.

[ATP] falls when energy demand outstrips supply (e.g. ischemia) but do not recover promptly with reperfusion because AMP is quickly catabolized and unavailable for recharging. [ATP] recovery is slow and limited by the availability of ribose-5-P. Ribose is not used as fuel and is channeled into nucleotide (ATP) synthesis allowing the effects of [ATP] recovery on function to be studied. Our hypothesis was that HF results from these responses of energy metabolism to stress which lower [ATP[ and affect the numerous myocardial reactions whose activity depends on [ATP]. The ability of ribose to quickly increase [ATP] allows these effects to be studied.

Methods
The effects of ribose infusion on two models of myocardial stress were studied. (1) An intact canine model of global ischemia which allowed serial myocardial biopsies and detailed function analysis. (2) A rat myocardial infarction model which allowed ECHO analysis of the remote myocardium.

Results

Table 1.

Global ischemia (20')

[ATP] recovery control w/ ribose infusion
9.9 ± 1.4 days  2.8 ± 0.6 days

Diastolic compliance recovery control w/ ribose infusion
9.4 ± 1.1 days  2.4 ± 0.9 days

Myocardial infarction

Pre MI

2 weeks post MI

LV systolic diameter (cm) control w/ ribose infusion
0.40 ± 0.03  0.39 ± 0.06
0.73 ± 0.10  0.47 ± 0.22

Remote LV wall thickness (cm) control w/ ribose infusion
0.13 ± 0.03  0.13 ± 0.02
0.10 ± 0.01  0.18 ± 0.12

LV ejection fraction control w/ ribose infusion
74 ± 5.9%  76 ± 2.3%
22 ± 6.3%  52 ± 5.0%

Conclusions
1) When energy demand outstripped supply (either from ischemia or increased work) myocardial [ATP] falls and recovery was slow. 2) Ribose is the limiting precursor for [ATP] recovery and infusion allowed the relationship between [ATP] and function to be studied. 3) In both myocardial ischemia recovery and increased load (from MI) ribose enhanced [ATP] and improved function. 4) The response of myocardial energy metabolism response to stress results in lower [ATP] which will decrease the activity of many function related reactions. 5) HF could begin and progress from these reactions of energy metabolism to cardiac stress.
Abstract P144: ER Stress-Mediated Reduction of SERCA2a Protein Is Not Involved in Increase in Lethal Heart Failure After Myocardial Infarction in Obese Type 2 Diabetes
Authors: Sato Tatsuya , Takada Akifumi , Miki Takayuki , Tanno Masaya , Kuno Atsushi , Kouzu Hidemichi , Itoh Takahito , Sunaga Daisuke , Miura Tetsuji
Published: March 2022

Rationale:
Our previous studies showed that increased endoplasmic reticulum (ER) stress in the myocardium impairs cytoprotective signaling in obese type 2 diabetes mellitus (T2DM).

Objective:
We examined roles of ER stress in alterations of ventricular function and mortality after myocardial infarction by T2DM.

Methods and Results:
SERCA2a protein level was significantly lower by 45% and Ser16- and Thr17-phosphorylated phospholamban levels tended to be lower in a rat model of T2DM (OLETF) than those in non-diabetic control rats (LETO). Treatment with an ER stress modulator, 4-phenylbutyric acid (4-PBA) or tauroursodeoxycholic acid (TUDCA), restored SERCA2a protein in OLETF to the level of that in LETO. Although left ventricular (LV) ejection fractions were comparable in OLETF and LETO, LV end-systolic elastance (1685±86 vs. 1953±68 mmHg/ml) and LVdP/dtmax (5886±337 vs. 7389±457 mmHg/s) were lower and tau was larger (12.1±0.9 vs. 8.9±0.8 msec) in OLETF. These impairments of LV function in OLETF were restored by treatment with 4-PBA (LV end-systolic elastance: 1813±58 mmHg/ml, LVdP/dtmax: 7053±546 mmHg/s and tau: 8.5±0.5 msec). Elevation of LV systolic pressure and dP/dtmax levels by dobutamine infusion was reduced, and inhibitory effects of verapamil on the dobutamine-induced positive inotropism were blunted in OLETF. In patch-clamp experiments using isolated cardiomyocytes, increase in L-type Ca
2+
channel current after isoproterenol treatment was markedly attenuated in OLETF as compared with the change in LETO. Mortality rate within 48 hrs after myocardial infarction was significantly higher in OLETF than in LETO (64.0% vs. 7.7%). Telemetric recording indicated that rapid progression of heart failure but not arrhythmia was responsible for the high mortality rate in OLETF. In contrast to the improvement of baseline ventricular dysfunction, neither 4-PBA nor TUDCA significantly reduced mortality rate after infarction in OLETF.

Conclusions:
ER stress plays a major role in reduction of SERCA2a by T2DM. Blunted response of cardiac L-type Ca
2+
channels to adrenoceptor activation, rather than reduced SERCA2a, may contribute to increase in lethal heart failure after myocardial infarction in T2DM.
Abstract P149: Mozobil Increases Myocardial Fibrosis via the Recruitment of Fibrocytes in the Angiotensin II--Treated Heart
Authors: Falkenham Alec G , Lee Tim , Legare Jean Francois
Published: March 2022

Background:
Fibroblast progenitor cells (fibrocytes) are important to the development of myocardial fibrosis and suggested to migrate to the chemokine CXCL12. CXCL12 has one primary receptor, CXCR4, to which the inhibitor Mozobil competitively binds. Mozobil has been shown to inhibit recruitment of fibrocytes to the lungs following bleomycin injury, but its affects on progenitor cells in the heart remains unclear. We hypothesized that if CXCL12 is necessary for fibrocyte recruitment, Mozobil would inhibit fibrocyte infiltration into the myocardium and consequently, decrease fibrosis.

Methods:
C57/Bl6 mice were treated with saline (control), AngII, or AngII+Mozobil via osmotic mini-pump. Mice were sacrificed at day 3 and tissues were collected for RNA, protein and histology. Heart sections were stained with H&E and Sirius Red and analyzed for cellular infiltrate and collagen deposition, respectively. Infiltrate was measured using standardized grid counts on a single heart section. The percent area affected was the number of grids containing cellular infiltrate over the total grids containing heart tissue (∼375 grids per section). Fibrocytes were cultured from mouse peripheral blood following mobilization with Mozobil. Fibrocytes were stained for collagen-1, CD133 and CXCR4.

Results:
Cultured fibrocytes were positive for markers CD133 and collagen-1, as well as CXCR4. AngII infusion resulted in significant cellular infiltration, affecting 30.0 ± 4.0% (n=8) of the myocardium, relative to saline (0.3% ± 0.4; n=8). In contrast, animals receiving AngII+Mozobil had 78.9±3.1% (n=5) of the myocardium affected (p<0.05). Increased infiltrate was also associated with significantly increased collagen deposition: Control (4.3% ± 0.1), AngII (11.9% ± 1.9), and AngII+Mozobil (29.0% ± 4.8). Furthermore, the infiltrate was positive for the progenitor marker CD133.

Conclusion:
Our findings confirm isolated fibrocytes express CXCR4. Unexpectedly however, our findings suggest CXCL12 is not required for the migration of fibrocytes to the myocardium, based on the failure of CXCR4 blockade to limit migration. Our findings suggest that increased migration into the heart may be through the increased mobilization of progenitor cells from the bone marrow.
Abstract P152: Cardiac mTOR Preserves Cardiac Function and Inhibits the Inflammatory Response After Transient Ischemia
Authors: Aoyagi Toshinori , Inouye Brendan T , Matsui Takashi
Published: March 2022

Recent reports strongly suggested that the mammalian target of rapamycin (mTOR) is necessary for cardiomyocyte survival in pathological cardiac hypertrophy. Using cardiac-specific transgenic mice overexpressing mTOR (mTOR-Tg), we reported that cardiac mTOR is sufficient to protect the heart against heart failure in pathological hypertrophy, accompanied by a decrease in production of proinflammatory cytokines. However, the role of cardiac mTOR in ischemic cardiac injury remains undefined. To assess the effects of cardiac mTOR in cardiac ischemia, we studied the response of hearts from mTOR-Tg mice to global ischemia-reperfusion injury (IRI) (20 min ischemia, 40 min reperfusion) in the ex vivo Langendorff perfusion model. mTOR-Tg mice demonstrated improved functional recovery compared with littermate control (WT) mice [percent recovery of LV developed pressure (LVDP); 44.6±7.3 vs. 24.7±5.3 %, mTOR-Tg vs. WT, n=9 each, p<0.05]. Biochemical analyses demonstrated that the level of mTOR protein decreased under IRI stress in WT while the level of mTOR protein in post-IRI mTOR-Tg mice was comparable to non-ischemic control mice. Both mTORC1 (p70S6K) and mTORC2 (Akt) activation in mTOR-Tg mice were increased by IRI, while these were not changed in WT mice after IRI. Quantitative RT-PCR demonstrated that expression of proinflammatory cytokines in mTOR-Tg mice after IRI was significantly lower than WT mice (IL-6, 7.1±1.5 vs. 14.6±1.6, p<0.005; TNF-alpha, 2.1±0.4 vs. 5.1±0.8, p<0.01; post-IRI mTOR-Tg mice vs. WT mice, fold change over non-ischemic control WT mice, n=9 each). To further characterize the inflammatory response, we measured cytokine in the effluent samples by ELISA. We observed that secretion of IL-6 in post-IRI mTOR-Tg mice was significantly lower than that in WT mice (50.1±11.5 vs. 95.6±23.8 pg/ml, post-IRI mTOR mice vs. post-IRI WT mice, n=9 each, p<0.05). IL-6 is known as a strong prognostic predictor in patients with heart failure. Taken together, these findings suggest that cardiac mTOR is sufficient to substantially preserve cardiac function and prevent the inflammatory response, which may have relevance for settings in heart failure.
Abstract P156: IRE1α Activity Is Abbreviated in Myocytes
Authors: Steiger DeAnna L , Wang Yibin
Published: March 2022

BACKGROUND:
A unifying feature of Ischemia/reperfusion injury, myocardial infarction and heart failure is disruption of endo(sarco)plasmic reticulum homeostasis and activation of the intracellular signaling pathway known as “ER Stress” or “Unfolded Protein Response” (UPR). UPR signaling is activated through three ER Stress sensor proteins, including IRE1α. IRE1α can, through direct interaction, induce adaptive or apoptotic signaling programs. Although ER stress signaling is known to be active following various cardiac injuries, it is unknown whether IRE1α contributes to an adaptive or apoptotic program. We sought to characterize the functional impact of IRE1-mediated UPR signaling in cardiomyocytes.

HYPOTHESIS:
IRE1α-mediated UPR signaling contributes to cardiac hypertrophy or cell death in response to pathological stresses. METHOD: NRVM were treated with adenovirus-mediated IRE1α-myc overexpression. Protein and RNA were harvested two and five days following treatment and IRE1α activation, activity and downstream signaling was monitored by PCR, qPCR, and immunoblot.

RESULTS:
Ectopic expression led to robust phosphor-IRE1α Ser724 and IRE1α RNase activity toward Xbp-1. BiP and EDEM expression was induced, indicative of adaptive UPR signaling. Apoptotic UPR signaling was not induced (CHOP expression). Surprisingly, IRE1α activity and downstream signaling was transient. After five days, both IRE1α RNase activity and downstream signaling were extinguished, despite continued robust IRE1α protein expression and activation (phospho-Ser724).

CONCLUSION:
NRVM possess uncharacterized intrinsic mechanisms to protect against unwarranted IRE1α signaling.
Abstract P173: Targeting α-1-Adrenergic Receptors for Heart Failure Therapy
Authors: Montgomery Megan D , Myagmar Bat-Erdene , Simpson Paul C
Published: March 2022

Alpha-1 adrenergic receptors (α1-ARs) have beneficial effects in the heart and cardiac myocytes. In pilot studies
in vivo
, a sub-hypertensive dose of the α1A subtype-selective agonist
A61603
has cardioprotective signaling and prevents and rescues cardiomyopathy in mouse models. These studies give a possible explanation for adverse cardiac effects of α1-antagonists in clinical trials and are a rationale to test α1-agonists in heart failure. Our objective was to establish models to screen α1-agonists for beneficial cardiac signaling. We tested the hypothesis that different α1-AR agonists have distinct beneficial cardiac effects at very low doses. To test this, we studied α1-AR-mediated cardioprotective and adaptive hypertrophic signaling in isolated neonatal rat ventricular myocytes (NRVM) and adult mouse ventricular myocytes (AMVM). We compared
A61603
to all clinically approved α1-agonists: epinephrine (EPI), norepinephrine (NE), phenylephrine (PE), dobutamine (DOB), methoxamine (MET), and midodrine (MID) at 5 doses, all in the presence of the beta-blocker propranolol (200 nM). Cardioprotective signaling was measured by α1-AR-ERK survival pathway activation and cell viability by MTT assay after doxorubicin (20 μM)-induced toxicity. Cellular adaptive hypertrophy was measured by protein synthesis in NRVM and fetal gene protein expression in AMVM. In NRVM,
A61603
was the most potent (EC
50
= 4 nM) and efficacious agonist for ERK activation. The rank-order potency was:
A61603
> NE > EPI > PE > MET > DOB > MID. For protein synthesis,
A61603
had high efficacy and potency (EC
50
= 12 nM). The rank-order potency was:
A61603
> NE > DOB > PE > EPI > MET > MID. In AMVM,
A61603
was potent for ERK (EC
50
= 58 nM) and increased cell viability 48% (1.48 vs. 1.00 control) with doxorubicin toxicity.
A61603
(200 nM) increased the number of myocytes expressing beta-myosin heavy chain and atrial natriuretic factor by immunocytochemistry. We conclude that myocyte culture models are predictive of α1-agonist efficacy and potency in adaptive and protective cardiac effects.
A61603
, an imidazoline with high affinity for the α1A-AR, is more potent and efficacious in myocyte models than are clinically available α1-agonists, and is a promising candidate for therapeutic testing
in vivo
.
Abstract P175: Selective Regional Injections of Alginate Hydrogel into Cardiac Veins Improve Left Ventricular Function and Reverse Chamber Remodeling in Dogs with Chronic Heart Failure
Authors: Sabbah Hani N , Wang Mengjun , Sabbah Michael S , Rastogi Sharad , Gupta Ramesh C , Helgerson Sam
Published: March 2022

Background:
Progressive LV enlargement and dysfunction are characteristics of heart failure (HF) and are associated with increased mortality and morbidity. We tested the hypothesis that selective regional injections of alginate hydrogel implants (AHI, Algisyl-LVR, Lonestar Heart, Inc.) into isolated segments of epicardial veins of the failing LV will increase LV wall thickness and lead to reduced LV size, lower LV wall stress and improved LV systolic and diastolic function.

Methods:
12 dogs with coronary microembolization-induced HF (LV ejection fraction, EF<30%) were studied. During an open-chest procedure, 1.5 to 2.0 cm segments of the 2
nd
diagonal branch and the 2
nd
marginal branch of the cardiac epicardial veins were isolated by ligation with silk sutures. In 6 dogs, the isolated segments were each injected with 1.0 ml of AHI and in 6 dogs with normal saline (controls). LV ED volume (EDV), ES volume (ESV), EF, stroke volume (SV), ED wall stress (EDWS), deceleration time of early mitral inflow velocity (DT), and severity of functional mitral regurgitation (MR) were measured before therapy (Pre) and at 6 months after AHI or saline delivery (Post). The treatment effect (Δ) was calculated as the difference between Pre and Post.

Results:
In preliminary we showed that an intravenous injection of 1.0 ml AHI increased regional LV end-diastolic (ED) and end-systolic (ES) wall thickness by 5.8 ± 0.7 % and 9.0 ± 1.0 %, respectively. Compared to saline controls, treatment of HF dogs with AHI prevented the increase in EDV and significantly decreased ESV, EDWS and severity of functional MR (Table). In addition, AHI significantly increased LV EF, SV and DT indicating improved LV systolic and diastolic function (Table). Treatment with Alginate implants also showed a safe and non-arrhythmogenic chronic profile.

Conclusion:
LV reconstruction using selective regional injections of AHI into epicardial cardiac veins represents a safe novel approach for the treatment of advanced chronic HF.

Table 1.

Hemodynamic and Ventriculographic Results

Control (n=6)
AHI (n=6)
P-Value

ΔEDV (ml)

2.5 ± 1.0
−1.1 ± 0.4
0.114

ΔESV (ml)

4.3 ± 1.7
−3.9 ± 0.4
0.001

ΔEF (%)

−3.8 ± 1.5
5.0 ± 0.4
0.001

ΔSV (ml)

−1.8 ± 0.8
0.11 ± 0.04
0.001

ΔEDWS (gm/cm²)

9.4 ± 6.5
−23.1 ± 4.6
0.006

ΔDT (msec)

−4.7 ± 0.4
27.0 ± 4.4
0.001

ΔMR (%)

1.7 ± 0.8
−3.6 ±1.3
0.004
Abstract P179: Human Peripheral Blood B Cells Improve Cardiac Function in Aged Rats After Myocardial Infarction
Authors: Goodchild Traci T , Sweet Michael B , Singh Jai Pal , Poznansky Mark C , Chronos Nicolas A
Published: March 2022

Background:
Aging is associated with increased mortality following acute myocardial infarction (AMI). Early cell therapy clinical trials for AMI have shown a modest improvement in cardiac function. Previous evidence suggest that B cells may be protective in tissue injury. Our initial studies showed that intramyocardial delivery of bone marrow derived B cells improves cardiac function in young rats.

Aim:
In this study, we determined the effect of B cell therapy in aged animals subjected to AMI using a clinically relevant protocol.

Methods:
Human PB B cells were isolated from buffy coats using CD19+ selection. Young (8-10wks) and aged (10-12mos) athymic rats had baseline echocardiography then coronary artery ligation (d1). At d7, rats had repeat echocardiography and received 1×10
7
B cells or vehicle intravenously. At d9 and d21, rats underwent follow-up echocardiography. Explanted hearts were measured for infarct size and B cell localization was determined in infarcted aged rat hearts after d8 in a subset of animals by IHC.

Results:
Fractional ventricular diameter shortening (FS) decreased at d7 compared to baseline in young (27.9 ±0.9 vs. 55.6±0.6%,
P
<0.001) and aged (31.6±0.9 vs. 53.4±0.5%,
P
<0.001) rats. At d9, FS was higher in B cell compared to control treated aged rats (44.9±2.6 vs. 29.3±3.0%,
P
<0.001). FS was minimally changed in B cell compared to control treated young rats (37.3±3.3 vs. 31.2±3.5%,
P
=NS). At d21, FS was higher in aged rats receiving B cells compared to controls (38.6±2.2 vs. 30.0±2.6%,
P
<0.001) and trended higher in B cell treated young rats compared to control (34.1±1.8 vs. 28.0±2.6%,
P
=0.058). FS was higher in aged rats receiving B cells stored for 1.5hrs at room temperature compared to freshly prepared B cells (35.1±3.2% vs. 27.1±2.4%,
P
=0.035). Infarct size was smaller in B cell treated aged rats compared to controls (12.9±2.3 vs. 22.4±2.4% LV,
P
=0.010); no difference in infarct size was detected in young rats. B cells were detected in the infarct of aged rats at d8.

Conclusion:
These data support that intravenous delivery of peripheral blood B cells is efficacious in improving cardiac function and limiting infarct size in aged rats, an experimental model more relevant to clinical population.
Abstract P186: IL-10 Mitigates Adverse Cardiac Remodeling and Improves Left Ventricle Function in Pressure Overload--Induced Hypertrophy
Authors: Verma Suresh K , Krishnamurthy Prasanna , Senger Neha , Thal Melissa , Hoxha Eneda , Lambers Erin , Thorne Tina , Qin Gangjian , Losordo Douglas W , Kishore Raj
Published: March 2022

Background:
Hypertrophy associated heart failure remain the leading cause of death worldwide. Inflammatory cytokines play critical role in adverse cardiac remodeling and heart failure. Therefore, approaches geared towards inhibiting cardiac inflammation may provide therapeutic benefits. Thus, we tested the hypothesis that anti-inflammatory cytokine, IL-10, therapy might inhibit pressure overload-induced cardiac remodeling.

Methods and Results:
Cardiac hypertrophy was induced in Wild-type (WT) and IL-10-knockout (KO) mice by isoproterenol (ISO; 45mg/kg b.wt./day, 14 days) infusion through mini-osmotic pumps. Control groups mice received saline. ISO-induced increase in heart weight/tibia length ratio in WT was further increased in KO mice. ISO-mediated cardiac dysfunction (%EF and %FS) in WT mice were exaggerated in KO mice. KO mice displayed higher expression of fetal genes (ANP and BNP) and inflammatory cytokines (TNFα and IL-1β). ISO-treated KO mice had notably higher fibrosis and apoptosis than the WT.
Interestingly, systemic recombinant IL-10 administration markedly attenuated effects of ISO and improved left ventricle function, reversed fetal gene expression, fibrosis and apoptosis both in WT and in KO mice.
To further understand the mechanism of IL-10 protection, NRCM and H9C2 cells were treated with ISO (10µM) and/or IL-10 (20ng/mL),
in vitro
. ISO significantly increased the mRNA expression of ANP and BNP as well as of inflammatory cytokines which was markedly reduced by IL-10. ISO treatment also induced the activation of p38, ERK1/2 MAP kinases and of NFkB while it inhibited STAT-3 phosphorylation. Interestingly, co-treatment with IL-10 suppressed p38 and ERK1/2 phosphorylation while enhancing STAT-3 phosphorylation. Interestingly, ISO-induced nuclear translocation of NFkB was mimicked by STAT3 specific inhibitor cucurbitacin, suggesting STAT3 as the downstream target of IL-10 effects.

Conclusion:
Our studies suggest that IL-10 treatment not only inhibits the progression but also reverses the pressure overload-induced adverse cardiac remodeling. Ongoing investigations will further provide a better understanding on the mechanistic and therapeutic aspects of IL-10 on hypertrophic remodeling and heart failure.
Abstract P187: Casein Kinase 2/Histone Deacetylase 2/Krúppel-like Factor 4 Is a Novel Axis of Development of Cardiac Hypertrophy
Authors: Eom Gwang Hyeon , Cho Young Kuk , Ko Jeong-Hyeon , Shin Sera , Choe Nakwon , Kim Yoojung , Joung Hosouk , Kee Hae Jin , Kook Hyun
Published: March 2022

Background.
Cardiac hypertrophy is characterized by transcriptional reprogramming of fetal gene expression, and histone modifiers are tightly linked to the regulation of those genes. We previously reported that activation of histone deacetylase (HDAC) 2, one of the class I HDACs, mediates hypertrophy. Here we suggest that disinhibiting of kruppel-like factor 4 (Klf4) by casein kinase-2α1 (CK2α1)-dependent phosphorylation of HDAC2 S394 develop the cardiac hypertrophy.

Methods and Results.
Hypertrophic stimuli phosphorylated Hdac2 S394, which was necessary for its enzymatic activation and thereby for the development of hypertrophic phenotypes. Transgenic mice overexpressing Hdac2-wild type exhibited cardiac hypertrophy, whereas those expressing phosphorylation-resistant Hdac2 S394A did not. Compared with that in age-matched normal human hearts, phosphorylation of Hdac2 S394 was dramatically increased in hypertrophic cardiomyopathy patients. Hypertrophy-induced phosphorylation of Hdac2 S394 and its enzymatic activity were completely blocked either by CK2-blockers or by
CK2a1 siRNA.
Hypertrophic stimuli led CK2α1 to be activated, and its chemical inhibitors blocked hypertrophy in both phenylephrine-treated cardiomyocytes and in isoproterenol-administered mice. However, by utilizing KLF4-binding element-disrupted
Nppa
promoter, treatment with either TBB or TBCA failed to reduce the mutant promoter activity. These results emphasized that CK2α1-induced hypertrophic events are dependent on both Hdac2 and KLF4. CK2α1-transgenic mice developed hypertrophy, which was attenuated by administration of trichostatin A, an HDAC inhibitor. Overexpression of CK2α1 caused hypertrophy in cardiomyocytes, whereas its chemical inhibitors as well as Hdac2 S394A blunted it. Hypertrophy in CK2α1-transgenic mice was exaggerated by crossing these mice with Hdac2-transgenic mice. By contrast, however, it was blocked when CK2α1-transgenic mice were crossed with Hdac2 S394A-transgenic mice.

Conclusions.
We have demonstrated a novel mechanism in the development of cardiac hypertrophy by which CK2 activates HDAC2 via phosphorylating HDAC2 S394 and consequence down-regulation of KLF4.
Abstract P190: Role of mTOR Signaling in LEOPARD-Syndrome-Associated Hypertrophic Cardiomyopathy
Authors: Krenz Maike , Schramm Christine , Fine Deborah , Edwards Michelle , Reeb Ashley
Published: March 2022

The identification of mutations in PTPN11 (encoding the protein tyrosine phosphatase Shp2) in families with congenital heart disease has facilitated mechanistic studies of various cardiovascular defects. However, the roles of normal and mutant Shp2 in the developing heart are still poorly understood. In particular, how Shp2 loss-of-function (LOF) mutations cause LEOPARD Syndrome, which is characterized by congenital heart defects such as pulmonary valve stenosis and hypertrophic cardiomyopathy (HCM), remains unclear. We employed both in vitro and in vivo models to investigate signaling mechanisms downstream of Q510E-Shp2, a particularly aggressive mutation leading to early-onset HCM. In cultured rat neonatal cardiomyocytes, Q510E-Shp2 expression significantly increased cell size. This increase was accompanied by hyperactivation of signaling through mammalian target of rapamycin (mTOR). mTOR inhibition with rapamycin reversed the pro-hypertrophic effects of Q510E-Shp2 in culture. Mice with cardiomyocyte-specific overexpression of Q510E-Shp2 starting before birth also showed up-regulation of signaling through Akt/mTOR/p70S6K and developed HCM in the early neonatal period. Q510E-Shp2 expression increased cardiomyocyte sizes and septum thickness, and caused cardiomyocyte disarray and fibrosis. Echocardiographically, hearts were hypocontractile and displayed increased wall thicknesses. Importantly, rapamycin administration rescued the Q510E-Shp2-induced phenotype in vivo. Our studies establish a role for mTOR signaling in HCM caused by the LOF mutation Q510E-Shp2. Pharmacological inhibition of mTOR was sufficient to rescue the HCM phenotype in both models. These findings have important implications not only for the development of novel therapeutic approaches for LEOPARD Syndrome patients, but also for closely related forms of congenital heart disease such as Noonan Syndrome.
Abstract P192: MicroRNA-199b Targets the Nuclear Kinase Dyrk1a in an Auto-Amplification Loop Promoting Calcineurin/NFAT Signaling
Authors: Salic Kanita , da Costa Martins Paula A , Gladka Monika M , Leptidis Stefanos , el Azzouzi Hamid , Hansen Arne , Condorelli Gianluigi , Arbones Maria L , Eschenhagen Thomas , De Windt Leon J
Published: March 2022

MicroRNAs (miRs) are a class of single-stranded, non-coding RNAs of ∼22 nucleotides in length, and growing evidence indicates that miRs are implicated in myocardial disease processes. A key pathway involved in heart failure consists of the phosphatase calcineurin and its downstream transcription factor Nuclear Factor of Activated T-cells (NFAT). We performed microRNA profiling in hearts from calcineurin transgenic mice and demonstrated that microRNA-199b (miR-199b) is a direct calcineurin/NFAT target gene. MiR-199b increases in expression in mouse and human heart failure, and targets the nuclear NFAT kinase dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1a (Dyrk1a), constituting a pathogenic feed forward mechanism with impact on calcineurin-responsive gene expression. Interestingly, cardiac miR-199b levels inversely correlated with cardiac Dyrk1a expression in biopsies of failing human hearts secondary to ischemic heart disease or non-ischemic dilated cardiomyopathy. Furthermore, mutant mice overexpressing miR-199b or haploinsufficient for Dyrk1a are sensitized to calcineurin/NFAT signaling or pressure overload and exhibit stress-induced cardiomegaly by reduced Dyrk1a expression. From a therapeutic point of view, in vivo inhibition of miR-199b by a specific antagomir normalized Dyrk1a expression, reduced nuclear NFAT activity, and caused marked inhibition and even reversal of pre-established hypertrophy and fibrosis in mouse models of heart failure. Our results reveal that microRNAs impact cardiac cellular signaling and gene expression, and implicate miR-199b as a therapeutic disease target in heart failure.