bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 374: Targeted Deletion of A
2B
Adenosine Receptors Attenuates the Protective Effects of Myocardial Postconditioning
Authors: Tan Xinglin , Tilley Stephen L , Krahn Thomas , Teng Bunyen , Mustafa S. J , Hofmann Polly A , Morrison R. R
Published: July 2021

Endogenous adenosine is an important ligand trigger for the cardioprotective effects of postconditioning (PostCon). To assess the hypothesis that A
2B
adenosine receptor (A
2B
AR) activation contributes to PostCon-induced protection, global ischemia-reperfusion was performed with and without PostCon or the selective A
2B
agonist, BAY 60 – 6583 (BAY), in isolated wild-type (WT) and A
2B
AR knockout (A
2B
KO) mouse hearts. In WT hearts, PostCon improved post-ischemic recovery of left ventricular developed pressure (LVDP) to 63.3±1.6 % of pre-ischemic baseline vs. 49.9±1.6 % in non-PostCon controls (CTL), lowered end diastolic pressure (EDP) to 15.8±1.5 mmHg vs. 27.9±1.6 mmHg in CTL, and reduced coronary efflux of cardiac troponin I (cTnI) to 2507±359 ng/g heart weight vs. 4693±343 ng/g in CTL (n=12 both groups,
p
<0.05 each comparison). Treatment with BAY in the first two min of reperfusion mimicked beneficial effects of PostCon in WT hearts (LVDP: 64.7±2.0 % baseline, EDP: 16.2±2.0 mmHg, cTnI: 3311±366; n=13, not significant compared to respective PostCon values). Real-time PCR confirmed absence of A
2B
AR in A
2B
KO hearts and demonstrated no changes in expression of other adenosine receptor subtypes compared with WT hearts. In A
2B
KO hearts, neither PostCon nor BAY improved recovery of LVDP (50.8±1.6 % baseline for CTL vs. 54.5±1.7 % with PostCon vs. 53.0±1.4 with BAY; n=6 each group), and neither affected EDP or release of cTnI. During reperfusion, both PostCon and BAY increased survival kinase signaling through Akt and ERK1/2 phosphorylation in WT but not A
2B
KO hearts. In non-ischemic WT hearts, Akt and ERK1/2 phosphorylation was increased by both BAY treatment and application of the PostCon stimulus. These data demonstrate that the protective effects of PostCon are attenuated by targeted deletion of A
2B
AR and are mimicked by selective A
2B
AR activation, suggesting A
2B
AR activation is an important trigger leading to PostCon-induced myocardial protection.
Abstract 512: Infiltrating CD4 Positive T cells may Support an Angiogenic Switch in Human Coronary Atheroma
Authors: Kolodgie Frank D , Nakazawa Aya , Yazdani Saami K , Pacheco Erica , Xu Xin , Cheng Qi , Ladich Elena , Virmani Renu
Published: July 2021

Background:
Pathologic angiogenesis has been implicated in the development of high-risk plaques where nascent immature leaky vessels become the primary source of intraplaque hemorrhage contributing to necrotic core expansion. The stage of lesion progression, however, favoring an angiogenic response has not been identified.

Methods and Results:
Multiple coronary lesions collected from 73 sudden coronary death victims at autopsy were examined. Plaque morphologies included pathologic intimal thickening (PIT), fibroatheromas whose cores were in an early stage (eFA) or late stage (lFA) of necrosis, thin-cap fibroatheromas (TCFA) and ruptures (pR). Lesions were characterized by immunostaining using specific markers directed against endothelial cells (CD31/CD34), smooth muscle cells (α-actin), macrophages (CD68), and T-lymphocytes (CD45RO, CD4). Mean intraplaque vessel densities per mm
2
between lesions with PIT and eFA increased significantly (PIT= 15.84±16.73 vs. eFA= 42.07±40.70, p=0.0054) where maximal numbers of vessels were found in pR (66.57±33.42). Overall, intraplaque vessels associated with actin
3
SMCs, were generally <10%, suggesting a more immature phenotype. Increased vessel densities coincided with greater numbers of inflammatory macrophages and T-cells. Notably, T-cell numbers in areas with high intraplaque vessel densities increased approximate 3-fold between lesions with PIT and eFA (PIT= 62.62±76.50 vs. eFA= 182.33±171.54, p=0.0043) with maximal numbers displayed in TCFAs and pRs, in particular at the intimal/medial border. Populations infiltrating T cells were predominantly CD4
3
and most of these appeared to be activated (human leukocyte antigen DR positive).

Conclusion:
Atherosclerotic coronary arteries express a latent proangiogenic phenotype coinciding with a localized increased in activated CD4
3
T-cells and early plaque necrosis.
Abstract 1380: Role of Copper Transport System for Extracellular Superoxide Dismutase in Angiotensin II-Induced Hypertension
Authors: Ozumi Kiyoshi , Kim Ha Won , McKinney Ronald D , Ushio-Fukai Masuko , Fukai Tohru
Published: July 2021

Extracellular superoxide dismutase (ecSOD) is a secretory copper enzyme highly expressed in the vasculature, and plays an important role in protecting angiotensin II (Ang II)-induced hypertension by reducing the extracellular levels of vascular superoxide. Copper-containing enzymes obtain cofactor copper through specific copper transport system including the copper uptake transporter Ctr1 and the copper chaperone/transcription factor, antioxidant-1 (Atox1). We previously demonstrated that Ang II upregulates ecSOD expression and activity; however, underlying molecular mechanisms remain unclear. Here we show that ecSOD expression and activity are markedly decreased in fibroblasts lacking Atox1 or Ctr1. Western and immunohistochemical analysis reveal that chronic Ang II infusion (500 ng/kg/min) for 7 days increases nuclear staining of Atox1 and ecSOD protein expression in mouse aorta, which are abolished in Atox1−/− mice. In cultured vascular smooth muscle cells, Ang II stimulates translocation of Atox1 from cytosol to nucleus with peak at 30 min, which is prevented in Ctr1−/− cells. Luciferase reporter gene assays show that Ang II increases ecSOD promoter by 4.1±0.2 fold, which is blocked by the mutation of Atox1 responsive elements (Atox1-RE) and by siRNA knockdown of Ctr1. DNA pull down assays show that Ang II markedly increases binding of Atox1 to the Atox1-RE including DNA segment in the ecSOD promoter. Of note, Atox1 co-immunoprecipitates with Ctr1 in a copper dependent manner. These findings suggest that Atox1 functions as a transcription factor for ecSOD in a Ctr1-dependent manner. Finally, Ang II infusion-induced increase in systolic blood pressure and vascular superoxide production, as measured by lucigenin assay, are further enhanced in Atox1−/− mice compared to those in WT mice (149±2.3 (KO) vs. 137±1.2 (WT) mmHg; 4.0±0.2 (KO) vs. 2.54±0.2 (WT) fold), both of which are reduced by treatment of the SOD mimetic tempol. In summary, copper-dependent transcription factor function of Atox1 plays an important role in Ang II-induced upregulation of ecSOD expression and activity through Ctr1-Atox1 pathway, which may contribute to preventing oxidant-stress-dependent Ang II-induced hypertension.
Abstract 1411: Doxorubicin Triggers Replicative Senescence and Death of Cardiac Progenitor Cells
Authors: Piegari Elena , De Angelis Antonella , Hosoda Toru , Cappetta Donato , Marino Laura , Carrillo-Infante Cynthia , Filippelli Amelia , Berrino Liberato , Urbanek Konrad , Kajstura Jan , Leri Annarosa , Rossi Francesco , Anversa Piero
Published: July 2021

Doxorubicin (DOXO) is a very effective drug for the treatment of neoplastic diseases. However, DOXO causes a cardiomyopathy that leads to congestive heart failure. To test whether DOXO affects the cardiac progenitor cell (CPC) compartment, CPCs were exposed to increasing doses of the anthracycline for 12, 24 and 48 hours. At 0.1
μ
M, DOXO did not alter CPC survival. However, 0.5 and 1
μ
M DOXO reduced progressively and dramatically CPC viability. Apoptosis measured by TdT assay, DNA laddering and caspase-3 activity increased markedly with time peaking at 48 hours. The impact of DOXO on CPC growth was determined by BrdU and phospho-H3 labeling. The percentage of BrdU-positive CPCs and the mitotic index decreased with increasing doses of DOXO and time. The expression of the molecular regulators of G1 and G1\S transition, cyclin D1 and cdk4, decreased progressively with increasing concentration of DOXO and time, and these changes were coupled with a severe reduction in phospho-Rb in CPCs. Also, DOXO resulted in a transient increase of the cell cycle inhibitor p21
Cip
and a persistent increase in p16
INK4a
in CPCs. To determine whether the generation of reactive oxygen species (ROS) was responsible for DNA damage in CPCs, the presence of 8-OH-deoxyguanosine (8-OHdG) was measured. DOXO treatment strikingly increased the fraction of CPCs positive for 8-OHdG. Moreover, the expression of the antioxidant enzymes manganese superoxide dismutase and catalase failed to increase and counteract ROS-mediated DNA damage. Severe telomeric shortening occurred in CPCs exposed to DOXO. Telomere attrition was also favored by a significant decrease in telomerase activity in DOXO-treated CPCs. Dysfunctional telomeres trigger a DNA damage response in which the major determinant is the expression of p53. Both the transcription factor p53 and its target gene p21
Cip1
were upregulated in DOXO-treated CPCs. These molecular adaptations of CPCs increased with the dose and time of exposure to the anthracycline. In conclusion, these observations support the notion that DOXO-induced heart failure is a stem cell cardiomyopathy.
Abstract 1439: Cytochrome P450 Genetic Variants Predict Cardiovascular Outcomes following Treatment with Clopidogrel but not with Prasugrel
Authors: Mega Jessica L , Shen Lei , Wiviott Stephen D , Walker Joseph R , Hockett Richard D , Brandt John T , Moser Brian A , Macias William , Antman Elliott M , Close Sandra L , Lilly Eli , Sabatine Marc S
Published: July 2021

Background
: Prasugrel and clopidogrel require transformation into active metabolites by cytochrome P450 (CYP) enzymes. Variants in CYP genes, particularly
CYP2C19
, are associated w/ reduced levels of metabolite and platelet inhibition w/ clopidogrel but not prasugrel. We investigated the impact of variants in CYP genes on clinical outcomes.

Methods
: TRITON-TIMI 38 randomized ACS patients w/ planned PCI to prasugrel (60 mg load, 10 mg qd) or clopidogrel (300 mg, 75 mg qd) for 6–15 mos. DNA was available in 2943 subjects who were genotyped for functional polymorphisms in
CYP2C19
,
2C9
,
3A4/5
,
2B6
, and
1A2
and classified for each gene as carriers or non-carriers of ≥1 reduced function allele.

Results
: Among subjects treated with clopidogrel,
CYP2C19
reduced function allele carriers (27% of population) had a 53% increased risk of the primary efficacy endpoint of CV death, MI, or stroke (HR 1.53, P=0.007, Fig

) and a 3-fold increased risk of ARC-defined definite or probable stent thrombosis (HR 3.09, P=0.038) compared w/ non-carriers. In contrast, with prasugrel
CYP2C19
reduced function allele carriers were not at increased risk of CV death, MI, or stroke (HR 0.89, P=0.86) or stent thrombosis (HR 0.60, P=0.57). No association between genotype and bleeding was observed w/ either treatment. There were no statistically significant associations between any other tested CYP genotypes and outcomes.

Conclusions
:
CYP2C19
genetic variants identify patients at significantly higher risk for CV death, MI, or stroke and for stent thrombosis among those treated with clopidogrel but not among those treated with prasugrel.
CYP2C19
genotyping offers the potential to help tailor antiplatelet therapy.
Abstract 1454: Differentiated Cardiomyocytes Divide in Response to Neuregulin1 by Disassembling The Contractile Apparatus
Authors: Kuhn Bernhard , Bersell Kevin , Mancarella Salvatore , Arab Shima
Published: July 2021

Introduction:
Cardiomyocytes carry the pump function of the heart. What molecular and cellular mechanisms control proliferation of cardiomyocytes is an unresolved question with high impact on regenerative medicine.

Hypothesis:
The growth factor neuregulin1 (NRG1) and its tyrosine kinase receptor ErbB4 control cardiomyocyte proliferation during prenatal development. NRG1 and ErbB4 are expressed in the adult heart. We hypothesized that activating NRG1 signaling stimulates cell cycle reentry and division of a subpopulation of differentiated cardiomyocytes.

Methods:
We determined cardiomyocyte cell cycle reentry and division
in vitro
using immunofluorescence microscopy and live cell imaging as read-out assays. We tested the
in vivo
proliferative effect of controlling NRG1 signaling at the level of its receptor using inducible cardiomyocyte-specific ErbB4 knockout mice and ErbB4 transgenic mice.

Results:
NRG1 induced cell cycle reentry of large, striated, and rod-shaped cardiomyocytes that express cardiac contractile proteins, consistent with a differentiated phenotype. Live cell imaging demonstrated in real time that NRG1 induces differentiated cardiomyocytes to undergo karyokinesis (nuclear division) and cytokinesis (cytoplasmic division), followed by separation into two differentiated daughter cardiomyocytes. During cell division, cardiomyocytes disassembled their contractile fibrils in the regions of the mitotic spindle and the cleavage furrow. NRG1 induced karyokinesis in 33% of mononucleated and in 1% of binucleated cardiomyocytes. Mononucleated cardiomyocytes completed cytokinesis, while binucleated cardiomyocytes did not.
In vivo,
postnatal genetic inactivation of the NRG1 receptor ErbB4 reduced cycling of differentiated mononucleated cardiomyocytes (Ctr. 5%
vs.
ko 0%,
P
< 0.01). In contrast, transgenic expression of ErbB4 increased cycling of differentiated mononucleated cardiomyocytes (Ctr. 7.2%
vs.
tg 22.6%,
P
< 0.05).

Conclusions:
ErbB4 is required and NRG1 and ErbB4 are sufficient to induce proliferation of a subpopulation of differentiated cardiomyocytes. NRG1 and the pathway that it regulates may provide new therapeutic targets to enhance mammalian cardiac regeneration.

This research has received full or partial funding support from the American Heart Association, AHA Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).
Abstract 1495: Existence of Hyperfiltration in an Early Diabetic Rat was Visualised by Multiphoton Microscopy
Authors: Nakamoto Hiroshi , Ogasawara Yasuo , Kajiya Fumihiko
Published: July 2021

To visualise glomerular filtration under physiological conditions in an early diabetic rat and to evaluate filtration molecularly and quantitatively. We used 4 week STZ (50 mg/kg) model as early diabetic rats. We measured diameters of afferent (aff) and efferent (eff) arterioles, glomerular red blood cell velocities and left renal blood flow with our intravital videomicro-scope. Glomerular filtration was visualised by multiphoton microscopy. Various sizes of dextran (3k, 10k, 40k and 70k dalton (Dal)) conjugated with Texas Red in 0.5 ml were administered intravenously by bolus shot and for the next 60 seconds glomerular filtration was observed. FITC conjugated 500k Dal dextran was administered to dye intravascular area. The time course of Texas Red intensity in Bowman’s space was measured from the recorded images. Intensity peak values were normalized by the peak value of 3k Dal as sieving coefficients since the intensity reached its peak within 10 seconds after administration of Texas Red conjugated dextrans. Afferent arteriolar diameters were larger than efferent arteriolar diameters in both control (n=6) and diabetic (n=7) rats. Increases of both afferent and efferent diameters were remarkable in diabetic rats (p<0.05) (aff/eff diameter, DM rats: 14.0 ± 1.9, 9.4 ± 0.7, Control rats: 11.9 ± 0.8, 8.8 ± 0.7 μm). Glomerular red blood cell velocity in diabetic rats was larger than that in control rats (p<0.05) (DM/Control rats: 0.236 ± 0.030, 0.196 ± 0.076 mm/s). Left renal blood flow in diabetic rats was larger than that in control rats (p<0.05) (DM/Control rats: 1.84 ± 0.16, 1.60 ± 0.17 ml/100 gBW). All of these findings suggest the existence of hyperfiltration in early diabetic rats. In fact, filtration of the larger dextrans (40k and 70k Dal) were clearly visualized by multiphoton microscopy in early diabetic rats indicating greater leakage even from the early stage of diabetes with a significant difference by about 20% for the 40k and 70k Dal sieving coefficients comparing with the control group (p<0.05). Both afferent and efferent arteriolar diameters were increased in early diabetic rats suggesting the existence of hyperfiltration and the hyperfiltration was clearly visualised by multiphoton microscopy.
Abstract 1499: Angiotensin And Endothelin Systems - Important Players In Urotensin II Induced Myocardial Distensibility
Authors: Fontes-Sousa Ana P , Pires Ana L , Monteiro-Cardoso Vera , Leite-Moreira Adelino F
Published: July 2021

Objective: Urotensin II (U-II) is a vasoactive peptide with important actions in the cardiovascular system. It was recently shown that it acutely decreases myocardial stiffness. Although angiotensin II (AngII) and endothelin-1 (ET-1) also modulate myocardial diastolic properties, their interaction with U-II at this level has not yet been investigated.

Methods: Effects of increasing concentrations of U-II (10 – 8–10 – 6M) were studied in rabbit papillary muscles immersed in a modified Krebs solution (0.6Hz;1.8mM Ca2+;35°C) in the absence (n=12) and in presence of losartan, a selective competitive antagonist of AngII receptor type 1 (10 – 6M;n=8) or PD-145065, a nonselective endothelin receptor antagonist (10 – 6M;n=6). Calculated parameters: passive tension (PT), active tension (AT), maximum velocity of tension rise and decline (dT/dtmax and dT/dtmin, respectively) and muscle length. Results presented as mean±SEM (p<0.05).

Results: U-II induced a concentration dependent negative inotropic and lusitropic effects, decreasing at 10 – 6M 15.8±5.6%AT, 13.5±5.4%dT/dtmax and 18.1±4.5%%dT/dtmin. This effect was inhibited by losartan or PD-145065. Additionally, U-II induced a concentration dependent increase in passive muscle length of 1.0081±0.002L/ Lmax. Restoring muscle length to Lmax decreased 19.5±3.5%PT, indicating increased myocardial distensibility. In the presence of PD-145065, the maximal concentration of U-II only increased muscle length to 1.004±0.002%L/Lmax, which corresponded to a decrease of PT of just 11.6±2.7%. With losartan these effects of U-II were abolished.

Conclusion: The acute increase in myocardial distensibility induced by U-II is attenuated by ET-1 receptor blockade and completely abolished in the presence of an AT1 antagonist. This interaction between U-II, AngII and ET-1 is a novel finding with potential pathophysiologic and therapeutic implications in heart failure that deserves further investigation.
Abstract 1511: Gap Junction Internalization and Autophagic Degradation in the Failing Heart
Authors: Hesketh Geoffrey G , Shah Manish H , Kass David A , Van Eyk Jennifer E , Tomaselli Gordon F
Published: July 2021

INTRODUCTION Gap junctions (GJs) mediate electrical coupling between cardiomyocytes, and are required for coordinated electrical activation and contraction of the heart. GJs are remodeled in diseased myocardium, resulting in impaired intercellular coupling and a predisposition to life threatening arrhythmias. To understand the mechanisms of GJ remodeling in heart failure (HF), we examined the ultrastructure of GJs in failing canine myocardium.

METHODS and RESULTS Using transmission electron microscopy, we observed the formation of GJs between lateral membranes of ventricular myocytes in rapid pacing induced heart failure (HF) in dogs. Lateral GJs exhibited complex membrane bending and extensive internalization. Lateral GJs were frequently adjacent to mitochondria and internalized GJs often formed concentric ring structures surrounding cellular debris. Internalized GJ membranes were associated with multi-lamellar membrane structures, with features characteristic of autophagosomes. Connexin43 (Cx43) and the autophagosome marker GFP-LC3 were extensively co-localized when expressed in HeLa cells. A hyperphosphorylated form of Cx43 previously associated with GJ degradation co-fractionated with LC3-II (autophagosome targeted form) in a Triton-insoluble and buoyant fraction of ventricular tissue. The hyperphosphorylated/buoyant population of Cx43 as well as LC3-II were both significantly increased (n=5 each, p=0.011 and p=0.016 respectively), and whole tissue levels of Cx43 were significantly decreased (n=5, p=0.01), in pacing-induced HF.

CONCLUSION We show that structurally heterogeneous GJs are present at the lateral membranes of cardiomyocytes in HF, suggesting aberrant targeting of GJs. Internalized GJs are degraded via autophagy, which involves a hyperphosphorylated form of Cx43, and is enhanced in HF. These findings reveal a novel pathway of GJ targeting and degradation in HF, and suggest potential therapeutic targets for HF-related conduction slowing and arrhythmias.
Abstract 1522: Local Ca Releases And Spontaneous Beating Rate Of Rabbit Sinoatrial Node Cells Are Controlled By Interaction Of Basal Phosphodiesterase 3 And 4 Activity
Authors: Vinogradova Tatiana M , Lyashkov Alexey E , Spurgeon Harold , Lakatta Edward G
Published: July 2021

A high basal phosphodiesterase (PDE) activity in sinoatrial node cells (SANC) regulates cAMP and modulates cAMP-mediated PKA-dependent phosphorylation of Ca
2+
cycling proteins that determines the characteristics of local subsarcolemmal Ca
2+
releases (LCR). LCRs occur during the terminal diastolic depolarization and activate the inward Na
+
-Ca
2+
exchange current, thus, regulating the SANC basal spontaneous beating rate. Though both PDE3 and PDE4 are present in rabbit SANC, a role of PDE4 or its interaction with PDE3 is not clear. To determine to what extent PDE4 controls LCRs and the SANC spontaneous beating rate, we used perforated patch to record spontaneous action potentials (APs) in conjunction with confocal linescan images or whole-cell patch clamp to measure L-type Ca
2+
current (I
Ca,L
) at 35
o
C. A specific PDE3 inhibitor, cilostamide (Cil, 0.3 μmol/L), increased the spontaneous beating rate by 28% (from 154 ± 21 to 191 ± 17 beat/min); in contrast a specific PDE4 inhibitor, rolipram (Rol, 2 μmol/L), had no effect on the firing rate. The substantial role of PDE4 in the control of basal spontaneous SANC firing was unmasked when PDE3 and PDE4 were simultaneously inhibited: the combination of Rol and Cil produced ~57% acceleration of the beating rate (from129 ± 10 to 200 ± 10 beat/min) equivalent to that produced by the broad-spectrum PDE inhibitor, IBMX (100μM), i.e. ~55% (from 145 ± 8 to 221 ± 6 beat/min). The combination of Cil and Rol increased LCR amplitude, size and decreased the LCR period similar to IBMX. Both IBMX and the combination of Cil and Rol produced a substantial increase in I
Ca,L
~135% (from 10 ± 2 to 22 ± 3 pA/pF) and ~125% (from 6 ± 1 to 13 ± 3 pA/pF) respectively. The efficiency of PDE3 inhibition alone on spontaneous beating rate might be ascribed to its lower K
m
for cAMP, than that of PDE4. When PDE3 is suppressed level of cAMP is sufficiently elevated to activate PDE4. Therefore, in the presence of PDE3 inhibition, PDE4 inhibition contributes to the increase in the spontaneous beating rate. Thus, an interaction of basal PDE3 and PDE4 activities in SANC modulates cAMP level and I
Ca,L
which controls Ca
2+
influx, cell Ca
2+
load, determining LCR characteristics. When both PDE3 and PDE4 are inhibited the maximum spontaneous beating rate of SANC is achieved.
Abstract 1587: Conditional Overexpression of the Oncogene c-Myc Produces Hypertrophic Cardiomyopathy and Results in Mitochondrial Dysfunction at Complex I
Authors: Lee Hyoung-Gon , Hoit Brian D , Liner Anna , Richardson Sandra , Chen Qun , Lesnefsky Edward J , Smith Mark A
Published: July 2021

c-Myc is transiently expressed early during cardiac pressure overload, but its pathological role is unclear. To address the contribution of c-myc in the genesis of cardiomyopathy, we established a tetracycline-inducible, cardiomyocyte-specific, c-myc-overexpressing bitrans-genic mouse. Doxycyline treatment (DOX) for 2 weeks (Myc-ON, n=5) increased cardiac mass two-fold with myocyte disarray, and increased fiber width and fibrosis compared to control mice (Myc-OFF, n=8); LV fractional shortening (15MHz, Sequoia, Siemens) was less (0.28±0.03(SEM) vs.0.53±0.04) and myocardial performance index was greater (0.91±0.15 vs. 0.36±0.03, both p<0.05) indicating ventricular dysfunction in Myc-ON vs. Myc-OFF. Death resulted from irreversible heart failure. The cell cycle markers PCNA, Ki-67 and cyclin D1 were strongly induced and BrdU incorporation confirmed cell cycle progression to S-phase. To determine if mitochondrial dysfunction contributed to the cardiomyopathy of c-Myc-overexpression, oxidative phosphorylation (OXPHOS) was studied with glutamate/malate (complex I), succinate (complex II) and TMPD-ascorbate (complex IV) as substrates. OXPHOS decreased with glutamate/malate (213±40 vs.329±25 nAO/min/mg, p<0.05) after two weeks of Myc-ON (n=4) vs. control (n=4), but was unaltered with other substrates, localizing a defect to complex I of the electron transport chain. The complex I defect was confirmed by a decrease in NADH:decylubiquinone oxidoreductase activity (561±49 vs.752±38 mU/mg). PGC-1a (a major regulatory mechanism of mitochondrial proliferation) was increased in Myc-ON, and ultrastructural analysis revealed a 2.5-fold increase in mitochondrial number, possibly in response to the OXPHOS defect. Taken together, these findings suggest:

cardiac restricted temporally-defined Myc overexpression turns on cell cycle machinery in cardiomyocytes and produces decompensated hypertrophic cardiomyopathy; and

activation of c-Myc leads to an OXPHOS defect at complex I.

Since complex I defects impair energy production and increase the production of reactive oxygen species, we speculate that mitochondrial dysfunction contributes to the cardiomyopathy that develops when adult cardiomyocytes overexpress c-Myc.
Abstract 1600: Endogenous Nox4 Is Protective During Ischemia/reperfusion In The Heart
Authors: Ago Tetsuro , Kuroda Junya , Hsu Chiao-Po , Zhai Peiyong , Pain Jayashree , Sadoshima Junichi
Published: July 2021

Although excessive production of reactive oxygen species (ROS) causes cellular injuries, ROS also act as signaling molecules and regulate physiological cellular functions. ROS are extensively generated during ischemia/reperfusion (I/R) and cause tissue damages. The NAD(P)H oxidases are major sources of ROS in the cardiovascular system. Among NAD(P)H oxidase proteins, Nox4 is expressed in the heart and cultured cardiac myocytes. To test whether ROS derived from Nox4 induce tissue damage following I/R in the heart, we made mice on FVB background with cardiac-specific overexpression of wild-type Nox4 (Tg-Nox4) and catalytically inactive Nox4 (Tg-DN-Nox4 (P437H)) in which NADPH is unable to bind to Nox4. We confirmed that ROS generation and oxidative stress in the heart are greater in Tg-Nox4 and is lower in Tg-DN-Nox4, compared with non-transgenic mice (NTg), as determined by dihydroethidium fluorescence and 8-hydroxyguanosine staining, respectively. Neither Tg-Nox4 nor Tg-DN-Nox4 exhibited obvious baseline phenotype at 3 months of age. Tg-Nox4, Tg-DN-Nox4, and NTg (n=5, each) at 3 months old were subjected to 45 minutes ischemia, by ligating the left anterior descending artery, followed by 24 hours reperfusion. Unexpectedly, myocardial infarction (MI) size/area at risk (AAR) determined by TTC staining was significantly greater in Tg-DN-Nox4 than in NTg (67% vs 30 %, p<0.05). On the other hand, MI size/AAR in Tg-Nox4 (35 %) was comparable to that in NTg. To elucidate the mechanism by which suppression of Nox4 deteriorates I/R injury, we examined expression of proteins involved in cardiac metabolism. We found that expression levels of peroxisome proliferator-activated receptor α (PPARα), an important transcription factor stimulating mitochondrial fatty acid oxidation, remained high in Tg-DN-Nox4 during I/R (3.0 fold vs NTg), whereas it was suppressed in NTg and Tg-Nox4. Consistently, overexpression of DN-Nox4 enhanced transcriptional activity of PPARα by 2-fold, as determined by a luciferase assay, in cultured myocytes. Taken together, endogenous Nox4 may protect the heart from I/R injury by suppressing fatty acid oxidation through downregulation of PPARα which would otherwise lead to excess ROS generation after I/R.
Abstract 1610: Repetitive Ischemia Elicits a Third Window of Ischemic Preconditioning
Authors: Yan Lin , Depre Christophe , Shen You-Tang , Jain Komal , Tian Bin , Vatner Dorothy E , Vatner Stephen F
Published: July 2021

Ischemic preconditioning (IPC), following 1 or 2 episodes of coronary occlusion (CO) is the most powerful mechanism of cardioprotection, but it remains unclear whether IPC occurs clinically. Since patients with coronary disease experience episodes of repetitive ischemia (RI), we hypothesized that a pattern of RI would provide more clinically relevant cardioprotection. Conscious, chronically instrumented pigs (6/group) were submitted to RI induced by 6 episodes, 12 hrs apart, of coronary stenosis for 90 min (RCS) and compared to pigs submitted to second window IPC (SWIPC) induced by 2 episodes of 10 min CO / 24 hr reperfusion (CR) and to shams. Lethal ischemia, induced by 60 min CO, resulted in an infarct size/area at risk (IS/AAR) of 42±4% in shams, and significantly less, p<0.05, after RCS (6±3%) and after SWIPC (16±4%). SWIPC showed 2–3-fold increases, p<0.05, in translocation of PKCepsilon and the inducible isoform of nitric oxide synthase (NOS), which were not found in RCS. Pretreatment with the NOS inhibitor L-NNA (35 mg/kg) prevented the protection by SWIPC but not by RCS. Microarrays were performed to further elucidate the differences in molecular mechanisms between the SWIPC and RCS models. The number of genes significantly regulated was greater in RCS (5739) than in SWIPC (2394). Of the 5739 genes regulated in RCS, only 31% were also regulated in SWIPC. Since another difference between the two models is that one involves CS and the other complete CO, we examined another group of RI pigs (n=5) with 6 episodes, 12 hrs apart, of 2–10 min periods of CO, which showed reduced IS/AAR with 60 min CO after RI (14±4%); similar to RCS the cardioprotection was still observed after L-NNA. Both RI models, in further contrast to SWIPC, demonstrated downregulation of genes involved in mitochondrial function, and upregulation of genes involving autophagy (such as cathepsins B and D), endoplasmic reticulum stress (Grp78) and cell cycle (E2F1). These results were confirmed at the protein level by western blotting. Therefore, RI, over a time course of several days, provides a NOS-independent “third window” of protection, which is as effective as traditional SWIPC, but is mechanistically distinct.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 1622: High salt Intake Superimposed On Left Ventricular Hypertrophy Activates Brain Enac And Leads To Sympathetic Hyperactivation And Ventricular Dysfunction
Authors: Ito Koji , Hirooka Yoshitaka , Sunagawa Kenji
Published: July 2021

Background:
High-salt intake often predisposes patients with left ventricular hypertrophy (LVH) to congestive heart failure. We hypothesized that high-salt intake superimposed on LVH activates brain epithelial Na channels (ENaC) and leads to sympathetic hyperactivation and LV dysfunction.

Methods and Results:
We used ICR mice and applied aortic banding (AB) to create LVH. Sham-operated mice served as control. Four weeks after AB, 24-h urinary norepinephrine excretion (U-NE) tended to increase rather than that in control (481±46 vs 354±42, n=10, p=0.09). AB hypertrophied LV wall thickness (WT) (2.2±0.6 vs 1.8±0.6 mm, n=8, p<0.05) without changing fractional shortening (%FS). We then fed the AB mice a high salt (8%) (AB-H) or regular salt (AB-R) diet for additional 4 weeks. AB-H depressed LV function (%FS: 27±2 vs 37±1%, n=8, p<0.05) and hypertrophied LVWT more than those in AB-R (WT: 2.8±0.5 vs 2.5±0.3 mm, n=8, p<0.05). U-NE was higher in AB-H than in AB-R (758±86 vs 459±35 ng/day, n=10, p<0.05). To examine the role of brain ENaC, we intracerebroventriculary (ICV) infused ENaC blocker (benzamil, 0.11μg/hr by osmotic pump) in AB-H for 4weeks. ENaC blocker decreased U-NE (567±35 vs 758±86 ng/day, n=10, p<0.05) and improved depressed LV function (%FS: 39±1 vs 28±3 %, n=10, p<0.05). To confirm the role of central Na in sympathetic hyperactivation and LV dysfunction, we administered, in AB with regular salt diet, high-Na (0.2 M) cerebrospinal fluid ICV (Na-ICV) (0.25μl/hr) for 2 weeks. Na-ICV significantly increased U-NE and deteriorates LV function (U-NE:987±103 ng/day, %FS: 31±1 %n=6)

Conclusions:
These results suggested that high-salt intake in the presence of LVH activates brain ENaC and leads to sympathetic hyperactivation and LV dysfunction.
Abstract 1636: Adverse Effect of Estrogen on Bone Morphogenetic Protein Receptor Signal Pathway in Pulmonary Arterial Endothelial Cells
Authors: Ichimori Hiroaki , Kogaki Shigetoyo , Ishida Hidekazu , Narita Jun , Uchikawa Toshiki , Nasuno Sayaka , Okada Yoko , Ozono Keiichi
Published: July 2021

Gender differences in the development of Pulmonary Artery Hypertension (PAH) have been documented in both human and animal studies. In human, idiopathic PAH is predominantly a disease of young women in their child-bearing years, which suggests a role of female sex hormones in the pathogenesis of PAH. However, the effect of sex hormones on pulmonary vasculatures and the development of PAH has not been fully understood. Recent researches have revealed genetic predisposition such as BMPR (Bone Morphogenetic Protein Receptor). The aim of the present study is to investigate the effect of β-estradiol (E2) and oxygen concentration upon BMPR signal pathway in pulmonary arterial endothelial cells (PAEC) in vitro. Human and rat PAEC were cultured and we examined the expression of BMPR2, BMP-regulated Smads, and Id1 under 21% or 1% O
2
with BMP2 stimulation. Then, we investigate changes in the expression of these molecules in the presence of E2 with or without estrogen receptor antagonist (ICI 182.780.). First, we confirmed that estrogen receptor α and β were expressed in both PAECs. Second, we demonstrated that the expression of mRNA transcripts for BMPR2 and Id1 in PAEC was reduced after exposure to 24 hours’ hypoxia. In addition, E2 decreased the expression of phosphorylated Smad (p-Smad)1/5/8 in a dose-dependent manner (10
−10
M-10
−7
M) and p-Smad1/5/8 expression were decreased about 80% by 10
−7
M of E2. These attenuation of p-Smad1/5/8 expression were rescued by ICI182.780. Third, under normoxic condition with cobalt chloride or deferoxamine to prevent the degradation of HIF (hypoxia-inducible factor)-1α, the presence of E2 decreased the expression of p-Smad1/5/8 like under hypoxia. Conversely, administration of HIF-1α inhibitor (YC-1) canceled the reduced expression of p-Smad1/5/8 like under normoxia. Under hypoxia, the presence of E2 attenuates the BMPR signal pathway in PAEC in vitro. Our data indicated that the advance effect of E2 on BMPR signal pathway was associated with HIF-1α and estrogen receptor. Our observations provide the first evidence that female sex hormone affects on BMPR signal pathway, which can offer new strategies for the treatment of PAH.
Abstract 1679: Nuclear Receptor Nur77 and 6-Mercaptopurine Protect against Atherosclerosis
Authors: Arkenbout E. Karin , Bonta Peter I , Pols Thijs W , de Vries Carlie J
Published: July 2021

Nur77 is an orphan nuclear receptor of the NR4A subfamily, which also comprises Nurr1 (NR4A2) and NOR-1 (NR4A3). We were the first to describe the expression of Nur77 in human atherosclerotic lesions both in macrophages and in activated, intimal smooth muscle cells. Here we demonstrate that Nur77 is expressed in human instent restenotic lesions that were obtained through coronary atherectomy. The generation of transgenic mice that overexpress Nur77 specifically in arterial smooth muscle cells allowed us to study the function of Nur77 in the injured vessel wall
in vivo
. Nur77 inhibits the formation of smooth muscle cell-rich lesions in the carotid artery ligation model, as well as in the femoral artery loosely-fitting cuff model. Both models relate to human (instent) restenotic lesions that are also composed predominantly of smooth muscle cells. Recently, it has been shown that 6-mercaptopurine (6-MP) enhances the activity of Nur77 and we demonstrated in a drug-eluting cuff mouse model that 6-MP reduces lesion formation in a Nur77-dependent way. In atherosclerosis, monocytes and macrophages are important in initiation and progression of the disease. We demonstrated that Nur77 downregulates the expression of inflammatory cytokines and chemokines and inhibits the uptake of oxidized LDL particles in macrophages. To extend our knowledge on Nur77 function in atherosclerosis we cross-bred our Nur77-transgenic mice with ApoE-/- mice and studied high-fat/cholesterol diet induced lesion formation in the aortic sinus. Nur77 overex-pression in smooth muscle cells reduces lesion formation in response to this diet. In conclusion, our data demonstrate that Nur77 and its agonist 6-MP inhibit vascular lesion formation and atherosclerosis.

Supported by The Netherlands Heart Foundation grants M93.007 and 2003B199.
Abstract 1681: Haploinsufficiency of MEF2A Increases Risk of Atherosclerosis In Mice
Authors: Wang Qing K , Bhagavatula Krishna M , Topol Eric J , Smith Jonathan D
Published: July 2021

MEF2A is a member of the myocyte enhancer factor-2 (MEF2) family of transcription factors. We showed that MEF2A was expressed in coronary arteries and mutations or rare variants of MEF2F were associated with risk of coronary artery disease (CAD) in humans. Both positive and negative replication studies were later reported. To further investigate the role of MEF2A in CAD and MI, we have characterized heterozygous
MEF2A
knockout mice and show here that haploinsufficiency of MEF2A in mice promotes atherosclerosis. Heterozygous MEF2A knockout mice were crossed to apoE-deficient mice, a widely used genetic model for atherosclerosis. The degree of atherogenesis was determined by the aortic root quantitative atherosclerosis assay.
In vivo
vascular permeability assay was performed using Evans blue dye upon stimulation with mustard oil in ears. Serum total cholesterol, HDL cholesterol, and triglyceride levels were measured with the Stanbio enzymatic kits. No significant differences were detected for total plasma cholesterol, HDL-C, VLDL-Cl and TG levels between MEF2A
+
ApoE
−/−
mice and their MEF2A
+/+
ApoE
−/−
littermate controls (P = 0.14 – 0.44). The glucose levels before and after fasting were also similar between the two lines of mice (fasting, 106.25 ± 20.7 vs. 104.8 ± 21.9, P = 0.87; non-fasting, 175.3 ± 82 vs. 179.9 ± 119, P = 0.092). Total aortic lesion area was twofold greater in MEF2A
+
ApoE
−/−
mice than in MEF2A
+/+
ApoE
−/−
littermate controls fed on western diet (83408 μm
2
vs. 46621 μm
2
in males, n = 11 vs. 15, P = 0.0003; 137822 μm
2
vs. 61568 μm
2
in females, n = 12 vs. 9, P = 0.00003). Permeability of the vasculature in response to a proinflammatory stimulus was 3.2 fold higher in MEF2A
+
ApoE
−/−
mice than in MEF2A
+
/
+
ApoE
−/−
littermate controls (P = 0.000012). MEF2A haploinsufficiency promotes atherosclerotic lesion formation in mice, which may be due to increased vascular permeability. Lipid or glucose levels do not contribute to the formation of atherosclerotic lesions in these mice.
Abstract 1693: Effect of Pitavastatin on Serum Concentrations of Endothelial Lipase and High-Density Lipoprotein Cholesterol in Humans
Authors: Sun Li , Ishida Tatsuro , Yoko Kojima , Yasuda Tomoyuki , Fukuda Akira , Kureha Fumie , Hirata Kenichi
Published: July 2021

[Purpose] Endothelial lipase (EL) is a novel phospholipase which regulates serum high-density lipoprotein cholesterol (HDL-C) levels. In the present study, we have measured serum levels of EL in human subjects, and examined the effect of statins on serum HDL levels and EL mass.

[Methods and Results] A sandwich ELISA for human EL was established using two monoclonal antibodies against amino- or carboxy-terminus of human EL. Serum EL concentrations were measured and compared with the serum lipid profile in 237 patients with cardiovascular diseases. There was no significant correlation between serum EL mass and age or gender. Serum EL levels were negatively associated with serum HDL-C levels, but not with serum LDL-C, total cholesterol, or triglyceride levels. Forty-eight patients with hypercholesterolemia were treated with pitavastatin for 6 months, and serum EL mass was evaluated before and after the pitavastatin treatment. Pitavastatin treatment significantly reduced serum EL levels by 15% and increased HDL-C levels by 12%. Complimentary cell culture experiments revealed that pitavastatin suppressed basal and cytokine-induced EL expression and phospholipase activities in endothelial cells, which is reversed by the concomitant treatment with mevalonate or geranylgeranylpyrophosphate. Also, overexpression of RhoA T19N, a dominant negative mutant of RhoA, decreased the EL expression.

[Conclusion] EL is a determinant of serum HDL-C levels in humans. Pitavastatin reduced EL expression through the protein isoprenylation and inhibition of Rho activity, and raised serum HDL-C levels. Thus, EL would be a target molecule for the HDL-raising pharmaceutical interventions.
Abstract 1696: Compound RVX-208 Modulates HDL-C Levels and Function in Non-human Primates and in Early (phase I) Human Trials
Authors: Krimbou Larbi , Jahagirdar Ravi , Bailey Dana , Hafiane Anouar , Ruel Isabelle , Wong Norman , Wagner Gregory S , Chiacchia Fabrizio , Gordon Allan , Hansen Henrik C , Johansson Jan , Genest Jacques
Published: July 2021

The novel compound RVX-208 is a small molecule that upregulates the gene expression of apoA-I and raises HDL-C in non-human primates. Here, we examined the effects of oral administration of RVX-208 on serum apoA-I and HDL-C levels , HDL size distribution, and HDL function. African green monkeys received RVX-208 (7.5, 15 and 30 mg/kg; twice daily and 60 mg/kg; once daily) or vehicle control for 28, 42, and 63 days. We report that RVX-208 chronic treatment resulted in a highly significant increase in the average of serum apoA-I and HDL-C levels (57% and 92%, respectively). Interestingly, RVX-208 treatment modified the distribution of HDL particle size causing a significant increase in preβ1-LpA-I and larger α1-LpA-I species. The ability of serum to promote cholesterol efflux via ABCA1, ABCG1 or SR-BI-dependent pathways in a cell culture model was significantly increased by RVX-208. The phase Ia safety and pharmacokinetic human study comprised of a total of 80 subjects. In the multiple ascending dose arm, 24 participants were randomly assigned to 3 cohorts of 8 healthy volunteers (6 active and 2 placebo), and received oral administration of RVX-208 at 2, 3 and 8 mg/kg per day or placebo for 7 days. The compound was well tolerated and had good oral absorption meeting the objectives of safety and pharmacokinetics. ApoA-I, HDL-C, HDL size distribution and ABCA1-dependent cholesterol efflux were assessed at days 1 (predose) and 7. The percent change from baseline to day 7 for apoA-I was 11% higher (P = 0.03) in the RVX-208 treated participants compared to placebo. Interestingly, preβ1-LpA-I change was 30% (P = 0.02) higher in the actively treated group and was found to strongly correlate with increased apoA-I levels (R2 = 0.72). Furthermore, ABCA1-dependent cholesterol efflux change was 10% higher (P = 0.03) and was found to correlate with increased preβ1-LpA-I . Taken together, these pharmacodynamic data from human healthy volunteers show consistent trends in apoA-I production and HDL functionality, supporting the findings in the African green monkey. Further investigation of the effect of RVX-208 on the HDL metabolic pathway is ongoing in humans and animals to establish the mechanisms of action and therapeutic potential in treating atherosclerotic cardiovascular disease.
Abstract 1710: Pharmacologic Profile of SCH 530348, a Novel Oral Antiplatelet Agent Selective for the Protease-Activated Receptor-1 (PAR-1)
Authors: Chintala Madhu , Ahn Ho-Sam , Foster Carolyn , Agans Jacqueline , Boykow George
Published: July 2021

Antiplatelet agents are a cornerstone of therapy for atherothrombotic disease. However, despite the proven efficacy of agents targeting the thromboxane A2 (aspirin) and P2Y12 ADP (clopidogrel, prasugrel) platelet activation pathways, the residual risk for ischemic events remains considerable. Binding of thrombin to the platelet PAR-1 is an important platelet activation pathway not targeted by existing agents. Inhibition of PAR-1 may thus provide incremental clinical benefits over aspirin and ADP receptor antagonists. PAR-1 receptors expressed on endothelial cells, smooth muscle cells, monocytes and neutrophils have been reported to mediate the pro-inflammatory and chemotactic responses to thrombin. In the present study, we report pharmacologic characterization of SCH 530348, a novel thrombin receptor antagonist (TRA) selective for PAR-1, using in vitro assays with human platelet membranes and cultured human coronary artery smooth muscle cells (HCASMC), and ex vivo platelet aggregation assays in cynomolgus monkeys. The affinity of SCH 530348 for the PAR-1 receptor was determined in human platelet membranes. Functional studies involving calcium transients, thymidine incorporation, and receptor kinetics were performed in HCASMC. The oral antiplatelet effects of SCH 530348 in cynomolgus monkeys were evaluated in ex vivo platelet aggregation assays. SCH 530348 exhibited high affinity for PAR-1 receptor. SCH 530348 potently inhibited thrombin-stimulated calcium transients and thymidine incorporation in HCASMC, and displayed slow dissociation from PAR-1 receptor. In cynomolgus monkeys, SCH 530348 administered orally at doses ranging from 0.1 mg/kg to 3 mg/kg, provided rapid, complete, and sustained inhibition of thrombin receptor agonist peptide (TRAP)-induced ex vivo platelet aggregation for 24 hours. Significant inhibition of TRAP-induced platelet aggregation was maintained at 48 hours after dosing of SCH 530348. SCH 530348 is a highly selective, potent, and orally active PAR-1 antagonist. Inhibition of PAR-1 by SCH 530348 may translate into beneficial clinical effects in patients with atherothrombotic disease, and this hypothesis is currently being evaluated in 2 large trials.