bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 281: Long-Term Therapy with a Partial Adenosine A1-Receptor Agonist Upregulates mRNA and Protein Expression of Glucose Transporters in Left Ventricular Myocardium of Dogs with Chronic Heart Failure
Authors: Sabbah Hani N , Gupta Ramesh C , Rastogi Sharad , Wang Mengjun , Zhang Keife , Mohyi Paula M , Albrecht-Kupper Barbara
Published: July 2021

Background:
Recruitment of glucose transport proteins GLUT-1 and -4 is a mechanism by which the heart increases glucose transport for metabolism in response to increased energy demands. In the failing LV, mRNA and protein levels of GLUT-1 and -4 are down-regulated and the response to increased demands is met by increased dependence on fatty acid oxidation. We previously showed that treatment with the partial adenosine A1-receptor agonist capadenoson (CAP) improves LV systolic function in dogs with advanced heart failure (HF). This study examined the effects of CAP on the regulation of GLUT-1 and -4 in LV myocardium of dogs with chronic HF.

Methods:
Studies were performed in LV tissue from 12 HF dogs randomized to 3 months oral therapy with the CAP (7.5 mg twice daily, n=6) or to no therapy at all (Control, n=6). LV tissue from 6 normal (NL) dogs was used for comparison. mRNA expression of GLUT-1 and -4 was measured in LV tissue homogenate using real time polymerase chain reaction (RT-PCR). Protein levels of GLUT-1 and -4 and calsequestrin (CSQ), a sarcoplasmic reticulum protein that does not change in HF (internal control), were measured in SDS-extract using Western blotting coupled with chemiluminescent method. Band intensity was quantified in densitometric units (du).

Results:
Compared to NL dogs, Control HF dogs showed a 3.6 fold decrease in mRNA expression of GLUT-1 and a 2.7 fold decrease in the expression of GLUT-4. CAP-treatment in HF dogs improved mRNA expression of GLUT-1 (1.38 fold decrease) and GLUT-4 (0.8 fold decrease). Similarly, compared to NL dogs, Control HF dogs showed a significant decrease of protein levels of GLUT-1 (0.64 ± 0.04 vs. 0.26 ± 0.04 du, p<0.05) and GLUT-4 (1.42 ± 0.06 vs. 0.40 ± 0.05 du, p<0.05). CAP treatment significantly increased protein levels of both GLUT-1 (0.49 ± 0.03 du) and GLUT-4 (0.82 ± 0.03) compared to Control HF dogs (p<0.05). CSQ levels were similar in all study groups.

Conclusions:
In dogs with chronic HF, long-term therapy with CAP improves mRNA and protein expression of GLUT-1 and -4. This improvement in expression of glucose transport proteins is likely to restore partial dependency of the failing heart to more efficient glucose metabolism, and therefore, lead to improved myocardial energetics and pump function.
Abstract 282: Chemically Tailoring Cardiovascular Drugs for Alzheimer's Disease Drug Discovery
Authors: Pasinetti Giulio , Ho Lap , Ono Kenjiro , Wang Jun
Published: July 2021

We previously reported that carvedilol, a brain bioavailable and bioactive antihypertensive agent, significantly reduces brain contents of Alzheimer’s disease (AD)-type β-amyloid (Aβ) oligomers experimentally. Currently, we are exploring clinical applications of carvedilol to prevent or attenuate the progression of AD dementia. However, the potential development of carvedilol for AD is complicated by its cardiovascular activities, including bradycardia and hypotension, particularly among patients already undergoing treatment with other cardiovascular medications. Based on this, using structure-activity relationship (SAR) medicinal chemistry we are developing novel modified carvedilol lead compounds with decreased cardiovascular features for treating AD. Testing commercially available compounds identified from a preliminary screening of a library of carvedilol analogs, we identified 6 bioactive small-molecule compounds that significantly reduce Aβ oligomerization in vitro. Anti-Aβ oligomerization activity was confirmed by circular diachroism and electron microscopy. One compound, (R)-(+)-4-hydroxyphenyl carvedilol, showed improved anti-oligomerization activity and reduced α-1 adrenergic receptor binding activity compared to carvedilol. Short-term proof-of-concept in vivo testing showed that treatment with 1.5 mg/kg/day (R)-(+)-4-hydroxyphenyl carvedilol significantly reduced levels of oligomeric Aβ and total Aβ in the brains of a transgenic mouse model of AD-type Aβ neuropathology without cardiovascular complications. We are currently evaluating the effects of (R)-(+)-4-hydroxyphenyl carvedilol on cardiovascular functions and the attenuation of Aβ-mediated cognitive dysfunction in experimental AD models. Our study provides the critical basis for exploring the bioavailability and pharmacokinetic characteristics of novel cardiovascular lead compounds with enhanced brain activities and reduced adverse events in IND-directed pre-clinical safety assessments and eventually Phase I clinical applications in AD.
Abstract 285: Cardiac Inflammation and Fibrosis Induced by Heart Failure Are Reversed by Short-Duration Estrogen Therapy
Authors: Iorga Andrea , Nadadur Rangarajan , Sharma Salil , Li Jingyuan , Eghbali Mansoureh
Published: July 2021

Heart failure is generally characterized by increased fibrosis and inflammation, which leads to functional and contractile defects. We have previously shown that short-term estrogen (E2) treatment can rescue pressure overload-induced decompensated heart failure (HF) in mice. Here, we investigate the anti-inflammatory and anti-fibrotic effects of E2 on reversing the adverse remodeling of the left ventricle which occurs during the progression to heart failure. Trans-aortic constriction procedure was used to induce HF. Once the ejection fraction reached ∼30%, one group of mice was sacrificed and the other group was treated with E2 (30 αg/kg/day) for 10 days. In vitro, co-cultured neonatal rat ventricular myocytes and fibroblasts were treated with Angiotensin II (AngII) to simulate cardiac stress, both in the presence or absence of E2. In vivo RT-PCR showed that the transcript levels of the pro-fibrotic markers Collagen I, TGFβ, Fibrosin 1 (FBRS) and Lysil Oxidase (LOX) were significantly upregulated in HF (from 1.00±0.16 to 1.83±0.11 for Collagen 1, 1±0.86 to 4.33±0.59 for TGFβ, 1±0.52 to 3.61±0.22 for FBRS and 1.00±0.33 to 2.88±0.32 for LOX) and were reduced with E2 treatment to levels similar to CTRL. E2 also restored in vitro AngII-induced upregulation of LOX, TGFβ and Collagen 1 (LOX:1±0.23 in CTRL, 6.87±0.26 in AngII and 2.80±1.5 in AngII+E2; TGFβ: 1±0.08 in CTRL, 3.30±0.25 in AngII and 1.59±0.21 in AngII+E2; Collagen 1: 1±0.05 in CTRL.2±0.01 in AngII and 0.65±0.02 (p<0.05, values normalized to CTRL)). Furthermore, the pro-inflammatory interleukins IL-1β and IL-6 were upregulated from 1±0.19 to 1.90±0.09 and 1±0.30 to 5.29±0.77 in the in vivo model of HF, respectively, and reversed to CTRL levels with E2 therapy. In vitro, IL-1β was also significantly increased ∼ 4 fold from 1±0.63 in CTRL to 3.86±0.14 with AngII treatment and restored to 1.29±0.77 with Ang+E2 treatment. Lastly, the anti-inflammatory interleukin IL-10 was downregulated from 1.00±0.17 to 0.49±0.03 in HF and reversed to 0.67±0.09 in vivo with E2 therapy (all values normalized to CTRL). This data strongly suggests that one of the mechanisms for the beneficial action of estrogen on left ventricular heart failure is through reversal of inflammation and fibrosis.
Abstract 288: Atheroprotective Effect of Probucol Is Related to Its Heme Oxygenase 1 Activation and Subsequent Suppression of Oxidized Low-Density Lipoprotein--Induced Dendritic Cell Maturation
Authors: Sun Aijun , Jin Xueting , Zhao Jingjing , Wang Keqiang , Xu Fang , Huang Wei , Liu George , Zou Yunzeng , Hu Kai , Ge Junbo
Published: July 2021

Aims: Probucol, an agent characterized by lipid-lowering and anti-oxidant property, retards atherosclerosis effectively. Our study aimed to test the hypothesis that probucol might act its anti-athersclerotic role by suppressing maturation of human monocyte-derived dendritic cells (h-monDC). Furthermore, we also used a LDLR-/- mice model fed a high-fat diet to detect whether probucol also perform its anti-atherosclerotic effect on suppressing DCs maturation in vivo.

Methods: H-monDCs were derived by incubating purified human monocytes with GM-CSF and IL-4. H-monDCs were pre-incubated with or without probucol and stimulated by oxidized low-density lipoprotein (ox-LDL) in the presence or absence of heme oxygenase (HO-1) siRNA. In vivo studies, streptozotocin (STZ) induced LDLR-/- mice were fed either a high-fat (HF) diet or added with 0.5% probucol for 4 months. Expression of h-monDC membrane molecules and mice splenic CD11c+DC membrane molecules were analyzed by FACS, cytokines were measured by ELISA and the STAT1/CIITA associated signaling pathway was determined by Western blotting. Mice aortic lesions were observed by En face staining and the expression of CD11c+DCs within atherosclerotic plaques were shown under confocal microscopy.

Results: Ox-LDL promoted h-monDC maturation and TNF-a production; and up-regulated STAT1 701 phosphorylation by activating HO-1 in STAT1/CIITA signaling pathway. These effects were inhibited by probucol. Knocking down HO-1 with specific siRNA blocked these effects of probucol. In LDLR-/- mice fed a high-fat diet, probucol treatment significantly regressed aortic atherosclerotic lesions, suppressed splenic CD11c+DCs maturation and IL-12p70 production; and resulted in absence of CD11c+DCs within atherosclerotic lesions.

Conclusions: Our study indicated that probucol effectively suppressed maturation of h-monDC induced by ox-LDL through HO-1 activation, and retarded atherosclerosis at least partly through inhibiting maturations of CD11c+DCs in LDLR-/- mice.
Abstract 29: Heme Levels Are Increased in the Hearts of Patients with End-Stage Heart Failure
Authors: Khechaduri Arineh , Bayeva Marina , Ardehali Hossein
Published: July 2021

Introduction: Diseases of iron imbalance can cause cardiomyopathy, as observed in hereditary hemochromatosis or Friedreich’s ataxia. However, the iron status of a failing heart has not been systematically examined.

Results: The iron levels were quantified in ten hearts from human patients with end-stage cardiomyopathy and ten unmatched healthy control hearts. Total cellular and cytosolic non-heme iron levels were significantly reduced in failing hearts; however, there was a 2-fold increase in cytosolic heme levels in heart failure, as measured by the total protoporphyrin IX (PPIX) content. The amount of iron-free PPIX was low and did not differ between the two groups, suggesting that the majority of PPIX is complexed with iron to form heme, and thus our assay accurately estimates heme levels in the heart.

To study the mechanism for the increased heme content in failing hearts, we examined the expression of genes involved in heme and iron homeostasis. Consistent with elevated heme, there was a decrease in the levels of the cellular iron importer (transferrin receptor 1 orTfR1) and the first enzyme in heme synthesis (δ-aminolevulinic acid synthase 1 or ALAS1), both of which are negatively regulated by heme. Other genes involved in heme synthesis, iron incorporation into PPIX, and heme degradation were not affected. However, we noted a significant increase in the mRNA and protein levels of ALAS2 in the failing hearts. ALAS2 is normally expressed in hemoatopoietic cells and displays very low expression in the heart. Its induction in the setting of heart failure provides a possible explanation for increased heme levels.

We then assessed whether increase in ALA and heme in failing hearts is associated with generation of reactive oxygen species (ROS). Addition of exogenous ALA led to a four-fold increase in ROS, while incubation with hemin have doubled ROS levels in these cells, suggesting that augmentation of heme synthesis may exacerbate heart failure by increasing the oxidative stress.

Conclusions: Our results suggest that heme levels are increased in human hearts with end-stage cardiomyopathy, possibly through the induction of ALAS2 expression. The elevated heme levels may exacerbate heart failure by increased production of ROS.
Abstract 302: Autophagy in the Aging Heart
Authors: Gurney Michael A , Gottlieb Roberta A , Linton Phyllis-Jean
Published: July 2021

Autophagy maintains cellular homeostasis by lysosomal degradation and recycling of damaged/dysfunctional organelles, removal of aggregated proteins, and the provision of energy to cells in times of stress. Recently, autophagy has been shown to decline with age in the liver and thymus, prompting us to ask if there was a corresponding age-related decrease in autophagy in the heart. The comparison of membrane-associated LC3-II to cytosolic LC3-I is indicative of autophagosome formation, and is one of several parameters used to assess autophagic activity. Basal autophagy in C57BL/6 mice across five age groups from 2.5 to 24 months, assessed by Western Blot, demonstrated little variance in LC3-I but a significant decrease in LC3-II (ANOVA; n=3/age group; p>0.05 and p<0.0002, respectively). Also, a Triton-X insoluble fraction containing p62/SQSTM-1 and ubiquitinated proteins were shown to correlate with changes in aggrephagy. We found a significant increase in p62 and ubiquitinated proteins with increasing age (p<0.01). Both findings suggest a decline in basal autophagy with age. When subjected to an acute stress, such as fasting, the levels of LC3-II but not LC3-I increased in young and old mice (n=4/age group; p<0.05 and p>0.05, respectively). To determine if flux was impaired in the aged, we treated fasted mice with chloroquine, which prevents autophagosome/lysosome fusion resulting in LC3-II accumulation. We found LC3-II accumulated only in the young (n=4; p<0.01). Thus, the aged can mount an acute response but flux declines. Moreover, RT-qPCR analysis of cardiac tissue showed a significant decrease in
atg5
and
atg7
and a significant increase in
atg8A
,
atg8B
,
lamp2A
,
atg4A
, and
atg4B
expression with advancing age (> 2.5 fold change). The dysregulated mRNA levels are suggestive of overcompensation of clearance, and the data support an overall decline in function with age. Although not manifest in the same manner, autophagy dysregulation also occurs with age in BALB/c mice, a shorter-lived strain. Our findings may be relevant to cardiac injury, such as in ischemia/reperfusion, given the emerging importance of autophagy in cardioprotection.
Abstract 303: Detrimental Effects of Prolonged AKT Activation in Hearts from Ozone-Exposed Healthy Adult Rats
Authors: Manchanda Shubham , Ramirez Magdalena , Manchanda Pranjal , Sethi Vishal , Dostal David , Sethi Rajat
Published: July 2021

Tropospheric ozone (O3) is one of six major air pollutants regulated by National Ambient Air Quality Standards (NAAQS) under the U.S. Clean Air Act. In the current study, we are trying to understand the effects of survival proteins, such as AKT and BCl-2, subsequent to chronic exposure of ozone in healthy adult rats. Twenty age/weight matched male Sprague Dawley rats were randomly divided into two groups that were exposed to air (0 ppm O3) or 0.8 ppm O3 for 8 hours/day for 28 days. Four rats were sacrificed, their hearts and lungs were extracted, and left ventricular tissue was used for various estimations. Western blot analysis was performed on lung and heart tissue from filtered air and O3-exposed rats to quantify the differential levels of P-p38 (activity) in the cytosolic fraction, Akt (expression) in the cytosolic fraction, p-Akt-Thr (activity) and p-Akt-Ser (activity) in the cytosolic fraction, Bcl-2 (expression) in the cytosolic fraction, and p-Bcl-2 (activity) in the cytosolic fraction. As a consequence of chronic O3 exposure, the significant findings of the current work are: 1) cytosolic fraction from 28 days O3-exposed group showed no significant change in P-p38MAPKα activity, but 56 days O3-exposed group showed increased P-p38MAPKα activity, 2) cytosolic fraction from 28 days and 56 days O3-exposed group showed increased expression of Akt, 3) cytosolic fraction from 28 days O3-exposed group showed decreased p-Akt-Ser activity, but 56 days O3-exposed group showed increased p-Akt-Ser activity, 4) cytosolic fraction from 28 days O3-exposed group showed increased p-Akt-Thr activity, but 56 days O3-exposed group showed decreased p-Akt-Thr activity, 5) cytosolic fraction from 28 days O3-exposed group showed decreased expression of Bcl-2, but 56 days O3-exposed group showed increased expression of Bcl-2, 6) cytosolic fraction from 28 days O3-exposed group showed increased p-Bcl-2 activity, but 56 days O3-exposed group showed decreased p-Bcl-2 activity. Based on these results we can conclude that prolonged Akt activation subsequent to chronic ozone exposure may be detrimental to hearts and have adverse effects over time. Roles of Bcl-2 and Bax in this injury are not yet clear and are being currently investigated in our laboratory.
Abstract 307: Phospholipase Cε Is Involved in Gq-Dependent Protein Kinase D Activation in Cardiac Myocytes
Authors: Smrcka Alan , Zhang Lianghui , Malik Sundeep
Published: July 2021

We previously demonstrated that PLCε is a critical mediator of endothelin-1 (ET-1)- and norepinephrine (NE)-dependent hypertrophy in neonatal rat ventricular myocytes (NRVMs). Both α-adrenergic and ET-1 receptors couple to Gq as well as other G proteins. To determine if PLCε is required for Gαq-dependent hypertrophy NRVMs were infected with adenoviruses expressing wtGαq and PLCε siRNA followed by measurement of hypertrophy. PLCε-siRNA significantly inhibited Gαq-induced increases in myocyte area and atrial natriuretic factor (ANF) mRNA expression. Similarly, disruption of PLCε association with perinuclear mAKAP inhibited Gαq-dependent hypertrophy. These data suggest that ET-1 and PE signal, at least in part, through Gαq and PLCε. To explore the functional role of PLCε in ET-1/Gq dependent hypertrophy, activation of protein kinase D (PKD) in NRVMs was assessed in response to ET-1. PLCε-siRNA significantly inhibited ET-1 induced PKD activation (∼50% inhibition). Disruption of PLCε-mAKAP interactions also significantly inhibited ET-1 induced PKD activation (∼50% inhibition). We propose that PLCε scaffolded to mAKAP at the nuclear envelope responds to Gαq-dependent, as well as other hypertrophic signals, to locally regulate PKD in a process that is critical for hypertrophy development.
Abstract 309: Long-Term Therapy with Ivabradine Increases ACE-2 Activity in Left Ventricular Myocardium of Dogs with Chronic Heart Failure
Authors: Gupta Ramesh C , Wang Mengjun , Rastogi Sharad , Zhang Keife , Sabbah Hani N
Published: July 2021

Background:
Angiotensin converting enzyme 2 (ACE2) is a homolog of ACE. ACE2 removes the terminal amino-acid from angiotensin-II (Ang II) to form Ang(1-7) which antagonizes the effects of Ang-II by activating the Mas receptor. We showed that ivabradine (IVA), a novel inhibitor of the cardiac pacemaker current I
f
, improves left ventricular (LV) function and prevents progressive LV remodeling in dogs with microembolization-induced heart failure (HF). Because RAAS system overactivity contributes to the progression of HF, we assessed changes in Ang-II level and ACE and ACE2 activity in LV of dogs treated with IVA.

Methods:
Studies were performed in LV tissue from 24 HF dogs randomized to 3 months therapy with high dose (HD) IVA (30 mg twice daily, n=8), low dose (LD) IVA (15 mg twice daily, n=8) or to no therapy at all (Control, CON, n=8). LV tissue from 6 normal (NL) dogs was used for comparison. A fluorogenic peptide, captopril (ACE inhibitor) and DX600 (ACE2 inhibitor) were used to quantify the activity of ACE and ACE2 in LV homogenate. Activity was expressed as fluorescence unit (FU)/μ g protein/hour. Ang II level was determined by ELISA.

Results:
IVA reduced heart rate (HR) and significantly increased LV ejection fraction in a dose-dependent manner. Ang II level and ACE and ACE2 activities were up-regulated in HF CON compared to NL dogs. Compared to CON, treatment with IVA reduced ACE activity and Ang II level but increased ACE2 activity in a dose-dependent manner (Table).

Conclusions:
In dogs with HF, therapy with IVA reduces activation of ACE and Ang II level but increases activation of ACE2. The latter may contribute to the observed beneficial effects of IVA on LV function and remodeling in HF.
Abstract 312: Troponin I Phosphorylation Crosstalk
Authors: Biesiadecki Brandon J , Nixon Benjamin R , Little Sean C , Brundage Elizabeth A , Davis Jonathan P
Published: July 2021

The troponin (Tn) complex is a critical regulatory and integrative hub for myofilament post-translational modifications that regulate cardiac contraction. Beta-adrenergic-induced protein kinase A (PKA) phosphorylation of cardiac troponin I (cTnI) at Ser-23/24 is a major myofilament cardiac contractile modulator. In addition to this PKA-induced phosphorylation, cTnI can be simultaneously phosphorylated at a number of other residues. To fully understand the molecular mechanisms that underlie cardiac muscle regulation at the myofilament level, cTnI phosphorylations must be studied as integrated events. As an initial step towards understanding the integration of these events, we employed a phosphomimetic approach to investigate the effect of cTnI Ser-150 pseudo-phosphorylation (TnI-S150D) on cTnI PKA pseudo-phosphorylation (TnI-S23/24D) function within the thin filament. The combinatorial effect of these phosphorylation events were determined by measuring the binding of calcium to troponin C in thin filaments reconstituted with human Tn. The steady-state calcium sensitivity of TnC was increased ∼2.3 fold in thin filaments reconstituted with TnI-S150D containing Tn, while TnI-S23/24D containing filaments exhibited an ∼3.9 fold decrease of calcium sensitivity. Importantly, in the presence of both cTnI pseudo-phosphorylations (TnI-S23/24/150D) calcium binding was not different from that of wild-type. Likewise, calcium dissociation from thin filaments reconstituted with TnI-S23/24D was increased by ∼3.5 fold, while TnI-S150D containing filaments demonstrated a decrease in calcium dissociation of ∼2.3 fold compared to wild-type filaments. Interestingly the combined pseudo-phosphorylations of TnI-S23/24/150D only blunted TnI-23/24D calcium dissociation kinetics to ∼1.2 fold of the TnI-S23/24D filaments alone. These findings demonstrate the integrated effects of cTnI site-specific phosphorylation crosstalk to affect the function of cTnI PKA-induced phosphorylation. These data further highlight the importance of understanding the integrated role of Tn phosphorylation crosstalk on cardiac contractile regulation.
Abstract 325: Brain Natriuretic Peptide During Coronary Intervention Prevents Endothelial Dysfunction Post PCI via NP-cGMP Activation
Authors: Peleg Aviva , Hasin Yonathan
Published: July 2021

Background:
Contrast media (CM) administrated during percutaneous coronary intervention (PCI) is associated with endothelial dysfunction (ED) and systemic vascular injury. Brain natriuretic peptide (BNP) administration 24 hours post PCI decreases ED.

Aims:
To evaluate 1.The ability of human BNP (hBNP) infusion during PCI, to prevent ED in acute coronary syndrome's (ACS) patients post the PCI. 2. The effect of CM on human coronary microvascular endothelial cells (HCMEC).3. Explain ED by invitro study.

Methods and results (in vivo):
Non-ST elevation ACS patients who underwent PCI (111) were randomized into 2 groups: an hBNP group who received hBNP infusion during the procedure (n=44), and control group who received nitroglycerin (n=67). Flow mediated dilatation (FMD) (by ≥2.5%), BNP, corin, serum creatinine (sCr) and estimate Glomerular Filtration Rate (eGFR), before and 24 hr after operative were recorded, starting with the same baseline. The post PCI FMD and eGFR were significantly reduced in the control group (p=0.05, 0.002) but not in the hBNP group (p=0.16, 0.4). BNP, corin and sCr increased significantly in the control group (p=0.001, 0.003, 0.0002 respectively) but not in hBNP group (p=0.09, 0.07, 0.18).

Methods and results (in vitro):
HCMEC were treated with CM (10%) in the presence and absence of BNP. eNOS, corin and cGMP levels were measured by ELISA and the results were compared to untreated cells. In both treatments eNOS was significantly reduced (p=0.001) and corin was significantly increased (p=0.002). cGMP was not affected by CM treatment (p=0.278), but was increased significantly (p=0.001) by hBNP combination. cGMP immuno-flourescence staining of HCMEC showed distorted cellular cGMP appearance by CM treatment, that was corrected in the combination with hBNP with accentuated subsarcolemmal staining.

Conclusions:
CM reduces eNOS level in HCMEC. Therefore, reduced in NO-cGMP pathway's products, probably is the mechanism that induces ED in-vivo. BNP treatment reduces FMD diminution and kidney injury post PCI. A compensatory rise in corin that increases BNP as well as the hBNP administration, invivo and invitro, maintains cytosolic cGMP via NP-cGMP pathway, and compensates for NO-cGMP loss, (reduced sGC) and thus prevents ED.
Abstract 329: δ-Opioid Receptor Mediated Regulation of Cortical MicroRNAs in the Rat Under Hypoxia
Authors: Yang Yilin , Zhi Feng , He Xiaozhou , Moore Meredith L , Kang Xuezhi , Chao Dongman , Wang Rong , Kim Dong H , Xia Ying
Published: July 2021

Cardiovascular disorders may reduce blood/oxygen supply to the brain, causing serious neurological disabilities. Activation of the delta-opioid receptor (DOR) induces cortical protection against hypoxic/ischemic insults. However, the mechanism underlying DOR protection is largely unclear yet. We have recently found that DOR activation modulates microRNA expression in the kidney exposed to hypoxic stress, suggesting that DOR protection may involve a microRNA mechanism. To determine if the microRNAs expressed in the cortex mediate DOR neuroprotection, we examined nineteen microRNAs, that are previously identified as hypoxia- and DOR-regulated microRNAs in the kidney, in the cortex treated with DOR activation in hypoxic condition. Total RNA for qPCR was isolated from the cortex of control or hypoxic rats as well as those animals treated with UFP-512, a potent and specific DOR agonist. Of the 19 microRNAs tested, 17 were significantly altered by hypoxia and/or DOR activation with the direction and amplitude dependent on hypoxic duration and times of DOR treatment. Several microRNAs such as miR-29b, 101b, -298, 324-3p, -347 and 466b were significantly depressed by hypoxia for 24 hours. The similar changes were seen in normoxic condition 24 hours after DOR activation with one time treatment of UFP-512. In contrast, some miRNAs were more tolerant to hypoxic stress and did not show any significant reduction until 5-days (e.g., miR-31 and -186) or 10-days (e.g., miR-29a, let-7f and -511), while DOR activation had differential effects on these miRNAs. Moreover, some miRNAs like miRs-363* and -370 were affected only by the combination of hypoxia and DOR treatment, with reductions greater than 70% in 5-day group. These data suggest that cortical microRNAs are significantly altered in response to hypoxia with differential hypoxia sensitivity among them and that DOR activation is able to modify, enhance or resolve the changes in microRNAs that are predicted to target ion transport, HIF, axonal guidance, free radical signaling, apoptosis and many other functions.
Abstract 335: Murine Model of Chronic Right Ventricular Diastolic Heart Failure Is Associated with Energetic Alterations and Extracellular Matrix Remodeling
Authors: Reddy Sushma , Zhao Mingming , Hu Dong-Qing , Fajardo Giovanni , Katznelson Ethan , Spin Joshua , Chan Frandics P , Bernstein Daniel
Published: July 2021

Introduction:
Pulmonary insufficiency (PI) is a common long-term complication after repair of congenital heart diseases such as tetralogy of Fallot, causing progressive right ventricular (RV) dilation and eventual RV failure. We characterize the physiologic and molecular characteristics of the first murine model of RV volume overload

Methods:
PI was created by entrapping the pulmonary valve leaflets with 2 sutures. Echo, MRI, catheterization and exercise testing were compared to sham controls at 1, 3 and 6 mos. RNA from RV free wall was hybridized to Agilent whole-genome microarrays.

Results:
Data is presented as sham vs. PI at 1, 3 and 6 mos (*p<0.05). PI resulted in increased RV end-diastolic volume (5.6±1.4 vs. 8.7±2.5, 9.8±1.3, 10.5±1.1μ l*) and diastolic heart failure: early reversal of E/A ratio followed by pseudonormalization (1.04±0.07 vs. 0.87±0.15, 0.82±0.17 and 0.96±0.16*), elevated RV end-diastolic pressure (2.7±0.5 vs. 7.6±1.3, 6.9±1.6 and 7±1mmHg*) and decreased exercise capacity (30±2.3 vs. 26±0.8, 26±2 and 22±2.6min*). Systolic function (RV outflow tract shortening) was relatively preserved (49±6 vs. 39±4, 44±3 and 41±3%, p=NS). At 1 mo, 372 genes were downregulated, with metabolic pathways, including Complex I, III and V, and G protein-coupled receptor signaling being the most enriched. At 3 mos, 434 genes were upregulated featuring numerous collagen genes, while 307 were downregulated, including fatty acid transport and Ca2+ signaling. During this time course, pathways involving TGFβ signaling, extracellular matrix and p53 signaling transitioned from down- to up-regulated. Key hubs in upregulated gene networks were TGFβ1, thrombospondin1, VEGFa, TNFα and GSK3β while NFκB1 was a downregulated hub. RV Fibrosis increased over time (0 vs. 0.6±0.5, 5.9±2.9, 3.92±0.2%*).

Conclusion:
We describe a novel murine model of chronic RV volume overload which recapitulates many aspects of clinical disease including RV dilation, impaired relaxation and decreased exercise capacity but preserved RV systolic function. Gene expression changes suggest mitochondrial bioenergetic dysfunction, ECM remodeling, enhanced TGF-β signaling and apoptosis. Many of these processes of RV diastolic heart failure are also known mechanisms of LV failure.
Abstract 338: Neuronal Responses to Hypoxic Stress in Mouse Cognitive Cortex
Authors: Ding Guanghong , Liu Ruqing , Chao Dongman , Xia Ying
Published: July 2021

Cardiovascular disorders may cause hypoxic/ischemic injury to the cortex, leading to dysfunction of cortical cognition with a worse outcome in the older population. Although damage to cognitive cortex has been implicated in cognitive dysfunctions, the exact pathophysiology of a hypoxic/ischemic insult to this region is not well understood yet at the cellular and molecular levels. We used the whole-cell patch clamp in mouse slice preparations to characterize the basic electrophysiological properties of pyramidal cells of prefrontal cortex (PFC) and determine the response of these neurons to hypoxic stress in young adult (24±4 days old, n=7) and relatively older (42±12 days old, n=17) mice. In relatively older adult neurons (42±12 days old, n=17), the currents evoked by stepping from -140 mV to 20 mV (IV, holding at -60 mV) were shifted upward by hypoxic stress, indicating an increase in membrane conductance. Whole cell currents (holding at -60 mV) were found to decrease with a 3-min hypoxia, while increased with 5-min and 7-min hypoxia, suggesting that prolonged hypoxia increased open probability of ion channels. Resting membrane potential (MP) shifted upward in all three hypoxic conditions with a larger change seen with the longer duration of hypoxia. Action potentials (APs) showed the same tendency with 7-min hypoxia, showing a significant reduction in the number of hypoxia-stimulated pulses. However, all hypoxic conditions blocked the APs generation, suggesting hypoxic inhibition of neuronal excitability. In contrast, hypoxia-induced IV was apparently reversed in the younger neurons as compared to the older neurons, especially after 5 minutes of hypoxia (99.26 pA vs. -14.91 pA at -100 mV, P<0.01). Interestingly, even in normoxic condition, MP was significantly larger in young adult neurons (-74.38 mV) than in the older ones (-64.67 mV) (P<0.001). These data suggest that younger pyramidal cells of PFC are less vulnerable to hypoxic stress. Moreover, our preliminary data showed that activation of the delta-opioid receptors (DOR) inhibited the hypoxic response in the older neurons. Since acupuncture induces DOR up-regulation in the cortex, it is likely that acupuncture protects cortical neurons against hypoxic/ischemic stress via the DOR mechanism.
Abstract 34: Statins Directly Activate Erk5 and Reduce Endothelial Dysfunction and Acute Allograft Rejection
Authors: Takei Yuichiro , Le Nhat-Tu , Lee Hakjoo , Heo Kyung-Sun , Hurley Cheryl , Smrcka Alan V , Miller Benjamin , Ko Kyung Ae , Morrell Craing , Fujiwara Keigi , Akaike Masashi , Abe Jun-ichi
Published: July 2021

Rationale:
The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, are principal therapeutic agents for the treatment of hypercholesteremia. Statins, however, appear to also exert cholesterol-independent pleiotropic effects, such as improvement of endothelial (EC) function, stabilization of fibrous plaques, and decrease vascular inflammation. It is now well established that statins are beneficial in the prevention of cardiovascular disease and also widely used for suppressing cardiac allograft rejection. Previously, we have reported anti-inflammatory effect of ERK5 kinase in ECs.

Methods and Results:
In this study, we screened small molecules that activate ERK5 using high throughput screening, and identified statins as strong activators of the transcriptional activity of ERK5. In particular, we have found that pitavastatin increases ERK5 transcriptional activity, KLF2 promoter activity, and eNOS mRNA expression in ECs. These effects are abolished by the depletion of ERK5, but not its direct upstream kinase, MEK5. In addition, pitavastatin directly and dose-dependently activates ERK5 kinase activity in an in vitro kinase reaction assay, suggesting that ERK5 is a direct target of this statin. To examine the functional role of EC ERK5 activation by the statin in vivo, we utilized inducible endothelial ERK5 knock out (EC-ERK5-KO) mice and evaluated the effect of pitavastatin on EC function and acute allograft rejection. Depletion of ERK5 in ECs resulted in significant EC inflammation and dysfunction in vivo. Although pitavastatin reduced leukocyte rolling and vascular reactivity in mesebteruc microvessels of diabetic mice and prolonged allograft survival in a full allomismatch combination model, these protective effects were lost in EC-ERK5-KO mice.

Conclusion:
These data suggest the crucial role of ERK5 in pleiotropic effects of statins on EC dysfunction and allograft rejection in vivo.
Abstract 345: Tissue-Printed Cardiac Progenitor Cells Improve Myocardial Function in a Mouse Model of MI
Authors: Gaetani Roberto , Feyen Dries , Verhage Vera , Slaats Rolf , Messina Elisa , Giacomello Alessandro , Doevendans Pieter , Sluijter Joost
Published: July 2021

Introduction:
Tissue engineering is emerging as a therapeutic approach to overcome the limitations of cell therapy and mechanically support the ventricular wall. Tissue printing technology (TP) allows for the construction of a defined and organized tissue engineered construct. Hypothesis: The aim of our study was to evaluate the combination of TP and cardiac-derived cardiomyocyte progenitor cells (CMPCs) to build a cardiogenic construct.

Methods:
Sca-1
+
CMPCs were printed in gelatin/hyaluronic acid matrix (30x10
6
cells/ ml) to form a biocomplex made of 6 perpendicularly printed layers with a surface of 2cm x 2cm and a structure 500 µm thick. After
in vitro
characterization, the printed biocomplex was transplanted in a mouse model of MI. Cardiac function was evaluated by MRI.

Results:
After printing, CMPCs retain cardiogenic gene expression for up to 4 weeks, as evaluated by PCR (n=4). Immunofluorescence analysis confirmed expression of Nkx2.5 and Ki67 with a slight decrease after 7 days in culture (77%
vs
52% and 79%
vs
64% respectively, n=5, p<0.05). After differentiation, hCMPCs increased MLC2a and TnI gene levels (n=4).
In vivo
engrafted hCMPCs expressed Nkx 2.5. TnI expression was also detected after 2 weeks (n=3) and 28 days (n=5). CD31 expression was detected in low number of transplanted hCMPCs. Endogenous CD45 expression was similar at 7 and 14 days, mainly localized at the border of the matrix (n=3). At 28 days, a reduction in EDV and ESV dilation was observed, as compared to control (EDV:107,87 ± 7,41
vs
136,6 ±9,6 and ESV:89 ± 6,6
vs
118 ±12, respectively, n=8 each group, p<0.05), although no significant differences could be observed with matrix only (EDV;107,87 ± 7,41
vs
120,8 ±9,5 and ESV;89 ± 6,6
vs
97 ±10, respectively, n=7). Infarct wall thinning was reduced in both groups compare to the control (457 ± 69,8 vs 357,8 ±18,6 and 201,1± 6,3 μ m, respectively; n=5 each group, p<0.05).

Conclusion:
Here, we showed that hCMPCs can be printed and cultured in Gel/ HA. Transplantation in a mouse model of MI contributed to an improvement in cardiac function and a better remodeling upon injury. Long-term follow up to address its effect on infarct size, cardiomyocytes survival and regeneration, revascularization, endogenous CPCs activation, and inflammation is ongoing.
Abstract 349: Functional Scaffold-Free 3D Cardiac Microtissues: A Novel Model for the Investigation of Heart Cells
Authors: Desroches Bethany , Zhang Peng , Choi Bum-Rak , King Michelle E , Maldonado Angel E , Li Weiyan , Rago Adam , Liu GongXin , Nath Nandan , Hartmann Kathryn M , Yang Bryant , Koren Gideon , Morgan Jeffrey , Mende Ulrike
Published: July 2021

To bridge the gap between 2D cell culture and tissue, various 3D cell culture approaches have been developed for the investigation of cardiac myocytes (CM) and fibroblasts (CF), but limitations still exist. The goal of this study was to develop a scaffold-free cardiac culture that could easily and inexpensively create many uniform cardiac microtissues that mimic the cellular distribution and functional behavior of CM and CF in tissue. Using micromolded non-adhesive agarose hydrogels containing 822 concave recesses (800 λm deep x 400 λm wide), we demonstrate that neonatal rat ventricular CM and CF, when seeded alone or in combination, self-assembled into viable (Live/Dead® stain) spherical-shaped microtissues. Importantly, when CM and CF were seeded simultaneously or sequentially, they self-sorted to be highly interspersed, reminiscent of their distribution in ventricular tissue, as shown by cell type-specific CellTracker
TM
or antibody labeling. Depending on their cellular composition, microtissues expressed extracellular matrix and calcium handling proteins (including SERCA2a), featured typical
I
K1
and
I
Ca-L
current densities and IV relationships, and formed functional cell-cell connections (as evidenced by spontaneous action potentials, contractions and connexin 43 expression). Microelectrode recordings and optical mapping showed characteristic triangular action potentials (AP) with a resting membrane potential of -66±7 mV (n=4) in spontaneously contracting CM microtissues. Under pacing, optically mapped AP duration at 90% repolarization and conduction velocity were 100±30 ms and 18.0±1.9 cm/s, respectively (n=5 each). The presence of CF led to a prolongation of both AP and calcium transients in CM:CF microtissues (1:1). Furthermore, cell-type-specific adenoviral gene transfer was achieved, with no impact on microtissue formation or cell viability. In conclusion, we have developed a novel scaffold-free cardiac 3D culture model, in which more than 800 homotypic (CM or CF) or heterotypic (CM:CF) microtissues can be formed in a 6-well with ease, analyzed functionally and subjected to cell-type-specific gene transfer, paving the way for future
in vitro
investigations of CM and CF behavior and their interactions in a 3D environment.
Abstract 350: Early Cell-Cell Coupling Contributes to Loss of Transplanted Stem Cell Retention and Efficacy
Authors: Chatchavalvanich Santipongse , Geenen David L
Published: July 2021

Bone marrow-derived mesenchymal stem cell (BM-MSC) replacement therapy is beneficial to the heart following ischemia but a significant loss of these cells within hours of administration could diminish their effect. We hypothesized that early coupling between BM-MSC and ischemic cardiomyocytes through gap junctions (GJ) may play a detrimental role in stem cell survival and retention in the acute phase of cell therapy. We seeded HL-1 cardiomyocytes in either normoxic (Nx) or ischemic (Isc) conditions for four hours. Subsequently, BM-MSC were seeded on the HL-1 monolayer and the co-cultures were returned to incubation either in their previous conditions (Nx, Isc) or switched from Isc to Nx condition (ischemia-reperfusion; Isc/Rep) for an additional two hours. Co-cultures were labeled with Annexin V, Sytox Red, and Sca-1 (BM-MSC), and subjected to flow cytometry. Ischemia induced a greater proportion of dead BM-MSC over the two-hour co-culture compared to the Nx group. Isc/Rep resulted in significantly higher early apoptotic but fewer dead BM-MSC. The presence of the GJ inhibitor carbenoxolone (CBX; 100 µM) in the co-culture reduced the number of dead and apoptotic cells in Isc and Isc/Rep groups by 3-5 fold (p<0.05). To determine the effect of GJ inhibition in vivo, we induced ischemia in mice by 90-minute LAD ligation followed by reperfusion for 24 hours. BM-MSC, CBX-treated BM-MSC, or CBX alone were injected at the end of the 90 min Isc period. Twenty-four hours after cell injection, left ventricular diastolic and systolic function was assessed by pressure-volume loop analysis. Isc/Rep caused impaired cardiac function which was attenuated by BM-MSC injection. CBX-treated BM-MSC further enhanced the cardiac function (MSC vs. MSC+CBX: Ees 7.3 ± 1.66 vs. 15.0 ± 5.81; Emax 18.8 ± 6.50 vs. 27.5 ± 9.33; PRSW 49.5 ± 9.89 vs. 99.4 ± 17.4; mean ± SD; p≤0.05; n = 6) while CBX alone did not. While long term integration of stem cells within the myocardium relies on functional GJ, early GJ communication may represent a novel paradigm whereby ischemic cardiomyocytes cause a “bystander effect” in newly transplanted stem cells and thus impair retention and functional benefits.
Abstract 360: Development of a Human Tissue-Engineered Blood Vessel from Adipose-Derived Stem Cells
Authors: Brennan Jaclyn A , Arrizabalaga Julien H , Nollert Matthias U
Published: July 2021

There exists a clear need for alternative sources of small-diameter vascular grafts for treating the millions of patients who suffer from cardiovascular disease each year. Bypass surgery or replacement of defective vessels is often required to treat coronary heart disease, but there is a limited supply of suitable autologous grafts, and synthetic grafts are ineffective for replacement of small-diameter vessels. Inherent thrombogenicity, compliance mismatch, and limited patency rates are all complications with current options. Tissue engineering has the potential to overcome these limitations by producing a readily-available vascular graft completely from biological material. It is the objective of this study to fabricate such a small-diameter tissue engineered blood vessel (TEBV) by using the human amniotic membrane as a mechanically-sound biological substrate. Our technology begins by differentiating adipose-derived stem cells into smooth muscle cells (SMCs) and seeding them onto a flat sheet of the amniotic membrane. We assessed our hypothesis that several types of SMCs can successfully attach and proliferate on this membrane by fluorescently staining cell nuclei with DAPI and characteristic SMC actin filaments with phallotoxins. After 7 days in static culture, the cell-seeded sheet was wrapped around a 3mm O.D. removable mandrel with 6-7 revolutions to develop a tubular construct with architecture akin to that of a muscular artery’s tunica media layer. After a 2 week static culture period, the TEBV was characterized for its biochemical and mechanical properties. We examined the contraction of the vessel in response to carbachol, a specific agonist for SMCs, and compared our results with the contraction of porcine coronary arteries. Burst pressure and elastic modulus tests were also performed. The mechanical integrity of this construct can be further improved upon its exposure to appropriate physiological conditions in a perfusion bioreactor. We show that adipose-derived endothelial cells (ECs) can be also be seeded into the lumen of this construct to prevent platelet adhesion. In conclusion, we have developed a small-diameter TEBV with off-the-shelf availability using a completely biological material seeded with patient-own stem cells.
Abstract 366: Engineered Human Cardiac Tissue from Skeletal Muscle Derived Cells and Induced Pluripotent Stem Cell Derived Cardiomyocytes
Authors: Tobita Kimimasa , Tchao Jason S , Kim Jong , Lin Bo , Huard Johnny , Salama Guy , Yang Lei
Published: July 2021

We have previously shown that rat skeletal muscle derived stem cells differentiate into an immature cardiomyocyte (CM) phenotype within a 3-dimensional collagen gel engineered cardiac tissue (ECT). Here, we investigated whether human skeletal muscle derived progenitor cells (skMDCs) can differentiate into a CM phenotype within ECT similar to rat skeletal muscle stem cells and compared the human skMDC-ECT properties with ECT from human induced pluripotent stem cell (iPSc) derived CMs. SkMDCs differentiated into a cardiac muscle phenotype within ECT and exhibited spontaneous beating activity as early as culture day 4 and maintained their activity for more than 2 weeks. SkMDC-ECTs stained positive for cardiac specific troponin-T and troponin-I, and were co-localized with fast skeletal muscle myosin heavy chain (sk-fMHC) with a striated muscle pattern similar to fetal myocardium. The iPS-CM-ECTs maintained spontaneous beating activity for more than 2 weeks from ECT construction. iPS-CM stained positive for both cardiac troponin-T and troponin-I, and were also co-localized with sk-fMHC while the striated expression pattern of sk-fMHC was lost similar to post-natal immature myocardium. Connexin-43 protein was expressed in both engineered tissue types, and the expression pattern was similar to immature myocardium. The skMDC-ECT significantly upregulated expression of cardiac-specific genes compared to conventional 2D culture. SkMDC-ECT displayed cardiac muscle like intracellular calcium ion transients. The contractile force measurements demonstrated functional properties of fetal type myocardium in both ECTs. Our results suggest that engineered human cardiac tissue from skeletal muscle progenitor cells mimics developing fetal myocardium while the engineered cardiac tissue from inducible pluripotent stem cell-derived cardiomyocytes mimics post-natal immature myocardium.
Abstract 38: A Decoy Peptide Selectively Inhibiting Dephosphorylation of Phospholamban
Authors: Park Woo Jin , Oh Jae Gyun , Jeong Dongtak , Hajjar Roger J
Published: July 2021

Cardiac sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) plays a crucial role in Ca2+ handling in cardiomyocytes. Phospholamban (PLB) is an endogenous inhibitor of SERCA2a and its inhibitory activity is enhanced by dephosphorylation by protein phosphatase 1 (PP1). Therefore, blocking PP1-mediated dephosphorylation of PLB would be an efficient strategy for restoration of the reduced SERCA2a activity in failing hearts. We sought to develop a decoy peptide that mimics the phosphorylated PLB and thus competitively inhibits the PP1-mediated dephosphorylation of PLB. The phosphorylation sites, Ser16 and Thr17, are located within the flexible extra-membrane loop (amino acids 14-22) of PLB. We therefore synthesized a 9-mer pseudo-phosphorylated peptide derived from this region with a replacement of Ser16 with Glu (ψ-PLB-SE). Two other 9-mer peptides with wild type PLB sequence (ψ-PLB) or with a replacement of Ser16 with Ala (ψ-PLB-SA) were also synthesized. These peptides were coupled to a cell-permeable peptide TAT to facilitate cellular uptake. Treatment of adult rat cardiomyocytes with TAT-ψ-PLB-SE, but not with TAT-ψ-PLB or TAT-ψ-PLB-SA, significantly elevated the phosphorylation level of PLB, concomitant with an increase in contractile parameters in vitro. Perfusion of isolated rat hearts with TAT-ψ-PLB-SE significantly restored the left ventricular developed pressure that was suppressed by ischemia-reperfusion (Fig. 1). These data indicate that ψ-PLB-SE prevented dephosphorylation of PLB by acting as a decoy for PP1 and it would provide effective modality to regulate SERCA2a activity in failing hearts.