bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 279: Inositol Triphosphate Receptor Activation Alters the Electrical Properties of Human Ventricular Myocytes
Authors: Signore Sergio , Ferreira-Martins João , Isobe Kazuya , Kannappan Ramaswamy , Sorrentino Andrea , Shafaie Mehrdad , Steadman Elisabeth , Goihberg Polina , Arranto Christian , Sanada Fumihiro , Del Ben Fabio , Cappetta Donato , Wybieralska Ewa , Ogorek Barbara , Del Monte Federica , DAlessandro David A , Michler Robert E , Rota Marcello
Published: July 2021

The recognition that ryanodine receptor (RyR) dysfunction is associated to arrhythmogenic diseases raises the possibility that alternative Ca2+ release channels alter the electrical properties of ventricular myocytes. The aim of this study was to determine whether inositol triphosphate receptor (IP3R)-mediated Ca2+ mobilization modulates Ca2+ cycling and electrical behavior in LV myocytes. Cells were obtained from human and mouse hearts and the expression and function of IP3Rs were evaluated. IP3Rs were identified in isolated myocytes by immunocytochemistry and Western blotting. In field-stimulated cells, IP3R activation via Gq-protein receptor agonists (ET-1, ATP) or enhancer of ligand affinity (thimerosal) increased diastolic Ca2+ and transient amplitude by 11% and 44%, respectively. Additionally, extra-systolic Ca2+ release and sustained Ca2+ elevations were detected. These effects were prevented by inhibition of IP3 production or by IP3R blockade. Importantly, myocytes obtained from mice infected in vivo with small hairpin RNA (shRNA) targeting IP3R type 2, failed to respond to Gq-protein receptor agonists. In patch-clamped human and mouse cells, changes in Ca2+ transient properties following IP3R activation were accompanied by a decrease in resting potential, action potential (AP) prolongation, and emergence of arrhythmic events. In mouse cardiomyocytes, assessment of excitation-contraction coupling gain and blockade of RyR channels under IP3R stimulation, excluded the contribution of RyRs to the effects induced by IP3R activation. In voltage-clamped cells, IP3R agonists promoted transient inward currents at the membrane potential of -70 mV and increased a nickel-sensitive current in the range of potentials corresponding to forward mode operation of the Na-Ca exchanger. To establish whether enhanced Ca2+ load was responsible for the altered electrical properties under IP3R activation, conditions buffering cytosolic Ca2+ levels were employed. Under these circumstances, IPR stimulation failed to prolong the AP and to induce arrhythmias. In conclusions, our observations demonstrate that Ca2+ mobilization via IP3Rs directly alters the electrical properties of human and rodent myocytes.
Abstract 290: Circulating miRNAs as Biomarkers in Acute Coronary Syndrome
Authors: Cordeddu Lina , Pilbrow Anna P , Cameron Vicky A , Troughton Richard W , Richards Mark A , Foo Roger S
Published: July 2021

Introduction
Acute coronary syndrome is characterized by the active, inflamed and unstable atherosclerotic plaque that is vulnerable to rupture, predisposing to lumen occlusion and varying extents of myocardial injury. Plasma microRNA have been examined in the hope of identifying an easily accessible biomarker for acute coronary syndrome. But so far studies have taken a candidate approach and screened only for miRNA that are potentially released from the injured myocardium. Even so, data from these studies are inconsistent and a consensus is yet to be reached.

Aim
We set out to screen for circulating plasma microRNAs to serve as biomarkers for patients with Acute Coronary Syndrome.

Methods and results
We selected patients who suffered from 2 ends of the severity spectrum of acute coronary syndrome vs age-matched healthy controls. These were 20 μsevere” (STEMI: troponin-positive with subsequent rapid deterioration in left ventricular function or death within 5 years), 20 μmild” (unstable angina: troponin-negative with sustained normal left ventricular function and survival at 5 years), and 20 normal healthy volunteers. Blood samples were obtained from patients within 2 weeks of the acute event. We took a non-constrained approach and screened using an array panel consisting of 379 miRNA. We found 32 miRNA that were significantly upregulated (29/32) or downregulated (3/32) in the comparison between patient vs. control (sum of t-statistic>2). We have made a preliminary analysis of these in relation to a full panel of other classical biomarkers and patient clinical details. We selected 4 candidate microRNAs (miR-27b, -103, -323-3p, -652) and proceeded to test the plasma levels of these in a validation cohort of 100 troponin-positive, 100 troponin-negative patients and 100 normal healthy volunteers. miR 27b, -323-3p, -652 were significantly upregulated in disease and hence robustly validated.

Conclusion
miR 27b, -323-3p, -652 strongly associates with the event of acute coronary syndrome. Further work will be required to determine the origin and physiological function of these candidate miRNA, and whether their plasma levels can be used for prognostication purposes.
Abstract 291: RAS Fingerprinting: Patient-Specific Evaluation of the Renin-Angiotensin-System in Plasma by LC-MS/MS-Based Multiplex Quantification of Angiotensin Peptides
Authors: Poglitsch Marko , Schwager Cornelia , Schuster Manfred , Loibner Hans
Published: July 2021

The Renin-Angiotensin-System (RAS) is critically involved in the regulation of important physiological functions including blood pressure and fluid balance. It is constituted by multiple enzymes giving rise to a meshwork of effector peptides, which mediate their functions through binding to specific receptor molecules. Therefore, the modification of angiotensin peptide levels represents a common strategy for the treatment of RAS-associated diseases and is frequently achieved by the pharmacologic regulation of enzymes taking part in angiotensin metabolism. We developed a highly sensitive method, which allows the simultaneous absolute quantification of up to 10 different angiotensin peptides in human plasma and whole blood (RAS-Fingerprinting). Either the measurement of in vivo RAS-Fingerprints (immediate sample stabilization) or heparin derived ex vivo RAS-Fingerprints in plasma or whole blood, provide unique insights into the biochemical constitution of the human RAS and therefore represent powerful tools for the patient specific evaluation of this physiologically important peptide hormone system. During the development and validation of a new LC-MS/MS based method for angiotensin quantification, comprehensive datasets for various applications of RAS-Fingerprinting have been generated. RAS-Fingerprints were measured in blood of healthy volunteers during the treatment with different RAS-blockers, which provided deep insights in the physiology and regulation of the human RAS and revealed surprising differences between individual patients. With the help of RAS-Fingerprinting, we were able to re-draw the picture of the human RAS at a previously unachieved level of detail and accuracy. Due to the availability of new selection criteria for pharmacologic screens, RAS-Fingerprinting could contribute to the development of innovative therapeutic approaches affecting the RAS. The extensive utilization of RAS-Fingerprinting in clinical studies will substantially enhance our understanding of the human RAS and could lead to the development of personalized treatment schemes in the management of RAS-associated diseases in the near future.
Abstract 3: Targeted Inhibition Of Transforming Growth Factor Beta In Cardiac Myocytes Prevents Myocardial Rupture After Infarction
Authors: Rainer Peter P , Koitabashi Norimichi , Hao Scarlett , Bedja Djahida , Kass David A
Published: July 2021

Introduction
Coronary artery disease is the leading cause of death and myocardial infarction (MI) its most severe manifestation.TGFb is a major switch governing the myocardial response to injury and global inhibition is detrimental following MI. Recently, cell-specific gene manipulation models have enabled investigation of cardiomyocyte (CM) versus non-CM TGFb signaling on maladaptive remodeling. We found CM TGFb to be central to pressure-overload disease. Here, we tested the hypothesis that CM TGFb modifies acute post-MI remodeling, and thus benefits rather than worsens outcome as observed with antibody-based inhibition that primarily targets interstitial/vascular cells.

Methods
Mice with cardiac-specific conditional knockdown of TgfbR1 or 2 (aMHC - MCM TgfbR fl/fl) or injection of TGFb-neutralizing antibody were subjected to proximal LAD-ligation and followed by echocardiography and continuous ECG monitoring. Expression and activity of the major matrix metalloproteinases MMP2/MMP9 and TIMP1 were assessed by qRT-PCR, immunoblot, and zymography, respectively. Only mice surviving >24h after surgery (>90%) were included in analyses.

Results
LAD ligation decreased fractional shortening on day 1 to 18±2% (61±1% in sham animals) with no differences between genotypes (n≥8). Similarly, echocardiographic parameters of myocardial dimensions and function did not differ at weeks 1 or 3. Early mortality (day 4 - 10) was strikingly lower in knockdown mice as compared to controls (10% vs 48%, p<0.05, n≥10). Remarkably, systemic TGFb inhibition resulted in 100% mortality (n=8) by day 5. The cause of death was established by necropsy and continuous ECG monitoring and was myocardial rupture in all cases. MMP9 expression and gelatinolytic activity was reduced by CM TGFbR knockdown at day 3 after MI, as was TIMP1 expression, while MMP2 expression/activity was not altered.

Conclusion
TGFb inhibition exhibits differential effects in wound healing following MI. Where global inhibition is deleterious, CM-targeted knockdown powerfully protects the heart from rupture. This highlights potent cross-talk between the CM TGFb signal and matrix remodeling and the importance of targeted inhibition to benefit cardiac disease.
Abstract 31: Regulation of Acetylation Restores Proteolytic Function in Diseased Myocardium
Authors: Wang Ding , Fang Caiyun , Zong Nobel , Kim Tae-Young , Liem David , Deng Mario , Ping Peipei
Published: July 2021

RATIONALE:
Proteasome complexes play essential roles in maintaining cardiac protein homeostasis in normal and stressed conditions. However, proteasomal function is often compromised in diseased myocardium; and the regulation of proteasomal dynamics remains poorly understood.

METHODS AND RESULTS:
We studied cardiac proteolytic function and its regulation by acetylation in murine and human models of heart disease. Proteasomes, from both normal and diseased myocardium, were treated with histone de-acetylases (HDACs) inhibitors and they exhibited enhanced proteolytic capacity in vitro and in vivo. This unique regulatory paradigm was first examined in a murine model of ischemic injury (n=5); where diminished proteolytic function in the ischemic hearts was restored by HDACs inhibition (either by SAHA or by Sodium Valproate) in a dose-dependent fashion. Importantly, this phenomenon was validated in human samples, where inhibition of HDACs augmented proteolytic functions in the diseased myocardium (n=5). Using high resolution LC-MS/MS coupled with a combined collision-induced dissociation and electron-transfer dissociation approach, the acetylome (N-terminal and lysine) of cardiac 20S proteasomes was delineated. Targeted enrichment strategy of the posttranslationally modified peptides enabled the capture of eight lysine and nine N-terminal acetylation sites in the murine heart, contributing to the first comprehensive acetylome map for the cardiac 20S proteasomes. Among them, at least six lysine acetylation sites were inducible by HDACs inhibitors. Furthermore, parallel investigations using cardiac 20S proteasomes pinpointed the functional impact of HDAC inhibitions to specific acetylation sites on the 20S proteasomal subunits.

CONCLUSIONS:
Proteasomal biology is modulated by acetylation modifications in the heart. This regulatory mechanism is punctual and potent; and it was observed both in an acutely pathological murine model of ischemia-reperfusion injury as well as in a chronic human disease of end-stage heart failure. These findings demonstrate the utility of pharmacological interventions (e.g., HDAC inhibition) to restore damaged proteolytic function in diseased myocardium.
Abstract 310: Inhibition of Class I Histone Deacetylase Activity Improves Left Ventricular Contractile Function and Cardiac Strain in Coronary Artery Occlusion-Induced Myocardial Infarction
Authors: Kasiganesan Harinath , Renaud Ludivine , Mani Santhosh K , James Chou C , Mukherjee Rupak , Menick Donald R
Published: July 2021

Protection from coronary heart disease-induced damage of the myocardium during myocardial infarction (MI) injury has been a target of investigation for the development of innovative cardioprotective therapies. Histone deacetylases (HDACs) are a class of enzymes that affect the transcriptional regulation of genes during pathological conditions. We observed that class I/IIb HDAC activity was nearly 5 times greater in the 7-day post-MI LV when compared to the sham ventricles.
In vitro
inhibition studies indicated that the majority of increased activity was due to class I HDACs. Therefore we hypothesized that suppression of class I HDACs would prevent the pathophysiological changes occurring during MI, thus improving LV pump function in post-MI myocardium. CD-1 mice were administered with the a class I HDAC inhibitor pimelic diphenylamide (PD106) or vehicle immediately after induction of MI and the treatment continued every other day for 7 days post MI. LV end-diastolic volumes, expressed as change from pre-MI values, was significantly lower in the PD106 treated mice compared to vehicle treated mice. Further, the post-MI reduction in LV ejection faction was significantly attenuated in the PD106-treated mice compared to the MI alone group. Similarly, echo cardiac strain analysis showed improved LV strain and better coherence in contractile function among infarct and border zones in PD106 MI group compared to MI only. These unique findings demonstrate that class I HDAC inhibitors may provide a novel therapeutic means to attenuate adverse post-MI LV remodeling.
Abstract 314: Infarct Size Reduction by Statins Requires Parkin and Mitophagy
Authors: Andres Allen M , Lee Pamela , Hernandez Genaro , Huang Chengqun , Ratliff Eric P , Thornton Christine A , Gottlieb Roberta A
Published: July 2021

The cardioprotective effects of statins are well known yet the mechanism is unclear. Previously we showed that autophagy is required for cardioprotection from ischemia/reperfusion injury. More recently, we reported that ischemic preconditioning involves Parkin-mediated mitophagy. We hypothesized that the molecular basis of statin-mediated cardioprotection may involve mitochondrial quality control through mitophagy. HL-1 cardiomyocytes treated with simvastatin for 24hr exhibited diminished Akt/mTOR signaling, increased activation of ULK1, and upregulation of autophagy (n=3, p<0.05). Similar findings were obtained in cardiac tissue in mice 4hr after i.p. administration of simvastatin. Mevalonate addition abolished statin’s effects on Akt/mTOR signaling and autophagy induction in HL-1 cells, indicating that the effects are mediated through inhibition of HMG-CoA reductase. Statin treatment in HL-1 cells triggered mitochondrial fragmentation, translocation of Parkin and p62/SQSTM1 to the mitochondria followed by mitophagy. To establish the requirement for statin-mediated mitophagy in cardioprotection, we investigated the ability of statins to reduce infarct size in Parkin knockout (KO) mice. While statin treatment reduced infarct size from 55% of area at risk to 30% in wild type mice, it had no protective benefit in Parkin KO mice (n=4-6, p<0.05). These findings indicate that cardioprotection by HMG-CoA reductase inhibitors involves suppression of mTOR signaling and induction of Parkin-dependent mitophagy.

Figure: Statin-induced cardioprotection against I/R injury: solid bars/diamonds = wild-type; open bars/diamonds = Parkin knockout mice.
Abstract 324: δ-Opioid Receptor Activation Modifies Hypoxic Expression of MicroRNAs in the Rat Kidney
Authors: He Xiaozhou , Yang Yilin , Zhi Feng , Moore Meredith L , Kang Xuezhi , Chao Dongman , Wang Rong , Kim Dong H , Xia Ying
Published: July 2021

Cardiovascular dysfunction often causes blood/oxygen insufficiency in the kidney that is very sensitive to changes in oxygen/blood delivery. Indeed, hypoxic/ischemic kidney stress is a frequent problem in clinical settings. There is, however, no promising strategy for prevention and treatment of such injury. Since recent studies suggest that microRNAs are differentially involved in hypoxic/ischemic events and delta-opioid receptor (DOR) activation increases antioxidant capacity and protects against hypoxic/ischemic injury, we asked if DOR activation regulates microRNA expression in the kidney under hypoxic condition. We isolated microRNAs from normoxic and hypoxic rat kidneys and used the paired microRNAs for microarray analysis. Thirty-one microRNAs were selected for quantitative PCR analysis based on the microarray data. Among them, 14 microRNAs were significantly altered in response to prolonged hypoxia for 1, 5 and 10 days, DOR activation with UFP-512 (1 mg/kg/day, ip, at days 0, 4 and 8) or a combination of both. Our novel data show that 1) DOR activation shifts miRNA expression profiles in normoxic conditions; 2) hypoxia differentially alters the miRNA expression profiles depending on hypoxic durations; and 3) DOR activation modifies hypoxia-induced changes in miRNA expression. For example, 10-day hypoxia reduced the level of miR-212 by >70% (p<0.001), while DOR activation could mimic such reduction even in normoxic kidney. In contrast, the same stress increased miR-29a by >100% (p<0.05), which could be attenuated by DOR activation. These results suggest that hypoxia comprehensively modifies the microRNA profile within the kidney, which can be mimicked or modified by DOR activation. Illumination of their targeted pathways, that regulate hypoxia sensitive transcription factors, antioxidant capacity, erythropoietin, Golgi-trafficking regulator, neurexophilin, ubiquitin-mediated degradation, intracellular trafficking, scaffolding, Wnt signaling, solute channels and ion homeostasis, may provide new insights into the potential therapeutics for hypoxic/ischemic injury of the kidney.
Abstract 333: Chemokine and Autophagy-Related Genes in Novel In Vivo and In Vitro Models for Viral Myocarditis
Authors: Kawai Eiichiro , Omura Seiichi , Sato Fumitaka , Martinez Nicholas E , Chaitanya Ganta V , Claycomb William C , Alexander J S , Tsunoda Ikuo
Published: July 2021

Picornavirus infection is one of leading causes of myocarditis in humans. Theiler's murine encephalomyelitis virus (TMEV) belongs to the genus
Cardiovirus
, the family
Picornaviridae
. While TMEV has been used as a mouse model for multiple sclerosis (MS), TMEV can also cause myocarditis in mice. We found that the susceptibilities of mouse strains to myocarditis differed from susceptibilities to the MS model. In the heart, we detected viral replication and inflammation. However, it is unclear whether virus itself or immune cells damaged the heart. To clarify the role of direct virus infection in myocarditis we used the cardiomyocyte cell line, HL-1, which retains a differentiated cardiomyocyte phenotype and contractile activity. We infected HL-1 with TMEV and monitored cytopathic effect (CPE) and cell viability. At 8 hours post infection (hpi), HL-1 cells developed CPE with decreased contractile activities, while cell death was not obvious. We detected 54% and 93% of cell death at 12 and 24 hpi, respectively, while no cell death was observed in mock-infection. We conducted microarray analyses to compare mRNA expression patterns between TMEV and mock-infected groups, using an Affymetrix GeneChip Mouse 1.0 ST Array. Principal component analysis (PCA) for microarray data clearly separated TMEV- and mock-infected groups (PC1 proportion of variance, 59%). TMEV infection induced high expression of several interferon-associated and chemokine genes, particularly
Cxcl10/IP-10
. Cxcl10 is a potent chemoattractant for activated T cells and NK cells, and its potential roles have been associated with immune cells in other myocarditis models. Our current results demonstrated that virus infection alone could induce Cxcl10 in cardiomyocytes without immune cells. We also found upregulation of autophagy-related 12 gene (
Atg12
). In general, autophagy can contribute to eradication of intracellular pathogens including viruses. However, since induction of autophagy by picornavirus has been shown to enhance virus replication, autophagy may also play a detrimental role in TMEV-induced myocarditis. Our novel
in viv
o and
in vitro
models of myocarditis are powerful tools to dissect the role of viral and immune pathogenesis of the heart, complementing each other.
Abstract 336: TAB1 Initiates p38α Autophosphorylation During Myocardial Ischemia
Authors: Bassi Rekha , Arabacilar Pelin , Marber Mike
Published: July 2021

The p38 mitogen-activated protein kinase (MAPK) family has been implicated in many aspects of cell biology including inflammation, gene expression, cell cycle progression, differentiation and apoptosis. The canonical pathway of p38 activation results in dual phosphorylation of p38 induced via TGF-beta activated kinase 1 (TAK1) which is an upstream Ser/Thr kinase of the MAPK kinases (MKK) of p38; MKK3/6. However, a MKK-independent mechanism of activation is proposed as TAK 1 binding protein 1 (TAB1) appears to induce autophosphorylation of p38 through a direct interaction during myocardial ischaemia, worsening injury. When human embryonic kidney cells are transfected with wild-type TAB1, there is a marked increase in activating dual phosphorylation of p38 compared to untransfected cells (5.3 ± 1.9 normalised to untransfected). The ATP-competitive p38 inhibitor SB203580 (1µM) markedly reduced the phosphorylation of transfected p38 (20.4 ± 9.7 vs 5.4 ± 1.5). An SB203580-resistant form of p38 (T106M) was also co-transfected with TAB1, and the resulting p38 phosphorylation was no longer reduced by the presence of SB203580 (12.4 ± 7.5 vs 12.0 ± 6.7) but was reduced by another pharmacological inhibitor, BIRB796 (10µM) that does not depend on T106 for binding (5.6 ± 1.1 vs 1.6 ± 0.4, P<0.05). In conclusion, these findings suggest that p38 activation by autophosphorylation is TAB1-mediated.
Abstract 340: From Stem Cells to Cardiomyocytes: HDAC1 Induces Cardiovascular Differentiation by Regulating SOX17-BMP2 Expression in Early Development.
Authors: Hoxha Eneda , Lambers Erin , Ramirez Veronica , Krishnamurthy Prasanna , Verma Suresh , Thal Melissa , Kishore Raj
Published: July 2021

Cardiomyocytes derived from embryonic and induced pluripotent stem cells (ES/iPS) provide an excellent source for cell replacement therapies following myocardial ischemia. However, some of the obstacles in the realization of the full potential of iPS/ES cells arise from incomplete and poorly understood molecular mechanisms and epigenetic modifications that govern their cardiovascular specific differentiation. We identified Histone Deacetylase 1 (HDAC1) as a crucial regulator in early differentiation of mES and iPS cells. We propose a novel pathway in which HDAC1 regulates cardiovascular differentiation by regulating SOX17 which in turn regulates BMP2 signaling in differentiating pluripotent cells.

Utilizing stable HDAC1 knock-down (HDAC1-KD) cell lines, we report an essential role for HDAC1 in deacetylating regulatory regions of pluripotency-associated genes during early cardiovascular differentiation. HDAC1-KD cells show severely repressed cardiomyocyte differentiation potential. We propose a novel HDAC1-BMP2-SOX17 dependent pathway through which deacetylation of pluripotency associated genes leads to their suppression and allows for early cardiovascular-associated genes to be expressed and differentiation to occur. Furthermore, we show that HDAC1 affects DNA methylation both during pluripotency and differentiation and plays a crucial, non-redundant role in cardiovascular specific differentiation and cardiomyocyte maturation.

Our data elucidates important differences between ES and iPS HDAC1-KD cells that affect their ability to differentiate into cardiovascular lineages. As varying levels of chromatin modifying enzymes are likely to exist in patient derived iPS cells, understanding the molecular circuitry of these enzymes in ES and iPS cells is critical for their potential therapeutic applications in regenerative medicine. Further research in the molecular mechanisms involved in this process will greatly aid our understanding of the epigenetic circuitry of pluripotency and differentiation in pluripotent cells.
Abstract 343: Inactivation of the P2Y2 Receptor Gene Reduces Atherosclerosis
Authors: Seye Cheikh , Agca Yucksel , Qian Shaomin
Published: July 2021

Rationale:
Nucleotides acting via P2Y
2
nucleotide receptors (P2Y
2
R) regulate both migration of vascular smooth muscle cells and endothelial recruitment of monocyte/macrophage in vivo.

Objective:
We aimed to determine whether inactivation of the P2Y
2
R gene protects against neointima hyperplasia and atherosclerosis.

Method and results:
We performed femoral artery cuff injury in wild-type and P2Y
2
R
-/-
mice and examined neointima formation 14 days after injury, the time corresponding to peak SMC accumulation in this model. Wild-type mice exhibited a 190% increase in cellular proliferation (
P
<0.001) in the arterial wall as assessed by BrdU incorporation, and a 150% increase in neointima formation (
P
=0.010) compared to wild-type mice. There was no significant difference in the medial area between wild type and P2Y
2
R
-/-
mice. To assess the contribution of P2Y
2
R to atherosclerosis, we first generated P2Y
2
R
-/-
/ApoE
-/-
mice by mating ApoE
-/-
mice with P2Y
2
R
-/-
mice. We then examined the development of atherosclerotic lesions 30 weeks after mice were fed with standard chow diet, the time corresponding to widespread atherosclerotic lesions in the ApoE
-/-
mouse model. P2Y
2
R
-/-
/ApoE
-/-
and

ApoE
-/-
exhibited no differences

in blood pressure, number of circulating leukocytes, differential

counts, or total blood cholesterol. The surface covered by atherosclerotic

lesions in the entire aorta was significantly reduced in P2Y
2
R
-/-
/ApoE
-/-
mice as compared to ApoE
-/-
. The total intimal lesion area in the aortic sinus was significantly

less in P2Y
2
R
-/-
/ApoE
-/-
mice than in ApoE
-/-
mice. Mac-3 staining revealed a significant reduction in the percentage of total plaque area occupied

by macrophages in P2Y
2
R
-/-
/ApoE
-/-
mice compared with Apo
E
-/-
mice.

Conclusion:
These findings suggest that unraveling the molecular mechanisms associated with P2Y
2
R may lead to potential drug targets for the treatment of coronary syndromes and percutaneous coronary interventions.
Abstract 344: Naturally Derived Cardiac Extracellular Matrix for Cardiac Progenitor Cell Therapy
Authors: French Kristin M , Boopathy Archana V , DeQuach Jessica A , Chingozha Loice , Christman Karen L , Lu Hang , Davis Michael E
Published: July 2021

Cardiovascular disease is the leading cause of death in the United States. Following acute myocardial infarction, there is dramatic myocyte loss, and the cells are replaced with a collagen scar. Cell therapy directed toward regenerating infarcted myocardium suffers from poor retention of cells, possibly because the conditions are not ideal to support injected cells. Many studies show the local microenvironment is a potent regulator of cell fate. Therefore, we hypothesize that culturing cardiac progenitor cells (CPCs) on a naturally derived, cardiac extracellular matrix (cECM) will increase their function. CPCs were isolated from rat cardiac tissue-homogenate with magnetic beads conjugated with c-kit antibodies. Porcine, cardiac extracellular matrix (cECM) was decellularized, digested, and lyophillized to be used as a coating material. CPCs were cultured on cECM and examined for cardiogenic lineage markers, survival, proliferation and adherence. Our results show that CPCs cultured on cECM proliferate better (2.9-fold) as compared to COL (2.3-fold; p<0.05). Additionally, CPCs demonstrate improved resistance to serum-starvation-induced apoptosis when cultured on cECM (40% ± 14%) as compared to COL (53% ± 14%; p<0.001). Furthermore, CPCs adhered more strongly to cECM than COL as measured using microfluidics. CPCs showed enhanced progression toward the cardiomyoctye lineage when cultured on cECM (nkx2.5: 2.3 ± 0.4-fold; α-myosin heavy chain: 14.6 ± 4.4-fold; troponinC: 2.4 ± 0.2-fold; p-values <0.05) as compared to COL. This was confirmed at the protein level with Gata-4 (1.8 ± 0.1-fold; p<0.001) and Nkx2.5 (1.6 ± 0.2-fold; p<0.05). A reduction in fibroblast specific protein-1 (0.5 ± 0.1-fold) was also seen on cECM as compared to COL and suggests the CPCs are less likely to contribute to scar formation. While these conditioned cells have not yet been examined in an in vivo setting, our comparison to COL mimics both the naked injection of these cells into an infarct zone, and comparison to a commonly used cell delivery vehicle. Our data suggest that CPCs delivered with cECM are more likely to survive and differentiate in the infarct zone than cells delivered by themselves or in collagen.
Abstract 348: Dietary Quercetin Prevents Warfarin-Induced Vascular Calcification in Rats
Authors: Nurminskaya Maria , Banyard Derek , Nurminsky Dmitry , Konoplyannikov Mikhail , Beazley Kelly
Published: July 2021

Medial arterial calcification contributes to the development of isolated systolic hypertension, the most frequent type of blood pressure elevation in the elderly; complicates a variety of prevalent disorders such as diabetes and renal disease; and can develop in response to warfarin - the staple in anticoagulant therapy to millions of people. Our in vitro studies have shown that the bioflavonoid quercetin effectively prevents warfarin-induced vascular calcification in VSMCs and identified b-catenin signaling as a specific target of this effect. Here, we have tested in vivo the association of b-catenin signaling with warfarin-induced calcification and the efficacy of quercetin in preventing warfarin-induced aortic calcium accrual. Vitamin K-warfarin treatment was used for 4 weeks to induce arterial calcification in male rats. Molecular analysis demonstrates activation of b-catenin signaling in the calcified areas and osteoblast-like transformation in vascular cells. Dietary quercetin (10 mg/kg body weight) (Quercegen Pharma, Newton, MA) efficiently prevents warfarin-induced calcification (3.04+/-0.45 ug Ca in vitamin K-warfarin animals vs 0.77+/-0.08 ug Ca in quercetin supplementation per mg dry arterial tissue (n=5; p<0.05). In parallel, quercetin inhibits b-catenin signaling and phenotypic transformation in vascular smooth muscle analyzed by expression of transcription factor Runx2, collagen type I and osteocalcin. Further, quercetin diet enhances expression of VSMC markers smooth muscle actin, myosin heavy chain, sm22a and calponin, suggesting a stabilized smooth muscle phenotype of these cells. No toxic or systemic effects of quercetin treatment were observed, identifying this bioflavonoid as a putative therapeutic for vascular calcification.
Abstract 351: Nonlinear Dynamics in Long-Term Left Ventricular Remodeling in Chagas Heart Disease and Adverse Outcomes: SEARCH-Rio Substudy
Authors: Benchimol-Barbosa Paulo R , Kantharia Bharat K , Carvalhaes Claudio G
Published: July 2021

Introduction:
Left ventricular (LV) remodeling in Chagas heart disease (CHD) depends on the severity of the initial insult and may have clinical implication. SEARCH-Rio study investigated nonlinear dynamics in long-term LV remodeling in CHD.

Methods:
Fifty stable outpatients with CHD were enrolled and followed at scheduled visits. Cardiac death (sudden, arrhythmic or heart failure) was ascertained by review of medical records. LV dimension was assessed by echocardiogram, and mass estimated by Devereux. Subjects were distributed in four equally spaced groups according to initial mass. Intra-group standard deviation (SD) was calculated throughout follow-up. A plot of LV mass (corrected to initial value) vs recurrences during follow-up assessed non-linear relationship. Lyapunov exponent (LE) assessed LV mass dynamics during follow-up.

Results:
Median follow-up was 95 months; 10 subjects died. LV mass groups were [mean; Figure 1]: I-110g, II-140g, III-170g, IV-200g. No inter-group SD differences at baseline were observed (p=NS; table inset). At end of follow-up, intra-group SD increased roughly proportional to initial mean LV mass (Figure 1; p=0.01). A logistic parabolic-shaped equation was fit to corrected LV mass data in the plot (r²=0.98) and alpha coefficient was 3.9 (p<0.01; Figure 2). In groups, median LE was: I: 0.22; II: 0.17; III: 0.22; IV: 0.15. Median LE was 0.7 for survivors and -0.45 for nonsurvivors. LE cutoff at 0.26 yielded 100% sensitivity and 88% specificity for cardiac death (AUC=0.89; p<0.01).

Conclusion:
In CHD, LV remodeling exhibits complex nonlinear dynamics. LV remodeling dynamics has prognostic implication in this population.
Abstract 36: Disrupting the Delta Protein Kinase C Interaction with the “d” Subunit of F1Fo ATP Synthase Protects Against Ischemia-Reperfusion Injury in Perfused Rat Hearts
Authors: Johnson John A , Ogbi Mourad , Caldwell Robert W
Published: July 2021

Cardiac ischemia / reperfusion (IR) injury is associated with severe energy deprivation and is the number one cause of death world-wide. Mitochondrial F1Fo ATP synthase produces >90% of cardiac energy in mammals, yet few studies have targeted its role in IR injury. Previously, we identified a hypoxia-induced interaction of delta protein kinase C (dPKC) with the “d” subunit of F1Fo ATP synthase (dF1Fo) in neonatal cardiac myocytes, which inhibits F1Fo function. In the present work we investigated the hypothesis that a novel peptide inhibitor of the dPKC-dF1Fo interaction would preserve ATP and reduce infarct-size in isolated rat hearts subjected to IR injury. This peptide [NH2-YGRKKRRQRRRMLATRALSLIGKRAISTSVC-COOH] contains HIV-Tat protein transduction and mitochondrial targeting domains, the dPKC-dF1Fo inhibitor sequence, and a FLAG epitope. In hearts exposed to global ischemia, or IR, dPKC co-immuno-precipitated with dF1Fo. Pretreatment with the dPKC-dF1Fo inhibitor exacerbated cardiac ATP loss by 1.9-fold (n=5, p<0.03) following 10 min of global ischemia. However; following a pro-longed IR exposure ATP levels were enhanced by 2.1-fold (p<0.02, n=5). These opposing effects of the[[Unable to Display Character: ]]dPKC-dF1Fo inhibitor on ATP levels are likely due to relief of dPKC inhibition of the different modes of the F1Fo complex during ischemia (ATPase) and oxygenated reperfusion (ATP synthase). We next used 2,3,5, tetrazolium chloride staining techniques to determine if the dPKC-dF1Fo inhibitor had infarct-sparing effects following prolonged IR. In hearts exposed to 30 min of global ischemia and 150 min of reperfusion the dPKC-dF1Fo inhibitor reduced infarct size, (expressed as the percentage of total LV area) from 45 + 3 % (n=6) to 22 + 3 % (n=6, p < 0.01). Delivery and stability of the dPKC-dF1Fo inhibitor in hearts was confirmed by FLAG immunoreactivity in western blots conducted on mitochondria isolated the left ventricle. This is the first demonstration that perfusion with the dPKC-dF1Fo inhibitor prior to IR improves ATP recovery and reduces infarction in intact mammalian hearts. Our results support the potential for this peptide as a first-in-class translational agent for combating cardiac IR injury.
Abstract 367: Exercising Exacerbates the Hypertrophic Response of Female Transgenic Mice Expressing Elevated Myocardial Na
+
/H
+
Exchanger Isoform 1
Authors: Mraiche Fatima , Fliegel Larry
Published: July 2021

Cardiac hypertrophy (CH) is heart growth in response to environmental demands, and a variety of hormonal, paracrine and autocrine stimuli. It is a means to reduce stress on the ventricular wall. The Na+/H+ exchanger isoform 1 (NHE1) has been implicated in the development and progression of CH. To better understand the involvement of NHE1, male and female transgenic mice that express cardiac specific active NHE1 expression were studied. N-line mice expressed wild-type NHE1, and K-line mice expressed activated NHE1. NHE activity of adult ventricular cardiomyocytes and protein expression were elevated by approximately 2 and 3-fold in the N- and K-line mice vs. control. The K-line female mice assessed by echocardiography demonstrated significant global cardiac dysfunction. Left ventricular fractional cell shortening and ejection fraction were significantly decreased in the K-line mice (23.1 ± 3.8% and 45.2 ± 6.9% K-line vs. 36.5 ± 1.1% and 66.4 ± 1.5% control, respectively; p<0.05). The K-line female mice also exhibit myocardial remodeling. The heart weight to body weight ratio was significantly greater in the K-line mice (143 ± 10.0% of control; P<0.05). Cross sectional area (K-line 195.6 ± 16.4% of control; p<0.05) and interstitial fibrosis (K-line: 275.4 ± 11.6% of control; p<0.05) were also elevated. Increased expression of active NHE1 protein in male mice was also much more detrimental than expression of the wild type protein as was seen with the female transgenic mice. Therefore, the NHE1 induced hypertrophic effect was not gender dependent. However, NHE1 expression induced gender specific differences with exercise. Exercising exaggerated the HW/BW ratio in female mice expressing activated NHE1 compared to males. These results suggest that gender specific activation of NHE1 may be critical in promoting hypertrophy in females in comparison with males.
Abstract 37: Genetic Profile and Modifiers of Transient Cardiomyopathy from Tamoxifen-Induced Cardiomyocyte Expression of Mutated Estrogen Receptor-Cre Fusion Protein
Authors: Rowell Janelle , Koitabashi Norimichi , Barth Andreas , Bedja Djahida , Gabrielson Kathleen L , Ren Shuxun , Wang Yibin , Kass David A
Published: July 2021

The Myh6-MerCreMer (MCM) transgenic mouse is used to conditionally augment or reduce targeted gene expression in cardiomyocytes in vivo. Studies report transient cardiomyopathy upon tamoxifen exposure in the MCM mouse itself; however, this varies among investigators despite their using seemingly similar protocols and mouse strain. To better understand this model and apparent variable response, we studied the transcriptome-function relationship during the initial days of tamoxifen therapy, and tested if a genetic background involving gene deletion for nicotinamide nucleotide transhydrogenase (Nnt) found in C57BL/6J (common strain in MCM studies) influences the severity of transient cardiac dysfunction. MCM
+/-
mice (pure C57BL/6J, all Nnt
-/-
) received intra-peritoneal injections of tamoxifen (40 mg/kg/day for 5 days). LV function remained normal until Day 2 after tamoxifen, yet qtPCR and microarray analysis in MCM
+/-
mice revealed markedly reduced expression of metabolic and calcium handling genes (
Pgc1a, Pgc1b, Plb, Atp2a2
) and upregulation of sarcomere protein skeletal isoforms by Day 2. This transcriptome subsequently evolved to one typical of cardiomyopathy (initial highlighted metabolic/Ca
2+
genes remaining low) that correlated with LV dysfunction. Purchased MCM
+/+
mice (sv129xC57BL/6J) were Nnt heterozygotes. After 7-10 days following tamoxifen dosing, offspring lacking Nnt developed worse cardiodepression than those with at least one Nnt allele. The MCM mouse exhibits marked changes in transcription profile 2 days after tamoxifen suggesting potential causal mechanisms for subsequent cardiomyopathy. The severity of dysfunction is influenced by background genetics in part related to Nnt.
Abstract 371: Mitochondrial Antiviral Signaling (MAVS) Protects Cardiomyocytes Under Oxidative Stress by Interfering with Bax-Mediated Cell Death
Authors: Yajima Toshitaka , Park Stanley , Zhou Hanbing , Nakamura Michinari , Machida Mitsuyo , Uyesugi Arthur , Pokala Vineel , Domenighetti Andrea
Published: July 2021

MAVS is a mitochondrial outer membrane protein that activates innate antiviral signaling by recognizing cytosolic viral RNAs and DNAs. While the discovery of MAVS is the first molecular evidence that links mitochondria to innate immune mechanisms, it is still unclear whether MAVS affects mitochondrial cell death as a member of caspase activation and recruitment domain (CARD)-containing proteins. We found that MAVS interacts with Bax through CARD by Yeast two-hybrid and a series of immunoprecipitation (IP) assay, which led us to hypothesize that MAVS functions not only in the innate antiviral mechanisms but also in the mitochondrial cell death pathway.

Methods:
1) We examined molecular interaction between MAVS and Bax under oxidative stress by IP using isolated myocytes with H2O2 stimulation and the heart post ischemia-reperfusion (I/R). 2) We evaluated the effect of MAVS on mitochondrial membrane potential and apoptosis under H2O2 stimulation using isolated myocytes with adenoviral MAVS knockdown. 3) We investigated the impact of MAVS on %myocardial infarction (%MI) post I/R using cardiac-specific MAVS knockout (cKO) and transgenic (cTg) mice which we have originally generated. 4) We examined the effect of MAVS on recombinant Bax (rBax)-mediated cytochrome c release using isolated mitochondria from wild type (WT) and MAVS KO mice.

Results:
1) The amount of Bax pulled down with MAVS was significantly increased in isolated myocytes with 0.2 mM H2O2 compared to those without stimulation (mean±SD; 1.808±0.14, n=5, p<0.001) and in the heart post I/R compared to sham (2.2±1.19, n=3, p=0.0081). 2) Myocytes with MAVS knockdown showed clear abnormalities in mitochondrial membrane potential and caspace-3 cleavage with 0.2 mM H2O2 compared to control cardiomyocytes. 3) MAVS cKO had significantly larger %MI than WT (81.9 ± 5.8% vs. 42.6 ± 13.6%, n=8, p=0.0008). In contrast, MAVS cTg had significantly smaller %MI that WT (30.0 ± 4.8% vs. 49.2 ± 4.8%, n=10, p=0.0113). 4) Mitochondria from MAVS KO exhibited cytochrome c release after incubation with 2.5 μ g of rBax while those from WT required 10 μ g of rBax.

Conclusion:
These results demonstrate that MAVS protects cardiomyocyte under oxidative stress by interfering with Bax-mediated cytochrome c release from mitochondria.
Abstract 43: Activation of the Nuclear Receptor Coactivator PGC-1α Mediates an Atheroprotective Effect
Authors: McCarthy Cathal , Mooney Declan , de Gaetano Monica , James William , Fitzgerald Desmond J , Belton Orina
Published: July 2021

Supplementing dietary chow with conjugated linoleic acid (CLA) induces marked regression of pre-established murine atherosclerosis, in contrast to other PPAR agonists. The finding suggests that there are unidentified endogenous pathways that suppress the progression or promote the regression of atherosclerosis. Identifying these pathways in the mouse and their homologues in humans may help elucidate the mechanisms of the disease and targets for future therapies. Here, we provide evidence that CLA inhibits foam cell formation
via
regulation of the nuclear receptor co-activator, PGC-1α in a manner that differs from PPAR activation.

Gene expression analysis was performed in the aorta of ApoE
-/-
mice following induction of atherosclerosis and dietary supplementation with/without CLA. CLA induced dramatic regression of the cholesterol-induced atherosclerosis. PGC-1α was identified as a ‘hub’ gene within a cluster of genes induced by CLA in the aorta of the ApoE
-/-
during regression. PGC-1α protein was also found in murine and human atherosclerotic plaque, where it was localised to macrophage/foam cells. In a mouse macrophage cell line exposed to oxLDL, CLA induced PGC-1α and several genes in the network in an isomer specific fashion, including RORαand ABCA1. CLA also induced the PGC-1α target genes Cyp7b1 and UCP-1, and PPAR. CLA inhibited foam cell formation in the same cells exposed to oxLDL and suppressed the expression of the scavenger receptors, SRA-1 and CD36. Expression of the PGC-1α in macrophages had similar effects. Thus, over-expression of PGC-1α limited the accumulation of oxLDL and subsequent foam cell formation, while deletion of the gene promoted foam cell formation in bone marrow derived macrophages upon exposure to oxLDL. Moreover, deletion of PGC-1α prevented the inhibition of macrophages/foam cell formation by CLA.

The nuclear receptor co-activator PGC-1α is a hub gene in a network of genes activated in the aorta during CLA-induced regression of atherosclerosis and mediates CLA’s inhibition of foam cell formation. PGC-1α is also is also expressed in human plaques where its expression is inversely associated with disease progression, raising the possibility that this pathway if activated could regulate human atherosclerosis.
Abstract 44: A Slowly Inactivating Sodium Current Contributes to the Prolongation of the Action Potential in Aging Cardiomyocytes
Authors: Isobe Kazuya , Signore Sergio , Sorrentino Andrea , Ferreira-Martins João , Shafaie Mehrdad , Steadman Elisabeth , Arranto Christian , Sanada Fumihiro , Kostyla James , Zheng Hanqiao , Del Ben Fabio , Fiorini Claudia , Wybieralska Ewa , Sanarico Anna , Goihberg Polina , Rota Marcello
Published: July 2021

Aging is associated with alterations in the electrical properties of the heart resulting in an increased incidence of arrhythmic events and perturbation of cardiac performance. The aim of the present study was to determine whether the late sodium current (I
NaL
), which presents slow inactivation kinetics, is upregulated in myocytes from old hearts contributing to the electrical remodeling of the senescent myocardium. For this purpose, the electrical profile of young (3-5 months) and old (24-30 months) C57BL6 mice was established at the organ and cellular levels. In comparison to young mice, old animals presented enlarged left ventricles and declined systolic and diastolic functions. The QRS complex and QT interval of the ECG were significantly prolonged in old animals, indicating that aging delays the electrical activation and recovery of the myocardium. The latter property was confirmed by slower repolarization phases of epicardial monophasic action potentials (AP) in old hearts, using perfused Langendorff preparations. Moreover, old hearts were more prone to develop arrhythmia by programmed electrical stimulation. By patch-clamp technique, myocytes from old mice showed a 2- and 1.7-fold increase in the time to 50% and 90% repolarization of the AP, respectively, than cells from young animals. In voltage-clamp mode, I
NaL
, which is operative during the repolarization phase of the AP, was 1.7-fold larger in cells obtained from old hearts with respect to young. Activation, steady state inactivation and time constants for I
NaL
were comparable in young and old myocytes, indicating that the enhanced late current was mediated by an increased maximal conductance. In old myocytes, blockade of I
NaL
with low doses of tetrodotoxin or mexiletine reduced by 24-48% the intermediate and late repolarization phases of the AP, whereas in young cells this intervention affected to a lesser extent only the late repolarization phase. Finally, administration of mexiletine in old mice shortened the QT interval by 25%, restoring ventricular electrical recovery. In conclusion, the late sodium current I
NaL
is upregulated with aging and contributes to the slower repolarization of the senescent myocardium.