bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P226: Small Heterodimer Partner Negatively Regulates Cardiac Hypertrophy Through Upregulation of GATA6
Authors: Eom Gwang Hyeon , Joung Hosouk , Kim Yoojung , Kee Hae Jin , Shin Sera , Kim Don-Kyu , Choe Nakwon , Kwon Duk-hwa , Nam Kwang-Il , Choi Hueng-Sik , Kook Hyun
Published: July 2021

Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that regulates a variety of cellular events such as cell proliferation, differentiation and metabolism in liver and bone. However, the role of SHP in heart has not yet been elucidated. In this study, we investigated the functional roles of SHP in cardiac hypertrophy. In rat neonatal cardiomyocytes model, phenylephrine (PE) down-regulated expression of SHP. Transient transfection of SHP decreased the promoter activity of
Nppa
(natriuretic polypeptide precursor type A). Adenovirus-mediated overexpression of SHP (Ad-SHP) blocked gene expressions of GATA4, GATA6, and serum response factor (SRF). The increase in [
3
H]-leucine incorporation induced by PE or fetal bovine serum (FBS) was dramatically reduced by Ad-SHP. Likewise, increases in cell size with those hypertrophic stresses were significantly attenuated by Ad-SHP. The expressions of atrial natriuretic factor (ANF), β-myosin heavy chain (βMHC), and skeletal α-actin were significantly higher in hearts of SHP null mice. SHP physically interacted with GATA6 in mammalian cells. SHP significantly decreased the activation of -3003
Nppa
promoter induced by GATA6. The action of SHP on
Nppa
promoter activity was partially recovered by GATA6. Taken together, these results suggest that SHP works as a novel anti-hypertrophic regulator by repressing GATA6.
Abstract P252: Loss of HMGB2 Induces Chromatin Remodeling and Hypertrophic Growth in Cardiomyocytes
Authors: Franklin Sarah , Chen Haodong , Mitchell-Jordan Scherise , Ren Shuxun , Ping Peipei , Wang Yibin , Vondriska Thomas M
Published: July 2021

Nuclear DNA is packaged around the octameric nucleosome core particle, constituting the basic building block of chromatin. Non-nucleosome chromatin structural molecules have been shown to induce higher order packaging of DNA into structurally compact and inactive heterochromatin, or loosely packed and active euchromatin. These chromatin remodeling events are thought to establish a cell type specific pattern of gene expression. During the development of cardiac hypertrophy and failure, genes normally only expressed during development are re-activated. While a number of transcription factors involved in these changes in fetal gene expression have been identified, the means for genome-wide structural remodeling of DNA are unknown. To identify factors controlling genomic plasticity in cardiomyocytes, we used mass spectrometry to quantify chromatin-associated proteins from cardiac nuclei during stages of hypertrophy and failure in the mouse. Adult mice were subjected to cardiac pressure overload by transverse aortic constriction. Chromatin was fractionated from cardiac nuclei and DNA-bound proteins were acid extracted and analyzed by mass spectrometry. We measured chromatin occupancy patterns for >300 proteins during distinct stages of heart failure. To explore the isoform specific roles of individual chromatin structural proteins, we used siRNA to knock-down expression of two high mobility group proteins (HMGB1 and 2) exhibiting altered expression in the hypertrophic heart. Loss of HMGB2 (but not HMGB1) induced robust hypertrophic growth in cardiomyocytes. qRT-PCR analyses demonstrated that HMGB2 is responsible for some but not all changes in the fetal gene program (ANF increased 150% and SERCA decreased 20%, whereas α- and β-MHC were unchanged). To further explore the endogenous regions of the genome under control of HMGB2 packing, we performed microarrays following HMGB2 knockdown. Hypertrophy or HMGB2 knock-down induced global chromatin remodeling conducive to gene expression, as measured by histone post-translational modifications and the ratio of core to linker histones. These studies reveal a novel role of HMGB2 to inhibit hypertrophic growth and provide insights into general principles for genome-wide chromatin remodeling.
Abstract P255: Protein Arginine Methyltransferase 5 and Methylosome Protein 50, Novel Components of P300/gata4 Complex, Suppress Hypertrophic Gene Transcriptions in Cardiomyocytes
Authors: Katanasaka Yasufumi , Nakagawa Yukiko , Sunagawa Yoichi , Wada Hiromichi , Shimatsu Akira , Fujita Masatoshi , Hasegawa Koji , Morimoto Tatsuya
Published: July 2021

Introduction:
One of intrinsic histone acetyltransferases (HAT), p300, serves as a coactivator of hypertrophy-responsive transcriptional factors such as a cardiac zinc finger protein GATA4 and is involved in its hypertrophic stimulus-induced acetylation and DNA binding. Disruption of this complex results in the inhibition of hypertrophic responses in cardiomyocytes. By tandem affinity purification and mass spectrometric analyses, we identified protein arginine methyltransferase 5 (PRMT5) and methylosome protein 50 (MEP50), an activator of PRMT5, as novel p300/GATA4-binding partners. However, the precise functional relationships among p300/GATA4, PRMT5 and MEP50 remain unknown.

Methods and Results:
To confirm the binding of GATA4 with PRMT5, expression plasmids encoding GATA4 and PRMT5 were co-transfected into HEK293 cells. Nuclear extracts from these cells were subjected to immunoprecipitation, followed by western blotting. We observed that GATA4 formed a complex with p300, PRMT5 and MEP50 in HEK293 cells. Next, to investigate whether PRMT5 is required for p300-mediated activation of the GATA4-dependent promoter activities, we performed reporter assays in HEK293 cells. While overexpression of either PRMT5 or MEP50 inhibited p300/GATA4-induced atrial natriuretic factors (ANF) and endotheline-1 (ET-1) promoter activities, knockdown of PRMT5 by RNAi enhanced these promoter activities in HEK293 cells. Immunoprecipitation followed by western blotting demonstrated that PRMT5 repressed p300-induced acetylation of GATA4 in HEK293T cells. Finally, PRMT5 or MEP50 repressed phenylephrine-induced promoter activation of the ANF and ET-1 in cardiomyocytes.

Conclusion:
These findings demonstrate that PRMT5 and MEP50 form a functional protein complex with GATA4/p300 and regulated hypertrophic responses in cardiomyocytes.
Abstract P280: Deletion of Cardiac Ankyrin Repeat Kinase Reduces Ischemia/Reperfusion Injury in the Heart in Vivo
Authors: Vagnozzi Ronald J , Gatto Gregory , Kallander Lara , Ballard Victoria , Lawhorn Brian , Stoy Patrick , Gao Erhe , Force Thomas
Published: July 2021

Ischemic heart disease impacts millions of Americans and can progress to heart failure. Current therapies do not address this progression and new therapeutic targets are needed. One novel potential target is cardiac ankyrin repeat kinase (CARK, also troponin I interacting kinase; TNNI3K). CARK is expressed only in the heart and is significantly up-regulated in failing human hearts. Beyond this, little is known about CARK’s biological roles. To determine CARK’s function in the injured heart we subjected transgenic (Tg) mice expressing wild-type or kinase-inactive (KI) CARK to 30 minutes of LV ischemia followed by 24 hours of reperfusion (I/R). CARK-Tg mice had significantly larger infarcts (32.2% AAR vs 16.1% in WT littermates, p<0.05) following I/R. Cardiac troponin I (cTnI) serum levels were also significantly elevated in CARK-Tg mice after 24h, consistent with increased injury. Conversely, infarct size was decreased in mice expressing KI CARK and levels of cTnI were reduced, suggesting that blocking CARK activity may protect against acute injury. To test this, we employed an inducible, cardiac-specific knockout mouse (CARK-KO). CARK-KO mice showed a significant reduction in infarct size (20.52% vs 32.9%, p=0.01) as well as cTnI levels post-I/R. To confirm these findings, wild-type mice were treated with a small molecule CARK inhibitor and then were subjected to I/R. CARK inhibition significantly reduced infarct size (10.92% vs 21.74% p<0.01) as well as serum levels of cTnI. These data indicate that loss of CARK reduces myocyte injury and death after I/R. To examine the mechanism of this effect, primary NRVM were either transduced with a CARK adenovirus or treated with one of two selective CARK inhibitors, and then subjected to oxidative stress using H2O2. CARK over-expression worsened, while CARK inhibition significantly blunted H2O2 - induced apoptosis. Taken together, these data suggest that CARK plays an adverse role in the heart’s response to ischemia, in part by increasing apoptosis. Furthermore, inhibition of CARK may protect the ischemic heart by limiting initial cell loss and thus reducing infarct size. These findings enhance understanding of CARK’s role in the heart and provide evidence for CARK as a novel therapeutic target for ischemic injury.
Abstract P287: Therapeutic Hypothermia Cardioprotection During Cardiac Arrest Alters Reperfusion-Associated mTORC1 and AMPK Signaling
Authors: Sharp Willard W , Beiser David , Shao Zuo-Hui , Han Mei , Li Chang-Qing , Li Jing , Wang Huashan , Hamann Kimm , Vanden Hoek Terry
Published: July 2021

Introduction:
Therapeutic hypothermia (TH) is highly cardioprotective in cellular and animal models of ischemia/reperfusion (I/R) and cardiac arrest and is dependent upon Akt. The mammalian target of rapamycin complex 1 (mTORC1) is a downstream target of Akt and an important regulator of cellular energy utilization and survival but its relationship to TH is not clear. We hypothesized that TH attenuates mTORC1 activation while simultaneously activating the cell survival kinases AMP activated protein kinase (AMPK) and Stat3. We further hypothesized that inhibitors of mTORC1 or activators of AMPK would replicate and/or enhance TH cardioprotection.

Methods:
Cardiomyocytes isolated from 1-2-day old C57BL6/J mice, were exposed to simulated I (90 min)/R (3 h). For TH, cells were cooled from 37C to 32C during ischemia and the first hour of reperfusion. Heart samples were also obtained from C57BL/6 mice that underwent an 8-min cardiac arrest. Following 6 min, the mice were randomized to normothermia (NT, 37C) or TH (30C) extended during CPR and for 1 h after resuscitation. Protein lysates were collected at serial time points for Western blot analyses.

Results:
Cardiomyocyte death during ischemia was minimal, but accelerated during reperfusion (4% vs. 46%,p<.05 n=5). TH attenuated this cell death (24% p<.05 n=5) and attenuated reperfusion-induced mTOR activation seen within 30 min as measured by differential phosphorylation of the mTOR targets 70S6K and eEF2. Compared to NT, TH-treated cardiomyocytes and hearts demonstrated increased p-AMPK after resuscitation, consistent with mTOR inhibition. Stat3 also showed enhanced phosphorylation on tyrosine 705. The mTOR inhibitor rapamycin (100nM) given during I/R similar to TH affected 70S6K and eEF2 and significantly reduced cell death (28% vs. 42%, p<.05 n=5). The AMPK activator metformin (100uM) also significantly reduced cell death (22%, p<.05 n=4).

Conclusion:
TH cardioprotection is associated with altered signaling events suggesting mTORC1 inhibition and activation of the cell survival kinases AMPK and Stat3. Pharmacologic strategies that mimic these TH effects are available and could be useful as TH adjuncts or mimetics that improve survival after cardiac arrest.
Abstract P290: RhoA Protects Against Myocardial Ischemia/Reperfusion Injury
Authors: Xiang Sunny Y , Miyamoto Shigeki , Vanhoutte Davy , Molkentin Jeffery D , Dorn Gerald W , Heller Brown Joan
Published: July 2021

The small GTPase RhoA has established effects on cytoskeletal dynamics and gene expression but its role in regulating cardiac physiology and disease remains elusive. To characterize the in vivo role of RhoA signaling in cardiomyocytes, we generated conditional cardiac-specific RhoA transgenic mice (CA-RhoA) with 2–5 fold increases in RhoA activation in the adult heart. CA-RhoA mice show no overt cardiomyopathy but when challenged by in vivo or ex vivo I/R, these mice exhibit strikingly increased tolerance to injury. Compared to control mice, myocardial infarct size in CA-RhoA mice is reduced by 60–70% (20% vs. 50%, ex vivo; 10% vs. 37%, in vivo) and recovery of contractile function is significantly improved. Protein kinase D (PKD) is robustly activated in CA-RhoA hearts and inhibiting PKD reverses the cardioprotection afforded by RhoA. Both RhoA and PKD are also activated during I/R and blocking PKD augments I/R injury in WT mouse hearts. To further confirm that RhoA and PKD play a protective role during I/R, cardiac-specific RhoA knockout mice generated in the Molkentin laboratory were tested and demonstrated to show decreased tolerance to I/R injury, manifests as increased infarct size (42% vs. 23%) and lactate dehydrogenase release relative to control mice. This was accompanied by attenuated PKD activation during I/R. Taken together, our data indicates that RhoA signaling in adult cardiomyocytes promotes survival and reveals an unexpected role of PKD as a downstream mediator of RhoA and on cardioprotection against I/R.
Abstract P299: Gender-Based Differences in Ozone-Induced Cardiac Dysfunction
Authors: Sethi Rajat , Perepu Rama , Garcia Carlos , Dostal David
Published: July 2021

Sex related differences have been noted in cardiovascular disease where females have a lower incidence of heart failure, and a higher rate of heart failure survival. On-the-other-hand, some studies have reported increased mortality in women compared with men. Recent data also suggest that women have an increased risk of death due to O
3
air pollution, which is in contrast to reports that short-term variations in gaseous pollutants are associated with an increase in hospitalization for cardiac disease that is not modified by gender. It has been speculated that because of the nature of the photochemical equilibrium of O
3
in the ambient environment and due to other confounding factors epidemiological investigations of the health effects of O
3
may be using O
3
as an exposure surrogate for other oxidants or co-pollutants, some of which may be interfering with response to O
3
. The present study in a controlled exposure protocol tested the hypothesis that female rats compared to male have decreased sensitivity to cardiac injury subsequent to O
3
exposure. Age matched male/female rats were exposed 8 hrs/day for 28 and 56 days to filtered air or 0.8 ppm O
3
. In order to assess the chronic effects to O
3
,
in-vivo
cardiac function was assessed, 24 hrs after termination of the O
3
exposure. Compared to female rats, LVDP values significantly decreased in O
3
exposed male rats. This enhanced attenuation of cardiac function in male animals was associated with increased myocardial TNF-alpha (TNF-α) levels and decreased myocardial activities of superoxidase dismutase (SOD). These novel findings suggest that decreased attenuation of cardiac function in O
3
exposed females compared to males exposed to similar conditions was associated with decreased inflammatory mediator production and decreased oxidative stress. Identifying the underlying factors for gender based variations in ozone response is very important to recognize at-risk groups who would benefit from preventive strategies. In addition identification of those at risk, their degree of sensitivity will assist with the cost-benefit analysis of “safe” exposure levels in the public health setting. The long-term goal of this study is in guiding regulatory policies for reduced environmental related health costs.
Abstract P300: A Novel Highly Selective Adenosine A
1
Receptor Agonist (VCP28) Reduces Ischemia/Reperfusion Injury at Concentrations that Do Not Affect Heart Rate
Authors: White Paul J , Urmaliya Vijay , Pouton Colin W , Devine Shane , Scammells Peter
Published: July 2021

Whilst adenosine A1 receptor agonists have repeatedly been shown to protect the ischaemic myocardium, the clinical use of these agents is limited by strong cardiodepressant effects. The cardioprotective effects of a novel adenosine A1 receptor agonist N6-(2,2,5,5-tetramethylpyrrolidin-1-yloxyl-3-ylmethyl) adenosine (VCP28) were compared with the selective adenosine A1receptor agonist N6-cyclopentyladenosine (CPA) in a H9c2(2–1) cardiac cell line-simulated ischemia (SI) model (12 hours) and a global ischemia (30 minutes) and reperfusion (60 minutes) model in isolated rat heart model. H9c2(2–1) cells were treated with CPA and VCP28 at the start of ischemia for entire ischemic duration, whereas isolated rat hearts were treated at the onset of reperfusion for 15 minutes. In a H9c2(2–1) cell SI model, CPA and VCP28 (100 nM) significantly (P , 0.05, n = 5–6) reduced the proportion of nonviable cells (30.88% 6 2.49% and 16.17% 6 3.77% of SI group, respectively) and lactate dehydrogenase efflux. In isolated rat hearts, CPA and VCP28 significantly (n = 6–8, P , 0.05) improved postischemic contractility (dP/dtmax, 81.69% 6 10.96%, 91.07% 6 19.87% of baseline, respectively), left ventricular developed pressure, and end diastolic pressure and reduced infarct size. The adenosine A1 receptor antagonist DPCPX abolished the cardioprotective effects of CPA and VCP28 in both models. At the concentrations used in the ischaemia models, VCP28 had no effect on heart rate, unlike CPA. In conclusion, the adenosine A1 receptor agonist VCP28 has cardioprotective equal effects to the prototype A1 agonist CPA at concentrations that have no effect on heart rate.
Abstract P305: Detrimental Effects of Chronic Activation of AKT in Hearts from Ozone-Exposed Healthy Adult Rats
Authors: Sethi Rajat , Bandla Ashoka , Ramirez Magdalena , Manchanda Shubham , Garcia Carlos , Dostal David
Published: July 2021

Death of cardiomyocytes results in decreased cardiac function, which correlates with overall morbidity and mortality in many different clinical settings. For these reasons, understanding the intracellular signaling pathways that control cardiomyocyte survival has significant clinical implications. Each year in the US 60,000 cardiovascular deaths have been linked to environmental pollutants such as ozone (O
3
). Earlier studies from our laboratory have shown that myocardial dysfunction, subsequent to chronic O
3
exposure, in normal adult rats. This attenuation of cardiac function was associated with increased myocardial TNF-alpha (TNF-α) levels. Progressive increases in the expression of myocardial TNF-α in 4 and 8 week O
3
exposed animals were followed by decreases in cardiac caveolin-1 levels. Acute activation of the serine-threonine kinase Akt is cardioprotective and reduces both infarction and dysfunction after ischemia/reperfusion injury (IRI). Akt is activated in samples from patients with chronic heart failure, however, less is known about the chronic effects of Akt activation in the hearts from ozone exposed patients. Although caveolin-1 has been shown to negatively regulate AKT expression; a cell survival pathway, regulatory role of AKT in hearts from ozone exposed subjects has never been studied. Sprague Dawley rats were exposed 8 hr/day for 28 and 56 days to filtered air or 0.8 ppm O
3
. In order to assess the chronic effects to O
3
,
expressions of AKT and p-AKT were determined by wetern blotting in hearts extracted from
rats, 24 hr after termination of air or O
3
exposure. Compared to rats exposed to filtered air, total AKT levels were significantly increased in all O
3
exposed animals. Similar results were seen with pAKT levels. Since earlier studies have determined detrimental effects of chronic activation of AKT in ischemia reperfusion injury, our novel findings suggest the interesting possibility of a mechanism by which chronic Akt activation can become maladaptive after exposure to chronic levels of ozone. It is possible that PI3K-dependent but Akt-independent effectors are required for full cardioprotection. Studies are currently in progress to prove this hypothesis and complete study will be presented at the meeting in New Orleans in 2011.
Abstract P315: S-Glutathiolation of Low-Molecular-Weight PTP Regulates VEGF-Angiogenic Signal
Authors: Abdelsaid Mohammed A , El-Remessy Azza
Published: July 2021

While reparative angiogenesis improves outcome of stroke and myocardial infarction; pathological angiogenesis aggravates tumors, psoriasis and diabetic retinopathy. We have previously shown that low levels of peroxynitrite (PN, 1µM) are required to mediate VEGF’s angiogenic signal. Prior studies showed that low molecular weight protein tyrosine phosphatase (LMW-PTP) can regulate activation of VEGFR2 and focal adhesion kinase (FAK), key proteins involved in VEGF angiogenic signal. Here, we test the hypothesis that VEGF-induced peroxynitrite regulates LMW-PTP activity via reversible S-glutathiolation. Our results using human microvascular endothelial (HME) cells showed that VEGF (20 ng/ml) caused immediate (1,5 min) and transient negative shift in redox-state assessed by reduced-glutathione (GSH) level that was restored back to baseline by 15–30 minutes. In parallel, total free thiols of LMW-PTP; assessed by 5-iodoacetamide; were immediately oxidized (1, 5 min) and recovered after 15–30 minutes. PN (1μM) mimicked VEGF’ actions by inducing oxidative inhibition of LMW-PTP and FAK phosphorylation that peaked at 15 min. Studies using anti-GSH antibody demonstrated strong s-glutathiolation of LMW-PTP thiols that peaked at (5–10min) under non-reducing conditions and were completely abrogated in DTT-treated samples. These effects were associated with impaired tyrosine phosphorylation of LMW-PTP. PN caused concentration-dependent significant inhibition of LMW-PTP phosphatase activity reaching maximum at 0.5mM. Under pathological conditions recapitulated by combining with peroxynitrite (0.5mM), VEGF caused immediate yet permanent negative shift in cellular redox-state, LMW-PTP inactivation and sustained FAK activation in HME cells. Modulating cellular redox-state to reductive stress using N-acetyl cysteine (NAC, 1mM) or FeTPPS (2.5μM) prevented VEGF-induced angiogenesis in vitro and in vivo using hypoxia-induced neovascularization mouse model. Taken together, shifting redox-state to oxidative stress or reductive stress can impair VEGF’s signal and achieving a balanced redox-state is critical to facilitate VEGF’ angiogenic signal.
Abstract P317: Acute Exercise Stress and ROS Signaling: Activation of Nrf2/ARE-Dependent Antioxidant Defense Mechanisms in the Mouse Myocardium
Authors: Miller Corey J , Ramachandran Kalavathy , Khanderao Gayatri D , Kannan Sankaranarayanan , Rajasekaran Vasanthi , Boheme Christoph , Symons J David , Davidson Christopher J , Namakkal Soorappan Rajasekaran
Published: July 2021

Background:
Cellular defense mechanisms are crucial for maintaining intracellular redox state and mitigating free radical accumulation with aging. Nuclear Erythroid 2 p45 related factor-2 (Nrf2) regulates basal and inducible expression of numerous cytoprotective/antioxidant genes. We hypothesize that acute exercise will induce ROS, which triggers Nrf2/ARE signaling and promotes myocardial defense mechanisms.

Methods:
Age-matched wild-type (WT) and Nrf2−/− (KO) mice at 2 and >20 months were subjected to acute exercise stress (AES) and then we assessed the activation of Nrf2/ARE-dependent transcriptional mechanisms in the heart. Myocardial ROS was measured by electron paramagnetic resonance (EPR) analysis.

Results:
Under basal conditions, total ROS and GSH levels were identical at 2 months, whereas they were significantly impaired in Nrf2-KO when compared to Wt myocardium at ∼20 months indicating that Nrf2-deficiency is coupled with blemished redox potential. Upon AES, the young WT and KO mice exhibited oxidative stress (OS), but the WT were able compensate for the stress by increasing Nrf2 nuclear translocation and subsequent upregulation of cytoprotective genes. However, the aged (WT & KO) mice developed OS in response to AES. The degree of OS was several fold higher in the aged Nrf2-KO mice when compared with WT, suggesting an important age dependent function for Nrf2 in the myocardium. Western blot analysis revealed significant down regulation of major antioxidants (GCS, Nqo1, Ho1, catalase, G6pd and Gsr) in KO mice, while WT mice exhibited compensatory antioxidant response to the AES. Gene expression (qPCR) analysis revealed profound upregulation of major antioxidants in WT, but there was no such response occurred in KO mice after AES, suggesting Nrf2 independent mechanisms are inadequate to protect the myocardium.

Conclusions:
Acute exercise induces ROS and thereby activates Nrf2 in the myocardium. However, disruption of Nrf2 increases susceptibility of the myocardium to OS induced damage. Thus Nrf2 signaling might be a potential therapeutic target to protect the heart from ROS and/or age dependent ischemia/reperfusion (I/R) injury and myocardial infarction (MI).
Abstract P333: A Study of Miniature Catheters' Spatial Electric Field Heterogeneity During Murine Cardiac Catheterizations
Authors: Angeli Stelios , Mean Richard , Constantinides Christakis
Published: July 2021

Conductance catheters allow real-time quantification of hemodynamics, allowing cardiac functional characterization, an important predictor of long-term prognosis in cardiac disease. The technique’s accuracy is, however, inherently limited by the signal contribution from surrounding structures (spatially and temporally varying). Despite prior attempts to quantify this effect (known as parallel conductance) no prior study assessed the spatial heterogeneity of the catheter's E-field. This study quantifies the E-field penetration pattern, accounting for tissue properties and geometry.

Ten C57BL/6J mice were induced and maintained with 1.5% isoflurane mixed in 100% O
2
. One C57BL/6J mouse underwent a right carotid catheterization for placement of a 1.4 Fr Pressure-Volume Millar catheter in the left ventricle (LV), followed by microCT imaging (80kV/160mA/10ms exposure/240 projections/rotation angle=1.5
o
). Multiphase MRI was performed using a 4D radial spiral pulse sequence (TE=300μs/TR=2.4ms/BW=125kHz/flip angle=45
o
/110μm
3
resolution). Segmentation allowed LV myocardial and blood region extractions from MRI and construction of finite element End-Diastolic and End-Systolic models. The catheter’s orientation in the LV was determined from micro-CT image data renditions. Generated LV and blood models were then imported in the software XFdtd and electrical properties were assigned for all materials. Simulations used a 20 μA, 20 kHz sinusoidal current, and ran for a lump component equivalent and a constructed blood-myocardial geometrical model. Specific absorption rate (SAR) maps yielded total tissue deposited power.

Simulations show that the catheter’s E-field in lump component and geometrical models, drops to 10% of its peak value at 0.4-0.6mm, and 1.1-2mm, respectively, away from the excitation electrodes. SAR maps yielded a <1% power leakage into surrounding structures at two different myocardial permittivity values of ε
r
=11844 and 38615.

Results from this study map the spatial dependence of the generated catheter E-field. Spatial E-field maps indicate that the field is primarily confined within the ventricular chamber with a relatively uniform spatial pattern, and with <1% of the input power leaking in surrounding structures.
Abstract P350: Interstitial Volume Regulation in the Heart: Mechanistic Insights from ex Vivo Fluid Dynamics in Pig Myocardia
Authors: McGee Maria P , Morykwas Michael , Jordan Jim , Argenta Louis
Published: July 2021

Developing and testing specific therapies for myocardial edema require understanding their mode and site of action. However, parsing determinants of interstitial volume/pressure relationships
in vivo
is difficult, particularly in the myocardium, where rhythmic contractions add to the confounding components of fluid-transfer driving pressures. Here, we describe a novel
ex-vivo
model system based on osmotic stress techniques and illustrate its application to analyses of local myocardial fluid dynamics that exclude systemic influences and systole/diastole compressive cycles. Freshly harvested ventricular explants were equilibrated in physiologic media at colloidosmotic pressures ranging from 3 to 219 mmHg, and fluid transfer in/out was measured gravimetrically as a function of time and pressure. The relationship between the change in explant volume and the imposed colloidosmotic pressure of the media was linear. The

hydration potential

, defined empirically as the pressure at which explant volume did not change, was calculated from the abscissa intercept at
volume change = 0
, and the

compliance

from the slope of volume/pressure regression lines. Fluid-transfer

rates
and energies

were derived from volume versus time trajectories measured at 4 and 37 °C.
Hydration potential
was 71.3 ± 10.6 and 23.0 ± 15.2 mmHg at 4 and 37°C (significant, P = 0.002), while
compliance
was 1.09 ± 0.3 and 1.22 ± 0.2 µl/g/ mmHg (not significant, P = 0.2, n = 5). Temperature-dependent differences between in/out flow rates were also significant, giving experimental

activation energies

of −5.9 ± 2.2 and 1.4 ± 0.7 kcal/mol for inflow and outflow, respectively. Results show that at physiologic temperatures, even without vascular hydrostatic pressure gradients and lymphatic drainage, the myocardial bias toward interstitial fluid efflux persists. These findings are consistent with local fluid-control mechanisms actuated by colloidosmotic and tensional forces rather than passive changes in flow resistance. This new approach allows quantitative evaluation of interstitial components of Starling’s forces and should help in mechanistic preclinical characterization of treatments to correct interstitial myocardial edema.
Abstract 125: Cardiac Myosin Binding Protein-C Phosphorylation Is Critical for Normal Diastolic Function
Authors: Tong Carl W , Abdalla Mohamed I , Liu Yang , Alluri Himakarnika , Dostal David E , Powers Patricia A , Moss Richard L
Published: July 2021

Heart failure with preserved ejection fraction (HFpEF)

accounts for approximately 50% of all cases of heart failure, but there is no effective treatment of this disease. Cardiac myosin binding protein-C (MyBPC3) is a thick filament protein that is thought to slow cross-bridge cycling by inhibiting acto-myosin interaction. When phosphorylated, MyBPC3 releases its inhibition thereby accelerating cross-bridge cycling. Thus, we hypothesize that MyBPC3 phosphorylation enhances heart’s ability to relax (lusitropy). To test this idea, we expressed wild type MyBPC3(tWT), non-phosphorylatable MyBPC3(t3SA), and constitutively

phosphorylated mimetic MyBPC3(t3SD) onto MyBPC3(-/-) mouse background. We used echocardiography, voluntary running, and measurements of brain natriuretic peptide (BNP) for comparison. MyBPC3(t3SA) and MyBPC3(t3SD) hearts had similar ejection fraction as MyBPC3(tWT) hearts. MyBPC3(t3SA) hearts show depressed diastolic function revealed by slower myocardial tissue Doppler relaxation velocity at the mitral valve annulus (Ea) and an increased mitral blood flow/myocardium relaxation ratio (E/Ea). In contrast, MyBPC3(t3SD) hearts exhibited enhanced lusitropy with increased Ea and smaller E/Ea. Moreover, the findings of shorter 7-day average voluntary running distances, increased lung/body weight ratios, and increased BNP levels indicated heart failure in MyBPC3(t3SA) mice. Conversely, MyBPC3(t3SD) mice did not show signs of heart failure. Since cardiac function in MyBPC3(t3SA) mice resembles HFpEF and MyBPC3(t3SD) mice exhibit enhanced lusitropy, we conclude that phosphorylation of MyBPC3 is crucial for normal diastolic function.
Abstract 127: Txnip-Mediated NLRP3 Inflammasome Activation as a Novel Mechanism of Myocardial Ischemia-Reperfusion Injury
Authors: Liu Yi , Zhang Lijian , Qu Yan , Gao Chao , Liu Jingyi , Yan Wenjun , Liu Peilin , Sun Lu , Yang Lu , Wang Haichang , Tao Ling
Published: July 2021

As an inhibitor of the antioxidant thioredoxin, thioredoxin-interacting protein (Txnip) is linked to insulin resistance. NLRP3 inflammasome, a major regulator of innate immunity, has been reported to be activated by Txnip, thus contributing to the pathogenesis of type 2 diabetes mellitus. However, the role of Txnip and its NLRP3 inflammasome activation in the myocardial ischemia/reperfusion (MI/R) injury has not been previously investigated. C57BL/6J mice were subjected to 30 min of ischemia and 3 or 24 hrs of reperfusion. The ischemic heart exhibited increased Txnip and NLRP3 expressions, increased interaction between Txnip and NLRP3 (by immunoprecipitation, 1.8-fold increase over sham), and increased IL-1β, IL-18 and caspase-1 expressions (%increase: 80%, 77% and 110%, respectively) (n=8, all
P
<0.05). Compared with vehicle group, those mice either receiving intramyocardial small-interfering RNA (siRNA) injection to specifically knockdown the myocardial NLRP3 or intraperitoneal injection of the inflammasome inhibitor (BAY 11-7082) exhibited significantly improved cardiac function (by 28% and 25%), decreased the infarct size (by 40% and 38%), and decreased the cardiomyocytes apoptosis (all
P
<0.05). NLRP3 knockdown or inflammasome inhibitor also decreased the inflammatory cells infiltration (macrophages and neutrophils) and cytokines (TNF-α, INF-γ and IL-6) production (all
P
<0.05). To elucidate the role of Txnip in the NLRP3 activation in MI/R, intramyocardial injection of Txnip siRNA was performed to specifically knockdown the myocardial Txnip expression. Compared with vehicle, the Txnip knockdown significantly decreased Txnip/NLRP3 interaction and NLRP3activation as evidenced by lower expressions of IL-1β and caspase-1, decreased inflammatory cells infiltration and cytokines expressions, and consequently decreased the myocardial infarct size and increased the heart function (all
P
<0.05). Collectively, we demonstrated for the first time that Txnip mediatedNLRP3 inflammasome activation is a novel mechanism of MI/R injury. Interventions targeted to blocking the activation of NLRP3 by inhibiting Txnip may have therapeutic potential for preventing MI/R injury.
Abstract 128: Nuclear Targeting of the α1A-Adrenergic Receptors Is Required for Cardiac Myocyte Contractility
Authors: Wu Steven C , Wright Casey D , Cypher Andrew L , O'Connell Timothy D
Published: July 2021

In the heart, α1-adrenergic receptors (α1-ARs) regulate physiologic hypertrophy, cardiac myocyte survival, and contractility. Conventionally, G protein-coupled receptors such as α1-ARs are thought to localize and signal at the plasma membrane. However, we demonstrated that endogenous α1-ARs localize to the nucleus in wild-type (WT) adult cardiac myocytes, and identified nuclear localization sequences (NLS) in the α1A- and α1B-subtypes. Mutation of the NLS in each subtype resulted in mis-localization of the receptors and loss of signaling. Here, we examined how nuclear α1-ARs induce inotropic responses through regulation of cardiac troponin I (cTnI). In unloaded WT cardiac myocytes, the α1-agonist phenylephrine (PE) reduced sarcomeric shortening and increased cTnI phosphorylation at the protein kinase C (PKC) site threonine 144 (Thr144). PE had no effect in cardiac myocytes lacking both α1A- and α1B-subtypes (α1ABKO), but reconstitution of the α1A-subtype restored PE effects. Conversely, reconstitution of an NLS-mutant-α1A-subtype blunted the effects of PE on shortening and cTnI phosphorylation. Furthermore, in WT cardiac myocytes, the nuclear export inhibitor leptomycin B also blunted the effects of PE. With nuclei isolated from WT cardiac myocytes, we found high levels of PKCδ with lower levels of PKCα and ε, and that PE induced PKCδ activation in isolated nuclei. We also examined endogenous PKC translocation induced by PE in membrane, cytosolic, nuclear, and myofilament fractions, and found that PKCδ translocated from the nuclear to the myofilament fraction. Finally, expression of a dominant-negative PKCδ in WT myocytes blunted PE-induced effects on shortening and cTnI phosphorylation. Our data show that α1A-subtype nuclear localization is required for activation of PKCδ in the nucleus, subsequent phosphorylation of cTnI at Thr144 at the sarcomere to regulate myocyte contractility.
Abstract 131: Differential Regulation of Pitx2 Expression in Pulmonary Vein Muscle Sleeves and Left Atrium with Aging: Implication for Pulmonary Vein Triggers
Authors: Wong Johnson , Verma Div , Selzman Craig , Chen Huei-sheng Vincent
Published: July 2021

Introduction:
Pitx2, Paired-like homeodomain transcription factor 2, is a homeobox transcription factor that regulates left-right asymmetry during cardiogenesis. Pitx2 insufficiency in the left atrium (LA) has been linked to LA remodeling and arrhythmogenesis in age-related atrial fibrillation (AF). However, the main triggers for AF reside in pulmonary vein muscle sleeves (PVs). Whether Pitx2 insufficiency in PVs also plays a pathogenic role in generating trigger foci remains to be determined. Also, changes in Pitx2 expression in PVs with age remain unclear. We use qRT-PCR and Illumina microarrays to determine age-related changes in Pitx2 expression of PVs in wild type (WT) & heterozygous Pitx2cnull+/- mice, and in humans with or without AF.

Methods:
PV and LA regions were obtained from 1-23 month-old WT and Pitx2cnull+/- mice. Human PVs were obtained from ~110D human fetal hearts (E110D aborted fetus), two groups of adult donor hearts [20-30s year-old (yo) and 40-50 yo] and two patients with persistent AF. These tissues were used for genetic expression analysis.

Results:
Pitx2c expression decreases slightly with age in mouse LA and PV tissues. The expression levels of Pitx2c of Pitx2cnull+/- mice in LA are consistently at 55.2±10.5% of WT but at near-equal levels in PVs over four different age groups. In contrast, during human development, Pitx2 expression in PV muscle sleeves first decreases from ~E110D to ~30 yo but increases from 30 to 50 yo. In patients with AF, Pitx2 expression in PVs is only lower by 15% while HCN4 expression is higher by 13.5% when compared to control PVs at >50 yo. Expressions of type 2 Ryanodine receptors and calsequestrin in human PVs increase with age but decrease by ~30% with AF, suggesting impaired calcium handling abilities may play more significant roles than Pitx2 in the generation of PV triggers of AF.

Conclusions:
Genetic analysis of Pitx2c expression in PV muscle sleeves and LA tissues of WT and Pitx2cnull+/- mice revealed differential expression patterns. In human PVs, Pitx2 expression shows a regulation pattern with age distinct from that in mice, and genes linked to intracellular calcium handling may play a more important role than Pitx2 in generating trigger foci in PVs to initiate AF.
Abstract 132: Alternative Splicing of Bridging Integrator 1 Is a Novel Regulatory Cue for Cardiomyocyte Proliferation
Authors: Zhang Shan-Shan , Hong Ting-Ting , Smyth James W , Wheeler Matthew L , Fong Tina S , Shaw Robin M
Published: July 2021

Expanding our knowledge of the genetic pathways of cardiomyocyte proliferation is important to understanding heart formation, disease, and regeneration. Few data exist on the role of multi-domain adaptor proteins in regulating the cardiomyocyte cell cycle. We have identified by RT-PCR a cardiac splice variant of the nucleocytoplasmic adaptor protein, bridging integrator 1 (cBIN1), which is expressed in cardiomyocytes but not in skeletal myocytes (Fig. 1A). We found cBIN1 to be the dominant BIN1 variant of developing mouse cardiomyocytes, and that it is also preferentially located in the nuclei of these immature cells. As the cells mature, the relative abundance of cBIN1 to constitutively expressed variants, by qRT-PCR, decreases from 2.0 in embryonic day 18.5 cardiomyocytes to 0.6 by postnatal day 2 (Fig. 1B). Coincident with altered expression, a majority of cBIN1 redistributes from the nucleus to the cytoplasm and plasma membrane after birth. Moreover, we found that overexpression of exogenous cBIN1 increases the number of actively cycling embryonic cardiomyocytes from 25% to 32%, as assayed by BrdU incorporation and FACS (Fig. 1C). Confirming this result, we found exon-specific siRNA knockdown of cBIN1 suppresses cell proliferation. These studies identify a BIN1 splice variant that exists in cardiac but not skeletal muscle, and that it facilitates cardiomyocyte cell cycle progression during heart maturation.
Abstract 133: Transcription Factor Early Growth Response 1 Promotes T-Type Calcium Channel Cav3.2 in Response to Hypertrophic Stimulation Through Evolutionary Conserved Promoter
Authors: Hsu Shao-Chun , Chen Chien-Chang
Published: July 2021

Introduction:
Although it is known that the Ca

v

3.2 T-type calcium channel is predominantly expressed in the embryonic stage and re-expressed in adult hearts during the cardiac hypertrophy. What does regulate the reexpression of Ca

v

3.2 in hearts?

Hypothesis:
Because the mRNA re-expression is mainly through the transcriptional regulation in the promoter or enhancer conserved in different species, we assessed to the hypothesis that the evolutionary conserved promoter (ECP) of Ca

v

3.2 carries important binding sites for transcription factors that regulate its re-expression in the hypertrophic hearts.

Methods and Results:
In this study, the ECP is gotten by aligning Ca

v

3.2 genes from different species. By fusing mouse ECP with the reporter gene firefly luciferase, we showed that the ECP drove high luciferase activity in the cells expressing endogenous Ca

v

3.2 but not in the one without Ca

v

3.2. To further validate ECP in vivo, Ca

v

3.2 reporter mice were generated by fusing the Ca

v

3.2 promoter with the reporter gene luciferase. ECP confers the reporter expressing as the endogenous Ca

v

3.2 in the tissue distribution, development of hearts, and most importantly, the inducibility of hypertrophic stimuli. By injecting reporters driven by different truncated promoters followed with the trans-aortic banding (TAB) surgery, the hypertrophic regulatory elements are identified_ -41 to -81 relative to the transcription start site (TSS) of Ca

v

3.2. At the end, we found the early growth response 1 (Egr1) is the important transcription factor to enhance Ca

v

3.2 gene expression. Our EMSA data suggested that Egr1 can bind to three regions of the hypertrophic regulatory elements.

Conclusion:
In conclusion, transcription factor early growth response 1 (Egr1) regulates the reexpression of Ca

v

3.2 T-type Calcium Channel in the cardiac hypertrophy.
Abstract 135: A Novel TGFb Selective Inhibitor Drives Cardiogenesis in Embryonic Stem Cells
Authors: Willems Erik , Teixeira Joaquim , Schade Dennis , Lanier Marion , Bushway Paul , Cashman John , Mercola Mark
Published: July 2021

The early signals that drive embryonic stem cells (ESC) to cardiomyocytes are well characterized, but little is known about the pathways that control the later stages of differentiation. Genetic studies in animals are however challenging to study these later steps of heart development and are thus incapable of providing useful information for controlling ESC differentiation. Therefore, we have implemented unbiased high throughput screens on mouse ESC (mESC) to discover small molecules that probe these steps of differentiation to unravel novel biological mechanisms that control cardiac fate. From over 17000 unique molecules, screened in a serum assay relying on a cardiac specific Myh6-GFP reporter to monitor cardiogenesis, we identified a dihydropyridine (DHP), which are well-known L-type calcium channel inhibitors. Subsequent pathway analysis in combination with a structure activity relationship study of over 200 DHP analogs pinpointed the activity specifically to the Activin A/TGFb pathway, with high inhibition efficiency for TGFb signals and poor to none for Activin A signals. Further functional characterization of DHP in the TGFb pathway demonstrated that DHP acts upstream of Smad2/3, positioning the activity at the receptor level, where DHP downregulates the amount of TGFb receptors on the cell surface and targets them for degradation. This TGFb selective inhibitor was then applied in mESC to probe the effects of inhibiting TGFb specifically at several stages of differentiation. When given early in differentiation DHP blocked all mesoderm, indicating that, in serum differentiated cultures, TGFb is essential for mesoderm induction. Secondly, when DHP was added during mesoderm patterning, cardiogenesis was enhanced specifically, whereas other cardiovascular lineages such as smooth muscle, blood and endothelial cells were not affected. In summary, we have identified a new probe of TGFb signaling that shows that TGFb signaling governs cardiopoietic fate by inhibiting formation of cardiomyocytes. Additionally, our molecule and its mechanism have important implications for congenital heart disease, since deficient TGFb signaling leads to vascular and cardiac defects, and elevated signaling underlies Loeys-Deitz syndrome.
Abstract 136: Novel Disease Analysis Using Long QT Syndrome-Specific Induced Pluripotent Stem Cells
Authors: Egashira Toru , Yuasa Shinsuke , Fukuda Keiichi
Published: July 2021

[Background]
Although previous reports have indicated that long QT syndrome-specific iPS cells (LQTS-iPSCs)-derived cardiomyocytes recapitulated disease phenotypes, those patients were previously diagnosed with mutated channel profiles. In reality, most patients have no such specific information. To address whether iPSCs could be used for personalized disease characterization, we generated iPSCs from a sporadic LQTS patient with unknown disease cause.

[Methods and Results]
1 We generated iPSCs from control (n = 2) and a patient with LQTS, and differentiated into cardiomyocytes through embryoid body (EB) formation. 2 Electrophysiological analysis of the LQTS-iPSCs-derived EBs using multi electrode array system revealed that the duration of the field potential (FPD) was markedly prolonged compared with the control (388.9 ± 44.3 msec vs 202.3 ± 16.3 msec, P<0.01). 3 We tested several drugs affecting QT prolongation to clarify the electrophysiological properties. The IKr blocker E4031 significantly prolonged FPD (% change 1.21 ± 0.02, P<0.01) and induced frequent severe arrhythmia, not only early-after depolarization (n = 8/16 vs n = 1/16) but also torsades de pointes-like arrhythmia, only in LQTS (n = 4/16 vs n = 0/16). The IKs blocker, chromanol 293B did not prolong FPD in LQTS but it significantly prolonged FPD in the control (% change 1.09 ± 0.04, NS vs 1.44 ± 0.07, P<0.01), suggesting the involvement of IKs disturbance in the patient. Isoproterenol induced ventricular tachycardia-like arrhythmia only in LQTS, which was blocked by propranolol. These data strongly suggested a functional impairment in the patient’s IKs channel; genotype analysis for KCNQ1 gene revealed a novel heterozygous mutation, 1893delC. 4 Patch clamp analysis and immunostaining confirmed a dominant-negative role for 1893delC in IKs channel through a trafficking deficiency.

[Conclusions]
LQTS-iPSCs-derived cardiomyocytes recapitulated the disease phenotypes, and they can be utilized for identification of the disease cause and genotype. This study demonstrated that iPSCs could be useful to characterize the disease, drug responses, diagnosis and genotyping in patients with sporadic LQTS, which in turn may facilitate medical therapies in the clinical settings.