bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 22: Calpain-Dependent Induction of Endoplasmic Reticulum Stress in Cardiomyocyte Apoptosis and Post--Myocardial Infarction Remodeling
Authors: Zheng Dong , Ma Jian , Wei Meng , Xu Huaxi , Peng Tianqing
Published: July 2021

Background: Calpain is up-regulated and implicated in cardiomyocyte apoptosis and myocardial remodelling post myocardial infarction. Endoplasmic reticulum (ER) stress is induced and contributes to myocardial injury in a variety of cardiac diseases. This study was to investigate whether calpain plays a role in ER stress, thereby mediating apoptosis in cardiomyocytes and myocardial remodelling after infarction.

Methods and Results: In rat cardiomyoblasts H9C2 cells, over-expression of calpain-1 increased the protein levels of Bip and CHOP, indicative of ER stress, and induced apoptosis determined by a decrease in Bcl-2 and increases in caspase-3 activation and DNA fragmentation. Inhibition of calpain activity or ER stress prevented apoptosis induced by calpain-1 over-expression. In contrast, over-expression of calpain-2 failed to induce apoptosis. The induction of ER stress by calpain-1 might be mediated through SERCA2a/Calcium signaling as up-regulation of calpain-1 reduced SERCA2a protein and elevated intracellular free Calcium in H9C2 cells. In a mouse model of myocardial infarction induced by coronary artery ligation, calpain activity was increased and ER stress was induced in the infracted heart. Up-regulation of calpastatin, the endogenous calpain inhibitor, inhibited calpain activation, prevented ER stress and apoptosis, reduced myocardial remodelling and improved myocardial function after infarction in calpastatin transgenic mice compared with their wild-type littermates.

Conclusion: Calpain-1 induces ER stress through the proteolysis of SERCA2a, thereby mediating apoptosis in cardiomyocytes. Inhibition of calpain prevents ER stress and apoptosis, reduces myocardial remodelling and dysfunction after infarction. Thus, ER stress may represent a novel mechanism by which calpain mediates myocardial injury in the infracted heart.
Abstract 228: Activation of the Free Fatty Acid Receptor GPR120 Prevents Fibrosis in NIH3T3 Cells and Primary Cardiac Fibroblasts
Authors: Qian Qingwen , O'Connell Tim D
Published: July 2021

Fibrosis significantly contributes to contractile dysfunction and pathologic ventricular remodeling in heart failure. Recently, we showed for the first time that ω3-polyunsaturated fatty acids (ω-PUFAs) prevent fibrosis and cardiac dysfunction in pressure-overload induced heart failure through inhibition of collagen expression, fibroblast proliferation, and fibroblast-to-myofibroblast transformation. In cultured cardiac fibroblasts, we found that ω3-PUFAs induced eNOS activity to prevent TGFβ1-Smad2/3 pro-fibrotic signaling. Mechanistically, ω3-PUFAs are thought to regulate ion channels or to incorporate into the membrane and alter the properties of lipid-rafts, but how a fatty acid might activate eNOS is unknown. Recently, GPR40 and GPR120 were identified as receptors for long-chain fatty acids (FFARs). Here, we examined GPR120 regulation of cardiac fibrosis. Among the FFARs, GPR40, 41, 43, 84, and 120, GPR120 mRNA was expressed at highest level in whole heart. Furthermore, we detected GPR120 expression in isolated cardiac myocytes and fibroblasts, as well as NIH3T3 mouse embryonic fibroblasts. To determine if activation of GPR120 was sufficient to prevent fibrosis, we tested the ability of the GPR120 agonist GW9508 to prevent TGFβ1-induced fibrosis in both NIH3T3 cells and primary cultures of cardiac fibroblasts. In both NIH3T3 cells and primary cardiac fibroblasts, TGFβ1 (1 ng/ml) induced a pro-fibrotic response indicated by increased fibroblast proliferation, α-smooth muscle actin expression, and collagen I expression. In both cell types, pre-treatment with GW9508 significantly inhibited TGFβ1-induced proliferation (63% in NIH 3T3 cells and 52% in cardiac fibroblasts, respectively), α-smooth muscle actin expression (78% in NIH 3T3 cells, 90% in cardiac fibroblasts), and collagen I expression. In summary, our results demonstrate for the first time, that GPR120 signaling in the heart prevents fibrosis.
Abstract 229: TNF Receptor 1 Signaling Is Critically Involved in Mediating Angiotensin II-Induced Cardiac Fibrosis and Dysfunction
Authors: Haudek Sandra B , Crawford Jeff , Reineke Erin , Allegre Alberto A , Taffet George E , Trial JoAnn , Entman Mark L
Published: July 2021

Angiotensin-II (Ang-II) plays a key role in the development of cardiomyopathies, as it is associated with many conditions involving heart failure and pathologic hypertrophy. Using a murine model of Ang-II infusion, we found that Ang-II induced the synthesis of monocyte chemoattractant protein 1 (MCP-1) that mediated the uptake of CD34
+
/CD45
+
monocytic cells into the heart. These precursor cells differentiated into collagen-producing fibroblasts and were responsible for the Ang-II-induced development of reactive fibrosis. Preliminary in vitro data using our monocyte-to-fibroblast differentiation model, suggested that Ang-II required the presence of TNF to induce fibroblast maturation from monocytes. In vivo, they indicated that in mice deficient of both TNF receptors (TNFR1 and TNFR2), Ang-II-induced fibrosis was absent. We now assessed the hypothesis that specific TNFR1 signaling is necessary for Ang-II-mediated cardiac fibrosis. Mice deficient in either TNFR1 (TNFR1-KO) or TNFR2 (TNFR2-KO) were subjected to continuous infusion of Ang-II for 1 to 6 weeks (n=6-8/group). Compared to wild-type, we found that in TNFR1-KO, but not in TNFR2-KO mouse hearts, collagen deposition was attenuated, as was cardiac α-smooth muscle actin protein (a marker for activated fibroblasts). When we isolated viable cardiac fibroblasts and characterized them by flow cytometry, we found that Ang-II infusion in TNFR1-KO, but not in TNFR2-KO, resulted in a marked decrease of CD34
+
/CD45
+
cells. Quantitative RT-PCR demonstrated a striking reduction of type 1 and 3 collagen, as well of MCP-1 mRNA expression in TNFR1-KO mouse hearts. Further measurements of cardiovascular parameters indicated that TNFR1-KO animals developed lesser Ang-II-mediated LV remodeling, smaller changes in E-linear deceleration times/rates over time, and displayed a lower Tei index (a heart rate independent marker of cardiac function), indicating less stiffness in TNFR1-KO hearts compared to wild-type and TNFR2-KO hearts. The data suggest that Ang-II-dependent cardiac fibrosis requires TNF and its signaling through TNFR1 which enhances the induction of MCP-1 and uptake of monocytic fibroblast precursors that are associated with reactive fibrosis and cardiac remodeling and function.
Abstract 240: Interruption of Ivabradine Treatment in Middle-Aged Post-MI Rats May Lead to Heart Failure
Authors: Dedkov Eduard I , Bogatyryov Yevgen , Oak Kunal , Christensen Lance P , Tomanek Robert J
Published: July 2021

Background:
We have previously demonstrated that heart rate reduction (HRR) with the selective cardiac pacemaker I

f

current inhibitor ivabradine (IVA) improved LV function and coronary perfusion reserve in post-MI middle-aged rats. The beneficial effects of IVA treatment were associated with attenuated collagen accumulation in the surviving LV myocardium and inhibition of the circulatory renin-angiotensin system. However, sustainability of these positive changes after a period of IVA treatment interruption remains to be determined.

Methods:
MI was induced in 12-month-old male Sprague-Dawley rats by left coronary artery ligation. Twenty four hours later, the rats with a confirmed large transmural MI were randomly assigned in two experimental groups. In a first group, rats were treated with IVA
i.p.
via osmotic pumps in a dose of 10.5 mg/kg/d for 4 weeks followed by a 4-week period without treatment (MI+IVA). In a second group, rats received placebo for 4 weeks (MI). Sham-operated rats served as a control. Eight weeks after MI, the various LV parameters were examined.

Results:
During 4 weeks of treatment, heart rate in MI+IVA rats was consistently reduced compared to untreated MI rats (mean of HRR was 27%). After IVA withdrawal, heart rate increased to the levels of the placebo group. Eight weeks after MI, the IVA treated group demonstrated smaller LV free wall infarct size (57.5 ± 2.7% vs. 72.5 ± 2.5%, p<0.01) and greater LV weight/body weight ratio (2.54 ± 0.06 vs. 2.26 ± 0.11 mg/g, p<0.05), indicating greater myocardial compensatory hypertrophy. Furthermore, the interstitial collagen content was significantly lower in the remote LV myocardium of MI+IVA rats compared to MI rats (7.5 ± 0.7% vs. 13.3 ± 1.0% in free wall and 7.3 ± 0.8% vs. 11.2 ± 0.9% in septum, p < 0.01). However, compared to non-infarcted controls, rats of both MI groups had markedly lower LV ejection fraction and coronary perfusion reserve.

Conclusions:
Our data demonstrate that although beneficial structural changes induced by IVA in post-MI myocardium can be sustainable for a long period of time they cannot prevent deterioration of LV function after withdrawal of treatment. Therefore, we believe that continuous treatment with IVA may be essential for attenuation of heart failure after a large MI.
Abstract 25: The Use of Voltage Mapping for Slow Pathway Visualization and Ablation of Atrioventricular Nodal Reentry Tachycardia in Pediatric and Young Adult Patients
Authors: Malloy Lindsey , Law Ian , Von Bergen Nicholas
Published: July 2021

Atrioventricular nodal reentry tachycardia (AVNRT) is a common arrhythmia in both pediatric and adult patients. Ablation of the arrhythmia substrate has typically been guided by anatomical location and electrogram morphology within the triangle of Koch. Using an anatomic approach can be challenging because of unusual pathway locations and anatomic variance. The use of voltage gradient mapping has been proposed in adults to aid in identification of the “slow pathway”, guiding placement of the ablation applications. The purpose of this study was to evaluate this novel technique of voltage guided ablation of AVNRT in a pediatric patient population, with a smaller triangle of Koch.

Patients with atrioventricular nodal reentry tachycardia at the University of Iowa Children’s Hospital who underwent voltage mapping within the slow pathway area were included. Using intracardiac electrical recordings, three-dimensional voltage maps of the right atrium were created. A voltage map identified a bridge of lower voltage signals surrounded by even lower voltage tissue. This bridge was used to guide cryoablation of the slow pathway. Patient demographics, appearance of the intracardiac voltage mapping, timing of procedure, lesions to success, and total number of lesions was obtained.

In this study there were 29 patients with an average age of 14 years (range 7 to 20 years) who underwent AVNRT ablation with voltage mapping. Ten were male. In these patients there was procedural success (no inducible AVNRT, single AV node echo beat or less) in all patients. In 25 of 29 patients, there was an adequate lower voltage saddle to allow guided ablation. The successful ablation site was within the first three lesions in 15/25 patients. Total lesions ranged from 5-34. There has been recurrence in 1 patient over an average follow-up period of one year (range five months - twenty months).

The use of voltage guided ablation of a low voltage saddle in atrioventricular nodal reentry tachycardia is a technique that appears to be effective and safe in the pediatric population and has the advantage of allowing an electrically guided ablation therapy. Voltage guided ablation of atrioventricular nodal reentry tachycardia is a safe and effective technique for ablating AVNRT.
Abstract 250: Haploinsufficiency of Muscle-Enriched A-Type Lamin Interacting Protein (MLIP) Manifests as Enlarged Dilated Hearts
Authors: Burgon Patrick , Lockwood Julia , Wells Glenn , Blais Alexandre
Published: July 2021

Approximately 116 unique mutations in the lamin A/C gene have been described to date that are associated with dilated cardiomyopathy. We recently reported the discovery of MLIP through its interaction with lamin A/C. MLIP is expressed ubiquitously and most abundantly in heart, skeletal and smooth muscle of amniotes (mammals, reptiles and birds) and has no paralogous homologue suggesting no functional redundancy. The MLIP gene encodes at least seven, alternatively spliced, LMNA-interacting factors that possess several structural motifs not found in any other protein. The MLIP isoforms pattern of expression differs between each of the tissues with heart being the most heterogeneous. Down-regulation of lamin A/C expression by shRNA results in the up-regulation and mis-localization of MLIP. In addition to interacting and co-localizing with lamin A/C we also demonstrated that MLIP localizes to micro-domains in the nucleus with promyelocytic leukemia protein (PML) in close proximity to chromatin. MLIP's biological function still remains elusive.

Eight week old hemizygous MLIP null mice develop enlarged hearts with a significant increase in heart to body weights (MLIP+/+ 5.62mg/g vs MLIP+/- 10.73mg/g, p<0.0001 n=7) with an overall 30% increase in the anterior-posterior ventricle length of MLIP hearts while maintaining a normal body weight (Figure). Echocardiographic analysis of MLIP+/- mice revealed that their hearts as having a significant (p3.93mm with a significant (p=0.011, n=12) reduction of left ventricular fractional shorting (LVFS) 31% when compare to littermate controls. Histological analysis of the hearts showed no overt phenotype other than an overall increase in the size of the MLIP+/- hearts. The cellular source for the increase in heart size and mass remains to be determined if it is the product of an increase in the number of cardiomyocytes due to aberrant hyperplasia or an increase in cardiomyocyte size through cellular hypertrophy.

In conclusion, MLIP is a newly discovered lamin interacting protein that may serve as a transcriptional regulator that impact genes involved in heart development, growth and function and provides a new signaling paradigm.
Abstract 251: Development of a Transgenic Goat Model with Cardiac-Specific Overexpression of Transforming Growth Factor-β1 to Study the Relationship Between Atrial Fibrosis and Atrial Fibrillation
Authors: Polejaeva Irina A , Hall Justin , Meng Qinggang , Zhou Xinchang , Sessions Benjamin R , Panter Kip , Stott Rusty , Rutigliano Heloisa M , Davies Christopher J , Wang Zhongde , Ranjan Ravi , Dosdall Derek , MacLeod Rob , Marrouche Nassir , White Kenneth L
Published: July 2021

Studies on patients, large animal models and transgenic mouse models have shown a strong association of atrial fibrosis with atrial fibrillation (AF). However, it is unclear whether there is a causal relationship between atrial fibrosis and AF or whether these events appear as a result of independent pathological changes in the heart. We are testing the hypothesis that goats that overexpress TGF-β1 (transforming growth factor beta1) specifically in cardiac myocytes will develop atrial fibrosis that in turn will lead to AF. Many aspects of AF-related remodeling have been studied comprehensively in goat models. However, these AF models are typically mechanically induced (eg, the rapid atrial pacing model). This unique transgenic goat model has the potential to offer insights into the role of fibrosis in AF initiation and progression without the confounding effects of mechanical AF induction.

Somatic cell nuclear transfer (SCNT or cloning) was used to produce TGF-β1 transgenic pregnancies. First, pcDNA3.1DV5-MHC-TGF-β1cys33ser vector was constructed by subcloning the MHC-TGF-β1 fragment from the plasmid pUC-BM20-MHC-TGF-β1 into the pcDNA3.1D V5 vector. The NeonTM transfection system was used to electroporate primary goat fetal fibroblasts. After two weeks of G418 selection, the resulting G418 resistant colonies were screened by PCR to confirm transgene integration into goat genomic DNA. PCR positive cells were used for SCNT. Cloned embryos (n=264) were cultured for 12-60 h in vitro and then transferred into synchronized recipient females (n=15). Confirmation of pregnancy was done by ultrasonography on day 30 post-transfer. At the time of this abstract submission, 40% (6/15) of recipients were confirmed to be pregnant as determined by the presence of a heartbeat. The range for the stage of gestation is between day-60 and day-120. The first delivery date is April 28, 2012. Several reports documented no pregnancy losses after 30 days of gestation in goats. Therefore, we expect that most if not all of these pregnancies will result in delivery of live offspring. To our knowledge, this will be the first transgenic goat model generated for cardiovascular research.
Abstract 253: The Tyrosine Kinase ErbB2 Inhibitor Lapatinib and the Anti-ErbB2 Antibody Trastuzumab Depress Cardiac Function Without Inducing Left Ventricular Dilation in Mice
Authors: Tocchetti Carlo G , Coppola Carmela , Rea Domenica , Barbieri Antonio , Palma Giuseppe , Piscopo Giovanna , Gala Marianna , Luciano Antonio , Giudice Aldo , Cipresso Clemente , Iaffaioli Rosario V , Scala Stefania , De Lorenzo Claudia , Arra Claudio , Maurea Nicola
Published: July 2021

Background:
ErbB2, tyrosine kinase receptor of the EGFR family, overexpressed in about 25% of breast cancers, modulates development and function in the myocardium. Lapatinib (LAP) is a small molecule that inhibits ErbB2-tyrosine kinase domain. The few existing trials report it to be associated with a low risk of LV dysfunction (2%), while the prototypical antibody Trastuzumab (TRAS) increases the frequency of asymptomatic ejection fraction decrease by 3-18%, and the risk of heart failure by 2-4%. Here, we characterize our LAP murine cardiotoxicity model in vivo by echocardiography, comparing it to TRAS and doxorubicin (DOX) cardiotoxicities.

Methods:
LV end-diastolic and end-systolic diameters (LVEDD and LVESD) were assessed. LV function was measured with fractional shortening (FS) by M-mode echo, and with radial myocardial strain (%) with speckle tracking (ST) in sedated C57BL6 mice (2-4 mo. old) at day 0, and after 2 and 7 days of daily administration of LAP (100mg/kg/day orally), and in control mice. For comparison we used our well-established models with TRAS (2.25 mg/kg/day, ip), and DOX (2.17 mg/kg/day ip) as positive control.

Results:
FS was already reduced with DOX at 2 days: 52±.2%, p<.001 vs 60±.4% (sham), while LAP and TRAS decreased FS only at 7 days (56±2 and 49±1.5%, respectively, both p<.05 vs 60±.5% for sham). Interestingly, FS reduction with LAP was milder than with TRAS (p=.01). At 7 days, both LAP and TRAS only increased LVESD: 1.23±.07 and 1.49±.07mm, respectively, both p<.05 vs 1.1±.03mm (sham). On the other hand, at 7 days DOX induced LV dilation, with significant enlargement of both LVESD (1.55±.08mm) and LVEDD (3.04±.06mm; sham=2.81±.03mm), both p<.005. Importantly, from a diagnostic point of view, myocardial strain was reduced early at 2 days with LAP and TRAS, predicting cardiotoxicity: 34±7% for LAP, 44±7% for TRAS, both p<.05 vs sham (66±.6%).

Conclusions:
Differently from DOX, ErbB2 inhibition in mice does not produce LV dilation, but only decreases LV function. In particular, specifically targeting ErbB2-tyrosine kinase domain with LAP confers less cardiotoxicity than TRAS. Myocardial strain identifies LV systolic dysfunction earlier than conventional echo and can be a useful tool to predict ErbB2 inhibitors cardiotoxicity.
Abstract 263: Defective Autophagy Plays a Role in the Development of Angiotensin-Induced Cardiac Hypertrophy
Authors: Johnson Simon C , Nguyen Vy , Castanza Anthony , Rabinovitch Peter S
Published: July 2021

While activated autophagy is a well reported feature of cardiac disease, the exact role of autophagy in the etiology and progression of cardiac hypertrophy and dysfunction is unclear; activation of autophagy appears to play either pro-survival or pro-apoptotic functions depending on the context. Here we demonstrate that dysfunctional autophagy plays a role in the development of angiotensin II-induced cardiac hypertrophy. Using a live, ex-vivo tissue imaging technique and transgenic LC3-GFP expressing mice we found that after short term (1 week) treatment, autophagic markers accumulate in angiotensin treated mouse hearts and that in a small population of cells this accumulation reaches a critical level where normal cell organization, specifically mitochondrial localization and contractile unit structure, and tissue architecture are disrupted. These observations are reminiscent of genetic diseases associated with dysfunctional autophagy, such as inclusion body myositis, where autophagic vesicles fail to properly clear and lead to cell death and tissue degeneration. We suggest that failed autophagy contributes to pressure-overload induced hypertrophy and that the cell death associated with dramatically dysfunctional autophagy in a subset of cardiomyocytes precedes fibrosis. This work provides a clear role for dysfunctional autophagy in promoting hypertrophy and fibrosis, and supports the notion that the promotion of successful autophagy is a clinically relevant target for therapeutic intervention.
Abstract 272: RBPJ Controls the Angiogenic Response of Mouse Adult Cardiomyocytes
Authors: Diaz Trelles Ramon , Scimia Maria Cecilia , Ruiz Lozano Pilar , Mercola Mark
Published: July 2021

Cardiac microvasculature density is critical for a correct cardiac function under normal and stress conditions. We found that the transcription factor RBPJ, downstream of the Notch signalling, can regulate angiogenic factors gene expression by repression (normal homeostasis) or activation (stress) and also by modulating the hypoxia induced angiogenic response.

Accordingly, in normal conditions cardiomyocyte specific RBPJ KO adult mice hearts show a denser microvasculature. Isolated mouse adult cardiomyocytes show increased gene expression and promoter hyperacetylation and hypermethylation of angiogenic factors and Notch target genes (like HES1).

Stress induced by myocardial infarction (MI) or cardiac overload (TAC) activate an angiogenic response to compensate the increased oxygen demand. Notch pathway is activated and RBPJ accumulated in the nucleus after MI and TAC. After TAC, deletion of RBPJ did not block hypertrophy induction, but prevented the increase in angiogenic factor production and microvessel density that normally occurs in response to increased workload. Remarkably, the KO preserved cardiac function and reduced cell death and fibrosis after myocardial infarction. Thus, RBPJ acts in cardiomyocytes as a master factor orchestrating homeostatic and disease-induced angiogenesis, and modulating RBPJ protects against ischemic injury.
Abstract 276: Cyclin D2 Is a Critical Mediator of Exercise-Induced Cardiac Hypertrophy
Authors: Luckey Stephen W , Haines Christopher D , Konhilas John P , Luczak Elizabeth D , Leinwand Leslie A
Published: July 2021

Exercise training activates a number of hypertrophic signaling pathways that can be distinct from those activated by pathologic stimuli. However, there must be some overlap in those pathways underlying myocyte cell growth. Using a number of mouse genetic models, we investigated the role of several molecules implicated in pathologic cardiac hypertrophy for their cardiac responses to exercise. We used three-month-old female transgenic mice expressing the anti-hypertrophic molecules, cardiac-specific constitutively active glycogen synthase kinase-3β (caGSK-3β), an inhibitor of Ca
2+
-calmodulin-dependent protein kinase (CaMKII Inh), and doubly transgenic mice expressing both caGSK-3β and CaMKII inhibition (caGSK-3β/CaMKII Inh). We also studied the exercise responsiveness of mice expressing the pro-hypertrophic cardiac-specific activated (myr)Akt. MAPK/ERK kinase kinase-1 (MEKK1) has been shown to be essential for pathologic cardiac hypertrophy and we therefore studied the requirement of MEKK1 for exercise-induced cardiac growth. Cell cycle regulators such as cyclin D2 have been shown to be required for pathologic cardiac hypertrophy; therefore we studied cyclin D2 null mice. Mice were divided into sedentary and 21 days of voluntary exercise on a cage wheel. Across the seven different mouse models, exercise capacity was similar with regards to running duration, distance, and speed. Importantly, we analyzed the impact of exercise on cardiac hypertrophy by measuring heart weight-to-body weight (HW/BW) ratios of sedentary and exercised mice. While exercise had no impact on body weight, heart weight increased significantly in all mouse models except the cyclin D2
-/-
mice. Overall there was a 3.5-fold range of percent increase in HW/BW ratios from the highest (caGSK-3β) to the lowest (cyclin D2
-/-
). In conclusion, genetic manipulation of these hypertrophic signaling pathways has little impact on exercise performance and only the loss of cyclin D2 attenuates exercise-induced cardiac growth. These data establish cyclin D2 as an important regulator of physiological hypertrophy and underscore the differences in pathologic and physiologic cardiac hypertrophy.
Abstract 292: Isoform-Specific Expression of Estrogen-Related Receptors Regulates Cardiomyocyte Metabolic Adaptation to Oxidative Stress
Authors: Cribben Kathryn , McDermott Paul
Published: July 2021

In the ischemic heart, metabolic adaptation of cardiomyocytes exposed to prolonged hypoxia is critical to sustain myocardial energy production. Metabolic gene expression is regulated by Estrogen-Related Receptor (ERR) transcription factors and Peroxisome Proliferator-Activated Receptor γ Coactivator-1 (PGC-1) co-activators. ERRs do not require ligand to achieve an active conformation and regulate an array of target genes involved in myocardial energy metabolism. These studies aim to determine the regulatory mechanisms governing expression of ERR isoforms (ERRα and ERRβ) in cardiomyocytes undergoing oxidative stress. We hypothesize that metabolic energy production in myocardium initially adapts to hypoxia by inducing transient ERRβ expression, and maintains this compensatory expression through induction of sustained ERRα expression.

Adult feline cardiomyocytes in primary culture were electrically stimulated to contract continuously at 1 Hz under normoxia (21% [O2]), hypoxia (0.5% [O2]), or hypoxia followed by reoxygenation. Expression of ERR, PGC-1 and target genes was measured by QRT-PCR (n≥5). QRT-PCR arrays were used to screen for prospective target genes.

In response to 24 hours of hypoxia, mRNA levels increased for ERRα (3-fold by 24 hours), ERRβ (11-fold by 12 hours), PGC-1α (16-fold by 24 hours) and PGC-1β (8-fold by 24 hours). Subsequent reoxygenation for 24 hours decreased ERRβ, PGC-1α and PGC-1β mRNA levels to that of normoxia controls. However, reoxygenation did not markedly decrease (∼25%; 0.7 of 3.0-fold) ERRα mRNA levels. Cytochrome C Oxidase Subunit VIc (Cox6c) and Fatty Acid Binding Protein 3 (Fabp3) were identified as potential ERRα target genes in adult cardiomyocytes based on correlation of expression. Moreover, hypoxia-induced increases in Cox6c and Fabp3 mRNA levels were partially blocked by treatment with XCT-790, an inhibitor of ERRα activity.

We conclude that hypoxia and subsequent reoxygenation elicit isoform-specific changes in ERR and PGC-1 mRNA expression in adult cardiomyocytes. This observed regulation of expression for ERR isoforms and their target genes may play a critical role in metabolic adaptation to oxidative stress.
Abstract 293: Mammalian Target of Rapamycin and Tristetraprolin Regulate Iron Homeostasis in Cardiac Cells
Authors: Bayeva Marina , Khechaduri Arineh , Ardehali Hossein
Published: July 2021

Introduction:
Iron is essential for normal heart function, and disruption of iron homeostasis can lead to cardiomyopathy. However, our understanding of iron regulation on a cellular level is incomplete, with a single model involving iron regulatory proteins (IRP) described to date. Here, we report the existence of a parallel iron regulatory pathway by energy sensor mTOR and inflammatory mediator trsitetraprolin (TTP).

Results:
To examine the role of energy metabolism in the regulation of cellular iron, we used rapamycin to inhibit mTOR pathway in H9c2 cardiac myoblasts and mouse embryonic fibroblasts (MEFs). Rapamycin treatment significantly elevated cellular iron content through a coordinated reduction in iron import (transferrin receptor, TfR1) and iron export (ferroportin, Fpn1), leading to deceleration of iron flux and net iron accumulation. We found that the primary action of rapamycin was to reduce TfR1 through destabilization of its mRNA. Surprisingly, this effect was not mediated by IRP1/2, the “classical” sensors of cellular iron levels, as TfR1 mRNA levels were significantly reduced by rapamycin even in cells with the genetic knockout of IRP1 and IRP2.

In yeast, a tandem zinc finger (TZF) protein Cth2 was found to conserve cellular iron in states of deficiency by preferentially degrading mRNA of non-essential iron-containing proteins thus reducing iron requirements and liberating iron for vital functions. We found that the mammalian TZF protein TTP, an established mediator of inflammation, was greatly induced by iron deficiency, enhanced degradation of iron-containing proteins, and complemented Cth2 deletion in yeast, thus establishing TTP as the functional homolog of Cth2 in mammalian iron regulation. Finally, TTP levels were increased by rapamycin in IRP1/2-independent manner, and genetic knockout of TTP in MEFs significantly reversed the effects of rapamycin on TfR1 mRNA levels and stability. These findings establish TTP as the mediator of iron-regulatory effects of mTOR and provide a novel link between energy metabolism, inflammation and iron regulatory pathways.

Conclusions:
We identified a novel pathway of cellular iron regulation by mTOR and TTP, which complements the “classical” IRP1/2 model.
Abstract 295: Hemodynamic Shear Stress Alterations Contribute to Calcific Aortic Valve Disease in a Frequency- and Magnitude-Dependent Manner
Authors: Sun Ling , LeCluyse John , Robillard Brian , Sucosky Philippe
Published: July 2021

INTRODUCTION:
Calcific aortic valve disease (CAVD) is an active process presumably triggered by interplays between atherogenic risk factors, molecular signaling networks and hemodynamic cues. While our earlier work demonstrated that progressive alterations in fluid wall-shear stress (WSS) on the fibrosa could trigger leaflet inflammation, the mechanisms of CAVD pathogenesis secondary to side-specific WSS abnormalities are poorly understood.

HYPOTHESIS:
Supported by our previous studies, we hypothesize that valve leaflets are sensitive to both WSS magnitude and pulsatility and that abnormalities in either promote CAVD development.

OBJECTIVE:
This study aims at elucidating
ex vivo
the contribution of isolated and combined alterations in WSS magnitude and pulsatility to valvular calcification.

METHODS:
The fibrosa and ventricularis of porcine leaflets were subjected simultaneously to different combinations of WSS magnitude and pulsatility (i.e., physiologic, sub- and supra-physiologic levels) for 48 hours in a double-sided shear stress bioreactor. Endothelial activation (ICAM-1, VCAM-1), paracrine expression (TGF-β and BMP-4), and proteinase/collagenase expression (MMP-2, cathepsin L) were detected by immunohistochemistry, while osteogenic differentiation (α-SMA) was assessed via western blot.

RESULTS:
Regardless of the magnitude or frequency, non-physiologic WSS conditions did not result in endothelial activation. Tissue exposure to either supra-physiologic WSS magnitude or pulsatility significantly upregulated paracrine (74-fold increase), proteinase (4-fold increase), collagenase (5-fold increase) and α-SMA (23-fold increase) expressions relative to the levels measured under physiologic WSS. In contrast, combined alterations in WSS magnitude and pulsatility downregulated those responses.

CONCLUSION:
This study demonstrates the sensitivity of aortic valve leaflets to both WSS magnitude and pulsatility and the ability of supra-physiologic WSS magnitude or pulsatility to trigger events involved in early CAVD pathogenesis. The results provide new potential insights into the mechanisms of CAVD secondary to hypertension and Paget’s disease, which are associated with abnormal blood flow and leaflet WSS.
Abstract 315: Coenzyme Q
10
Protects Against Diabetes-Induced Diastolic Dysfunction and Adverse Cardiac Remodeling in a Mouse Model of Type 1 Diabetes
Authors: Huynh Karina , Kiriazis Helen , Du Xiao-Jun , Love Jane E , Jandeleit-Dahm Karin , McMullen Julie R , Ritchie Rebecca H
Published: July 2021

Diastolic dysfunction is often the earliest manifestation of diabetic cardiomyopathy, usually accompanied by adverse cardiac remodeling and increased oxidative stress. We tested the hypothesis that administration of Coenzyme Q
10
(CoQ) attenuates type 1 diabetes-induced left ventricular (LV) dysfunction and remodeling. Further, we aimed to compare the efficacy of CoQ to the ACEI, ramipril. Male 6-week old mice received either streptozotocin (STZ, 55mg/kg/day for 5 days) to induce diabetes, or citrate buffer. After 4 weeks, mice were treated with either CoQ dissolved in olive oil (10mg/kg/day), olive oil alone, ramipril (3mg/kg/day) or left untreated for 8 weeks (n=11-14/group). Diabetic mice had increased blood glucose levels compared with non-diabetic controls. Superoxide (O
2
-
) production was enhanced in untreated diabetic mice, and attenuated with CoQ treatment. Diastolic function was impaired in diabetic mice, on Doppler echocardiography (E/A ratio, deceleration time DT) and catheterization (LV end diastolic pressure EDP and LV-dP/dt). Administration of CoQ ameliorated diastolic dysfunction on E/A ratio, DT and LVEDP in diabetic mice, with a similar trend on LV-dP/dt. Although DT and LVEDP were improved with ramipril treatment, E/A ratio was not. Diabetic mice also exhibited cardiomyocyte hypertrophy (H&E staining), cardiac fibrosis (Sirius red staining) and increased apoptosis. Both CoQ and ramipril reduced these markers of adverse LV remodeling. In conclusion, these data suggest that both CoQ and ramipril can attenuate diabetic cardiomyopathy. Addition of CoQ to standard care may offer improved treatment of diastolic dysfunction in diabetic patients.
Abstract 319: Adrenergic Hormones Regulate Cardiac Metabolic Health During Embryogenesis
Authors: Baker Candice , Osuala Kingsley , Ebert Steven
Published: July 2021

The adrenergic hormones, norepinephrine (NE) and epinephrine (EPI), are critically important during embryogenesis and cardiovascular development, yet their specific mechanisms of action remain mysterious. Disruption of the
dopamine β-hydroxylase (Dbh)
gene results in an inability to produce NE and EPI, which leads to death in utero between embryonic days 10.5 (E10.5) and E15.5 from apparent heart failure. To determine genes differentially expressed by the loss of adrenergic hormones, Affymetrix® GeneChip microarray analysis revealed 23 genes (of the 22,000 analyzed) that are significantly altered 2-fold or greater in E10.5
Dbh
-/-
hearts. Of these altered genes, 31% (7 of 23) are linked to metabolic regulation. The gene encoding for NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 2,
Ndufb2
, for example, is a subunit of the mitochondrial complex 1, and displayed a 2.1-fold change in
Dbh
-/-
embryonic hearts. This led us to investigate the metabolic health of these
Dbh
-/-
embryos. Initially, we measured adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels in E9.5, E10.5, and E11.5 embryos from
Dbh
-/-
,

Dbh
+/-

, and

Dbh
+/+

mice. At E9.5, ATP concentrations were similar in
Dbh
-/-
,

Dbh
+/-

, and

Dbh
+/+

embryos (
p
>0.7, n=6), but began to decline in the
Dbh
-/-
embryos at E10.5 compared to

Dbh
+/-

and

Dbh
+/+

embryos (
p
<0.02, n=5). By E11.5, ATP concentrations were markedly decreased in
Dbh
-/-
embryos, while ADP levels were increased 5-fold relative to

Dbh
+/+

controls. This resulted in a significant (
p
<0.01, n=4) and drastic (nearly 50-fold) difference in ATP/ADP ratio (0.4 ± 0.1) in
Dbh
-/-
compared with

Dbh
+/+

control embryos (20.7 ± 4.1) at E11.5. Despite this dramatic difference in energy levels, all embryos appeared grossly normal, had similar heart rates, and displayed no significant differences in caspase 3 or 7 cleavage activity. Thus, the energy deficiency observed in

Dbh
-/-

embryos did not appear to be associated with preceding signs of distress or demise. These results suggest that adrenergic hormones play an important role in regulating energy production during a critical phase of early development that roughly coincides with the transition from embryonic to fetal stages when increased energy utilization is needed.
Abstract 32: Investigating the Autoactivation of p38α Mitogen-Activated Protein Kinase Mediated by Transforming Growth Factor-β-Activated Protein Kinase Binding Protein 1
Authors: Felice De Nicola Gian , Martin E D , Bassi Rekha , Verma Sharwari , Conte Maria , Marber Michael S
Published: July 2021

Activation of p38α MAPK (p38α), by phosphorylation of two residues in the TGY motif of the activation loop, can occur independently of upstream kinases. One such mechanism involves the scaffolding protein Transforming Growth Factor-β-activated protein kinase binding protein 1 (TAB1). Under certain circumstances, such as myocardial ischemia, this activation can aggravate lethal injury. It is one of a few examples of activating autophosphorylation and poses a conundrum. How does an inactive kinase, and therefore with low affinity for ATP, phosphorylate its own activation loop when ATP binding is a prerequisite step for phosphotransfer? The aim of this study was to characterize the TAB1 binding of p38α. The binding characteristics of p38α and TAB1 were determined by Isothermal Titration Calorimetry, followed by the binding of p38α and ATPγS, a slowly hydrolysable form of ATP, in the presence and absence of TAB1. The binding of TAB1 to p38α increased significantly the affinity of p38α for ATP. Following the identification of a key region in TAB1 responsible for p38α binding, a synthetic peptide encompassing this region was used to analyze the biophysical and biological consequences of TAB1 binding.
In vitro
kinase assays were used to test the biochemical characteristics using a combination of wildtype kinase, kinase dead (K53M) or both in the absence or presence of TAB1(371-416). Using an antibody specific to the dual phosphorylation of the TGY motif as a readout, TAB1 binding to p38α increased p38α autophosphorylation
in cis
. NMR was employed to map the interaction surfaces between of p38α and TAB1 and to analyze the effect of TAB1-binding on p38α. The residues identified as important for the interaction between TAB1 and p38α were mutated and tested in cell free and biological systems to confirm their role as critical determinants for binding. In conclusion, we have further elucidated a mechanism whereby TAB1 binding to p38α alters the conformation of p38α, increasing its affinity for ATP and thereby facilitating autophosphorylation. We have identified the binding contacts of TAB1 and p38α that may be important in the design of therapeutics enabling selective and circumstance-specific inhibition of p38α activation.
Abstract 321: Cardiometabolic Recovery Kinetics Post Apheresis in a Model of Accelerated Atherothrombosis
Authors: Bhatia Neal K , Patel Aalok , Ramjee Vimal , Epperson Monica , Le Ngoc-Anh , Vaughn Louette , Nell-Dybdahl Christine , Baer Jefferson , Wilson Peter , Sperling Laurence
Published: July 2021

Background:
Atherothrombotic disease is a chronic, smoldering multifaceted process of inflammatory, lipid, and oxidative insult. Familial hypercholesterolemia (FH) serves as a model of accelerated disease in which these pathways may be better characterized.

Methods:
Eight FH patients were assessed before and after LDL apheresis at multiple time points for levels of: Non-esterified fatty acids (NEFA), lipoprotein-associated phospholipase A2 mass (PLAC), thiobarbituric acid reactive substances (TBARS), C-reactive protein (CRP), triglycerides (TG), insulin, and glucose.

Results:
Mean pre- and post-apheresis plasma levels were: 2.76 and 1.05 for CRP, 16.64 and 7.11 for insulin, 98.7 and 81.1 for glucose, 156 and 110 for TG, 177 and 97 for PLAC, and 0.5 and 0.72 for NEFA (p < 0.10, all). Plasma glucose, insulin, and TG levels showed concurrent post-prandial peaks at 12 and 36 hours post-apheresis (Fig 1). NEFA peaked during fasting states at 24 and 48 hours post-apheresis (Fig 1). Maximum mean % gain was: 33.7%[(1.98-1.05)/2.76] for CRP from 6 to 12 hours, followed by 24.1%[(0.48-0.35)/0.54] for TBARS and 15.2%[(123.2-96.29)/177.12] for PLAC from 12 to 24 hours.

Conclusion:
Our data show that LDL apheresis significantly decreases inflammatory, metabolic and oxidative burden in FH patients. Peaks in glucose, insulin and TG were within normal range arguing against post-prandial dysmetabolism in FH patients. NEFA increased with apheresis and fasting, suggestive of hormone-sensitive lipase upregulation. The temporal progression of recovery shows a predominant inflammatory process acutely, followed by high oxidative stress as a result of PLAC preponderance.
Abstract 322: Incubation With Histone Deacetylases 3 and 4 or p300/CREB Binding Protein Associated Factor Does Not Significantly Alter Cardiac Sarcomere Function
Authors: Walker John S , Li Xiaotao , Walker Lori A , Horn Todd , McKinsey Timothy , Buttrick Peter
Published: July 2021

Recent studies have suggested that treatment of skinned cardiac trabeculae with a histone deacetylase (HDAC) inhibitor increases calcium sensitivity, measured as the calcium required for half maximal force development (pCa50). The proposed mechanism is the acetylation of sarcomeric proteins by an endogenous histone acetyl transferase, p300/CREB binding protein associated factor (pCAF). As a test of this proposed mechanism, we treated skinned cardiac myocytes from 8 rat left ventricles (LV) with supra-physiological concentrations of pCAF, with Acetyl COA as an acetyl group donor, and supra-physiological concentrations of constitutively active forms of HDACs 3 and 4, and examined the force-calcium relation. Myocytes were prepared from frozen rat LV samples by gentle homogenization in rigor solution (mM: 50 Tris, 1 EGTA, 100 KCl, 2 MgCl2, 2 DTT and a protease inhibitor cocktail) containing 0.1% Triton X-100. The myocyte homogenates were washed and resuspended in relaxing solution (mM: 5 MgATP, 10 Creatine phosphate, 40 K-Propionate and 10-6 Ca-EGTA) divided into four 300 µl aliquots, One was an untreated control and three were treated with either i) HDAC3/Ncor or HDAC4 , ii) pCAF + 1mM Acetyl CoA, or ii) 1mM Acetyl CoA. Force-calcium relationships were determined in mixtures of relaxing and activating solution (mM: 5 MgATP, 10 Creatine phosphate, 0.5 K-Propionate, 10-1.5 Ca-EGTA) to give 6 calcium concentrations between 10-9 and 10-3.5 mM. While HDAC 3 or 4 treatment tended to decrease calcium sensitivity, and pCAF+Acetyl COA, and Acetyl COA alone, tended to increase calcium sensitivity compared to controls, we found no statistically significant effects of HDAC3 or 4, or pCAF on the calcium sensitivity, maximum developed force or co-operativity of the myocytes (ANOVA, n=8, P<0.05).
Abstract 323: Growth Differentiation Factor-15 Is Upregulated in Profibrotic States of Dilated Cardiomyopathy and Not Hypertrophic Cardiomyopathy
Authors: Chang Stephen , Christodoulou Danos , Gorham Joshua , Wakimoto Hiroko , Eminaga Seda , Conner Dave , Depalma Steve , Sparks Libby , Seidman Jonathan , Seidman Christine
Published: July 2021

Introduction:

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are leading causes of morbidity and mortality in the adult population. Both DCM and HCM arise from structural perturbations and remodeling of the heart, and many cases have been found to result from underlying familial mutations. The signaling pathways by which these mutations lead to pathological ventricular remodeling, fibrosis, and heart failure remain unknown.

Methods:

In this study we attempt to identify molecular pathways in a DCM mouse model and compare and contrast the results with those previously observed in HCM mouse models. We use a transgenic mouse expressing an arginine-to-cysteine (R9C) mutation in phospholamban (PLN), previously identified in DCM patients. To define molecules involved in disease progression, we generated expression profiles using high-throughput sequencing and assessed genes of interest by immunostaining.

Results:

PLN
R9C/+
hearts exhibit increasing fibrosis, with proliferation of non-myocyte cells occurring throughout the disease spectrum. Losartan treatment, which effectively blocks the HCM phenotype, does not block the emergence of DCM and neither reduces fibrosis nor increases lifespan in PLN
R9C/+
mice. Growth differentiation factor-15 (GDF15) RNA and protein levels are significantly upregulated in the left ventricles of PLN
R9C/+
mice during DCM and heart failure. GDF15 is predominantly expressed in non-myocytes in WT mice, but in PLN
R9C/+
mice, is upregulated almost 25-fold in myocytes and less than 1.5-fold in non-myocytes.

Conclusion:

Both fibrosis and the proliferation of non-myocytes in PLN
R9C/+
mouse hearts increase as the DCM phenotype worsens, similar to that observed in HCM mouse models. However, losartan has no effect on the DCM phenotype suggesting that the molecular pathways of DCM and HCM may be different. This study identifies GDF15 as a signaling molecule that may play a unique role in DCM. GDF15 is upregulated in myocytes during DCM, but remains relatively unchanged in HCM. Understanding the genetic mechanisms underlying the progression of disease and fibrosis in both DCM and HCM will help us to characterize the distinct pathways of both diseases, as well as identify new, more specific targets for therapy.
Abstract 331: HCN Overexpression in Failing Heart Possibly Contributes to Ventricular Arrhythmias
Authors: Kuwabara Yoshihiro , Kuwabara Koichiro , Takano Makoto , Kinoshita Hideyuki , Nakagawa Yasuaki , Usami Satoru , Nakao Kazuhiro , Minami Takeya , Yamada Yuko , Yamada Chinatsu , Shibata Junko , Nishikimi Toshio , Yasuno Shinji , Ueshima Kenji , Nakao Kazuwa
Published: July 2021

Accumulating evidence suggests increased ventricular expression of hyperpolarization-activated cation channels (HCNs) in hypertrophied and failing hearts contributes to the induction of arrhythmias. In this study, we addressed the capacity of HCNs blockade to prevent lethal arrhythmias associated with heart failure.

Transgenic mice expressing a dominant-negative mutant of neuron-restrictive silencer factor in a cardiac-specific manner (dnNRSF-Tg) exhibited dilated cardiomyopathy and sudden arrythmic death with an increase in ventricular HCNs expression, which are potentially responsible for the observed lethal arrhythmias. Ivabradine (Iva), a specific HCN channel inhibitor, significantly improved the survival among dnNRSF-Tg mice. Though echocardiographic, hemodynamic, and histological analyses showed no significant difference between Iva and control, ECG telemetric monitoring showed the significant reduction of arrhythmias in dnNRSF-Tg mice treated with Iva (VT; Iva 19/h, control 92/h ; p<0.05), suggesting that Iva improved the survival by preventing lethal arrhythmias.

We also found that the transgenic mice overexpressing HCN2 specifically in the heart (HCN2-Tg) are susceptible to ventricular arrhythmias induced by chronic isoproterenol infusion. In isolated ventricular myocytes from HCN2-Tg, but not in those from wild type mice, isoproterenol induced abnormal spontaneous action potentials, which were suppressed with Iva.

Our findings suggest that increased ventricular expression of HCN channels possibly contributes to the ventricular arrhythmias, and HCN channels blockade may represent a new and effective means of preventing sudden arrhythmic death in patients with heart failure.