bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 88: Small Molecule Inhibition of Mixed Lineage Kinase 3 Attenuates Cardiac Fibroblast Activation and Pathologic Cardiac Remodeling
Authors: Martin Melissa L , Dewhurst Stephen , Gelbard Harris A , Goodfellow Val , Blaxall Burns C
Published: July 2021

Heart failure (HF) is a manifestation of most cardiovascular diseases whose increasing prevalence highlights the need for novel therapeutics. The extent of pathologic cardiac remodeling is correlated with clinical outcome. Importantly, cardiac injury enhances cardiac fibroblast (CF) activation, producing myofibroblasts that release pro-fibrotic/inflammatory mediators which target cardiomyocytes (CM), CFs, and local inflammatory cells to exacerbate remodeling. Mixed Lineage Kinases (MLKs) are a family of stress-activated MAPKKKs whose functional role(s) in the heart remain largely unknown. MLK3 has been implicated in HIV-associated neurocognitive disorder (HAND), where it mediates deleterious cross-talk between microglia and neurons, suggesting an analogous mechanism of pathologic intercellular CF-CM communication in HF. We hypothesize that MLK3 exacerbates cardiac remodeling through enhanced CF activation that contributes to pathologic cardiac intercellular communication. We have synthesized a series of MLK3-specific small molecule inhibitors, one of which (URMC-099) was found to attenuate microglial-mediated neurotoxicity in murine models of HAND. To investigate the role of MLK3 in CF activation, neonatal rat ventricular CFs were stimulated with isoproterenol (Iso) or angiotensin II (AngII). Concurrent treatment with URMC-099 attenuated both α-smooth muscle actin expression, indicative of myofibroblast transition, and proinflammatory cytokine production. Further, therapeutic efficacy and specificity of URMC-099 were tested in iso-infused and myocardial infarction (MI) models of HF using wild-type (WT) and MLK3-/- mice (shown to have no overt cardiac phenotype). URMC-099 significantly attenuated cardiac hypertrophy (HW:BW) and reduced interstitial fibrosis (assessed by Masson's Trichrome staining) in an acute iso-pump model of HF in WT mice. Current echocardiographic data post-MI suggest cardioprotection in the MLK3-/- and URMC-099 treated mice. In conclusion, our collaborative data not only suggest a role for MLK3 in cardiac remodeling through pathologic CF activation but indicate a possibly novel paradigm of pathologic intercellular communication in multiple disease states.
Abstract 92: The Protective Effect of β3-Adrenergic Signaling Occurs via Differential Phosphorylation of nNOS in Cardiac Myocytes
Authors: Watts Vabren L , Sepulveda Fernando M , Cingolani Oscar H , Niu Xiaolin , Miller Karen L , Kim Rosa , Vandegaer Konrad , Barouch Lili A
Published: July 2021

Objective:

The aim of this study is to identify the mechanisms involved in β3-adrenoreceptor (β3-AR)-dependent cardiac protection via nNOS signaling in cardiomyocytes in the setting of hypertrophy. Background: β3-AR and its downstream signaling pathways are recognized as novel modulators of heart function. Unlike β1- and β2-ARs, β3-ARs are stimulated at high catecholamine concentrations and induce negative inotropic effects, it serves as a “brake” to protect the heart from catecholamine overstimulation. We previously showed that β3-agonism restored left ventricular function, generated nitric oxide, and suppressed reactive oxygen species in mouse myocardium after pressure overload. Interestingly, cardioprotection was lost after acute nNOS inhibition and in nNOS
-/-
animals.

Methods:

Neonatal rat ventricular cardiomyocytes (NRVMs) were isolated from 2-4 day old Sprague-Dawley pups. Cells were treated with hypertrophic agents (angiotensin II, endothelin-1, and norepinephrine), the specific β3-AR agonist (BRL-37433),and phosphometic Sindbis viruses for nNOS.

Results:

Forty-eight hours of ET-1 (100nM) or 72 hours of NE (100μ M) treatment increased cell size and β3-AR mRNA expression vs. untreated cells. In hypertrophied cardiomyocytes, BRL (75nM) reduced cell size and induced NOS activity, nNOS phosphorylation at stimulatory site Ser1412, dephosphorylation of deactivation site Ser847, and ROS suppression. BRL-dependent NOS activity and ROS suppression were both attenuated by the nNOS inhibitor L-VNIO. NOS activity was also attenuated by phosphomemetic mutants Ser1412A (constitutively dephosphorylated) and Ser847D (constitutively phosphorylated). In addition, G
i/o
and Akt inhibition suppressed BRL-induced nNOS-Ser1412 phosphorylation and NOS activity.

Conclusion:

Our data suggest that BRL regulates β3-specific myocardial NOS activity via alterations in nNOS phosphorylation in isolated hypertrophied myocytes and failing hearts of murine animals. This is the first study to demonstrate a role for nNOS phosphorylation as a key factor in cardiac myocyte and β3-AR signaling. These results contribute significantly to our understanding of the cardiac protection.
Abstract 97: Ablation of Calpain-Targeted Site in Cardiac Myosin Binding Protein C Is Cardioprotective
Authors: Sadayappan Sakthivel , Ji Xiang , Barefield David , Sarkey Jason , Govindan Suresh , Aitmou Younss , Zhu Guangshuo , Takimoto Eiki , Luther Pradeep K , Dobrev Dobromir , Kass David A , de Tombe Pieter P
Published: July 2021

Rationale:
Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function. We recently demonstrated a direct correlation between cMyBP-C dephosphorylation and its degradation during ischemia-reperfusion (I-R) injury. Strikingly, cMyBP-C phosphorylation protects the heart from I-R injury. However, the mechanism of cMyBP-C-mediated cardioprotection is unknown.

Objective:
To determine if preventing cMyBP-C proteolysis and cleavage confers cardioprotection during I-R injury.

Methods and Results:
cMyBP-C is a substrate for calpains and dephosphorylation makes cMyBP-C more susceptible to proteolysis. In patients with heart failure, such proteolysis leads to the cleavage and release of the predominant N'-fragment (40 kDa) in association with increased calpain activity. MS/MS analysis identified a cardiac-specific phosphorylation motif responsible for the release of the 40 kDa fragment, 272-TSLAGAGRR-280, which we term the calpain-targeted site (CTS). Next, we utilized cardiac-specific transgenic mice expressing cMyBP-C[[Unable to Display Character: △]]CTS in which CTS motif was ablated and bred into cMyBP-C null (t/t) mice. We hypothesized that ablation of the CTS motif would confer resistance to calpain-mediated proteolysis and thus protect the heart from I-R injury. An animal that transgenically expressed the normal cardiac isoform, cMyBP-CWT, served as a control. Histological, electron microscopic and immunofluorescence analyses confirm that cMyBP-C[[Unable to Display Character: ∆]]CTS incorporates normally into the cardiac sarcomere and results in complete rescue of the cMyBP-Ct/t phenotype, compared control groups. Moreover, echocardiography and in vivo hemodynamic studies revealed that cMyBP-C[[Unable to Display Character: ∆]]CTS:t/t mice have normal cardiac function, similar to control mice. Altogether, these data suggest that cMyBP-C[[Unable to Display Character: ∆]]CTS is benign. Compared to cMyBP-CWT, cMyBP-C[[Unable to Display Character: ∆]]CTS protein is protected from calpain-mediated degradation. Finally, we determined that ablation of the CTS reduced infarct size and preserved cMyBP-C stability and cardiac function during I-R injury.

Conclusion:
Our data suggest that preserving cMyBP-C stability by thwarting calpain-mediated proteolysis protects cardiac structure and function from I-R injury, as a supportive therapy.
Abstract 011: Transcriptional Basis Of Epicardial Progenitor Cell Activation During Heart Development, Regeneration And Repair
Authors: Huang Guo N , Thatcher Jeffrey E , McAnally John , Kong Yongli , Qi Xiaoxia , Tan Wei , DiMaio J. Michael , Amatruda James F , Gerard Robert , Hill Joseph A , Bassel-Duby Rhonda , Olson Eric N
Published: July 2021

The epicardium encapsulates the heart and functions as a source of multipotent progenitor cells and paracrine factors essential for cardiac development and repair. Injury of the adult heart results in reactivation of epicardial progenitor cells, which reengages a developmental gene program, but the underlying transcriptional basis has not been delineated. We established a mouse embryonic heart organ culture and gene expression system that facilitated the identification of epicardial enhancers activated during heart development and injury. Epicardial activation of these enhancers depends on a combinatorial transcriptional code centered on C/EBP, HOX, MEIS, and GRAINYHEAD families of transcription factors. Furthermore, disruption of C/EBP signaling in the adult epicardium reduced injury-induced neutrophil infiltration and improved cardiac function. These findings reveal a transcriptional basis for epicardial progenitor cell activation during heart development and injury, providing a platform for enhancing cardioprotection and regeneration.
Abstract 027: Therapeutic Potential of a Novel Necrosis Inhibitor in Myocardial Ischemia-Reperfusion Injury
Authors: Cho Hyun-Jai , Hwang In-Chang , Kim Ju-Young , Kim Ji-Hyun , Kwon Yoo-Wook , Park Jonghanne , Yang Han-Mo , Park Kyung-Woo , Kim Soon-Ha , Kim Hyo-Soo
Published: July 2021

Background:
Reperfusion, although essential for salvage of myocardium in the myocardial infarction, paradoxically causes a wide variety of injuries. The opening of the mitochondrial permeability pore and Ca
2+
overload contribute to myocardial ischemia-reperfusion (I/R) injury.

Objectives:
Necrosis, the main mechanism of cell death during I/R injury to the myocardium, is an uncontrolled cell death, a pathologic condition accompanying inflammatory responses. We aimed to examine the protective role of this novel necrosis inhibitor against myocardial I/R injury using in vitro and in vivo models through anti-necrosis pathway.

Methods and Results:
Rat cardiomyocytes were exposed to hypoxia-reoxygenation injury after pretreatment with dimethyl sulfoxide (vehicle), necrosis inhibitor (NecX), antioxidant (vitamin C) or apoptosis inhibitor (Z-VAD-fmk). NecX-treated cells, compared with vehicle, showed fewer necrosis (Annexin-V/PI) (13.5±1.9% versus 44.1±3.1%; P=0.049) and more viable cells (fluorescein diacetate) (98.0±0.5% versus 51.3±2.1%; P=0.021). We next analyzed the mechanisms of cell death, mitochondrial membrane potential and mitochondrial Ca
2+
level. NecX-treated group showed higher mitochondrial membrane potential and lower Ca
2+
level, resulting in the prevention of mitochondrial swelling and necrosis. In the rat model of myocardial ischemia for 45 minutes followed by reperfusion, we compared the therapeutic efficacy of NecX and cyclosporine A (CsA) with 5% dextrose (control), each administrated 5 minutes before reperfusion. Pretreatment with NecX markedly inhibited myocardial necrosis (NecX, 7.8±7.8%; control, 65.4±2.4%, P=0.017; CsA, 32.3±5.1%, P=0.041) and reduced the area of fibrosis (NecX, 4.8±0.9%; control, 25.7±1.6%, P=0.011; CsA, 18.8±1.3%, P=0.006). Additionally, it preserved systolic function and prevented pathologic remodeling of left ventricle.

Conclusion:
The novel necrosis inhibitor demonstrates a significant protective effect against myocardial I/R injury and has advantages over CsA, based more on the direct necrosis inhibition on cardiomyocytes, indicating that it is a promising candidate for cardioprotective adjunctive therapy with reperfusion in patients with myocardial infarction.
Abstract 037: Transient Cardiac Expression Of Constitutively Active G alpha Q Activates Renin-angiotensin System, Leading To Progressive Heart Failure And Ventricular Arrhythmias In Transgenic Mice
Authors: Matsushita Naoko , hirose Masamichi , Taeishi Yasuchika , Suzuki Satoshi , Kashihara Toshihide , Mende Ulrike , Taira Eiichi , Yamada Mitsuhiko
Published: July 2021

Introduction:
Transgenic mice with transient cardiac expression of constitutively active Galpha q (Gαq-TG) caused progressive heart failure and ventricular arrhythmias after the initiating stimulus becomes undetectable. However, the mechanisms are still unknown. Renin-angiotensin system plays a critical role in the development of cardiac hypertrophy and heart failure. We examined the effects of chronic administration of olmesartan on ventricular function, the number of premature ventricular contractions (PVC), and ventricular remodeling in Gαq-TG mice.

Methods and Results:
Olmesartan (1 mg/kg/day) or vehicle was chronically administered to Gαq-TG from 6 to 32 weeks of age, and all experiments were performed in mice at the age of 32 weeks. Chronic olmesartan treatment prevented the severe reduction of left ventricular fractional shortening and inhibited ventricular interstitial fibrosis and ventricular myocyte hypertrophy in Gαq-TG. Electrocardiogram demonstrated that premature ventricular contraction (PVC) was frequently (more than 20 beats/min) observed in 9 of 10 vehicle-treated Gαq-TG but in none of 10 olmesartan -treated Gαq-TG. The QT interval was significantly shorter in olmesartan-treated Gαq-TG than vehicle-treated Gαq-TG. CTGF, collagen type 1, ANP, BNP, and β-MHC gene expression was increased in vehicle-treated Gαq-TG. Olmesartan significantly decreased these gene expressions in Gαq-TG. Moreover, protein expressions of canonical transient receptor potential (TRPC) channels 3 and 6 increased in vehicle-treated Gαq-TG hearts. Olmesartan significantly decreased TRPC6 expressions in Gαq-TG. Angiotensin converting enzyme (ACE) 1 and 2 gene expressions were also increased in vehicle-treated Gαq-TG and was not decreased to the control level in olmesartan-treated Gαq-TG.

Conclusions:
These findings suggest that renin-angiotensin system has an important role in the development of cardiac hypertrophy and heart failure even if the initiating stimulus is different from the activation of renin-angiotensin system.
Abstract 044: Transient Receptor Potential Channel 6 (TRPC6) Is An Important Mediator Of Mechanical Stretch Responses In The Atrial Endocardial Endothelium.
Authors: Nikolova-Krstevski Vesna , Wagner Soeren , Friedrich Oliver , Fatkin Diane
Published: July 2021

Atrial fibrillation (AF) is the most common heart arrhythmia and a major risk factor for thromboembolic stroke and hearth failure. Atrial pressure and/or volume overload that cause increased mechanical stretch in the atrium are the common features of the diseases that cause AF. The latter suggests that mechanical stress has a major role in the pathogenesis of AF. The atrial endocardial endothelium (AE) is the interface between the myocardium and the circulating blood but its role in the mechanotransduction in the atrium is unknown. Endothelial cells (EC) are very sensitive and responsive to external mechanical stimuli and AE dysfunction has been reported in individuals with AF. We therefore hypothesized that the AE is an important mediator of stretch responses in the atrium and that stretch-induced AE dysfunction may be critical for initiation and/or maintenance of AF. We investigated the AE responses to increased mechanical stress by subjecting novel primary AE cells to mechanical stretch. The mechanical stretch conditions included cyclic stretch of 30 cycles/min at 10 percent displacement. The AE cells were stretched for 1-10 min, and 1, 4, 8, and 24h. Changes in the cell shape with elongation, hypertrophy and re-alignment of the cells and their stress fibres in a direction perpendicular to the lines of stretch were first observed after 1h of stretch. TRPC6 is a stretch sensitive ion channel and important regulator of Ca2+-signalling in the EC cells. Immunostaining and Western blot analysis showed redistribution and changed levels of expression of TRPC6 in the stretched AE cells, respectively. The latter suggested changed TRPC6 activity which was evaluated by measuring extracellular Ca2+ influx through the TRPC6 channels in the AE cells under baseline and stretch conditions. We found that short-stretch (1-10min) increased TRPC6 activity, while the long stretch (1-24h) silenced it. Based on these findings it could be concluded that TRPC6 is an important mediator of the mechanical stretch responses in the AE; the functional consequences of the altered channel activity are predetermined by the duration of the stretch-stimulus; and TRPC6 changes in the stretched AE may cause AE dysfunction through altered Ca2+ signalling that contributes to AF development.
Abstract 046: A Potential MicroRNA Biomarker for Atherosclerotic Lesions in Baboons
Authors: Karere G M , Glenn J P , Birnbaum S , Kuhn N , Lange K , Christensen C , Rice K , Mahaney M , Havill L , VandeBerg J L , Cox L A
Published: July 2021

Introduction:
Cardiovascular disease is the leading cause of death in developed countries and commonly due to atherosclerosis. Atherosclerosis is a resultant of interplay of gene and environmental factors including diet. Liver is the primary target for lipid metabolism and circulatory microRNAs (miRNAs) are potential biomarkers for atherosclerosis. However, little information is available on miRNAs for early prediction and diagnosis of severity of atherosclerosis.

Hypothesis:
We hypothesized that peripheral blood mononuclear cell (PBMCs) miRNA(s) are potential biomarkers for atherosclerosis. We tested this by first identifying PBMCs and liver miRNAs with discordant expression in baboons with low and high serum low density-lipoprotein cholesterol (LDL-C) concentrations and then determined if the miRNAs correlated with atherosclerotic lesions in baboons fed a high fat diet for 2 years.

Methods and Results:
Using Next Gen methods, we sequenced PBMCs and liver small RNA libraries from baboons discordant for LDL-C concentrations (low LDL-C, n = 3; high LDL-C, n = 3); fed a high-cholesterol, high-fat (HCHF) diet for 7 weeks. We identified 47 and 27 novel baboon miRNAs, and 494 and 490 human homologues in PBMCs and in liver, respectively. Seventy-one and 30 miRNAs showed differential response to HCHF diet in PBMCs and in liver, respectively. Comparison of miRNA expression profiles from PBMCs and liver showed concordant expression for miR-221/222 family; it was down-regulated in high LDL-C baboons in response to HCHF diet. Using qRT-PCR, we quantified miR-221 abundance in PBMCs and in livers of high LDL-C baboons (n = 109) on 2-year HCHF challenge and revealed, for hepatic miR-221, a positive correlation with severity of lesions in descending aorta (Pearson correlation coefficient, r = 0.18, p = 0.05) and aortic arch (r = 0.22, p = 0.03).

Conclusions:
Liver miR-221 abundance is associated with extent of lesions in baboons fed a HCHF diet for 2 years and may be a biomarker for atherosclerosis.
Abstract 063: Rapid Induction of Aldosterone Synthesis in Cultured Neonatal Rat Cardiomyocytes under High Glucose Conditions
Authors: Nagoshi Tomohisa , Fujisaki Masami , Nishikawa Tetsuo , Date Taro , Yoshimura Michihiro
Published: July 2021

Background:
In addition to the classical adrenal cortical biosynthetic pathway, there is increasing evidence that aldosterone is produced in extra-adrenal tissues. Although we have previously reported that aldosterone is produced in the heart, the concept of cardiac aldosterone synthesis remains controversial. This is partly due to the lack of established experimental models representing the aldosterone synthase gene (CYP11B2) expression in a robustly reproducible fashion. Therefore, in this study, we investigated suitable conditions in a neonatal rat cardiomyocytes culture system producing CYP11B2 with considerable efficacy.

Methods and Results:
Neonatal rat cardiomyocytes were cultured in serum-free medium containing various doses of glucose for two to 24 hours. The CYP11B2 mRNA expression in the cardiomyocytes was determined using real-time PCR, and the aldosterone concentrations secreted from the cardiomyocytes into the culture medium were measured using an enzyme immunoassay. We found that suitable conditions for inducing the cardiac aldosterone gene expression included four hours of incubation with high glucose conditions (>25 mM). Under these particular conditions, the CYP11B2 expression (2.18±0.37-fold, p<0.01), in accordance with aldosterone secretion, was significantly increased compared to that observed in the cells cultured under standard glucose condition (5.5 mM).

Conclusions:
The suitable conditions for detecting the CYP11B2 expression and aldosterone secretion in the neonatal rat cardiomyocytes culture system are now clarified: high glucose conditions with a relatively brief period of culture promote the CYP11B2 expression as well as aldosterone production/secretion in cardiomyocytes. The current system will help to accelerate further progress in research on cardiac tissue aldosterone synthesis.
Abstract 083: Macrophages Dictate the Progression and Manifestation of Hypertensive Heart Disease in Rat
Authors: KAIN DAVID , Yagil Chana , Landa-Rouben Natalie , Yagil Yoram , Leor Jonathan
Published: July 2021

Introduction:
Inflammation has been implicated in the initiation, progression and manifestation of hypertension. However, the role of macrophages in hypertension-induced LV remodeling has yet to be determined.

Hypothesis:
We assessed the hypothesis that macrophages play a significant role in the initiation and progression of hypertension-induced LV remodeling.

Methods and Results:
Hypertension was induced in male Sabra salt-sensitive (SBH/y) rats with high salt diet (8%NaCl) over 6 weeks. Hypertensive SBH/y developed LV hypertrophy, hyper-contractility and a higher LV ejection fraction (EF). Notably, the number of macrophages, particularly cardiac macrophages was significantly greater in hypertensive SBH/y than in controls. Macrophage depletion was induced after induction of hypertension by intravenous administration of clodronate liposomes at 3-day intervals over 4 weeks (n=9) and was validated by FACS analysis and ED1 (CD68) staining of heart sections. Control hypertensive rats (n=9) were treated with PBS liposomes. Surprisingly, macrophage depletion attenuate the progression of hypertension in the clodronate-treated group (from 177±2 to 179±2 mmHg) compared with controls (from 179±2 to 184 ± 5mmHg; p=0.01).Serial echocardiography studies before and 30 days after initiation of macrophage depletion or control showed that LV systolic diameter and volume were smaller; anterior wall thickening, fractional shortening and EF were higher in the macrophage-depletion group (p<0.05). LV strain and strain rate were significantly higher in the macrophage depletion group (p=0.04). Furthermore, 1 month after BP elevation, the expression of miR-31, which has been implicated in the pathogenesis of cardiac hypertrophy, was decreased (4.6-fold) in the macrophage-depleted hearts compared to controls, suggesting that macrophages control miR-31 expression in LV hypertrophy.

Conclusion:
Our findings suggest a significant role of macrophages in the initiation and progression of salt-sensitive hypertension and the related heart disease. Targeting macrophages, therefore, could be a potential therapeutic target for the treatment of hypertension and prevention of LV remodeling and dysfunction.
Abstract 084: Dkk1 Knock out Leads To Augmentation Of Cardiac Hypertrophy Following Aortic Banding
Authors: Riffel Johannes H , Bernhold Elmar , Hagenmueller Marco , Zhang Min , Niehrs Christof , Katus Hugo A , Hardt Stefan E
Published: July 2021

Background:
Myocardial hypertrophy is an important risk factor for cardiac morbidity and mortality. In the normal adult heart, Wnt signaling remains quiescent. However, recent studies have demonstrated reactivation of Wnt signaling in hypertrophic growth of cardiomyocytes. Under such conditions Wnt signaling may be beneficial or maladaptive depending on the context. The Wnt coreceptors LRP5 and LRP6 are important for signal transmission via the β-catenin pathway and are negatively regulated by Dkk1, a member of a small family of secretory proteins. Dkk1 binds to LRP6 and thereby acts as a Wnt antagonist. In our study we investigated the cardiovascular phenotype of Dkk1 knock-out mice following aortic banding.

Study Design and Results:
Dkk1 (+/-) knock-out mice were subjected to aortic banding (AB) or sham operation. After 4 weeks echocardiographic and invasive measurements were performed. After that the mice were euthanized, heart weight was measured and myocardial samples were snap frozen for biochemical measurements or fixed in formalin for further histological evaluation. Under baseline conditions there were no differences in cardiomyocyte size, heart weight and cardiac function in Dkk1 knock-out animals compared to wild type animals. 4 weeks after aortic banding we observed a significant increase in heart weight/body weight ratio in Dkk1 knock out animals compared to the control group (7.3 ± 0.3 mg/g vs. 6.4 ± 0.3 mg/g, p < 0.05). Furthermore cardiomyocyte size was highly elevated in Dkk1 knock out mice compared to control animals, suggesting an augmentation in cardiac hypertrophy. Transcription levels of the pro-hypertrophic markers atrial natriuretic factor (ANF) and beta-MHC were increased in Dkk1 knock out animals. Interestingly echocardiographic data revealed an aggravation of cardiac function in Dkk1 knock out mice following aortic banding (Ejection fraction (EF): 62 ± 3 % vs. 74 ± 1 %, p < 0.05).

Summary:
Our findings suggest that Dkk1 knock out aggravates cardiac hypertrophy following aortic banding. The underlying molecular mechanisms remain to be further explored.
Abstract 093: Adrenergic Stimulation Induces Mitochondrial Fragmentation and Cell Injury through PKD1-dependent Phosphorylation of DLP1 in H9c2 Cardiac Myoblasts.
Authors: Jhun Bong Sook , O-Uchi Jin , Hurst Stephen , Sheu Shey-Shing
Published: July 2021

Introduction:
Regulation of mitochondrial morphology and dynamics is crucial for the maintenance of various cellular functions in cardiac myocytes. Abnormal mitochondrial morphologies concomitant with mitochondrial dysfunction are frequently observed in various pathophysiological states of human heart such as heart failure, where the catecholamine level is elevated. However, it is still unclear what kinds of cardiac signaling pathways regulate mitochondrial morphology and function under pathophysiological conditions.

Hypothesis:
Adrenergic signaling induces cardiac mitochondrial morphology changes and mitochondrial dysfunction, which simultaneously contribute to cardiac injury.

Methods:
H9c2 cardiac myoblasts were stimulated by α
1
-adrenoceptor (α
1
-AR) agonist phenylephrine and mitochondrial morphology was monitored by confocal microscopy. Translocation and phosphorylation of a mitochondrial fission protein, dynamin-like protein 1 (DLP1) was observed from whole cell lysates, cytosolic proteins and mitochondrial proteins by western blotting.

Results:
We found that persistent α
1
-AR stimulation induced mitochondrial fragmentation, followed by an increase in the production of mitochondrial reactive oxygen species (ROS) and the release of cytochrome c from mitochondria to the cytosol in H9c2 cardiac myoblasts. These effects were abolished by the treatment of α
1
-AR antagonist, prazosin. Further, mitochondrial fragmentation by α
1
-AR stimulation was inhibited by expression of the dominant-negative fission mutant DLP1-K38A, suggesting that the mitochondrial fission is required for mitochondrial fragmentation observed in α
1
-AR stimulation. We also found that DLP1 was translocated from cytosol to mitochondria under α
1
-AR stimulation. In addition, activation of protein kinase D1 (PKD1), a protein kinase downstream of α
1
-AR signaling, led to the phosphorylation of DLP1 at serine 637 which lies within a putative PKD phosphorylation consensus motif.

Conclusion:
α
1
-AR signaling induces mitochondrial fragmentation and cell injury, possibly through PKD1-dependent phosphorylation of DLP1.
Abstract 119: PRMT5 Is A New Epigenetic Regulator In Cardiovascular Disease
Authors: Merkel Tobias S , Hassel David , Krüger Marcus , Weichenhan Dieter , Plass Christoph , Backs Johannes
Published: July 2021

Protein arginine methylation is an abundant posttranslational modification involved in various cellular events such as DNA repair, splicing, and transcription. Protein arginine methyltransferases (PRMTs) catalyze the symmetric and asymmetric dimethylation of arginines and can be subdivided into two distinct families: type I enzymes catalyze the formation of asymmetric dimethylation whereas type II enzymes enhance the addition of symmetric methyl groups.

Here, we report evidence that PRMT5 a type II enzyme serves as an important epigenetic regulator in myocardial disease.

We identified PRMT5 via mass spectrometry as an interactant of class IIa histone deacetylases (HDACs). Additional cardiac interaction partners of PRMT5 were identified by a human cDNA library based yeast-two-hybrid screen. Thereby we elucidated Interleukin enhancer binding factor 3 (ILF3) as a putative interacting protein. ILF3 is a member of the NFAT family. NFAT signalling is known to be a potent driver of cardiomyocyte hypertrophy. Accordingly, we detected a 40% decrease in the size of neonatal rat ventricular myocytes in response to siRNA mediated knockdown of
prmt5
. A dominant-negative version of PRMT5 is capable of interfering with the Calcineurin-NFAT axis and blocks NFAT activation.
In vivo
knockdown of endogenous
prmt5
in the zebrafish led to a distinct cardiac phenotype with altered morphology and impaired function. To further elucidate
prmt5
function
in vivo
we use mice with floxed
prmt5
alleles. As a loss of
prmt5
is lethal in the ES cell stadium we use an inducible α myosin heavy chain Mer-Cre-Mer system. Knockout mice display a complete loss of PRMT5 in cardiomyocytes. In line with our
in vitro
studies these mice are viable and show no phenotype under baseline conditions.

Our work in progress investigates the hypothesis that class IIa HDACs serve as a scaffold to recruit PRMT5 to the DNA, forming a complex with ILF3 to block NFAT activity and the pro-hypertrophic gene program.
Abstract 140: Knockdown of Nucleosome Assembly Protein 1-like 1 Promotes Cardiomyocytes Differentiation by Mesoderm induction through Notch Signaling in Mouse Induced Pluripotent Stem Cells
Authors: Gong Hui , Yan Yuan , Xue Yuanyuan , Yin Peipei , Zhang Guoping , Chen Zhidan , Ye Yong , Yang Chunjie , Yuan Jie , Ge Junbo , Zou Yunzeng
Published: July 2021

Recently, we used a functional proteomic analysis to screen out nucleosome assembly protein 1-like 1 (Nap1l1) which was downregulated during the differentiation of P19CL6 cells into cardiomyocytes. Here, we attempted to study the role of Nap1l1 in the cardiogenesis of mouse iPSCs. We observed Nap1l1 was downregulated during the differentiation of iPSCs. Knockdown of Nap1l1 dramatically enhanced the differentiation of iPSCs to cardiomyocytes characterized by the increased number of beating embryonic bodies (EBs), the larger alpha-myosin heavy chain (α-MHC)-stained area and the upregulation of cardiac transcription factors (Nkx2.5, GATA4, Mef2c, Tbx5). The effects were sharply inhibited by Nap1l1 overexpression in iPSCs. Cardiomyocytes derived from Nap1l1-knockdown-iPSCs exhibited proper cell biological characteristics. Further study revealed that Nap1l1 knockdown in iPSCs promoted mesoderm (Flk-1, Brachyury and Mesp1) development, but Nap1l1 overexpression inhibited the effect. To explore whether Nap1l1 knockdown in iPSCs enhances cardiomyocytes differentiation by mesoderm induction. Mesoderm cells (Flk-1 positive cells) from iPSCs development were sorted by fluorescent-assisted cell sorting (FACS) and recultured to induce cardiomyocytes differentiation. The result revealed that the same number of Flk-1(mesodermal marker) positive cells from Nap1l1 knockdown, Nap1l1 overexpression or their control iPSCs didn’t show obvious difference in cardiomyocyte differentiation. Loss of Notch signaling in ES cells has been reported to favor commitment to a mesoderm and to induce cardiogenesis. The present study revealed that NICD and downstream genes (Hes1, Hes5, Hey1 and Hey 2) were positively regulated by Nap1l1 expression during differentiation of iPSCs. Notch signaling inhibitor greatly rescued the inhibitory effects of Nap1l1 overexpression on mesoderm induction and cardiogenesis. These findings demonstrate that downregulation of Nap1l1 significantly enhances mesodermal induction and subsequently promotes cardiogenesis from mouse iPSCs via regulating Notch signaling, which will facilitate application of iPSCs for heart diseases.
Abstract 144: An Intact NEDD8 Pathway Protects Cardiomyocytes against Proteotoxic Stress
Authors: Su Huabo , Li Jie , Ma Wenxia
Published: July 2021

NEDD8 is a ubiquitin (Ub)-like protein whose modification on cellular proteins is crucial to various cellular processes. Conjugation of NEDD8 to target proteins (neddylation) is mediated by its unique set of E1, E2 and E3, and can be reversed by the actions of deneddylase such as the COP9 signalosome (CSN) and NEDP1, a process termed deneddylation. Canonical neddylation involves NEDD8 E1 (NAE1) and directly attaches NEDD8 to the substrate, whereas atypical neddylation utilizes a Ub activating enzyme (UBE1) to incorporate NEDD8 to the Ub chain of target proteins. The physiological significance of such atypical neddylation remains unknown in any cell types. Here we report a protective role of atypical neddylation to proteotoxic stress in cardiomyocytes. Proteasome inhibitor Bortezomib (BZM) increases high molecular weight species of neddylated proteins in a dose- and time- dependent manner. Immunoprecipitation analysis reveals mixed modification of these proteins by Ub and NEDD8. Silencing of UBE1, but not inhibition of NAE1, diminishes BZM-induced neddylation response and aggravates the BZM-induced cell death. Interestingly, forced expression of deneddylase NEDP1 also reduces BZM-induced neddylation but surprisingly attenuates cell injury. The protective effect of NEDP1 is dependent on its deneddylation activity. We thus propose that atypical neddylation is an adaptive response and that dynamic cycling of neddylation and deneddylation is essential to cardiomyocytes to antagonize proteotoxic stress, possibly through preventing depletion of ubiquitin. Additionally, imposing proteotoxic stress by H2O2, simulated ischemia reperfusion or expression of misfolded proteins all elicits such atypical neddylation in cardiomyocytes. Furthermore, increased neddylated proteins are also evident in mouse hearts suffering from proteotoxic stress, which is caused by either expression of misfolded proteins or deficiency of a 19S proteasome subunit. These findings, combined with our previous reports that the CSN regulates proteasomal and autophagic proteolysis in the heart, strongly suggest that a fine-tuned NEDD8 system is critical to protein quality control and is essential to cardiomyocyte survival and functioning.
Abstract 150: FAK/Pyk2 Inhibitor Prevents Mitochondrial Ca
2
+ Overload and Cardiac Injury during Adrenergic Stimulation.
Authors: O-Uchi Jin , Jhun Bong Sook , Hurst Stephen , Sheu Shey-Shing
Published: July 2021

Introduction:
Proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK) are abundantly
expressed in cancer
cells. In addition, these kinases are the potent therapeutic targets for cancer treatment and currently several selective FAK/Pyk2 inhibitors are in clinical trials. Recent study revealed that Pyk2 is also highly expressed in heart tissue and significantly activated during human heart failure. We have recently reported that α
1
-adrenoceptor (α
1
-AR) stimulation accelerates mitochondrial Ca
2+
uptake through Pyk2-dependent phospholylation of mitochondrial Ca
2+
uniporter (MCU). However, the roles of Pyk2 in cardiac mitochondrial physiology and pathophysiology have not been well established.

Hypothesis:
Persistent adrenergic signaling activates cell death signaling via Pyk2-dependent MCU activation and mitochondrial Ca
2+
overload.

Methods:
Using H9C2 cardiac myoblasts, mitochondrial

Ca
2+
and reactive

oxygen species (ROS) were measured using mitochondrial matrix-targeted Ca
2+
-sensitive inverse pericam and MitoSOX, respectively. Mitochondrial permeability transition pore (mPTP) activity was observed by measuring the amount of cytochrome c in cytosol by Western blotting or by monitoring the release of GFP-tagged mitochondrial protein, Smac-GFP using confocal microscopy.

Results:
Pyk2 was not only expressed in cytosol, but also in cardiac mitochondria. α
1
-AR agonist phenylephrine activated mitochondrial Pyk2 and enhanced mitochondrial Ca
2+
uptake via Pyk2-dependent MCU phosphorylation. In addition, persistent α
1
-AR stimulation increases ROS, activity of mPTP. These effects were abolished by co-expression of dominant-negative MCU or kinase-dead Pyk2, suggesting that Pyk2-dependent MCU activation followed by mitochondrial Ca
2+
overload are critical for this mechanism. Moreover, pretreatment of a potent FAK/Pyk2 inhibitor PF-431396 also effectively inhibited α
1
-AR-mediated ROS generation and mPTP activation.

Conclusion:
FAK/Pyk2 inhibitor prevents mitochondrial Ca
2+
overload, oxidative stress and mitochondrial injury under persistent adrenergic stimulation. Thus, Pyk2 may become a novel potent therapeutic target for preventing cardiac cell injury and death during heart failure.
Abstract 152: Intrinsic Vascular Tone may be Necessary for Successful Resuscitation from Experimental Cardiac Arrest
Authors: Ramadeen Andrew , Dashi Gerhard , Hu Xudong , Tsui Albert K , Zou Lily , Baig Ehtesham , Hare Gregory M , Dorian Paul
Published: July 2021

Introduction:
Survival rates from out-of-hospital cardiac arrest are often <10% despite performance of good quality CPR. We assessed the hypothesis that the effect of CPR on cardiac and brain perfusion may be dependent on factors other than thoracic compression force and ventilation.

Methods:
Eighty healthy Yorkshire pigs (29±3 kg) were anesthetized and underwent 2-4 minutes of untreated ventricular fibrillation (VF), followed by guideline based CPR, then defibrillation. “Survivors” were pigs in sinus rhythm with aortic systolic pressure ≥30 mmHg 30 minutes after defibrillation; all others were “non-survivors”. Hemodynamic, ventilatory and defibrillation parameters were measured and tested for association with survival.

Results:
Thirty four pigs survived (43%). During baseline and untreated VF, hemodynamic and blood gas parameters were not different between survivors and non-survivors. During CPR, compressions generated adequate left ventricular pressures in both groups (99±21 mmHg vs. 106±28 mmHg, survivors vs. non-survivors, P=ns). Compressions produced 28% higher peak aortic pressures in survivors than non-survivors (respectively 73±21 mmHg vs. 57±17 mmHg, P<0.005). During the decompression phase, nadir aortic pressures were 41% higher in survivors than non-survivors (respectively 24±7 mmHg vs. 17±5 mmHg, P<0.0001). Controlled manual bag ventilation during CPR resulted in significantly higher minute ventilation being delivered to survivors compared to non-survivors (4.8±2.3 L/min vs. 3.9±1.4 L/min, P<0.05). Coronary perfusion pressure, carotid blood flow, cerebral O
2
tension, and end tidal CO
2
were also higher in survivors.

Conclusions:
Guideline based CPR in a uniform population of pigs undergoing a structured cardiac arrest and resuscitation protocol does not produce consistent results. Intravascular pressures, intrathoracic pressure and critical organ flow correlate with survival. During cardiac arrest, more emphasis may need to be placed on vascular support rather than powerful compressions.
Abstract 158: What’s the Nrf Got To Do With Alcoholic Cardiomyopathy?
Authors: Haddad Georges E , Walker Robin K , Jeffress Miara , AlRubaiee Mustafa , Umoh Nsini , Ross Tiorra , Douglas Stephanie
Published: July 2021

The beneficial cardiovascular effects of low alcohol consumption are becoming more clinically evident. This is in sharp contrast with the detrimental ones associated with heavy alcohol drinking. We have previously shown that cardiac inotropy is closely related to the activation of the survival PI3K/Akt pathway. The objective of this study was to assess the role of the PI3K/Akt pathway in mediating the inotropic effects of chronic alcohol consumption. Littermate adult rats were put on a 3-months isocaloric Lieber-Decarli liquid diet with either low alcohol (LA: 5mM) or high alcohol (HA: 100mM) levels. The measured final blood alcohol concentrations were 0.02% and 0.46% respectively.
In vivo
intra-ventricular catheterization (Scisense/iWorks) of the left ventricle (LV) showed that an improved LV contractility (ESPVR and PRSW) of the LA group while the opposite occurred with the HA group. The alterations in contractility were independent of the afterload as
in vitro
cellular and sarcomeric inotropic measurements (Ionoptix) showed similar profile to the LV
in vivo
data. These functional LA effects were correlated with reduced H
2
O
2
levels (as well as SOD-3 activity) and enhanced Akt, NFκB, FOXO-1 and NRF1/2 gene expressions which translated into higher NRF2 protein levels and cardiocyte survival. On the contrary, HA increased H
2
O
2
level (as well as SOD-3 activity) and reduced the Akt, NFκB, FOXO-1 and NRF1/2 gene expressions which translated into lower NRF2 protein levels and cardiocyte survival. In addition, we observed dampening of caspase-3/7 activity with HA which was not affected with LA. We conclude that the improved low alcohol-induced cardiac inotropic function may be mediated through reduction of oxidative stress via Akt/NRF2 activation, which showed opposite expressions with the detrimental high alcohol effects. We also note that cell death occurrences may turn to necrotic pathways as alcohol exposure increases beyond LA levels.
Abstract 168: TMEM8A 1080A>G Polymorphism in a 3’UTR MicroRNA Target Site Influences Aerobic Capacity in Health Individuals: the HUNT Study
Authors: Silva Gustavo J , Afonso de Almeida Marcio A , Bye Anja , el Azzouzi Hamid , Blangero John , Wisløff Ulrik
Published: July 2021

Large-scale epidemiological studies demonstrate that aerobic fitness, measured as maximal oxygen uptake (VO
2max
), is the single best predictor for cardiovascular mortality in both healthy individuals and patients with cardiovascular disease. However, the genetic basis for aerobic capacity has remained unknown. A genome-wide associative study was conducted in 1500 healthy men from the Nord-Trøndelag Health (HUNT3) study in order to identify SNPs located at microRNA target sites associated to VO
2max
. VO
2max
was directly measured using a treadmill ramp protocol. The following bioinformatics approach were adopted: first, 2,393,543 conserved microRNA binding sites were initially selected from the TargetScan database out of 25,847,058 already identified in the human genome; second, among 60,480,978 SNPs described in the dbSNP, 388,110 SNPs are located in 3’UTRs and only 0.26% (1000 SNPs) were represented in the Illumina Human Cardio-Metabo (200k) chip. We identified a significant (p=0.003142) SNP (rs540, MAF=0.4131), located in the 3’UTR of the transmembrane protein 8A (TMEM8A) gene, associated with VO
2max
. TMEM8A is a five membrane-spanning domain, non-globin gene entrapped in the globin pathway. Accordingly, two microRNAs (hsa-miR-190 and hsa-miR-190b) can potentially bind the original variant of the TMEM8A 3’UTR. Interestingly, in the polymorphic variant (G(1080)) these microRNAs are not predicted anymore, and, curiously, five other microRNAs can now bind it (hsa-let-7a-3p, hsa-let-7f-2-3p, hsa-let-7b-3p, hsa-miR-300 and hsa-miR-381). Our data shows for the first time a SNP that can potentially influence microRNA gene expression regulation associated to aerobic capacity in a large population study.
Abstract 176: Intravenous Infusion Of Glycated End Products Induces Cardiac Remodeling And Fibrosis Through RAGE Mediated Oxidative Stress
Authors: Subramanian Umadevi , Vellaichamy Elangovan
Published: July 2021

Advanced glycation end products (AGEs) play a major role in the development of cardiovascular disorders in diabetic patients. Growing evidence has shown that the western diet is a plentiful source of exogenous AGEs. However, the direct impact of these AGEs on cardiac remodeling process is not well understood. Thus, the present study was intended to scrutinize the long term effects of circulating AGEs on cardiac extracellular matrix alterations both
in vivo
, Wistar rats, and
in vitro
, H9C2(2-1) cardiac myofibroblast cells.
In vivo
Rats (n=8/group) were administered with an intravenous infusion of
in vitro
prepared glycated-RSA (AGEs-RSA) (50 mg/kg /day for 30 days), while control animals received the non-glycated-RSA.
In vitro
, H9C2 (2-1) cardiac myofibroblast cells were exposed to AGEs (100µg/ml) for 24 hours. Upon AGEs infusion, a significant increase in fibrosis (3-fold, p<0.01) with increased expression of matrix genes MMP-2 and -9 (P<0.01, respectively), CTGF (p<0.01) and TGF-β (P<0.01) was found in the heart tissues compared to non-glycated-RSA infused rats. Furthermore, an increase in mRNA and protein expression of RAGE (receptor for AGE), and NADPH oxidase (NOX-p47 phox subunit), iNOS and NF-κB was observed in the heart tissue of AGEs infused rats. Furthermore, induction of lipid peroxides (p<0.001) were also found in the heart tissue of AGEs-RSA infused rats. Similarly, H9C2 (2-1) cells exposed to AGEs-RSA demonstrated a significant increase in ROS release (DCF fluorescence, p<0.001) with augmented expression of RAGE (
P
<0.01) and NOX-p47 phox (
p
<0.001) compared to untreated cells. Whereas, cells pre-treated with N-Acetyl cysteine and RAGE neutralizing antibody significantly impeded the NOX expression (
p
<0.01) and attenuated the up-regulation of RAGE and NF-κB in AGEs-RSA treated H9C2 cells. Thus, the results of the present study demonstrated the deleterious effect of AGEs that directly induces oxidative stress and matrix derangement in heart tissue even when ingested through exogenous sources.
Abstract 179: EGFR Transactivation Triggered by Alpha1a-Adrenergic Receptor Genetic Variant is Generalizable Phenomenon in Cardiovascular Cells
Authors: Oganesian Anush , Gradinaru Irina , Babaeva Ekaterina , Schwinn Debra A
Published: July 2021

Alpha
1
-Adrenergic Receptors (α
1
AR) are G protein-coupled transmembrane receptors (GPCRs) that mediate actions of the sympathetic nervous system through binding of endogenous catecholamines epinephrine and norepinephrine. Three subtypes of α
1
ARs (α
1a
, α
1b
, α
1d
) are expressed within most sympathetic tissues and in the human heart and they couple predominantly to the G
q/11
family of G proteins. α
1a
ARs play a major role in regulating vascular tone, in blood vessel repair and in cardiovascular diseases like hypertension, myocardial hypertrophy, ischemia and heart failure. Recently, we discovered a novel, unique mechanism of hypertension triggered by naturally occurring human α
1a
AR-G247R (247R) genetic variant, identified in the 3
rd
intracellular loop (3iL) of the receptor in hypertensive patient. 247R signals in Gq-independent but βarrestin1-dependent manner leading to transactivation of epidermal growth factor receptor (EGFR) via constitutive active coupling to the βarrestin1 /MMP7/EGFR pathway. We also demonstrated that expression of 247R in cardiomyoblasts leads to phenotypical and morphological changes of the cells to novel fibroblast-like phenotype. Here we examine if the 247R-triggered hyperproliferation is generalizable in other cell types such as smooth muscle cells (SMC). Our preliminary results reveal that constitutive expression of 247R in human coronary artery SMCs and in A-10 rat cell line leads to ~2-fold increased hyperproliferation and ERK-kinase activation. The increased proliferation of 247R cells is reduced by EGFR specific inhibitor AG1478 and general MMP inhibitor GM6001, indicating involvement of EGFR transactivation pathway in 247R-triggered hyperproliferation. We elucidate molecular mechanisms and signal transduction pathways responsible for increased cell proliferation and hypertrophy in SMCs. Search of SNP databases revealed several additional SNPs in the same 3iL of human α
1a
AR. The structure modeling reveals that 247R is a representative of these SNPs, and novel EGFR transactivation pathway triggered by 247R may represent general mechanism for SNPs identified in the 3iL of α
1a
AR involved in development of hypertension.