bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 134: Low Dose Particle Radiation Affects Long-Term Survival of Bone Marrow Progenitor Cell Populations
Authors: Sasi Sharath , Park Daniel , Zuriaga Maria A , Walsh Kenneth , Yan Xinhua , Goukassian David A
Published: July 2021

Radiation-induced decreases in the number of bone marrow (BM)-derived endothelial progenitor cell (BM-EPCs) and their lineage precursors which include Early- and Late-Multi-Potent Progenitor cells (E-MPP and L-MPP) could contribute to the pathogenesis of ischemic and vascular diseases. We examined the effect of full-body single dose of proton (1H) at 0.5 Gy, 1 GeV and 0.15 Gy, 1 GeV/nucleon of iron (56Fe) - ionizing radiation (IR) on survival and proliferation of BM-EPCs. The survival of E-MPPs and L-MPPs in the BM after particle IR in C57BL/6 mice were determined at 1, 2, 4, 8, 12, 28 and 40 weeks post-IR. BM-derived mononuclear cells were triple-stained with RAM34 (CD34, c-kit, and Sca1), AC133, and hematopoietic lineage negative cocktail, then sorted by FASC for E- and L-MPP. BM EPCs ex-vivo - There was a transient 2.5-3.5-fold increase in BM-EPC apoptosis, with 3.5-fold increases for 56Fe and 1H at 5hrs and 24hrs, respectively that was no longer detected by day 7. Subsequently, there was a 3-fold increase in BM-EPC apoptosis on day 28 for both ion-IR mice. Compared to 24 hrs, there was a ~20% (1H) and ~45% (56Fe) increase in the rate of EPC proliferation on day 14 that returned to control levels on day 28. BM E-MPP and L-MPP in vivo - Compared to control mice, 1H-IR increased the number of both E-MPPs (665%) and L-MPPs (203%), whereas 56Fe-IR decreased E-MPP (74%) and L-MPPs (65%) at 1 week post-IR, suggesting stimulation by 1H but overt damage by 56Fe in the BM milieu. In 56Fe-IR mice, E-MPPs recovered between 4 and 12 weeks, followed by declines at later time points. In 1H-IR mice, E-MPPs were near control levels up to 4 weeks, but declined at later time points. The long-lasting and cyclical effects of IR on the BM E- and L-MPPs after a single 1H or 56Fe IR dose suggests the presence of prolonged and non-targeted effects in BM milieu, that occur in cells that were not traversed by IR, rather induced by signals from IR cells. Our studies showed that, both 1H- and 56Fe-IR has profound and long-lasting (28-40 months) negative effects on the number of E- and L-MPPs. Future longitudinal studies are necessary to determine whether BM progenitor cells may be affected after terrestrial IR exposure, such as cancer radiotherapy, CT and PET scans, and in astronauts after exploration-type space missions.
Abstract 149: Parkin Translocation And Mitophagy Occur Independently of PINK1 in the Myocardium
Authors: Kubli Dieter A , Lee Youngil , Moyzis Alexandra G , Gustafsson Åsa B
Published: July 2021

We recently reported that Parkin plays a crucial role in preserving myocardial tissue following a myocardial infarction (MI) by promoting removal of damaged mitochondria via autophagy (mitophagy). Parkin is thought to function downstream of the serine/threonine kinase PINK1 for mitophagy of mitochondria that have lost membrane potential (ΔΨm). Here, we describe the ability of Parkin to translocate to dysfunctional mitochondria in the absence of PINK1. We discovered that after 4 hours of MI, Parkin accumulated on mitochondria in the infarct border zone in both wild type (WT) and PINK1 knockout mice (PINK1
-/-
). Additionally,
ex vivo
perfusion of PINK1
-/-
hearts with the mitochondrial uncoupler FCCP caused rapid translocation of Parkin to mitochondria, equivalent to the response in WT hearts. We further confirmed Parkin translocation and subsequent mitophagy by fluorescence microscopy in isolated PINK1
-/-
adult cardiac myocytes treated with the mitochondrial complex I inhibitor rotenone. Rotenone-stimulated Parkin translocation resulted in increased co-localization of GFP-LC3-labeled autophagosomes with mitochondria in both WT and PINK1
-/-
myocytes. Thus, PINK1 is dispensable for Parkin translocation and mitophagy in cardiac myocytes. In contrast, we found that Drp1-mediated mitochondrial fission was a prerequisite for Parkin translocation and mitophagy activation. Drp1 translocation to mitochondria preceded Parkin translocation in WT and PINK1
-/-
hearts perfused with FCCP. Moreover, overexpression of the dominant negative Drp1-K38E mutant prevented Parkin translocation in rotenone-treated isolated adult rat cardiac myocytes. Interestingly, while Drp1 was able to rapidly translocate to mitochondria in FCCP-perfused Parkin
-/-
hearts, Drp1 translocation to border zone mitochondria was only observed in WT hearts after MI and not Parkin
-/-
. This suggests that Parkin stabilizes Drp1 that has translocated to mitochondria upon damage. We conclude that while Parkin translocation to cardiac mitochondria is independent of PINK1 activity, Drp1-mediated fission is required for Parkin translocation. Parkin and PINK1 are therefore components of distinct but overlapping pathways for mitochondrial maintenance in the heart.
Abstract 154: Interaction Between Nitroso-Redox Balance and Temperature Influences SR Ca
2+
Leak In Cardiomyocytes: Role of Neuronal NOS Signaling
Authors: Dulce Raul A , Mayo Vera , Rangel Erika B , Balkan Wayne , Hare Joshua M
Published: July 2021

Sarcomeric reticulum (SR) Ca
2+
leak contributes to impaired Ca
2+
cycling and contractile dysfunction of the heart. Leak is thought to be regulated by post-translational modification of RyR2 associated with the nitroso-redox state. Ca
2+
cycling is affected by both nitric oxide (NO) signaling and temperature. We studied the effect of temperature and nitroso-redox state on Ca
2+
leak. We hypothesized that nitroso-redox balance modulates leak in a temperature–dependent manner. Tetracaine was used to estimate the leak in cardiomyocytes (CMs) in mouse models of nitroso-redox imbalance (NOS1
-/-
), hyper S-nitrosylation (GSNOR
-/-
) and in WT mice. Reactive oxygen species (ROS) were measured. Experiments were carried out at 23°C – 37°C.

In WT CMs, Ca
2+
leak was inversely proportional to temperature whereas, in NOS1
-/-
CMs, the leak suddenly increased when the temperature surpassed 30°C. Supplementing the media with NO (1 microM SNAP), reduced leak in NOS1
-/-
CMs at physiologic temperature while at low temperatures, there was a trend toward an increase, a pattern similar to that of GSNOR
-/-
rather than WT CMs. The xanthine oxidase inhibitor, oxypurinol (100 microM), diminished the leak in NOS1
-/-
CMs. SR Ca
2+
content in NOS1
-/-
CMs was reduced as the temperature decreased, whereas it was not affected in WT cells. Cooling from 37°C to 23 °C increased ROS generation in WT CMs; however there was an opposite effect observed in NOS1
-/-
CMs, which was further reduced by oxypurinol. Expression of the NOS isoforms (by RT-PCR) was unaffected by temperature in WT.

Therefore, Ca
2+
leak is temperature dependent, is affected by the absence of NOS1 activity and increases with the rise of ROS production. Defective denitrosylation activity keeps leak low, suggesting a protective effect of S-nitrosylation against the cooling-induced increase in SR Ca
2+
leak. These results suggest that Ca
2+
leak from the SR is regulated by a temperature-dependent balance between NO and ROS.
Abstract 161: Kcne2 Gene Deletion Combines With A Western Diet To Cause Early-onset Diabetes Mellitus And Atherosclerosis In Mice
Authors: Lee Soo-Min , Kant Ritu , Abbott Geoffrey W
Published: July 2021

The KCNE2 single transmembrane domain protein regulates multiple types of cardiac voltage-gated potassium (Kv) channel
in vivo
and its disruption causes the ventricular arrhythmia, Long QT Syndrome. Interestingly, a SNP near the human KCNE2 locus was also previously linked to early-onset myocardial infarction (MI). Because KCNE2 is also expressed outside the heart, we are interested in understanding extracardiac defects caused by
Kcne2
deletion in mice, and also the potential contribution of these extracardiac defects to cardiac dysfunction. Thus, recently, we found that
Kcne2
deletion creates a multifactorial substrate for sudden cardiac death (SCD) that includes diabetes mellitus and dyslipidemia. Because atherosclerosis is the most common cause of MI, here, using
Kcne2
-/-
mice, we investigated atherosclerosis and other potential predisposing factors in MI and SCD, and quantified the impact of a Western diet (high fat/high cholesterol) on these processes. Previously, we discovered impaired glucose tolerance in adult
Kcne2
-/-
mice using a standard glucose tolerance assay in which plasma glucose is quantified 0-2 hours following glucose injection. Here, we found that in
Kcne2
-/-
mice as young as 5 weeks, just 2 weeks on a Western diet induced impaired glucose tolerance, whereas 5-week-old wild-type mice regardless of diet, or
Kcne2
-/-
mice on a control diet, had normal glucose tolerance. This indicates a strong interaction between diet and
Kcne2
deletion in creating early-onset diabetes mellitus. Atherosclerosis was also investigated, using Sudan IV staining of plaques in the aorta of wild-type and
Kcne2
-/-
mice fed on either standard (control) or Western diet. Strikingly,
Kcne2
-/-
mice as young as 4.5 months exhibited aortic plaques, and this was accelerated by a Western diet. In contrast, wild-type littermates did not exhibit plaques at this age, regardless of diet. Ongoing studies to determine potential metabolic defects in the liver and pancreas are aimed at understanding the molecular mechanisms underlying these findings. Our data provide further evidence of the unexpected complexity of monogenic cardiovascular syndromes caused by disruption of genes associated with cardiac arrhythmias.
Abstract 165: The Transcriptional Cofactor Eyes Absent 4 Is A Critical Regulator In Cardiac Physiology
Authors: Williams Tatjana , Hundertmark Moritz , Nordbeck Peter , Voll Sabine , Elsner Ines , Schoenberger Jost , Ritter Oliver
Published: July 2021

Introduction:
Eyes absent 4 (Eya4) is a transcription cofactor involved in a number of cellular and developmental processes. We have previously shown that a truncating mutation in Eya4, E193, causes hearing impairment followed by terminal heart failure suggesting Eya4 is a regulator in cardiac physiology.

Methods and Results:
Transgene- or adenovirus mediated overexpression of Eya4 or E193 altered the expression of p27kip1, a critical mediator of cardiac hypertrophy in adult cardiomyocytes.

Luciferase reporter and EMSA assays revealed that Eya4 directly binds to and suppresses p27kip1 promoter activity, while E193 exerts an opposing effect, respectively. Activity and phosphorylation of downstream molecules were significantly altered in Eya4 and E193 transgenic (TG) mice in a contradictory manner. Cardiac phenotypes evolved in both TG models already under basal conditions. Eya4 TG hearts developed hypertrophy as judged by increases in heart weight and cross-sectional cell surfaces and re-activation of fetal genes as well as fibrosis. E193 TG animals showed onset of DCM along with wall thinning, ventricular dilation, fibrosis and slightly compromised cardiac function. These two distinct cardiac phenotypes were even more aggravated upon pressure overload or Angiotensin II infusion.

Finally, we just recently identified a new mutation in Eya4, E215, which also causes hearing impairment and heart failure.

Conclusion:
Our data indicate that Eya4, in a physical complex with Six1, plays a critical role in regulating normal cardiac function via p27/CK2-α/HDAC2 and allude that mutations within the Exa4/Six1 transcriptional complex interfere with this newly established signalling pathway, finally leading to age-related onset of cardiomyopathy.

Clinical Perspective:
Gaining a better understanding of this disease mechanism could help identify new treatment options for heart failure patients.
Abstract 176: Saxagliptin and Tadalafil Differentially Alter Global and Cellular Cardiac Function in a Translational Miniature Swine Model of Left Ventricle Hypertrophy
Authors: Hiemstra Jessica A , Gibson Anne K , Ivey Jan R , Cobb Melissa S , Baines Christopher P , Krenz Maike , Domeier Timothy L , Emter Craig A
Published: July 2021

Left ventricular (LV) hypertrophy is a common characteristic of heart failure with preserved ejection fraction (HFpEF). Our lab recently characterized a mini-swine model of LV hypertrophy induced by aortic banding (AB) that displays clinical features associated with HFpEF including LV hypertrophy, diastolic dysfunction, and depressed contractile reserve. Disrupted cGMP signaling, a result of impaired production or enhanced catabolism, may play a role in development of HFpEF. We hypothesized preservation of cGMP signaling would attenuate pathological remodeling and improve cardiac function. The purpose of this study was to promote cGMP signaling via two mechanisms: 1) the DPP4 inhibitor saxagliptin; and 2) the PDE5 inhibitor tadalafil. We assessed whole heart and individual cardiomyocyte function 6 months post-AB in: control non-banded (CON; n=6), AB-control (AB; n=7), AB saxagliptin-treated (AB-SAX; n=7), and AB tadalafil-treated (AB-TAD; n=8) swine. Heart weight:body weight ratio increased to a similar extent in all AB groups. However, changes in cardiomyocyte morphology were variable. Cardiomyocyte length was increased only in the AB-TAD group, while cell width increased in both AB and AB-TAD animals. Cardiomyocyte length:width ratio decreased in the AB and AB-TAD groups, commensurate with decreased LV end diastolic (ED) and end systolic (ES) volumes. These changes were prevented in AB-SAX animals, as LV volumes and cell morphology were similar to CON. Pressure-volume analysis showed resting LV wall stiffness (ED pressure volume relationship [EDPVR] slope) was increased similarly in all AB groups. Increased resting LV contractility (ESPVR and preload recruitable stroke work) was observed in AB and AB-TAD animals. Interestingly, cardiomyocyte shortening was reduced in the AB-TAD group contrasting findings observed at the whole heart level. Saxaglitpin attenuated hypercontractile LV function at rest and preserved normal cardiomyocyte shortening. In conclusion, LV and cardiomyocyte function was distinctly altered in response to separate methods of pharmacological cGMP regulation. Our data suggest different pharmacological approaches to augment cGMP signaling promote distinct LV functional adaptations to developing HF.
Abstract 178: Exercise Augments Cardiac Akt Activity And Reverses Aging-related Systolic Dysfunction.
Authors: Kawase Haruya , Bando Yasuko K , Aoyama Morihiko , Monji Akio , Cheng Xieng W , Kuzuya Masafumi , Murohara Toyoaki
Published: July 2021

Purpose:
Aging is one of the primary factors causing left-ventricular (LV) remodeling and susceptibility to heart failure (HF). Clinical evidences demonstrate a sustained exercise (EX) ameliorates HF; however, the molecular mechanism underlying the aging-related LV remodeling remains uncertain and few data have demonstrated whether EX may be beneficial for the aging-related LV remodeling and contractility. Because preclinical studies indicate the pivotal role of protein kinase Akt in aging, we thus hypothesized whether EX may exert benefits on HF induced by aging in which Akt may play an essential role.

Methods:
Male aged (40 w/o) and young (14 w/o) C57BL6 mice were subjected to the EX (45-min running (10~20m/s, 5-degree) on treadmill every second day for 15 weeks; agedC57EX and agedC57CON). To elucidate the role of Akt in aging heart, age- and gender-matched Akt knockout mice were also enrolled.

Results:
Aging impairs both systolic and diastolic function without any changes in cardiac geometry. The systolic dysfunction of agedC57 reversed by EX with concomitant Akt activation; however, its diastolic dysfunction remained unaffected. EX enhanced cardiac Akt activity independently of aging. Aged AktKO exhibited systolic dysfunction to the more severe extent, which was reversed by EX.

Conclusions:
Our study demonstrates that #1 Akt is essential for adaptive cardiac contractility both to EX and aging. #2 Aging promotes diastolic dysfunction independently of Akt axis.
Abstract 188: Regulation Of Acetylation Of Histone Deacetylase 2 By P300/CBP-associated Factor/histone Deacetylase 5 In The Development Of Cardiac Hypertrophy
Authors: Kook Hyun , Eom Gwang Hyeon , Nam Yoon Seok , Choe Nakwon , Min Hyun- Ki
Published: July 2021

Rationale:
Histone deacetylases (HDACs) are closely involved in cardiac reprogramming. Although the functional roles of the class I and class IIa HDACs are well established, the significance of interclass crosstalk in the development of cardiac hypertrophy remains unclear.

Objective:
Recently, we suggested that casein kinase-2α1-dependent phosphorylation of HDAC2 leads to enzymatic activation, which in turn induces cardiac hypertrophy. Here we report an alternate posttranslational activation mechanism of HDAC2 that involves acetylation of HDAC2 mediated by p300/CBP-associated factor (pCAF)/HDAC5.

Methods and Results:
Hdac2 was acetylated in response to hypertrophic stresses in both cardiomyocytes and a mouse model. The acetylation was reduced by a histone acetyltransferase inhibitor but was increased by a nonspecific HDAC inhibitor. The enzymatic activity of Hdac2 was positively correlated with its acetylation status. pCAF bound to Hdac2 and induced acetylation. The HDAC2 K75 residue was responsible for hypertrophic stress-induced acetylation. The acetylationresistant Hdac2 K75R showed a significant decrease in phosphorylation on S394, which led to the loss of intrinsic activity. Hdac5, one of class IIa HDACs, directly deacetylated Hdac2. Acetylation of Hdac2 was increased in Hdac5 null mice. When an acetylation-mimicking mutant of Hdac2 was infected into cardiomyocytes, the anti-hypertrophic effect of either nuclear tethering of Hdac5 with leptomycin B or Hdac5 overexpression was reduced.

Conclusions:
Taken together, our results suggest a novel mechanism by which the balance of HDAC2 acetylation is regulated by pCAF and HDAC5 in the development of cardiac hypertrophy.
Abstract 189: βAR Resensitization At The Plasma Membrane Provides Beneficial Cardiac Remodeling
Authors: Martelli Elizabeth E , Vasudevan Neelakantan T , George John , Mohan Maradumane L , Gupta Manveen K , Naga Prasad Sathyamangla V
Published: July 2021

βAR downregulation and desensitization are hallmarks of heart failure. Agonist occupied βARs undergo desensitization through phosphorylation by G-protein coupled receptor kinases leading to βAR internalization. Phosphorylated βAR becomes resensitized following dephosphorylation by PP2A in the endosomes. In contrast to this paradigm our recent studies have shown that resensitization of βARs can occur at the plasma membrane through the inhibition of PP2A activity by I2PP2A. We generated a stable HEK cell line expressing short hairpin RNA targeting I2PP2A (shRNA-I2PP2A). Confocal microscopy studies show cells expressing shRNA-I2PP2A resulted in significant loss of receptor phosphorylation despite the presence of the agonist. Radioligand binding and confocal imaging also showed marked inhibition of receptor internalization upon depletion of I2PP2A with significant PP2A activation. We also observed preservation of βAR function despite the presence of agonist measured by cAMP generation and adenylyl cyclase activity. To further dissect the interaction of I2PP2A-PP2A we generated mutants of PP2A from amino acids 263-309. Over expression of these PP2A mutant peptides significantly reversed receptor phosphorylation upon isoproterenol (ISO) stimulation compared to full length PP2A. Also, expression of PP2A mutant showed marked increase in adenylyl cyclase activity in response ISO stimulation suggesting that this mutant I2PP2A competes out inhibitory I2PP2A interaction with PP2A. To test whether alteration in βAR resensitization contributes to cardiac dysfunction with stress we generated transgenic (Tg) mice with cardiac specific over expression of wt I2PP2A and mutant I2PP2A (phopspho- and dephospho-). ISO treatment of wt I2PP2A Tg and littermate controls showed that wt I2PP2A Tg mice had significant βAR dysfunction and cardiac hypertrophy. Assessment of age dependent cardiac function of these mice showed that wt I2PP2A Tg and phospho-I2PP2A Tg mice have cardiac hypertrophic response followed by dilated cardiomyopathy; expression of dephospho-I2PP2A mutant reversed this phenotype. These studies suggest targeting I2PP2A alters receptor function and may have implications in cardiac remodeling and hypertrophy with cardiac stress.
Abstract 196: DDAH1 Knockout Exacerbates Hypoxia-induced Pulmonary Arterial Hypertension In Mice
Authors: Kwak Dongmin , Xu Dachun , Atzler Dorothee , Hu Xinli , Xu Xin , Wang Huan , Fassett John , Li Hailing , Yan Shuo , Guo Haipeng , Chen Yingjie
Published: July 2021

Pulmonary arterial hypertension (PAH) is a progressive disease with a very poor prognosis. Recent studies have demonstrated that PAH is associated with diminished nitric oxide bioavailability, increased levels of endogenous nitric oxide synthase (NOS) inhibitor ADMA, and decreased lung dimethylarginine dimethylaminohydrolase (DDAH) activity. We have demonstrated that DDAH1 is essential for degradation of endogenous NOS inhibitor ADMA, and is important for optimal vascular endothelial NO production. However, it is not clear whether decreased DDAH activity and ADMA accumulation exacerbates development or progression of PAH and right ventricular hypertrophy. In addition, the impact of cardiomyocyte specific DDAH1 dysfunction on right ventricular hypertrophy is unknown. Using global DDAH1 gene deficient mice, we demonstrate that chronic ADMA accumulation by global DDAH1 gene deletion did not cause spontaneous PAH under control conditions, but significantly exacerbated chronic hypoxia-induced PAH, as indicated by significantly increased right ventricular (RV) pressure, more RV hypertrophy, and enhanced pulmonary vascular remodeling in DDAH1 deficient mice as compared to wild type mice. Chronic hypoxia resulted in reduced lung DDAH activity and increased circulating ADMA content in wild type mice. Cardiomyocyte specific DDAH1 gene deletion did not exacerbate hypoxia-induced increases in RV pressure or lung vascular remodeling, but significantly exacerbated hypoxia-induced RV hypertrophy in comparison to wild type littermates, indicating DDAH1 distributed in cardiomyocytes protect the heart against RV hypertrophy independent to pressure overload. Collectively, our data indicate that lung DDAH1 plays a critical role in protection against hypoxia-induced PAH and right ventricular hypertrophy.
Abstract 197: Determining the Potential Role of cTnT Isoform Switching In the Development of Early Childhood Tropomyosin-Linked DCM
Authors: Lynn Melissa , Tal-Grinspan Lauren , Jin J.P. , Tardiff Jil
Published: July 2021

An oft-noted component of sarcomeric HCM and DCM is the observation that patients within families, carrying the same primary mutation, often exhibit significant phenotypic variability. This lack of a distinct link between genotype and phenotype has greatly complicated clinical management. In a recent study of two large unrelated multigenerational families carrying the tropomyosin (Tm) mutation Asp230Asn (D230N), a striking “bimodal” distribution of severity was observed. In these families, many children (<1 year) with the mutation presented with a severe form of DCM that led to sudden, often fatal congestive heart failure, while adults developed a mild to moderate DCM in mid-life. Of note, children who survived the initial presentation often recovered significant systolic function in adolescence and young adulthood. Therefore, to better understand the mechanism of this “bimodal” phenotype, we began to investigate the potential modulating role of isoform switching by other sarcomeric components. We hypothesize that the age-dependent remodeling seen in children with D230N Tm is a result of temporal isoform switches involving a closely linked Tm binding partner cardiac Troponin T (cTnT). Initial biophysical studies (circular dichroism and regulated
in vitro
motility, R-IVM) show that while D230N does not alter Tm’s thermal stability it does have a profound impact on myofilament activation. Both maximal velocity of filament sliding and calcium sensitivity were decreased. Furthermore, an additive decrease was observed in these parameters for R-IVM solutions containing cTnT
1
(fetal)+D230N Tm filaments as compared to cTnT
3
(adult)+D230N. Preliminary
in vivo
studies utilizing our novel double transgenic Tm-D230N x cTnT
1
mice show profound changes in wall thickness and chamber dilation, as compared to age-matched non-transgenic mice and D230N Tm mice. Further studies aim to model the “bimodal” clinical phenotype seen in families with D230N Tm and assess the potential for disease reversibility using a cardiac specific inducible cTnT
1
transgenic mouse model. Our goal is to use a translational approach to better understand the mechanism by which primary mutations lead to distinct clinical phenotypes in order to improve clinical management.
Abstract 198: Noninvasive Approach Assessing Atrial Mechanics and Serum Biomarkers of Collagen Turnover Provides a Surrogate for Fibrosis and Atrial Fibrillation
Authors: Mirza Mahek , Rizvi Farhan , Albrecht Melissa , Strunets Anton , Emelyanova Larisa , Holmuhamedov Ekhson , Werner Paul H. , Kress David C. , Khandheria Bijoy K. , Sra Jasbir , Jahangir Arshad
Published: July 2021

Introduction:
Atrial fibrosis alters myocardial electrophysiological properties, increasing susceptibility to postoperative atrial fibrillation (PoAF); however, its estimation is problematic. We hypothesized that a noninvasive approach using history of AF (HxAF), LA mechanics and serum biomarkers of collagen turnover provides a surrogate for the extent of interstitial atrial fibrosis to identify patients at risk for PoAF.

Methods:
In patients undergoing cardiac surgery from April-Dec 2013, concentrations of biomarkers reflecting collagen synthesis/degradation and extracellular matrix remodeling were determined in serum from preoperative blood using an enzyme-linked immunosorbent assay, and echocardiographic evaluation was performed using M-mode, 2D, Doppler and 3D speckle tracking.

Results:
Of 66 patients (68 ±11 y, 67% men), 15 had HxAF and 11 of 51 with no HxAF (22%) developed new onset PoAF. In patients with HxAF, biomarkers for collagen turnover were elevated (Fig A) and correlated with a reduction in LA ejection fraction and global and regional relaxation of the LA wall (p=0.01, Fig B). In patients with no HxAF, procollagen type III (PIIINP) was significantly different in those who developed PoAF (p=0.01) and correlated with reduction in contractility in the posterior LA roof (p=<0.001) with a prolonged time to peak end-diastolic volume (p=0.03). LA size or ventricular structure and function were not different between groups.

Conclusion:
Surrogate serum and imaging biomarkers correlate with the substrate abnormality that promotes AF. These results need to be validated in larger cohorts to assess the power of these parameters in predicting new onset PoAF.
Abstract 200: Novel Interaction of Spinophilin with Alpha1a-Adrenergic Receptor and its Genetic Variant in Cardiovascular Cells
Authors: Babaeva Ekaterina , Gradinaru Irina , Schwinn Debra A , Oganesian Anush
Published: July 2021

Activation of α
1
-Adrenergic Receptors (α
1
ARs), members of the G protein-coupled receptor (GPCR) superfamily, in response to stimulation of the sympathetic nervous system by catecholamines plays a major role in regulating cardiovascular (CV) function. Among three α
1
AR subtypes (α
1a
,α
1b
,α
1d
), α
1a
ARs predominate in human resistant vessels and in heart. Recently, we discovered that naturally occurring human α
1a
AR-G247R (247R) genetic variant, identified in the 3
rd
intracellular loop (3iL) of the receptor in highly hypertensive patient, triggers constitutive hyperproliferation in CV cells (cardiomyoblasts, smooth muscle cells (SMC) and fibroblasts), which may lead to myocardial fibrosis and remodeling. In fibroblasts and cardiomyoblasts 247R triggered hyperproliferation is due to constitutive active coupling to Gq-independent βarrestin1/MMP/EGFR/ERK dependent pathway, while in SMC it is Gq- and MMP/EGFR/ERK-dependent. Here we report that α
1a
AR-WT (WT) and 247R differentially interact with ubiquitous multi-domain scaffold protein spinophilin (SPL) that binds to 3iL of several GPCRs competing with arrestin thereby prolonging their signaling. The role of SPL in CV regulation is poorly studied. We hypothesized that SPL mediates constitutive signaling of 247R and examined whether SPL directly interacts with α
1a
AR-WT or 247R. Our preliminary results reveal a direct interaction of SPL with WT and 247R: the SPL-WT interaction appears to be stronger as determined by co-immunoprecipitation. Different domains of SPL differentially interact with WT or 247R. SPL 1-480aa fragment interacts stronger with WT indicating interaction with 3iL, while SPL 480-817 fragment interacts stronger with 247R. Our preliminary results also demonstrate that 247R expression in all three cell types elevates endogenous SPL protein levels. Importantly, inhibition of SPL expression with specific siRNA reduces 247R-triggered hyperproliferation in SMC and cardiomyoblasts to near normal levels, while SPL knockdown has no effect in WT cells. Thus, we identified SPL as a novel protein involved in interacting and signaling of α
1a
AR and its genetic variant in CV cells and that SPL could be considered as a potentially novel target in α
1a
AR-mediated cardiovascular disorders.
Abstract 205: Mitochondrial Sirt3 Is Upregulated By Glucagon-like Peptide-1 Receptor Activation And Contributes To Reversal Of Cardiac Mitochondrial Remodeling Induced By Type 2 Diabetes.
Authors: Monji Akio , Bando Yasuko K , Kawase Haruya , Aoyama Morihiko , Murohara Toyoaki
Published: July 2021

RATIONALE:
Sirtuin 3 (SIRT3) is a mitochondrial protein deacetylase that maintains basal ATP yield and its expression level is increased by fasting, exercise, and some NAD+ intermediates. We recently reported that the glucagon-like peptide-1 (GLP-1) receptor agonist exendin-4 (Ex-4) ameliorated cardiac mitochondrial remodeling in diabetic cardiomyopathy via increase in cardiac cyclic AMP (AJP2013). Because changes in cyclic AMP level is regulated by adenylyl cyclase which is one of the downstream target of Ex-4, we hypothesized that SIRT3 may involve in the Ex-4-mediated myocardial reverse remodeling of mitochondria in diabetic mice.

METHODS:
Type 2 diabetic Mice (16-week old male) were allocated into experimental groups as follows: Ex-4 (24 nmole/kg/day, subcutaneously administrated by osmotic pump for 40 days, DIO/Ex-4) and vehicle control (DIO/CON). Heart samples and cultured rat neonatal cardiomyocytes were subjected to mitochondrial fractionation using density gradient.

RESULTS:
Cardiac cyclic AMP concentration and phosphorylation of CREB were elevated in DIO-ex4, suggesting successful administration of Ex-4. Electron microscopic analysis revealed that Ex-4 reversed destroyed cristae structure and defragmented mitochondria of DIO/CON heart. The ratio of expression levels of Mitofusin-1 (Mfn1) and mitofusin-2 (Mfn2) was consistently suppressed by Ex-4 treatment, suggesting normalization of mitochondrial morphology. Of note, DIO-CON exhibited marked decrease in cardiac SIRT3 level compared to lean/nondiabetic counterpart, which was reversed by exendin-4 treatment. Pharmacological production of intracellular cAMP levels (Isoprotelenol (10 microM) and 8-bromo-cyclic AMP (1 mM)) increased SIRT3 mRNA in cultured primary cardiomyocytes. The AMP-activated protein kinase (AMPK) inhibitition blocked the increase in SIRT3 mRNA, indicating that the SIRT3 expression was regulated by AMPK-dependent manner.

CONCLUSIONS:
Our results indicate that Ex-4 reversed abnormal suppression of SIRT3 in mitochondria via activating the PKA/AMPK-dependent pathway.
Abstract 208: Mitochondrial-targeted Grk2 Increases Superoxide Formation And Impairs Cardiomyocyte Respiratory Reserve Capacity
Authors: Sato Priscila Y , Chuprun J K , Ibetti Jessica , Elrod John W , Koch Walter J
Published: July 2021

β-adrenergic receptors (βARs) are powerful regulators of cardiovascular function and are impaired in heart failure (HF). Signal transduction of βARs is canonically shut down by phosphorylation via G protein-coupled receptor kinase 2 (GRK2) and the subsequent binding of β-arrestins. This process of receptor desensitization is enhanced in HF via the up-regulation of GRK2 and contributes to disease progression. We have recently reported non-canonical actions of GRK2, which contribute to the development of HF independent of βAR desensitization. We have previously shown that GRK2 can act as a pro-death kinase in cardiomyocytes bytranslocating to mitochondria and activating mitochondria permeability transition. This study was designed to gain more understanding of the mitochondrial function of GRK2. We isolated adult cardiomyocytes from cardiac-specific transgenic mice overexpressing GRK2 at levels found in human HF (TgGRK2), and examined superoxide production using the redox sensitive reporter MitoSox Red. Confocal imaging revealed a 4.6 fold increase in superoxide levels in cardiomyocytes overexpressing Grk2 as compared to non-transgenic (NLC) cardiomyocytes (corrected total cell fluorescence 11.59±1.06, TgGRK2 (n= 3 hearts, 88 cells) vs 2.54±0.02 NLC (n=3 hearts, 52 cells), (p<0.001). This indicates that the chronic elevation of GRK2 induces mitochondrial oxidative stress priming the myocyte for enhanced injury. To further explore the mitochondrial actions of GRK2 and consequences of redox stress we examined oxidative phosphorylation by performing oxygen consumption measurements in neonatal rat ventricular myocytes overexpressing GRK2 or GFP-expressing control myocytes. Seahorse analysis showed that cells overexpressing GRK2 have a significant decrease in spare respiratory capacity indicating that cells with elevated GRK2 levels have an impaired capacity to generated ATP during times of stress. Further studies with mutants that limit GRK2 kinase activity or mitochondrial localization demonstrate that mitochondrial GRK2 may be a significant contributor to cellular dysfunction as seen in heart failure.
Abstract 209: Different Reaction To Lactate Between The Atrial And The Ventricular Muscle In Excitation-contraction Coupling
Authors: Shimura Daisuke , Kusakari Yoichiro , Goda Nobuhito , Minamisawa Susumu
Published: July 2021

Background:
The effect of a metabolic change on excitation-contraction (EC) coupling is poorly understood in the atrial myocardium, while the previous studies have mainly focused on EC coupling in the ventricular myocardioum. Although the myocardium mostly uses fatty acid as an energy source, we have reported that metabolic substrate, includes lactate, can be used for energy production and that the metabolomic profile is different between the atria and the ventricles in mouse heart. In addition, it is still under discussion whether lactate can be an energy source for muscles. In the present study, we aimed to investigate the effect of lactate exposure on EC coupling in the atrial and the ventricular myocardium.

Methods:
We micro-injected aequorin (a Ca2+-sensitive photoprotein) into superficial cells of the left atrium and/or the left ventricular papillary muscle isolated from mice (C57/BL6, 12 - 17 weeks of age), and simultaneously measured intracellular Ca2+ concentration and tension (1 Hz at 36 °C). We added lactate (~10 mM) into HEPES-Tyrode solution (pH was adjusted at 7.4) and observed the changes in the peak tension and the peak Ca2+ concentration.

Results and Conclusion:
Lactate at a concentration of 10 mM significantly decreased the peak tension (61.7±6.0%; n = 3; P < 0.05.) and the peak Ca2+ concentration (78.8±4.8%; n = 3; P < 0.05.) in the atrial myocardium. Although we observed similar effect of lactate on the ventricular papillary muscle, it was modest compared with the atrial myocardium (72.8±5.6%, 91.6±13.5%, peak tension and Ca2+, respectively; n = 3; no significance). Our results suggest that the atrium has different characteristic of EC coupling from the ventricles in response to an increase in lactate, of which condition is often observed in myocardial ischemia. Moreover, lactate did not seem to contribute to make energy in terms of the tension in the heart. Simultaneous measurement of tension and intracellular Ca2+ concentration can be useful to analyze the atrial physiological property.
Abstract 215: The Redox Activation of DUSP4 by N-Acetyl Cysteine Protects Endothelial Cells and the Myocardium against Oxidative Damage
Authors: Chen Chun-An , Barajas-Espinosa Alma , Basye Ariel , Yan Haixu , Jesse Erin , Quan David
Published: July 2021

Background:
Redox imbalance is the primary cause for endothelial dysfunction (ED), obstructed blood flow, and subsequent heart attack and failure. Under oxidant stress, many critical proteins regulating endothelial function undergo oxidative modifications that lead to ED. Cellular levels of glutathione (GSH), the primary reducing source, can significantly regulate cell function via reversible protein thiol modification. N-Acetyl cysteine (NAC), a precursor for GSH biosynthesis, is beneficial for many vascular diseases; however, the detailed mechanism of these benefits is still not clear.

Methods:
We employed EPR spin-trapping, HPLC, fluorescent microscopy, immunoblotting, and qPCR of both in vitro and ex vivo experiments using either cultured cells or the Langendorff heart preparation.

Results:
NAC treatment increases NO generation from endothelial cells, as well as the enzyme and cofactor responsible for its production, ie eNOS and BH4. Interestingly, NAC treatment also increased the expression of DUSP4, an inducible nuclear dual-specificity phosphatase implicated in cardiovascular function.

We hereby establish that DUSP4 redox modulates two important kinases (p38 and ERK1/2) of MAPK signaling pathways and provides protection against Cd2+-induced ROS damage as well as hypoxia-reoxygenation insult to endothelial cells. Furthermore, a four week oral NAC pre-treatment promotes DUSP4 both protein and mRNA expression in the rat myocardium and renders the heart less susceptible to ex vivo ischemia-reperfusion injury. Protein expression profiles in the myocardium closely mimic those observed with cultured endothelial cells. The infarct size of NAC-treated hearts is significantly reduced. The myocardial rate pressure product is much improved above vehicle treated rats.

Conclusion:
NAC serves as a direct antioxidant and as a regulator of transcription and translation of DUSP4, modulating the activity of its downstream effectors: ERK1/2 and p38, and thus protects against ROS-induced damage both in vitro and ex vivo. As such, NAC-derived drugs can provide a novel therapy to oxidant-induced diseases via the specific up-regulation of protective proteins such as DUSP4 and eNOS.
Abstract 219: Cardiac Autophagy in Response to Fasting is Impaired in Mice with Metabolic Syndrome
Authors: Ito Bruce R , Ravindran Nandini , Andres Allen M , Gottlieb Roberta A , Mentzer Robert M
Published: July 2021

Introduction:
Macroautophagy (autophagy) is highly responsive to nutrient/energy status and cellular stress. It plays an adaptive role in limiting ischemia/reperfusion (I/R) injury in the heart. Nutrient depletion is a potent inducer of autophagy and is mediated by reciprocal regulation of AMPK and mTOR pathways which in turn regulate autophagy initiation via ULK1/Atg1. Autophagy is reduced in the setting of obesity, dyslipidemia, and insulin resistance associated with metabolic syndrome (MetS). The purpose of this study was determine whether impaired autophagy in MetS is due to altered upstream signaling or a disruption in the autophagic pathway.

Methods:
Mice were ad-lib fed a chow (Lean) or a high fat diet (HFD) (D12492, 60% fat) for 18 weeks. In both groups, half of the mice were freely fed with the other half fasted for 24 hours prior to cardiac harvest. Protein levels were determined with Western blotting and gene expression with quantitative PCR.

Results:
Consistent with previous data, HFD animals exhibited obesity and insulin resistance as reflected by increased body weight, and elevated serum insulin/HOMA compared to Lean animals. Fasting resulted in a marked reduction in serum insulin in both Lean (p=0.013) and HFD (p =0.002) animals. This was accompanied by marked increases in cardiac AMPK activation as reflected by increased phosphorylation in both Lean (p =0.002) and HFD (p=0.002) groups with parallel reciprocal suppression of mTOR activity as reflected by decreased pS6K and pS6. As expected, the Lean group exhibited a fasting-induced increase in LC3-II (p=0.020) indicating an increase in autophagy. This did not occur in the HFD group despite a similar induction in LC3B and p62 gene expression. This lack of autophagy stimulation with fasting in HFD animals was not explained by differences in autophagy initiation as the phosphorylation status of ULK1 decreased similarly with fasting in both groups.

Conclusions:
These data indicate that the impairment of cardiac autophagy in the presence of MetS is not attributable to abnormal upstream nutrient sensing pathways. The findings suggest a MetS-associated defect in the autophagy/lysosomal fusion pathway. This could explain the increased vulnerability of the heart to I/R injury in the setting of MetS.
Abstract 225: Chronic Alcohol Consumption Alters the Epigenetic Fingerprint of Cardiac Cell Types
Authors: Mackie Alexander R , Vaughan Erin E , Khan Mohsin , Verma Suresh K , Garikipati Srikanth , Krisnamurthy Prasanna , Ramirez Veronica , Abramova Tatiana , Ellingson Kenneth , Misener Sol , Ito Aiko , Kishore Raj
Published: July 2021

The cardiac influence of chronic ethanol (EtOH) consumption in humans is dictated by the frequency of ethanol ingestion. Whereas moderate consumption (i.e. 1-2 alcoholic drinks/day) imparts a cardiac benefit to patients by reducing adverse cardiovascular event (ACE) occurrences, consumption below or beyond these moderate levels significantly increases the incidence of ACEs. Despite these observations, little is known regarding the functional impact of chronic EtOH consumption on post-myocardial infarct repair or the cellular mechanisms involved in this process. Thus, we investigated the post-AMI functional consequences of chronic ethanol consumption in mice. Mice received chronic ethanol via the Lieber-DeCarli paradigm (i.e. 0%, 1% (moderate) or 5% (high) ethanol v/v) in an isocaloric fashion for 8 weeks. After 8 weeks, mice underwent a 60-minute ischemic/reperfusion injury and the subsequent assessment of their cardiac function for 4 weeks post-AMI. As early as two weeks post-AMI, mice fed the 1% EtOH displayed modest yet significant improvements in ejection fraction and systolic ventricular volumes as compared to control mice. Conversely, the 5% EtOH diet group displayed diminished ejection fraction and increases in both systolic chamber volume and infarct size. To explore the cellular basis of these observed functional changes, primary cardiac cell types (fibroblasts and endothelial cells) treated chronically (5 days) with ethanol in vitro (i.e. 0%, 0.1% (moderate) or 0.5% (high) ethanol v/v) displayed robust changes in their epigenetic histone-modification profiles indicating a high likelihood for changes in cell specific gene expression. In addition, conditioned media from ethanol treated primary cardiac fibroblasts (PCFBs) altered the tube forming capacity of endothelial cells in a matrigel tube-formation assay in a dose pattern akin to what was observed in vivo. This data suggests that chronic ethanol directly invokes epigenetic changes in PCFBs that can modify their contribution to cardiac repair processes following AMI. Lastly, we are actively investigating whether ethanol-induced changes to endothelial cell epigenetic patterns alter the cells responses in the face of an ischemic insult.
Abstract 226: GGF2 Requires Activation Of PI3 Kinase Pathway To Mediate Cytoprotective Effects In The Mouse Atrial-derived Hl-1 Cardiomyocyte-like Cell Line
Authors: Srinivas Maya , Cao Jing , Zilinski Roni , Caggiano Anthony O , Button Donald
Published: July 2021

Glial growth factor (GGF2) is a Neuregulin-1 (NRG-1), which binds ErbB receptors and activates downstream cell signaling processes. NRG-1 ligands and their receptors are required for cardiac development and adult cardiac function. GGF2 has been shown to improve left ventricular function in several models of heart failure though the mechanisms by which GGF2 is efficacious are poorly understood. GGF2 is currently in clinical trials for treatment of congestive heart failure. Many studies have focused on GGF2 regulation of PI3 kinase (PI3K) as measured by formation of pAKT
in vivo
and in numerous cell lines, including HL-1 cells, neonatal rat ventricular myocytes (NRVMs) and human iPSC-derived cardiomyocytes. NRG-1s also activate the MAP kinase (MAPK) pathway as measured by pERK1/2 formation. To better understand GGF2 action in the stressed heart, we have developed
in vitro
systems to study GGF2 protection of cardiomyocytes from doxorubicin induced toxicity. After serum starvation, cells were pre-incubated with GGF2 (0.15 pM to 9.5 nM) for 1 hr followed by exposure to 1 μM doxorubicin for 18 hrs. After doxorubicin treatment MTT labeling index of cultures was used as an endpoint measurement of cell metabolic status. Doxorubicin decreases MTT labeling index by 90% while GGF2 prevented this toxicity by almost 50% compared to untreated cells, with an EC
50
of 83.3±19.4 pM . This effect appears to be dependent upon AKT signaling and not MAPK as assessed using inhibitors of both pathways. Inhibition of MAPK increases pAKT formation without changing extent of GGF2 protection. Blocking ErbB4 receptor, highly expressed in cardiomyocytes, causes a decrease in pAKT formation, suggesting that this receptor is important for mediating GGF2 action in these cells. Our results demonstrate that, in HL-1 cells, GGF2 mediates cytoprotective responses which require PI3K activation. Similar studies are being pursued in doxorubicin-treated NRVMs and human iPS derived cardiomyocytes. Initial results indicate GGF2 also mediates cytoprotective effects in these cell types. Future efforts will be aimed at elucidating potential roles for PI3K-dependent GGF2 regulation of mitochondrial function, Ca
2+
signaling or REDOX regulation in the observed cytoprotective actions.