bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 801: Characterization of Cardiac Myeloid Cells in Aged Mice and Their Role in Cardiac Dysfunction During Bacterial Infection
Authors: Saljoughian Noushin , Wu Qian , Kashef Cameron , Patel Sonali , Ganesan Latha P. , Lafuse William P. , Rajaram Murugesan V.S.
Published: October 2019

Biological aging is a risk factor for the development of cardiovascular disease. The changes in the normal immune cell phenotype and function in the myocardium due to immunosenescence is the major source for cardiac distress. Cardiac resident macrophages in the heart play a critical role in the maintenance of homeostatic functions and tissue repair during myocarditis. Recently, we have demonstrated that non-tuberculosis
Mycobacterium avium
(NTM- common in lung infections in the elderly population in the U.S.) infection predisposes the aged mice to cardiac dysfunction; however the mechanism is not known. Here, we investigated the various subsets of myeloid cells and their contribution to the heart function in old mice. We infected young (10 weeks) and old mice (18 months) with NTM and characterized the resident and infiltrated myeloid cells. Our results revealed that before infection old mice showed elevated numbers of myeloid cells in heart tissue compared to young mice. In contrast, NTM infection (30 day post infection) significantly increased the frequency of different myeloid cell subsets in the young mice. Although, the number of cardiac macrophages in young and old mice are not significantly different in infected and uninfected mice, we found that MHCII
low
macrophages (cardiac resident) are significantly higher in the heart tissue of old mice than young mice before infection which significantly decreased after infection. Interestingly, we observed that MHCII
high
and MerTK
+
macrophage (infiltrated macrophages) populations are increased in young and old mice but with a higher rate in young mice. Notably, we found that the number of myeloid-derived suppressor cells (MDSCs) in the heart of aged mice is decreased by infection, which was increased in young mice. In conclusion our data indicates that the NTM infection alters the myeloid cell population and function in the aged heart and failed to maintain the normal heart function, where as in young mice, infection leads to the recruitment of myeloid cells which help to confront the infection and maintain the normal heart function. Further MDSCs accumulation in the heart of young mice after infection is responsible for anti-inflammatory effects to control the hypertrophy, fibrosis and inflammation.
Abstract 802: Cardiomyocyte-derived Exosomal MicroRNAs Regulates Post-Infarction Inflammation and Myofibroblast Phenoconversion
Authors: Santulli Gaetano , Sardu Celestino , Shu Jun , Matarese Alessandro , Wang Xujun
Published: October 2019

Background:
The inclusion of microRNAs (miRNAs) in
extracellular microvesicles/exosomes
(named cardiosomes when deriving from cardiomyocytes) allows their active transportation and ensures cell-cell communication.

Aim:
We hypothesize that cardiosomal miRNAs play a pivotal role in the activation of myofibroblasts following ischemic injury.

Methods:
Using bioinformatic approaches and an established murine model of myocardial infarction (MI, achieved via ligation of the left anterior descending coronary artery), we tested our hypothesis by measuring in isolated fibroblasts and cardiosomes the expression levels of a set of miRNAs, upregulated in cardiomyocytes post-MI and involved in myofibroblast phenoconversion.

Results:
We found that miR-92a and miR-195 were significantly upregulated (absolute values) in cardiosomes as well as in fibroblasts isolated at different time points after MI compared with SHAM conditions. These miRs were demonstrated to be both predicted (bioinformatics) and biologically validated (luciferase assays) targets of SMAD7. Moreover, primary isolated cardiac fibroblasts were activated both when incubated with cardiosomes isolated from ischemic cardiomyocytes and when cultured in conditioned medium of post-MI cardiomyocytes, whereas no significant effects were observed following incubation with cardiosomes or medium from sham cardiomyocytes. Myofibroblast activation was assessed measuring the expression of aSMA, periostin, collagen I/III, FAP, fibronectin ED-A. Both miRs also regulated the inflammatory response (evaluated in terms of IL-6 and CXCL1). The addition of an inhibitor of exosome release (GW4869 10 μM for 12 h) significantly attenuated all these responses. The mechanistic contribution of miR-92a and miR-195 was further confirmed using specific miR mimics and inhibitors.

Conclusions:
Taken together, our findings indicate for the first time that two cardiomyocyte-specific miRNAs (miR-92a and miR-195), transferred to fibroblasts in form of exosomal cargo, are essential in the activation of myofibroblasts in MI.
Abstract 803: Sepsis-induced Acute Heart Failure is Driven by MEF2D-dependent Transcription
Authors: Schell Richard , Alban Felix , Haslinger Clemens , Theis Philipp , Katus Hugo A , Backs Johannes
Published: October 2019

Rationale:
Our recent work unmasked an unexpected link between inflammation and adverse cardiac remodeling via activation of the transcription factor myocyte enhancer factor 2 (MEF2). We identified a signaling pathway consisting of PGE2, EP3-receptors, PKD and Rac1 leading to a nucleo-cytoplasmic shuttling of HDAC5 with subsequent activation of MEF2. Here, we aimed to elucidate the in vivo role of MEF2 in a model of acute inflammatory cardiomyopathy.

Results:
Exposure of BALB/c-mice to 7 mg/kg from E. coli derived lipopolysaccharide (LPS) led to severe depression of left-ventricular function and was accompanied by considerably increased levels of PGE2. Strikingly, cardiac myocyte-specific MEF2D deficient mice (MEF2D-KO) were largely protected from LPS-induced acute heart failure, as documented by preserved ejection fraction (LVEF after 24 hours: 49% in contrast to 31% in LPS-treated littermates). Moreover, expression of typical target genes of MEF2 (e.g. Myomaxin) were attenuated in MEF2D-KO, confirming that the loss of MEF2D contributed to reduced MEF2 activity. A cardiomyocyte-specific unbiased transcriptome-sequencing in the early stage of inflammation-induced acute heart failure revealed new MEF2D-dependent genes.

Conclusion:
Thus, we provide new conceptual insights into inflammation-induced acute heart failure and provide evidence that the transcription factor MEF2D mediates acute contractile dysfunction. We therefore propose that transcriptional therapies might represent a novel approach to combat not only chronic but also acute heart failure.
Abstract 823: The Extracellular Matrix Component, Hyaluronan, Provokes a Pro-Inflammatory Phenotype in Macrophages
Authors: Audam Timothy N , Dassanayaka Sujith , Jurkovic Andrea , Long Bethany , Brittian Kenneth R , Wysoczynski Marcin , Jones Steven P
Published: October 2019

Background:
The extracellular matrix (ECM) provides structural and functional support for the myocardium, but myocardial infarction (MI) changes the composition of the ECM. One of the chief components of the ECM, hyaluronan (HA), is elevated after MI; however, specific biological actions of HA—particularly at the level of infiltrating immune cells and implications of such interactions on ventricular remodeling—have not been explored.

Goals:
Because upregulation of HA coincides with macrophage infiltration after MI, we determined whether hyaluronan interacts with macrophages and investigated the implication of such interactions on macrophage function.

Methods:
WT mice were subjected to non-reperfused MI to determine changes in hyaluronan synthases (HAS), hyaluronidases (HYAL), and HA levels in the heart. Interaction of HA with macrophages was studied by polarizing bone marrow derived macrophages and analyzing cells by flow cytometry. Next, we characterized the ability of macrophages to metabolize HA by profiling polarized macrophages for HA-metabolizing enzymes, HA receptors, HA-binding proteins, hyaluronidase activity, and phagocytosis.

Results:
Compared to Sham hearts, MI (n=5/group) augmented the expression of HAS-2 (10-fold, p=0.002) and HYAL-2 (2 to 4-fold, p=0.0004) in the infarct and remote regions of the heart at 5 d post-MI. HA levels (n=8/group) were elevated in the infarct (1 to 2-fold, p=0.0200) and remote (2 to 3-fold, p=0.0007) regions of the heart compared to sham hearts. Polarizing macrophages (n=3/group) in the presence fluorescein-conjugated HA (HA-FL) showed that naïve (M0), pro-inflammatory (M1), and pro-resolving (M2) macrophages interact with HA-FL; M1 showed the highest FITC intensity. Interestingly, exposing macrophages (n=5/group) to HA provoked an inflammatory phenotype, as reflected by enhanced expression of TNFα (4-fold, p=0.0001) and IL-1β (7-fold, p=0.0094) mRNA; HA also enhanced macrophage phagocytosis (0.5-fold, p= 0.0476).

Conclusion:
Hyaluronan is elevated following MI and can influence macrophage function. Because of the accumulation of hyaluronan and macrophages in the post-MI heart, macrophage-hyaluronan interactions may be a nexus regulating ventricular remodeling.
Abstract 836: Regulation of
Bmp7
and
Ctgf
Suppresses Dilated Cardiomyopathy and Cardiac Fibrosis
Authors: Jiang Jianming , Tan Chia Yee
Published: October 2019

Introduction:
Cardiac
Lmna
insufficiency causes dilated cardiomyopathy (DCM) coupled with cardiac fibrosis and inflammation. TGFβ family members are key mediators for cardiac performance, cardiac fibrosis and inflammation. However, how TGFβ family members are regulated in heart is unknown.

Hypothesis:
Modulation of downstream pathways could be served as a therapeutic strategy.

Methods and Results:
We demonstrate that regulation of
Bmp7
and
Ctgf
suppresses
Lmna
DCM and cardiac fibrosis. Upregulation of
Bmp7
alone was not sufficient to suppress DCM and cardiac fibrosis. Importantly, upregulation of
Bmp7
together with
Ctgf
silencing significantly suppressed DCM and cardiac fibrosis. Mechanistically, downregulation of
Ctgf
inhibited TGFβ/Smad signaling pathway in DCM hearts. Co-modulation of
Bmp7
and
Ctgf
reduced infiltration of T cells in hearts. A screen of cardiac related factors identified
Yy1
enhanced
Bmp7
promoter activity and prevent the upregulation of
Ctgf
promoter activity.
Yy1
suppressed
Lmna
DCM by inducing
Bmp7
expression and reduced
Ctgf
upregulation in cardiomyocytes. Importantly, knockdown of upregulated
Bmp7
attenuated the suppressive effect of
Yy1
on DCM and cardiac fibrosis.

Conclusions:
Our findings demonstrate that DCM caused by
LMNA
insufficiency can be treated with co-modulation of
Bmp7
and
Ctgf
.
Abstract 839: Sphingosine 1-Phosphate Receptor 1 in Cardiomyocytes Protects Against Cardiac Fibrosis
Authors: Leach Desiree F , Wolfe Jayne , Jorgensen Ryan , Lavelle Bianca , Shah Sanjiv , Wilsbacher Lisa
Published: October 2019

Sphingosine 1-phosphate receptor 1 (S1P
1
, encoded by
S1pr1
) is a G protein-coupled receptor that binds the ligand S1P and signals in multiple cell types including endothelium and cardiomyocytes. We previously demonstrated that excision of
S1pr1
in cardiomyocytes during embryonic development leads to ventricular noncompaction and perinatal death in most but not all mutant mice. To investigate roles for S1P
1
in adult cardiomyocytes, we used three conditional
S1pr1
deletion strategies. Mice that survived early embryonic deletion of
S1pr1
in cardiomyocytes using
Mlc2a-
Cre demonstrated interstitial cardiac fibrosis by 4 months of age. Animals with later embryonic deletion of cardiomyocyte
S1pr1
using αMHC-Cre escaped the ventricular noncompaction phenotype, but they displayed significantly increased interstitial fibrosis after isoproterenol infusion. Likewise,
S1pr1
excision from cardiomyocytes in adult mice using αMHC-MerCreMer led to increased cardiac fibrosis after angiotensin II infusion. In all conditional
S1pr1
excision strategies, systolic function in mutant mice remained similar to control mice. Finally, we conditionally deleted the sphingosine kinases
Sphk1
and
Sphk2
in developing cardiomyocytes and found no reduction in survival for cardiomyocyte
Sphk1/2
mutant mice. These results suggest that S1P from non-cardiomyocyte sources support cardiac development, and that cardiomyocyte S1P
1
activity protects against cardiac fibrosis.
Abstract 840: Epicardial Prestrained Confinement and Residual Stresses: A Newly Observed Heart Ventricle Confinement Interface
Authors: Shi Xiaodan , Liu Yue , Copeland Katherine M , McMahan Sara R , Zhang Song , Butler J. Ryan , Hong Yi , Cho Michael , Bajona Pietro , Gao Huajian , Liao Jun
Published: October 2019

The heart epicardial layer, with elastin as the dominant component, has not been well investigated, specifically on how it contributes to ventricular biomechanics. In this study, we revealed and quantitatively assessed the overall status of prestraining and residual stresses exerted by the epicardial layer on the heart left ventricle (LV). During porcine heart wall dissection, we discovered that bi-layered LV surface strips, consisting of an epicardial layer and cardiac muscle, always curled towards the epicardial side due to epicardial residual stresses. We hence developed a curling angle characterization technique to intuitively and qualitatively reveal the location-dependency and direction-dependency of epicardial residual stresses. Moreover, by combining prestrain measurement and biaxial mechanical testing, we were able to quantify the epicardial prestrains and residual stresses on the unpressurized intact LV. To investigate the potential mechanical effect of epicardial prestraining, a finite-element (FE) model has been constructed, and we demonstrate that it is the prestraining of the epicardial layer, not the epicardial layer alone, providing an additional resistance mechanism during LV diastolic expansion and ventricular wall protection by reducing myocardial stress. In short, our study on healthy, native porcine hearts has revealed an important phenomenon—the epicardial layer, rich in elastin, acts like a prestrained ‘balloon’ that wraps around the heart and functions as an extra confinement and protection interface. The obtained knowledge fills a gap in ventricular biomechanics and will help design novel biomimicking materials or prosthetic devices to target the maintenance/recreation of this ventricle confinement interface.
Abstract 845: Distinctive Inflammatory and Fibrotic Signature of Extracellular Vesicles from Epicardial Fat of Patients with Atrial Fibrillation
Authors: Shaihov-Teper Olga , Ram Eilon , Levin-Kotler La-Paz , Naftali-Shani Nili , Leor Jonathan
Published: October 2019

Background and Aim:
Epicardial fat (eFat) has been linked to atrial remodeling and fibrillation (AF). We aimed to determine whether extracellular vesicles (EVs) derived from eFat play a role in the pathogenesis of AF.

Methods and Results:
We collected small specimens of eFat from patients (pts) with and without (w/o) AF undergoing heart surgery. eFat specimens were incubated as organ cultures and EVs were isolated from the culture medium by ultra-centrifugation. We used immunoblotting, electron microscopy, and nanoparticle tracking analysis to characterize the EVs (
Fig. A-C
). Significantly, eFat specimens from AF pts secreted greater amounts of EVs compared with patients w/o AF (
Fig. D
). Moreover, eFat EVs from AF pts secreted a higher concentration of inflammatory and fibrotic cytokines but less anti-inflammatory cytokines, compared with patients w/o AF (
Fig. E-H
). Notably, EV cytokines reflected the inflammatory and fibrotic status of eFat in AF pts better than free cytokines (
Fig. I-L
). Next, we tested several miRNA that could influence cardiac fibrosis. For example, miR-133 inhibits TGF-β, decreases collagen content and inhibits atrial remodeling. Expression of EV miR-133 was lower in eFat of AF pts than in patients w/o AF (
Fig. M
). Eventually, “wound healing” scratch assay show that fibroblast migration was greater after incubation with eFat EVs from AF patients, compared with pts w/o AF. (
Fig. N
).

Conclusions:
eFat from AF patients secretes a higher number of EVs with an inflammatory and fibrotic profile. Our findings suggest that eFat EVs contribute to inflammation and fibrosis, both of which contribute to the pathogenesis of atrial remodeling and fibrillation.
Abstract 847: Adipocyte-specific Pharmacological Inhibition of Adipose Triglyceride Lipase (ATGL) Ameliorates Cardiac Fibrosis in Heart Failure
Authors: Takahara Shingo , Soni Shubham , Byrne Nikole J , Parajuli Nirmal , Maayah Zaid H , Ferdaoussi Mourad , Levasseur Jody L , Migglautsch Anna K , Breinbauer Rolf , Dyck Jason R
Published: October 2019

Objectives:
ATGL is a key enzyme that regulates triglyceride lipolysis in most cells, including the adipocyte and the cardiomyocyte. Recent studies indicate that accelerated adipose tissue lipolysis contributes to worsening heart failure (HF), suggesting that ATGL inhibition specifically in the adipocyte may prevent worsening HF. Indeed, we have shown that the ATGL inhibitor, Atglistatin, which appears to be largely adipocyte-specific, ameliorates cardiac dysfunction and hypertrophy in HF induced by transverse aortic constriction (TAC). However, the mechanisms by which Atglistatin treatment prevents worsening HF in mice remains unknown.

Methods:
Eight week-old C57BL/6N mice underwent TAC or sham surgery. After 1 week, mice with HF were assigned into 4 groups: 1) vehicle (standard chow)-sham, 2) Atglistain-sham, 3) vehicle-TAC and 4) Atglistatin-TAC. Atglistain was administered in food pellets containing 2mmol Atglistatin/kg for 2 weeks. Thereafter, mice were euthanized and hearts were used for subsequent histological and quantitative PCR analysis.

Results:
Picro-sirius red staining demonstrated that hearts from the Atglistatin TAC mice had significantly less cardiac fibrosis than the vehicle TAC mice (1.6±0.4 vs 5.3±1.1%, in Atglistatin TAC vs vehicle TAC; p<0.01). Consistent with this finding, Atglistatin treatment of the TAC group reduced transcript levels of fibrosis-related genes, including
Col1a1
(-58.7%, p=0.03) and
Postn
(-54.7%, p<0.01), compared to vehicle-treated hearts. Furthermore, Atglistatin decreased the transcript levels of the macrophage polarization-related genes, including
Lgals3
(-55.3%, p<0.01) and
Spp1
(-38.7%, p=0.03)
,
in the hearts from TAC mice compared to controls. Moreover, immunofluorescent analysis showed that Atglistatin reduced infiltration of Galectin-3 positive macrophages in the heart compared to vehicle-TAC mice, suggesting reduced macrophage infiltration and activation and less cardiac fibrosis.

Conclusion:
Our data indicate that reducing triglyceride lipolysis in the adipose tissue via ATGL inhibition may ameliorate HF via modulation of HF-induced macrophage and fibroblast activation in the heart.
Abstract 849: Drp1-dependent Altered Mitochondrial Dynamics Contribute to Protein Aggregation and Mitochondrial Dysfunction in R120G αB-crystallin-induced Proteotoxicity
Authors: Alam Shafiul , Abdullah Chowdhury S , Aishwarya Richa , Morshed Mahboob , Miriyala Sumitra , Panchatcharam Manikandan , Bhuiyan Md. S
Published: October 2019

Introduction:
αB-crystallin (CryAB) is a chaperone protein that plays a pivotal role in the maintenance of the structural and mechanical integrity of the sarcomere. Mutation in αB-crystallin (CryAB R120G) causes sarcomere disorganization leading to protein aggregation. Accumulation of toxic protein aggregates causes cardiac contractile dysfunction and perturbed mitochondrial spatial organization. However, the role of mitochondria in the pathogenesis of protein aggregation-induced cardiomyopathies remain obscure.

Objective:
In this study, we investigated the role of mitochondrial dynamics and function in the development of R120G αB-crystallin induced cardiac proteotoxicity.

Methods and Results:
CryAB R120G Tg hearts showed accumulation of toxic protein aggregates resulting in disruption of the sarcomere structure, development of cardiomyocyte hypertrophy, and contractile dysfunction. Hearts obtained from CryAB R120G Tg mice showed increased expression and localization of Drp1 on the mitochondrial membrane. Alteration in the expression of mitochondrial dynamics regulatory proteins was observed at two months age when these mice exhibited normal cardiac morphometry and systolic function. Adenoviral-mediated expression of CryAB R120G in cardiomyocytes also showed increased mitochondrial fission, expression dependent inhibition of mitochondrial respiration and activation of cellular toxicity. Inhibition mitochondrial fission by Drp1 partial knockdown in CryABR120G expressing cardiomyocytes significantly decreased protein aggregation and improved mitochondrial respiration.

Conclusion:
Drp1-dependent excessive mitochondrial fission results in defects in mitochondrial respiration and accumulation of protein aggregates in CryABR120G expressing cardiomyocytes. Therefore, our study shows that maladaptive aberrant mitochondrial fission causes CryABR120G -induced mitochondrial dysfunction and cardiac proteotoxicity-associated cellular dysfunction.
Abstract 853:
GPX4
Gene Expression is Dose-responsive to Doxorubicin Exposure in iPSC-Cardiomyocytes and Correlated With Mitochondrial Function
Authors: Reyes Monica E , Callender Rashida A , Ma Jianzhong , Grove Megan L , Morrison Alanna C , Hildebrandt Michelle A
Published: October 2019

Impaired mitochondrial function has been implicated as a mechanism of doxorubicin-induced cardiotoxicity, however the precise genes that regulate this process in human cardiomyocytes remain to be elucidated. We hypothesized that doxorubicin significantly alters expression of genes involved in mitochondrial function in human cardiomyocytes, which in turn impairs mitochondrial respiration. Towards this, we treated human inducible pluripotent stem cell (iPSC)-cardiomyocytes with doxorubicin at 15 different time and dose conditions. Gene expression was assessed by RNAseq for each condition and 169 genes involved in mitochondrial function were analyzed for differential expression between control and treated conditions. Mitochondrial respiration (basal respiration, ATP production, maximal respiration, and spare respiratory capacity) was measured using the Seahorse Bioscience XFe96 Cell Mito Stress Test kit and correlated to gene expression levels. Of the 169 genes analyzed, 25 were significantly differentially expressed (P < 0.05) including
GPX4
(P = 5.70 x 10
-3
). Expression of
GPX4
remained significant in pairwise comparisons by dose for day 2 and qRT-PCR validation confirmed a dose-dependent decrease in
GPX4
expression. Maximal respiration (r = -0.62; P = 0.031) and spare respiratory capacity (r = -0.67; P = 0.017) correlated with
GPX4
expression in doxorubicin-treated iPSC-cardiomyocytes.
GPX4
encodes a glutathione peroxidase that is responsible for protecting the cell against oxidative damage. Damage due to reactive oxygen species (ROS) is one of the established mechanisms of anthracycline-induced cardiac damage. Our findings underscore a role for mitochondrial function and ROS in the development of doxorubicin-induced cardiotoxicity and implicates
GPX4
in this process. The assessment of doxorubicin-altered gene expression in iPSC-cardiomyocytes may provide insight into how impaired mitochondrial respiration leads to cardiotoxicity and heart failure in cancer survivors treated with doxorubicin and other anthracyclines.
Abstract 854: The Role of Mitochondrial Permeability Transition in the Regulation of ETC Supercomplexes Assembly by OPA1
Authors: Jang Sehwan
Published: October 2019

Optic atrophy type 1 (OPA1), a dynamin-related GTPase is the inner mitochondrial membrane (IMM) protein, which, in addition to mitochondrial fusion, participates in maintaining the structural integrity of the IMM. The function and topography of the IMM is also regulated by changes in the matrix volume. Opening of the mitochondrial permeability transition pores (PTP) is the leading cause of mitochondrial matrix swelling. However, the cause-and-effect relationship between OPA1 proteolytic cleavage and PTP induction remains unknown. Here, we elucidated the role of PTP in OPA1-mediated regulation of electron transport chain (ETC) supercomplexes (SCs) assembly. First, intact mitochondria isolated from the rat heart were used to analyze the effect of PTP induction on the cleavage of long (L)-OPA1. Calcium-induced mitochondrial swelling increased L-OPA1 cleavage (66%, P<0.001) associated with accumulation of short (S)-OPA1. Inhibition of swelling by sanglifehrin A (a PTP inhibitor) prevented L-OPA cleavage whereas tert-butyl hydroperoxide (TBH) had no effect on L-OPA levels. Second, OPA1 knockdown (KD) by siRNA in HEK cells resulted in a 74% decrease (P<0.001) in OPA1 protein expression but did not change the proportion of L-OPA1 to total OPA. Mitochondria isolated from OPA1 KD cells showed reduced calcium-stimulated swelling with no changes in protein levels of cyclophilin D, a major PTP regulator. OPA1 KD cells demonstrated a 20% increase (P<0.05) in calcium-induced ROS generation. Analysis of individual ETC complexes revealed significant reduction of protein levels of the complexes I, III, and IV in OPA1 KD cells. Likewise, activities of complexes I and IV were reduced (48% and 37%, respectively, P<0.01 for both). OPA1 KD cells contained 38% (P<0.05) less ATP whereas the citrate synthase activity reflecting mitochondria mass was not affected. Blue native PAGE analysis demonstrated a 35% decrease (P<0.01) of high molecular weight proteins (>100 kDa) and a 24% decrease (P<0.05) of the respirasome, the main SC, levels in OPA1 KD mitochondria. We conclude that mitochondrial swelling by PTP opening stimulates OPA1 cleavage, and reduced OPA1 levels result in cristae malformation including reduced respirasome levels and the ETC complexes activity.
Abstract 856: Mitochondrial Protein Kinase B (akt) Translocation Mediates Insulin-stimulated Cardiac Glucose Oxidation
Authors: Karwi Qutuba G , Wagg Cory S , Altamimi Tariq R , Lopaschuk Gary D
Published: October 2019

Introduction:
Insulin stimulates glucose oxidation, an effect which is associated with simulating pyruvate dehydrogenase (PDH). However, how the insulin signal is transduced from the cell membrane to the mitochondria to stimulate PDH is not known. Protein kinase B (Akt), protein kinase C-delta (PKCδ) and glycogen synthase kinase-3beta (GSK-3β) are main components of the cytosolic insulin signalling pathway and it has been suggested that they can be translocated to the mitochondria following insulin receptor activation in noncardiac tissue. Therefore, we investigated whether any of these kinases has a role mediated cardiac insulin-stimulated glucose oxidation in the heart.

Methods and Results:
Male and female C57BL/6 mice were anesthetized and hearts were collected and perfused in the isolated working heart mode. Hearts were perfused with [5-
3
H] glucose and [U-
14
C] glucose to simultaneously measure glycolysis and glucose oxidation rates, respectively, in the presence and absence of insulin. Insulin enhanced the phosphorylation and translocation of Akt
Ser473
, PKCδ
Tyr311
and GSK-3β
Ser9
to the cardiac mitochondria along with enhancing PDH activity by decreasing its phosphorylation. Pharmacological inhibition of Akt using AktiVIII completely abolish the stimulatory effect of insulin on cardiac glucose oxidation rates (354 ± 145 vs 2307 ± 185 nmol. g dry wt
-1
. min
-1
in vehicle-treated hearts, p<0.05). However, pharmacological inhibition of either PKCδ or GSK-3β using Bisindolylmaleimide I or 3F8, respectively, did not have a significant effect on insulin-stimulated glucose oxidation rates. None of the pharmacological inhibitors had any significant effect on cardiac glycolysis.

Conclusion:
Insulin mediates its stimulatory effect on cardiac glucose oxidation via a mechanism which involve the activation and translocation of Akt to the mitochondria.
Abstract 857: MCUB Regulates the Molecular Composition of the Mitochondrial Calcium Uniporter Channel During Cardiac Stress to Limit Mitochondrial Calcium Overload
Authors: Lambert Jonathan P , Luongo Timothy S , Tomar Dhanendra , Jadiya Pooja , Gao Erhe , Zhang Xueqian , Lucchese Anna M , Kolmetzky Devin W , Shah Neil , Elrod John W
Published: October 2019

The mitochondrial calcium uniporter (mtCU) is a ~700 kD multi-subunit channel residing in the inner mitochondrial membrane required for mitochondrial Ca
2+
(
m
Ca
2+
) uptake. Mitochondrial Calcium Uniporter B (
MCUB
) is reported to negatively regulate
m
Ca
2+
uptake, but its precise functional role and contribution to cardiac physiology remain unresolved. Size exclusion chromatography of ventricular mitochondria revealed MCUB was absent from high-molecular weight (MW) mtCU complexes in sham animals, but present 24 hours following myocardial ischemia-reperfusion injury (IR). To investigate
MCUB
’s contribution to mtCU regulation we created a
MCUB
-/-
cell line by CRISPR-Cas9n.
MCUB
deletion increased histamine-mediated [
m
Ca
2+
] transient amplitude by ~50% vs. WT controls (mito-R-GECO1).
MCUB
deletion increased mtCU capacitance (mitoplast patch-clamp) and rate of [
m
Ca
2+
] uptake. Size-exclusion chromatography revealed loss of MCUB increased MCU incorporation into high-MW mtCU, suggesting stoichiometric replacement and overall more functional mtCU’s. To examine MCUB’s role in cardiac physiology we generated a cardiac-specific, tamoxifen-inducible MCUB mouse model (CAG-CAT-MCUB x MCM; MCUB-Tg). FPLC revealed MCUB was undetected in high-MW mtCU complexes of Cre controls, but enriched in MCUB-Tg hearts. MCUB incorporation decreased the presence of channel gatekeepers, MICU1/2, and decreased the MW of the mtCU complex. Immunoprecipitations suggest MCUB interacts with MCU but not MICU1/2. MCUB-Tg adult cardiomyocytes (ACMs) expressing AAV9-mitycam (
m
Ca
2+
reporter) were paced and displayed a ~30% decrease in
m
Ca
2+
transient peak amplitude with significantly reduced
m
Ca
2+
uptake rates vs controls. A reduction in OxPhos reserve capacity correlated with a severe impairment in cardiac contractile reserve (LV invasive hemodynamics during isoproterenol infusion). MCUB-Tg cardiac mitochondria were resistant to Ca
2+
-induced permeability transition and MCUB-Tg mice displayed ~50% decrease in infarct size per area-at-risk after in vivo IR-injury. These data suggest MCUB regulation of the mtCU is an endogenous compensatory mechanism to decrease
m
Ca
2+
overload during ischemic injury, but maladaptive to cardiac energetic responsiveness.
Abstract 866: Cardiac-specific Deletion of General Control of Amino-Acid Synthesis 5-like 1 Regulates Fatty Acid Oxidation in Diet Induced Obesity
Authors: Thapa Dharendra , Manning Janet R , Stoner Michael W , Zhang Manling , Scott Iain
Published: October 2019

Background:
Healthy hearts use several fuel substrates for energy metabolism, with fatty acid oxidation (FAO) accounting for ~70% of total ATP production. Lack of flexibility to utilize different substrates, and a shift towards increased fatty acid utilization, is a key feature of obesity and diabetes. We previously reported that increased acetylation of several mitochondrial FAO enzymes, regulated in part via increased abundance of the mitochondrial acetyltransferase GCN5L1, correlated with increased enzymatic activity and FAO in the heart. The focus of the current study was to investigate whether decreased acetylation, via cardiac depletion of GCN5L1, regulates cardiac energy metabolism following exposure to a high fat diet (HFD).

Hypothesis:
We hypothesize that decreased acetylation of metabolic enzymes in GCN5L1 cardiac KO (cKO) mice may limit excessive cardiac FAO activity and improve glucose utilization under HFD conditions. Furthermore, we postulate that this metabolic phenotype would result in improved mitochondrial bioenergetic output and cardiac function.

Methods:
To examine the role of GCN5L1 in regulating mitochondrial protein acetylation, immunoprecipitation with acetylated lysine antibodies was performed to assess the acetylation status of metabolic proteins. Western blots and qPCR were performed to quantitate protein and mRNA expression levels. Further, metabolic enzyme activities were analyzed
in vitro
. Finally, cardiac workload was measured
ex vivo
to assess changes in cardiac function.

Results:
We found that increased mitochondrial protein acetylation under HFD conditions was significantly decreased in GCN5L1 cKO mice relative to WT. This decrease correlated with reduced acetylation of the FAO enzymes SCAD and HADHA in GCN5L1 cKO mice. HFD exposure led to increased cardiac FAO enzyme activity in WT animals, which was reduced in GCN5L1 cKO animals. Finally, a significant decrease in cardiac workload observed in WT animals was not present in GCN5L1 cKO animals under HFD conditions.

Conclusions:
These findings indicate that decreased acetylation of FAO proteins via cardiac depletion of GCN5L1 results in decreased cardiac FAO utilization and enzymatic activity under HFD conditions.
Abstract 867: NAD
+
Repletion Reverses HFpEF by Attenuating Myocardial Metabolic Dysfunction
Authors: Tong Dan , Schiattarella Gabriele G , Jiang Nan , Altamirano Francisco , Szweda Pamela A , Elnwasany Abdallah , Lee Dong I , Szweda Luke I , Kass David A , Gillette Thomas G , Hill Joseph A
Published: October 2019

Background:
Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent clinical condition associated with significant morbidity, mortality and health care expenses. Yet, no effective treatment has been identified. We recently demonstrated that concomitant metabolic and hypertensive stress in mice elicited by a combination of high fat diet (HFD) and constitutive nitric oxide synthase inhibition by N
[w]
-nitro-l-arginine methyl ester (L-NAME) faithfully recapitulates the numerous and myriad features of human HFpEF (paper in press at
Nature
).

Methods:
Wild type C57Bl6 mice were fed with HFD and L-NAME via drinking water for 5-8 weeks. Myocardial mitochondrial morphology was assessed by electron microscopy. Mitochondrial function was assessed by measuring oxygen consumption rates using an oxygen electrode. Protein expression and modification were assessed by Western blotting and immunoprecipitation.

Results:
Using this novel mouse model, we discovered significant impairment of mitochondrial fatty acid oxidation (FAO) associated with hyperacetylation of key FAO enzymes in HFpEF myocardium. Mechanistically, downregulation of Sirtuin3, the major mitochondrial deacetylase, and deficiency of its co-substrate nicotinamide adenine dinucleotide (NAD
+
), culminated in mitochondrial protein hyperacetylation. Strikingly, supplementation with nicotinamide riboside (NR), a NAD
+
precursor, led to dramatic improvement of mitochondrial function, and importantly, amelioration of the HFpEF phenotype.

Conclusion:
In summary, we have unveiled that protein hyperacetylation-mediated mitochondrial dysfunction is a crucial mechanism of HFpEF pathogenesis. This is, to our knowledge, the first study identifying a specific signature of metabolic remodeling in HFpEF heart. We also demonstrated the therapeutic effect of NAD
+
repletion in a preclinical HFpEF model. In next steps, confirming this benefit in a clinical trial is warranted.
Abstract 870: Cardiac Gene Therapy With Relaxin Receptor 1 Overexpression Protects Against Acute Myocardial Infarction and Associated Adverse Remodeling
Authors: Devarakonda Teja , Kohlbrenner Erik , Mauro Adolfo G , Cain Chad , Das Anindita , Hajjar Roger J , Salloum Fadi N
Published: October 2019

Background:
Relaxin signaling confers cardioprotection against acute myocardial infarction (MI) and mitigates adverse cardiac remodeling. We investigated the role of relaxin receptor (RXFP1) overexpression via cardiotropic AAV9 vectors in attenuating MI-associated adverse remodeling.

Methods and Results:
Murine RXFP1 cDNA with CMV promoter was introduced into AAV cis plasmids for generation of AAV9-RXFP1 viral vectors. 1.0 x 10
11
viral genomes in 100μL saline were administered i.v. to 8-week-old CD1 male mice. Four weeks later, overexpression was confirmed via qPCR of RXFP1 mRNA in whole cardiac tissue lysates (
Fig A
) and primary cardiomyocytes (
Fig B
) versus control. Overexpressing (AAV9-RXFP1) mice were subjected to MI (30 minutes of coronary artery ligation followed by reperfusion for 24h or 7d). Immunohistochemical staining of cardiac sections 7d post-MI shows increased RXFP1 expression in AAV9-RXFP1 mice, as shown in
Fig C
. Infarct size was reduced at 24h post-MI (TTC staining,
Fig D
) and fractional shortening (M mode echo) was preserved at 24h and 7d post-MI in AAV9-RXFP1 mice (
Fig E
); endocardial strain rate assessment (speckle tracking analysis) showed preserved function in the affected ventricular segments of AAV9-RXFP1 mice 7d post-MI (
Fig F
). Picrosirius staining showed reduced LV fibrosis at 7d post-MI in AAV9-RXFP1 mice (
Fig G
).

Conclusion:
Administration of AAV9-RXFP1 successfully upregulates RXFP1 in cardiac tissue and primary cardiomyocytes, reduces infarct size and fibrosis, and preserves cardiac function post MI. Targeting cardiac RXFP1 via gene therapy could be a novel strategy to prevent MI-related adverse remodeling.
Abstract 874: Role of Ginsenoside Rb1 in Resistin-Induced Vascular Smooth Muscle Cell Dysfunction
Authors: Lu Weifeng , Haider Nezam , Moly Pricila , Zhou Wei
Published: October 2019

Background:
Acetyl-LDL and resistin have been proved to play a critical role in inducing VSMC malfunction in the process of atherosclerosis. Ginsenoside Rb1, a major consitituent of Ginseng that has been used for thousands of years, was reported to have powerful vascular-protective effect. Here we aim to explore its protective effect on Acetyl-LDL and resistin induced dysfunction in VSMCs.

Methods:
Human coronary artery smooth muscle cells (HCASMC) were used at passage 5 to 8 for experiments. Cells were treated for various time points with or without resistin at 40 ng/mL and Acetyl-LDL in the presence or absence of Rb1. TBHP was also used as a positive control. Cell migration was analyzed using scratch wound assay, and cytosolic ROS (H2DCFDA as a dye probe) was measured by microplate reader and compared among groups.

Results:
Resistin time-dependently increased HCASMC migration, which could be significantly attenuated by Rb1 at 20μM. Resistin and Act-LDL increased ROS production in HCASMCs to a similar level, while pretreatment with Rb1 reversed the effects by resistin and acetyl-LDL. In addition, we demonstrated that both resistin and Rb1 had dose-dependent effect on ROS production in HCSMCs and that Rb1 at 40μM pre-treatment significantly reduced resistin (40ng/ml) induced cytosolic ROS in VSMCs.

Conclusion:
We demonstrated the protective effect of Rb1 on HCASMC and showed, for the first time, that Rb1 pre-treatment being more effective than co-treatment, suggesting that ginsenoside Rb1 has the potential of protecting against atherosclerosis. More in-dept study is needed.
Abstract 877: Thioredoxin-1 Maintains Cardiac Function and Metabolic Gene Expression via mTOR Signaling
Authors: Oka Shinichi , Mizushima Wataru , Huan Chun-yang , Sadoshima Junichi
Published: October 2019

Thioredoxin-1 (Trx1) is a 12 kDa oxidoreductase that reduces proteins with disulfide bonds through thiol disulfide exchange reactions. Several lines of evidence show that Trx1 protects cardiomyocytes against stress, such as ischemia and pressure overload. However, due to the embryonic lethality of systemic Trx1 knockout mice, the role of endogenous Trx1 in cardiac function has not been tested with loss-of-function mice. In this study, we generated cardiac-specific Trx1 knockout (Trx1cKO) mice, using Trx1floxflox and αMHC-Cre transgenic mice, to elucidate the physiological role of Trx1 in the postnatal heart. The Trx1cKO mice were viable but started dying at 13 days of age. The median survival age was 27 days. Echocardiographic analyses indicated cardiac dysfunction in Trx1cKO mice, evidenced by reduced ejection fraction (WT: 0.78; Trx1cKO: 0.50*; p<0.05 vs. WT). RNA-sequencing and pathway analyses revealed that metabolic genes involved in energy production were most prominently downregulated in Trx1cKO mice. Mitochondrial morphological abnormality and increased oxidative stress were evident in Trx1cKO mice. Among Trx1 substrates possibly involved in metabolic gene expression, mechanistic target of rapamycin (mTOR) was oxidized and inhibited in Trx1cKO mice. In cultured cardiomyocytes, Trx1 knockdown inhibited mTOR signaling, as evidenced by reduced phosphorylation of mTOR substrates, such as S6K and 4EBP1. Trx1 knockdown in cardiomyocytes inhibited mitochondrial protein expression, respiration and ATP production (relative ATP production in isolated mitochondria; control: 1; Trx1 knockdown: 0.69*; p<0.05 vs. control), suggesting that Trx1 maintains mitochondrial function in a cell autonomous manner. Importantly, mTOR-C1483F mutant is resistant against oxidation and inactivation induced by downregulation of Trx1. Trx1 knockdown suppressed reporter gene activity driven by the promoter sequences of endogenous metabolic genes, such as Ndufa1 and Ndufs1, which was rescued by mTOR-C1483F. These results suggest that Trx1 is essential for cardiac function and metabolism, possibly through reduction of mTOR at Cys1483.
Abstract 883: Nadph Oxidase Nox1-dependent Endocytosis and Activation is Regulated by a Conserved Trafficking Motif in Vascular Smooth Muscle Cells
Authors: Schickling Brandon M , Streeter Jennifer , Miller Francis J
Published: October 2019

We previously reported that TNF-α-mediated Nox1-dependent redox signaling of vascular smooth muscle cells (SMC) requires endocytosis. However, the molecular mechanisms regulating Nox1 endocytosis are not known. The goal of this study was to identify motifs in the Nox1 protein sequence necessary for Nox1 endocytosis and reactive oxygen species (ROS) generation. Immunoprecipitation of Nox1, after treatment of SMC with TNF-α, showed it to be associated with Early Endosome Antigen 1 (EEA1) and Ras-related protein Rab-5A (Rab5), markers of early endosomes. Analysis of the Nox1 protein sequence identified two conserved putative trafficking motifs, Leucine-Leucine (LL), and Valine-Leucine-Valine (VLV). Biotinylation of SMC plasma membrane proteins under non-stimulated conditions suggested increased levels of Nox1 VLV mutant in the plasma membrane, whereas levels of Nox1 LL mutant where comparable to wildtype (WT) Nox1. Furthermore, cells expressing the VLV mutant showed impaired endocytosis of Nox1 following TNF-α as compared to cells expressing WT Nox1 or the LL mutant. SMC were treated with TNF-α in the presence of Oxyburst, a non-cell permeable redox-sensitive fluorophore whose internalization requires endocytosis. Cells expressing WT or Nox1 LL, but not Nox1 VLV, exhibited punctate intracellular fluorescence, indicating endosomal ROS production. Next, we examined whether the VLV motif is required for Nox1-dependent SMC migration. Transwell migration of SMC was similar in cells expressing WT or the LL Nox1 mutant; however, migration was decreased in cells expressing the Nox1 VLV mutant. Finally, we identified proteins whose association with Nox1 during endocytosis were dependent on the VLV motif. Nox1 WT and VLV mutant were cloned in frame with the biotin ligase BioID2. Following stimulation with TNF-α, Western blotting for anti-biotin, and subsequent mass spectrometry, identified several proteins directly associated with WT Nox1 but not Nox1 VLV. These findings suggest that endosomal compartmentalization of Nox1 and its redox signaling is regulated by a specific motif on Nox1 that is necessary for protein interactions. Targeted inhibition of Nox1 activation is a novel therapeutic strategy in the prevention of vascular disease.
Abstract 884: Expression of Anti-mir-33a-5p in Cultured Endothelial Cells Increases Macrophage and Smooth Muscle Cell Cholesterol Efflux by Exosome-mediated Transfer of Anti-mir-33a-5p
Authors: Stamatikos Alexis , Vojtech Lucia , Knight Ethan , Bi Lianxiang , Wacker Bradley , Tang Chongren , Dichek David
Published: October 2019

Background:
The microRNA miR-33a-5p impairs apoAI-mediated cholesterol efflux by silencing ABCA1 expression. Systemic injection of antagomirs to miR-33a-5p reduces atherosclerosis but can cause hepatic steatosis and hypertriglyceridemia. In contrast, local delivery of miR-33a-5p antagomirs to lipid-laden intimal cells [smooth muscle cells (SMC) and macrophages (Mϕ)] could potentially treat atherosclerosis without causing metabolic abnormalities. Helper-dependent adenoviral vectors (HDAd) provide durable transgene expression in endothelial cells (EC) in vivo; however, HDAd does not cross an intact large vessel EC layer and therefore cannot deliver antagomirs directly to SMC and Mϕ. We hypothesized that transducing EC with an HDAd expressing anti-miR-33a-5p (HDAdAnti) could result in packaging of anti-miR-33a-5p into EC-derived exosomes that, in turn, deliver anti-miR-33a-5p to neighboring SMC and Mϕ. Inhibition of miR-33a-5p in SMC and Mϕ would upregulate ABCA1, and enhance apoAI-mediated cholesterol efflux.

Methods:
We transduced cultured EC with either HDAdAnti or a control HDAd expressing a “scrambled” shRNA (HDAdScr), harvested conditioned medium (CM) from the EC, and treated SMC and Mϕ (some of which were cholesterol-loaded) with the CM. We measured levels of anti-miR-33a-5p and miR-33a-5p, ABCA1 protein, and apoAI-mediated cholesterol efflux in the treated SMC and Mϕ.

Results:
Exosomes containing anti-miR-33a-5p were present only in CM from HDAdAnti-transduced EC. Compared to Mϕ incubated with CM from EC transduced with HDAdScr, Mϕ incubated with CM from HDAdAnti-transduced EC had 80% lower levels of miR-33a-5p, a 2.2-fold increase in ABCA1 protein, and a 1.6-fold increase in apoAI-mediated cholesterol efflux (all P<0.001). Similar results were obtained with SMC: 65% lower levels of miR-33a-5p expression, 1.6-fold increase in ABCA1 protein, and 1.4-fold increase in apoAI-mediated cholesterol efflux (all P≤0.01).

Conclusions:
EC transduced with HDAdAnti release exosomes containing anti-miR-33a-5p. These exosomes transfer anti-miR-33a-5p to SMC and Mϕ, increasing cholesterol efflux. Application of this approach in vivo may accelerate vessel wall cholesterol efflux, preventing and reversing atherosclerosis.