bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 504: AAV9-Mediated Overexpression of TRPM4 Increases the Incidence of Ventricular Arrhythmias in Living Mice
Authors: Pironet Andy , Vandewiele Frone , Syam Ninda , Vande Velde Greetje , Gijsbers Rik , Vennekens Rudi
Published: October 2019

Cardiac conduction disorders are a common cause of lethal cardiac arrhythmias and can be caused by mutations in ion channels. Several studies suggest that mutations in the Transient Receptor Potential subfamily M member 4 (TRPM4) are responsible for hereditary forms of cardiac conduction disorders. The majority of mutations that are described in TRPM4 lead to gain-of-function of channel activity. Analysis of the first identified gain-of-function mutation, p.E7K in a cellular overexpression system suggests that attenuated deSUMOylation of the protein, leads to impaired endocytosis and consequently an increase in the number of functional channels in the plasma membrane. To test the idea that an increased number of TRPM4 channels leads to cardiac arrhythmias, we created a functional overexpression model of the TRPM4 channel in living mice. To this end, we overexpressed TRPM4 in mice using adeno-associated virus serotype 9 (AAV9) particles, which has been described as the most cardiotropic of AAV serotypes. Overexpression of TRPM4 was achieved via tail vein injection of AAV9 particles. Subsequently, we performed telemetric ECG-measurements in freely-moving mice to determine their
in vivo
cardiac phenotype. In baseline conditions, the heart rate and ECG-parameters were similar between TRPM4-overexpressing and WT mice. Additionally, the number of arrhythmic incidents in resting mice was not different between TRPM4-overexpressing mice and WT mice. Instead, WT mice overexpressing TRPM4 exhibited more premature ventricular ectopic beats during exercise-induces β-adrenergic stress. Conduction abnormalities were rare and was not increased in mice overexpressing TRPM4. Taken together, mice overexpressing TRPM4 were more prone to develop premature ventricular ectopic beats during exercise-induced β-adrenergic stress.
Abstract 512: Extracellular Matrix Components Isolated From Diabetic Mice Alters Cardiac Fibroblast Function Through the AGE/RAGE Signaling Cascade
Authors: Burr Stephanie , Stewart James A
Published: October 2019

Roughly 30 million Americans suffer from diabetes and these individuals are at an increased risk of developing cardiovascular complications. A common complication is heart failure which occurs due to the stiffening of the left ventricle brought on by cardiac fibroblast “activation” that results in the remodeling the extracellular matrix (ECM). Fibroblast “activation” can be triggered by the AGE/RAGE signaling cascade. Advanced Glycated End products (AGEs) are produced and accumulate in the ECM overtime, but under hyperglycemic conditions this process is accelerated. We aim to investigate how the presence of AGEs in the either diabetic or non-diabetic ECM can affect fibroblast ECM remodeling as well as determine the role of AGE/RAGE signaling during this process. In order to assess this question diabetic and non-diabetic fibroblasts were embedded in 3D matrices composed of collagen isolated from either diabetic or non-diabetic mice. Non-diabetic fibroblasts displayed similar matrix contraction and α-SMA expression to diabetic fibroblasts when embedded in diabetic collagen. In addition, increasing the AGE/RAGE cascade leads to increase gel contraction indicating increase in fibroblast “activation”. These results indicate 1) the ECM from diabetic and non-diabetic mice differ from one another, 2) diabetic ECM can impact fibroblast function and shift them towards an “active” state, and 3) that fibroblasts can modify the ECM through activation of the AGE/RAGE signaling cascade. These results suggest the importance of understanding the impact diabetes has on the ECM and fibroblast function.
Abstract 516: GRK5 Regulates Cardiac Fibroblast Differentiation and Fibrosis
Authors: Eguchi Akito , Koch Walter J
Published: October 2019

Pathological remodeling of the heart is a hallmark of heart failure (HF) and these structural changes further perpetuate the disease. Cardiac fibroblasts are the critical cell type that is responsible for maintaining the structural integrity of the heart. Stress conditions, such as a myocardial infarction (MI), can activate and induce the transdifferentiation of quiescent fibroblasts into synthetic and contractile myofibroblasts. G protein-coupled receptor (GPCR) kinases (GRKs) are important mediators of cardiovascular homeostasis through dampening of GPCR signaling. GRK5 is one isoform of these kinases that is expressed in the heart and has been shown to be up-regulated in human HF. Of note, GRK5 has been demonstrated to translocate to the nucleus in cardiomyocytes in a calcium-calmodulin (Ca
2+
-CAM)-dependent manner, promoting hypertrophic gene transcription through activation of NFAT and NFκB. Interestingly, these transcription factors are also involved in fibroblast activation. GRK5 is highly expressed and active in cardiac fibroblasts (CFs), however its pathophysiological role in these crucial cardiac cells is unknown. Utilizing adult mouse CFs
in vitro
, our preliminary data suggests a relevant role for GRK5 in the activation of fibroblasts. We observed that genetic deletion of GRK5 (GRK5KO) in CFs attenuated α-smooth muscle actin (α-SMA) expression, a myofibroblast marker, after stimulation with Angiotensin II (AngII). In addition, deletion of GRK5 diminished the expression of myofibroblast genes and proteins such as Collagen I and III after AngII stimulation. GRK5KO CF’s were also refractory to AngII mediated collagen gel contraction and protein translation.
In vivo
, mice with fibroblast-specific deletion of GRK5 were protected against cardiac fibrosis and hypertrophy after AngII infusion and post-MI compared to wild type mice. Mechanistically, GRK5 translocates to the nucleus after AngII stimulation and pharmacological inhibition of this event (via inhibition of Ca
2+
-CAM) prevented AngII mediated fibroblast transdifferentiation. These data support the hypothesis that GRK5 is an essential regulator of fibroblast activation and could be a novel therapeutic target to decrease adverse myocardial remodeling after injury.
Abstract 522: IL-10 Knockout Bone Marrow Fibroblast Progenitor Cells-derived Exosomes Activate Cardiac Fibroblast and Exaggerate Pressure Overload-induced Fibrosis in Mice Heart
Authors: Kumari Rajesh , Ranjan Prabhat , Krishnamurthy Prasanna , Kishore Raj , Verma Suresh K
Published: October 2019

Background:
Inflammatory mediators play important role in cardiac remodeling. In heart, fibrosis is mainly mediated by activated fibroblasts (myofibroblasts), however, origin of myofibroblasts in diseased heart remains unclear. Recently, we have shown that bone marrow fibroblast progenitor cells (FPCs) significantly contribute in cardiac fibrosis in IL10 KO (KO) mice, but its molecular mechanism is not known. Here, we hypothesize that BM-FPCs activate resident cardiac fibroblast via their paracrine mediators (packaged in exosomes) to exaggerate the cardiac fibrosis in KO mice post- transverse aortic constriction (TAC).

Method and Results:
Cardiac hypertrophy and fibrosis was induced in Wild-type (WT) and IL10-knockout (IL10KO) mice by TAC surgery. TAC-induced left ventricular (LV) dysfunction and fibrosis were further exaggerated in KO mice. Recombinant IL10 administration markedly improved LV function and inhibited PO-induced cardiac fibrosis. PO enhanced FPC mobilization and homing in KO mice compared to WT mice. Furthermore, to identify the paracrine signaling, exosomes were isolated from WT and KO-FPCs culture media and characterized for fibrotic miRNA and proteins. Our data suggests that KO-FPC-exosomes are enriched with profibrotic miRs (miR21 and miR27) and proteins (RhoA). To explore whether KO-FPC-exosomes modulates resident fibroblasts biology and activation, adult cardiac fibroblasts (WT) were isolated and treated with WT and IL10KO-BMFPC-exosomes for 48 hrs. myoFB-associated gene (Collagen1α, Collagen3α and α- SMA) expression was notably increased in KO-FPC-exosome treated cells. Finally, immunostaining data further corroborated with our gene expression data and showed significantly enhanced α-SMA expression after KO-FPCs exosome treatments in cFBs.

Conclusion:
In conclusion, our data suggest that fibrotic remodeling factors (miRs and/or proteins) packaged in KO-FPC exosomes are sufficient to enhance the resident cardiac fibroblast activation and mediate cardiac fibrotic remodeling.

Acknowledgement:
NIH (RO1-135060) and AHA (14SDG20480104) SKV
Abstract 523: Myocardial Hypertrophy and Fibrosis Regulated by Nogo-ROCK-norepinephrine Transporter Signaling Pathway in Spontaneously Hypertensive Rats
Authors: Li Shijun
Published: October 2019

Objective:
To investigate the regulation of Nogo/ROCK- norepinephrine transporter (NET) signaling pathway on myocardial hypertrophy and fibrosis in spontaneously hypertensive rats (SHR).

Methods:
Twenty male SHR rats aged 7-week-old were randomly divided into four groups: control group (n=5), NEP1-40 group (n=5), fasudil group (n=5) and fluoxetine group (n=5), and fed for 7 weeks. Left ventricular mass (LVW) was separated and weighed. Left ventricular mass index (LVMI) was calculated. Real-time quantitative PCR was used to detect the gene expression of myocardial hypertrophy and fibrosis and NET.

Results:
Compared with the control group, the left ventricular mass index (LVMI), the gene expression of ANP, BNP andβ-MHC were significantly increased in NEP1-40 group, and were significantly decreased in Fasudil group. Compared with the control group, the gene expression of CTGF, Collagen I and Collagen III increased significantly in NEP1-40 group, while decreased significantly in Fasudil group. Compared with the control group, the gene expression of norepinephrine transporter (NET) significantly increased in fasudil group. Compared with the control group, the gene expression of ANP, BNP, β-MHC, CTGF and Collagen I were significantly increased in fluoxetine group.

Conclusion:
Nogo/ROCK-NET signaling pathway is involved in the regulation of myocardial hypertrophy and fibrosis in spontaneously hypertensive rats.
Abstract 525:
In vitro
Reverse Remodeling After Mechanical Unloading With Engineered Heart Tissue
Authors: Shen Shi , Sewanan Lorenzo R , Campbell Stuart G
Published: October 2019

LVADs serve as a bridge to transplantation for many dilated cardiomyopathy (DCM) patients with advanced heart failure. In rare cases, mechanical unloading can lead to reverse myocardial remodeling sufficient for the removal of LVAD. We recreated the first reverse remodeling process
in vitro
in order to examine the role of active actin-myosin contraction and fibroblasts in contributing to this recovery, and to test the efficacy of drugs commonly prescribed for concurrent use with LVAD implantation. Engineered heart tissue (EHT) were prepared from decellularized porcine ventricular scaffolds in a proof-of-concept animal model with neonatal rat cardiomyocytes and subsequently with human induced pluripotent stem cell-derived cardiomyocytes and adult human cardiac fibroblasts. Mechanical stretch and myosin inhibition were used to simulate the pathological remodeling of DCM. Preliminary animal data showed that our model was able to capture the key features of DCM and reverse remodeling process including improved slack length, tissue stiffness, and active kinetics following mechanical unloading. β-adrenergic receptor response, protein assay, immunohistochemistry, and genetic analysis are being carried out to comprehensively investigate reverse remodeling in this artificial human system. Additional drug studies aim to investigate how reverse remodeling is affected by concurrent administration of β
1
antagonists and β
2
agonists (typical drug regimen for heart failure) and may inform new potential treatment during mechanical unloading to promote beneficial reverse remodeling for patients.
Abstract 529: Circulating Pro Fibrotic Protein Promotes Fibrosis in Liver and Heart
Authors: Tsukano Yuki , Shimizu Ippei , Yoshida Yohko , Ikegami Ryutaro , Hayashi Yuka , Suda Masayoshi , Katsuumi Goro , Wakasugi Takayuki , Nakao Masaaki , Minamino Tohru
Published: October 2019

Analyzing two sets of DNA micro array data with bioinformatics, we identified a secreted form pro-fibrotic protein (sPFP) expressed in dysfunctional brown adipose tissue (BAT) in mice. Testing our biobank samples, we found this protein increased in plasma of non-alcoholic steatohepatitis (NASH) patients or aged individuals. We generated a murine obese NASH model by imposing a high fat diet in C57BL/6NCr mice for 9-10 months since 4 weeks of age, and found that sPFP is produced predominantly by BAT. In this model, we also found that sPFP increased in plasma. We generated a murine systemic sPFP knockout (KO) model and found that liver fibrosis ameliorated in sPFP-KO model. We also suppressed circulating sPFP with a peptide vaccine targeting this molecule, and found that sPFP vaccination therapy inhibited liver fibrosis. Next, we generated sPFP gain of function (GOF) model by the administration of plasmid encoding sPFP into skeletal muscle. Liver fibrosis augmented in sPFP-GOF model, and these results suggested that sPFP has causal role for the progression of fibrotic response in liver. In the obese NASH model, we found that cardiac fibrosis also developed and it ameliorated in sPFP-KO model, indicating that sPFP may have pathological roles for heart failure with preserved ejection fraction (HFpEF) related with age-related disorders. In addition to an increase in circulating sPFP in aged individuals, we found that sPFP increased in BAT of chronological aged mice model. In vitro studies with differentiated brown adipocytes showed that c-Fos upregulated sPFP in transcript level. Our results suggest that sPFP contributes for the progression of fibrotic responses in obese or aged models. Inhibition of sPFP may become a therapy for NASH or HFpEF.
Abstract 540: Sigma 1 Receptor-dependent Regulation of Mitochondrial Respiration and Function in Cardiomyocytes
Authors: Aishwarya Richa , Bhuiyan Md. Shenuarin
Published: October 2019

Background:
Sigma 1 receptor (Sigmar1) is an abundantly expressed molecular chaperone protein in cardiomyocytes. Sigmar1 ligands have been shown to have protective function in animal models of cardiac hypertrophy, heart failure, and many other cardiovascular disorders. Despite extensive studies, the physiological and molecular functions of Sigmar1 in cardiomyocytes remains elusive.

Objective:
We investigated the molecular function of Sigmar1 in regulating mitochondrial respiration and function in cardiomyocytes.

Methods and Results:
To define the molecular function of Sigmar1 in regulating mitochondrial respiration and function in cardiomyocytes, we used small interfering RNA-mediated Sigmar1 knockdown and adenovirus-mediated overexpression in cultured neonatal rat ventricular cardiomyocytes. Sigmar1 knockdown in neonatal cardiomyocytes showed significantly decreased mitochondrial respiration measured by Seahorse Analyzer. Mitochondria stained with mitoTracker green showed a significantly increased number of hyperfused mitochondria in Sigmar1 knockdown cardiomyocytes. Similarly, mitochondria isolated from Sigmar1 global knockout mouse hearts also showed decreased mitochondrial respiration and the accumulation of larger size mitochondria measured by flowcytometry. Sigmar1 overexpression in cardiomyocytes significantly increased mitochondrial respiration and also showed increased mitochondrial fragmentation.

Conclusions:
Our findings suggested that Sigmar1 plays an essential role in maintaining normal mitochondrial morphology, respiration, and function in cardiomyocytes.
Abstract 543: The Metabolites of Human Cardiac Precursors Enhanced Cardiomyogenic Differentiation of hiPSC
Authors: Chen Wen-Pin , Li You-Yi
Published: October 2019

Background:
Wnt signaling pathways are involved in cardiomyogenic differentiation. Besides that, nutrients and metabolism may also influence cell differentiation. However, it is still unclear whether the cell lineage commitment could be controlled by the metabolites of its precursor cells. The aim of this study is to examine whether the metabolites of cardiac precursors could determine cardiomyogenic differentiation fate of hiPSC.

Methods:
hiPSC was maintained in serum-free and feeder-free monolayer condition. After CHIR99021-induced mesendodermal differentiation for two days, IWP4 was added to inhibit Wnt signaling and to induce myogenic differentiation for another two days. After wash, the culture medium was collected during cell differentiation period of day 5-10 (d5-10-diff-medium). ExoQuick-TC kit was used to extract the exosomes in the medium. Cardiomyocytes were identified by immunocytochemistry staining with mouse monoclonal antibody against cardiac sarcomeric actinin alpha, and were counted the percentage by flow cytometry.

Results:
The exosome-free d5-10-diff-medium was found to be able to dramatically induce cardiomyogenic differentiation only in CHIR99021-induced mesendodermal cells, rather than directly in hiPSC, in the absence of Wnt inhibitor. The metabolic profiling of the exosome-free d5-10-diff-medium was further characterized by LC-MS. After functional validation, the effective metabolites for the induction of cardiomyogenic differentiation were identified and the possible signaling pathways were analyzed.

Conclusion:
This study demonstrated that the metabolites of cardiac precursors could control cardiac cell commitment by driving the development of cardiac metabolic system.
Abstract 560: Differences in GCN5L1 Expression Between Male and Female Hearts Are Associated With Increased Mitochondrial Protein Acetylation
Authors: Manning Janet R , Thapa Dharendra , Zhang Manling , Stoner Michael , Scott Iain
Published: October 2019

Introduction:
Historically, most preclinical heart research has relied predominantly on male subjects, despite the identification of significant differences in signaling, metabolism, and pathology between males and females. Delineating the mechanisms behind these sex differences is critical to the effective application of novel findings to the general population as a whole. GCN5L1 is a mitochondrial-targeted protein that drives the acetylation and activation/deactivation of several metabolic proteins, and which has recently been reported to regulate cardiac metabolism and recovery from ischemia-reperfusion. It is unknown whether GCN5L1 plays a role in sex-specific differences in the myocardium.

Hypothesis:
Differential regulation of myocardial GCN5L1 expression between male and female animals results in differences in the acetylation and activity of proteins that regulate glucose and fatty acid oxidation.

Methods:
Heart lysates from male and female mice were probed for the expression of GCN5L1, and the acetylation status of whole cell and mitochondrial protein fractions was analyzed. Proliferating cardiac cells were treated with 17β-estradiol, and the expression of GCN5L1 was evaluated by immunoblotting. The acetylation status of mitochondrial proteins was measured using immunoblotting and immunoprecipitation.

Results and Conclusions:
Significant differences in cardiac GCN5L1 protein expression were found between male and female mice. In contrast, there was no difference in the protein expression of SIRT3, the mitochondrial deacetylase enzyme previously shown to counter GCN5L1 activity. Mitochondrial acetylation levels were found to be different between male and female mice. Treatment of cardiac cells with estradiol resulted in increased GCN5L1 expression, with a concurrent increase in the acetylation status of GCN5L1 targets localized to mitochondria. We conclude that sex-based differences in mitochondrial protein acetylation may be mediated by hormone-induced changes in GCN5L1 expression.
Abstract 569: Functional Benefits of Muscle PGC-1alpha in Aged Animals
Authors: Yang Steven , Loro Emanuele , Wada Shogo , Khurana Tejvir S , Arany Zoltan
Published: October 2019

Metabolic homeostasis requires a complex network of transcriptional programs. PGC-1 (peroxisome-proliferator-activated receptor-γ coactivator-1) alpha is a potent transcriptional coactivator that coordinates the activation of a large number of nuclear-encoded genes, which, in turn, regulate numerous metabolic processes. Exercise strongly induces muscle PGC-1alpha in both humans and rodents. Over-expression of PGC-1alpha in skeletal muscle activates mitochondrial oxidative metabolism, fast-to-slow fiber-type switching, and neovascularization, leading to markedly increased endurance. In light of these findings, PGC-1alpha has been proposed to protect from age-associated sarcopenia, bone loss, and whole-body metabolic dysfunction, although these findings have been controversial. We therefore comprehensively evaluated muscle and whole-body function and metabolism in 24 month-old transgenic mice that over-express PGC-1alpha in skeletal muscle. We find that the powerful effects of PGC-1alpha on promoting muscle oxidative capacity and protection from muscle fatigability persist in aged animals, although at the expense of muscle strength. However, skeletal muscle PGC-1alpha does not prevent bone loss and in fact accentuates it, nor does it have long-term benefit on whole-body metabolic composition or insulin sensitivity. Interestingly, we see protection from sarcopenia in male animals with over-expression of PGC-1alpha in skeletal muscle but not in female animals. In summary, muscle-specific expression of PGC-1alpha into old age has beneficial effects on muscle fatigability and may protect from sarcopenia in males, but does not improve whole-body metabolism and appears to worsen age-related trabecular bone loss.
Abstract 583: Single Cell Analysis of Monocytes and Macrophages in the Infarcted Heart
Authors: Ng Richard P , Calcagno Dave , Toomu Avinash , Fu Zhenxing , Aguirre Aaron , Weissleder Ralph , King Kevin
Published: October 2019

Introduction:
Ischemic Heart Disease (IHD), the leading cause of death in the world, is often initiated by myocardial infarction (MI). In mice, death typically occurs on day 4 after acute MI is induced by coronary artery ligation. At this time the interferon regulatory factor 3 (IRF3)/type I interferon (IFN) pathway is activated almost exclusively in mononuclear cells in the infarct. We have shown that the genetic or pharmacologic disruption of critical components of this innate immune pathway reduces inflammation, limits ventricular dilation and contractile dysfunction, and improves survival.

Hypothesis:
Treatment with the IFNAR neutralizing antibody will bias the differentiation of cardiac macrophages and in doing so establish a cardioprotective environment in the infarct.

Methods:
We conducted single cell RNA-sequencing analysis of mononuclear cells isolated from infarct tissue collected on day 4 after MI from mice that had been treated with an IFNAR neutralizing antibody (Ab) as well as untreated mice.

Results:
Analyses demonstrate the presence of 3 core cell populations in the untreated sample: monocytes expressing
Plac8
and
Ms4a4c
, phagocytic macrophages expressing
Mrc1
and
Ccl8
, and reparative macrophages expressing
Arg1
and
Fn1
. In addition, the dramatic co-expression of related genes in response to environmental stimuli, such as secreted type I IFNs or reactive oxygen species (ROS), resulted in the formation of two more clusters of cells. One cluster was marked by the expression of interferon stimulated genes. The other was marked by the expression of an established anti-oxidant transcriptional program. Anti-interferon therapy with IFNAR Ab inhibited both the interferon stimulated genes and the anti-oxidative transcriptional program.

Conclusion:
We have defined the differentiation patterns of monocytes and macrophages within the infarcted heart on day 4 after MI. Treatment with the IFNAR Ab after MI alters the composition of macrophage subsets.
Abstract 585: Role of Splice Variant Clic5B in Cardiac Mitochondrial Localization and Function
Authors: Ponnalagu Devasena , Hussain Ahmed T , Gao Erhe , Singh Harpreet
Published: October 2019

Blocking chloride channels (Cl
-
) with indanyloxyacetic acid (IAA-94) abrogated the cardio-protective effects of ischemic preconditioning (IPC) and increased myocardial infarction suggesting the possible contribution of IAA-94 sensitive Cl
-
channels in IPC mediated cardio-protection. Unlike cation channels, insights into the role of Cl
-
channels in cardioprotection is limited due to lack of information on their molecular identity. Chloride intracellular channel (CLIC) proteins are one of the known targets of IAA-94. They exist in both soluble and integral membrane form. Amongst the six known mammalian paralogs, CLIC1, CLIC4 and CLIC5 are most abundant in heart. Previously, we showed differential localization of CLIC4 and CLIC5 to outer and inner mitochondrial membrane respectively where as CLIC1 is present in the endoplasmic reticulum. Both CLIC4 and CLIC5 are important for mitochondrial functional integrity as well as cardioprotection from ischemia-reperfusion injury. Interestingly, none of the CLICs possesses conventional mitochondrial targeting sequence, which intrigued us to understand the mechanism of their localization to mitochondria. In this study we demonstrate that the mitochondrial localization of CLIC5 is related with the presence of splice variant CLIC5B (~50 kDa) in heart but not in lung lysates. Two different bands of CLIC5 at ~30 kDa (CLIC5A) and ~50 kDa, were observed in the cardiac mitochondrial lysates. Lung lysates showed the presence of only CLIC5A. In addition, mass spectrometric analysis revealed a peptide coverage of ~30% corresponding to CLIC5B at 50 kDa in heart but not in lung lysates further confirming its presence in heart. Moreover CLIC5 localized to mitochondria in p3 neonatal cardiomyocytes isolated from mouse but not in mouse lung epithelial (11 ± 2.1 %) cells which lacked CLIC5B indicating that the splice variant CLIC5B has a role in IMM localization. Interestingly, cardiac mitochondria from

clic5
-/-

mice exhibited increased ROS (p< 0.05, n=3) production whereas the

clic5
-/-

lung mitochondria did not show any change, suggesting the role of CLIC5B in regulating the mitochondrial function as well. Our study establishes splice variation as a mechanism for targeting ion channels to mitochondria.
Abstract 589: Left Ventricular Unloading and Delayed Coronary Reperfusion Protects the Structural and Functional Integrity of Mitochondrial Complex 1 in a Preclinical Model of Acute Myocardial Infarction
Authors: Swain Lija , Qiao Xiaoying , Reyelt Lara , Annamalai Shiva , Crowley Paige , Boggins Courtney , Kapur Navin K
Published: October 2019

Background:
Mitochondrial Complex 1 (MC1) plays a key role in energy metabolism during myocardial ischemia-reperfusion injury (M-IRI). Disruption of MC1 promotes reactive oxygen species (ROS) generation and myocardial damage. We recently reported that first reducing left ventricular (LV) workload using a trans-valvular pump and delaying coronary reperfusion, known as Primary Unloading (PU), reduces infarct size in preclinical models of M-IRI. The Door to Unload Pilot Trial recently demonstrated the safety and feasibility of PU in patients with ST-segment elevation myocardial infarction (STEMI), however the cardioprotective mechanisms of PU remain poorly understood. We hypothesized that compared to immediate reperfusion (IR); PU preserves mitochondrial function in M-IRI.

Results:
M-IRI was induced by balloon occlusion of the left anterior descending artery for 120 minutes in adult swine followed by reperfusion for 180 minutes. After 90 minutes of occlusion, animals were assigned to 30 minutes of continued occlusion (IR, n=5) or 30 minutes of support with an Impella CP pump (PU, n=5) with persistent occlusion before reperfusion. Compared to IR, PU reduced LV stroke work by 38.4% (p=0.047) and infarct size by 41.3±5.6% (p=0.0052). Compared to IR, PU preserved gene and protein expression of specific MC1 subunits (NDUFA8 and NDUFS3). Using the Agilent Seahorse Platform, we identified that compared to IR, PU preserved function of MC1 and ATP production in mitochondria isolated from the infarct zone by 82.45±12.8% (p=0.0040, IR vs PU). Compared to IR, PU reduced levels of ROS and preserved mitochondrial membrane potential. Next, we studied the activation state of MC1 and observed that compared to IR alone, PU increased the ratio of activated A form versus de-activated D form from 2.79 ± 0.29 to 7.58 ± 0.80 (p=0.005) levels of MC1 within the infarct zone.

Conclusion:
We report for the first time that compared to IR, mechanically unloading the LV and delaying reperfusion protects the structural and functional integrity of mitochondrial complex 1. These findings provide new insight into the cardioprotective mechanisms of Primary Unloading and support the development of novel therapeutic approaches to improve clinical outcome for patients with STEMI.
Abstract 590: EphrinA1-Fc Attenuates Dysfunction and Fibrosis in Nonreperfused Myocardium at 4 weeks Post-MI in WT B6 but Not EphA2-R Mutant Mice
Authors: Whitehurst K'Shylah S , Estes Heather K , Chase Robert C , Sharma Uma M , Virag Jitka A
Published: October 2019

Each year, an estimated 805,000 US citizens have a new or recurrent myocardial infarction (MI), causing over 360,000 deaths. We have previously reported that a single intramyocardial administration of recombinant ephrinA1-Fc, a member of the ephrinA/EphA family of membrane-anchored receptor tyrosine kinases, significantly reduced ischemic injury and remodeling 4 days in nonreperfused WT B6 mice via reduced apoptosis and inflammation and increased autophagic flux. After acute ischemia/reperfusion (30 min I/24hr or 4 days R) in WT B6 mice, ephrinA1-Fc demonstrated anti-inflammatory effects coupled with complete functional preservation due to modulation of cardiomyocyte ultrastructure and metabolism. The goal of this study is to assess the capacity of a single intramyocardial administration of ephrinA1-Fc at the time of permanent coronary artery ligation to maintain the structural and functional salvage effects through the wound healing process to stable scar formation in both B6 and EphA2-R-M (EphA2 receptor null mutant) mice. At 4 weeks post-MI in WT B6 mice, ephrinA1-Fc improved ejection fraction by 11%, attenuated cardiomyocyte hypertrophy by 38%, and decreased interstitial fibrosis by 35%. In contrast, these parameters were not different or worsened in EphA2-R-M mice treated with ephrinA1-Fc, suggesting that this receptor plays an important role in ephrinA1-Fc-mediated mitigation of the deleterious ventricular remodeling caused by permanent coronary artery occlusion. To further investigate the effects of ephrinA1-Fc directly on fibroblast function, isolated primary mouse cardiac fibroblasts were cultured with ephrinA1-Fc both with and without pro-fibrotic TGF-β stimulation to determine the direct effects of ephrinA1-Fc on col I and III production, remodeling mediators MMP-2, MMP-9, and TIMP-1, as well as signaling molecules DDR2 and SMADs2/3 as potential mechanistic pathways involved. The results of these assays are forthcoming. This study demonstrates the potential of a single administration of ephrinA1-Fc to not only attenuate acute ischemic injury but also mitigate pathological accumulation of interstitial fibrosis and remodeling, thereby reducing ventricular stiffening and the resultant progression to heart failure.
Abstract 603: Differential Proteoform Regulation in Cardiac Aging
Authors: Han Erin Yu , Bagchi Rushita , Lau Edward , Lam Maggie PY
Published: October 2019

Background:
The aging heart is prone to diseases and injuries, and is associated with changes in gene expression and alternative splicing. The goal of our study is to identify changes in protein isoforms in cardiac aging and explore their roles in age-related processes.

Methods:
Left ventricles (LV) from young (12 weeks) and old (78 weeks) C57BL/6J mice (male and female) were collected and stored at -80 °C until use. LV proteins were extracted, reduced, and alkylated. Proteins were digested with trypsin or chymotrypsin and the tryptic or chymotryptic peptides were desalted using C18 spin columns followed by injection into an Easy-nLC 1200 liquid chromatography system coupled to a high-resolution Q-Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer for LC-MS/MS analysis. To quantify isoform expression, MS spectra were searched against a custom peptide isoform database generated from a publicly available ENCODE RNAseq dataset (accession ENCSR000BYQ) on normal C57BL/6J heart (8 weeks) through an in-house computational workflow. Protein isoform expression was compared between the young and old heart samples.

Results:
Our experiments identified over 1,200 proteins (FDR < 0.05) in young and aged mouse LV tissue, including 65 isoform-specific tryptic and chymotryptic peptide sequences originating from 45 alternatively spliced genes that are not currently documented in the commonly utilized protein sequence database SwissProt. The detected protein isoforms include TMED2, TOM1 and TACC2. Our preliminary analysis suggests widespread proteome changes between young and aged hearts, which may have implication on energetics and fibrotic changes in cardiac aging.

Conclusion:
We report the first large-scale survey on the expression of canonical and alternative protein isoforms in the aging LV proteome. The identification of age-associated alternative isoforms suggests a potential mechanism for protein function regulation in the aging heart and may have implications for age-related disease processes.
Abstract 606: Stimulation of Cardiomyocyte a- and b-Adrenoceptors Uniquely Regulates Exosome Generation and miRNA Content
Authors: Kwon Jin-Sook , Koch Walter J
Published: October 2019

Background and Purpose:
Influence of adrenoceptor (AR) signaling for the regulation of exosomes secreted from cardiomyocytes is unknown and since catecholamines are increased in heart failure (HF), there is interest in uncovering whether α- or β-ARs can induce specific changes in circulating blood exosomes in HF. In this study, we have evaluated whether α- or β-AR stimulation of neonatal rat ventricle myocytes (NRVMs) can alter the number, size or microRNA (miR) content of secreted exosomes.

Methods and Results:
The number and size distribution of exosomes from NRVMs were evaluated by Nanosight NS300. Phenylephrine (PE, 10 uM for 3 days), an α-AR agonist, increased exosome number significantly of NRVM compared than vehicle (PBS) treatment group (Veh: 7.82X10
8
± 5.06X10
7
and PE: 10.09X10
8
± 15.0 X10
7
/ 1X10
6
NRVMs, n=3) but Isoproterenol (ISO, 1uM), a β-AR agonist, did not. Both agonists had no effect on exosome size distribution. After purifying total RNA from secreted exosomes, we evaluated the expression level of 37 candidate miR’s, which were selected from previous microarray data. ISO treatment decreased 14 miR’s (miR-222, -106a, -292a, -181b, -210, -489, -214, -1947, -195, -17, -7b, -93, -532 and -19a) and PE increased 5 miR’s (miR-20a, -210, -17-1, -Let7a, and 146b). Importantly, PE or ISO regulated any identical miR’s. As a control for treatment effects, we found that PE and ISO elevated myocyte mRNA levels of ANP and BNP to similar degrees.

Conclusions:
Both α- and β-AR agonists are can stimulate cardiomyocyte hypertrophy to similar degrees, however they have distinct regulatory patterns for secreted exosomes. This includes the number produced and more importantly, miR content. These miR’s and exosomes found in circulating blood may be able to distinguish and be biomarkers for different modes of maladaptive cardiac hypertrophy and HF.
Abstract 608: Long Non Coding RNA Miat Contributes to Cardiac Hypertrophy and Regulates Ribosomal Genes
Authors: Wang He , Wang Zhihua , Wang Yifan , Rau Christopher , Ren Shuxun , Wang Yibin
Published: October 2019

Cardiac hypertrophy is characterized by elevated protein synthesis and pathological cardiomyocyte enlargement. The mechanisms underlying the stress response in ribosomal remodeling at organelle level during cardiac hypertrophy is not fully established. We have identified a long non-coding RNA, Myocardial Infarction Associated Transcript (MIAT), that is significantly associated with pathological cardiac hypertrophy through a systems genetic analysis. MIAT expression is induced by hypertrophic signal, and silencing of the MIAT gene significantly blocks myocyte hypertrophy in response to hypertrophic stimuli associated with diminished global protein synthesis activities. Furthermore, MIAT inactivation significantly reduces ribosomal genes at mRNA and protein levels, and reduces protein synthesis activity at basal and post-hypertrophic stimulation
in vitro
and
in vivo
. Using a MIAT knockout mouse model, we investigated the in vivo pathophysiological impact of MIAT in pressure-overload induced cardiac hypertrophy and heart failure. Remarkably, we find that the MIAT knockout mice show significantly blunted cardiac hypertrophy and preserved cardiac function following pressure-overload. This study highlights a novel mechanism of translational regulation by a lncRNA MIAT. The outcome will expand functional role of lncRNAs in heart diseases and advance our current understanding of the complexity of regulatory circuits in cardiac hypertrophy and heart failure. It will provide novel insights to protein synthesis and ribosomal regulation and reveal potential therapeutic targets for pathological hypertrophy.
Abstract 615: The Cardiac Myosin Inhibitor, CK-3773274, Reduces Contractility in the R403q Mouse Model of Hypertrophic Cardiomyopathy
Authors: Hwee Darren T , Wu Yangsong , Cremin Peadar , Morgan Bradley P , Malik Fady I , Chin Eva R
Published: October 2019

Cardiac sarcomere hypercontractility appears to underlie pathological hypertrophy and fibrosis in select genetic hypertrophic cardiomyopathies. The small molecule, CK-3773274, is a novel cardiac myosin inhibitor that decreases contractility
in vitro
and in healthy animals
in vivo
. The objective of this study was to evaluate the effect of CK-3773274 in the genetic R403Q mouse model of hypertrophic cardiomyopathy. At approximately 40 weeks of age, left ventricular wall dimensions were determined by echocardiography in male wild type (WT) and heterozygous R403Q mice. As an indicator of cardiac hypertrophy, R403Q mice had significantly greater septal and posterior wall thickness than WT mice (septal wall WT: 0.93 ± 0.03 mm vs. R403Q: 1.22 ± 0.08 mm; posterior wall WT: 0.84 ± 0.04 mm vs. R403Q: 1.09 ± 0.04 mm; mean ± SEM, p< 0.05). R403Q mice were treated with single oral doses of CK-3773274 ranging from 0.25 to 1.5 mg/kg and fractional shortening (FS) and heart rate were assessed at select time points over a 24-hour period. One hour after dose administration, CK-3773274 significantly reduced FS in a dose-related fashion relative to pre-dose baseline values (FS % R403Q baseline: 55.5 ± 2%; 0.25 mg/kg: 43.9 ± 2%; 1 mg/kg: : 27.3 ± 2%; 1.5 mg/kg: 13.7 ± 1%; mean ±SEM, p<0.05 vs. baseline at all doses) without any changes to heart rate. At all dose levels, fractional shortening returned to baseline values by 24 hours. The plasma concentration at 10% and 50% reduction of FS relative to baseline (IC
10
and IC
50
) was 0.11 and 0.78 μM, respectively. In summary, single oral dose administration of CK-3773274 reduced fractional shortening in a dose and concentration-dependent manner in the genetic R403Q mouse model of hypertrophic cardiomyopathy. Cardiac myosin inhibition may be a viable approach to reduce underlying hypercontractility of the cardiac sarcomere in hypertrophic cardiomyopathies.
Abstract 619: Cardiac Function in Electronic Cigarette-Exposed Adolescent Mice
Authors: Neczypor Evan W , Tanwar Vineeta , Adelstein Jeremy M , Muffler Michael D , Pellegrini Anaruma Chadi , Wold Loren E
Published: October 2019

Objective:
Electronic cigarettes (e-cigs) are the most commonly used tobacco product by teens and young adults. The aim of the present study was to determine the potential effects of e-cig vapor exposure on cardiac function in adolescent mice.

Materials and Methods:
Adult FVB/NJ male and female mice were bred in our housing facility. Following birth, male offspring were raised in room air until weaning. At 3 weeks of age, mice were separated into two exposure groups: 1) E-cigarette exposure with nicotine (EC(+), N=5) (10.1 mg nicotine/1mL 50:50 propylene glycol:vegetable glycerin), or 2) E-cigarette exposure without nicotine (EC(-), N=4) and exposed for two months. A puff profile consisting of one 70-mL vapor puff per minute of exposure was administered for 4 h/day, 5 days/week over the course of two months. Cotinine concentration in the urine of mice from either group was determined using a cotinine ELISA kit. Echocardiography was used to examine global cardiac function after 1 and 2 months of exposure.

Results:
The average cotinine concentration in the urine of the EC(+) group after two months of exposure was 113.33 ng/mL, while there was not a detectable concentration of cotinine in the urine of EC(-) mice. Echocardiography showed no significant alterations in ejection fraction (%EF), fractional shortening (%FS), left ventricular end systolic diameter (LVES
d
), or posterior wall thickness (PWT) between the EC(+) and EC(-) groups, nor any significant alterations between the first month and the second month timepoints for either group.

Future Directions:
Exposures will continue for longer durations until a cardiac phenotype becomes apparent. Once in vivo cardiac dysfunction becomes evident, we will investigate in vitro cardiomyocyte function.
Abstract 620: Molecular Mechanisms and Therapeutic Approaches to Myofilament Glycation as a Result of Diabetes
Authors: Papadaki Maria , Theerachat June , Kirk Jonathan A
Published: October 2019

Methylglyoxal (MG), a glycolysis byproduct, is significantly elevated in the blood of patients with diabetes and modifies proteins by reacting with arginine and lysine residues to form irreversible adducts. We previously found that patients with diabetes and heart failure (dbHF) exhibited an increase in MG-modifications on cardiac myofilament proteins compared to non-failing hearts. We discovered these modifications reduced myofilament calcium sensitivity and maximal calcium-activated force, and despite being primarily on actin and myosin, did not affect their direct binding. The aim of this study was to elucidate MG’s molecular effects on muscle mechanics as well as to identify effective therapeutic options for dbHF patients. We first utilized a custom loaded fiber system to simultaneously measure isometric force and ATPase activity. When mouse fibers were treated with 100 μM Mg for 20 mins, similar to previous studies, there was a decrease in force production, but there was a concomitant decrease in ATPase activity as well. Thus, the tension cost was not altered by MG, supporting our hypothesis that MG modifications on myosin do not alter myosin function, instead altering its interactions with thin filament proteins. MG modifications are irreversible so therapeutic approaches should compensate for the functional defects induced. We pre-treated mouse skinned myocytes with MG, and then measured force-calcium relationships before and after treatment with different drugs. We first used 1 mM aminoguanidine (AG), an MG scavenger that has been efficacious in basic science studies but failed in clinical trials. Agreeing with the clinical trial, we show AG cannot reverse MG’s effects. We then treated the myocytes with omecamtiv mecarbil (OM), a myosin activator currently in clinical trials. In myocytes not pretreated with MG, OM increased calcium sensitivity and decreased maximum force, as reported previously. Interestingly, OM had no effect of calcium sensitivity but still decreased Fmax, suggesting OM may have less effect in patients with diabetes. Overall, in this study we further elucidate the mechanism of MG effect’s on the myofilament, and also show these modifications may reduce the efficacy of OM, a small molecule currently in clinical trials.
Abstract 621: Cardiac Myosin Binding Protein-C Phosphorylation in Ser-273 and Ser-282 is Critical to Maintain Cardiac Function During Aging
Authors: Rosas Paola C , Warren Chad M , Batra Ashley , Creed Heidi , Solaro R J , Tong Carl W
Published: October 2019

Background/hypothesis:
Heart failure (HF) afflicts 6.2M Americans as of 2019 and its prevalence is expected to increase due to a growing aging population. HF progression involves depression in contractility and relaxation, which are strongly controlled by phosphorylation of cardiac myosin binding protein-C (cMyBP-C). We hypothesize that phosphorylated cMyBP-C supports normal heart function during aging.

Methods:
We aged mouse models of cMyBP-C de-phosphorylation mimetic (t3SA: S273A/S282A/S302A mutations), phosphorylation mimetic (t3SD: S273D/S282D/S302D mutations) and WT equivalent expressed onto cMyBP-C(-/-) background (tWT).

Results:
Western blotting (N=16, 2-24 months) showed that cMyBP-C phosphorylation decreases with aging at S273 and S282, showing strong Pearson correlations with age (see figure; S273P r= -0.931, p<0.0001; S282P r=-0.868, p<0.00001). Simultaneous force and [Ca
2+
]
i
measurements on intact papillary muscles showed that phosphorylation-mimetic t3SD has greatest dFR=(-dF/dt)min/(+dF/dt)max, signifying better lusitropy with similar calcium decay rate constant (k
Ca
) as others. Echocardiography at 3, 12, 15, 18 months showed that t3SD has preserved ejection fraction, faster peak myocardial relaxation velocity e’ and preserved posterior ventricular wall thickness with aging. Moreover, t3SD demonstrated superior 600-days survival than others (t3SD 87.5% n=96; tWT 69.3% n=122; t3SA 53.2% n=97; overall LogRank p=0.0002244).

Conclusions:
De-phosphorylation of cMyBP-C at S273 and S282 contributes to worsening of cardiac function with aging. Conversely, phosphorylated cMyBP-C mitigates aging-related cardiac dysfunction.
Abstract 628: Diabetes-Mediated Vascular Calcification is RAGE-Dependent
Authors: Kay Amber M , Stewart James A
Published: October 2019

Type II diabetes mellitus (DM) is characterized by chronic hyperglycemia, and medial vascular calcification is a common cardiovascular complication of DM. This leads to aortic stiffening, which can leave patients at an increased risk for heart attack or stroke. Advanced Glycation End-Products (AGEs)/Receptor for AGEs (RAGE) signaling cascade has been implicated as a potentiator of diabetes-mediated vascular calcification, but it is not well understood. AGE/RAGE signaling influences both cellular and systemic responses to increase bone matrix proteins in hyperglycemic and calcification conditions and has also been shown to increase oxidative stress by promoting diabetes-mediated vascular calcification. This causes a phenotypic switch of vascular smooth muscle cells (VSMCs) to osteoblast-like cells and the hypothesized activation of adventitial fibroblasts (AFBs) to a myofibroblast phenotype. The purpose of this research is to understand AGE/RAGE mediated vascular calcification as a complication of diabetes. Calcification was induced for 7 days in primary mouse VSMCs and AFBs of non-diabetic, diabetic, non-diabetic RAGE knockout (RKO), and diabetic RKO, and then treated with AGEs to activate RAGE. Alizarin Red S staining was utilized to visualize calcification. Intracellular calcium levels were quantified and normalized to cell number (DAPI). Pronounced calcification was observed in non-diabetic VSMCs and the loss of RAGE resulted in decreased calcification in the non-diabetic RKO VSMCs. AFBs were exposed to the same experimental conditions as the VSMCs and calcification was increased in the diabetic AFBs while calcification was significantly decreased in the diabetic RKO AFBs. These data demonstrated that diabetes-mediated vascular calcification was RAGE-dependent in both cell types. Literature has cited the VSMC as the primary mediator for vascular calcification, but we have shown that the AFBs in the outer layer of the aorta have the ability to calcify and this is mediated by RAGE signaling, which elucidates a role for RAGE in diabetes-mediated vascular calcification. Understanding the role of AGE/RAGE signaling in diabetes-mediated vascular calcification will allow for possible targets for pharmacological intervention.