bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 225: Evaluating Pro-Inflammatory and Pro-Resolving Lipid Modulations in an Oral 15-HETE Model of Pulmonary Hypertension
Authors: Oconnor Ellen I , Ruffenach Gregoire , Grijalva Victor , Meriwether David , Papesh Jeremy , Dorreh Nasrin , Dorfman Anna , Eghbali Mansoureh , Reddy Srinivasa
Published: October 2019

Pulmonary arterial hypertension (PH) is a chronic lung disease with a mortality rate of 50% at 5 years after diagnosis. PH is characterized by progressive increase in pulmonary arterial pressure leading to right ventricular (RV) increase in afterload, hypertrophy, and failure. Despite the availability of multiple animal models of PH, finding relief or cure for PH patients has been elusive to date. Recent studies in our laboratory have shown that levels of hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic acids (HODEs) are elevated in the lungs and plasma of patients with PH as well as in animal models of PH. In testing for causality, we showed that administration of 5 μg of 15-HETE/day to wild type male mice for 3 weeks was sufficient to cause increase in pulmonary pressure, vascular resistance, plasma levels of HETEs and HODEs, right ventricle hypertrophy, and right ventricle systolic pressure (RVSP), consistent with PH. We set out to utilize this new mouse model of PH to understand the mechanisms in PH pathology by first examining the role of lipid mediators of inflammation in the development of PH. Our lab has developed a mass spectrometry method for measuring the levels of 39 inflammatory pathway lipid mediators from the cyclooxygenase (COX) and lipoxygenase (LOX) pathways that has been validated for use in tissue, plasma, and cell supernatant. Utilizing this panel with samples from mice fed 15-HETE and supernatant from an intestinal epithelial cell line; IEC-6 cells treated with 15-HETE, we have observed significant changes in multiple lipid mediators of inflammation. In addition to seeing increases in HETEs and HODEs with 15-HETE treatment, we observed significant increases in leukotriene C4 (LTC4) and 14(S)-hydroxy docosahexaenoic acid (14S-HDHA) and trending increases in thromboxane B2 (TXB2), and leukotriene E4 (LTE4) in the plasma, significant increases in 20 different lipids in lung tissue, including LTC4, TXB2, and LTE4, and significant increases in LTC4 and 5-oxo-eicosatetraenoic acid (5-oxoETE) in the supernatant of IEC-6 cells incubated with 15-HETE for 12 hours. These results indicate that novel inflammatory lipids and pathways may play an important in the development of hypertension and help identify new targets for therapies.
Abstract 260: The ER Unfolded Protein Response Effector, ATF6, Reduces Fibrosis and Moderates Activation of Cardiac Fibroblasts
Authors: Stauffer Winston T , Blackwood Erik A , Azizi Khalid , Stephens Haley N , Doroudgar Shirin , Glembotski Christopher C
Published: October 2019

Fibroblasts in the heart respond to myocardial injury by infiltrating the affected area and differentiating into new cell types called myofibroblasts. These cells are characterized both by the induction of contractile proteins and the secretion of extracellular matrix proteins which form fibrotic scar tissue. Investigating the factors governing fibroblast activation is key to understanding how these cells function in the heart and may be key to future therapeutic strategies. Activating transcription factor 6 (ATF6), an effector of the endoplasmic reticulum unfolded protein response, plays critical roles in development, as well as in the differentiation of certain cell types, though it has not been studied in this regard in the heart. Our lab has demonstrated that ATF6 in cardiac myocytes is cardioprotective
in vivo
during heart disease. However, ATF6 has not been studied in cardiac fibroblasts and its effect on fibrosis in the heart is unknown. We hypothesized that ATF6 in fibroblasts is an important regulator of their function. Fibroblast activation markers including αSMA were increased in infarcted hearts with global ATF6 deletion. Additionally, hearts with pressure overload showed increased fibrosis staining in global ATF6-null mice relative to WT hearts. In isolated adult murine ventricular fibroblasts (AMVF), loss of ATF6 induced myofibroblast markers with and without the activation stimulus TGFβ. ATF6 loss of function also enhanced the effect of TGFβ on fibroblast contraction. These effects were associated with an increase in Smad phosphorylation, a crucial step in the TGFβ pathway. Interestingly, the effect of ATF6 loss of function in AMVF was erased when treated with a TGFβ receptor inhibitor. Additionally, when ATF6 was overexpressed or when endogenous ATF6 was chemically activated, myofibroblast markers were reduced and activation by TGFβ was blunted. ATF6 activation was associated with induction of several TGFβ/Smad pathway negative regulators including SMURF1, SMURF2, and PMEPA1, though none of these are known to be ATF6 target genes. These data suggest that ATF6 plays an important role in moderating fibroblast activation and this may contribute to previously reported roles for ATF6 in preserving cardiac function post-injury.
Abstract 263: MYH14 Protects Against of Isoproterenol-induced Left Ventricular Hypertrophy and Fibrosis
Authors: Wang Jessica J , Chang Sunny C , Tran Alex , Sanchez Jessica , Yang Max , Omi Kazuhiro
Published: October 2019

Objectives:
We had previously identified
Myh14
as a genetic modifier of isoproterenol-induced left ventricular hypertrophy through a genome-wide association study performed among Hybrid Mouse Diversity Panel strains. Whether left ventricular hypertrophy due to
Myh14
deficiency is compensatory or maladaptive is not well established. Moreover, though MYH14 had been previously reported in the intercalated disc of cardiomyocytes, the mechanisms by which
Myh14
deficiency leads to left ventricular hypertrophy is unknown.

Methods:
We performed RNA-seq on cardiac tissues from 12-week old male
Myh14
+/+ and -/- mice that had been treated under control versus isoproterenol conditions for 3 weeks. We examined the differentially expressed genes to identify enriched gene ontology (GO) terms altered by
Myh14
deficiency at the transcriptional level.

Findings:
Differential expression analysis showed an enrichment of transcripts coding for extracellular matrix proteins in isoproterenol treated versus control mice, many of which were further enriched in
Myh14
-/- versus +/+ mice. Transcripts uniquely differentially expressed due to
Myh14
deficiency were enriched for GO terms such as inorganic cation import across plasma membrane, inner ear receptor cell / neuron development, response to purine-containing compound, and sensory perception of mechanical stimulus, supporting the roles these pathways play in mediating the impact of
Myh14
deficiency on cardiac remodeling.

Conclusions:
Our RNA-seq results suggest that MYH14 plays a protective role against mechanical stress. Future directions include elucidation of specific roles that the affected pathways play in mediating mechanical stress as a result by
Myh14
deficiency as well as impact on the observed phenotype of left ventricular hypertrophy and fibrosis.
Abstract 271: Reduced Expression of the Cardiac Sodium Channel Nav1.5 Triggers Enhanced Fatty Acid Metabolism and Oxidative Stress
Authors: Boudreau Ryan L , Zhang Xiaoming , McLendon Jared M , Kutschke William , Anderson Ethan J , London Barry
Published: October 2019

SCN5A
encodes the voltage-gated Na+ channel Nav1.5 that is best known for its role in cardiac conduction. We recently identified a microRNA (miR-24) binding site within the
SCN5A
coding region and found that its activity is modulated by an adjacent synonymous SNP (rs1805126). In humans, we linked the rs1805126 minor allele with decreased cardiac
SCN5A
expression and increased non-arrhythmic death in HF patients. To understand how lower
SCN5A
expression may lead to worse HF, we evaluated
SCN5A
+/-
haploinsufficient mice, which develop cardiac fibrosis after 12 months of age. Given that oxidative stress often precedes fibrosis and is increased in HF, we assessed ROS levels in
SCN5A
+/-
mouse hearts and found a 2.5-fold increase relative to wildtype (WT) hearts. We also performed co-expression analyses using human cardiac mRNA profiling data and unexpectedly found that
SCN5A
is linked to
PPARA
, a key driver of fatty acid oxidation (FAO, a predominant source of cardiac ROS), and to gene networks related to glucose metabolism and oxidative phosphorylation (OXPHOS). Along these lines, we found that
SCN5A
+/-
mouse hearts show mRNA changes indicative of increased FAO/OXPHOS and decreased glycolysis, with a coincident broad up-regulation of PPAR target genes. Metabolomics data further indicated that glycolytic flux is perturbed in
SCN5A
+/-
mouse hearts, and cardiac myofiber respiration assays showed that these hearts exhibit enhanced FAO. To test if
SCN5A
+/-
mice suffer worse HF outcomes, we subjected young adult male mice to thoracic aortic constriction (TAC), a model of cardiac hypertrophy progressing to HF. While WT mice showed typical hypertrophic responses and signs of HF,
SCN5A
+/-
hearts were resistant to TAC-induced hypertrophy, which based on prior reports, may be the result of elevated FAO. Overall, our data support the notion that lower cardiac
SCN5A
expression leads to an overreliance on FAO and accumulation of ROS in heart, which may exacerbate HF in patients. Together, our studies point to unforeseen roles for Nav1.5 in cardiac metabolism, opening several new paths of investigation. Future studies will interrogate 1) if human hearts with low
SCN5A
expression show signs of oxidative stress, and 2) if
SCN5A
+/-
mice suffer worse HF after myocardial infarction.
Abstract 274: Mitochondrial Translation Machinery Defects Causes Cardiomyopathy and i
n vivo
Functional Screening of Therapeutic Targets
Authors: Gao Feng , Liang Tian , Zhang Feng , Liu Ning , Fu Xuyang , Dong Xiaoxuan , Wang Da-zhi , Chen Jinghai
Published: October 2019

More than 95% of ATP consumed in the heart is derived from oxidative phosphorylation (OxPhos) in the mitochondria. Besides over a thousand proteins encoded by nuclear DNA, mitochondrial gene expression machinery generates 13 proteins for the OxPhos system that employs mitochondrial ribosomal proteins for translation. Mitochondrial ribosome protein S5 (MRPS5) encoded by MRPs gene family, is located at the mammalian mitochondrial ribosome 28S subunit and its importance has not been explored in the heart. Western blotting showed that MRPS5 expression was decreased in TAC-induced hypertrophic mouse hearts (
in vivo
), and in phenylephrine or isoproterenol-stimulated cardiomyocytes (
in vitro
). In the failing hearts of patients, Mrps5-mediated mt-CO1 was markedly decreased as well. To determine the functional role of MRPS5 in the heart, we generated an inducible cardiac-specific knockout mouse and showed that Mrps5-deficient mice developed time-dependent cardiac hypertrophy, fibrosis and heart failure. Mitochondria in the Mrps5-deficient adult cardiomyocytes exhibited membrane swelling and cristae collapsed together with decreased oxygen consumption and ATP production. Mitochondrial calcium homeostasis was also disrupted. Combined analysis of transcriptomics and metabolomics spatiotemporally identified 20 potential target genes associated with Mrps5 deficiency-mediated cardiac pathological processes. Through
in vivo
functional screening via adeno-associated virus (AAV) mediated gene delivery, we further narrowed down and confirmed that Kruppel-like factor 15 (Klf15) remarkably rescued cardiac phenotype in the Mrps5-deficient mice with maintained mitochondrial homeostasis, suppressed cardiac hypertrophy and fibrosis, as well as preserved cardiac function. Further RNA-sequencing and biochemical analysis mechanistically revealed that genes involved in glycolysis/ gluconeogenesis, PPAR signaling pathway and biosynthesis of amino acids, markedly altered when exploited AAV-Klf15 gene therapy in Mrps5-deficient mice heart. Our results demonstrates Mrps5 is essential for mitochondrial homeostasis and cardiac function and uncovers Klf15 as a potential therapeutic target for treating cardiac mitochondrial diseases.
Abstract 276: Maternal High Fat, High Sucrose Diet-induced Cardiac Mitochondrial Abnormalities in the Offspring are Transmitted via the Oocyte Nucleus
Authors: Ferey Jeremie L , Ruebel Meghan , Reid Michaela , Latham Keith E , Moley Kelle H , Diwan Abhinav
Published: October 2019

Introduction:
Maternal obesity correlates with higher cardiovascular disease risk in human offspring. We found high fat, high sucrose (HF/HS) diet results in accretion of abnormal mitochondria in mouse oocytes, and chow-fed offspring of HF/HS-fed dams exhibit transgenerational transmission of abnormal cardiac mitochondria through male and female germlines. Nuclear or mitochondrial inheritance can occur via the female germline, whereas male germline inheritance implicates nuclear epigenetic changes. However, recent reports indicate sperm mitochondria transmission is possible. Whether the mechanism is via effects on the nucleus or germ cell mitochondria is unknown.

Hypothesis:
Maternal HF/HS-feeding results in cardiac mitochondrial dysfunction in offspring due to effects on the oocyte nucleus.

Methods:
C57BL/6 female mice were chow- or HF/HS-fed, and mated to chow-fed C57BL/6 males. Female pronuclei were exchanged between embryos obtained from chow- and HF/HS-fed dams. Embryos without pronuclear transfer were additional controls. All embryos were transferred into chow-fed ICR dams. Cardiac mitochondrial structure (transmission electron microscopy) and function (high-resolution respirometry) were evaluated in 12-week-old offspring.

Results:
Adult offspring born of embryos having maternal pronuclei from chow-fed dams and ooplasm from chow- or HF/HS-fed dams had mitochondrial structure or oxygen consumption levels comparable to offspring born of control chow-fed dam embryos. In contrast, offspring born from embryos with maternal pronuclei from HF/HS-fed dams and ooplasm from chow-fed dams had abnormal cardiac mitochondrial structure (round shape, cristal rarefaction) and reduced oxygen consumption (~40%), comparable to embryo transfer offspring of HF/HS-fed dams.

Conclusion:
Shown for the first time, abnormal cardiac mitochondria in obese dam offspring result from HF/HS diet effects on the oocyte nucleus (likely epigenetic changes during oogenesis). Elucidating diet/obesity-induced oocyte epigenetic changes, and the mechanisms for induction and transgenerational transmission, could enable therapies to target inheritable heart disease risk in humans.
Abstract 282: Mitochondrial Complex I Induced Myocardial Stunning Following Cardiopulmonary Resuscitation
Authors: Piao Lin , Fang Yonghu , Hamanaka Robert B , Mutlu Gökhan M , Dezfulian Cameron , Archer Stephen L , Sharp Willard W
Published: October 2019

Background:
Cardiogenic shock following cardiopulmonary resuscitation (CPR) for sudden cardiac arrest is common, occurring even in the absence of acute coronary artery occlusion, and contributes to high rates of post-CPR mortality. The pathophysiology of this shock is unclear and effective therapies for improving clinical outcomes are lacking.

Methods and Results:
Using a murine model of asystolic cardiac arrest, we investigated the pathophysiology of post-CPR cardiogenic shock and discovered that duration of cardiac arrest (4, 8, 12 or 16-minute) prior to CPR determined post-resuscitation success rates, degree of neurological injury, and severity of myocardial dysfunction. Post-CPR cardiac dysfunction was not associated with myocardial necrosis, apoptosis, inflammation, or mitochondrial permeability transition pore opening and recovered within several days, indicative of myocardial stunning. Post-CPR myocardial stunning was associated with increases in ventricular and mitochondrial reactive oxygen species (ROS,
P
<0.001 vs Sham, respectively). Seahorse micropolarimetry of isolated post-CPR cardiac mitochondria revealed decreased rates of maximal oxygen consumption rates (OCR) for both Complex I and Complex II vs controls (
P
<0.01 vs Sham, respectively), indicating inhibition of mitochondrial oxidative phosphorylation. Paradoxically, in the presence of ADP stimulated coupled respiration, post-CRP mitochondria demonstrated increased OCR (
P
<0.05 vs Sham) and increased rates of proton leak (
P
<0.05 vs Sham), suggesting Complex I as the site of ROS generation. These findings were not observed at complex II. S1QEL, a complex I-specific superoxide inhibitor, administered during CPR, decreased myocardial ROS generation while improving post-CPR myocardial function (
P
<0.01 vs CPR control), neurological injury (
P
<0.01 vs CPR control), and survival (
P
<0.01 vs CPR control).

Conclusions:
Our results demonstrate that cardiogenic shock following resuscitation from cardiac arrest is consistent with myocardial stunning mediated by mitochondrial complex I injury and ROS generation. Targeting this mechanism represents a novel and practical therapy for improving sudden cardiac arrest resuscitation outcomes.
Abstract 288: Mitoq Regulates Redox-related Non-coding Rnas to Improve Mitochondrial Network in Pressure Overload Heart Failure
Authors: Song Jiajia , Kim Seulhee , Latimer Mary N , Goh Kah Yong , Prabhu Sumanth D , Qin Gangjian , Darley-Usmar Victor , Liu Xiaoguang , Wende Adam R , Young Martin E , Zhou Lufang
Published: October 2019

Background:
Previous studies show that mitochondrial network excitability, or the propagation of ROS signals, is impaired in cardiomyocytes from failing hearts. While oxidative stress has been implicated in heart failure (HF)-associated mitochondrial network abnormality, the effect of mitochondrial-targeted antioxidant, such as mitoquinone (MitoQ), on mitochondrial network in pressure overload hearts has not been demonstrated. We hypothesize that MitoQ improves mitochondrial networks in HF via regulation of redox-related cardiac remodeling-associated non-coding RNAs.

Methods and results:
To test the hypothesis, C57BL/6J mice were subjected to ascending aortic constriction (AAC) to induce left ventricular (LV) pressure overload, followed by 7 days of MitoQ treatment (2 μmol). Doppler echocardiography revealed severe LV dilation and decreased ejection fraction following AAC, which were attenuated by MitoQ. Electron microscopy and immunostaining showed that inter-mitochondrial and mitochondria-sarcoplasmic reticulum (SR) network structure were altered in HF myocardium, in parallel with reduced expression of mitofusin proteins (e.g., MFN1 and MFN2) compared to sham-operated animals. MitoQ blunted mitofusin protein downregulation and improved mitochondrial networks. Our data also identified a MitoQ-mediated mechanism of mitofusin expression in HF by ameliorating the dysregulation of redox-related cardiac remodeling-associated long non-coding RNAs and microRNAs (i.e. Plscr4-miR-214 axis).

Conclusion:
The present study indicates that MitoQ improves inter-mitochondrial and mitochondrial-SR structural organization in pressure overload hearts by attenuating the dysregulation of cardiac remodeling-associated non-coding RNAs.
Abstract 290: Lpp3 Deficiency Impairs Mitochondrial Function And Enhances Myocardial Lpa Mediated Signaling
Authors: Miriyala Sumitra , Panchatcharam Manikandan , Bhuiyan, Md.Shenuarin , Alcalde Diana Escalante , McCarthy Kevin , Kevil Christopher
Published: October 2019

The bioactive lysophosphatidic acid (LPA) plays a well-known role in atherosclerotic disease, whereas, its role in myocardial function remains virtually unexplored. Following acute myocardial infarction, serum LPA concentration rises by six-fold over control human subjects, suggesting LPA may contribute to the pathogenesis of myocardial infarction. LPA production involves hydrolysis of lysophosphatidylcholine by the secreted enzyme autotaxin, whereas lipid phosphate phosphatase-3 (LPP3) catalyzes LPA dephosphorylation to generate lipid products that are not receptor active. We present the first evidence that cardiac ischemia/reperfusion (I/R) injury enhances myocardial autotaxin levels and decreases myocardial LPP3 expression, and this is associated with increased serum LPA levels. Upon reperfusion, reactive oxygen species production arises as a burst of superoxide from mitochondria following I/R injury. The redox-sensitive transcription factor NFAT has been shown to bind to the autotaxin promoter and induce its expression. Therefore, we looked at the autotaxin and LPP3 regulation in mice following I/R injury in the myocardium. After 1h ligation followed by 3h reperfusion in the myocardium, we observed a 3-fold increase in the autotaxin protein levels, whereas LPP3 protein levels were significantly downregulated as observed through Western blot analysis in these myocardial ischemic tissues. Autotaxin and miR-92a mRNA expression levels were significantly upregulated, whereas KLF2 and LPP3 mRNA expressions were significantly downregulated following I/R injury at 24 hours. Western blot analysis showed a 3-fold increase autotaxin protein levels and immunohistochemistry of human infarct tissues at 24 hours showed disruption of the sarcomere with decreased LPP3 staining. We found that I/R injury transactivates miR-92a, and inhibit KLF2, an upstream activator of LPP3. Taken together, us in vivo data, from the myocardial I/R injury and human infarct tissues, suggest that regulation of autotaxin and LPP3 activity might cause the rise in serum LPA levels as reported with acute myocardial infarct patients
Abstract 306: DJ-1 Preserves Mitochondrial Function in the Ischemic Heart by Reducing the Glycation of Complex I
Authors: Pantner Yvanna , Shimizu Yuuki , Polavarapu Rohini , Li Lian , Chin Lih-Shen , Calvert John
Published: October 2019

Background:
DJ-1 is a cytoprotective protein implicated in many cellular processes, including the oxidative stress response and the glycative stress pathway. DJ-1 has proteolytic activity when cleaved under conditions of oxidative stress. We recently found that the cleaved form of DJ-1 attenuates ischemia-reperfusion (I/R)-induced heart failure by reducing glycative stress. Mitochondrial electron transport proteins are susceptible to glycative stress. Here, we sought to determine if DJ-1 regulates the glycation of Complex I after I/R injury.

Methods and Results:
Initial studies found that compared to wild-type control mice, mitochondria from DJ-1 deficient (DJ-1 KO) hearts showed increased levels of advanced glycation end products and carboxymethyl-lysine (CML) following I/R.Additionally, CML was shown to be bound to complex I at higher levels in DJ-1 KO hearts. This corresponded with reduced Complex I activity and reduced mitochondrial oxygen consumption in the presence of Complex I substrates. To confirm an association between DJ-1 and Complex I, wildtype animals were subjected to I/R injury and co-immunoprecipitation revealed higher levels of DJ-1 bound to Complex I after I/R. To further determine if DJ-1 influences the glycation of Complex I, an adenoviral approach (AAV9-CMV-DJΔc) was used to over-express the cleaved form of DJ-1. Under I/R conditions, mitochondrial CML levels were attenuated in hearts treated with AAV9-CMV-DJΔc compared to hearts treated with a control virus. Moreover, mitochondria from AAV9-CMV-DJΔc treated hearts showed improved Complex I activity and oxygen consumption.

Conclusion:
These data demonstrate that DJ-1 plays a role in maintaining Complex I efficiency when under ischemic conditions. In elucidating a specific mechanism for DJ-1’s role in attenuating ischemia-reperfusion-induced heart failure, these data break new ground for potential therapeutic strategies using DJ-1 as a target.
Abstract 320: MicroRNA-133a Attenuates the Development of Thoracic Aortic Aneurysm
Authors: Akerman Adam W , Collins Elizabeth N , Harrison Jessica K , Oh Joyce , Collins Lauren B , Riopedre Jessica , DeVaughn Amari , Raybuck Charles M , Ikonomidis John S
Published: October 2019

Rationale:
Thoracic aortic aneurysms (TAA) result from dysregulated remodeling of the vascular extracellular matrix, and may occur as result of altered resident cellular phenotype. MicroRNA-133a is reduced in clinical TAA specimens and plays an inhibitory role in regulation of pathological phenotypic switch of vascular cells. Accordingly, this study tested the hypothesis that miR-133a replacement attenuates the development of TAA.

Methods and Results:
TAA was induced in wild type mice (0.5M CaCl
2
application, 15 minutes). Following 4 weeks, aortic diameter was increased compared to sham operated controls (1094 ± 38 vs 654 ± 8 μm; p<0.05, n=7). Copy number of miR-133a, quantitated by ddPCR, was reduced in TAA tissue (0.28 ± 0.04 vs 0.68 ± 0.11 copy per U6; p<0.05 vs control, n=7). While no change in smooth muscle cell specific Desmin nor α-smooth muscle actin were detected, an increase in copy number of the fibroblast specific discoidin domain receptor 2 (DDR2) and smooth muscle cell myosin heavy chain 11 (MYH11) were observed in TAA tissue. Combined, these results are consistent with the emergence of a population of myofibroblasts with TAA development. Accordingly, fibroblasts were isolated from TAA and sham operated control tissues (n=10). Copy number of miR-133a was reduced in TAA fibroblasts (0.41 ± 0.14 vs 1.60 ± 0.43 copy per U6; p < 0.05 vs control). TAA fibroblast phenotype was compared to controls; adhesion was reduced (-0.85±0.02 vs -0.77±0.02), migration was increased (63±4% vs 47±4%), and rate of collagen disk contraction over 7 hours was increased (-12.2±1.6 vs -8.5±1.0) (all values, p<0.05 vs control). Finally, after TAA induction in mice, a single tail vein injection of either a miR-133a overexpression or scrambled sequence (control) lentivirus was performed. Following 4 weeks, miR-133a replacement attenuated TAA development (800 ± 18 μm vs 1016 ± 16 μm; p < 0.05 vs control; n=8).

Conclusion:
Aortic fibroblast phenotype is altered during TAA progression, and miR-133a replacement attenuates the development of TAA in a murine model. These unique findings suggest stable alterations in aortic fibroblasts may be associated with pathological extracellular matrix remodeling, and regulation by miR-133a may lead to a novel therapeutic strategy.
Abstract 325: Characterization of Circular RNAs in Vascular Smooth Muscle Cells With Vascular Calcification
Authors: Kook Hyun , Ryu Juhee , Jeong Geon , Lim Yeong-Hwan , Kwon Duk-Hwa , Choe Nakwon , Shin Sera , Kim Yongsook , Ahn Youngkeun , Kim Young-Kook
Published: October 2019

Circular RNAs (circRNAs) are generally formed by back splicing, and are expressed in various cells. Vascular calcification, a common complication of chronic kidney disease, is often associated with cardiovascular disease. The relationship between circRNAs and vascular calcification has not yet been studied. Inorganic phosphate was used to treat rat vascular smooth muscle cells to induce vascular calcification. CircRNAs were identified by analyzing RNA sequencing data, and their expression change during vascular calcification was validated. The selected circRNAs, including circSamd4a, circSmoc1-1, circMettl9, and circUxs1 were resistant to RNase R digestion, and mostly localized in the cytoplasm. Silencing circSamd4a promoted vascular calcification, while overexpressing it reduced vascular calcification. In addition, microRNA (miRNA) microarray and luciferase reporter assay suggested that circSamd4a could act as miRNA suppressor. Our data shows that circSamd4a have anti-calcification roles by functioning as miRNA sponges. Since circSamd4a is conserved in humans, they can serve as novel therapeutic targets in resolving vascular calcification.
Abstract 339: Biomechanics and Calcium Handling of Thin Filament Hypertrophic Cardiomyopathy Variants
Authors: Repetti Giuliana G , Toepfer Christopher N , Garfinkel Amanda C , Venturini Gabriela , Seidman Jonathan G , Seidman Christine E
Published: October 2019

The mechanism by which genetic variants of the thin filament cause hypertrophic cardiomyopathy (HCM) has not been fully elucidated. Induced pluripotent stem cell (iPSC)-derived cardiomyocytes can provide a rapidly generatable disease modeling tool to study HCM in these variants. While variants in the thick and thin filament both cause HCM, cardiomyocytes that harbor pathogenic variants in the thin filament genes troponin I (
TNNI3
) and troponin T (
TNNT2
) display a distinct molecular phenotype, which we hypothesize differs from the thick filament HCM phenotype. Both thin and thick filament mutant cardiomyocytes display increased measured oxygen consumption rate assessed by metabolic assays, however we hypothesize that the mechanism that drives metabolic change is not common between thick and thin filament variants. Mutations in the thick filament typically shift myosin conformations during relaxion towards a higher energy utilizing a disordered relaxed state (DRX) that enables ATP hydrolysis with more free myosin heads, and this likely accounts for their increased oxygen consumption. However thin filament mutations cause myosin heads to preferentially adopt a conformation in which the myosin heads are sequestered and unable to bind actin. This super relaxed state (SRX) is associated with energy conservation, which would predict reduced contractility. Yet contractility data from thin filament mutants replicate the classic HCM phenotype of hypercontractility and disturbed sarcomere relaxation. To further probe the mechanism by which thin filament variants drive HCM pathophysiology we have employed methodologies to assess calcium transients in iPSC-derived cardiomyocytes harboring thin filament variants.
TNNT2
variants recapitulate this disrupted calcium handling.
Abstract 349: Beclin1 as a Critical Regulator of the Endosomal Degradation Pathway
Authors: Lampert Mark
Published: October 2019

Effective cellular quality control is crucial for cellular homeostasis and cardiac health. Defects in the pathways that regulate the removal of damaged proteins and organelles contribute to heart failure. Autophagy is well known for its role in removing protein aggregates and organelles; however, we recently found that the small GTPase Rab5 and early endosomes also participate in Parkin-mediated mitochondrial clearance. Beclin1 is a scaffolding protein which can form distinct PI3K protein complexes to regulate different processes such as autophagy. Here, we have investigated the role of Beclin1 in regulating the endosomal degradation pathway in mice. We have generated inducible cardiac-specific Beclin1 KO mice to investigate the
in vivo
role of Beclin1 in myocytes. We found significant contractile dysfunction as early as one week after deletion of Beclin1, which led to increased mortality. Loss of Beclin1 led to rapid development of cardiac hypertrophy as characterized by increased
Myh7, Nppa, Nppb
, myocyte size, and heart weight/body weight. Furthermore, we found that loss of Beclin1 led to increased inflammatory markers
Il6
and
Tgfb1
, as well as increased DNA damage/apoptosis in the heart. Ultrastructural analysis revealed Beclin1 deficiency led to significant myocardial disarray, demonstrating the importance of Beclin1 for proper myocyte alignment. These mice also had reduced autophagic flux but increased IGFR-beta receptor protein levels, indicating defects in both the autophagy and endosomal degradation pathways. Additionally, immunofluorescence analysis revealed that Beclin1 colocalizes with Rab5 at the early endosome in cells, and coimmunoprecipitation experiments confirmed their interaction. Together, these data establish the essential role of Beclin1 in cellular homeostasis as a central regulator of endosomal activity.
Abstract 350: Bcl-xL-Ser14 Phosphorylation is Critical for Compensatory Cardiac Hypertrophy
Authors: Nakamura Michinari , Fefelova Nadezhda , Liu Tong , Ikeda Shohei , Zhai Peiyong , Del Re Dominic P , Li Hong , Xie Lai-Hua , Sadoshima Junichi
Published: October 2019

Cardiac hypertrophy is an adaptive response at least initially since it reduces wall stress. Phosphorylation of Bcl-xL at Serine (Ser) 14, which dissociates Bcl-xL from Bax and promotes apoptosis, is increased in the heart within one hour after transverse aortic constriction (TAC)-induced pressure overload (PO). Here, we investigated how the increased Ser14-phosphorylation affects hypertrophy during PO. The Bcl-xL knock-in (KI) mice, in which Ser14 was replaced with Ala (Ser14Ala), exhibited a significantly greater mortality than wild-type (WT) mice (p=0.001) after TAC, with elevated end diastolic pressure (LVEDP, 34.6 vs 16.5 mmHg, p<0.05), impaired systolic function (EF, 38.2% vs 67.5%, p<0.001), and increased fibrosis (1.6-fold, p<0.001). The level of apoptosis was similar between the KI and WT mice one week after TAC, as assessed by TUNEL staining. The KI mice showed less cardiomyocyte and cardiac hypertrophy (cardiomyocyte size, 0.71-fold; heart weight/tibia length, 0.88-fold, both P<0.001). Adult cardiomyocytes isolated from the KI mice two days after TAC showed significantly lower contractility compared to those isolated from WT mice (0.32-fold, p<0.001). Mechanistically, gene set enrichment analysis using the RNA-seq data obtained from one-day TAC hearts showed that ion channel activity-related gene sets enriched in WT mice are downregulated in the KI mice. In line with this result, angiotensin II (Ang II) increased Ser14-phosphorylation and cytosolic Ca
2+
level in WT-MEFs
in vitro
, whereas MEFs isolated from the KI mice showed a significantly lower elevation of cytosolic Ca
2+
against Ang II. Proteomics analysis showed that Ankyrin, an anchoring protein that targets and stabilizes ion channels on the membrane, interacts with endogenous Bcl-xL in the heart. Taken together, these data suggest that phosphorylation of Bcl-xL at Ser14 is critical for augmenting Ca
2+
release from the sarcoplasmic reticulum by modulating the ion channel activity in part via Ankyrin against PO or AngII, thereby developing compensatory hypertrophy and maintaining contractile function. Our findings indicate that increasing the Bcl-xL-Ser14 phosphorylation during acute phase PO could be a potential therapeutic strategy for maintaining cardiac function.
Abstract 408:
Mir148a
Family Regulates Cardiac Differentiation of Human Embryonic Stem Cells by Inhibiting The
Dll1
-mediated Notch Signaling Pathway
Authors: Miao Shumei , Fang Xing , Yu You , Han Xinglong , Wu Hongchun , Zhao Zhen-Ao , Wang Yongming , Lei Wei , Hu Shijun
Published: October 2019

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can differentiate into spontaneously beating cardiomyocytes in vitro, and hold great promise for cardiovascular disease modeling, therapy and drug discovery. However, these applications were hampered by low yield and purity of the in vitro differentiated cardiomyocytes. Although miRNAs represent a type of potential candidates to promote cardiac differentiation, most of cardiomyocyte-enriched miRNAs have not been functionally investigated until now. This study investigated the roles of
MIR148A
family in cardiac differentiation from hESCs. The
MIR148A
family is composed of
MIR148A
,
MIR148B
and
MIR152
, three highly conserved miRNAs sharing same seed sequences. The expression levels of all
MIR148A
family members were progressively increased during cardiac differentiation in vitro. By using CRISPR-Cas9-mediated knockout, we demonstrated that triple knockout of
MIR148A
family (
MIR148A
-TKO), rather than individual knockout of each members, could grossly inhibited TNNT2
+
cardiomyocyte generation. Ectopic expression of
MIR152
significantly restored the cardiac differentiation efficiency of
MIR148A
-TKO hESCs. The transcriptome analysis identified a total of 1071 upregulated genes and 766 down-regulated genes in the
MIR148A
-TKO hESC-derived cells compared to wild-type hESC-derived cells at day 4 of cardiac differentiation. Gene Ontology analysis revealed that most genes down-regulated in
MIR148A
-TKO hESC-derived cells were involved in the events of lateral/cardiac mesoderm development, while the up-regulated genes were mainly involved in the events of paraxial/somitic development. Furthermore, the NOTCH ligand Delta-like1 (
DLL1
) was validated as the target gene of
MIR148A
family, and the knockdown of
DLL1
could inhibit target gene expression of Notch signaling pathway, and promote cardiac differentiation of
MIR148A
-TKO hESCs. Our findings demonstrate a new function of
MIR148A
family during cardiac differentiation. Synergistic inhibition of
DLL1
-mediated Notch signaling represents a major mechanism for the
MIR148A
family to inhibit undesired lineage formation and promote hESC differentiation into cardiomyocytes.
Abstract 409: Regulation of Cardiomyocyte Maturation by an RNA Splicing Regulator Rbfox1
Authors: Lee Josh Zixi , Huang Jijun , Rau Christoph , Gao Chen , Yang Ziyue , Wang He , Pushkarsky Ivan , Parikh Shan , Di Carlo Dino , Knollmann Bjorn , Wang Yibin
Published: October 2019

The maturation of stem cell derived cardiomyocytes (iCMs) is incomplete relative to the fully matured adult myocytes. Lack of maturation represents a major limitation to the applications of iCMs as heart disease models or heart failure therapies. Current attempts to promote iCMs maturation, such as prolonged culture, mechanical stretching and electronic pacing, are often based on empirical methods with poor reproducibility or little mechanistic basis. In order to better understand the molecular mechanisms driving cardiomyocyte maturation, we performed extensive transcriptome analyses in neonatal vs. adult hearts. In addition to metabolic and cell cycle regulatory pathways, Gene Ontology analysis revealed RNA splicing regulation is significantly enriched in the transcriptome reprogramming during postnatal maturation in heart. Specifically, we find a cardiomyocyte enriched RNA splicing factor Rbfox1 is dramatically induced in the perinatal maturating mouse hearts. Ectopic expression of Rbfox1 in neonatal cardiomyocytes markedly promotes the cellular and molecular features of adult cardiomyocyte, including contractility, calcium handling, sarcomere organization, morphology, electrophysiology and gene expression. Most remarkably, expression of RBFox1 in human iPSC derived cardiomyocytes promotes similar maturation process as observed in the neonatal rat myocytes. At mechanistic level, RBFox1 expression in the iCMs enhances transcriptome maturation as indicated by targeted RNA splicing in genes involved in muscle contraction, gene expression, RNA processing and sarcomere organization. In summary, we have uncovered a novel molecular path towards neonatal myocyte maturation in perinatal murine hearts by targeted modulation of cardiomyocyte transcriptome via RNA-splicing. This approach has potential to be employed as a molecular approach to promote human iCMs maturation.
Abstract 414: Postnatal Cardiac Tissue Harbors Progenitor Cells With Unique Metabolic Profile
Authors: Kurian Justin , Yuko Antonia , Busch Kelsey , Harlamova Daria , Kasatkin Nicole , Wagner Marcus , Mohsin Sadia , Houser Steven , Wang Hong , Khan Mohsin
Published: October 2019

Rationale:
The developmental cardiac tissue is a proliferative organ capable of regeneration that extends briefly into the postnatal period. Studies show that the heart undergoes alterations in metabolism that coincide with cessation of regenerative processes during postnatal development. Whether cardiac cells in the early postnatal heart exhibit metabolic properties that favor regeneration remains unknown.

Objective:
To determine whether postnatal cardiac tissue harbors CPCs with unique metabolic profile.

Methods and Results:
CPCs were isolated from C57BL/6 mice aged 2-day (NCPC), 2-month (adult CPC) and 2-year old (ACPC). Morphological assessment showed NCPCs were smaller and rounded compared to CPCs and ACPCs and expressed putative stem cell markers and were negative for hematopoietic marker CD45. Interestingly, NCPCs expressed elevated levels of pluripotency markers such as LIN28 compared to ACPCs and CPCs. Increased viability, proliferation rate, metabolic activity and reduced doubling times were observed in NCPCs compared to CPCs and ACPCs measured by CyQuant, MTT, and Resazurin assays. NCPCs demonstrated a youthful phenotype and were resistant to H2O2 induced stress compared to ACPCs and CPCs as measured by β-Galactosidase and TUNEL labeling respectively together with a unique expression of paracrine factors as assessed by proteome profiler array. Interestingly, NCPCs demonstrated increased ATP generation, glycolytic activity and reduced oxidative phosphorylation compared to CPCs and ACPCs measured by seahorse assay parallel with enhanced expression of glycolytic enzymes measured by qRT-PCR and western blot. NCPCs showed increased lactate production and pyruvate kinase activity and low mitochondrial membrane potential. Interestingly, mitochondrial fuel dependency assay showed increased glutamine dependency as the fuel for mitochondrial oxidative phosphorylation. RNA-sequencing analysis demonstrated increased expression of metabolic signaling in NCPCs compared to CPCs and ACPCs.

Conclusions:
Postnatal cardiac tissue possesses progenitor cell population with unique metabolic profile that coincides with enhanced functional properties of NCPCs suggesting their potential therapeutic value for cardiac repair.
Abstract 415: Electrical Stimulation of Pediatric Cardiac-derived c-kit
+
Progenitor Cells Improves Retention and Cardiac Function in Right Ventricular Heart Failure
Authors: Maxwell Joshua T , Trac David , Shen Ming , Brown Milton , Davis Michael E , Zaladonis Carly , Baker Emily , Li Martin L , Jacobs Daniel I
Published: October 2019

Nearly 1% of babies born in the US will be diagnosed with a congenital heart defect (CHD). While surgical therapy has improved survival, many of these children go on to develop right ventricular heart failure (RVHF). The emergence of cardiovascular regenerative medicine as a potential therapeutic strategy for pediatric HF has provided new avenues for treatment with a focus on repairing or regenerating the diseased myocardium to restore cardiac function. While primarily tried using adult cells and adult disease models, stem cell therapy is relatively untested in the pediatric population. Cardiac-derived c-kit
+
progenitor cells (CPCs) have been widely studied as a cell-based therapy for cardiac pathologies; however, unless these cells are extracted at a very young age their therapeutic efficacy has been shown to be diminished. Finding novel ways to enhance the reparative potential of these cells is thus of critical significance. We previously reported pediatric CPCs (isolated from patients between 1-5 years of age) respond to electrical stimulation (ES) by initiating intracellular calcium oscillations. Here, we investigate the ability of ES to enhance the retention and therapeutic function of pediatric CPCs in an animal model of RVHF. Human CPCs isolated from pediatric patients were exposed to chronic ES and implanted into the RV myocardium of rats. Cardiac function and cellular retention analysis showed electrically stimulated CPCs (ES-CPCs) were retained in the heart at a significantly higher level and longer time than control CPCs and also significantly improved right ventricular functional parameters. ES also induced upregulation of extracellular matrix and adhesion genes, such as integrins β1 and β5, and significantly increased
in vitro
survival and adhesion of cells. Lastly, we show that ES induces CPCs to release higher levels of reported pro-reparative factors
in vitro,
including angiogenin, FGF, HGF, and VEGF. These findings suggest electrical stimulation can be utilized to increase the retention, survival, and therapeutic effect of human c- kit
+
progenitor cells. Furthermore, our insights into the mechanisms of increased retention and paracrine function of ES-CPCs can have implications on a variety of cell-based therapies.
Abstract 416: Approach of mRNA Delivery by Using Nanoparticles to Recover Endocardial Notch Signaling
Authors: Messerschmidt Victoria L , Kuriakose Aneetta , Nguyen Kytai , Lee Juhyun
Published: October 2019

Notch signaling plays a significant role in cardiac development and morphogenesis, especially for cardiac chamber development. Failure of mechanotransduction of endocardial Notch signaling in ventricular chamber causes lack of trabeculation. Advent of nanoparticle (NP) technology offers a therapeutic approach of targeted delivery. Although delivering mRNA is an effective method, it is challenging due to stability and cell defense mechanisms. To overcome the limitations and effectively deliver mRNA to endocardium, we used Poly(lactic-co-glycolic acid) (PLGA) synthesized NPs, which have been used in various drug delivery systems due to its biocompatibility, efficient encapsulation of hydrophilic and hydrophobic materials, specific targeting effects, and lower payload concentration. We hypothesized our PLGA NPs are able to deliver
NICD
mRNA to target endocardium to overexpress Notch signaling in a Notch inhibited zebrafish animal model. Using double emulsion, we synthesized PLGA NPs at 55–65 kDa and performed
in vitro
test on HUVECs. 69.8 ± 4.3% of Rhodamine B was successfully loaded into the NPs, and after incubation with HUVECs the signal lasted for 28 days after burst release. Hemolysis and whole blood clotting assays proved that NPs did not interfere with the blood clotting process or lyse blood cells. We characterized mRNA incorporated PLGA nanoparticles and incubated with HUVECs after antibody conjugation. To test
in vivo
study, we injected
gata1a
morpholino oligomer (MO) to
Tg(fli1:gfp)
transgenic line to inhibit hematopoiesis, which reduces wall shear stress (WSS) of ventricular chamber leading lack of trabeculae. At 2 days post fertilization (dpf),
NICD
mRNA incorporated nanoparticles were injected into the circulatory system of
gata1a
MO injected zebrafish to rescue trabeculation. At 5 dpf, zebrafish hearts were harvested and analyzed via qRT-PCR to compare Notch related gene expression among control,
gata1a
MO injected, and rescue group. The
NICD
mRNA injected group successfully rescued the expression level of Notch components despite a lack of WSS and mechanosensitive Notch signaling via
gata1a
MO injection. This finding will provide an initial therapeutic approach of
NICD
mRNA for trabecular related congenital heart disease.
Abstract 426: Single Cell RNA-seq Analysis of Dynamic LIF Control of Adipose Derived Stem Cells Transition to Cardiomyocytes
Authors: Yao Jiayi , Yao Jiayi
Published: October 2019

Heart failure continues to be a leading cause of human mortality worldwide and is the consequence of various forms of myocardial injuries that compromise the pumping ability of the heart. Cellular fate transition through manipulation of modifying factors holds great promise for large-scale production of cardiomyocytes required for therapy and for revealing principles of gene regulation in the process. In this study, we found leukemia inhibitory factor (LIF) acts directly on adipose derived cells to induce cardiomyogenesis by JAK/STAT3. LIF induces adipose-cardiac transdifferentiation in adipose derived stem cells, which exhibits morphology and function typical of beating cardiomyocytes. However, the cell population in adipose tissue that generates cardiomyocyte is not from adipocytes or stromal cells, as examined by Cre-Loxp system and FACs. Here, using single cell RNA sequencing, we identified a cell population in adipose tissue that undergoes a gradual and simultaneous switch form relatively quiescent fate to proliferate cardiomyocyte like cells. By trajectory alignment, we compared the gene expression kinetics across fresh adipose cells and LIF induced cardiac transition process to identify crucial genes involved in the transition. Our findings may provide a better understanding of the mechanism underlying LIF-induced adipose-cardiac transdifferentiation, therefore reveal new therapeutic strategies for the treatment of myocardial injuries, and provide crucial information for regenerative medicine.