bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 732: Attenuation of Tnf-α Induced Injury in Cardiomyocytes by Chlorogenic Acid via Inhibiting Nf-κB and Jnk Signals
Authors: Guo Shuzhen , Tian Lei , Su Cong Ping , Wang Qing , Wu Fu Jian , Bai Rui , Zhang Hui Min , Liu Jin Ying , Lu Wen Jing , Wang Wei , Lan Feng
Published: October 2019

The traditional Chinese herb
Lonicerae Japonicae Flos
has been used to treat various diseases including heart failure, showing significant benefits in the clinical practice. However, the mechanism of its cardioprotective effects remains unclear. As the main active ingredient found in the plasma after oral administration of
Lonicerae Japonicae Flos
, chlorogenic acid (CGA) has been reported to possess anti-inflammatory, anti-oxidant, anti-apoptosis and analgesic function. To investigate whether the cardioprotective effects of
Lonicerae Japonicae Flos
is through CGA, we applied Transverse Aortic Constriction (TAC)-induced heart failure mouse model, to access the effects of CGA. The results of in vivo experiments show that CGA has cardioprotective effects and could mitigate the TNF-α(Tumor necrosis factor-alpha) induced toxicity in TAC heart failure mouse model. Therefore, we further used TNF-α-induced cardiac injury in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to explore whether CGA have cardioprotective effects and elucidate the underlying mechanism(s). CGA pretreatment could reverse TNF-α induced cellular injuries, including improved cell viability, increased mitochondrial membrane potential and inhibited cardiomyocytes apoptosis. We then examined the NF-κB/p65 and major MAPKs signaling pathways involved in TNF-α induced apoptosis of hiPSC-CMs. Importantly, both CGA and NF-κB inhibitor (QNZ) can reverse the cell viability impairment and apoptosis phenotypes. Moreover CGA can directly inhibit NF-κB signal by suppressing the phosphorylation of NF-κB/p65. As for the MAPKs, CGA suppressed the activity of JNK only, but enhanced ERK1/2 and had no effect on p38. In summary, our study revealed that CGA has profound cardioprotective effects through inhibiting the activation of NF-κB and JNK pathway, providing a novel therapeutic alternative for prevention and treatment of heart failure.
Abstract 748: Circulating Extracellular Vesicles as Novel Biomarkers of Cardiovascular Risk in Older Women With Different Sitting Time Patterns
Authors: Chang Ya-Ju , Avitia Yesenia , Godbole Suneeta , Bellettiere John , Rock Cheryl L , Patterson Ruth E , Jankowska Marta M , Kerr Jacqueline , Allison Matthew A , Natarajan Loki , Sears Dorothy D
Published: October 2019

Background and Hypothesis:
Extracellular vesicles (EVs) are membrane-bound particles shed from a variety of cell types. They contain molecular cargo from the parent cell including metabolites, proteins, and nucleic acids. EVs are present in biofluids and could be promising biomarkers for monitoring wellness. Total sedentary time is linearly associated with cardiovascular and coronary heart disease mortality risk among older women, indicating the intensive impact of sedentary behavior on the circulatory system. Endothelial cells (ECs) provide a barrier and maintain circulatory homeostasis in response to physical and biochemical stimuli. We hypothesize that EC-derived EV count and content changes with EC health and that EC-EVs play a role in bridging sedentary lifestyle with cardiovascular disease risk.

Materials and Results:
Archival parent study data and plasma samples from a combined cohort of 518 women aged ≥55 years and BMI ≥25kg/m
2
enrolled in the Metabolic, Exercise, and Nutrition at UCSD, Reach for Health, and Community of Mine studies were available to examine EV levels and contents associated with sitting time. Physical activity was measured objectively using accelerometers. Women in the lowest quartile of moderate-vigorous physical activity and who had the highest and lowest mean sitting bout duration across quartiles were indicated as Super Sitters and Interrupted Sitters, respectively. For EC-EV characterization, CD144 was shown to be a specific marker of EC-derived EVs and an anti-CD144 antibody specifically recognized EC-derived EVs from human plasma. EV biochemical markers (e.g., Hsp70, LAMP1, and CD63) were detected on the CD144
+
EVs. Immune-gold staining identified CD81, CD63, LAMP1, and CD144 on individual EVs with a typical toroidal shape featuring phospholipid-bilayers using transmission electron microscopy. A protection assay showed CD144
+
EVs protect miR-126, an EC-enriched miRNA, from RNase degradation. Study of EV levels and contents in plasma from Super Sitter and Interrupted Sitter groups is underway.

Conclusion:
CD144
+
EVs carrying molecular cargo from ECs. Hence, EVs and their cargo (e.g., miRNAs) are valuable for examination as novel biomarkers associated with sedentary behavior-induced cardiovascular risk.
Abstract 749: Circulating miRNAs and Apoptosis Genes With Hyperglycemia in Ischemic Stroke Patient
Authors: Hsieh Yi-Chen , Chi Nai-Fang , Chiou Hung-Yi
Published: October 2019

Stroke is the third cause of death and the leading cause of adult disability in Taiwan. More than half of acute ischemic stroke patients had hyperglycemia, which is one of the major reason for unfavorable outcomes. However, the physiopathology is remain unclear and better knowledge of underlying genetic mechanisms is needed to improve prognosis. This study aimed to identify possible genes and its regulated miRNA involving in hyperglycemia-induced unfavorable stroke outcomes. This is a two-stage study design. In the first screening stage, 3 hyperglycemia ischemic stroke patients and 3 normal-glycemia cases were recruited. MiRNA sequencing and PCR profile arrays were conducted. Messenger RNA with significant differences and miRNA were analyzed using miRSystem database and Cytoscape. The results showed that apoptosis regulatory pathway genes (FAS and BCL2L1) and its related microRNAs (has-let-7i-5p,has-let-7b-5p,has-let-7a-5p,has-let-7f-5p,has-let-7g-5p,has-let-7e-5p,has-miR-98-5p) were highly associated with acute hyperglycemia ischemic stroke. In the second validation stage, we then verified the above mentioned apoptosis regulatory pathway genes and its related miRNAs using middle cerebral artery occlusion (MCAO) mice model and validate in 143 ischemic stroke patients. From the animal model, the results showed that the FAS gene of hyperglycemic rats exhibited a higher performance than that of normal glycemic rats. The results of miRNAs indicated that the expression of has-let-7a-5p, has-let-7b-5p, has-let-7e-5p, and has-let-7i-5p was lower in hyperglycemia rats than those belonged to normal glycemic rats. For ischemic stroke patients, unfavorable outcome patients had higher FAS expression. Furthermore, there is a significant group-by-time interactions in FAS expression, with unfavorable outcome patients having increasing expression in FAS gene (p=0.0208). A remarkable group-by-time interaction in has-let-7e-5p expression was also observed, with unfavorable outcome patients having decreasing expression in has-let-7e-5p (p=0.0128). Our study uncovers FAS gene play an important role in hyperglycemia resulting in poor outcomes in ischemic stroke patients, and might provide new direction for treatment in the future.
Abstract 750: Gene Signatures to Distinguish Amyloid Cardiomyopathy Risk in Multiple Myeloma Patients
Authors: Jha Alokkumar , Morgado Isabel , Lee Dian J , Alexander Kevin , Tsai Cheng-Yu , Schimmel Katharina , Ward Jennifer , Witteles R , Liedtke Michaela , Liao Ronglih , Dangwal Seema
Published: October 2019

Amyloid light chain (AL) amyloidosis results from tissue deposition of clonal light chains, most commonly produced by clonal plasma cells. AL amyloidosis is closely associated with multiple myeloma (MM), another disease which arises from clonal plasma cell proliferation. Here we aim to identify gene signatures to distinguish AL cardiomyopathy (AL-CM) risk in MM patients.

We utilized publicly available data sets and applied Graph Cluster Perturbation approach to cluster the co-expression networks based on pathways in MM and AL-CM utilizing 225 samples from four datasets: GSE42955 study (12 dilated CM, 12 ischemic CM and 5 control LV human heart tissues), GSE95077 study (16 amiloride drug treated/untreated myeloma cell line samples), GSE24128 study (16 newly diagnosed AL with monoclonal plasma cell samples over- or under-expressing cyclin D1), and GSE6477 study (76 primary bone marrow samples from hyper-diploid myeloma patients and 80 non-hyperdiploid MM patients). From these data sets, the networks for extracellular matrix organization, immune system, innate immune system, metabolism, and neutrophil degranulation pathways for MM and amyloid CM were extracted.

Summary:
Ranking of the perturbed genes based on pathways similarity index in MM and AL resulted in a panel of genes: CD44, NRAS, GRAP2, CTLA4, GSN, CCND1, NFKB1, and IRF1 (p= <0.01). As shown in

Figure

, the high hazard ratio of this gene cluster in MM with AL-CM (3.76; p=0.021) compared to MM without CM (0.96; p=0.032) suggests the potential of this gene panel to distinguish high or low risk groups in MM based on survival.
Abstract 774: Gene-Environment Regulatory Circuit of Right Ventricular Pathology in Cyanotic Congenital Heart Defects
Authors: Yan Zhao , Kang Xuedong , Biniwale Reshma , Shahoud Fadi , Glen Van Arsdell , Quintero Fabiola , Stanley F Nelson , Touma Marlin
Published: October 2019

Introduction:
The phenotypic spectrum of congenital heart defects (CHDs) is contributed by both genetic and environmental factors. Their contributions are profoundly heterogeneous but may operate on common pathways that are not well understood. Elucidating the gene-environment interplays in CHDs pathogenesis is essential to identify chamber specific therapies.

Methods:
Following the UCLA CHD-BioCore approved protocol, right ventricle outflow tracts (RVOTs) samples were collected from infants with tetralogy of fallot (TOF) or Ventricular Septal Defect (VSD). Total RNA was used for RNA-seq. Systemic hypoxia was induced using hypoxia chamber. Weighted gene network co-expression analysis was performed using R package.

Results:
Genome-wide transcriptome analysis of RVOT samples from cyanotic and non-cyanotic CHDs uncovers disease-associated differences in gene expression. Co-expression network analysis reveals that individuals with CHDs show diagnosis-specific and environment-specific gene modules. In particular, hypoxia-dependent induction of proliferation and E2F1 dependent cell cycle reprograming are inversely associated with WNT11 signaling in the cyanotic TOF. In addition, epithelial mesenchymal transition (EMT), fibrosis and apoptosis are repressed. Importantly, perinatal hypoxia attenuates the baseline differences between ventricular chambers in neonatal mouse heart. Remarkably, the observed attenuation of ventricular patterning (AVP) genes is more robust in the right ventricle and highly concordant in hypoxemic hearts in mouse and human. Transcriptional dysregulation of the P53 network is a shared hallmark among AVP genes and cyanotic TOF modules. Importantly, P53 network analysis suggests CREB1, a hypoxia sensor, and LEF1, an EMT regulator during cardiogenesis, as potential mediators of hypoxia-Wnt11 circuit. Finally, the data demonstrate that genetic subtypes of TOF may influence hypoxia-induced regulation leading to phenotypic heterogeneity at molecular and cellular levels in cyanotic TOF hearts.

Conclusions:
Gene-environment interrelations may influence changes in regional gene expression leading to phenotypic heterogeneity at the molecular cellular level in complex cyanotic CHDs.
Abstract 784: Eicosapentaenoic Acid, Unlike Other Omega-3 Fatty Acids, Inhibits Membrane Cholesterol Crystalline Domains Under Conditions of Hyperglycemia
Authors: Sherratt Samuel C , Mason R. P
Published: October 2019

Prescription eicosapentaenoic acid (EPA) reduced cardiovascular events in the REDUCE-IT study population, including in patients with diabetes. During hyperglycemia, there is abnormal membrane cholesterol aggregation which has been linked to oxidative stress and plaque instability. EPA at a pharmacologic dose may preserve normal cholesterol distribution as compared to other omega-3 fatty acids (FAs), including docosahexaenoic acid (DHA), eicosatriaenoic acid (ETE) and α-linolenic acid (ALA). Membrane vesicles were prepared from dilinoleoylphosphatidylcholine (DLPC) at a cholesterol-to-phospholipid mole ratio of 0.6:1 and treated with the omega-3 FAs under conditions of hyperglycemia (200 mg/dL). Changes in membrane lipid organization and width were measured using small angle X-ray diffraction approaches. Cholesterol domains were identified by the presence of diffraction peaks corresponding to a unit cell periodicity or width of 34 Å. Results showed that only EPA was able to prevent the formation of cholesterol domains and had a membrane structure characterized by a normal width (54 Å) and cholesterol distribution despite hyperglycemia. But samples containing the other FAs had a biphasic membrane structure containing prominent cholesterol crystalline domains. The relative size of the cholesterol domains was greatest in the order of ALA>DHA>ETE. Thus, EPA preserved normal cholesterol distribution under conditions of hyperglycemia in a manner that was not reproduced with other omega-3 FAs. These data indicate differences between these long chain FAs and support a potential benefit for EPA in reducing atherosclerotic disease.
Abstract 792: Core Cell Survival Requirements of Hypoxia Inducible Factors in Macrophages Dominate Isoform-Specific Function during Cardiac Repair
Authors: Deberge Matthew , Lantz Connor , Wilsbacher Lisa , Thorp Edward B
Published: October 2019

Objective:
Hypoxia inducible factors (HIF) regulate the adaptive response to ischemic injury and enhancement of HIF levels has been proposed as a therapeutic target to improve tissue repair after acute myocardial infarction (MI). HIFs also regulate the inflammatory function of macrophages, the main protagonists of tissue repair after MI, but it is unknown whether changes in macrophage HIF levels will affect outcomes after MI. Using a murine model of acute MI and conditional knockouts, we investigated a role for macrophage HIFs in tissue repair after MI.

Methods and Results:
In mice subjected to permanent occlusion MI, we observed an accumulation of hypoxic cardiac macrophages and sequential stabilization of HIF-2α followed by HIF-1α, which persisted into the development of heart failure. In patients with ischemic cardiomyopathy, flow cytometry and single-cell transcriptomics revealed a parallel stabilization of HIFs and hypoxia signaling in cardiac macrophages. Since HIFs were stabilized early after MI in cardiac macrophages, we selectively targeted macrophage HIFs using conditional knockouts. Selective deletion of macrophage HIF-2α promoted cardiac macrophage production of IL-10, activating fibroblasts and stimulating collagen deposition, culminating in improved ventricular remodeling and preservation of systolic function. Similarly, selective deletion of macrophage HIF-1α improved cardiac repair; however, this was mechanistically distinct from HIF-2α, inhibiting HIF-1α-dependent proteolytic destruction of MerTK and leading to improved efferocytosis by cardiac macrophages. Contrary to our expectations, loss of both HIF-1α and HIF-2α in hypoxic macrophages enhanced mitochondrial reactive oxygen species and activated necroptosis, leading to increased cell death and cardiac rupture after acute MI. Inhibition of necroptosis with necrostatin-1 attenuated hypoxic macrophage cell death and significantly improved survival after acute MI.

Conclusions:
These data imply strategies that broadly enhance or inhibit HIFs will lead to catastrophic outcomes after MI, while selective inhibition of macrophage HIF-1α or HIF-2α may enhance tissue repair, preserve systolic function, and limit progression to heart failure.
Abstract 793: Macrophage Transcription Factor EB Attenuates Left Ventricular Remodeling Via Lysosomal Lipolysis
Authors: Javaheri Ali , Bajpai Geetika , Picataggi Antonino , Mani Smrithi , Foroughi Layla , Evie Hosannah , Kovacs Attila , Weinheimer Carla , Hyrz Krzystztof , Evans Trent , Xiao Qingli , Ballabio Andrea , Lee Jin-Moo , Matkovich Scot , Razani Babak , Schilling Joel , Lavine Kory J , Diwan Abhinav
Published: October 2019

Background:
Autophagy, lipid metabolism, and inflammation are interrelated cellular processes that implicate lysosomes in human disease. After ischemia reperfusion (IR) injury, inflammasome activation and interleukin 1-beta (IL1-beta) secretion promote heart failure progression. Whether macrophage autophagy and lysosomal biogenesis can attenuate post-IR remodeling and inflammation is unknown. We hypothesized that macrophages exhibit lysosome dysfunction and autophagic impairment after IR injury, and that augmentation of macrophage lysosomal biogenesis via macrophage-specific overexpression of transcription factor EB (Mf-TFEB), a master regulator of autophagy and lysosomal biogenesis, would attenuate myocardial remodeling and inflammation in ischemic cardiomyopathy.

Methods and Results:
In mice subject to IR injury and humans with ischemic cardiomyopathy, we observed evidence of lysosomal and autophagic impairment. To ameliorate post-IR macrophage lysosomal injury, we expressed Mf-TFEB in a closed-chest IR murine model using a tamoxifen-inducible CX3CR1erCre and TFEB overexpression cassette bearing a Cre-excisable STOP codon. Compared to Cre-only controls, Mf-TFEB mice had significantly increased left ventricular (LV) ejection fraction 28-days post-IR (40% relative increase, p=0.002, n=15-17 per group), decreased abundance of pro-inflammatory macrophages, and reduced levels of IL1-beta in myocardial tissue. Surprisingly, neither inflammasome suppression nor TFEB-mediated attenuation of post-IR remodeling required intact macro-autophagy as evidenced by Mf-TFEB-mediated rescue of post-IR LV dysfunction in mice with concomitant inducible ATG5 ablation. RNA sequencing of flow-sorted macrophages from post-IR mice identified lysosomal acid lipase amongst other lipases regulated by TFEB. Mechanistically, pharmacologic inhibition of lysosomal acid lipase specifically abrogated the in vivo effects of TFEB on post-IR remodeling.

Conclusions:
Our findings suggest that macrophage TFEB regulates lysosomal lipolysis to attenuate inflammasome activity and protect against post-IR LV dysfunction, suggesting an alternative paradigm for how lysosome function may impact acute inflammation in vivo.
Abstract 814: Trpv4 Deletion in Endothelium Protects Heart Against Pressure Overload Induced Hypertrophy
Authors: Adapala Ravi K , Kanugula Anantha K , Ohanyan Vahagn , Lenkey Nina , Paruchuri Sailaja , Chilian William M , Thodeti Charles K
Published: October 2019

Coronary microvascular dysfunction has been identified as one of the underlying causes for progression of heart failure following hypertrophy or myocardial infarction. However, the mechanisms underlying microvascular dysfunction during the progression of heart failure are unknown. Recently, we found that transient receptor potential vanilloid 4 (TRPV4) channel acts as mechanosensor in endothelial cells and negatively regulates angiogenesis. Therefore, to determine the role of TRPV4 in coronary microvascular function during pathological hypertrophy, we have induced pressure-overload in WT and TRPV4KO mice via transverse aortic constriction (TAC). We found that TAC-induced cardiomyocyte hypertrophy and reduced cardiac function in WT mice, after 28 days. In contrast, both myocyte structure and cardiac function were preserved in TRPV4KO-TAC compared to WT-TAC mice. Notably, WT-TAC hearts exhibited increased ECM deposition and reduced microvascular density compared to TRPV4KO-TAC hearts, suggesting that the absence of TRPV4 may protect myocardium from pressure-overload-induced stress. To evaluate the specific role of endothelial TRPV4 in coronary microvascular function, endothelial specific TRPV4KO (TRPV4
ECKO
) mice were generated by crossing TRPV4
lox/lox
mice with Tie2-Cre mice. After confirming endothelial deletion of TRPV4 through RT-PCR and immunostaining, we have subjected TRPV4
lox/lox
and TRPV4
ECKO
mice to TAC. Immuno-histological analysis revealed that TRPV4
ECKO
hearts exhibited increased microvascular density compared to TRPV4
lox/lox
mice, 28 days post TAC. Further, we found preserved cardiac structure (myocyte cross sectional area) and cardiac function (% ejection fraction and fractional shortening) with reduced cardiac fibrosis in TRPV4
ECKO
mice compared to TRPV4
lox/lox
, post TAC. Thus, our results suggest that endothelial TRPV4 channels are key regulators of coronary microvasculature function and deletion of endothelial TRPV4 offers cardio-protection via increased coronary angiogenesis following pressure overload-induced by TAC.
Abstract 828: The Functional Role of Myofibroblast-Expressed Smooth Muscle Alpha Actin in Post-Myocardial Infarction Tissue Healing
Authors: Fu Xing , Liu Qianglin , Wang Leshan , Molkentin Jeffery
Published: October 2019

After myocardial infarction (MI), dead cardiomyocytes are permanently replaced by scar tissue due to the lack of cardiomyocyte regeneration. In response to MI, cardiac fibroblasts (CFs) differentiate into myofibroblasts which possess highly organized smooth muscle α-actin (αSMA) stress fibers and secrete a large amount of extracellular matrix (ECM) proteins, essential for the post-MI infarct scar formation. It is well known that the accumulated ECM in the infarct scar stabilize the scar and prevents cardiac rupture. However, the functional role of αSMA stress fiber itself in CF-derived myofibroblasts is still largely unknown besides the wide use of it as a marker of myofibroblast differentiation. Our recent work demonstrated that during myofibroblast differentiation of CFs post-MI, the expression of αSMA reaches a peak a few days earlier than genes encoding ECM proteins. Myofibroblasts then lose αSMA expression and differentiate to the newly identified matrifibrocytes as the infarct scar becomes increasingly stable. These facts suggest a more important role of αSMA stress fibers than ECM proteins in the tissue healing process in early phase post-MI, possibly through providing a support lattice which in turn strengthens the infarcted myocardium. To test our hypothesis, we generated a mouse line with
Acta2
(the gene encoding αSMA) flanked by LoxP sites (

Acta2
fl/fl

) to enable cre-mediated
Acta2
knockout. Here we show that the loss of
Acta2
in CFs leads to enhanced proliferation and reduced contractility upon TGFβ-induced myofibroblast differentiation
in vitro
. By using a mouse line with tamoxifen-inducible CF-specific
Acta2
knockout and CF lineage tracing (

Tcf21
MerCreMer
;Acta2
fl/fl
;R26
GFP

) and a WT control mouse line (

Tcf21
MerCreMer
;R26
GFP

), we identified that CF-specific
Acta2
knockout results in significantly exacerbated post-MI cardiac function, increased cardiac rupture incidence, and enhanced fibrosis. More lineage tracing experiments and transcriptome analyses are being conducted to better decipher the impact of
Acta2
knockout on CF physiology and post-MI tissue healing.
Abstract 835: Development of Molecular Targeted Therapy Against Right Ventricular Failure: Evaluation by Transcriptome and
i
n vivo Analysis
Authors: Ito Shogo , Yuasa Shinsuke , Komuro Jin , Katsuki Toshiomi , Kimura Mai , Kishino Yoshikazu , Kusumoto Dai , Hashimoto Hisayuki , Fukumoto Yoshihiro , Fukuda Keiichi
Published: October 2019

Backgrounds:
Right ventricular (RV) failure is a final common pathway in heart failure. But there is no specific therapy for RV failure. Moreover, it remains unclear how RV failure is developed. To develop a novel therapy for RV failure, we focused on the RV specific character and elucidate the function in RV failure.

Methods:
Microarray analysis using several parts of adult murine heart was conducted and differentially expressed genes (DEGs) were applied to pathway analysis. Molecular mechanism was examined by using rat cardiomyocytes in vitro. To understand the function of target molecule in vivo, we induced RV failure by pulmonary artery constriction (PAC) in mice and the pathway was specifically blocked in the RV failure model.

Results:
In microarray analysis for upper RV, RV free wall, LV and ventricular septum, 995 genes were statistically extracted as DEG in upper RV. An alternative complement pathway was significantly activated in upper RV and complement factor, and C3a was a potential upstream factor attributable to unique feature of upper RV. Because C3a plays a central role in alternative complement pathway, we examined the direct role of C3a in cardiomyocytes and RV function. Administration of C3a recombinant protein to primary-cultured cardiomyocytes activated the several MAP kinases including ERK, p38 and JNK, via C3a receptor. Mice developed severe RV failure around 14 days after PAC. Surprisingly, administration of C3a receptor antagonist dramatically improved right ventricular contractile dysfunction in PAC mice. C3 (substrate of C3a) deficient PAC mice also attenuated RV contractile dysfunction, fibrotic change and fetal gene expressions. There results indicated that complement factor C3a regulates the unique phenotypes in RV. Chemical or genetical blockade of C3a ameliorates RV dysfunction in PAC mice.

Conclusion:
We revealed that alternative complement pathway was activated in RV and C3a had a crucial role in the pathogenesis of RV failure. Accordingly, the blockade of C3a pathway would be a potential therapeutic target for RV disorders.
Abstract 848: The Iron Chelator Deferiprone Clears Hemorrhagic Byproducts Following Acute Myocardial Infarction in a Swine Model of Ischemia-Reperfusion
Authors: Weyers Jill J , Thomas Reuben , Qi Xiuilng , Barry Jennifer , Rabadia Vraj , Manca Dino , Connelly John , Spino Michael , Strauss Bradley H , Wright Graham A , Ghugre Nilesh R
Published: October 2019

Current treatments for myocardial infarction (MI) aim to remove the vascular occlusion and reperfuse the infarcted tissue. While reperfusion can salvage myocardium, it can also increase damage to the unsalvageable tissue, one mechanism of which is hemorrhage; the resulting blood and iron deposits increase edema and inflammation. We hypothesize that removing these iron deposits with the iron chelating drug deferiprone (DFP) will reduce inflammation and lead to faster healing post-MI.

Using a swine model of ischemia-reperfusion, pigs were administered either DFP or saline for four weeks post-MI. Cardiac MRI tracked heart function, infarct progression (via late gadolinium enhancement), hemorrhage (via T2* imaging), and edema (via T2 imaging) at baseline and 1 day, 1 week, and 4 weeks post-MI. Animals were then sacrificed and hearts were processed for histology.

Treatment with DFP decreased the presence of iron in the infarct compared to saline by one week post-MI, as shown by increased T2* values. DFP treatment also decreased end diastolic wall swelling, suggesting less inflammation post-MI. To support this, T2 values and histology both revealed trends toward less edema with DFP. Histology also showed trended increases in fibroblast proliferation and collagen deposition at four weeks post-MI in the treatment group, consistent with a faster resolution to scar. Ventricular remodeling (by ventricular volumes), infarct size (by MRI, histology and blood Troponin I levels), and infarct transmurality were all unaffected, but contractile function improved with DFP treatment: ejection fraction and wall thickening both improved faster.

In conclusion, DFP treatment successfully reduced hemorrhagic iron within the infarct as compared to saline, indicating a clearing of the iron byproducts. Treatment also decreased wall swelling, suggesting reduced inflammation, and improved contractile function. In the infarct, reduced swelling could indicate faster remodeling and a faster resolution to scar, and histology confirms a more progressed scar structure with DFP treatment. Overall, iron chelation appears to be a promising therapy that could reduce the negative effects of hemorrhage and speed the functional recovery of patients post-MI.
Abstract 858: NAD(H) Redox Imbalance in the Heart Accelerates Diabetic Cardiomyopathy
Authors: Chiao Ying Ann , Light Christine , Tian Rong , Sadoshima Junichi , Lee Chi Fung
Published: October 2019

Diabetes is linked to NAD(H) redox imbalance and lowered NAD
+
levels, but the roles of NAD
+
metabolism in diabetic cardiomyopathy are not established. We induced diabetes in C57BL6 wild type mice (WT) by streptozotocin (STZ) injections to cause depletion in serum insulin levels and elevation in fasting glucose levels. Hyperglycemic, male WT mice showed gradual declines in systolic and diastolic functions (FS, 50% vs 30%; E’/A’ ratio, 1.5 vs 1.0; 16-week after STZ), without causing cardiac hypertrophy. Dysfunctions in diabetic hearts were associated with lowered NAD
+
/NADH ratio and protein hyperacetylation, suggesting the roles of NAD(H) redox imbalance in diabetic cardiomyopathy. To test if NAD(H) redox imbalance promotes dysfunctions of diabetic hearts, we employed cardiac-specific Ndufs4-KO mice (cKO), which exhibit lowered NAD
+
/NADH ratio without overt cardiac dysfunction at baseline. Changes in cardiac functions at 2-, 4-, and 8-week after STZ injections, insulin and fasting glucose levels of control and cKO male mice were measured. Similar insulin depletion and hyperglycemia (~450 mg/dL) were observed in diabetic control and cKO mice. Systolic and diastolic dysfunctions were worsened in diabetic cKO mice (control vs cKO: FS 37% vs 22%; E’/A’ 1.25 vs 0.95, 8-week after STZ). Cardiac fibrosis levels were slightly elevated in diabetic control and cKO hearts, but were not different between the two groups (~4% in both group of mice). The results suggested that the accelerated declines in cardiac functions contributed by NAD(H) redox imbalance are not due to the altered extracellular matrix environment. To restore the NAD(H) redox balance in diabetic hearts, we elevated NAD
+
levels in male control and cKO mice with cardiac-specific NAMPT overexpression. Elevations in cardiac NAD
+
levels alleviated systolic and diastolic dysfunctions in diabetic control and cKO mice, despite similar levels of hyperglycemia among groups. Similar exacerbation of cardiac dysfunction was observed in female diabetic cKO mice, while NAMPT overexpression similarly improved cardiac function in female diabetic control and cKO mice. Our findings showed that NAD(H) redox imbalance promotes the progression of diabetic cardiomyopathy, via a fibrosis-independent mechanism.
Abstract 859: Sepsis-Induced Cardiomyopathy is Caused by Mitochondrial Dysfunction Due to Protein Kinase C (PKC) Delta Activation
Authors: Morrow John P , Joseph Leroy C , Reyes Michael V , Drosatos Konstaninos
Published: October 2019

Objective:
Sepsis-induced cardiomyopathy (SIC) is a frequent complication of sepsis that is associated with increased mortality. There are no effective therapies. Oxidative stress and mitochondrial dysfunction are part of the pathophysiology of SIC but the upstream molecular pathways involved are unclear.

Methods:
We used PKCdelta knock-out (KO) mice and WT littermates for surgically-induced polymicrobial sepsis from cecal ligation and puncture (CLP). Isolated cardiomyocytes were used to evaluate oxidative stress, mitochondrial function, contractility, and calcium transients.

Results:
PKCdelta is activated in cardiomyocytes in sepsis models. In vivo, PKCdelta KO mice do not have decreased systolic function after sepsis, unlike WT littermates. WT cardiomyocytes show increased mitochondrial oxidative stress, partial depolarization of the mitochondrial inner membrane, decreased contractility, and decreased calcium transient amplitude. PKCdelta KO cardiomyocytes are protected from all of these abnormalities. Further, mitotempo improves contractility and calcium transients in WT cardiomyocytes, supported the role of mitochondrial dysfunction as a mechanism of SIC.

Conclusion:
PKDdelta KO mice are protected from SIC. Cardiomyocyte experiments indicate that mitochondrial dysfunction promotes oxidative stress and a decrease in contractility during sepsis. Translational impact: PKCdelta inhibition could be effective treatment for SIC.
Abstract 860: Functional Characterization of a Novel Human Heart-specific Microprotein With a Potential Mitochondrial Localization and Role in Sarcomere Dynamics
Authors: Muecke Michael Benedikt B , Schulz Jana F , Schneider Valentin , Sharma Arun , Toepfer Christopher N , van Heesch Sebastiaan , Seidman Jonathan G , Seidman Christine E , Hübner Norbert
Published: October 2019

Cardiac microproteins are an emerging class of important regulators of cellular homeostasis, often encoded by presumed long noncoding RNAs. With only few described so far, their prevalence and biological significance largely remains unclear. To create an atlas of cardiac-expressed microproteins, we performed ribosome profiling on 80 human heart samples. This revealed the translation of hundreds of previously undetected microproteins, which we validated
in vivo
by targeted mass spectrometry. A single heart-specific microprotein caught our attention as it showed strong expression coregulation with components of mitochondrial OXPHOS pathway. We next deleted this microprotein's lncRNA host gene in hiPSCs using CRISPR/Cas9, and, upon differentiation into 30-d old cardiomyocytes, find a strong expression upregulation of genes involved in oxidative phosphorylation and calcium handling. To investigate the impact of the microprotein KO on cardiomyocyte mechanical function, we assessed sarcomere dynamics using an endogenous TTN-GFP tag, enabling live cell imaging analysis with the SarcTrack algorithm. Preliminary analysis sowes a putative increase in sarcomere contraction and relaxation times, suggesting a repressive regulatory function of the eliminated microprotein encoding lncRNA.
Abstract 861: Inner Mitochondrial Collapsing in Response to Acute Overstretch of Rat Ventricular Papillary Muscles
Authors: Nishioka Naritomo , Kusakari Yoichiro , Tanihata Jun , Minamisawa Susumu
Published: October 2019

Introduction:
The effect of acute ventricular volume overload on organelles such as myofibrils and mitochondria are incompletely understood.

Purpose:
We performed acute overstretch on cardiac papillary muscle to investigate the functional and morphological changes.

Methods:
We used male SD-rats (10-12 weeks old) and dissected papillary muscles from the right ventricle. A papillary muscle preparation was stretched to Lmax. We overstretched it within 2 seconds up to 110% of Lmax (110%Overstretch; 110%OS) or 120% of Lmax (120%OS), and stimulated it (1Hz, 36°C) with tension measurement, compared with Lmax group. After the measurement of tension, we analyzed the morphological changes in muscle fibers and organelles using a Transmission electron microscopy (TEM).

Results:
The active tension after overstretch was immediately decreased to 67.8±3.8% in 110%OS, and to 17.3±4.0% in 120%OS. In TEM study, muscle fibers kept their well-organized structures and the sarcomere length was stretched to 2.4 μm in 110%OS, and to 2.6 μm in 120%OS. In contrast, in 110%OS, inner mitochondrial density was lower than that in Lmax, indicating swelled mitochondria. In 120%OS, inner mitochondrial empty space with vacuolation was found in the large area of myocardium, indicating that inner mitochondria cristae were susceptible to mechanical stress-induced deterioration.

Conclusions:
Acute overstretch of rat papillary muscles causes inner mitochondrial collapsing with preserved the sarcomere structure. It could account for the mechanisms of acute volume-overloaded heart failure.
Abstract 862: SGK-1 Metabolically Reprograms VSMC Energy Metabolism by Modulation of Substrate Utilization and Mitochondrial Structure
Authors: Roggerson Krystal , Zhong Wei , Babayewa Oguljahan , Francis Sharon
Published: October 2019

Obesity is associated with vascular remodeling which increases the risk of developing heart disease; the leading cause of death world-wide. The mechanisms underlying vascular remodeling during obesity are not completely understood, but impaired cellular energy metabolism is thought to play an important role. Oxidative phosphorylation (OXPHOS) and glycolysis are the cell’s two main energy generating pathways that rely on energy substrate availability. Glucose is the primary substrate for glycolysis, whereas, pyruvate which is metabolized from glucose, long chain fatty acids (LCFAs) and glutamine are the substrates metabolized for OXPHOS. Previously, we reported that serum and glucocorticoid-inducible kinase 1 (SGK-1) is up-regulated in the vasculature of diet-induced obese mice, implicating a role for this serine/threonine kinase in obesity-related vascular remodeling. Indeed, our current data demonstrate that deletion of serum and glucocorticoid-inducible kinase 1 (SGK-1) specifically in vascular smooth muscle cells (VSMC) protects against obesity-induced vascular remodeling. Further, protection from remodeling was concomitant with metabolic reprogramming of VSMCs due to a metabolic shift from glycolysis towards OXPHOS in SGK-1 knockout (KO) VSMCs compared to wildtype (WT) VSMCs. Interestingly, metabolic reprogramming was also evident in SGK-1 KO VSMC under basal conditions, suggesting a novel role for SGK-1 in cellular energy metabolism. Inhibition of glycolysis was due to an overall reduction in glucose uptake, whereas, an increase in OXPHOS was associated with redistribution of mitochondria and altered levels of the mitochondrial fusion and fission regulatory proteins, suggesting that SGK-1 reprograms energy metabolism through modulation of energy substrate utilization and mitochondrial structure. Altogether, our data point to SGK-1 inhibition as a novel therapy for obesity-related vascular pathology.
Abstract 863: Pathological Roles of Senometabolites in Cardiovascular Disorders
Authors: Shimizu Ippei , Yoshida Yohko , Ikegami Ryutaro , Minamino Tohru
Published: October 2019

Mechanisms contributing for the synchronization of aging are yet to be defined. Here, we define “senometabolite” as circulating metabolites having causal roles for the synchronization and progression of aging. Analyzing metabolomic studies in coronary plaque corrected with directional coronary atherectomy (DCA), we found that hydroxyl-metabolite significantly increased in patients with unstable angina compared with stable angina. We generated murine diet induced obese model and found this metabolite increased in aorta and plasma. Administration of this metabolite into human umbilical vein endothelial cells (HUVECs) induced cellular senescence, and immunofluorescence study showed that putative transhydrogenase increased in patients with atherosclerotic disorders. We did metabolomics studies in aged individuals or patients with heart failure and identified another metabolite, oxidized choline, increased under these conditions compared to respective controls. We generated murine left ventricular (LV) pressure overload model and found that oxidized choline increased both in plasma and failing heart. Administration of oxidized choline deteriorated cardiac function, in contrast, genetic model showed suppression of this metabolite ameliorated systolic dysfunction in LV pressure overload model. Proteomic study showed that oxidized choline reduced the expression of cytochrome c oxidase subunit1, and metabolomics study showed that both ATP and phosphocreatine level significantly reduced in cardiac tissues of wild type mice administrated with this metabolite. Administration of oxidized choline also reduced muscle strength, induced fibrosis in skeletal muscle, and electron microscopy showed an increase in dysfunctional mitochondria both in the heart and skeletal muscle. In aged wild type mice, metabolomic study showed oxidized choline increased in plasma. Aging process is still mysterious and continues to be an interesting topic to be explored. Our findings indicate that circulating senometabolites contribute for the progression of pathologies in age related disorders.
Abstract 876: Chronic GPER1 Cardioprotection in H9c2 Myoblasts is Mediated by an MST1-YAP Pathway and Mitochondrial Function and Dynamics
Authors: Madungwe Ngonidzashe , Feng Yansheng , Bopassa Jean C
Published: October 2019

Introduction:
We recently showed that cardiac tissue treated acutely with estrogen (17β-estradiol; E2), pre- and post-ischemia, exhibited reduced myocardial infarct size and better functional recovery after ischemia /reperfusion via the G protein-coupled estrogen receptor 1 (GPER1). These E2-GPER1 cardioprotective effects have been found to involve the preservation of mitochondrial structure and function. Here, we investigate the impact and mechanisms underlying chronic GPER1 activation in H9c2 cardiac myoblasts treated with a cytotoxic agent, H
2
O
2
.

Methods:
H9c2 cells were treated with 200 μM H
2
O
2
, for 24 h and reoxygenated in no-serum conditions for 4 and 8 days with the addition of E2 (80nM) and GPER1 agonist (G1; 100nM) and GPER1 antagonist (G15; 1μM). We measured cell death and proliferation, as well as gene expression, mitochondrial morphology and function using flow cytometry, cell assays, qRT-PCR, along with fluorescence and electron microscopy. We also analyzed cell lysates by Western blot to assess the mechanisms involved.

Results:
We found that chronic treatment of cardiac myoblasts with E2 and G1 results in reduced cell death. The surviving cells showed improved mitochondrial function as evidenced by better cristae morphology, increased ATP production and increased resistance to opening of the mitochondrial permeability transition pore. Particularly chronic GPER1 activation led to reduced cell cycle activity along with changes in mitochondrial dynamics of fusion and fission. Additionally, we found that chronic GPER1 activation signaling involves decreased phosphorylation of mammalian sterile-20-like kinase (MST1) and increased translocation of the transcription coactivator, yes-associated protein (YAP), to the nucleus for upregulation of pro-survival genes. These effects were mainly prevented by the G15, suggesting a GPER1-dominant mechanism.

Conclusion:
Chronic GPER1 activation in H9c2 cardiac myoblasts treated with oxidative stress results in reduced cell death by preserving mitochondrial structural integrity and function by increasing mitochondrial dynamics. This chronic GPER1 cardioprotection involves activation of MST1/YAP signaling pathways as well as upregulation of pro-survival gene.
Abstract 879: Aldehyde Dehydrogenase Activator 1 (Alda-1) Attenuates Coronary Endothelial Dysfunction-Mediated Cardiac Damage in ALDH2*2 Diabetic Mice
Authors: Pan Guodong , Pang Haiyan , Deshpande Mandar , Palaniyandi Suresh
Published: October 2019

Background:
Endothelial dysfunction plays a critical role in the pathogenesis of diabetic complications. Diabetes-mediated reactive aldehydes like 4-hydroxy-2-nonenal (4HNE) are associated with endothelial cell impairment. Aldehyde dehydrogenase (ALDH) 2, a mitochondrial enzyme which detoxifies 4HNE, protects heart function against diabetic cardiomyopathy. Nearly 600 million East Asians have a point mutation of ALDH2, termed as ALDH2*2, which causes an intrinsic low ALDH2 activity. Previously, we reported an aggravated coronary endothelial dysfunction in ALDH2*2 mutant knock-in mice (ALDH2*2 mice) with diabetes. Aldehyde dehydrogenase activator 1 (Alda-1) is the only known activator for both normal ALDH2 and mutant ALDH2*2. We hypothesized that Alda-1 could protect endothelial cells (ECs) from hyperglycemia induced coronary endothelial dysfunction in diabetic ALDH2*2 mice.

Methods and Results:
Diabetes was induced by streptozotocin injection (150 mg/kg) to ALDH2*2 mice. Alda-1 (10 mg/kg/d, DM-A) or vehicle (DM-V) was delivered through mini-pumps for 3 weeks after diabetes. We found an increased left ventricular pressure (59.9±18.7 vs 66.4±20.1 mmHg) and a decreased perfusion pressure (89.4±8.1 vs 83.6±12.1 mmHg) in Langendorff hearts from DM-A (DM-V vs DM-A, p<0.05, respectively). In heart sections, CD31+ (127.2±22.3 vs 270.5±53.7 /mm
2
) and eNOS+ cells (136.2±22.8 vs 366.7±33.6 /mm
2
) were increased while apoptosis (1117.5±303.6 vs 727.1±254.8 /mm
2
) and 4HNE+ (515.8±8.3 vs 181.7±72.5 /mm
2
) cells were reduced in DM-A (DM-V vs DM-A, p<0.05, respectively). Direct perfusion of 4HNE to Langendorff hearts from control ALDH2*2 mice (4HNE) increased perfusion pressure (Δ3.18±0.5 vs Δ36.6±7.7 mmHg), arteriole closure (8.3±0.4 vs 39.8±3.7 /mm
2
), and decreased eNOS+ cells (2877.1±117.3 vs 37.5±5.6 /mm
2
) (Sham vs 4HNE, p<0.05). One-day Alda-1 (10 mg/kg) pre-treatment before 4HNE perfusion (A-4HNE) decreased perfusion pressure (Δ7.1±1.7 mmHg), number of closed arterioles (22.4±4.8 /mm
2
), and increased eNOS+ (878.0±121.3 /mm
2
) cells (4HNE vs A-4HNE, p<0.05, respectively).

Conclusion:
Alda-1 protects hyperglycemia mediated 4HNE induced coronary endothelial dysfunction and thereby cardiac tissue in ALDH2*2 diabetic mice.
Abstract 880: The Role of Sialidase Neu3 in the Cardiac Response to Ischemia and Reperfusion Injury
Authors: Piccoli Marco , Canali Maria Elena , Ghiroldi Andrea , Cirillo Federica , Anastasia Luigi
Published: October 2019

Reperfusion strategies are life-saving approaches to restore the blood flow in the cardiac tissue after acute myocardial infarction (AMI). However, they come with the drawback that they inevitably induce ischemia/reperfusion injury (IRI), resulting in increased cardiomyocytes damage and heart failure. In this context, the physiological activation of several pro-survival kinases, such as Akt and Erk, as well as of the hypoxia inducible factor (HIF) has been recognized to be critical during IRI. Along with this line, we recently discovered a novel, PHDs independent, mechanism of HIF-1; activation mediated by sialidase Neu3. Interestingly, Neu3 is upregulated under chronic hypoxia in cyanotic congenital cardiac patients. Moreover, induced activation of Neu3 increased myoblast resistance to hypoxic stress. Thus, in this study, we further investigated the possible role of Neu3 in protecting cardiac myoblasts during IRI. In particular, we set-up an in-vitro model of IRI on H9C2 rat cardiomyoblasts. Results showed a modulation of Neu3 during IRI, with a progressive down-regulation during the ischemic phase, followed by a reactivation during the reperfusion phase. Remarkably, overexpression of Neu3 significantly increased cardiomyoblasts resistance to IRI, both in terms of cell proliferation and resistance to apoptosis. Treatment with Akt and Erk inhibitors, as well as with a Neu3 specific inhibitor completely reverted the beneficial effects mediated by Neu3 upregulation. In conclusion, our results show that Neu3 activation has a cardioprotective effect during IRI, calling for further studies to unveil its full potential as a therapeutic target to treat cardiac ischemia and reperfusion injury and to improve patients recover after AMI.