bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 24: Bnip3 Provokes ROS Production and Maladaptive Autophagy by Displacing Uncoupling Protein3 (UCP3) From Cytochrome c Oxidase of Respiration Chain Complex in Cardiotoxicity
Authors: Dhingra Rimpy , Margulets Victoria , Jassal Davinder , Dorn II Gerald , Kirshenbaum Lorrie A.
Published: July 2021

Doxorubicin is known for its cardiotoxic effects and inducing cardiac failure, however, the underlying mechanisms remain cryptic. Earlier we established the inducible - death protein, Bcl-2-like Nineteen- Kilodalton- Interacting - Protein 3 (Bnip3) to be crucial for disrupting mitochondrial function and inducing cell death of cardiac myocytes. Whether Bnip3 underlies cardiotoxic effects of doxorubicin toxicity is unknown. Herein we demonstrate a novel signaling pathway that functionally links activation and preferential mitochondrial targeting of Bnip3 to the cardiotoxic properties of doxorubicin. Perturbations to mitochondria including increased calcium loading, ROS, loss of αΨm and mPTP opening were observed in cardiac myocytes treated with doxorubicin. In mitochondria, Bnip3 forms strong association with Cytochrome c oxidase subunit1 (COX1) of respiratory chain and displaces uncoupling protein 3 (UCP3) resulting in increased ROS production, decline in maximal and reserved respiration capacity and cell viability. Impaired mitochondrial function was accompanied by an accumulated increase in autophagosomes and necrosis demonstrated by increase release of LDH, cTnT and loss of nuclear High Mobility Group Protein 1 (HMGB-1) immunoreactivity. Interestingly, pharmacological or genetic inhibition of autophagy with 3-methyl adenine (3-MA), or Atg7 knock-down suppressed necrotic cell death induced by doxorubicin. Loss of function of Bnip3 restored UCP3-COX complexes, mitochondrial respiratory integrity and abrogated necrotic cell death induced by doxorubicin. Mice germ-line deficient for Bnip3 were resistant to doxorubicin cardiotoxicity displaying normal mitochondrial morphology, cardiac function and survival rates comparable to vehicle treated mice. The findings of the present study demonstrate that doxorubicin provokes maladaptive autophagy and necrotic cell death of ventricular myocytes that is mutually dependent and obligatorily linked to Bnip3.
Abstract 25: Polyphenolic - Ellagic Acid Suppresses Mitophagy- Induced Necrotic Cell Death During Doxorubicin Cardiotoxicity
Authors: Dhingra Rimpy , Dhingra Abhinav , Jayas Rahul , Kirshenbaum Lorrie A.
Published: July 2021

Reactive oxygen species (ROS) play a major role in cardiac dysfunction during myocardial ischemia. ROS production has been linked to oxidative stress injury and mitochondrial perturbations including permeability transition pore opening (mPTP), loss of mitochondrial membrane potential ([[Unable to Display Character: ∆]]Ψm) and necrotic cell death. Previously we identified the inducible Bcl-2 protein, Bnip3 as critical regulator of mitochondrial function and cell death of ventricular myocytes. Polyphenolic compounds including ellagic acid from pomegranate, have strong anti-oxidant properties. The effects of ellagic acid on oxidative stress injury in the heart has not been explored. In this report, we provide new compelling evidence that ellagic acid suppressed mitochondrial ROS production, loss of [[Unable to Display Character: ∆]]Ψm and necrotic cell death of cardiac myocytes induced by doxorubicin (DOX) or hypoxia. We further show mechanistically that the cytoprotective effects of ellagic acid were related to the transcriptional repression of Bnip3. In contrast to vehicle treated cells, cells treated with DOX or hypoxia displayed a marked increase in Bnip3 expression and mitochondrial association, concordant with increased ROS, mPTP, and loss of [[Unable to Display Character: ∆]]Ψm. Consistent with these mitochondrial defects there was a marked increase in mitophagy as confirmed by the dual emission Mitokeima probe that detects autophagic degradation by labelling mitochondria containing autophagosomes fused with lysosome. Mitophagy was accompanied by a marked increase in LDH release, loss of nuclear HMGB1 immunostaining and cell death. Interestingly, cells treated with ellagic acid were resistant to mitochondrial and the cytotoxic effects of DOX displaying reduced ROS production, mitophagy and were indistinguishable from vehicle treated control cells with respect to cell viability. Notably, Dox-induced Bnip3 expression was dramatically reduced in cells treated with ellagic acid. Hence, the findings of the present study demonstrate that ellagic acid suppresses mitochondrial perturbations and cell death of cardiac myocytes by mechanism that links to the repression of mitochondrial Bnip3.
Abstract 26: Fibroblast Growth Factor 21 Prevents Diabetic Cardiomyopathy by Attenuating Cardiac Lipotoxicity via Erk1/2-dependent Signaling Pathway in Mice
Authors: Tan Yi , Zhang Chi , Yan Xiaoqing , Huang Zhifeng , Gu Junlian , Zhou Shanshan , Shao Minglong , Feng Wenke , Li Xiaokun
Published: July 2021

The role of FGF21 plays in the development and progression of diabetic cardiomyopathy (DCM) has not been addressed. Here we demonstrated that type 1 diabetes decreased FGF21 levels in the blood, but up-regulated cardiac fgf21 expression about 40 fold at 2 months and 3-1.5 fold at 4 and 6 months after diabetes, which indicated a cardiac specific FGF21 adaptive up-regulation. To define the critical role of FGF21 in DCM, type 1 diabetes was induced in FGF21 knock out (FGF21KO) mice. At 1, 2 and 4 months after diabetes onset, no significant differences between FGF21KO and wild type (WT) diabetic mice in blood glucose and triglyceride levels were observed. But FGF21KO diabetic mice showed earlier and more severe cardiac dysfunction, remodeling and oxidative stress, as well as greater increase in cardiac lipid accumulation than WT diabetic mice. Mechanistically, FGF21 reduced palmitate-induced cardiac cell death, which was accompanied by up-regulation of cardiac Erk1/2, p38 MAPK and AMPK phosphorylation. Inhibition of each kinase with its inhibitor and/ or siRNA revealed that FGF21 prevents palmitate-induced cardiac cell death via up-regulating the Erk1/2-dependent p38 MAPK/AMPK signaling pathway. In vivo administration of FGF21, but not FGF21 plus ERK1/2 inhibitor, to diabetic mice significantly prevented cardiac cell death and reduced inactivation of Erk1/2, p38 MAPK and AMPK, and prevented cardiac remodeling and dysfunction at late-stage. Our results demonstrate that cardiac FGF21 decompensation may contribute to the development of DCM and FGF21 may be a therapeutic target for the treatment of diabetic cardiac damage via activation of Erk1/2-P38 MAPK-AMPK signaling.
Abstract 261: Foxo4 Promotes Early Inflammatory Response Upon Myocardial Infarction via Endothelial Arg1
Authors: Zhu Min , Wang Zhaoning , Luo Robert , Goetsch Sean , Schneider Jay , Hill Joseph , Morris Jr. Sidney , Liu Zhi-Ping
Published: July 2021

Myocardial infarction (MI) can result in a maladaptive remodeling of the heart that leads to heart failure. The post-MI inflammation is necessary for wound healing. Unfortunately it is also a key component of subsequent heart failure pathology. Thus, understanding the mechanism of post-MI inflammation and identifying targets for intervention are of translational interests. To understand the role of FoxO4 in post-MI remodeling, we induced MI in WT and
FoxO4
global knockout (
gKO
) mice by surgical ligation of the left anterior descending coronary artery.
FoxO4-
inactivation resulted in a significantly higher post-MI survival, better cardiac function, and reduced fibrosis and infarct size compared to WT mice.
FoxO4 gKO
mouse hearts had significantly less mount of neutrophils, and reduced expressions of cytokines, MMP9, and Arginase 1 (Arg1). To determine the contribution of cellular FoxO4 to the post-MI phenotype, we generated cardiomyocyte and endothelial cell specific
FoxO4
knockout mice (
FoxO4 cKO
and
eKO
). While
FoxO4
cKO
mice showed similar post-MI phenotype to those of WT littermates,
FoxO4
eKO
mice had better cardiac function with reduced inflammation, neutrophil infiltration, and Arg1 expression similar to those of
FoxO4 gKO
mice. Since Arg1 is a competitive inhibitor of nitric oxide (NO) synthase and NO inhibits lymphocyte adhesion and transmigration across the endothelial barrier, downregulation of neutrophils in post-MI
FoxO4-
null mouse hearts could be due to reduced endothelial Arg1 and thus increased NO. Consistent with this hypothesis, we show that Arg1 is a direct transcriptional target of FoxO4. Endothelial FoxO4 is necessary and sufficient to activate Arg1 expression in response to hypoxia. Knockdown of FoxO4 in human aortic endothelial cells suppressed leukocyte adhesion. Furthermore, chemical inhibition of Arg1 in WT mice had similar cardioprotection following MI as
FoxO4-
inactivation and administration of NOS inhibitor to
FoxO4
gKO
mice reversed the beneficial effects of
FoxO4
-deletion on post-MI cardiac function. Our studies showed that FoxO4 promotes early inflammatory response via endothelial Arg1 and suggest a potential therapy to reduce the early post-MI inflammation and enhance cardiac function in MI-injured hearts.
Abstract 268: Myofilament Tyrosine Phosphorylation by Src Kinase is upregulated in ErbB2 Transgenic Mice
Authors: Xu Mingguo , Ramirez-Correa Genaro A , Fu Zongming , Shah Polina S , Belmonte Frances , Gabrielson Kathleen , Murphy Anne M
Published: July 2021

Objectives:
Tyrosine (Tyr) phosphorylation of the myofilament is an emerging, and potentially important, post-translational modification in cardiomyopathy. ErbB2, a Tyr receptor kinase, was overexpressed in transgenic mice (ErbB2-Tg) resulting in significant cardiac hypertrophy. We hypothesize that the development of cardiac hypertrophy in ErbB2-Tg is associated with increased myofilament Tyr phosphorylation and may implicate myofilament Tyr phosphorylation in cardiac hypertrophy.

Methods:
Proteins were isolated from ErbB2-Tg and Ntg heart homogenates (
n
=4 per group). Reduction/alkylation was followed by trypsinization. Resulting peptides were desalted in C
18
columns and lyophilized. Phosphorylated Tyr (p-Tyr) enrichment was performed on 20 mg of peptides using a p-Tyr Mouse mAb kit (Cell Signaling). Immuno-precipitated and desalted peptides were analyzed by LC-MS/MS (Orbitrap Elite, Thermo). Raw data were searched with Mascot 2.3. Label-free quantification with MS1 extracted ion chromatograms was performed using Skyline. Western blot analysis for total and phosphorylated Src kinase was performed per manufacturer’s protocol (Cell Signaling).

Results:
We found a total of 286 p-Tyr modified peptides in ErbB2-Tg compared to 226 in control NTg mice. Over 70 p-Tyr sites on myofilament protein were up-regulated in ErbB2-Tg, including troponin I, myosin heavy chain, titin, α-tropomyosin, myosin-binding protein-C3, myosin regulatory light chain-2 and myosin light chain-1. We used PhosphoMotif Finder to search the potential responsible kinase. Most of the p-Tyr sites were consistent with Src kinase motifs. Furthermore, Western blot analysis showed that total, and phospho-Src (Y416) expression was increased in ErbB2-Tg mice.

Conclusion:
We concluded that these novel p-Tyr sites on myofilament proteins are increased in ErbB2-Tg mice and correlate with up-regulated Src kinase activity. Thus increased tyrosine myofilament phosphorylation may be involved in the development of cardiac hypertrophy. Since ErbB2 is a therapeutic target of trastuzumab therapy this may also have translational implications to ameliorate off target effects of cancer treatment.
Abstract 283: Chaer Lncrna Negatively Regulates Polycomb Repressive Complex 2 During Cardiac Hypertrophy
Authors: Wang Zhihua , Wang Xinghua , Chen Iris , Gao Chen , Yokota Tomohiro , Wang He , Ren Shuxun , Cass Ashley , Xiao Xinshu G , Li Guangping , Wang Yibin
Published: July 2021

Long non-coding RNAs (lncRNAs) emerge to be critical regulators of cellular processes, but only a few out of thousands have been functionally characterized. We identified a novel heart-specific lncRNA, named cardiac hypertrophy associated epigenetics regulator (Chaer), which was both necessary and sufficient for hypertrophy of neonatal rat ventricular cardiomyocytes. RNA deep-sequencing revealed that Chaer contributed to the global transcriptome reprogramming during phenylephrine (50 μM)-induced hypertrophy, and regulated imprinted gene H19 expression independent of DNA methylation but dependent on histone tri-methylation at H3K27 (H3K27me3). RNA immunoprecipitation assay found that Chaer directly interacting with and negatively regulating PRC2 function on H3K27me3. Tagged RNA pull-dwon and RNA EMSA assays confirmed that Chaer directly bound to the catalytic subunit Ezh2 with a conserved 66-mer motif near its 5’ end in competition with and functionally interrupting other PRC2-binding lncRNAs. Interestingly, Chaer-PRC2 interaction was transiently enhanced at the onset of hypertrophy and responsible for hypertrophy fetal gene induction which was sensitive to Ezh2 inhibitor GSK126 (1 μM). Moreover, mTOR inhibitor rapamycin (20 nM) completely blocked the enhanced Chaer-PRC2 interaction, reversed the decrease of global H3K27me3, and abolished phenylephrine-induced expression of hypertrophy fetal genes. Finally, Chaer silence in vivo using chemically modified siRNA and nanoparticle transfection reagents significantly reversed the development of cardiac hypertrophy, pathological remodeling and H3K27m3-modification-mediated fetal gene induction under transaortic-constriction-induced pressure overload. The findings unveil Chaer as an epigenetic determinant of cardiac hypertrophy, and shed a light into the early molecular events under cardiac stress.
Abstract 30: 4-PBA Prevents TAC-induced Myocardial Hypertrophy and Interstitial Fibrosis by Attenuating ER Stress in Mice
Authors: Luo Tao , Chen Baihe , Wang Xianbao
Published: July 2021

Background:
Recently, 4-phenylbutyric acid (4-PBA) has been recognized as a potent ER stress inhibitor and a histone deacetylase inhibitor, but its therapeutic effect in cardiovascular diseases is still not fully understood. Our previous study indicated that attenuation of ER stress by administration of low dosage of 4-PBA (20 mg/kg/d) prevented post-infarction-induced cardiac rupture and remodeling through modulating both cardiac apoptosis and fibrosis in the mouse model of myocardial infarction. However, little is known whether the administration of 4-PBA is effective for hypertrophic heart disease. The aim of this study is therefore to test the therapeutic effect of 4-PBA on pressure-overload induced myocardial hypertrophy.

Methods and Results:
Transverse aortic constriction (TAC) was used to produce pressure-overload in C57BL/6 male mice for 4 weeks. After surgery, 4-PBA (20mg/kg/d) or 0.9% NaCL was intraperitoneally injected daily. At the end of 4 weeks, the survivals were underwent high-resolution echocardiographic imaging observation. The results showed that the left ventricular posterior wall thickness at end systole (LVPWs) and left ventricular posterior wall thickness at end diastole (LVPWd) were increased in TAC group. Administration of 4-PBA ameliorated this hypertrophic effect. Autopsy also confirmed the anti-hypertrophic effect of 4-PBA. Masson-staining found that there was no difference in perivascular fibrosis between TAC and TAC+4-PBA groups. However, myocardial interstitial fibrosis and collagen deposition were significant decreased by 4-PBA. We finally detected the ER stress and histone acetylation using western blotting. Our results showed that 4-PBA, at the dosage of 20 mg/kg/day, decreased the expression of CHOP and had no effect on histone 3 acetylation.

Conclusion:
These findings indicate that attenuating ER stress by 4-PBA maybe a promising therapeutic strategy to prevent pressure-overload induced myocardial hypertrophy and interstitial fibrosis.
Abstract 305: Single Cell RNA Sequencing of Adult Cardiac c-kit+ Cells in a Murine Lineage Tracing Model
Authors: Maliken Bryan D , Kanisicak Onur , Molkentin Jeffery D
Published: July 2021

A subset of adult cardiac resident cells defined by the stem cell factor tyrosine kinase receptor termed c-kit, are believed to have myogenic potential and are now being delivered via intracoronary infusion to presumably promote cardiac regeneration and improve ventricular function after ischemic cardiac injury. However, recent studies have shown that despite these benefits, c-kit+ progenitor cells in the adult murine heart are more inclined to take on an endothelial rather than cardiomyocyte lineage. To better define the factors involved in early differentiation of these resident cardiac progenitor cells and to distinguish distinct cell subpopulations, we performed single cell RNA sequencing on c-kit+ cells from Kit-Cre lineage traced GFP reporter mice versus total mesenchymal cells from the heart that were CD31- and CD45-. Cells were isolated by cardiac digestion and FACS was performed, positively sorting for the c-kit+ lineage while negatively sorting for CD31 and CD45 to eliminate endothelial and leukocyte progenitor contamination, respectively. Following this isolation, cells were examined to determine GFP reporter status and then submitted for single cell RNA sequencing using the Fluidigm A1 system. Clustering of 654 genes from this data identified 6 distinct subpopulations indicating various stages of early differentiation among CD31- and CD45-negative cardiac interstitial cells. Furthermore, comparison of GFP+ c-kit cells to the total non-GFP mesenchymal cells identified 75 differentially expressed transcripts. These unique gene signatures may help parse the genes that underlie cellular plasticity in the heart and define the best molecular lineages for transdifferentiation into cardiac myocytes.
Abstract 306: Sympathetic Reinnervation is Required for Mammalian Cardiac Regeneration.
Authors: White Ian A , Gordon Julie , Balkan Wayne , Hare Joshua M
Published: July 2021

Rationale:
Established animal models of limb and tissue regeneration with re-vascularization demonstrate a critical dependence on concurrent reinnervation by the peripheral nervous system.

Objective:
Considering the significant abundance of autonomic nerves in the mammalian heart we tested the hypothesis that reinnervation is required for neonatal mouse cardiac regeneration.

Methods and Results:
Crossing Wnt1:cre transgenic mice with a double-tandem (td) tomato reporter strain identifies all neural crest-derived cell lineages including the peripheral autonomic nerves in the heart. Whole mount epi-fluorescence microscopy facilitated the clear resolution of subepicardial autonomic nerves in the mouse ventricles providing unprecedented detail of the subepicardial neuroanatomy of the mouse heart. Sympathetic nerve bundles envelop the entire heart and extend to the tip of the ventricular apex. Our data demonstrate that during regeneration of the resected ventricular apex of the neonatal mouse heart, sympathetic nerves fibers undergo concurrent re-growth into the injury site resulting in complete sympathetic reinnervation of the regenerated tissue. Sympathectomy of the heart, induced by administration of 6-OHDA, was sufficient to block innate cardiac regeneration in the neonatal mouse.

Conclusions:
We report that the innate ability of the neonatal mouse heart to undergo regeneration in response to injury is dependent on sympathetic innervation of the ventricular myocardium. Ablation of post-ganglionic sympathetic nerves blocks the innate regenerative capacity of neonatal mouse hearts suggesting that sympathetic reinnervation is critical for ventricular regeneration. This finding has significant implications for adult regeneration following myocardial infarction where nerve growth is hindered by age related influences and scar tissue.
Abstract 31: High-density Lipoprotein, a Feed-forward Forechecking Loop
Authors: Behnke A.-l. , Mockenhaupt F. , Willy K. , Winkler K. , Hetzer R. , Ermilov E. , Siegel G.
Published: July 2021

Background:
The protective effect of HDL is projected into the cholesterol back-transport of breakdown products of lipid metabolism to the liver. This is a feed-back circuit which controls cholesterol exit. Cholesterol entry could be safeguarded in a feed-forward forechecking loop at the proteoglycan (PG) receptor sites (syndecan, perlecan). HDL is the counter partner in the system of internalization and diffusion control for lipoprotein entry into the vascular wall. Based on stereochemical and chiral conformity with its PG receptor and the much higher negative charge density, HDL owns by far a higher affinity compared with LDL.

Methods:
Flow-dependent isometric tension, intracellularly recorded membrane potential and cAMP-cGMP were measured in segments of 25 coronaries from heart transplantations.

Results and Discussion:
Compared to Krebs solution, LDL (100 mg/dL) impaired flow-dilatation and caused a relative contraction by 18.5% (Table). In contrast, HDL (50 mg/dL) and HDL+LDL stimulated flow-dilatation by 31.1% and 41.4%, respectively (p < 0.96). Thus, the contractile effect of LDL was absent in the Krebs-HDL-LDL solution. The same effects were apparent in the membrane potential and cAMP-cGMP-concentrations of the VSM cells. These results are confirmed by ellipsometry measurements, where nanoplaque formation by LDL is suppressed by preincubation with HDL [Siegel, Malmsten, Ermilov: Adv Coll Interface Sci 205 (2014) 275-318].

Conclusion:
LDL-cholesterol entry into the vascular wall is effectfully controlled by HDL in a feed-forward forechecking loop (HDL 4х higher affinity constant to the PG receptor). The interaction between both is dominated by competition.
Abstract 311: Single-Dose Isoproterenol does not Depress Cardiac Function in Mice
Authors: Wallner Markus , Duran Jason M , Koller Sarah , Mohsin Sadia , Lis Steffen , Sharp Thomas E , Berretta Remus M , Houser Steven R
Published: July 2021

Rationale:
Myocardial injury after repeated or continuous administration of isoproterenol (ISO) in preclinical models has been widely described in the literature by our lab and others. Recent controversial reports using a one-time dose of ISO, to mimic a Takotsubo-like cardiomyopathy, demonstrated pronounced and reversible depression of cardiac function at one-week post injection with widespread myocyte death followed by robust myocardial regeneration and recovery of cardiac function.

Hypothesis:
Single-dose ISO does not produce depression of cardiac function

Methods and Results:
C57Bl/6 mice were given a single subcutaneous injection of vehicle (saline) or 5, 200, or 300 mg/kg of ISO. Cardiac function was measured using transthoracic echocardiography with cardiac strain analysis at baseline prior to ISO injection and after 1, 7, 14, and 28 days post-injection. Animals were sacrificed after 1, 7, and 28 days post-injection for evaluation of gross heart weight (HW), HW/body weight (BW) and HW/tibia length (TL). Left ventricular (LV) functional measurements revealed no significant differences in global systolic function (ejection fraction and fractional shortening) between vehicle- or ISO-treated animals at any concentration. Additionally, no significant differences were detected between vehicle- or ISO-treated animals in any cardiac dimensions measured by echocardiography (LV cross-sectional area, internal diameter, end-diastolic or end-systolic volumes) or in gross HW, HW/BW or HW/TL. LV global cardiac strain was also not significantly different between vehicle and ISO-treated animals at any time point. When apical regions of the LV endocardium (the area most predominantly affected by Takotsubo cardiomyopathy) were specifically examined using strain analysis, no significant differences could be detected between vehicle and ISO-treated animals at any time point.

Conclusion:
Single-dose ISO injury in a mouse model does not produce any depression of cardiac function at 1 week post injection.
Abstract 312: Molecular Characterization and Prospective Isolation of Human Secondary Heart Field Progenitors From Pluripotent Stem Cells
Authors: ghazizadeh zaniar , Fattahi Faranak , Sharifi Mehdi , Tale Ahmad Sara , Shabani Parisa , Mirshahvaladi Shahab , Baharvand Hossein , Aghdami Nasser , Salekdeh Ghasem H
Published: July 2021

The secondary heart field (SHF) progenitors ultimately contributes to diverse cardiovascular cell types through the formation of an early, multipotent heart progenitor pool and are marked by expression of ISL1, a LIM-homeodomain transcription factor. Human SHF can be derived from human pluripotent stem cells but their characterization has been limited due to the inefficiency of the differentiation protocols and lack of a proper reporter or surface marker based purification system. Using genetic tools and antibiotic selection we were able to purify ISL1+ cells for global gene expression analysis to identify key pathways that SHF identity. Genetic and small molecule based manipulation of these pathways alter ISL1 induction in differentiating cultures. Further proteomic analysis of enriched ISL1+ cells identified a hit surface marker that enables prospective isolation of ISL1+ secondary heart field progenitor cells with more than 90% purity. Purified SHF cells were multipotent and differentiate into pacemaker cells, endothelial and smooth muscle cells as well as mature beating cardiomyocytes. Finally transplantation of hPSC-derived purified SHF progenitors using this surface marker, restored myocardial function and regenerated infarcted area in mice myocardial infarction model.
Abstract 321: Macrophage Elavl1 Regulates Fibrogenesis Through Post-transcriptional Mechanism Under Diabetic Conditions
Authors: Jeyabal Prince , Thandavarayan Rajarajan A , Joladarashi Darukeshwara , Suresh Babu Sahana , Krishnamurthy Shashirekha , Verma Suresh K , Garikipati Venkata N , Kishore Raj , Krishnamurthy Prasanna
Published: July 2021

Patients with diabetes are predisposed to increased risk of cardiovascular diseases. Persistent interaction of infiltrating macrophages and resident fibroblasts play a critical role in cardiac fibrosis. However, the signaling mechanism is not clear. We hypothesized that macrophage ELAV1 (mRNA stabilizing protein) modulates profibrotic mediators and extracellular matrix turnover by binding to 3′UTR and regulating the mRNA stability of TGF-beta and MMP-9 in hyperglycemic conditions. Mice receiving intramyocardial injection of HuR-specific shRNA showed significant reduction in infarct size and fibrosis area. Reduced fibrosis was associated with decrease in TGF-beta and MMP-9 expression in the myocardium. Conditioned media (CM) from high glucose (HG) treated macrophages significantly increased profibrogenic response (increased mRNA expression of Col1a1, Col3a1 and fibronectin) in fibroblast cell line as compared to fibroblasts incubated with CM from low glucose (LG)-treated macrophages. Knockdown of ELAV1 in HG-treated macrophages abrogated the profibrotic effects in fibroblasts. Indirect immunofluroscence of bone marrow-derived macrophages (BMM) demonstrated that HG increases nuclear ELAV1 export to the cytoplasm. Pharmacological inhibition of Protein kinase C-delta (PKCd) blocked HG-induced ELAV1 nuclear to cytoplasmic translocation. In vitro, stable knockdown of ELAV1 in mouse macrophage cell line RAW 264.7 reduced mRNA expression of TGF-beta and MMP-9 following LPS challenge, accompanied by a marked reduction in the mRNA stability of these genes. Our study here establishes an ELAV1/TGF-beta/MMP-9/PKC-delta signaling axis in the macrophages controlling the profibrogenic responses in fibroblasts, the major contributor in the pathogenesis of fibrosis. Therefore, targeting this signaling pathway might be of therapy value for cardiac fibrosis in diabetic patients.
Abstract 323: MicroRNA208a Silencing Attenuates Doxorubicin Induced Cardiac Toxicity and Dysfunction
Authors: Tony Hasahya , Zeng Qiutang , Yu Kunwu
Published: July 2021

Aims:
Salvaging GATA4 expression mitigates doxorubicin-induced myocyte apoptosis and cardiac dysfunction. We investigated if therapeutic silencing of miR-208a, a heart specific microRNA known to target GATA4, could attenuate doxorubicin-induced myocyte apoptosis and improve heart function.

Methods:
Eight weeks old female Balb/C mice were randomly assigned to Sham, antagomir and Control groups. Antagomir group mice were pre-treated with 50nmols of miR-208a antagomir 4 days prior to giving doxorubicin. At day 0, control and antagomir group got 20mg/kg of doxorubicin while sham mice received phosphate buffered solution. Echocardiography was done at day 7, after which animals were sacrificed, and hearts assessed for apoptosis and expression of miR-208a, GATA4 and Bcl-2 by quantitative PCR.

Results:
Doxorubicin significantly upregulated miR-208a P=0.008 , downregulated GATA4 P=0.025 , and increased myocyte apoptosis P=0.001. Therapeutic silencing of miR-208a mitigated the doxorubicin-induced increase in miR-208a, P= 0.003 and salvaged GATA4 expression, with noted increase in Bcl-2 levels compared to controls, P=0.033. Doxorubicin significantly increased cardiomyocyte apoptosis P= 0.001, and this effect was attenuated by pretreatment with miR-208a antagomir, P=0.002 (Figure 1A and B). Doxorubicin also caused significant cardiac dysfunction, P=0.005, while antagomir treatment attenuated doxorubicin-induced cardiac dysfunction as assessed by fractional shortening P=0.011 (Figure 1Cand D)

Conclusion:
Therapeutic silencing of miR-208a salvages GATA4 and attenuates doxorubicin-induced myocyte apoptosis with subsequent improvement in cardiac function.
Abstract 329: The Human Pericardial Fluid is Enriched With Cardiovascular-expressed Micrornas and Exosomes Able to Elicit Therapeutic Responses
Authors: Beltrami Cristina , Shantikumar Saran , Shearn Andrew , Laftha Abas , Rajakaruna Cha , Angelini Gianni D , Emanueli Costanza
Published: July 2021

Cells release functionally active microRNAs (miRs) into extracellular vesicles (EVs: exosomes and microparticles). In vitro, EVs shuttle miRs and other molecules between cells. The functional relevance of EVs in the context of human physiopathology is still debated.

We hypothesize that the pericardial fluid (PF) mediates myocardium cell-to-cell communication through EVs exchanges. Here, we aimed to (1) characterize human PF EVs and exosomal miRs; (2) investigate if PF EVs exert a biological function.

PF, plasma, thoracic aorta (TA) and right atrium appendage (RAA) samples were collected as leftovers from aortic valve replacement surgery. A miR array identified PF miRs expression. The top expressed cardiovascular miRs were measured (RT-qPCR) in TA and RAA (to validate expression in patients cardiovascular tissues), PF and plasma, and exosomes enriched from PF and plasma. EVs concentration and size distribution in PF and plasma were quantified by a nanoparticle tracking analysis system. The exosome functional activity was tested in human endothelial cells (ECs) and in a mouse model of limb ischemia (LI).

A pool of cardiovascular miRs (including miR-21 and -29a), were enriched in the PF in comparison to plasma, suggesting that the PF/plasma concentration gradients of cardiovascular miRs were caused by the trafficking of these miRs from the myocardium and thoracic vessels to the PF. This gradient was reverted for the negative control miR-122 (produced by the liver and undetectable in the PF). Exosomes were abundant in the PF, even if less concentrated than in plasma. PF-exosome treatment of ECs exposed to hypoxia significantly decreased cell apoptosis and increased cell proliferation (P<0.01 vs PBS). Local PF-exosomes delivery increased post-LI ischemic blood flow recovery (P<0.01 vs PBS). By contrast, plasma-exosomes were not effective (P=NS vs PBS).

We have provided the first characterization of EVs and exosomal miRs in the human PF and for first identified a therapeutic potential of PF- exosomes from cardiovascular patients. Future studies will be tailored to exploit the properties PF-exosome in regenerative medicine.
Abstract 334: An Inotropic S100A1-derived Peptide Enhances Contractile Performance and Survival of Post-ischemic Failing Hearts
Authors: Siragusa Mauro , Spaich Kristin , Völkers Mirko , Gao Erhe , Ritterhoff Julia , Katus Hugo A , Most Patrick
Published: July 2021

S100A1 gene therapy was suggested as therapeutic for cardiomyopathies since myocardial levels of S100A1 are decreased in heart failure. S100A1 increases systolic and diastolic performance of cardiomyocytes through enhanced Ca2+-induced SR Ca2+ release and augmented SR Ca2+ re-uptake. These effects are due to enhanced systolic Type 2 Ryanodine Receptor (RyR2) and diastolic Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2a (SERCA2a) activity. The S100A1 C-terminus (S100A1ct) was recently shown to be the bioactive domain of the protein. The aim of this study was therefore to characterize the effects of an S100A1ct-derived peptide on cardiomyocyte function and to test its therapeutic action in a mouse model of heart failure. An S100A1ct-derived peptide encompassing aa75-85 of the human S100A1 protein and preceded by a hydrophilic motif (DKDDPP) was named S100A1ct6/11. S100A1ct6/11 was cell permeable and accumulated in the intracellular space of intact rat ventricular cardiomyocytes in a striated pattern, similar to endogenous S100A1. S100A1ct6/11 exerted a time- and dose-dependent positive inotropic effect in electrical field stimulated rat ventricular cardiomyocytes. This effect was associated with the regulation of the SR calcium content. Peptides encompassing aa75-85 derived from S100 paralogs A4 and B did not mimic S100A1ct6/11-mediated inotropy. S100A1ct6/11 protected cardiomyocytes from pro-arrythmic store overload-induced calcium leak as well as from chronic caffeine exposure-induced apoptotic cell death. Mice intravenously injected with S100A1ct6/11 exhibited a significant enhancement of LV contractile performance under basal and βAR-stimulated conditions. Daily intraperitoneal administration of S100A1ct6/11 for two weeks to mice with post-ischemic contractile dysfunction resulted in significantly improved contractile performance and survival due to reduced myocardial apoptosis. Moreover, this treatment protected failing hearts with caffeine-induced leaky RyR2 channels from βAR-triggered lethal ventricular tachyarrhythmias. In conclusion, administration of the S100A1ct6/11 peptide may represent a novel option for the treatment of cardiomyopathies without apparent side effects.
Abstract 340: Single Nucleotide Polymorphism of MLX Gene Plays a Crucial Role in the Pathogenesis of Takayasu Arteritis Through Facilitating Inflammasome Formation of the Aorta
Authors: Maejima Yasuhiro , Tamura Natsuko , Ito Yusuke , Isobe Mitsuaki
Published: July 2021

We have identified that single nucleotide polymorphisms (SNPs) of both
IL12B
gene which encodes IL-12p40 and
MLX
gene, which encodes a basic helix-loop-helix leucine zipper transcription factor, were significantly associated with clinical manifestations of Takayasu arteritis (TA), an autoimmune systemic arteritis, by genome-wide association study (GWAS). Recently, we reported that SNP of
IL12B
plays an important role in mediating the development of TA through modulating the level of IL-12p40-related cytokines, including IL-12 and IL-23. However, it remains unknown whether
MLX
mutation is associated with the pathogenesis of TA. Here, we elucidate that whether SNP of the
MLX
(rs665268), a missense mutation of MLX that alters the 139
th
glutamine to arginine (Q139R), upregulates the factor(s) associated with the development of TA. Pull-down assays with recombinant proteins demonstrated that mutation of Q139R on MLX protein enhanced the heterodimer formation of MLX with MondoA, a binding partner of MLX that promotes thioredoxin-interacting protein (TXNIP) expression. As Gln is a neutral amino acid, we speculated that alteration from Gln
139
to Arg
139
, which confers a positive charge to the DNA binding site of MLX, should enhance formation of the MLX-MondoA-DNA complex. Consistent with our hypothesis, luciferase reporter gene assays showed that the TXNIP promoter activity of human aortic smooth muscle cells (hASMCs) co-transfected with TXNIP promoter-containing reporter, MondoA and MLX-Q139R plasmids was significantly higher than those with the wild type MLX (MLX-WT) (MLX-Q139R: 63138 ± 2557 RLU* vs. MLX-WT: 31263 ± 3433 RLU, *
p
< 0.05). The protein levels of both NLRP3 and procaspase-1, major components of inflammasome upregulated by TXNIP, was significantly increased in hASMCs transfected with MLX-Q139R than compared to those in transfected with MLX-WT. Immunohistochemical analyses of the human aortas revealed that NLRP3 in the SMCs accumulated more prominently in the aortas of TA than those in normal ones.

In conclusion, Q139R mutation on MLX plays a crucial role in the pathogenesis of TA through facilitating NLRP3 accumulation, which in turn facilitating inflammasome formation in the aorta.
Abstract 359: Tripartite Motif Protein 58 (trim58) is a Novel Regulator of Membrane Repair With Increased Expression in Failing Cardiac Muscle
Authors: Krishnamurthy Karthikeyan , Alloush Jenna , Xu Zhaobin , Beck Eric X , Mohler Peter J , Janssen Paul , Weisleder Noah
Published: July 2021

In recent years, members of the tripartite motif-containing (TRIM) family of E3 ubiquitin ligases have been shown to be both positively and negatively regulated in various disease pathologies. TRIM72 (MG53) has been directly linked with regulation of sarcolemmal membrane repair in striated muscle cells, including cardiomyocytes. Recently, we were first to identify that a novel tripartite motif family member, TRIM58, is a negative regulator of the cell membrane repair process in striated muscle cells. Overexpression of TRIM58 decreases the membrane repair capacity of cultured myoblasts as measured by dye occlusion following laser-mediated disruption of the sarcolemmal membrane. We also find that TRIM58 can directly interact with TRIM72/MG53 and alter signaling through phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). Protein profiling experiments show that TRIM58 expression increases during the development of heart failure suggesting that TRIM58 may be relevant in the development of cardiac failure. Western blot and histological examination in both human (Fig.1) and mouse transverse aortic constriction (TAC) heart failure samples clearly show an increased expression of TRIM58 in cardiac tissue. Our results suggest that TRIM58 levels might serve as a potential prognostic marker and that TRIM58 may be a therapeutic target for the management of cardiovascular disease.
Abstract 37: Nrip Deficiency Leads to Dilated Cardiomyopathy
Authors: Chen Show-Li
Published: July 2021

Previously, we demonstrate a gene, nuclear receptor interaction protein (NRIP, also named DCAF6 or IQWD1) as a Ca2+- dependent calmodulin binding protein that can activate calcineurin phosphatase activity. Here, we found that α-actinin-2 (ACTN2), is one of NRIP-interacting proteins from the yeast two-hybrid system using NRIP as a prey. We further confirmed the direct bound between NRIP and ACTN2 using in vitro protein-protein interaction and in vivo co-immunoprecipitation assays. To further map the binding domain of each protein, the results showed the IQ domain of NRIP responsible for ACTN2 binding, and EF hand motif of ACTN2 responsible for NRIP bound. Due to ACTN2 is a biomarker of muscular Z-disc complex; we found the co-localization of NRIP and ACTN2 in cardiac tissues by immunofluorescence assays. Taken together, NRIP is a novel ACTN2-interacting protein. To investigate insights into in vivo function of NRIP, we generated conventional NRIP-null mice. The H&E staining results are shown in the hearts of NRIP KO mice are enlarged and dilated and the cell width of NRIP KO cardiomyocyte is increased. The EM of NRIP KO heart muscles reveal the reduction of I-band width and extension length of Z-disc in sarcomere structure; and the echocardiography shows the diminished fractional shortening in heart functions. Additionally, the calcium transient and sarcomere contraction length in cardiomyocytes of NRIP KO is weaker and shorter than wt; respectively. In conclusion, NRIP is a novel Z-disc protein and has function for maintenance of sarcomere integrity structure and function for calcium transient and muscle contraction.
Abstract 373: Spatial Expression Analysis of the Ischemic Heart Identifies Novel Factors Involved in Cardiac Remodeling and Disease
Authors: Lacraz Gregory , Junker Jan Philipp , Scholman Koen , Versteeg Danielle , van Oudenaarden Alexander , van Rooij Eva
Published: July 2021

Cardiac ischemic injury is associated with expressional changes in a broad range of coding and non-coding genes. While genome-wide expression techniques like RNA sequencing, give valuable information about the transcriptional activity of genes, spatial clues are lost.

Here, we use a novel method (TomoSEQ) based on traditional histological techniques with low-input RNA sequencing to generate a spatial gene expression atlas for the infarcted heart. Transcriptome profiling of 50 consecutive sections from hearts exposed to ischemia reperfusion or sham surgery, allowed us to trace differential gene regulation throughout the infarct, borderzone and remote area. Fourteen days post-injury, we clearly observed differential expression curves specific for genes known to be relevant for cardiac function and disease, like
Nppa
(remodeling/hypertrophy),
Col3a1
(fibrosis) and
Atp2a2
(calcium handling), with the first two being more abundantly expressed in the infarct, and the latter more dominant towards the remote. Interestingly, the exact spatial information on these 3 reference genes enabled us to identify a set of genes that show an identical regulation, potentially linking these transcripts to the aforementioned biological function of the reference genes. The validity of using this method to identify genes that are expressionally linked was underscored by the observation that the correlation between the expression level of the
Nppa
,
Col3a1
,
Atp2a2
and the newly identified genes could be confirmed in human cardiac tissue samples from patients suffering from ischemic heart disease. We next used an
in silico
approach to identify common transcription factors among the co-regulated genes. TomoSEQ analysis revealed a strong spatial correlation between these identified transcription factors and their target genes. This provides further support for our methodology, but also provides an opportunity to identify novel transcription factors relevant for cardiac function and disease.

Our study shows the power of spatial transcriptome profiling throughout the infarcted heart by giving local clues on changes in gene expression and allowing for the identification of novel genes and key transcription factors that could be relevant for cardiac repair upon injury.
Abstract 38: Simultaneous Optical Pacing and Optical Voltage Mapping in Optogenetic Neonatal Rat Ventricular Myocyte Cultures
Authors: Li Qince , Goh KahYong , Kong Wei , Ni Ruby R , Fast Vladimir , Zhou Lufang
Published: July 2021

Optogenetics is an emerging technology allowing remote and precise control of cell activity in living tissues. Despite its rapid advancements, application of this innovative technology to cardiovascular research is still limited, in part due to shortage of optogenetic cardiac tissue models and compatible imaging methods. The present study aimed to develop an optogenetic culture model using neonatal rat ventricular myocytes (NRVM) expressing light-gated Channelrhodopsin-2 (ChR2) and characterize activation spread during optical stimulation using optical mapping of membrane potential (Vm). Primary NVRM cultures were infected with lentivirus containing ChR2 gene. Cultures were paced electrically or optically with pulses of blue (470 nm) LED light. Activation spread was simultaneously mapped using Vm-sensitive dye (RH-237) and a photodiode mapping system. Results showed that ChR2 could be readily transduced to NRVMs by the lentiviral method; however, high-level ChR2 expression was associated with substantial cell toxicity. Lower ChR2 expression, achieved by administration of bromodeoxyuridine, had minor effects on cell morphology and function while allowing optical pacing at frequencies of 0.5-3 Hz. Simultaneous Vm mapping showed that conduction velocity, APD80, and dV/dtmax were similar in optogenetic and control cultures. Finally, the optogenetic cultures could be optically paced at multiple sites, leading to significantly reduced overall activation time. In summary, we demonstrated that ChR2 expression can cause cell toxicity in NRVM cultures but the toxicity can be mitigated allowing optical pacing and simultaneous optical activation mapping without significant impairment of electrophysiological function. This optogenetic cardiac culture model expands the availability of optogenetic tools for cardiac research.
Abstract 388: High-throughput Single Cell Tracking of Mitochondrial Function in Cardiomyocytes
Authors: Fajardo Giovanni , Bezold Kristi , Meyer Tobias , Mochly-Rosen Daria , Bernstein Daniel
Published: July 2021

Mitochondria play a significant role in the regulation of multiple functions in the heart, ranging from metabolism to cell death. Most mitochondrial assays require either isolating the organelle, thus disrupting the intracellular signaling or using intact cells with average measurements for the entire population neglecting the ability to distinguish cell heterogeneity. Here we describe a novel method for tracking single cell mitochondrial function in cell populations over time. Adult mouse myocytes were exposed to the mitochondrial uncoupler FCCP and hydrogen peroxide to induce changes in membrane potential and oxidative stress, respectively. TMRM and mitosox fluorescence were used to quantify mitochondrial membrane potential and ROS production, Fluo-4 fluorescence was used to assess intracellular calcium. Tracking of single cells was performed using Matlab and Imaris software. After FCCP exposure TMRM signal intensity decreased before there was a significant change in myocyte length that led to hypercontracture and cell death within 10 minutes. To better understand the dynamics of mitochondrial function and its relationship with cell death a dose response curve was established for hydrogen peroxide at 100, 500 and 1000 μM. The higher doses of hydrogen peroxide induced hypercontracture faster than lower doses. For further studies 100 μM was used to assess how homogenous the response to hydrogen peroxide was in cell populations. We found 3 distinct populations of cells responding at different times, a population of cells hypercontracted between 7-10 min, another between 10-15 min and a third population only after 15 min. Changes in membrane potential, oxidative stress and intracellular calcium were simultaneously assessed for every single cell during these time points. In addition, given the organized structure of the mitochondria in the myocyte we have adapted our technique to track individual mitochondria to study heterogeneous responses at the single mitochondrion level. This method provides a unique tool to simultaneously assess multiple parameters of mitochondrial function in single cells over time. In doing so, we have unmasked a complex heterogeneity of single cell behavior that is lost in methods than only average cell populations.