bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 362: Dysregulation of CPT2 Splicing in Diabetic Heart Disease
Authors: Jaworski Elizabeth , Nutter Curtis , Verma Sunil , Deshmukh Vaibhav , Kuyumucu-Martinez Muge
Published: July 2021

Diabetes mellitus is a group of metabolic diseases that are caused by elevated blood glucose levels. Individuals with diabetes have an increased risk of cardiovascular complications that include diabetic cardiomyopathy, hypertension, and coronary artery disease. Research has shown that hyperglycemia causes metabolic abnormalities in the heart such that cardiomyocytes are unable to utilize glucose for energy production due to reduced glucose intake, instead they solely depend on fatty acid oxidation for energy. Eventually, fatty acids accumulate and cause cardiac lipotoxicity, a presumed factor in the development of diabetic cardiomyopathy. Carnitine Pamitoyl Transferease 2 (CPT2) is one of the enzymes responsible for the transport of long-chain fatty acids into the mitochondria for fatty acid oxidation and energy production. CPT2 activity is elevated in diabetic hearts by mechanisms that are unclear. CPT2 is composed of 5 exons; the largest, exon 4 contains the transferase domain and is alternatively spliced in diabetes. In normal hearts, half of the CPT2 transcripts include exon 4 representing the active form of the enzyme. Through RNA sequencing analysis assay, we discovered that CPT2 is mis-spliced in diabetic hearts in a way that 70% of total CPT2 transcripts include the functional domain exon 4. The splicing change in CPT2 results in increased expression of the active CPT2 isoform in diabetic hearts. In summary, we identified a functionally important alternative splicing event in the CPT2 gene that may contribute to increased fatty acid oxidation and lipotoxicity in the diabetic heart.
Abstract 67: GSK-3 Dephosphorylation by PP2A Modulates Age-dependent Cardiac Growth and Hypertrophic Response
Authors: Mohan Maradumane L , Martelli Elizabeth E , Naga Prasad Sathyamangla V
Published: July 2021

Glycogen synthase kinase-3 (GSK-3) is a known negative regulator of cardiac hypertrophic response and much remains unknown about its dephosphorylation process that activates GSK-3. Since GSK-3 function is regulated by PI3K, age based assessment of cardiac hypertrophic response was measured in PI3Kγ knockout (PI3Kγ KO) mice. Interestingly, we observed marked reduction in heart size in PI3Kγ KO mice compared to littermate controls. Consistent with the reduced size, we observed elevated PP2A activity and dephosphorylation mediated activation of GSK-3 in PI3Kγ-KO hearts. Mechanistically, we found that higher PP2A activity in PI3Kγ-KO was due to PP2A methylation mediated by elevated PP2A methyl transferase (PPMT-1) activity. To test in vivo whether PI3K activity regulates GSK-3 dephosphorylation mediated cardiac growth, we generated mice with cardiac specific overexpression of inactive PI3Kγ (PI3Kγinact) and constitutively active PI3Kγ (myrPI3Kγ) in the PI3Kγ-KO background. Age based hypertrophic response and cardiac function was assessed by heart weight/body weight ratios and echocardiography. The heart size was significantly increased in PI3Kγinact (LVEDD- 3.41+/- 0.12) and myrPI3Kγ (LVEDD- 3.73 +/- 0.14) overexpressing mice, when compared to WT (LVEDD- 3.00 +/- 0.15) mice at 18 months age, indicating that kinase activity of PI3Kγ is inconsequential for age-dependent regulation of cardiac growth. Similar cardiac growth due to overexpression of active and inactive form of PI3Kγ suggests that the kinase independent function potentially overrides the kinase activity of PI3Kγ. Correspondingly, PP2A activity was normalized and was associated with decreased GSK-3 dephosphorylation with overexpression of PI3Kγinact. This led to normalization of heart size in these mice compared to PI3Kγ-KO littermates correlating with the decreased GSK-3 dephosphorylation and consequent inhibition of GSK-3 activity. Regulation of PP2A mediated dephosphorylation of GSK-3 by PI3Kγ combined with similar level of increased cardiac growth in PI3Kγ-KO mice with cardiac overexpression of PI3Kγinact or myrPI3Kγ, suggests a non-canonical role of PI3Kγ in hypertrophy of the heart due to age related increase in mechanical demand.
Abstract 69: Angiotensin II- Canonical TGF-β Signaling Downregulates Apelinergic Pathway in Hypertension
Authors: Rai Rahul , Nagpal Varun , Boe Amanda E , Vaughan Douglas E
Published: July 2021

Background:
Apelin is a novel peptide which along with its receptor, APJ, mediates apelinergic signaling. Apelinergic signaling plays a critical role in cardiovascular homeostasis, including regulation of blood pressure. While exogenous apelin blocks Angiotensin II (AngII) mediated nuclear signaling, the role and regulation of endogenous apelin in hypertension (HTN) remains obscure. We hypothesize that apelinergic pathway is downregulated in HTN, which is primarily mediated by aberrant AngII signaling.

Approach:
To test our hypothesis we utilized two mouse models of HTN, including AngII infusion and oral administration of N (ω)-nitro-l-arginine methyl ester (L-NAME). Blood pressure was monitored via noninvasive tail-cuff device. To determine the signaling involved we investigated the effect of AngII on apelinergic pathway in vitro.

Results:
Cardiac apelin was decreased significantly in both murine models of HTN. Downregulated apelin also corresponded with increased deposition of collagen, and up-regulation of senescence markers including PAI-1. Meanwhile, APJ levels were unaffected in both these hypertensive models. In our in vitro studies AngII downregulated apelin expression in human aortic endothelial cells (HAECs) and human cardiac fibroblasts (HCFs). Furthermore, our studies in AngII infused mice and in HCFs highlight the role of TGF-β-pSMAD signaling, independent of MEK involvement, in AngII induced apelin downregulation.

Conclusion and Significance:
Our studies demonstrate that aberrant AngII signaling downregulates apelin in HTN. This downregulation involves canonical Tgf-β1 signaling and affects apelin transcription. Importantly, we propose that AngII mediates its hypertensive pathology by decreasing apelinergic regulation. Since exogenous apelin blocks AngII signaling, further knowledge and negation of AngII induced apelin downregulation could result in the development of novel anti-hypertensive therapies.
Abstract 74: The Inflammatory Phenotype Of Mesenchymal Fibroblasts And Its Role In Aging Dependent Cardiac Fibrosis- A Target For Statins?
Authors: Cieslik Katarzyna A , Trial JoAnn , Entman Mark L
Published: July 2021

In the aging mouse (C57BL/6) myocardium fibrosis steadily increases after 14 months of age and is accompanied by elevated numbers of myeloid derived fibroblasts. Recently, we proposed a mechanism by which inflammatory mesenchymal fibroblasts (IMF) derived from mesenchymal stem cells secrete monocyte chemoattractant protein-1 (MCP-1) necessary for myeloid fibroblast induction in the aging heart. The current study extends the characterization of this inflammatory phenotype by describing elevated interleukin-6 (IL-6) secretion and increased expression of IL-6 receptor (IL-6R) in IMF. Since IL-6R lacks an intracellular domain it requires a co-receptor gp130 (generally expressed) to induce an intracellular signal. Thus, generation of an IL-6R soluble receptor allows IL-6 signaling on cells that do not express IL-6R (or expression is low), such as endothelial cells. We investigate the function of IL-6 and IL-6R in the promotion of transendothelial migration of monocytes through cardiac endothelium and their maturation into myeloid fibroblasts in in vitro assay. Treatments with IL-6 and more extensively IL-6+IL-6R resulted in a 3-5 fold increase (above the control level) in myeloid cell migration and maturation into myeloid fibroblasts. Thus IMF can contribute both IL-6 and IL-6R to endothelial cells and facilitate myeloid cell transendothelial migration. In agreement with these data, analysis of the aged mouse heart revealed the presence of fibroblasts expressing IL-6 (procollagen type I
+
IL-6
+
cells), M1 macrophages (CD86
+
cells) and M2 macrophages (CD301
+
procollagen type I
+
cells) that were absent in hearts from young mice. The mechanisms by which expression of these factors is upregulated in IMF are being investigated; our data suggest that MCP-1 and IL-6 expression are controlled by the farnesyltransferase (FTase)-Ras-Erk1/2 pathway. Interestingly, since atorvastatin interferes with farnesyl synthesis it also reduced MCP-1 and IL-6 expression in IMF. These data may introduce a new use of this class of drugs in the prevention of the age-related fibrosis.
Abstract 79: Mitochondrial Mechanisms of Structural and Electrical Remodeling in Atrial Fibrillation
Authors: Ozcan Cevher , Battaglia Emily , Young Rebeccah , Suzuki Gen , Canty John M
Published: July 2021

Background:
The mechanisms responsible for atrial electro-anatomic remodeling during the development of atrial fibrillation (AF) are not known. Mitochondria determine atrial myocyte cell survival and function by regulating cellular oxidative and metabolic stress. We therefore postulated that mitochondria play a central role in the remodeling of atrial myocardium and the pathogenesis of AF.

Methods:
Our hypothesis was tested in a novel murine model (cardiac specific LKB1 knockout (KO)) where there is a high rate of sinus rhythm progressing to AF (95% of mice). Structural and electrophysiological properties of atria were studied in sinus rhythm and AF. Multiple parameters including the ECG, cardiac MRI, echocardiography, histopathology and immunoblotting in KO mice were compared with age- and gender-matched wild type controls.

Results:
LKB1 KO mice developed biatrial enlargement while in sinus rhythm that progressively increased as persistent AF developed. Apoptotic and necrotic myocyte death produced a substantial reduction in atrial cardiomyocytes (44%) that was replaced by fibrosis (34% vs 4.9%, p<0.05) and non-myocyte cells. In KO atria, there was a 6 fold increase in TUNEL positive nuclei and significant elevations of caspase 3 (10.4±1 vs 3.6±0.5 AU, p<0.05) and caspase 9 (1.4±0.1 vs 0.7±0.06 AU, p<0.05) activity. Mitochondrial reactive oxygen species were increased in KO atria (13.6±0.7 vs. 4.2±1 AU, p<0.05). In addition to extracellular matrix remodeling, cell-to-cell coupling was disrupted in KO atria with reduced gap junction proteins such as connexin 40 (0.24±0.03 vs 0.82±0.01 AU, p<0.05) and connexin 43 (1.4±0.1 vs 14.4±1 AU) (p<0.05). Electron microscopy showed impaired ultrastructure of mitochondrion and myofibrils in atrial myocytes.

Conclusions:
We conclude that mitochondria play a pivotal role in the genesis of AF. Mitochondrial ROS generation and caspase activation lead to progressive cardiomyocyte loss and the transition of sinus rhythm to paroxysmal and persistent AF. These findings raise the possibility that therapies directed at maintaining atrial mitochondrial function may prevent the development of chronic AF.
Abstract 85: Long-term Monotherapy with Bendavia (MTP-131), A Novel Mitochondria-Targeting Peptide, Restores H11 Kinase Protein Levels in Mitochondrial Fractions of Left Ventricular Myocardium of Dogs with Advanced Heart Failure
Authors: Gupta Ramesh C , Szekely Kristina , Wang Mengjun , Rastogi Sharad , Zhang Kefei , Mohyi Paula , Sabbah Hani N
Published: July 2021

Background:
The heat shock protein H11 kinase (H11K) is expressed in the heart of dogs, rodents and humans and rapidly increases in response to increased contractile workload. Deletion of H11K in pressure overload transgenic mice accelerates LV dysfunction and remodeling by deactivating STAT3, a stress-inducible transcription factor. Deactivation of STAT3 has been shown to adversely impact mitochondrial respiration and consequently oxidative phosphorylation. We previously showed that chronic therapy (3 months) with Bendavia (BEN, MTP-131), a novel mitochondria-targeting peptide, improves LV systolic function in dogs with heart failure (HF) and normalizes mitochondrial respiration and rate of ATP synthesis in LV myocardium of dogs with HF. This study tested the hypothesis that H11K is reduced in mitochondria of the failing heart and that long-term therapy with BEN normalizes levels of this key heat shock protein.

Methods:
LV tissue was obtained from 12 dogs with microembolization-induced HF (LV ejection fraction ~30%) randomized to 3 months therapy with subcutaneous injections of BEN (0.5 mg/kg once daily, n=6) or saline (Control, n=6). LV tissue from 6 normal dogs was used for comparison. Tissue samples were used to extract mitochondrial fractions (MF). Protein levels of H11K and Porin, a mitochondrial protein not altered in HF, were determined in MF by Western blotting and protein band were quantified in densitometric units (du).

Results:
Protein level of Porin in MF was similar among NL, Control and BEN-treated dogs (0.24±0.0 vs. 0.22±0.01 vs. 0.23±0.01 du, respectively). Level of H11K was 0.45±0.03 du in NL, decreased to 0.17±0.02 in Controls (p<0.05vs. NL) and was normalized by BEN (0.37±0.05, p<0.05 vs. Control). H11K normalized to Porin was 1.93±0.23 du in MF of NL dogs, decreased to 0.78±0.0.09 du in Controls (p<0.05 vs. NL) and was restored to near normal levels after treatment with BEN (1.61±0.20 du, p<0.05 vs. Control).

Conclusions:
H11K protein levels are reduced in MF from LV myocardium of dogs with HF and normalized after chronic therapy with BEN. Restoring H11K with BEN likely contributed to the observed improvement in mitochondrial respiration and rate of ATP synthesis previously reported after long-term therapy with BEN.
Abstract 88: AMP-activated Protein Kinase Signaling Mediates the Cardioprotective effect of Antithrombin against Myocardial Ischemia and Reperfusion Injury
Authors: Li Ji , Ma Yina , Wang Jinli , Yang Hui , Wang Yanqing , Masnithody Chandrashekhara , Rezaie Alireza R
Published: July 2021

Background:
Antithrombin (AT) is an endogenous protein of the serpin superfamily involved in regulation of the proteolytic activity of the serine proteases of the coagulation system. AT is known to exhibit anti-inflammatory and cardioprotective properties when it binds to distinct heparan sulfate proteoglycans (HSPGs) on vascular endothelial cells. The energy sensor AMP-activated protein kinase (AMPK) plays an important cardioprotective role during myocardial ischemia and reperfusion (I/R). The objective of this study was to investigate whether the cardioprotective signaling function of AT against I/R injury is mediated through the AMPK pathway.

Methods and Results:
The cardioprotective activities of wild-type (WT) AT and its two recombinant derivatives, one having high affinity and the other no affinity for heparin, were evaluated in an acute I/R (20 min/4 h) injury model in which the left anterior descending coronary artery (LAD) was occluded. The serpin derivatives were given 5 min before reperfusion. The results showed that AT-WT can activate the protective AMPK signaling pathway in both in vivo and ex vivo conditions. Blocking AMPK activity abolished the cardioprotective function of AT against I/R injury. The AT derivative having high affinity for heparin was more effective in activating AMPK, but the derivative lacking any affinity for heparin was inactive in eliciting AMPK-dependent cardioprotective activity. The activation of AMPK by AT inhibited the inflammatory c-Jun N-terminal protein kinase (JNK) pathway during I/R. Further studies revealed that the AMPK activity of AT also modulates cardiac substrate metabolism by increasing glucose oxidation but inhibiting fatty acid oxidation during I/R.

Conclusions:
These results suggest that AT binds to vascular HSPGs to invoke a cardioprotective function by triggering cardiac AMPK activation, thereby attenuating JNK inflammatory signaling pathways and modulating substrate metabolism during I/R.
Abstract 10: MicroRNA 17 in Angiogenesis: Lessons Learned From Immobilized Vascular Endothelial Growth Factor
Authors: Aday Sezin , Besnier Marie , Zoldan Janet , Saif Jaimy , Santos Tiago , Carreto Laura , Bernardino Liliana , Langer Robert , Emanueli Costanza , Ferreira Lino
Published: July 2021

Several pre-clinical and clinical studies are exploring the therapeutic effect of cell-based therapies in ischemic diseases, including Peripheral Artery Disease (PAD). Unfortunately, most of the cells (more than 80%) die few days after delivery. We postulate that better understanding of VEGF Biology might be important for the design of more effective pro-survival strategies. Release of VEGF from ECM activates a carcinogenic program since it triggers vasculogenesis, tumor growth and metastasis. Contrarily, immobilized VEGF might have all the properties of soluble VEGF without inducing a carcinogenic program. Thus, identification of downstream players such as miRNAs mediating this process might be important. Herein, we evaluated therapeutic effect of miR-17 downregulation, which mediates the effect of immobilized VEGF both in vitro and in vivo.

We recently showed that conjugated VEGF modulates cell activity by decreasing the expression miR-17 both in vitro and in vivo. In the present study, cell survival and angiogenesis were evaluated firstly in vitro using endothelial cells (ECs) transfected with antagomiR-17 to mimic the down-regulation of miR-17 by conjugated VEGF. AntagomiR-17 increased EC survival at least 1.5 times (n=6) compared to pro-angiogenic miRNAs reported in the literature (e.g. miR-424 and miR-132) and sprout formation on Matrigel at least 2 times (n=5) compared to all groups. The effect of antagomiR-17 was more pronounced under hypoxia conditions. In vivo, antagomiR-17 accelerated hemodynamic recovery of the whole limb (n=12) in unilateral limb ischemia obtained by occlusion of the left femoral artery. Blood flow recovery evaluated by Laser Doppler analysis was significantly higher 21 days after surgery in antagomiR-17 group compared to all other groups. Immunohistochemical analyses showed an increase in the capillary density of skeletal muscle in antagomiR-17 condition. In order to determine the gene target and potential pathway involved in the biological effect of antagomiR-17, next generation mRNA sequencing was performed.

In conclusion, here we show the potential and underlying molecular mechanism of antagomiR-17 treatment in endothelial cell survival and angiogenesis both in vitro and in vivo.
Abstract 102: Cardiac-specific Overactivation of the Mechanistic Target of Rapamycin Complex 1 Induces Metabolic, Structural and Functional Remodeling
Authors: Davogustto Giovanni , Salazar Rebecca , Vasquez Hernan , Taegtmeyer Heinrich
Published: July 2021

The heart remodels metabolically and structurally before it fails. Metabolically, the heart increases its reliance on carbohydrates for energy provision. Structurally, the heart hypertrophies to sustain increased hemodynamic stress. There is evidence suggesting that the activation of the mechanistic Target Of Rapamycin Complex 1 (mTORC1) pathway is closely tied to glucose uptake by the heart to drive the metabolic and structural remodeling. We have previously shown that with insulin stimulation or increases in workload, the glycolytic intermediate glucose 6-phosphate (G6P) is required to activate mTORC1. Sustained mTORC1 activation leads, in turn, to ER stress and contractile dysfunction. Studies by others in the kidney have shown that mTORC1 activation upregulates glucose transporter 1 (Glut1) expression and glucose uptake. We therefore test the hypothesis that chronic mTORC1 overactivation results in G6P accumulation, and precedes structural and functional remodeling in the heart. We developed mice with inducible, cardiac-specific deficiency of the protein tuberin (TSC2), a member of the tuberous sclerosis complex, the principal inhibitor of mTORC1. Intracellular G6P concentrations were measured enzymatically. Immunoblotting was performed on protein markers to confirm activation of mTORC1 downstream targets and of the unfolded protein response. Histologic analysis were performed to assess structural changes. Serial echocardiograms were performed to evaluate cardiac function. The results indicate that chronic mTORC1 activation through inducible, cardiac-specific deletion of TSC2 is accompanied by G6P accumulation and metabolic remodeling. Metabolic remodeling precedes structural and functional remodeling. We suggest that in the heart, sustained mTORC1 activation is a key driver of metabolic and structural remodeling.
Abstract 103: MiR-4261 Controls Endothelial Cells Angiogenesis
Authors: Zhou Qiulian , Lv Dongchao , Sun Qi , Chen Ping , Bei Yihua , Xiao Junjie
Published: July 2021

Myocardial infarction (MI) is among major causes of morbidity and mortality associated with coronary artery disease. Angiogenesis improves tissue perfusion and cardiac repair after MI. Therefore, angiogenesis is considered to be a novel therapeutic way for ischemic heart diseases. MicroRNAs (miRNAs, miRs) have been reported to play important roles in regulating post-ischemic neovascularization. The current study aims at investigating the role of miR-4261 in angiogenesis. We found that miR-4261 mimics increased, while miR-4261 inhibitors decreased the proliferation of human umbilical vein endothelial cells (HUVEC) using EdU incorporation assay (17.25%±1.31% vs 30.91%±0.92% in nc-mimics vs mir-4261-mimics, 17.91%±1.36% vs 8.51%±0.82% in nc-inhibitor vs mir-4261-inhibitor, respectively) and CCK-8 assays (0.84±0.04 vs 1.38±0.04 in nc-mimics vs mir-4261-mimics, 0.80±0.02 vs 0.72±0.01 in nc-inhibitor vs mir-4261-inhibitor, respectively). The wound healing assay showed that miR-4261 mimic transfection resulted in a significant increase in the migration of HUVEC compared to that of the negative controls while miR-4261 inhibition had the opposite effects. Tube formation assays showed that HUVEC transfected with miR-4261 mimics increased the number of tubes formed (57.25±2.56 vs 81.5±2.53 in nc-mimics vs mir-4261-mimics, respectively), while miR-4261 inhibitor-transfected cells had the opposite effect (56.55±0.45 vs 41.38±0.52 in nc-inhibitor vs mir-4261-inhibitor, respectively). These results indicate that miR-4261 play an important role in regulating angiogenesis. However, it remains unknown which target gene mediated the effects of miR-4261. Thus, it will be of great interest to further investigate the molecular mechanisms of miR-4261 in the proliferation, migration, and tube formation of HUVEC in vitro. MiR-4261 could be a potential therapeutic target to enhance angiogenesis.
Abstract 105: Increased Myocardial Sensitivity to Ischemic Injury in an Animal Model of Posttraumatic Stress Disorder
Authors: Rorabaugh Boyd R , Bui Albert , Seeley Sarah L , Krivenko Anna , Eisenmann Eric D , Fry Megan E , Lawson Joseph D , Stoner Lauren E , Zoladz Phillip R
Published: July 2021

Background:
Posttraumatic stress disorder (PTSD) is a psychological disorder characterized by the formation of traumatic memories following exposure to a life threatening event. In addition to psychological manifestations, PTSD promotes atherosclerosis and increases the incidence of myocardial infarction. However, it is unknown whether the effects of PTSD are limited to increasing the incidence of myocardial infarction or if PTSD also increases infarct severity. Therefore, we used an animal model of PTSD to determine whether posttraumatic stress influences infarct size and postischemic recovery of cardiac contractile function.

Methods:
Rats were subjected to a well-established animal model of PTSD that is based on predator exposure and psychosocial stress (Zoladz et al., Stress 11:259-281). Rats subjected to this model exhibit many PTSD-like characteristics including the formation of traumatic memories, increased anxiety, increased startle reflex, hypertension, and alterations in the hypothalamic-pituitary adrenal axis. Male rats (7 weeks of age) were either subjected to psychosocial stress (n = 9) or continuously housed in their home cages (n = 8) for 31 days. Hearts were subsequently isolated and subjected to 20 minutes of ischemia and 2 hours reperfusion on a Langendorff isolated heart system.

Results:
Stressed rats exhibited significantly elevated corticosterone concentrations and anxiety-like behavior in the elevated plus maze. Infarct sizes were significantly larger in hearts from stressed rats (44.7 ± 1.7 % of area at risk) compared to nonstressed rats (31.0 ± 5.4 % of area at risk). Consistent with increased myocardial injury, postischemic recovery of rate pressure product (stressed = 16,922 ± 1,554 mmHg*bpm; nonstressed = 26,407 ± 2,977 mmHg*bpm) and +dP/dT (stressed = 1,901 ± 189 mmHg/sec; nonstressed =3,259 ± 498 mmHg/sec) were significantly attenuated in hearts from stressed rats. Furthermore, postischemic end diastolic pressure was significantly elevated in hearts from stressed (57 ± 6 mmHg) compared to nonstressed (32 ± 7 mmHg) rats.

Conclusion:
This animal model suggests that PTSD may make the myocardium more sensitive to ischemic injury through a mechanism that is independent from its ability to promote atherosclerosis.
Abstract 107: Mitochondrial Dysfunction Mediated Myocardial Stunning Following Resuscitation From Cardiac Arrest
Authors: Sharp Willard W , Piao Lin , Fang Yong , Beiser David G , Liao James K , Archer Stephen L
Published: July 2021

Rationale:
Severe myocardial contractile dysfunction following resuscitation from cardiac arrest (CA) is a major contributor to CA mortality. The pathophysiology and etiology of this dysfunction is not known and there are no pharmacological therapies known to improve outcomes. Previously, we demonstrated that Dynamin related protein 1 (Drp1) is activated and recruited to the mitochondria during CA and that the Drp1 inhibitor Mdivi-1 improves post CA survival.

Objective:
To determine the effects of CA length on myocardial and mitochondrial function. We also sought to determine the effects of Mdivi-1 on post CA outcomes.

Methods and Results:
Asystolic cardiac arrest (CA) was induced in mice by IV injection of 0.08 mg/g KCL. CPR begun at 4, 8, 12, and 16 minutes post-cardiac arrest had rates of return of spontaneous circulation (ROSC) of 100%(12/12), 93%(14/15), 71%(10/14), and 44% (4/9) and 2-hour survival of 100%(12/12), 67%(10/15), 50%(7/14), and 11%(1/9). Transthoracic echocardiography 15 min post-resuscitation demonstrated percent fractional shortening of 36±4% (Sham,n=6), 30±4% (4 minCA,n=11), 24±5% (8minCA,n=10), 15±2% (12minCA,n=12). In surviving animals, myocardial dysfunction persisted for 2 hours post-resuscitation, but slowly recovered to baseline by 72 hours. No evidence of myocardial necrosis, inflammation, or apoptosis was noted following resuscitation. Progressive increases in mitochondrial derived reactive oxygen species (ROS) during CA was observed by MitoSOX red myocardial tissue staining. Mitochondria isolated from 12 min CA hearts demonstrated decreased substrate coupled and uncoupled respiration. Mdivi-1, a mitochondrial inhibitor of division (fission), improved survival and neurological scores in mice following an 8 min cardiac arrest compared to controls.

Conclusions:
Severe, time dependent myocardial stunning (contractile dysfunction in the absence of irreversible injury) was observed following asystolic cardiac arrest. This myocardial stunning was associated with mitochondrial injury and improved by an inhibitor of Drp1. Strategies targeting ischemia/reperfusion-induced changes in mitochondrial dynamics hold promise for improving myocardial function and survival following cardiac arrest.
Abstract 113:
Klf5
and
Ppara
Expression is Increased at the Early Stage and Reduced at the Late Stage of Myocardial Ischemia/Reperfusion in Mice
Authors: Pol Christine J , Valenti Mesele-Christina , Schumacher Sarah M , Yuan Ancai , Gao Erhe , Goldberg Ira J , Koch Walter J , Drosatos Konstantinos
Published: July 2021

Inhibition of cardiac fatty acid oxidation (FAO) is considered beneficial after ischemia reperfusion (I/R). Krüppel-like factors (KLF) have an important role in metabolism. In a model of cardiac energetic deficiency (LPS-treated mice),
in silico
promoter analysis and whole genome array analysis indicated cardiac KLF5 as important regulator of
Ppara
. Gene expression analysis in human ventricular myocytes (AC16) treated with Ad-Klf5 followed by ChIP analysis confirmed that KLF5 is a positive transcriptional regulator of
Ppara
. Mice with cardiomyocyte-specific
Klf5
ablation

(αMHC-Klf5
-/-
)

that we made had reduced cardiac
Ppara
expression and other FAO-related genes. The role of PPARα activation in I/R injury is unclear as both beneficial and detrimental effects have been reported. We aimed to gain insight in the effects of KLF5 and PPARα on acute I/R injury.

To mimic I/R
in vitro
, AC16 cells were subjected to hypoxia (9.5 h) followed by short (1h) or prolonged (14h) normoxia.
Ppara
expression was initially (1h) increased (p<0.05) and then (14 h) decreased (p<0.05). Klf5 expression pattern showed a trend of similar changes
.
Infarction was performed by 30 min of left coronary artery occlusion followed by 2 or 24 hours of reperfusion in wild type mice. I/R resulted in a trend for an initial increase in
Klf5
(2-fold, p=0.05),
Ppara
(3-fold, p=0.08), and
Pdk4
(8-fold, p<0.01) mRNA at 2h post-surgery.
Klf5
(2-fold; p<0.01) and
Ppara
(9-fold; p<0.05) expression were decreased 24h post-surgery. Consistent with
Ppara
changes, there was a 2-fold decrease of
Cpt1
(p<0.01) and
Vlcad
(p<0.01) and a trend for decreased
Aox
(2-fold),
Lcad
(2-fold), and
Pdk4
(6-fold) compared to sham. Echocardiography indicated normal cardiac function after 24h post-surgery. Despite reduced FAO,
αMHC-Klf5
-/-
mice subjected to I/R had a marked increase in mortality; 40% (4 of 10) of
αMHC-Klf5
-/-
mice died within the first 24h of reperfusion while no mortality was observed in wild type mice that underwent I/R.

In conclusion, I/R is associated with an increase in
Klf5
and
Ppara
in the first hours of reperfusion followed by a decrease in
Klf5
and
Ppara
. Increased mortality for

αMHC-Klf5
-/-

mice with I/R injury suggests that the initial increase may be an adaptive response that is critical for survival.
Abstract 116: Myelopoiesis Following Myocardial Ischemia (MI) Involves Activation of the Nlrp3 Inflammasome by Neutrophil-derived S100a8/a9
Authors: Nagareddy Prabhakara R , Annabathula Rahul , Ye Saojing , Klyachkin Yuri , Abdel-Latif Ahmed , Smyth Susan
Published: July 2021

Ischemic myocardial damage triggers leukocytosis particularly the production of monocytes and neutrophils from the bone marrow and spleen (myelopoiesis). These cells infiltrate the evolving myocardial wound, degrade extracellular matrix and aid in the clearance of dead cardiac myocytes and their debris. Although this inflammatory process is a prerequisite for tissue healing, it is non-specific and often blunt. If unchecked, excessive production of monocytes and neutrophils may result in abnormal ventricular remodeling and heart failure. The myocardial cellular and molecular events that orchestrate with the BM/spleen to regulate myelopoiesis remain unclear. We report here that the number of circulating monocytes and neutrophils peak within 24 hours following coronary artery ligation (LAD) in mice. This is due to expansion and proliferation of hematopoietic stem and multi-potential progenitor cells (HSPC) in the BM as well as extra medullary hematopoiesis in the spleen. MI induced -myelopoiesis was associated with a dramatic increase in the expression of S100a8/a9 (a damage associated molecular pattern), its receptor (Tlr4), the Nlrp3 inflammasome and pro-IL1β in the heart. Cell separation studies revealed that the infiltrating neutrophils and cardiac fibroblasts are the predominant source of S100a8/a9 and the Nlrp3 inflammasome respectively in the heart. Further, deletion of s100a8/a9 not only reduced MI -induced myelopoiesis but also significantly improved the mortality and cardiac function in mice following LAD. These data supports our hypothesis that neutrophil-derived S100a8/a9 interact with Tlr4 on cardiac fibroblasts to induce the Nlrp3 inflammasome and produce IL1β, which in turn stimulates IL-1R on HSPCs to promote myelopoiesis. Pharmacological strategies aimed at inhibition of S100a8a/9 or the Nlrp3 inflammasome-mediated production of IL1β may be a promising approach to limit inflammation following acute coronary syndrome.
Abstract 120: Epidermal Growth Factor Receptor (EGFR) Family Dimeric Partners Switch During Pathological Stress in microRNA-7 Transgenic Mice
Authors: Gupta Manveen , Wang Xi , Martelli Elizabeth , Naga Prasad Sathyamangla V
Published: July 2021

miRNA-7 is known to target epidermal growth factors receptor 1(EGFR1) in cancer cells. EGFR family members (EGFR 1, 2, 3 and 4) are known to form homo- and/or hetero-dimers to mediate downstream signals . Our previous study in human heart failure (Naga Prasad etal., JBC, 2009) showed that EGFR2 (ERBB2) was targeted by miRNA-7. Based on this study, we developed transgenic (Tg) mice with cardiomyocyte-specific overexpression of miRNA-7. miRNA-7 Tg mice have age dependent deterioration in cardiac dysfunction and is associated with cardiac dilation as measured by echocardiography (3 months - 60% FS, 6 month -52% FS &12 months - 24%FS) and yet, they survive well for more than a year. To investigate whether pathological stress would accelerate the deterioration in cardiac function, miRNA-7 Tg mice were subjected to transverse aortic constriction (TAC) for two weeks. In contrast to the wild type littermates which undergo hypertrophic response following TAC, miRNA-7 Tg mice have accelerated cardiac dysfunction and dilation within two weeks. Biochemical analysis interestingly showed differential switching of dimeric EGFR partners in hearts of the miRNA-7 Tg mice compared to their sham controls. Our presentation will discuss the differential downstream signaling induced by changing of EGFR dimeric partners induced by pathological stress like TAC in the wildtype and miRNA-7 Tg mice.
Abstract 124: Sam68 Impedes the Recovery of Arterial Injury by Augmenting Inflammatory Response
Authors: Han Shuling , Zhou Junlan , Arnone Baron T , Biyashev Dauren , Boriboun Chan , Kishore Raj , Losordo Douglas W , Qin Gangjian
Published: July 2021

Background:
The role of Src-associated in mitosis 68 kDa (Sam68) in cardiovascular biology has not been studied. A recent report suggests that Sam68 suppresses TNF-α-induced NF-κB activation. Since NF-κB plays a critical role in vascular inflammation and injury via generation of inflammatory cytokines and recruitment of inflammatory cells, we sought to dissect the mechanism by which Sam68 regulates NF-κB signaling and its functional significance during vascular injury.

Methods & Results:
The endothelial denudation injury was induced in the carotid arteries of Sam68-/- and WT mice. Sam68-/- mice displayed an accelerated re-endothelialization and attenuated neointima hyperplasia, which was associated with a reduced number of macrophages and lowered expression of pro-inflammatory cytokines (i.e., TNF-α, IL-1β and IL-6) in the injured vessels. Importantly, the ameliorated vascular remodeling was recapitulated in WT mice after transplantation of bone marrow (BM) from Sam68-/- mice, suggesting beneficial role was attributed largely to BM-derived inflammatory cells. In cultured Raw264.7 macrophages, knockdown of Sam68 resulted in a significant reduction in the TNF-α-induced expression of TNF-α, IL-1β, and IL-6 and in the level of nuclear phospho-p65, indicating an attenuated NF-κB activation. These results were confirmed in peritoneal macrophages and macrophages differentiated from BM mononuclear cells of Sam68-/- and WT mice. To identify molecular mechanisms, Raw264.7 cells were treated with TNF-α and Vehicle, followed by Sam68 co-immunoprecipitation and mass-spec identification of Sam68-interacting proteins. Specifically, TNF-α treatment results in altered interactions of Sam68 with Filamin A (FLNA), a cytoskeleton protein known to be involved in NF-κB activation. Loss- and gain-of-function of Sam68 and FLNA suggest their mutual dependence in NF-κB activation and pro-inflammatory cytokine expression, and Sam68 is required for TRAF2-FLNA interaction.

Conclusions:
Our results for the first time suggest that Sam68 promotes pro-inflammatory response in injured arteries and impedes recovery, and this effect is attributed, in part, to the exaggerated NF-κB activity via Sam68-FLNA interaction and consequent TRAF2 stabilization.
Abstract 152: In vitro Modeling of the Pacemaker/working Myocardial Fate Decision by the Shox2-nkx2-5 Antagonistic Mechanism
Authors: Song Yingnan , Ye Wenduo , Chen Yiping
Published: July 2021

Our recent studies have identified a Shox2-Nkx2-5 antagonistic mechanism that regulates pacemaker/working myocardial cell fate decision in the developing venous pole in mice, particularly in the junction domain of the sinoatrial node (SAN) and pulmonary vein myocardium where Shox2 and Nkx2-5 are co-expressed. In such regulatory mechanism, the stronger transcription output by Shox2 promotes pacemaker cell fate, while stronger Nkx2-5 output favors working myocardial fate. Interestingly, we also found that a majority of Hcn4-positive cells derived from mouse embryonic stem (mES) cells co-express Shox2 and Nkx2-5, suggesting that in vitro differentiated pacemaker-like cells also adopt such an antagonistic mechanism to regulate cell fate. To establish an in vitro modeling system to further dissecting the functional importance of the Shox2-Nkx2-5 antagonism and for future in vitro differentiation of pacemaker cells, we established induced pluripotent stem (iPS) cell lines from mice carrying allelic series of Shox2 and Nkx2-5, as well as lineage specific Cre and RosamTmG reporter alleles. These iPS cell lines are being tested to prove the principle that the Shox2-Nkx2-5 antagonistic mechanism also functions in iPS cell-derived cardiomycytes to regulate cell fate, and are being used for lineage tracing of differentiated cardiomycytes. Furthermore, these cell lines could also be applied to modeling human diseases with Nkx2-5 mutations in vitro.
Abstract 161: Induced Pluripotent Stem Cell-based Model of Cardiac Arrhythmia: New Platform for Drug Screen
Authors: Song LouJin , Yazawa Masayuki
Published: July 2021

Human induced pluripotent stem cell (iPSC)-based model of cardiac diseases has been proved to be useful and valuable for identifying new therapeutics. However, the use of human iPSC-based model of cardiac diseases for drug screen is hampered by the high-cost and complexity of methods used for reprogramming, in vitro differentiation, and phenotyping. To address the limitations, we first optimized a protocol for reprogramming of human fibroblasts and keratinocytes into pluripotency using single lipofection and the episomal vectors in a 24-well plate format. This method allowed us to generate multiple lines of integration-free and feeder-free iPSCs from seven patients with cardiac diseases and three controls. Second, we differentiated human iPSCs derived from Timothy syndrome patients into cardiomyocytes using a monolayer differentiation method. We found that Timothy syndrome cardiomyocytes showed slower, irregular contractions and abnormal calcium handling compared to controls, which were consistent with previous reports using a retroviral method for reprogramming and using an embryoid body-based method for cardiac differentiation. Third, we developed an efficient approach for recording action potentials and calcium transients simultaneously in control and patient cardiomyocytes using genetically encoded fluorescent indicators, ArcLight and R-GECO1. The dual optical recordings enabled us to observe prolonged action potentials and abnormal calcium handling in Timothy syndrome cardiomyocytes. We confirmed that roscovitine rescued the phenotypes in Timothy syndrome cardiomyocytes and these findings were consistent with previous studies using conventional electrophysiological recordings and calcium imaging with dyes. The approaches using our optimized methods and dual optical recordings will improve iPSC applicability for disease modeling to test potential therapeutics. With those new approaches in hand, next we plan to use the iPSC-based model of Timothy syndrome to investigate novel molecules involved in the pathogenesis of Timothy syndrome and to screen and identify new therapeutic compounds for Timothy syndrome patients.
Abstract 162: Influence of Age and Ischemia on Cardiac Subsarcolemmal and Interfibrillar Mitochondria in a Novel Model of Estrogen Deficiency
Authors: Garvin Alexandra M , Aurigemma Nicole C , Korzick Donna H
Published: July 2021

Altered mitochondrial respiration (MR) and calcium retention capacity (CRC) are proposed cardiac cell death mechanisms exacerbated by aging in males. The present study aimed, for the first time, to determine changes in mitochondrial subpopulation function with age and ischemia/reperfusion (I/R) injury in the female heart. A novel model to recapitulate human menopause/age interactions was used in F344 female rats ovariectomized (OVX) at 15mo and studied at 24mo (MO OVX; n=15), vs adult (6mo; n=18). MR and CRC were assessed in isolated subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria following in vivo coronary artery ligation (CAL; 31 min I and 10 min R) or sham. State 3 MR energized by either complex I (CI) or complex II (CII) substrates was selectively reduced by age in SSM (p<.02), and by I/R in IFM (p<.05). The I/R-dependent decrease in CRC was 64% (18 vs 29.5) greater in MO OVX vs. adult IFM, suggesting earlier mitochondrial permeability transition pore (MPTP) opening. At CI, but not CII, cyclosporine A (CsA) enhanced CRC 20% (103 vs 86) more in SSM and 75% (98 vs 56) more in IFM from adult compared to MO OVX, suggesting reduced protective efficacy with age and MPTP involvement. Additionally, mitochondrial cyclophilin D increases with age, while cytoplasmic RIP1 is increased with age and I/R further implicating the MPTP mechanism and link with programmed necrosis in the aged female heart. In contrast to males, our data suggest a sex-specific phenotype whereby reductions in both SSM and IFM dynamics may play an additive role in the enhanced susceptibility to I/R injury and myocardial infarction in the aged female heart, which remains the leading cause of death in older women.
Abstract 190: Serum HDL-C Decreases in MicroRNA-33b Knock-in Mice for an Intron of Sterol Regulatory Element-binding Factor 1 (Srebf1)
Authors: Horie Takahiro , Nishino Tomohiro , Kuwabara Yasuhide , Baba Osamu , Kimura Takeshi , Ono Koh
Published: July 2021

Background:
MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports, including ours, indicated that miR-33 (miR-33a) located within the intron of sterol regulatory element-binding factor (SREBF) 2 controls cholesterol homeostasis. Primates, but not rodents, express a second miR-33 gene (miR-33b) from an intron of SREBF1. To address miR-33b function in vivo, we developed humanized mice, in which a miR-33b transgene is inserted within a Srebf1 intron.

Methods:
The human miR-33b sequence was introduced into intron 16 of mouse Srebf1 because miR-33b is located in intron 16 of human SREBF1 and there are high homologies in exons 16 and 17 between human and mouse. The expression of serum miRNA was normalized with cel-miR-39 as a spike-in control.

Results:
We successfully established miR-33b knock-in (KI) mice with C57BL/6 background and this miR-33b KI strategy did not alter Srebf1 intron 16 splicing, which was confirmed by RT-PCR and sequencing. An LXR agonist T0901317, which induces Srebf1 expression, enhanced miR-33b expression in primary hepatocytes and the liver of miR-33 KI mice. The protein levels of known miR-33a target genes, such as ABCA1, ABCG1, and SREBP-1, were reduced compared with those in wild-type mice. Peritoneal macrophages from the miR-33b KI mice had a reduced cholesterol efflux capacity via apoA-I and HDL-C. Serum HDL-C levels were reduced by almost 35% in miR-33b KI mice. HPLC elution analysis showed that the decreased HDL-C levels were mainly composed of very large-, large-, medium sized HDL-C, which was compatible with the previous results of miR-33a deficient mice. Next, we measured miR-33b levels in serum of human. The expressions of miR-33b-3p were inversely correlated with serum HDL-C levels (P=0.025, R=0.336).

Conclusions:
miR-33b KI mice for an intron of Srebf1 showed reduced HDL-C level and serum miR-33b-3p levels were inversely correlated with HDL-C levels in human. These results indicate that miR-33b can be a potential target for raising HDL-C and may account for lower HDL-C levels in humans than those in mice. These mice will aid in elucidating the roles of miR-33s and screening of the drugs that can alter miR-33s levels and activities.
Abstract 192: High Fat Diet Promotes Haplotype Dependent Differential Transcriptional Regulation of the Human Angiotensinogen Gene
Authors: Rana Anita , Puri Nitin , Jain Sudhir , Kumar Ashok
Published: July 2021

Angiotensinogen (AGT) is the only known precursor to angiotensin II. Systemic renin angiotensin aldosterone system (RAAS) is activated in human and experimental models of obesity. RAAS activation in obesity is linked to the development of cardiovascular pathophysiologies. We have identified polymorphisms in 2.5 Kb promoter of human angiotensinogen gene (hAGT) that forms two haplotype (Hap) blocks: -6A/G (-1670A/G, -1562C/T, -1561T/C) and -217A/G (-532T/C, -793A/G, -1074T/C,& -1178G/A). Hap -6A/-217A (Hap -6A) is associated with human hypertension whereas, Hap -6G/-217G (Hap -6G) reduces cardiovascular risk. Here, we examine high fat diet-mediated allele-specific transcriptional regulation of the hAGT gene in adipose tissue, in vivo, in transgenic (TG) mice engineered with either haplotype of the hAGT gene. Twelve-week-old male TG mice with Hap –6A or –6G were fed normal diet (10% kcal as fat) and high fat diet (60% kcal as fat) for 10 weeks. Using Q - RT PCR and western blot we show increased hAGT expression in adipose tissue of the Hap -6A-TG mice after high fat diet than control diet (Hap -6A-0.68±0.04 vs. Hap -6G- 0.33±0.03 A.U., p<0.05). ChIP assay shows greater chromatin binding of GR, MR, CEBPβ and STAT3 transcriptional factors (Hap -6A- 0.80±0.04 vs. Hap -6G- 0.26±0.06 A.U., p<0.05) to the hAGT transgenes in Hap -6A TG mice after high fat diet. No significant change was observed in the endogenous mouse AGT gene. In addition, after high fat diet, change ([[Unable to Display Character: ∆]]) in inflammatory and redox markers was significantly (p<0.05) greater in TG mice with Hap I including, IL1 (4.6±0.8 vs. 2.1±0.49 fold), IL6 (4.0±0.69 vs. 2.1±0.2 fold) and NOX1 (8.3±0.4 vs. 2.5±0.6 fold). This is accompanied by reduction in levels of antioxidant defenses (SOD1: 0.97±0.0 vs. 1.4±0.1 fold; HO1: 0.77±0.1 vs. 1.3±0.2 fold) & activation of MAPK14 and ERK1/2 signaling. Taken together, our results show that SNPs in the hAGT Hap -6A favor high fat diet induced binding of transcriptional factors GR, MR, CEBP-β and STAT3 that lead to elevated expression of the hAGT in expanded mass of the adipose tissue. This also activates the RAAS with pathophysiological implications including, phosphorylation of kinases such as MAPK14 and ERK2, increase in tissue pro-inflammatory and oxidative stress molecules.