bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P336: Changes in Arterial Pulse Wave Velocity, Effective Arterial Elastance, and Contractility in Hypertension Induced Cardiovascular Disease in Rats: Independent Prognostic Values
Authors: Roof Steve R , del Rio Carlos
Published: June 2021

Hypertension is an established comorbidity in cardiovascular (CV) disease. Changes in aortic pulse-wave velocity (PWV) have been shown to predict the longitudinal development of hypertension, its underlying vascular alterations, and associated CV risks. However, as the heart and arterial tree are coupled, it has been shown that the successful monitoring of not only CV disease progression, but of its therapies, requires the evaluation of ventriculo-arterial uncoupling and its determinants. This study aimed to establish the relationships between PWV and changes in the estimated effective arterial elastance (Ea) and/or contractility over a wide-range of hypertension-induced CV pathologies in a controlled rodent model/environment.

In a large series (n = 96) of anesthetized (pentobarbital) male Sprague-Dawley rats, PWV (carotid-to-femoral), systolic pressures, contractility (preload recruitable stroke work; PRSW), and Ea were evaluated using echocardiography, invasive hemodynamics, and left ventricular (LV) pressure-volume relationships. Data were collected in healthy animals (n = 37) and those with hypertension-induced chronic systolic/diastolic left-ventricular dysfunction (n = 59) (chronic beta-adrenergic stimulation and/or renoprival). A subset of each group, were also studied under common clinical cardio/vasoactive pharmacological interventions.

Disease animals had significantly (P < 0.001) higher systolic pressures (141 ± 4 vs 116 ± 4 mmHg), faster PWV (9.0 ± 0.8 vs 5.1 ± 0.5 m/s), and depressed contractility (PRSW: 39 ± 1 vs. 49 ± 1 mmHg*). Both PWV (7.6 ± 0.7 to 4.3 ± 0.4 m/s) and Ea (95 ± 10 to 63 ± 8 mmHg/mL) decreased in response to vascular therapies in the diseased animals; only PWV was reduced in healthy rats. More interestingly, over all conditions, PWV was a poor predictor of both systolic pressures (R
2
=0.00009) and Ea (R
2
= 0.024). but yet was a moderate predictor of PRSW (R
2
=0.23, P < 0.001), suggesting a contractility-dependent velocity of propagation.

Taken together, these data suggest that indices of ventriculo-arterial efficacy, cardiac afterload, and peripheral arterial stiffness while all are affected by CV disease, each cannot be used to predict one another, and all provide independent prognostic information in disease.
Abstract P603: Lifetime Increase In Cerebral Angiotensin-(1-7) Induces Neuroprotection In A Model Of Brain Ischemia And Reperfusion
Authors: Kangussu Lucas M , Almeida-Santos Ana F , Bader Michael , Massensini Andre R , Santos Robson A , Campagnole-Santos Maria J
Published: June 2021

Recent studies showed that angiotensin-(1-7) [Ang-(1-7)] has cerebroprotective actions in ischemic and hemorrhagic stroke. Here we tested the hypothesis that transgenic rat (TGR-7371), which overexpress Ang-(1-7) in the brain, would exhibit neuroprotection in a model of brain ischemia/reperfusion by bilateral common carotid arteries occlusion (BCCAo). Evaluation of neurological deficit scores and bilateral asymmetry test (BAT) were performed 7 days after transient (25 min) BCCAo in TGR-7371 and Sprague-Dawley (SD) rats. The integrity of the blood-brain barrier (BBB) was assessed by the degree of extravasation of Evans blue dye (EB) intravenously injected and expressed as μg/100 mg of tissue. Cytokine levels were quantified in the whole brain through Elisa assay and expressed as pg/100 mg of tissue. Neurological deficits, such as ptosis palpebral, walking in circles, and/or ataxia, were observed in both SD-BCCAo and TGR-BCCAo, contrasting to sham-operated groups. However, TGR-BCCAo showed a significant lower neurological score and latency in BAT when compared with SD-BCCAo. SD-BCCAo showed greater extravasation of EB (13.5 ± 2; n=4) than sham group (2.4 ± 0.07; n=4). TGR-BCCAo had a significant reduced amount of EB (8 ± 1.5; n=5), indicating attenuation in loss of integrity of the BBB. As expected, levels of pro-inflammatory cytokines were increased in SD-BCCAo (IL-1β: 225 ± 16, IL-6: 290 ± 24, TNF-α: 404 ± 39) when compared to SD control rats (IL-1β: 24 ± 1, IL-6: 24 ± 3, TNF-α: 50 ± 4). Interestingly, TGR-BCCAo presented lower levels of cytokines (IL-1β: 125 ± 15; IL-6: 205 ± 27; TNF-α: 286 ± 6) when compared to SD-BCCAo. Levels of IL-10 were higher in SD-BCCAo than in SD control (112 ± 5
vs
29 ± 5) and even higher in TGR-BCCAo (172 ± 27). The present study shows that lifetime increase in cerebral expression of Ang-(1-7) induces neuroprotection in experimental global cerebral ischemia and reperfusion.
Abstract P605: Continuous Blood Pressure Parameter Calculated By The Optical Sensor Follows The Blood Pressure Value Changes Accompanied With The Treadmill Exercise
Authors: Yamazaki Madoka , EDA Hideo
Published: June 2021

Introduction:
Recently, it has been increased need for development and validation of non-mercury blood pressure (BP) measuring system with consideration for the environments. We developed a BP measuring system using an optical sensor and reported that estimated BP value by the optical sensor (Opt BP) highly correlated with measured value of the BP with a mercury sphygmomanometer at rest (SBP r=0.93, DBP r=0.84) (Yamazaki and Eda, European society of Hypertension 2016). Purpose of this study is to validate whether the optical sensor can capture change in the blood pressure.

Methods:
We conducted treadmill testing using Bruce protocol with 13 university students (male/female: 8/5). During the exercise and recovery stage, BP, heart rate and cardiac rhythm were recorded. The BP was measured with a cuff using auscultatory K sound every 1 or 2 minute automatically (Tango+, Sun Tech, U.S.A). The optical sensor was attached the left index finger fixed with medical tape during the exercise. The data was sampled and stored in Windows PC within a second and the estimated BP value was displayed on the screen simultaneously. We compared the automatically measured BP (Auto BP) value and the Opt BP.

Results:
Opt BP continuously showed the data also between the Auto BP measurements (

Figure

). Diastolic Opt BP was significantly higher (23mmHg) than the Auto BP in 10 of 13 subjects.

Conclusion:
Continuous BP estimation by our optical system detected the BP change. The BP estimation at the higher sampling rate enables us to check the intermitted BP measurement value. Moreover, it is possible to store the estimated value more than 24 hours with a large memory device.
Abstract P607: Nitric Oxide (NO) Regulates Paracellular Permselectivity in Isolated, Perfused Rat Thick Ascending Limbs through Claudin-19
Authors: Monzon Casandra M , Garvin Jeffrey
Published: June 2021

About 50% of the Na reabsorbed in thick ascending limbs (TALs) traverses the paracellular pathway. The ionic selectivity of this route is regulated by claudins in the tight junctions. TALs express claudin-19 which has been reported to regulate TAL Na permeability. We showed that nitric oxide (NO) decreases Na/Cl permeability ratio (PNa/PCl) in TALs by increasing the absolute permeabilities of both ions though PCl increased more. However, whether NO affects paracellular permeability via claudin-19 is unknown. We hypothesize that NO regulates the paracellular permselectivity in TALs through this claudin. To test this we perfused TALs from Sprague Dawley rats and measured dilution potentials (a measure of permselectivity) with and without exogenously-added or endogenously-produced NO in the presence or absence of an antibody against an extracellular domain of claudin-19 or Tamm-Horsfall protein (control). Dilution potentials were generated by reducing bath NaCl from 141 to 32 mM. For the NO donor spermine NONOate (SPM): during the control period, the dilution potential was -9.3 ± 1.8 mV. After SPM (200 μM), it was -6.7 ± 1.6 mV (n = 6; p < 0.003). In the presence of the claudin-19 antibody, SPM had no significant effect on dilution potentials (claudin-19 antibody alone: -12.7 ± 2.1 mV vs claudin-19 antibody + SPM: -12.9 ± 2.4 mV; n = 6). The claudin-19 antibody alone had no effect on dilution potentials. In the presence of the Tamm-Horsfall protein, the effect of SPM was still present (Tamm-Horsfall protein antibody alone: -9.7 ± 1.0 mV; Tamm-Horsfall protein antibody + SPM: -6.3 ± 1.1 mV, p<0.006, n = 6). For experiments with endogenously-produced NO, L-arginine the substrate for NO synthase was added. During the control period, the dilution potential was -11.0 ± 1.1 mV. After L-arginine (500 μM) treatment, they were -9.0 ± 1.2 mV (n = 9; p < 0.05). In the presence of the claudin-19 antibody, L-arginine had no significant effect on dilution potentials (claudin-19 antibody alone: -10.1 ± 0.9 mV vs claudin-19 antibody + L-arginine: -10.1 ± 1.0 mV; n = 9). In the presence of the Tamm-Horsfall protein, the effect of L-arginine was still present. We conclude that the actions of NO on the paracellular permselectivity in thick ascending limbs are at least in part mediated by claudin-19.
Abstract P608: Obesity Induced Hypertension Exacerbated Through Upregulation Of Renal Naci Cotransporter, Reversed By Increasing Pgc-1α-ho-1 Gene Expression To Restore Mitochondrial Function
Authors: Bamshad David , Cao Jian , Schragenheim Joseph , Stier Charles T , Abraham Nader G
Published: June 2021

Introduction:
Hypertension caused by chronic obesity as a result of high calorie food intake or in leptin receptor deficient db/db mice may be linked to mitochondrial dysfunction. Previously we and others have shown that an epoxyeicosatrienoic acid agonist (EET-A), reduced adiposity and ROS resulting in normalization of BP by unknown mechanisms. We hypothesize that EET-A will attenuate BP by restoring mitochondrial function through increasing the PGC-1α-HO-1 axis and increasing urinary sodium excretion by downregulating NCC channels.

Methods:
Db/db mice at 16-wks of age were divided into 3 treatment groups and for an additional 16-wks received: A) control, B) EET-A 1.5mg/100g BW i.p. 2x/week and C) EET-A and lentiviral (Ln)- PGC-1α shRNA (to suppress PGC-1α protein). Oxygen consumption (VO
2
), visceral fat and blood glucose were determined. Additionally, renal tissues were harvested to measure the type 2 Na-K-Cl cotransporters (NKCC2), epithelial Na channels- (ENaC), NaCl cotransporters (NCC), PGC-1α, HO-1, insulin receptors, and mitochondrial biogenesis markers.

Results:
At the conclusion of 32 weeks:

Group A, developed hypertension and presented with decreased urinary Na excretion, decreased VO
2
, decreased downstream PGC-1α signaling, and mitochondrial dysfunction. There were increased levels of NCCs but not of NKCC2s or ENaCs. Renal PGC-1α, HO-1, pAMPK, and mitochondrial fusion protein Mfn 1/2, and Opa1 were decreased, p<0.05.

Group B, exhibited restoration of renal levels of PGC-1α, HO-1, pAMPK, and mitochondrial biogenesis proteins Mfn 1/2 and Opa1. NCC expression was reduced and was associated with an increase in urinary Na excretion; (p<0.05).

The beneficial effect of EET-A observed in group B was suppressed in group C using Ln- PGC-1α shRNA which suppressed PGC-1α expression in renal tissue > 50% and was accompanied by the onset of even more severe suppression of urinary Na excretion than in Group A.

Conclusion:
Treatment of obese mice with EET-agonists leads to the recruitment of PGC-1α-HO-1 which enhances mitochondrial function and induces the downregulation of NCC channels and increased sodium excretion. EET may serve as a powerful therapeutic agent for the treatment of obesity induced hypertension.
Abstract P634: Aerobic Training Prevents The Development Of Metabolic Abnormalities Induced By Chronic Stress, But Not Abnormal Circulating Levels Of Noradrenaline And Serotonin
Authors: Sanches Andrea , Perez Juliana D , Barankevicz Gizele B , Vanzella Emeriele C , Casarini Dulce E , Marcondes Fernanda K , Cunha Tatiana S
Published: June 2021

The chronic mild and unpredictable stress (CMS) protocol induces insulin resistance, dyslipidemia, oxidative stress and endothelial dysfunction in rats. Regular physical exercise is an effective non-pharmacological tool for the treatment of disorders induced by stress. The aim was to evaluate the role of physical training on hormonal and metabolic changes triggered by CMS. Forty male Sprague-Dawley rats were randomized into: Control, Stress, Exercise, Exercise + Stress, submitted to CMS protocol or to 8-week treadmill training (50-70% of the maximal exercise test). In the 4
th
, 5
th
and 6
th
wk, the animals were submitted to CMS protocol over seven days, repeating the procedures for 3 consecutive weeks. Two weeks after last stressor stimulus, blood and left ventricle were collected. Physical performance of animals submitted to the CMS was lower when compared to control animals, and physical training has not been able to alleviate this loss (p<0.05). The exercise prevented the development of metabolic changes induced by CMS, reducing hyperinsulinemia (Stress: 1.7±0.1
vs.
Exercise: 1.4±0.1; Exercise + Stress: 1.3±0.1; Control: 1.4±0.0 ng/mL), insulin resistance index (Stress: 9.2±0.3
vs.
Exercise: 7.1±0.2; Exercise + Stress: 5.8±0.2; Control: 8.1±0.2) and serum free fatty acids (Stress: 311.9±10.0
vs.
Exercise: 193.0±16.7; Exercise + Stress: 251.6±14.16; Control: 190.1±17.3 mg/dL, p<0.05). In addition, it was also capable of reducing the cardiac concentration of serotonin (Stress: 3.1±0.1
vs.
Exercise: 0.6±0.1; Exercise + Stress: 0.5±0.0; Control: 1.3±0.1 pg/g, p<0.05) of stressed animals (CMS). Physical training did not reduce the circulating concentration of noradrenaline (Stress: 603.7±52.2; Exercise: 83.8±10.8; Exercise + Stress: 748.9±46.1
vs.
Control: 165.1±27.1 pg/mL) and serotonin (Stress: 1296.0±47.0; Exercise: 1196.0±68.1; Exercise + Stress: 1736.0±60.12
vs.
Control: 619.8±79.6), which remained high in the groups submitted to the CMS (p>0.05). The results show that exercise improves metabolic losses triggered by CMS, but not the but not abnormal circulating levels of noradrenaline and serotonin, and suggest that physical training must be prescribed with caution to stressed individuals.
Abstract P635: Effect of Patient Characteristics and
DPP4
genotype on Dipeptidyl Peptidase IV Activity and Inhibition by Sitagliptin
Authors: Wilson Jessica R , Shuey* Megan M , Brown Nancy J , Devin Jessica K
Published: June 2021

Dipeptidyl peptidase IV (DPP4) inhibitors are a class of oral antihyperglycemic agents commonly used to treat type 2 diabetes mellitus (T2DM). Evidence suggests that these medications also have cardiovascular effects.

We tested the hypothesis that subject characteristics and genetic variability in
DPP4
, the gene encoding DPP4, affect response to DPP4 inhibition with sitagliptin.

We studied 56 subjects undergoing trials of sitagliptin therapy. DPP4 activity was measured during placebo and after sitagliptin and percent inhibition calculated. Subject characteristics were: 41.1% men (n=23), 19.6% African American (n=11), 30.4% with both T2DM and hypertension (HTN) (n=17), mean age 39.16±15.70 years, and mean BMI 26.71±6.24 kg/m
2
. Sitagliptin doses were 100mg daily for 4-7 days (n=31) or a single dose of 200mg (n=25).

Baseline DPP4 activity decreased significantly with age (r=-0.36, p<0.01) and was lower in T2DM HTN (22.54±5.20 vs 26.08±5.88U in non-diabetics, p=0.02). There was no effect of gender, race, weight, or BMI on baseline or inhibited DPP4 activity in T2DM or non-T2DM. Baseline DPP4 activity was significantly lower in rs4664446 (p=0.03; lower activity GG). These characteristics were not significant in a multivariable model. During sitagliptin, DPP4 activity was significantly higher (13.06±5.56 vs 6.85±3.34U, p<0.01) and percent inhibition lower (43±13% vs 74 ±11.2%, p<0.01) in T2DM HTN. Inhibited DPP4 activity declined with age in non-diabetics but not in T2DM. The variant rs2909451 was significantly associated with DPP4 activity during sitagliptin (p<0.01; higher activity TT). In multivariable analysis, inhibited DPP4 activity was associated with T2DM (p=0.02), rs2909451 genotype (p<0.01), but not age (p=0.08) or sitagliptin dose regimen (p=0.24). Inhibition of DPP4 by sitagliptin is decreased in T2DM HTN. Future studies are needed to determine if DPP4 inhibitor dose should be increased in T2DM HTN.
Abstract 117: Poly (i:c) Attenuates Myocardial I/R Injury via Glycolytic Dependent Yap Activation and by Suppression of MiR-143
Authors: Hu Yuanping , Wang Xiaohui , Ha Tuanzhu , Kalbfleisch John , Kao Race , Williams David , Li Chuanfu
Published: June 2021

Glycolytic metabolism plays a critical role in ischemia/reperfusion (I/R) injury. The Yes associated protein (YAP) is a core effector of the Hippo pathway that regulates cell proliferation and apoptosis. We observed that poly (I:C) enhanced glycolysis and Yap activation in neonatal cardiomyocytes. This study investigated whether poly (I:C) will attenuate myocardial I/R injury via a glycolytic dependent mechanism. Mice (n=6/group) were treated with poly (I:C) (10 μg/25g body weight) one h before the hearts were subjected to ischemia (45 min) followed by reperfusion (24 h). Sham surgery served as sham control. Poly (I:C) treatment significantly reduced infarct size by 35% and enhanced EF% by 20.5% and FS% by 24.9% compared with I/R group. The expression of miR-143 was markedly reduced and Yap levels were significantly increased in poly (I:C) treated hearts.
In vitro
data show that poly (I:C) treatment enhanced extracellular acidification rate (ECAR) and lactate production in HL-1 cardiomyocytes.
In vivo
inhibition of hexokinase 2 abolished poly (I:C)-induced cardioprotection. To determine the role of miR-143 in regulation of glycolysis, we transfected HL-1 cardiomyocytes with anti-miR-143 mimics before the cells were subjected to hypoxia/reoxygenation. We observed that anti-miR-143 significantly enhanced cell viability, reduced LDH release, and increased hexokinase 2 levels and extracellular acidification rate (ECAR). To determine whether suppression of miR-143 will induce protection against myocardial I/R injury, we loaded anti-miR-143 on exosomes (Exo-antimiR-143) by transection of bone marrow stromal cells with anti-miR-143 mimics. MiR-control mimics served as control (Exo-miR-control). Exo-miR-143 was delivered into the myocardium through the right carotid artery immediately before the hearts (n=6/group) were subjected to I/R. We observed that delivery of Exo-antimiR-143 significantly enhanced EF% by 20.5% and FS% by 26.4% and decreased infarct size by 42.2%, when compared with untreated I/R group. Delivery of Exo-miR-control did not alter I/R-induced cardiac dysfunction and infarct size. We conclude that poly (I:C) attenuates myocardial I/R injury via glycolytic dependent YAP mechanism and suppression of miR-143 expression.
Abstract 121: Metabolic Regulation of Ischemia/Reperfusion: Role of the Unfolded Protein Response
Authors: Wang Zhao
Published: June 2021

Myocardial infarction is a leading cause of death worldwide. The most effective approach to mitigate cardiac damage is timely and efficient restoration of occlusion in coronary artery. This process, ischemia/reperfusion (I/R), triggers additional damage (I/R damage), which may substantially contribute to the final cardiac infarction. Despite extensive efforts and paramount clinical interests, effective therapeutics remains vacant. A better understanding of the pathology of I/R damage is therefore urgently required. Most processes in I/R are potent inducers of the unfolded protein response (UPR), a cellular adaptive process to accommodate protein-folding stress. We have shown that the most conserved branch of the UPR, the Xbp1s pathway, is strongly induced by I/R. Using both gain- and loss-of-function approaches, we revealed that Xbp1s is necessary and sufficient to protect heart against I/R injury. We went further to discover that Xbp1s is a direct upstream transcriptional factor for multiple, key enzymes of the hexosamine biosynthetic pathway (HBP). The final production of HBP, UDP-GlcNAc, is the obligate substrate for O-GlcNAc protein modification, a prominent post-translational modification. Acute induction of O-GlcNAc modification confers strong protection against various stresses. Upregulation of Xbp1s therefore induces the HBP, UDP-GlcNAc production, consequent O-GlcNAc protein modification, and more importantly, cardioprotection against I/R damage. We found that modulation of the necroptosis pathway participates in Xbp1s-mediated cardioprotection. Elucidation of the pathology of I/R injury will greatly advance our understanding of underlying mechanisms and help identify potential therapeutic targets to tackle the devastating coronary heart disease.
Abstract 136: Cytoplasmic Histone Deacetylase 4 Promotes Vascular Calcification
Authors: Abend Alon , Shkedi Omer , Fertouk Michal , Caspi Lilac , Kehat Izhak
Published: June 2021

Background:
Arterial calcification and valvular calcifications are associated with significant morbidity and mortality. Studies have now conclusively showed that an osteochondrogenic differentiation of vascular smooth muscle cells (VSMC) is a key mechanism in the development of vascular calcification, opening a direction for therapeutic interventions.

Objective:
To identify the role, subcellular location and mechanism of action of class IIa Histone deacetylase 4 (HDAC4) in vascular calcification

Methods and Results:
We used VSMCs, ex-vivo mouse aortic rings and human calcified aortic valves to show that HDAC4 is upregulated in vascular and valve calcification. Despite being exclusively cytoplasmic in VSMCs we show using both gain- and loss- of function approaches that HDAC4 is a positive regulator of the process. The cytoplasmic location of HDAC4 is controlled by the activity of salt inducible kinase (SIK) and the inhibition of SIK sends HDAC4 to the nucleus and inhibits the ossification process. In the cytoplasm HDAC4 binds to the LIM domains of the adaptor protein ENIGMA (Pdlim7) to promote vascular ossification of VSMCs.

Conclusions:
Here we identified a new role and a new mechanistic paradigm for HDAC4. We provide multiple lines of evidence that the cytoplasmic activity of HDAC4 promotes vascular calcification, and identify HDAC4, SIK and ENIGMA as mediators of this pathological process
Abstract 201: A Novel Contrast Agent - Magnetic Iodixanol
Authors: Arokiaraj Mark C
Published: June 2021

Aims:
The study was performed in search of a novel contrast agent, which has non-renal excretion. Contrast induced nephropathy and cardiogenic shock resulting in renal failure are common problems. A novel contrast with non-renal excretion is very useful.

Materials and Methods:
Magnetic nanoparticles were synthesized and it was conjugated with commercially available Iodixanol. The particle size measurement showed size of about 220nm. The conjugation was performed to result in covalent bonding. Final concentration of nanoparticles was 1mg in 1 ml. Thereafter the contrast was tested for radio-opacity. Magnetic properties of the contrast were tested with magnets. A drop of contrast was tested over magnets and over a glass slide. The droplet on the magnet fixes and retains the shape and the bubble structure in various positions of the magnet manipulation. Over a glass slide it showed free movement and loses the bubble structure. Figure shows bubble evaluation and cine-fluoroscopy.

Conclusion:
Magnetic nanoparticle conjugated Iodixanol shows potentials as a novel contrast agent for use in the future.
Abstract 202: Early Reprogramming Derived Cocktail FIJs Supports Cardiomyocyte Proliferation for Heart Regeneration
Authors: Cheng Yuan-Yuan
Published: June 2021

Although remnant cardiomyocytes (CMs) possess a certain degree of proliferative ability, their regenerative efficiency is too low for cardiac regeneration after injury. As terminally differentiated cells, CMs are considered difficult to successfully reprogram into iPSCs. In this study, a modified reprogramming method was employed to avoid damage to the CMs, and the resulting CM-derived iPSCs were characterized their pluripotency and differentiative abilities. During the early stages of reprogramming, we observed sequential morphological changes in both CMs and non-CMs, but alkaline phosphatase assay revealed that CMs demonstrate a time-delayed sequence of events compared to non-CMs. In order to identify the specific events preceding colony formation, total RNA was purified for microarray analysis on day 0, 2, 4, and 6 of the reprogramming process. There were several interesting changes revealing such as up-regulation of sterol synthesis in non-CMs and down-regulation of chemokines in CMs on day 2. Most importantly, we detected significant enhanced mitosis for CMs on day 2. Nevertheless, typical iPSC-like colonies were clearly observed after 6 days of reprogramming in both cell types. In the purpose of bringing CM back to regain proliferative ability for heart regeneration, the candidates were selected from the microarray results on day 2 of the reprogramming. Triple combined gene cocktail FIJs was determined supporting CM proliferation with 7 times higher Ki-67
+
or H3P
+
population % in neonatal murine CMs
in vitro
. The same supportive role of FIJs was also confirmed in adult mice showing higher H3P
+
adult CMs
in vivo
. Furthermore, heart function detected by echocardiography was significantly improved and less fibrosis shown by trichrome staining after the triple gene cocktail treatment in adult mice suffering from myocardial infarction, and this improvement was owing to the enhanced CM proliferation. In conclusion, triple gene cocktail selected from CM reprogramming day 2 provides valuable information for inducing remnant CM proliferation for heart regeneration.
Abstract 204: Serine Threonine Kinase-35 Regulates Diabetes-induced Endothelial Progenitor Cells Dysfunction
Authors: Joladarashi Darukeshwara , Thandavarayan Rajarajan A. , Suresh Babu Sahana , Jeyabal Prince , Kishore Raj , Krishnamurthy Prasanna
Published: June 2021

The completion of the sequencing of the human genome has revealed that up to ~20% of the human genes encode proteins involved in signal transduction. Among these, there are >500 protein kinases. Protein kinases play a central role in the intracellular signal transduction systems involved in cell proliferation, differentiation, metabolism, and other responses to external stimuli. Serine Threonine Kinase 35 (STK35), a novel kinase that binds to nuclear actin, has been shown to regulate important cellular functions such as cell migration, proliferation, survival, and angiogenesis. However, the role of STK35 in Endothelial Progenitor Cells (EPC) function and in cardiac regeneration under diabetic condition is unknown. The purpose of this study was to determine the role of STK35 in diabetes-induced EPC dysfunction. The expression of STK35 in response to high glucose was measured by RT-PCR. STK35 expression was assessed in myocardial tissue from both human patients and mice with diabetes. To enable detailed study of STK35 biological effects in EPCs, we performed loss-of-function experiments by STK35 inhibition in EPCs. EPC from bone marrow of mice and exposed to high glucose level, showed decreased STK35 expression (vs normoglycemia-treated cells). In addition, STK35 knockdown in EPCs reduces their migration and vascular tube formation. Moreover, STK35 knockdown in EPCs have shown decreased in the expression of proangiogenic cytokines and growth factors. Results from both diabetic human heart and diabetic mice heart have also shown lower STK35 expression as compared to respective non-diabetic hearts. Taken together, these data suggest that diabetes decreases STK35 expression in EPCc thereby impairs EPC function under diabetic condition. Our future studies will test whether
ex-vivo
STK35-overexpression in EPCs rescues diabetes-induced dysfunction and whether their transplantation into myocardium of diabetic mice promotes angiogenesis and cardiac repair after injury.
Abstract 214: TLR3 Mediates Neonatal Heart Repair and Regeneration Through Glycolysis-dependent YAP/TAZ Mediated miR-152 Expression
Authors: Wang Xiaohui , Hu Yuanping , Ha Tuanzhu , Kalbfleisch John , Kao Race , Williams David , Li Chuanfu
Published: June 2021

The neonatal heart possesses the capability of regenerating and repairing damaged myocardium which is lost when cardiac metabolism switches from predominate glycolysis to oxidative phosphorylation seven days after birth. We have observed that Toll-like receptor 3 (TLR3) deficient neonatal hearts exhibit impaired cardiac function and larger infarct size after myocardial infarction (MI). We also found that stimulation of neonatal cardiomyocytes with the TLR3 ligand, poly (I:C) significantly enhances glycolytic capacity. Our observation suggests that TLR3 is required for neonatal heart repair and regeneration of damaged myocardium. This study investigated the mechanisms by which TLR3 mediates neonatal heart regeneration and repair. Neonatal cardiomyocytes were isolated from one day old WT mice and treated with poly (I:C) (1μg/ml) for 12-36 hours. We observed that poly (I:C) treatment: i) significantly enhances glycolytic metabolism; ii) increases YAP/TAZ activation: iii) increases miR-152 expression; iv) suppresses expression of DNMT1 and p27kip1, and v) promotes cardiomyocyte proliferation. However, inhibition of glycolysis with 2-Deoxyglucose (2-DG) prevented poly (I:C)-induced YAP/TAZ activation and miR-152 expression in neonatal cardiomyocytes. Similarly, inhibition of YAP/TAZ activation with Verteprofin (VP) abolished poly (I:C) induced miR-152 expression and neonatal cardiomyocyte proliferation. To investigate the role of miR-152 in neonatal cardiomyocyte proliferation, we transfected neonatal cardiomyocytes with miR-152 mimics and observed that increased miR-152 levels significantly promotes neonatal cardiomyocyte proliferation. We also observed that transfection of neonatal cardiomyocytes with miR-152 mimics markedly suppresses the expression of DNMT1 and p27kip1. Inhibition of DNMT1 with 5Azcytidine significantly promotes neonatal cardiomyocyte proliferation. Finally, we observed that treatment of neonatal mice (n=6) with 2-DG abolished cardiac functional recovery 3 weeks after MI. We conclude that TLR3 is required for neonatal heart regeneration and repair after MI. The mechanisms involve glycolytic dependent activation of YAP/TAZ mediated by miR-152 which represses DNMT1/p27kip1 expression.
Abstract 221: Sam68 Interacts With mTORC1 and Contributes to Myocardial Hypertrophy
Authors: Zhou Junlan , Boriboun Chan , Qin Gangjian
Published: June 2021

Introduction:
The Src-associated in mitosis 68 kDa (Sam68) belongs to the STAR (signal transducer and activator of RNA) family of RNA-binding proteins. Our recent work revealed its critical role in the regulation of adipose thermogenesis. However, whether Sam68 regulates cardiomyocyte biology and heart disease is currently unknown.

Methods and Results:
Echocardiography analyses were performed in Sam68
-/-
mice and WT littermates; the baseline left-ventricular wall thickness (LVPWs and LVPWd), chamber size (LVDs and LVDd), and contractility (LVEF and FS) are similar between the 2 groups of animals. However, after transverse aortic constriction (TAC) for 2 and 4 weeks, Sam68
-/-
mice exhibited a significantly lesser increase in LV wall thickness (at 2 and 4 weeks), a closer to normal LV chamber size (at 4 weeks), and a lower heart-weight (HW)/body-weight (BW) ratio (at 4 weeks), as compared to WT controls. Consistently, after continuous administration of angiotensin II (by subcutaneous osmotic minipumps) for 2 weeks, Sam68
-/-
mice displayed a similarly ameliorated LV wall thickness, chamber size, and HW/BW ratio. The attenuated hypertrophic responses of Sam68
-/-
mice to both TAC and Ang II treatment was supported by a lowered expression of fetal genes (β-myosin heavy chain, atrial and brain natriuretic peptides) in the heart (qRT-PCR) and excitingly, by the decreased activity of mTORC1, as indicated by the lowered levels of phosphorylated S6K1 and 4E-BP1 (Western blotting). The cardiomyocyte-specific role of Sam68 was further confirmed in cultured H9c2 cells with lentivirus-mediated Sam68 knockdown, which confirmed the reduction of mTORC1 activity. Furthermore, co- immunoprecipitation assays revealed that the endogenous Sam68 protein interacts Raptor and mTOR, but not Rictor, in the mTOR complex. Experiments are underway to establish the functional significance of the Sam68-mTOR pathway in cardiac hypertrophy and failure in vivo.

Conclusion:
Collectively, our data suggest that Sam68 is a novel cofactor of mTORC1. It interacts with Raptor to augment mTOR signaling and contribute to the development of cardiac hypertrophy.
Abstract 232: Restless Leg Syndrome and its Association with Iliofemoral Venous Disease
Authors: Reddy Navya , Reddy Nagender
Published: June 2021

Background:
Restless leg syndrome (RLS) is one of the most common sleep disorders, affecting between 5% and 10% of the U.S. population (1). However it has an unclear etiology, and is often over-medicated without a resulting qualitative improvement in lifestyle. It has not been determined whether iliofemoral or iliocaval venous disease, resulting from either thrombotic lesions or external compression, has an association with RLS.

Methods:
A total of 510 patients treated for iliofemoral venous compression syndrome (IVCS) with angioplasty and stenting were documented to have concomitant RLS per NIH screening criteria,2 prior to endovenous revascularization. 367 of the following patients were selected for retrospective research analysis based on pre-procedural selection criteria which included: IVCS with > 60% luminal stenosis, confirmed RLS > 1 year with or without current treatment, diminished quality of life, and clinical signs and symptoms of chronic venous disease. Patients with Diabetes, End stage renal disease, current pregnancy, and Rheumatic disease were excluded from this study. Patients with thrombotic lesions of the iliofemoral veins were also excluded. Following revascularization, patients were followed at 6 weeks, 3 months, and 6 months with doppler evaluation and asked to rate the severity of their RLS using a scale developed by the International Restless Leg Syndrome study group (3).

Results:
Of the 367 patients studied, 52% of patients had improvement of their RLS symptoms at 3 months, 90% reported significant relief with cessation of medication(s) within 6 months, and 10% did not have any statistically significant improvement by 6 months (p < 0.001).

Conclusion:
This study demonstrates a strong relationship between deep vein outflow obstruction, with either iliocaval or iliofemoral stenosis, and RLS. It is suspected that a large population suffers from iliofemoral venous disease, and including this subset of patients in the differential diagnosis for RLS can allow for earlier treatment of IVCS and prevention of RLS.
Abstract 237: Transient Activation of Pkc Results in Long-lasting Detrimental Effects on Systolic [ca
2+
]
I
in Cardiomyocytes by Altering Actin Cytoskeletal Dynamics and T-tubule Integrity
Authors: Guo Ang , Song Long-Sheng , Chen Biyi , Chen Rong , Huang Chunkai , Wang Yihui
Published: June 2021

Protein kinase C (PKC) family isozymes contribute to the development of heart failure through dysregulation of Ca
2+
handling properties and disruption of contractile function in cardiomyocytes. However, the majority of studies have examined either the acute or chronic effects of PKC activation or inhibition, yet PKC is likely only transiently activated under pathological conditions. Herein, we report that transient activation of PKC in cultured cardiomyocytes promotes long-term deleterious effects on the integrity of the transverse (T)- tubule system, resulting in a significant decrease in the amplitude and increase in the rising kinetics of Ca
2+
transients. Treatment with a PKCα/β inhibitor restored the synchronization of Ca
2+
transients and maintained T-tubule integrity in cultured cardiomyocytes. Studies using a dominant negative PKCα identified PKCα as the mediator of T-tubule remodeling in cardiomyocytes. Supporting these data, PKCα/β inhibition protected against T-tubule remodeling and cardiac dysfunction in a mouse model of pressure overload-induced heart failure. Mechanistically, transient activation of PKC resulted in biphasic actin cytoskeletal rearrangement. Transient inhibition of actin polymerization or depolymerization resulted in severe T-tubule damage, recapitulating the T-tubule damage induced by PKC activation. Moreover, inhibition of stretch activated channels (SAC) protected against T-tubule remodeling and E-C coupling dysfunction induced by transient PKC activation and actin cytoskeletal rearrangement. These data identify a key mechanistic link between transient PKC activation and long-term Ca
2+
handling defects through PKC-induced actin cytoskeletal rearrangement and resultant T-tubule damage.
Abstract 239: Sex and 17-beta Estradiol Regulate the Nuclear Translocation of the Cardiac L-type Calcium Channel C-terminus
Authors: Mahmoodzadeh Shokoufeh , Haase Hannelore , Sporbert Anje , Morano Ingo
Published: June 2021

The cardiac L-type Ca
2+
channel (Cav1.2) constitutes the main entrance gate for Ca
2+
that triggers cardiac contraction. It has been shown that the distal C-terminus fragment of Cav1.2 α1C subunit (α1C-dCT) is proteolytically cleaved and shuttles between the plasma membrane and the nucleus, which is regulated both developmentally and by Ca
2+
. However, the effects of sex and sex hormone 17β-Estradiol (E2) on α1C-dCT nuclear translocation are still unexplored. To analyze the sexual disparity in the α1C-dCT nuclear translocation, we first generated an antibody directed against a synthetic peptide (GRRASFHLE) located in α1C-dCT, and used it to probe ventricular myocytes from adult female and male mice. Immunocytochemistry of isolated mouse primary adult ventricular myocytes revealed both nuclear staining and cytosolic punctuate staining around the T-tubules. The ratio of nuclear to cytosolic intensity (I
nuc
/I
cyt
) was significantly higher in isolated female cardiomyocytes (1.42±0.05) compared to male cardiomyocytes (1.05±0.02). Western blot analysis of nuclear fraction confirmed these data. Furthermore, we found a significant decrease in nuclear staining intensity of α1C-dCT in both female and male cardiomyocytes upon serum withdrawal for 18h (I
nuc
/I
cyt
1.05±0.02 and 0.89± 0.02, respectively). Interestingly, subsequent E2 treatment (10
-8
M) for 8h normalized the intracellular distribution of α1C-dCT in male cardiomyocytes (I
nuc
/I
cyt
1.04±0.02), but not in female cardiomyocytes. Acute treatment of male cardiomyocytes with E2 for 45min revealed a similar effect. This effect of E2 was revised by ICI 182780, an estrogen receptor (ER) inhibitor, (10
-5
M), indicating the involvement of ER in this signaling pathway. Taken together, we showed for the first time that the shuttling of α1C-CT in cardiomyocytes is regulated in a sex-dependent manner, and E2-activated ER may play a role in the nuclear shuttling of α1C-dCT in male cardiomyocytes. We suggest that this identified novel signaling mechanism may explain, at least partly, the observed sex differences in the regulation of cardiac Cav1.2 channel activity.
Abstract 240: Differential Impact of RBFox Family Proteins in Adult Murine Heart
Authors: Gao Chen , Ren Shuxun , Wang Yibin
Published: June 2021

Background:
The complexity of cardiac transcriptome and proteome is significantly contributed by alternative splicing of mRNA. Alternative splicing is regulated by the cis-regulatory elements located in pre-mRNA together with the trans-activating factors guiding the assembling and function of the spliceosome. In our earlier study, we have observed global changes of alternative splicing events during pressure-overload induced heart failure, and identified RBFox1 as a key regulator for cardiac RNA splicing regulation during postnatal development and pathological remodeling. Both RBFox1 and RBFox2 are highly enriched in cardiomyocytes, and their expression are both significantly repressed in response to pathological stress. Loss-of-function studies for RBFox1 and RBFox2 are achieved using cardiac specific but constitutively active Cre. Therefore, the isoform specific contribution of RBFox1 vs. RBFox2 in maintaining cardiac physiology and homeostasis in adult heart is unknown.

Methods and Results:
We generated mouse models of cardiac specific and inducible knockout of RBFox1 and RBFox2 individually in adult hearts by breeding the individual floxed alleles with the αMHC-Mer-Cre-Mer mice. At baseline, inactivating RBFox1 in adult heart caused a slight but significant decrease of cardiac function without activating hypertrophy gene expression. However, following myocardial infarction, the RBFox1 deficient hearts showed enhanced global fibrosis in non-infarcted areas comparing to the control animals. In contrast, inactivating RBFox2 in adult mouse heart caused overt heart failure associated with chamber dilation without external stress as early as 2 weeks post tamoxifen administration.

Conclusion:
We have identified differential impact of RBFox1 and RBFox2 deficiency in adult mouse heart. Our in vivo study illustrate the functional importance of the RBFox family RNA splicing regulators in normal physiology of adult heart, and support the pathogenic contribution of loss of RBFox expression to heart failure. Further analysis focusing on the underlying molecular mechanisms for their differential impact would yield new insights on transcriptome regulation and complexity in cardiac physiology and diseases.
Abstract 245: Cxcl12 Chemokine Signaling Regulates Synchronous Development of Coronary Vessels and Myocardium
Authors: Lien Ching-Ling , Harrison Michael R
Published: June 2021

Introduction:
The coronary vasculature plays critical roles in heart functions. Coronary heart disease is a major cause of myocardial infarction and heart failure, which continues to be the leading cause of mortality. However, the mechanisms underlying coronary vessel development and myocardial vascularization are not fully understood. Furthermore, it is not known if myocardial developmental defects caused by lack of vascularization will lead to decreased regenerative capacity of the adult heart.

Hypothesis:
Synchronous development with coronary vessels is essential for normal morphogenesis and regenerative capacity of the myocardium of zebrafish heart.

Methods and Results:
Using transgenic reporter fish lines, we observed that during zebrafish heart development, coronary vessels form a close association with a specific subpopulation of cortical cardiomyocytes marked by the transcription factor Gata4. When
gata4+
cardiomyocytes emerge onto the surface of the juvenile zebrafish hearts, they follow the tracks of coronary endothelial cells. We previously showed that
cxcr4a
mutant zebrafish fail to form a vascular network, whereas ectopic expression of Cxcl12b ligand induces coronary vessel formation. In
cxcr4a
mutant hearts,
gata4+
cardiomyocytes fail to associate with coronary vessels. Importantly,
cxcr4a
mutant zebrafish fail to undergo heart regeneration following injury. Gene expression analysis using RNAseq was performed to dissect the molecular mechanisms underlying synchronous development of coronary vessels and
gata4+
cardiomyocytes.

Conclusion:
Our results suggest that Cxcl12/Cxcr4 chemokine signaling has an essential role in directing cardiomyocyte migration guided by endothelial cells. Poorly developed vasculature in
cxcr4a
mutants likely underlies defects in myocardial development and decreased regenerative potential of zebrafish hearts.
Abstract 253: Complete Reversal of Xylazine-induced Bradycardia With Intralipid in Female Mice
Authors: Motayagheni Negar , Eghbali Mansoureh
Published: June 2021

Sudden cardiac arrest
accounts for 300 000 to 400 000
deaths
annually in united states both in men and women. Cardiac arrest could be due to abnormally slow heart rate known as bradycardia. Bradycardia is a catastrophic event which is associated with significant mortality and morbidity. We have previously shown that Intralipid, an emulsion of soy bean oil, egg yolk phospholipids and glycerol, protects the heart against ischemia/reperfusion injury as well as Bupivacaine induced cardiotoxicity. Here we examined whether intralipid can protects the heart against bradycardia. Wild type female mice C57/Bl6 (2-4 month old) were anesthetized by isoflurane after heparinization. The heart was removed immediately and placed in cold Krebs-Henseleit buffer. The aorta was cannulated and the isolated heart (Langendorff) was perfused with Krebs-Henseleit at 37°C for 15 min for stabilization. Xylazine (100-300 mg) was directly applied to the heart surface for 1-2 min until bradycardia was achieved. The heart was then perfused with either Krebs-Henseleit (KH) solution (control group), or 1% ILP (intralipid group). Hemodynamic parameters and heart rates were recorded with a catheter directly inserted into left ventricle (n=5-8 per group). The heart rates at the baseline before inducing bradycardia was 224±7 beats/min and the left ventricular pressures was 64±4 mmHg. Administration of Xylazine decreased the heart rate significantly to 81±9 beats/min and left ventricular pressure to 5±2 mmHg (p<0.001). Perfusion of the heart with intralipid rapidly restored the heart rate to 209±30 and left ventricular pressure to 59±4 which were not significantly different than their values before inducing bradycardia at the baseline. In the hearts that received Krebs-Henseleit after bradycardia, the heart rate (81±10 beats/min) and left ventricular pressure (20±8 mmHg) were significantly lower than intralipid group. In conclusion Intralipid has the ability to rapidly reverse bradycardia in female mice.
Abstract 268: Advanced Glycation End Products Effect on the Polarization of Macrophages Cardiac After Acute Myocardial Infarction in Wistar Rats
Authors: Rangel Juliana O , Fracasso Bianca , Phaelante Amanda , Curuja Fernanda , Sturza Daniel , Clausell Nadine , Biolo Andreia , Rohde Luis E , Andrades Michael
Published: June 2021

Macrophage phenotypes play an important role in post-MI cardiac remodeling. M1 macrophages have a proinflammatory phenotype and are found in the heart early after MI whereas M2 macrophages arise after and replace M1 macrophages in order to mediate the resolution of inflammation and angiogenesis. Advanced glycation end products (AGE) may modulate macrophage polarization but its functional role in the context of post-MI remains incompletely understood. Our goal is to assess whether AGE can polarize macrophages in post-MI and interfere in cardiac remodeling. We have collected experimental data from 68 male Wistar rats (2-3 month-old) divided in 4 groups: sham, MI + 0.9% NaCl (i.p.), MI + methylglyoxal (AGE inducer, 17 mg/kg/day; i.p.); and MI + aminoguanidine (anti-AGE agent, 0.5 g/L in drinking water). Subgroups were euthanized on days 2, 6, and 10 days post-MI. The echocardiographic analysis did not show differences in ejection fraction or akinetic/hypokinetic area among the MI groups. Cytokine levels were assessed by multiplex analysis in cardiac tissue homogenate. There was an increase of proinflammatory cytokine IL-6 on 10th day after surgery in MI group at the remote area, while IL-1β and IL-12 showed reduction compared with the sham group. Anti-inflammatory cytokine IL-10 did not show any difference among the groups. Until now, it is not possible to conclude whether AGE can induce macrophage polarization in vivo. More analyses are necessary to respond our objective.