bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2: Sodium-Hydrogen Exchanger Inhibition Attenuates Traumatic Hemorrhage Induced Acute Lung Injury in Pigs
Authors: Wu Dongmei , Qi Jiansong , Abraham William
Published: July 2021

Background:
Acute lung injury is a complication of traumatic hemorrhagic shock, and contributes to the high mortality rate. This study examined the effects of Na+/H+ exchanger inhibition on traumatic hemorrhage induced acute lung injury in pigs.

Methods:
Tibia fractures with soft tissue injury were induced in 12 anesthetized instrumented male pigs. The animals underwent hemorrhage of 25ml/kg for 20min, followed by a 4mm abdominal aortic tear with surgical repair after 30minutes. Animals then underwent initial fluid resuscitation with either 15ml/kg Hextend (n=6) or 3mg/kg BIIB513 +15ml/kg Hextend (n=6). Resuscitation occurred over 40 minutes. A second resuscitation was given 4 hours later. Hemodynamics, arterial and mix-venous blood gases were measured throughout the experiment, which was terminated 6 hours after initial resuscitation.

Results:
Hemorrhage and resuscitation with Hextend alone resulted in a significant increase in pulmonary artery pressure (PAP) and pulmonary vascular resistance (PVR). These changes were accompanied by a significant decrease in mix-venous oxygen saturation, decreased oxygen delivery and an increased oxygen extraction ratio. BIIB513 + Hextend treatment significantly attenuated the increase in PAP by 26% and PVR by 50%, as well as significantly improving blood oxygen delivery by 30%. BIIB513+ Hextend treatment group had a 70% higher blood oxygen content and mixed venous blood oxygen saturation than did the control group (61% vs. 42%). NHE-1 inhibition with BIIB513 reduced lung tissue levels of IL-6 by 80%, TNF-á by 37%, and myeloperoxidase activity by 38%. Nuclear factor (NF)-ê B DNA binding activity in the lung was slightly attenuated in BIIB513 treated animals), compared to those treated with hextend alone.

Conclusion
: The present study shows that the response to fluid resuscitation after controlled hemorrhage is improved by the addition of the NHE-1 inhibitor BIIB513. NHE-1 inhibition resulted in attenuation of traumatic hemorrhage and resuscitation induced acute lung injury by improving pulmonary vascular function, possibly by reducing NF-κB activation and neutrophil infiltration, thereby limiting the tissue inflammatory injury.
Abstract P16: The Decreases of Left Ventricle Volume and Area During VF Affect Resuscitation Outcomes
Authors: Cho Jun Hwi , Ristagno Giuseppe , Yu Tao , Li Yongqin , Sun Shijie , Castillo Carlos , Weil Max Harry , Tang Wanchun
Published: July 2021

Introduction:
Dynamic changes in dimensions of left ventricle (LV) during cardiac arrest might influence the efficacy of CPR. However, the relationships between change in LV dimensions during cardiac arrest and CPR hemodynamic and outcomes remain to be studied. In the present study, we investigated changes of left ventricular volume and area during prolonged untreated ventricular fibrillation (VF) and related those change to coronary perfusion pressure (CPP) during CPR and resuscitation outcomes.

Hypothesis:
Decreased LV volume and area during prolonged VF interval would yield low CPP and poor resuscitation outcomes.

Methods:
In 12 domestic male pigs weighing 40±3 kg, VF was electrically induced and untreated for 15 min. CPR was performed for a 5 min interval prior to defibrillation. CPR was continued until return of spontaneous circulation (ROSC) or for a max of 15 minutes. Diastolic aortic and right atrial pressures were measured and CPP was calculated. LV dimensions were continuously assessed by echocardiographic measurement during the 15 minutes of untreated VF.

Results:
6 animals were successfully resuscitated. During the 15 minute interval of VF, LV area and volume reduced in all the animals. In animals that were not resuscitated LV dimensions were significantly lower compared to those in resuscitated animals (p=0.01). These differences were observed after 10 minutes of untreated VF. The animals with greater reduction in LV dimension during VF, achieved significantly lower CPP during CPR in contrast to animals with larger LV during VF (Table

).

Conclusion:
Greater decrease in LV volume and area during cardiac arrest are associated with low CPP during CPR and poor resuscitation outcome. LV dimensions prior to start CPR might therefore affect the effectiveness of resuscitation interventions.
Abstract P78: Teaching Basic Life Support to School Children in Northern Ireland Using the ’ABC for life’ Programme
Authors: Toner Philip , Connolly Michael , McGrath Patrice , Laverty Leanne , Connolly David , McCluskey David
Published: July 2021

Aims: Northern Ireland has one of the highest incidences of ischaemic heart disease in the world. Teaching BLS to school children has previously been recommended. Our aim was to assess if eleven year old children can aquire and retain knowledge of basic life support (BLS) skills six months after a course of instruction (Study 1) and to assess the effectiveness of a three tier ‘peer training’ model using medical students, teachers and pupils (Study 2). This would be a cost effective way of significantly decreasing mortality from out-of-hospital cardiac arrests.

Methods: The ‘ABC for Life’ programme is a regional course in BLS training in Northern Ireland specifically designed for 10 –12 year old school children. Medical students are trained as instructors in BLS by cardiac resuscitation officers at a major teaching hospital. Children were given a 22 point questionnaire before training and immediately after training to assess acquisition of BLS, and again six months after training to assess retention of knowledge (Study 1). In study 2, medical students instructed teachers how to teach BLS to their pupils. Pupils were given the same questionnaire to assess knowledge of BLS immediately before and after a teacher led training session at their school.

Results: Study 1: Children showed a highly significant increase in knowledge following the training session (46.8% vs 82.7%, p<0.001). Six months later their knowledge remained significantly higher than that of a control group who had never been trained (61.1% vs 46.8%, p<0.01). Study 2: Thirty eight teachers were trained from one education board. The first five children trained at each school were randomly selected. Questionnaires were returned for 44.7% pupils at baseline and 42.1% pupils after training. Mean baseline scores improved significantly after training (57.2% vs 77.7%, p<0.001).

Conclusion
These studies demonstrate that by using medical students and teachers, a large number of children can be trained in a short time period and at relatively low cost. Children can acquire and retain knowledge of BLS for at least six months. The initiative provides manikins and training resources to all schools. To date, the programme has trained 350 schools. We have commenced research of physical skills using a recordable mainikin.
Abstract P85: Out-of-hospital Cardiac Arrests With Extracorporeal Cardiopulmonary Resuscitation In Japan: Systematic Literature Review
Authors: Sakamoto Tetsuya , Asai Yasufumi , Nagao Ken , Tahara Yoshio , Atsumi Takahiro , Morimura Naoto , Nara Satoshi
Published: July 2021

Background: In Japan, extracorporeal cardiopulmonary resuscitation (ECPR) became popular for cardiac arrest patients who resist conventional advanced life supports. Regardless of many clinical experiences, there has been no previous systematic literature review.

Methods: Case series, reports and proceedings of scientific meeting about ECPR for out-of-hospital cardiac arrest written in Japanese between January 1, 1983 and July 31, 2007 were collected with Japana Centra Revuo Medicina (medical publication database in Japan) and review by experts. The outcome and characteristics of the patients were investigated, and the influence of publication bias of the case series study was also examined by the Funnel Plot method.

Results: There were 951 out-of-hospital cardiac arrest patients who received ECPR in 92 reports (including 59 case series and 33 case reports) during the period. The average of age was 38.1 (4 – 88) years old and 76.1% was male. Three hundreds and eighty-one cases (40.1%) were arrests of cardiac etiology, and 212 were non-cardiac (22.3%). The cause of arrest was not described in other 37.6%. Excluding reports for only one case, weighted survival rate at discharge of 792 cases those were clearly described the outcome was 39.5±10.0%. When the relationship between the number of cases and the survival rate at discharge in each 59 case series study was shown in figure

by the Funnel Plot method, the plotted data presented the reverse-funnel type that centered on the average of survival rate of all.

Conclusions: The influence of publication bias of previous reports in Japan was relatively low. ECPR can greatly contribute to improve the outcome of out-of hospital cardiac arrests.
Abstract P97: Standardized Post Resuscitation Care including Therapeutic Hypothermia for Patients with Non-VF Arrests
Authors: Tomte Oystein , Draegni Tomas , Jacobsen Dag , Mangschau Arild , Sunde Kjetil
Published: July 2021

Introduction: Whether therapeutic hypothermia (TH) improves survival after out-of-hospital cardiac arrest (OHCA) with initial asystole (AS) or pulseless electrical activity (PEA) is still controversial. At our hospital, revived OHCA patients follow the same standardized post resuscitation care protocol, if active treatment is desired. This includes TH, irrespectively of initial rhythm. Thus, we wanted to report the outcome for our OHCA patients of non-VF origin.

Methods: Since September 2003 all OHCA patients admitted to our hospital have been registered in a database. All patients with initial AS or PEA admitted to the Intensive Care Unit (ICU), or ward, between September 2003 and July 2007 were included. Favorable outcome is defined as a Cerebral Performance Category (CPC) of 1–2.

Results: Altogether 58 patients, all comatose, with a median age of 55 (range 19 –90 years), were registered. Cardiac etiology was present in 24 patients (41%), half of which had a myocardial infarction. Among the 34 patients (59%) with non-cardiac etiology, 15 were intoxications (44%), seven had asphyxia after respiratory failure (21%), five had suffered a cerebral event (15%), three drowned (9%), one had a pulmonary embolism (3%), and three causes remained unidentified. Overall, 37 patients (67%) were treated with TH; 16 (43%) received external cooling (Arctic Sun, Medivance), 13 (35%) endovascular cooling (Coolgard, Alsius Corporation) and eight (22%) other external cooling, all methods combined with initial ice cold fluids/ice packs. Median time in ICU was three days (range 0 –19 days). Eleven patients (19%) were discharged alive from hospital (cardiac etiology in four, 36%). Nine patients (16%) had a favorable outcome, and two patients (3%) had CPC 3.

Conclusion: After implementation of standardized post resuscitation care including TH, we found a 16% favorable survival among those admitted after non-VF OHCA, demonstrating a survival potential also for these patients.
Abstract P101: Comparison of Ventilation Rate Measured with Thoracic Bioimpedance and with Capnography during Out-of-Hospital Cardiopulmonary Resuscitation
Authors: Idris Ahamed H , Beadle Sarah , Daya Mohamud , Zive Dana
Published: July 2021

Objective:
To determine the ability of thoracic bioimpedance to measure ventilation rate during cardiac arrest and CPR.

Methods:
Philips MRx devices monitored 32 patients during out-of-hospital cardiac arrest and CPR. The devices recorded chest compressions with an accelerometer, continuous 1-lead EKG, thoracic bioimpedance, and continuous capnography. Of the 32 files, 4 were not used in this study because of incomplete recording. Two reviewers manually annotated ventilation waveforms independently using Laerdal QCPR software, which also automatically annotated ventilation through the bioimpedance channel. Reviewers manually measured ventilation rate (number of breaths/min) recorded with capnography for each 1 minute epoch, which were matched and compared with those measured through bioimpedance for each patient file (N = 28). A total of 585 1-minute epochs were measured and compared. We assessed intra-class correlation for 2 individual raters for ventilation rates measured with capnography and with annotated bioimpedance to establish inter-user reliability of measurements. Ventilation rate measured with capnography vs. bioimpedance was compared with simple regression.

Results:
The majority (60%) of ventilation rates measured with capnography and with automated software bioimpedance were within 2 breaths/min of each other. After manual annotation of the bioimpedance channel, 81% of 1-min epochs were within 2 breaths/min of rates measured with capnography. Ventilation rate measured with capnography had good correlation with bioimpedance (r = .82, p < .0001). Inter-rater agreement is estimated to be 0.96 for ventilation rate measured with capnography and 0.93 for rate measured with bioimpedance.

Discussion:
The software occasionally missed obvious ventilation waveforms and occasionally annotated waveforms obviously caused by chest compressions. Manual review and annotation improved the accuracy of ventilation rates measured with bioimpedance. Approximately 75% to 90% of recordings made with the Philips MRx device are expected to be useful for measurement.

Conclusion:
Ventilation rate measured with thoracic bioimpedance alone is acceptable using the Philips MRx device. Inter-rater agreement for measurements is excellent.
Abstract P112: Precordial Thump Ineffective in Terminating Ventricular Fibrillation but May Have a Role in Asystole
Authors: Madias Christopher , Maron Barry J , Alsheikh-Ali Alawi A , Murphy Barbara , Egerhei Lara , Mark Estes N. A. , Link Mark S
Published: July 2021

BACKGROUND. The efficacy of the chest thump in cardiac arrest is controversial. In an animal model of cardiac arrest we assess the efficacy of precordial thump in ventricular fibrillation (VF) and asystole.

METHODS. Under general anesthesia, 10 swine were induced to VF using a RV pacing lead and an AC fibrillator. In random order, animals received chest thumps with a 30 or 40 mph lacrosse ball or a chest thump with a fist. If VF continued after 3 thumps, animals were externally defibrillated. All animals underwent 6 VF inductions and received a total of 18 chest thumps; 2 sets of 3 thumps each with a fist, 30 mph, and 40 mph lacrosse balls. If asystole followed defibrillation, chest thumps with a fist were given to induce premature ventricular contractions (PVCs) until resumption of sinus rhythm. LV pressures of all thumps were recorded.

RESULTS. None of the 180 chest thumps terminated VF. The LV pressure generated by thumps during VF was 263 ± 52 mmHg for manual thumps, 392 ± 179 mmHg for 30 mph thumps, and 616 ± 182 mmHg for 40 mph thumps (p<0.001). All VF episodes were terminated by a 200J shock. Defibrillation was followed by asystole after 46/60 (77%) of VF inductions. During asystole, 1533/1856 (83%) chest thumps induced PVCs. LV pressure was higher for thumps that induced PVCs than those that did not (113 ± 27 vs 75 ± 23 mmHg, p<0.001). For each 10 mmHg rise in LV pressure, the OR of inducing a PVC was 1.9 (95% CI 1.8 –2.1; Figure

).

CONCLUSIONS. Despite achieving high LV pressures, precordial thumps were not effective in terminating VF, but did produce PVCs in asystole. Based on this data, a precordial thump cannot be recommended for VF, but its use in asystole should be further explored.
Abstract P120: Effect of Age on Hemodynamics, Heart Rate Variability, and Outcome in a Murine Model of Sepsis
Authors: Hollenberg Steven , Zanotti Sergio , Skaf Jad , Lichaa Hady , Gupta Anupam , Weinstock Josh , Parrillo Joseph E
Published: July 2021

Background:
The incidence and mortality of sepsis increase with aging, but animal models often use young animals whose biological ages do not reflect patient populations. We compared old and young mice in a murine model of resuscitated sepsis.

Methods:
C57Bl/6 mice (Young 2–3 months, n=32; Old 15–20 months, n=24) were made septic by cecal ligation and puncture (CLP) and resuscitated with fluids and antibiotics q 6 hr; controls underwent sham ligation. Serial echocardiography using a 30MHz probe was performed under light isoflurane anesthesia for measurement of stroke volume (SV), fractional shortening (FS), and cardiac output (CO). Blood pressure was measured using implantable radiotelemeters. From the waveforms, heart rate volatility (% of 5 minute intervals with standard deviation < the lowest 5% of baseline), an index of heart rate variability less susceptible to artifact than other measures, was derived for the entire experiment.

Results:
After CLP, heart rate and blood pressure did not differ significantly between old and young mice. SV decreased early in both groups (old 54.4 to 25.6 μL, young 57.2 to 24.0 p<0.01 vs baseline), as did CO. With resuscitation, SV and CO improved in both groups (old 48.0 μL and 24.5 ml/min, young 44.0 μL and 20.7 ml/min). Old animals were larger than young animals, but normalized values (% change from baseline) for SV, CO, and LVEDV were similar after CLP. Old septic mice had more periods of low heart rate volatility than young septic mice or old controls (62% vs 37% vs 3% of the experimental period). Mortality tended to be higher in old than young mice (46% vs 22% at 72 hr, p=0.09, 54% vs 22% at 144 hr, p=0.06).

Conclusion:
In a clinically relevant model of murine sepsis, age did not have a significant impact on hemodynamics, but decreased heart rate variability was more prominent in aged mice, and age was associated with increased mortality. This suggests the potential for nonlinear hemodynamic parameters to provide insights into both the pathogenesis of disease and the effects of aging.
Abstract P188: Impact of Metabolic Crisis on Fatal Outcome in Patients with Cardiogenic Shock Complicating ST-segment Elevation Myocardial Infarction
Authors: Suzuki Makoto , Shimizu Hideaki , Fujita Shinpei , Sasaki Yasuhiro , Miyoshi Akihito , Takagi Yaemi , Habara Hirokazu , Sato Sumiko , Kakishita Mikio , Nakamura Youichi
Published: July 2021

We investigated the relation of initial metabolic acidemia to in-hospital mortality in patients treated with emergency coronary angioplasty for shock complicating first anterior ST-segment elevation myocardial infarction (STEMI).

Methods
A total of 23 consecutive patients (17 men, 73±12 years) with Killip class IV class due to anterior STEMI were studied. Using median levels of arterial base excess (BE, −5.8 mmol/L), the patients were divided into high and low BE groups, and both groups were compared regarding microvascular revascularization and clinical outcomes. To evaluate myocardial tissue-level reperfusion, severe microvascular injury was defined by the presence of both angiographic myocardial blush grade 0/1 and less than 30 % resolution of ST-elevation after angioplasty.

Results
In-hospital mortality was 92 % in the high BE group (−12.0±4.9 mmol/L) as compared with 9 % in the low BE group (−0.9±2.4mmol/L, p=0.0001 vs. high BE group). Baseline clinical and angiographic characteristics were not different between the two groups. Arterial gas analysis showed lower pH and higher levels of lactate in the high BE group than in the low BE group (7.22±0.16 vs. 7.42±0.06, p=0.006, 8.52±4.43 vs. 2.42±1.33, p=0.016). Despite successfully culprit angioplasty in all cases, the incidence of severe microvascular injury was significantly high in the high BE group as compared with the low BE group (83 vs. 36 %, p=0.018). Initial levels of BE showed a significant negative relation to ST-segment resolution (r=0.61, p=0.002). A multivariate regression analysis demonstrated a potent association of initial levels of BE with severe microvascular injury (r
2
=0.341, p=0.015).

Conclusions
We identified the pivotal association of initial metabolic crisis with severe microvascular reperfusion injury leading to high in-hospital mortality in patients with cardiogenic shock complicating STEMI.
Abstract P192: What is the Role of Chest Compression Depth during Out-of-Hospital CPR?
Authors: Stiell Ian G , Everson-Stewart Siobhan , Christenson James , Cheskes Sheldon , Powell Judy , Bigham Blair , Morrison Laurie , Larsen Jonathan , Nichol Graham , Hess Erik , Vaillancourt Christian , Davis Daniel P , Callaway Clifton W
Published: July 2021

Introduction:
Current CPR compression depth guidelines were empirically derived. We sought to study patterns of CPR compression depth and their associations with patient outcomes in out-of-hospital cardiac arrest (OOHCA).

Methods:
We studied OOHCA patients from the Resuscitation Outcomes Consortium Epistry - Cardiac Arrest for whom electronic CPR data were available from proprietary accelerometer technology attached to the prehospital defibrillators. We calculated anterior chest wall depression in millimeters and the period of active CPR (chest compression fraction [CCF]) for each minute of CPR. We controlled for covariates including compression rate and calculated adjusted odds ratios (OR) for any return of circulation (ROSC) and 24 hour survival. We calculated unadjusted OR for hospital discharge.

Results:
We included 615 adult patients from 6 U.S. and Canadian cities with these characteristics: mean age 68.9; male 62 %; witnessed 43%; bystander CPR 31%; initial rhythms - VF/VT 25%, PEA 17%, asystole 43%, unspecified non-shockable 15%; mean compression rate 103/min; mean compression depth <38mm 51%, 38–51mm 39%, >51mm 11%; median CCF 0.66; outcomes - ROSC 57%, 24 hour survival 18%, discharge 5%. We found an inverse association between depth and compression rate (P<0.0001; see Table

), no association between depth and CCF (P=0.30), and a positive association between CCF and rate (P<0.0001). ORs with 95% CIs for each 5mm increment in compression depth and the outcomes were: 1) ROSC - adjusted OR 1.06 (0.97–1.16), 2) 24 hour survival - adjusted OR 1.01 (0.98–1.03), 3) discharge - unadjusted OR 1.13 (0.95–1.34).

Conclusions:
We found half of patients received suboptimal compression depth, an inverse association between compression depth and rate, and no clear association between survival and increased compression depth. Further study is required to determine the optimum chest compression depth and its interaction with rate and CCF in order to maximize outcomes.

Compression Rate versus Compression Depth
Abstract 220: Detection of Cysteine Sulfenic Acid Formation: Role in Reactive Oxygen Species-dependent VEGF signaling Linked to Endothelial Migration
Authors: Nakamura Yoshimasa , Kaplan Nihal , Poole Leslie B , Fukai Tohru , Ushio-Fukai Masuko
Published: July 2021

NADPH oxidase Nox2-derived reactive oxygen species (ROS) have been implicated in VEGF receptor type2 (VEGFR2)-mediated signaling linked to endothelial cell (EC) migration. ROS-mediated protein modification involves oxidation of reactive cysteine residues to form a cysteine sulfenic acid (Cys-SOH) and subsequent oxidation products. However, the role of Cys-SOH formation in VEGF signaling and EC migration remains unknown. Using a newly-developed Cys-SOH trapping reagent, here we show that VEGF stimulation significantly increases the total Cys-SOH formation of proteins in HUVECs. VEGFR2 immunoprecipitates show significant increase in Cys-SOH within 5 min (90.3%), which remained elevated at least for 30 min (30.1%). This VEGF-induced Cys-SOH formation of VEGFR2 is prevented by pretreatment with a thiol donor antioxidant, N-acetylcysteine, which is associated with inhibition of VEGFR2 autophosphorylation (80.2%). The trapping of endogenous Cys-SOH by dimedone blocks VEGFR2 autophosphorylation (50.3%), its downstream phosphorylation of PLCgamma (70.4%) and ERK1/2 (90.1%) as well as EC migration (80.3%) without affecting p38MAPK phosphorylation. Wound scratch assay and immunofluorescence analysis reveal that Cys-SOH modified proteins are accumulated at the leading edge where they colocalize with Nox2, phospho-VEGFR2, F-actin and IQGAP1, a novel VEGFR2- and actin-binding scaffold protein, in actively migrating ECs. A significant increase in Cys-SOH is also seen in IQGAP1 coprecipitates (~250 kDa) following VEGF stimulation with peak at 2 min (50.1% increase). Knockdown of IQGAP1 using siRNA, or trapping of Cys-SOH block wound-induced Cys-SOH formation at the leading edge as well as EC migration toward the injured site. In summary, these results suggest that VEGFR2 and IQGAP1-associated proteins are targets for Cys-SOH formation at the leading edge in actively migrating ECs. The present study provides a novel mechanism of redox regulation in VEGF signaling linked to EC migration, and a rationale for the importance of identification of novel proteins that form Cys-SOH during repair process after injury and angiogenesis in vivo.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 306: Engineered Neonatal Rat Cardiac Tissue Models Immature
in vivo
Myocardium
Authors: Ralphe John C , Witt Michelle R , Kirk Jonathan A , Clause Kelly M , Keller Bradley B , Tobita Kimimasa
Published: July 2021

Correlating contractile function, cellular, and molecular events in the immature heart is hampered by the limited availability of easily studied models. 2D cultures provide molecular data but can not replicate
in vivo
mechanical events. Isolated perfused hearts provide contractile data, but gene expression manipulation and molecular studies are limited. Here we demonstrate the utility of a 3D neonatal cardiomyocyte culture to model immature native myocardium. Isolated cardiomyocytes from day-old Sprague-Dawley rat pups were mixed with collagen and matrigel, and cast into cylindrical constructs (termed NECT). After seven days in culture NECT force-strain, force-frequency, and calcium transient data were collected. Identical data were collected from aged matched intact papillary muscle. RNA and protein were isolated from NECT and papillary muscle to assess sarcomeric proteins and quantitate developmentally regulated gene expression. Transcription of β-actin and cardiac α-actin were lower in NECT vs. native (
P
<0.001; N=8). Myosin heavy chain α and β isoform ratios and content were similar between NECT and native while metabolic genes GLUT1 and MCAD were increased, and GLUT4 and PDK4 decreased (
P
<
.05)
. Major sarcomeric proteins were well represented in NECT while regulatory proteins MyBPC and TnI were decreased. Peak developed force and calcium transients were similar between NECT and native, while the rate of force development and relaxation were accelerated in the NECT (Table

). 3D culture of neonatal cardiomyocytes, NECT, replicates important structural and functional features of immature native myocardium. The in vitro investigation of 3D NECT represents a unique opportunity to study electromechanical, metabolic, and endocrine factors that influence myocardial maturation.

This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).

Comparison of Force and Calcium Transients in NECT
Abstract 312: Selective Inhibition of Myocardial NADPH Oxidase Significantly Decreases Heart Size in Transgenic (Tg) Mice with Cardiac-Specific Overexpression of a Dominant-Negative (DN) Mutant of p67
phox
Authors: Zhao Qiong , Ornatowska Magdalena , Urboniene Dalia , McGee Kristopher , Solaro R J , Franks Roberta , Christman John W , Xiao Lei
Published: July 2021

The role of NADPH oxidase (NOX) was recently suggested in cardiac diseases including hypertrophy. Overexpression of a DN mutant of p67
phox
, a cytosolic subunit of NOX, DN-p67 with a V204A point mutation completely inhibited NOX enzymatic function of superoxide generation. We found that overexpression of DN-p67 in cultured adult rat cardiac myocytes attenuated α
1
-adrenoceptor-induced hypertrophy
,
we thus hypothesized that NOX/p67
phox
plays a critical role in cardiac hypertrophy and remodeling
in vivo
. Accordingly, we recently generated a Tg mouse model in C57BL/6 strain with cardiac-specific overexpression of this DN-p67 mutant along with an IRES-initiated EGFP tag protein using the cardiac-specific α-MHC promoter. Hearts from 1.5-, 3- and 6-month (M) old heterozygous Tg mice and their Non-Tg (NTg) littermates were harvested and compared. The heart weight/body weight (HW/BW) ratio in Tg group was significantly smaller (overall: 14%↓; 1.5-M: 10%↓; 3-M: 16%↓; 6-M: 12%↓) vs. the NTg group (
p
< 0.01, n=23). This result was confirmed by 2D echocardiography (2D-echo) measuring the left ventricular myocardial area (LVMA) determined by subtracting endocardial area from epicardial area in short axis view at the end diastole. The LVMA (mm
2
) in 3-M and 6-M mice was significantly (
p
< 0.05, n=21) smaller in Tg (0.44±0.02) than the NTg (0.50±0.02) with equal average BW in both groups. LV internal dimensions and systolic function were preserved without difference in Tg vs. NTg. The LV wall thickness is smaller in Tg vs. NTg by both 2D-echo and hematoxylin/eosin staining of LV cross-sections, but no change in fibrosis was detected in both groups by trichrome staining (n=4). Interestingly, the expression of NOX catalytic subunit gp91
phox
in Tg hearts was significantly decreased (~3-fold↓, n=5) vs. NTg by Western blot, whereas the cardiac expression of other NOX subunits p47
phox
and p22
phox
remained the same in both groups. In summary, we have generated a viable Tg mouse model with cardiac-specific overexpression of a DN p67
phox
, which shows significant reduction in both myocardial mass/size and gp91
phox
expression with normal cardiac function and development. Our results first show the novel and critical role of NOX/p67
phox
in normal cardiac growth and heart development.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 351: Role of Ketoaldehyde Protein Adducts in Ischemic Cardiomyopathy
Authors: Hwang Hyun S , Davies Sean S , Hill Michael F , Pessah Isaac N , Allen Paul D , Roberts L. J , Knollmann Bjorn C
Published: July 2021

Background:
Oxidative damage is an important component of ischemic heart injury. Isomeric ketoaldehydes, isoketals (IsoKs), are produced by the isoprostane pathway of lipid peroxidation. IsoKs, which are among the most reactive products of lipid oxidation, form irreversible lactam adducts with lysine residues and cross-link proteins. We have identified a specific IsoK scavenger, salicylamine (SA), which prevents IsoKs from adducting to proteins
in vitro
. Here, we tested whether IsoK adducts are increased in ischemic cardiomyopathy and whether SA is cardioprotective.

Methods:
IsoK-lysyl adducts were measured by mass spectrometry in human hearts with ischemic cardiomyopathy and in mouse hearts after MI from coronary ligation. The effect of SA was tested in the mouse MI model.

Results:
IsoK adduct levels (ng/g tissue) were significantly increased in human hearts with ischemic cardiomyopathy compared with normal donor hearts (2.3±0.4 vs 0.7±0.1, p<0.05) and also in mouse hearts 7 days after MI.
In vitro
, IsoKs (0.5–5μM) dose dependently enhanced binding of [3H]ryanodine to cardiac SR preparations (RyR2). In mice, pre-treatment with SA for 3 days prior to coronary ligation significantly reduced overall mortality (Fig.

), improved cardiac contractility (fractional shortening: SA 36% vs placebo, 27%, p<0.01) and reduced infarct size (SA 18% vs placebo 36%, p<0.05) measured 7 days post MI.

Conclusion:
IsoK adducts are increased in ischemic cardiomyopathy and a specific scavenger of IsoKs exerts remarkable cardioprotective effects in a mouse model of MI. IsoKs potently activate RyR2 channels, which may explain in part the cardioprotective action of IsoK scavengers.

This research has received full or partial funding support from the American Heart Association, AHA Greater Southeast Affiliate (Alabama, Florida, Georgia, Louisiana, Mississippi, Puerto Rico & Tennessee).
Abstract 354: Host Immune Factors and Donor Adult Stem Cells Mediate Myocardial Ischemia Repair
Authors: Tolar Jakub , Braunlin Elizabeth , McElmurry Ron T , Lees Christopher J , Riddle Megan , Mansoor Abdul M , Zhang Jianyi , Blazar Bruce R
Published: July 2021

Cellular therapy with non-hematopoietic adult stem cells (ASC) has emerged as a novel strategy for treatment of myocardial infarction (MI). Cells from non-identical donors, however, are typically rejected by the immune system of recipient. To overcome this barrier to transplantation we induced MI by left coronary artery ligation followed by direct intramyocardial injection of ASC or saline in cohorts of syngeneic C57BL/6 (B6) immune competent versus B6 Rag/IL2Rγc (Rag/γc) T-, B- and natural killer-cell immune deficient mice. We made an unexpected observation that heart function was significantly improved in ASC-treated B6 mice, but not in ASC-treated Rag/γc mice; and that the beneficial effect of ASC was inversely correlated with ASC persistence (i.e., ACS were rejected by B6 mice but engrafted in Rag/γc mice). As the donor cells were rejected within several weeks by B6 animals we hypothesized that the host immune system and ASC provided trophic signals for cardiac repair early after injury. In search for anatomical correlation, we first assessed changes in capillary density. Four weeks after MI, significantly more capillaries (normalized to number of nuclei) were observed in the border zone of B6 animals treated with ASC versus saline (p = 0.01), and significantly fewer capillaries were found in the infarct zone when B6 animals (treated with ASC) were depleted of T-cells and NK-cells (p = 0.03). To gain mechanistic insight into the early events in the ASC-mediated repair, next we have assessed one week after MI ± ASC messenger RNA expression of genes known to be engaged in angiogenesis. In B6 mice the most prominent change in response to MI injury was 17-fold increased expression of tissue inhibitor of metalloproteinases 1. The ASC mediated response to MI injury was defined by increased expression of fibroblast growth factor 2, integrin αV, phophorylated form of vascular endothelial growth factor receptor 2 and endothelial-specific receptor tyrosine kinase (all p < 0.01). These data are the first to define the paracrine tissue repair factors necessary to produce therapeutic benefit in response to donor ASC after MI. Importantly, our data also suggest that MI is not solely a heart lesion, but rather a systemic inflammatory condition controlled by host immune system.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 364: Cardioprotective Effects Of Carbon Monoxide Mediated By Hypoxia-inducible Factor (HIF) 1-alpha
Authors: Hayasaki Takanori , Liao Hui , Hyman Matthew C. , Pinsky David J.
Published: July 2021

Until recently, carbon monoxide (CO) was thought of a toxic gaseous waste product of heme catabolism. Although recent evidence following left anterior descending artery (LAD) ligation in rats has suggested a potential cardioprotective effect of low dose inhaled CO, the molecular mechanism underlying this protection are obscure. The purpose of this study was to elucidate the mechanistic basis for this effect. Myocardial ischemia-reperfusion injury (LAD ligation) was performed in mice which were exposed to room air (RA) or low dose CO (250 ppm) 24 hrs before ischemia. Myocardial infarct size was significantly diminished (by 38%; p<0.01, n=6) in the CO group compared with the RA group. As one pathway of ischemic preconditioning is believed to be mediated by cycloxygenase (COX)-2, we hypothesized that CO might upregulate COX-2 to elicit ischemic cardioprotection. CO increased COX-2 expression in the hearts of mice in a time-dependent manner, peaking at 24 hrs with 4.8-fold protein induction. Additional experiments, done to test a potential separate mechanism of ischemic cardioprotection by CO, showed that CO increased HIF-1α 5.2 fold. The expression of HIF-1α and COX-2 were also higher in CO-exposed cardiomyocytes (HL-1 cells) compared with RA-exposed HL-1 cells. Treatment of HL-1 cells with the COX-2 specific inhibitor (NS398) and prostaglandin (PGE2) had no effect on HIF-1α mRNA levels. However, NS398 significantly abrogated (by 62%, p<0.05, n=3) the increase of HIF-1α expression induced by CO, and PGE2 significantly increased (by 34%, P<0.05, n=3) the HIF-1α expression of RA-exposed inhaled HL-1 cells. Moreover, treatment of mice with NS398 almost completely abolished the cardioprotective effect of CO against ischemia-reperfusion injury. This finding suggests that CO stabilizes HIF-1α via COX-2 activation of prostaglandin E2 synthesis in the heart, and implies that CO protects the heart in ischemia-reperfusion injury via stabilization of HIF-1α.
Abstract 392: Increased Oxidative Stress and Apoptosis as Mechanisms for Adverse Effects of Chronic Pressure Overload and Catecholamine Stress in Mice with Cardiac Overexpression of Type 5 Adenylyl Cyclase
Authors: Lai Lo , Yan Lin , Hu Che-Lin , Gao Shumin , Hong Chull , Vatner Stephen F
Published: July 2021

Studies with cardiac overexpression of type 6 adenylyl cyclase (AC6) suggest it protects against cardiac stress. Less is known about cardiac overexpression of AC5. We generated transgenic (Tg) mice with cardiac specific overexpression of AC5 and examined both the effects of pressure overload induced by 1 wk of transverse aortic banding (TAC) and 1 wk of chronic isoproterenol (ISO) delivered by osmotic mini-pump (n=8/group). TAC induced greater, p<0.05, left ventricular (LV) hypertrophy, as assessed by LV/body weight, in AC5 Tg (4.9±0.2) compared with wild type (WT) (4.0±0.1) and exhibited a significant decrease, p<0.05, in LV ejection fraction (LVEF) (58±4%) where LVEF was maintained in WT (75±1%). TUNEL analysis shows that apoptosis in AC5 Tg was twice that in WT, p<0.05. This was confirmed by a 2-fold increase in activity of caspase 3 in AC5 Tg compared to WT mice, p<0.05. The AC5 Tg began to develop heart failure as reflected by increased, p<0.05, lung/body weight (10.2±1.6) vs WT (6.4±0.2). In the heart, the antioxidant manganese superoxide dismutase (MnSOD), quantified by western blotting, was decreased by 40% in AC5 Tg compared to WT mice, p<0.05, after 1 wk of aortic banding. Chronic ISO also induced a greater, p<0.05, decrease in LVEF (48±4%) compared to WT (60±2%). 100 microM of ISO stimulation increased cell viability by 36% and decreased apoptosis by 34% (p<0.05 vs LacZ control). MnSOD was upregulated by 2.9-fold in AC5 knockdown myocytes after treatment with ISO for 48 hrs. These results demonstrate that cardiac overexpression of AC5 exerts adverse effects on oxidative stress and apoptosis in response to both chronic pressure overload and catecholamine stress.
Abstract 409: Effects of Angiotensin II Receptor Blocker (ARB) and PPAR-gamma Agonist on Vascular Failure in Patients with Hypertension, Diabetic Mellitus and Chronic Kidney Disease
Authors: Watanabe Hideki , Nakagawa Kunio , Kakihana Masaaki
Published: July 2021

Background:
Hypertension (HT), diabetes mellitus (DM), and chronic kidney disease (CKD) are known to be associated with “vascular failure”, which is defined as the integration of endothelial dysfunction and metabolic abnormalities of the vessel wall including inflammation, oxidative stress. We evaluated effects of ARB, calcium channel blocker (CCB), and PPAR-γ agonist on vascular failure in patients with HT, DM and CKD.

Methods:
Forty eight patients with untreated HT, type 2 DM (HbA1c 6.0 – 6.5%), and CKD (stage 3 or 4) were randomized to receive telmisartan (
Tel. group
, n=12), telmisartan and pioglitazone (
Tel. and Pio. group
, n=12), amlodipine (
Aml. group
, n=12), or amlodipine and pioglitazone (
Aml. and Pio. group
, n=12). We measured flow-mediated dilatation (FMD) and nitroglycerin-induced dilatation (NID) of brachial artery, and pulse wave velocity (PWV). We also measured high sensitivity C-reactive peptide (hsCRP) and TBARS levels as indexes of inflammation and oxidative stress. Measurements were performed at baseline, and then at every 6 months after the treatments.

Results:
Blood pressure was significantly decreased in these groups, and there was no difference among four groups at baseline and during the study. FMD was significantly increased, and PWV, hsCRP, and TBARS were significantly decreased in four groups. However, these improvements were much better in
the Tel. and Pio. group
than the other groups. NID did not change during the study.

Conclusion:
Combination therapy with ARB and PPAR-γ agonist was much better in the improvement of vascular failure in patients with HT, DM and CKD as compared with the therapy with ARB alone, CCB alone, or CCB and PPAR-γ agonist.
Abstract 1361: Leukocyte Antigen-Related Phosphatase Regulates Insulin Sensitivity by Interaction with Snapin
Authors: Vendrov Aleksandr E , Tchivilev Igor , Niu Xi-Lin , Li Juxiang , Runge Marschall S , Madamanchi Nageswara R
Published: July 2021

Several protein tyrosine phosphatases including leukocyte antigen-related (LAR) phosphatase have been implicated in insulin resistance, which is a risk factor for atherosclerosis. We showed previously that LAR negatively regulates insulin-like growth factor-1 (IGF1) signaling in vascular smooth muscle cells (VSMC) leading to increased proliferation and migration. Absence of LAR also enhanced neointima formation in response to arterial injury in mice. However, the role of LAR-modulated signaling in the development of insulin resistance has not been elucidated. Here, we investigated the function of LAR in regulating glucose uptake and insulin sensitivity. We identified snapin, a SNARE-associated protein involved in glucose transporter Glut4 vesicle fusion with plasma membrane, as a LAR-interacting protein using a yeast two-hybrid screen. IGF1-induced serine phosphorylation of snapin, its translocation to membrane and association with SNARE complex were enhanced in VSMC lacking LAR. Similarly, PI3K-PDK1-PKCζ signaling pathway was more active in LAR-/- cells after IGF1 treatment. This resulted in enhanced Glut4 activation, its membrane translocation and association with snapin. Glut4 membrane translocation and association with snapin after IGF1 treatment were impaired in snapin+/− VSMC. IGF1 treatment also increased serine phosphorylation of GSK3
β
in LAR−/− VSMC leading to increased activation of glycogen synthase. Consistent with this, enhanced glucose uptake was observed in LAR−/− VSMC compared to wild-type cells after IGF1 treatment. Basal and IGF1-induced glucose uptake were significantly lower in snapin+/− VSMC than in wild-type cells. Snapin+/− mice had higher levels of blood glucose, lower quantitative insulin sensitivity check index (QUICKI) and impaired response to insulin in insulin tolerance test (ITT) compared to wild-type mice. Decrease of QUICKI and impairment of IIT were more pronounced in snapin+/− mice fed a high-fat diet. In addition, Doppler ultrasonography indicated increased arterial stiffness in snapin+/− mice. Together, these data indicate that LAR negatively regulates snapin phosphorylation which in turn affects glucose uptake leading to the development of insulin resistance and vascular pathology.
Abstract 1363: Novel Role of Early Endosome Antigen 1 in Reactive Oxygen Species-dependent AT1 Receptor-mediated Signaling Linked to Hypertrophy in Vascular Smooth Muscle Cells
Authors: Nazarewicz Rafal R , Patrushev Nikolay A , Hilenski Lu , Ushio-Fukai Masuko , Alexander R. W
Published: July 2021

Angiotensin II (Ang II) stimulates hypertrophy in vascular smooth muscle cells (VSMCs) primarily through the G protein-coupled receptor (GPCR) Ang II type 1 receptor (AT1R) at the cell membrane and after its internalization. Major outputs of AT1R leading to vascular hypertrophy are triggered by reactive oxygen species (ROS)-dependent transactivation of the EGF receptor (EGFR), followed by activation of p38MAPK, Akt, p70S6K, and ERK1/2. However, underlying regulatory mechanisms are largely unknown. The small GTPase Rab5 and its downstream effector early endosome antigen 1 (EEA1) regulate endosomal internalization of GPCR. We hypothesized that EEA1 could be a scaffold facilitating the multiple outputs of AT1R signaling including those involved in hypertrophy. We found that Ang II (0.1
μ
M, 5 min) and H2O2 (100
μ
M, 5 min) induce formation of a complex composed of EEA1, Rab5, AT1R and EGFR that is inhibited by PEG-catalase (100 U/ml) and PEG-superoxide dismutase (SOD, 200 U/ml), inferring the redox sensitivity of the process. To explore this possibility further, we showed that Ang II and H2O2 stimulate EEA1 threonine phosphorylation (3.0- and 2.0-fold, respectively) co-temporaneously with Ang II- and H2O2-induced complex formation. Inhibition of p38MAPK by inhibitors (SB202190, PD169316; 1
μ
M) or treatment with PEG-catalase and PEG-SOD prevents EEA1 phosphorylation and its recruitment to the complex. To gain further insight, we used EEA1 siRNA to knock down EEA1 (80%). EEA1 siRNA significantly inhibits (79% control) Ang II-stimulated [3H]leucine incorporation in VSMC and is associated with decrease in Akt phosphorylation and its downstream p70S6K (80% and 50%, respectively) without affecting ROS-independent Erk1/2 phosphorylation. EEA1 siRNA does not affect Ang II-induced EGFR phosphorylation.

Conclusion: ROS- and p38MAPK-dependent Ang II-induced EEA1 phosphorylation is involved in formation of a signaling complex (AT1R, EGFR, EEA1, Rab5) that is related to Ang II stimulated, AT1R- and EGFR-mediated vascular hypertrophy. Akt-dependent phosphorylation of the p70S6K kinase is also EEA1-dependent. These findings provide insight into a novel role for EEA1 in Ang II signaling in VSMC hypertrophy.
Abstract 1372: Sirt3 Promotes Survival of Cardiac Myocytes under oxidative stress and Upregulates Mitochondrial Genes Through Nuclear Respiratory Factor
Authors: Odewale Ibrahim , Alcendor Ralph , Sadoshima Junichi
Published: July 2021

Silent information regulator 1 (Sirt1), the prototype member of the sirtuin family, has been implicated in longevity and stress resistance. Sirt3, another member of the sirtuin family, is localized primarily in mitochondria and involved in metabolism in both white and brown adipose tissues. However, the cardiac role of Sirt3 is not well understood. Here, we examined the cellular function of Sirt3 in cardiac myocytes. Western blot analysis revealed that Sirt3 is highly expressed in cardiac myocytes, at the level comparable to Sirt3 in white and brown adipose tissues. Although Sirt1 in the heart was upregulated in response to glucose deprivation, high-fat diet, or pressure overload, expression of Sirt3 was unchanged in these conditions. Expression of Sirt3 was increased two-fold, however, after H
2
O
2
(100
μ
M) treatment in primary cardiac myocytes and two-fold by aging (18 vs 3 months) in the heart. Sirt3 is localized primarily in the mitochondria in myocytes under normal conditions, but it translocates into the nucleus when cells are treated with H
2
O
2
. Adenovirus-mediated overexpression of Sirt3 significantly reduced myocyte cell death in serum free media or in the presence of H
2
O
2
as determined by cytoplasmic accumulation of mono- and oligonucleosomes (p<0.05), suggesting that Sirt3 protects myocytes from apoptosis under oxidative stress. Furthermore, Sirt3 increased expression of NRF1, a transcriptional modulator of metabolic genes and mitochondrial DNA transcription, by 2.5-fold. There was a 3.4-fold increase in Glut4 expression as well as a 4-fold increase in COX IV. On the other hand, Sirt1 failed to upregulate NRF1. Sirt3 neither interacted with PGC-1
α
nor stimulated PGC-1
α
-mediated transcription. Thus, although both Sirt1 and Sirt3 exert their function by regulating metabolic genes, their mechanism of action is different. In summary, cardiac myocytes abundantly express Sirt3 in mitochondria. Sirt3 is upregulated by oxidative stress and translocates into the nucleus, where Sirt3 regulates expression of mitochondrial genes, such as Glut4 and COX IV, through upregulation of NRF1 and plays a protective role against oxidative stress in cardiac myocytes.