bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P2083: Erk-mediated Phosphorylation Of Grk2 Leads To Reduced Pyruvate Dehydrogenase Activity And Alters Pyroptotic Signaling
Authors: Zhai Ruxu , Montgomery Sarah , Yuzhen Tian , Alnemri Emad , tang Hsin Yao , Sato Priscila
Published: November 2022

G protein-coupled receptors (GPCRs) are important regulators of cellular functions where agonist binding leads to receptor conformational changes and downstream activation of g-protein-mediated signaling cascades. Signal transduction is terminated by receptor phosphorylation mediated by G-protein coupled receptor kinases (GRKs). GPCR kinase 2 (GRK2) is a main GRK in the heart and is upregulated in heart failure (HF) patients. Recently, we and others have shown that GRK2 can translocate to cardiac mitochondria where it regulates metabolism. Notably, we found that phosphorylated GRK2 at S670 post- ischemia/reperfusion (IR) decreases glucose mitochondrial utilization and pyruvate dehydrogenase (PDH) activity. Furthermore, 2D-SDS PAGE followed by LC/MS/MS revealed a potential link between mitochondrial GRK2 and PDH phosphorylation. Using a novel PDHα KI CRISPR Cas HEK cell line, we tested the hypothesize that PDH phosphorylation of this novel site was important for PDH activity. Utilizing cytosolic and mitochondrially-targeted pyruvate indicators we measured pyruvate levels in WT and KI HEK cells. In parallel, using a novel mouse model GRK2-S670A, we investigated the role of ERK in mediating GRK2 mitochondrial translocation and pyroptotic signaling. Further experiments will determine long-term impact of mitochondrial GRK2 in cell survival in cardiac pathological conditions.
Abstract P2089: Sex Differences In Racism And Renal Injury Biomarker Association In African Americans
Authors: Pointer Mildred A , Holanda Thais , Muhammad Dwayne , Alberico Claudia , Laurie Nikia , Kumar Deepak
Published: November 2022

Among the general population of African Americans (AAs) the risk for kidney disease is approximately 4-fold greater than Caucasians. However, the risk for kidney disease can be as high as 10-fold greater among hypertensive young African American (AA) males. The reason for this disparity remains elusive, but environmental stress has been identified as a contributor to disease risk among Blacks. Male (n=78) and female (n=91) AAs were administered the perceived racism scale (PRS) to investigate the relationship between perceived racism and biomarkers of stress (cortisol), hypertension (systolic and diastolic blood pressure; SBP/DBP, respectively), obesity (body mass index, BMI), and diabetes (CRP, and C peptide). Resting heart rate (HR) and BMI were measured; a blood sample collected to measure cortisol as well as C reactive protein (CRP) and C peptide (both biomarkers of diabetes); and urine was collected for measuring albumin to creatinine ratio (A:C, index of renal injury). There were no sex differences in age (36 ± 2 vs 33 ± 2), SBP (127± 13 vs 124 ± 2), DBP (72 ± 1 vs 69 ± 1), but A:C was higher in males (28 ± 12 vs 21 ± 4; p<0.003; males vs females, respectively). Males had higher job domain PRS scores (20 ± 2 vs 14 ± 1, p<0.01 male vs female, respectively) and PRS was positively correlated with cortisol (r=0.54, p<0.05) in males only. Furthermore, job PRS was positively correlated with biomarkers of obesity (BMI: r=0.419, p<0.004), hypertension (SBP, DBP: r=0.521, r=0.480; p<0.002, <0.009, respectively), and diabetes (C peptide: r=0.468, p<0.03) in males but not females. Interestingly, job PRS scores (r=0.498, p<0.005) as well as hypertension (SBP/DBP: r=0.398, r=0.611; p<0.03, p<0.001, respectively) and diabetes (CRP: r=0.812, p<0.001) biomarkers were positively correlated with A:C in males but not females. These findings support the idea that the increased risk for kidney injury in young hypertensive AA males results from environmental stress of racism that activates biological pathways leading to cardiovascular and kidney disease. More studies are needed to identify the underlying pathways between perceived racism and promotion of cardiometabolic disease.
Abstract P2091: Age-dependent Remodeling Of The Perinexus Modulates The Phenotype And Arrhythmic Risk Of Long Qt Syndrome Type 3
Authors: Wu Xiaobo , King David , Hoeker Gregory , Johnstone Scott R , Gourdie Robert G , Weinberg Seth , Poelzing Steven
Published: November 2022

Background:
The Long-QT Syndrome Type 3 (LQT3) is characterized by action potential duration (APD) prolongation due to cardiac voltage-gated sodium channel (Nav1.5) gain of function. Arrhythmia risk increases with age in LQT3 patients, but it is unknown whether this is due to progressive APD prolongation. Recent studies have shown that the LQT3 phenotype can be exacerbated by widening the perinexus, an intercalated disc nanodomain adjacent to gap junctions. However, it remains unclear if the perinexus mediates the LQT3 phenotype during aging.

Objective:
To investigate the role of the perinexus during aging in LQT3.

Methods:
Ventricular epicardial APD was obtained by optically mapping Langendorff-perfused adult (3-month) and aged (15-month) guinea pig hearts. LQT3 phenotype was induced with anemone toxin (ATXII), and the perinexus was widened with an intercalated disc adhesion inhibitor (βadp1). Perinexal width (Wp) was quantified by transmission electron microscopy. Susceptibility to arrhythmia was assessed with burst pacing in the absence and presence of βadp1 and evaluated from electrocardiograms (ECG).

Results:
APD was not different between adult and aged hearts at baseline, with ATXII, or ATXII+βadp1. Wp was significantly wider in adult LQT3 hearts either without or with βadp1 relative to aged hearts (without βadp1: 29.6±2.5 vs 22.9±3.8 nm; with βadp1: 38.3±1.7 vs 29.9±1.2 nm). These data suggest the perinexus narrows with aging and attenuates previously reported cellular APD prolongation such that there is no difference in whole-heart, epicardial APD between adult and aged LQT3 hearts. However, ECG Tpeak-Tend interval was not prolonged in adult, but significantly prolonged in aged hearts after treatment with ATXII+βadp1 (18.7±5.6 vs 35.5±11.5 ms), suggesting that βadp1 increases transmural dispersion of repolarization during aging in LQT3. Furthermore, ATXII+βadp1 did not induce arrhythmias in adult (0/6) but was arrhythmogenic in aged hearts (3/5).

Conclusions:
Perinexal narrowing associated with aging attenuates APD prolongation and reduces arrhythmic risk in aged LQT3 hearts. Loss of perinexal adhesion in aged LQT3 hearts results in significant and heterogeneous APD prolongation and increased risk of arrhythmia.
Abstract P2097: Hypochloremia Alters Cardiac Physiology After Myocardial Ischemia-Reperfusion Injury
Authors: Sanghvi Shridhar , Sridharan Divya , Trovato Vincenzo , Smith Sakima , Khan Mahmood , Singh Harpreet
Published: November 2022

Ischemic heart disease (IHD) is one of the leading causes of death related due to heart failure (HF). It is mainly characterized by impairment of heart contraction-relaxation (rhythmicity) and cardiac output. While a myriad of cellular processes is associated with IHD, one of the major cellular aspects strongly associated is the electrolyte and ionic imbalance. Serum sodium levels have been a well-established adverse prognostic marker for patients with chronic heart failure. Recent evidences have suggested that the lower serum chloride (hypochloremia, <90mM) is associated with increased mortality risk in patients with chronic HF independent of serum sodium levels. Serum chloride (Cl
-
) is a major anion in physiology but its correlation to IHD has not been studied extensively. Hence, to better understand the role of hypochloremia in HF patients, we reviewed patients who received a left ventricular assist device (LVAD) placement at The Ohio State University Medical Center. Survival analysis demonstrated that patients with hypochloremia before LVAD placement had decreased survival at one year compared to patients with normal serum Cl
-
(p=0.0055). Moreover, our e
x vivo
studies with isolated rat hearts indicate hypochloremia aggravates myocardial infarction post ischemia-reperfusion (IR) injury. Hence, to elucidate Cl
-
mediated signaling mechanism we incorporated highly translational human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Our cellular studies demonstrate that physiological alteration of extracellular Cl
-
affects the function of beating hiPSC-CM. Hypochloremia significantly increases the beating rate by reducing the calcium cycling durations in hiPSC-CM. Studies are ongoing to determine the involvement of the chloride channel in maintaining chloride homeostasis. Taken together, our results for the first time establish the novel role and mechanism of chloride homeostasis in IR injury. These results will advance the need to search for effective therapeutic approaches to target novel Cl
-
channels in IHD.
Abstract P2101: Time Dependence Of Esophageal Injury With And Without Proactive Cooling During High-power Short-duration Radiofrequency Ablation
Authors: Mercado-Montoya Marcela , Gomez-Bustamante Tatiana , Kulstad Erik B , Berjano Enrique , Mickelsen Steven R , Hernandez-Arango Pablo , Schieber Jay , Daniels James D
Published: November 2022

Background:
Recent data suggest that luminal esophageal temperature (LET) monitoring during high-power short-duration (HPSD) ablation may be inadequate to prevent esophageal thermal injury during radiofrequency (RF) ablation. This is due in part to delayed responses to thermal latency and the continued temperature rise that occurs in tissues even after the cessation of RF energy with HPSD settings. Proactive esophageal cooling has been shown to reduce severe esophageal thermal injury during medium-power medium-duration RF ablation, but less is known about the effects in HPSD ablation.

Objective:
We aimed to quantify the time course of esophageal damage in scenarios with and without proactive esophageal cooling, hypothesizing a reduction in the resulting tissue damage with proactive esophageal cooling.

Methods:
Using a computer model of RF ablation in the left atrium adjacent to the esophagus, we calculated temperature over time using ablation power of 50 W for 10 s and 90 W for 4 s. We then determined the fraction of resulting damage to esophageal tissue via the Arrhenius equation and by the percentage of tissue above lethal isotherm temperature of 50°C. Transmurality of injury was then plotted over time, and results were compared between ablation with and without proactive esophageal cooling.

Results:
Esophageal damage increased even after the cessation of RF energy, suggesting significant thermal latency effects under HPSD ablation. Lesion growth continued for >10 s after cessation of 50 W ablation energy, and for >12 s after cessation of 90 W ablation energy without cooling. With proactive cooling, lesion growth after cessation of ablation energy was essentially halted. Without esophageal cooling, maximum injury transmurality reached 79% with 50 W, and 58% with 90 W. With esophageal cooling, injury transmurality remained below 20% with 50 W, and 10% with 90 W, representing reductions of 75% and 83%, respectively.

Conclusions:
RF ablation using HPSD settings demonstrates significant latency of effect, with tissue damage and lesion growth continuing beyond the cessation of application of RF energy. Proactive esophageal cooling significantly dampens this effect, reducing esophageal injury by up to 83%.
Abstract P2107: Improving Stem Cell Models Of Duchenne Muscular Dystrophy-related Cardiomyopathy By Incorporation Of Physiologic Mechanical Stress
Authors: Fullenkamp Dominic E , Willis Alexander B , Curtin Jodi L , Amaral Ansel P , Harris Sloane I , Burridge Paul W , Demonbreun Alexis R , McNally Elizabeth M
Published: November 2022

Background:
Cardiomyopathy in Duchenne muscular dystrophy (DMD) is a significant driver of disease morbidity and mortality. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer the opportunity to study patient-specific mutations in the dish, however they generally suffer from immaturity. DMD is caused by mutations in the gene
DMD
, which codes for the protein dystrophin. Dystrophin localizes to the sarcolemma, forming part of a critical transmembrane mechanical and signaling complex that bridges the sarcomere to the extracellular matrix. Disruption of this axis leads to membrane fragility and mechanical stress is a main driver of DMD pathology. New therapies for DMD are in clinical development, including exon skipping, micro-dystrophin gene therapy, CRISPR-based gene editing, and membrane resealants, with some exon skipping strategies receiving FDA approval. The main outcomes of these therapies have been tied to improvement in skeletal muscle function with cardiac efficacy remaining understudied, despite its clear importance for patient outcomes.

Results:
Equibiaxial strain was employed as a physiologic mechanical stressor to simulate the in vivo mechanism of myocyte injury in an in vitro format using patient-derived DMD iPSC-CMs. By titrating strain, we determined that application of 10% strain for 2 hours robustly differentiates DMD and control iPSC-CMs using the clinically relevant biomarker LDH release as a proxy for membrane damage (control LDH release fold change: no significant change; DMD LDH release fold change: 2.51, p<0.0001). We evaluated DMD biomarkers released into the media using aptamer-based proteomics profiling and identified a striking strong correlation with serum biomarkers from DMD patients. Longer duration strain (24 hours) led to cell injury in both control and DMD iPSC-CMs. A membrane resealant was evaluated in this platform, which demonstrated enhanced recovery following treatment for both DMD and control iPSC-CMs with concomitant reduction in biomarker release.

Conclusions:
iPSC-CM models can be improved by incorporating physiologic mechanical stress, which is a main driver pathology. This platform is especially useful for evaluating dystrophic cardiomyopathy therapeutics.
Abstract P2118: Modeling Arrhythmia Risk In Human Stem Cell Derived Cardiomyocytes
Authors: Blancard Malorie , Fetterman K. A , Lyra Leite Davi M , Dellefave-Castillo Lisa , Webster Gregory G , Puckelwartz Megan , McNally Elizabeth M , George Alfred L , Burridge Paul W
Published: November 2022

In older (45+ years) populations, sudden cardiac death (SCD) is primarily caused by ischemic heart disease while in younger populations, SCD is often due to genetic causes. Despite recent advances in risk stratification based on clinical evaluation and genetic testing, our ability to predict SCD risk in the younger population remains inadequate. Implantable cardioverter defibrillators (ICDs) have been an essential intervention to reduce the number of out-of-hospital cardiac arrests in patients determined to be at risk of SCD. Nevertheless, one third of patients with an ICD receive inappropriate shocks and are exposed to infection without clear benefit. In addition, some children and young adults receive an ICD that will never deliver a shock even after decades of follow-up. No clear guidelines have been established to detect SCD-predisposing conditions due to cost, sensitivity, and specificity of testing modalities. Human iPSC-derived cardiomyocytes (hiPSC-CMs) are a widely recognized and reliable
in vitro
platform to study clinical phenotypes but have never been used in the context of SCD risk prediction. We hypothesize that we can establish an hiPSC-CMs cellular model to predict SCD risk. Sixty individuals were recruited and classified in one of five categories, ranging from healthy controls with no known cardiac disease (category 1) to individuals that show the highest SCD risk (category 5). These 5 categories are defined by 3 main parameters: 1) absence or presence of ICD, 2) whether the ICD was placed as primary or secondary prevention and 3) the number of shocks received after ICD implantation. hiPSCs were reprogrammed using blood samples and then differentiated into cardiomyocytes. Molecular and electrophysiology measurements were performed for the different risk categories using high-throughput systems. hiPSC-CMs were assayed by single-cell calcium imaging, microelectrode array and automated action potential and current recordings.
In vitro
measurements revealed that specific parameters such as field potential duration correlate with the SCD risk and outcome. In conclusion, this study will provide a cellular-based tool to determine patient-specific SCD risk in order to advance medical care for at-risk patients and their family members.
Abstract P3007: Exposure To Particulate Air Pollution Dysregulates Splenic Inflammation Resolution And Erythrocyte Processing
Authors: Asplund Haley , Haberzettl Petra , Hellmann Jason , Conklin Daniel J , Jones Steven , Bhatnagar Aruni , Sansbury Brian E
Published: November 2022

Chronic exposure to ambient particulate matter (PM) increases the risk of cardiovascular disease (CVD). One proposed mechanism linking PM exposure and CVD is the induction of low-grade systemic inflammation that underlies disease progression. However, whether exposure to fine PM (PM
2.5
) directly affects the endogenous pathways that resolve the inflammatory response is not known. Resolution of inflammation is distinct from anti-inflammation in that rather than blunting the magnitude of the initial inflammatory response, it is the active promotion and acceleration of its decay. Defective or insufficient resolution is a hallmark of many chronic inflammatory diseases including atherosclerosis and diabetes. Therefore, the overall goal of this study was to elucidate how PM exposure impacts the resolution of inflammation and how this relates to the subtle, but extensive pathological changes brought about by PM exposure. We found that in mice exposed PM
2.5
for 30 days there was a significant decrease in the abundance of specialized lipid mediators that promote resolution (SPMs) in the spleen. The changes in spleen are particularly critical due to its central role in regulating immune response dynamics and as a filter to remove senescent and damaged erythrocytes. While SPMs are known to enhance macrophage erythrophagocytosis
ex vivo
, how decreased
in vivo
abundance affects splenic function remains unclear. Therefore, we performed RNA sequencing of PM
2.5
-exposed spleens and found that numerous pathways involved in both erythrophagocytosis and myelopoiesis were significantly upregulated, including those related to myeloid cell homeostasis and development, erythrocyte differentiation, iron ion homeostasis, and heme biosynthesis and metabolism. Additionally, we identified significant alterations in circulating red blood cells, including decreased expression of CD47, a prominent marker of self and “don’t eat me signal”, on the surface of circulating erythrocytes after exposure to PM
2.5
, as well as increased red cell distribution width. Together, these data suggest that PM exposure alters red blood cells and may stimulate enhanced erythrophagocytosis in the spleen, increasing red blood cell turnover.
Abstract P3012: Syndecan 1 Regulates Lung Endothelial Barrier Function In Homeostatic And Injury Conditions
Authors: Thota Lakshmi , Chignalia Andreia
Published: November 2022

Introduction:
Acute lung injury (ALI) is an inflammatory syndrome that may occur secondarily to bacterial infection. Pulmonary edema is a severe complication of ALI and it may result from endothelial barrier disruption. The mechanisms that initiate ALI during bacterial infection remains unresolved. Syndecan 1 (Sdc1) is a heparan sulfate proteoglycan that contributes to endothelial barrier stability and is cleaved during inflammatory processes. Whether Sdc1 contributes to ALI progression is unresolved. Herein we investigated if Sdc1 mediates LPS-induced ALI in mice.

Methods:
Male and Female Sdc1 knockout (Sdc1KO) and wild type (WT) mice, with 5-8 weeks of age, were injected with lipopolysaccharide (LPS, 10mg/Kg, IP) or PBS and euthanized after 6hr. Bronchoalveolar lavage fluid (BAL) and lung tissue were collected and assessed for myeloperoxidase (MPO) activity, albumin content, RhoA activity using commercially available spectrometric and ELISA kits. Lung edema was assessed by lung wet/dry ratio (W/D).

Results and Conclusions:
LPS increased lung W/D by 1.5-fold (p=0.0042, n≥5/group) in WT but not Sdc1KO mice. Sdc1KO showed higher basal lung W/D
vs
WT (Sdc1KO=3.55
vs
WT=2.21, p=0.044, n≥5/group). Similarly, a trend increase in basal lung MPO activity was seen in Sdc1KO when compared to WT (p=0.33, n≥5/group). A significant increase in MPO activity was seen in LPS-treated WT (LPS=0.47±0.03
vs.
PBS=0.26±0.03, p=0.003) but not LPS-treated Sdc1KO mice (p=0.11; n≥5/group) when compared to matched PBS-treated strain controls. Albumin content was increased by LPS only in WT BAL (LPS=106.9±8.9
vs
PBS=63.65±6.15, p=0.03, n≥5/group). LPS increased RhoA activity exclusively in WT lungs. (LPS=0.04±0.002
vs
PBS= 0.02±0.003, p=0.04, n≥5/group). A trend increase in RhoA activity was seen in PBS-treated Sdc1KO when compared to PBS-treated WT (p=0.06, n≥4/group). Collectively, our data suggest that Sdc1KO show basal lung endothelial barrier failure. This phenotype is associated with decreased response to LPS. The mechanisms whereby Sdc1 regulate barrier function may involve RhoA-dependent pathway. Sdc1 may participate in lung edema development during ALI.
Abstract P3024: Mitochondrial Dysfunction Underlies Sinoatrial Node Dysfunction In Heart Failure With Preserved Ejection Fraction
Authors: Mesquita Thassio , Miguel-dos-Santos Rodrigo , Stotland Aleksandr , Jones Xaviar , Motawakel Jacob , Cho Jae H , Fournier Mario , Liu Weixin , Marban Eduardo , Cingolani Eugenio
Published: November 2022

Background:
Metabolic comorbidities are frequent in patients with heart failure with preserved ejection fraction (HFpEF). We recently demonstrated that chronotropic incompetence and exercise intolerance are associated with intrinsic sinoatrial (SAN) dysfunction in animal models of HFpEF. However, there are no studies investigating whether metabolic dysfunction in HFpEF can lead to mitochondrial dysfunction in the SAN.

Hypothesis:
Our recent findings uncovering SAN pacemaker dysfunction in HFpEF upon metabolic stress led us to test the hypothesis that mitochondrial dysfunction may underlie this condition.

Methods:
Male C57Bl6 mice fed high fat diet (HFD) plus nitric oxide inhibitor (L-NAME) or regular chow served as HFpEF and control, respectively. Optical mapping, transcriptomics, targeted quantitative proteomics of mitochondrial proteins, mitochondrial respiration and reactive oxygen species (ROS) assays were conducted using explanted mouse SAN tissue or isolated pacemaker cells.

Results:
SAN from HFpEF-verified animals after 20 weeks of HFD+LNAME exhibited prolonged SAN recovery time after pacing (100%; P<0.05). RNA sequencing revealed augmentation of gene clusters related to metabolism (i.e.
ucp
and
pdk4
) and extracellular matrix expansion (i.e.
col, postn
and
Cilp1
). Targeted proteomics validated SAN RNA-sequencing findings, showing upregulation of proteins related to ROS scavenging, fatty acid transport and TCA/OXPHOS (i.e. SOD, CPT2 and DHSA/B). Depressed maximal mitochondrial respiration was seen in isolated mitochondria from SAN of HFpEF animals compared to controls (-42%; P<0.05). This was supported by increased generation of mitochondrial ROS in single pacemaker cells from HFpEF SAN (70%; P<0.05). Acute mitochondrial dysfunction induced by a mitochondrial uncoupler (FCCP) elicited a pronounced prolongation of SAN recovery time in HFpEF compared to controls (165%; P<0.05).

Conclusion:
Our results indicate that mitochondrial function is depressed in SAN from HFpEF animals, and is associated with SAN pacemaker dysfunction. Further studies are required to test cause-and-effect and to evaluate potential therapeutic strategies targeting mitochondrial pathways for HFpEF associated abnormalities of sinus rhythm.
Abstract P3027: Intermittent Fasting Improves High Fat Diet Mediated Heart Failure With Preserved Ejection Fraction
Authors: Pan Guodong , Roy Bipradas , Rattan Ramandeep , Palaniyandi Suresh S
Published: November 2022

Intermittent fasting (IF) has improved metabolic aberrations in metabolic diseases, however, the mechanism is unclear. Our lab focuses on 4-hydroxy-2-nonenal (4HNE), an oxidative stress byproduct which accumulates in metabolic diseases, in cardiac damage and activity of aldehyde dehydrogenase 2 (ALDH2), a 4HNE detoxifying enzyme, which was implicated in cardioprotection. We reported that the ALDH2*2 knock-in mutant mice (AL) with low intrinsic ALDH2 activity that were fed with high-fat diet (HFD) exhibited severe heart failure with preserved ejection fraction (HFpEF) compared to HFD fed control mice (C57). We hypothesized that IF can contribute to the improvement of HFpEF even in AL mice by reducing 4HNE. C57 and AL mice that were fed HFD and L-NAME (nitric oxide inhibitor) in drinking water were grouped as
C57-free
and
AL-free
meaning taking food freely or
C57-IF
and
AL-IF
meaning daily 16 hours fasting with 8-hr free access to food. After 2-months of HFD feeding, the body weights of C57-IF (35.5±3.8g) and AL-IF (36.7±3.4g) groups were significantly lower than C57-free (43.5±4.2g, p<0.05 vs C57-IF) and AL-free (45.8±5.6g, p<0.05 vs AL-IF) groups. The 24-hour food intakes of C57-IF (8.6±0.9 KCal) and AL-IF (8.8±1.1 KCal) were significantly decreased, compared to C57-free (12.3±1.7 KCal, p<0.05 vs C57-IF) and AL-free (13.3±1.5 KCal, p<0.05 vs AL-IF) groups. The respiratory exchange ratio (RER) of AL-free group (0.62±0.12) was significantly lower than three other groups (p<0.05 vs C57-free, C57-IF and AL-IF, respectively). The E/A ratio (E/A), a diastolic parameter, was decreased in C57-free (1.14±0.21) and AL-free (0.89±0.17) groups, not in C57-IF (2.15±0.31, p<0.05 vs C57-free) and AL-IF (1.97±0.25, p<0.05 vs AL-free) groups. The E/A in AL-free group was also lower than C57-free group (p<0.05). Another diastolic parameter, isovolumic relaxation time (IVRT) was also prolonged in C57-free (27.3±3.3 ms) and AL-free (29.4±3.7 ms) mice, not in C57-IF (13.3±1.8 ms, p<0.05 vs C57-free) and AL-IF (15.2±2.1 ms, p<0.5 vs AL-free) mice. In conclusion, IF decreased food intake and obesity, and improved metabolism and heart function in both C57 and ALDH2*2 mice with HFpEF, and also diminished the differences in RER and E/A between them via lowering the 4HNE adducts.
Abstract P3040: Mitochondrial Membrane Protein Sigmar1 Regulates Mitochondrial Ultrastructure And Respiratory Functions In The Heart
Authors: Abdullah Chowdhury S , Aishwarya Richa , Alam Shafiul , Remex Naznin Sultana , Nitu Sadia S , Orr A. Wayne , Hartman Brandon , King Judy A. , Bhuiyan Md Shenuarin
Published: November 2022

Background:
Sigma-1 receptor (Sigmar1) is multifunctional, ubiquitously expressed chaperone protein in mammalian cell systems. Earlier biochemical and immuno-electron microscopic analysis revealed Sigmar1’s cellular localization depends on cell and tissue systems, explaining its involvement in regulating diverse cellular and molecular functions. However, Sigmar1’s localization at organelle levels, membrane tropism, and direct molecular functions in cardiomyocytes remain elusive.

Objective:
We conducted systematic biochemical, microscopic, and functional assays to define Sigmar1’s subcellular localization, membrane topology, and functions in the heart.

Methods and Results:
Immuno-labeled quantum dots (QDs) in transmission electron microscopy revealed anti-Sigmar1 labeled QDs on the mitochondrial membranes, lysosomes, and at the sarcoplasmic reticulum-mitochondria interface. Subcellular fractionation of heart lysates also confirmed Sigmar1’s distribution in purified mitochondrial and lysosomal fractions. We further confirmed Sigmar1’s mitochondrial localization by Sigmar1’s colocalization with Mito-Tracker Red labeled isolated cardiac mitochondria. Biochemical experiments constituting alkaline extraction, detergent solubilization, and dose-dependent Proteinase K digestion on isolated heart mitochondria demonstrated Sigmar1 as an integral mitochondrial membrane protein. Sigmar1’s structural requirement for mitochondrial localization by expressing Flag-tagged Sigmar1 fragments demonstrated N-terminal required for mitochondrial localization. High-resolution mitochondrial respirometry studies showed that genetic loss of Sigmar1 in primary cardiomyocytes reduces mitochondrial respiration. Sigmar1 knock-out mice hearts also showed the accumulation of abnormal, irregular-shaped giant mitochondria and defects in mitochondrial respiration.

Conclusions:
Overall, our studies demonstrated that Sigmar1 is an integral mitochondrial membrane protein and is essential in maintaining mitochondrial ultrastructure and respiratory functions in the heart.
Abstract P3055: Combined Metabolomic And Transcriptomic Profiling Approaches Reveal The Cardiac Response To High-fat Diet
Authors: Morrow John P , Joseph Leroy C , Nguyen Quynh , Shi Jianting , Zhang Hanrui
Published: November 2022

The response of vital organs to different types of nutrition or diet is a fundamental question in physiology. It is unclear how different diets can alter cardiac metabolism and function. We examined the cardiac response to 4 weeks of high-fat diet in adult mice, measuring cardiac metabolites and mRNA. Metabolomics showed dramatic differences after high-fat diet, including increases in several acyl-carnitine species. The RNAseq data showed changes consistent with adaptations to use more fatty acid as substrate and an increase in the anti-oxidant protein catalase. Changes in mRNA were correlated to changes in protein level for several highly responsive genes, including catalase and HMGcs2. We also found significant sex differences in both metabolomics and RNAseq datasets, both at baseline and after high fat diet. This work reveals the response of a vital organ to dietary intervention at both metabolomic and transcriptomic levels, which is a fundamental question in physiology that may have relevance for cardiovascular diseases. This work also reveals significant sex differences in cardiac metabolites and gene expression.

Figure 1: Metabolomic changes after HFD; electron microscopy shows changes in mitochondrial morphology.
A. Principal component analysis (loading) of metabolites demonstrates that the first two principal components (PC) cluster the samples into 4 groups. PC1 + PC2 account for 85.47% of the proportion of variance in this metabolomic dataset.B. Graph of acyl-carnitine species, dots with lines indicated mean +/- SEM. “C#” refers to the number of carbon atoms in the lipid, that is C16 is palmitoylcarnitine, C14 is myristoylcarnitine, etc. N= 10 hearts for each group. The comparison of control (chow) to HFD is highly statistically significant by 2-way ANOVA, p<0.0001.C. Representative examples of electron microscopy from cardiac ventricular samples.D. Graphs of average mitochondrial area (um
2
) and average number of mitochondria per high-power image. N= 4 hearts for each group, * indicates significantly different by t-test.
Abstract P3061: A Monoclonal Antibody Blocking Interleukin-1 Receptor Accessory Protein Reduces Acute Viral Myocarditis Severity
Authors: Lema Diego A , Talor Monica , Rattik Sara , Gronberg Caitriona , Liberg David , Cihakova Daniela
Published: November 2022

IL-1, IL-33 and IL-36 have complementary, pro-inflammatory roles during the immune response that promote autoimmune diseases, particularly myocarditis. While antibodies that neutralize circulating cytokines exist, we hypothesized that blockade of their shared co-receptor, IL-1 receptor accessory protein (IL1RAP), would exhibit a more potent and broader anti-inflammatory profile. The CAN10 antibody blocks IL1RAP and is currently under development for treating severe autoimmune and inflammatory diseases. To investigate if IL1RAP blockade is an effective treatment of cardiac inflammation, we induced autoimmune viral myocarditis in 8-week-old Balb/c male mice with Coxsackievirus B3 and treated them with mCAN10, an anti-mouse IL1RAP monoclonal antibody. Mice were euthanized at day 10, the peak of acute inflammation in the heart. IL1RAP blockade reduced the severity of myocarditis at this timepoint when compared to isotype, saline or IL-1 receptor antagonist (IL1RA) treatment (the gold standard in IL-1 receptor blockade), without affecting viral clearance from the heart. Spectral flow cytometry of heart immune populations revealed that mCAN10 significantly reduced the number of infiltrating leukocytes of broad populations, especially effector CD4
+
and CD8
+
T cells, inflammatory Ly6C
+
CCR2
+
monocytes, neutrophils and eosinophils. We also observed early echocardiographical parameters suggesting improved heart function, including ejection fraction, fractional shortening and interventricular septum diameter. Interestingly, mCAN10-treated mice demonstrated reduced proliferation of bone marrow precursors of inflammatory myeloid and lymphoid lineage leukocytes. Splenic immune cell populations were not affected, indicating that mCAN10 does not cause global immune suppression in these animals. Altogether, our data show that a single monoclonal antibody targeting three inflammatory cytokine systems, IL-1, IL-33 and IL-36, potently reduces acute viral myocarditis severity in a broader fashion that is not recapitulated by IL-1α/β blockade, and shows translational promise for therapeutics of inflammatory disease. Phase I clinical studies of the CAN10 antibody are in preparation.
Abstract P3064: Immunoengineered Tantalum Carbide Mxene Quantum Dots For Prevention Of Transplant Vasculopathy
Authors: Dhingra Sanjiv , Yan Weiang , RAFIEERAD ALIREZA , Alagarsamy Keshav Narayan , Arora Rakesh C
Published: November 2022

Transplant vasculopathy is an aggressive form of atherosclerosis that manifests uniquely in transplanted hearts, lungs, and kidneys. Recently, immunoengineered transition metal carbides, or MXenes, have demonstrated unique immunomodulatory properties and this can potentially be leveraged to prevent transplant vasculopathy. Here, we present the synthesis, characterization and application of novel zero-dimensional tantalum carbide (Ta
4
C
3
T
x
) MXene quantum dots (MQDs) for immunomodulation. A facile hydrofluoric acid-free etching protocol was developed to synthesize zero-dimensional Ta
4
C
3
T
x
MQDs from Ta
4
AlC
3
MAX phase. The resultant MQDs are 3 to 7 nm in diameter and are surface modified with carboxyl, hydroxyl, and amine functional groups for biological interactions. Immunomodulation was assessed
in vitro
through co-cultures of human umbilical vein endothelial cells (ECs) and peripheral blood mononuclear cells. We found that MQDs interact with activated human ECs
in vitro
to reduce activation and pro-inflammatory Th1 polarization of allogeneic CD4
+
lymphocytes. Mechanistically, we showed using quantitative PCR that treatment with MQDs significantly increased endothelial surface expression of the T-cell co-inhibitory molecule PD-L1 and decreased the co-stimulatory molecule CD86. Furthermore, exploratory bulk RNA-sequencing of co-cultured lymphocytes showed decreases in the immunologic signatures of naïve T-cells and increases in the immunologic signatures of CD25
+
regulatory T-cells, suggesting activation of this suppressive T-cell population. Finally, when applied in an
in vivo
rat model of transplant vasculopathy, treatment with MQDs reduced lymphocyte infiltration and preserved medial smooth muscle cell integrity within transplanted aortic segments. These findings suggest that these novel MQDs have potential as an effective treatment to ameliorate transplant vasculopathy.
Abstract P3070: Distinct Inflammatory Milieu In Patients With Right Heart Failure
Authors: Yang Bin Q , Liu Jason , Schilling Joel D
Published: November 2022

Introduction:
Right heart failure (RHF) is associated with worse clinical outcomes in patients with heart failure (HF) and pulmonary hypertension. Previous studies have shown elevated circulating proinflammatory cytokines in patients with HF; however, no study has compared the inflammatory signature in patients with RHF to those with HF and preserved right ventricular (RV) function.

Hypothesis:
We predicted that patients with RHF would have a distinct inflammatory milieu compared to normal and HF patients.

Methods:
We recruited adult patients undergoing right heart catheterization (RHC) and excluded those with a history of transplant, autoimmune disease, malignancy, or infection. Patients underwent blood sampling during RHC and were classified into normal, HF, and RHF cohorts
a priori
. We then performed multiplex protein assay on serum targeting cytokines, chemokines, and growth factors.

Results:
A total of 43 patients were included. HF and RHF patients had similar demographics and cardiac indices, but the RHF cohort had elevated markers of congestion characterized by higher right atrial pressure and creatinine, and a trend towards higher bilirubin (Figure). Patients with RHF had elevated levels of select proinflammatory cytokines, chemokines, and growth factors. Of the factors measured, VEGF-A, soluble CD163, and CXCL12 were predictive of all-cause mortality and LVAD/transplant-free survival.

Conclusion:
Patients with RHF have a distinct proinflammatory signature compared to those with preserved RV function. VEGF-A, soluble CD163, and CXCL12 predicted clinical outcomes in an advanced HF population.
Abstract P3078: Stress-induced Early Recruitment Of Neutrophils To The Infarcted Hearts Influence Cardiac Outcomes.
Authors: Dahdah Albert , Park Ki H , Sreejit Gopalkrishna , Jaggers Robert , Johnson Jillian , Nagareddy Prabhakara R
Published: November 2022

Myocardial infarction (MI)-induced heart failure is one of the leading causes of death in the modern world. This is in part due to abnormal cardiac remodeling, instigated by a large influx of neutrophils immediately following MI. These activated neutrophils in the heart release a plethora of proteolytic enzymes and exacerbate tissue injury leading to cardiac failure and death. The role of neutrophils during MI is complex with the intensity and duration of their residency dictating the ensuing remodeling process.

We have previously shown that the recruitment of neutrophils to heart post-MI is mirrored by an increased proliferation of progenitors (granulopoiesis) in the bone marrow (BM). However, the increase in the number of neutrophils at the infarct begins much earlier (< 6 hours post MI) than the peak granulopoiesis (24 hours post-MI) in the BM. These finding suggest that sources other than granulopoiesis may contribute heavily to the initial neutrophil burden of the heart. One such source is the marginated pool. In the circulation, the marginated pool of neutrophils represents the same size as that of the circulating pool. We hypothesize that “
demargination of neutrophils from the vascular wall contribute to the neutrophil burden of the heart particularly during the early hours after MI
”. To test this hypothesis, we created a mouse model of MI by permanent ligation of the left anterior descending coronary artery and studied the impact of early recruitment of neutrophil to ischemic heart.

We found that the initial wave of neutrophils recruited to the ischemic heart were exclusively sourced from the vasculature and not granulopoiesis in the BM/ Spleen. The neutrophils that were recruited to the heart exhibit distinct hallmarks of demargination including decreased F-actin, CD62L and increased Adam17 expression. This early recruitment of neutrophils was orchestrated by catecholamine stress as strategies aimed at inhibition of catecholamine biosynthesis or blockade of β adrenergic receptors (β-AR) significantly reduced neutrophil burden and improved cardiac function. Interestingly, this protective effect was lost when neutrophil recruitment was suppressed over a longer period. In conclusion we show that while short-term blockade of β-AR signaling suppresses the early neutrophil swarming, suppression beyond the reparative phase (> 3 days post-MI) adversely impact the cardiac function. Together these data suggest that catecholamine stress induced-demargination constitute the major source of neutrophils during the early hours of MI. Understanding the precise signaling mechanisms that drive demargination and optimizing the right dose/duration of a specific β-AR blocker may help suppress the initial onslaught of neutrophils without compromising their overall beneficial function during the reparative phase of inflammation.
Abstract P3081: Glypican 3 Regulates Diabetes Induced Mesenchymal Stromal Cells Dysfunctions
Authors: Joladarashi Darukeshwara , Gurrala Charan T , Magadum Ajit , Cimini Maria , Mallaredy Vandana , Gonzalez Carolina , Lucchese Anna Maria , Truongcao May , Wang Chunlin , Benedict Cindy , Cheng Zhongjian , Kishore Raj
Published: November 2022

Mesenchymal stromal cells (MSC) based therapies are considered as an ideal stem cell-based treatment for cardiovascular diseases due to their immunosuppressive characteristics, anti-inflammatory properties, and differentiation potential. Diabetic microenvironment encompassing high glucose, inflammation, hypoxic conditions, and reactive oxygen species content has deleterious effects on the functionality of MSCs. The purpose of this study was to understand the molecular basis of diabetes-induced MSC dysfunction and potentially reversing these dysfunctions to enhance cell-based therapeutics for myocardial repair in diabetic patients. we isolated MSCs from bone marrow of mice from db/+ non-diabetic (WT-MSC) and diabetic mice (db/db-MSC) and examined the effect of diabetes on MSC differentiation, proliferation, angiogenesis, and immunomodulation
in vitro,
and their reparative functions on myocardial injury repair post-MI in mice. Compared to WT-MSC, MSCs isolated from bone marrow of diabetic mice showed impaired differentiation, decreased proliferation, reduced immunomodulatory activities and impairment to promote endothelial cell tubulogenesis. Our data also shows diminished functional activity of diabetic MSCs to improve post-MI cardiac functions. Furthermore, we found that glypican-3 (GPC3), a heparan sulfate proteoglycan, is highly upregulated in diabetic MSCs when compared to WT-MSC. GPC3 overexpression in WT-MSC showed decreased proliferation, lowers the immunosuppression activity, and reduced vascular tube formation. Interestingly, GPC3 knockdown in WT-MSC showed increased proliferation, improved the immunosuppression activity, and enhanced vascular tube formation. Our data also showed that, knockdown of GPC3 in diabetic MSCs restore their functions. In conclusion, these data suggest that diabetes increases GPC3 expression in MSC thereby impairs MSC functions under diabetic condition and knockdown of GPC3 in diabetic MSC rescued diabetes-induced dysfunctions. Ongoing studies are testing whether ex vivo GPC3 knockdown in db/db MSCs will rescue their defective cardiac reparative activities, post-MI.
Abstract P3082: Emergency Granulopoiesis Increases Neutrophil Phenotypic And Functional Diversity
Authors: Stephan Jonah , Gu Zhen , Taylor Zoubul , Wysoczynski Marcin
Published: November 2022

Ischemic injury due to myocardial infarction (MI) elicits immune cell infiltration to remove necrotic tissue and facilitate scar formation. Neutrophils are among the first infiltrating immune cells, and neutrophilia has been commonly accepted as a biomarker of adverse prognoses post-MI. The increased demand for neutrophils is met by an accelerated granulocyte production in a process known as emergency granulopoiesis. However, the impact of emergency granulopoiesis on neutrophil function remains cryptic. These experiments seek to determine the impact of emergency granulopoiesis on neutrophil phenotype and secretory function. To activate emergency granulopoiesis, C57BL/6 mice were injected with G-CSF for 3 d (100 μg/kg/day, s.c.). Neutrophil phenotype and function were evaluated 24 h after the last dose of G-CSF. We found that G-CSF injections resulted in increased neutrophils in peripheral blood, spleen, and bone marrow. Flow cytometric analysis revealed that neutrophils produced via emergency granulopoiesis showed lower expression of the classical neutrophil marker Ly6G and an increased number of CD101
Neg
neutrophils in peripheral blood and spleen (n=3-6/group, p<0.05). Functional analysis
in vitro
showed that neutrophils produced via emergency granulopoiesis are 2-3 times more susceptible to PMA- and fMLF-induced degranulation of primary granule content (neutrophil elastase and myeloperoxidase), but release of secondary (NGAL, CHI3L1), and tertiary granules (MMP9) remained unchanged (n=5/group, p<0.05). Based off these observations, we conclude that emergency granulopoiesis results increased diversity of neutrophil phenotype and function. Future studies will elucidate the impact of emergency granulopoiesis neutrophils on post-MI scar formation and chronic ventricular remodeling.
Abstract P3091: Transgenic Expression Of Nrf2 Induces A Pro-reductive Stress And Adaptive Cardiac Remodeling In Mice
Authors: Jyothidasan Arun , Murugesan Saravanakumar , Quiles Justin , Challa Anil Kumar , Cinghu Senthil Kumar , Nanda Vivek , Namakkal-Soorappan Rajasekaran N
Published: November 2022

Background:
NFE2L2, nuclear factor, erythroid 2 like 2 (Nrf2), is a transcription factor that protects cells through maintaining a homeostatic redox state during stress. Constitutive expression of Nrf2 (CaNrf2-TG) was previously shown to be pathological to the heart over time. We tested a hypothesis that the cardiac-specific expression of full length Nrf2 (mNrf2-TG) would moderately increase the basal antioxidant defense, triggering a pro-reductive environment leading to adaptive cardiac remodeling.

Methods:
Cardiac-specific full-length Nrf2 transgenic (mNrf2-TG) and non-transgenic (NTG) mice hearts on the C57/BL6 background at 7-8 months of age were used to analyze the transcriptome and cardiac structure/function. Next generation sequencing (NGS) for RNA profiling (n=3/group) and qPCR-based validation (n=5/group) of the NGS data were accomplished. Myocardial redox levels and imaging (echocardiography) were performed.

Results:
Transcriptomic analysis revealed that out of 14665 identified mRNAs, 679 were differently expressed (DEG) in TG hearts. Of 679 DEGs, 429 were upregulated and 251 were downregulated significantly (FC>2, P<0.05). Gene Set Enrichment Analysis revealed that the top altered pathways were (a) Nrf2 signaling, (b) glutathione metabolism and (c) ROS scavenging. A comparative analysis of glutathione redox state in the hearts demonstrated significant differences between pro-reductive vs. hyper-reductive conditions (233 ± 36.7 and 380 ± 68.7 vs. 139 ± 8.6 μM/mg protein in mNrf2-TG and CaNrf2-TG vs NTG). Expression of fetal genes and hypertrophic biomarkers, cytoskeletal genes, histone deacetylases (HDACs), and GATA transcription factors were moderately increased in mNrf2-TG compared to CaNrf2-TG hearts. Non-invasive echocardiography analysis revealed an increase in systolic function (Ejection Fraction) in mNrf2-TG, suggesting an adaption, as opposed to pathological remodeling in CaNrf2-TG mice experiencing a hyper-reductive stress, leading to reduced survival (40% at 60 weeks).

Conclusion:
The effects of excess Nrf2-driven antioxidant transcriptome revealed a pro-reductive condition in the myocardium leading to an adaptive cardiac remodeling. While pre-conditioning the myocardial redox with excess antioxidants (i.e. pro-reductive state) could be beneficial against oxidative stress, a chronic pro-reductive environment in the myocardium might transition the adaptation to pathological remodeling.