bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P3032: Liver-directed Mitochondrial Uncoupling Attenuates The Progression Of Early And Late-stage Atherosclerosis In Mice
Authors: Goedeke Leigh A , Zhang Xinbo , Sun Jonathan , Canfran-Duque Alberto , Rotllan Noemi , Nasiri Ali , Kahn Mario , Zhang Xian-Man , Fernandez Hernando Carlos F , Shulman Gerald
Published: November 2022

Atherosclerotic cardiovascular disease (CVD) remains a leading cause of morbidity and mortality in people with type 2 diabetes (T2D) and new therapies are urgently needed. We recently developed an orally available, liver-directed controlled-release mitochondrial protonophore (CRMP) and showed that this agent safely reverses hypertriglyceridemia, fatty liver, and T2D in rodent and nonhuman primate models of obesity by promoting a subtle sustained increase in hepatic mitochondrial inefficiency. To determine the effect of CRMP on atherogenesis, we performed intervention studies in which 12-week Western-diet fed
Ldlr
-/-
mice with established atherosclerosis were treated with CRMP (30 mg/kg-day) or vehicle control for an additional 12 weeks. CRMP-treated mice displayed a significant reduction in fasting plasma and hepatic triglyceride content, independently of changes in body weight, fasting plasma glucose, insulin or cholesterol levels. Morphometric analysis of the aortic root and brachiocephalic artery revealed that CRMP treatment significantly reduced total plaque area and neutral lipid accumulation. CRMP treatment also decreased markers of plaque vulnerability, as evidenced by a reduction in necrotic core area and increase in fibrous cap area (
P
<0.05 vs veh). To determine the mechanisms by which CRMP attenuated atherosclerosis, we performed progression studies in

Ldlr
-/-

mice fed a Western diet containing CRMP or vehicle control for 12 weeks. Similarly to the intervention studies, CRMP significantly reduced aortic root plaque area, neutral lipid accumulation and necrotic core area. These changes were associated with a reduction in plasma triglyceride content and improvement in whole-body insulin sensitivity as assessed by hyperinsulinemic-euglycemic clamps (all
P
<0.05 vs veh). Single-cell RNA sequencing of whole aortas from CRMP and vehicle-treated
Ldlr
-/-
mice revealed that CRMP reduced the frequency of inflammatory macrophages compared to vehicle (~19% vs ~2%), consistent with an immune-dampening effect of CRMP. Taken together, these data support an anti-atherogenic role for CRMP and suggest the therapeutic potential of liver-directed mitochondrial uncouplers for the treatment of early and late-stage atherosclerosis.
Abstract P3033: The Pathogenic R120G Mutant Of Alpha-B-crystallin Accumulates Within Cardiac Mitochondria
Authors: Quiles Justin M , Chi Liguo , Najor Rita H , Gustafsson Åsa B
Published: November 2022

Alpha-B-crystallin (CryAB) is an abundant molecular chaperone comprising nearly 5% of total cardiac mass. While predominately cytosolic, some studies note mitochondrial translocation of CryAB during stress. The autosomal dominant R120G mutation leads to CryAB misfolding and protein aggregation resulting in cardiomyopathy. Although mitochondrial dysfunction has been reported in cardiac-specific CryAB
R120G
transgenic mice, it is unclear whether these effects are secondary to the formation of cytotoxic aggregates or directly relate to mitochondrial damage. Using proteinase K protection assays in isolated mitochondria from mouse hearts and embryonic fibroblasts (MEFs), we have discovered that wild-type (WT) CryAB is present in the mitochondrial matrix indicating that this protein likely functions as a chaperone within the organelle. Interestingly, however, compared to the WT protein, CryAB
R120G
was significantly more enriched in MEF mitochondria. Furthermore, treatment of mice with adeno-associated virus serotype 9 (AAV9) encoding CryAB
R120G
under the control of a cardiac-specific promoter resulted in time-dependent intramitochondrial CryAB
R120G
accumulation across 2 and 4-weeks post injection. These data are the first to indicate that misfolded CryAB
R120G
resides specifically within the mitochondrial matrix. Knockdown of the mitochondrial matrix protease LonP1 significantly increased intramitochondrial as well as total CryAB
R120G
protein levels, and sensitized MEFs to cell death indicating that Lonp1 functions to proteolytically process imported CryAB
R120G
. While this response may acutely preserve mitochondrial function, sustained accumulation of intramitochondrial CryAB
R120G
likely exhausts proteolytic machinery within the organelle leading to induction of mitochondrial autophagy (mitophagy). Indeed, mitochondrial respiration was unchanged in both AAV9-treated and homozygous CryAB
R120G
knock-in mice at early time-points, whereas mitophagy-deficient Parkin
-/-
mice displayed significantly exacerbated mitochondrial CryAB
R120G
protein accumulation. Altogether, these data indicate aberrant accumulation of misfolded proteins within cardiac mitochondria underlies the pathogenicity of CryAB
R120G
.
Abstract P3035: Time-of-intake Regulates Glucocorticoid Effects On Heart Metabolism And Function Through Cardiomyocyte-autonomous Mechanisms
Authors: Quattrocelli Mattia , Wintzinger Michelle , Miz Karen , Panta Manoj , Durumutla Hima B , Prabakaran Ashok D
Published: November 2022

Glucocorticoids are circadian regulators of energy balance. However, the cardiomyocyte-autonomous mechanisms of glucocorticoid action on heart metabolism remain unresolved. Also, little attention is given to the impact of circadian time of intake on the effects of these drugs in heart. Here, we investigated whether circadian time of exposure gates the effects of synthetic glucocorticoids on heart metabolism and function. In single pulse experiments, we compared the effects of the glucocorticoid prednisone in wildtype (WT) hearts through light-phase- versus dark-phase-specific injections. Light-phase pulse - but not dark-phase pulse - increased NAD
+
and ATP levels in murine hearts in vivo for one circadian cycle post-pulse. To ask whether this effect was replicated in chronic treatment, we used the intermittent once-weekly prednisone regimen that we previously used to boost muscle metabolism without the wasting and metabolic toxicity caused by once-daily dosing. In WT mice treated for 12 weeks with once-weekly prednisone, light-phase-specific dosing gated the positive effects of treatment on NAD
+
, mitochondrial respiration and abundance in heart, as compared to dark-phase-specific dosing. These processes were cardiomyocyte-autonomous as the circadian-specific treatment effects were blocked by inducible cardiomyocyte-specific ablation of the glucocorticoid receptor. To probe relevance of this treatment for the injured heart, we treated mice after induction of ischemic injury. Light-phase-specific prednisone treatment significantly improved NAD
+
, mitochondrial function and systolic function in injured hearts. RNA-seq showed that treatment elicited a virtuous gene program of circadian metabolism in the myocardium. Indeed, the effects of treatment on heart metabolism gene cascades were dependent on the intact heart peripheral clock, as shown by mice with inducible cardiomyocyte-specific ablation of the clock factor BMAL1. In summary, our data indicate that glucocorticoids regulate cardiomyocyte metabolism in a cell-autonomous fashion hinging upon circadian-time of intake and interaction with the peripheral clock. Our work supports a role for chrono-pharmacology of ischemic heart disease with glucocorticoids.
Abstract P3046: Deletion Of Sarm1 Nad Hydrolase Ameliorates Diabetic Cardiomyopathy
Authors: Nizami Hina Lateef , Light Christine , Gu Haiwei , Lee Chi Fung
Published: November 2022

Diabetes is a risk factor for heart disease. NAD depletion has emerged as a hallmark of cardiometabolic diseases, and hence, elevating NAD level alleviates diabetic cardiomyopathy. On the other hand, inhibition of NAD consumption also maintains NAD homeostasis. SARM1 is a novel intracellular NAD hydrolase that contributes to diabetic neuropathy; however, its role in heart disease has never been described.

We assayed 243 plasma metabolites from wild-type (WT) and SARM1-KO (KO) mice by quantitative metabolomics, and found that SARM1 deficiency caused changes in more (67) metabolites in male mice than in female mice (28). Only 5 of these metabolites overlapped between male and female, indicating sex-dependent effect of SARM1 deletion on plasma metabolites. SARM1 deficiency may alter susceptibility of male mice to metabolic stress, since metabolites associated with diabetes and impaired metabolism were altered, e.g. pyridoxic acid, alloisoleucine, LPC/PC, ceramide etc. Despite the differences in plasma metabolite levels, KO mice from either sex did not show changes in cardiac structure and function at baseline.

We next tested a hypothesis that SARM1 deficiency protects mice against diabetic cardiomyopathy. WT and KO mice were challenged with streptozotocin (STZ) to induce diabetic stress. 16-week diabetes promoted progressive systolic (fractional shortening) and diastolic dysfunction (E’/A’, e/E’) in WT male mice. SARM1 deficiency attenuated diabetes-induced cardiac dysfunction, although fasting glucose levels were similarly elevated in diabetic WT and KO mice. Diabetic WT hearts showed declined NAD level, which was elevated by SARM1 deficiency. The restored NAD level in KO hearts did not change expression of other NAD hydrolases (Bst1 and CD38). Diabetic WT hearts up-regulated cardiomyopathy markers such as nppb and Nox4 mRNAs, and myh7/myh6 ratio, that were normalized by SARM1 deficiency. Chronic diabetes promoted cardiac fibrosis and upregulation of profibrotic gene expression such as ctgf and Inhbb, that were suppressed by SARM1 deficiency.

Our data suggest a protective effect of SARM1 deficiency on diabetic cardiomyopathy in male mice. The effects of SARM1 deficiency on diabetic hearts could be sex-dependent and remain to be determined.
Abstract P3047: Identification Of Role Of Intracellular Chloride Channel Clic4 In Diabetic Cardiomyopathy.
Authors: Ponnalagu Devasena , Wright Katherine , Antelo Diego , Bindra Sahej , Stanford Kristin I , Edwards John C , Singh Harpreet
Published: November 2022

Cardiovascular diseases are the most common cause of morbidity and mortality in Diabetic patients. Mitochondrial dysfunction is the major contributor of the myocardial structural, functional, and metabolic defects in diabetic cardiomyopathy. Activation of mitochondrial ion channel, K
ATP
have been shown to facilitate protection from the adverse effects of diabetic cardiomyopathy via modulating mitochondrial function. In this study we delineate the impact of mitochondrial associated membrane (MAM) chloride channel-CLIC4 in diabetic cardiomyopathy. Our results show that pharmacological inhibition of chloride intracellular channel (CLIC) with IAA-94 abrogated the cardio-deleterious effects of hyperglycemia in neonatal cardiomyocytes, thus, implicating role of targets of IAA-94 in the onset and development of diabetic cardiomyopathy. Amongst the six mammalian paralogs of CLICs, we identified CLIC4 and CLIC5 are most abundant in the heart. In addition, we observed over expression and localization changes of MAM-CLIC4 but not mitochondrial CLIC5 in neonatal cardiomyocytes upon high glucose treatment. Furthermore, cardiomyocytes isolated from CLIC4 knock out mice under hyperglycemia do no exhibit disruption of sarcomere structures, reduction in contractility, and enhanced ROS production in comparison to wild type cardiomyocytes. These observations suggest that an absence of CLIC4 revokes the structure-function changes in cardiomyocytes upon hyperglycemia. Moreover, we demonstrate that

clic4
-/-

mice were protected from body weight gain and impaired glucose tolerance in comparison to wild type mice in the presence of a high fat diet. Interestingly, we also observed that

clic4
-/-

mice are resistant to increase in blood glucose levels upon streptozocin administration. Thus, suggesting a regulatory role of CLIC4 in glucose metabolism. Overall, this study suggests a probable novel role of intracellular chloride channel CLIC4 in diabetic cardiomyopathy via regulating glucose metabolism.
Abstract P3049: Age-independent Cardiac Protection By Pharmacological Activation Of Beclin-1 During Endotoxemia And Its Association With Energy Metabolic Reprogramming In Myocardium-A Targeted Metabolomics Study
Authors: Kim Matthew , nikouee azadeh , Zou Raymond , Ren Di , He Zhibin , Li Ji , Wang Lu , Djukovic Danijel , Raftery Daniel , Purcell Hayley J , Promislow Daniel , Sun Yuxiao , Goodarzi Mohammad , Zhang Qing-Jun , Liu Zhi-Ping , Zang Qun S
Published: November 2022

Background:
We previously showed that Beclin-1-dependent autophagy is cardiac protective in a rodent model of endotoxemia using young adult mice. In this report, we compared the potential therapeutic effects of pharmacological Beclin-1 activating peptide, TB-peptide, on the cardiac outcomes of young adult and aged mice during endotoxemia. We further examined alterations in myocardial metabolism induced by lipopolysaccharide (LPS) challenge with and without the TB-peptide treatment.

Methods:
C57BL/6J mice of 10-week and 24-month-old were challenged by LPS at doses at which cardiac dysfunction occurred. Following the treatment of TB-peptide or control vehicle, heart contractility, circulating cytokines, and myocardial autophagy were evaluated. A targeted metabolomics assay was applied to analyze cardiac metabolism.

Results:
TB-peptide boosted autophagic response, attenuated cytokine production, and improved cardiac performance in both young and aged mice during endotoxemia. A targeted metabolomics assay was designed to detect a pool of 361 known metabolites, of which 156 were detected in at least one of the heart tissue samples. LPS-induced impairments were found in glucose and amino acid (AA) metabolisms in mice of all ages, and TB-peptide provided ameliorative effects to rescue these alterations. However, lipid metabolites were upregulated in the young group but moderately downregulated in the aged by LPS, suggesting an age-dependent response. TB-peptide mitigated LPS-mediated trend of lipids in the young mice but provided little effect on the aged ones.

Conclusion:
Pharmacological activation of Beclin-1 by TB-peptide protects the heart in both young and aged population during endotoxemia, suggest a therapeutic potential for sepsis-induced cardiomyopathy. Metabolomics analysis suggests that this age-independent protection by TB-peptide is associated with reprograming of energy production via glucose and AA metabolisms.
Abstract P3051: Yap Regulates Serine Biosynthesis During Compensatory Cardiac Hypertrophy
Authors: Hu Chengchen , Sadoshima Junichi
Published: November 2022

Introduction:
In response to increased workload, cardiomyocytes increase in size, and the heart undergoes hypertrophy. The initial hypertrophy is a compensatory process that adds contractile units but reduces wall stress to help maintain heart function. Yes-associated protein (YAP), a major effector of the Hippo pathway, is transiently activated in response to acute pressure overload (PO). Cardiac-specific heterozygous downregulation of YAP inhibits hypertrophy but promotes heart failure, suggesting that endogenous YAP is salutary and mediates compensatory hypertrophy during the acute phase of PO. However, the mechanism by which YAP mediates compensatory hypertrophy is not well understood. We have shown previously that 3-phosphoglyceric acid and serine accumulate in the heart during acute PO in a YAP-dependent manner. The serine biosynthetic pathway provides precursors for the TCA cycle and antioxidant and purine biosynthesis.

Hypothesis:
YAP induces serine synthesis, thereby promoting compensatory cardiac hypertrophy in the presence of acute PO.

Methods and Results:
We evaluated the levels of intermediate enzymes in the serine synthesis pathway in YAP-overexpressing neonatal rat ventricular myocytes (NRVMs). Furthermore, we investigated whether YAP-induced cardiomyocyte hypertrophy is inhibited by siRNA targeting serine biosynthesis enzymes. Immunofluorescent staining of NRVMs with cardiac troponin T was conducted to evaluate cardiomyocyte size, which was assessed using ImageJ software. YAP upregulated protein levels of phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), phosphoserine phosphatase (PSPH), and serine hydroxymethyltransferase 1 (SHMT1) in NRVMs. siRNA-mediated downregulation of PHGDH, PSAT1, PSPH, or SHMT1 significantly inhibited YAP-induced cardiomyocytes hypertrophy.

Conclusions:
YAP upregulates the expression of enzymes in the serine biosynthesis pathway, thereby inducing hypertrophy in NRVMs. Metabolites of the serine biosynthesis pathway may play an important role in mediating YAP-induced compensatory cardiac hypertrophy.
Abstract P3052: Dysregulation Of The Ampk-drp1 Axis Promotes Pathophysiological Remodeling Of Mitochondrial Ultrastructure And Ventricular Electrophysiological Function
Authors: Rudokas Michael W , Cacheux Marine , Wu Xiaohong , Young Lawrence H , Akar Fadi G
Published: November 2022

AMP-activated protein kinase (AMPK), a master metabolic sensor, plays a central role in the regulation of mitochondrial (mito) function. AMPK activation inhibits mito fission potentially by modulating mito dynamics proteins, including dynamin-related protein 1 (DRP1). The role of the AMPK-DRP1 axis in electrophysiological (EP) function is unknown.

Methods:
We examined the impact of myocardial AMPK inactivation on mito ultrastructure by electron microscopy, mito dynamics proteins by western blot, and EP function by optical mapping in mice expressing an AMPK kinase dead isoform (AMPK-KD, N=3-7) vs littermate controls (Ctrl, N=3-6).

Results:
Quantification of mito morphology revealed increased mito area consistent with swelling (0.53 μm
2
vs 0.80 μm
2
, p<0.0001) and decreased mito count (0.68 mito/μm
2
vs 0.44 mito/μm
2
, p=0.13) in AMPK-KD vs Ctrl. Immunoblots revealed decreased DRP1 phosphorylation at S637 (a fission inhibiting site) with no major change in total DRP1, S616 phospho-DRP1, MFN1/2 or OPA1 levels in AMPK-KD. High resolution optical mapping of
ex vivo
perfused hearts uncovered marked (by 39.5%, p<0.001) conduction slowing (
Figure
) and a moderate increase in AP duration (43.8 ms vs 49.7 ms, p<0.05) reflecting widespread ventricular EP remodeling in AMPK-KD. Of note, discrete lines of functional conduction block were observed in 4/7 AMPK-KD but not Ctrl hearts consistent with pro-arrhythmic remodeling.

Conclusions:
Myocardial AMPK pathway inactivation causes mito ultrastructural abnormalities and pathological EP remodeling. The AMPK-DRP1 axis may be a novel target for arrhythmia suppression in metabolic disorders with defective AMPK downstream signaling.
Abstract P3056: Influence Of Maternal Diabetes And High Fat Diet On Myocardial Ketone Body Metabolism In The Offspring
Authors: Ayyappan Prathapan , Larsen Tricia D , Gandy Tyler C , Baack Michelle L
Published: November 2022

Alteration in myocardial substrate utilization and energy metabolism is a well-known contributor to the pathogenesis of heart disease. Recent studies suggest the failing heart may benefit from ketone bodies as an energy source. Infants exposed to diabetic pregnancy are at higher risk of cardiomyopathy at birth and early onset cardiovascular disease (CVD) as adults. We showed fetal exposure to excess circulating fuels, especially to the combination of maternal high fat diet (HFD) and diabetes, exaggerates CVD risk through fuel-mediated mitochondrial dysfunction including complex I dysfunction. Diabetic pregnancy increases ketone body exposure in utero, but whether prenatal exposures impair myocardial metabolism of ketones postnatally remains unknown. The objective of this study was to determine whether neonatal rat cardiomyocytes (NRCM) exposed to maternal diabetes and HFD in utero can increase ketone oxidation as a preferred fuel source over glycolysis. To test our hypothesis, we developed a ketone stress test (KST) or modified extracellular flux analyses to compare real-time ß-hydroxy butyrate (ßOHB) metabolism in NRCM and myocardial expression of genes responsible for ketone and lipid metabolism. While control and combination exposed NRCM had similar basal respiration, the presence of ketone (4.5 mM) increased maximal respiration, spare respiratory capacity, and ATP-limited glycolytic rate in the combination exposed group. Interestingly, baseline proton efflux rate (PER) from lactate (anaerobic glycolysis) decreased while PER from CO
2
(aerobic glycolysis) increased in response to ketones in the combination exposed group. Expression of genes responsible for ketone body metabolism (
Hmgcs2
and
Bdh1
) was not different. The study uses a novel KST to analyze real-time ketone body metabolism and demonstrates myocardial ketone body metabolism is preserved and may be protective in the NRCM exposed to maternal overnutrition.
Abstract P3059: Hydrogen Sulfide Inhibits Aortic Valve Fibrosis, Inflammation And Calcification By Inducing Pro-autophagy Effect In Human Valvular Interstitial Cells
Authors: Song Naaleum , Ji Eunhye , Choi Kyoung-Hee , Yu Jeong-Eun , Lee Sahmin
Published: November 2022

Background:
As of now, there is no pharmacological treatment for aortic valve stenosis (AS) except for valve replacement. In the previously studied proteomics analysis, we demonstrated that the expression of sulfide:quinone oxidoreductase (SQOR) was significantly lower in aortic valve interstitial cells (VICs) isolated from AS patients than those of normal patients. However, the role of SQOR in AS pathophysiology has not been clarified yet.

Methods:
To elucidate the cellular mechanism of SQOR and hydrogen sulfide (H2S) as therapeutic aspects in AS, we studied the effects of NaHS (H2S-releasing compound) on diseased aortic VICs using real-time PCR and western blots.

Results:
NaHS treatment led to increased SQOR expression in diseased aortic VICs and induced an increase of the Nrf2-target genes, especially NQO1. It showed that the effects of NaHS decreased not only fibrosis and inflammation markers (MMP9, TNF-α, IL6) but also calcification markers (ALP, osteocalcin, RUNX2, COL1A1). In addition, AMPK and Beclin-1 significantly increased by NaHS while Akt and mTOR decreased which is expected to have pro-autophagy effect.

Conclusion:
H2S releasing compound enhances the expression of SQOR and activates AMPK/mTOR mediated pro-autophagy function resulting in inhibition of fibrosis, inflammation and calcification in human VICs. Therefore, H2S can be a potential therapeutic target in AS.
Abstract P3065: Myeloid YAP Inhibition Improves Cardiac Phenotype During Pressure Overload Stress
Authors: Francisco Jamie J , Zhang Yu , Nakada Yasuki , Hu Che-Ming , Oka Shinichi , Zhai Peiyong , Del Re Dominic
Published: November 2022

Inflammation is an integral component of cardiovascular disease and is thought to contribute to cardiac dysfunction in ischemic and non-ischemic models of heart failure. While ischemia-induced inflammation has been extensively studied in the heart, relatively less is known regarding cardiac inflammation during non-ischemic stress. Recent work has implicated a role for Yes-associated protein (YAP), a transcription co-factor, in modulating cardiac inflammation in response to ischemic injury; however, whether YAP influences inflammation in the heart during non-ischemic stress is not described. We hypothesized that YAP mediates a pro-inflammatory response during pressure overload (PO)-induced non-ischemic injury, and that targeted YAP inhibition in the myeloid compartment is cardioprotective. In mice, PO elicits an immune response characterized by infiltration of myeloid cells that precedes cardiac dysfunction. Myeloid cells isolated from the heart after 7 days PO showed evidence of increased YAP activity. Myeloid-specific YAP knockout mice (YAP
F/F
;LysM
Cre
) were subjected to PO stress. After 4 weeks cardiac hypertrophy was similar between myeloid YAP KO mice and controls. However, systolic dysfunction, cardiac fibrosis, apoptosis, “fetal gene” induction, and additional indicators of pathological remodeling were attenuated in myeloid YAP KO mice compared to controls. Additionally, inflammatory gene expression and macrophage infiltration in the myocardium were significantly attenuated in myeloid YAP KO mice after PO indicating reduced inflammation compared to controls. Conversely, angiogenic indicators and pro-resolving genes were enhanced in myeloid YAP KO hearts. Experiments using primary bone marrow-derived macrophages (BMDMs) from YAP KO and control mice demonstrated that YAP suppression attenuated “M1-like” inflammatory gene expression, while “M2-like” resolving genes were augmented following stimulation. The inflammasome is a multiprotein complex and important facilitator of cytokine processing that mediates inflammation in the injured heart. We observed attenuated inflammasome priming and function in YAP deficient BMDMs, as well as in myeloid YAP KO hearts following PO, indicating disruption of inflammasome induction. Finally, we leveraged nanoparticle-mediated delivery of the YAP inhibitor verteporfin to test the translational potential of targeting YAP to prevent heart failure. An alternate-day dosing regimen of drug-loaded nanoparticles attenuated inflammation and fibrosis, and improved myocardial capillary density after PO compared to DMSO nanoparticle control treatment. Together these data implicate myeloid YAP as an important molecular nodal point that facilitates cardiac inflammation and pathology during PO stress and suggest that selective inhibition of YAP may prove a novel therapeutic target in non-ischemic heart disease.
Abstract P3072: Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (iPSC-CMs) Respond To Inflammatory Stimuli Through Innate Immune Responses
Authors: Selgrade Daniel F , Gacita Anthony , Fullenkamp Dominic , Puckelwartz Megan , Demonbreun Alexis , Nobrega Marcelo A , George Alfred L , McNally Elizabeth M
Published: November 2022

Introduction:
Cells respond to inflammatory stimuli in settings of sterile and pathologic cardiac inflammation. Nuclear factor Kappa B (NFKB) is a transcriptional mediator of innate immune responses. Lipopolysaccharide (LPS) promotes NFKB activation through toll-like receptors (TLR) to initiate cell autonomous processes that resist infection. High-mobility box group 1 (HMGB1) is a chromatin associated protein released upon nuclear rupture that similarly promotes cytokine release in response to injury. Few studies have interrogated multiple inflammatory stimuli and their impacts on iPSC-CM function. We examined innate immune activation using distinct TLR agonists and assessed functional responses in both 2D iPSC-CM monoculture and 3D collagen-based engineered heart tissues.

Methods and Results:
We measured electric field potential duration (Nanion CardioExcyte 96) of iPSC-CM monolayers after exposure to LPS and HMGB1. We observed a four fold increase in downstroke velocity after exposure to HMGB1 (512.7±126.9 V/s; p=0.0002 n=12) and a 30% decrease in downstroke velocity after LPS treatment (-188.9±61.4 V/s; p=0.004 n=12). This response was associated with nuclear localization of phosphorylated NFKB. Collagen-based engineered heart tissues demonstrated contractile defects in response to 48h of exposure to TLR agonists. A 50% reduction in fractional shortening was observed following HMGB1 treatment (-0.0060±0.0009; p<0.0001 n=6) and an 80% reduction was associated with LPS exposure (-0.0088±0.0006; p<0.0001 n=6). To identify gene expression regulatory regions associated with this response, we examined transcriptional activity of three candidate enhancer regions implicated in cytokine production in iPSC-CMs. These regions demonstrated a 0.5- to 20-fold increase in enhancer activity in iPSC-CMs. One enhancer demonstrated a 22% increase in activity following 48h of LPS, consistent with a direct role for NFKB in innate immune activation in human iPSC-CMs; this enhancer regulates a gene with increased expression in dilated cardiomyopathy.

Conclusion:
These data indicate that cardiomyocytes respond directly to inflammatory stimuli and demonstrate the utility of 2D and 3D human tissue culture models for cardiac inflammation.
Abstract P3073: Altered Mitochondrial Mg
2+
Homeostasis In Liver Links Lipid Metabolism, Inflammation And Atherogenesis
Authors: PONNUSAMY THIRUVELSELVAN , Velusamy Prema , Santhanam Shanmughapriya
Published: November 2022

Although evidences show the role of Mg
2+
in lipid metabolism and atherosclerosis, the molecular mechanism by which loss of Mg
2+
homeostasis promotes cardiovascular disease (CVD) is not known. Mg
2+
homeostasis is regulated by mitochondria and Mrs2 is the only known molecular machinery associated with mitochondrial Mg
2+
influx. To begin elucidating the importance of Mg
2+
homeostasis in lipid metabolism and CVD we made liver-specific Mrs2 KO mouse (Mrs2
Δhep
). Liver being the central hub of lipid metabolism, we asked whether loss of Mrs2 alters lipid metabolism and lipoprotein profile. Our results show increased accumulation of hepatic triacylglycerol (TG) and cholesterol in Mrs2
Δhep
. Consistent with increased TG/cholesterol, plasma cholesterol and oxidized low density lipoprotein (ox-LDL) levels significantly differed between control and KO. Because ox-LDL contribute to atherosclerosis, we hypothesized altered hepatic lipid metabolism to induce inflammation, immune imbalance and accelerate lesions through modified atherogenic lipoprotein and cytokine profile. Quantification of plasma cytokines and chemokines were performed using the proteome profiler. Because T cells are critical drivers and modifiers of atherosclerosis, we quantified the lymphocyte and monocyte subsets by flow cytometry. Because T regulatory cells have anti-antherogenic roles, we studied the effect of ox-LDL on Tregs. Our results show increased and decreased levels of inflammatory and anti-inflammatory cytokines respectively in Mrs2
Δhep
. In Mrs2
Δhep
stimulation with ox-LDL showed an increased antigen specific proliferation of T-cells. We also observed increased and decreased population of Th17 and Tregs respectively. The decrease in Tregs population was attributed to increased apoptosis. In conclusion our observation suggests that high levels of ox-LDL in Mrs2
Δhep
to induce inflammation and inflammatory cell expansion accompanied by apoptosis in Tregs. Our results conclude dysregulated Mg
2+
homeostasis to alter lipid metabolism and atherogenesis by inducing inflammation and compromised Tregs population. Our study will be the first of its kind to establish a triangular molecular link between Mg
2+
homeostasis, lipid metabolism, and atherogenesis.
Abstract P3074: Immune Checkpoint Inhibition Reshapes Cardiac Immune Landscape Through Crosstalk Between Macrophage And T-cell
Authors: Ma Pan , Liu Jing , Lai Lulu , Qin Juan , Sultan Debbie , Bredemeyer Andrea , Bajpai Geetika , Feng Guoshuai , Amrute Junedh , Lokshina Inessa , Liu Yongjian , Lin Chieh-Yu , Moslehi Javid , Lavine Kory
Published: November 2022

Immune checkpoint inhibitors (ICI) targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein 1(PD-1)/ programmed death-ligand 1(PD-L1) have revolutionized cancer management since the approval. However, they are associated with immune-related adverse events (irAEs), including myocarditis. ICI-associated myocarditis is often fulminant and fatal. The associated major risk factor is the combination of CTLA-4 and PD-1 inhibition. The mechanistic basis of ICI-associated myocarditis is unclear and mechanism-based treatment strategies are needed. Crosstalk between T-cells and macrophages may represent one pathogenic mechanism underlying ICI-associated myocarditis. We employed an established mouse model

Ctla4
+/-
Pdcd1
-/-

to study the crosstalk between T-cells and macrophage in the heart and its impact on reshaping cardiac immune landscape using unbiased single cell-RNA-seq technology. We discovered a more inflammatory cardiac microenvironment in ICI-associated myocarditis mouse, characterized by CD68
+
cells infiltration, robust CCR2 signal, enhanced T-cells activation with interferon gamma (IFN-γ) secretion and increased frequency of

Cxcl9
+
Cxcl10
+

macrophages with an activated phenotype enriched in response to IFN-γ. Importantly, the existence of

Cxcl9
+
Cxcl10
+

macrophages (characterized by CD16α and
CXCL9/CXCL10
expression) was confirmed in human ICI-associated myocarditis specimens which establishes a connection between mouse model and human disease. Analysis of cell communication between myeloid cells and T-cells predicted that

Cxcl9
+
Cxcl10
+

macrophages communicate with T-cells via IFN-γ-IFN-γR and Cxcl9/Cxcl10-CXCR3 signals. Pseudotime time analysis suggests

Cxcl9
+
Cxcl10
+

macrophages were derived from monocytes. Blocking IFN-γ signal prolonged the survival time of ICI-associated myocarditis mouse and reduced emergence of

Cxcl9
+
Cxcl10
+

macrophages in the heart suggesting specific blocking the mediator of crosstalk between T-cells and macrophages attenuated ICI-associated myocarditis. This study will provide support to the usage of IFN-γ as a potential target to prevent ICI associated myocarditis during or at the end of ICI therapy in cancer patients.
Abstract P3077: The Role Of Neutrophils In Initiating Type I Interferon Response In Myocardial Ischemia/reperfusion
Authors: Brockman Maegan L , Wang Lanfang , Calvert John W , Levit Rebecca D
Published: November 2022

Background:
Inflammation succeeding myocardial ischemia/reperfusion (MI/R) plays a significant role in damaging cardiomyocytes and influencing infarct size, with neutrophils being the most rapid and most numerous cells in the post-MI/R heart. While neutrophils play a role in wound healing, they can cause further damage through the generation of reactive oxygen species, proteolytic enzymes, and neutrophil extracellular traps (NETs). We thus hypothesize that double stranded DNA stemming from these NETs activates cGAS-STING signaling in a murine model of MI/R, leading to the production of type I interferons by neutrophils, which worsens reperfusion injury.

Methods:
Single-cell RNA sequencing was performed on neutrophils isolated from the hearts of mice 24 hours post-MI/R. Additionally, mice underwent MI/R and heart tissues were harvested 6- and 24- hours after reperfusion for ELISA and qPCR analysis.

Results:
Utilizing single-cell RNA sequencing to better characterize the role of neutrophils and NETs in MI/R, we identified a significant population of neutrophils with a strong type I interferon (IFN) signature recruited to the area of infarct 24 hours post MI/R in a murine model. To further investigate the IFN response seen in this population of neutrophils, we preformed protein and gene expression analysis on heart tissue samples isolated from mice at timepoints in which neutrophils would be present in the area of infarct. Analysis by qPCR shows an increased fold change in gene expression of IFNα (7.22
±
6.05, n = 3; 3.83
±
2.22, n = 4) and IFNβ (6.74
±
5.39, n = 3; 3.64
±
2.25, n = 4) at 6- and 24- hours post MI/R respectively, compared to sham mice. Furthermore, analysis by ELISA shows a significant increase in IFNβ protein levels in the heart after 6- hours (0.41pg/mg
±
0.04, n =8) compared to sham mice (0.16 pg/mg
±
0.06, n = 7; p ≤ 0.01, Student’s t-test).

Conclusions:
These data show a rapid increase in type I IFN in the hearts of mice post-MI/R, which implicates neutrophils may be participating in the IFN signaling pathway. Identifying neutrophils as an early source of IFN can provide a potentially new therapeutic target in treating MI/R.
Abstract P3083: Beta 1.3 1.6 D Glucan Immunomodulatory Activity Of Indonesian Ganoderma Lucidum Mycelium Extract In Repairing Heart Tissue And Function On Myocardial Infarction Patients
Authors: Sugita Peter , sargowo djanggan
Published: November 2022

Introduction:
Immune response plays a significant role in heart repair and regeneration after myocardial infarction and/or ischemia-reperfusion injury (MI/RI). Prolonged inflammation response contributes to adverse cardiac remodeling and reduction in pump function. In stable angina pectoris and patients with high-risk for heart disease, Beta 1.3 1.6 D Glucan has demonstrated reduced inflammation parameters including IL-6, TNF-α and CRP.

Hypothesis:
Beta 1.3 1.6 D Glucan modulates inflammation responses related to myocardial infarction or MI/RI, therefore preventing adverse heart remodeling measured by Left Ventricle Mass Index (LVMI) and Left Ventricle Ejection Fraction (LVEF).

Method:
In May - August 2018 a randomized double-blind placebo-controlled trial was conducted comprising of 50 STEMI/NSTEMI patients at the Saiful Anwar General Hospital, Malang, Indonesia. Next to standard therapy, 25 patients received Beta 1.3 1.6 D Glucan, 540 mg/day for 90 days, while the other 25 patients received placebo.

Results:
During the study duration, 5 patients dropped out. The mean value of pre-test LVMI for all patients is 114.07 ± 59.61 g/m
2
. After 90 days, there was a significant difference (p=0.012) in LVMI between the Beta 1.3 1.6 D Glucan and placebo group. The Beta 1.3 1.6 D Glucan group had a LVMI reduction of 4.55 ± 14.46 g/m
2
compared to the placebo group with an increase of 11.48 ± 23.94 g/m
2
in LVMI. The mean value of pre-test LVEF for all patients is 57.2 ± 11.39%. Post-test results demonstrated a significant difference (p=0.042) between both groups, with an increase in LVEF of 2.3 ± 9.77% for Beta 1.3 1.6 D Glucan group and a decrease in LVEF of 2.08 ± 3.35% for placebo group.

Conclusion:
Beta 1.3 1.6 D Glucan as an adjuvant therapy may have a beneficial effect to repair the vascular, heart tissues and function in myocardial infarction patients.
Abstract P3085: The Human Myocardium Harbors A Population Of B Cells With A Distinctive Gene Expression Signature Conserved Across Species
Authors: Bermea Kevin , Kostelecky Nicolas , Rousseau Sylvie , Lin Chieh-Yu , Adamo Luigi
Published: November 2022

Introduction:
Investigations in murine models have identified myocardial B cells with a characteristic gene expression signature and have pointed at B cells as a promising target to develop novel therapies for heart failure. However, there is almost no data on B cells in the human heart.

Methods and Results:
We performed immunostaining to characterize the amount and distribution of B cells in human hearts. In 12 healthy hearts collected at autopsy, we found 1.4 ± 1.6 CD20+ B cells per high power field. The number of CD3+ T cells was almost 20 times higher (26.8 ± 28.6, P<0.001). Utilizing CD31 (endothelial marker), we found that human myocardial B cells were both in intravascular and interstitial locations, with a slight prevalence for the interstitium (P=0.006). Parallel analysis of tissue collected post-mortem or at the time of VAD placement showed that analysis of post-mortem tissue did not introduce bias. To gain insight into the biology of human myocardial B cells we first analyzed publicly available single cell sequencing datasets of myocardial (Heart Cell Atlas) and peripheral blood cells (10X Genomics). We found that the human myocardium harbored a minor population of B1 cells and a larger population of B2 cells, and that myocardial B cells had a gene expression signature distinct from that of peripheral blood B cells. We validated these findings on primary B cells sorted from the heart and peripheral blood of 2 patients subjected to LVAD implant (binomial for concordance p=4.13e-32). To identify biological pathways upregulated in myocardial B cells across species, we compared differential gene expression in myocardial vs peripheral blood B cells across the studied human datasets and published rodent datasets. We identified 703 genes with consistent differential gene expression (binomial p=2.9e-48). KEGG pathway analysis of these genes highlighted “B cell receptor signaling pathway”, “Antigen processing and presentation” and “Cytokine-cytokine receptor interaction” among the top conserved pathways upregulated in cardiac B cells (FDR <0.001).

Conclusions:
The human heart harbors a small population of B cells that is comprised of both B1 and B2 cells. Human myocardial B cells have a distinct gene expression signature that is conserved across species.
Abstract P3086: Regulation Of Rhoa In Myeloid Cells On Remodeling Of The Infarcted Heart
Authors: Xu Bing
Published: November 2022

Small GTPase RhoA is critically involved in myocardial injury, repair, and fibrosis. However, the role of RhoA signaling in regulating of macrophage is poorly understand. In this study, we investigated the role of RhoA in macrophage in modulating cardiac function after myocardial infarction and dissected the mechanisms mediating RhoA dependent modulation of macrophage function.

RhoA deletion in myeloid cells worsen the cardiac function and lead an increase in post-infarction scar and cardiac fibrosis. RhoA deletion in myeloid cells decreased CCR2
+
infiltration in heart, however, increase the circulation of Ly6c
hi
monocytes in blood expression. RhoA deletion macrophage produce more pro-inflammatory cytokines IL-1β and IL-12b compared to control mice. Mechanistically, CCR2/RhoA axis is critical for CCl2 induced monocytes infiltration. We demonstrated RhoA critically regulates function of infarct macrophage by mediating CCL2 inducing macrophage infiltration and by contributing to proinflammatory transition.
Abstract P3088: The Histone Lysine Methyltransferase SMYD1a Protects Heart From Ischemic Injury By Regulating OPA1 Mediated Cristae Remodeling And Supercomplex Formation
Authors: Szulik Marta W , Valdez Steven , Walsh Maureen , Davis Kathryn , Bia Ryan , Horiuchi Emilee , O'Very Sean , Laxman Anil , Nicolova Linda , Sheikh Hanin , Hickenlooper Samuel , Miller Mickey R , Wang Li , Garcia-Llano June , Tracy Christopher , Drakos Stavros G , Funai Katsuhiko , Chaudhuri Dipayan , Boudina Sihem , Franklin Sarah
Published: November 2022

The histone lysine methyltransferase SMYD1 has been shown to be critical for embryonic cardiac development and in maintaining cardiomyocyte homeostasis in adult mice. Subsequently, we reported that loss of
Smyd1
in the adult mouse myocardium leads to progressive cardiac hypertrophy and heart failure, which is accompanied with downregulation of mitochondrial proteins involved in oxidative phosphorylation, including
Ppargc1a
, and reduction of mitochondrial respiration capacity. To build upon these results and evaluate if SMYD1a can attenuate disease-induced remodeling in an animal model, we generated transgenic mice which inducibly express SMYD1a (the human ortholog) in cardiomyocytes and subjected them to permanent occlusion (PO) of the LAD. This lead to >50% reduction in infarct size and preserved cardiac function, as compared to littermate controls. Additionally, we demonstrated that under physiological conditions SMYD1a maintains metabolic homeostasis by regulating expression of
Ppargc1a
and its downstream targets, including components of the electron transport chain. Our molecular analysis shows that observed protection from ischemic injury results from enhanced mitochondrial respiration through Complex I and II as well as increased ATP production. This is associated with increased mitochondria cristae, and formation and stabilization of respiratory chain supercomplexes within the cristae. These changes in cristae structure occur concomitant with enhanced OPA1 expression, a major regulator of mitochondrial fusion and cristae morphology. Through this work we have established that OPA1 is a novel, functionally important downstream target of SMYD1a by which cardiomyocytes upregulate energy efficiency, protecting them from ischemic injury. These results also highlight SMYD1a as the only known epigenetic regulator of cristae morphology and provide broad implications for understanding the epigenetic mechanisms driving cardiac metabolism. Ultimately this work has identified a novel signaling pathway by which cardiomyocytes regulate energy efficiency, protecting them from ischemic injury.
Abstract P3093: Gender-specific Functional Dimorphism Of Bone Marrow Endothelial Progenitor Cells: Estrogen Independent Epigenetic Mechanisms
Authors: Gurrala Charan T , Garikipati Venkata , Cheng Zhongjian A , Mallaredy Vandana , Cimini Maria , Joladarashi Darukeshwara , Truongcao May , Wang Chunlin , Lucchese Anna Maria , Huang Grace , Gonzalez Carolina , Magadum Ajit , Roy Rajika , Ghosh Jayashri , Benedict Cindy , Koch Walter J , Kishore Raj
Published: November 2022

Introduction:
Several studies, including our labs, have previously determined the role of estrogen in augmenting EPC-based cardiac repair; however, a direct comparison of therapeutic efficacy of gender-specific stem cells or estrogen-independent mechanisms of gender-specific dimorphism in the reparative properties of BM-EPCs, has not been established.

Hypothesis:
We hypothesized that epigenetic mechanisms contribute to the sex-specific functional dimorphism of Sca-1+/CD31+ BM-EPCs in regulating cell-homing, pro-angiogenic and anti-inflammatory functions in the ischemic myocardium leading to the enhanced reparative function of female EPCs.

Methods:
To evaluate our hypothesis, we sorted the male, female and ovariectomized (OVX) mice derived Sca-1+/CD31+ BM-EPCs using MACS multi-sort method. We then subjected the BM-EPCs through a series of cytokine quantifications and epigenetic screening followed by assessment of their therapeutic function
in vitro
and
in vivo
.

Results:
Female and ovariectomized (OVX) female BM-EPCs secrete high levels of pro-angiogenic factors and low levels of pro-inflammatory cytokines compared to male BM-EPCs. Further evaluation of the secretome showed that the male EPCs secreted high levels of interleukins and chemokines compared to female and OVX EPCs. We found that male EPCs exclusively secreted CCL3/Mip-1α. Functional
in vitro
angiogenic evaluation of the EPC secretome showed higher propensity of female and OVX EPCs than the male EPCs. Post-MI injection of BM-EPCs resulted in remarkable preservation of cardiac structure and functions in both female BM-EPC groups compared to the male EPCs. Male EPC injection resulted in high inflammation in the heart tissue. Epigenetic sequencing of the BM-EPCs for the H3K9me3 mark showed high methylation in male EPCs compared to female and OVX EPCs. The inhibition of histone methyltransferase, Ehmt2/G9a using BIX-01294 upregulated the secretion of inflammatory factors in all EPCs. The conditioned medium from all EPCs with high levels of CCL3 inhibited angiogenesis
in vitro
. Neutralizing CCL3 in the same medium restored
in vitro
angiogenesis.

Conclusion:
Estrogen-independent epigenetic mechanisms govern the enhanced cardiac reparative properties of female BM-EPC.