bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P3037: Metabolic Transitions In The Developing Mouse Heart
Authors: Beutner Gisela , Burris Jonathan , Collins Michael P , Fangnibo Hanvi Denise , Cohen Ethan D , O'Reilly Michael , Brookes Paul S , Bentley Karen , Porter George A
Published: November 2022

During cardiac development, cellular energy production evolves dramatically. Mechanisms that control these changes and activation of electron transport chain (ETC) remain unclear. Our aim was to provide a comprehensive analysis of mitochondrial biology and bioenergetics in the developing wild type heart for use in experimental models, such as mice, in which the mitochondrial protein, cyclophilin D (CyPD), was deleted. Mouse hearts were harvested from wild type and CyPD null hearts throughout development and examined by electron microscopy, liquid chromatography/mass spectrometry, oxygen consumption and enzyme assays, gel electrophoresis/immunoblotting, and calcium retention capacity (CRC). As the heart develops, myocyte size and mitochondrial number, size, and complexity increases with transitions at the end of the embryonic period and immediately after birth. Metabolomic profiling demonstrated changes in glycolysis, the TCA cycle, and redox state during development and these changes may facilitate proliferation in the neonatal heart. Activation of ETC complexes I, III, IV, and V occurs in the mid-embryonic period, while assembly of ETC supercomplexes begins at the late embryonic period. In contrast, complex II activity is relatively stable during development. CRC assays demonstrated a gradual decrease in the probability of mPTP opening during development, and while CyPD expression increased, its specific enzymatic activity decreased. For most assays tested, these metabolic transitions occurred earlier in development in CyPD null mice. In conclusion, the developing heart undergoes changes in cellular bioenergetics with two transitions at the end of the embryonic period and immediately after birth. Deletion of CyPD causes these transitions to occur earlier in development, suggesting that CyPD and closure of the mPTP controls mitochondrial activation and bioenergetics during cardiac development.
Abstract P3038: Upregulation Of The Mitochondrial Calcium Uniporter Abolishes Mitochondrial Dysfunction Associated With Obesity In Vascular Smooth Muscle Cells
Authors: Avery Jade , Francis Sharon
Published: November 2022

Obesity is a growing national epidemic that is associated with an increased prevalence of arterial disease. We found that aortic medial hypertrophy during obesity was accompanied by upregulation of serum and glucocorticoid-inducible kinase 1 (SGK1) and reduced mitochondrial oxidative capacity of vascular smooth muscle cells (VSMC). Interestingly, VSMC-specific deletion of SGK1 attenuated obesity-related aortic remodeling and restored mitochondrial oxidative capacity in VSMC implicating a mediator role for SGK1 in mitochondrial dysfunction during obesity. The mechanistic basis for impaired mitochondrial respiration during obesity in VSMC is unknown. Thus, due to its fundamental role in stimulation of mitochondrial oxidative phosphorylation, the current study will investigate the mitochondrial calcium uniporter (MCU) in obesity-related mitochondrial dysfunction in VSMC. As SGK1 is known to regulate the expression and/or activity of ion channels, we hypothesize that upregulation of SGK1 during obesity negatively regulates MCU expression, mitochondrial oxidative activity, and ATP production. MCU protein levels were assessed by western blot analysis. Mitochondrial oxidative activity and ATP production rate were measured using the Agilent Seahorse XF Cell Mito Stress Test and the ATP Rate Assay, respectively in cultured wildtype and SGK1 knockout aortic VSMC from low-fat (LF) control and high-fat (HF) obese mice. To assess the importance of MCU, both assays were performed in the presence or absence of the MCU inhibitor, Ru360. Obesity caused a significant decrease in MCU abundance, but SGK1 deletion resulted in enrichment of MCU in VSMC. Ru360 treatment reversed the increase in basal mitochondrial respiration. We found that obesity reduced overall ATP production by diminishing ATP production from glycolysis and mitochondrial respiration. However, SGK1 deletion enhanced ATP production from glycolysis and mitochondrial respiration during obesity and this effect was attenuated by Ru360. These findings demonstrate that SGK1 negatively regulates MCU expression and activity and imply that augmentation of MCU-driven mitochondrial oxidative activity may underlie the protective effects of SGK1 inhibition on the vascular during obesity.
Abstract P3039: NAD Redox Imbalance Exacerbates Diabetic Cardiomyopathy, Regulates Nmrk2 Expression And Alters NAD Synthesis
Authors: Minor Keaton , Gu Haiwei , Lee Chi Fung , Light Christine
Published: November 2022

Heart failure occurs at twice the rate in diabetic patients compared to normal subjects. NAD redox imbalance or depletion has emerged as a hallmark of diabetes and heart disease. We showed that NAD redox imbalance or depletion promotes heart failure progression, but its role in diabetic cardiomyopathy has not been established. We used two mouse models with altered NAD metabolism. 1. Cardiac-specific NDUFS4-KO (cKO) mice which have mitochondrial complex I deficiency, decreased NAD/NADH ratio, but normal baseline cardiac function and energetics. 2. Cardiac-specific overexpression of nicotinamide phosphoribosyltransferase (NAMPT) in mice which elevates cardiac NAD levels. Diabetic stress induced by streptozotocin (STZ) promoted systolic (fractional shortening) and diastolic (E’/A’, e/E’) dysfunction, which were exacerbated in cKO mice. Overexpression of NAMPT to reverse NAD redox imbalance ameliorated cardiac dysfunction in both diabetic control and diabetic cKO mice. We measured expression levels of 58 genes related to NAD metabolism in these diabetic hearts. Transcript levels of genes for NAD consumption (sirtuins, PARPs and hydrolases) and NAD synthesis pathways (including NMRK1) did not change, except Nicotinamide Riboside Kinase 2 (NMRK2). NMRK2 transcript was upregulated in diabetic cKO hearts, associated with a decline in NAD levels. Expression of NAMPT to elevate NAD levels downregulated NMRK2 expression in both treated and untreated mice. The data indicate that upregulation of NMRK2 transcript is negatively correlated with cardiac function and NAD levels. NMRK2 is an enzyme involved in the Preiss-Handler and Salvage pathways of NAD synthesis by phosphorylating the NAD precursor Nicotinamide Riboside (NR) into Nicotinamide Mononucleotide (NMN). NAD metabolite analysis in the heart tissue showed a trended increase in the substrate of NMRK2, NR, and a trended decrease in the products, NMN or NAMN in diabetic cKO hearts with a decline in NAD levels. Our results suggest that NAD redox imbalance drives the progression of diabetic cardiomyopathy, and NMRK2 upregulation may regulate disease progression and NAD synthesis through mechanisms to be determined.
Abstract P3043: Functional Role Of Endothelial Mitochondrial Oxidative Stress In Cognitive Impairment: Effects Of L-arginine In Hypertensive Frail Older Adults
Authors: Mone Pasquale , Jankauskas Stanislovas S , Varzideh Fahimeh , Kansakar Urna , Lombardi Angela , Santulli Gaetano
Published: November 2022

Background:
Hypertension is common in older adults, particularly in frailty. The association of hypertension and frailty leads to a high-risk of cognitive impairment in older adults. Endothelial dysfunction has been shown to underlie both hypertension and cognitive impairment. L-Arginine is an amino acid involved in many biological processes and is a substrate of two enzymes: nitric oxide (NO) synthase (NOS) and arginase (NOA). L-Arginine is fundamental for NO production by endothelial cells, regulating vascular tone and cardiovascular homeostasis, improving inflammation and oxidative stress. On these grounds, we hypothesized that L-Arginine could improve cognitive impairment via its effect on endothelial mitochondria.

Methods:
To assess the effects of L-Arginine on endothelial oxidative stress and cognitive function, we used an
in vitro
and a clinical approach. First, we treated with L-Arginine human umbilical vein endothelial cells (HUVECs). HUVECs were cultured in EGM-2 medium and incubated at 37°C and 5% CO
2
. HUVECs were plated on glass bottom culture dishes and treated with Angiotensin II (1 μM) and Angiotensin II with L-Arginine (500 μM) in EGM-2 medium for 24h. The generation of reactive oxygen species (ROS) was quantified using MitoSOX™ Red (an established ROS probe that accumulates in mitochondria), incubating cells for 10 min at 37°C and 5% CO
2
, as we previously described. We then designed a single-blind, placebo-controlled, parallel-group, randomized trial to observe the effects of 4-weeks oral supplementation of L-Arginine on global cognitive function of hypertensive frail older patients.

Results:
In vitro
, we demonstrated that L-Arginine significantly attenuated Angiotensin II-induced oxidative stress in endothelial cells. Co-treatment of HUVECs with Ang II and L-Arginine significantly attenuated mitochondrial ROS production (p<0.001). In the clinical study, we evaluated 35 frail hypertensive elderly patients assigned to L-Arginine and 37 assigned to placebo: at follow-up, we found a significantly improved Montreal Cognitive Assessment (MoCA) test score in the L-Arginine treated arm compared to placebo (p<0.05).

Conclusions:
Taken together, our findings indicate for the first time that oral L-Arginine supplementation improves cognitive impairment in frail hypertensive older adults through a mechanism that involves endothelial mitochondrial ROS.
Abstract P3048: Establishing The Mitochondrial Citrate Transporter As A Regulator Of Cardiac Development
Authors: Ghazal Nasab , Peoples Jessica N , Rodgers Allie A , Park Austin S , Kwong Jennifer Q
Published: November 2022

Introduction:
Mitochondria are essential to provide oxidative energy to fuel excitation-contraction coupling of the postnatal heart. The role of mitochondrial energy production systems in the hypoxic environment of the embryonic heart is less clear, but defects in mitochondrial energy production machinery are associated with ventricular wall defects such as left ventricular noncompaction. In developing models to study mitochondrial function specifically in the postnatal mouse heart, we uncovered an unexpected embryonic-lethal knockout model that suggested roles for mitochondrial function in heart development. Uncovering mechanisms connecting mitochondrial function to the tightly regulated process of ventricular wall morphogenesis could reveal novel roles for mitochondria during development and guide further studies of developmental heart defects.

Hypothesis:
The mitochondrial citrate carrier (SLC25A1), a mitochondrial inner membrane transporter implicated in regulating mitochondrial function in neurons, is essential for ventricular wall morphogenesis and cardiac development.

Methods and Results:
We investigated the role of the mitochondrial citrate carrier (SLC25A1) in cardiac development. Slc25a1 knockout mice display impaired growth and do not survive past embryonic day (E) 18.5. Hearts from E18.5 Slc25a1 knockout embryos display a striking array of cardiac malformations including persistence of an expanded zone of trabeculated myocardium, reduced compact myocardium, and ventricular septal defects. Analysis of mitochondrial structure and function revealed that loss of Slc25a1 causes mitochondrial ultrastructural defects and depressed mitochondrial respiration.

Conclusions:
Our results highlight a novel role for SLC25A1 in cardiac development with specific roles in regulating both ventricular wall development and mitochondrial energetics during heart morphogenesis. Ongoing work will identify the mechanisms by which SLC25A1—a mitochondrial transporter that is not a primary component of the mitochondrial oxidative phosphorylation system—regulates mitochondrial energetics, which will help us delineate new pathways connecting mitochondria to heart development.
Abstract P3053: Mitochondrial Calcium Accumulation Drives The Progression Of Non-ischemic Heart Failure: Integrated Lessons From Genetic Mouse Models
Authors: Garbincius Joanne F , Luongo Timothy , Lambert Jonathan P , Mangold Adam S , Kolmetzky Devin , Murray Emma , Hildebrand Alycia , Jadiya Pooja , Roy Rajika , Nwokedi Mary , Ibetti Jessica , Koch Walter J , Elrod John W
Published: November 2022

Acute mitochondrial calcium (
m
Ca
2+
) uptake stimulates bioenergetics to meet increased ATP demand, but when excessive predisposes to necrotic cell death. A major unresolved controversy is whether chronic alterations in cardiomyocyte
m
Ca
2+
homeostasis contribute to maladaptive remodeling and contractile dysfunction in non-ischemic heart disease. We hypothesized that cardiomyocyte
m
Ca
2+
accumulation drives cardiac maladaptation in response to stressors that chronically increase workload and cytosolic Ca
2+
cycling. We subjected mice with adult, cardiomyocyte-specific manipulation of
m
Ca
2+
uptake through the mitochondrial calcium uniporter (
Mcu
deletion,
Mcu
-cKO; MCU overexpression, MCU-Tg) or
m
Ca
2+
efflux through the mitochondrial sodium-calcium exchanger, NCLX (NCLX overexpression, NCLX-OE), to chronic pressure or neurohormonal overload. Fractional shortening failed to increase in
Mcu
-cKO mice over the first days of isoproterenol (Iso) infusion. Mortality was increased in
Mcu
-cKO mice over this period, and this effect was recapitulated in NCLX-OE mice infused with angiotensin II + phenylephrine (PE), although contractility did not decline in either case. Hypertrophic responses to chronic stress were attenuated in NCLX-OE but not
Mcu
-cKO hearts, and adenoviral NCLX expression limited mitochondrial metabolism, protein synthesis, and cell growth in neonatal rat cardiomyocytes treated with PE. These data indicate that
m
Ca
2+
accumulation is required for cardiac hypertrophy, but MCU is not. MCU-Tg hearts decompensated towards failure with 1-2 weeks of Iso. Although these hearts exhibited increased cardiomyocyte necrosis, deletion of the mPTP regulator cyclophilin D failed to rescue contractility, suggesting that
m
Ca
2+
overload causes cardiac failure, even independent of permeability transition. Fitting with this view, NCLX-OE attenuated the decline in contractile function that occurred with 12-week pressure overload. We conclude that despite initial adaptive effects, sustained
m
Ca
2+
elevation drives the progression of non-ischemic heart disease triggered by a chronic increase in cardiac workload. Our findings raise concern over proposed therapeutic strategies aiming to augment
m
Ca
2+
accumulation in heart failure.
Abstract P3057: Heat Shock Protein Is Involved In Gja1-20k Transport To Mitochondria
Authors: Shimura Daisuke , Hong Tingting , Shaw Robin M
Published: November 2022

GJA1-20k is an internally translated, N-terminus truncated isoform of the gap junction protein Connexin 43 (Cx43). We have identified essential roles of GJA1-20k for Cx43 trafficking to cell membrane, actin stabilization, and regulation of mitochondrial dynamics. Recently, we found that GJA1-20k works with actin to induce a “protective” mitochondrial fission, protecting cells and hearts against ischemia-reperfusion (I/R) injury. This GJA1-20k mediated mitochondrial fission does not require the canonical fission mediator, Dynamin-Related Protein 1 (DRP1) pathway. At present, however, little is known how GJA1-20k occurs at mitochondrial membrane. Our
in silico
analysis indicates that the N-terminus of GJA1-20k contains a potent binding site to Heat Shock Protein 70 (HSP70). Since HSP70 is a well known mitochondrial transporter, we hypothesized that HSP70 interacts with GJA1-20k to support its translocation to mitochondria. To explore the involvement of HSP70, in HeLa cells we knocked down HSP70 in the presence of exogenous GJA1-20k. An absence of HSP70 inhibited GJA1-20k localization to mitochondrial membrane. In fact, in the absence of HSP70, most GJA1-20k was aggregated, suggesting a failure to distribute. Given that the expression of both HSP70 and GJA1-20k are upregulated during I/R injury, these data indicate that both stress-response proteins cooperate to localize GJA1-20k to mitochondrial membrane, initiating protective mitochondrial fission.
Abstract P3058: Cardiac-specific Deletion Of Ptp1b Induces A Global Metabolic And Lean Phenotype
Authors: Sun Yan
Published: November 2022

About 13% of US adults have diabetes, and more than half of them exhibitcardiac hypertrophy and fibrosis, resulting in cardiac diastolic dysfunction,systolic dysfunction and heart failure. Obesity is the leading risk factor for type2 diabetes. The Global Burden of Disease 2019 listed the high BMI as the fifthrisk of preterm deaths for females, and the sixth for males worldwide. Thecurrent treatments for these diseases are ineffective providing an urgent needof new therapeutic targets. Protein tyrosine phosphatase 1B (PTP1B) is themaster regulator of insulin signaling and its expression in brain, heart, liver andadipose tissues are elevated in obese mice and humans. It has been reportedthat neuronal deletion of PTP1B decreased food intake, increased energyexpenditure and protected mice from diet-induced obesity. However, whetherPTP1B in peripheral tissues would also contribute to diet-induced obesityremains unclear. To test if cardiac-PTP1B plays roles in the development ofobesity, we generated cardiomyocyte-specific PTP1B knockout mice (PTP1BCM-KO mice) and fed them with high-fat diets. Our preliminary datademonstrated that, compared to control mice, the PTP1B CM-KO mice hadreduced body weight and improved glucose homeostasis. Further analysisrevealed that deletion of PTP1B in cardiomyocytes increased energyexpenditure but didn’t affect the food intake. Metabolomics data suggested fattyacid oxidation is elevated in PTP1B CM-KO mice heart, whereas the glycolysispathway is suppressed, the amount of AMP (Adenosine Monophosphate) inPTP1B CM-KO mice is elevated 86 times than control mice on HFD. Thesefindings were further verified by qPCR analysis and ELISA. PTP1B knockoutupregulated not only NAD+ (nicotinamide adenine dinucleotide) but alsoNADPH in the heart. Nicotinamide phosphoribosyltransferase (Nampt) is therate-limiting enzyme of the NAD
+
salvage pathway and exists in an intracellular(iNampt) and an extracellular (eNampt) form. iNampt plays a regulatory role inNAD
+
biosynthesis and affects energy homeostasis. eNampt has been reportedto act as a cytokine and associated with obesity and diabetes. In the PTP1BCM-KO mice, we observed a higher level of both iNampt and eNampt,suggesting a mechanism through iNampt locally and/or through eNamptsystematically. Based on these observations, we hypothesize that the deficiency of PTP1B in cardiomyocytes increases energy expenditure,protects mice from diet-induced obesity and attenuates cardiacmalfunction induced by obesity, and cardiac-PTP1B governs whole bodyenergy homeostasis through Nampt and NAD
+
. We will test our hypothesisin the following specific Aims: Aim1: To elucidate the role of PTP1B in cardiac function and glucosehomeostasis.Aim2: To determine if and how the deletion of cardiac-PTP1B affectenergy balance and metabolic status through iNampt locally and/oreNampt systemically.
Abstract P3060: Circulating Gut Microbial Peptides Exacerbate Cardiac Dysfunction In IL10KO Mice Following Transaortic Constriction
Authors: Ranjan Prabhat , Goswami Sumanta , Dutta Roshan Kumar , Prasad Ram , zhang qinkun , Lal Hind , Verma Suresh
Published: November 2022

Background:
Heart failure is the leading cause of morbidity and mortality in the USA. Recently, impaired intestinal permeability is known to augment cardiac dysfunction. Though, the cardioprotective role of IL10 is well known, its role in reducing gut dysbiosis and maintaining intestinal permeability during hypertrophic heart failure is not known. We hypothesized that IL10 maintains intestinal permeability and inhibits the release of gut microbial peptides (GMPs) which ultimately improves cardiac function.

Methods:
WT & IL10KO mice were subjected to TAC or sham surgery. Heart and intestine were collected to evaluate inflammation genes expression, immune cell infiltration (flow cytometry), and intestinal permeability (WB and immunohistochemistry). Blood was collected to quantify peptidoglycan (PGN) and other bacterial metabolites (TMAO) levels. The effect of PGN on mitochondrial functions was evaluated in neonatal cardiomyocytes.

Results:
Cardiac functions were significantly reduced in IL10KO mice following TAC. IL10 KO mice showed exacerbated inflammation (IL1β, IL6 and TNFα genes expression) following TAC. TAC induced leukocytes (monocyte/macrophage, T-cell and neutrophils) recruitments in the intestine in IL10KO mice. TAC mice showed gut dysbiosis (16S rRNA sequencing) in IL10KO mice. Further, IL10KO mice exhibited higher intestinal permeability as shown by increase in PV1 level and reduction in zonulin-1 (ZO-1) protein. Plasma PGN level was significantly increased in IL10KO mice following TAC. Pathway-based cardiotoxicity array showed a significant increase in uncoupler protein 2 (UCP2) and phospholamban (PLN; a calcium handling protein) genes expression in cardiomyocytes following PGN treatment. Finally, IL10 treatment restores PGN-induced decrease in mitochondrial membrane potential (TMRE assay) in cardiomyocytes.

Conclusion:
Taken together, data suggest that IL-10 helps to maintain gut wall permeability and reduced gut microbial peptide circulation to the heart and thus improves cardiac function by improving mitochondrial function. Ongoing investigations using molecular approaches will provide a better understanding of gut-heart signaling and its impact on the progress of heart failure.
Abstract P3062: T-cell Myd88 Regulates Cardiac Fibrosis In Heart Failure By Modulating T-cell Survival And Pro-inflammatory Signaling
Authors: Bayer Abraham L , Smolgovsky Sasha , Ngwenyama Njabulo , Aronovitz Mark , kaur Kuljeet , Alcaide Pilar
Published: November 2022

Background:
Despite the association of heart failure (HF) with cardiac and systemic inflammation, anti-inflammatory therapies have shown limited success. Cardiac inflammation is characterized by the presence of damage associated molecular patterns (DAMPs), myeloid and T-helper cell infiltration, and the activation of cardiac fibroblasts (CFB). The adaptor “Myeloid differentiation response 88” (MyD88) is central to DAMP sensing in myeloid cells, but its role in T-cell function and cardiac inflammation is largely unknown. We
hypothesized
that T-cell signaling through MyD88 modulates cardiac inflammation and fibrosis in HF.

Methods and Results:
We reconstituted T-cell deficient (
Tcra
-/-
) mice, normally protected from cardiac fibrosis, with WT or

Myd88
-/-

Type 1 helper T-cells (Th1) in the onset of transaortic constriction (TAC). We found mice given

Myd88
-/-

Th1 cells exhibited significantly higher levels of cardiac T-cell infiltration, increased cardiac fibrosis, and lower fractional shortening than mice given WT Th1 cells. T-cell specific MyD88 deficient mice (
Myd88
fl/fl
CD4
Cre
) also developed more fibrosis and increased cardiac T-cells compared to Cre- littermate controls in response to TAC. RNA-seq analysis of WT and

Myd88
-/-

Th1 cells revealed enrichment of pro-inflammatory signaling and pro-survival genes in

Myd88
-/-

Th1 cells, which we validated by immunoblotting for phospho-ERK, NF-kB, and AKT. In contrast, IFNγ, the Th1 signature cytokine, was similar by qPCR and flow cytometry in WT and

Myd88
-/-

cells. Moreover, we discovered that

Myd88
-/-

Th1 cells have increased survival
in vitro,
by monitoring propidium iodide incorporation in real time, and
in vivo
by adoptive transfer of equal numbers of WT and

Myd88
-/-

cells together into
Tcra
-/-
mice and quantifying both cell types in lymphoid organs over time. Lastly, more

Myd88
-/-

Th1 cells adhered to immobilized VCAM-1 and CFB
in vitro
compared to WT, resulting in enhanced CFB transformation.

Conclusion:
T-cell MyD88 limits cardiac fibrosis in TAC through modulation of T-cell inflammatory signaling and cell survival. Disruption of this pathway in T-cells results in enhanced transformation of fibroblasts and worsened systolic function.
Abstract P3069: The Impact Of Lymphatic Vasculature Post-Cardiac Transplantation On Allograft Function, Fibrotic Remodeling, And Lymphatic Drainage
Authors: Ginn Sydney C , Levit Rebecca D
Published: November 2022

Lymphatic drainage inherently modulates cardiac function by maintaining the immune response and tissue-fluid homeostasis. During cardiac transplantation, the lymphatic collecting vessels are severed at the time of donor heart excision and not surgically reconstructed in the recipient. We hypothesize severed cardiac lymphatics contribute to transplant rejection and reduced cardiac function by influencing myocardial edema and immune cell transit.

Methods:
A heterotopic abdominal heart transplant (HAHT) rodent model permitted the immunologic evaluation of transplant rejection as supported in past literature. Echocardiography confirmed allograft survival via active contraction, while histologic quantification of lymphatic vessels and fibrosis were obtained for native and transplanted rodent hearts (n=3). Lymphatic drainage was assessed via intramyocardial injection of fluorescein isothiocyanate (FITC)-dextran.

Results:
Electrocardiographic impulses detected during the echocardiograms revealed additional QRS complexes attributed to contractions of the transplanted hearts. Histologic analysis of fibrosis in rodent allografts at an early timepoint exhibited increases in total tissue accumulation (1.48 – 5.15 fold) and significant increases in various focal regions (left ventricle, ***p < 0.001; interventricular septum, ****p < 0.0001) compared to the native hearts. Furthermore, large amounts of cellular infiltrate and marginal increases in cardiac diameter (1.10 – 1.42 fold) were observed in the transplants. While the scarcity of immunofluorescence signal for lymphatic markers has caused cardiac lymphatic quantification to be inconclusive, evaluation of lymphatic transport revealed fluctuating time-dependent circulating FITC levels (0.002 - 0.010 mg/mL; *p < 0.05) suggesting multi-teared compartmental release properties between the microcirculatory system, lymphatic system, and cardiac tissue.

Conclusion:
The HAHT rodent model provides a promising modality to assess both lymphatic transport and transplant rejection. Moreover, these studies will progress understanding of transplant rejection’s pathological intricacies and provide the groundwork for lymphangiogenic-based therapeutic intervention.
Abstract P3075: Hippo Pathway In Cardiac Fibroblast Regulates Macrophage Recruitment Following Injury
Authors: Meng Fansen , Li Xiao , Li Rich Gang , Tsai Chang-Ru , Martin James F
Published: November 2022

Background:
The evolutionary conserved Hippo signaling pathway (HSP) is an inhibitor of tissue regeneration, including the Lats1/2 kinases that phosphorylate and inactivate YAP and TAZ. Emerging data indicate that HSP regulates innate immunity, inflammation, and autoimmune diseases. However, the role of HSP in post-MI inflammation is poorly understood.

Methods:
Single-cell RNA-sequencing (scRNA-Seq) and spatial transcriptomics were utilized in cardiac fibroblast (CF)-specific-Lats1/2 knockout (Lats CKO) mouse heart. Flow cytometry, immunofluorescence (IF), and echocardiogram (Echo) were used to describe the phenotype in ventricle of Lats CKO mouse heart with/out Ccr2 knockout.

Results:
We integrated our scRNA-Seq data in Lats CKO mouse heart with three published data sets of MI mouse heart. A CF subcluster with elevated YAP activity and expressing chemokines Ccl2 and Ccl7 (Ccl2/Ccl7+ CFs) and macrophages (Macs) expressing Ccr2, the receptor for Ccl2/Ccl7 (Ccr2+ Macs), were identified, which suggests a signaling transduction from Ccl2/Ccl7+ CFs to Ccr2+ Macs in post-MI hearts. To figure out the function and mechanism of this specific cell communication, we employed a Lats CKO mice model which mimics the post-MI heart and exhibits high YAP activity and conformed the cell communication between Ccl2/Ccl7+ CFs and Ccr2+ Macs by spatial transcriptomics. Furthermore, Ccr2 loss of function in Lats CKO mice suppressed Mac expansion and CF proliferation. Consistently, Ccr2 knockout reversed the phenotype presenting smaller left ventricle chamber in Lats CKO mouse heart because of CF proliferation.

Conclusion:
YAP target genes Ccl2 and Ccl7 in CFs are responsible for Ccr2+ Macs recruitment and the recruited Ccr2+ Macs in turn promote CF proliferation post-MI.
Abstract P3079: Hypoxia Stimulates Neonatal Hypoxia-inducible Factor 1α Dependent Macrophage Secretion Of Cardiomyocyte Mitogens
Authors: Becker Amanda C , Lantz Connor , Thorp Edward B
Published: November 2022

Background:
The neonatal murine heart can regenerate after injury. This regenerative capacity is dependent on macrophages and lost shortly after birth. How macrophages facilitate regeneration remains vague, and the neonatal myeloid response to hypoxia as well as the role of myeloid Hypoxia-Inducible Factors (HIFs) in cardiac regeneration are unknown. We hypothesized that post-ischemic regulation of regenerative programs are confined to neonatal cardiac macrophages and are lost in the adult. We also hypothesized hypoxia is a stimulus for neonatal macrophages to secrete cardiomyocyte mitogens in a
HIF
dependent manner.

Methods:
Single-cell sequencing was performed on cardiac myeloid cells post myocardial infarction (MI) in wildtype neonatal and adult mice to explore differences in regenerative vs reparative responses to injury. Neonatal cardiomyocytes harvested from wildtype mice were co-cultured with supernatant from hypoxia stimulated neonatal bone marrow-derived macrophages from myeloid
HIF-1
α
expressing vs
HIF-1
α
genetically depleted mice. Immunofluorescent staining with antibodies to Troponin T, Ki67, and DAPI was performed to quantify cardiomyocyte proliferation.

Results:
High-resolution clustering identified 3 distinct macrophage populations present in neonatal and adult mice post MI. Gene Ontology identified significantly upregulated genes in biological pathways associated with regeneration and HIF-1α stabilization in
Timd4+Mrc1+
macrophages in the neonate but not the adult. Immunofluorescent staining of neonatal cardiomyocytes at steady state revealed an average of 6.0% Ki67+ cardiomyocytes compared to 35.4% Ki67+ cardiomyocytes exposed to supernatant from hypoxia stimulated
HIF-1
α
+
macrophages (N=10/group; SEM
+
1.96;
p
value <0.0001) and 10.1% Ki67+ cardiomyocytes exposed to supernatant from hypoxia stimulated
HIF-1

α
-

macrophages (N=10/group; SEM
+
1.54;
p
value 0.0162).

Conclusions:
We have newly identified enrichment of regenerative and HIF-1α stabilization pathways specific to neonatal resident cardiac macrophages after ischemic injury. We also newly conclude hypoxia is a stimulus for neonatal macrophages to secrete
HIF-1
α
dependent mitogenic factors that induce cardiomyocyte proliferation.
Abstract P3080: S100A8/9 Is A Key Regulator For Normal Heart Function During
Pseudomonas Aeruginosa
Infection
Authors: Kumar Naresh , Pestrak Matthew J , Wu Qian , Dellos-Nolan Sheri , Shukla Rajnikant , Mitchem Cortney F. , yu Lianbo , Lafuse William , Nagareddy Prabhakara R , Ganesan Latha P. , Wozniak Daniel , Rajaram Murugesan V
Published: November 2022

Rationale:
P. aeruginosa
(
P.a
) infection can cause severe pneumonia in ICU patients that leads to cardiac inflammation and cardiac dysfunction. Growing evidence indicates that a large number of neutrophils are recruited to the site of
P.a.
infection and releases S100A8/9 into the microenvironment. S100A8/9 has been shown to activate both the immune system (TLR4/RAGE) and has bactericidal properties. Often, hyperactivation of the inflammatory responses by S100A8/9 causes cardiac inflammation and dysfunction. Thus, we hypothesize that excessive production of S100A8/9 restrict bacterial growth and enhances the host immune response in order to control the infection and preserve heart function.

Objective:
To identify the mechanism of S100A8/9 mediated cardiac inflammation and cardiac dysfunction during
P. aeruginosa
infection.

Methods and Results:
To investigate the role of S100A8/9 in cardiac inflammation and cardiac dysfunction, we infected wild type (WT) and S100A8/9KO mice with
P.a.
intranasally. We monitored bodyweight at 24h intervals and measured weight loss, survival, and bacterial burden in the lungs and heart. We performed multicolor flow cytometry on BAL cells and heart cells for immune cell phenotyping and ELISA for cytokine levels. Also, we measured cardiac electric activity (EKG) and heart function (echocardiography). We found that bacterial burden in the lungs and heart was higher in S100A8/9KO mice than in WT, which correlated with increased mortality. We found severe cardiac electrical abnormalities (second-degree AV block) in S100A8/9KO mice and left ventricular dysfunction. However, there was no significant difference in the BALF immune cell phenotypes of both S100A8/9KO and WT infected mice. Finally, the S100A8/9 inhibitor enhanced the survival of
P.a.
infected mice.

Conclusion:
Our data demonstrate that neutrophil recruitment and release of S100A8/9 is critical to control bacterial growth in the lungs, however, uncontrolled neutrophil accumulation leads to excessive production of S100A8/9. Hyperactivation of the immune system by excessive S100A8/9 leads to systemic inflammation and bacterial dissemination of the heart. Furthermore, our studies revealed that blocking S100A8/9 receptor binding enhances the survival of mice during
P.a.
infection.
Abstract P3084: T Helper Cell Polarity Determines Salt-sensitivity And Development Of Hypertension
Authors: KIM InKyeom , Kim Jee Young , Lee Soyung
Published: November 2022

Background:
Sodium chloride drives the stable induction of pathogenic T helper (Th) 17 cells and development of hypertension. However, high-salt intake induces hypertension in salt-sensitive persons but not in salt-resistant persons. Pathogenic Th17 cell plays an important role in the development of hypertension whereas suppressive Treg cell plays an important role in the prevention of hypertension.

Objective:
We tested the hypothesis that .

Methods and Results:
Eight-week-old male SS and SR rats were fed chow (NS, 0.4% NaCl) or high-salt (HS, 4% NaCl) diet for 4 weeks. Systolic blood pressure was measured by the tail-cuff method. Cultured splenic T cells from Dahl salt-sensitive (SS) and salt-resistant (SR) rats were exposed to hypertonic salt solution with or without IL-6 or TGFβ. T helper cell (Th17 and Treg) polarity was determined via flow cytometry. The expression of Th17-related genes such as interleukin (IL)-17A, IL-23R and retinoic acid receptor-related orphan receptor (ROR) γt) as well as Treg-related genes such as IL-10, CD25 and forkhead box (Fox) P3 were measured by ELISA or qRT-PCR. Adoptive transfer of Th17 cells isolated from SS or SR donors fed HS were performed intraperitoneally into SS and SR recipients. Upon chow diet, SR has higher Treg cells than SS does. HS intake induced hypertension and increased proinflammatory Th17 cells in SS but not in SR. Hypertonic salt solution induced differentiation of cultured splenic T cells preferentially into proinflammatory Th17 cells and anti-inflammatory Treg cells in SS and SR, respectively. Polarizing cytokine IL-6 also induced differentiation of cultured splenic T cells preferentially into proinflammatory Th17 cells and anti-inflammatory Treg cells in SS and SR, respectively, which were further potentiated by hypertonic salt solution. Adoptive transfer of activated Th17 cells isolated from hypertensive SS or normotensive SR, both of which had been fed HS diet, induced hypertension in SS recipients, but not SR recipients.

Conclusions:
These results indicate that T helper cell polarity after HS intake determines salt-sensitivity and development of hypertension in SS and SR rats. Th17 cell polarity predicts salt-sensitivity and development of hypertension, whereas Treg cell polarity predicts salt-resistance and maintenance of normotension. SR rats protect against hypertension after HS intake through anti-inflammatory Treg cell polarity.
Abstract P3092: Transcriptomic Landscape Of The Postnatal Atrioventricular Conduction System
Authors: Oh Yena , Cook David , Park Jin G. , Vanderhyden Barbara , Munshi Nikhil , Kim Kyoung-Han
Published: November 2022

The atrioventricular conduction system (AVCS) is a network of specialized cardiomyocytes responsible for coordinated spreading of electrical impulses throughout the heart to allow synchronized contractions. Specifically, it is composed of the AV node (AVN), which delays the electrical signal, and the ventricular conduction system (VCS) (i.e.
His
bundle, bundle branches and
Purkinje
fibers), which rapidly propagates the signal throughout the ventricles. Although these AVCS structures were discovered >100 years ago, its molecular constituents are not fully known. In this study, AVCS cells, making up ~0.05% of myocytes (~500 cells/heart), were isolated from early postnatal hearts (Postnatal day 1 to 4) of fluorescent reporter mice (Cntn2- Cre;Rosa26TdTomato), purified using fluorescence-activated cell sorting, and were subjected to single-cell RNA-sequencing. Unbiased cluster analysis on ~7000 AVCS cells demonstrated distinct transcriptomic profiles in the AVN, proximal-VCS (i.e.
His
bundle and bundle branches) and distal-VCS (i.e.
Purkinje
fibers). Conforming to previous studies, global conduction markers, such as
Etv1
and
Kcne1,
were present throughout the entire AVCS, whereas a VCS-specific TF,
Irx3
, was expressed only in the VCS. Additionally,
Tbx3, Pcp4, Nppa
and
Lyz2
were enriched in the proximal, not distal, VCS. Sub-clustering analysis of AVN and proximal-VCS further identified unique molecular profiles, such as
Shox2
(in compact AVN) and
Rspo3
(likely in lower nodal region connecting AVN and proximal-VCS). Moreover, we uncovered a potential regulatory relationship between
Irx3
and
Tbx3
in the AVCS, mediating their downstream targets,
Gja5
and
Gja1
. In
Irx3
and
Tbx3
-positive proximal-VCS,
Gja5
and
Gja1
expressions were lower compared to only
Irx3
-positive distal-VCS, whereas in only
Tbx3
-positive AVN, both
Gja5
and
Gja1
expressions were undetected. Importantly, this regional difference was recapitulated in cultured neonatal mouse atrial and ventricular myocytes overexpressing
Irx3
and/or
Tbx3
. Overall, our study provides a comprehensive view of the cellular and molecular heterogeneity within the postnatal AVCS at a high-resolution and reveals novel gene regulatory mechanisms that may govern the development of the AVCS.
Abstract P3094: Mir-181c-5p As Detrimental Player In A Mouse Model Of Cardiorenal Syndrome With Thrombotic Microangiopathy
Authors: Boen Jente R , Krüger Dustin , Dendooven Amélie , Bruyns Tine , Van Fraeyenhove Jens , Feyen Eline , Gevaert Andreas B , Segers Vincent F , Van Craenenbroeck Emeline M
Published: November 2022

Background:
MiR-181c-5p is described to induce heart failure (HF), while its role in renal pathology is undetermined. Renal dysfunction is present in 40-60% of HF patients and associated with poor prognosis. This study is the first of its kind to investigate the role of miR-181c-5p in a mouse model of cardiorenal disease (CRD). Our hypothesis states a protective effect of miR-181c-5p inhibition on HF development.

Methods:
CRD was induced by feeding male C57BL/6J mice (n=20) a high-fat diet (HFD) and L-NAME in drinking water (5g/L) for 6 weeks, angiotensin-II was co-administered via osmotic minipumps (1000ng/kg/min) during the final 2 weeks. Healthy controls (n=16) underwent sham-surgery. Mice were randomly assigned to weekly injections (40mg/kg) with miR-181c-5p antagomiR (INH) or scrambled control for the duration of the study. We assessed cardiac function (echocardiography, hemodynamics), renal function (plasma creatinine), target expression (RT-qPCR), and histology.

Results:
CRD animals showed mild systolic and diastolic cardiac dysfunction compared to healthy controls, with cardiac fibrosis (2.5±0.3 vs 1.8±0.2% area; p=0.0004) and hypertrophy (0.11±0.03 vs 0.08±0.01g/cm; p=0.005). Renal dysfunction presents with kidney atrophy (0.07±0.006 vs 0.09±0.01g/cm; p=0.02), increased plasma creatinine (21±6 vs 10±5; p=0.01), renal fibrosis (0.26±0.22 vs 0.005±0.21 % area; p=0.036) and glomerular abnormalities (glomerulosclerosis, hypertrophy/atrophy, mesangial matrix expansion, reduced podocyte number). CRD+INH animals had comparable cardiac phenotype to CRD (p>0.05). Their renal phenotype was exacerbated with elevated glomerular damage (26±3 vs 18±9;p=0.04) and significantly increased mortality rate (50%) (Kaplan-Meier p=0.01) compared to healthy controls (0%) or CRD (20%), associated with increased occurrence of tubular atrophy, endothelial swelling and systemic thrombotic microangiopathy (TMA). qPCR analysis identified
Vegf
as potential target of miR-181c-5p.

Conclusion:
This study demonstrates a detrimental effect of miR-181c-5p inhibition on renal function in a CRD mouse model, driven by glomerular damage and TMA through Vegf signaling. Cardiac function was unaffected.
Abstract P3098: Histone Modifying Genes In Heart Development And Disease
Authors: Zhu Junyi , Lee Hangnoh , van de Leemput Joyce , Han Zhe
Published: November 2022

Sequencing of Congenital Heart Disease (CHD) patients identified variants in many histone modifying genes, suggesting that histone modification plays important roles in heart development and disease. However, how these histone modifying genes perform their epigenetic regulation function during heart development remain elusive, and whether these novel genetic variants identified from CHD patients are causal remain unclear. We found that most histone modifying genes are highly conserved from
Drosophila
to humans, and many are expressed in the developing heart in both systems. Using a high-throughput functional validation system that we generated for providing the “gene-level” disease association for candidate CHD genes, we screened lots of histone-modifying genes and identified many that are required for heart development in Drosophila. Among them, genes involved in histone H3K4 mono- and dimethylation are particularly important for heart development and appear to regulate evolutionarily conserved targets during heart development from
Drosophila
to mammals. We also generated Drosophila models for CHD patient-derived variants in histone-modifying genes to study how these variants affect the function of the human genes in the context of the fly heart development, to provide the “variant-level” evidence for their causal association with CHD.
Abstract P3104: Extracellular Vesicles From The Infarcted Heart Facilitate Tumor Growth
Authors: Caller Tal , Shaihov–Teper Olga , Schary Yeshai , Lendengolts Daria , Rotem Itai , Peled Racheli , Shai Ruty , Amit Uri , Glick-Saar Efrat , Dominissini Dan , Boomgarden Alex , D'Souza-Schorey Crislyn , Naftali-Shani Nili , Leor Jonathan
Published: November 2022

Background and Aim:
Heart disease might be an independent risk factor for cancer (reverse cardio-oncology). The cellular and molecular mechanisms that link heart disease to cancer remain elusive, and specific therapies are limited. We hypothesized that cardiac extracellular vesicles (cEVs) secreted by diseased hearts carry and disseminate factors that promote tumor growth.

Methods & Results:
We subjected female mice to myocardial infarction (MI) or sham-MI and 28 days of follow-up. Left ventricular remodeling was confirmed by echocardiography. To determine the role of cEVs in tumor growth, we focused on cardiac mesenchymal stromal cells (cMSCs), which play a central role in cardiac repair, remodeling, and fibrosis. Thus, we isolated cMSCs from mice hearts 10 or 28 days after MI or sham MI. cMSCs after MI secreted more small EVs than cMSCs from sham-MI. Proteomic analysis revealed a distinctive profile of cEVs after MI (
Fig A
). Purified cMSC-EVs targeted both breast and lung cancer cells in vitro. A scratch assay showed that MI-cEVs facilitated cancer cell proliferation and migration two times faster than sham-MI cEVs (
Fig B
, p=0.0002). Finally, lung or breast cancer cells were inoculated into the hind limb or mammary pad 10 days before or after MI. Tumor growth was monitored by serial ultrasound examinations. While MI stimulated tumor growth, EV inhibition by GW4869 markedly attenuated this effect (
Fig C
).

Conclusions:
We show, for the first time, that cMSCs from the infarcted and remodeling heart secret EVs that target tumor cells and facilitate tumor growth. We propose cEVs as potential mediators and therapeutic targets in patients with concomitant heart disease and cancer.
Abstract P3106: Trametinib Decreases Electron Transport Chain Activity In The Uninjured Mouse Heart
Authors: Sandroni Peyton B
Published: November 2022

Trametinib (Trm) is a highly selective inhibitor of the tyrosine kinases MEK1/2 used to treat patients with melanoma and non-small cell lung cancer featuring BRAF mutations. Although Trm is generally well tolerated, it is associated with adverse cardiovascular effects including heart failure. Furthermore, the mechanisms underlying this cardiotoxicity are unclear. Here, we assessed the hypothesis that Trm decreases oxidative phosphorylation in the mouse heart. Female FVB mice were treated with Trm (3 mg/kg/d) or vehicle (DMSO) via oral gavage (n = 10, each group). We assessed cardiac contractility on Day 0 and Day 7 using echocardiography. We then performed electron transport chain (ETC) enzyme assays in isolated cardiac mitochondria and RNA sequencing of cardiac tissue to assess ETC Complex I-IV enzyme activity and ETC transcript abundance, respectively. Additionally, we measured citrate synthase (CS) activity as a marker for mitochondrial abundance. Mice treated with Trm had decreased fractional shortening on Day 7 compared to Day 0 (Panel A, 47.7 ± 1.6% vs. 55.5 ± 0.8%, p < 0.001) (mean ± SEM), confirming the cardiotoxicity of our treatment model. Additionally, mice treated with Trm had increased lung weight to heart weight (LW/HW) ratios compared to vehicle controls (Panel B, 1.69 ± 0.11 vs. 1.39 ± 0.03, p = 0.01), indicating heart failure. Mice treated with Trm had decreased CS (7,235 ± 586 vs. 8,562 ± 192 nmol/mg/min, p = 0.024), Complex II (1,659 ± 207 vs. 2,239 ± 239, p = 0.04), and Complex IV (3,979 ± 460 vs. 5,576 ± 615, p = 0.03) activities with a trend towards decreased Complex III (2,703 ± 452 vs. 3,554 ± 434, p = 0.09) activity (Panel C). Mice treated with Trm had decreased transcript abundance of Complex I (41 of 43), Complex II (6 of 6), Complex III (8 of 8), and Complex IV (18 of 23) transcripts (Panel D). Gene Ontology cellular compartment analysis of transcriptomic data revealed that Trm treatment primarily alters abundance of transcripts related to the mitochondrion (Panel E, p
adj
= 3.2 x 10
-113
). KEGG Pathway analysis revealed that oxidative phosphorylation was the most significantly altered process following Trm treatment (Panel F, p
adj
= 2.9 x 10
-27
). Collectively, these findings suggest that Trm causes widespread decreases in ETC activity that may hinder cardiomyocyte oxidative phosphorylation and contractility.