Background: Chronic reductive stress (cRS) contributes to adverse cardiac remodeling and diastolic dysfunction; however, the mechanisms remain unclear. Here, we examined how cRS progressively disrupts proteostasis, causing proteotoxicity overtime leading to cardiac remodeling and dyskinesia. Methods: Cardiac-specific constitutively active Nrf2 transgenic (CaNrf2-TGL/TGH) and NTG mice (n=4–6/group) at ~3 and 6 months of age were studied. NGS-RNA sequencing, speckle tracking strain echocardiography, Proteostat ® histochemistry, and TEM assessed cRS effects on the cardiac transcriptome, endoplasmic reticulum (ER) disintegration, myocardial mechanics, and proteotoxicity. Statistical analyses were performed using Student's t -test. Results: Protein aggregation in TGH/TGL hearts (≥6 months) indicates cRS-induced misfolding. Altered ER transcriptome and subsarcolemmal ER dilation (TEM analysis) suggest ER stress, leading to myocardial structural and functional disintegration. Longitudinal strain showed moderate to severe endocardial deformation in TGL (p>0.01) and TGH (p>0.0001) vs. NTG at 3 months, while severe segmental dyssynchrony was evident in both TGL and TGH at 6 months. Increased global radial strain and decreased global longitudinal strain in TGH at 3 months (p>0.01) suggest the onset of early dyssynchrony. While TGL at 3 months revealed a moderate myocardial segmental deformation. By 6 months, strain differences emerged in TGL vs. NTG (p>0.001) and worsened in TGH (p>0.0001). TEI index, a measure of global cardiac function, was significantly elevated in both caNrf2-TGL and caNrf2-TGH (2 and 4-fold; P>0.0001) compared to NTG mice (normal index: 0.3-0.6) at 6 months, suggesting impaired systolic and diastolic functions in the RS hearts. Conclusion: We established a strong correlation between non-amyloid proteotoxicity and cardiac dysfunction under chronic reductive stress, challenging the previous belief that antioxidants are always beneficial.

Background: Functional HDL exhibiting antioxidant and anti-inflammatory properties and supports cardiovascular health by regulating cellular cholesterol. Oxidative stress impairs HDL function, leading to its dysfunction ( d HDL). Here, we tested whether reductive stress (RS), characterized by excess antioxidants, promotes atherosclerosis. Methods: HDL from CVD patients with RS was tested for cholesterol efflux, Ly6c expression, and MMP9 release in RAW macrophages. Next, we assessed whether RS trigger foam cell formation in the bone marrow derived macrophages (BMDMs) of CAG-caNrf2-TG mice (RS) using oil Red-O staining. 2D-aortic echocardiography was conducted to examine the aortic dimension in CAG-caNrf2-TG (TG) vs WT mice. Results: We found that RS-CVD subjects had lower total HDL cholesterol, reduced Apo A1 content, and decreased Paraoxonase activity. Sulforaphane treatment (2µM; 24 hr) enhanced foam cell formation in RAW macrophages treated with RS-CVD HDL, suggesting RS worsens atheroma. Elevated MMP9 release and LY6C expression indicate increased matrix remodeling and macrophage susceptibility to foam cell formation under RS. Notably, BMDMs from the TG/RS-mice showed significantly increased foam cell formation and elevated Ly6C expression, a pro-inflammatory marker. In vivo echocardiography demonstrated decreased aortic root diameter (AoR: 1.5 vs 0.94 mm, n=3-5/group)) and increased pulse wave velocity in TG/RS-mice, suggesting vascular remodeling. Conclusion: This study shows that RS imapirs HDL function and aggravates atherosclerosis progression.

Chronic reductive stress (cRS), induced by constitutive activation of Nrf2 in transgenic (TG) mouse hearts leads to pathological cardiac remodeling and diastolic dysfunction. Transcriptomic analysis revealed that both pro-reductive (PR) and reductive stress (RS) conditions disrupt ER-associated gene expression in a dose-dependent manner, with pronounced dysregulation in high-expressing TG (TGH) mice. These shifts were associated with persistent activation of the unfolded protein response (UPR), impaired ER function, and redox imbalance marked by elevated glutathione and reduced ROS levels. Proteostasis disruption under cRS led to protein misfolding, ER dilation, and aggregation of mis/unfolded proteins. TGH mice showed increased ubiquitination and accumulation of aggregated proteins, alongside inadequate proteasome activity, indicating inadequate protein quality control (PQC) mechanisms. RNA-seq data revealed transcriptional upregulation of ubiquitin-proteasome genes and downregulation of key chaperones, suggesting a failed compensatory response. Speckle-tracking echocardiography (STE) detected myocardial dyssynchrony and progressive strain abnormalities in TGH mice, correlating with increased proteotoxic burden and impaired redox homeostasis. Elevated TEI index values confirmed systolic and diastolic dysfunction. Time- and dose-dependent upregulation of Nogo/Reticulon4 transcripts and proteins further supported maladaptive cardiac remodeling. Collectively, these findings highlight that chronic RS disrupts ER homeostasis, induces proteotoxicity, and impairs cardiac structure and function, particularly in high transgene-expressing hearts.

Background: Viscerosensory stimuli are transmitted from the peripheral nervous system to the central nervous system for processing, integration, and adequate motor response. Proper signal transmission is guarded by bidirectional interaction between neurons and glia and their dysfunction has been associated with sensory deficits in both aging and visceral disorders. However, compared to the central nervous system, the bidirectional interactions and pathways of communication between glia and neurons remain relatively unknown. The nodose ganglion (NG) houses the cell bodies of visceral sensory nerves, traveling from the periphery, via the vagus nerve, to the central nervous system. Autonomic remodeling in this ganglion is associated with many of the visceral diseases that increase in incidence with age. Given the increasing recognition of the importance of glia in neuronal function and neurotransmission, understanding their inter-cell dynamics are vital to appropriately interpret the neurophysiology of aging. Objectives: This study aimed to better understand subtypes of neurons and glia most involved in neuro-glial communication, the underlying genetic pathways involved in this crosstalk, and how these pathways change with age. Methods: Single-cell RNA sequencing (scRNAseq) was performed on NG of young (12 weeks) and old (16 months) mice (C57BL/6; N = 6 for both groups). Distinct satellite glial cell (SGC) and neuronal populations were clustered based on transcriptomic similarities using dimensionality reduction. Marker gene analyses were used for cell-type identification. Specific pathways responsible for glial-glial, neuron-neuron, and glial-neuronal cross talk, including secreted signaling, extracellular-matrix receptor interactions, and cell-cell contact, were assessed between the different glial and neuronal sub-clusters in young and aged NG. The R-packages Seurat and CellChat were used for cell clustering and cell-cell communication signaling pathway identification, respectively. Results: SGC ( N = 6835 cells total) were identified by high expression of glial-specific transcripts, S100b and Fabp7. Neurons ( N = 2027 neurons total) were identified by expression of Tubb3, Snap25, and Uchl1. Five distinct glial clusters and six distinct neuronal clusters were identified by expression of known functional marker genes. While neuron-to-neuron communication was limited, significant communication within glial subtypes and between glia and sensory neurons was noted, with residential SGCs being most central to this network. Residential SGC interactions were noted most significantly for excitatory, multimodal neuronal subtypes ( Vglut2+, TRPV1/2+, Piezo1/2+). The primary pathways driving this communication were trophic pathways (e.g., vascular endothelial growth factor , Tenascin C, and Thrombospondin-1). Surprisingly, while glia-glia signaling did not change with aging, glial-neuronal communication seemed to increase. Conclusion: These data suggest that, while there is limited communication between neurons in the nodose ganglia, there is extensive glial-glial and glial-neuronal communication. Resident SGCs and excitatory multimodal neurons, which are known to be critical in sensory neurotransmission, are central to this crosstalk. Aging was associated with increased glial-neuronal crosstalk, though the cause and effect of this increased glial-neuronal communication with age requires further investigation. Dr. Vaseghi is supported by NIH R01HL148190 and AHA 970217. VvW is supported by the NWO Rubicon grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: Protein half-life and turnover are crucial for cellular function, especially under basal and stress conditions, often contributing to proteinopathies. While the impact of oxidative stress (OxS) on proteostasis is well-documented, the role of reductive stress, an overabundance of antioxidant status, in proteotoxic cardiac disease remains elusive. Hypothesis: Tested whether chronic reductive stress (cRS) impairs protein turnover and induce proteotoxic cardiac disease. Methods: In transgenic mice expressing constitutively active Nrf2 (caNrf2-TG) and non-transgenic controls (n=6/gp.), we examined the half-life and turnover rates of the myocardial proteome using D2O labeling and mass spectrometry. Results: We observed significant changes in the half-life of over 1,700 proteins, with approximately 1,200 proteins exhibiting increased half-life at 3 months, despite no noticeable defects in cardiac structure and function. Under OxS induced by isoproterenol (ISO), about 700 proteins showed reduced half-life, underscoring distinct regulatory mechanisms in protein turnover between cRS and OxS. Proteins with altered half-lives were involved in key cellular functions, including metabolism, signal transduction, immune response, transport, and cell cycle regulation under cRS, revealing novel targets undetected in an OxS context. Notably, distinct positive adaptive compensatory (59; p<0.05) and maladaptive pathologic (58; p<0.05) responses were observed, resulting in unusual protein stabilization and aggregation, leading to proteotoxic stress under cRS. Comparing transcript levels and protein half-life/turnover (HL/TnO) revealed non-linear metrics for subsets of proteins under cRS. Furthermore, bioinformatic predictions identified potential post-translational modification sites in critical proteins with extended half-lives, correlating with the onset of myocardial remodeling in cRS hearts at 3 months, as indicated by altered Tie index and irregular vector velocities of regional wall motions. Conclusion: Our findings highlight the importance of prolonged protein half-life and reduced turnover, along with unchanged or decreased transcription, as predictive markers for proteotoxic cardiac disease.

Background: The co-occurrence of cardiac and neurological complications is becoming increasingly common among individuals diagnosed with either of these disorders. Hypothesis: We tested the hypothesis that elevating glutathione (GSH) levels through N-acetyl cysteine (NAC), a precursor for GSH synthesis, leads to behavioral abnormalities in a mouse model with proteotoxic heart disease. Approach: We used a transgenic mouse model expressing a cardiac-specific human mutant R120G-αB-crystallin (hR120GCryAB-TG), which develops progressive heart failure due to proteotoxicity. Our investigation aimed to understand the effects of NAC on brain redox and behavior in this model. Results: Administering NAC for a period of 12 weeks, we observed an increase in glutathione levels and its redox state (GSH/GSSG) in both heart (1.0 vs. 3.0 Fold in NTG vs. TG; p>0.001) and brain (1.0 vs. 4.0 Fold in NTG vs. TG; p>0.01) tissues. Interestingly, this increase was associated with exacerbated cardiac dysfunction and abnormal behavior. The administration of NAC worsened both structural and functional defects in the hearts of the transgenic (TG) mice, indicating that enhancing antioxidant defense mechanisms could trigger proteotoxic insults through reductive stress (RS). Additionally, the brain of TG mice exhibited enhanced reductive capacity (p>0.01), accompanied by significant alterations in the levels of Tau and α-Synuclein proteins, when compared to non-transgenic (NTG) mice. While NTG mice, with or without NAC treatment, displayed normal behavior, TG mice treated with NAC developed severe behavioral abnormalities, including cognitive impairment (p>0.01). Conclusions: These findings suggest that increased GSH levels (i.e. RS) in progressive heart failure could cause behavioral issues. Thus, further research into the underlying redox mechanisms in other organs, including the brain, is necessary to better understand the use of antioxidant supplements for heart failure treatment. This study will aid in developing personalized therapeutic strategies. National Institute of Health - NHLBI R01 (R01-2RHL118067; Namakkal-Soorappan) American Heart Assocaition (20POST35200085; Sunny). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Parasympathetic dysfunction after chronic myocardial infarction (MI) is known to predispose ventricular tachyarrhythmias (ventricular tachycardia/ventricular fibrillation [VT/VF]). VT/VF after MI is more common in males than females. The mechanisms underlying the decreased vagal tone and the associated sex difference in the occurrence of VT/VF after MI remain elusive. In this study, using optogenetic approaches, we found that responses of glutamatergic vagal afferent neurons were impaired following chronic MI in male mice, leading to reduced reflex efferent parasympathetic function. Molecular analyses of vagal ganglia demonstrated reduced glutamate levels, accompanied by decreased mitochondrial function and impaired redox status in infarcted males versus sham animals. Interestingly, infarcted females demonstrated reduced vagal sensory impairment, associated with greater vagal ganglia glutamate levels and decreased vagal mitochondrial dysfunction and oxidative stress compared with infarcted males. Treatment with 17β-estradiol mitigated this pathological remodeling and improved vagal neurotransmission in infarcted male mice. These data suggest that a decrease in efferent vagal tone following MI results from reduced glutamatergic afferent vagal signaling that may be due to impaired redox homeostasis in the vagal ganglia, which subsequently leads to pathological remodeling in a sex-dependent manner. Importantly, estrogen prevents pathological remodeling and improves parasympathetic function following MI.

The endoplasmic reticulum (ER) regulates protein folding and maintains proteostasis in cells. We observed that the ER transcriptome is impaired during chronic reductive stress (RS) in cardiomyocytes. Here, we hypothesized that a prolonged moderate treadmill exercise mitigates the RS-induced ER dysfunction and cardiac remodeling in cardiac-specific constitutively active Nrf2 mice (CaNrf2-TG). RNA sequencing showed notable alterations in the ER transcriptome of TG hearts at 4, 12, and 24 weeks (16, 28, and 35 genes, respectively). Notably, the downregulation of ER genes was significant at 12 weeks, and further pronounced at 24 weeks, at which the cardiac pathology is evident. We also observed increased levels of ubiquitinated proteins in CaNrf2-TG hearts across all ages, along with VCP, a marker of ERAD function, at 24 weeks. These findings indicate that constitutive Nrf2 activation and RS impair protein-folding activity and augments ERAD function over time. Exercise intervention for 20 weeks (beginning at 6 weeks of age), reduced cardiomyocyte hypertrophy (from 448μm2 to 280μm2) in TG mice, through adaptive remodeling, and preserved the cardiac function. However, while exercise did not influence antioxidants or ER stress protein levels, it significantly improved ERAD function and autophagy flux (LC-I to LC-II) in the TG-EXE hearts. Collectively, our findings underscore the prophylactic potential of exercise in mitigating RS-associated pathology, highlighting its essential role in maintaining cellular proteostasis through ER-independent mechanisms.

Background: Vagal dysfunction after chronic myocardial infarction (MI) is associated with an increased risk of ventricular tachycardia (VT)/ventricular fibrillation (VF) and increased mortality in heart failure. The incidence and outcomes of VT/VF are influenced by sex, with women having a lower incidence of VT/VF associated with ischemic heart disease. However, the mechanisms behind these sex differences remain unclear and could be due to sex differences in autonomic remodeling post-MI that is mediated by estrogen. Hypotheses: We hypothesized that estrogen improves vagal function after MI by reducing oxidative stress in vagal sensory neurons and improving vagal afferent/efferent reflexes and function. Methods: Mice with expression of channelrhodopsin (ChR2) in specific sensory afferent neurons were created by crossing vesicular glutamate transporter 2 (VGlut2)-IRES-Cre mice with ChR2-EYFP mice, and their offspring (Vglut2-ChR2-EYFP) used for optogenetic stimulation studies. Sham surgery (n=6) or MI (n=6) was performed in two separate groups of male mice. A third group of male mice (n=8) underwent 17β-Estradiol (E2) pellet (0.5 milligrams/pellet) implantation followed by MI (E2+MI) after 2.5 weeks. Terminal studies were performed 2-3 weeks post-MI/sham procedures by placing a blue light (473 nm) laser probe over the left cervical vagus in vivo to stimulate vagal sensory neurons (20 Hz, 10 & 20 msec stimulations for 5 sec). Heart rate (HR) and blood pressure (BP) responses were measured in response to optogenetic stimulation. Plasma E2 and nodose ganglion 4-hydroxynaneol (4HNE) levels, a marker of lipid peroxidation, were evaluated. Results: In response to optogenetic vagal stimulation, MI had reduced reflex heart rate responses vs. sham animals, while E2+MI mice demonstrated significantly better responses vs. MI and similar responses to sham animals (20 Hz, 10 msec: sham -59 ± 3%, MI -24 ± 3%, E2+MI -58 ± 4%, MI vs. E2+MI, P < 0.001; 20 Hz, 20 msec: sham -59 ± 4%, MI -31 ± 3%, E2+MI -60 ± 3%, MI vs. E2+MI, P < 0.001). In addition, while BP responses post-MI were reduced vs. sham, E2+MI male mice also demonstrated greater BP responses to optogenetic stimulation (20 Hz, 10 msec: sham -28 ± 4%, MI -12 ± 1%, E2+MI -27± 1%, MI vs. E2+MI, P < 0.001; 20 Hz, 20 msec: sham -32 ± 4%, MI -13 ± 1%, E2+MI -27 ± 5%, MI vs. E2+MI, P < 0.001). 4HNE levels were increased in MI vs. sham animals, while E2+MI mice demonstrated significant reductions in 4HNE levels (Sham 0.17 ± 0.09 μg/protein, MI 0.77 ± 0.11 μg/protein, E2±MI 0.26 ± 0.06 μg/protein, MI vs. E2+MI, P < 0.001). Finally, plasma measurements confirmed higher levels of estrogen in E2-implanted mice (MI 48 ± 10 pg/mL, E2+MI 850 ± 170 pg/mL, MI vs. E2+MI, P < 0.001). Conclusions: MI causes pathological vagal remodeling that is reflected in decreased sensory vagal neurotransmission and increased oxidative stress in the vagal ganglia. Parasympathetic dysfunction and oxidative stress are mitigated by estrogen, suggesting that estrogen may be an important factor in mitigating the resulting pathological autonomic remodeling post-MI that predisposes to VT/VF. NIHR01HL148190. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Parasympathetic dysfunction after chronic myocardial infarction (MI) is known to predispose ventricular tachyarrhythmias (ventricular tachycardia/ventricular fibrillation [VT/VF]). VT/VF after MI is more common in males than females. The mechanisms underlying the decreased vagal tone and the associated sex difference in the occurrence of VT/VF after MI remain elusive. In this study, using optogenetic approaches, we found that responses of glutamatergic vagal afferent neurons were impaired following chronic MI in male mice, leading to reduced reflex efferent parasympathetic function. Molecular analyses of vagal ganglia demonstrated reduced glutamate levels, accompanied by decreased mitochondrial function and impaired redox status in infarcted males versus sham animals. Interestingly, infarcted females demonstrated reduced vagal sensory impairment, associated with greater vagal ganglia glutamate levels and decreased vagal mitochondrial dysfunction and oxidative stress compared with infarcted males. Treatment with 17 beta-estradiol mitigated this pathological remodeling and improved vagal neurotransmission in infarcted male mice. These data suggest that a decrease in efferent vagal tone following MI results from reduced glutamatergic afferent vagal signaling that may be due to impaired redox homeostasis in the vagal ganglia, which subsequently leads to pathological remodeling in a sex-dependent manner. Importantly, estrogen prevents pathological remodeling and improves parasympathetic function following MI.

Background: Vagal dysfunction after chronic myocardial infarction (MI) exacerbates heart failure and predisposes to ventricular tachyarrhythmias (VT/VF). VT/VF after MI is more common in males than females. The mechanisms behind these sex differences are unknown. Hypothesis: We hypothesized that differences in autonomic remodeling in the vagal ganglia, which in a healthy state, provide beat-to-beat sensory neurotransmission and modulate efferent cardiac vagal tone, may serve as the mechanisms behind the sex differences in the incidence of VT/VF. Methods: MI was created (left anterior descending artery ligation) in male and female mice expressing channel rhodopsin in glutamatergic vagal neurons (VGlut2-ChR2-EYFP mice). Sham animals underwent thoracotomy only. Two weeks post-MI, heart rate (HR) responses were assessed following in vivo vagal afferent nerve activation via optogenetic stimulation (5 sec duration, 20 Hz, 10 ms and 20 ms stimulations). Ventricular fibrosis was quantified. Mitochondrial oxygen consumption rates (OCR), glutamate levels, and the levels of oxidative stress marker, 4-hydroxy nonenal (4HNE), were measured in the vagal ganglia. Results: Male MI animals demonstrated significantly reduced HR responses to optogenetic stimulation than female MI animals. No sex differences in the degree of ventricular fibrosis was observed. A decrease in glutamate content accompanied by decreased mitochondrial OCR and increased 4HNE levels was observed in the vagal ganglia of male infarcted vs. female infarcted mice, figure. There were no differences in the HR responses, glutamate content, mitochondrial OCR, and 4HNE levels between sham males vs. sham females. Conclusion: This study demonstrates significant sex differences in vagal remodeling post-MI, with decreased efferent vagal tone in males mediated at least in part by reduced vagal afferent glutamatergic signaling associated with impaired neuronal mitochondrial function.

Background: Progressive vagal dysfunction occurs with age and predisposes to pathologies in multiple visceral organs. The nodose ganglia (NG) comprises cell bodies of vagal afferent (sensory) neurons, which are crucial for interoception of cardiovascular, pulmonary, and gastro-intestinal function. Satellite glial cells (SGC) envelope and interact with vagal neurons, modulating their activity. With aging, viscero-sensory perception becomes impaired, which might simultaneously increase cardiovascular risk. What subpopulations of SGC are present in murine NG and if their activity and function similarly change during aging remains unknown. Therefore, we explored the transcriptomic profile of SGC in murine NG and characterized changes herein between young and old mice. We hypothesized that with aging SGC shift towards a more senescent and pro-inflammatory phenotype. Methods: Single-cell RNA sequencing (scRNAseq) was performed on NG of young (11.5 weeks) and old (16 months) mice (C57BL/6; N = 6/group). SGC (n = 4046 cells for young, n = 2789 cells for old) were identified by high expression of glial-specific transcripts, S100b and Fabp7. Distinct SGC populations were clustered based on transcriptomic similarity using dimensionality reduction and marker gene analyses were used for cell-type identification. Results: Cluster analysis was represented by t-distributed stochastic neighbor embedding and revealed five distinct transcriptomic subtypes. Marker genes analyses identified cluster 0 as immature SGCs based on increased expression of genes involved in development and cytoskeletal production such as Klf2 and Stmn2, respectively. Cluster 1 was enriched in genes involved in cholesterol syntheses (i.e. Me1 and Scd1), characteristic of mature, functional SGC. Cluster 2 on the other hand had high expression of genes involved in immune responses, such as Igtp and Gbp2. Cluster 3 was identified as ‘resident SGC’ based on their enrichment in genes associated with cell adhesion and extracellular matrix-related genes (i.e. Lum and Dcn), whereas cluster 4 showed high expression of early inflammatory markers and microglial markers, including C1qb and C1qa. Interestingly, with aging, the proportional size of these clusters shifted; whereas immature SGC from cluster 0 comprised 79% in young mice, they made up merely 46% in elderly mice. On the contrary, immune responsive SGC in cluster 2 only contributed to 1.4% of the SGC population in young mice but made up 37% of all SGC in old mice. Relative size of cluster 1,3 an 4 remained similar with aging. Conclusion: Using scRNAseq we demonstrated that aging causes a shift in SGC population in murine NG. Aging was characterized by a relative decrease in undifferentiated SGC and a relative increase in immune related SGC. A better understanding of aging-induced changes in SGC residing in NG could aid in preventing age-related autonomic dysfunction and optimizing vagal therapies targeted at elderly populations. NIH R01 HL148190 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Significant cardiorespiratory coordination is required to maintain physiological function in health and disease. Sensory neuronal “cross-talk” between the heart and the lungs is required for synchronous regulation of normal cardiopulmonary function and is most likely mediated by the convergence of sensory neural pathways present in the autonomic ganglia. Using neurotracer approaches with appropriate negative control experiments in a mouse model, presence of cardiorespiratory neurons in the vagal (nodose) ganglia are demonstrated. Furthermore, we found that convergent neurons represent nearly 50% of all cardiac neurons and approximately 35% of all respiratory neurons. The current findings demonstrate a pre-existing neuronal substrate linking cardiorespiratory neurotransmission in the vagal ganglia, and a potentially important link for cardiopulmonary cross-sensitization, which may play an important role in the observed manifestations of cardiopulmonary diseases.

Introduction: Parasympathetic dysfunction after myocardial infarction (MI) predisposes to arrhythmias and heart failure. Mechanisms behind this dysfunction are unclear. It is known that cardiac sensory afferent neurons in the vagal ganglia sense beat-to-beat changes at the level of the heart. This vagal afferent signaling/activation then increases central cardiac vagal efferent drive. Hypothesis: We hypothesized that MI causes a functional reduction in vagal afferent signaling that subsequently decreases efferent vagal tone, resulting in parasympathetic dysfunction. Methods: A transgenic mouse line was created by crossing mice expressing excitatory vesicular glutamate transporter 2, VGlut-ires-cre, with those expressing channel rhodopsin 2 EYFP (ChR2), resulting in mice with VGlut and ChR2 expression in afferent-specific vagal neurons and fibers. MI was created by left anterior descending artery ligation. Sham animals underwent thoracotomy only. Control animals did not undergo a thoracotomy. Two weeks post-MI or sham, the left cervical vagus nerve was isolated and optically stimulated in vivo using blue light laser (473 nm, 20 Hz, 10 & 20 msec). Heart rate (HR), respiratory rate (RR), and time to activation of efferent vagal effects (time to nadir HR/RR) were assessed. Results: In response to optical stimulation, MI animals (n=5) demonstrated significantly reduced HR responses vs. sham (n=4) and control (n=7) animals at both 10 ms and 20 ms, figure. Time to nadir HR was also increased in MI animals (P<0.05 vs. sham/control). RR decreased in all animals, without significant differences between groups. Conclusions: MI is associated with decreased efferent vagal responses to similar levels of parasympathetic afferent activation. These results suggests that mechanisms behind decreased vagal tone post-MI may be due altered vagal afferent neurotransmission and signaling capabilities. MI selectively affects cardiac, not respiratory, neural responses.

Abstract Background: Membrane lipids/cholesterol determines the fluidity, clustering of receptors and other protein interactions that delineate signaling pathways for cell cycle/proliferation. Emerging evidences suggest that highly proliferative cancer cells show a high lipid and cholesterol avidity and are considered as hallmarks of cancer aggressiveness including ovarian cancer. Paraoxonase 1 (PON1) is a high density lipoprotein (HDL) associated enzyme with a plethora of functions. PON1 deficiency aggravates HDL inflammatory index, systemic inflammation, and results in lipid retention associated diseases including atherosclerosis and hepatic steatosis. Meta-analysis studies documented that PON1 polymorphisms are associated with various cancers including ovarian cancer and PON1 activity is negatively correlated with human ovarian tumor size, providing clinical association between PON1 and ovarian cancers. Yet, the role and mechanism of action of PON1 in ovarian cancer development has not been elucidated. Hypothesis: We hypothesized that PON1 may inhibit the ovarian tumorigenesis by modulating HDL function. Methods and Results: In this report, in patients with ovarian cancer, we observed that plasma PON1 levels are higher, but with lower activity compared to healthy control. Immunoprecipitation of plasma PON1, followed by ELISA against oxidized lipids suggest that PON1 is oxidatively modified, specifically by 4-HNE. In addition, PON1 activity is negatively correlated with HDL pro-inflammatory index. Using a xenograft mouse model, we demonstrate that overexpression of PON1 prevents the development of ovarian cancer. HDL from xenograft transgenic mice shows less pro-inflammatory properties with increased cholesterol efflux efficiency from ovarian cancer cells. Mechanistically, PON1 impairs VEGF signaling in ovarian cancer cells by increasing cholesterol (Mitogenic factor) efflux, resulting in reduced dimerization of vascular endothelial growth factor receptor 2 (VEGF-2R) and VEGF-2R phosphorylation (angiogenic inducer) as well as downstream signaling pathways, including Akt and ERK1/2 which in turn reduce the cell proliferation and angiogenesis. Furthermore, cholesterol and VEGF levels are significantly lowered in the tumor microenvironment in PON1 transgenic mice but there are no changes in the gene expressions that are associated with either cholesterol synthesis or its degradation pathways. Conclusion: Taken together, we report for the first time that PON1 acts as a tumor suppressor of ovarian cancer by possibly reducing VEFG signaling and enhancing the HDL function, suggesting that activation of HDL associated PON1 might be a fruitful strategy to inhibit the ovarian tumor formation. Citation Format: Asokan Devarajan, Victor Grijalva, Dawoud Sulaiman, Feng Su, Ellen O'Connor, Robin Farias-Eisner, Srinivasa Reddy. HDL associated Paraoxonase 1 reduces the ovarian tumorigenesis by enhancing the HDL function [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3946.

Paraoxonase 1(PON1) is an HDL-associated protein, which metabolizes inflammatory, oxidized lipids associated with atherosclerotic plaque development. Because oxidized lipid mediators have also been implicated in the pathogenesis of rheumatoid arthritis (RA), we evaluated the role of PON1 in murine inflammatory arthritis. K/BxN serum transfer (STIA) or collagen antibody transfer (CAIA) was used for arthritis induction in B6 mice homozygous for the PON1 human transgene [PON1Tg], PON1 knock-out mice [PON1KO], and wild type littermate control mice [WT]. Experiments were also performed in K/BxN mice with chronic arthritis, and in RA patients and healthy controls. Arthritis activity in K/BxN mice was associated with a marked dyslipidemia, lower PON1 activity and higher bioactive lipid mediators (BLM), as well as a dysregulated hepatic lipid gene expression profile. Higher serum PON1 activity correlated with lower BLM and lower arthritis activity in both K/BxN mice and RA patients. Overexpression of the human PON1 transgene was associated with reduced inflammatory arthritis, which correlated strongly with higher circulating PON1 activity, upregulation of the hepatic glutathione pathway, and reduction of circulating BLM. These results implicate PON1 as a potential novel therapeutic target for joint disease in RA with potential for vascular benefit, which warrants further investigation.

Abstract Background: Immune system can recognize the neoplastic cells and target them for destruction through tumoricidal process. Further, immune cell dysfunction leads to escape of cancer cells from immune recognition, lead to the initiation and progression of tumor formation. Clinical shreds of evidence suggest that the presence of intratumoral immune cells correlates with improved progression free and overall survival among patients with ovarian carcinoma and provide evidence of activation of antitumor mechanisms. Hence identification of drugs that target the cancer cells as well as activate the immune cells is a sound strategy for cancer therapy. Paraoxonase 2 (PON2) is a membrane-associated lactonase with lipid transport, anti-oxidant and phagocytosis/efferocytosis properties. PON2 deficiency aggravates systemic inflammation, and causes the metabolic associated diseases. Recently we have reported that overexpression of PON2 reduces the ovarian cells proliferation and tumor formation by reducing IGF-1 production and its signaling pathway. Further, using microarray based experiment, we have identified several pathways that modulate the immune cells function such as activation of Natural Killer (NK) cells, dendritic cell maturation, B cells activation, and communication between innate and an adaptive cells signaling pathway were regulated by PON2. Hypothesis: PON2 enhances tumoricidal activity in a paracrine fashion. Methods and Results: NK cells were isolated from control C57BL/6 and purity was assessed with flow cytometry using FITC anti mouse CD49B and results revealed that 95 % of NK cells population. NK cells treated with condition medium from ID8hPON2 (human PON2 overexpressed in mouse ovarian cancer cell line) show an increase in tumoricidal activity compared to condition medium from ID8EV. Neither ID8hPON2 nor ID8EV condition medium affects the viability of NK cells. Moreover, there is no difference in CD49B positive staining in the tumor microenvironment between ID8EV and ID8hPON2 cells receiving mice. Condition medium obtained from ID8hPON2 ID8EV group shows increases the interleukin 18 level and neutralizing antibody against IL18 fail to induce the tumoricidal activity indicating mechanistically PON2 enhances the tumoricidal activity via IL18 dependent manner. Furthermore, IL18 levels are increased in tumor micro environment. PON2 increase the IL18 through transactivation of c-Myc gene that are independent of mitochondrial function. Conclusion: Taken together, our study for the first time showed that PON2 enhances the tumerocidal activity via paracrine manner by upregulating IL18 and PON2 could be used as conventional as well as immunotherapy for ovarian cancer. Citation Format: Asokan Devarajan, Dawoud Sulaiman, Feng Su, Ekambaram Ganapathy, Robin Farias-Eisner, Srinivasa Reddy. Cardioprotective paraoxonase 2 enhances the tumoricidal activity of natural killer cells in a paracrine manner [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3309.