Sham Ligation Research Articles

Sotagliflozin attenuates cardiac dysfunction and remodeling in myocardial infarction rats

ObjectiveSotagliflozin is a dual sodium-glucose co-transporter-1 and 2 (SGLT1/2) inhibitor with selectivity towards SGLT2. Previous studies showed that SGLT2 inhibitors can improve cardiac function and reduce myocardial infarction size in animal models of myocardial infarction (MI). However, it remains unknown whether the dual inhibition of SGLT1/2 by sotagliflozin has beneficial effects in this context. In this study, we investigated the potential cardioprotective effects of sotagliflozin in an animal model of MI. MethodsSprague Dawley (SD) rats underwent left anterior descending coronary artery ligation or sham ligation then were randomly assigned to receive either sotagliflozin (10 mg/kg) or vehicle via intraperitoneal injection. Fourteen days post-MI, we assessed cardiac function using echocardiography and evaluated histological and molecular markers of cardiac remodeling and inflammation in the left ventricle. ResultsOur findings indicate that sotagliflozin treatment resulted in improved cardiac function and reduced infarct size compared with the vehicle-treated group. Additionally, sotagliflozin improved cardiac remodeling as shown by the decreased cardiac hypertrophy and cardiac apoptosis in the post-MI heart. Mechanistically, an apparent reduction in the cardiac inflammatory response in sotagliflozin-treated hearts was observed in the post-MI rats. ConclusionOverall, our results suggest that sotagliflozin may have cardioprotective effects against myocardial infarction.

Non-invasive tragus stimulation improves cardiac post-ischemic remodeling by regulating cardiac parasympathetic activity.

Our previous study proved that low-level tragus nerve stimulation (LL-TS) could improve left ventricular remodelling by cardiac down-stream mechanisms. However, the cardiac up-stream mechanisms remain unknown. Twenty-eight adult beagle dogs were randomly divided into an MI group (myocardial infarction was induced by permanent ligation of the left coronary artery, n=10), an LL-TS group (MI plus intermittent LL-TS treatment, n=10), and a control group (sham ligation with the same stimulation as the LL-TS group, n=8). Auricular tragus nerve was bilaterally delivered to the tragus via ear-clips connected to a custom-made stimulator. The voltage slowing sinus rate was used as the threshold to set the LL-TS 80% below this level. At the end of 4weeks post-MI, LL-TS could significantly increase atrial ganglion plex (GP) activity, decreased left stellate ganglion (LSG) activity, reduced LV dilation, and improved ventricular functions. Chronic intermittent LL-TS treatment significantly attenuated left ventricular remodelling via the up-regulation of α7nAChR expression and the down-regulation of MMP-9 level in post-MI LV tissue. The elevated protein and mRNA of MMP-9 levels in remote areas were significantly ameliorated by LL-TS treatment. Chronic LL-TS increased GP neural activity and improved ventricular remodelling possibly via α7nAChR/MMP-9 axis.

Fetal pulmonary hypertension: dysregulated microRNA-34c-Notch1 axis contributes to impaired angiogenesis in an ovine model.

Persistent pulmonary hypertension of the newborn (PPHN) occurs when pulmonary vascular resistance (PVR) fails to decrease at birth. Decreased angiogenesis in the lung contributes to the persistence of high PVR at birth. MicroRNAs (miRNAs) regulate gene expression through transcript binding and degradation. They were implicated in dysregulated angiogenesis in cancer and cardiovascular disease. We investigated whether altered miRNA levels contribute to impaired angiogenesis in PPHN. We used a fetal lamb model of PPHN induced by prenatal ductus arteriosus constriction and sham ligation as controls. We performed RNA sequencing of pulmonary artery endothelial cells (PAECs) isolated from control and PPHN lambs. We observed a differentially expressed miRNA profile in PPHN for organ development, cell-cell signaling, and cardiovascular function. MiR-34c was upregulated in PPHN PAECs compared to controls. Exogenous miR34c mimics decreased angiogenesis by control PAEC and anti-miR34c improved angiogenesis of PPHN PAEC in vitro. Notch1, a predicted target for miR-34c by bioinformatics, was decreased in PPHN PAECs, along with Notch1 downstream targets, Hey1 and Hes1. Exogenous miR-34c decreased Notch1 expression in control PAECs and anti-miR-34c restored Notch1 and Hes1 expression in PPHN PAECs. We conclude that increased miR-34c in PPHN contributes to impaired angiogenesis by decreasing Notch1 expression in PAECs. Adds a novel mechanism for the regulation of angiogenesis in persistent pulmonary hypertension of the newborn. Identifies non-coding RNAs that are involved in the altered angiogenesis in PPHN and thus the potential for future studies to identify links between known pathways regulating angiogenesis. Provides preliminary data to conduct studies targeting miR34c expression in vivo in animal models of pulmonary hypertension to identify the mechanistic role of miR34c in angiogenesis in the lung vasculature.

Protein Kinase C Epsilon Contributes to the Exaggerated Mechanoreflex in Rats with Heart Failure

BackgroundDuring exercise, skeletal muscle contraction stimulates mechanically sensitive channels on the sensory endings of thin fiber muscle afferents. Stimulation of these mechanically sensitive channels initiates a reflex, termed the mechanoreflex, which contributes importantly to reflex increases in sympathetic nerve activity (SNA) and mean arterial blood pressure (MAP). In patients and animals with heart failure with reduced ejection fraction (HF‐rEF), mechanoreflex activation contributes to exaggerated increases in SNA and is linked to decreased exercise tolerance and increased mortality. The exaggerated mechanoreflex in HF‐rEF is partially attributed to an increased responsiveness of thin fiber skeletal muscle afferents to mechanical stimulation. Several lines of evidence demonstrate that signaling within sensory neurons mediated by the non‐traditional protein kinase c epsilon (PKCε) isoform augments mechanically activated channel function and produces mechanical hyperalgesia. Whether PKCε‐mediated signaling within sensory neurons contributes the exaggerated mechanoreflex in HF‐rEF is unknown.Purpose & HypothesisWe determined the role played by PKCε within sensory endings of thin fiber muscle afferents in evoking the mechanoreflex in rats with HF‐rEF and healthy control rats. We tested the hypothesis that injection of the selective PKCε inhibitor PKCε141 (44μg) into the arterial supply of the hindlimb would reduce the reflex increase in renal SNA (RSNA) and MAP evoked during 30 seconds of 1 Hz dynamic hindlimb muscle stretch (a model of mechanoreflex activation) in HF‐rEF rats, but not in healthy control rats.MethodsExperiments were performed on Sprague‐Dawley rats subjected to coronary artery ligation surgery to produce myocardial infarction and HF‐rEF or a sham ligation. At least six weeks after the initial surgery, in decerebrate, unanesthetized rats we compared (Student’s t‐tests) the mechanoreflex evoked by hindlimb skeletal muscle stretch before and after injection of PKCε141 into the arterial supply of the hindlimb.ResultsEjection fraction (determined via echocardiography) was significantly reduced in HF‐rEF rats (48±1%) compared to sham rats (80±2%, P<0.01). Intra‐arterial PKCε141 injection reduced the pressor and RSNA response to hindlimb muscle stretch in HF‐rEF rats (n=6, 4M/2F, peak ΔMAP pre: 34±8; post: 19±7 mmHg; P=0.04; peak ΔRSNA pre: 121±32; post: 70±24%; P=0.04), but not SHAM rats (n=4, 3M/1F, peak ΔMAP pre: 14±4; post: 14±3 mmHg; P=0.87; peak ΔRSNA pre: 62±32; post: 58±26%; P=0.80) rats. In control experiments (n=3 HF‐rEF rats, 2M/1F), injection of PKCε141 into the jugular vein had no effect on the pressor or RSNA response to muscle stretch demonstrating a lack of central nervous system effects.ConclusionsThese data suggest that that PKCε signaling within the sensory neuron endings contributes to the exaggerated mechanoreflex in HF‐rEF and enhance the understanding of the sympathetic and cardiovascular adjustments to exercise in the ~6 million Americans with HF‐rEF.

Bile Duct Ligation Differently Effects Plasma Osmolality but not Blood Pressure in Male vs Female Rats

Dilutional hyponatremia is known to increase morbidity in patients with advanced liver disease due to the inappropriate release of arginine vasopressin (AVP). Our previous studies show that bile duct ligation (BDL), an animal model of liver disease, increase circulating copeptin in male rats but a decrease in osmolality as compared to the shams. Female BDL rats did not demonstrate the same increase in circulating copeptin or decrease in plasma osmolality as compared to sham females. In this study, we tested if male and female BDL rats showed differences in their blood pressure responses to BDL. Previous studies indicate that male BDL rats become hypotensive while females have not been tested. Adult male and female Sprague Dawley rats had either BDL or sham ligation surgery and were implanted with radio telemetry transmitters for measuring Mean arterial pressure (MAP) and heart rates (HR). Rats were housed in a commercial metabolic cage for one week prior to euthanasian to measure water intake and urine output. No change was seen in daily water intake between the male BDL and sham groups, however the female BDL animals had a significant increase in their daily water intake (p < 0.05, n =6) when compared to the shams. Despite the increase in water intake seen only in BDL females, both the male and female BDL groups saw a large increase in daily urine output (p < 0.001, n = 4‐9) compared to the shams. Analysis of variance showed that there was a main effect of treatment (F(3, 20)= 6.7, P < 0.05) that was due to the MAP of both male and female BDL groups being significantly lower than female shams (P < 0.05). Heart rate was not different among either sexes or treatment groups. Liver weight to body weight ratio was significantly increased in both male and female BDL rats. These results show that both male and female BDL rats became hypotensive when compared to their shams, however the differences in development of hyponatremia among the male and female rats are not due to difference in blood pressure changes associated with BDL.

Bile Duct Ligation Changes the Inhibitory Control of Vasopressin Neurons in Male Rats

Dilutional hyponatremia typically is caused by increased Arginine Vasopressin (AVP) secretion. This increases the risk of complications and mortality in patients with liver cirrhosis. Liver cirrhosis in male rats increases AVP release but the underlying mechanisms are still under investigation. In other models of chronic AVP release, changes in chloride homeostasis alters the inhibitory control of AVP neurons in the supraoptic nucleus (SON) causing GABA mediated excitation instead of inhibition. In the bile duct ligation (BDL) model of cirrhosis, changes in either intracellular chloride concentration or cell firing activity have not been directly tested. We hypothesize that liver cirrhosis increases intracellular chloride concentration leading to a decrease in GABA‐mediated inhibition of SON neurons from male rats.To test this hypothesis, we injected an adeno‐associated virus with a vasopressin promoter and a ratiometric chloride indicator, AAV2‐0VP1‐ClopHensorN into the SON of adult male Sprague‐Dawley rats. The rats either received BDL surgery, which was used to model liver cirrhosis, or a sham ligation surgery. All BDL rats had significantly higher liver weight to body weight ratio (n = 10‐20, p < 0.05), lower plasma osmolality (n = 10‐20, p < 0.001) and lower hematocrit (n = 10‐20, p < 0.05) as compared to sham rats. All rats were anesthetized with isoflurane (2‐3%), their brains were collected, and slices containing the SON were prepared. Loose‐cell patch recordings were made at 31±1oC using standard artificial cerebrospinal fluid. Labeled AVP cells and unlabeled SON cells were tested with focal application of muscimol (100µM), a GABAA receptor agonist.Cells from BDL rats showed impaired GABAA‐mediated inhibition in response to muscimol, as compared to those from sham rats. All putative AVP SON neurons from sham rats (4/4) showed decreased activity following muscimol while half of the putative AVP SON neurons (7/14) from BDL rats were excited (p < 0.001). Similar results were observed in experiments were the injections missed the SON. In these experiments, all SON neurons from sham rats were inhibited by muscimol (26/26) while thirty‐five percent of SON neurons from BDL rats (16/45) were excited by muscimol (p = 0.001). ClopHensorN based chloride imaging showed that muscimol application was associated with decreased intracellular chloride concentrations in putative AVP cells from BDL rats as compared to sham rats (n = 31‐32, p = 0.001). The results show that liver cirrhosis impairs the normal inhibitory response of SON neurons to GABAA receptor activation which could contribute to increased cell activity and dilutional hyponatremia in male rats.

Examination of testicular lumicrine regulation of activins and immunoregulatory genes in the epididymal caput.

Immunoregulatory genes encoding activin A (Inhba) and B (Inhbb), and indolamine 2,3-dioxygenase-1 (Ido1) are highly expressed in the murine caput epididymidis, which also has a network of intraepithelial mononuclear phagocytes. This environment is postulated to promote immunological tolerance to epididymal sperm. The factors regulating the immunoregulatory agents in the epididymal caput are poorly understood. This study aimed to investigate the potential role of testicular lumicrine factors in regulating activin and other immune-related genes in the caput epididymidis. The efferent ducts in adult C57/Bl6 mice were exposed and ligated bilaterally. Serum and tissues were collected seven days later. Animals with bilateral sham ligation and animals with no ligations (collectively referred to as the "intact" group) were used as controls. Pressure-induced seminiferous epithelial damage due to intratubular fluid accumulation was observed in all ligated testes. Testicular inhibin was significantly increased and testosterone was elevated in some animals following bilateral ligation, but serum testosterone, serum LH, and serum inhibin were normal. Ligation caused epithelial regression in the initial segment, with similar but less severe effects in other caput segments. Activin A staining by immunohistochemistry in the epithelium was reduced in bilateral ligation, particularly in the initial segment, with moderately reduced staining intensity in the rest of the caput. Inhba expression within the caput was not significantly affected by bilateral ligation, but Inhbb was reduced by more than 60%. Transcripts encoding the macrophage-specific receptor Cx3cr1 were significantly reduced following bilateral ligation, but other immune cell markers, Ido1, and inflammatory genes were unaffected. These data indicate that testicular lumicrine secretion regulates several genes that are preferentially expressed in the initial segment, but has marginal effects on genes such as those encoding activin A and IDO1, which are expressed more widely in the caput.

Cholinergic stimulation with pyridostigmine modulates a heart-spleen axis after acute myocardial infarction in spontaneous hypertensive rats

The mechanisms regulating immune cells recruitment into the heart during healing after an acute myocardial infarction (AMI) have major clinical implications. We investigated whether cholinergic stimulation with pyridostigmine, a cholinesterase inhibitor, modulates heart and spleen immune responses and cardiac remodeling after AMI in spontaneous hypertensive rats (SHRs). Male adult SHRs underwent sham surgery or ligation of the left coronary artery and were randomly allocated to remain untreated or to pyridostigmine treatment (40 mg/kg once a day by gavage). Blood pressure and heart rate variability were determined, and echocardiography was performed at day six after MI. The heart and spleen were processed for immunohistochemistry cellular analyses (CD3+ and CD4+ lymphocytes, and CD68+ and CD206+ macrophages), and TNF levels were determined at day seven after MI. Pyridostigmine treatment increased the parasympathetic tone and T CD4+ lymphocytes in the myocardium, but lowered M1/M2 macrophage ratio towards an anti-inflammatory profile that was associated with decreased TNF levels in the heart and spleen. Treatment with this cholinergic agent improved heart remodeling manifested by lower ventricular diameters and better functional parameters. In summary, cholinergic stimulation by pyridostigmine enhances the parasympathetic tone and induces anti-inflammatory responses in the heart and spleen fostering cardiac recovery after AMI in SHRs.

Thromboxane A 2 Receptors Contribute to the Exaggerated Exercise Pressor Reflex in Rats with Heart Failure

Background Mechanical and metabolic signals associated with skeletal muscle contraction stimulate the sensory endings of thin fiber skeletal muscle afferents. During exercise, stimulation of these afferents initiates a reflex, termed the exercise pressor reflex (EPR), which contributes importantly to reflex increases in sympathetic nerve activity (SNA) and mean arterial blood pressure (MAP). In patients and animals with heart failure with reduced ejection fraction (HF-rEF), activation of the EPR contributes to exaggerated increases in SNA and is linked to decreased exercise tolerance and increased mortality. The exaggerated EPR in HF-rEF is attributed, at least in part, to an increased responsiveness of thin fiber skeletal muscle afferents produced by cyclooxygenase (COX) products of arachidonic metabolism. However, the COX metabolite receptors on the sensory endings that mediate the COX-induced afferent sensitization in HF-rEF are unknown. Purpose & Hypothesi The purpose of this investigation was to determine the role played by one of the major receptors for products of COX metabolism present on sensory neurons, namely thromboxane A2 receptors (TxA2-Rs), in the exaggerated EPR in HF-rEF rats. We tested the hypothesis that injection of the TxA2-R antagonist daltroban (80μg) into the arterial supply of the hindlimb would reduce the reflex increase in renal SNA (RSNA) and MAP evoked in response to 30 seconds of 1 Hz dynamic hindlimb muscle contraction in HF-rEF rats, but not control rats. Methods Experiments were performed on male Sprague-Dawley rats subjected to either a coronary artery ligation surgery to produce myocardial infarction and HF-rEF or a sham ligation. At least six weeks after the initial surgery, in decerebrate, unanesthetized rats we compared the EPR evoked by electrically induced hindlimb skeletal muscle contraction before and after injection of daltroban into the arterial supply of the hindlimb. Results Ejection fraction was significantly reduced in HF-rEF (47±3%) compared to sham (84±1%, p<0.01). We found that daltroban reduced the EPR response in HF-rEF (n=8; peak ΔMAP pre: 17±4; post: 12±2mmHg; P=0.04; peak ΔRSNA pre: 135±55; post: 46±13%; P=0.07), but not in SHAM rats (n=10; peak ΔMAP pre: 13±3; post: 11±2mmHg; P=0.48; peak ΔRSNA pre: 64±17; post: 63±15%; P=0.96;) rats. In a separate group of HF-rEF rats (n=4), we determined whether systemic circulation of daltroban accounted for the attenuating effects on EPR activation seen when daltroban was injected into the hindlimb arterial supply. We found that intravenous injection of daltroban had no effect on the EPR (peak ΔMAP pre: 26±7; post: 25±7mmHg; P=0.50). In these same four HF-rEF rats we also found that hindlimb arterial injection of the vehicle for daltroban (0.4mL saline containing 1% DMSO) had no effect on the EPR (peak ΔMAP pre: 23±8; post: 20±6mmHg; P=0.40). Conclusions These data suggest that that TxA2-R receptors on the sensory endings of muscle afferents contribute to the exaggerated EPR in HF-rEF. Our data enhance the understanding of the sympathetic and cardiovascular adjustments to exercise in the ~6 million Americans with HF-rEF.

NAC and Vitamin D Restore CNS Glutathione in Endotoxin-Sensitized Neonatal Hypoxic-Ischemic Rats.

Therapeutic hypothermia does not improve outcomes in neonatal hypoxia ischemia (HI) complicated by perinatal infection, due to well-described, pre-existing oxidative stress and neuroinflammation that shorten the therapeutic window. For effective neuroprotection post-injury, we must first define and then target CNS metabolomic changes immediately after endotoxin-sensitized HI (LPS-HI). We hypothesized that LPS-HI would acutely deplete reduced glutathione (GSH), indicating overwhelming oxidative stress in spite of hypothermia treatment in neonatal rats. Post-natal day 7 rats were randomized to sham ligation, or severe LPS-HI (0.5 mg/kg 4 h before right carotid artery ligation, 90 min 8% O2), followed by hypothermia alone or with N-acetylcysteine (25 mg/kg) and vitamin D (1,25(OH)2D3, 0.05 μg/kg) (NVD). We quantified in vivo CNS metabolites by serial 7T MR Spectroscopy before, immediately after LPS-HI, and after treatment, along with terminal plasma drug concentrations. GSH was significantly decreased in all LPS-HI rats compared with baseline and sham controls. Two hours of hypothermia alone did not improve GSH and allowed glutamate + glutamine (GLX) to increase. Within 1 h of administration, NVD increased GSH close to baseline and suppressed GLX. The combination of NVD with hypothermia rapidly improved cellular redox status after LPS-HI, potentially inhibiting important secondary injury cascades and allowing more time for hypothermic neuroprotection.

Endoperoxide 4 receptor inhibition has no effect on the pressor response evoked during isolated mechanoreflex activation in rats with heart failure

BackgroundThe stimulation of primarily mechanically sensitive group III and primarily metabolically sensitive group IV muscle afferents during skeletal muscle contractions evokes reflex increases in blood pressure (i.e., the exercise pressor reflex) and facilitates exercise performance. Heart failure patients with reduced ejection fraction (HF‐rEF) exhibit an exaggerated exercise pressor reflex, leading to excessive peripheral vasoconstriction and impaired limb blood flow during exercise. The exaggerated exercise pressor reflex in HF‐rEF is attributed, at least in part, to an exaggeration of the mechanically sensitive portion of the reflex (i.e., the mechanoreflex). Furthermore, multiple lines of evidence suggest that, in HF‐rEF, the mechanoreflex sensitization is attributable to the cyclooxygenase products of arachidonic acid metabolism. For example, recent evidence in a rat model of HF‐rEF induced by myocardial infarction demonstrated that the exaggerated rise in renal sympathetic nerve activity during hindlimb muscle stretch (a model of mechanoreflex activation) was significantly reduced following inhibition of the COX‐2 isoform that predominately produces prostaglandin E2 (PGE2). However, the COX metabolite receptor on sensory neurons that mediated the PGE2‐induced mechanoreflex sensitization in HF‐rEF is not known.Purpose &amp; HypothesisOur purpose was to determine the effect of inhibition of the PGE2 receptor endoperoxide 4 (EP4) on mechanoreflex activation in rats with HF‐rEF. We hypothesized that hindlimb arterial injection of the EP4 receptor antagonist L‐161,982 would reduce the exaggerated increase in MAP during 30 s of static hindlimb muscle stretch in rats with HF‐rEF, but not in healthy control rats.MethodsWe utilized a rat model of HF‐rEF in which the left main coronary artery is ligated with 5‐0 suture to induce myocardial infarction (n=7). Control rats (n=3) were subjected to a sham ligation in which the suture was passed under the artery but not tied. At least four weeks after the initial surgery, in decerebrate, unanesthetized rats we compared (paired Student’s t‐tests) the pressor response (peak ΔMAP) evoked during muscle stretch before and after the injection of L‐161,982 (1 μg) into the arterial supply of the hindlimb circulation.ResultsEjection fraction determined via echocardiography was significantly reduced in HF‐rEF rats (35±6%) compared to sham rats (82±2%, p&lt;0.01, unpaired Student’s t‐test). We found that EP4 receptor inhibition had no effect during hindlimb muscle stretch on the peak ΔMAP in either HF‐rEF rats (pre: 24±4; post: 26±6mmHg; p=0.74) or sham rats (pre: 13±3; post: 11±2mmHg; p=0.23). Moreover, western blot analysis revealed no difference between HF‐rEF and sham rats in protein expression of EP4 receptor within the L4 and L5 dorsal root ganglia (unpaired Student’s t‐test; p=0.25).ConclusionsThe present data suggest that that EP4 receptors do not contribute to the exaggerated mechanoreflex evoked in rats with HF‐rEF. Our data enhance the understanding of the blood pressure control during exercise in the ~6 million Americans currently diagnosed with HF‐rEF.Support or Funding InformationNational Institutes of Health Grant HL‐142877 to SWC

Urolithin B improves cardiac function and reduces susceptibility to ventricular arrhythmias in rats after myocardial infarction

Cardiac fibrosis and inflammation play critical roles in ventricular remodelling after myocardial infarction (MI). Urolithin B (UB), a metabolite of ellagitannin-rich foods, has various biological activities, but its effect on ventricular remodelling after MI has not been determined. The present study evaluated whether UB inhibited ventricular structural remodelling and decreased the occurrence of ventricular arrhythmias after MI. Sprague-Dawley (SD) rats underwent ligation of the left anterior descending coronary artery before randomization to receive phosphate-buffered saline (PBS) or UB at doses of 2.5 mg/kg/day and 5 mg/kg/day via intraperitoneal administration or sham ligation. Cardiac function was assessed using echocardiography, haemodynamic detection and brain natriuretic peptide (BNP) levels 2 weeks post-MI. Hearts were used for electrophysiological testing and molecular and histological analyses. UB (5 mg/kg/day) significantly protected against post-MI cardiac dysfunction. UB markedly reduced infarct areas and myocyte size and attenuated cardiac fibrosis and inflammation post-MI. UB decreased the incidence of ventricular tachycardia and ventricular fibrillation compared to the MI group. We determined that UB inhibited the phosphorylation of JAK2/STAT3 and Smad2/3 signalling molecules. Our data suggest that UB reduces the occurrence of malignant ventricular arrhythmias after MI, which is likely associated with attenuation of ventricular structural remodelling via inactivation of the JAK2/STAT3 and Smad2/3 signalling pathway.

OR06-4 Brain Derived Neurotrophic Factor in the Regulation of Supraoptic Vasopressin Neurons in Development of Hyponatremia

Dilutional hyponatremia due to elevated arginine vasopressin (AVP) secretion increases mortality in patients with liver failure. The current conventional therapies are palliative and frequently ineffective. The neural mechanisms causing dysregulation of AVP secretion are not completely understood. Based on previous studies, we hypothesis that inappropriate AVP release associated with liver failure is due to increased Brain Derived Neurotrophic Factor (BDNF) in the supraoptic nucleus (SON). BDNF diminishes GABAA inhibition in SON AVP neurons by increasing intracellular chloride ([Cl]i) through tyrosine receptor kinase B (TrkB) activation and downregulation of K+/Cl- co-transporter 2 (KCC2). This loss of inhibition could increase AVP secretion. To test this hypothesis, chronic bile duct ligated (BDL) rats, a model of dilutional hyponatremia associated with liver failure, were injected with shRNA to prevent increased BDNF in the SON. Adult male Sprague Dawley rats were bilaterally injected in the SON with AAV2- shRNA-BDNF-mCherry or AAV2- shRNA-SCR-mCherry. After two weeks, the rats had BDL or sham ligation surgery. Four weeks later, SON regions were collected by punches for western blot analysis. Fluid accumulation, liver and body weight were recorded to verify if the rats develop liver failure. Blood samples were collected to measure plasma osmolality, hematocrit, and circulating AVP concentration. Data were analyzed by one-way ANOVA with Bonferroni comparisons. The BDL rats had significant increases in liver to body weight ratio (p< 0.05; 6-9) compared to sham rats indicating liver failure and ascites with bile duct ligation. BDL rats with scrambled injections (BDL SCR) developed hyponatremia with significant decrease in plasma osmolality and hematocrit along with significant increase in plasma AVP concentration (all p< 0.05; 6-9) compared to sham groups. This is the first study to show that phosphorylation of TrkB (pTrkBY515) is significantly increased along with significant decrease in phosphorylation of KCC2 (pKCC2S940) in BDL SCR rats compared to the sham rats (p< 0.05; 6-8). Knockdown of BDNF in the SON of BDL rats (BDL shBDNF) prevented the rats from developing hyponatremia and caused significant increase in plasma osmolality and hematocrit compared to BDL SCR rats (p< 0.05; 6-9). The BDL shBDNF rats had significant (p< 0.05; 6-9) decreases in plasma AVP concentration compared to BDL SCR rats. shBDNF injections in the SON of BDL rats significantly blocked the increase in TrkB phosphorylation and decrease in KCC2 phosphorylation (p< 0.05;6-8) associated with bile duct ligation. mCherry expression was used to verify the specificity of stereotaxic injections. Rats that did not had successful virus injections in the SON were analyzed separately. The results indicate that BDNF produced in the SON contributes to increased AVP secretion and hyponatremia during liver cirrhosis.

Sex Difference and Hormones in the Regulation of Vasopressin Secretion during Dilutional Hyponatremia

Dilutional hyponatremia due to elevated arginine vasopressin (AVP) secretion increases mortality in patients with liver failure. No previous studies have addressed sex difference in dilutional hyponatremia in animal models of liver failure. The aim of this study is to investigate sex difference in dilutional hyponatremia and the effect of ovarian hormones in AVP secretion. We have used chronic bile duct ligated (BDL) rats, a model of dilutional hyponatremia associated with liver failure.Adult male Sprague Dawley rats, female Sprague Dawley rats and ovariectomized (OVX) female rats were used. After two weeks, the rats had BDL or sham ligation surgery. Four weeks later, the brains were processed for immunohistochemistry analysis of FosB and AVP expression. Blood samples were collected to measure plasma osmolality, hematocrit, circulating estradiol and copeptin concentration. Copeptin was used as biomarker for plasma AVP. Liver weight, body weight, and fluid accumulation were recorded to verify if the rats developed liver failure and ascites. Data were analyzed by one ANOVA with Bonferroni post hoc comparisons.All the BDL rats had significant increase in liver to body weight ratio compared to sham rats indicating liver failure and ascites with bile duct ligation. Male BDL rats had hyponatremia along with significant increases in plasma copeptin and FosB expression in Supraoptic AVP neurons compared to male shams (all p&lt;0.05; 5–7). Unlike male BDL rats, the female BDL rats did not become hyponatremia and did not had increases Supraoptic AVP neuron activation and copeptin secretion compared to female sham.Ovariectomy significantly decreased plasma estradiol concentration in sham rats compared to intact female sham (p&lt; 0.05; 6–10). However, circulating estradiol concentration was significantly elevated in ovariectomized BDL (OVX BDL) rats compared to the ovariectomized sham (OVX sham) and female sham rats (p&lt;0.05; 6–10). To identify the source of estradiol contributing to the observed increase in OVX BDL rats, adrenal glands were collected at the end of protocol. Adrenal gland steroids were extracted to measure estradiol and its precursors, testosterone and DHEA concentration.The OVX BDL rats had significantly increased adrenal estradiol along with significant decrease in adrenal testosterone and DHEA compared to OVX sham rats (all p&lt; 0.05; 6–7). Plasma osmolality, hematocrit and copeptin concentration were not different between OVX BDL and OVX sham rats. Our results show that unlike male BDL rats, female BDL rats did not develop hyponatremia, supraoptic AVP neuron activation, or increased copeptin secretion compared to sham ligated females. Female OVX BDL rats did not become hyponatremia or increased copeptin secretion compared to female OVX sham rats. It is possible that the increase in adrenal estradiol compensated for the lack of ovarian estrogens in OVX BDL rats. Additional experiments will be required to determine the effects of estrogen in this model.Support or Funding InformationSupported by R01 HL119458 and 18PRE34060035This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dual inhibition of sodium\u2013glucose linked cotransporters 1 and 2 exacerbates cardiac dysfunction following experimental myocardial infarction

BackgroundInhibiting both type 1 and 2 sodium–glucose linked cotransporter (SGLT1/2) offers the potential to not only increase glucosuria beyond that seen with selective SGLT2 inhibition alone but to reduce glucose absorption from the gut and to thereby also stimulate glucagon-like peptide 1 secretion. However, beyond the kidney and gut, SGLT1 is expressed in a range of other organs particularly the heart where it potentially assists GLUT-mediated glucose transport. Since cardiac myocytes become more reliant on glucose as a fuel source in the setting of stress, the present study sought to compare the effects of dual SGLT1/2 inhibition with selective SGLT2 inhibition in the normal and diseased heart.MethodsFischer F344 rats underwent ligation of the left anterior descending coronary artery or sham ligation before being randomized to receive the dual SGLT1/2 inhibitor, T-1095, the selective SGLT2 inhibitor, dapagliflozin or vehicle. In addition to measuring laboratory parameters, animals also underwent echocardiography and cardiac catheterization to assess systolic and diastolic function in detail.ResultsWhen compared with rats that had received either vehicle or dapagliflozin, T-1095 exacerbated cardiac dysfunction in the post myocardial infarction setting. In addition to higher lung weights, T-1095 treated rats had evidence of worsened systolic function with lower ejection fractions and reduction in the rate of left ventricle pressure rise in early systole (dP/dtmax). Diastolic function was also worse in animals that had received T-1095 with prolongation of the time constant for isovolumic-pressure decline (Tau) and an increase in the end-diastolic pressure volume relationship, indices of the active, energy-dependent and passive phases of cardiac relaxation.ConclusionsThe exacerbation of post myocardial infarction cardiac dysfunction with T-1095 in the experimental setting suggests the need for caution with the use of dual SGLT1/2 inhibitors in humans.

Abstract 89: Differences in Orexin-related Gene Expression in the Brain in Early versus Late Stage Heart Failure in Male Rats

Orexin is a neuropeptide produced exclusively by neurons in the lateral hypothalamus. Orexinergic neurons send projections throughout the brain and the system has been hypothesized to be critical for the coordination of “survival related-processes. Emerging evidence has identified that the orexin system is dysregulated in several forms of cardiovascular disease. In hypertension for example, upregulation of the orexin system contributes to elevated sympathetic drive. In heart failure, genomic changes in the orexin type 2 receptor have been linked to reduced cardiac function and pretreatment with an orexin type 2 receptor agonist prior to cardiac stress was identified to be cardio-protective. These data suggest changes in orexin system activation before or during cardiac stress may have therapeutic potential. The present study was undertaken to evaluate changes in the brain orexin system at 4 weeks following myocardial injury compared to the chronic changes previous documented at 16 weeks post infarction. Methods: Adult male rats underwent either sham operation (n=9) or ligation of the left main coronary artery (n=9) while under anesthesia and were allowed to recover either 4 weeks or 16 weeks. Results: The infarct size ranged from 21 to 39% of the left ventricle (28+/-6%, mean+/-STDEV). At 4 weeks of age hypothalamic orexin gene expression was upregulated 54% (P&lt;0.07) when compared to sham operated animals. This was in contrast to a 78% reduction in gene expression at 16 weeks following myocardial infarction. No significant difference in orexin receptor expression (type 1 or type 2) within the hypothalamus was identified at either time point. These findings demonstrate that the orexin system undergoes dynamic changes following myocardial insult. Since orexin supplementation has been reported to be both neuroprotective and cardioprotective, it is possible that early orexin upregulation is an adaptive/protective response and sustained stimulation of the orexin system over a longer time period may improve health outcomes associated with heart failure.

Combined Intraperitoneal and Intrathecal Etanercept Reduce Increased Brain Tumor Necrosis Factor-Alpha and Asymmetric Dimethylarginine Levels and Rescues Spatial Deficits in Young Rats after Bile Duct Ligation

Background: Rats subjected to bile duct ligation (BDL) exhibit increased systemic oxidative stress and brain dysfunction characteristic of hepatic encephalopathy (HE), including fatigue, neurotransmitter alterations, cognitive and motor impairment, and brain inflammation. The levels of tumor necrosis factor-alpha (TNF-α) and asymmetric dimethylarginine (ADMA) are both increased in plasma and brain in encephalopathy induced by chronic liver failure. This study first determined the temporal profiles of TNF-α and ADMA in the plasma, brain cortex, and hippocampus in young BDL rats. Next, we examined whether etanercept was beneficial in preventing brain damage.Methods: Young rats underwent sham ligation or BDL at day 17 ± 1 for 4 weeks. Treatment group rats were administered etanercept (10 mg/kg) intraperitoneally (IP) three times per week with or without etanercept (100 μg) intrathecally (IT) three times in total.Results: We found increased plasma TNF-α, soluble tumor necrosis factor receptor 1 (sTNFR1), soluble tumor necrosis factor receptor 2 (sTNFR2), and ADMA levels, increased cortical TNF-α mRNA and protein and ADMA, and hippocampal TNF-α mRNA and protein, and spatial defects in young BDL rats. The increase in cortex TNF-α mRNA and ADMA were reduced by IP etanercept or combined IP and IT etanercept. Dually IP/IT etanercept administration reduced the increased cortical and hippocampal TNF-α mRNA and protein level as well as spatial deficits.Conclusions: We conclude that combined intraperitoneal and intrathecal etanercept reduce increased brain TNF-α and ADMA levels and rescues spatial deficits in young rats after BDL.

Myocardial infarction sensitizes medial prefrontal cortex to inhibitory effect of locus coeruleus stimulation in rats.

Anxiety is a common comorbidity that develops after myocardial infarction and is now an established independent risk factor for cardiovascular mortality. Here, we assessed anxiety and mapped neural activity of forebrain regions that regulate anxiety in a rat model of myocardial infarction in order to identify sites of dysregulation. Anxiety responses to novel (open field) or aversive stimuli (discriminative auditory fear conditioning) were assessed in rats subjected to coronary artery ligation (CAL) or sham ligation. Forebrain metabolic activity was measured by cytochrome oxidase (CO) histochemistry. Changes in CO activity and the incidence of ventricular arrhythmias were also assessed during modulation of fear circuitry induced by electrical stimulation of the locus coeruleus. Coronary artery ligation had negligible effects on open-field behavior, but increased expression of learned fear and impaired fear cue discrimination. Cytochrome oxidase activity was increased in the medial prefrontal cortex and in the lateral amygdala after CAL. Locus coeruleus stimulation reduced CO activity in the infralimbic medial prefrontal cortex only in rats subjected to CAL. Stimulation of the LC also elicited new ventricular arrhythmias in rats subjected to CAL. Coronary artery ligation sensitizes the infralimbic medial prefrontal cortex to the inhibitory effects of locus coeruleus stimulation. Suppression of infralimbic medial prefrontal cortical activity may impair the ability of rats subjected to CAL to discriminate between cues that signal aversive and neutral events which, in turn, may promote excessive sympathetic activation of the cardiovascular system in response to innocuous stimuli.

Endoplasmic Reticulum (ER) Stress Induces p44/42 Mitogen‐Activated Protein Kinase (MAPK) Signaling, Inflammation and Renin‐Angiotensin System (RAS) Activity in Forebrain and Contributes to Sympathetic Nerve Activity in Heart Failure Rats

The unfolded protein response to ER stress, induced by an excessive accumulation of unfolded proteins in the ER lumen, activates a series of downstream signal transduction pathways including MAPK signaling. ER stress has been implicated in the pathophysiology of a variety of chronic disease states including diabetes mellitus, obesity and cardiovascular diseases. A role for brain ER stress in angiotensin II ‐induced hypertension in mice was recently reported. We have found that ER stress is upregulated in the subfornical organ (SFO) and hypothalamic paraventricular nucleus (PVN) of rats with ischemia‐induced heart failure (HF). We have also found that MAPK signaling is upregulated in the SFO and PVN of these HF rats and contributes to sympathetic excitation. The present study sought to determine whether brain ER stress induces p44/42 MAPK signaling and the upregulation of excitatory/inflammatory mediators in SFO and PVN and increased sympathetic nerve activity in HF. HF and SHAM rats were produced by coronary artery ligation or sham ligation, respectively. HF rats received a 4‐week intracerebroventricular (ICV) infusion (0.25 μl/hr) of vehicle (VEH) or the ER stress inhibitor tauroursodeoxycholic acid (TUDCA, 10μg/day). SHAM rats received ICV VEH for 4 weeks. Total and phosphorylated p44/42 MAPK in the SFO and PVN were measured by Western blot. Immunofluorescence for phosphorylated p44/42 MAPK in the SFO and PVN was visualized by confocal microscopy. The mRNA levels of inflammatory mediators tumor necrosis factor (TNF) ‐α, interleukin (IL)‐1β, cyclooxygenase (COX) ‐2, COX‐1 and RAS components renin angiotensin converting enzyme (ACE) and angiotensin II type 1 receptor (AT1‐R) in SFO and PVN were measured by real‐time PCR. Plasma norepinephrine (NE) was measured by ELISA. Compared with SHAM rats, VEH‐treated HF rats had higher (*p&lt;0.05, n=6 in each group) levels of phosphorylated p44/42 MAPK (normalized to total p44/42 MAPK) in SFO and PVN. In HF rats treated with TUDCA, phosphorylated p44/42 MAPK was lower in the SFO and PVN by Western blot (~45 %*) and by immunofluorescent imaging. VEH‐treated HF rats had significantly elevated (2.2–3.6 fold*) mRNA levels for TNF‐α, IL‐1β, COX ‐2, ACE and AT1‐R in the SFO and PVN, compared with VEH‐treated SHAM rats, but these levels were markedly reduced (37–51%*, n=6 in each group) in the TUDCA‐treated HF rats. The plasma NE level (ng/ml) was higher in VEH‐treated HF (38.86 ± 2.35*) than SHAM (18.64 ± 2.59) rats; this increase was significantly attenuated in the HF rats treated with TUDCA. These data demonstrate that brain ER stress induces MAPK signaling, inflammation and RAS activity in key cardiovascular regulatory regions of the brain and contributes to the sympathetic activation in HF. Brain ER stress is a potential target for therapeutic intervention in HF.Support or Funding InformationSupported by NIH RO1 HL096671 and R01 HL073986

Abstract 13920: Impact of Diabetes on Acute Kidney Injury After Myocardial Infarction: Possible Involvement of Toll-like Receptor in the Kidney

Background: Comorbid acute kidney injury (AKI) predicts poor prognosis in patients with acute myocardial infarction (MI). Although type 2 diabetes (T2D) is a well-known risk factor of AKI after MI, the mechanism of the increased risk remains unclear. Here we hypothesized that T2D increases AKI after MI via toll-like receptor (TLR)-mediated inflammation. Methods and Results: OLETF, a rat model of obese T2D, and LETO, non-diabetic controls, at 25-30 weeks of age were randomized into sham and permanent coronary ligation (MI) groups. At baseline, body weight (617±23 vs. 537±13 g), fasting plasma glucose (267±32 vs. 153±15 mg/dl) and urinary protein level (6.4 vs. 0.6 g/gCr), but not serum creatinine, were significantly higher in OLETF than in LETO. Histologically, glomerular size was increased by 17% without mesangial proliferation in OLETF compared to that in LETO, indicating that OLETF developed early-stage nephropathy by this age. At 12 h after MI, mRNA levels of TLR2, TLR4, IL-6 and TNF-α in the kidney were increased by 1.6-, 1.2-, 2.6-, 1.5-fold, respectively, in OLETF but not in LETO. Furthermore, immunoblot analyses showed that phosphorylation levels of p38 MAPK and JNK, downstream mediators of the TLR signal, were significantly elevated by MI in OLETF. Histological abnormalities in the kidney or increase in serum creatinine were not detected in either LETO or OLETF 12 h after MI. However, in immunohistochemical analyses, areas positive for neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) were significantly increased by 4.0- and 5.3-fold, respectively, and NGAL mRNA level was increased by 1.8-fold after MI in OLETF but not in LETO. In sham-operated LETO and OLETF, areas positive for NGAL and KIM-1 were barely detected. Infarct sizes were similar and cardiac BNP mRNA levels in the non-infarcted left ventricle were equally elevated at 12 h after MI in LETO and OLETF, suggesting that MI-induced cardiac loads were comparable in the two groups. However, mortality at 48 h after MI was significantly higher in OLETF than in LETO (68% vs. 18%, P&lt;0.05). Conclusion: The results suggest that AKI after MI is enhanced in T2D via TLR-mediated inflammation. The cardio-renal interaction may underlie increased post-MI mortality in T2D.