Role In Cardiovascular Regulation Research Articles

Molecular Dissection of the Role of ACE2 in Glucose Homeostasis.

Angiotensin Converting Enzyme 2 (ACE2) was discovered 25 years ago as a negative regulator of renin-angiotensin system, opposing the effects of angiotensin-II. Beyond its well-demonstrated roles in cardiovascular regulation and Covid-19 pathology, ACE2 is involved in a plethora of physio-pathological processes. In this review, we summarize the latest discoveries on the role of ACE2 in glucose homeostasis and regulation of metabolism. In the endocrine pancreas, ACE2 is expressed at low levels in β cells, but loss of its expression inhibits glucose-stimulated insulin secretion and impairs glucose tolerance. Conversely, overexpression of ACE2 improved glycemia and suggests that recombinant ACE2 might be a future therapy for diabetes. In the skeletal muscle of ACE2-deficient mice, a progressive triglyceride accumulation was observed, whereas in diabetic kidney the initial increase in ACE2 is followed by a chronic reduction of expression in kidney tubules and impairment of glucose metabolism. At the intestinal level, dysregulation of the enzyme alters the amino acid absorption and intestinal microbiome, while at the hepatic level, ACE2 protects against diabetic fatty liver disease. Not least, ACE2 is upregulated in adipocytes in response to nutritional stimuli and administration of recombinant ACE2 decreased body weight and increased thermogenesis. In addition to tissue-specific regulation of ACE2 function, the enzyme undergoes complex cellular posttranslational modifications which are changed during diabetes evolution, with at least proteolytic cleavage and ubiquitination leading to modifications in ACE2 activity. Detailed characterization of ACE2 in specific cellular- and tissue-manner holds promise for improving therapeutic outcomes in diabetes and metabolic disorders.

Resting beat-to-beat blood pressure variability in humans: role of alpha-1 adrenergic receptors.

Resting beat-to-beat blood pressure variability is a strong predictor of cardiovascular events and mortality. However, its underlying mechanisms remain incompletely understood. Given that the sympathetic nervous system plays a pivotal role in cardiovascular regulation, we hypothesized that alpha-1 adrenergic receptors (the main sympathetic receptor controlling peripheral vasoconstriction) may contribute to resting beat-to-beat blood pressure variability. Beat-to-beat heart rate (electrocardiography) and blood pressure (photoplethysmography) were continuously measured before and 2 h following, selective blockade of alpha-1 adrenergic receptors via oral administration of prazosin (1mg/20kg) in ten young healthy adults (two women). Cardiac output and total peripheral resistance were estimated using the ModelFlow method. Selective blockade of alpha-1 adrenergic receptors was confirmed by the marked reduction in the pressor response to intravenous infusion of phenylephrine hydrochloride (-80 ± 15%, P = 0.001 versus pre-prazosin). The blockade significantly decreased the standard deviation of the systolic (pre-prazosin versus post-prazosin: 5.6 ± 1.4 versus 3.8 ± 0.7mmHg, P = 0.002), diastolic (3.2 ± 1.2 versus 2.2 ± 0.5mmHg, P = 0.022), and mean blood pressure (3.7 ± 1.2 versus 2.5 ± 0.5mmHg, P = 0.009), as well as total peripheral resistance (0.8 ± 0.5 versus 0.5 ± 0.1mmHg/L/min, P = 0.047), but not cardiac output (521 ± 188 versus 453 ± 160mL/min, P = 0.321). Similar results were found using different indices of variability. These findings indicate that alpha-1 adrenergic receptors play a significant role in regulating resting beat-to-beat blood pressure variability in young, healthy adults.

Potential role of signal transducer and activator of transcription 3 in the amygdala in mitigating stress-induced high blood pressure via exercise in rats.

Chronic stress elevates blood pressure, whereas regular exercise exerts antistress and antihypertensive effects. However, the mechanisms of stress-induced hypertension and preventive effects through exercise remain unknown. Thus, we investigated the molecular basis involved in autonomic blood pressure regulation within the amygdala. The effects of a 3-week restraint stress and daily voluntary exercise against stress on cardiovascular parameters and gene expression profiles in the amygdala were examined using a microarray method. Candidate genes were selected from differentially expressed genes; the localization of their expression within the central nucleus of the amygdala and their roles in cardiovascular regulation were examined using small-interfering RNA transfection and radiotelemetry. Chronic restraint stress caused an increase in blood pressure levels; however, with voluntary exercise, the blood pressure levels remained comparable to those of the controls. Compared with the controls, chronic restraint stress decreased signal transducer and activator of transcription 3 expression in the amygdala, whereas voluntary exercise improved its expression to normal levels. Immunohistochemical staining revealed the expression of signal transducer and activator of transcription 3 in neurons of the amygdala; inhibition of this expression using small-interfering RNA increased the arterial pressure. However, spontaneous baroreflex gain and low- and high-frequency components of heart rate variability remained unaffected by the inhibition of signal transducer and activator of transcription 3. In the amygdala, signal transducer and activator of transcription 3 regulates the blood pressure levels and is possibly involved in blood pressure elevation in response to chronic stress and its improvement by voluntary exercise.

Cardiac cGMP Regulation and Therapeutic Applications.

cGMP plays a central role in cardiovascular regulation in health and disease. It is synthesized by NO or natriuretic peptide activated cyclases and hydrolyzed to 5'GMP by select members of the PDEs (phosphodiesterase) superfamily. The primary downstream effector is cGMP-dependent protein kinase, primarily cGK-1a also known as protein kinase G 1a in the heart and vasculature. cGMP signaling is controlled in intracellular nanodomains to regulate myocyte growth, survival, metabolism, protein homeostasis, G-protein-coupled receptor signaling, and other critical functions. The vascular effects of cGMP signaling have been dominated by its lowering of smooth muscle tone, but other cellular processes are also engaged. Localization of cyclases and corresponding PDEs within intracellular domains, along with their varying expression across different cell types, adds multiorgan complexity to cGMP signaling. This diversity can be leveraged therapeutically by targeting selective pathway components to impact some but not other cGMP signaling effects. Here, we review the generation and regulation of cGMP by PDEs and cyclases, focusing mainly on their role in cardiac physiology and pathophysiology. Current therapeutic uses of cGMP modulation and ongoing trials testing new potential applications are discussed.

Structural Rearrangement of the AT1 Receptor Modulated by Membrane Thickness and Tension.

Membrane-embedded mechanosensitive (MS) proteins, including ion channels and G-protein coupled receptors (GPCRs), are essential for the transduction of external mechanical stimuli into biological signals. The angiotensin II type 1 (AT1) receptor plays many important roles in cardiovascular regulation and is associated with diseases such as hypertension and congestive heart failure. The membrane-mediated activation of the AT1 receptor is not well understood, despite this being one of the most widely studied GPCRs within the context of biased agonism. Here, we use extensive molecular dynamics (MD) simulations to characterize the effect of the local membrane environment on the activation of the AT1 receptor. We show that membrane thickness plays an important role in the stability of active and inactive states of the receptor, as well as the dynamic interchange between states. Furthermore, our simulation results show that membrane tension is effective in driving large-scale structural changes in the inactive state such as the outward movement of transmembrane helix 6 to stabilize intermediate active-like conformations. We conclude by comparing our simulation observations with AlphaFold 2 predictions, as a proxy to experimental structures, to provide a framework for how membrane mediated stimuli can facilitate activation of the AT1 receptor through the β-arrestin signaling pathway.

Involvement of D1 dopamine receptor in the nucleus of the solitary tract of rats in stress-induced hypertension and exercise.

Chronic stress can cause hypertension, whereas daily exercise promotes healthy well being through destressing. Although the nucleus of the solitary tract (NTS) is involved in the development of hypertension, the molecular and physiological mechanisms of stress and exercise remain unclear. In this study, we tested whether gene expression in the NTS is altered by stress and daily exercise and whether this is involved in cardiovascular regulation. We have performed RT 2 Profiler PCR arrays targeting a panel of neurotransmitter receptor genes in the NTS of Wistar rats subjected to chronic restraint stress (1 h a day over 3 weeks) with or without voluntary wheel exercise. We also performed immunohistochemistry to determine whether the identified molecules were expressed at the protein level. Additionally, microinjection studies in anesthetized rats were performed to examine whether validated molecules exhibit physiological roles in cardiovascular regulation of the NTS. We observed that blood pressure was significantly increased by stress and the increase was suppressed by exercise. Using PCR analysis, we determined that the expression levels of four genes in the NTS, including the dopamine receptor D1 gene ( Drd1 ), were significantly affected by stress and suppressed by exercise. We also examined dopamine D1 receptor (D1R) expression in NTS neurons and found significantly greater expression in the stressed than nonstressed animals. Furthermore, the microinjection of a D1R agonist into the NTS in anesthetized rats induced hypotensive effects. These results suggest that NTS D1R plays a role in the counteracting processes of stress-induced hypertension.

Exploring Brainstem Structural Abnormalities: Potential Biomarkers for Panic Disorder.

Panic disorder (PD), characterized by recurrent and intense panic attacks, presents a complex interplay between psychological and neurobiological factors. Although the amygdala and hippocampus have been studied extensively in the context of PD, the brainstem's involvement remains relatively underexplored. This study aims to address this gap by examining structural abnormalities within specific brainstem regions, including the medulla, pons, and midbrain. The study sample population comprised twenty-one adult patients diagnosed with PD and an age-gender-education-matched control group. Utilizing rigorous inclusion and exclusion criteria, confounding factors related to comorbid psychiatric conditions and brain structure abnormalities were minimized. Our findings revealed a significant reduction in medulla volume among PD patients, a finding that persisted even after correcting for individual differences in total intracranial volume. The medulla's role in cardiovascular regulation and autonomic function, coupled with its involvement in fear responses, underscores its potential significance in the pathophysiology of PD. This study elucidates the medulla's structural abnormalities as a potential biomarker for PD. Understanding the role of the brainstem in PD could pave the way for more targeted and effective interventions for this condition.

A biodegradable calcium sulfite nanoreactor for pH triggered gas therapy in combination with chemotherapy.

As a gasotransmitter, endogenous sulfur dioxide (SO2) plays an important role in cardiovascular regulation. In addition, excessive SO2 can react with overexpressed hydrogen peroxide (H2O2) in tumor cells to generate toxic radicals, which can induce severe oxidative damage to tumor cells and result in cell apoptosis. This highlights the potential of SO2 in oncotherapy. However, the limited availability of endogenous H2O2 and uncontrolled release of SO2 gas significantly impede the effectiveness of SO2 gas therapy. To address this challenge, a biodegradable calcium sulfite (CS) nanocarrier loaded with 10-hydroxycamptothecin (HCPT) was developed for tumor pH-triggered SO2 gas therapy in combination with chemotherapy. This nanoreactor could be degraded in an acidic tumor microenvironment to release SO2 gas and the HCPT drug. The released SO2 gas induced serious oxidative damage to tumor cells by depleting glutathione (GSH) and generating toxic radicals through a reaction with intracellular H2O2. Simultaneously, the HCPT drug promoted tumor cell apoptosis through chemotherapy and boosted SO2 gas therapy by elevating the H2O2 level within the tumor cells. Consequently, the combination of SO2 gas therapy and chemotherapy provided a promising approach for effective tumor treatment.

A selective mineralocorticoid receptor blocker, esaxerenone, attenuates vascular dysfunction and inhibits atherogenesis in apolipoprotein e-deficient mice

Abstract Introduction Pharmacological blockade of mineralocorticoid receptors (MR) is a potential therapeutic approach to reduce cardiovascular complications because MR play a crucial role in cardiovascular regulation. Recent studies suggest that MR antagonists affect several extrarenal tissues, including vascular function. We investigated the effects of a novel non-steroidal selective MR blocker, esaxerenone (ESAX), recently approved for hypertension treatment in Japan, on vascular function and the development of atherosclerosis. Methods and results ESAX (3mg/kg/day) or vehicle was orally administered to western type diet-fed apolipoprotein E-deficient mice for 20 weeks or for 8 weeks to investigate the effects on atherogenesis or vascular dysfunction. Administration of ESAX for 20 weeks suppressed the development of atherosclerotic plaques in the aortic arch compared with the vehicle group (p<0.001) without alteration of blood pressure. In addition, ESAX reduced the expression of intercellular cell adhesion molecule-1 (p<0.05), macrophage infiltration (p<0.01), lipid deposition (p<0.05) and tended to decrease the expression of vascular cell adhesion molecule-1 as determined by immunohistochemical analysis. Moreover, phosphorylation of eNOSSer1177 (p<0.05) and Akt (p<0.05) were increased in the aorta of ESAX-treated mice. Administration of ESAX for 8 weeks attenuated western type diet-induced endothelial dysfunction (p<0.05) accompanied with the decrease in expression of ICAM1 (p<0.05) and phosphorylation of JNK (p<0.05) in the descending aorta. In in vitro experiment, aldosterone decreased the phosphorylation of eNOSSer1177 and increased the phosphorylation of eNOSThr495 in a dose dependent manner, which were attenuated by ESAX in HUVECs. In ex vivo vascular reactivity assay, ESAX diminished the aldosterone-induced endothelial dysfunction. Conclusion ESAX ameliorated hyperlipidemia-induced atherosclerosis and vascular dysfunction in mice, suggesting that ESAX is a promising therapeutic approach for cardiovascular complications.

Orthostatic Stress and Baroreflex Sensitivity: A Window into Autonomic Dysfunction in Lone Paroxysmal Atrial Fibrillation.

The abnormal neural control of atria has been considered one of the mechanisms of paroxysmal atrial fibrillation (PAF) pathogenesis. The baroreceptor reflex has an important role in cardiovascular regulation and may serve as an index of autonomic function. This study aimed to analyze the baroreceptor reflex's role in heart rate regulation during upright tilt (HUT) in patients with lone PAF. The study included 68 patients with lone PAF and 34 healthy individuals who underwent baroreflex assessment. Parameters such as baroreflex sensitivity (BRS), number of systolic blood pressure (BP) ramps, and the baroreflex effectiveness index (BEI) were evaluated. The study found that PAF patients had comparable resting BPs and heart rates (HRs) to healthy individuals. However, unlike healthy individuals, PAF patients showed a sustained increase in BP with an upright posture followed by the delayed activation of the baroreceptor function with a blunted HR response and lower BEI values. This indicates a pronounced baroreflex impairment in PAF patients, even at rest. Our data suggest that together with BRS, BEI could be used as a marker of autonomic dysfunction in PAF patients, making it important to further investigate its relationship with AF recurrence after ablation and its involvement in cardiovascular autonomic remodeling.

Angiotensin II type 1a receptor knockout ameliorates high-fat diet-induced cardiac dysfunction by regulating glucose and lipid metabolism.

Obesity-related cardiovascular diseases are associated with overactivation of the renin-angiotensin system (RAS). However, the underlying mechanisms remain elusive. In this study, we investigate the role of angiotensin II (Ang II) in high-fat diet (HFD)-induced cardiac dysfunction by focusing on cardiac glucose and lipid metabolism and energy supply. Ang II plays a role in cardiovascular regulation mainly by stimulating angiotensin II type 1 receptor (AT1R), among which AT1aR is the most important subtype in regulating the function of the cardiovascular system. AT1aR gene knockout (AT1aR ‒/‒) rats and wild-type (WT) rats are randomly divided into four groups and fed with either a normal diet (ND) or a HFD for 12 weeks. The myocardial lipid content, Ang II level and cardiac function are then evaluated. The expressions of a number of genes involved in glucose and fatty acid oxidation and mitochondrial dynamics are measured by quantitative polymerase chain reaction and western blot analysis. Our results demonstrate that AT1aR knockout improves HFD-induced insulin resistance and dyslipidemia as well as lipid deposition and left ventricular dysfunction compared with WT rats fed a HFD. In addition, after feeding with HFD, AT1aR ‒/‒ rats not only show further improvement in glucose and fatty acid oxidation but also have a reverse effect on increased mitochondrial fission proteins. In conclusion, AT1aR deficiency ameliorates HFD-induced cardiac dysfunction by enhancing glucose and fatty acid oxidation, regulating mitochondrial dynamics-related protein changes, and further promoting cardiac energy supply.

Renal vascular conductance is unrelated to leg vascular conductance or muscle sympathetic nerve activity at rest and during stress in humans.

The sympathetic nervous system is important for cardiovascular regulation, particularly during acute stress. Efferent sympathetic outflow can be regulated in an organ-dependent manner, but whether renal and leg vasoconstriction are associated at rest or during sympathetic stressors is unknown. Therefore, we sought to determine the relationships between muscle sympathetic nerve activity (MSNA), leg vascular conductance (LVC), and renal vascular conductance (RVC) at rest and during common laboratory-based sympathoexcitatory stimuli in a cohort of young healthy adults. Beat-to-beat arterial pressure (photoplethysmography), MSNA (microneurography), superficial femoral artery blood flow, and renal artery blood velocity (Doppler ultrasound) were measured at rest and during static handgrip exercise (30% maximal voluntary contraction), postexercise circulatory occlusion (PECO), and cold stress (hand in 3.8 ± 1.3°C water) in 37 young healthy adults (16 females, 21 males). At rest, RVC was unrelated to LVC (r = -0.11, P = 0.55) or MSNA burst frequency (ρ = -0.22, P = 0.26). Static handgrip, PECO, and cold stress each induced an increase in mean arterial pressure and MSNA and a reduction in RVC (all P < 0.001). LVC was unaltered during stress (all P ≥ 0.16), with the exception of a reduction during the second minute of cold stress (P = 0.03). During stress, changes in RVC were not associated with changes in LVC (handgrip: r = -0.24, P = 0.21; PECO: ρ = -0.04, P = 0.82; cold stress: r = -0.17, P = 0.38) or MSNA (handgrip: ρ = -0.14, P = 0.48; PECO: r = 0.27, P = 0.15; cold stress: r = -0.27, P = 0.16). Furthermore, MSNA was not associated with LVC at rest or during stress (all P ≥ 0.12). The present findings highlight the differential control of regional sympathetic vasoconstriction at rest and during stress in young healthy humans.NEW & NOTEWORTHY The sympathetic nervous system plays a critical role in cardiovascular regulation at rest and during stress. We demonstrate that renal artery vascular conductance is unrelated to superficial femoral artery vascular conductance or muscle sympathetic nerve activity at rest or during laboratory-based sympathetic stressors in young healthy adults. These findings support the concept of differential control of peripheral sympathetic outflow at rest and during stress in humans.

A Selective Mineralocorticoid Receptor Blocker, Esaxerenone, Attenuates Vascular Dysfunction in Diabetic C57BL/6 Mice

Pharmacological blockade of mineralocorticoid receptors (MRs) is a potential therapeutic approach to reduce cardiovascular complications since MRs play a crucial role in cardiovascular regulation. Recent studies suggest that MR antagonists affect several extrarenal tissues, including vessel function. We investigated the effect of a novel nonsteroidal selective MR blocker, esaxerenone, on diabetes-induced vascular dysfunction. Diabetes was induced by a single dose of streptozotocin in 8-week-old male C57BL/6 mice. Esaxerenone (3 mg/kg/day) or a vehicle was administered by gavage to diabetic mice for 3 weeks. Metabolic parameters, plasma aldosterone levels, and parameters related to renal function were measured. Endothelium-dependent or -independent vascular responses of the aortic segments were analyzed with acetylcholine or sodium nitroprusside, respectively. Human umbilical vein endothelial cells (HUVECs) were used for the in vitro study. Induction of diabetes elevated plasma aldosterone level (P＜0.05) and impaired endothelium-dependent vascular relaxation (P＜0.05). The administration of esaxerenone ameliorated the endothelial dysfunction (P＜0.01) without the alteration of metabolic parameters, blood pressure, and renal function. Esaxerenone improved the eNOSSer1177 phosphorylation in the aorta obtained from diabetic mice (P＜0.05) compared with that in the vehicle-treated group. Furthermore, a major MR agonist, aldosterone, decreased eNOSSer1177 phosphorylation and increased eNOSThr495 phosphorylation in HUVECs, which recovered with esaxerenone. Esaxerenone ameliorated the endothelium-dependent vascular relaxation caused by aldosterone in the aortic segments obtained from C57BL/6 mice (P＜0.001). Esaxerenone attenuates the development of diabetes-induced endothelial dysfunction in mice. These results suggest that esaxerenone has potential vascular protective effects in individuals with diabetes.

Erythrocytes from patients with ST-elevation myocardial infarction induce cardioprotection through the purinergic P2Y13 receptor and nitric oxide signaling

Red blood cells (RBCs) are suggested to play a role in cardiovascular regulation by exporting nitric oxide (NO) bioactivity and ATP under hypoxia. It remains unknown whether such beneficial effects of RBCs are protective in patients with acute myocardial infarction. We investigated whether RBCs from patients with ST-elevation myocardial infarction (STEMI) protect against myocardial ischemia–reperfusion injury and whether such effect involves NO and purinergic signaling in the RBCs. RBCs from patients with STEMI undergoing primary coronary intervention and healthy controls were administered to isolated rat hearts subjected to global ischemia and reperfusion. Compared to RBCs from healthy controls, RBCs from STEMI patients reduced myocardial infarct size (30 ± 12% RBC healthy vs. 11 ± 5% RBC STEMI patients, P < 0.001), improved recovery of left-ventricular developed pressure and dP/dt and reduced left-ventricular end-diastolic pressure in hearts subjected to ischemia–reperfusion. Inhibition of RBC NO synthase with L-NAME or soluble guanylyl cyclase (sGC) with ODQ, and inhibition of cardiac protein kinase G (PKG) abolished the cardioprotective effect. Furthermore, the non-selective purinergic P2 receptor antagonist PPADS but not the P1 receptor antagonist 8PT attenuated the cardioprotection induced by RBCs from STEMI patients. The P2Y13 receptor was expressed in RBCs and the cardioprotection was abolished by the P2Y13 receptor antagonist MRS2211. By contrast, perfusion with PPADS, L-NAME, or ODQ prior to RBCs administration failed to block the cardioprotection induced by RBCs from STEMI patients. Administration of RBCs from healthy subjects following pre-incubation with an ATP analog reduced infarct size from 20 ± 6 to 7 ± 2% (P < 0.001), and this effect was abolished by ODQ and MRS2211. This study demonstrates a novel function of RBCs in STEMI patients providing protection against myocardial ischemia–reperfusion injury through the P2Y13 receptor and the NO–sGC–PKG pathway.

Abstract MP19: Endogenous 5-hydroxytryptamine May Regulate Skeletal Muscle Vascular Resistance In Normal Healthy Rats

Infusing low doses of 5-hydroxytryptamine (5-HT) into normal rats causes chronic (weeks to months) hypotension and a fall in total peripheral resistance. These effects are mediated by activation of 5-HT 7 receptors. Therefore, we generated 5-HT 7 receptor knockout rats (5-HT 7 KO) to explore possible cardiovascular effects of 5-HT 7 receptors under normal and pathophysiological conditions. We previously reported that healthy 5-HT 7 KO rats have normal blood pressure and total peripheral resistance at rest. This suggested that 5-HT 7 receptors plays no role in cardiovascular regulation under normal conditions. But total peripheral resistance is determined by multiple vascular beds that differ in their sensitivity to 5-HT. Others have indicated that 5-HT 7 receptors in the skeletal muscle vasculature are particularly sensitive to the effects of 5-HT. Therefore, we hypothesized that 5-HT 7 KO rats would show both reduced responsiveness to exogenous 5-HT and increased resting skeletal muscle vascular resistance. Experiments were performed in isoflurane-anesthetized, male Sprague-Dawley (SD) (n=6), 5-HT 7 wild-type (5-HT 7 WT) (n=5) and 5-HT 7 KO (n=6) rats at 7-8 months of age. Arterial pressure was measured with an aortic catheter. Blood flow to the hindquarters (mostly skeletal muscle) was measured with transit-time, ultrasound flowmetry. After 10 minutes of baseline hemodynamic measurements were obtained, 5-HT was infused iv at a rate of 25 μg/kg/min for 15 minutes, followed by a 15-minute recovery period. As expected, 5-HT 7 KO rats did not show a significant fall in hindquarter vascular resistance (HQVR) during 5-HT infusion, while SD and 5-HT 7 WT did. More importantly, HQVR at baseline was significantly (p &lt; 0.05) higher in 5-HT 7 KO rats (16.0 ± 2.0 mmHg/ml/min) than in 5-HT 7 WT rats (10.9 ± 0.06 mmHg/ml/min) or SD rats (7.0 ± 0.03 mmHg/ml/min). These results support our hypothesis that in healthy (albeit anesthetized) rats, 5-HT 7 receptors reduce skeletal muscle vascular resistance.

Temporal Profiling of Molecular Alterations in the Hypothalamic Paraventricular Nucleus During Angiotensin‐II Hypertension

The hypothalamic paraventricular nucleus (PVN) plays a central role in cardiovascular regulation including hypertension development in response to elevations in angiotensin-II (Ang-II). Upstream circumventricular nuclei located outside of the blood-brain-barrier sense circulating Ang-II and influence PVN endocrine and autonomic regulation via efferent synaptic projections. Despite progress in unraveling the function of circumventricular-PVN networks in neurogenic hypertension development, the underlying PVN mechanisms responsible for hypertension development remain unclear. In particular, most investigations have examined PVN alterations during established hypertension, which limits insight into molecular changes that may occur prior to hypertension development. Based on this, we performed a detailed temporal profiling of key molecular targets in the PVN during Ang-II hypertension progression. Male C57Bl/6J mice were implanted with subcutaneous osmotic minipumps for chronic delivery of low dose Ang-II (600 ng/kg/min). Brains were harvested for immunohistochemistry analysis of target molecules across rostral to caudal subregions of the PVN (n=3-4/group) at baseline (day 0), pre-hypertensive (days 3 and 7), and established-hypertension (day 14) timepoints. Previous work has suggested a role for arginine vasopressin (AVP) in Ang-II-induced hypertension. In line with this, AVP immunoreactivity in the PVN was elevated within 3 days of Ang-II infusion; this was evident only in the caudal PVN (day 3 fold change: 2.5±0.2, density/cell, p<0.05), whereas AVP expression in the rostral and medial portions was unchanged. Interestingly, caudal AVP immunoreactivity returned to basal levels by day 7 and remained low for the remainder of the protocol. In contrast to AVP, oxytocin expression was unchanged across the rostral to caudal PVN during 2-week Ang-II hypertension development. We also measured preproenkephalin (PPE) immunoreactivity, and found a progressive increase throughout the entire PVN with elevations initiated at pre-hypertensive times (day 7 fold change: 2.0±0.1, density/area, p=0.08) that were sustained during established hypertension (day 14 fold change: 2.6±0.3, density/area, p<0.05). To test possible involvement of the arcuate nucleus, which transmits circulating information from the median eminence to the PVN, in Ang-II hypertension, we measured alpha-melanocortin stimulating hormone (alpha-MSH) levels. Histological analysis for alpha-MSH immunoreactivity in the PVN, as an indicator of synaptic innervation strength from arcuate nucleus proopiomelanocortin neurons, revealed no changes during Ang-II infusion. Collectively, these findings indicate elevations in PVN AVP and PPE, but not oxytocin and alpha-MSH, in response to elevations in circulating Ang-II. Furthermore, the temporal responses suggest that different PVN molecules may be responsible for the initiation (e.g. AVP) versus maintenance (e.g. PPE) of Ang-II hypertension.

Role of the glutamatergic system of ventrolateral periaqueductal gray (vlPAG) in the cardiovascular responses in normal and hemorrhagic conditions in rats.

Objective(s):Periaqueductal gray (PAG) is a mesencephalic area divided into four columns including ventrolateral periaqueductal gray (vlPAG). vlPAG plays a role in cardiovascular regulation during normal and hemorrhagic (Hem) conditions. Due to presence of glutamate in this area, we evaluated the effect of glutamatergic receptors of this area on cardiovascular activity in normotensive and hypovolemic Hem rats.Materials and Methods:Animals were divided into twelve groups: saline (vehicle), Glutamate, GYK52466 (non-NMDA receptor antagonist), and MK801 (NMDA receptor antagonist) with and without Glu microinjected into vlPAG in normal and Hem conditions. Following the femoral artery cannulating and microinjecting, changes (Δ) of heart rate (HR), systolic blood pressure (SBP), and mean arterial pressure (MAP) were recorded via a PowerLab unit.Results:In normotensive conditions, microinjection of Glu increased ΔMAP, ΔSBP, and ΔHR (P<0.001). MK-801 and GYKI-52466 nonsignificant reduced cardiovascular responses than vehicle while their changes were significant compared with glutamate (P<0.001). Co-injection of GYKI- 52466 with Glu did not significantly reduce ΔSBP and ΔMAP induced by Glu (P>0.05) but co-injection of MK-801 with Glu significantly attenuate these effects(P<0.01). In Hem, Glu increased ΔSBP, ΔMAP, and ΔHR (P<0.05). GYKI-52466 alone did not change cardiovascular responses but MK-801 decreased ΔSBP than Hem (P<0.01). Co-injection of GYKI-52466 with Glu had significant(P<0.05) but MK-801 with Glu had no significant effect compared with Hem (P>0.05).Conclusion:The glutamatergic system of vlPAG increases cardiovascular values that are mostly mediated through the NMDA receptor. Since vlPAG is well known as an inhibitory region, it seems that glutamate does not have a noteworthy cardiovascular role in vlPAG during Hem and normal conditions.

Chapter 13 - The subfornical organ and organum vasculosum of the lamina terminalis: Critical roles in cardiovascular regulation and the control of fluid balance

In this chapter, we review the extensive literature describing the roles of the subfornical organ (SFO), the organum vasculosum of the terminalis (OVLT), and the median preoptic nucleus (MnPO), comprising the lamina terminalis, in cardiovascular regulation and the control of fluid balance. We present this information in the context of both historical and technological developments which can effectively be overlaid upon each other. We describe intrinsic anatomy and connectivity and then discuss early work which described how circulating angiotensin II acts at the SFO to stimulate drinking and increase blood pressure. Extensive studies using direct administration and lesion approaches to highlight the roles of all regions of the lamina terminalis are then discussed. At the cellular level we describe c-Fos and electrophysiological work, which has highlighted an extensive group of circulating hormones which appear to influence the activity of specific neurons in the SFO, OVLT, and MnPO. We highlight optogenetic studies that have begun to unravel the complexities of circuitries underlying physiological outcomes, especially those related to different components of drinking. Finally, we describe the somewhat limited human literature supporting conclusions that these structures play similar and potentially important roles in human physiology.

Cholecystokinin octapeptide reduces myocardial fibrosis and improves cardiac remodeling in post myocardial infarction rats.

Myocardial infarction (MI) increases myocardial fibrosis (MF) and subsequent cardiac remodeling. Cholecystokinin octapeptide (CCK-8) is expressed in cardiomyocytes and plays an important role in cardiovascular regulation. In this study, we intend to use a rat model of myocardial infarction to evaluate the effects of CCK-8 on myocardial fibrosis and cardiac remodeling. Male Sprague-Dawley rats were separated into 3 groups: sham operation, MI + NaCl, and MI + CCK-8. All rats were subjected to left coronary artery ligation to induce MI or sham operation and then treated with CCK-8 or saline for 28 days. After 4 weeks, echocardiography was performed to assess cardiac function and myocardial fibrosis was evaluated using H&E and Masson's Trichrome-stained sections. The levels of BNP, CCK-8 in the plasma of all rats were detected by ELISA; RNA sequencing (RNA-seq) analysis was also adapted to detect differentially expressed genes in myocardial tissues of each group. Myocardial expression of fibrosis markers was analyzed by western blotting, immunohistochemistry and qRT-PCR. CCK-8 was demonstrated to improve left ventricular function and results of H&E staining, Masson's trichrome staining, immunohistochemistry and western blotting showed that CCK-8 attenuated MF. Gene expression profiles of the left ventricles were analysed by RNA-seq and validated by qRT-PCR. Cardiac fibrosis genes were downregulated by CCK-8 in the left ventricle. CCK-8 can alleviate fibrosis in the noninfarcted regions and delay the left ventricular remodeling and the progress of heart failure in a MI rat model.

Potential Role of the Amygdala and Posterior Claustrum in Exercise Intensity-dependent Cardiovascular Regulation in Rats

Tuning of the cardiovascular response is crucial to maintain performance during high-intensity exercise. It is well known that the nucleus of the solitary tract (NTS) in the brainstem medulla plays a central role in cardiovascular regulation; however, where and how upper brain regions form circuits with NTS and coordinately control cardiovascular responses during high-intensity exercise remain unclear. Here focusing on the amygdala and claustrum, we investigated part of the mechanism for regulation of the cardiovascular system during exercise. In rats, c-Fos immunostaining was used to examine whether the amygdala and claustrum were activated during treadmill exercise. Further, we examined arterial pressure responses to electrical and chemical stimulation of the claustrum region. We also confirmed the anatomical connections between the amygdala, claustrum, and NTS by retrograde tracer injections. Finally, we performed simultaneous electrical stimulation of the claustrum and amygdala to examine their functional connectivity. c-Fos expression was observed in the amygdala and the posterior part of the claustrum (pCL), but not in the anterior part, in an exercise intensity-dependent manner. pCL stimulation induced a depressor response. Using a retrograde tracer, we confirmed direct projections from the amygdala to the pCL and NTS. Simultaneous stimulation of the central nucleus of the amygdala and pCL showed a greater pressor response compared with the stimulation of the amygdala alone. These results suggest the amygdala and pCL are involved in different phases of exercise. More speculatively, these areas might coordinately tune cardiovascular responses that help maintain performance during high-intensity exercise.