bluuebunny/crossref_metadata_2025 huggingface dataset:
Deficiency of c‐Jun‐N‐terminal kinase prevents angiotensin II‐induced inward remodeling but not hypertrophy in cerebral arterioles (856.13)
Authors: Umesalma Shaikamjad , Gerhold Thomas , Baumbach Gary
Published: June 2021

Angiotensin II (Ang II) is an important determinant of inward remodeling in cerebral arterioles. We examined the effect of c‐Jun‐N‐terminal kinase (JNK) deficiency on structural alterations in cerebral arterioles induced by Ang II. Three month old JNK1‐deficient (JNK1‐/‐) and wild‐type (WT, C57BL/6J) mice were administered Ang II (1000 ng/kg/day) or saline for 4 weeks via osmotic minipumps. Systolic arterial pressure (SAP) was measured by a tail‐cuff method. External diameter (ED) of maximally dilated cerebral arterioles was measured in vivo through an open cranial window in anesthetized mice. Cross‐sectional area (CSA) of the vessel wall was measured histologically. Infusion of Ang II significantly increased SAP in both WT (148±7 vs 120±4 [saline infused] mmHg, P<0.05) and JNK1‐/‐ (148±3 vs 118±5 mmHg, P<0.05) mice; reduced cerebral arteriolar ED in WT (57±4 vs 68±3 μm, P<0.05), but not in JNK1‐/‐ mice (65±4 vs 66±4 μm, P<0.05); and increased CSA of the arteriolar wall in JNK1‐/‐ (501±41 vs 388±49 μm2, P<0.05), as well as WT (519±74 vs 393±30 μm2, P<0.05). In conclusion, our findings suggest that JNK1 may play an important role in Ang II‐induced inward remodeling, but not hypertrophy, in cerebral arterioles.

Grant Funding Source: NIH
Effects of treatment with aliskiren and l‐arginine in baroreflex sensitivity in renovascular hypertensive rats (856.5)
Authors: Mengal Vinícius , Silva Paulo , Tiradentes Renata , Santuzzi Cintia , Gouvea Sonia , Abreu Glaucia
Published: June 2021

In some pathological conditions such as hypertension, there is an impairment in the autonomic control of blood pressure resulting in changes in baroreflex sensitivity. In this context, this work aims to elucidate the benefits of the combination of aliskiren and L‐arginine in the control of blood pressure and the restoration of reduced baroreflex sensitivity in renovascular hypertension. Renovascular hypertension (2K1C) was produced in Male Wistar rats (140‐160 g). Seven days after was performed by plethysmography to measure systolic blood pressure. The rats were divided in groups: Sham (6), 2K1C (7), 2K1C aliskiren 50 mg/kg (8), 2K1C L‐arg 300 mg/kg (8) and 2K1C ALK + L‐arg (7). The treatment was performed for 21 days by gavage. The animals were anesthetized and fitted with femoral venous and arterial catheters for drug injection and arterial pressure recordings. We performed the direct recording of blood pressure and heart rate compared to variations in blood pressure induced with vasoactive drugs phenylephrine and sodium nitroprusside before and after administration of superoxide dismutase (SOD) mimetic tempol. At the end of the protocol, the expression of GPphox and SOD in aorta were analyzed by Western blotting. Statistical analysis was determined using 1‐way ANOVA and Tukey test. Values are expressed as mean ± SEM and the significance of P <0.05. Only the treatment with the combination of ALK + L‐arg was able to normalize systolic blood pressure. The 2K1C showed a reduction in baroreflex sensitivity after intravenous bolus of phenylephrine when compared to sham group as well as a reduction when administered in this modulation sodium nitroprusside. This reduction was restored in 2K1C after treatment with aliskiren and with the combination of aliskiren and L‐arginine in both intravenous administrations. The combination of ALK and L‐ARG was able to reduce the arterial pressure levels and increase in baroreflex sensitivity. These effects could be related a reduction in oxidative stress.

Grant Funding Source: CNPq
Two‐photon microscopy enables non‐invasive, in vivo, longitudinal study of arterial Ca2+ and diameter in optical biosensor mice during the development of hypertension (856.7)
Authors: Mauban Joseph , Fairfax Seth , Zhang Jin , Rizzo Mark , Wier W.
Published: June 2021

Here, we report that use of two‐photon microscopy of ear arteries in exMLCK biosensor mice enables non‐invasive, in vivo, serial examination of vascular smooth muscle Ca2+ during the development of hypertension. ExMLCK mice express a FRET‐type biosensor of Ca2+/Calmodulin based on smooth muscle myosin light chain kinase (MLCK) and its ratiometric properties permit direct quantification of intracellular Ca2+ concentration. Anesthetized biosensor mice were positioned on a custom platform, facilitating imaging of selected arterial branches within the intact depilated ear, and studied before and during chronic infusion of angiotensin‐II (ANGII; 800 µg/g/day) via mini‐osmotic pump. Acute responsiveness to i.p. phenylephrine (PE; 30µg/g) was determined in a separate group of mice equipped with arterial blood pressure radio telemetry. Eight days of chronic ANGII increased intracellular Ca2+ from 294±47nM to 501±92nM (P<0.05) and decreased artery diameter by ‐14±2% (P<0.05). Acute PE induced robust vasomotion and 2‐3 fold oscillations in spatially averaged Ca2+. In summary, this new methodology permits for the first time the simultaneous study of Ca2+ signaling, structural remodeling and vascular reactivity in the intact living mouse during hypertension development.

Grant Funding Source: Supported by NHLBI RO1091969, RO1HL107654, RO1DK077140, and T32HL072751.
Radiofrequency renal nerve ablation decreases blood pressure in SHR accompanied by increased urinary sodium excretion (857.4)
Authors: Gao Juan , Smart Frank , Katsurada Akemi , Navar Gabriel , Kapusta Daniel
Published: June 2021

We have shown that radiofrequency catheter ablation of the renal arteries (RF‐RDN) decreases blood pressure (BP) in spontaneously hypertensive rats (SHR). Here, we studied the effects of RF‐RDN on changes in water and sodium excretion and urinary angiotensinogen levels in SHR with established hypertension. Methods Nineteen‐week old male SHR were instrumented with telemetry probes for measurement of BP. After 1‐week, control BP was measured. The next day SHR randomly received either bilateral Sham‐RDN (n=5) or RF‐RDN (n=6) of the renal arteries (Biosense Webster Stockert 70 generator and RF‐probe). BP (Systolic, SBP; Diastolic, DBP) was then measured for 2 weeks. Rats were then placed in metabolic cages for collection of urine and measurement of 24‐hr water and sodium balance. Results BP was significantly decreased in RF‐RDN SHR (2 weeks; ΔSBP, ‐12±5; ΔDBP, ‐9±4 mmHg) as compared to sham SHR (2 weeks; ΔSBP, ‐2±2; ΔDBP, 2±2 mmHg). RF‐RDN SHR increased urinary sodium excretion (RF‐RDN, 0.33±0.02; Sham, 0.25±0.03 mEq/day) and food intake (RF‐RDN 21±1; Sham 17±1 g/day). However, there were no significant differences between groups in 24‐hr daily water (RF‐RDN, 21±1; Sham, 19±2 ml) or sodium balance (RF‐RDN, 0.59±0.04; Sham, 0.48±0.06 mEq), or urinary angiotensinogen, protein and creatinine excretion rates. At the end of week 2, kidney norepinephrine levels in RF‐RDN rats (left, 0±0; right, 10±10 ng/g kidney) were markedly decreased compared with levels from sham SHR (left, 130±10; right, 110±10 ng/g kidney). Conclusions These data demonstrate that in hypertensive SHR, bilateral RF‐RDN produces an increase in urinary sodium excretion, which may potentially contribute to the concurrent decrease in BP.

Grant Funding Source: Biosense Webster IIS‐175 to DRK & FS; P30 GM103337 to LGN; P20 GM103514 to DRK
A deficiency in the formation of 20‐HETE impairs the myogenic and TGF responses of the Af‐Art of Dahl salt sensitive rats (857.8)
Authors: Ge Ying , Murphy Sydney , Fan Fan , Williams Jan , Falck John , Roman Richard
Published: June 2021

Previously, we demonstrated that transfer of chromosome 5 containing the CYP4A genes from Brown Norway (BN) rat onto the Dahl S (SS) genetic background (SS.5BN strain) increases the formation of 20‐HETE in renal microvessels and contributes to the antihypertensive effect. Therefore, we hypothesize that a deficiency in the formation of 20‐HETE in the renal vasculature impairs myogenic and TGF responses in the afferent arteriole (Af‐Art) in SS rats. The production of 20‐HETE was 54% lower in renal microvessels of SS rats than in SS.5BNrats. The luminal diameter of the Af‐Art decreased by 15±2% (n=6, p<0.05) in SS.5BN rats when the perfusion pressure was increased from 60 to 120 mmHg, whereas it remained unaltered in SS rats. Administration of an adenosine type 1 receptor agonist (CCPA, 1 µM) reduced the diameter of the Af‐Art in the SS.5BN rats by 44±2%(n=6, p<0.05)., whereas the Af‐Art of SS rats was unaltered SS rats exhibited poor autoregulation of RBF and PGC in vivo, whereas they were well regulated in SS.5BN rats. Administration of a 20‐HETE synthesis inhibitor, HET0016 (1 µM, n=6) completely blocked the myogenic and adenosine responses in the Af‐Art and autoregulation of RBF and PGC in SS.5BN rats but not in SS rats. These data indicated that a deficiency in the formation of 20‐HETE in renal microvessels contributes to the lack of renal autoregulation and development of hypertension in SS rats.

Grant Funding Source: This studies were supported by Grant HL36279 and AHA 13POST14220024.
Effects of intermittent heat acclimation on thirst and hypothalamic angiotensin II receptor levels in rats (858.1)
Authors: Johnson Jacob , Barney Christopher
Published: June 2021

The effects of heat acclimation on thermal dehydration and subsequent water intake are not well understood. In this study, male Sprague‐Dawley rats were exposed to 37.5°C (Intermittent Heat Acclimation ‐ IHA) with or without water or to 25°C with water for 4 hours/day for 7 consecutive days. On day 8, rats from each group were exposed to either 25°C with water or 37.5°C without water. Blood samples were taken for estimation of cellular and extracellular dehydration and the hypothalamus was removed to quantify angiotensin II receptor (AT1 and AT2) levels. Water losses and intakes were measured on days 1 and 7 during the exposure period and for 3 hours following heat exposure. Heat exposure increased water intake during and after exposure, and having access to water during heat exposure increased evaporative water loss but did not alter urine output. IHA decreased evaporative water loss during heat exposure, while not altering urine output during or after heat exposure. IHA also increased water intake in the heat and decreased both water intake following heat exposure and total water intake without changing blood indicators of cellular or extracellular dehydration. Neither IHA nor acute heat exposure altered the levels of AT1 or AT2 receptors in the hypothalamus. IHA reduces evaporative water loss during heat exposure leading to a subsequent reduction in total water intake without altering physiological thirst drives.

Grant Funding Source: Supported by a NSF REU Site Award (DBI‐0754293)
Copeptin and norepinephrine during graded hypovolemic stress in women prone to vasovagal syncope (858.6)
Authors: Lindenberger Marcus , Skoog Johan , Alehagen Urban , Länne Toste
Published: June 2021

Objective: Vasovagal syncope is the most common form of syncope in young individuals, with increased incidence in women. Despite being common, the aetiology is not elucidated. The aim was to study various biomarkers involved in blood pressure homeostasis in women prone to vasovagal syncope.

Design and Methods: Graded lower body negative pressure (LBNP) to presyncope was used determine LBNP tolerance index (LTI) in 13 young women prone to vasovagal syncope (VVS) and 15 matched controls (C). Plasma levels of Norepinephrine (P‐NE), Copeptin, a surrogate marker of vasopressin (Cop) and midregion proadenomedullin (MR‐ProADM) was measured before and during graded LBNP.

Results: LTI was lower in VVS (163±8) compared to controls (235±8) (P < 0.0001). VVS presented with increased levels of P‐NE at rest as well as LBNP of 30 mmHg, with similar levels at presyncope. P‐NE increase from rest to presyncope was smaller in VVS than in C (P = 0.01). Cop was greater in VVS during LBNP of 30 mmHg, but similar during presyncope. MR‐ProADM was unaffected by graded hypovolemia.

Conclusions: Women prone to VVS present with increased levels of P‐NE and Cop during early LBNP, but with similar levels at presyncope, possibly as a result of greater hypovolemic stress at a given LBNP level. To elucidate its role in VVS, these data must be interpreted together with other data during hypovolemia.
Vascular endothelin (ET‐1) mediates skin Na+ storage in response to chronic increases in salt intake (860.13)
Authors: Speed Joshua , Pollock Jennifer , Titze Jens , Pollock David
Published: June 2021

We have recently implicated extrarenal vascular ET‐1 in the body’s integrated response to a chronic high salt intake by mediating storage of Na+ within the skin. We hypothesize that upregulation of vascular ET‐1 in response to a high salt diet leads to the recruitment of macrophages and lymphocytes to the interstitium of the skin, which promote lymph hyperplasia, all of which provide a buffer for extracellular Na+ homeostasis. In the present study, we determined if vascular endothelial cell ET‐1 knockout mice (VEET KO) have reduced vascular cell adhesion molecule expression and increased skin Na+ storage in response to a high salt diet. After 2 weeks of a high NaCl (4%) diet, VEET KO mice have higher total skin Na/water ratio than floxed controls (0.112±0.007 vs. 0.096±0.003 mmol/ml respectively). Floxed control mice have elevated ET‐1 mRNA in the aorta compared to animals on low salt (1.38±0.21 vs. 1.05±0.14 fold change respectively, n=6), a response that was completely blunted in VEET KO mice (1.07±0.12 vs. 1.05±0.16 fold change, n=5). There was no change in vascular cell adhesion molecule‐1 between groups. These data support our hypothesis that vascular endothelial derived ET‐1 plays a critical role in mediating skin storage of Na+ in response to a high salt intake.

Grant Funding Source: Supported by NHLBI and AHA
Oxidative stress signaling in response to CD4+T cells stimulated via placental ischemia play an important role in hypertension during preeclampsia (860.14)
Authors: Scott Jeremy , Wallace Kedra , Cornelius Denise , Amaral Lorena , Moseley Janae , Chapman Krystal , LaMarca Babbette
Published: June 2021

Preeclampsia (PE) is associated with hypertension, oxidative stress, and chronic inflammation. The RUPP rat model of PE and normal pregnant (NP) recipient rats of RUPP CD4+T cells exhibit much of the disease pathology. Reactive oxygen species (ROS) could be released from monocytes or neutropils, however the objective of this study was to determine if RUPP CD4 T cells are a cellular source of ROS and hypertension (MAP) and during pregnancy. CD4+Tcells from RUPP rats were injected into NP recipients on gestational day (GD)13; GD19 MAP, urine and tissues were analyzed. Prior to injection RUPP CD4+ T cells secreted more myleoperoxidase compared to NP (116.9+15.2 vs. 48.5+8.3 ng/mg/mL; p=0.003). Placental NADPH oxidase mediated ROS in RUPP rats was 830.6+68, 726.2+43.73 in NP + RUPP CD4+ T cells and 420.7+51.4 RLU/min/mg in NP rats; p=0.009 and p=0.004, respectively. MAP was 100+2 in NP rats, 126+4 in RUPP rats (p=0.0001) and 118+4mmHg in NP+RUPP CD4+ T cell rats (p=0.002) which was attenuated with apocynin; 111+2 in RUPP + apocynin; 102+3 mmHg in NP + RUPP CD4+ T cells (p=0.01). These data suggest that CD4+Tcells are an important source of ROS mediating hypertension in response to placental ischemia.

Grant Funding Source: NIH NICHD; APS STRIDE
IL‐10 supplementation suppressed hypertension and inflammation in response to placental ischemia during pregnancy (860.17)
Authors: Harmon Ashlyn , Paige Adrienne , Cornelius Denise , Wallace Kedra , Thomas Alexia , Scott Jeremy , Moseley Janae , LaMarca Babbette
Published: June 2021

Women with preeclampsia (PE) develop hypertension, proteinuria, increased pro‐inflammatory and decreased regulatory immune responses (Tregs, IL‐10) contributing to increased local ET‐1. The RUPP rat model of PE exhibits a similar increase in MAP, immune imbalance and ET‐1 as women with PE. We hypothesized that IL‐10 supplementation in RUPP rats will restore T cell balance, decrease MAP and inflammation. IL‐10 was administered intraperitoneally via osmotic pumps to RUPP rats on day 14 of gestation following the RUPP procedure. On day 18 carotid catheters are inserted, and on day 19 MAP, pup weights, serum and tissues are collected. MAP in NP rats was 91+/‐2, RUPP was 122+/‐2 which decreased to 106+/‐2 mmHg in RUPP+IL‐10. Circulating CD4+ T cells were 14% gated cells in RUPP and 8% gated cells RUPP+IL‐10. IL‐6 was 33+/‐5 NP, 238+/‐96 RUPP, 40+/‐10 pg/mL RUPP+IL‐10; TNF‐α was 14.73+/‐3 NP, 69+/‐ 19 RUPP, 20+/‐11 pg/mL RUPP+IL‐10, and placental ET‐1 was 2.5 fold higher in RUPP but only 1.4 fold higher in RUPP+IL‐10 compared to NP rats. These data suggest an important role for IL‐10 supplementation to balance the inflammatory response and improve blood pressure and ET‐1 production associated with placental ischemia of pregnancy.

Grant Funding Source: Supported by RO1HD067541 & T32HL105324
Stimulation of a neuroimmune pathway protects from hypertension (860.22)
Authors: Mathis Keisa
Published: June 2021

Vagus nerve stimulation can suppress cytokine release from splenic immune cells and therefore reduce systemic inflammation through a pathway involving nicotinic acetylcholine receptors (α7nAChR). We tested whether activation of this ‘cholinergic anti‐inflammatory pathway’ protects from hypertension during systemic lupus erythematosus (SLE), a chronic autoimmune inflammatory disorder with autonomic dysfunction, renal injury, and hypertension. A female mouse model of SLE that develops hypertension (NZBWF1) was infused with nicotine hydrogen tartrate salt (2 mg/kg/day, subcutaneously) or saline for 7 days via osmotic minipumps. Splenic TNF‐α protein expression was higher in SLE mice (1.37±0.06) than controls (0.44±0.08; p<0.001), nicotine‐treated controls (0.48±0.05; p<0.001), and nicotine‐treated SLE mice (1.09±0.06; p=0.041). Albumin excretion rate (μg/day) was increased in SLE mice compared to controls (157±62 vs. 59±15; p=0.038). Nicotine prevented the rise in albuminuria in SLE mice (112±42; p=0.013), but had no effect in controls (46±8). Blood pressure (mmHg) was increased in SLE mice compared to controls (140±4 vs. 114±2; p<0.001). Nicotine prevented the rise in blood pressure in SLE mice (129±4; p=0.022), but did not alter blood pressure in controls (121±3). These data suggest the anti‐inflammatory effects of nicotine may prevent hypertension in mice with SLE and that the novel cholinergic anti‐inflammatory pathway may be an important target in patients with hypertension associated with chronic inflammatory diseases.

Grant Funding Source: Supported by F32HL114272 and P01HL051971
Hypothalamic PVN Gαi2 protein‐mediated regulation of renal NE turnover and sodium homeostasis: implications for the pathophysiology of salt‐sensitive hypertension (860.23)
Authors: Wainford Richard , Mahne Sarah , Kuwabara Jill
Published: June 2021

Aim: These studies examined the direct role of PVN Gαi2 proteins, which are up‐regulated during high‐salt intake in Sprague‐Dawley (SD) rats, in the regulation of water & electrolyte homeostasis, MAP and CNS/renal norepinephrine (NE) levels during high salt‐intake.

Methods: Groups of male SD rats received a bilateral PVN infusion of a scrambled (S) or Gαi2 oligodeoxynucleotide (ODN‐300ng/side/day) and a 7‐day normal 0.4% (NS) or high 8% NaCl (HS) diet. On day‐7 24h Na+ balance was assessed. In sub‐groups MAP, estimated blood volume (EBV), estimated plasma volume (EPV), plasma NE levels and PVN and kidney NE turnover were assessed (N=6/gp/study).

Results: In S infused rats HS did not alter sodium homeostasis (24h Na+ balance [meq] NS 0.7±0.3 vs HS 0.9±0.3), MAP (MAP [mmHg] NS 126±3 vs HS 127±3) or EBV and EPV. In S infused rats HS intake suppressed plasma NE content (plasma NE [nmol/L] NS 72±4 vs HS 40±5, P<0.05), kidney NE content (NE [pg/mg] NS 586±36 vs HS 496±31, P<0.05) and NE turnover in the PVN. HS intake in rats receiving a Gαi2 infusion evoked sodium retention (24h Na+ balance [meq] NS 0.6±0.2 vs HS 2.5±0.4 P<0.05), hypertension (MAP [mmHg] NS 127±3 vs HS 146±4, P<0.05) and increased blood and plasma volume (EPV [ml] NS 10.9±0.4 vs HS 11.8±0.4, P<0.05; EBV [ml] NS 21.1±0.5 vs HS 22.4±0.3, P<0.05). In Gαi2 infused rats HS intake increased plasma NE content (plasma NE (NE [nmol/L]; NS 67±7 vs HS 105±11, P<0.05), kidney NE content (NE [pg/mg] NS 614±48 vs HS 823±59, P<0.05) and kidney NE turnover (NE turnover rate [pg/mg/hr] NS 7.5±0.6 vs HS 20.3±1, P<0.05) while suppressing PVN NE turnover.

Conclusion: Impairment of PVN Gαi2 signal transduction during high salt‐intake results in dysregulation of renal NE content and turnover. This imbalance likely drives sodium & fluid retention stimulating the development of hypertension. These data highlight the interaction between the CNS and kidney, and the role of the sympathetic nervous system, in the long‐term regulation of MAP.

Grant Funding Source: Support by R01HL107330 & K02HL112718A1
Src‐kinase mediates angiotensin II‐induced potentiation in TRPV4 agonist‐evoked calcium transients in hypothalamic immortalized neuronal cell line 4B (860.25)
Authors: Saxena Ashwini , Bachelor Martha , Carreno Flavia , Cunningham J
Published: June 2021

We have previously demonstrated that in bile duct ligated rats, an animal model of inappropriate vasopressin (AVP) release, TRPV4 protein expression and membrane trafficking is increased in AVP neurons. Here, we used an in vitro approach with an immortalized neuronal AVP expressing cell line (4B neurons) to investigate the possible regulation of TRPV4 by angiotensin II (Ang II). We characterized the presence of TRPV4 mRNA and protein in 4B cells. After Ang II (100nM;1 hr) treatment significantly increased TRPV4 levels in crude membrane fractions (p<0.001) and tyrosine phosphorylation of TRPV4 (p<0.001). Using calcium sensitive dye Fura‐2AM, we noted that Ang II treated cells exhibited increased calcium transients in response to TRPV4 agonist, GSK1016790A (20nM, p<0.05). This increase was blocked by the Losartan (Ang II receptor antagonist) and Src‐kinase inhibitor, PP2, but not by its analog PP3. Our data indicate that Ang II may alter the phosphorylation status of TRPV4 through Src‐kinases.

Grant Funding Source: Supported by: R01 HL62569
VAMP8 knockout mice show enhanced renin release and content, kidney damage and salt sensitivity (860.5)
Authors: Mendez Mariela , Haque Mohammed , Ortiz Pablo
Published: June 2021

The vesicle fusion SNARE protein VAMP8 mediates trafficking of aquaporin 2 in kidney collecting ducts. Gene deletion of VAMP8 in mice (V8‐KO) resulted in polyuria and enhanced plasma renin. Renin is stored in granules in juxtaglomerular (JG) cells and its release is mediated by SNARE proteins. In other endocrine cells, VAMP8 mediates granule maturation and constitutive exocytosis. We found that VAMP8 is expressed in JG cells. However, it is not known whether VAMP8 regulates renin release from JG cells or the enhanced plasma renin in V8‐KO is due to compensation for volume loss. We hypothesized that VAMP8 mediates renin granule maturation and constitutive renin release from JG cells. Primary cultures of JG cells from V8‐KO showed enhanced renin and pro‐renin protein expression compared to wild type (WT) (n=5). Baseline renin release from JG cells was 5‐fold higher in V8‐KO than WT (WT=0.06±0.01; KO=0.3±0.05 μgAngI/hr/mg prot.; p<0.05). Immunolabeling of renin and confocal imaging showed enlarged granules and vacuoles in JG cells from V8‐KO. Similar results were observed in V8‐KO JG cells by electron microscopy. Kidney histology showed enhanced renal fibrosis and glomerular sclerosis in V8‐KO (n=4). Despite the higher release of renin and kidney damage, baseline blood pressure in V8‐KO was similar to WT (mmHg: WT=122±3.4; V8‐KO=121±4.5). A high salt diet (1% NaCl drinking water) increased blood pressure in V8‐KO mice by 16±5 mmHg (p<0.05) but not in age matched WT controls. We concluded that VAMP8 plays an inhibitory role in renin release, most likely by mediating normal granule maturation. VAMP8 may regulate blood pressure by inhibiting renin release during a high salt diet. VAMP8 is protective against kidney damage, but the mechanism is unclear.

Grant Funding Source: Supported by AHA and NIH
Endothelin, Ang II, vasopressin and osmolality regulate hyaluronan turnover by renomedullary interstitial cells suggesting an involvement of this matrix component in fluid handling (860.9)
Authors: Hansell Peter , Palm Fredrik , Takahashi Tomoko , Ikegami‐Kawai Mayumi , Stridh Sara
Published: June 2021

The content of the matrix component hyaluronan (HA) change in the medullary interstitium in relation to hydration status in vivo: elevated during hydration, reduced during dehydration suggesting a role in fluid handling by affecting interstitial fluid flux. We examined the effect of osmolality and fluid regulating hormones on HA‐turnover by RMIC in culture. Reducing media osmolality to simulate hydration, increased supernatant HA 4‐fold. Cell HA synthase (HAS) 2 and Hyaluronidase (Hyal) 2 mRNA expressions were unaltered while Hyal 1 mRNA was reduced by 35%. Supernatant Hyal activity was reduced by 44%. An inhibitor of Hyal activity (Asc‐P) elevated HA to similar high levels as low osmolality. AngII reduced HA by 58% and combined treatment with vasopressin reduced HA even further (69%). HAS and Hyal mRNA were stable while Hyal activity increased by 38% by AngII and 67% by the combination. Low dose endothelin (ET, 10‐10M) increased HA 3‐fold which was inhibited by ET‐B receptor blocker BQ788. On the contrary, high dose ET (10‐6M) reduced HA by 47%. The ET‐A receptor antagonist BQ123 not only reversed the reducing effect of high dose ET on HA but elevated it to the same level as low dose ET suggesting importance of ET‐B receptors. The study demonstrates a regulatory influence of osmolality and fluid regulating hormones on HA turnover, supporting the concept of an involvement of medullary interstitial HA in renal fluid handling by affecting interstitial permeability.

Grant Funding Source: Supported by the Swedish Research Council‐Medicine
Maintenance and termination of atrial fibrillation in a patient‐specific model (861.1)
Authors: Gonzales Matthew , Vincent Kevin , Rappel Wouter‐Jan , Narayan Sanjiv , McCulloch Andrew
Published: June 2021

Atrial fibrillation (AF) is the most common arrhythmia in the United States and is a risk factor for stroke and cardiac dysfunction. Radiofrequency (RF) ablation has shown great promise for improving outcomes for drug‐resistant AF. However, readmission rates remain high, and improving surgical strategies for targeted RF ablation is subject of intense research. In this study a patient‐specific biatrial finite element model of cardiac electrophysiology is used to explore mechanisms of AF maintenance and termination. Results demonstrate that with normal physiological anisotropy ratios and effective refractory periods (ERPs) of atrial myocytes, free wavelets do not attach to small (~1 cm), inexcitable regions and become rotors. With the depressed excitability, depressed anisotropy, and shorter ERPs present in AF, stationary rotors form around large (>1 cm) inexcitable regions and have a small spatial excitable gap; in contrast, meandering rotors are stabilized by small (<1 cm) inexcitable regions but anchor only transiently to them, colliding with their waveback and drifting until a region of space recovers and allows them to pivot. Based on these results, simulated RF ablation scars could terminate AF by two mechanisms: One, RF lesions increase the inexcitable region size for both meandering and stationary rotors, creating or widening a spatial excitable gap and allowing impingement and extermination of the rotor by an external wave. Two, the RF lesions increase the AF cycle length by increasing the path length of a rotor, in turn both regularizing the atrial activation sequence and decreasing global fractionation, promoting spontaneous termination.

Grant Funding Source: Supported by NIH grants HL96544 (ADM), HL083359 (SMN, WJR), and EB009380
Neonatal dexamethasone exposure causes the early maturation of cardiomyocytes in rats (862.4)
Authors: Gay Maresha , Zhang Lubo
Published: June 2021

Dexamethasone (Dex) is a synthetic glucocorticoid commonly administered to preterm infants for the maturation of the lung. However, it is also associated with adverse effects in increasing cardiovascular disease later in life. In the present study we investigated the involvement of Dex in phenotypical and functional changes in the developing heart. The hypothesis of this study is that neonatal Dex treatment will result in an early terminal differentiation of the heart marked by the transition of mononucleate cardiomyocytes to binucleate phenotype and reduced proliferation in cardiomyocytes. To test this hypothesis neonatal rats were treated with tapered doses of Dex on day 1 (0.5 µg/g), 2 (0.3 µg/g) and 3 (0.1 µg/g) by intraperitoneal injection. Control animals received either saline or no injections. Primary cardiomyocytes were isolated at day 4, 7, and 14. The results revealed that Dex‐treated animals tended to have higher the heart to body ratio compared to controls. Immunocytochemistry analysis revealed Ki‐67, a proliferation marker, was unchanged at day 4 then significantly decreased at day 7 and 14. A significant increase in binucleation of cardiomyocytes was noted in day 4 animals with no subsequent changes for later days. These findings suggest that Dex treatment caused a premature exit of the cell cycle and terminal differentiation of cardiomyocytes in the developing heart, which may lead to a reduction in cardiomyocyte number and ultimately compromise cardiac health later in life.
Akt/PI3K signaling sustains the chronic alcoholic effects on the heart (862.6)
Authors: Umoh Nsini , Jeffress Miara , Haddad Georges
Published: June 2021

Cardiovascular diseases are the leading causes of increased mortality and morbidity rates. Specifically, alcohol induced cardiomyopathy is a preventable dysfunction that is associated with premature deaths. High alcohol (HA) intake has been shown to negatively impact the heart function into having reduced contractility with dilated cardiomyopathy. However, low alcohol (LA) intake has been clinically shown to be beneficial. Prior studies in our lab have linked the Akt/PI3K pathway to cardiac dysfunction. In this study, we investigate the role of Akt activity (western blot) that contributes to changes in cardiac contraction and calcium mobilization (Ionoptix), cell survival (Cell Viability Assay), caspase activity (caspase 3/7 assay) and oxidative stress associated with chronic LA (0.02%) and HA (0.45%) consumption using Lieber‐DeCarli alcohol diet (4 months). Our results demonstrate that rats on a LA liquid diet experienced decreased caspase activity and increased calcium mobilization and cellular/sarcomere contraction. However, the above Akt‐mediated effects are diminished with rats on a HA diet. Our results also show that the inhibition of NFkB causes further aggravation of alcohol‐induced cardiomyopathy. Our data suggests that Akt signaling plays an important role in supporting the beneficial effects of LA which is diminished with HA exposure having detrimental effects on cardiac function.

Grant Funding Source: This work was supported in part by grants NIH/NIAAA/1R15AAA019816‐01A1, and 2G12 RR003048 RCMI/NCRR/
The effect of acute exercise on circulating serum retinol binding protein 4 in seniors with and without impaired glucose tolerance (863.4)
Authors: Young Laura , Marcus Robin , LaStayo Paul , Drummond Micah , Graham Timothy , Symons J. David
Published: June 2021

Retinol binding protein 4 (RBP4) is an adipokine that contributes to insulin resistance and impaired glucose tolerance. There are no published reports concerning the influence of acute exercise on circulating RBP4. We sought to characterize this response in (1) healthy, normoglycemic males (CON; 23±4 y, BMI 22.3±9.4), (2) seniors with normal fasting glucose and glucose tolerance (S‐NGT; 70±5 y, BMI 24.2±6.5), and (3) seniors with impaired glucose tolerance (S‐IGT; 73±8 y, BMI 26.4±5.3; n=7 per group). Glucose tolerance was based on a 75 g oral glucose tolerance test wherein 2 h glucose values (mg/dL) were: 97±21 (CON), 106±24 (S‐NGT), and 159±13 (S‐IGT; p<0.05 vs. CON and S‐NGT). 48 h after determining VO2max (ml/kg/min) in CON (56±10, p<0.05 vs. S‐NGT and S‐IGT), S‐NGT (33±8), and S‐IGT (29±8) subjects, participants completed 20‐min of cycling at 85% VO2max. RBP4 was assessed (ELISA) in venous samples obtained before exercise and 50‐min and 24 h post exercise. Pre‐exercise RBP4 concentrations (ug/mL) were greater (p<0.05) in S‐IGT (76±23) vs. S‐NGT (61±7) or CON (44±7) subjects. Values obtained at 50‐min and 24 h post exercise showed further increases after exercise (p<0.05) in the S‐IGT (99±23 and 77±25, respectively) vs. both S‐NGT (56±7 and 39±9) and CON subjects (40±7 and 47±21). These data indicate that in senior subjects with IGT, circulating RBP4 is elevated before exercise, with persistent elevation in recovery (50‐min and 24 h) from exercise. Ongoing studies will determine whether skeletal muscle RBP4 regulates insulin‐glucose homeostasis.
Cardiac mitochondrial disruption in response to hypothermia and rewarming (864.1)
Authors: Schaible Niccole , Han Young Soo , Arteaga Grace , Tveita Torkjel , Sieck Gary
Published: June 2021

Recovery from accidental hypothermia followed by rewarming (H/R) is often unsuccessful due to heart failure. Although the causes for H/R‐induced heart failure remain elusive, studies at the cellular level suggest alterations in both cytoplasmic and mitochondrial Ca2+ regulation. We hypothesize that H/R is associated with mitochondrial Ca2+ overload, which leads to: 1) excessive reactive oxygen species (ROS) generation, 2) mitochondrial membrane depolarization, and 3) mitochondrial cytochrome c release. To test this hypothesis, isolated cardiomyocytes from adult male Sprague‐Dawley rats were subjected to an in vitro H/R protocol (1 h cooling to 15oC held for 30 min followed by 1 h rewarming to 37oC). Cytoplasmic (CM‐H2DCFDA fluorescence) and mitochondrial (MitoSOX fluorescence) ROS generation increased during cooling and remained elevated throughout the H/R protocol. Mitochondrial membrane potential (TMRM fluorescence) depolarized during rewarming suggesting mitochondrial permeability transition pore opening. After rewarming, cytochrome c content (Western blot) in cytoplasmic to mitochondrial fractions increased indicating release. These results suggest that Ca2+ overload associated with H/R induces ROS generation leading to mitochondrial disruption.

Grant Funding Source: Supported by NIH Grant 5T32HL105355‐02 (GCS)
Mutation of Kvβ2 active site attenuates cardiac injury: role of repolarization reserve (864.2)
Authors: Chapalamadugu Kalyan , Panguluri Siva , Tur Jared , Tipparaju Srinivas
Published: June 2021

Cardiac Kvβ2 subunit alters gating and expression of potassium channels. However, the physiological significance remains unknown. Therefore, we evaluated the role of Kvβ2 in cardiac injury. We used 14 week male wild type (Wt) or mutant Kvβ2:PM (active site mutant; Y90F) mice, infused with saline or isoproterenol (Iso, 30mg/kg; bw/day) for 14 days. Iso treatment resulted in cardiac hypertrophy in both mice as noted by histomorphological analysis. Echocardiographic assessment revealed significant decrease (p <0.05) in EF% and FS% upon Iso stress in both Wt (71.1±1.7 vs. 55.9±1.7; 40±1.4 vs. 29.0±1.2) and PM (66.3±2.7 vs. 57 ± 2.8; 36.2 ± 2.0 vs. 30.0 ± 1.9), mice. Ex vivo recording of action potential durations (APD) showed repolarization depletion in Wt saline vs. Iso infused mouse hearts at APD60 (11.9±0.7 vs. 31.6±1.1) and APD80, 43.1 ± 1.9 vs. 51.9 ± 1.9), respectively. However, in PM mice Iso did not induce APD prolongation (APD60, 19.7±1.1 vs. 24.2±1.8 or APD80, 49.8±1.8 vs. 49.3±2.0) suggesting protection. Similarly, ECG analysis showed QTc interval (59.7±2.4 vs. 68.6±2.0, ms) change in Wt saline vs. Wt Iso groups, which is not observed in PM mice (70±1.7 vs. 68.9±0.6). Expression of key ion channels (Kv4.2, Kv1.4 and Kv1.5) was unaltered in PM mice, but significantly depleted in Wt with Iso treatment. Together, Kvβ2 active site mutant restores repolarization reserves and offers protection against cardiac injury.

Grant Funding Source: HL102171
Cellular and molecular characterization of type II diabetic bladder dysfunction (865.12)
Authors: Klee Nicole , Moreland Robert
Published: June 2021

In a rat model of Type II diabetes (T2D), cellular and molecular alterations in muscarinic and purinergic receptor stimulation and associated downstream signaling molecules in bladder smooth muscle was examined over time. T2D was induced in Sprague Dawley rats using the combination of a high fat diet and low dose streptozotocin (HFD/STZ). A 40% HFD was initiated in 14 week old rats; after one month, rats were administered two low doses of STZ (30 mg/kg) one week apart. This model results in obesity, hyperglycemia, insulin resistance, and has been shown to induce diabetic bladder dysfunction (DBD); although this model has rarely been used to characterize DBD or determine mechanistic changes in the regulation of the bladder. DBD is a common complication of T2D, a non‐life threatening complication of diabetes that has a significant negative impact on quality of life. Bladder smooth muscle was isolated and isometric force was recorded in response to the cumulative and non‐cumulative addition of carbachol, bethanechol, ATP, and KCl in both control and HFD/STZ rats. Urodynamic profile, body weight, and glucose measurements were recorded over time pre and post STZ injections. The results of these studies can then be used to determine the efficacy of current and novel therapeutic interventions aimed specifically at the site of dysfunction induced by T2D as well as potential sites of dysfunction affected by aging.

Grant Funding Source: NIH DK 85734
Physiological parameters and signaling proteins in compensation and decompensation of rat urinary bladder activities (865.8)
Authors: Choi Bo‐Hwa , Kang Ju‐Hee , Lee Tack , Suh Chang Kook , Han Su‐Mi , Park Chang‐Shin
Published: June 2021

We conducted this study to determine whether the degree of detrusor contractility is associated with the compensation or decompensation of bladder function depending on the residual volume (RV) during the first two weeks after the onset of partial bladder outlet obstruction (BOO ). Moreover, we also examined whether the degree of the phosphorylation and expression of signaling proteins [AMP‐activated kinase (AMPK), extracellular signal舉regulated protein kinases 1 and 2 (ERK1/2) and protein kinase C (PKC)] is associated with the prevalence of compensation or decompensation of bladder function. Twenty‐seven female Sprague‐Dawley (SD) rats were randomly assigned to either the sham‐operated group (n=7) or the group with partial bladder outlet obstruction (BOO ) (n=20). We then measured cystometric parameters from three reproducible micturition cycles and averaged the results for a comparison between the two groups. Based on a cut‐off value of a mean RV% of 25%, we subdivided our experimental animals into two subgroups: the subgroup with bladder compensation (mean RV%, <25%) and the subgroup with bladder decompensation (mean RV%, >25%). Our results indicated that the degree of detrusor overactivity (DO ) was associated with the compensation or decompensation of bladder function depending on the RV during the first two weeks after the onset of BOO in an animal experimental model of partial BOO . Moreover, we also demonstrate that AMPK and ERK1/2 are involved in the compensation or decompensation of bladder function. Furthermore, our results suggest that PKC is not involved in two‐phase bladder contraction. Alterations in the activities of signaling proteins, such as AMPK and ERK1/2 may prove to be helpful in the treatment of patients with voiding difficulty.
Response and mast cell activation to tolterodine in protamine sulfate‐ and lipopolysaccaride‐treated rat urinary bladder (865.9)
Authors: Choi Bo‐Hwa , Kang Ju‐Hee , Lee Tack , Suh Chang Kook , Han Su‐Mi , Park Chang‐Shin
Published: June 2021

We aimed to use an animal model of chronic interstitial cystitis in rats to investigate the histology and function of the bladder, with a special focus on mast cell degranulation and response of detrusor overactivity to tolterodine. A total of 18 female Sprague‐Dawley rats were used in this study. In 12 rats, lipopolysaccharide was intravesically instilled after the induction of interstitial cystitis by protamine sulfate; 6 rats were subjected to sham instillations. After 1 month, cystometry was performed in all rats with simultaneous registrations of intravesical and intraabdominal pressures. The effects of tolterodine on bladder function were tested in half of the interstitial cystitis animals. All rats in the interstitial cystitis group showed detrusor overactivity during the filling phase and no significant changes in frequency or pressure compared with that after tolterodine injection. Histological examination revealed a significant increase in total numbers of infiltrated mast cells in the interstitial cystitis rats compared with the sham rats (p<0.05). Eighty percent of the interstitial cystitis rats showed degranulated mast cells, whereas the sham rats did not. Urinary bladder inflammation similar to that in human interstitial cystitis in terms of degranulated mast cells and bladder function was induced in Sprague‐Dawley rats. Our animal model should provide major insights into pathophysiologic mechanisms in the bladder, such as the mechanisms underlying the ineffectiveness of anticholinergics in patients with overlapping interstitial cystitis and overactive bladder.
Smooth muscle‐targeted overexpression of peroxisome proliferator‐activated receptor gamma disrupts vascular wall structure and function (866.4)
Authors: Sutliff Roy , Kleinhenz Jennifer , Murphy Tamara , Pokutta‐Paskaleva Anastassia , Gleason Rudolph , Lyle Alicia , Taylor W. , Yang Qinglin , Hart C.
Published: June 2021

Objective: To explore the role of PPARγ in vascular regulation, smooth muscle VP16‐PPARγ‐overexpressing mice were subjected to studies of systemic vascular function, biomechanical testing, histological analysis, and molecular characterization. Methods: Mice with a flox‐controlled VP16‐PPARγ gene were crossed with mice containing a tamoxifen‐inducible Cre recombinase driven by a smooth muscle myosin heavy chain promoter. All mice received tamoxifen injections at 6 weeks of age. Experiments were performed at 10‐16 weeks of age. Results: Compared to littermate controls, transgenic mice displayed: 1) aortic dilation (by histology, echocardiography, and microCT), 2) increased aortic compliance, 3) reduced blood pressure, 4) 80% reduction in aortic ring contractile forces in response to KCl or phenylephrine, 5) lipid accumulation in the aortic media (Oil Red O staining), and 6) reduction in aortic contractile markers and extra‐cellular matrix components with increased expression of adipose markers and selected mitochondrial proteins (by qPCR).Conclusion: These results demonstrate that targeted overexpression of activated PPARγ in smooth muscle disrupts vascular wall structure and function in part by driving medial smooth muscle towards an adipocyte phenotype.

Grant Funding Source: Supported by grants from NIH (R01 HL102167) and VA (Merit Review Award 1I01BX001910)
miR‐9 mediates Nox4‐dependent regulation of myocardin and serum response factor in human smooth muscle cells (866.6)
Authors: Schickling Brandon , Devor Eric , Miller Francis
Published: June 2021

Myocardin is a transcriptional coactivator of serum response factor (SRF) responsible for vascular smooth muscle cell (SMC) differentiation. Changes in Nox4 NADPH oxidase expression are associated with changes in myocardin and SRF levels and have been implicated in the phenotypic switching of SMCs. However, the mechanisms underlying this molecular switch in response to vascular injury are poorly understood. We examined the role of microRNAs (miR) in the expression of Nox4, SRF and myocardin in human SMCs. Analysis of the 3’UTR of Nox4 revealed a single binding site for miR‐25, and two putative miR‐9 binding sites in the 5’ region of the 3’UTR. Treatment of human SMCs with a miR‐25 or a miR‐9 mimic suppressed mRNA expression of Nox4. We created a Nox4 3’UTR luciferase construct and found that the miR‐25 mimic decreased relative light units (RLU) by 40% which was prevented with mutation of the single miR‐25 site. The miR‐9 mimic decreased Nox4 3’UTR RLU by 60% and mutation of the two miR‐9 sites had a synergistic effect in restoring RLU to wild type levels. In addition, both miR‐9 and miR‐25 mimics decreased mRNA expression of myocardin and SRF. Finally, the miR‐25 mimic increased levels of miR‐9, but the miR‐9 mimic had no effect on miR‐25 levels. We conclude that miR‐25‐dependent regulation of Nox4 regulates SMC differentiation in part via miR‐9 dependent regulation of SRF and myocardin.
Nuclear factor kappa B inhibition promotes closure of the rat ductus arteriosus (866.8)
Authors: Kajimura Ichige , Akaike Toru , Yokoyama Utako , Ishikawa Yoshihiro , Minamisawa Susumu
Published: June 2021

Background: The ductus arteriosus(DA) is necessary for fetuses and closes after birth. Patent DA(PDA) often causes serious conditions in premature babies. On the other hand, some patients with congenital heart disease need PDA for living. Prostaglandin E2 (PGE2) is involved in both vasodilation and structural remodeling such as intimal thickening through cAMP pathway. However, DA remodeling is not completely prevented by inhibition of cAMP pathway, suggesting that there is a cAMP‐independent PGE2 signal pathway. Methods and Results: We found the nuclear factor kappa b (NFkB) pathway was activated more in EP4‐stimulated SMCs than in Forskolin‐treated SMCs, which was shown by differential DNA microarray analyses. The NFkB inhibitor IMD‐0354 reduced HA production in EP4‐ stimulated SMCs. DA explants from 19th fetuses were cultured for 48 hours in the presence or absence of IMD‐ 0354. IMD‐0354 treatment facilitated closure of DA explants. When IMD‐ 0354 was injected into 21st rat fetuses, the DA significantly contracted. Furthermore, IMD‐ 0354 constricted the DA dissected from 21st rat fetuses in a dose‐dependent manner. Conclusion: The present data demonstrated that NFkB inhibition facilitated DA closure. NFkB may play a role in a cAMP‐independent PGE2 signal pathway of the rat DA.
Less intimal thickening and impaired elastic fiber formation in the chicken ductus arteriosus (867.14)
Authors: Omori Eriko , Akike Toru , Kajimura Ichige , Minamisawa Susumu , Goda Nobuhito
Published: June 2021

Object: We investigate the vascular remodeling of the chick ductus arteriosus in order to investigate the new mechanism of its closure.. Experiments and Results: We isolated the DAs from chicken embryos and neonates. Histological analysis with Elastica van‐Gieson staining demonstrated that the sparse and fragmented elastic fibers in the medium from embryo day 16, and impairment became more apparent with development. Intimal cushion formation was not clearly observed even after external hatching. Real‐time PCR analysis revealed that the expression levels of elastin and lysyl oxidase mRNAs were significantly lower in the DA than in the aorta. Genes related with the PGE2‐EP4 signal pathway were not predominantly expressed in the chicken DA.

Conclusion: Internal cushion formation was less prominent in the chicken DA than in the mammalian DA. On the other hand, elastic fiber formation was severely impaired in the chicken DA, which was unlikely due to the PGE2‐EP4 signal pathway.