bluuebunny/crossref_metadata_2025 huggingface dataset:
Contractions of rat vas deferens are mediated by α1A‐ and α1D‐adrenoceptors
Authors: Docherty James R
Published: June 2021

The subtypes of α1‐adrenoceptor mediating contractions of rat vas deferens to exogenous noradrenaline have been examined employing the α1A‐adrenoceptor antagonist RS100329 and α1D‐adrenoceptor antagonist BMY7378. In the presence of cocaine (3 μM) to block the noradrenaline transporter, the potency of noradrenaline at producing contractions was significantly increased in epididymal but not prostatic portions, and the effect was mainly in terms of phasic contractions. RS100329 produced approximately parallel shifts in the potency of noradrenaline at producing tonic contractions in epididymal portions in the absence of cocaine, but produced non‐parallel shifts in the presence of cocaine. In epididymal portions, BMY7378 showed high potency against phasic contractions to noradrenaline in the presence but not the absence of cocaine. It is concluded that the predominant tonic contraction to noradrenaline in epididymal portions of rat vas deferens is mediated by α1A‐adrenoceptors, but α1D‐adrenoceptors are additionally involved in phasic contractions. These phasic contractions are amplified by cocaine, suggesting that blockade of the noradrenaline transporter makes α1D‐adrenoceptors available to exogenous noradrenaline.
Desferrioxamine Attenuates Doxorubicin‐Induced Acute Cardiotoxicity Through TGF‐β/Smad P53 pathway
Authors: Al-Shabanah Othman A
Published: June 2021

Doxorubicin (DOX) is an anthracycline antibiotic with broad spectrum antitumour activity against different types of human malignancies. Unfortunately, the optimal clinical usefulness of DOX is usually associated with both acute and chronic cardiotoxicity. Interaction of DOX with iron with the consequent generation of reactive oxygen species (ROS) is a major player in DOX‐induced cardiomyopathy. Accordingly, this study has been initiated to investigate, on gene expression level, the preventive effect of the iron chelator, desferrioxamine (DFX), against DOX‐induced cardiotoxicity in rat. Male Wistar albino rats were divided into four groups. Group 1 was injected intra‐peritoneally (I.P.) with normal saline and served as control. Group 2 (DOX group) was injected with a single dose of DOX (15 mg/kg, I.P.). Rats in group 3 were given a single dose of DXF (250 mg/kg, I.P.). Animals in group 4 were injected with DFX (250 mg/kg) 30 min prior to a single dose of DOX (15 mg/kg, I.P.). Twenty four hours later, animals were sacrificed and hearts were isolated for studying mRNA expression of genes involved in TGF‐β/Smad P53 pathway using Real‐Time PCR. A single dose of DOX significantly increased mRNA expression of TGF‐β, Smad2, Smad4, CDKN2A and P53 and significantly decreased the expression of Samd7 and Mdm2 gene expression levels. Administration of DXF prior to DOX‐resulted in complete reversal of DOX‐induced alteration in the studied genes expression to normal levels. The protective effect of DXF was showed in restoring the normal levels of the cardiac enzymes and isoenzymes after treatment. Data from this study study suggest that: (1) DOX induces its acute cardiotoxicity secondary to increasing the expression of TGF‐β/Smad pathway. (2) DOX increases apoptosis through up‐regulation of CDKN2A and P53 and down‐regulation of Mdm2 gene expression. (3) The preventive effect of DXF against DOX‐induced cardiotoxicity is mediated via the TGF‐β1/Smad pathway.
Increased Phosphodiesterase 4B (PDE4B) and decreased cellular cAMP regulate LPS‐inducible TNF‐α in glucose‐primed monocytes
Authors: Chambers Elaina , Gobejishvili Leila , Ghare Smita , McClain Craig , Barve Shirish
Published: June 2021

Diabetics experience gut permeability & subsequent elevated levels of serum endotoxin. Therefore, lipopolysaccharide (LPS)‐ inducible cytokines, specifically TNFα, are highly relevant in this disease state & are causal in the development of diabetic complications. Our earlier work demonstrated that cAMP & monocyte/macrophage‐specific PDE4B play a key role in LPS‐inducible TNFα expression. The current data demonstrates that human monocytes exposed to high glucose (HG) exhibit a “primed phenotype”; wherein they express elevated levels of TNFα following LPS stimulation. The data obtained shows for the first time that HG exposure up‐regulates PDE4B expression & activity leading to a consequent increase in TNFα following LPS stimulation. Our data clearly demonstrates that HG mediated priming is the direct result of epigenetic changes involving increased histone acetylation & enhanced transcription factor:DNA binding. These data strongly implicate that PDE4B plays a significant mechanistic role in the development of diabetic inflammation. Moreover, these data also suggest that PDE4B could serve as a therapeutic target in the treatment of Diabetes.
KS‐370G ameliorates renal fibrosis, oxidative stress and inflammation induced by unilateral ureteral obstruction
Authors: Chuang Sung-Ting , Su Ming-Jai
Published: June 2021

Renal fibrosis is a common morphological feature of progressive chronic renal diseases including unilateral ureteral obstruction (UUO). In this study, we evaluated the role of KS‐370G, a caffeamide derivative, in the kidney fibrosis induced by UUO. Adult ICR mice were subjected to UUO and then administered with either KS‐370G (10mg/kg/day, gavage) or vehicle from 1day after UUO. UUO was maintained for 14 days. KS‐370G administration daily for 13 days significantly decreased the UUO‐induced kidney fibrotic changes including collagen deposition and fibronectin protein expression. KS‐370G also attenuated renal weight loss which result from UUO. KS‐370G reduced iNOS expression that contribute to renal inflammation. KS‐370G attenuated the increases of oxidative stress by the prevention of UUO‐induced decreases of manganese superoxide dismutase (MnSOD). Furthermore, UUO‐induced AMP‐activated protein kinase (AMPK), p42/p44 mitogen‐activated protein kinase (MAPK), Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (stat3) phosphorylation was markedly blocked by KS‐370G. In conclusion, KS‐370G prevented renal fibrosis following UUO by the inhibition of inflammation, oxidative stress and JAK2/Stat3 signaling pathway. The source of research support: National Science Council.
Resveratrol Reduces Cisplatin Mediated Oxidative Modifications of Proteins and Oxidative Stress Enzyme Activity in Renal Rat Tissue
Authors: Valentovic Monica A , Mills Amber , Lamyaithong Andre Benja , Ball John G , Wright Michael S , Van Meter Stephanie
Published: June 2021

Nephrotoxicity is an adverse effect associated with the cancer chemotherapeutic agent cisplatin. (RES) is a phytochemical found in grapes which possesses anticancer and antioxidant properties. This study investigated RES attenuation of cisplatin renal cytotoxicity and reduction in oxidative stress mediated by cisplatin. Kidneys were isolated from male Fischer 344 rats and renal slices were pre‐incubated with 30 ul ethanol (VEH) or 30 ug/ml resveratrol (RES, final concentration) for 30 min. Tissue were immediately incubated for a maximum of 120 min with 0–75 ug/mL cisplatin. Loss of membrane integrity was measured as lactate dehydrogenase (LDH) leakage. Cisplatin associated oxidative stress was evaluated by measuring total, Cu/Zn and Mn Superoxide dismutase (SOD), glutathione peroxidase and catalase enzyme activity in renal cortical slices. LDH leakage required a 120 min exposure to cisplatin. Cisplatin alter GSH peroxidase and SOD within 60 min when compared to VEH group. RES reduced oxidative stress by maintaining GSH peroxidase and SOD enzyme activity. An increase in protein carbonyls as detected by Oxyblot, was increased by cisplatin and totally prevented by RES. Our findings showed RES diminished cisplatin renal toxicity and early changes in oxidative stress. (Supported by NIH Grants INBRE 3P20RR016477‐09S4 and 5P20RR016477 to the West Virginia IDeA Network for Biomedical Research Excellence).
Sirtuin 1 enzyme activity and autophagy proteins are increased in the kidney during murine sepsis
Authors: Holthoff Joseph Hunter , Pathak Elina , Wang Zhen , Basnakian Alexei G. , Mayeux Philip R.
Published: June 2021

Sepsis affects over 750,000 patients annually and the development of acute kidney injury (AKI) during sepsis nearly doubles the mortality rate to 75%. We have recently published that the dietary antioxidant, resveratrol (RSV), can improve kidney function and survival in the cecal ligation and puncture (CLP) model of sepsis in the mouse. Since RSV has been reported to lead to the activation of sirtuin1 (SIRT1), which may be protective, we investigated the relationship between SIRT1 and acetylation of p53. SIRT1 enzyme activity was increased at 10 and 18h post CLP as were the autophagy‐related proteins Atg5 and LC3. Moreover, acetylated p53 levels were significantly increased at 6h and 10h, but not 18h. By immunostaining, the number of TUNEL‐positive cells was increased by sepsis and acetylated p53 was localized mainly to the cytoplasm of proximal tubular epithelial cells in the cortex and cortico‐medullary junction. While RSV (10 mg/kg, ip) did not increase SIRT1 activity, RSV did block the increase in acetylated p53. These data suggest that CLP causes both autophagy and apoptosis in the kidney and that RSV may dampen the apoptotic response. Supported by F30 DK085705 (JHH) and R01 DK075991 (PRM).
Ubiquitination of Neuronal Nitric Oxide Synthase in the P450 Oxygenase and Calmodulin‐binding Domain
Authors: Clapp Kelly M , Peng Hwei-Ming , Jenkins Gary J , Ford Michael , Morishima Yoshihiro , Lau Miranda , Osawa Yoichi
Published: June 2021

Nitric oxide synthase (NOS), a cytochrome P450‐like hemoprotein enzyme, catalyzes the synthesis of nitric oxide, a critical signaling molecule in a variety of physiological processes. Our lab has discovered that certain drugs suicide‐inactivate neuronal NOS (nNOS) and lead to the preferential ubiquitination of the inactivated nNOS by an Hsp70‐ and CHIP (C‐terminus of Hsp70‐interacting protein)‐dependent process. Conversely, calmodulin and certain reversible inhibitors stabilize the nNOS. We wish to understand the process by which altered nNOS is recognized and ubiquitinated. We developed an in vitro ubiquitination system that contains purified E1, E2 (UbcH5a), and CHIP that recapitulate the cells’ ability to selectively recognize and ubiquitinate the altered forms of nNOS. For example, the slowly reversible inhibitor, NG‐nitro‐L‐arginine, but not the D‐isomer, protects nNOS from ubiquitination. We identified 12 ubiquitination sites through LC‐MS/ MS analysis of the tryptic peptides obtained from the ubiquitinated nNOS. Nine sites are located in the oxygenase domain, two in the calmodulin‐binding domain, and one in the reductase domain. Mutational analyses of residues 739 and 743 in the calmodulin‐binding domain are underway. The sites of ubiquitination are consistent with the importance of the Oxy‐CaM domain in regulating stability of nNOS. Supported in part by NIH grant GM077430.
Cardiomyocyte Geometry and Stretch Effects on Longitudinal Conduction Velocity
Authors: Pfeiffer Emily , Stowe Jennifer , Tan Justin , McCulloch Andrew
Published: June 2021

The effects of cardiac myocyte ultrastructure geometry and deformation on conduction properties are not well understood, and in fact, reports of conduction acceleration and deceleration under cardiac loading are in conflict. Changes in cell capacitance with longitudinal deformation have been reported. In addition to geometrical changes during routine cardiac loading, cardiomyocytes have been observed to adopt changing aspect ratios during maturation and disease. Using a micropatterned elastic substrate and custom biaxial stretch devices, we have measured a longitudinal conduction velocity slowing near 30% with anisotropic strain, from 2.64 ± 0.45 cm/s before loading to 1.96 ± 0.46 cm/s under 14% strain parallel to the cell longitudinal axis and 3.6% perpendicular to it, recovering to 2.31 ± 0.32 cm/s after unloading, for n=5. These measurements were performed on cells cultured on micropatterned structures 5 μm deep, 10 μm wide and 10 μm apart, and will be compared with similar studies on cells controlled to adopt different cross‐sectional dimensions.
Three‐month omega‐3 fatty acid supplementation does not improve cardiac diastolic function in healthy older adults
Authors: Gao Zhaohui , Feehan Robert P. , Sinoway Lawrence I. , Monahan Kevin D.
Published: June 2021

Cardiac diastolic function declines with age. Animal studies suggest that the magnitude of the decline can be attenuated by omega‐3 fatty acid (n‐3) supplementation. Whether n‐3 supplementation improves cardiac diastolic function in healthy older humans is unknown. The aim of this study was to determine whether 3 months of n‐3 supplementation (1.9 and 1.5 g EPA and DHA/day) improved echocardiographic indices of cardiac diastolic function (including traditional Doppler, tissue Doppler imaging, and 2D speckle tracking imaging) in healthy older adults. Echocardiographic examinations were performed on 11 healthy older volunteers (average age: 66±3 yrs, 5 females) before and after n‐3 supplementation. Diastolic functional parameters, including mitral inflow velocities (E and A), myocardial diastolic velocity (Em and Am) and longitudinal early and late diastolic strain rates (E‐sr and A‐sr), were measured at rest. n‐3 supplementation had no effect on blood pressure and heart rate. Moreover, measures of diastolic function were unchanged (all P>0.05). These data suggest that daily ingestion of high doses of n‐ 3 supplements for 3 months is not effective in improving measures of cardiac diastolic function in healthy older adults.

Supported by P01 HL096570 (LIS); R01 HL070222 (LIS); HL092309 (KDM); AG024420 (KDM); UL1 RR033184 (LIS).
Speckle Tracking Imaging in Evaluation of Left Ventricular Mechanics in a Swine Model of Atrioventricular Block during Cardiac Pacing: A Comparison Study with Conventional Right Ventricular Apical Pacing Therapy
Authors: Zhou Wei , Benharash Peyman , Yamakawa Kentaro , Mahajan Aman
Published: June 2021

The purpose of the study was to evaluate the LV mechanics during various cardiac pacing using 2D speckle tracking echocardiography (STE). In eight open‐chest pigs, cardiac pacing was performed at different sites namely right ventricular (RV) apex, RV outflow tract (RVOT) and left ventricular (LV) lateral as well as biventricular (Bi‐V: RV apex + LV lateral) and triangle (Tri‐V: RV apex + RVOT +LV lateral). Changes in LV synchrony and mechanics (longitudinal shortening and twist) were assessed by STE before and after atrioventricular (A‐V) block induced by chemical ablation. During RV apical and LV lateral pacing, a more dyssynchronous LV contraction was observed together with impairment in LV longitudinal shortening and in LV twist before and after A‐V block as compared to RVOT pacing. RVOT and Tri‐V pacing similarly improved the LV longitudinal shortening and twist and decreased the LV dissynchrony as compared to RV apical and bi‐V pacing after A‐V block. The activation pattern of the LV during RVOT and Tri‐V pacing was more similar to the activation pattern of normal heart than that during RV apical and Bi‐V pacing. LV mechanical dissynchrony, LV strain and LV torsion during different cardiac pacing can be assessed by speckle tracking imaging. Our data suggest that RVOT and Tri‐V pacing may serve as a better cardiac resynchronization therapy.
Contribution of Voltage‐Dependent Potassium & Calcium Channels to Coronary Pressure‐Flow Autoregulation
Authors: Berwick Zachary C , Moberly Steven P , Kohr Meredith C , Morrical Ethan B , Kurian Michelle M , Goodwill Adam G , Tune Johnathan D
Published: June 2021

The goal of this investigation was to test the hypothesis that voltage‐sensitive potassium (KV) and calcium (CaV1.2) channels contribute to coronary pressure‐flow autoregulation in vivo. This hypothesis was tested in open‐chest, anaesthetized Ossabaw swine during step changes in coronary perfusion pressure (CPP) from 140‐40 mmHg before/during inhibition of KV channels with 4AP (0.3 mM, ic) or CaV1.2 channels with diltiazem (10 μg/min, ic). 4AP significantly decreased vasodilatory responses to H2O2 (0.3 – 10 μg/min) and blood flow at CPPs = 140‐60 mmHg. This decrease in coronary flow was associated with diminished ventricular contractile function (dP/dT) and myocardial oxygen consumption. However, the overall sensitivity to changes in CPP from 120 to 60 mmHg (i.e. autoregulatory gain; Gc) was unaltered by 4‐AP administration (Gc = 0.35 ± 0.11 control vs. 0.39 ± 0.07 4‐AP). In contrast, diltiazem markedly increased coronary blood flow at CPPs > 80 mmHg and decreased coronary autoregulatory gain (Gc = −0.21 ± 0.08) relative to control (P < 0.01), with no effect on contractile function or oxygen consumption. Taken together, our findings demonstrate that: 1) KV channels tonically contribute to the control of vascular resistance over a wide range of CPPs; 2) CaV1.2 channels play a critical role in coronary pressure‐flow autoregulation.
Effect of Aortic Stenosis on Coronary Perfusion Studied with Wave Intensity Analysis
Authors: Siebes Maria , Rolandi M. Cristina , Yong Ze-Yie , Spaan Jos A.E. , Baan Jan
Published: June 2021

Background

An aortic stenosis (AS) can cause angina despite normal coronary arteries. We used wave intensity (WI) to elucidate mechanisms underlying adaptation of the coronary microcirculation to AS. WI distinguishes between contributions of left ventricular (forward, FW) and microcirculatory (backward, BW) origin and between compression waves during contraction (BCW and FCW resp.) and expansion waves (FEW and BEW resp.) during relaxation.

Methods

During cardiac catheterization we simultaneously measured aortic pressure, coronary pressure (Pd) and flow velocity (U) at rest and at maximal hyperemia in 12 patients without and in 10 patients with severe AS. WI was calculated as (dPd/dt)(dU/dt) and the energy of separated BW and FW contributions was derived.

Results

U at rest was about 20 cm/s in both groups. Compared to no AS, Pd was 17% lower (p< 0.01) with AS, while microvascular resistance (MR=Pd/U) was 30% lower (p<0.05). Hyperemic Pd and MR did not differ, while U was 20% lower (p<0.05) in AS. Compression wave energy remained similar. However, FEW in AS was 43% lower at rest and 66% lower at hyperemia (p<0.05). The BEW in AS was 50% higher at rest (p<0.05), correlating with the lower MR (p<0.05, r=0.45), and not different at hyperemia.

Conclusion

AS primarily affects the expansion waves and hence hampers coronary perfusion during cardiac relaxation.

Support: FP7‐ICT 2007224495 (euHeart)
Cardiac Pressure Overload Elicits Coronary Circulatory Dysfunction in Mice
Authors: Lu Guangrong , Hein Travis W. , Xu Xin , Kuo Lih
Published: June 2021

Chronic pressure‐overload evokes cardiac hypertrophy and diminished flow reserve, but its effects on coronary vasomotor function remain unclear. Herein, we evaluated hemodynamic changes in vivo by color Doppler echocardiography, and studied coronary vasomotor function in vitro in a pressure‐overload mouse model generated by transaortic constriction (TAC). The constriction of aorta (~45%) for 3–5 wks caused an increase in heart‐body weight ratio by 60±15% and right coronary blood flow by 120±27%, but a reduction in left ventricular fractional shortening by 50±6%. The coronary septal arterioles showed significant wall thickening and exhibited vasoconstriction to acetylcholine compared to vasodilation in sham control mice. Coronary arterioles from TAC mice also showed reduced dilation to adenosine and enhanced constriction to endothelin‐1. A higher concentration of Rho‐kinase (ROCK) inhibitor H‐1152 was required to reverse ET‐1‐induced vasoconstriction. Both ROCK‐1 and ‐2 isoform expressions in coronary arterioles were significantly upregulated in TAC mice. We conclude that TAC for 3–5 wks induces coronary vasomotor dysfunction and wall thickening possibly due to endothelial impairment and ROCK upregulation, which diminish the vasodilation and accentuate the vasoconstriction and thus may explain the compromised flow reserve in response to stimulation or myocardial activation.
Perioperative Administration of Statins has Cardioprotective Effects in Streptozotocin‐induced Diabetic Rats
Authors: Crespo Maria J. , Cruz Nildris , Quidgley Jose , Creagh Osmar , Torres Hector , Rivera Karines , Hernandez Cesar
Published: June 2021

Cardiovascular (CV) function improves postoperative outcomes for patients receiving statins in the perioperative period. The optimal time for administering statins to obtain cardioprotective effects during this period in diabetes is unknown. Using echocardiography, we compared the effects of 24‐hour vs. one‐week administration of simvastatin (SV, 10 mg/kg/d); pravastatin (PV, 10 mg/kg/d) and atorvastatin (AV, 10 mg/kg/d) on the CV status of STZ‐induced diabetic rats. Untreated diabetic rats were used as controls. 24‐hour treatment increased ejection fraction (AV: 57.67±10.47%; PV: 51±8.44%; SV 65±9.5%), when compared to non‐treated diabetic rats (44±0.1%, N=4, P<0.05). Cardiac output index (COI, ml/min × 100 gBW) (AV: 88.55±29.54; PV: 81.58±29.31; SV: 90.39±18.86), and stroke volume (mL) (AV: 0.63±0.23; PV: 0.58±0.17; SV: 0.60±0.16) were also improved, when compared to non‐treated diabetic rats (COI: 52±15.76; Stroke Volume: 0.31±0.03 mL; N=4, P<0.05). After one‐week administration of AV and SV, cardiac parameters were similar to those observed after 24 hours. In the PV group, by contrast, the beneficial effects were not maintained after one‐week. These results indicate that cardioprotective effects of statins appear as early as 24 hours after administration. This study has strong clinical implications for diabetic patients who are subjected to surgical procedures. MBRS‐RISE Grant R25‐GM061838
Diabetes Abolishes the Cardioprotective Effect of Estrogen on Systolic Cardiac Function
Authors: Santiago Nivia , Melo Marcos B , Chappell Mark , Varagic Jasmina , Santos Robson AS , Campagnole-Santos Maria J
Published: June 2021

The present study evaluated the role of female sex hormones on the adverse effects of diabetes in cardiac function. Adult female Sprague‐Dawley rats (10 weeks‐old) were submitted to ovariectomy. One week later, diabetes was induced with a single dose of streptozotocin (65 mg/kg, i.p) without insulin replacement. After 4 weeks cardiac function was evaluated by echocardiography (Vevo 2100, VisualSonics, Toronto, Ontario, Canada) in intact controls (CTL); diabetics (STZ); ovariectomized (OVX); and OVX with diabetes (OVX‐STZ). Diabetes and OVX alone or in combination did not affect systolic blood pressure. While OVX animals exhibited a higher LV weight: tibia length ratio (LVW/TL; 18%; vs CTL; p<0.05), LVW/TL was particularly reduced in the diabetic animals (STZ: 14%; OVX‐STZ: 11%; vs CTL; p<0.05). However, the relative wall thickness was decreased in OVX and diabetic animals (STZ: 21%; OVX: 18%; OVX‐STZ: 21%; vs CTL; p<0.05). The ejection fraction (EF) and fractional shortening (FS) were decreased in OVX, respectively (65% ± 1% vs 72 % ± 2% in CTL; 37± 1% vs 43 ± 2% in CTL). The systolic function was markedly reduced by the same magnitude in both STZ (EF: 66± 1%; FS: 38 % ± 1%) and OVX‐STZ (EF: 65± 2%; FS: 37± 1%) relative to CTL. These data indicate that diabetes removes the cardioprotective effect of estrogen on cardiac function independently of alterations in blood pressure.
Estrogen Protects Female Akita Mice Against Type I Diabetes and Parasympathetic Dysfunction
Authors: Zhang Yali , Georgescu Serban Paul , Welzig Charles Michael , Naggar Jack , Aronovitz Mark , Park Ho-Jin , Galper Jonas Bernard
Published: June 2021

Male Akita mice with a mutation in the Ins2 gene develop hyperglycemia, hypoinsulinemia and secondary effects of diabetes (DM). Female Akitas develop mild hyperglycemia after puberty. To determine whether estrogen might protect against the development of hyperglycemia and secondary effects of DM, ovariectomized (Ovx) female Aktia mice implanted with placebo (P) or 17β‐estradiol (E2) pellets were compared with sham‐operated female Akita mice (Sham) or wild type (WT) females. OVx+P resulted in a time dependent increase of glucose levels: 300 to 600 mg/dl over 60 days with increasing polydipsia, glycosuria, proteinuria and HbA1c. Ovx+E2 mice did not develop secondary effects of DM and glucose levels decreased to those seen in WT females. Using implantable EKG transmitters we demonstrated the development of a diabetic autonomic neuropathy in Ovx+P mice measured by a marked attenuation in the magnitude of the heart rate response to carbamylcholine and a decrease in the duration of bradycardia: 4±1 min, ±SEM, n=10 in Ovx mice vs 12±2 min, n=10 p<0.01 in Sham, which was reversed in E2 treated mice. Tamoxifen, a specific activator of the estrogen‐receptor α partially reversed Ovx induced hyperglycemia, HbA1c and parasympathetic dysfunction. Thus estrogen may protect female Aktia mice from development of hyperglycemia and secondary effects of DM. This model might be useful in future studies of gender specific mechanisms in DM.
The role of enhanced cardiac fibroblast glycosylation products on collagen synthesis
Authors: Ramirez-Sanchez Israel , Aguilar Hugo , Maya-Ramos Lisandro , Villarreal Francisco
Published: June 2021

A prominent effect of diabetes on the heart is the development of cardiac fibrosis. High blood glucose levels are known to alter the enzymatic glycosylation of molecules. Enhanced protein (i.e. amino acid) glycosylation levels are known to alter the function of proteins such as transcription factors by interfering in many cases with their regulatory control sites (e.g. enhanced glycosylation at phosphorylation residues). This study was pursued to examine the role that enhanced glycosylation levels has on cardiac fibroblast (CF) collagen production. For this purpose, rat CF were cultured in low and high glucose (HG) conditions. The exposure of CF to HG led to significant increases in collagen production that were associated with alterations in key regulators of extracellular matrix production including TGF‐beta, SMADs and ERK. Exposure to HG also led to increases in total levels of protein glycosylation. The glycosylation levels of the transcription factor SP1 (that importantly regulates collagen promoters) were also enhanced by HG. An adenovirus coding for glycnacase was used to examine the effects of diminished protein glycosylation on CF collagen production and signaling pathways. Results indicate that the reversal of excess glycosylation levels can restore CF collagen production to levels similar to controls. Studies are currently being pursued to identify which alterations of key signaling pathways underlie these beneficial effects.
Smooth muscle glutathione depletion contributes to increased myogenic tone in resistance arteries of type‐2 diabetic mice
Authors: Velmurugan Gopal V , White Carl
Published: June 2021

Oxidative stress associated with increased reactive oxygen species and depleted levels of antioxidants such as glutathione (GSH) are hallmarks of diabetes. In the present study, we tested the hypothesis that GSH depletion contributes to the increased myogenic tone observed in type‐2 diabetes. Mesenteric arteries isolated from 12–14 week old diabetic (db/db) and nondiabetic (db/m) mice were denuded of endothelium, cannulated, and myogenic tone measured as percentage of a passive vessel diameter. At physiological pressures, myogenic tone was greater in vessels from the db/db compared to db/m. GSH levels were experimentally elevated in db/db vessels by incubating overnight with GSH ethyl ester. Increased GSH levels in the diabetic db/db vessels reduced myogenic tone compared to untreated db/db controls. In the reciprocal experiment, GSH levels in nondiabetic db/m vessels were depleted by overnight treatment with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor. The BSO treated db/m vessels showed increased myogenic tone compared to untreated controls. In conclusion, these data suggest that depletion of the vascular smooth muscle GSH pool contributes to increased myogenic tone in mesenteric vessels from type 2 diabetic animals. Funded by the American Heart Association.
Increased TGFβ Signaling in Hearts of Type I Diabetic Mice May Result in Diabetic Cardiac Autonomic Dysfunction
Authors: Georgescu Serban Paul , Naggar Jack , Aronovitz Mark , Welzig Charles Michael , Picard Kristen , Vaikus Celestia , Zhang Yali , Park Ho-Jin , Karas Richard , Galper Jonas B.
Published: June 2021

Diabetic Autonomic Neuropathy (DAN), a complication of diabetes is associated with cardiac parasympathetic dysfunction, severe arrhythmias and sudden death. Increased TGFβ signaling has been implicated in the mechanism of end organ damage in type I diabetes. We had shown that in atrial myocytes TGFβ attenuates the negative chronotropic response of the heart to the acetylcholine analogue Carbamylcholine (Carb). To determine whether TGFβ signaling was increased in the diabetic heart we compared PAI‐1 and TGFβRII levels in atria of Akita Type I diabetic mice (DM) and WT mice. PAI‐1 mRNA determined by qPCR increased 1.6±0.33 fold, n=4 p<0.03. Western blots demonstrated increased pSmad2/3 and a 3.4±0.5fold, n=3 p<0.03 increase in the TGFβRII. Treatment of mice with insulin pellets reversed these effects. Using EKG transmitters we compared carb‐induced bradycardia in WT and DM. Duration of bradycardia was 5.1 min in DM vs 12.6 min in WT. SB‐505124 (SB, an inhibitor of TGFβ signaling) treatment for 10d restored the level of PAI‐1 mRNA in the DM to WT levels, increased duration of bradycardia in response to carb to 11.1 min. In whole cell patch clamp studies of Akita atrial myocytes SB increased IKAch, the K current which mediates the decrease in heart rate to levels similar to those in WT cells: −248±15 pA/pF to 341±15 pA/pF n=15, p<0.01. Thus elevated TGFβ signaling in type I diabetic mouse appears to be associated with cardiac DAN.
Cardiac Myocyte Exosome Stability and Function: Role of HSP60
Authors: Kuo Tiffany , Malik Zulfiqar , Kott Kayleen S. , Chen Le , Knowlton Anne A.
Published: June 2021

Exosomes are small lipid vesicles, which are released from cells, and have been implicated as mediators of intracellular signaling, as well as a mechanism to remove material from the cell. We have shown that heat shock protein (HSP)60 is released from cardiac myocytes via exosomes. We investigated the stability of exosomes, as free HSP60 causes apoptosis.

Methods

Exosomes were isolated from cardiac myocyte media and purified with Exoquick. Acetyl choline esterase activity, protein content and exosome sizing were used to verify the quality of preparation.

Results

Exosomes were incubated 18 h under different conditions: heat (4, 25, 37, and 42C), ethanol (21 and 65 mM) and acidosis (pH 6.6, 7.1, 7.4). Exosome stability was unchanged under these conditions based on the lack of release of HSP60. To further characterize the role of HSP60, we used biotinylation to determine whether HSP60 was on the intact exosome surface. Biotinylated protein was purified by affinity column, and then tested by western for HSP60. The results show that there is surface HSP60.

Conclusion

Exosomes are stable under physiologic variations seen with disease. HSP60, which is toxic to myocytes when extracellular and is released by cardiac myocytes through exosomes, is present on the surface of the exosome. Ongoing work is investigating whether the exosomes are toxic to cells. Supported by HL077281.
Anticancer effects of α, β Momorcharin and its mechanism of action on different cancer cell lines
Authors: Singh Jaipaul , Manoharan Gunasekar
Published: June 2021

The protein α β Momorcharin extracted from the tropical plant, Momordica charantia, was shown to possess anti‐cancer and anti‐HIV properties. This study investigated both time and dose dependent effects of α β Momocharin on the viability of 1321N1, Gos‐3, U87‐MG, Sk Mel, Corl‐23 and Weri Rb‐1, 6 cancer cell lines and L6 cells in the absence and presence of either temozolomide or vinblastine. The study also determined the mechanism(s) via which α β Momorcharin can exert its effect on cell death measuring the activities of caspase‐3 and −9, cytochrome c release and intracellular free calcium concentrations [Ca 2+]i.

The results show that α β Momocharin can evoke both time (800 μM) and dose‐dependent (200 μM ‐ 800 μM) decreases in cell viability over a period of 24 h following incubation compared to untreated cells or healthy L6 cells. Combining α β Momocharin with either temozolomide (80 μM‐320 μM) or vinblastine (10 μg‐40 μg) resulted in significant (p< 0.05) decreases in cell viability. Incubation of the different cancer cell lines with α β Momocharin (800 μM) for 24 h resulted in significant (p< 0.05) increases in the activities of caspase‐3 and −9, cytochrome c release and elevation in [Ca2+ ]i in all the cell lines compared to untreated and L6 cells. The results indicate that α β Momocharin can evoke cancer cell death via apoptosis and cellular Ca2+ homeostasis.
Impaired Regulation of Renal K Elimination in Mice Lacking SGLT1
Authors: Gerasimova Maria , Rose Michael , Cunard Robyn , Koepsell Hermann , Rieg Timo , Vallon Volker
Published: June 2021

Sodium‐glucose transporter SGLT1 is expressed in the luminal membrane of late proximal tubules and further distal segments. We report impaired renal K excretion in Sglt1 knockout (−/−) mice as evidenced by renal K excretion that is not different from wild‐type (WT) mice despite of higher plasma concentrations of aldosterone and K (940±143 vs 277±42 pg/ml; 4.45±0.09 vs. 4.15±0.07 mM; P<0.05), which are major stimuli of renal K excretion. Significant differences in plasma aldosterone and K were maintained on high K diet but prevented by low K diet. Sglt1−/− mice lost body weight compared with WT in response to a low and high K diet given for 6 days (−8.5±0.5 vs −4.4±0.7% of bw and −1.2±0.4 vs. 1.7±0.6% of bw; P<0.05). NADPH oxidase 2 (NOX2)‐derived reactive oxygen species (ROS) can inhibit ROMK channels in the distal nephron to lower renal K excretion. We found that urinary excretion of the more stable ROS, H2O2, was 10fold greater on low vs high K diet in WT but only varied by 3fold in Sglt1−/− mice which, compared with WT, maintained higher levels under high K diet (25±4 vs 12±3 nmol H2O2/mg creatinine, P<0.05) but tended to have lower levels on low K diet. Renal Nox2 mRNA expression was 42% higher in Sglt1−/− vs WT mice under control diet (P<0.05). The studies link SGLT1 to renal H2O2 excretion and K handling. Renal K excretion is not impaired in Sglt2−/− mice supporting a specific role for SGLT1.
Microparticle enlargement is a key point of vascular injury caused by decompression stress
Authors: Yang Ming , Milovanova Tatyana N , Bogush Marina , Bhopale Veena , Thom Stephen R
Published: June 2021

Objective

Studies in a mouse model have shown that decompression stress increases the number of circulating microparticles (MPs), which precipitate neutrophil activation and vascular damage. Our objective was to discern whether their size was a strong contributor to pathophysiological changes caused by decompression stress.

Methods

MPs were isolated from mice subjected to 790 kPa air pressure for 2 hours. MPs size was measured by a multi‐photon microscope. MPs were injected into naïve mice via tail vein. Vascular injury was documented as elevations of vascular permeability and cleaved caspase 3.

Results

In control animals, 0.6±0.1 % of annexin V positive particles had diameters > 2.0 μm, while in 2 hours post‐decompression group this fraction increased to 9.8±1.8 % (p<0.05, n=5) and in 24 hours group 14.6±4.9 % (p<0.05, n=3). When 2 hours post‐decompression MPs samples were hydrostatically recompressed at 790 kPa pressure for 1 hour, the fraction of diameters > 2.0 μm was significantly reduced compared to without hydrostatic recompression group (p<0.05, n=5). When MPs with or without recompression were injected into naïve mice, both neutrophil activation and vascular damage were much less in those injected with recompression MPs.

Conclusion

we conclude that enlargement of annexin V positive microparticles plays an important role in tissue injury caused by decompression stress.

This work was supported by a grant from the U.S. Office of Naval Research.
The Skeletal Muscle Metaboreflex is Attenuated in Healthy Older Adults
Authors: Greaney Jody L , Edwards David G , Fadel Paul J , Farquhar William B
Published: June 2021

A previous study reported that healthy aging attenuated the exercise pressor reflex (EPR); however, whether this is due to alterations in the metabolic or mechanical component of the EPR remains unclear. Thus, the goal of this study was to comprehensively assess the influence of age on the metabolic component of the EPR using graded intensities of muscle metaboreflex activation. In young (n=18; 23±1 yr; resting blood pressure (BP) 111±3/69±3 mmHg) and older (n=23; 60±1 yr; resting BP 112±1/69±2 mmHg) normotensive subjects graded isolation of the muscle metaboreflex was achieved by post exercise ischemia (PEI) following moderate (PEI‐M) and high (PEI‐H) intensity static handgrip performed at 30% and 40% maximum voluntary contraction, respectively. BP (Finometer) and muscle sympathetic nerve activity (MSNA; peroneal microneurography) were measured continuously. The increases in mean BP and MSNA burst frequency were significantly less in older subjects compared to younger subjects during both PEI‐M (15±2 young v 10±1 older mmHg and 13±3 young v 2±2 older bursts/min; both P<0.05) and PEI‐H (20±2 young v 13±1 older mmHg and 12±3 young v 4±2 older burst/min; both P<0.05). These data indicate that older normotensive adults have blunted neurocirculatory responses to PEI compared to young adults, suggesting that the metaboreflex is attenuated in healthy aging.

Supported by NIH Grant HL‐074851‐02 and 2 P20 RR016472‐11 (to W.B.F.); HL‐093167‐03 (to P.J.F.); AHA 11PRE7580029 (to J.L.G.)
Cardiac output and total vascular conductance responses to simulated carotid hypertension in young women: exercise and ovarian hormones
Authors: Kim Areum , Fisher James P , Deo Shekhar H , Vianna Lauro C , Fadel Paul J
Published: June 2021

Previously we reported that compared to men, young women during the early follicular (EF) phase of the menstrual cycle exhibited an enhanced carotid baroreflex (CBR) control of blood pressure (BP) to hypertensive stimuli at rest due to a predominant contribution of cardiac output (CO). Whether such changes are altered during exercise or by fluctuations in endogenous ovarian hormone concentrations is unknown. Thus, 5s pulses of neck suction (NS; −80Torr) were applied at rest and during steady‐state cycling at 50% heart rate (HR) reserve to activate carotid baroreceptors in 10 young women at 3 different menstrual cycle phases (EF, late follicular (LF), and mid luteal (ML)). Beat‐to‐beat HR and BP were measured. Stroke volume was estimated with Modelflow, and CO and total vascular conductance (TVC; CO/meanBP) were calculated. At rest, the change in CO was the major contributor in mediating BP changes to NS during all menstrual cycle phases (CO vs. TVC; 99±15% vs. −3±16%, EF; 105±22% vs. −10±25%, LF; 108±19% vs. −14±22%, ML; P>0.05). This effect was lost during exercise in which a greater contribution of TVC was observed (CO vs. TVC; 14±34% vs. 83±131%, EF; 16±10% vs. 84±9%, LF; 34±14% vs. 66±13%, ML; P>0.05). Our findings suggest that in contrast to rest, exercising women exhibit a greater involvement of TVC in the CBR control of BP to hypertension; an effect that is independent of menstrual cycle phase.

Supported by R01HL093167
Endothelin‐1 induces NR1 phosphorylation in NG 108‐15 cells
Authors: Liu Yuzhen , Li Xianghong , Weiss J. Woodrow
Published: June 2021

We previously reported that: 1) treatment with ET‐1 of carotid bodies from rats exposed for 3 weeks to cyclic intermittent hypoxia (CIH) increases carotid sinus nerve activity (CSNA) more than treatment of carotid bodies from sham‐exposed rats; and, 2) administration of the NMDA receptor antagonist, MK801, reduces ET‐1 induced CSNA in CIH‐exposed rats. Thus ET‐1 modulates carotid chemoreflex activity at least in part through NMDA receptor activation but the mechanism by which ET‐1 modulates NMDA receptor activation is poorly understood. To explore possible mechanisms we used differentiated NG108‐15 cells treated with ET‐1. ET‐1 treatment induced dose‐ and time‐dependent phosphorylation of NR1, a subunit of NMDA receptor, at two sites, Ser896 and Ser897. ET‐1 treatment of NG108‐15 cells also activates PKC and PKA in a time dependent manner. The PKC inhibitor chelerythrine prevented ET‐1 induced NR1 phosphorylation at Ser896 and the PKA inhibitor H‐89 blocked NR1 phosphorylation at Ser897. The ETA receptor antagonist BQ123, but not the ETB receptor antagonist BQ788, blocked ET‐1‐induced NR1 phosphorylation. Importantly, ET‐1 enhanced the recruitment of NR1 into the plasma membrane. Our result demonstrates a novel mechanism by which ET‐1 may modulate NMDA receptor function and may provide insight into the molecular mechanisms by which ET‐1 contributes to hypoxic acclimatization to CIH.
Gap junctions in the neonatal mouse pre‐Bötzinger complex: Insights using fluorescence recovery after photobleaching
Authors: Kelty Jonathan , Shields Edward
Published: June 2021

Whether interneuronal coupling via gap junctions is essential to the generation/patterning of rhythmic activity by an inspiration‐related network in the medulla's pre‐Bötzinger complex remains unresolved. We used a fluorescence recovery after photobleaching approach in medullary slice preparations from neonatal mice to determine whether diffusion of carboxyfluorescein from neuron to neuron is blocked by the gap junction uncoupler carbenoxolone (CBX). Tissues incubated in standard artificial cerebrospinal fluid (aCSF) regained a greater percentage of their initial fluorescence than did those incubated in aCSF + CBX. Regardless of whether slices were incubated in aCSF or in aCSF + CBX, almost all bleached somata regained some fluorescence within 20 min following photobleaching. Presumably this recovery was due, at least in part, to movement of carboxyfluorescein into somata from adjacent portions of neurites. Only a small fraction of bleached somata directly abutted other neuronal somata exhibiting fluorescence suggesting that either a) carboxyfluorescein moved into recovering somata from adjacent glia and/or b) that CBX somehow interferes with the redistribution of carboxyfluorescein within cells.
Microglia attenuate the opioid‐induced depression of preBötzinger Complex (preBötC) inspiratory rhythm in vitro via a TLR4‐independent pathway
Authors: Zwicker Jennifer D , Zhang Yong , Hutchinson Mark R , Rice Kenner C , Watkins Linda R , Funk Gregory D
Published: June 2021

Opioids activate neurons via opioid receptors (Rs) but also activate microglia via toll like receptors (TLR). Activation of microglial TLR4 impairs the ability of opioids to suppress pain and is hypothesized as pivotal in opioid‐mediated reward and tolerance. Here we test the contribution of microglia and TLR4 to the opioid‐induced respiratory depression. Using rhythmic medullary slices from neonatal rats we compared the duration of apneas evoked by locally injecting DAMGO (μ‐opiate R agonist; 50μM) into the preBötC before and after 40 min incubation in minocycline (inhibits microglial activation, 500 nM). Minocycline increased the duration of DAMGO‐evoked apnea 9.3±4.7‐fold from 33±7 to 295±76 s (n=3). In time‐matched controls, the second DAMGO‐evoked apnea was only 0.5±0.4‐fold greater than the first. The TLR4 antagonist LPS‐RS (90sec; 2000 ng/mL) had no effect on the frequency (f) depression evoked by DAMGO in the preBötC. Similarly, bath application of (+) naloxone (10μM; TLR4selective antagonist) had no effect on the f depression evoked by DAMGO (500nM) or fentanyl (1μM) in the bath. In contrast, (−) naloxone (500nM; TLR4 and μ‐opioid antagonist) reversed the depression. These data suggest that microglia attenuate the opioid‐induced respiratory depression via a mechanism that does not involve TLR4 activation. Supported by CIHR, WCHRI, AIHS, NIDA & NIAAA.
Developmental changes in NMDA receptor subunits alter burst frequency of neonatal rat brainstem slices containing the preBötzinger Complex
Authors: Getsy Paulina Marie , Kaufman Daniel , Anagnostos Kate , Wilson Christopher Glenn
Published: June 2021

N‐methyl‐d‐aspartate receptors (NMDARs) are crucial in controlling synaptic excitation and plasticity at glutamatergic synapses in the CNS. NMDARs are composed of multiple NR1 and at least one of four NR2 subunits and previous studies have shown development changes in NMDARs. In the perinatal brain, NR2B subunits predominate, however these subunits are supplemented with NR2A/NR2C subunits. We hypothesized that respiratory rhythm in the brainstem of postnatal rats (P0–P3), is developmentally modulated by NMDAR subunit changes in the preBötzinger Complex (pBC). We prepared pBC‐containing slices from Sprague Dawley rat pups (P0 to P5) and measured changes in hypoglossal (XII) rootlet output and characterized the switch from NR2B‐ to NR2A‐containing NMDARs by bath application of 10 μM ifenprodil, a noncompetitive inhibitor of the NR2B subunit. Our results show a significant decrease (p < 0.05; compared to baseline) in inter‐burst interval after application of ifenprodil in P0–P3 (n=3) slices with no significant change in animals > P3. This suggests that NR2B‐containing NMDARs play a role in the modulation of respiratory rhythm early in life. Support: This work was supported by NIH/NHLBI grant HL081622
Collateralizaton of projections of rostral ventrolateral medulla (RVLM) neurons to levels of the thoracic spinal cord that regulate upper‐ and lower‐body blood flow
Authors: Gowen Michael F , Weber Sarah A , Suzuki Takeshi , Sugiyama Yoichiro , Yates Bill J
Published: June 2021

We have demonstrated that vestibular stimulation results in distinct changes in blood flow in the upper and lower body, indicating that the sympathetic nervous system has the capacity to elicit regionally‐specific changes in blood flow. Since the RVLM plays a major role in regulating blood flow, the present study tested the hypothesis that distinct populations of RVLM neurons project to the T4 segment (which controls blood flow in the upper body) and the T10 segment (which regulates lower body blood flow). For this purpose, multiple injections of the fluorescent dye Fast Blue were made in cats in the vicinity of the intermediolateral cell column (IML) in T4, whereas multiple injections of the dye Fluoro‐Ruby were made near the IML in T10. RVLM neurons that were single‐labeled for one of the tracers (indicating that they projected to only one segment) were approximately four times more prevalent than those that were double‐labeled (had collateralized projections to both T4 and T10), including catecholaminergic neurons identified through immunohistochemistry. A χ2 test confirmed that double‐labeled cells were less prevalent than single‐labeled RVLM neurons (p<0.001). Our conclusion from these data is that the RVLM has the capacity to independently regulate sympathetic nervous system influences on blood flow in different body regions.