bluuebunny/crossref_metadata_2025 huggingface dataset:
Aging increases the amplitude of inward‐rectifier K+ channel currents in murine resistance artery smooth muscle cells (677.8)
Authors: Hayoz Sebastien , Pettis Jessica , Bradley Vanessa , Segal Steven , Jackson William
Published: June 2021

Elevating extracellular K+ ([K+]o) dilates resistance arteries by activating smooth muscle cell (SMC) inward‐rectifier K+ channels (KIR). The rise in [K+]o of exercising skeletal muscle can thereby contribute to functional hyperemia. As muscle blood flow is diminished with aging, we tested the hypotheses that aging impairs K+‐induced activation of KIR in SMCs of resistance arteries. Superior epigastric arteries (SEAs) were dissected from abdominal skeletal muscle of Young (Y; 蠅3 mo) or Old (O; 蠅24 mo) male C57BL/6 mice and either dissociated to yield SMCs for perforated patch clamp recording or cannulated and pressurized for myography. Elevation of [K+]o from 5 to 15 mM activated Ba2+ (100 μM)‐sensitive inward K+ currents between ‐120 and ‐60 mV that were smaller in SMCs from Y than in O: at ‐120 mV, KIR current density was ‐0.2±0.3 pA/pF in Y vs ‐0.8±0.2 pA/pF in O; n=26; p<0.05. In contrast, elevating [K+]o from 5 to 15 mM evoked similar dilation in SEAs of Y (61±6%; n=12) and O (60±7%; n=13); responses in both were abolished by 100 μM [Ba2+]o (n=5). Thus, aging does not impair KIR currents in SMCs of resistance arteries and their dilation via KIR activation is maintained. These findings indicate that compensatory adaptations with aging maintain SEA function in the presence of increased KIR activity.

Grant Funding Source: Supported by NIH R01 HL086483 and R25 HL103156
Roles for neutrophils and monocytes in murine cytomegalovirus‐induced microvascular dysfunction (678.16)
Authors: Khoretonenko Mikhail , Leskov Igor , Stokes Karen
Published: June 2021

One of the first responses of the vasculature to cardiovascular disease (CVD) risk factors, such as hypercholesterolemia (HC), is endothelial dysfunction, characterized by impaired arteriolar dilation and venular inflammation and platelet recruitment. Pathogens have been implicated in CVD, and we have recently shown murine cytomegalovirus (mCMV), also impairs endothelium‐dependent vasodilation. Although mCMV did not significantly increase blood cell recruitment, mCMV extended the first wave of inflammation (neutrophils) due to HC. Here we used spinning disk intravital microscopy to determine the identity of these recruited leukocytes, and light intravital microscopy to determine if these leukocyte subpopulations contributed to arteriolar dysfunction in mCMV‐infected mice on normal diet (ND) or high cholesterol diet (HC). We demonstrated that mCMV shifted populations of adherent leukocytes in microvasculature of HC mice from predominantly neutrophils towards ~1:1 GR‐1+ monocytes:neutrophils. Depletion of neutrophils using an anti‐Ly‐6G Ab restored arteriolar vasodilation in the mCMV‐HC, but not mCMV‐ND mice. It was also effective at abrogating leukocyte and platelet adhesion. Depletion of GR‐1+ cells (neutrophils and monocytes) protected against the arteriolar dysfunction in both mCMV‐ND and mCMV‐HC mice. These results suggest that mCMV causes a phenotypic shift in the inflammatory leukocytes recruited to venules during HC towards a larger monocyte component, although neutrophils remain key players in the arteriolar dysfunction. Furthermore, our data implicate a role for inflammatory GR‐1+ monocytes in the arteriolar dysfunction associated with mCMV infection.

Grant Funding Source: Supported by COBRE GM103433‐NIGMS & Malcolm Feist Cardiovascular Research Endowment Fellowship, LSU
NOS uncoupling in oxidative stress‐associated neurogenic hypertension induced by the high fructose diet (682.2)
Authors: Chan Julie , Wu Kay
Published: June 2021

The reactive oxygen species (ROS) produced from uncoupled nitric oxide synthase (NOS) leads to oxidative stress. In the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons located, oxidative stress is involved in neural mechanism of hypertension. We investigate in this study the significance of NOS uncoupling in oxidative stress‐associated neurogenic hypertension induced by the high fructose diet (HFD). In normotensive Wistar‐Kyoto rats, oral ingestion of the HFD for 8 weeks resulted in insulin resistance, hyperglycemia, hypertriglyceridemia, sympathoexcitation and hypertension, accompanied by an increase in tissue level of superoxide but a decrease in NO level in the RVLM. Protein expression of the neuronal NOS (nNOS) and protein inhibitor of nNOS (PIN) was increased, but NOS activity and ratio of dimer over monomer of nNOS were decreased. Gene knocking down of PIN in the RVLM by the small interfering RNA (siRNA) or intracisternal infusion of L‐arginine increased NOS activity, maintained tissue NO level and ameliorated the heightened tissue ROS level in the RVLM of the HFD‐fed animals. The same treatments also significantly reduced the high blood pressure and sympathoexcitation induced by the HFD. In addition, intracisternal infusion of ROS scavenger, tempol, notably blunted sympathoexcitation and hypertension but not hyperglycemia induced by the HFD. Together these results indicate that NOS uncoupling because of an increase in PIN activity may contribute to oxidative stress in the RVLM, resulting in sympathoexcitation and neurogenic hypertension induced by the HFD.

Grant Funding Source: Supported by NSC102‐2321‐B182A‐003, National Science Council, Taiwan
Obesity related hypertension: an altered downstream regulation of leptin signaling (682.5)
Authors: Lim Kyungjoon
Published: June 2021

Background: Short term (3weeks) high fat diet (HFD) consumption leads to an increase in blood pressure (BP) and renal sympathetic nerve activity (RSNA) in rabbits. We suggest there is an altered down‐stream sensitivity of leptin in the hypothalamus which involves altered responses to α‐melanocyte stimulating hormone (α‐MSH) and neuropeptide Y (NPY) when rabbits are fed a HFD.

Aim: To investigate the role of α‐MSH and NPY in the hypothalamus in HFD‐fed rabbits.

Methods: New Zealand White Rabbits were fed with a normal or a 13.3% HFD for 3 weeks. A bilateral guide cannula into the dorsomedial (DMH) or ventromedial hypothalamus (VMH) and an RSNA recording electrode was implanted. After 3 weeks on the diet, α‐MSH (0.1, 0.3nmol), NPY (0.1, 0.5nmol) or Ringer’s were injected directly into the DMH or VMH.

Results: Rabbits exhibited higher BP, HR and RSNA when fed a HFD compared to controls (n=6‐8). α‐MSH injection into the DMH increased BP (5.7%, P<0.05 and 8.3%, P<0.01 with 0.1nmol and 0.3nmol respectively) and RSNA (40%, P<0.05 with 0.3nmol) in HFD rabbits. When α‐MSH was injected into the VMH, BP was unchanged but RSNA was increased (28%, P=0.01 and 38%, P<0.01) in HFD rabbits. NPY injection had no effect in both control and HFD rabbits.

Conclusion: Elevated blood pressure and sympatho‐excitation in HFD rabbits are due to increased sensitivity of the leptin signalling pathway which involves a dorsal and ventral hypothalamic projection of α‐MSH activated second order neurons.
Kappa opioid receptor therapy post pressure overload induced hypertrophy preserved cardiac function (682.6)
Authors: Zemljic‐Harpf Alice , Deussen Daniel , Vaishnav Yash , Headrick John Headrick , Peart Jason , Niesman Ingrid , Roth David , Patel Hemal
Published: June 2021

Introduction: The loss of contractility and myocardial function are major aspects of heart failure. Obesity is an independent risk factor for developing hypertensive heart disease. The incidence of heart failure is currently increasing and novel therapeutics are needed. Opioid receptor stimulation is protective against myocardial ischemic‐reperfusion‐injury; whether this protection can be translated to heart failure is unclear. Hypothesizing that stimulation of κ‐opioid receptors preserves cardiac function during left ventricular hypertrophy, we performed the first study of OR‐stimulation in mice exposed to left ventricular pressure overload.

Methods: Transverse aortic constriction (TAC) was performed on male, 8‐10 week old, C57Bl/6J mice. Two weeks after TAC, treatment was started with either an irreversible δ‐opioid receptor agonist (ORA) FIT, the selective κ‐ORA U50488H or a vehicle control. Drugs were administered intraperitoneally, FIT (10μg/kg) every 72h and U50488H (1.25mg/kg) daily. Echocardiography was performed before TAC and consecutively at weeks 2, 4, 6, 8 and 10 to determine cardiac function and left ventricular dimensions.

Results: The physiological hypertrophic response post TAC was not altered in δ‐ and κ‐ORA treated TAC animals. Heart weight/tibia length ratios were significantly higher in TAC (TAC > 9.74 to Sham < 7.01, p<0.037), with no difference between the three groups. In the vehicle TAC group cardiac function started to diminish from 4 weeks onward. Cardiac function was preserved in TAC mice treated with U50488H, when compared to vehicle TAC. The ejection fraction and fractional shortening were significantly (p<0.04) higher in U50488H group from the six week time point onward, when compared to vehicle TAC and showed no significant difference when compared to U50488H treated Sham group.

Conclusions: κ‐ORA (U50488H) stimulation may offer a novel treatment option to preserve heart function in pressure overload induced left ventricular hypertrophy.
The functional changes in the basilar artery due to overpressure blast injury (685.1)
Authors: Toklu Hale , Muller‐Delp Judy , Yang Zhihui , Ghosh Payal , Strang Kevin , Scarpace Philip , Wang Kevin , Tumer Nihal
Published: June 2021

Overpressure blast‐wave induced brain injury (OBI) leads to progressive pathophysiological changes resulting in a reduction in brain blood flow, blood brain barrier breakdown, edema and ischemia. The aim was to evaluate cerebral vascular function after single and repeated OBI. Male Sprague Dawley rats (250‐300 g) were divided into 3 groups: Control (Naive), single OBI [30 psi peak pressure, 1‐2 msec duration], and repeated (every three days) OBI (r‐OBI). Rats were sacrificed 24 h post injury (8th day) and the basilar artery was cannulised in the pressurized system (90cm H2O). Vascular responses to KCl (30‐50‐100 mM) endothelin (10‐11‐10‐7 M), acetylcholine (ACh) (10‐9‐10‐4 M) and diethylamine (DEA)‐NONO‐ate (10‐9‐10‐4 M) were evaluated.

OBI resulted in an increase in the contractile responses to endothelin and a decrease in the relaxant responses to ACh in both single and repeated injury groups. However, impaired DEA‐induced dilation and increased wall thickness to lumen ratio were observed only in the r‐OBI group.

These findings indicate that single OBI causes endothelium‐dependent and ‐independent alterations in cerebral vascular function and repeated injury results in increased arterial wall thickness.

Grant Funding Source: This work was supported by the Medical Research Service of the Department of Veterans Affairs, McKni
Characterization of a novel transgenic rat model for imaging brain vascular dynamics in vivo using confocal endomicroscopy (686.27)
Authors: Mayorov Dmitry , Roloff Eva , Alenina Natalia , Bader Michael , Teschemacher Anja , Paton Julian , Kasparov Sergey
Published: June 2021

We evaluated the feasibility of imaging brain vasculature in a transgenic rat model (L7543) which expressed a construct carrying the eGFP under the control of CAG promoter (Popova et al., Transgen. Res. 2008) using fiber endomicroscopy (CellVizio, Mauna Kea Technologies). eGFP expression were first evaluated in cortical and brainstem slices from L7543 rat using confocal microscopy (Leica SP5). Expression of eGFP in the cortex and brainstem was largely restricted to endothelial cells, although astrocytes in younger rats (up to P35) were also fluorescent. Next, the brainstem and cortex were imaged, without a contrasting agent, in anaesthetized P21‐P40 rats using a fiber probe‐based confocal laser endomicroscopy. Pial microvessels of the ventral or dorsal brainstem, or cortex were imaged using S‐1500 and Mini‐Z probes (1.5 and 0.94 mm tip diameter, respectively). The vascular contractility was visualized after application of a thromboxane agonist U46619 and sodium nitroprusside. Arterioles and veins were identified based on the eGFP expression profile. In addition, beveled probes S‐300/B and S‐650/B (0.3 and 0.65 mm tip diameter, respectively) were used to visualize vascular tree in Z‐dimension. Currently we are working to adapt this method for imaging in unanaesthetized rats. This approach should be useful to investigate changes in cerebral microcirculation in hypertension and other vascular pathologies.

Grant Funding Source: Supported by British Heart Foundation
Brain‐derived neurotrophic factor overexpression in the paraventricular nucleus increases blood pressure via angiotensin type‐1 receptor‐mediated mechanisms (686.3)
Authors: Erdos Benedek , Backes Iara , Hayward Linda , Sumners Colin , Scheuer Deborah
Published: June 2021

BDNF expression increases in the PVN during hypertensive stimuli including stress and hyperosmolarity, and we have shown previously that overexpression of BDNF in the PVN leads to significant increases in mean arterial blood pressure (MAP). Here, we tested the hypothesis that BDNF‐induced pressor responses are mediated by angiotensin signaling in the central nervous system. MAP was recorded in male Sprague‐Dawley rats using telemetry. At 11wks of age, adeno‐associated viral vectors (AAV2‐GFP or AAV2‐BDNF) were injected bilaterally into the PVN, and losartan (Los; 15µg/ 0.5µL/ hour) or saline was infused icv for 4 wks using osmotic pumps. During the 4 wks after vector injections, MAP remained unchanged in the GFP+saline (dMAP=2.5±0.4 mmHg) and GFP+Los (dMAP=2.6±1.2 mmHg) groups. MAP increased markedly in both BDNF groups, but the increase was significantly reduced in BDNF+Los (dMAP=15.0±3.2 mmHg) compared with BDNF+saline (dMAP=28.7±2.2 mmHg, p<0.05). RT‐PCR from PVN tissue samples of AAV2‐GFP and AAV2‐BDNF‐injected rats indicated that BDNF significantly upregulated AT1R mRNA (2‐ddCt was 1.1±0.2 in GFP and 2.0±0.4 in BDNF rats using 18s as internal control, p<0.05). In contrast, mRNA levels of angiotensinogen, angiotensin converting enzyme (ACE), ACE2 and AT2R were unaffected by BDNF. We conclude that the BDNF‐induced MAP increase is partially mediated by AT1R, which is upregulated by BDNF within the PVN.

Grant Funding Source: Supported by AHA 11SDG7560022, an AFAR research grant, and by NIH #HL76807, #HL93186.
Exercise augments but sinoaortic denervation blocks noradrenergic signaling to PVN in trained normotensive rats (686.5)
Authors: Michelini Lisete , Santos Carla , Ruggeri Adriana , Braga Douglas , Ceroni Alexandre
Published: June 2021

Exercise training (T) increases ascending NORergic signaling from NTS to PVN, improves baroreflex sensitivity (BrS) and reduces basal heart rate (HR). We evaluate the effects of sinoaortic denervation (SAD) and T on cardiovascular parameters and expression of PVN oxytocinergic (OTergic) and vasopressinergic (VPergic) neurons. SAD and SHAM rats were submitted to low‐intensity T or kept sedentary (S) for 3 month. After hemodynamic measurements, rats were euthanized; perfused brains were post‐fixed and cryoprotected. Sequential PVN slices (30 μm) were processed for OT, VP and dopamine β‐hydroxylase (DBH) immunoreactivity (ir); images were acquired and analyzed by Image J. In SHAM rats T reduced basal HR, increased BrS (328±8 b/min, 2.4±0.2 b/min/mmHg, ‐13% and +25% vs S controls), augmented the density of DBH terminals and OTir in ventromedial PVN and reduced VPir in the magnocelular PVN, without changing DBH signaling to this area. SAD markedly reduced basal OTir and VPir in both PVN areas and blocked functional and T‐induced effects on OTir and VPir. T‐induced afferent signaling, directed preferentially to preautonomic areas, increases the expression of OTergic neurons, contributing to the appearance of beneficial effects in intact T rats. SAD blocks these effects confirming that PVN signaling conveyed by baro‐ and chemoreceptors is essential to maintain neuronal tonicity and to mediate T‐induced effects

Grant Funding Source: Supported by FAPESP, CNPq
The type 2 diabetic patient and cardiac mitochondrial dysfunction: a new perspective (688.10)
Authors: Shepherd Danielle , Croston Tara , Holden Anthony , Tveter Kevin , Thapa Dharendra , Nichols Cody , Olfert I. Mark , Hollander John
Published: June 2021

Cardiac complications are the leading cause of mortality among diabetic patients, with the mitochondrion being implicated in the etiology. We have previously observed enhanced dysfunction to cardiac subsarcolemmal mitochondria (SSM) in db/db mice as a result of type 2 diabetes mellitus. The goal of this study was to determine whether hyperglycemia or hemoglobin A1c (HbA1c) level correlates with the extent of SSM dysfunction observed in type 2 diabetic patients. Using linear spline models to assess correlative risks we determined that diabetic patients display dysfunctional SSM state 3 respiration rate and ETC complex I activity irrespective of the absolute blood glucose or HbA1c level. In addition, we found that SSM dysfunction observed in type 2 diabetic patients with comorbidities, such as coronary artery disease (CAD) and hypertension is not due to either CAD or hypertension independently. Further, a scatter plot analysis of patients’ body mass index (BMI) indicated that BMI is not an effective predictor of mitochondrial dysfunction. These findings suggest that independent of comorbidities cardiac SSM dysfunction is present in the type 2 diabetic patient heart. Interestingly, the degree of mitochondrial dysfunction in diabetic patients remains consistent despite the extent of elevated blood glucose or HbA1c level. (Support: AHA 13PRE16850066; NIH DP2DK083095; NSF DGE1144676; NIH T32HL090610)
Anti‐inflammatory effects of Hedyotis diffusain ob/ob mice (688.2)
Authors: Kang Min‐Jung , Kim Jung‐In , Yim Jung‐Eun
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) is the most common liver disease and is associated with insulin resistance, oxidative stress, and inflammation. Insulin resistance might play an important role in the pathogenesis of NAFLD. Proinflammatory cytokines are thought to be associated with the development of insulin resistance. It has been reported that Hedyotis diffusa was effective at improving insulin resistance and fatty liver in an animal model. This study investigated the anti‐inflammatory effects of Hedyotis diffusa in leptin‐deficient ob/ob mice. Five week‐old ob/ob mice (C57BL/6J, n=16) were fed AIN‐93G diet or diet containing 1% fermented alcohol extract of Hedyotis diffusa for 8 weeks. The serum TNF‐α, IL‐6, and monocyte chemotactic protein‐1 (MCP‐1) levels were measured using enzyme‐linked immunosorbent assay (ELISA) kits. Body weight and food intake of the Hedyotis diffusa group were not significantly different from those of the control group. The serum TNF‐α level of the Hedyotis diffusa group was significantly lower than in the control group. Hedyotis diffusa extract supplementation reduced the serum IL‐6, as compared to the control group. The serum MCP‐1 levels were significantly reduced by the consumption of Hedyotis diffusa extract. Therefore, Hedyotis diffusa could be beneficial for alleviating NAFLD by reducing inflammation.

Grant Funding Source: This research was supported by NRF in Korea (20100011980)
Effect of vitamin D3 supplementation on thyroid hormones and deiodinase 2 expression in streptozotocin‐induced diabetic rats (688.9)
Authors: Alrefaie Zienab
Published: June 2021

Aim: The present study aimed to assess the effect of vitamin D3 administration on deiodinase 2 (D2) mRNA expression in various tissues of diabetic rats.

Methods: Thirty male Wistar rats (200‐250 g) were included into; control, diabetic and diabetic supplemented with vitamin D3 groups. Diabetes was induced by single intraperitoneal injection of streptozotocin 45 mg/kg. Vitamin D3 was administered orally in a dose of 500 IU/kg/day in olive oil for 10 weeks. Serum levels of free T4, free T3 and TSH were measured. Tissue homogenates from liver, kidney, muscle, femur bone, heart and brain were obtained and assessed for expression of D2 mRNA.

Results: Diabetic rats demonstrated significant increase in free T4 and significant decrease in free T3 with non significant increase in TSH compared to control rats. The changes in thyroid profile were ameliorated by vitamin D3 administration to diabetic rats. D2 mRNA expression was significantly reduced in all tissue homogenates of diabetic rats, however vitamin D3 did enhance D2 mRNA expression especially in liver and brain of diabetic rats.

Conclusion: Diabetes mellitus inhibited peripheral conversion of T4 into T3 secondary to reduction in the expression of D2 in almost all body tissues. Vitamin D3 greatly corrected the alterations in thyroid profile and D2 expression in diabetic rats and protected against the subclinical hypothyroid state that could associate diabetes mellitus.

Grant Funding Source: No
Isoform‐specific interactions between meprin metalloproteases and the catalytic subunit of protein kinase A (690.13)
Authors: Niyitegeka Jean‐Marie , Bastidas Adam , Newman Robert , Taylor Susan , Bond Judith , Ongeri Elimelda
Published: June 2021

Meprin metalloproteases are abundantly expressed in the brush border membranes of kidney proximal tubules. Meprins are also expressed in podocytes, skin, and leukocytes. Meprins have been implicated in the pathology of acute and chronic kidney injury. To understand how meprins impact kidney disease, it is important to characterize interactions between meprins and their substrates. This study evaluated isoform‐specific interactions between the catalytic subunit of protein kinase A (PKA C) and meprins. Kidney proteins from meprin αβ double knockout mice, as well as purified recombinant mouse PKA Cα, Cβ1, and Cβ2, were incubated with activated forms of either meprin A (α‐α) or meprin B (β‐β). Cleavage products were analyzed by Coomassie staining or Western blot analysis. To identify meprin cleavage sites on PKA C isoforms, full length PKA C and meprin cleaved fragments were trypsin digested, separated by C18 nanoflow liquid chromatography‐MALDI, and analyzed by ProteinPilot. To characterize the kinetics of meprin B cleavage of PKA Cα, the Km and kcat were determined using Michaelis‐Menten enzyme kinetics. Meprin A only cleaved PKA Cβ1 while meprin B cleaved all three PKA C isoforms. Moreover, meprin cleavage of PKA Cα and Cβ2 significantly decreased their kinase activity, suggesting that the impact of meprins on kidney injury may be due, in part, to modulation of PKA signaling pathways.

Grant Funding Source: Supported by NIH grants SC3GM102049, GM19301, GM19301, F31GM099415, and DK19691
Gelsolin expression modulates TGF‐beta induced cell differentiation and epithelial to mesenchymal transition in breast cancer cells (694.3)
Authors: Liou Ying‐Ming , Chen Zhi‐Yuan , Shieh Dar‐Bin , Chiu Kung‐Ying
Published: June 2021

Gelsolin (GSN) is one of actin filament‐severing proteins that play a key role in the actin cytoskeleton rearrangement and the modification of tumor cell proliferation and metastasis. TGF‐beta has been shown to initiate the Epithelial to Mesenchymal Transition (EMT) in breast cancer cells. However, the relationship between the expression level of GSN and the TGF‐beta signaling for cell differentiation and EMT progression is not clear. In this study, MDA‐MB‐231 human breast cancer cells were treated with 2 ng/ml TGF‐beta1 for 3 days, and the population of CD44+/CD24‐ cells were sorted out by flow cytometry. These cells were found to increase the gene markers for stem cell pluripotency (i.e. Oct4, Sox2 and Nanog), associated with increased expression for mesenchymal cell markers such as N‐cadherin, and vimentin, and with a concomitant decrease for epithelial marker such as E‐cadherin. In addition, TGF‐beta1 induced an increased gene expression for GSN but decreased expressions for the two DNA methyltransferases, DNMT1and DNMT3B. Methylation specific PCR analysis also showed that TGF‐beta1 caused a 50% decrease in methylation with concomitant 3‐fold increases in unmethylation on the CpG island at the GSN promotor in the CD44+ cells. These results indicated that TGF‐beta1 induced epigenetic modification of GSN might be responsible for the cancer stem cell signaling in breast cancer cells.

Grant Funding Source: Supported by NSC 102‐2320‐B‐005‐002
The effect of doxorubicin accumulation on interstitial nitric oxide production in the rat skeletal muscle (698.8)
Authors: Fabris Sergio , MacLean David
Published: June 2021

The purpose of the present study was to examine the effect of DOX accumulation on interstitial nitric oxide (NO) following the administration of 1.5 or 4.5 mg/kg DOX after 24, 48, 72, 96, 120, 144, 168 and 192 hrs. Interstitial dialysate samples of anaesthetized rats (n=3 control, n=6 treatment) were collected via microdialysis probes (n=4 per rat) and analyzed. Following the 1.5 mg/kg dose, NO increased (P<0.05) after 24 (5.48±0.33 µM), 48 (7.06±0.17 µM), 72 (5.17±0.2 µM), 96 (10.11±0.62 µM), 120 hrs (6.77±0.26 µM) and decreased (P<0.05) at 168 hrs (1.69±0.2 µM) compared to control (2.94±0.36 µM). A dose of 4.5 mg/kg increased NO (P<0.05) after 24 (4.8±0.2 µM), 48 (4.44±0.27 µM), 96 (9.69±1.06 µM), 168 (3.95±0.2 µM) and 192 hrs (4.13±0.16 µM) compared to control. Interestingly NO was decreased (P<0.05) with the 4.5 mg/kg dose after 48 and 120 hrs compared to the 1.5 mg/kg, though NO increased (P<0.05) after 168 and 192 hrs compared to the 1.5 mg/kg dose. These data demonstrate for the first time that DOX accumulation in the SM increases interstitial NO and this increase is dependent on the administered dose, suggesting that DOX administration stimulates interstitial NO production.

Grant Funding Source: Supported by NSERC
Directional dependent stretch‐induced response in neonatal mouse cardiomyocytes (699.3)
Authors: Buchholz Kyle , Zambon Alexander , McCulloch Andrew , Omens Jeffrey
Published: June 2021

Static, mechanical stretch of neonatal cardiomyocytes induces changes in gene expression and can be used as a model to study cardiac hypertrophy. It has been hypothesized that the location of mechanosensors in relation to the cytoskeletal alignment may cause a directionally dependent response.

To investigate the directionally‐dependent cellular response to stretch, neonatal mouse cardiomyocytes are cultured on a deformable silicone membrane, which is micropatterned to create parallel grooves that align the cardiomyocytes. By adjusting the groove orientation, the primary direction of stretch can be applied along or transverse to the cell alignment.

RNA sequencing (RNA‐Seq) results show a more robust gene response to longitudinal than to transverse stretch. After 30 minutes of stretch, 349 and 1161 genes are considered differentially expressed (DE) from transverse and longitudinal stretch, respectively. After 4 hours, the number of DE genes increases to 962 and 3925. Pathway enrichment analysis indicates that MAPK signaling pathways are induced by both directions of stretch; whereas only longitudinal stretch induces PI3K/Akt signaling. These gene expression and signaling results provide evidence that cardiomyocytes vary in their response to stretch direction and will help identify the mechanosensors responsible for transducing the two modes of stretch.

Grant Funding Source: 5P01HL46345
Acute alcohol intoxication antagonizes contraction‐induced mTOR signaling in skeletal muscle (702.1)
Authors: Steiner Jennifer , Navaratnarajah Maithili , Lang Charles
Published: June 2021

Acute alcohol (ethanol; EtOH) intoxication impairs basal protein synthesis and mTOR signaling in skeletal muscle but whether EtOH also blunts contraction‐induced anabolic signaling is unknown. Fasted male mice were injected IP with 3g/kg EtOH or saline (Con), after which they were anesthetized and the right hindlimb was electrically stimulated (10 sets of 6 contractions) via the sciatic nerve. The gastrocnemius (GAS) was collected 30 min, 4 h or 12 h after stimulation. In vivo protein synthesis (PS) determined by the flooding dose technique was decreased 17% at 30min and increased 14% at 4 h in Con mice but was unchanged in EtOH‐treated mice. EtOH blunted the stimulation‐induced increase in phosphorylation of S6K1 T389 (45‐57%), S6K1 T421/S424 (15‐52%), rpS6 S240/244 (37‐72%) and 4E‐BP1 (37‐56%) at 30min, 4 h and 12 h, and eIF4B S422 at 30min. Akt S473 was unchanged by alcohol and/or stimulation as were S6K1 downstream signaling factors eEF2 T56 and PDCD4 S67. Injection of Torin2 in separate mice suggested that stimulation‐induced increases were mTOR‐mediated. Therefore, acute alcohol intoxication (i.e. binge drinking) attenuated the stimulation‐induced increase in protein synthesis and related mTOR signaling and this anabolic resistance may contribute to chronic alcohol induced muscle wasting and myopathy.

Grant Funding Source: R37 AA011290
Effects of Pueraria lobata/Rehmannia glutinosa combination with exercise on metabolic changes in ovariectomized rats (702.6)
Authors: Oh Sang A , Kim Haein , Lim Yeni , OK Hyang Mok , Do Gyeong‐Min , Kwon Oran
Published: June 2021

There is a fair amount of evidence showing that increased risks of obesity and insulin resistance are associated with postmenopausal state, but can be modulated by diet and exercise. In this study, we evaluated the effects of Pueraria lobata/Rehmannia glutinosa combination (PR), exercise, or both on metabolic changes in ovariectomized (OVX) Sprague Dawley rats for 8 weeks: Sham control, OVX control, OVX+PR (200 and 400 mg/kg bw), OVX+EX, OVX+EX+PR (200 and 400 mg/kg bw) (n=10/group). PR was fed by gavage and incremental treadmill exercise was used. Oral glucose tolerance test was measured at week 4 and week 8. After 8 weeks, fasting plasma levels of glucose, insulin, C‐peptide, total cholesterol, free fatty acids, leptin, and adiponectin were measured. mRNA expressions (Glut 4 and FOXO) and western blotting (p‐IRS and p‐AKT) were also studied in the muscle. Together, the results suggest the additive effects of PR supplementation and exercise in the management of postmenopausal metabolic changes.

Grant Funding Source: This research was supported by MOTIE/KIAT (N0000697) and MEST/BK 21 PLUS (22A20130012143).
Influence of intensity on acute aortic pulse pressure and central arterial stiffness in long‐distance runners (705.4)
Authors: Durocher Bridget , Harwood Morton , Edwards Jenna , Drum Scott , Durocher John
Published: June 2021

Aortic pulse pressure (APP) and central pulse wave velocity (cPWV) tends to decrease 60 min after distance running when compared to pre‐race values. However, it is unclear if changes in these two measures of cardiovascular health 24 hours post‐race are related to exercise intensity during competition. We sought to determine if changes in central pulse pressure and central arterial stiffness would be associated with differences in pacing. Specifically, we hypothesized that changes in APP and cPWV from 24 hours pre‐ to post‐race would be directly correlated with the percentage of vigorous activity during competition. We quantified intensity and pacing in long‐distance runners during marathon (n=4) and ultramarathon (n=8) as percentages of moderate (3‐6 METs) and vigorous (>6 METs) activity obtained from wrist accelerometry. We performed applanation tonometry to asses APP and cPWV 24 hours pre‐ and post‐race. Changes in APP (r=0.77) and cPWV (r=0.61) were directly correlated to the percentage of vigorous activity (range: 60 to 99%), and inversely correlated (r=‐0.76 and r=‐0.60, respectively) with percentage of moderate activity (range: 1 to 40%). We conclude that long‐distance runners who pace themselves with intermittent moderate intensity activity (i.e. slow jogging or walking) during competition appear to have lower central pulse pressure and lower central arterial stiffness 24‐hours post‐race.
The influence of two different menstrual cycle phases on the relationship between DNA oxidation and hydration status following exercise (705.5)
Authors: Yasuda Nobuo
Published: June 2021

The purpose of this study was to determine the relationship between urinary 8‐hydroxy‐2’‐deoxyguanosine (8‐OHdG) and urine specific gravity (USG) following two hours of cycling exercise over the follicular and luteal phases. Twelve recreationally active eumenorrheic women served as the subjects [age: 20.9±0.3 year; height: 160.1±5.6 cm; body weight: 54.7±5.8 kg; body mass index: 21.3±1.8 kg/m2; body fat: 21.9±3.1 %; peak oxygen uptake (VO2peak): 44.4±5.0 ml/kg/min (mean±SD)]. Before and after 2 hours of cycling exercise performed at 60%VO2peak, 8‐OHdG and USG were determined with high performance liquid chromatography and a handheld refractometer, respectively. All subjects performed the same exercise protocol during the follicular (F: 5~8 days after the onset of the menses) and luteal (L: 22~25 days after the onset of the menses) phase. In order to delay fatigue and prevent hypoglycemia and dehydration, carbohydrate‐electrolyte solution were consumed every twenty minutes (2 ml/kg body mass) during each exercise protocol. No menstrual cycle differences were observed in the relationship between urinary 8‐OHdG and USG following exercise, assessed with an ANCOVA. The findings of the present study indicate that the follicular and luteal phases appear to be similar in terms of the relationship between oxidative DNA damage and hydration status following exercise.

Grant Funding Source: Supported partly by funds from the Grant‐in‐Aid for Scientific Research (C) in Japan (No.23500867)
Cardiothoracic impedance does not reflect beat‐to‐beat changes in cardiac stroke volume during mild lower body negative pressure (707.1)
Authors: Holme Nathalie , Elstad Maja , Rein Erling , Walløe Lars
Published: June 2021

Background: Variability in cardiac stroke volume (SVV) has been proposed as a clinical tool to assess the magnitude of acute hemorrhage. We investigated how three non‐invasive methods represent the beat‐to‐beat changes of cardiac stroke volume (SV).

Methods: Ten healthy subjects underwent mild simulated hemorrhage through lower body negative pressure (LBNP) at ‐30 mmHg after a five minutes resting period at baseline. SV beat‐to‐beat was estimated simultaneously by three non‐invasive methods: Ultrasound Doppler (SV_usd), finger arterial blood pressure curve (SV_bpc) and cardiothoracic impedance (SV_imp). SVV was calculated at respiratory frequency between 0.15 and 0.40 Hz from spectral analysis. The median and 95% confidence intervals (CI) were calculated by Hodges‐Lehman’s estimates from epochs of 120 seconds at baseline and at ‐30 mmHg of LBNP. Statistical differences were calculated by Wilcoxon matched‐pairs signed rank test.

Results: SVV_usd and SVV_bpc decreased at ‐30 mmHg LBNP to 32% (18%, 47%) and 32% (21%, 43%) of baseline variability. In contrast, SVV_imp was unchanged at ‐30 mmHg (88% (42%, 182%), p=0.82) relative to baseline.

Conclusion: Cardiac stroke volume estimated by Ultrasound Doppler and finger arterial blood pressure curve reflect the same variations beat‐to‐beat during simulated hemorrhage. Cardiothoracic impedance does not reflect beat‐to‐beat changes in SV.

Grant Funding Source: The Research Council of Norway
Intermittent hypoxia conditioning restricts iNOS and eNOS expression in rat myocardium: a mechanism for protection from ischemia and reperfusion injury (710.4)
Authors: Manukhina Eugenia , Goryacheva Anna , Belkina Ludmila , Terekhina Olga , Mallet Robert , Downey H
Published: June 2021

Recently we demonstrated that intermittent, normobaric hypoxia conditioning (IHC) prevented injuries of myocardium and coronary blood vessels induced by myocardial ischemia and reperfusion (IR). This cardio‐ and vasoprotection of was associated with alleviation of nitric oxide overproduction. The aim of this study was to identify specific NO synthase(s) responsible for the IR‐induced NO overproduction and to determine the effect of IHC on these NO synthases. IHC of rats was performed in a normobaric chamber (5‐8 cycles/d for 20 d, FIO2 9.5 ‐ 10% for 5 ‐ 10 min/cycle, with intervening 4‐min normoxia). IR was produced by ligation of the left anterior descending coronary artery for 30 min followed by 60‐min reperfusion. The protein nitration marker, nitrotyrosine (3‐NT) and neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) nitric oxide synthases were measured by immunoblot. IR induced appreciable 3‐NT accumulation in the left ventriclular free wall, increasing the 3‐NT content by 42% (p<0.01), but not in septum. In IHC rats, 3‐NT after IR was similar to that of control rats without IR. IHC decreased iNOS by 71% (p<0.05) and eNOS by 41% (p<0.05) in the left ventricular myocardium; the myocardial content of nNOS remained unchanged. Therefore, IHC prevents IR‐induced NO overproduction in myocardium by restricting myocardial expression of iNOS and eNOS.

Grant Funding Source: Supported by the Russian Foundation for Basic Research grant 10‐04‐00980
Chronic intermittent hypoxia alters blood pressure and mesenteric small resistance arterial responsiveness in mice (710.9)
Authors: Wang Youhua , Chen Ling , Zhang Jin
Published: June 2021

Blood pressure (BP) is elevated in rats and mice exposed to chronic intermittent hypoxia (CIH). It is unclear if altered vascular function, such as myogenic reactivity (MR), contributes to BP elevation in CIH. We aimed to characterize the vascular alterations in small resistance arteries from CIH‐exposed mice. C57BL/6J mice were exposed to either CIH (1 min alterations of 5% and 21% O2) or normoxia (sham control, constant 21% O2), 8 hrs/day for 5 weeks. Compared to sham controls, CIH‐exposed mice had significantly higher BP, measured by radio telemetry. Mesenteric small resistance arteries were isolated and pressurized for diameter and wall thickness measurement. Myogenic constriction in response to intraluminal pressure increase (i.e., MR) was significantly attenuated, especially at pressures 蠅 70 mm Hg. At 70 mm Hg, vasodilations to acetylcholine and sodium nitroprusside, but not to external Ca2+ removal, were significantly impaired in CIH, whereas vasoconstrictions to phenylephrine (PE), endothelin‐1, U46619, and high [K+]o were similar between sham and CIH arteries. When intraluminal pressure was increased to 110 mm Hg, however, PE‐induced vasoconstriction was significantly reduced, and nitric oxide‐dependent vasodilation was further attenuated in CIH arteries. Arterial remodeling was not obvious since wall thickness, wall to lumen ratio, and passive diameters in Ca2+‐free solution were similar between two groups. Thus, CIH 1) elevates BP; 2) reduces MR; 3) reduces arterial responsiveness to vasodilators, and, at higher intraluminal pressure, to vasoconstrictors; 4) has no effect on arterial remodeling in mouse mesenteric small resistance arteries. It is likely that the altered arterial responsiveness is involved in the regulation of BP in CIH.

Grant Funding Source: NIH/NHLBI R01 HL107654
Regulation of inflammatory gene expression by diabetes induced long noncoding RNA in macrophages (711.1)
Authors: Reddy Marpadga , Chen Zhuo , Park Jung‐Tak , Wang Mei , Lanting Linda , Zhang Qian , Wu Xiewie , Putta Sumanth , Natarajan Rama
Published: June 2021

Long noncoding RNAs (lncRNAs) regulate gene expression in diverse patho‐physiological conditions, but their role in diabetes complications is not clear. We hypothesized that diabetes induced lncRNAs could mediate macrophage dysfunction implicated in vascular complications. We profiled gene expression in bone marrow derived macrophages (BMM) from type 2 diabetic db/db and control db/+ mice, after differentiation with GMCSF or MCSF. RT‐QPCR showed dysregulated polarization of BMM from db/db mice. RNA‐Seq revealed differential expression of 2000 transcripts including lncRNAs in BMM from db/db mice relative to db/+ mice. Differentially expressed genes were enriched in networks relevant to fibrosis, adhesion and inflammation, suggesting altered regulation of macrophage function. We validated the increased expression of a lncRNA E330013P06 (E33) in BMM and peritoneal macrophages from db/db and diet induced type 2 diabetic mice as well as macrophages treated with high glucose and palmitic acid. Furthermore, overexpression and siRNA mediated gene silencing demonstrated that E33 can increase inflammatory genes and foam cell formation in macrophages, establishing a novel role for a diabetes induced lncRNA in key macrophage functions involved in diabetic complications. Our findings suggest that understanding of lncRNA dependent mechanisms in macrophages could lead to novel therapeutic interventions.

Grant Funding Source: NIH‐NIDDK
Protease‐activated receptor‐2 regulates BK channel activity in rat vagal bronchopulmonary sensory neurons (712.4)
Authors: Moss Charles , Gabriel Sabry , Gu Qihai
Published: June 2021

Activation of protease‐activated receptor‐2 (PAR2) by endogenous or exogenous agonists contributes to airway inflammation and airway hyperresponsiveness, the hallmark features of allergic asthma; and a neurogenic mechanism involving PAR2 activation‐induced hypersensitivity of bronchopulmonary sensory nerves has been indicated. Large‐conductance Ca2+‐activated potassium (BK) channels are known to play an important role in shaping the neuronal excitability. The aim of this study was to investigate the role of BK channels in contributing to the PAR2‐induced neuronal hyperexcitability. Our immunohistochemical and RT‐PCR experiments demonstrated the expression of BK channels in rat bronchopulmonary sensory neurons. In whole‐cell perforated patch‐clamp recordings, BK currents were isolated from total K+ currents with an application of selective BK channel blocker paxilline. Our results showed that pretreatment with PAR2‐activating peptide (100 µM, 2 min) significantly reduced the BK current density in these sensory neurons. Our data suggest that the depression of BK channel activity and subsequent elevation of neuronal excitability of bronchopulmonary afferents may contribute to the pro‐inflammatory effects of PAR2 activation in the lungs and airways.

Grant Funding Source: Supported by funds from MEDCEN and MUSM
Differential contributions of M2 and M3 muscarinic receptors to breathing and neurokinin and amino acid concentration in the ventral respiratory column (712.8)
Authors: Muere Clarissa , Neumueller Suzanne , Olesiak Samantha , Miller Justin , Langer Thomas , Hodges Matthew , Pan Lawrence , Forster Hubert
Published: June 2021

Previously, it has been shown that dialysis of the non‐selective muscarinic receptor (mAChR) antagonist, Atropine (ATR, 50 mM), in vivo into the ventral respiratory column (VRC) of goats elicits an increase in breathing frequency associated with increases in substance P (SP), glycine, and GABA (Muere et al., J Appl Physiol, 114: 694‐704, 2013). To determine the specific mAChR subtype(s) mediating these effects, the specific M2 mAChR antagonist, Methoctramine (ME, 50 μM), and the specific M3 mAChR antagonist, 4‐DAMP (5 mM), were individually dialyzed into various sites in the VRC of adult goats chronically implanted with microtubules. Breathing was continuously measured and the effluent dialysate collected for quantification of neurochemical content. Preliminary results show that dialysis of ME tended to increase breathing frequency (n=4). ME tended to decrease levels of the amino acids glutamine, glycine, and GABA, while having no effect on SP concentration. Dialysis of 4‐DAMP had no effect on breathing frequency (n=2) and had variable effects on amino acid concentration. However, 4‐DAMP increased SP concentration to levels comparable with dialysis of ATR at a lower dose of 5 mM. These data suggest that the effect of ATR on breathing and neurochemical content is not due to a predominant effect on a specific mAChR subtype. Rather, a combination of mAChR subtypes differentially regulates multiple neuromodulators that affect respiration.

Grant Funding Source: Supported by NHLBI Grants HL‐25739 & HL‐007852 & Department of Veterans Affairs
Enhanced phrenic long‐term facilitation in rats with motor neuron death from intrapleural CTB‐saporin injections (713.1)
Authors: Nichols Nicole , Mitchell Gordon
Published: June 2021

Amyotrophic lateral sclerosis (ALS) is a devastating disease leading to progressive motor neuron degeneration and death by ventilatory failure. In a model of ALS (SOD1G93A), pLTF following acute intermittent hypoxia is enhanced at disease end‐stage by unknown mechanisms (Nichols et al., ibid, 2010). Intrapleural injections of cholera toxin B fragment conjugated to saporin (CTB‐SAP) selectively kills respiratory motor neurons and mimics motor neuron death observed in ALS rats (Nichols et al., ibid, 2013). This CTB‐SAP model allows us to study the impact of respiratory motor neuron death on breathing without many complications attendant to ALS. Here, we tested the hypothesis that phrenic motor neuron death is sufficient to enhance pLTF. pLTF was assessed in anesthetized, paralyzed and ventilated Sprague Dawley rats 7 and 28 days following bilateral intrapleural injections of: 1) CTB‐SAP (25μg), or 2) un‐conjugated CTB and SAP (control). CTB‐SAP enhanced pLTF at 7 (CTB‐SAP: 145±22%, n=10 vs. Control: 54±5%; n=15; p<0.05), but not 28 days post‐injection (CTB‐SAP: 61±14%, n=11 vs. Control: 37±9; n=5; p>0.05). Thus, pLTF at 7 (not 28) days post‐CTB‐SAP more closely resembles pLTF in end‐stage ALS rats, suggesting that processes unique to the period of active motor neuron death enhance pLTF. This project increases our understanding of respiratory plasticity and its implications for breathing in motor neuron disease.

[Supported by NIH HL080209, HL69024 and the Francis Families Foundation]

Grant Funding Source: Supported by NIH HL080209, HL69024 and the Francis Families Foundation
Inactivity‐induced phrenic motor facilitation does not require new protein synthesis (713.8)
Authors: Baertsch Nathan , Strey Kristi , Baker‐Herman Tracy
Published: June 2021

Reduced respiratory neural activity elicits a TNFα‐ and atypical PKC‐dependent form of spinal plasticity in phrenic motor output termed inactivity‐induced phrenic motor facilitation (iPMF), a long‐lasting increase in phrenic nerve burst amplitude apparent once respiratory neural activity is restored. Many forms of neuroplasticity require protein synthesis for long‐term expression, including the most widely studied form of spinal respiratory plasticity, phrenic long‐term facilitation (pLTF; Baker‐Herman and Mitchell, 2002) elicited following acute intermittent hypoxia. We hypothesized that iPMF would also require de novo protein synthesis. Briefly, Sprague Dawley rats were urethane anesthetized, vagotomized, paralyzed, and prepared for electrophysiological recording. Rats received intrathecal injection (C4) of vehicle (aCSF; n=5) or the protein synthesis inhibitor emetine (n=6; 1ug/kg; Baker‐Herman and Mitchell, 2002) ~15 min prior to a prolonged (30min) period of neural apnea. As expected, in aCSF treated rats, phrenic nerve amplitude was significantly increased compared to baseline 5 min following reversal of neural apnea (53±11, %baseline) and was maintained for >60 min (54±16, %baseline), indicating long‐lasting iPMF. Contrary to our hypothesis, rats receiving emetine also expressed long‐lasting iPMF such that phrenic nerve burst amplitude was significantly increased from baseline at both 5 (41±8, %baseline) and 60 min (42±12,%baseline) following neural apnea (p<0.05). Importantly, phrenic nerve burst amplitude in rats receiving intrathecal emetine was not significantly different from rats receiving aCSF at any time point following neural apnea (p>0.05). Together, these data suggest that iPMF is a form of long‐lasting plasticity that does not require new protein synthesis.

Grant Funding Source: Supported by NIH HL105511