bluuebunny/crossref_metadata_2025 huggingface dataset:
Disruption of endothelial cell/pericyte adhesion by PAI‐1 in ischemic limb of diabetic mouse
Authors: Wu Jianbo , Strawn Tammy L , William Fay P
Published: June 2021

Background

Disruption of pericytes and acellular capillariesare increased in the feet of human diabetic patients. Selectiveloss of adhesion of pericytes to endothelial cells is known toearly event in diabetic microangiopathy. In diabetes, increased levels of PAI‐1 are strongly associated with endothelial dysfunction and progression. Based on these findings, we investigated whether PAI‐1 plays a role in modulating endothelial/pericyte adhesion inischemic hindlimbs of diabetic mice.

Methods

Type I diabetes was induced by intraperitoneal administrationof streptozocin in both PAI‐1 deficient mice (PAI‐1−/−) and WT mice. Ligation of femoral arterywas conducted to develop hindlimb ischemia. Plasma was collected for measurement of PAI‐1 and advanced glycated end products (AGE) levels by ELISA. Ischemic gastroceminus muscles were isolated. Adhesion ofpericytes to endothelial cells was assessed by scanning confocal and electron microscopy.

Results

Plasma total and active PAI‐1 levels were significantly increased in WT mice with STZ‐induced diabetes. PAI‐1−/−‐STZ was found to suppress plasma AGE level compared to WT‐STZ mice (basal level: 4.74 ± 1.19 Vs. 4.28 ± 1.03, n=8; STZ status: 5.54 ± 0.28 Vs. 9.75 ± 1.73, n=8, p< 0.05). Attachment between endothelial cell andpericyte from schemic gastroceminus muscles in PAI‐1−/−‐STZ was markedly increased compared with that in WT‐STZ mice.

Conclusions

Increased PAI‐1 levels disrupt endothelial cell/pericyte adhesionin ischemic limb of the diabetic mouse. These results identify an important role for PAI‐1 in diabetic microangiopathy.
MiR‐133 As An Epigenetic Regulator Of Diabetic Heart Failure
Authors: Chavali Vishalakshi , Tyagi Neetu , Tyagi Suresh C , Mishra Paras Kumar
Published: June 2021

The miRNA has emerged as a therapeutic target for diabetic cardiomyopathy (DCM). Although epigenetic regulation of specific genes associated with DCM has been documented, the role of miRNAs in epigenetic regulation is nebulous. We tested the hypothesis that miR‐133 inhibits DNA methylation by down regulating two major genes: DNA methyltransferase (DNMT3a) and methyl‐CpG‐binding domain (MBD2) in diabetes. To prove the hypothesis, we transfected HL1 cardiomyocytes with miR‐133 and scrambled. The successful transfection was validated by GFP marker and qPCR of miR‐133 (sno234 was endogenous control). We used five treatment groups‐ a) control (CT), b) high glucose (HG‐25mM for 24 h), c) scrambled (Scr), d) miR‐133 transgenic (miR), and e) miR‐133 transgenic treated with high glucose (miR+glu) to analyze the expression of DNMT3a and MBD2 using multiplex RT‐PCR. The results revealed that DNMT3a and MBD2 are induced in HG group as compared to that of CT. However, the expression of these genes are attenuated in miR and miR+glu as compared to HG. These results suggest that high dose of glucose induces DNA methylation by up regulating DNMT3a and MBD2, whereas miR‐133 mitigates them. Our findings provide a novel insight of miR‐133 as an epigenetic regulator of DCM.
Brain‐Derived Neurotrophic Factor Intensifies the Early Inflammatory Response After Myocardial Infarction
Authors: Halade Ganesh V , Ramirez Trevi A , Zhang Jianhua , Hensler Julie G , Jin Yu-Fang , Lindsey Merry L
Published: June 2021

Plasma brain‐derived neurotrophic factor (BDNF) levels correlate positively with indices of metabolic and cardiovascular disease, but role of BDNF in the cardiac response to myocardial infarction (MI) has not been investigated. BDNF heterozygous (HET) mice become obese and insulin resistant with age. We hypothesized that 50% reduced levels of BDNF would impair post‐MI cardiac remodeling. We compared 7–9 month old male C57BL/6J wild type (WT) and HET mice (n=11/group) at day 0 and days 1,3,5 and 7 post‐MI (n=11–18/group). WT and HET mice had similar infarct sizes (43–50±2% for 1–7 days MI). Plasma proteomic profiling at day 1 post‐MI revealed that, monocyte chemotactic protein (MCP)‐1 (177±pg/ml in WT and 228±25pg/ml in HET) and MCP‐5 (38±4pg/ml in WT and 49±5pg/ml in HET) levels were enhanced in the HET mice, compared to day 0 controls and WT day 1 levels (p<0.05). By inflammatory RT2‐PCR array, the mRNA expression of MCP‐1 and MCP‐5 were also increased in HET compared to WT at day 1 (p<0.05). Interestingly, matrix metalloproteinase‐9 (129±11ng/ml in WT and 96±4ng/ml in HET) and myeloperoxidase (183±13ng/ml in WT and 147±7ng/ml in HET) levels increase in WT plasma post‐MI, and these increases were attenuated in HET mice at day 1 (p<0.05). Our finding demonstrates that reduced BDNF in the post‐MI setting modifies the early inflammatory response to MI, potentially by altering neutrophil and macrophage kinetics. Funding support ‐ NIH/NHLBI/NCCAM and the VA.
DUAL ROLE OF STAT3 IN HYPERTENSION‐INDUCED CARDIAC REMODELING
Authors: Zouein Fouad Anthony , Zgheib Carlos , Hamza Shereen , Hall John E. , Ruiz Arnaldo , Juncos Luis , Booz George W.
Published: June 2021

STAT3 has a role in innate immunity and inflammation, but involvement of this transcription factor in cardiac remodeling is controversial. While heart‐targeted STAT3 knockout mice have heart failure with age, unconstrained STAT3 activity was linked to heart dilation in the infarcted myocardium. STAT3‐induced gene expression requires Y705 phosphorylation and is enhanced by S727 phosphorylation. This study assessed the role of STAT3 in heart remodeling induced by hypertension. Four month old SASA mice having a STAT3 S727A mutation that precludes phosphorylation were treated with angiotensin (Ang) II (1000 ng/kg/min) or vehicle for 17 days. Systolic blood pressure (mm Hg) increased in both SASA (169 ± 3) and wild type (WT) mice (166 ± 3) with Ang II. Cardiac levels of inflammatory cytokines were increased with Ang II in both WT and SASA mice, but less so in SASA: Fold increases in IL‐6, TGFβ, VEGF, and MCP1 for WT/SASA mice were respectively: 9/4, 10/4, 3/2, and 16/4. Modest impairments in systolic (ejection fraction & fractional shortening) and diastolic (mitral valve E:A ratio) function were seen with Ang II in both WT and SASA mice, but more so in SASA. Interstitial fibrosis and loss of myofibrillar integrity with Ang II was more pronounced in SASA mice. Our findings show that STAT3 contributes to inflammation of the heart that occurs with hypertension‐induced cardiac remodeling, but also has a role in cardiac protection.
Communication between Cardiomyocytes and Cardiac Fibroblasts: Mechanistic Role of ROCK1 in Fibrotic Cardiac Remodeling
Authors: Yang Xiangsheng , li qi , Lin Xi , yue xiaojing , chang jiang
Published: June 2021

Study objective

Our previous study found that Rho associated coiled‐coil protein kinase 1 (ROCK1) was cleaved into an active isoform ROCKΔ1 in failing human hearts. The study is to investigate the pathological consequence of ROCKΔ1 accumulated in heart and the associated molecular mechanism.

Methods and Results

We generated transgenic mice expressing ROCKΔ1 in cardiomyocytes to mimic human heart disease. Mutant mice developed fibrotic cardiomyopathy with diastolic dysfunction. Transgenic heart displayed the activation of TGFβ1 and NF‐κB signaling. Treatment with Rho kinase inhibitor attenuated the phenotype. Cardiac fibroblasts were differentiated into myofibroblasts by co‐culturing with transgenic cardiomyocytes but not with wild‐type cardiomyocytes. Inhibitors of Rho kinase, TGFβR1 and NF‐κB diminished the process. A new cis‐regulatory element of TGFβ1, the serum response factor‐dependent TGFβ1 regulation, was responsible for Rho kinase‐mediated TGFβ1 signaling activation.

Conclusions

The results demonstrate that ROCKΔ1 is a novel and potent fibrogenic factor. Activation of TGFβ1 and NF‐κB signaling along with a subset of cytokines released from cardiomyocytes induces cardiac fibroblast maturation, facilitates collagen deposition and macrophage infiltration. Both pathways synergistically contribute to Rho kinase‐mediated pathological fibrosis.

Supported by NIH R01HL102314.
S1P induces CCN1 expression through RhoA/MRTF‐a activation and protects cardiomyocytes against cell death
Authors: Ding Eric , Miyamoto Shigeki , Zhao Xia , Xiang Sunny Yang , Neubig Richard R , Brown Joan Heller
Published: June 2021

The GPCR agonist sphingosine 1‐phosphate (S1P) protects heart against ischemia/reperfusion (I/R), but the mechanisms are not fully understood. The matricellular protein CCN1 expression is induced by GPCR agonists and subsequently secreted from the cell, where it activates integrin signaling. We determined whether CCN1 contributes to S1P mediated cardioprotection. In neonatal rat ventricular myocytes (NRVMs), S1P treatment induces CCN1 expression as early as 30 min and the response is sustained over 24hours. S1P is a robust activator of RhoA in the heart and CCN1 induction by S1P is blocked when RhoA function is inhibited by C3 toxin. In addition RhoA overexperssion is sufficient to induce CCN1 expression. We tested the role of MRTF‐a, a coactivator of SRF, in S1P mediated CCN1 expression. S1P induces nuclear accumulation of MRTF‐a which is inhibited by C3 toxin. Pharmacological inhibitors of MRTF‐a or knockdown of MRTF‐a largely diminishes S1P mediated CCN1 induction. To determine the functional significance of this pathway, simulated I/R (sI/R) induced apoptosis was measured. S1P treatment protects NRVMs against sI/R and this protection is decreased by MRTF‐a inhibition or CCN1 knockdown. These results suggest that CCN1 is an important mediator of S1P induced cardioprotection and regulated by RhoA/MRTF‐a signaling.
First determination of ATP in alveolar epithelium in situ, effect of mesenchymal stem cells
Authors: Islam Mohammad Naimul , Sun Li , Das Shonit Ranjan , Bhattacharya Jahar
Published: June 2021

The ATP probe, GlnKII‐GFP (Perceval®; Berg, 2009) enables quantification of ATP in single cells. Here, we used the probe for the first time in situ to determine whether mitochondrial transfer from bone marrow‐derived mesenchymal stem cells (MSCs) increases ATP in pulmonary alveoli. We transfected mouse lungs with the probe, then 24 hours later we gave mice LPS by airway instillation, followed a further 4 hours later with instillation of MSCs. After another 24 hours, we isolated and blood‐perfused the lungs and viewed alveoli by live confocal microscopy. In control lungs, Perceval fluorescence was well evident in the alveolar epithelium (AE), indicating successful transfection. The fluorescence decreased by 25% after LPS (P<0.05), but was restored to control levels by MSC instillation (P<0.05). Concomitantly, induced surfactant secretion by alveolar type 2 cells, an ATP‐dependent response, was blocked by LPS (P<0.05), but restored by subsequent MSC instillation (P<0.05). We conclude, the Perceval probe enables ATP assays in intact cells of organ systems amenable to live confocal microscopy. Our findings indicate that in the AE, LPS decreased ATP and blocked surfactant secretion. However, MSCs rescued ATP production and ATP‐dependent surfactant secretion in this pneumonitis model. Thus, by restoring alveolar mitochondrial function, MSCs may rescue overall lung function in sepsis (support HL64896).
Src Family Kinases collaborate with distinct TNF‐alpha‐induced signaling pathways to regulate actin dynamics at cell‐cell junctions and barrier function in endothelial cells
Authors: Adam Alejandro Pablo , Lowery Anthony , Vincent Peter
Published: June 2021

We previously showed that activation of Src Family Kinases (SFK) by expression of dominant negative C‐terminal Src Kinase (DN‐CSK) is not sufficient to decrease endothelial AJ integrity. However, SFK activation by expression DN‐CSK results in the formation of very dynamic lamellae that show a distinct reticular pattern of adherens junction (AJ) proteins, localization of cortactin at the leading edge of the lamellae and activation of SFK at the cell‐cell junctions. DN‐Csk also induced β3 integrin activation and phosphorylation at the AJ. TIRF live imaging showed actin polymerization preceding AJ formation at the borders of lamellae in close contact with the extracellular matrix. Interestingly, low doses of TNF‐α that do not decrease TEER or cortical actin in control cells did produce a decrease in TEER in cells expressing DN‐Csk, together with a loss in the cortical actin band and the formation of short, thick bundles of actin. These actin changes were prevented by inhibition of ROCK activity or β3 integrin binding, even though TEER was still decreased. Preliminary data show that TEER decrease can be prevented by inhibiting p38 activity. These results suggest that the loss of endothelial barrier function induced by TNF‐α and SFK activation utilizes a p38‐dependent pathway, while a β3 integrin‐ and Rho‐ROCK‐dependent pathway is required for the cytoskeletal changes. Supported by NHLB‐HL077870.
Development of complex model systems for analysis of cell‐cell and cell‐microenvironment interactions in breast cancer
Authors: Andersen Anne Poder , Ronnov-Jessen Lone , Hulikova Alzbeta , Swietach Pawel , Pedersen Stine Falsig
Published: June 2021

To study cancer development it is essential to employ models that recapitulate the tumor microenvironment. We previously showed that the Na+/H+ exchanger NHE1 and the Na+, HCO3− cotransporter NBCn1 are essential for pH regulation in MCF‐7 breast cancer cells and their activity increased by ΔNErbB2 expression. Furthermore, NHE1 inhibition strongly sensitized ΔNErbB2‐ expressing MCF‐7 cells to chemotherapy‐induced death. Here, we explore the impact of specific aspects of the tumor microenvironment on pH‐regulatory and survival/motility signaling programs. Simulation of the tumor microenvironment (pHe 6.5, 1% O2, 7.5 mM lactate) elicited marked upregulation of Akt activity (Akt S473 phosphorylation) in MCF‐7 cells ± ΔNErbB2. In 2D co‐culture with primary human breast fibroblasts, MCF‐7 cells appeared to upregulate the lactate‐H+ cotransporter MCT1 and EMT markers such as α‐smooth muscle actin. Finally, in MCF‐7 spheroids (3D multicellular growths), the expression patterns of NHE1, NBCn1 and pAkt/Akt were altered compared to 2D mono‐culture controls. In conclusion, the tumor microenvironment profoundly impacts on pH regulatory ion transport proteins and survival signaling in breast cancer cell models. Funding: Novo Foundation.
ERK1/2‐dependent bestrophin‐3 (Best‐3) expression prevents ER‐stress‐induced cell death of renal epithelial cells by reducing CHOP
Authors: Lee Wing-Kee , Chakraborty Prabir K , Roussa Eleni , Wolff Natascha A , Thévenod Frank
Published: June 2021

Upon ER stress induction, cells endeavor to survive by engaging the adaptive stress response, known as the unfolded protein response, or by removing aggregated proteins. Chronic ER stress leads to apoptosis through induction of pro‐apoptotic CHOP. Here, we show that Best‐3, a Ca2+‐activated Cl−channel, is upregulated by the ER stressors, thapsigargin (TG, 3 μM), tunicamycin (TUN, 6 μM) and the toxic metal Cd2+ (25 μM). In cultured rat kidney proximal tubule cells, ER stress after 6 h by Cd2+, TG and TUN was downstream of reactive oxygen species formation and attenuated by Ca2+ chelator BAPTA‐AM (10 μM) or general antioxidant α‐tocopherol (100 μM). Immunofluorescence staining localized Best‐3 to the plasma membrane, nuclei and intracellularly, though not in the ER, in cultured cells and rat kidney cortex sections. ER stress augmented Best‐3 mRNA and was decreased by α‐tocopherol, BAPTA‐AM, calmodulin kinase inhibitor calmidazolium (40 μM), ERK1/2 inhibitor U0126 (10 μM), and ERK1/2 RNAi. Knockdown of Best‐3 resulted in decreased cell number consequentially of cell death, as determined by nuclear staining and PARP‐1 cleavage. Furthermore, Best‐3 overexpression reduced cell death by Cd2+, TG and TUN, attributed to diminished CHOP. From our novel data, we conclude that ERK1/2‐dependent Best‐3 activation regulates cell fate decisions during ER stress by suppressing CHOP induction and death.

Funded by DFG TH345/10‐1
Specific targeting of pancreatic β‐cells for imaging and therapy using multivalent targeting of receptor combinations
Authors: Hart Nathaniel , Vagner Josef , Chung Woo Jin , Weber Craig , Limesand Sean , de Silva Channa , Lynch Ronald
Published: June 2021

Perturbations in pancreatic β‐cell mass (BCM) and function underlie the development of Diabetes Mellitus. Current treatments and analysis of BCM are limited by inadequate specificity of β‐cell targeting agents. We propose improved specificity of β‐cell agents can be obtained by targeting unique combinations of cell surface proteins that distinguish β‐cells from other cells within the abdomen. We synthesized a multivalent ligand composed of two different receptor binding moieties: Glucagon Like Peptide‐1 (GLP‐1) and Glibenclamide (Glb, Sulfonyurea ligand, SUR1). Both GLP1R and SUR1 are expressed in the βTC3 cell line. The bivalent ligand binds to βTC3 cells with an apparent Km of ~10nM, and binding is right‐shifted by competition with monomeric GLP‐1 or Glb, indicating higher affinity binding is due to multivalent interactions. GLP‐1 elevates cAMP while Glb increases Ca2+in β‐cells. We observe that the GLP‐1/Glb elicits a half maximal Ca 2+response at ~10nM reflecting its Km but the magnitude of the Ca 2+ response was at least 10X less than monomeric Glb. Unlike the Ca 2+response, cAMP production was depressed by only ~20% compared to monomeric GLP‐1. Although signaling is depressed, glucose activated insulin secretion is potentiated to similar levels by both monomeric GLP‐1 and GLP1/Glb. Our findings indicate that multivalent ligands may provide cell type specific agents with tunable functional behavior.

Supported by Juvenile Diabetes Research Foundation.
Obscurins: Giant proteins with tumor suppressing activities in breast cancer
Authors: Perry Nicole Alana , Shriver Marey , Mameza Marie , Grabias Bryan , Balzer Eric , Kontrogianni-Konstantopoulos Aikaterini
Published: June 2021

Obscurins (~70–870 kDa) are cytoskeletal proteins originally identified in striated muscles where they hold structural and regulatory roles. Recently, analysis of 13,023 genes in breast and colorectal cancers identified OBSCN as one of the most frequently mutated genes, implicating it in cancer formation and progression. Herein, we studied the expression profile of obscurins in breast, colon and skin cancer cell lines, and their involvement in cell growth. Immunoblot analysis demonstrated significant reduction of obscurin proteins in cancer cell lines. MCF10A breast epithelial cells treated with shRNAs targeting obscurins exhibited significantly increased survival and reduced apoptosis accompanied by moderate anchorage independent growth, following exposure to the DNA damaging agent etoposide. Quantitative RT‐PCR indicated that the anti‐apoptotic genes Bag‐4 and HAX‐1 were up‐regulated in MCF10A obscurin‐null cells, whereas initiator caspase‐9 and death caspase‐3 transcripts were down‐regulated. Our findings are the first to pinpoint critical roles for obscurins in cancer development by contributing to the regulation of cell survival and apoptosis.
Caveolin isoform switch in differentiating C2C12 cells is not prevented by palmitate
Authors: Pinz Ilka M. , Vittorioso Melissa , Knowles Catherine J.
Published: June 2021

Caveolae are membrane invaginations and important for cellular signaling. In skeletal muscle the expression of caveolin‐3 (cav3) is required for normal muscle fiber orientation and function, and caveolin‐1 (cav1) is constitutively expressed. Palmitate (palm) decreases membrane localization of cav3 in cardiomyocytes. Thus, we tested the hypothesis that palm impairs myotube formation in differentiating C2C12 cells. Two time points were tested: 1) After myotube formation: C2C12 myoblasts and myotubes were treated with 0.4 mM palm for 18 hours and 2) during differentiation: C2C12s were treated at 50–60% confluency. Western blot, IP and confocal microscopy determined expression and localization of cav1 and 3. In controls an isoform switch from cav1 in myoblasts to cav3 in myotubes occurred. Palm exposure after tube formation suppressed cav3 expression in confocal images. During differentiation, C2C12s form myotubes in the presence of palm, however the morphology was changed to long and spindly compared to controls. Palm increased the number of myotubes, but considering the changed morphology this suggests inhibition of fusion to larger myotubes. These data suggest that a caveolin isoform switch occurs in the differentiation of C2C12s from cav1 to cav3, and that cav3 expression is important for the correct morphology of myotubes formed.

Support by AHA SDG 0930260N to IMP
Prolactin‐stimulated ubiquitination of the zinc transporter ZnT2 regulates zinc secretion
Authors: Seo Young Ah , Kelleher Shannon L
Published: June 2021

Zinc (Zn), an essential divalent cation, is required for over 300 biological processes including DNA and protein synthesis and intracellular signaling. The Zn transporter ZnT2 regulates Zn export from mammary epithelial cells (MECs) into milk during lactation. Tight regulation of ZnT2 is critical to ensure adequate Zn transfer to the infant yet maintain Zn homeostasis in MECs. Prolactin (PRL) regulates nutrient production and secretion from MECs. PRL signaling is deactivated through ubiquitination and degradation of its receptor (PRLr). Herein, we report that after an initial increase, PRL decreased Zn secretion in endogenous and ZnT2‐overexpressing MECs. This resulted from ZnT2 internalization and ubiquitination, which targeted ZnT2 for proteasome‐dependent degradation. Mutagenesis identified two N‐terminal lysine residues (K4 and K6) necessary for ubiquitin attachment. Mutation of these lysine residues to arginine (K4R and K6R) inhibited ubiquitination and degradation, which confined ZnT2 to the cell membrane and maintained Zn secretion. These findings indicate that PRL‐mediated activation of ubiquitin‐dependent ZnT2 degradation represses Zn secretion from MECs. This suggests that PRL‐stimulated ubiquitination regulates ZnT2 function to maintain optimal cell function in mammary cells and provides insight into novel mechanisms through which ubiquitination regulates ion transport.
Forskolin stimulation promotes urea transporter UT‐A1 ubiquitination, endocytosis and degradation in MDCK cells
Authors: Chen Guangping , Su Hua , Carter Conner B , Fröhlich Otto , Sands Jeff M
Published: June 2021

Adenylyl cyclase stimulator forskolin (FSK) stimulates UT‐A1 phosphorylation, membrane trafficking as well as urea transport activity. Here, we found that forskolin stimulation induces UT‐A1 ubiquitination in UT‐A1 MDCK cells. This suggests that phosphorylation by FSK also activates the UT‐A1 protein degradation machinery. UT‐A1 MDCK cells were treated with 100μg/ ml cycloheximide to inhibit proteins synthesis, with or without 10 μM FSK. Total UT‐A1 protein abundance was significantly reduced after FSK treatment. We then specifically examined the degradation of cell surface UT‐A1 by cell surface biotinylation. UT‐A1 MDCK cells were pretreated with 2 μM brefeldin A (BFA) for 2 h to block the newly produced protein from trafficking to the cell membrane. FSK treatment accelerated UT‐A1 removal from the cell plasma membrane. Proteasome inhibitor MG132 reduced FSK‐induced membrane UT‐A1 reduction. The reduced cell membrane UT‐A1 is due to an increase in UT‐A1 endocytosis as judged by biotinylation and MesNa treatment. We further found that UT‐A1 activation/phosphorylation by FSK promotes its binding to 14–3‐3γ and ubiquitin E3 ligase MDM2. Our study shows that FSK activates UT‐A1 urea transporter and the activation/phosphorylation subsequently triggers the downregulation mechanism of UT‐A1 which is important for the cells to limit the reaction in response to stimulation and enable the cells back to basal conditions.
Estradiol increases the density and open probability of epithelial sodium channels in alveolar cells
Authors: Greenlee Megan M , Duke Billie Jeanne , Eaton Douglas C
Published: June 2021

Female sex hormones stimulate the expression of the lung epithelial sodium channel (ENaC); however, the effect of female sex hormones on channel number (N) and open probability (Po) has not been examined. We hypothesized that ENaC activity (NPo) will increase in lung alveolar cells treated with β‐estradiol and progesterone. Rat L2 alveolar‐type II‐like cells were cultured on transwells with apical air interfaces and steroid‐free basolateral media supplemented with vehicle (0.02% ethanol), β‐estradiol (200 pg/mL), progesterone (20 ng/mL), or a combination (200 pg/mL β‐estradiol and 10 ng/mL progesterone). We used cell‐attached patch clamp to measure ENaC NPo 18–28 hours after treatment. Combined hormone treatment increased the number of patches with channels from 38% to 88% (n=8, P<0.05) and increased ENaC NPo from 0.011 ± 0.008 to 0.47 ± 0.22 (n=8, P<0.01). The channel mean open time increased from 20 ± 2.5 ms (vehicle) to 73 ± 30 ms with hormone treatment (n=3–7, P<0.05). β‐estradiol alone activated ENaC (NPo=0.31 ± 0.18); progesterone alone did not (NPo= 0.001 ± 0.001). These results show that β‐estradiol stimulates ENaC activity in alveolar cells. Considering recent data showing greater ENaC activity in lungs from CFTR‐null mice, these results may have important implications for female cystic fibrosis patients at the onset of puberty.

Funding: NIH 5R37DK037963‐25 and 5K12GM000680‐12 to DCE
Manipulating cytosolic ATPases alters both energetics and p38 MAPK activation in contracting fast‐twitch muscles from the mouse
Authors: Wiseman Robert W , Pizzimenti Natalie , Brault Jeffrey
Published: June 2021

Force generation is not responsible for activation of p38 MAPK in contracting muscle (Dentel et al, AJP: 2004), but energetic demands and/or calcium cycling may still be involved. We tested the hypothesis that inhibiting the major contraction‐induced cytosolic ATPases would attenuate the exercise‐induced MAPK activation. Energetic demands were inhibited using 75 μM N‐benzyl‐ p‐toluene sulphonamide (BTS, actomyosin) and 50 μM cyclopiazonic acid (CPA, SERCA). Extensor digitorum longus muscles from male Swiss Webster mice were incubated in Ringer's solution (37°C) with or without inhibitors and stimulated at 10 Hz for 15 min. Muscles were immediately freeze‐clamped for western blot and metabolite analysis. As expected, BTS decreased force by 95%, CPA potentiated force consistent with SERCA inhibition and combined treatment eliminated force after a transient increase from CPA treatment. Surprisingly, independent inhibition of the ATPases had no affect on phosphorylation, but combined treatment decreased phosphorylation of the γ isoform but α/β were unchanged. Given that ATPase inhibition differentially activates MAPK isoforms as well as metabolite levels of the freeze clamped muscles, our data suggest that an energetic signal triggers the phosphorylation of the γ isoform but other factors activate the α/β isoforms, perhaps mediated by cytosolic calcium flux. Supported by NSBRI MA 00210.
Phytoecdysteroid treatment increases fiber size and PI3k‐Akt signaling in aged skeletal muscle
Authors: Lawrence M. M. , Zwetsloot K. A. , Arthur S. T. , Nieman D. C. , Lila M. A. , Grace M. , Ray B. A. , Via J. R. , Romoda C. P. , Westbrook D. K. , Hargrove K. E. , Lederer P. M. , John C. S. , Cooley I. , Tessener P. C. , Shanely R. Andrew
Published: June 2021

Protein synthesis through the PI3k‐Akt pathway is reduced in aged skeletal muscle, leading to a decrease in fiber size and number. Phytoecdysteroids, in particular 20‐hydroxyecdysone (20E), from the plant Ajuga turkestanica increases protein synthesis in C2C12 skeletal muscle cells and muscle strength in young rats. The objective of this study was to determine if an extract from A. turkestanica (ATE), enriched in phytoecdysteroids, affects muscle fiber size and PI3k‐Akt signaling in skeletal muscle of aged mice. Aged male C57BL/6 mice (20 mo) received ATE, 20E, or vehicle (CT) only for 28 days. H&E staining revealed 41% and 30% larger fiber cross‐sectional area (CSA) in the triceps brachii (p=0.014) and plantaris (p=0.018) muscles of 20E‐treated sedentary mice, respectively, compared to CT. ATE treatment resulted in larger, but non‐significant differences in CSA of the triceps brachii (26%; p=0.088) and plantaris (8%; p=0.497) muscles, compared to CT. Western blotting performed on gastrocnemius muscles showed a significant increase in phosphorylation of Akt in 20E‐treated sedentary aged skeletal muscle, compared to CT (p=0.013). In conclusion, these data suggest that 20E rescues the loss of muscle fiber size and increases activation of the PI3k‐Akt pathway in sedentary aged skeletal muscle. Supported in part by a USDA Kannapolis Scholars Fellowship.
Dietary nitrate dramatically increases force in mouse skeletal muscle
Authors: Hernández Andrés , Schiffer Tomas A. , Ivarsson Niklas , Bruton Joseph D. , Lundberg Jon O. , Weitzberg Eddie , Westerblad Håkan
Published: June 2021

Nitrate supplementation has beneficial effects on exercise performance, with both improved mitochondrial efficiency and reduced ATP cost of muscle force production implicated. It was the purpose of this study to examine if nitrate supplementation could improve Ca2+ handling and contractile function using skeletal muscles from C57bl/6 male mice given 0.1 mmol • kg−1 sodium nitrate in drinking water for 7 days (Nitrate). Age matched controls were provided water without added nitrate (Control). Myoplasmic free [Ca2+] ([Ca2+]i) was significantly elevated from 20–150 Hz stimulation in fast‐twitch muscle fibers dissected from Nitrate (n = 5) compared to Control mice (n = 7). This resulted in increased contractile force at stimulation frequencies up to 50 Hz. At 100 Hz stimulation, the rate of force development was ~35% faster in the Nitrate group. These changes were accompanied by increased expression of the Ca2+ handling proteins calsequestrin and the dihydropyridine receptor in fast‐twitch muscles of Nitrate mice. In conclusion, these results show a striking and novel effect of nitrate supplementation on intracellular Ca2+ handling and force production. These data provide a new mechanism by which nitrate can exert effects on muscle performance, and provide a potential new therapeutic role for nitrate in conditions with muscle weakness.
The effect of nitric oxide on the detection of superoxide generated by mouse skeletal muscle fibres in vitro
Authors: Pearson Tim , Jackson Malcolm J
Published: June 2021

Superoxide is generated from several pathways within muscle cells, including NADPH oxidase and the mitochondrial electron transport chain. Muscle derived superoxide has been claimed to exert multiple effects on muscle fibres including influencing force production, adaptive responses to contractions and in excess contributes to loss of function and cell death.

Muscle superoxide activities were examined in single isolated fibres from the flexor digitorum brevis muscles of C57Bl6 mice using fluorescent confocal microscopy. Fibres were loaded with either dihydroethidium (5μM) or MitoSox Red (125nM) to determine cytoplasmic or mitochondrial superoxide respectively whilst in the presence or absence of L‐NAME (100μM), a nitric oxide synthase inhibitor. Images were acquired using Ex405 and Em605/15nm from fibres either at rest or subjected to two 9 minute contraction protocols at ten and forty minutes.

2‐hydroxyethidium fluorescence increased acutely by 20–25% (1st, P<0.02) and 17–26% (2nd, P<0.001) after the first and second contraction period compared with fibres at rest (n=8) in both the presence and absence of L‐NAME. In contrast, the presence of L ‐ NAME significantly increased MitoSox fluorescence as compared with untreated fibres. The pattern of fluorophore oxidation argued that the contraction‐induced increase in cytoplasmic superoxide was not due to release from the mitochondria.

Supported by MRC.
ERK1/2 are possible mediators of the anti‐proteolytic effects of β2‐adrenoceptor/cAMP signaling in skeletal muscle
Authors: Gonçalves Dawit Albieiro Pinheiro , Silveira Wilian A. , Graça Flávia A. , Kettelhut Ísis C. , Navegantes Luiz Carlos C.
Published: June 2021

The present study was undertaken to identify the role of ERK1/2 kinases in the antiproteolytic effects of β2‐adrenoceptor (β2‐AR)/cAMP signaling in skeletal muscles. For that, we examined the effects of Formoterol (FOR), a newer generation β2‐agonist, on the phosphorylation (p) levels of ERK1/2, cAMP content and the mRNA expression of atrophy‐ (Atrogin‐1, MuRF1 and cathepsin L) and autophagy‐ (LC3b and Gabarapl1) related genes in C2C12 myoblast and myotubes cells and adult skeletal muscles. In C2C12 cells, FOR significantly increased p‐ERK1/2 from 5 to 30 min indicating that these kinases can be directly activated by β2‐AR signaling under basal conditions. Following, gastrocnemius and tibialis anterior muscles from 2‐d fasted C57Bl6/J mice were analyzed after 1, 4, and 12h of a single s.c. injection of FOR (3μg/kg; s.c.) Fasting reduced cAMP content (36%) and p‐ERK1/2 (~40%), effects that were associated with the upregulation (6‐ to 10‐fold) of atrophic and autophagic genes. At 1h, FOR reestablished cAMP levels and p‐ERK1/2 and downregulated (~40%) Gabarapl1 mRNA. At 4h, FOR reduced Atrogin‐1 mRNA to basal levels and MuRF1 and Gabarapl1 mRNAs by ~40% but did not alter LC3b and cathepsin L mRNAs. These data suggest the involvement of ERK1/2 kinases in the anti‐proteolytic effects of β2‐AR/cAMP signaling in skeletal muscle. Supported by CNPq & FAPESP (08/06694‐6, 09/07584‐2).
Effects of pharmacologic manipulation of the brainstem serotonergic system on duration of the laryngeal chemoreflex—implications for Sudden Infant Death Syndrome
Authors: Donnelly William Thomas , Bartlett Donald , Leiter James C.
Published: June 2021

Sudden infant death syndrome (SIDS) occurs when a sleeping infant fails to mount an effective response to a challenge to cardiorespiratory homeostasis. Serotonin receptor binding in the nucleus of the solitary tract (NTS) is reduced in infants who die of SIDS. The laryngeal chemoreflex (LCR) causes apnea and may initiate the process leading to SIDS. We tested the hypothesis that altering serotonergic signaling in the NTS would alter the duration of LCR apnea. Rat pups (P5 to P20) were anesthetized and tracheostomized. Tracheal tubing was inserted rostrally up to the larynx, and water was instilled to elicit the LCR. The EKG and intercostal EMG activity were recorded. ICV serotonin shortened apnea duration, and ICV WAY100635 (a 5HT‐1A receptor antagonist) prolonged it. ICV ketanserin (a 5HT‐2A receptor antagonist) had no consistent effect on apnea duration. Focal injection of WAY100635 in the cNTS also had no consistent effect on apnea duration, but injection of serotonin in this area shortened it. We conclude that serotonin within the NTS shortens the duration of LCR apnea, but this effect cannot be attributed solely to activation of 5HT‐1A receptors. Support: NIH grants HL36379 & HL42707
AGE‐DEPENDENT EFFECTS OF PROGESTERONE ON VENTILATION AND APNEA IN RATS
Authors: Joseph Vincent , Julien Cécile , Kinkead Richard , Bairam Aida
Published: June 2021

Progesterone has been proposed as an alternative for apnea treatment in neonates but relevant data are rare. In rats aged 1, 4, 7, and 12 days we investigated the effects of progesterone (Prog, 4 mg/kg, i.p.) on baseline ventilation, hypoxic ventilatory response (HVR, 12% O2, 20 minutes), apnea frequency (for spontaneous and post‐sigh apneas), and arterial oxygen desaturation and bradycardia during apneas (Mouse‐Ox device). Control rats received a saline injection (2 ul/g i.p.). Prog had no effect on baseline ventilation, increased the peak HVR in 4 and 7 d‐old and the steady state HVR in 1, 4 and 7 d‐old, but had no effects in 12 d‐old rats. About 23% of all apneas were associated with desaturation and/or bradycardia independently of their types. Prog reduced the frequency of those apneas particularly in 1‐d‐old rats. These preliminary observations suggest that acute Prog injection stimulates HVR in rats less or equal to 7 d‐old and reduces apnea independently to its effect on HVR. Supported by Foundation of Stars.
Thermal prolongation of the Hering‐Breuer inflation reflex in neonatal rats depends on an adenosinergic mechanism in the brainstem
Authors: Arnal Ashley V. , Gore Julie L. , Bartlett Donald , Leiter J. C.
Published: June 2021

The Hering‐Breuer (HB) inflation reflex prolongs expiratory duration (TE), and elevated body temperature increases this prolongation in neonatal rats. The neurophysiological mechanisms of this thermal sensitivity have not been studied, but the thermal sensitivity of the HB reflex is similar to that of the laryngeal chemoreflex (LCR). Therefore, we tested the hypothesis that blocking adenosine receptors with caffeine, which shortens the LCR, would blunt the thermal prolongation of apnea associated with the HB reflex. Neonatal rats (P4 to P20) were anesthetized briefly to administer intracisternal caffeine (2 microL of 10 mM) or vehicle, allowed to wake up and studied in a head out plethysmograph. The HB reflex was elicited at body temperatures of 32 and 36 degrees C by decreasing the plethysmographic pressure (−5 cm H2O) at the end of inspiration. The inflation was maintained until the next inspiratory effort, and we measured the apneic interval between inspiratory efforts. Intracisternal injection of caffeine shortened the HB reflex apnea at the higher body temperature, and the shortening was greater in younger animals.

Support: grants 36379 & 42707 from the NICHD.
The effects of chronic nicotine exposure on chemosensitivity of medullary 5‐HT neurons
Authors: Avraam Joanne , Wu Yuanming , Richerson George B
Published: June 2021

5‐HT neurons are chemoreceptive and involved in respiratory control. SIDS has been linked to brainstem 5‐HT abnormalities, fetal nicotine exposure and impaired responses to changes in O2 and CO2. We tested the hypothesis that nicotine may increase the risk of SIDS by impairing 5‐HT neuronal function. We used perforated patch clamp to record changes in firing rate (FR) in response to acidosis and to acute nicotine exposure of 5‐HT neurons cultured for >12 days from the medullary raphé of 2 day old mice. An osmotic minipump was implanted into pregnant mice at E10 supplying 6 or 60 mg kg−1 day−1 of nicotine, and/or nicotine was subsequently added to the cultures at 0.05 μM or 0.5 μM respectively. Neurons were bathed in aCSF at 5% CO2 (pH 7.4) containing GABA and glutamate receptor blockers until FR stabilized at 1 Hz, then were exposed to two‐ 3 min episodes of 9% CO2 (pH 7.2) with wash out each time. This was then repeated with aCSF containing 10μM nicotine.

Neurons exposed chronically to nicotine (n=92) exhibited a reduced response to acute nicotine exposure and to acidosis. These effects were found to be attributed to both prenatal and postnatal exposure and were more pronounced in mature neurons and in neurons exposed to a higher dose of nicotine. In the developing neonate such changes in 5‐HT neuronal function and chemoreception may impair responses to changes in O2 and CO2 and potentially increase the incidence of SIDS.
Carotid body growth and the critical period for hyperoxia‐induced developmental plasticity in rats
Authors: Bavis Ryan W , Fallon Sarah C , Tobin Kristina E , Dmitrieff Elizabeth F
Published: June 2021

Chronic postnatal hyperoxia elicits both transient and long‐lasting plasticity in the control of breathing, primarily through effects at the carotid body. We hypothesized that the critical period for long‐lasting changes to the hypoxic ventilatory response (HVR) would correspond to the period of postnatal carotid body growth (i.e., morphological plasticity), while transient attenuation of carotid body O2 sensitivity would occur independent of postnatal age. Although the critical period for long‐lasting attenuation of the HVR is limited to the first two postnatal weeks (Bavis et al., J. Appl. Physiol. 92: 1013–1018, 2002), we observed a substantial increase in carotid body volume through 12 wk of age; growth rates and glomus cell division (BrdU labeling) appeared to slow by 8–10 wk of age. In rats exposed to 7 d of 60% O2 beginning at ~4 wk of age, normoxic ventilation was reduced but HVR was unchanged; normoxic ventilation recovered within 7 d of returning to room air. We conclude that (1) carotid body growth is not the only factor determining the critical period for long‐lasting changes to the HVR and (2) transient changes to the HVR are also limited to exposures during early postnatal development (i.e., prior to P28). Supported by NIH grant P20 RR‐016463 (Maine INBRE).
Immunoreactivity for the NR1 subunit of the NMDA receptor occurs in spinally‐projecting catecholamine and serotonin neurons of the rat ventral medulla
Authors: Llewellyn-Smith Ida J , Mueller Patrick J
Published: June 2021

Bulbospinal neurons in the ventral medulla play important roles in sympathetic nervous regulation. Current evidence suggests that these neurons are activated by NMDA and non‐NMDA subtypes of glutamate receptors. In this study, we examined bulbospinal neurons for the NR1 subunit of the NMDA receptor. Rats received bilateral injections of cholera toxin B (CTB) into spinal segment T9 to label bulbospinal neurons. Triple immunofluorescence was used to detect CTB (blue, AMCA), NR1 (red, Cy3) and either tyrosine hydroxylase (TH) or tryptophan hydroxylase (TpTH) (green, FITC). In the rostral ventrolateral medulla, NR1 occurred in both CTB+/TH+ neurons and CTB+ neurons. CTB+/TpTH+ neurons in raphé pallidus and the parapyramidal region also contained NR1. In addition, NR1 was present in occasional CTB+ neurons in raphé pallidus. These results show that the obligatory subunit of the NMDA receptor is expressed by spinally‐projecting C1 and non‐C1 neurons as well as spinally‐projecting serotonin neurons in raphé pallidus and the parapyramidal region. These receptors likely mediate at least some of the neuronal excitation in these regions in normal and pathological conditions. (R01‐ HL096787, NHMRC #480414).
Paradoxical relationship between alpha‐adrenergic tone and muscle sympathetic nerve activity in human metabolic syndrome
Authors: Mckenna Edward Joseph , Limberg Jacqueline , Sebranek Joshua , Walker Benjamin , Hagen Scott , Morgan Barbara , Schrage William
Published: June 2021

Animal models of metabolic syndrome (MS) exhibit greater basal adrenergic tone that may contribute to impaired blood flow. We hypothesized humans with MS would exhibit greater α‐adrenergic tone that correlated with higher muscle sympathetic nerve activity (mSNA). Phentolamine (PH, α‐adrenergic antagonist) was infused intra‐arterially into the forearm of MS adults (n=11) and healthy controls (n=16). mSNA was measured (microneurography) in a subset of subjects (n=15). Forearm blood flow (Doppler ultrasound) was normalized for higher blood pressure (BP) in MS and relative changes in vascular conductance (VC = blood flow/BP) were assessed. VC increased with PH infusion similarly between groups. MS exhibited greater mSNA compared to controls (66±9 vs 35±4 bursts/100 heart beats, p<0.01). Greater α‐adrenergic tone was inversely related to mSNA (r = 0.5, p= 0.07), thus adults with higher mSNA had less increase in VC with PH infusion. In summary, despite higher mSNA, adults with MS do not exhibit greater α‐adrenergic tone. These results suggest physiological adaptations occur with higher mSNA that blunt sympathetically‐mediated vasoconstriction at rest. This may be due to reduced adrenergic responsiveness or norepinephrine release.

Support: AHA 10PRE3870000, NIH HL091397, APS Summer Undergraduate Research Fellowship.
Carotid baroreflex control of blood pressure to simulated hypotension in young and older women
Authors: Kim Areum , Fisher James P , Fadel Paul J
Published: June 2021

Limited studies have examined the influence of aging on cardiovascular control in women. The arterial baroreflex plays a critical role in beat‐to‐beat regulation of arterial blood pressure (BP). However, the effect of aging on baroreflex control in women remains incompletely understood. Thus, we examined baroreflex control of BP, heart rate (HR), and muscle sympathetic nerve activity (MSNA) in 9 young (YW, 22±1 yr) and 8 older (OW, 62±2 yr) women using 5s pulses of neck pressure (+40 Torr) to simulate carotid sinus hypotension. Mean BP and MSNA burst frequency and incidence were higher at rest in OW compared to YW (P<0.05). In response to neck pressure, OW exhibited attenuated BP (+14±2 YW vs. +6±1 OW %baseline; P<0.05) and HR (+18±3 YW vs. +5±1 OW %baseline; P<0.05) responses. In contrast, increases in total MSNA to neck pressure were similar between groups (+88±26 YW vs. +95±20 OW %baseline; P>0.05). Our preliminary findings suggest that older women exhibit blunted pressor responses to carotid hypotension. These attenuated BP responses do not seem to be explained by an impairment in baroreflex control of MSNA, but may be related to reduced cardiac responsiveness. However, a potential alteration in the vascular response to increases in MSNA between young and older women requires investigation.

Supported by R01HL093167
Macrophage inhibitory factor (MIF) in the nucleus of tract solitary (NTS) improves baroreflex function in spontaneously hypertensive rats (SHR)
Authors: Freiria-Oliveira Andre H. , Blanch Graziela T. , Li Hongwei , Colombari Debora S. A. , Sumners Colin , Colombari Eduardo
Published: June 2021

SHR have impaired baroreflex function. Previously we demonstrated that increased expression of MIF in the NTS of SHR can reduce resting mean arterial pressure (MAP). Thus, in this study we investigated whether MIF over expression in the NTS of SHR can alter baroreflex function. Male SHR (9 weeks old) or normotensive (NT) control rats were injected at 5 sites in the NTS with (1×109 gc in 150 nl) adenoassociated viruses for over expression of MIF or eGFP. Thirty days after the microinjections the femoral artery and vein were cannulated under anesthesia to measure MAP and heart rate (HR), and to perform drug infusions in awake animals the next day. During the experiment, we used phenylephrine to elicit a pressor response (5 μg/kg) and sodium nitroprusside (30 μg/kg) to produce a depressor response, and analyzed the HR reflex response to each 10 mmHg change in MAP. The results showed that the SHR‐eGFP group exhibited a significant impairment of the baroreflex compared with the NT‐eGFP rats, as expected. Further, the SHR‐MIF rats exhibited improvement in the baroreflex curves as to tachycardia (1/slope: − 2.4 ± 0.38) and bradycardia responses (1/slope: −3 ± 0.28) when compared to SHR‐eGFP rats (1/slope bradycardia: −1.1 ± 0.28; tachycardia: −1.2 ± 0.72). The results showed that MIF over expression in the NTS improves the cardiac baroreflex in SHR, helping to decrease the MAP in these animals.

Supported by: CNPq, FAPESP, NIH
Estrogen regulation of ion channel expression mediating neuronal excitability in rat presympathetic PVN neurons
Authors: Lee Seul Ki , Ryu Pan Dong , Lee So Yeong
Published: June 2021

The hypothalamic paraventricular nucleus (PVN) is an important site in regulating the autonomic nervous system. Specifically, the PVN neurons projecting the rostral ventrolateral medulla (PVN‐RVLM) play a regulatory role in sympathetic outflow and their neuronal activities are influenced by A‐type potassium currents and T‐type calcium currents. It has been demonstrated that 17β‐estradiol (E2) regulates neuronal excitability and estrogen receptor β is expressed in PVN‐RVLM neurons. We hypothesized that E2 may regulate neuronal activity via the transcriptional regulation of ion channels expressed in PVN‐RVLM neurons. We investigated changes in firing rates and relative mRNA expressions of Kv4.2, Kv4.3, and Cav3.1 using whole cell patch clamp recording and single cell analysis. The results demonstrated that E2 increased firing rates, and altered mRNA expressions of Kv4.2 and Cav3.1 in PVN‐RVLM neurons. These results suggest that E2 increased the neuronal excitability by mediating Kv4.2 and Cav3.1 expressions in PVN‐RVLM neurons. This study received support from the National Research Foundation of Korea (2010‐0017216).