bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of the rigidity of pulmonary capillaries in avian lung
Authors: Watson Rebecca R. , Fu Zhenxing , West John B
Published: June 2021

We have previously shown that the pulmonary capillaries in chicken lungs are extraordinarily rigid in that large alterations in the capillary transmural pressure result in only small changes in capillary diameter. For example, the capillaries remain wide open even when the pressure outside them is raised 35 cmH2O above the pressure inside them. Since the walls of the capillaries are exceedingly thin (mean thickness ca. 0.27 μ m) it seems unlikely that the rigidity can be explained by the walls themselves. However the capillary walls are tethered by epithelial bridges that form the walls of the surrounding air capillaries and these could provide the necessary support. The bridges themselves are composed of two epithelial sheets enclosing a small amount of tissue, presumably extracellular matrix, and their total thickness is as little as 0.1 μ m in some places. Therefore at first sight they do not seem strong enough to offer sufficient support. However because of the topographical arrangement of the bridges it is possible that the mechanical loads are shared in such a way that even these thin structures can provide the necessary support just as the thin spokes of a bicycle wheel confer remarkable rigidity to the wheel.

Supported by NIH grants 5 RO1 HL60968 and 5 T32 HL07212.
Contributions of PKC, RhoA and ERK signaling to serotonergic contractility of pulmonary arteries from chronic hypoxic fetal and adult sheep
Authors: Goyal Ravi , Nguyen Dan , Loftin Matthew , Derrick Allison , Pearce William J , Longo Lawrence D , Wilson Sean M
Published: June 2021

Serotonin (5‐HT) regulates pulmonary arterial (PA) contractility through calcium and kinase dependent signaling pathways and this activity may change during postnatal development as well as in response to chronic hypoxia (CH). Although 5‐HT activates numerous kinase pathways, protein kinase C (PKC), Rho kinase, and extracellular regulated kinase (ERK) are important to arterial reactivity. Among these, Rho kinase is upregulated by CH in PA of fetal sheep and adult rat. The hypothesis that there is preferential increase in Rho kinase over PKC and ERK pathways in response to CH and maturation was therefore tested by measuring contractility responses in PA rings isolated from CH fetal and adult sheep. PA rings were stimulated with 10 μM 5‐HT and comparative analyses of the kinase pathways made in the absence and then presence of inhibitors of PKC (1 μM BIM I), RhoA (10 μM Y 27632), and ERK (10 μM U0126). All three kinase pathways contributed equally to 5‐HT elicited contractility in adult whereas RhoA was augmented in fetus, while PKC and ERK had negligible roles. In conclusion, the studies provide evidence for selective changes in kinase signaling pathways that may be due to either maturation or CH. This study supports a role for altered kinase signaling in the development of pulmonary arterial disease and illustrates the therapeutic importance of RhoA pathways in hypertension of the newborn. (Support from UM, NIH and Sigma Xi)
Thromboxane analog U46619 redistributes pulmonary microvascular perfusion in isolated rat lungs, but does not retain 3 μm diameter particles in lung capillaries
Authors: Conhaim Robert L. , Watson Kal A. , Dovi William F. , Harms Bruce A.
Published: June 2021

U46619 is a potent vasoconstrictor that increases vascular resistance 2–4 fold in isolated, perfused rat lungs. We wondered if any of this constriction occurred in lung capillaries. To test this, we infused 3.0 μm diameter fluorescent latex particles into the pulmonary arteries of isolated rat lungs perfused with U46619. We then measured the percentage of the infused particles that flowed out of the left atrium over 5 minutes. This percentage averaged 14.2±10.0% in vasoconstricted lungs, and 11.5±10.2% in flow‐matched controls (n.s.) (mean flow, 4.9±1.0 ml/min). To further investigate the effects of U46619 vasoconstriction, we imaged trapping patterns of the particles within the lungs and quantified these using dispersion index (DI) analysis. LogDI, a measure of latex particle distribution, averaged 0.81±0.43 in vasoconstricted lungs and 0.54±0.28 in flow‐matched controls (p = 0.03). The larger logDI value in the U‐46619‐perfused lungs demonstrates that the trapped latex particles were more clustered (non‐uniform) than those in the flow‐matched controls. Our results suggest that U46619 redistributes pulmonary microvascular perfusion, but most likely not through constriction of lung capillaries. Supported by a grant from the Department of Veterans Affairs.
Dynamic pressure‐flow relationships in the human cerebral circulation at rest with and without alpha1‐adrenoreceptor blockade
Authors: Ogoh Shigehiko , Brothers R. Matthew , Eubank Wendy L. , Raven Peter B.
Published: June 2021

Dynamic pressure‐flow relationships observed within the cerebral circulation indicate that when the critical closing pressure (CCP; a functional index of cerebral vascular tone) is reached cerebral blood flow stops. The purpose of this study was to identify the influence of the alpha1‐adrenoreceptors on cerebral vascular tone at rest. This was accomplished by continuously measuring middle cerebral artery blood velocity (MCA V) and arterial pressure from the radial artery before and after Prazosin ingestion (1mg/20kg BW) in 6 healthy subjects. Subsequent calculations of the CCP were preformed for each condition by linear extrapolation from the steady‐state beat‐to‐beat waveforms of velocity and pressure. Prazosin decreased mean arterial pressure (Control: 92±4 mm Hg vs. Prazosin: 87±5 mm Hg; P=0.021), however, Prazosin had no effect on mean MCA V (Control: 61±5 cm/s vs. Prazosin: 58±5 cm/s; P=0.511) or cerebral vascular resistance index (Control: 1.72±0.14 mm Hg s/cm vs. Prazosin: 1.88±0.15 mm Hg s/cm; P=0.82). The correlation between the dynamic pressure‐flow relationship without Prazosin was r=0.96±0.01 while with Prazosin it was r=0.95±0.01. Prazosin did not significantly change CCP (Control:16.3±4.3 mm Hg vs Prazosin:14.4±3.1 mm Hg, P=0.32). These findings suggest that basal sympathetic activity has no effect on cerebral vascular tone at rest.

Supported in part by NIH grant #HL045547.
Epoxyeicosatrienoic acid‐dependent cerebral vasodilation evoked by metabotropic glutamate receptor activation
Authors: liu xiaoguang , Hwang Sung Hee , Jones Paul D , Hammock Bruce D , Roman Richard J , Harder David R , Koehler Raymond C
Published: June 2021

Activation of metabotropic glutamate receptors (mGluRs) on astrocytes participates in neurovascular coupling. Epoxyeicosatrienoic acids (EETs) may be involved in downstream signaling. 3,5‐Dihydroxyphenylglycine (DHPG) was used as a type I mGluR agonist for pharmacological activation. Cerebral blood flow (CBF), measured by laser‐Doppler flowmetry in isoflurane anesthetized rats, increased by 30±7% (±SD) at 20 min of subarachnoid superfusion of DHPG (1 mM) over the cortical surface. The increase was blocked by the mGluR antagonists MPEP and LY367385 but was unaffected by inhibitors of NO synthase, heme oxygenase, or 20‐HETE synthesis. However, the CBF response to DHPG was markedly reduced by prior superfusion of the EET antagonist 14,15‐EEZE (5±11%) or the EET synthesis inhibitor MS‐PPOH (8±7%). EETs are metabolized by soluble epoxide hydrolase (sEH). The maximal CBF increase induced by DHPG was augmented from 30±7% to 43±6% by superfusion of the sEH inhibitor t‐AUCB. Furthermore, the CBF response to DHPG alone subsided to 12±12% at 60 min of superfusion, but was sustained by co‐superfusion of the sEH inhibitors t‐AUCB (34±14%) and MPTPU (21 ± 5%). These results demonstrate that EETs are a major contributor to the cerebral vasodilation evoked by type I mGluR activation and that inhibiting EETs metabolism augments the CBF response during prolonged activation. (Supported by NIH grant HL59996)
Differential NAD(P)H Oxidase Expression and Activity in Rat Pulmonary and Coronary Arteries
Authors: Csiszar Anna , Labinskyy Nazar , Xiangmin Zhao , Olson Susan , Wolin Michael S , Ungvari Zoltan
Published: June 2021

Previous studies have characterized NAD(P)H oxidases (NOX) in bovine pulmonary and coronary arteries in the context of examining their differential role in responses associated with changes in PO2. In the present study we compared NOX expression and activity in rat intrapulmonary (RPA) and coronary (RCA) arteries. In RPA there was an elevated production of NOX‐derived O2.‐ and H2O2, as compared to RCA. Microarray gene expression profiling revealed that among the factors known to influence NOX activity, gp91phox, glucose‐6‐phosphate dehydrogenase (G6PD) and bone morphogenetic protein‐4 (BMP4) were enriched in RPA, as compared to RCA. Protein expression of gp91phox in RPA was also increased. BMP4 (3 ng/mL) elicited significant NOX‐dependent increases in ROS production in RCA and human CAECs (∼250%). However, BMP4 ‐induced increases in ROS generation in RPA and PAECs (∼120%) were significantly lower. Incubation with an anti‐BMP4 antibody did not decrease ROS generation significantly in RPA. Thus, both rat and bovine PAs exhibit increased NOX activity, likely due to an increased expression/activity of gp91phox and G6PD, whereas our data does not support a major role for BMP4 in this phenomenon.
Simultaneous quantitative detection of mitochondrial superoxide production and apoptosis in live cells.
Authors: Mukhopadhyay Partha , Rajesh Mohanraj , Yoshihiro Kashiwaya , Hawkins Brian J , Madesh Muniswamy , Pacher Pal
Published: June 2021

Experiments with isolated mitochondria have established that these organelles are pivotal intracellular sources of superoxide in a variety of pathophysiological conditions. Recently, a novel fluoroprobe MitoSOX Red was introduced for selective detection of superoxide in the mitochondria of live cells. Here we show approximately 3–7 fold dose‐ and time‐dependent increase in mitochondrial superoxide production (measured by MitoSOX using flow cytometry and confocal microscopy) in rat cardiac derived H9c2 myocytes and/or in human coronary artery endothelial cells triggered by Antimycin A, Paraquat, Doxorubicin or high glucose. We also describe simultaneous measurements of mitochondrial superoxide generation with apoptotic markers (Annexin V and Sytox Green) by both flow cytometry and confocal microscopy in endothelial cell lines. The advantages of the described flow cytometry method over other cell‐based techniques are the tremendous speed (1–2 h), exquisite precision and the possibility of simultaneous quantitative measurements of mitochondrial superoxide generation and apoptotic (and other) markers, with maximal preservation of cellular functions. This method combined with fluorescent microscopy may be very useful to reveal important spatial‐temporal changes in mitochondrial superoxide generation and execution of programmed cell death in virtually any cell type.
Control of VWF secretion in human endothelial cells by local production of NO
Authors: Qian Jin , Zhang Qian , Fulton David
Published: June 2021

Endothelium‐derived nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS) exerts control over vascular function via two distinct mechanisms, the activation of soluble guanylate cyclase (sGC)/cGMP‐dependent signaling or through S‐nitrosylation of proteins. The respective contribution of eNOS subcellular localization to sGC dependent or independent pathways is not known. The ability of local production of NO to influence non‐sGC dependent mechanisms will be tested by monitoring the secretion of Von Willebrand factor (vWF). The secretion of vWF from Weibel‐Palade Bodies is tonically inhibited by NO which nitrosylates N‐ethylmaleimide sensitive factor (NSF). eNOS “knockdown” HAEC were reconstituted with eNOS fusion proteins targeted to PM or Golgi. vWF production was greatly attenuated in HAEC expressing PM versus Golgi localized eNOS. To further investigate the mechanisms underlying reduced vWF secretion, we incubated HAECs with the sGC inhibitor ODQ to block the sGC‐cGMP pathway. vWF secretion was not significantly modified by ODQ in cells expressing either PM or Golgi localized eNOS and PM eNOS induced greater S‐nitrosylation of NSF versus Golgi eNOS. Furthermore, reconstitution of HAEC with Golgi or PM –targeted Ca2+/CaM ‐insensitive eNOS constructs, which produce equal amounts of NO regardless of location, resulted in equivalent inhibition of vWF secretion.
Combined Peroxynitrite Decomposition Catalyst and Vasopressin therapy in Methicilin Resistant Staphylococcus aureus (MRSA) Sepsis
Authors: Jonkam Collette Changie , Enkhbaatar Perenlei , Traber Lillian , Bansal Kamna , Esechie Aimalohi , Traber Daniel
Published: June 2021

Sepsis is associated with excessive production of reactive nitrogen species such as peroxynitrite, a potent inducer of cellular damage. Currently we tested the effects of peroxynitrite decomposition catalyst (INO‐4885) on MRSA sepsis. METHODS: Eves were surgically prepared for study: Control (injured, non‐treated, n=6), INO‐4885 (injured, treated with INO‐4885, n=5) and INO + Vasopressin (injured, treated with INO‐4885 and Vasopressin, n=3). Injured animals were given 48 breaths of cotton smoke and ~2.5 x 1011 CFU of live MRSA into the lungs. INO‐4885 bolus was given at 1h post injury followed by continuous infusion. 3 sheep also received Vasopressin when the mean arterial pressure (MAP) decreased by 10mmHg below baseline value. RESULTS: The severe hypotension and fall in systemic vascular resistance seen in control animals was partially alleviated by INO, and totally reversed by combined INO and AVP. AVP alone failed to attenuate these variables. The control group also developed severe signs of vascular leakage (fluid retention, decreased plasma protein and oncotic pressure), which was partially improved by INO alone and reversed by combined therapy. AVP alone had no effect. CONCLUSION: Peroxynitrite may decrease the sensitivity of AVP receptors. Inhibition of peroxynitrite restores the AVP receptor sensitivity and reduces the requirement of AVP.
Elucidating the Genomic and Proteomic Parameters for Restenotic Patients
Authors: Wu Chieh‐Hsi , Wen Cheng‐Hao , Weng Jing‐Ru
Published: June 2021

Restenosis has still been a limiting factor for the clinical outcome of coronary angioplasty. The incidence of restenosis after six months is 10 to 50 %. The variance of restenosis incidence among patients remains elucidated. We aimed to identify the liable factors leading to restenosis. The single nucleotide polymorphism (SNP) and LC/MS‐MS have been applied to accomplish these specific aims. We have pooled the genomic DNA from 75 patients with restenosis for Affimetrix 500K SNP Microarray analysis. Statistic analysis has been used to determine the linkage differentiation (LD) for the significant difference between patients. We have also collected the serum proteins from 75 patients with or without restenosis for 2‐D Gel Analysis followed by LC/MS‐MS identification. Preprohaptoglobin, alpha‐2‐macroglobulin precursor and anti‐rabies immunoglobulin have been found to be related to restenosis. Western blot analysis for each individual patient was carried out to confirm the finding of 2‐D results. Of most interest, the genotypes of haptoglobins with 1‐1 form predominate to the other forms in patients with restenosis yet the 2‐2 form was found to be the risk factor for restenosis in Western Europe. This discrepancy may be due to the ethnic factor. These data provide a basis for improving the risk stratification of patients thereby a possible individualized therapy for percutaneous coronary intervention.
Role of protein kinase C (PKC) δ in angiotensin II induced collagen expression in cardiac myofibroblasts
Authors: Katwa Laxmansa C , Chintalgattu Vishnu
Published: June 2021

Previous studies have demonstrated that myofibroblasts (myoFb) play a key role in cardiac collagen expression and remodeling at the site of myocardial infarction (MI). Angiotensin II (AngII) is known inducer of cardiac collagen expression and fibrosis. However, the role of PKC‐δ in AngII induced collagen expression is unclear. The present study was designed to investigate the role of PKC‐δ in AngII induced type I collagen expression in cardiac myoFb isolated from the site of 28 day post‐MI. AngII (10−7M) treatment significantly (p<0.05) increased myoFb type I collagen expression by 188%, whereas PKC‐δ siRNA treatment or rottlerin (3μM) a PKC‐δ inhibitor abrogated the AngII induced type I collagen expression. MyoFb transfected with PKC‐δ over expression vector showed increase (178%) in the type I collagen gene expression as compared to control. In vivo rat data from 2‐ week of chronic AngII infusion (450ng/min/kg) showed an increase in type I collagen expression (172 %, p<0.05 AngII vs rottlerin or control) as compared to sham animals; this increase in type I collagen expression was abrogated by rottlerin (300μg/day/kg). In conclusion, both in vitro (using isolated myoFb) and in vivo (a chronic AngII infusion model) data strongly suggest a role for PKC‐δ in AngII induced cardiac collagen expression and fibrosis.
Sarcolipin Ablation Increases Ca2+ Pump Efficiency in Mouse Skeletal Muscle
Authors: Bombardier Eric , Vigna Chris , Babu Gopal , Backx Peter H. , Periasamy Muthu , MacLennan David H. , Gramolini Anthony O. , Tupling A. Russell
Published: June 2021

Reconstitution experiments have shown that sarcolipin (SLN) uncouples ATP hydrolysis from Ca2+ transport by SR Ca2+ pumps and increases the amount of heat released per mol of ATP hydrolyzed by causing an increased rate of “slippage” on Ca2+ pumps. In this study, we analyzed skeletal muscle Ca2+ pump activity in the presence and absence of the Ca2+ ionophore A23187 (iono) and Ca2+ uptake without oxalate in SLN null mice (KO) to determine whether SLN uncouples ATP hydrolysis from Ca2+ transport in vivo. In 3 muscles analyzed (soleus (Sol), EDL, gastrocnemius (G)), there were no differences in maximal Ca2+ pump activity measured in homogenates (hom) with iono between KO and wildtype mice (WT). In Sol, but not EDL or G, there was an increased Ca2+ pump affinity for Ca2+ in KO as demonstrated by a leftward shift in the Ca2+ pump activity‐pCa curves. Ca2+ pump activity measured in the absence of iono was ~15–25% lower in all KO muscles compared with WT which is consistent with the idea that SLN increases “slippage” and reduces the extent of back‐inhibition on Ca2+ pumps. In the only muscle analyzed (Sol), Ca2+ uptake measured in hom without oxalate was not different between KO and WT which means Ca2+ transport efficiency in KO was increased ~19%. These results show that at a physiological SLN:Ca2+ pump ratio, SLN uncouples ATP hydrolysis from SR Ca2+ uptake in skeletal muscle. Supported by NSERC (ART), HSFO (DHM, PHB, AOG) and CIHR (DHM, PHB).
The Role of Per1 in the Aldosterone‐Mediated Regulation of the Epithelial Sodium Channel
Authors: Gumz Michelle Louise , Stow Lisa R , Cain Brian D , Wingo Charles S
Published: June 2021

The mineralocorticoid aldosterone is a major regulator of sodium (Na+) transport and contributes to the control of blood pressure and cardiac function. We previously used microarray technology to identify the immediate transcriptional targets of aldosterone in a mouse inner medullary collecting duct cell line, mIMCD‐3 (Gumz et al. AJP Renal 2003 285:F664); two of the induced transcripts were circadian clock genes, Per1 and Per2. The goals of the present study were to characterize the transcriptional regulation of Per1 and Per2 by aldosterone and to define the role of Per1 in the aldosterone‐mediated regulation of Na+ transport. We confirmed that aldosterone up‐regulates Per1 and Per2 mRNA in mIMCD‐3 cells as well as a second murine cell line, IMCD‐K2. RNA silencing was used to test the hypothesis that Per1 mediates the downstream effects of aldosterone on Na+ transport. Interestingly, knockdown of Per1 with any of four independent Per1 siRNA sequences dramatically decreased mRNA levels of the alpha subunit of the epithelial Na+ channel (ENaCα) in the presence of aldosterone. Importantly, Per1 knockdown had no effect on the serum and glucocorticoid induced kinase 1 (Sgk1) mRNA levels. These data indicate that Per1 plays a role in the aldosterone‐mediated regulation of Na+ transport via ENaCα, and this effect appears to be independent of Sgk1.
Flow‐induced shear stress reduces K content of MDCK cells by a Ca and TEA‐dependent K efflux mechanism
Authors: Holtzclaw J. David , Grimm P. Richard , Irsik Debra L. , Sansom Steven C.
Published: June 2021

High tubular flow induces K+ secretion by large conductance, Ca2+‐activated K+ channels (BKCa) in mammalian connecting tubules (CNTs) and cortical collecting ducts (CCDs). Although expressed in CNTs and PCs, BKCa are also highly expressed in intercalated cells (IC), which may not have the capacity to sustain K+ secretion because of minimal Na‐K‐ATPase. We used MDCK cells, a cell line with properties consistent with both PCs and ICs, in a parallel plate flow chamber (PPFC) to support the hypothesis that high flow activates BKCa in ICs to reduce IC size thereby increasing tubule internal diameter and decreasing resistance to flow. Using immunocytochemistry and RT‐PCR, we found that MDCKs contain BK‐α, BK‐β1, BK‐β2 and BK‐β4 subunits. MDCKs were exposed to static (control) or 10 dynes/cm2 shear stress in a PPFC at 37ºC for 6 hrs. Perfusate was similar to mammalian CNT luminal fluid. After incubation, K+content in MDCKs was measured by flame photometry and normalized by DNA. With high flow, intracellular K+ decreased by 40% compared to static conditions (4.2 vs. 7.1 μg K+/μg DNA, N=4). This decrease was attenuated by removal of luminal Ca2+ with EGTA (10.5 vs. 9.2, N=3) or addition of 5 mM TEA (11.0 vs. 11.6, N=2), a blocker of BKCa. These data show that MDCKs contain the BKCa molecular components and suggest that the elevated [Ca2+]i due to shear stress may result in activation of BKCa, which reduces cell K+ content and volume.
Exercise Stimulates Transcription Factors (Nrf2 & NFkB), Increases Antioxidant Defenses, Decreases Oxidative Stress, and Restores Renal Dopamine D1 Receptor Function in Aging
Authors: George Liza , Asghar Mohammad , Lokhandwala Mustafa F
Published: June 2021

We reported that oxidative stress associated with aging contributes to diminished natriuretic response to dopamine in old (24‐month) Fischer 344 rats. Here, we explored whether 3‐month exercise in old rats reduces oxidative stress and restores natriuretic response to D1 receptor activation. In old rats, 3‐month treadmill exercise decreased oxidative stress marker malondialdehyde in renal proximal tubules (RPTs) compared to sedentary controls. In the exercised animals, western blotting analyses showed an increase in the expression of antioxidant enzymes, hemeoxygenase 1 and superoxide dismutase. Moreover, nuclear expression of transcription factors, NFkB and Nrf2, was higher in exercised compared to sedentary rats. Electrophoretic mobility shift assay (EMSA) showed an increased nuclear NFkB‐DNA binding in exercised rats. Also, exercise improved D1 receptor expression as evidenced by increased receptor protein content and receptor numbers and restored D1 receptor agonist (SKF 38393) induced natriuresis and diuresis compared to controls. These results suggest that long‐term exercise increases antioxidant defenses involving NFkB and Nrf2 in RPTs, which contributes to decrease in oxidative stress and restores D1 receptor function in old rats. We conclude that exercise as an intervention may be beneficial in attenuating age‐related decline in renal function. [NIH/NIA, AG25056 funded the study].
Fatty Acid Import and Esterification in Adipocytes: lipid‐based regulation of AMP kinase
Authors: Bernlohr David A , Wiczer Brian M , Lobo Sandra
Published: June 2021

Fatty acid influx in adipocytes is mediated, in part, via the concerted actions of CD36 and FATP1 found within caveolin‐1 rich microdomains of the plasma membrane. The influx reaction is stimulated ∼ 2‐fold by insulin and involves the translocation of FATP1 from intracellular sites to the plasma membrane analogous to GLUT4 translocation. FATP1 is an acyl CoA synthetase that facilitates the insulin‐stimulated component of long and very long chain FFA influx. Concomitant with uptake at the plasma membrane is the acyl CoA dependent esterification of fatty acids in a process termed vectoral acylation. ATP provides the energy for vectoral acylation and in a FATP1‐dependent process produces AMP as a product. AMP in turn activates the AMP‐dependent protein kinase (AMPK) and leads to the phosphorylation/inactivation of acetyl CoA carboxylase providing regulatory control of triglyceride synthesis from either nutritionally derived fatty acids or from de novo lipogenesis. Importantly, FATP4 and ACSL1, two other acyl CoA synthetases in adipocytes also regulate AMPK via their activities that function in fatty acid re‐esterification as part of the triglyceride‐diglyceride cycle in fat cells. Supported by ADA‐RA‐12
Regulation of Renin Gene Expression by Oxidative Stress
Authors: Itani Hana A , Liu Xuebo , Sigmund Curt D
Published: June 2021

Angiotensin‐II (AII) is involved in the pathogenesis of hypertension and mediates its effects in part by causing oxidative stress. AII feedback inhibits renin (REN) gene expression. To examine the effect of ROS on REN expression, we measured endogenous REN mRNA levels by RNase Protection in REN‐expressing As4.1 cells in response to increasing doses of hydrogen peroxide (HP). HP caused a dose dependent decrease in steady state REN mRNA levels. The effect of HP was most pronounced after 24hrs of treatment. ROS generated by xanthine and xanthine oxidase also significantly decreased REN mRNA. Blocking of transcription revealed that HP effect is transcriptionally mediated yet a post‐transcriptional component may be involved. To measure HP effect on REN promoter activity, we transiently transfected As4.1 cells with luciferase constructs consisting of different lengths of the REN promoter. HP caused a dose‐dependent reduction in transcriptional activity of the REN promoter. Constructs containing individual mutations in the CREB, HRE, CBF1, and HOXD. PBX binding sites display a reduced baseline activity in some mutants, yet the dose response to HP was preserved, although at a lower magnitude than the original construct. This data suggests that REN gene expression is regulated by oxidative stress. Our results suggest that intercellular calcium levels and TNFα may be involved in mediating HP effect on REN gene expression.
Role of FosB in the supraoptic nucleus of rats with hepatic cirrhosis
Authors: Ji Lisa L. , Matthews Kristen , Carreno Flavia R. , Hinojosa‐Laborde Carmen , Chakravarty Sumana , Nestler Eric J. , Cunningham J. Thomas
Published: June 2021

Bile duct ligation (BDL), a model of hepatic cirrhosis, is associated with inappropriate vasopressin release and increased FosB expression in vasopressin neurons in the supraoptic nucleus (SON). BDL rats were injected bilaterally in the SON (n = 5) with 400 nl of an adeno‐associated virus (AAV) vector expressing a dominant negative construct for FosB (AAV‐ΔJunD) plus GFP. AAV injected BDL rats, control BDL rats, and sham ligated rats were instrumented with radio telemetry transmitters for measuring blood pressure and heart rate. Three weeks after BDL surgery, rats were individually housed in metabolism cages for 1 week. Daily average mean arterial pressures and heart rates were not different among the groups. Average daily water intake was significantly greater in AAV‐BDL rats (55 ± 7 ml) and BDL rats (47 ±4) compared to sham ligated controls (35 ± 1). Urine output was significantly greater in AAV‐treated BDL rats (38 ± 5 ml) than control BDL rats (21 ± 2) or sham controls (23 ± 1). Urine sodium was significantly lower in AAV‐treated BDL rats (47 ± 13 mmol/L) than the other groups (BDL 89 ± 12; Sham 136 ± 22) although average daily Na excretion was not different among the groups. SON injections of the FosB dominant null produced a diuresis in BDL rats that may be related to decreased circulating levels of vasopressin. FosB expression in vasopressin SON neurons may contribute to inappropriate vasopressin release in BDL rats. (HL62579)
Streptozotocin‐Induced Diabetic Animal Model Recapitulates Many Features of Frailty in Older Adults with Diabetes
Authors: Anderson‐Antle Lisa L , Bauer David J , Gopalakrishnan Sandeep , Lark Daniel S , Sandoval Christopher L , Eells Janis T , Snyder Ann C
Published: June 2021

Frailty is defined as a decline in the neuromuscular system, abnormal aging, and loss of physiological reserve. It is difficult to conduct research in this population, therefore we sought to identify an animal model that would recapitulate many of the characteristics of frailty. Purpose: To test the hypothesis that streptozotocin‐induced diabetes in the Wistar rat would exhibit characteristics similar to those reported in frail diabetic older adults. Methods: Male Wistar rats were rendered diabetic with streptozotocin (STZ, 50 mg/kg, ip). Animals were weighed weekly and blood glucose values were determined twice a week for 14 weeks. At week 15, animals were euthanized, perfused with cold saline and tissues (heart, muscle, kidneys) were rapidly dissected and tissue and organ weights were determined on an analytical balance. Results: Tissue weights of the heart and rectus femoris muscle were significantly reduced in STZ‐treated animals compared to control. Heart: STZ = 1.43 ± 0.11 g vs CON 1.75 ± 0.05 g (p < .05); Rectus femoris: STZ = 0.85 ± 0.31 g vs CON 1.62 ± 0.27 g (p < .0001). In contrast, kidney weights were significantly greater in STZ treated animals [STZ = 2.20 ± 0.24 g vs CON = 1.69 ± 0.12 (p < .0001)]. Conclusions: These data support our hypothesis and are indicative of the induction of myocardial dilatation, sarcopenia and glomerulopathy in chronic STZ‐induced diabetes.

Funding Source: BTA Award and UWM RGI.
Evaluation of whole muscle apoptotic susceptibility in young and old animals
Authors: Kazak Lawrence , Hood David A.
Published: June 2021

Apoptosis regulates muscle mass during disease and aging. However, the signaling pathways leading to apoptosis need to be established. We developed an in vitro muscle incubation and fractionation protocol to assess the activation and localization of apoptotic proteins in response to various stimuli. Soleus strips (∼10–15mg) from young (6 mo) and old (36 mo) F344XBN rats were used. These strips contain ∼30 fibers/mg weight with <1% fiber damage and they can be incubated in vitro for 4–6 hrs to assess their responsiveness to apoptotic agents. Following incubation, the strips were fractionated into mitochondrial and cytosolic fractions. Subsequently, indices of apoptotic susceptibility were measured. Incubation of muscle strips in vitro with the apoptosis‐inducing agent camptothecin for 4 hours resulted in an increase in mitochondrial Bax in both young and old animals. JNK phosphorylation and DNA fragmentation were elevated by 3‐fold in old, compared to young animals. Bax localization to mitochondria was increased by ∼40% in old animals, and there was a 30% increase in AIF localized to the cytosol. Surprisingly, we detected no differential localization of Cyto c to the cytosol between young and old animals. We conclude that this in vitro muscle strip protocol is useful for the assessment of apoptotic susceptibility in whole muscle.
Neuromuscular junctions are more sensitive to onset of aging than myofibers
Authors: Roby Mackenzie Anne , Eason Meg , Harris Brennan , Deschenes Michael
Published: June 2021

Sarcopenia has been linked to a host of health problems including osteoporosis, accidental falls, and type II diabetes. Denervation may precipitate the loss in muscle size and change in fiber type distribution characterizing sarcopenia. This study aimed to determine whether remodeling of the neuromuscular junction (NMJ) preceded alterations in myofiber profile commonly observed with aging. Ten young (5 mo) and 10 moderately aged (22 mo) Fischer 344 rats were killed and plantaris muscles were removed, and frozen until analysis. To visualize NMJs, longitudinal muscle sections were doubly stained with rhodamine conjugated α‐bungarotoxin and fluorescein labeled RT‐97 antibody, which bind to postsynaptic acetylcholine receptors and presynaptic nerve terminal branches, respectively. Myofiber profiles were assessed with histochemical staining for mATPase activity of muscle cross‐sections. Compared to NMJs of young rats, those of moderately aged ones displayed significant (P<0.05) remodeling including greater nerve terminal branch length and complexity of arborization, along with amplified perimeter length and area of staining at post‐synaptic endplates. In contrast, myofibers of aged rats showed no evidence of atrophy or changes in fiber type composition. These data show that age‐related alterations of the neuromuscular system are observed sooner in NMJs than in myofibers.
The Impact of Long‐Term Physical Activity on Age‐Related Changes in Protein and Gene Expression
Authors: Lanza Ian R , Short Daniel K , Short Kevin R , Asmann Yan W , Raghavakaimal Sreekumar , Bigelow Maureen L , Joyner Michael J , Nair K Sreekumaran
Published: June 2021

Age‐related mitochondrial dysfunction is paralleled by reductions in gene transcripts and expression of proteins involved in cellular ATP production. Short‐term endurance exercise has been shown to enhance muscle protein synthesis in the elderly. We determined the impact of long‐term endurance exercise on gene expression and protein abundance of numerous proteins involved in cellular energetics. Vastus lateralis tissue was biopsied from 11 young sedentary (YS), 10 old sedentary (OS), 11 young trained (YT), and 11 old trained (OT) subjects. Trained subjects vigorously exercised ¡Ý 1 hr/d, ¡Ý five d/wk for ¡Ý 4 years. Large‐scale measurement of relative protein abundance was performed using isobaric tags for relative and absolute quantitation (iTRAQ) reagents and tandem mass spectrometry. Gene transcript levels were measured by high‐density oligonucleotide microarrays. Ingenuity Pathway Analysis was used to determine canonical pathways that were altered by age. Of the 33 proteins involved in cellular ATP production 27 were less abundant in OS vs. YS, while only 4 of these proteins were less abundant in OT vs. YT. Genes involved in glycolysis and oxidative phosphorylation were underexpressed in OS vs. YS, with no effects of age in OT vs. YT. CONCLUSIONS: Long‐term vigorous endurance exercise ameliorates the detrimental effects of old age on muscle gene and protein expression in humans.
Sarcalumenin plays a critical role in age‐dependent reduction in cardiac function and SERCA2a activity
Authors: Jiao Qibin , Shimura Miei , Akaike Toru , Ishikawa Yoshihiro , Minamisawa Susumu
Published: June 2021

Sarcalumenin (SAR), a Ca2+‐binding protein located in the longitudinal sarcoplasmic reticulum (SR), interacts with cardiac SR Ca2+‐ATPase(SERCA2a) to modulate its activity and expression. We have demonstrated that SAR deficiency resulted in mild cardiac dysfunction in young mice (JBC 2005). Since it is well known that SERCA2a activity is decreased with aging, we examined the effects of aging on cardiac function in SAR knockout mice (SAR‐KO). Cardiac function assessed by in vivo hemodynamic study and the expression levels of SERCA2a protein were progressively decreased in senescent SARKO (n=13) when compared with senescent wild‐type (WT) (n=12) or young SARKO (4month old, n=12). LV dp/dtmax (mmHg/s) was 3897 ± 134, 7091 ± 657, and 6836 ± 298 in senescent SARKO, senescent WT, and young SARKO, respectively. The expression levels of SERCA2a protein (% of young WT) were 28 ± 8, 84 ± 17, and 44 ± 17 in senescent SARKO, senescent WT, and young SARKO, respectively. Interestingly, downregulation of SAR preceded downregulation of SERCA in aging cardiac muscles of wild‐type mice. These findings indicated that SAR plays a critical role in age‐dependent reduction in cardiac function and SR Ca2+ reuptake.
Effects of IL‐10 and age on IL‐6, IL‐1β, and TNFα responses to an acute inflammatory insult in mouse skeletal muscle
Authors: Huey Kimberly A. , Meador Benjamin M. , Krzyszton Cara P. , Johnson Rodney W.
Published: June 2021

Pro‐inflammatory cytokine responses can increase with aging and reduced IL‐10—an anti‐inflammatory cytokine—may contribute to these changes. IL‐10 can reduce cytokine expression in non‐muscle tissues; however, no studies have tested the effects of both IL‐10 and age on cytokine responses in skeletal muscle. Thus, we tested the hypothesis that the absence of IL‐10 is associated with greater IL‐6, IL‐1β, and TNFα responses to an inflammatory insult in muscle, and that aging exaggerates these responses. We compared IL‐6, IL‐1β and TNFα mRNA and protein in muscle of young and mature wildtype (IL‐10+/+) and IL‐10 deficient (IL‐10−/−) mice after lipopolysaccharide (LPS)‐induced inflammation. Cytokine mRNA and protein were quantified with real time PCR and Elisa, respectively, 4 h after LPS or saline. IL‐6 mRNA and protein were significantly elevated by LPS for IL‐10+/+ and IL‐10−/−mice with a greater response in IL‐10−/− mice and these effects were greater in mature than young mice. LPS significantly increased IL‐1β mRNA relative to saline and this response was reduced only in the young IL‐10−/− relative to IL‐10+/+ mice. TNFα mRNA and protein levels were unchanged. The major results provide in vivo evidence that the absence of IL‐10 is associated with a greater IL‐6 response to LPS in skeletal muscles, and that aging further exaggerates these responses. This work was supported by NIH AR049855 to KAH and AG 023580 to RWJ.
Accumulation of dMHC positive fibers contributes to sarcopenia
Authors: Wright Kathryn Jean , Huba Elaine , Hepple Russell T
Published: June 2021

Sarcopenia is associated with accumulation of very small fibers amongst fibers of normal size. Previously, it was observed that some of these fibers in soleus muscle expressed a marker of regenerating muscle, developmental myosin heavy chain (dMHC). The purpose of this study was to determine the incidence of dMHC positive fibers across the range of muscle phenotypes from slow oxidative (soleus muscle; Sol), to fast oxidative (red region of gastrocnemius muscle; Gr) to fast glycolytic (white region of gastrocnemius muscle; Gw) muscle in senescent rats. Mean fiber size was 54% smaller in Sol, 43% smaller in Gr, and 67% smaller in Gw versus young adult rats. Whereas the fraction of dMHC positive fibers in Sol (12.6 +/− 2.8%) and Gas (8.5 +/− 1.5%) was similar, Gr (19.4 +/− 6.4%) had a higher dMHC expression than Gw (4.4 +/− 1.4%). In all muscle regions, the fiber cross‐sectional area of dMHC positive fibers was smaller than dMHC negative fibers. These results show that dMHC positive fibers are present across the range of metabolic and contractile phenotypes in aged muscles, and their small size shows that the accumulation of these fibers contributes to the decline in muscle mass with aging.
The effect of heat shock on acute hypertrophic signaling following skeletal muscle damage
Authors: Touchberry Chad D. , Bomhoff Greg L , Gupte Anisha , Geiger Paige , Gallagher Phil
Published: June 2021

Heat shock proteins (Hsp's) are molecular chaperones that are critical for the maintenance of cellular homeostasis and have been shown to protect cells and tissues from a large variety of protein damaging insults. While Hsp's have been implicated in reducing damage and preventing apoptosis, it has recently been suggested that heat induction of Hsp's may inhibit hypertrophy and the rate of muscle regeneration. The purpose of this study was to determine if a single heat shock (HS) treatment influences Akt & p70s6k, potent regulators of cell growth. Wistar rats were randomized into two groups: control (core temp: 35.4°C) or heat stressed (core temp: 41°C, 20 min) 48 hours prior to a bout of downhill treadmill running (18 m/min, −16% grade). Soleus (Sol), red vastus lateralis (VLR) and white vastus lateralis (VLW) were assessed for Hsp70, Hsp27, Akt, and p70s6k 150 min following exercise. Heat shock significantly (p ≥ 0.05) increased core temperature and Hsp content in skeletal muscle, as well as significantly (p ≥ 0.05) reduced plasma creatine kinase levels. Heat shock resulted in non‐significant changes (p ≤ 0.05) in phosphorylation of the hypertrophic proteins Akt (Sol: −1.5% VLR: 61.1%, VLW: 8.1% ) & p70s6k (Sol: 6.5%,VLR: 52.7% , VLW: 35.42% ). Contrary to previous literature, our data suggests that the induction of Hsp's does not impair the acute hypertrophic response in skeletal muscle as measured by Akt & p70s6k.
Hypothalamic Paraventricular Nucleus Contributes to the Sympathoexcitatory Effects of Hyperinsulinemia
Authors: Stocker Sean D , Bardgett Megan E
Published: June 2021

Hyperinsulinemia increases sympathetic nerve activity (SNA) and contributes to cardiovascular dysfunction in obesity and diabetes. PVH neurons regulate SNA through mono and poly‐synaptic connections to preganglionic neurons in the spinal cord. The present study determined whether PVH neurons contribute to the increase in SNA during hyperinsulinemia. Hyperinsulinemic‐euglycemic clamps were performed in anesthetized male Sprague‐Dawley rats (370–420 g) by an infusion of insulin (7.5 mU/kg/min) and 50% dextrose (0.75–2.0 ml/h) for 90 min. Insulin significantly increased lumbar SNA (101 ± 13%, P<0.01) and arterial blood pressure (baseline: 111 ± 2 mmHg vs 60 min: 124 ± 3 mmHg, P<0.01) without any change in renal SNA (8 ± 9%) or plasma glucose levels (baseline: 79 ± 14 vs 60 min: 82 ± 10 mg/dl). Inhibition of the PVH with bilateral injection of the GABAA agonist muscimol (500 pmol/100 nl, n=4) significantly reduced the increase in lumbar SNA (93 ± 20% to 32 ± 19%) and arterial blood pressure (13 ± 4 to 2 ± 3 mmHg). Injection of isotonic saline into the PVH (n=3) did not alter lumbar SNA (131 ± 24% versus 143 ± 2%) or arterial blood pressure (10 ± 3 vs 10 ± 3 mmHg). Similarly, injection of muscimol outside of the PVH (n=3) did not effect the increase in lumbar SNA (113 ± 16% vs 126 ± 27%) or arterial blood pressure (11 ± 3 vs 9 ± 3 mmHg). These findings indicate that PVH neurons contribute to the sympathoexcitatory effects of hyperinsulinemia.
The brain renin‐angiotensin system promotes a negative energy balance in mice
Authors: Grobe Justin L , Grobe Connie L , Gall Andrew J , Thedens Dan R , Sakai Koji , Rahmouni Kamal , Blumberg Mark S. , Johnson A. Kim , Sigmund Curt D.
Published: June 2021

The role of the renin‐angiotensin system (RAS) in the control of energy homeostasis has long been debated, and evidence suggests opposing actions of the central versus the circulating and adipose RAS. We previously developed double‐transgenic (sRA) mice consisting of the human renin gene under the transcriptional control of the neuron‐specific synapsin promoter, and human angiotensinogen under the control of its own endogenous promoter, which results in a CNS‐specific over‐activation of the RAS. Here, we investigated the hypothesis that a centrally overactive RAS would increase energy utilization, and possibly decrease food intake. sRA mice (n=8) exhibited a sustained significant (15%, P<0.001) decrease in body mass compared to littermate controls, which was accompanied by a significantly increased metabolic rate during both sleep (20%, P<0.001) and stationary wakefulness (18%, P=0.002) when assessed indirectly through oxygen consumption (ages 10–12 weeks). sRA mice paradoxically exhibited increased food consumption (20%, P=0.005). Together, these results indicate a negative energy balance in sRA mice, which is predominantly mediated through increased energy expenditure. These findings prompt further studies into the CNS‐RAS in metabolic disorders such as diabetes and the metabolic syndrome. This work was supported by grants (CDS) and postdoctoral fellowships (JLG, CLG) from the NIH and the APS.
The enhanced hyperglycemic response to hemorrhage hypotension in obese rats is related to an impaired baroreflex.
Authors: de Lima Daniel Carvalho , de Andrade Silveira Simonton , Haibara Andréa Siqueira , Coimbra Cândido Celso
Published: June 2021

We assessed the hyperglycemic response to hemorrhage in rats fed with hypercaloric diet. Male wistar rats (4 weeks) were fed a chow (CD, n=6) or hypercaloric diet (HD, n=7) for 19 weeks. After 19 weeks, the animals were submitted to implantation of silastic cannula into jugular vein for drugs administration, blood collecting and hemorrhage (1.2 mL/100g body wt/2 min). A polyethylene catheter was inserted into abdominal aorta for cardiovascular monitoring. An utonomic balance was evaluated by testing baroreflex sensitivity (with phenylephrine and sodium nitroprusside). Our data showed that hypercaloric diet induced obesity. We observed a reduction in baroreflex sensitivity (50%) and an increase in hemorrhagic hyperglycemia response (35%) in HD compared to CD group (p<0.01). We found a negative correlation between the change in baroreflex sensitivity and the increase in hyperglycemic response to hemorrhage (r=0.72, p<0.01) and in the Lee index (r=0.72, p<0.01). A positive correlation between the increased Lee index and hemorrhage hyperglycemia (r=0.90, p<0.01) was also observed. Our data showed that obesity induced by hypercaloric diet promotes an autonomic imbalance characterized by enhanced hemorrhagic hyperglycemia response, which is related to impaired baroreflex sensitivity. Supported by: CNPq and FAPEMIG.
Gene Expression Networks in Rostral Ventrolateral Medulla (RVLM) of Rats with Angiotensin‐salt Hypertension
Authors: Yuan Lihui , Li Xia , Sved Judith , Sved Alan , Raizada Mohan , Liu Li
Published: June 2021

The rostral ventrolateral medulla (RVLM) is essential in neural control of cardiovascular function and changes in RVLM may play an important role in hypertension. In this study expression profiling was used to identify RVLM genes that may contribute to hypertension produced by the interaction of angiotensin II (AngII) and high dietary salt. Hypertension was established in male Sprague Dawley rats by infusion of AngII (150 ng/kg/hr, sc) in combination with a 2% NaCl diet; control rats were fed a 0.1% NaCl diet and did not receive AngII. After 13 days of treatment, which increased blood pressure by ~30 mm Hg, RVLM were collected and analyzed using Agilent gene array chips. Significantly regulated genes (ANOVA, p<0.05) were evaluated using PathwayStudio® software to reveal functional network relationships. A large gene network was revealed with regulatory hubs centered around several genes including CASP3 (caspase 3), HSPA1A (heat shock 70kD protein 1B), EGFR (epidermal growth factor receptor), and NR3C1 (nuclear receptor). In addition, many genes in the network, including transcription factors TP73L, RUNX2 and DAXX, oxidoreductase CYP4A11 and CYP21A2, have previously been implicated either directly or indirectly in hypertension. These results provide a framework to begin to understand molecular changes in the RVLM associated with hypertension. (Supported by HL76083)