bluuebunny/crossref_metadata_2025 huggingface dataset:
Exercise restores β-adrenergic vasorelaxation in aged rat carotid arteries
Authors: Leosco Dario , Iaccarino Guido , Cipolletta Ersilia , De Santis Domenico , Pisani Eliana , Trimarco Valentina , Ferrara Nicola , Abete Pasquale , Sorriento Daniela , Rengo Franco , Trimarco Bruno
Published: March 2015

Aging is associated with alterations in β-adrenergic receptor (β-AR) signaling and reduction in cardiovascular responses to β-AR stimulation. Because exercise can attenuate age-related impairment in myocardial β-AR signaling and function, we tested whether training could also exert favorable effects on vascular β-AR responses. We evaluated common carotid artery responsiveness in isolated vessel ring preparations from 8 aged male Wistar-Kyoto (WKY) rats trained for 6 wk in a 5 days/wk swimming protocol, 10 untrained age-matched rats, and 10 young WKY rats. Vessels were preconstricted with phenylephrine (10-6 M), and vasodilation was assessed in response to the β-AR agonist isoproterenol (10-10-3 × 10-8 M), the α2-AR agonist UK-14304 (10-9-10-6 M), the muscarinic receptor agonist ACh (10-9-10-6 M), and nitroprusside (10-8-10-5 M). β-AR density and cytoplasmic β-AR kinase (β-ARK) activity were tested on pooled carotid arteries. β-ARK expression was assessed in two endothelial cell lines from bovine aorta and aorta isolated from a 12-wk WKY rat. β-AR, α2-AR, and muscarinic responses, but not that to nitroprusside, were depressed in untrained aged vs. young animals. Exercise training restored β-AR and muscarinic responses but did not affect vasodilation induced by UK-14304 and nitroprusside. Aged carotid arteries showed reduced β-AR number and increased β-ARK activity. Training counterbalanced these phenomena and restored β-AR density and β-ARK activity to levels observed in young rat carotids. Our data indicate that age impairs β-AR vasorelaxation in rat carotid arteries through β-AR downregulation and desensitization. Exercise restores this response and reverts age-related modification in β-ARs and β-ARK. Our data support an important role for β-ARK in vascular β-AR vasorelaxation.
Assessment of the ultrastructural and proliferative properties of human embryonic stem cell-derived cardiomyocytes
Authors: Snir Mirit , Kehat Izhak , Gepstein Amira , Coleman Raymond , Itskovitz-Eldor Joseph , Livne Erella , Gepstein Lior
Published: March 2015

Assessment of early ultrastructural development and cell-cycle regulation in human cardiac tissue is significantly hampered by the lack of a suitable in vitro model. Here we describe the possible utilization of human embryonic stem cell (ES) lines for investigation of these processes. With the use of the embryoid body (EB) differentiation system, human ES cell-derived cardiomyocytes at different developmental stages were isolated and their histomorphometric, ultrastructural, and proliferative properties were characterized. Histomorphometric analysis revealed an increase in cell length, area, and length-to-width ratio in late-stage EBs (>35 days) compared with early (10–21 days) and intermediate (21–35 days) stages. This was coupled with a progressive ultrastructural development from an irregular myofibrillar distribution to an organized sarcomeric pattern. Cardiomyocyte proliferation, assessed by double labeling with cardiac-specific antibodies and either [3H]thymidine incorporation or Ki-67 immunolabeling, demonstrated a gradual withdrawal from cell cycle. Hence, the percentage of positively stained nuclei in early-stage cardiomyocytes ([3H]thymidine: 60 ± 10%, Ki-67: 54 ± 23%) decreased to 36 ± 7% and 9 ± 16% in intermediate-stage EBs and to <1% in late-stage cardiomyocytes. In conclusion, a reproducible temporal pattern of early cardiomyocyte proliferation, cell-cycle withdrawal, and ultrastructural maturation was noted in this model. Establishment of this unique in vitro surrogate system may allow to examine the molecular mechanisms underlying these processes and to assess interventions aiming to modify these properties. Moreover, the detailed characterization of the ES cell-derived cardiomyocyte may be crucial for the development of future cell replacement strategies aiming to regenerate functional myocardium.
PAF- and bradykinin-induced hyperpermeability of rat venules is independent of actin-myosin contraction
Authors: Adamson R. H. , Zeng M. , Adamson G. N. , Lenz J. F. , Curry F. E.
Published: March 2015

We tested the hypothesis that acutely induced hyperpermeability is dependent on actin-myosin contractility by using individually perfused mesentery venules of pentobarbital-anesthetized rats. Venule hydraulic conductivity ( Lp) was measured to monitor hyperpermeability response to the platelet-activating factor (PAF) 1- O-hexadecyl-2-acetyl- sn-glycero-3-phosphocholine or bradykinin. Perfusion with PAF (10 nM) induced a robust transient high Lp[24.3 ± 1.7 × 10-7cm/(s·cmH2O)] that peaked in 8.9 ± 0.5 min and then returned toward control Lp[1.6 ± 0.1 × 10-7cm/(s·cmH2O)]. Reconstruction of venular segments with the use of transmission electron microscopy of serial sections confirmed that PAF induces paracellular inflammatory gaps. Specific inhibition of myosin light chain kinase (MLCK) with 1–10 μM 1-(5-iodonaphthalene-1-sulfonyl)-1 H-hexahydro-1,4-diazepine hydrochloride (ML-7) failed to block the PAF Lpresponse or change the time-to-peak Lp. ML-7 reduced baseline Lp50% at 40 min of pretreatment. ML-7 also increased the rate of recovery from PAF hyperpermeability measured as the decrease of half-time of recovery from 4.8 ± 0.7 to 3.2 ± 0.3 min. Inhibition of myosin ATPase with 5–20 mM 2,3-butanedione 2-monoxime also failed to alter the hyperpermeability response to PAF. Similar results were found using ML-7 to modulate responses. These experiments indicate that an actin-myosin contractile mechanism modulated by MLCK does not contribute significantly to the robust initial increase in permeability of rat venular microvessels exposed to two common inflammatory mediators. The results are consistent with paracellular gap formation by local release of endothelial-endothelial cell adhesion structures in the absence of contraction by the actin-myosin network.
Blood-brain barrier tight junctions are altered during a 72-h exposure to λ-carrageenan-induced inflammatory pain
Authors: Huber J. D. , Hau V. S. , Borg L. , Campos C. R. , Egleton R. D. , Davis T. P.
Published: March 2015

In this study, we examined the effect of λ-carrageenan-induced inflammatory pain on the functional and structural properties of the rat blood-brain barrier (BBB) over a 72-h time period. Systemic inflammation was induced by an intraplantar injection of 3% λ-carrageenan into the right hind paw of female Sprague-Dawley rats. In situ brain perfusion and Western blot analyses were performed at 1, 3, 6, 12, 24, 48, and 72 h. In situ brain perfusion showed λ-carrageenan significantly increased brain uptake of [14C]sucrose at 1, 3, 6, and 48 h (139 ± 9%, 166 ± 19%, 138 ± 13%, and 146 ± 7% compared with control, respectively). Capillary depletion analysis insured the increased brain uptake was due to increased BBB permeability and not vascular trapping. Western blot analyses for zonula occludens-1 (ZO-1) and occludin were performed on isolated cerebral microvessels. ZO-1 expression was significantly increased at 1, 3, and 6 h and returned to control expression levels by 12 h. Total occludin expression was significantly reduced at 1, 3, 6, 12, and 48 h. This investigation demonstrated that λ-carrageenan-induced inflammatory pain elicits a biphasic increase in BBB permeability with the first phase occurring from 1–6 h and the second phase occuring at 48 h. Furthermore, changes in BBB function are correlated with altered tight junctional protein expression of occludin and ZO-1. Changes in the structure of tight junctions may have important clinical ramifications concerning central nervous system homeostasis and therapeutic drug delivery.
Native LDL and minimally oxidized LDL differentially regulate superoxide anion in vascular endothelium in situ
Authors: Stepp David W. , Ou Jingsong , Ackerman Allan W. , Welak Scott , Klick David , Pritchard Kirkwood A.
Published: March 2015

Low-density lipoprotein (LDL) and its oxidized derivatives are hypothesized to impair vascular function by increasing superoxide anion (O[Formula: see text]·). To investigate mechanisms in situ, isolated carotid arteries were incubated with native LDL (nLDL) or minimally oxidized LDL (mmLDL). With the use of en face fluorescent confocal microscopy and hydroethidine, an oxidant-sensitive fluorescent probe, we found that nLDL increased O[Formula: see text]· in vascular endothelium greater than fourfold by an Nω-nitro-l-arginine methyl ester (l-NAME)-inhibitable mechanism. In contrast, mmLDL increased O[Formula: see text]· in vascular endothelium greater than eightfold by mechanisms that were partially inhibited byl-NAME and allopurinol and essentially ablated by diphenyleneiodium. These data indicate that both nLDL and mmLDL uncouple endothelial nitric oxide synthase (eNOS) activity and that mmLDL also activates xanthine oxidase and NADPH oxidoreductase to induce greater increases in O[Formula: see text]· generation than nLDL. Western analysis revealed that both lipoproteins inhibited A-23187-stimulated association of heat shock protein 90 (HSP90) with eNOS without inhibiting phosphorylation of eNOS at serine-1179 (phospho-eNOS), an immunological index of electron flow through the enzyme. As HSP90 mediates the balance of ·NO and O[Formula: see text]· generation by eNOS, these data provide new insight into the mechanisms by which oxidative stress, induced by nLDL and mmLDL, uncouple eNOS activity to increase endothelial O[Formula: see text]· generation.
Changes in amino-terminal portion of human B2receptor selectively increase efficacy of synthetic ligand HOE 140 but not of cognate ligand bradykinin
Authors: Schroeder Christian , Breit Andreas , Böning Hilke , Dedio Jürgen , Gera Lajos , Stewart John , Müller-Esterl Werner
Published: March 2015

Recently, we have shown that a widely used antagonist of the human bradykinin B2receptor (B2R) HOE 14O acts as a full agonist of the chicken ornithokinin receptor (BoR). To understand the molecular mechanisms underlying differential efficacy of HOE 140 for the various kinin receptors, we have constructed chimeric kinin receptors (CKR) in which the amino-terminal portion including the first two transmembrane regions and the first extracellular loop (CKR-2) or only the second transmembrane region and the first extracellular loop (CKR-1) of B2R were substituted with the corresponding segments of BoR. Ligand efficacy of synthetic ligand HOE 140 decreased in the order BoR > CKR-2 > CKR-1 > B2R, whereas the efficacy of the endogenous kinin ligand was unchanged. Enhanced HOE 140 efficacy was not due to a structural change in the ligand binding site or to an enhanced receptor expression level. Rather, heterologous binding competition studies indicated that structural change(s) introduced into the engineered receptors caused a selective reduction in apparent affinity of HOE 140 for the uncoupled inactive receptor state R but not for the active G protein-coupled state R* , thereby increasing the ratio of R* over R for a given ligand concentration. Our results may help explain the unusually broad efficacy spectrum of HOE 140, which varies from inverse to full agonism, depending on kinin receptor subtype, tissue origin, or species.
A recombinant polymeric hemoglobin with conformational, functional, and physiological characteristics of an in vivo O2transporter
Authors: Bobofchak Kevin M. , Mito Toshiaki , Texel Sarah J. , Bellelli Andrea , Nemoto Masaaki , Traystman Richard J. , Koehler Raymond C. , Brinigar William S. , Fronticelli Clara
Published: March 2015

With the objective of developing a recombinant oxygen carrier suitable for therapeutic applications, we have employed an Escherichia coli expression system to synthesize in high-yield hemoglobin (Hb) Minotaur, containing α-human and β-bovine chains. Polymerization of Hb Minotaur through S-S intermolecular cross-linking was obtained by introducing a Cys at position β9 and substituting the naturally occurring Cys. This homogeneous polymer, Hb Polytaur, has a molecular mass of ∼500 kDa and was resistant toward reducing agents present in blood. In mice, the circulating half-time (3 h) was fivefold greater than adult human Hb (HbA). The half-time of autooxidation measured in blood (46 h) exceeded the circulating retention time. Hypervolemic exchange transfusion resulted in increased arterial blood pressure similar to that with albumin. The increase in pressure was less than that obtained by transfusion of cross-linked tetrameric Hb known to undergo renovascular extravasation. The nitric oxide reactivity of Hb Polytaur was similar to HbA, suggesting that the diminished pressor response to Hb Polytaur was probably related to diminished extravasation. Transfusion of 3% Hb Polytaur during focal cerebral ischemia reduced infarct volume by 22%. Therefore, site-specific Cys insertion on the Hb surface results in uniform size polymers that do not produce the large pressor response seen with tetrameric Hb. Polymerization maintains physiologically relevant oxygen and heme affinity, stability toward denaturation and oxidation, and effective oxygen delivery as indicated by reduced cerebral ischemic damage.
Developmental regulation of Na+/H+exchanger expression in fetal and neonatal mice
Authors: Rieder Carmen V. , Fliegel Larry
Published: March 2015

We examined the hypothesis that Na+/H+exchanger expression is regulated during fetal and neonatal development and differentiation. To examine transcriptional regulation of the NHE1 isoform of the Na+/H+exchanger, transgenic mice were created that contained the mouse NHE1 promoter driving expression of green fluorescent protein. The level of NHE1 transcription varied between tissues and with the stage of embryonic development. The highest expression was in the heart and liver of 12- to 15-day-old mice, and this declined with age. To examine Na+/H+exchanger protein levels, we immunoblotted mouse tissues from 18-day-old embryos, neonates, and adults. Protein levels increased after embryonic day 18 and peaked at 14 days of age in the heart, lung, liver, kidney, and brain. The greatest rise in NHE1 protein expression occurred in the heart, whereas the smallest increase was in the brain. The results suggest that Na+/H+exchanger transcription and protein levels are controlled in a tissue-specific and time-dependent manner during development.
Neutralization of IL-18 attenuates lipopolysaccharide-induced myocardial dysfunction
Authors: Raeburn Christopher D. , Dinarello Charles A. , Zimmerman Michael A. , Calkins Casey M. , Pomerantz Benjamin J. , McIntyre Robert C. , Harken Alden H. , Meng Xianzhong
Published: March 2015

Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) have been implicated in cardiac dysfunction during endotoxemia. Because IL-18 is a proinflammatory cytokine known to mediate the production of TNF-α and IL-1β and to induce the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), we hypothesized that neutralization of IL-18 would attenuate lipopolysaccharide (LPS)-induced cardiac dysfunction. Mice (C57BL/6) were injected with LPS (0.5 mg/kg ip) or vehicle (normal saline), and left ventricular developed pressure (LVDP) was determined by the Langendorff technique. LVDP was depressed by 38% at 6 h after LPS. LPS-induced myocardial dysfunction was associated with increased myocardial levels of TNF-α and IL-1β as well as increased expression of ICAM-1/VCAM-1. Pretreatment with neutralizing anti-mouse IL-18 antibody attenuated LPS-induced myocardial dysfunction (by 92%) and was associated with reduced myocardial IL-1β production (65% reduction) and ICAM-1/VCAM-1 expression (50% and 35% reduction, respectively). However, myocardial TNF-α levels were not influenced by neutralization of IL-18. In conclusion, neutralization of IL-18 protects against LPS-induced myocardial dysfunction. IL-18 may mediate endotoxemic myocardial dysfunction through induction of and/or synergy with IL-1β, ICAM-1, and VCAM-1.
Presence of a calcium-activated chloride current in mouse ventricular myocytes
Authors: Xu Yanfang , Dong Pei Hong , Zhang Zhao , Ahmmed Gias Uddin , Chiamvimonvat Nipavan
Published: March 2015

The properties of several components of outward K+ currents, including the pharmacological and kinetics profiles as well as the respective molecular correlates, have been identified in mouse cardiac myocytes. Surprisingly little is known with regard to the Ca2+-activated ionic currents. We studied the Ca2+-activated transient outward currents in mouse ventricular myocytes. We have identified a 4-aminopyridine (4-AP)- and tetraethyl ammonium-resistant transient outward current that is Ca2+ dependent. The current is carried by Cl−and is critically dependent on Ca2+ influx via voltage-gated Ca2+ channels and the sarcoplasmic reticulum Ca2+ store. The current can be blocked by the anion transport blockers niflumic acid and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid. Single channel recordings reveal small conductance channels (∼1 pS in 140 mM Cl−) that can be blocked by anion transport blockers. Ensemble-averaged current faithfully mirrors the transient kinetics observed at the whole level. Niflumic acid (in the presence of 4-AP) leads to prolongation of the early repolarization. Thus this current may contribute to early repolarization of action potentials in mouse ventricular myocytes.
Effect of lower limb exercise on forearm vascular function: contribution of nitric oxide
Authors: Green Daniel , Cheetham Craig , Mavaddat Louise , Watts Katie , Best Matthew , Taylor Roger , O'Driscoll Gerard
Published: March 2015

We examined vascular function in an inactive muscle bed, the forearm, during lower limb exercise and determined the contribution of endothelium-derived nitric oxide (NO) to the hyperemic response. Eight young males were randomized to participate in two studies, each consisting of two bouts of lower limb exercise, separated by a 30-min recovery. Peak forearm blood flow (PFBF) and mean blood flow (MFBF) were continuously recorded at baseline and during exercise using continuous high-resolution vascular ultrasound and Doppler flow velocity measurement. During one session, the brachial artery was cannulated to allow continuous infusion of saline or N G-monomethyl-l-arginine (l-NMMA), an inhibitor of NO synthase. The alternate session was performed to control for possible effects of repeated exercise. At 60, 100, and 160 W, l-NMMA significantly decreased both PFBF and MFBF compared with the saline infusion. These results suggest that systemic production of NO occurs during exercise in resting vessel beds, which do not feed metabolically active tissue. This finding provides a plausible explanation for the antiatherogenic benefits of exercise.
Metabolic and functional consequences of cytosolic 5′-nucleotidase-IA overexpression in neonatal rat cardiomyocytes
Authors: Sala-Newby Graciela B. , Freeman Nicola V. E. , Curto Maria A. , Newby Andrew C.
Published: March 2015

Adenosine exerts a spectrum of energy-preserving actions on the heart negative chronotropic effects. The pathways leading to adenosine formation have remained controversial. In particular, although cytosolic 5′-nucleotidases can catalyze adenosine formation in cardiomyocytes, their contribution to the actions of adenosine has not been documented previously. We recently cloned two closely related AMP-preferring cytosolic 5′-nucleotidases (cN-IA and -IB); the A form predominates in the heart. In this study, we overexpressed pigeon cN-IA in neonatal rat cardiomyocytes using an adenovirus. cN-IA overexpression increased adenosine formation and release into the medium caused by simulated hypoxia and by isoproterenol in the absence and presence of inhibitors of adenosine metabolism. Adenosine release was not affected by an ecto-5′-nucleotidase inhibitor, α,β-methylene-ADP, but was affected by a nucleoside transporter, dipyridamole. The positive chronotropic effect of isoproterenol (130 ±3 vs. 100 ±4 beats/min) was inhibited (107 ±3 vs. 94 ±3 beats/min) in cells overexpressing cN-IA, and this was reversed by the addition of the adenosine receptor antagonist 8-( p-sulfophenyl)theophilline (120 ± 3 vs. 90 ± 4 beats/min). Our results demonstrate that overexpressed cN-IA can be sufficiently active in cardiomyocytes to generate physiologically effective concentrations of adenosine at its receptors.
Knockout of Kir6.2 negates ischemic preconditioning-induced protection of myocardial energetics
Authors: Gumina Richard J. , Pucar Darko , Bast Peter , Hodgson Denice M. , Kurtz Christopher E. , Dzeja Petras P. , Miki Takashi , Seino Susumu , Terzic Andre
Published: March 2015

Although ischemic preconditioning induces bioenergetic tolerance and thereby remodels energy metabolism that is crucial for postischemic recovery of the heart, the molecular components associated with preservation of cellular energy production, transfer, and utilization are not fully understood. Here myocardial bioenergetic dynamics were assessed by18O-assisted31P-NMR spectroscopy in control or preconditioned hearts from wild-type (WT) or Kir6.2-knockout (Kir6.2-KO) mice that lack metabolism-sensing sarcolemmal ATP-sensitive K+(KATP) channels. In WT vs. Kir6.2-KO hearts, preconditioning induced a significantly higher total ATP turnover (232 ± 20 vs. 155 ± 15 nmol · mg protein−1· min−1), ATP synthesis rate (58 ± 3 vs. 46 ± 3%18O labeling of γ-ATP), and ATP consumption rate (51 ± 4 vs. 31 ± 4%18O labeling of Pi) after ischemia-reperfusion. Moreover, preconditioning preserved cardiac creatine kinase-catalyzed phosphotransfer in WT (234 ± 26 nmol · mg protein−1· min−1) but not Kir6.2-KO (133 ± 18 nmol · mg protein−1· min−1) hearts. In contrast with WT hearts, preconditioning failed to preserve contractile recovery in Kir6.2-KO hearts, as tight coupling between postischemic performance and high-energy phosphoryl transfer was compromised in the KATP-channel-deficient myocardium. Thus intact KATPchannels are integral in ischemic preconditioning-induced protection of cellular energetic dynamics and associated cardiac performance.
HSC73-tubulin complex formation during low-flow ischemia in the canine myocardium
Authors: Decker Robert S. , Decker Marlene L. , Nakamura Sakie , Zhao Yu-Sheng , Hedjbeli Sascha , Harris Kathleen R. , Klocke Francis J.
Published: March 2015

Canine myocardium was exposed to bouts of low-flow ischemia to identify the interactions that develop between the microtubule-based cytoskeleton and the heat shock protein 70 (HSP70) family of heat shock proteins in viable cardiomyocytes. “Moderate” or “severe” low-flow ischemia was produced in chronically instrumented dogs by reducing circumflex coronary flow by 50% for 2 h or by 75% for 5 h followed by reperfusion for 2 and 24 h, respectively. Electron and immunofluorescence microscopy demonstrated either partial or nearly complete depolymerization of the intermyofibrillar microtubules in areas of myofibril disruption and partial dissolution of the perinuclear microtubule girdle. In contrast, centrosomal tubulin arrays appeared to remain intact following low-flow ischemia. In cardiomyocytes displaying myofibril disruption, constitutively expressed HSP73 (HSC73) colocalized with intact but not disrupted microtubules and with perinuclear and centrosomal tubulin following moderate ischemia. Microtubule depolymerization and high molecular weight tubulin-HSC73 complexes were present in more severely ischemic tissue. These results suggest that HSC73 directly interacts with tubulin and may protect selected elements of the microtubule network and limit myofibril disruption during reversible low-flow ischemia.
Oxygen transport across vasa recta in the renal medulla
Authors: Zhang Wensheng , Edwards Aurélie
Published: March 2015

In this model of oxygen transport in the renal medullary microcirculation, we predicted that the net amount of oxygen reabsorbed from vasa recta into the interstitium is on the order of 10−6 mmol/s, i.e., significantly lower than estimated medullary oxygen requirements based on active sodium reabsorption. Our simulations confirmed a number of experimental findings. Low medullary Po 2results from the countercurrent arrangement of vessels and an elevated vasa recta permeability to oxygen, as well as high metabolic needs. Diffusional shunting of oxygen between descending vasa recta (DVR) and ascending vasa recta also explains why a 20-mmHg decrease in initial Po 2 at the corticomedullary junction only leads to a small drop in papillary tip Po 2 (<2 mmHg with baseline parameter values). Conversely, small changes in the consumption rate of DVR-supplied oxygen, in blood flow rate, in hematocrit, or in capillary permeability to oxygen, beyond certain values sharply reduce interstitial Po 2. Without erythrocytes, papillary tip Po 2 cannot be maintained above 10 mmHg, even when oxygen consumption is zero.
Decreased blood pressure and vascular smooth muscle tone in mice lacking basolateral Na+-K+-2Cl−cotransporter
Authors: Meyer Jamie W. , Flagella Michael , Sutliff Roy L. , Lorenz John N. , Nieman Michelle L. , Weber Craig S. , Paul Richard J. , Shull Gary E.
Published: March 2015

The basolateral Na+-K+-2Cl−cotransporter (NKCC1) functions in the maintenance of cellular electrolyte and volume homeostasis. NKCC1-deficient ( Nkcc1−/−) mice were used to examine its role in cardiac function and in the maintenance of blood pressure and vascular tone. Tail-cuff measurements demonstrated that awake Nkcc1−/−mice had significantly lower systolic blood pressure than wild-type ( Nkcc1+/+) mice (114.5 ± 2.2 and 131.8 ± 2.5 mmHg, respectively). Serum aldosterone levels were normal, indicating that extracellular fluid-volume homeostasis was not impaired. Studies using pressure transducers in the femoral artery and left ventricle showed that anesthetized Nkcc1−/−mice have decreased mean arterial pressure and left ventricular pressure, whereas myocardial contraction parameters were not significantly different from those of Nkcc1+/+mice. When stimulated with phenylephrine, aortic smooth muscle from Nkcc1+/+and Nkcc1−/−mice exhibited no significant differences in maximum contractility and only moderate dose-response shifts. In phasic portal vein smooth muscle from Nkcc1−/−mice, however, a sharp reduction in mechanical force was noted. These results indicate that NKCC1 can be important for the maintenance of normal blood pressure and vascular tone.
Mechanisms of decreased leukocyte localization in the developing host
Authors: Mariscalco M. Michele , Vergara Wilfredo , Mei Jia , Smith E. O'Brian , Smith C. Wayne
Published: March 2015

Delays in leukocyte localization likely contribute to diminished host defense in neonates. Understanding the processes that may be affected has been hampered by the lack of suitable developmental models. Using intravital microscopy, we directly examine leukocyte recruitment in a rabbit pup model. In response to intraperitoneal interleukin (IL)-1β, there were one-third as many leukocytes that arrested in pup mesenteric vessels and emigrated compared with adult vessels, although leukocyte flux was not different. Leukocyte rolling velocity in pups was one-half that in adults. In response to surgical trauma alone, the number of arrested pup cells was 15% that of adult cells, although again leukocyte flux was not different. An anti-L-selectin antibody inhibited rolling significantly by 60 min for both pups and adults. The effect on arrest and emigration occurred at significantly earlier times, although the effect was less in rabbit pups. A primary defect in leukocyte emigration in the rabbit pup appears to be a failure of the cell to transition efficiently from rolling to arrest. L-selectin-dependent adhesion and emigration are decreased, rolling is not, suggesting that at least part of the defect is due to events downstream of the initial tether.
Subdiaphragmatic murine electrophysiological studies: sequential determination of ventricular refractoriness and arrhythmia induction
Authors: Gutstein David E. , Danik Stephan B. , Sereysky Jedd B. , Morley Gregory E. , Fishman Glenn I.
Published: March 2015

Programmed electrical stimulation (PES) is a crucial aspect of the evaluation of the risk of arrhythmias in cardiac patients and provides a powerful tool for understanding the mechanisms of arrhythmia in experimental models. Whereas PES in the mouse is well characterized, the procedures allowing for follow-up studies in the same animal have not been developed. In this report, we describe a novel subdiaphragmatic approach that allows for repeat electrophysiological studies in the mouse. Under inhaled anesthesia, PES was performed in 36 wild-type mice via a stimulating electrode introduced through an epigastric incision and placed directly into the diaphragmatic surface of the heart. The procedure was repeated 7 days later. Ventricular effective refractory periods (VERP) did not change significantly between the initial and follow-up trials. Chronic treatment with amiodarone, however, was associated with a 70% prolongation in VERP from initial to follow-up studies ( P ≤ 0.001). In addition, PES of a genetically modified strain with sudden cardiac death, the connexin43 conditional knockout mouse consistently induced lethal polymorphic ventricular tachycardia. Thus sequential PES in mice is feasible with the use of a subdiaphragmatic approach, yields reproducible VERP values, and can be used to follow pharmacologically induced changes in VERP and identify mice at risk of lethal ventricular arrhythmias.
Noninvasive remote ischemic preconditioning for global protection of skeletal muscle against infarction
Authors: Addison Patrick D. , Neligan Peter C. , Ashrafpour Homa , Khan Asim , Zhong Anguo , Moses Michael , Forrest Christopher R. , Pang Cho Y.
Published: March 2015

The aim of this study was to investigate the efficacy and mechanism of action of a noninvasive remote ischemic preconditioning (IPC) technique for the protection of multiple distant skeletal muscles against ischemic necrosis (infarction). It was observed in the pig that three cycles of 10-min occlusion and reperfusion in a hindlimb by tourniquet application reduced the infarction of latissimus dorsi (LD), gracilis (GC), and rectus abdominis (RA) muscle flaps by 55%, 60%, and 55%, respectively, compared with their corresponding control ( n = 6, P < 0.01) when they were subsequently subjected to 4 h of ischemia and 48 h of reperfusion. This infarct-protective effect of remote IPC in LD muscle flaps was abolished by an intravenous bolus injection of the nonselective opioid receptor antagonist naloxone (3 mg/kg) 10 min before remote IPC and a continuous intravenous infusion (3 mg/kg) during remote IPC and by an intravenous bolus injection of the selective δ1-opioid receptor antagonist 7-benzylidenealtrexone maleate (3 mg/kg). However, this infarct-protective effect of remote IPC was not affected by an intravenous bolus injection of the ganglionic blocker hexamethonium chloride (20 mg/kg) or the nonspecific adenosine receptor antagonist 8-( p-sulfophenyl)theophylline (10 mg/kg) or by a local intra-arterial injection of the adenosine1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (3 mg/muscle flap) given 10 min before remote IPC. It was also observed that this remote IPC of skeletal muscle against infarction was associated with a slower rate of muscle ATP depletion during the 4 h of sustained ischemia and a reduced muscle neutrophilic myeloperoxidase activity after 1.5 h of reperfusion. These observations led us to speculate that noninvasive remote IPC by brief cycles of occlusion and reperfusion in a pig hindlimb is effective in global protection of skeletal muscle against infarction. This infarct-protective effect is most likely triggered by the activation of opioid receptors in the skeletal muscle, and remote IPC is associated with an energy-sparing effect during sustained ischemia and attenuation of neutrophil accumulation during reperfusion.
Interaction between ATP and catecholamines in stimulation of platelet aggregation
Authors: Birk Alex V. , Leno Endri , Robertson Hugh D. , Bolotina Victoria M. , Szeto Hazel H.
Published: March 2015

Platelets, on activation by endothelial damage, release ADP, ATP, serotonin, epinephrine, and norepinephrine. Although ATP is known to augment the action of norepinephrine in cardiovascular and endocrine systems, the possible interaction between ATP and catecholamines in regulation of platelet reactivity has not been reported. The addition of ATP (1–5 μM) to human platelet-rich plasma did not induce platelet aggregation; however, it selectively augmented the aggregatory response to norepinephrine and epinephrine, but not to serotonin. This potentiating action of ATP was dose dependent and was not due to contamination by, or hydrolysis to, ADP. The action of ATP was blocked by 10 μM of adenosine 3′-phosphate 5′-phosphosulfate, a selective P2Y1receptor antagonist. ATP alone did not cause release of intracellular Ca2+, but produced a significant Ca2+response in the presence of norepinephrine. In contrast, the P2X1receptor agonists P1,P6-diadenosine-5′ hexophosphate and α,β-methylene-ATP had no effect on norepinephrine-induced platelet aggregation even when added at 100 μM. This synergistic interaction between ATP and norepinephrine in stimulating platelet aggregation may have significant clinical implications and suggests a prothrombotic role for ATP in stress.
Direct measurement of cardiac sympathetic efferent nerve activity during dynamic exercise
Authors: Tsuchimochi Hirotsugu , Matsukawa Kanji , Komine Hidehiko , Murata Jun
Published: March 2015

The assumption that tachycardia during light to moderate exercise was predominantly controlled by withdrawal of cardiac parasympathetic nerve activity but not by augmentation of cardiac sympathetic nerve activity (CSNA) was challenged by measuring CSNA during treadmill exercise (speed, 10–60 m/min) for 1 min in five conscious cats. As soon as exercise started, CSNA and heart rate (HR) increased and mean arterial pressure (MAP) decreased; their time courses at the initial 12-s period of exercise were irrespective of the running speed. CSNA increased 168–297% at 7.1 ± 0.4 s from the exercise onset, and MAP decreased 8–13 mmHg at 6.0 ± 0.3 s, preceding the increase of 40–53 beats/min in HR at 10.5 ± 0.4 s. CSNA remained elevated during the later period of exercise, whereas HR and MAP gradually increased until the end of exercise. After the cessation of exercise, CSNA returned quickly to the control, whereas HR was slowly restored. In conclusion, cardiac sympathetic outflow augments at the onset of and during dynamic exercise even though the exercise intensity is low to moderate, which may contribute to acceleration of cardiac pacemaker rhythm.
Aldosteronism: an immunostimulatory state precedes proinflammatory/fibrogenic cardiac phenotype
Authors: Gerling Ivan C. , Sun Yao , Ahokas Robert A. , Wodi Linus A. , Bhattacharya Syamal K. , Warrington Kenneth J. , Postlethwaite Arnold E. , Weber Karl T.
Published: March 2015

Chronic inappropriate (relative to dietary Na+intake) elevations in circulating aldosterone (ALDO), termed aldosteronism, are associated with remodeling of intramural arteries of the right and left heart. Lesions appear at week 4 of treatment with ALDO and 1% dietary NaCl in uninephrectomized rats (ALDOST) and include invading monocytes, macrophages and lymphocytes with intracellular evidence of oxidative and nitrosative stress, myofibroblasts, and perivascular fibrosis. In this study, we tested the hypothesis that an immunostimulatory state with activated circulating peripheral blood mononuclear cells (PBMCs) precedes this proinflammatory and profibrogenic cardiac phenotype and is initiated by reduction in the cytosolic free Mg2+concentration ([Mg2+]i). At 1 and 4 wk of ALDOST (preclinical and clinical stages, respectively), we monitored serum Mg2+, PBMC [Mg2+]iand cytosolic free [Ca2+] (via fluorimetry), and expressed genes (via microchip array) as well as markers of oxidative and nitrosative stress in plasma [α1-antiproteinase activity (α1-AP)] and cardiac tissue (immunohistochemical detection of gp91phoxsubunit of NADPH oxidase and 3-nitrotyrosine). Age- and gender-matched unoperated and untreated (UO) rats and uninephrectomized salt-treated (UN) rats served as controls. Serum [Mg2+] was unchanged by ALDOST. In contrast with UO and UN, [Mg2+]iand plasma α1-AP were each reduced ( P < 0.05) at weeks 1 and 4. The decline in PBMC [Mg2+]iwas accompanied by Ca2+loading. Differential (twofold and higher) expression (up- and downregulation) in PBMC transcriptomes was present at week 1 and progressed at week 4. Involved were genes for the α1-isoform of Na+-K+-ATPase, the ATP-dependent Ca2+pump, antioxidant reserves, inducible nitric oxide synthase, and PBMC activation with autoimmune responses. Expression of 3-nitrotyrosine and activation of gp91phoxwere seen in inflammatory cells that invaded intramural arteries. Thus early in aldosteronism (preclinical stage), an immunostimulatory state featuring activated circulating PBMCs with reduced ionized [Mg2+]iand oxidative and nitrosative stress precedes and may even predispose to coronary vascular lesions that first appear at week 4.
Localization of the permeability barrier to solutes in isolated arteries by confocal microscopy
Authors: van Haaren Paul M. A. , VanBavel Ed , Vink Hans , Spaan Jos A. E.
Published: March 2015

Endothelial cells are covered by a surface layer of membrane-associated proteoglycans, glycosaminoglycans, glycoproteins, glycolipids, and associated plasma proteins. This layer may limit transendothelial solute transport. We determined dimension and transport properties of this endothelial surface layer (ESL) in isolated arteries. Rat mesenteric small arteries (diameter ∼150 μm) were isolated and cannulated with a double-barreled θ-pipette on the inlet side and a regular pipette on the outlet side. Dynamics and localization of intra-arterial fluorescence by FITC-labeled dextrans (FITC-Δs) and the endothelial membrane dye DiI were determined with confocal microscopy. Large FITC-Δ (148 kDa) filled a core volume inside the arteries within 1 min but was excluded from a 2.6 ± 0.5-μm-wide region on the luminal side of the endothelium during 30 min of dye perfusion. Medium FITC-Δ (50.7 kDa) slowly penetrated this ESL within 30 min but did not permeate into the arterial wall. Small FITC-Δ (4.4 kDa) quickly passed the ESL and accumulated in the arterial wall. Prolonged luminal fluorochrome illumination with a bright mercury lamp destroyed the ∼3-μm exclusion zone for FITC-Δ148 within a few minutes. This study demonstrates the presence of a thick ESL that contributes to the permeability barrier to solutes. The layer is sensitive to phototoxic stress, and its damage could form an early event in atherosclerosis.
Troponin I serines 43/45 and regulation of cardiac myofilament function
Authors: Pyle W. Glen , Sumandea Marius P. , Solaro R. John , De Tombe Pieter P.
Published: March 2015

We studied Ca2+dependence of tension and actomyosin ATPase rate in detergent extracted fiber bundles isolated from transgenic mice (TG), in which cardiac troponin I (cTnI) serines 43 and 45 were mutated to alanines (cTnI S43A/S45A). Basal phosphorylation levels of cTnI were lower in TG than in wild-type (WT) mice, but phosphorylation of cardiac troponin T was increased. Compared with WT, TG fiber bundles showed a 13% decrease in maximum tension and a 20% increase in maximum MgATPase activity, yielding an increase in tension cost. Protein kinase C (PKC) activation with endothelin (ET) or phenylephrine plus propranolol (PP) before detergent extraction induced a decrease in maximum tension and MgATPase activity in WT fibers, whereas ET or PP increased maximum tension and stiffness in TG fibers. TG MgATPase activity was unchanged by ET but increased by PP. Measurement of protein phosphorylation revealed differential effects of agonists between WT and TG myofilaments and within the TG myofilaments. Our results demonstrate the importance of PKC-mediated phosphorylation of cTnI S43/S45 in the control of myofilament activation and cross-bridge cycling rate.
Reperfusion, not simulated ischemia, initiates intrinsic apoptosis injury in chick cardiomyocytes
Authors: Vanden Hoek Terry L. , Qin Yimin , Wojcik Kim , Li Chang-Qing , Shao Zuo-Hui , Anderson Travis , Becker Lance B. , Hamann Kimm J.
Published: March 2015

Although ischemia-reperfusion (I/R) can initiate apoptosis, the timing and contribution of the mitochondrial/cytochrome c apoptosis death pathway to I/R injury is unclear. We studied the timing of cytochrome c release during I/R and whether subsequent caspase activation contributes to reperfusion injury in confluent chick cardiomyocytes. One-hour simulated ischemia followed by 3-h reperfusion resulted in significant cell death, with most cell death evident during the reperfusion phase and demonstrating mitochondrial cytochrome c release within 5 min after reperfusion. By contrast, cells exposed to prolonged ischemia for 4 h had only marginally increased cell death and no detectable cytochrome c release into the cytosol. Caspase activation could not be detected after ischemia only, but it significantly increased after reperfusion. Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, Ac-Asp-Gln-Thr-Asp-H, or benzyloxycarbonyl-Leu-Glu (Ome)-His-Asp-(Ome)-fluoromethyl ketone given only at reperfusion significantly attenuated cell death and resulted in return of contraction. Antixoxidants decreased cytochrome c release, nuclear condensation, and cell death. These results suggest that reperfusion oxidants initiate cytochrome c release within minutes, and apoptosis within hours, significant enough to increase cell death and contractile dysfunction.
Chronic atrial fibrillation does not further decrease outward currents. It increases them.
Authors: Dun Wen , Chandra Parag , Danilo Peter , Rosen Michael R. , Boyden Penelope A.
Published: March 2015

Rapid atrial pacing causes electrical remodeling that leads to atrial fibrillation (AF). AF can further remodel atrial electrophysiology to maintain AF. Our previous studies showed that there was a marked difference in the duration of AF in dogs that have been atrial paced at 400 beats/min for 6 wk. We hypothesized that this difference is based on the changes in the degree of electrical remodeling caused by rapid atrial pacing versus that by AF. Right atrial cells were isolated from control dogs (Con, N = 28), from dogs with chronic AF (cAF dogs, N = 13, episodes lasting at least 6 days), or from dogs with nonsustained or brief episodes of AF (nAF dogs, N = 10, episodes lasting minutes to hours). Both transient outward ( Ito) and sustained outward K+ current ( Isus) densities/functions were determined using whole cell voltage-clamp techniques. In nAF cells, Ito density was reduced by 69% at +40 mV: from 7.1 ± 0.5 pA/pF (Con, n = 59) to 2.2 ± 0.2 pA/pF (nAF, n = 24) ( P < 0.05). The voltage dependence of inactivation of Ito was shifted positively and decay kinetics were changed; however, recovery from inactivation was not altered in nAF cells. In contrast, Ito density in cAF cells was both significantly different from Con cells and larger than that in nAF cells [at +40 mV, 3.5 ± 0.3 pA/pF (cAF, n = 29), P < 0.05]. In cAF cells, recovery from inactivation and decay of Ito were both slow; yet, voltage dependence inactivation of Ito approached that of Con cells. Furthermore, “recovered” Ito of cAF cells was more sensitive to tetraethylammonium than currents of Con and nAF cells. Isus densities of nAF and cAF cells did not differ. Both nAF and cAF cells have reduced Ito versus Con cells, but Ito remodeling of nAF cells differed from that of cAF cells. Ito in cAF dogs was likely remodeled by AF per se, whereas that in nAF dogs was likely the consequence of the rapid rate in the absence of sustained AF.
Preconditioning with ethanol prevents postischemic leukocyte-endothelial cell adhesive interactions
Authors: Yamaguchi Taiji , Dayton Catherine , Shigematsu T. , Carter Patsy , Yoshikawa Toshikazu , Gute Dean C. , Korthuis Ronald J.
Published: March 2015

Long-term ethanol consumption at low to moderate levels exerts cardioprotective effects in the setting of ischemia and reperfusion (I/R). The aims of this study were to determine whether 1) a single orally administered dose of ethanol [ethanol preconditioning (EtOH-PC)] would induce a biphasic temporal pattern of protection (early and late phases) against the inflammatory responses to I/R and 2) adenosine and nitric oxide (NO) act as initiators of the late phase of protection. Ethanol was administered as a bolus to C57BL/6 mice at a dose that achieved a peak plasma concentration of ∼45 mg/dl 30 min after gavage and returned to control levels within 60 min of alcohol ingestion. The superior mesenteric artery was occluded for 45 min followed by 60 min of reperfusion beginning 10 min or 1, 2, 3, 4, or 24 h after ethanol ingestion, and the numbers of fluorescently labeled rolling and firmly adherent (stationary) leukocytes in single postcapillary venules of the small intestine were quantified using intravital microscopic approaches. I/R induced marked increases in leukocyte rolling and adhesion, effects that were attenuated by EtOH-PC 2–3 h before I/R (early phase), absent when assessed after 10 min, 1 h, and 4 h of ethanol ingestion, with an even more powerful late phase of protection reemerging when I/R was induced 24 h later. The anti-inflammatory effects of late EtOH-PC were abolished by treatment with adenosine deaminase, an adenosine A2(but not A1) receptor antagonist, or a NO synthase (NOS) inhibitor during the period of EtOH-PC. Preconditioning with an adenosine A2(but not an A1) receptor agonist in lieu of ethanol 24 h before I/R mimicked the protective actions of late phase EtOH-PC. Like EtOH-PC, the effect of preconditioning with an adenosine A2receptor agonist was abrogated by coincident NOS inhibition. These findings suggest that EtOH-PC induces a biphasic temporal pattern of protection against the proinflammatory effects of I/R. In addition, our observations are consistent with the hypothesis that the late phase of EtOH-PC is triggered by NO formed secondary to adenosine A2receptor-dependent activation of NOS during the period of ethanol exposure.
Time-domain representation of ventricular-arterial coupling as a windkessel and wave system
Authors: Wang Jiun-Jr , O'Brien Aoife B. , Shrive Nigel G. , Parker Kim H. , Tyberg John V.
Published: March 2015

The differences in shape between central aortic pressure (PAo) and flow waveforms have never been explained satisfactorily in that the assumed explanation (substantial reflected waves during diastole) remains controversial. As an alternative to the widely accepted frequency-domain model of arterial hemodynamics, we propose a functional, time-domain, arterial model that combines a blood conducting system and a reservoir (i.e., Frank's hydraulic integrator, the windkessel). In 15 anesthetized dogs, we measured PAo, flows, and dimensions and calculated windkessel pressure (PWk) and volume (VWk). We found that PWk is proportional to thoracic aortic volume and that the volume of the thoracic aorta comprises 45.1 ± 2.0% (mean ± SE) of the total VWk. When we subtracted PWk from PAo, we found that the difference (excess pressure) was proportional to aortic flow, thus resolving the differences between PAo and flow waveforms and implying that reflected waves were minimal. We suggest that PAo is the instantaneous summation of a time-varying reservoir pressure (i.e., PWk) and the effects of (primarily) forward-traveling waves in this animal model.