bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotypic consequences of β1-tubulin expression and MAP4 decoration of microtubules in adult cardiocytes
Authors: Takahashi Masaru , Shiraishi Hirokazu , Ishibashi Yuji , Blade Kristie L. , McDermott Paul J. , Menick Donald R. , Kuppuswamy Dhandapani , Cooper George
Published: March 2015

In pressure-overload cardiac hypertrophy, microtubule network densification is one cause of contractile dysfunction. Cardiac transcriptional upregulation of β1-tubulin rather than the constitutive β4-tubulin and of microtubule-associated protein (MAP)4 accompanies hypertrophy, with extensive microtubule decoration by MAP4. Because MAP4 stabilizes microtubules, and because the isoform-variable carboxy terminus of β-tubulin binds to MAP4, we wished to determine whether one or both of these proteins has etiologic significance for cardiac microtubule network densification. Recombinant adenoviruses encoding β1-tubulin, β4-tubulin, and MAP4 were used to infect isolated cardiocytes. Overexpressed MAP4 caused a shift of tubulin dimers to the polymerized fraction and formation of a dense, stable microtubule network. Overexpressed β1- or β4-tubulin had neither any independent effect on these variables nor any effect additive to that of simultaneously overexpressed MAP4. Results from transgenic mice with cardiac overexpression of β1-tubulin or MAP4 were confirmatory, but unlike the effects of brief adenovirus-mediated MAP4 overexpression in isolated cardiocytes, MAP4 transgenic hearts showed a marked increase in total α- and β-tubulin. Thus MAP4 overexpression caused increased tubulin expression, formation of stable microtubules, and altered microtubule network properties, such that MAP4 upregulation may be one cause for the dense, stable microtubule network characteristic of pressure-overloaded, hypertrophied cardiocytes.
NovelN6-substituted adenosine 5′-N-methyluronamides with high selectivity for human adenosine A3receptors reduce ischemic myocardial injury
Authors: Tracey W. Ross , Magee William P. , Oleynek Joseph J. , Hill Roger J. , Smith Andrew H. , Flynn David M. , Knight Delvin R.
Published: March 2015

We recently reported the identification of a novel human adenosine A3receptor-selective agonist, (2 S,3 S,4 R,5 R)-3-amino-5-{6-[5-chloro-2-(3-methylisoxazol-5-ylmethoxy)benzylamino]purin-9-yl}-4-hydroxytetrahydrofuran-2-carboxylic acid methylamide (CP-608,039), with 1,260-fold selectivity for the human A3versus human A1receptor (DeNinno et al., J Med Chem 46: 353–355, 2003). However, because the modest (20-fold) rabbit A3receptor selectivity of CP-608,039 precludes demonstration of A3-mediated cardioprotection in rabbit models, we identified another member of this class, (2 S,3 S,4 R,5 R)-3-amino-5-[6-(2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylic acid methylamide (CP-532,903), which both retained human A3receptor selectivity (210-fold; human A3/human A1Ki: 23/4,800 nM) and had improved rabbit A3receptor selectivity (90-fold; rabbit A3/rabbit A1Ki: 23/2,000 nM). Infarct size was measured in Langendorff hearts or in vivo after 30 min of regional ischemia and 120 min of reperfusion. Five-minute perfusion with CP-532,903 before ischemia-reperfusion elicited a concentration-dependent reduction in infarct size in isolated hearts (EC50: 0.97 nM; maximum reduction in infarct size: 77%, P < 0.05 vs. control). Furthermore, administration of CP-532,903 (150 nM) at reperfusion also significantly reduced infarct size by 64% ( P < 0.05 vs. control), which was not different ( P ≥ 0.05) from the cardioprotection provided by the same concentration of drug given before ischemia. The selective rabbit A1receptor antagonist BWA1433 did not affect CP-532,903-dependent cardioprotection. In vivo, CP-532,903 (1 mg/kg) reduced infarct size by 50% in the absence of significant hemodynamic effects (mean arterial pressure, heart rate, rate-pressure product). CP-532,903 and CP-608,039 represent a novel class of human A3receptor-selective agonists that may prove suitable for investigation of the clinical cardioprotective efficacy of A3receptor activation.
Cardioprotection involves activation of NF-κB via PKC-dependent tyrosine and serine phosphorylation of IκB-α
Authors: Zhang Jun , Ping Peipei , Vondriska Thomas M. , Tang Xian-Liang , Wang Guang-Wu , Cardwell Ernest M. , Bolli Roberto
Published: March 2015

Previous studies indicated that activation of PKC and Src tyrosine kinases by ischemic preconditioning (PC) may participate in the activation of NF-κB. However, the molecular mechanisms underlying activation of NF-κB during ischemic PC remain unknown. In the hearts of conscious rabbits, it was found that ischemic PC (6 cycles of 4-min coronary occlusion and 4-min reperfusion) significantly induced both tyrosine (+226.9 ± 42%) and serine (+137.0 ± 36%) phosphorylation of the NF-κB inhibitory protein IκB-α, concomitant with increased activation of the IκB-α kinases IKKα (+255.0 ± 46%) and IKKβ (+173.1 ± 35%). Furthermore, both tyrosine and serine phosphorylation of IκB-α were blocked by pretreatment with either the nonreceptor tyrosine kinase inhibitor lavendustin-A (LD-A) or the PKC inhibitor chelerythrine (Che) (both given at doses previously shown to block ischemic PC). Interestingly, Che completely abolished PC-induced activation of IKKα/β, whereas LD-A had no effect. In addition, IκB-α protein level did not change during ischemic PC. Together, these data indicate that ischemic PC-induced activation of NF-κB occurs through both tyrosine and serine phosphorylation of IκB-α and is regulated by nonreceptor tyrosine kinases and PKC.
KATP channels and myocardial preconditioning: an update
Authors: Gross Garrett J. , Peart Jason N.
Published: March 2015

Ischemic or myocardial preconditioning (IPC) is a phenomenon whereby brief periods of ischemia have been shown to protect the myocardium against a more sustained ischemic insult. The result of IPC may be manifest as a marked reduction in infarct size, myocardial stunning, or incidence of cardiac arrhythmias. Whereas many endogenous neurotransmitters, peptides, and hormones have been proposed to play a role in the signal transduction pathways mediating the cardioprotective effect of IPC, nearly universal evidence indicates the involvement of the ATP-sensitive potassium (KATP) channel. Initial evidence suggested that the surface or sarcolemmal KATP (sarcKATP) channel triggered or mediated the cardioprotective effects of IPC; however, more recent findings have suggested a major role for a mitochondrial site or possibly a mitochondrial KATP channel (mitoKATP). This review presents evidence that supports a role for these two channels as a trigger and/or downstream mediator in the phenomenon of IPC or pharmacologically induced PC as well as recent evidence that suggests the involvement of a mitochondrial calcium-activated potassium (mitoKca) channel or the electron transport chain in mediating the beneficial effects of IPC or pharmacologically induced PC.
Opening of mitochondrial KATP channel occurs downstream of PKC-ε activation in the mechanism of preconditioning
Authors: Ohnuma Yoshito , Miura Tetsuji , Miki Takayuki , Tanno Masaya , Kuno Atsushi , Tsuchida Akihito , Shimamoto Kazuaki
Published: March 2015

We examined whether the mitochondrial ATP-sensitive K channel (KATP) is an effector downstream of protein kinase C-ε (PKC-ε) in the mechanism of preconditioning (PC) in isolated rabbit hearts. PC with two cycles of 5-min ischemia/5-min reperfusion before 30-min global ischemia reduced infarction from 50.3 ± 6.8% of the left ventricle to 20.3 ± 3.7%. PC significantly increased PKC-ε protein in the particulate fraction from 51 ± 4% of the total to 60 ± 4%, whereas no translocation was observed for PKC-δ and PKC-α. In mitochondria separated from the other particulate fractions, PC increased the PKC-ε level by 50%. Infusion of 5-hydroxydecanoate (5-HD), a mitochondrial KATP blocker, after PC abolished the cardioprotection of PC, whereas PKC-ε translocation by PC was not interfered with 5-HD. Diazoxide, a mitochondrial KATP opener, infused 10 min before ischemia limited infarct size to 5.2 ± 1.4%, but this agent neither translocated PKC-ε by itself nor accelerated PKC-ε translocation after ischemia. Together with the results of earlier studies showing mitochondrial KATP opening by PKC, the present results suggest that mitochondrial KATP-mediated cardioprotection occurs subsequent to PKC-ε activation by PC.
Interaction of myeloperoxidase with vascular NAD(P)H oxidase-derived reactive oxygen species in vasculature: implications for vascular diseases
Authors: Zhang Chunxiang , Yang Jian , Jacobs Jonathan D. , Jennings Lisa K.
Published: March 2015

Vascular NAD(P)H oxidase-derived reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) have emerged as important molecules in the pathogenesis of atherosclerosis, hypertension, and diabetic vascular complications. Additionally, myeloperoxidase (MPO), a transcytosable heme protein that is derived from leukocytes, is also believed to play important roles in the above-mentioned inflammatory vascular diseases. Previous studies have shown that MPO-induced vascular injury responses are H2O2 dependent. It is well known that MPO can use leukocyte-derived H2O2; however, it is unknown whether the vascular-bound MPO can use vascular nonleukocyte oxidase-derived H2O2 to induce vascular injury. In the present study, ANG II was used to stimulate vascular NAD(P)H oxidases and increase their H2O2 production in the vascular wall, and vascular dysfunction was used as the vascular injury parameter. We demonstrated that vascular-bound MPO has sustained activity in the vasculature. MPO could use the vascular NAD(P)H oxidase-derived H2O2 to produce hypochlorus acid (HOCl) and its chlorinating species. More importantly, MPO derived HOCl and chlorinating species amplified the H2O2-induced vascular injury by additional impairment of endothelium-dependent relaxation. HOCl-modified low-density lipoprotein protein (LDL), a specific biomarker for the MPO-HOCl-chlorinating species pathway, was expressed in LDL and MPO-bound vessels with vascular NAD(P)H oxidase-derived H2O2. MPO-vascular NAD(P)H oxidase-HOCl-chlorinating species may represent a common pathogenic pathway in vascular diseases and a new mechanism involved in exacerbation of vascular diseases under inflammatory conditions.
Nascent EDHF-mediated cerebral vasodilation in ovariectomized rats is not induced by eNOS dysfunction
Authors: Xu H. L. , Santizo R. A. , Baughman V. L. , Pelligrino D. A.
Published: March 2015

In estrogen-depleted [i.e., ovariectomized (Ovx)] animals, an endothelium-derived hyperpolarizing factor (EDHF)-like mechanism may arise to, at least partially, replace endothelial nitric oxide (NO) synthase (eNOS)-derived NO in modulating cerebral arteriolar tone. Additional findings show that eNOS expression and function is restored in estrogen-treated Ovx female rats, while the nascent EDHF-like activity disappears. Because NO has been linked to repression of EDHF activity in the periphery, the current study was undertaken to examine whether the nascent EDHF role in cerebral vessels of Ovx females relates to a chronically repressed eNOS-derived NO-generating function. We compared the effects of chronic NOS inhibition with Nω-nitro-l-arginine-methyl ester (l-NAME; 100 mg · kg–1 · day–1 for 3 wk) on EDHF-mediated pial arteriolar vasodilation in anesthetized intact, Ovx, and 17β-estradiol-treated (0.1 mg · kg–1 · day–1 ip, 1 wk) Ovx (OVE) female rats as well as in male rats that were prepared with closed cranial windows. In the chronic NOS inhibition groups, pial arteriolar responses were monitored in the absence (all groups) and presence (females only) of indomethacin (Indo; 10 mg/kg iv). Finally, the gap junction inhibitory peptide Gap 27 (300 μM) was applied to block EDHF-related vasodilation. NO donor ( S-nitroso- N-acetyl-penicillamine) responses were similar in all rats studied. Acetylcholine (ACh) reactivity was virtually absent in control Ovx rats and chronically NOS-inhibited intact female, OVE, and male rats. However, a partial recovery of ACh reactivity was seen in l-NAME-treated Ovx females. In addition, in the presence of l-NAME, a normal CO2 reactivity was observed in all females, whereas a 50% reduction in CO2 reactivity was seen in males. In intact and OVE rats, both chronic and acute ( NG-nitro-l-arginine suffusion) NOS inhibition, combined with Indo, depressed ADP-induced dilation by ≥50%, and subsequent application of Gap 27 had no further effect on ADP-induced vasodilation. ADP reactivity was retained in Ovx rats after combined chronic NOS inhibition and acute Indo, but was attenuated significantly by Gap 27. In males, Gap 27 had no effect on arteriolar reactivity. Taken together, our data demonstrate that in the cerebral microcirculation, NO does not have an inhibitory effect on EDHF production or action. The increased EDHF-like function in chronic estrogen-depleted animals is not due to eNOS deficiency, suggesting a more direct effect of estrogen in modulating EDHF-mediated cerebral vasodilation.
Actin cytoskeletal modulation of pressure-induced depolarization and Ca2+influx in cerebral arteries
Authors: Gokina Natalia I. , Osol George
Published: March 2015

The objective of this study was to examine the role of the actin cytoskeleton in the development of pressure-induced membrane depolarization and Ca2+influx underlying myogenic constriction in cerebral arteries. Elevating intraluminal pressure from 10 to 60 mmHg induced membrane depolarization, increased intracellular cytosolic Ca2+concentration ([Ca2+]i) and elicited myogenic constriction in both intact and denuded rat posterior cerebral arteries. Pretreatment with cytochalasin D (5 μM) or latrunculin A (3 μM) abolished constriction but enhanced the [Ca2+]iresponse; similarly, acute application of cytochalasin D to vessels with tone, or in the presence of 60 mM K+, elicited relaxation accompanied by an increase in [Ca2+]i. The effects of cytochalasin D were inhibited by nifedipine (3 μM), demonstrating that actin cytoskeletal disruption augments Ca2+influx through voltage-sensitive L-type Ca2+channels. Finally, pressure-induced depolarization was enhanced in the presence of cytochalasin D, further substantiating a role for the actin cytoskeleton in the modulation of ion channel function. Together, these results implicate vascular smooth muscle actin cytoskeletal dynamics in the control of cerebral artery diameter through their influence on membrane potential as well as via a direct effect on L-type Ca2+channels.
Oxidative stress-induced dysregulation of arteriolar wall shear stress and blood pressure in hyperhomocysteinemia is prevented by chronic vitamin C treatment
Authors: Bagi Zsolt , Csekö Csongor , Tóth Erika , Koller Akos
Published: March 2015

We aimed to test the hypothesis that an enhanced level of reactive oxygen species (ROS) is primarily responsible for the impairment of nitric oxide (NO)-mediated regulation of arteriolar wall shear stress (WSS) in hyperhomocysteinemia (HHcy). Thus flow/WSS-induced dilations of pressurized gracilis muscle arterioles (basal diameter: ∼170 μm) isolated from control (serum Hcy: 6 ± 1 μM), methionine diet-induced HHcy rats (4 wk, serum Hcy: 30 ± 6 μM), and HHcy rats treated with vitamin C, a known antioxidant (4 wk, 150 mg · kg body wt–1· day–1; serum Hcy: 32 ± 10 μM), were investigated. In vessels of HHcy rats, increases in intraluminal flow/WSS-induced dilations were converted to constrictions. Constrictions were unaffected by inhibition of NO synthesis by Nω-nitro-l-arginine methyl ester (l-NAME). Vitamin C treatment of HHcy rats reversed the WSS-induced arteriolar constrictions to l-NAME-sensitive dilations but did not affect control responses. Similar changes in responses were obtained for the calcium ionophore A-23187. In addition, diastolic and mean arterial blood pressure and serum 8-isoprostane levels (a marker of in vivo oxidative stress) were significantly elevated in rats with HHcy, changes that were normalized by vitamin C treatment. Taken together, our data show that in chronic HHcy long-term vitamin C treatment, by decreasing oxidative stress in vivo, enhanced NO bioavailability, restored the regulation of shear stress in arterioles, and normalized systemic blood pressure. Thus our study provides evidence that oxidative stress is an important in vivo mechanism that is primarily responsible for the development of endothelial dysregulation of WSS in HHcy.
DITPA stimulates bFGF, VEGF, angiopoietin, and Tie-2 and facilitates coronary arteriolar growth
Authors: Wang Xinguo , Zheng Wei , Christensen Lance P. , Tomanek Robert J.
Published: March 2015

Previous studies from our laboratory and those of others have shown thyroxine to be a stimulator of coronary microvascular growth. The present study tested the hypothesis that 3,5-diiodothyropropionic acid (DITPA), a thyroid hormone analog with inotropic but not chronotopic characteristics, is angiogenic in the nonischemic heart. Daily injections (3.75 mg/kg sc) of DITPA to Sprague-Dawley rats affected protein increases in vascular endothelial growth factor (VEGF)164, VEGF188, basic fibroblast growth factor (bFGF) (FGF-2), angiopoietin-1, and Tie-2 during the first few days of treatment. After 3 wk of treatment, arteriolar length density and the relative number of terminal arterioles (<10 μm diameter) increased in the left ventricle as determined by image analysis of perfuse-fixed hearts. These findings occurred in hearts that did not undergo changes in mass nor in increases in capillary length density. We conclude that DITPA, which is known to improve ventricular function after infarction, is angiogenic in normal nonischemic hearts.
Stimulation of brain Na+channels by FMRFamide in Dahl SS and SR rats
Authors: Wang Hao , White Roselyn , Leenen Frans H. H.
Published: March 2015

Stimulation of brain Na+channels by Phe-Met-Arg-Phe-NH2(FMRFamide) increases sympathetic nerve activity and blood pressure (BP) in Wistar rats. Blockade of brain ouabain-like compounds (OLC) by specific antibody Fab fragments prevents these responses to intracerebroventricular FMRFamide. In the present study, we evaluated the effects of high-salt intake on brain FMRFamide levels and the responses of BP and brain OLC to intracerebroventricular infusion of FMRFamide in Dahl salt-sensitive (SS) and salt-resistant (SR) rats. FMRFamide and OLC content was measured with the use of RIA and ELISA, respectively. A high-salt diet (1,370 μmol Na+/g) for 2 wk significantly increased BP in Dahl SS but not in SR rats. On a regular salt diet, Dahl SS and SR rats showed similar FMRFamide levels in the whole hypothalamus, pons and medulla, and spinal cord. A high-salt diet for 2 wk did not affect FMRFamide levels in these tissues in both Dahl SS and SR rats. In Dahl SS but not in SR rats, chronic intracerebroventricular infusion of FMRFamide (200 nmol · kg–1· day–1) for 2 wk significantly increased BP (mean arterial pressure: 116 ± 5 vs. 100 ± 2 mmHg; P < 0.01). Chronic intracerebroventricular infusion of FMRFamide significantly increased hypothalamic and pituitary OLC in Dahl SS but not SR rats. These results indicate that Dahl SS rats exhibit enhanced central responses to FMRFamide. In Dahl SS but not in SR rats on a high-salt diet, enhanced Na+entry through FMRFamide-activated brain Na+channels may increase brain OLC release, thereby leading to hypertension.
Aortic leucine-to-glutamate pathway: metabolic route and regulation of contractile responses
Authors: Schachter David , Sang James C.
Published: March 2015

Rat aortic endothelium is differentiated regionally for three signal pathways capable of regulating the cGMP content of the underlying smooth muscle. Formation of nitric oxide (NO) from l-arginine and of glutamate from l-leucine increase cGMP; however, formation of prostaglandin H2(PGH2) decreases cGMP. All three have peak activity in the windkessel area just distal to the aortic arch and decrease peripherally. We report evidence that the biochemical route of the leucine-to-glutamate (Leu→Glu) pathway is via metabolism of leucine to acetyl CoA, that the controlling reaction of the pathway is mediated by the branched chain α-ketoacid dehydrogenase complex (BCDC), and that glutamate formation via the Leu→Glu pathway is a major source of aortic segment free glutamate in vitro. Interruption of the pathway by treatment of precontracted rat aortic rings in vitro with each of three classes of inhibitors (leucine analogs, competitors for the BCDC reaction, or inhibitors of l-glutamate transport) enhances contractile responses. The enhancement requires an intact endothelium and is not owing to reductions in NO formation. The results support the hypothesis that the Leu→Glu pathway functions in the regulation of aortic contractility and compliance.
PTK, MAPK, and NOC/oFQ impair hypercapnic cerebrovasodilation after hypoxia/ischemia
Authors: Jagolino Amanda L. , Armstead William M.
Published: March 2015

This study characterized the contributions of protein tyrosine kinase (PTK) and mitogen-activated protein kinase (MAPK) in nociceptin/orphanin FQ (NOC/oFQ)-induced impairment of hypercapnic pial artery dilation (PAD) after hypoxia/ischemia (H/I) in piglets equipped with a closed cranial window. NOC/oFQ (10−10 M cerebrospinal fluid H/I concentration) impaired hypercapnic PAD (21 ± 2% vs. 13 ± 1%). Coadministration of either of the PTK inhibitors genistein or tyrphostin A23 or the MAPK inhibitors U-0126 or PD-98059 with NOC/oFQ (10−10 M) partially prevented the inhibition of hypercapnic PAD compared with that observed in their absence (21 ± 2% vs. 17 ± 1% for genistein). After exposure to H/I, PAD in response to hypercapnia was impaired, but pretreatment with either genistein, tyrphostin A23, U-0126, or PD-98059 partially protected such impairment (17 ± 1% vs. 4 ± 1% vs. 9 ± 1% for sham control, H/I, and H/I + genistein pretreatment, respectively). These data show that PTK and MAPK activation contribute to NOC/oFQ-induced impairment of hypercapnic PAD. These data suggest that activation of PTK and MAPK is also involved in the mechanism by which NOC/oFQ impairs hypercapnic PAD after H/I.
gp91phox Contributes to NADPH oxidase activity in aortic fibroblasts but not smooth muscle cells
Authors: Chamseddine Ali H. , Miller Francis J.
Published: March 2015

Reactive oxygen species (ROS) derived from vascular NADPH oxidase are important in normal and pathological regulation of vessel growth and function. Cell-specific differences in expression and function of the catalytic subunit of NADPH oxidase may contribute to differences in vascular cell response to NADPH oxidase activation. We examined the functional expression of gp91 phox on NADPH oxidase activity in vascular smooth muscle cells (SMC) and fibroblasts (FB). As measured by dihydroethidium fluorescence in situ, superoxide [Formula: see text] levels were greater in adventitial cells compared with medial SMC in wild-type aorta. In contrast, there was no difference in [Formula: see text] levels between adventitial cells and medial SMC in aorta from gp91 phox-deficient (gp91 phox KO) mice. Adventitial-derived FB and medial SMC were isolated from the aorta of wild-type and gp91 phox KO mice and grown in culture. Consistent with the observations in situ, basal and stimulated ROS levels were reduced in FB isolated from aorta of gp91 phox KO compared with FB from wild-type aorta, whereas ROS levels were similar in SMC derived from gp91 phox KO and wild-type aorta. There were no differences in expression of superoxide dismutase between gp91 phox KO and wild-type FB to account for these observations. Because gp91 phox is associated with membranes, we examined NADPH-stimulated [Formula: see text] production in membrane-enriched fractions of cell lysate. As measured by chemiluminescence, NADPH oxidase activity was markedly greater in wild-type FB compared with gp91 phox KO FB but did not differ among the SMCs. Confirming functional expression of gp91 phox in FB, antisense to gp91 phox decreased ROS levels in wild-type FB. Finally, deficiency of gp91 phox did not alter expression of the gp91 phox homolog NOX4 in isolated FB. We conclude that the neutrophil subunit gp91 phox contributes to NADPH oxidase function in vascular FB, but not SMC.
Mechanical alternans and restitution in failing SHHF rat left ventricles
Authors: Dumitrescu Cristian , Narayan Prakash , Efimov Igor R. , Cheng Yuanna , Radin M. Judith , McCune Sylvia A. , Altschuld Ruth A.
Published: March 2015

We examined mechanical alternans and electromechanical restitution in normal and failing rat hearts. Alternans occurred at 5 Hz in failing versus 9 Hz in control hearts and was reversed by 300 nM isoproterenol, 6 mM extracellular Ca2+, 300 nM −BAY K 8644, or 50 nM ryanodine. Restitution curves comprised phase I, which was completed before relaxation of the steady-state beat, and phase II, which occurred later. Phase I action potential area and developed pressure ratios were significantly reduced in the failing versus control hearts. Phase II was a monoexponential increase in relative developed pressure as the extrasystolic interval was increased. The plateau of phase II was significantly elevated in failing hearts. Thapsigargin (3 μM) plus ryanodine (200 nM) potentiated phase I to a significantly greater extent in control versus failing hearts and abolished phase II in both groups. The results suggest that both regulation of Ca2+ influx across the sarcolemma and Ca2+ release by the sarcoplasmic reticulum may contribute to altered excitation-contraction coupling in the failing spontaneously hypertensive heart failure prone rat heart.
An electrodiffusion model for effects of surface glycocalyx layer on microvessel permeability
Authors: Fu Bingmei M. , Chen Bin , Chen Wenhao
Published: March 2015

To investigate the charge effect of the endothelial surface glycocalyx on microvessel permeability, we extended the three-dimensional model developed by Fu et al. ( J Biomech Eng 116: 502–513, 1994) for the interendothelial cleft to include a negatively charged glycocalyx layer at the entrance of the cleft. Both electrostatic and steric exclusions on charged solutes were considered within the glycocalyx layer and at the interfaces. Four charge-density profiles were assumed for the glycocalyx layer. Our model indicates that the overall solute permeability across the microvessel wall including the surface glycocalyx layer and the cleft region is independent of the charge-density profiles as long as they have the same maximum value and the same total charge. On the basis of experimental data, this model predicts that the charge density would be 25–35 meq/l in the glycolcalyx of frog mesenteric capillaries. An intriguing prediction of this model is that when the concentrations of cations and anions are unequal in the lumen due to the presence of negatively charged proteins, the negatively charged glycocalyx would provide more resistance to positively charged solutes than to negatively charged ones.
Transfection of human endothelial cells with HIV-1tatgene activates NF-κB and enhances monocyte adhesion
Authors: Pieper Galen M. , Olds Cara L. , Bub Jeffrey D. , Lindholm Paul F.
Published: March 2015

Human immunodeficiency virus (HIV)-1 Tat released from HIV-1-infected monocytes is believed to enter other cells via an integrin-facilitated pathway, resulting in altered gene expression. Indeed, exogenous Tat protein can increase cell adhesion molecule gene expression in human endothelial cells. Signaling pathways initiated by Tat in endothelial cells are not known. We evaluated the ability of endogenous tat to stimulate monocyte adhesion via activation of nuclear factor-κB (NF-κB) within human umbilical vein endothelial cells. Transfection with pcTat, but not control vector DNA, increased NF-κB binding activity, NF-κB luciferase reporter activity, and monocyte adhesion. pcTat also increased κB-dependent HIV-1-LTR-CAT reporter activity 28-fold compared with a 3-fold increase produced by transfection with an equivalent amount of pcTax (from human leukemia virus). The pcTat-induced increase in pNF-κB-Luc activity and monocyte adhesion to endothelial cells was blocked by cotransfection with dominant-negative mutant IκBα and by incubation with 10 mM aspirin. We conclude that monocyte adhesion to human endothelial cells stimulated by pcTat is mediated via an NF-κB-dependent mechanism. Furthermore, inhibition studies using aspirin suggest that pcTat-stimulated NF-κB activation and monocyte adhesion occur via a redox-sensitive mechanism.
Alteration of mitochondrial function in a model of chronic ischemia in vivo in rat heart
Authors: Boudina Sihem , Laclau Muriel N. , Tariosse Liliane , Daret Danièle , Gouverneur Gérard , Bonoron-Adèle Simone , Saks Valdur A. , Dos Santos Pierre
Published: March 2015

The aim of this study was to investigate mitochondrial alterations in an animal model of chronic myocardial ischemia in rats obtained by surgical constriction of the left coronary artery. Resting coronary blood flow was measured using the fluorescent microsphere technique. Contractile function, defined by rate-pressure product, and myocardial oxygen consumption were measured in a Langendorff preparation. The mitochondrial function was evaluated on permeabilized skinned fibers. Three weeks after surgery, ischemic hearts showed a significant decrease in coronary blood flow compared with sham. Hemodynamic measurements showed a significant systolic and diastolic dysfunction. Alterations in mitochondrial function in ischemic hearts were mainly characterized by a significant decrease in the maximal velocity and apparent half-saturation constant for ADP, loss of the stimulatory effect of creatine, and a stimulatory effect of exogenous cytochrome c. These functional alterations were supported by structural alterations characterized by mitochondrial clustering and swelling associated with membrane rupture. We conclude that the alterations in systolic function after chronic ischemia are supported by severe modifications of mitochondrial structure and function.
Dual role of endothelin-1 via ETA and ETB receptors in regulation of cardiac contractile function in mice
Authors: Piuhola Jarkko , Mäkinen Markus , Szokodi István , Ruskoaho Heikki
Published: March 2015

An increase in coronary perfusion pressure leads to increased cardiac contractility, a phenomenon known as the Gregg effect. Exogenous endothelin (ET)-1 exerts a positive inotropic effect; however, the role of endogenous ET-1 in the contractile response to elevated load is unknown. We characterized here the role of ETA and ETB receptors in regulation of contractility in isolated, perfused mouse hearts subjected to increased coronary flow. Elevation of coronary flow from 2 to 5 ml/min resulted in 80 ± 10% increase in contractile force ( P < 0.001). BQ-788 (ETB receptor antagonist) augmented the load-induced contractile response by 35% ( P < 0.05), whereas bosentan (ETA/B receptor antagonist) and BQ-123 (ETA receptor antagonist) attenuated it by 34% and 56%, respectively ( P < 0.05). CV-11974 (ANG II type 1 receptor antagonist) did not modify the increase in contractility. These results show that endogenous ET-1 is a key mediator of the Gregg effect in mouse hearts. Moreover, ET-1 has a dual role in the regulation of cardiac contractility: ETA receptor-mediated increase in contractile force is suppressed by ETB receptors.
Time-dependent transients in an ionically based mathematical model of the canine atrial action potential
Authors: Kneller James , Ramirez Rafael J. , Chartier Denis , Courtemanche Marc , Nattel Stanley
Published: March 2015

Ionically based cardiac action potential (AP) models are based on equations with singular Jacobians and display time-dependent AP and ionic changes (transients), which may be due to this mathematical limitation. The present study evaluated transients during long-term simulated activity in a mathematical model of the canine atrial AP. Stimulus current assignment to a specific ionic species contributed to stability. Ionic concentrations were least disturbed with the K+ stimulus current. All parameters stabilized within 6–7 h. Inward rectifier, Na+/Ca2+ exchanger, L-type Ca2+, and Na+-Cl− cotransporter currents made the greatest contributions to stabilization of intracellular [K+], [Na+], [Ca2+], and [Cl−], respectively. Time-dependent AP shortening was largely due to the outward shift of Na+/Ca2+exchange related to intracellular Na+(Na[Formula: see text]) accumulation. AP duration (APD) reached a steady state after ∼40 min. AP transients also occurred in canine atrial preparations, with the APD decreasing by ∼10 ms over 35 min, compared with ∼27 ms in the model. We conclude that model APD and ionic transients stabilize with the appropriate stimulus current assignment and that the mathematical limitation of equation singularity does not preclude meaningful long-term simulations. The model agrees qualitatively with experimental observations, but quantitative discrepancies highlight limitations of long-term model simulations.
Neuronal NOS-dependent dilation to flow in coronary arteries of male eNOS-KO mice
Authors: Huang An , Sun Dong , Shesely Edward G. , Levee Ellen M. , Koller Akos , Kaley Gabor
Published: March 2015

Flow-induced dilation was examined in isolated coronary arteries of endothelial nitric oxide (NO) synthase knockout mice (eNOS-KO) and wild-type (WT) mice. The basal tone of arteries (percentage of passive diameter) was significantly greater in eNOS-KO than in WT mice; their flow-induced dilations, however, were similar. Endothelial removal eliminated the dilations in vessels of both strains of mice. In arteries of WT mice, N ω-nitro-l-arginine methyl ester (l-NAME) (10−4 M) or indomethacin (10−5 M) alone, inhibited flow-induced dilation by ∼50%, whereas their simultaneous administration abolished the responses. In arteries of eNOS-KO mice, flow-induced dilation was inhibited by ∼40% with l-NAME. The residual portion (60%) of the response was eliminated by the additional administration of indomethacin. 7-Nitroindazole (10−4 M) attenuated flow-induced dilation by ∼40% in arteries of eNOS-KO mice, but did not affect responses in those of WT mice. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (3 × 10−5 M) inhibited thel-NAME/7-nitroindazole-sensitive portion of the responses in arteries of eNOS-KO mice. Immunohistochemical evidence confirms the presence of neuronal NOS (nNOS) in the arterial endothelium of eNOS-KO mice. In conclusion, nNOS-derived NO, via activation of cGMP, together with prostaglandins, maintains flow-induced dilation in coronary arteries of male eNOS-KO mice.
Effect of in vivo gene transfer of nNOS in the PVN on renal nerve discharge in rats
Authors: Li Yi-Fan , Roy Shyamal K. , Channon Keith M. , Zucker Irving H. , Patel Kaushik P.
Published: March 2015

The paraventricular nucleus (PVN) of the hypothalamus is known to be involved in the control of sympathetic outflow. Nitric oxide (NO) has been shown to have a sympathoinhibitory effect in the PVN. The goal of the present study was to examine the influence of overexpression of neuronal NO synthase (nNOS) within the PVN on renal sympathetic nerve discharge (RSND). Adenovirus vectors encoding either nNOS (Ad.nNOS) or β-galactosidase (Ad.β-Gal) were transfected into the PVN in vivo. Initially, the dose of adenovirus needed for infection was determined from in vitro infection of cultured fibroblasts. In Ad.nNOS-treated rats, the local expression of nNOS within the PVN was confirmed by histochemistry for NADPH-diaphorase-positive neurons. There was a robust increase in staining of NADPH-diaphorase-positive cells in the PVN on the side injected with Ad.nNOS. The staining peaked at 3 days after injection of the virus. In α-chloralose- and urethane-anesthetized rats, microinjection of N G-monomethyl-l-arginine (l-NMMA), a NO antagonist, into the PVN produced a dose-dependent increase in RSND, blood pressure, and heart rate. There was a potentiation of the increase in RSND, blood pressure, and heart rate due to l-NMMA in Ad.nNOS-injected rats compared with Ad.β-Gal-injected rats. These results suggest that the endogenous NO-mediated effect in the PVN of Ad.nNOS-treated rats is more effective in suppressing RSND compared with Ad.β-Gal-treated rats. These observations support the contention that an overexpression of nNOS within the PVN may be responsible for increased suppression of sympathetic outflow. This technique may be useful in pathological conditions know to have increased sympathetic outflow, such as hypertension or heart failure.
Calpains and cytokines in fibrillating human atria
Authors: Goette Andreas , Arndt Marco , Röcken Christoph , Staack Thorsten , Bechtloff Roland , Reinhold Dirk , Huth Christof , Ansorge Siegfried , Klein Helmut U. , Lendeckel Uwe
Published: March 2015

Atrial fibrillation (AF) is accompanied by intracellular calcium overload. The purpose of this study was to assess the role of calcium-dependent calpains and cytokines during AF. Atrial tissue samples from 32 patients [16 with chronic AF and 16 in sinus rhythm (SR)] undergoing open heart surgery were studied. Atrial expression of calpain I and II, calpastatin, troponin T (TnT), troponin C (TnC), and cytokines [interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-10, transforming growth factor (TGF)-β1, and tumor necrosis factor-α] were determined. Expression of calpain I was increased during AF (461 ± 201% vs. 100 ± 34%, P < 0.05). Amounts of calpain II and calpastatin were unchanged. Total calpain enzymatic activity was more than doubled during AF (35.2 ± 17.7 vs. 12.4 ± 9.2 units, P < 0.05). In contrast to TnC, TnT levels were reduced in fibrillating atria by 26% ( P < 0.05), corresponding to the myofilament disintegration seen by electron microscopy. Small amounts of only IL-2 and TGF-β1 mRNA and protein were detected regardless of the underlying cardiac rhythm. In conclusion, atria of patients with permanent AF show evidence of calpain I activation that might contribute to structural remodeling and contractile dysfunction, whereas there is no evidence of activation of tissue cytokines.
Mutation of COOH-terminal lysines in overexpressed αB- crystallin abrogates ischemic protection in cardiomyocytes
Authors: Martin Jody L. , Bluhm Wolfgang F. , He Huaping , Mestril Ruben , Dillmann Wolfgang H.
Published: March 2015

High levels of αB-crystallin are present in the cardiomyocyte, yet little is understood about the function and importance of this protein. Like many other small heat shock proteins, αB-crystallin forms large oligomeric complexes whose size can be regulated by posttranslational modifications. The size of these complexes can modify the function of the protein. A naturally occurring COOH-terminal mutant has many detrimental effects in the lens of the eye and altered oligomerization. Therefore, we mutated the two COOH-terminal lysines of αB-crystallin to glycines (K174/175G) and adenovirally mounted them to transduce cardiomyocytes. We analyzed the effect of this mutation on oligomerization, microtubular stabilization, and ischemic outcome. A nearly 45% downward shift in complex size was observed with the mutant by native PAGE followed by immunoblotting. The overexpressed protein no longer protected the tubulin cytoskeleton against ischemic stress by confocal analysis. The mutant caused a 30% increase in cytosolic enzyme release with ischemia compared with control, whereas a 33% decrease was associated with wild-type αB-crystallin overexpression. We conclude that the COOH terminus of αB-crystallin is crucial to its proper function.
Effect of increased expression of cytoskeletal protein vinculin on ischemia-reperfusion injury in ventricular myocytes
Authors: Wei Hongguang , L'Ecuyer Thomas , Vander Heide Richard S.
Published: March 2015

The transition from reversible to irreversible ischemic injury (ischemia-reperfusion, I/R) occurs coincident with the loss of vinculin, a cytoskeletal protein involved in the attachment of the myofibrils to the sarcolemmal membrane. If the loss of vinculin were critical to the development of I/R, then increased levels of vinculin would be predicted to delay the onset of irreversible injury assuming that the protein is functional and localized to the proper subcellular site. The present study determined whether increased expression of vinculin, specifically in the cytoskeletal compartment, would provide protection from I/R injury. Neonatal rat myocytes were cultured and infected with a newly created replication-deficient adenovirus driving the expression of vinculin. I/R was induced with chemical inhibitors of glycolysis and mitochondrial respiration. Irreversible cell injury was assessed with lactate dehydrogenase (LDH) release. Virus-infected myocytes expressed significantly more vinculin in the cytoskeletal fraction and increased the expression of paxillin but sustained the same amount of injury in response to simulated I/R as control cells ( n = 4; P = not significant, paired t-test). Hypothermic I/R (ischemia at 25°C) resulted in a significant reduction in LDH release ( P ≤ 0.02; n = 4). Virus-mediated overexpression of vinculin does not appear to represent a rational approach to overcoming I/R injury.
Arterial distensibility: acute changes following dynamic exercise in normal subjects
Authors: Naka Katerina K. , Tweddel Ann C. , Parthimos Dimitris , Henderson Andrew , Goodfellow Jonathan , Frenneaux Michael P.
Published: March 2015

The time course of acute changes in large artery distensibility immediately and for 60 min following maximum treadmill exercise in normal subjects was characterized by simultaneously measuring upper and lower limb pulse wave velocity (PWV). A new oscillometric technique was used, which has proven to be sensitive to changes in distensibility induced by acute changes in vascular tone independently of blood pressure. The observed changes in PWV are attributable to changes in vascular tone corresponding to recovery from a systemic net constrictor response and a local net dilator response to exercise with persisting postexercise vasodilatation. They are inadequately explained by associated changes in blood pressure and cannot be attributed to changes in heart rate or viscosity. Modeled as a system of n coupled linear differential equations, the minimum (and adequate) order required to reproduce these patterns was n = 1 for the upper and n = 2 for the exercising lower limb. The economy of the solution suggests entrainment among the multiple interactive mechanisms governing vasomotor control.
Orthogonal properties of the redox siblings nitroxyl and nitric oxide in the cardiovascular system: a novel redox paradigm
Authors: Wink David A. , Miranda Katrina M. , Katori Tatsuo , Mancardi Daniele , Thomas Douglas D. , Ridnour Lisa , Espey Michael G. , Feelisch Martin , Colton Carol A. , Fukuto Jon M. , Pagliaro Pasquale , Kass David A. , Paolocci Nazareno
Published: March 2015

Endogenous formation of nitric oxide (NO) and related nitrogen oxides in the vascular system is critical to regulation of multiple physiological functions. An imbalance in the production or availability of these species can result in progression of disease. Nitrogen oxide research in the cardiovascular system has primarily focused on the effects of NO and higher oxidation products. However, nitroxyl (HNO), the one-electron-reduction product of NO, has recently been shown to have unique and potentially beneficial pharmacological properties. HNO and NO often induce discrete biological responses, providing an interesting redox system. This article discusses the emerging aspects of HNO chemistry and attempts to provide a framework for the distinct effects of NO and HNO in vivo.
Energy requirements for the Na+gradient in the oxygenated isolated heart: effect of changing the free energy of ATP hydrolysis
Authors: Jansen Maurits A. , Shen Hai , Zhang Li , Wolkowicz Paul E. , Balschi James A.
Published: March 2015

This study tests the hypothesis that a decrease of the free energy of ATP hydrolysis (Δ GATP) below a threshold value will inhibit Na+-K+-ATPase (Na+pump) activity and result in an increase of intracellular Na+concentration ([Na+]i) in the heart. Conditions were designed in which hearts were solely dependent on ATP derived from oxidative phosphorylation. The only substrate supplied was the fatty acid butyrate (Bu) at either low, 0.1 mM (LowBu), or high, 4 mM (HighBu), concentrations. Escalating work demand reduced the Δ GATPof the LowBu hearts.31P,23Na, and87Rb NMR spectroscopy measured high-energy phosphate metabolites, [Na+]i, and Rb+uptake. Rb+uptake was used to estimate Na+pump activity. To measure [Na+]iusing a shift reagent for cations, extracellular Ca2+was reduced to 0.85 mM, which eliminated work demand Δ GATPreductions. Increasing extracellular Na+([Formula: see text]) to 200 mM restored work demand Δ GATPreductions. In response to higher [Na+]e, [Na+]iincreased equally in LowBu and HighBu hearts to ∼8.6 mM, but Δ GATPdecreased only in LowBu hearts. At lowest work demand the LowBu heart Δ GATPwas –53 kJ/mol, Rb+uptake was similar to that of HighBu hearts, and [Na+]iwas constant. At highest work demand the LowBu heart Δ GATPdecreased to –48 kJ/mol, the [Na+]iincreased to 25 mM, and Rb+uptake was 56% of that in HighBu hearts. At the highest work demand the HighBu heart Δ GATPwas –54 kJ/mol and [Na+]iincreased only ∼10%. We conclude that a Δ GATPbelow –50 kJ/mol limits the Na+pump and prevents maintenance of [Na+]ihomeostasis.
Cyclooxygenase inhibitor blocks rebound response after NO inhalation in an endotoxin model
Authors: Chen Luni , He Hao , Mondejar Enrique Fernandez , Hedenstierna Göran
Published: March 2015

This study addressed the possible role of cyclooxygenase (COX) and its products in the rebound response to inhaled nitric oxide (INO). Anesthetized, mechanically ventilated piglets were exposed to endotoxin alone, endotoxin combined with INO, or endotoxin with INO plus the COX inhibitor diclofenac (3 mg/kg iv) ( n = 8 piglets/group). A control group of healthy pigs ( n = 6) was also studied. Measurements were made of blood gases, hemodynamic parameters, lung tissue COX expression, and plasma concentrations of thromboxane B2 (TxB2), PGF2α, and 6-keto-PGF1α. Endotoxin increased lung inducible COX (COX-2) expression and circulating prostanoids concentrations. Inhalation of NO during endotoxemia increased the constitutive COX (COX-1) expression, and the circulating TxB2 and PGF2α increased further after INO withdrawal. The combination of COX inhibitor with INO blocked all these changes and eliminated the rebound reaction to INO withdrawal, which otherwise was seen in endotoxemic piglets given INO only. We conclude that the rebound response to INO discontinuation is related to COX products.
Bradykinin-induced proinflammatory signaling mechanisms
Authors: Shigematsu Sakuji , Ishida Shuji , Gute Dean C. , Korthuis Ronald J.
Published: March 2015

Intravital microscopic techniques were used to examine the mechanisms underlying bradykinin-induced leukocyte/endothelial cell adhesive interactions (LECA) and venular protein leakage (VPL) in single postcapillary venules of the rat mesentery. The effects of bradykinin superfusion to increase LECA and VPL were prevented by coincident topical application of either a bradykinin-B2receptor antagonist, a cell-permeant superoxide dismutase (SOD) mimetic or antioxidant, or inhibitors of cytochrome P-450 epoxygenase (CYPE) or protein kinase C (PKC) but not by concomitant treatment with either SOD, a mast cell stabilizer, or inhibitors of nitric oxide synthase, cyclooxygenase, xanthine oxidase, NADPH oxidase, or platelet-activating factor. Immunoneutralizing P-selectin or intercellular adhesion molecule-1 (ICAM-1) completely prevented bradykinin-induced leukocyte adhesion and emigration but did not affect VPL. On the other hand, stabilization of F-actin with phalloidin prevented bradykinin-induced leukocyte emigration and VPL but did not alter leukocyte adhesion. These data indicate that bradykinin induces LECA in rat mesenteric venules via a B2-receptor-initiated, CYPE-, oxidant- and PKC-mediated, P-selectin- and ICAM-1-dependent mechanism. Bradykinin also produced VPL, an effect that was initiated by stimulation of B2receptors and involved CYPE and PKC activation, oxidant generation, and cytoskeletal reorganization but was independent of leukocyte adherence and emigration.