bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Rat Cardiac Muscle Contraction and Mitochondrial Respiration by Endogenous Peroxynitrite Formation During Posthypoxic Reoxygenation
Authors: Xie Yi -Wu , Kaminski Pawel M. , Wolin Michael S.
Published: June 2012

Abstract
—This study was designed to investigate the potential role of endogenous peroxynitrite (ONOO
−
) formation in the inhibition of cardiac muscle contractility and mitochondrial respiration during posthypoxic reoxygenation. Isometric contraction of isolated rat left ventricular posterior papillary muscle was virtually eliminated at the end of an exposure to 15 minutes of hypoxia and remained 40±5% depressed an hour after the reintroduction of O
2
. O
2
uptake by rat left ventricular cardiac muscle, measured by a Clark-type O
2
electrode, was also inhibited by 24±2% at 10 minutes after reoxygenation. The inhibition of contractility and respiration during posthypoxic reoxygenation was markedly attenuated by the NO synthase inhibitor nitro-
l
-arginine, exogenous superoxide dismutase, and the ONOO
−
scavenger urate but not by the hydroxyl radical scavenger mannitol. Generation of ONOO
−
with the NO donor
S
-nitroso-
N
-acetylpenicillamine (SNAP) plus the superoxide-releasing agent pyrogallol caused an irreversible inhibition of cardiac contractile and respiratory function. Unlike ONOO
−
, exogenous (SNAP) and endogenous (bradykinin) sources of NO inhibited contractility in a reversible manner. Under conditions of comparable amounts of respiratory inhibition in unstimulated incubated muscle, the NO-dependent agents and the mitochondrial antagonist NaCN produced a smaller degree of suppression of contractility compared with ONOO
−
and posthypoxic reoxygenation. These results are consistent with a contributing role for endogenous ONOO
−
formation in the inhibition of cardiac muscle contractility and mitochondrial respiration during posthypoxic reoxygenation.
Spatial Changes in the Transmembrane Potential During Extracellular Electric Stimulation
Authors: Zhou Xiaohong , Knisley Stephen B. , Smith William M. , Rollins Dennis , Pollard Andrew E. , Ideker Raymond E.
Published: June 2012

Abstract
—The purpose of this study was to determine the spatial changes in the transmembrane potential caused by extracellular electric field stimulation. The transmembrane potential was recorded in 10 guinea pig papillary muscles in a tissue bath using a double-barrel microelectrode. After 20 S1 stimuli, a 10-ms square wave S2 shock field with a 30-ms S1–S2 coupling interval was given via patch shock electrodes 1 cm on either side of the tissue during the action potential plateau. Two shock strengths (2.1±0.2 and 6.5±0.6 V/cm) were tested with both shock polarities. The recording site was moved across the tissue along fibers with either 200 μm (macroscopic group [n=5], 12 consecutive recording sites over a 2.2-mm tissue length in each muscle) or 20 μm (microscopic group [n=5], 21 consecutive recording sites over a 0.4-mm tissue length in each muscle) between adjacent recording sites. In the macroscopic group, the portion of the tissue toward the anode was hyperpolarized, whereas the portion toward the cathode was depolarized, with 1 zero-potential crossing from hyperpolarization to depolarization present near the center of the tissue. In the microscopic group, only 1 zero-potential crossing was observed in the center region of the tissue, whereas, away from the center, only hyperpolarization was observed toward the anode and depolarization toward the cathode. Although these results are consistent with predictions from field stimulation of continuous representations of myocardial structure, ie, the bidomain and cable equation models, they are not consistent with the prediction of depolarization-hyperpolarization oscillation from representations based on cellular-level resistive discontinuities associated with gap junctions, ie, the sawtooth model.
High-Affinity Arginine Transport of Bovine Aortic Endothelial Cells Is Impaired by Lysophosphatidylcholine
Authors: Kikuta Ken-ichiro , Sawamura Tatsuya , Miwa Soichi , Hashimoto Nobuo , Masaki Tomoh
Published: June 2012

Abstract
—The mechanisms of endothelial dysfunction characterized by the impaired nitric oxide (NO) release have not yet been clarified. Because the phenomenon is mimicked in vitro by the application of oxidized LDL and its major lipid constituent, lysophosphatidylcholine (LPC), we analyzed their effects on the arginine-NO system, especially on the arginine transport system. LPC inhibited NO release induced by ADP in cultured bovine aortic endothelial cells. The inhibition was attenuated by the excess amount of extracellular arginine. LPC was found to inhibit the arginine transport in bovine aortic endothelial cells, which is mediated by high- and low-affinity components. LPC predominantly impaired the high-affinity component. In the presence of a high concentration of arginine, LPC showed apparently no inhibition of arginine transport, because the low-affinity transporter compensated for the activity. Taken together, the impairment of the high-affinity transport system might account for the inhibition of NO release by LPC. LPC also inhibited arginine transport in the intima of intact bovine aorta. Furthermore, LPC inhibited the activity of the high-affinity arginine transporter in endothelial cells, in the cationic amino acid transporter-1 expressed in COS-7 cells. The activity of cationic amino acid transporter-1 might be important for the prevention of endothelial dysfunction.
Mechanisms of Stretch-Induced Changes in [Ca
2+
]
i
in Rat Atrial Myocytes
Authors: Tavi Pasi , Han Chunlei , Weckström Matti
Published: June 2012

Abstract
—To study the effects of stretch on the function of rat left atrium, we recorded contraction force, calcium transients, and intracellular action potentials (APs) during stretch manipulations. The stretch of the atrium was controlled by intra-atrial pressure. The Frank-Starling behavior of the atrium was manifested as a biphasic increase of the contraction force after increasing the stretch level. The development of the contraction force after step increase of the stretch (intra-atrial pressure from 1 to 3 mm Hg) was accompanied by the increase in the amplitude of the calcium transients (
P
<0.05, n=4) and decrease in the time constant of the Ca
2+
transient decay. The APs of the individual myocytes were also affected by stretch; the duration of the AP was decreased at positive voltages (AP duration at 15% repolarization level,
P
<0.001; n=13) and increased at negative voltages (AP duration at 90% repolarization level,
P
<0.01; n=13). To study the mechanisms causing these changes we developed a mathematical model describing [Ca
2+
]
i
and electrical behavior of single rat atrial myocytes. Stretch was simulated in the model by increasing the troponin (TnC) sensitivity and/or applying a stretch-activated (SA) calcium influx. We mimicked the Ca
2+
influx by introducing a nonselective cationic conductance, the SA channels, into the membrane. Neither of the 2 plausible mechanosensors (TnC or SA channels) alone could produce similar changes in the Ca
2+
transients or APs as seen in the experiments. The model simulated the effects of stretch seen in experiments best when both the TnC affinity and the SA conductance activation were applied simultaneously. The SA channel activation led to gradual augmentation of Ca
2+
transients, which modulated the APs through increased Na
+
/Ca
2+
-exchanger inward current. The role of TnC affinity change was to modulate the Ca
2+
transients, stabilize the diastolic [Ca
2+
]
i
, and presumably to produce the immediate increase of the contraction force after stretch seen in experiments. Furthermore, we found that the same mechanism that caused the normal physiological responses to stretch could also generate arrhythmogenic afterpotentials at high stretch levels in the model.
Quinidine Interactions With Human Atrial Potassium Channels
Authors: Nenov Neviana I. , Crumb William J. , Pigott John D. , Harrison Lynn H. , Clarkson Craig W.
Published: June 2012

Abstract
—Clinical studies have suggested that quinidine is less effective when used for the treatment of atrial arrhythmias in pediatric patients compared with its clinical effectiveness in the adult patient population. Age-related changes in the cardiac actions of quinidine on action potential duration and interaction with potassium channels in several mammalian species also have been reported. We investigated the effects of postnatal development on quinidine’s interaction with major repolarizing currents (
I
to
,
I
Kur
,
I
ns
, and
I
K1
) in human atrial myocytes, using the whole-cell configuration of the voltage-clamp technique. Our results indicate that there are age-related changes in both the IC
50
for quinidine blockade of
I
to
, as well as the mechanism of quinidine unblocking. In contrast, quinidine was found to inhibit both adult and pediatric
I
K1
and
I
Kur
in an age-independent manner, whereas the nonselective cation current (
I
ns
), which contributes to the sustained outward current (
I
sus
), was insensitive to quinidine. The results from this study help to clarify the electrophysiological mechanism by which quinidine elicits its antiarrhythmic effect in the pediatric and adult human population.
Hypoxia Induces High-Mobility-Group Protein I(Y) and Transcription of the Cyclooxygenase-2 Gene in Human Vascular Endothelium
Authors: Ji Yan-Shan , Xu Qing , Schmedtje John F.
Published: June 2012

Abstract
—Cyclooxygenases catalyze a rate-limiting step in the synthesis of vascular endothelial prostaglandins. Expression of the inducible cyclooxygenase-2 (COX-2) gene is increased by hypoxia in human vascular endothelial cells via the nuclear factor (NF)-κB p65 transcription factor, which is necessary but not sufficient to fully induce COX-2 transcription in response to hypoxia. After finding that cytoplasmic NF-κB p65 and IκBα (an inhibitory protein that binds NF-κB p65 precursors) levels are not changed by hypoxia, we hypothesized that other factors might play a role in regulating the COX-2 promoter, like the high-mobility-group (HMG) I(Y) family of proteins, which features multiple A·T hooks and is associated with NF-κB–mediated transactivation. Nuclear protein obtained from human umbilical vein endothelial cells (HUVECs) was supplemented with HMG I(Y) during electrophoretic mobility shift assays using an NF-κB-3′ element probe. These data suggested that HMG I(Y) proteins interact with NF-κB p65 to induce COX-2 promoter activity. We also found that TATA-box DNA demonstrated increased electrophoretic shifting indicative of DNA binding after incubation with either hypoxic HUVEC nuclear protein or normoxic nuclear protein supplemented with HMG I(Y). Transfection of HUVECs with an expression vector containing the COX-2 promoter ligated to HMG I(Y) cDNA demonstrated positive feedback on COX-2 promoter activity in hypoxia. We confirmed that COX-2 is transcriptionally regulated by hypoxia using a nuclear runoff assay. Hypoxia increased steady-state cellular levels of HMG I(Y) mRNA as an early event, corresponding with increases in HMG I(Y) protein. Overexpression of HMG I(Y) was associated in a dose-response relationship with increasing prevalence of the COX-2 protein in hypoxic HUVECs. Furthermore, sense (and antisense) HMG I(Y) overexpression caused stimulation (or inhibition) of COX-2 promoter activity as measured by luciferase reporter gene expression. The physiological significance of these findings was demonstrated by cyclooxygenase-dependent release of prostaglandin E
2
by HUVECs in hypoxia. We concluded that hypoxia increases expression of HMG I(Y) proteins while facilitating transactivation of the COX-2 promoter. The HMG I(Y) family of proteins may therefore function as part of a hypoxia-induced enhanceosome that helps to promote transcription of COX-2.
Heparan Sulfate Proteoglycans Mediate a Potent Inhibitory Signal for Migration of Vascular Smooth Muscle Cells
Authors: Koyama Noriyuki , Kinsella Michael G. , Wight Thomas N. , Hedin Ulf , Clowes Alexander W.
Published: June 2012

Abstract
—Migration of vascular smooth muscle cells (SMCs) is a key step in vascular remodeling and formation of pathological lesions in diseased arteries and may be controlled by extracellular matrix (ECM) and by factors that regulate ECM composition, such as platelet-derived growth factor (PDGF). In culture, PDGF-AB and -BB enhance but PDGF-AA (although having no effect alone) suppresses SMC migration stimulated by other PDGF isoforms. To determine whether the migration-inhibitory mechanism of PDGF-AA was mediated by ECM composition, we examined baboon SMC migration in a Boyden chamber assay using filters coated with different ECM proteins. PDGF-AA suppressed the PDGF-BB–induced migration of baboon SMCs on a filter coated with basement membrane proteins (Matrigel) and fibronectin but failed to inhibit cell migration on a type I collagen (Vitrogen)-coated filter. Fibronectin and fibronectin fragments that contain heparin-binding domains permitted PDGF-AA inhibition of cell migration, but a fragment lacking heparin-binding domains did not. Treatment of SMCs with heparin lyases II and III, but not with chondroitin ABC lyase, diminished the PDGF-AA–mediated inhibition of migration. PDGF-AA stimulated accumulation of proteoglycan (PG) in the cell layer more potently than did PDGF-BB, whereas the turnover of cell layer PG was unaffected by either PDGF-AA or -BB. Northern blot analysis revealed that PDGF-AA increased syndecan-1 mRNA expression more than did PDGF-BB, whereas both PDGF isoforms decreased perlecan expression. The changes in cell migration and PG synthesis induced by PDGF-AA were accompanied by changes in the morphology of SMCs. PDGF-AA dramatically induced the spreading of SMCs, whereas the heparin lyase treatment of PDGF-AA–stimulated cultures diminished cell spreading. The data suggest that PDGF-AA selectively modifies heparan sulfate PG accumulation on SMCs and thereby influences the interactions of SMCs with heparin-binding ECM proteins. These interactions, in turn, generate signals that suppress SMC migration.
LDLs Impair Vasomotor Function of the Coronary Microcirculation
Authors: Hein Travis W. , Kuo Lih
Published: June 2012

Abstract
—Oxidized LDLs (Ox-LDLs) inhibit endothelium-dependent dilation of isolated conduit arteries in a manner comparable to the impairment demonstrated in atherosclerotic vessels. However, it is not known whether the microvessels, which do not develop atherosclerotic lesions, are susceptible to Ox-LDL. Since endothelial release of NO plays an important role in vasodilation and since its dysfunction associated with atherosclerosis has been shown to extend into the coronary microcirculation, we hypothesized that Ox-LDLs impair endothelium-dependent vasodilation of coronary arterioles by reducing the synthesis and/or release of NO. To test this hypothesis, porcine subepicardial vessels (50 to 100 μm) were isolated, cannulated, and pressurized to 60 cm H
2
O without flow for in vitro study. Isolated vessels developed basal tone and dilated in a dose-dependent manner to the endothelium-dependent vasodilators serotonin, ATP, and ionomycin. These vasodilatory responses were inhibited by the NO synthase inhibitor
N
G
-monomethyl-
l
-arginine and were subsequently reversed by extraluminal administration of the NO precursor
l
-arginine (3 mmol/L), suggesting the involvement of NO in these vasomotor responses. Intraluminal incubation of the vessels with native LDL (N-LDL) or Ox-LDL (1 mg protein/mL) significantly attenuated dilations to serotonin, ATP, and ionomycin. Ox-LDL produced more severe inhibition than did N-LDL, and the inhibitory effect was comparable to that of
N
G
-monomethyl-
l
-arginine. The inhibitory effects of N-LDL and Ox-LDL were reversed by exogenous
l
-arginine (3 mmol/L) and were prevented by sodium dihydroxybenzene disulfonate (Tiron), a cell-permeable superoxide scavenger. In contrast, administration of the cell-impermeable superoxide scavenger superoxide dismutase prevented the inhibitory effect of N-LDL but not of Ox-LDL. In addition, the inhibitory effects of LDL were not restored by
d
-arginine or by removal of intraluminal LDL. Neither N-LDL nor Ox-LDL altered endothelium-independent vasodilation to sodium nitroprusside. These results indicate that coronary arterioles are susceptible to LDLs that specifically impair endothelium-dependent vasodilation by reducing NO synthesis. It is suggested that the initiation of superoxide anion production and the subsequent
l
-arginine deficiency may be responsible for the detrimental effect of LDL.
Novel Myofilament Ca
2+
-Sensitizing Property of Xanthine Oxidase Inhibitors
Authors: Pérez Néstor Gustavo , Gao Wei Dong , Marbán Eduardo
Published: June 2012

Abstract
—Antioxidants are known to mitigate the cardiac contractile dysfunction that follows brief periods of ischemia (“myocardial stunning”). Stunning decreases contractility at the level of the contractile proteins; therefore, we asked whether antioxidant treatment preserves myofilament Ca
2+
responsiveness after global ischemia and reflow. Right ventricular trabeculae were dissected from rat hearts subjected either to 20 minutes ischemia and reperfusion in the absence of drugs (stunned group) or to the same protocol in the presence of allopurinol, an inhibitor of xanthine oxidase (XO), and mercaptopropionylglycine (MPG), a hydroxyl radical scavenger (antioxidant group). At 20 minutes of reflow, isovolumic developed pressure recovered completely in the antioxidant group, but in the stunned group it recovered by only 57%. [Ca
2+
]
i
and contractile force measurements in trabeculae revealed the expected depression of myofilament function in the stunned group, with no change in Ca
2+
transients relative to nonischemic controls. In contrast, Ca
2+
transients were smaller, but force was greater, in the antioxidant group relative to both the stunned group and to nonischemic controls. Steady-state [Ca
2+
]
i
-force relationships revealed a striking increase of maximal force and a modest shift of activation to a lower range of [Ca
2+
]
i
. The increase in maximal force was reproduced by allopurinol+MPG or by allopurinol alone under nonischemic conditions and also by oxypurinol (100 μmol/L), a potent inhibitor of XO. We conclude that allopurinol and oxypurinol sensitize the cardiac myofilaments to Ca
2+
. This Ca
2+
-sensitizing effect underlies the preservation of contractility observed with an allopurinol+MPG antioxidant cocktail in a model of stunned myocardium. These serendipitous findings identify allopurinol and oxypurinol as the lead compounds of a novel class of inotropic agents.
Expression of Costimulatory Molecule CD40 in Murine Heart With Acute Myocarditis and Reduction of Inflammation by Treatment With Anti-CD40L/B7-1 Monoclonal Antibodies
Authors: Seko Yoshinori , Takahashi Naoyuki , Azuma Miyuki , Yagita Hideo , Okumura Ko , Yazaki Yoshio
Published: June 2012

Abstract
—Evidence has accumulated that antigen-specific T cells infiltrate the heart and play an important role in the pathogenesis of viral myocarditis. Costimulatory molecules such as B7s and CD40 expressed on antigen-presenting cells are known to play a critical role for antigen-specific T-cell activation to occur. To investigate the role for a costimulatory molecule, CD40, in the development of acute viral myocarditis, we first analyzed the expression of CD40 in the hearts of mice with acute viral myocarditis induced by Coxsackievirus B3. We also evaluated the induction of CD40 in cultured cardiac myocytes treated with interferon gamma in vitro. Second, we analyzed the cytokine production by cultured cardiac myocytes by stimulation with anti-CD40 monoclonal antibody (mAb) in vitro. Third, we examined the effects of in vivo administration of anti–CD40L/B7-1 mAbs on the development of acute viral myocarditis. We found that Coxsackievirus B3–induced murine acute myocarditis results in enhanced expression of CD40 on cardiac myocytes. The expression of CD40 on cardiac myocytes could be induced by interferon gamma in vitro. We also found that the production of interleukin-6 by cardiac myocytes was stimulated with anti-CD40 mAb and that in vivo anti–CD40L/B7-1 mAb treatment significantly decreased the myocardial inflammation. Our findings strongly suggest that CD40 plays an important role in the development of acute viral myocarditis and raise the possibility of immunotherapy with anti–CD40L/B7-1 mAbs to prevent T cell–mediated myocardial damage in viral myocarditis.
Familial Hypertrophic Cardiomyopathy
Authors: Bonne Gisèle , Carrier Lucie , Richard Pascale , Hainque Bernard , Schwartz Ketty
Published: June 2012

Abstract
—Hypertrophic cardiomyopathy is characterized by left and/or right ventricular hypertrophy, which is usually asymmetric and involves the interventricular septum. Typical morphological changes include myocyte hypertrophy and disarray surrounding the areas of increased loose connective tissue. Arrhythmias and premature sudden deaths are common. Hypertrophic cardiomyopathy is familial in the majority of cases and is transmitted as an autosomal-dominant trait. The results of molecular genetics studies have shown that familial hypertrophic cardiomyopathy is a disease of the sarcomere involving mutations in 7 different genes encoding proteins of the myofibrillar apparatus: β-myosin heavy chain, ventricular myosin essential light chain, ventricular myosin regulatory light chain, cardiac troponin T, cardiac troponin I, α-tropomyosin, and cardiac myosin binding protein C. In addition to this locus heterogeneity, there is a wide allelic heterogeneity, since numerous mutations have been found in all these genes. The recent development of animal models and of in vitro analyses have allowed a better understanding of the pathophysiological mechanisms associated with familial hypertrophic cardiomyopathy. One can thus tentatively draw the following cascade of events: The mutation leads to a poison polypeptide that would be incorporated into the sarcomere. This would alter the sarcomeric function that would result (1) in an altered cardiac function and then (2) in the alteration of the sarcomeric and myocyte structure. Some mutations induce functional impairment and support the pathogenesis hypothesis of a “hypocontractile” state followed by compensatory hypertrophy. Other mutations induce cardiac hyperfunction and determine a “hypercontractile” state that would directly induce cardiac hypertrophy. The development of other animal models and of other mechanistic studies linking the genetic mutation to functional defects are now key issues in understanding how alterations in the basic contractile unit of the cardiomyocyte alter the phenotype and the function of the heart.
Functional Evidence for a Novel Suramin-Insensitive Pyrimidine Receptor in Rat Small Pulmonary Arteries
Authors: Hartley S. Anthony , Kato Kenichi , Salter Katie J. , Kozlowski Roland Z.
Published: June 2012

Abstract
—Uridine nucleotides are known to cause constriction of pulmonary arterial smooth muscle. However, the P2 receptor subtypes underlying the contractile effects of these nucleotides in the pulmonary circulation have not been determined. We have used myography and the patch-clamp recording technique to compare the effects of UTP and UDP in isolated small pulmonary arteries (diameter 100 to 400 μm) and their constituent smooth muscle cells. In endothelium-denuded arteries, both UTP and UDP (0.01 to 3 mmol/L) induced concentration-dependent increases in tension that were independent of P2X receptor stimulation. The UDP-mediated increase in tension was significantly less sensitive to the nonselective P2 receptor blocker suramin than the UTP-mediated increase in tension. In single isolated arterial myocytes, voltage-clamped at −50 mV (close to the resting membrane potential of these cells), application of both UTP and UDP evoked periodic oscillations of inward current primarily because of a Ca
2+
-activated Cl
−
current (
I
Cl,Ca
). Oscillations of
I
Cl,Ca
evoked by UTP were reversibly inhibited by suramin, although those evoked by UDP were insensitive to the antagonist. In addition to confirming the presence of classical P2Y
2
receptors, these results also provide functional evidence for the existence of a novel UDP receptor in pulmonary arterial myocytes, which may contribute to pyrimidine-evoked vasoconstriction. This notion is supported by molecular evidence that demonstrates the presence of P2Y
6
receptor transcripts in rat pulmonary arterial smooth muscle.
A High Level of CCAAT-Enhancer Binding Protein-δ Expression Is a Major Determinant for Markedly Elevated Differential Gene Expression of the Platelet-Derived Growth Factor-α Receptor in Vascular Smooth Muscle Cells of Genetically Hypertensive Rats
Authors: Kitami Yutaka , Fukuoka Tomikazu , Hiwada Kunio , Inagami Tadashi
Published: June 2012

Abstract
—Platelet-derived growth factor-α receptor (PDGF-αR) expression is markedly elevated in cultured vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) when compared with normotensive rat strains, Sprague-Dawley, Wistar, and Wistar-Kyoto rats (WKY). This “almost-all-or-none” type of differential expression strongly suggests that PDGF-αR or its transcription-regulating mechanisms or factors are significantly related to genetic hypertension. To evaluate the role of PDGF-αR in vascular remodeling and hypertension, we have investigated the underlying molecular mechanism. We have recently shown that the regulatory domain responsible for this difference is localized to the PDGF-αR promoter region between –246 and –139, which contains an enhancer core sequence specific for CCAAT-enhancer binding proteins (C/EBPs). We defined the roles of this element for hypertensive strain-specific PDGF-αR gene transcription. DNA-protein binding studies by competition in electromobility shift and supershift assays revealed that 2 members, C/EBP-β and C/EBP-δ, are mainly responsible for DNA-protein complex formation; the former acts as a transcriptional repressor and the latter as an activator of the PDGF-αR gene, respectively. Western or Northern blot analyses supported evidence for high expression of C/EBP-δ seen only in SHR-derived VSMCs. Furthermore, forced expression of C/EBP-δ transactivated the transcriptional efficiency of the PDGF-αR gene even in WKY-derived VSMCs, whereas that of C/EBP-β had an opposite effect in SHR-derived VSMCs. These findings indicate that differential expression of members of the C/EBP family, mainly C/EBP-δ and possibly C/EBP-β, are responsible for the strain-specific gene transcription of PDGF-αR in VSMCs.
Effect of Recombinant Soluble P-Selectin Glycoprotein Ligand-1 on Leukocyte-Endothelium Interaction In Vivo
Authors: Scalia Rosario , Hayward Reid , Armstead Valerie E. , Minchenko Alexander G. , Lefer Allan M.
Published: June 2012

Abstract
—Traumatic shock induces profound pathophysiological alterations and initiates inflammatory reactions in many tissues, thus resulting in acute multiple organ damage (eg, intestine, pancreas, and liver). In the rat, Noble-Collip drum trauma increases P-selectin expression on the vascular endothelium as a result of loss of endothelium-derived NO. Here we postulated that blockade of the earliest steps in leukocyte adhesion (ie, leukocyte rolling) via administration of a recombinant soluble form of P-selectin glycoprotein ligand-1 (PSGL-1; the recombinant soluble form is rsPSGL.Ig) would attenuate selectin-mediated events observed in the rat during traumatic shock. Using intravital microscopy of the rat mesenteric microvasculature, we found that intravenous infusion of rsPSGL.Ig significantly inhibited leukocyte-endothelium interaction (ie, leukocyte rolling, adherence, and transmigration) induced by traumatic shock as well as by activation of the microvascular endothelium with 50 μmol/L
N
G
-nitro-
l
-arginine methyl ester. Immunohistochemical detection of P-selectin on the mesenteric venular endothelial surface demonstrated that rsPSGL.Ig functionally neutralizes effects of P-selectin on the endothelial cell surface rather than attenuating P-selectin expression. Systemic administration of rsPSGL.Ig to traumatized rats prolonged survival time and survival rate, significantly attenuating ileal myeloperoxidase activity and significantly preserving mesenteric endothelial function. Furthermore, PSGL-1 mRNA levels were significantly increased in the blood of traumatized rats and were reduced after systemic administration of rsPSGL.Ig. Thus, soluble recombinant forms of PSGL-1 are able to ameliorate acute shock states by suppressing selectin-mediated leukocyte-endothelium interaction at both the functional and molecular levels.
Hetero-Domain Interactions as a Mechanism for the Regulation of Connexin Channels
Authors: Stergiopoulos Kathleen , Alvarado José Luis , Mastroianni Marta , Ek-Vitorin José F. , Taffet Steven M. , Delmar Mario
Published: June 2012

Abstract
—Previous studies have shown that chemical regulation of connexin43 (Cx43) depends on the presence of the carboxyl terminal (CT) domain. A particle-receptor (or “ball-and-chain”) model has been proposed to explain the mechanism of gating. We tested whether the CT region behaved as a functional domain for other members of the connexin family. The pH sensitivity of wild-type and Ct-truncated connexins was quantified by use of electrophysiological and optical techniques and the
Xenopus
oocyte system. The CT domain of Cx45 had no role in pH regulation, although a partial role was shown for Cx37 and Cx50. A prominent effect was observed for Cx40 and Cx43. In addition, we found that the CT domain of Cx40 that was expressed as a separate fragment rescued the pH sensitivity of the truncated Cx40 (Cx40tr), which was in agreement with a particle-receptor model. Because Cx40 and Cx43 often colocalize and possibly heteromerize, we tested the pH sensitivity of Cx40tr when coexpressed with the CT domain of Cx43 (hetero-domain interactions). We found that the CT domain of Cx43 enhanced the pH sensitivity of Cx40tr; similarly, the CT domain of Cx40 restored the pH sensitivity of the truncated Cx43. In addition, the CT domain of Cx43 granted insulin sensitivity to the otherwise insulin-insensitive Cx26 or Cx32 channels. These data show that the particle-receptor model is preserved in Cx40 and the regulatory domain of one connexin can specifically interact with a channel formed by another connexin. Hetero-domain interactions could be critical for the regulation of heteromeric channels.
GC Factor 2 Represses Platelet-Derived Growth Factor A-Chain Gene Transcription and Is Itself Induced by Arterial Injury
Authors: Khachigian Levon M. , Santiago Fernando S. , Rafty Louise A. , Chan Onza L.-W. , Delbridge Gabrielle J. , Bobik Alex , Collins Tucker , Johnson Alfred C.
Published: June 2012

Abstract
—Platelet-derived growth factor (PDGF) is a mitogen and chemoattractant for a wide variety of cell types. The genes encoding PDGF A chain (PDGF-A) and PDGF B chain (PDGF-B) reside on separate chromosomes and are independently regulated at the level of transcription. Regulatory events underlying inducible PDGF-A expression have been the focus of much investigation. However, mechanisms that inhibit transcription of this gene are not well understood. In this study, we report the capacity of a newly cloned DNA binding factor, GC factor 2 (GCF2), to repress expression driven by the human PDGF-A promoter. 5′ Deletion and transient cotransfection analysis in vascular endothelial cells revealed that GCF2 repression is mediated by a nucleotide region located in the proximal region of the PDGF-A promoter. Electrophoretic mobility shift assays demonstrate that GCF2 binds to this region in a specific and dose-dependent manner. Interestingly, the site bound by GCF2 overlaps those for specificity protein-1 (Sp1) and early growth response factor-1 (Egr-1), zinc finger transcription factors that direct basal and inducible expression of the PDGF-A gene. Gel shift experiments revealed that GCF2 competes with these factors for interaction with the PDGF-A promoter. Overexpression of GCF2 suppressed endogenous PDGF-A expression in vascular endothelial cells and smooth muscle cells. GCF2 was induced on mechanical injury of cells in culture as well as after balloon injury of the rat carotid artery wall. Time course studies revealed the sustained induction of GCF2 after injury while PDGF-A levels sharply returned to baseline. Smooth muscle cell proliferation was inhibited by GCF2, an effect reversed by the addition of exogenous PDGF-AA. These findings demonstrate negative regulation of PDGF-A expression by GCF2. This is the first report of the induction of an endogenous transcriptional repressor in the rat vessel wall.
Cardioprotection of Preconditioning by Metabolic Inhibition in the Rat Ventricular Myocyte
Authors: Wu S. , Li H. Y. , Wong T. M.
Published: June 2012

Abstract
—To determine whether opioid receptors (ORs) are involved in the delayed cardioprotection of ischemic preconditioning (IP), the effect of severe metabolic inhibition (MI) with a glucose-free buffer that contained sodium cyanide and 2-deoxy-
d
-glucose on the viability of isolated rat ventricular myocytes was first determined 20 hours after preconditioning with a sublethal metabolic inhibition (MIP) with a glucose-free buffer that contained 2-deoxy-
d
-glucose and lactate for 30 minutes in the presence of OR antagonists. With the use of trypan blue exclusion as an index of cell viability, severe MI killed >60% of the cells and the value increased significantly after MIP. In the presence of 5×10
−6
mol/L nor-binaltorphimine (nor-BNI), a selective κ-OR antagonist, but not 5×10
−6
mol/L CTOP, a selective μ-OR antagonist, or 5×10
−6
mol/L naltrindole, a selective δ-OR antagonist, the cardioprotection of MIP was significantly attenuated. To verify the role of κ-OR, we studied the effects of severe MI after pretreatment with the κ-OR agonist U50,488H (UP) for 30 minutes. U50,488H at 3×10
−6
to 1×10
−4
mol/L increased cell viability concentration-dependently with an EC
50
of 3.311×10
−6
mol/L. In the presence of 5×10
−6
nor-BNI, the cardioprotection of UP (3×10
−5
mol/L) was blocked. A time course study showed that UP-induced cardioprotection occurred in 2 windows: the first occurred ≈1 hour later and the other occurred 16 to 20 hours later. Additional studies on cell contraction and intracellular Ca
2+
([Ca
2+
]
i
) revealed that both UP and MIP attenuated the inhibitory effects of severe MI on contractility and electrically induced [Ca
2+
]
i
transient in single ventricular myocytes. On blockade of protein kinase C, the delayed cardioprotections of UP and MIP were significantly attenuated. In conclusion, the results of the present study have provided evidence that κ-OR mediates the cardioprotection of MIP, which may involve protein kinase C and [Ca
2+
]
i
.
Adenovirus-Mediated Local Expression of Human Tissue Factor Pathway Inhibitor Eliminates Shear Stress–Induced Recurrent Thrombosis in the Injured Carotid Artery of the Rabbit
Authors: Nishida Takahiro , Ueno Hikaru , Atsuchi Nobuhiko , Kawano Ryuji , Asada Yujiro , Nakahara Yo , Kamikubo Yu-ichi , Takeshita Akira , Yasui Hisataka
Published: June 2012

Abstract
—The main cause of acute coronary syndrome may be recurrent thrombosis, which is initiated by the activation of the extrinsic coagulation pathway. Tissue factor (TF) pathway inhibitor (TFPI) efficiently inhibits an early step in this pathway by the formation of a complex with factor VIIa, TF, and factor Xa. We determined whether local TFPI gene transfer can inhibit thrombosis in an injured artery without inducing systemic side effects. Balloon-injured rabbit carotid arteries were infected with an adenoviral vector that expressed either human TFPI (AdCATFPI) or bacterial β-galactosidase (AdCALacZ). Two to 6 days after gene transfer, thrombosis was induced by the production of constant stenosis of the artery, and blood flow was measured continuously with an electromagnetic flow probe. A cyclic flow variation, which is thought to reflect the recurrent formation and dislodgment of mural thrombi, was observed in all AdCALacZ-infected arteries as well as in saline-infused arteries. In contrast, no cyclic flow variation was detectable in AdCATFPI-transfected arteries, even in the presence of epinephrine (1 μg · kg
−1
· min
−1
infusion). Prothrombin time, activated partial thromboplastin time, and the ex vivo platelet aggregation induced by either adenosine diphosphate or collagen were unaltered in AdCATFPI-infected rabbits. We found that in vivo TFPI gene transfer into an injured artery completely inhibits the recurrent thrombosis induced by shear stress even in the presence of catecholamine, without affecting systemic coagulation status. Adenovirus-mediated local expression of TFPI may have the potential for the treatment of human thrombosis.
Establishment of β-Adrenergic Modulation of L-Type Ca
2+
Current in the Early Stages of Cardiomyocyte Development
Authors: Maltsev Victor A. , Ji G. J. , Wobus Anna M. , Fleischmann Bernd K. , Hescheler Jürgen
Published: June 2012

Abstract
—β-Adrenergic modulation of the L-type Ca
2+
current (
I
CaL
) was characterized for different developmental stages in murine embryonic stem cell-derived cardiomyocytes using the whole-cell patch-clamp technique at 37°C. Cardiomyocytes first appeared in embryonic stem cell-derived embryoid bodies grown for 7 days (7d).
I
CaL
was insensitive to isoproterenol, forskolin, and 8-bromo-cAMP in very early developmental stage (VEDS) cardiomyocytes (from 7+1d to 7+2d) but highly stimulated by these substances in late developmental stage (LDS) cardiomyocytes (from 7+9d to 7+12d), indicating that all signaling cascade components became functionally coupled during development. In early developmental stage (EDS) cells (from 7+3d to 7+5d), the stimulatory response to forskolin and 8-bromo-cAMP was relatively weak. The forskolin effect was strongly augmented by ATP-γ-S. At this stage, basal
I
CaL
was stimulated by the nonselective phosphodiesterase (PDE) inhibitor isobutylmethylxanthine, by PDE inhibitors selective for the PDE II, III, and IV isoforms, as well as by the phosphatase inhibitor okadaic acid. Stimulation of
I
CaL
by the catalytic subunit of the cAMP-dependent protein kinase A (PKA) was found to be similar (about 3 times) throughout development and in adult mouse ventricular cardiomyocytes, indicating that no structural changes of the Ca
2+
channel related to phosphorylation occurred during development.
I
CaL
was stimulated by isoproterenol in the presence of a PKA inhibitor and GTP-γ-S in LDS but not VEDS cardiomyocytes, suggesting the development of a membrane-delimited stimulatory pathway mediated through the stimulatory GTP binding protein, G
s
. We conclude that uncoupling and/or low expression of G
s
protein accounted for the
I
CaL
insensitivity to β-adrenergic stimulation in VEDS cardiomyocytes. Furthermore, in EDS cells at the 7+4d stage, the reduced β-adrenergic response is due, at least in part, to high intrinsic PDE and phosphatase activities.
Genomic Organization of the
KCNQ1
K
+
Channel Gene and Identification of C-Terminal Mutations in the Long-QT Syndrome
Authors: Neyroud Nathalie , Richard Pascale , Vignier Nicolas , Donger Claire , Denjoy Isabelle , Demay Laurence , Shkolnikova Maria , Pesce Ricardo , Chevalier Philippe , Hainque Bernard , Coumel Philippe , Schwartz Ketty , Guicheney Pascale
Published: June 2012

Abstract
—The voltage-gated K
+
channel KVLQT1 is essential for the repolarization phase of the cardiac action potential and for K
+
homeostasis in the inner ear. Mutations in the human
KCNQ1
gene encoding the α subunit of the KVLQT1 channel cause the long-QT syndrome (LQTS). The autosomal dominant form of this cardiac disease, the Romano-Ward syndrome, is characterized by a prolongation of the QT interval, ventricular arrhythmias, and sudden death. The autosomal recessive form, the Jervell and Lange-Nielsen syndrome, also includes bilateral deafness. In the present study, we report the entire genomic structure of
KCNQ1
, which consists of 19 exons spanning 400 kb on chromosome 11p15.5. We describe the sequences of exon-intron boundaries and oligonucleotide primers that allow polymerase chain reaction (PCR) amplification of exons from genomic DNA. Two new (CA)
n
repeat microsatellites were found in introns 10 and 14. The present study provides helpful tools for the linkage analysis and mutation screening of the complete
KCNQ1
gene. By use of these tools, five novel mutations were identified in LQTS patients by PCR–single-strand conformational polymorphism (SSCP) analysis in the C-terminal part of
KCNQ1
: two missense mutations, a 20-bp and 1-bp deletions, and a 1-bp insertion. Such mutations in the C-terminal domain of the gene may be more frequent than previously expected, because this region has not been analyzed so far. This could explain the low percentage of mutations found in large LQTS cohorts.
Induction of Serotonin Transporter by Hypoxia in Pulmonary Vascular Smooth Muscle Cells
Authors: Eddahibi S. , Fabre V. , Boni C. , Martres M. P. , Raffestin B. , Hamon M. , Adnot S.
Published: June 2012

Abstract
—The increased delivery of serotonin (5-hydroxytryptamine, 5-HT) to the lung aggravates the development of hypoxia-induced pulmonary hypertension in rats, possibly through stimulation of the proliferation of pulmonary artery smooth muscle cells (PA-SMCs). In cultured rat PA-SMCs, 5-HT (10
–8
to 10
–6
mol/L) induced DNA synthesis and potentiated the mitogenic effect of platelet-derived growth factor-BB (10 ng/mL). This effect was dependent on the 5-HT transporter (5-HTT), since it was prevented by the 5-HTT inhibitors fluoxetine (10
–6
mol/L) and paroxetine (10
–7
mol/L), but it was unaltered by ketanserin (10
–6
mol/L), a 5-HT
2A
receptor antagonist. In PA-SMCs exposed to hypoxia, the levels of 5-HTT mRNA (measured by competitive reverse transcriptase–polymerase chain reaction) increased by 240% within 2 hours, followed by a 3-fold increase in the uptake of [
3
H]5-HT at 24 hours. Cotransfection of the cells with a construct of human 5-HTT promoter-luciferase gene reporter and of pCMV-β-galactosidase gene allowed the demonstration that exposure of cells to hypoxia produced a 5.5-fold increase in luciferase activity, with no change in β-galactosidase activity. The increased expression of 5-HTT in hypoxic cells was associated with a greater mitogenic response to 5-HT (10
–8
to 10
–6
mol/L) in the absence as well as in the presence of platelet-derived growth factor-BB. 5-HTT expression assessed by quantitative reverse transcriptase–polymerase chain reaction and in situ hybridization in the lungs was found to predominate in the media of pulmonary artery, in which a marked increase was noted in rats that had been exposed to hypoxia for 15 days. These data show that in vitro and in vivo exposure to hypoxia induces, via a transcriptional mechanism, 5-HTT expression in PA-SMCs, and that this effect contributes to the stimulatory action of 5-HT on PA-SMC proliferation. In vivo expression of 5-HTT by PA-SMC may play a key role in serotonin-mediated pulmonary vascular remodeling.
An Alternative Transcript of the Rat Renin Gene Can Result in a Truncated Prorenin That Is Transported Into Adrenal Mitochondria
Authors: Clausmeyer Susanne , Stürzebecher Raphaela , Peters Jörg
Published: June 2012

Abstract
—Characterization of the local renin-angiotensin system in the rat adrenal zona glomerulosa indicated a dual targeting of renin both to the secretory pathway and mitochondria. To investigate the transport of renin into mitochondria, we constructed a series of amino-terminal deletion variants of preprorenin. One of these variants, lacking the complete signal sequence for the endoplasmic reticulum and 10 amino acids of the profragment, was transported efficiently into isolated mitochondria. The transport was further shown to be dependent on mitochondrial membrane potential and ATP synthesis. Analysis of adrenal RNA revealed the existence of 2 renin transcripts. While one of the transcripts corresponds to the known full-length transcript, the other one lacks exon 1; instead, exon 2 is preceded by a domain of 80 nucleotides originating from intron 1. This domain, as well as the following region of intron 1 being excised, shows all essential sequence elements defining an additional, so-far-unknown exon. The second mRNA possibly derives from an additional transcription start in intron 1 and an alternative splicing process. Translation of this mRNA could result in a truncated prorenin representing a cytosolic form of renin, which is required for transport into mitochondria. This truncated prorenin corresponds exactly to the deletion variant being imported into mitochondria in vitro.
Evidence for a Vasopressin System in the Rat Heart
Authors: Hupf Harald , Grimm Daniela , Riegger Günter A. J. , Schunkert Heribert
Published: June 2012

Abstract
—Traditionally, a hypothalamo-neurohypophysial system is thought to be the exclusive source of arginine vasopressin (AVP), a potent antidiuretic, vasoconstricting, and growth-stimulating neuropeptide. We have identified de novo synthesis of AVP in the heart as well as release of the hormone into the cardiac effluents. Specifically, molecular cloning of sequence tags amplified from isolated, buffer-perfused, and pressure-overloaded rat hearts allowed the detection of cardiac AVP mRNA. Subsequent experiments revealed a prominent induction of AVP mRNA (peak at 120 minutes, 59-fold,
P
<0.01 versus baseline) and peptide (peak at 120 minutes, 11-fold,
P
<0.01 versus baseline) in these isolated hearts. Newly induced vasopressin peptide was localized most prominently to endothelial cells and vascular smooth muscle cells of arterioles and perivascular tissue using immunohistochemistry. In addition to pressure overload, nitric oxide (NO) participated in these alterations, because inhibition of NO synthase by
N
ω
-nitro-
l
-arginine methyl ester markedly depressed cardiac AVP mRNA and peptide induction. Immediate cardiac effects related to cardiac AVP induction in isolated, perfused, pressure-overloaded hearts appeared to be coronary vasoconstriction and impaired relaxation. These functional changes were observed in parallel with AVP induction and largely prevented by addition of a V
1
receptor blocker (10
−8
mol/L [deamino-Pen
1
, O-Me-Tyr
2
, Arg
8
]-vasopressin) to the perfusion buffer. Even more interesting, pressure-overloaded, isolated hearts released the peptide into the coronary effluents, offering the potential for systemic actions of AVP from cardiac origin. We conclude that the heart, stressed by acute pressure overload or NO, expresses vasopressin in concentrations sufficient to cause local and potentially systemic effects.