bluuebunny/crossref_metadata_2025 huggingface dataset:
The Early and Late Phases of Ischemic Preconditioning
Authors: Qiu Yumin , Tang Xian-Liang , Park Seong-Wook , Sun Jiang-Zhong , Kalya Anantharam , Bolli Roberto
Published: June 2012

Abstract
The effectiveness of the late phase of ischemic preconditioning (PC) in protecting against myocardial infarction and the concomitant contractile dysfunction after sustained ischemia remains unclear. The early and late phases of PC have not been compared using the same protocol in the same experimental model; furthermore, the late phase of PC has not been assessed in the conscious state in a large animal preparation. The goal of this study was to directly compare the effects of early and late PC on myocardial infarct size and postischemic dysfunction in chronically instrumented, conscious pigs. Four groups of pigs were subjected to a 40-minute coronary occlusion followed by 3 days of reperfusion. Group 1 (n=7) served as control. Group 2 (n=6) was subjected to ten 2-minute occlusion/2-minute reperfusion cycles 25 minutes before the 40-minute occlusion (early PC). Groups 3 (n=7) and 4 (n=4) were subjected to 10 and 25 cycles, respectively, of 2-minute occlusion/2-minute reperfusion 24 hours before the 40-minute occlusion (late PC). Infarct size averaged 45.1±5.9% of the region at risk in control pigs, was reduced by 79% (to 9.4±3.2%) in group 2, but did not differ in groups 3 (33.3±4.8%) and 4 (38.8±8.2%) versus group 1. Power analysis demonstrated that there was an 80% probability of detecting a 40% decrease in infarct size in groups 3 and 4 versus group 1. The recovery of systolic wall thickening (measured with ultrasonic crystals) after the 40-minute occlusion was poor in groups 1, 3, and 4 but markedly enhanced in group 2 throughout the 3 days of reperfusion; this beneficial effect could have been due to limitation of infarct size, alleviation of stunning, or both. Thus, a series of ten 2-minute coronary occlusions had a profound (≈80%) early infarct–limiting effect, which was associated with a marked functional benefit. This protection, however, disappeared 24 hours later and could not be reinstituted by increasing the number of PC coronary occlusions to 25. The incidence and duration of ventricular tachycardia after reperfusion was not changed by either early or late PC; no conclusions could be drawn regarding ventricular fibrillation or ischemia-induced ventricular tachycardia, since these arrhythmias did not occur in control animals. Taken together, the present results demonstrate striking differences between the early and late effects of PC: In conscious swine subjected to a sustained coronary occlusion, a PC protocol that induces powerful protection during the early phase of PC fails to induce any protection during the late phase, indicating either that a late protective effect of PC does not exist or that, if it exists, it must be weaker than the early protective effect.
Compartment-Selective Sensitivity of Cardiovascular Morphogenesis to Combinations of Retinoic Acid Receptor Gene Mutations
Authors: Lee Robin Y. , Luo Jiangming , Evans Ronald M. , Giguere Vincent , Sucov Henry M.
Published: June 2012

Abstract
Several aspects of normal cardiovascular development require signaling by the vitamin A metabolite retinoic acid. We have previously established germ-line mutations in mice in the genes that encode the RARα1, RARβ, and RXRα retinoic acid receptors as a means of studying the function of these receptors in vivo. Although mutation of RXRα results in fetal ventricular defects, the RARα1 and RARβ mutations are apparently nonphenotypic in the heart and elsewhere. In this study, we have established and analyzed combinations of these receptor gene mutations. Malformations of the ventricular chamber (chamber hypoplasia and muscular ventricular septal defects), conotruncus (double-outlet right ventricle, transposition, and membranous ventricular septal defects), aortic sac (persistent truncus arteriosus and aorticopulmonary window), and aortic arch–derived arteries were recovered in various combinations of the RARα1, RARβ, and RXRα gene mutations. Depending on the combination of receptor mutations, selective defects were obtained in specific cardiovascular compartments, suggestive of differential expression or function of each receptor within domains of the developing heart.
Cyclic Strain–Induced Monocyte Chemotactic Protein-1 Gene Expression in Endothelial Cells Involves Reactive Oxygen Species Activation of Activator Protein 1
Authors: Wung B. S. , Cheng J. J. , Hsieh H. J. , Shyy Y. J. , Wang D. L.
Published: June 2012

Abstract
Endothelial cells (ECs) are constantly exposed to blood pressure–induced mechanical strain. We have previously demonstrated that cyclic strain can induce gene expression of monocyte chemotactic protein-1 (MCP-1). The molecular mechanisms of gene induction by strain, however, remain unclear. Recent evidence indicates that intracellular reactive oxygen species (ROS) can act as a second messenger for signal transduction and thus affect gene expression. The potential role of ROS in strain-induced MCP-1 expression was investigated. ECs under cyclic strain induced a sustained elevated production of intracellular superoxide. ECs under strain or pretreated with either H
2
O
2
or xanthine oxidase/hypoxanthine induced MCP-1 expression. Strain- or oxidant-induced MCP-1 mRNA levels could be inhibited by treating ECs with catalase or antioxidant
N
-acetyl-cysteine (NAC). Functional analysis of MCP-1 promoter and site-specific mutations indicates that the proximal tissue plasminogen activator–responsive element (TRE) in the −60-bp promoter region is sufficient for strain or H
2
O
2
inducibility. Electrophoretic mobility shift assays demonstrated an increase of nuclear proteins binding to TRE sequences from ECs subsequent to strain or H
2
O
2
treatment. NAC or catalase pretreatment of ECs inhibited the strain- or H
2
O
2
-induced AP-1 binding. These results clearly indicate that cyclic strain inducibility of MCP-1 in ECs uses the interaction of AP-1 proteins with TRE sites via the elevation of intracellular ROS levels in strained ECs. These findings emphasize the importance of intracellular ROS in the modulation of hemodynamic force–induced gene expression in vascular ECs.
Hypoxia-Induced Inhibition of Adenosine Kinase Potentiates Cardiac Adenosine Release
Authors: Decking Ulrich K. M. , Schlieper Georg , Kroll Keith , Schrader Jürgen
Published: June 2012

Abstract
To elucidate the physiological role of the AMP-adenosine metabolic cycle and to investigate the relation between AMP and adenosine formation, the O
2
supply of isolated guinea pig hearts was varied (95% to 10% O
2
). The net adenosine formation rate (AMP→adenosine) and coronary venous effluent adenosine release rate were measured; free cytosolic AMP was determined by
31
P-nuclear magnetic resonance. Switching from 95% to 40% O
2
increased free AMP and adenosine formation 4-fold, whereas free cytosolic adenosine and venous adenosine release rose 15- to 20-fold. In the AMP range from 200 to 3000 nmol/L, there was a linear correlation between free AMP and adenosine formation (
R
2
=.71); however, adenosine release increased several-fold more than formation. At 95% O
2
, only 6% of the adenosine formed was released; however, this fraction increased to 22% at 40% O
2
, demonstrating reduced adenosine salvage. Selective blockade of adenosine deaminase and adenosine kinase indicated that flux through adenosine kinase decreased from 85% to 35% of adenosine formation in hypoxia. Mathematical model analysis indicated that this apparent decrease in enzyme activity was not due to saturation but to the inhibition of adenosine kinase activity to 6% of the basal levels. The data show (1) that adenosine formation is proportional to the AMP substrate concentration and (2) that hypoxia decreases adenosine kinase activity, thereby shunting myocardial adenosine from the salvage pathway to venous release. In conclusion, because of the normal high turnover of the AMP-adenosine metabolic cycle, hypoxia-induced inhibition of adenosine kinase causes the amplification of small changes in free AMP into a major rise in adenosine. This mechanism plays an important role in the high sensitivity of the cardiac adenosine system to impaired oxygenation.
Hemodynamic Regulation of Tumor Necrosis Factor-α Gene and Protein Expression in Adult Feline Myocardium
Authors: Kapadia Samir R. , Oral Hakan , Lee Joseph , Nakano Masayuki , Taffet George E. , Mann Douglas L.
Published: June 2012

Abstract
Tumor necrosis factor-α (TNF-α) mRNA and protein biosynthesis were examined in adult feline myocardium in the presence and absence of superimposed hemodynamic pressure overloading. A brief period of hemodynamic pressure overloading ex vivo resulted in de novo TNF-α mRNA expression within 30 minutes and de novo TNF-α protein production within 60 minutes; neither TNF-α mRNA nor protein was detected in hearts perfused at normal perfusion pressures. Moreover, TNF-α mRNA and protein biosynthesis were observed in myocyte and nonmyocyte cell types in the pressure-overloaded hearts. To determine whether a simple passive stretch of the myocardium was a sufficient stimulus for TNF-α biosynthesis, we examined TNF-α mRNA expression in stretched and unstretched papillary muscles. This study showed that myocardial stretch was a sufficient stimulus for the induction of TNF-α mRNA biosynthesis. The functional significance of the intramyocardial production of TNF-α was determined by examining cell motion in isolated contracting cardiac myocytes treated with superfusates from pressure-overloaded and control hearts. These studies showed that cell motion was depressed in myocytes treated with superfusates from the pressure-overloaded hearts but was normal with the superfusates from the control hearts. Finally, hemodynamic pressure overloading in vivo under physiological conditions was also shown to result in de novo intramyocardial TNF-α mRNA biosynthesis. In conclusion, this study constitutes the initial demonstration that the adult mammalian myocardium elaborates biologically active TNF-α, both ex vivo and in vivo, in response to hemodynamic pressure overloading.
Endothelin-1 Inhibits the Slow Component of Cardiac Delayed Rectifier K
+
Currents via a Pertussis Toxin–Sensitive Mechanism
Authors: Washizuka Takashi , Horie Minoru , Watanuki Masato , Sasayama Shigetake
Published: June 2012

Abstract
Endothelin-1 (ET-1) is a 21–amino acid peptide hormone released from myocardial and endothelial cells, whose receptors (both ET
A
and ET
B
) are expressed in the myocardium. We report here that ET-1 inhibits the cardiac delayed rectifier K
+
current (I
K
) via a pertussis toxin (PTX)–sensitive mechanism. Ventricular myocytes enzymatically isolated from guinea pig hearts were voltage-clamped by the conventional whole-cell and nystatin–perforated patch technique (intrapipette and extrapipette K
+
concentrations, 150 and 5.4 mmol/L, respectively) in the presence of nifedipine (2 μmol/L). Amplitudes of tail and steady state (2-second pulse) currents were measured as I
K
. ET-1 suppressed the basal I
K
by 20.9±2.3% in a concentration-dependent manner, with an IC
50
of 1.1±0.3 nmol/L (n=19), although it did not suppress the basal I
K
using the nystatin method. E-4031 (5 μmol/L), a blocker of the rapid component of I
K
(I
Kr
), did not prevent the inhibitory action of ET-1. ET-1 reduced by 63.4±6.5% the slow component of I
K
(I
Ks
) that had been enhanced to ≈2-fold by isoproterenol (ISO, 20 nmol/L). The action was concentration dependent, with an IC
50
of 0.7±0.4 nmol/L (n=22), and was also observed using the nystatin method. The effect of ET-1 appeared to be mediated by an ET
A
receptor, because it was prevented by FR139317, an ET
A
-selective antagonist (1 μmol/L, n=4), and sarafotoxin S6c, an ET
B
-selective agonist (100 nmol/L, n=4), could not inhibit the ISO-enhanced I
K
. ET-1 antagonized I
Ks
enhanced by histamine (250 nmol/L, n=7) and forskolin (500 nmol/L, n=7) but did not inhibit I
Ks
enhanced by the internal application of cAMP (100 μmol/L, n=6). Preincubation of myocytes with PTX (5 μg/mL for >60 minutes at 36°C) completely abolished the inhibitory action of ET-1 on the ISO-enhanced I
Ks
(n=4). Thus, nanomolar ET-1 inhibits I
Ks
via the ET
A
receptor/PTX–sensitive G protein/PKA pathway.
Phosphatase-Mediated Enhancement of Cardiac cAMP-Activated Cl
−
Conductance by a Cl
−
Channel Blocker, Anthracene-9-Carboxylate
Authors: Zhou Shi-Sheng , Takai Akira , Tominaga Makoto , Okada Yasunobu
Published: June 2012

Abstract
An aromatic carboxylate, anthracene-9-carboxylic acid (9-AC), is known as a Cl
−
channel blocker. However, variable 9-AC effects have hitherto been reported on the cardiac cAMP-activated Cl
−
conductance, when applied extracellularly. We have reexamined the 9-AC effect on the Cl
−
conductance activated by isoproterenol or forskolin in guinea pig ventricular myocytes under whole-cell patch-clamp conditions. The inward current was blocked by 9-AC at ≥0.5 mmol/L, but in contrast, the outward current was enhanced at much lower concentrations (ED
50
, ≈13 μmol/L). 9-AC applied by the intracellular perfusion technique increased both the inward and outward currents. In the presence of intracellular 9-AC, deactivation of the conductance after washout of isoproterenol or forskolin was largely prevented. 9-AC produced an enhancing effect, even after inhibiting the deactivation process by okadaic acid (OA), whereas it failed to produce additional effects in the presence of orthovanadate. Intracellular application of 9-AC together with OA virtually abolished the current deactivation. The 9-AC effects on the Cl
−
conductance were not dependent on intracellular Ca
2+
or pH. Putative inhibitors of alkaline (bromotetramisole) and acid phosphatases (tartrate) were without effect. 9-AC failed to inhibit the activities of purified protein phosphatase (PP)-1, -2A, and -2C. In the extract of guinea pig ventricle, 9-AC (≥10 μmol/L for full action) significantly inhibited a fraction of endogenous phosphatase activity that was sensitive to orthovanadate but not to OA, bromotetramisole, and tartrate. It is concluded that 9-AC blocks cardiac cAMP-activated (cystic fibrosis transmembrane conductance regulator) Cl
−
conductance from the extracellular side but enhances the conductance from the intracellular side by inhibiting an orthovanadate-sensitive phosphatase distinct from PP-1, -2A, -2B, or -2C and alkaline or acid phosphatase.
Line Stimulation Parallel to Myofibers Enhances Regional Uniformity of Transmembrane Voltage Changes in Rabbit Hearts
Authors: Knisley Tamara C. Baynham Stephen B.
Published: June 2012

Abstract
The sign of transmembrane voltage (V
m
) change (ΔV
m
) in the heart during unipolar point stimulation is nonuniform, which introduces dispersion of states of V
m
-dependent ion channels that depends on fiber orientation. We hypothesized that line stimulation parallel to cardiac fibers increases regional uniformity of the ΔV
m
sign. To test this, we evaluated electrode current distribution and ΔV
m
produced by unipolar line stimulation in isolated rabbit hearts. The V
m
-sensitive fluorescent dye, di-4-ANEPPS, and a laser scanner provided ΔV
m
measurements at 63 spots in an 8×8-mm epicardial region. Line stimulation was tested at specific angles with respect to the fiber direction. Current peaks occurred at electrode ends. For electrodes parallel to fibers (0°), epicardium in regions beyond the ends exhibited a nonuniform ΔV
m
sign, whereas epicardium between the ends exhibited a uniform ΔV
m
sign that was essentially negative (hyperpolarized) during anodal pulses and positive (depolarized) during cathodal pulses. The ΔV
m
sign between the ends became less uniform when the stimulation angle was increased relative to the long axis of the fibers. At 90°, the ΔV
m
sign between the ends was nonuniform and was frequently opposite, near versus away from the electrode. Spatial distributions of ΔV
m
during line stimulation were qualitatively predictable from anisotropic effects of point stimulation provided that combined effects of points along the electrode and points with higher current near ends were considered. For biphasic line stimulation, ΔV
m
during the second phase was weakly correlated with the temporal sum of effects of phases given individually, indicating limited ability of summation to predict ΔV
m
. Thus, uniformity of the ΔV
m
sign during stimulation is enhanced in the region between the ends of a line electrode parallel to fibers. This may lessen arrhythmogenic dispersion of V
m
-dependent ion channel states in the region.
Potentiation of Endothelium-Dependent Relaxation by Epoxyeicosatrienoic Acids
Authors: Weintraub Neal L. , Fang Xiang , Kaduce Terry L. , VanRollins Mike , Chatterjee Papri , Spector Arthur A.
Published: June 2012

Abstract
Epoxyeicosatrienoic acids (EETs) are potent endothelium-derived vasodilators formed from cytochrome P-450 metabolism of arachidonic acid. EETs and their diol products (DHETs) are also avidly taken up by endothelial cells and incorporated into phospholipids that participate in signal transduction. To investigate the possible functional significance of EET and DHET incorporation into cell lipids, we examined the capacity of EETs and DHETs to relax porcine coronary arterial rings and determined responses to bradykinin (which potently activates endothelial phospholipases) before and after incubating the rings with these eicosanoids. 14,15-EET and 11,12-EET (5 μmol/L) produced 75±9% and 52±4% relaxation, respectively, of U46619-contracted rings, whereas 8,9-EET and 5,6-EET did not produce significant relaxation. The corresponding DHET regioisomers produced comparable relaxation responses. Preincubation with 14,15-EET, 11,12-EET, 14,15-DHET, and 11,12-DHET augmented the magnitude and duration of bradykinin-induced relaxation, whereas endothelium-independent relaxations to aprikalim and sodium nitroprusside were not potentiated. Pretreatment with 2 μmol/L triacsin C (an inhibitor of acyl coenzyme A synthases) inhibited [
3
H]14,15-EET incorporation into endothelial phospholipids and blocked 11,12-EET– and 14,15-DHET–induced potentiation of relaxation to bradykinin. Exposure of [
3
H]14,15-EET–labeled endothelial cells to the Ca
2+
ionophore A23187 (2 μmol/L) resulted in a 4-fold increased release of EET and DHET into the medium. We conclude that incorporation of EETs and DHETs into cell lipids results in potentiation of bradykinin-induced relaxation in porcine coronary arteries, providing the first evidence that incorporated EETs and DHETs are capable of modulating vascular function.
Low-Dose
N

ω

-Nitro-
l
-Arginine Methyl Ester Treatment Improves Survival Rate and Decreases Myocardial Injury in a Murine Model of Viral Myocarditis Induced by Coxsackievirus B3
Authors: Mikami Shuji , Kawashima Seinosuke , Kanazawa Kenji , Hirata Ken-ichi , Hotta Hak , Hayashi Yoshitake , Itoh Hiroshi , Yokoyama Mitsuhiro
Published: June 2012

Abstract
Recent reports demonstrated the expression of inducible-type NO synthase in the heart of viral myocarditis. Since NO has multiple biological actions, a substantial amount of NO produced in the diseased heart may act either as a cytotoxic or as a cytoprotective molecule in the process of myocarditis. In the present study, we examined the effect of inhibition of NO synthesis on the mortality and the extent of myocardial injury in a murine model of coxsackievirus B3–induced myocarditis. We fed the infected mice drinking water containing a relatively low concentration (0.37 mmol/L) of
N
ω
-nitro-
l
-arginine methyl ester (L-NAME) for 14 days after virus inoculation. This dose of L-NAME did not change virus titers in the heart. However, L-NAME–fed mice showed a significant reduction in mortality compared with those fed normal drinking water (nontreated mice). On the contrary, mice given a higher concentration of L-NAME (3.7 mmol/L) exhibited increased mortality. In addition, mice fed a low concentration of L-NAME showed reductions in the severity of heart failure and in the area of myocardial necrosis. Although systemic blood pressure was reduced in nontreated mice, in mice fed a low concentration of L-NAME, it was maintained at a level similar to that in uninfected control mice. L-NAME–treated mice also exhibited a reduction in the degree of inflammatory cell infiltration associated with decreased production of tissue prostaglandin E
2
levels in the heart compared with nontreated mice. Therefore, NO is likely to be involved in the pathogenic mechanisms of myocardial injury and resultant cardiac dysfunction in a murine model of coxsackievirus B3–induced viral myocarditis.
Leukemia Inhibitory Factor, a Potent Cardiac Hypertrophic Cytokine, Activates the JAK/STAT Pathway in Rat Cardiomyocytes
Authors: Kodama Hiroaki , Fukuda Keiichi , Pan Jing , Makino Shinji , Baba Akiyasu , Hori Shingo , Ogawa Satoshi
Published: June 2012

Abstract
Leukemia inhibitory factor (LIF) is a member of the interleukin-6 family of cytokines, which induces a wide range of responses in a variety of cells. The aim of this study was to investigate whether LIF induces cardiomyocyte hypertrophy and transmits signals through the JAK/STAT (indicating just another kinase/signal transducer and activator of transcription) pathway in primary cultured neonatal rat cardiomyocytes. LIF increased protein content and [
3
H]phenylalanine uptake in cardiomyocytes in a dose-dependent manner. LIF (10
3
U/mL) induced rapid tyrosine phosphorylation of gp130, JAK1, JAK2, STAT1, and STAT3 but not Tyk2 or STAT2. LIF also induced autokinase activity of JAK1 in a time-dependent manner. Gel shift assays for interferon gamma activation site/interferon-stimulated responsive element and
sis
-inducible element (SIE) revealed that LIF induced dimerization of STAT1 and STAT3 and formation of
sis
-inducing factor complexes, which subsequently interacted with SIE in the promoter. Preincubation with anti-STAT1 and anti-STAT3 antibodies inhibited the binding of SIF complexes. In conclusion, LIF induces cardiac hypertrophy and directly stimulates the JAK/STAT pathway in cardiomyocytes.
Attachment of Meandering Reentrant Wave Fronts to Anatomic Obstacles in the Atrium
Authors: Ikeda Takanori , Yashima Masaaki , Uchida Takumi , Hough Dustan , Fishbein Michael C. , Mandel William J. , Chen Peng-Sheng , Karagueuzian Hrayr S.
Published: June 2012

Abstract
Acetylcholine chloride (ACh) induces nonstationary meandering reentrant wave fronts in the atrium. We hypothesized that an anatomic obstacle of a suitable size prevents meandering by causing attachment of the reentrant wave front tip to the obstacle. Eight isolated canine right atrial tissues (area, 3.8×3.2 cm) were mounted in a tissue bath and superfused with Tyrode’s solution containing 10 to 15 μmol/L ACh. Holes with 2- to 10-mm diameters were sequentially created in the center of the tissue with biopsy punches. Reentry was induced by a premature stimulus after eight regular stimuli at 400-ms cycle length. The endocardial activation maps and the motion of the induced reentry were visualized dynamically before and after each test lesion using 509 bipolar electrodes. In the absence of a lesion (n=8), the induced single reentrant wave front, in the form of a spiral wave, meandered irregularly from one site to another before terminating at the tissue border. Holes with 2- to 4-mm diameters (n=6) had no effect on meandering. However, when the hole diameters were increased to 6 mm (n=8), 8 mm (n=8), and 10 mm (n=6), the tip of the spiral wave attached to the holes, and reentry became stationary. Transition from meandering to an attached state converted the irregular and polymorphic electrogram to a periodic and monomorphic activity with longer cycle lengths (101±11 versus 131±9 ms for no hole versus 10-mm hole, respectively;
P
<.001). Regression analysis showed a significant positive linear correlation between the cycle length of the reentry and the hole diameter (
r
=.89,
P
<.01) and between the cycle length of the reentry and the excitable gap (
r
=.89,
P
<.05). We conclude that a critically sized anatomic obstacle converts a nonstationary meandering reentrant wave front to a stationary one. This transition converts an irregular “fibrillation-like” activity into regular monomorphic activity.
Expression, Function, and Regulation of E-Type Prostaglandin Receptors (EP
3
) in the Nonischemic and Ischemic Pig Heart
Authors: Hohlfeld Thomas , Zucker Tom-Philipp , Meyer Jutta , Schrör Karsten
Published: June 2012

Abstract
The action of prostacyclin, prostaglandin E
1
(PGE
1
), and their mimetics on myocardial function includes changes in contractility, electrophysiological properties, and protection from injury caused by transient myocardial ischemia. This study was undertaken to investigate the basic properties of myocardial E-type prostaglandin (EP) receptors. Ligand binding studies using an enriched preparation of sarcolemmal membranes prepared from pig hearts revealed a single class of binding sites for [
3
H]PGE
1
, with a
K
d
of 3.7 nmol/L and a B
max
of 92 fmol/mg protein. Competition experiments indicated highest affinity for EPs, suggesting an EP receptor. In addition, the EP receptor subtype–selective agonists sulprostone (EP
1
and EP
3
) and M&B 28.767 (EP
3
) were active, suggesting the presence of an EP
3
receptor subtype. PGE
1
stimulated sarcolemmal GTPase and inhibited sarcolemmal adenylyl cyclase activity, indicating EP
3
receptor coupling to an inhibitory G protein (G
i
). Additional in vivo experiments showed that intracoronary infusion of PGE
1
(1 nmol/min) decreased isoprenaline-stimulated left ventricular contractile activity without altering systemic vascular resistance. This inhibition of β-adrenergic effects is compatible with the known myocardial anti-ischemic action of prostaglandins. Further experiments examined EP
3
receptor density and G-protein coupling in sarcolemma from ischemic and reperfused ischemic myocardium. In anesthetized open-chest minipigs, occlusion of the left anterior descending coronary artery for 60 minutes increased EP
3
receptor density by 50%, whereas receptor affinity was unchanged. This upregulation was prevented by pretreatment with colchicine (2 mg/kg IV), indicating microtubule-dependent receptor externalization. Northern hybridization showed comparable EP
3
receptor mRNA expression in control and ischemic myocardium. The increase of receptor protein was reversed during 60 minutes of reperfusion. G-protein coupling proved to be intact in ischemic and reperfused ischemic myocardial tissue, as shown by preserved GTP-γ-S–induced decrease of [
3
H]PGE
1
binding. These data demonstrate for the first time that myocardial receptors for PGE
1
belong to the EP
3
subtype. The properties of this receptor include inhibition of adenylyl cyclase and upregulation during regional myocardial ischemia, suggesting an involvement in the anti-ischemic activity of E- and I-type prostaglandins.
Nitric Oxide Inhalation Inhibits Platelet Aggregation and Platelet-Mediated Pulmonary Thrombosis in Rats
Authors: Nong Zengxuan , Hoylaerts Marc , Van Pelt Natasha , Collen Désiré , Janssens Stefan
Published: June 2012

Abstract
Endothelium-derived nitric oxide (NO) inhibits in vitro platelet aggregation via a cGMP-dependent mechanism. The effect of inhaled NO on platelet-mediated pulmonary thrombosis following intravenous thrombotic challenge with collagen was examined in rats and compared with the effect of G4120, a cyclic Arg-Gly-Asp–containing synthetic pentapeptide that binds to the platelet glycoprotein IIb/IIIa receptor. Intraplatelet cGMP dose-dependently increased from 39±6 fmol/10
8
platelets in control to 46±6, 68±13, and 81±13 fmol/10
8
platelets after inhalation with 20, 40, and 80 ppm NO, respectively (
P
<.05 for 40 and 80 ppm). Ex vivo platelet aggregation of platelet-rich plasma induced by 1 μg/mL collagen was reduced from 75±4% in control rats to 22±10% and 20±7% in rats ventilated with 40 and 80 ppm NO, respectively, and to 30±9% in G4120-treated rats (each
P
<.05 versus control). Circulating platelet counts 3 minutes after collagen injection were significantly higher in the inhaled NO and G4120 groups compared with control rats (250 000±18 000 and 223 000±10 000/μL versus 160 000±18 000/μL, each
P
<.05). The rise in pulmonary arterial pressure after collagen injection was significantly reduced in NO- and G4120-treated rats (26±1 and 27±1 versus 32±1 mm Hg in control rats, each
P
<.05). The number of pulmonary resistance vessels containing platelet thrombi was significantly smaller after inhaled NO and G4120 treatment compared with control (56±3% and 50±3% versus 68±3%, respectively;
P
<.05). Thus, NO inhalation reduces in vivo activation of circulating platelets and platelet-rich thrombosis in thromboembolic pulmonary hypertension. Inhalation of NO may be useful in cardiovascular diseases associated with platelet activation.
Lipopolysaccharide Depresses Cardiac Contractility and β-Adrenergic Contractile Response by Decreasing Myofilament Response to Ca
2+
in Cardiac Myocytes
Authors: Yasuda Satoshi , Lew Wilbur Y. W.
Published: June 2012

Abstract
Lipopolysaccharide (LPS) plays a key role in the pathogenesis of sepsis. Cardiac function and the inotropic response to β-adrenergic stimulation are impaired in sepsis. We hypothesized that LPS, in clinically relevant levels (1 ng/mL), directly depresses contractility and β-adrenergic responses in cardiac myocytes. Cardiac myocytes were isolated from the left ventricle of adult rabbits using digestive enzymes (collagenase and protease). We depyrogenated the enzymes (LPS contamination lowered from 100 to 300 ng/mL to <0.7 ng/mL) to minimize development of LPS tolerance during cell isolation. After 6 hours of incubation with 1 ng/mL LPS, there was a decrease in the extent of active cell shortening with no change in Ca
2+
transients (measured with indo 1 fluorescence), indicating decreased myofilament responsiveness to Ca
2+
. This was related to NO pathways, since cGMP (a second messenger of NO) increased in cardiac myocytes and LPS effects were completely reversed with a 1 mmol/L
N
G
-monomethyl-
l
-arginine (L-NMMA, a NO synthase inhibitor). LPS did not alter the intracellular Ca
2+
response to β-adrenergic stimulation with isoproterenol but attenuated the contractile response (maximal cell shortening, 15.5±1.0% versus 23.3±1.1% in control myocytes;
P
<.001). LPS attenuation of the contrac-tile response to isoproterenol was restored completely by L-NMMA and almost completely restored (to 86% of the control response) by an inhibitor of cGMP-dependent protein kinase. We conclude that LPS depresses cardiac contractility and the contractile response to β-adrenergic stimulation by a NO-cGMP–mediated decrease in myofilament responsiveness to Ca
2+
. The direct effects of low levels of LPS on cardiac myocytes may contribute to cardiac depression and hemodynamic decompensation during sepsis.
Increased Pressure Induces Sustained Protein Kinase C–Independent Herbimycin A–Sensitive Activation of Extracellular Signal–Related Kinase 1/2 in the Rabbit Aorta in Organ Culture
Authors: Birukov Konstantin G. , Lehoux Stéphanie , Birukova Anna A. , Merval Régine , Tkachuk Vsevolod A. , Tedgui Alain
Published: June 2012

Abstract
The 42- and 44-kD mitogen-activated protein kinases, also referred to as extracellular signal–related kinase (ERK) 2 and 1, respectively, may be transiently activated by stretching vascular smooth muscle cells (VSMCs). Using an organ culture model of rabbit aorta, we studied short- and long-term ERK1/2 activation by intraluminal pressure (150 mm Hg). Activation of ERK1/2 was biphasic: it reached a maximum (217.5±8.4% of control) 5 minutes after pressurizing and decreased to 120.7±5.1% of control after 2 hours. Furthermore, after 24 hours of pressurizing, ERK1/2 activity was as high (241.8±14.7% of control) as in the acute phase. Long-term pressure-induced ERK1/2 activation correlated with stimulation of tyrosine phosphorylation of proteins in the 125- to 140-kD range. Neither protein kinase C inhibitors (1 μmol/L staurosporine or 50 μmol/L bisindolylmaleimide-I) nor tyrosine kinase inhibitors (50 μmol/L tyrphostin A48 or 50 μmol/L genistein) affected pressure-induced ERK1/2 activation. However, the Src-family tyrosine kinase inhibitor herbimycin A (500 nmol/L) did reduce both 5-minute (by 92±8%) and 24-hour (by 63±7%) pressure-induced ERK1/2 activation. Thus, our results demonstrate a sustained activation of ERK1/2 and tyrosine kinases by intraluminal pressure in the arterial wall. Pressure-induced ERK1/2 activation is PKC independent and Src-family tyrosine kinase dependent and possibly includes activation of extracellular matrix–associated tyrosine kinases.
Cardiac Myosin Binding Protein C Gene Is Specifically Expressed in Heart During Murine and Human Development
Authors: Fougerousse Françoise , Delezoide Anne-Lise , Fiszman Marc Y. , Schwartz Ketty , Beckmann Jacques S. , Carrier Lucie
Published: June 2012

Abstract
—Cardiac myosin binding protein C (MyBP-C) is a substantial component of the sarcomere, with both structural and regulatory roles. The gene encoding cardiac MyBP-C in humans is located on chromosome 11p11.2, and mutations that are most predicted to produce truncated proteins have been identified in this gene in unrelated families with familial hypertrophic cardiomyopathy (FHC). To understand better the pathophysiology of FHC and with a view to the development of animal models for this disease, we have investigated by in situ hybridization the pattern of expression of the cardiac MyBP-C gene during human and mouse development using species-specific oligonucleotide probes. From 4 weeks of human development, a strong labeling of cardiac MyBP-C mRNAs was unambiguously detected in all heart compartments, and no signal could be visualized in somites. In murine embryos, from embryonic day 9.5 until birth, a strong signal was detected exclusively in the heart. Our results showed that during both human and murine development, in contrast to chicken development, the cardiac MyBP-C gene is abundantly and specifically expressed in the heart.
Oscillatory and Steady Laminar Shear Stress Differentially Affect Human Endothelial Redox State
Authors: De Keulenaer Gilles W. , Chappell David C. , Ishizaka Nobukazu , Nerem Robert M. , Alexander R. Wayne , Griendling Kathy K.
Published: June 2012

Abstract
—Atherosclerotic lesions are found opposite vascular flow dividers at sites of low shear stress and oscillatory flow. Since endothelial proinflammatory genes prominent in lesions are regulated by oxidation-sensitive transcriptional control mechanisms, we examined the redox state of cultured human umbilical vein endothelial cells after either oscillatory or steady laminar fluid shear stress. Endothelial oxidative stress was assessed by measuring activity of the superoxide (O
2
·
−
)–producing NADH oxidase (a major source of reactive oxygen species in vascular cells), intracellular O
2
·
−
levels, induction of the redox-sensitive gene heme oxygenase-1 (HO-1), and abundance of Cu/Zn superoxide dismutase (Cu/Zn SOD), an antioxidant defense enzyme whose level of expression adapts to changes in oxidative stress. When cells were exposed to oscillatory shear (±5 dyne/cm
2
, 1 Hz) for 1, 5, and 24 hours, NADH oxidase activity and the amount of HO-1 progressively increased up to 174±16% (
P
<0.05) and 505±111% (
P
<0.05) versus static conditions, respectively, whereas levels of Cu/Zn SOD remained unchanged. This upregulation of HO-1 was completely blocked by the antioxidant
N
-acetylcysteine (NAC, 20 mmol/L). In contrast, steady laminar shear (5 dyne/cm
2
) induced NADH oxidase activity and NAC-sensitive HO-1 mRNA expression only at 1 and 5 hours, a transient response that returned toward baseline at 24 hours. Levels of Cu/Zn SOD mRNA and protein were increased after 24 hours of steady laminar shear. Furthermore, intracellular O
2
·
−
, as measured by dihydroethidium fluorescence, was higher in cells exposed to oscillatory than to laminar shear. These data are consistent with the hypothesis that continuous oscillatory shear causes a sustained activation of pro-oxidant processes resulting in redox-sensitive gene expression in human endothelial cells. Steady laminar shear stress initially activates these processes but appears to induce compensatory antioxidant defenses. We speculate that differences in endothelial redox state, orchestrated by different regimens of shear stress, may contribute to the focal nature of atherosclerosis.
Effect of Vacuolar Proton ATPase on pH
i
, Ca
2+
, and Apoptosis in Neonatal Cardiomyocytes During Metabolic Inhibition/Recovery
Authors: Karwatowska-Prokopczuk Ewa , Nordberg Judith A. , Li Hai Ling , Engler Robert L. , Gottlieb Roberta A.
Published: June 2012

Abstract
—Recently, we found that vacuolar proton ATPase (VPATPase) operates in cardiomyocytes as a complementary proton-extruding mechanism. Its activity was increased by preconditioning with resultant attenuation of intracellular acidification during ischemia. In this study, we examined whether VPATPase-mediated proton efflux during metabolic inhibition/recovery may spare Na
+
overload via Na
+
-H
+
exchange, attenuate Na
+
-Ca
2+
exchange, and decrease apoptosis. Neonatal rat cardiomyocytes were subjected to 2- to 3-hour metabolic inhibition with cyanide and 2-deoxyglucose and 24-hour recovery. The effect of VPATPase inhibition by 50 nmol/L bafilomycin A
1
on apoptosis, pH
i
, and [Ca
2+
]
i
was studied by flow cytometry with propidium iodide, seminaphthorhodafluor (SNARF)-1-AM, and indo-1-AM staining, respectively. VPATPase inhibition increased the amount of apoptosis measured after 24 hours of recovery and abrogated the protective effect of inhibition of Na
+
-H
+
exchange by (5-
N
-ethyl-
N
-isopropyl)amiloride (EIPA). Dual blockade of VPATPase and Na
+
-H
+
exchange was additive in effect with EIPA on pH
i
during metabolic inhibition/recovery and recovery from the acid challenge with sodium propionate. VPATPase blockade increased the rate of accumulation of intracellular Ca
2+
at the beginning of metabolic inhibition and abrogated the delaying effect of EIPA on intracellular Ca
2+
accumulation. These results indicate that VPATPase plays an important accessory role in cardiomyocyte protection by reducing acidosis and Na
+
-H
+
exchange–induced Ca
2+
overload.
Simulation Study of Cellular Electric Properties in Heart Failure
Authors: Priebe Leo , Beuckelmann Dirk J.
Published: June 2012

Abstract
—Patients with severe heart failure are at high risk of sudden cardiac death. In the majority of these patients, sudden cardiac death is thought to be due to ventricular tachyarrhythmias. Alterations of the electric properties of single myocytes in heart failure may favor the occurrence of ventricular arrhythmias in these patients by inducing early or delayed afterdepolarizations. Mathematical models of the cellular action potential and its underlying ionic currents could help to elucidate possible arrhythmogenic mechanisms on a cellular level. In the present study, selected ionic currents based on human data are incorporated into a model of the ventricular action potential for the purpose of studying the cellular electrophysiological consequences of heart failure. Ionic currents that are not yet sufficiently characterized in human ventricular myocytes are adopted from the action potential model developed by Luo and Rudy (LR model). The main results obtained from this model are as follows: The action potential in ventricular myocytes from failing hearts is longer than in nonfailing control hearts. The major underlying mechanisms for this prolongation are the enhanced activity of the Na
+
-Ca
2+
exchanger, the slowed diastolic decay of the [Ca
2+
]
i
transient, and the reduction of the inwardly rectifying K
+
current and the Na
+
-K
+
pump current in myocytes of failing hearts. Furthermore, the fast and slow components of the delayed rectifier K
+
current (
I
Kr
and
I
Ks
, respectively) are of utmost importance in determining repolarization of the human ventricular action potential. In contrast, the influence of the transient outward K
+
current on APD is only small in both cell groups. Inhibition of
I
Kr
promotes the development of early afterdepolarizations in failing, but not nonfailing, myocytes. Furthermore, spontaneous Ca
2+
release from the sarcoplasmic reticulum triggers a premature action potential only in failing myocytes. This model of the ventricular action potential and its alterations in heart failure is intended to serve as a tool for investigating the effects of therapeutic interventions on the electric excitability of the human ventricular myocardium.
Activation of Cardiac Tissue by Extracellular Electrical Shocks
Authors: Fast Vladimir G. , Rohr Stephan , Gillis Anne M. , Kléber André G.
Published: June 2012

Abstract
—This study investigated the activation of cardiac tissue by “secondary sources,” which are localized changes of the transmembrane potential (V
m
) during the application of strong extracellular electrical shocks far from the shock electrodes, in cultures of neonatal rat myocytes. Cell monolayers with small intercellular clefts (length, 45 to 270 μm; width, 20 to 70 μm [mean±SD, 54±13 μm]; n=46) were produced using a technique of directed cell growth. Changes in V
m
relative to the action potential amplitude (ΔV
m
/APA) were measured using a fluorescent voltage-sensitive dye and a 10×10 photodiode array. Shocks with voltage gradients of 4 to 18 V/cm were applied across the clefts during either the action potential (AP) plateau or diastole. During the AP plateau, shocks induced secondary sources in the form of localized hyperpolarizations and depolarizations in the regions immediately adjacent to opposite sides of the clefts. The strength of the secondary sources, defined as the difference of ΔV
m
/APA across a cleft, increased with increasing cleft length or increasing electrical field gradient. For shocks with a gradient of 8.5 V/cm, the estimated critical cleft length necessary to reach a V
m
level corresponding to the diastolic threshold of excitation was 171±7 μm. Accordingly, shocks with average strength of 8.2 V/cm applied during diastole produced secondary sources that directly excited cells adjacent to the clefts when the cleft length was 196±53 μm (n=14) and that failed when the cleft length was 84±23 μm (n=9,
P
<001). The area of earliest excitation in such cases coincided with the area of maximal depolarization induced during the plateau phase. These data suggest that small inexcitable obstacles may contribute to the V
m
changes during the application of strong extracellular electrical shocks in vivo.
Aspirin Increases Ferritin Synthesis in Endothelial Cells
Authors: Oberle Stefanie , Polte Tobias , Abate Aida , Podhaisky Hans-Peter , Schröder Henning
Published: June 2012

Abstract
—Aspirin has recently been shown to increase endothelial resistance to oxidative damage. However, the mechanism underlying aspirin-induced cytoprotection is still unknown. Using cultured cells, the present study investigates the effect of aspirin on the expression of ferritin, a cytoprotective protein that sequesters free cytosolic iron, the main catalyst of oxygen radical formation. In bovine pulmonary artery endothelial cells, aspirin at low antithrombotic concentrations (0.03 to 0.3 mmol/L) induced the synthesis of ferritin protein in a time- and concentration-dependent fashion up to 5-fold over basal levels, whereas ferritin H (heavy chain) mRNA remained unaltered. Aspirin-induced cytoprotection from hydrogen peroxide toxicity was mimicked by exogenous iron-free apoferritin but not iron-loaded ferritin, demonstrating the antioxidant function of newly synthesized ferritin under these conditions. Ferritin induction by aspirin was specific in that other nonsteroidal anti-inflammatory drugs such as salicylic acid, indomethacin, or diclofenac failed to alter ferritin protein levels. Aspirin-induced ferritin synthesis was abrogated in the presence of the iron chelator desferrioxamine, pointing to an interaction of aspirin with iron-responsive activation of ferritin translation. Together, our results suggest induction of ferritin as a novel mechanism by which aspirin may prevent endothelial injury in cardiovascular disease, eg, during atherogenesis.
Cardiac and Extracardiac Expression of Csx/Nkx2.5 Homeodomain Protein
Authors: Kasahara Hideko , Bartunkova Sonia , Schinke Martina , Tanaka Makoto , Izumo Seigo
Published: June 2012

Abstract
—
Csx/Nkx2.5
is an evolutionary conserved homeobox gene related to the
Drosophila tinman
gene, which is essential for the dorsal mesoderm formation. Expression of
Csx/Nkx2.5
mRNA is the earliest marker for heart precursor cells in all vertebrates so far examined. Previous studies have demonstrated that
Csx/Nkx2.5
mRNA is highly expressed in the heart and at lower levels in the spleen, tongue, stomach, and thyroid in the murine embryo. Since some developmental genes are regulated by posttranscriptional mechanisms, we analyzed the developmental pattern of Csx protein expression at the single-cell level using Csx-specific antibodies. Immunohistochemical analysis of murine embryos at 7.8 days post coitum revealed that Csx protein is strongly expressed in the nucleus of endodermal and mesodermal cells in the cardiogenic plate. Subsequently, in the heart, Csx protein was detected only in the nucleus of myocytes of the atrium and the ventricle through the adult stage. During the fetal period, Csx protein expression in the nucleus was also noted in the spleen, stomach, liver, tongue, and anterior larynx. Unexpectedly, confocal microscopy revealed that Csx immunoreactivity was detected only in the cytoplasm of a subset of cranial skeletal muscles. Csx protein was not detected in the thyroid glands. The expression of Csx protein in all organs was markedly downregulated after birth except in the heart. These results raise the possibility that
Csx/Nkx2.5
may play a role in the early developmental process of multiple tissues in addition to its role in early heart development.
Sequential Injury of the Rabbit Abdominal Aorta Induces Intramural Coagulation and Luminal Narrowing Independent of Intimal Mass
Authors: Courtman David W. , Schwartz Stephen M. , Hart Charles E.
Published: June 2012

Abstract
—We hypothesized that activation of the coagulation cascade is involved in arterial remodeling in response to sequential injury. An active site–inhibited recombinant human factor VIIa (FVIIai) was used to inhibit tissue factor, the primary cofactor in the extrinsic pathway of coagulation, in a sequential balloon injury model of the rabbit abdominal aorta. Single balloon injury produced limited intimal thickening at 3 weeks (intimal area, 0.40±0.05 mm
2
) and no loss in luminal area (12.2±0.9 mm

2

before injury and 12.1±0.9 mm

2

at 6 weeks after injury). Sequential balloon injury, 3 weeks after the first balloon denudation, produced a progressive loss of lumen, with 22% and 47% loss of luminal area, respectively, at 3 and 6 weeks. Luminal loss could not be accounted for by intimal growth (at 3 weeks after sequential injury, the intimal area was 0.47±0.08 mm
2
, <4% of the initial luminal area). Sequential injury acutely produced extensive mural and intramural fibrin deposition. Treatment with FVIIai inhibited both the fibrin deposition and the chronic loss of lumen. At 3 weeks after sequential injury, luminal cross-sectional areas were 9.8±0.6 mm

2

for control rabbits and 14.3±1.4 mm

2

for FVIIai-treated rabbits. Neither neointimal area nor cell proliferation was reduced by FVIIai treatment. The intimal cell proliferation index 3 days after injury was 7.6±1.1% in control rabbits versus 5.8±1.1% in treated rabbits (
P
>0.05). These results indicate that tissue factor is an important mediator of coagulation in repeat injury and implicate the extrinsic coagulation cascade in a blood vessel remodeling response that is independent of neointimal growth but leads to extensive loss of lumen.