bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of β1 and β3 Integrins in Human Smooth Muscle Cell Adhesion to and Contraction of Fibrin Clots In Vitro
Authors: Yee Karen O , Rooney Michael M. , Giachelli Cecilia M. , Lord Susan T. , Schwartz Stephen M.
Published: June 2012

Abstract
—The degree of lumen narrowing in advanced lesions correlates poorly with the amount of intimal mass accumulated in the atherosclerotic plaque. As an alternate mechanism of stenosis, we propose that human smooth muscle cells bind to fibrin deposited in the matrix and exert contractile forces to cause a narrowing of the lumen. In the present study we demonstrated in vitro that human newborn aortic smooth muscle cell lines can contract and adhere to fibrin clots composed of either fibronectin-depleted plasma (“plasma”) or recombinant fibrin. By using neutralizing antibodies and RGD peptides, we showed that members of the integrin family mediated the interaction between human newborn smooth muscle cells and fibrin. Neutralizing antibodies against the integrin αvβ3 (c7E3 Fab and LM609) did not inhibit either plasma clot contraction or recombinant fibrin clot contraction by human newborn smooth muscle cells. In contrast, antibodies against α5, β1, and α5β1 inhibited contraction of clots composed of either plasma or recombinant fibrin. Anti-αvβ3, anti-αv, anti-α5, anti-β1, and anti-α5β1 antibodies inhibited human newborn smooth muscle cell adhesion to plasma clots; however, only anti-α5, anti-β1, and anti-α5β1 antibodies significantly inhibited adhesion to recombinant fibrin. While the linear RGD peptides had no effect, the cyclic peptide penRGD inhibited adhesion to plasma clots and recombinant fibrin. However, it did not block contraction of recombinant fibrin clots. These results suggest that during the interaction of human newborn smooth muscle cell lines with fibrin, α5β1 plays a significant role. This interaction is of potential interest as a target for efforts to block vascular contraction.
Role of Pectinate Muscle Bundles in the Generation and Maintenance of Intra-atrial Reentry
Authors: Wu Tsu-Juey , Yashima Masaaki , Xie Fagen , Athill Charles A. , Kim Young-Hoon , Fishbein Michael C. , Qu Zhilin , Garfinkel Alan , Weiss James N. , Karagueuzian Hrayr S. , Chen Peng-Sheng
Published: June 2012

Abstract
—To determine the role of pectinate muscle (PM) bundles in the formation of intra-atrial reentry, 10 isolated canine right atrial tissues were perfused with Tyrode’s solution containing 1 to 2.5 μmol/L acetylcholine (ACh). The endocardium was mapped using 477 bipolar electrodes with 1.6-mm resolution. Reentry was induced by a premature stimulus (S
2
). Computer simulation studies were used to investigate the importance of regional myocardial thickness in reentry formation. A total of 40 episodes of reentry were induced; 28 episodes were stationary, and the remaining 12 were nonstationary. The stationary reentry was induced either immediately after the S
2
stimuli (n=9) or after an initial period of irregular activations that lasted 1460±1077 ms (n=19). Of 28 episodes, 20 were initiated by conduction block along large PM ridges, leading to wave break and the initiation of reentry. The reentrant wave fronts remained stationary and rotated around these ridges as anchoring sites. During the transition from the initial irregular activations to stationary reentry, the electrogram morphology converted from “fibrillation-like” to “flutter-like” activity. In 8 episodes, initially stationary reentry converted to irregular activations because of interference with outside wave fronts (n=5) or spontaneous separation of waves from the ridges (n=3). Compared with stationary reentry, nonstationary reentry always occurred over an area without large PMs, and the mean life span was much shorter (102±151 versus 3.8±1.1 rotations,
P
<0.001). Computer simulation studies showed that a critical ridge thickness is needed for reentry to anchor, thereby converting fibrillation to flutter. We conclude that PM ridge forms an area where wave break occurs, allowing the initiation of reentry. It also provides a natural anchor to the reentrant wave front, lengthening the life span of reentry. The attachment and detachment of the reentrant wave front to and from the ridge determine “flutter-like” or “fibrillation-like” activity.
Immune Sources of Transforming Growth Factor-β
1
Reduce Transplant Arteriosclerosis
Authors: Koglin Jörg , Glysing-Jensen Troels , Räisänen-Sokolowski Anne , Russell Mary E.
Published: June 2012

Abstract
—Activated CD4-positive T cells are essential in the early stages of arteriosclerotic lesion development after cardiac transplantation. Besides its parenchymal effects, transforming growth factor-β
1
(TGF-β
1
) mediates immunosuppressive effects on proliferation and activation of CD4 cells. This study was designed to assess immune contributions of TGF-β
1
to arteriosclerosis by comparing the effect of TGF-β
1
–deficient and –competent infiltrating inflammatory cells on the development of intimal thickening in a heterotopic mouse transplant model (CBA to C57B6). Transplant arteriosclerosis was evaluated in cardiac grafts placed into knockout recipients heterozygous for TGF-β
1
(n=7) and was compared with those placed into wild-type recipients (n=11). At 55 days, allografts in TGF-β
1
–deficient recipients had increased concentric intimal thickening. Computer-assisted analysis of all elastin-positive vessels (n=173) showed significantly increased luminal occlusion (67.8±5.6%) in grafts from TGF-β
1
–deficient recipients compared with wild-type recipients (47.4±4.1%,
P
=0.003). To determine whether TGF-β
1
deficiency altered CD4 activation patterns, we studied intragraft cytokine expression. Using
32
P-reverse-transcriptase polymerase chain reaction assays, we show that TGF-β
1
–deficient recipients had an increased expression of the transcription factor STAT 4, interferon gamma, and interleukin-2 (Th1-type response) and unaltered or reduced expression of the transcription factor STAT 6, interleukin-4, and interleukin-10 (Th2-type response). Hence, when present, immune sources of TGF-β
1
attenuate transplant arteriosclerosis. This effect is associated with attenuation of Th1 forces.
Transgenic Mice With Increased Copper/Zinc–Superoxide Dismutase Activity Are Resistant to Hepatic Leukostasis and Capillary No-Reflow After Gut Ischemia/Reperfusion
Authors: Horie Yoshinori , Wolf Robert , Flores Sonia C. , McCord Joe M. , Epstein Charles J. , Granger D. Neil
Published: June 2012

Abstract
—The objectives of this study were to (1) determine whether transgenic (Tg) mice overexpressing copper/zinc–superoxide dismutase (CuZn-SOD) are protected from the deleterious effects of gut ischemia/reperfusion (I/R) and (2) compare the effectiveness of Tg SOD overexpression in attenuating I/R injury to intravascularly administered CuZn-SOD or manganese (Mn)-SOD. The accumulation of fluorescently labeled leukocytes and number of nonperfused sinusoids were monitored by intravital microscopy in livers of wild-type mice (C57BL/6), CuZn-SOD Tg mice, and wild-type mice receiving either CuZn-SOD or Mn-SOD. All parameters were measured for 1 hour after release of the occluded (for 15 minutes) superior mesenteric artery. Gut I/R in wild-type mice led to an increased number of stationary leukocytes, while reducing the number of perfused sinusoids (capillary no-reflow). All of these responses were significantly blunted in CuZn-SOD Tg mice, with a corresponding attenuation of liver enzyme release into plasma. Exogenously administered SOD had little or no effect on gut I/R-induced leukostasis or capillary no-reflow in the liver. These observations suggest a role for superoxide in gut I/R-induced leukostasis and hypoxic stress in the liver. Furthermore, the findings suggest that cellular localization of SOD activity is an important determinant of the protective actions of this enzyme in experimental models of I/R injury.
Regulation of Angiopoietin-2 mRNA Levels in Bovine Microvascular Endothelial Cells by Cytokines and Hypoxia
Authors: Mandriota Stefano J. , Pepper Michael S.
Published: June 2012

Abstract
—Angiopoietin-2 (Ang2) is a ligand for the endothelial cell tyrosine kinase receptor Tie2 and counteracts blood vessel maturation/stability mediated by angiopoietin-1 (Ang1), the other known ligand of Tie2. Using degenerate oligonucleotides and reverse transcriptase–polymerase chain reaction, we have screened bovine microvascular endothelial (BME), aortic, lymphatic, pulmonary artery, and transformed fetal aortic endothelial cells, as well as rat smooth muscle cells for Ang1 and Ang2 expression. Except for high Ang2 mRNA levels found in BME cells, none of the endothelial cell types studied expressed appreciable levels of Ang1 or Ang2 mRNAs, whereas smooth muscle cells expressed both Ang1 and Ang2. BME cell Ang2 mRNA levels were increased by vascular endothelial growth factor (1.9- to 2.9-fold), basic fibroblast growth factor (1.6- to 2-fold), both cytokines in combination (2.9- to 4-fold), and hypoxia (3.1- to 5.6-fold) and were decreased by Ang1 (31% to 70%) or transforming growth factor-β
1
(64% to 81%). Ang2 also decreased (60% to 82%) BME cell Ang2 mRNA. mRNA levels for the Tie1 or Tie2 receptors were only slightly modulated under the conditions described above. These findings suggest that the angiogenic effect of a number of regulators may be achieved in part through the regulation of an autocrine loop of Ang2 activity in microvascular endothelial cells.
Heparin Proteoglycans Released From Rat Serosal Mast Cells Inhibit Proliferation of Rat Aortic Smooth Muscle Cells in Culture
Authors: Wang Yenfeng , Kovanen Petri T.
Published: June 2012

Abstract
—Mast cells are present in the human arterial intima. To study whether mast-cell degranulation influences the rate of proliferation of smooth muscle cells, we cocultured sensitized (IgE-bearing) rat serosal mast cells and rat aortic smooth muscle cells (SMCs). When sensitized mast cells were stimulated to degranulate with antigen, the rate of proliferation of the cocultured SMCs decreased sharply. This inhibitory effect was found to be due mainly to the very high molecular weight (
M
r
) heparin proteoglycans (average
M
r
750 000) released from the stimulated mast cells. When the heparin proteoglycans were purified from mast-cell granule remnants and added to the SMC culture, they were found to block the cell cycle at the G
0
→S transition and the exit from the G
2
/M phase, their inhibitory effect resembling that of commercial heparin. However, in contrast to the reported dependence of the inhibitory effect of commercial heparin on the release of transforming growth factor-β from serum, the inhibitory effect of the mast cell–derived heparin proteoglycans in the presence of serum was not transforming growth factor-β dependent. Moreover, the effect of the mast cell–derived heparin proteoglycans was more efficient than that of commercial heparins of high (average
M
r
15 000) and low (average
M
r
5000) molecular weight. We also purified heparin glycosaminoglycans (average
M
r
75 000) from the mast cell–derived heparin proteoglycans and found that they also inhibited SMC growth efficiently, although less strongly than their parent heparin proteoglycans. These results reveal, for the first time, that mast cells are able to regulate SMC growth. Thus, activated mast cells, by releasing heparin proteoglycans, possibly participate in the regulation of SMC growth in the human arterial intima, the site of atherogenesis.
Adenylate Kinase–Catalyzed Phosphotransfer in the Myocardium
Authors: Dzeja Petras P. , Vitkevicius Konradas T. , Redfield Margaret M. , Burnett John C. , Terzic Andre
Published: June 2012

Abstract
—Although the downregulation of creatine kinase activity has been associated with heart failure, creatine kinase–deficient transgenic hearts have a preserved contractile function. This suggests the existence of alternative phosphotransfer pathways in the myocardium, the identity of which is still unknown. In this study, we examined the contribution of adenylate kinase–catalyzed phosphotransfer to myocardial energetics. In the isolated mitochondria/actomyosin system, which possesses endogenous adenylate kinase activity in both compartments, substrates for adenylate kinase promoted the rate and amplitude of actomyosin contraction that was further enhanced by purified adenylate kinase. Inhibition of adenylate kinase activity diminished both actomyosin contraction and mitochondrial respiration, which indicated reduced energy flow between mitochondria and myofibrils. In intact myocardium, the net adenylate kinase–catalyzed phosphotransfer rate was 10% of the total ATP turnover rate as measured by
18
O-phosphoryl labeling in conjunction with gas chromatography and mass spectrometry. In pacing-induced failing heart, adenylate kinase–catalyzed phosphotransfer increased by 134% and contributed 21% to the total ATP turnover. Concomitantly, the contribution by creatine kinase dropped from 89% in normal hearts to 40% in failing hearts. These phosphotransfer changes were associated with reduced levels of metabolically active ATP but maintained overall ATP turnover rate. Thus, this study provides evidence that adenylate kinase facilitates the transfer of high-energy phosphoryls and signal communication between mitochondria and actomyosin in cardiac muscle, with an increased contribution to cellular phosphotransfer in heart failure. This phosphotransfer function renders adenylate kinase an important component for optimal myocardial bioenergetics and a compensatory mechanism in response to impaired intracellular energy flux in the failing heart.
Role of the Ets Transcription Factors in the Regulation of the Vascular-Specific Tie2 Gene
Authors: Dube Antoinise , Akbarali Yasmin , Sato Thomas N. , Libermann Towia A. , Oettgen Peter
Published: June 2012

Abstract
—The Tie2 gene encodes a vascular endothelium-specific receptor tyrosine kinase that is required for normal vascular development and is also upregulated during angiogenesis. The regulatory regions of the Tie2 gene that are required for endothelium-specific gene expression in vivo have been identified. However, the transcription factors required for Tie2 gene expression remain largely unknown. We have identified highly conserved binding sites for Ets transcription factors in the Tie2 promoter. Mutations in 2 particular binding sites lead to a 50% reduction in the endothelium-specific activity of the promoter. We have compared the ability of several members of the Ets family to transactivate the Tie2 promoter. Our results demonstrate that 1 of 3 distinct isoforms of the novel Ets transcription factor NERF, NERF2, is expressed in endothelial cells and can strongly transactivate the regulatory regions of the Tie2 gene in comparison to other Ets factors, which have little or no effect. NERF2 can bind to the Tie2 promoter Ets sites in electrophoretic mobility shift assays. These studies support a role for Ets factors in the regulation of vascular-specific gene expression and suggest that the novel Ets factor NERF2 may be a critical transcription factor in specifying the expression of the Tie2 gene in vascular endothelial cells.
Soluble Transforming Growth Factor-β Type II Receptor Inhibits Negative Remodeling, Fibroblast Transdifferentiation, and Intimal Lesion Formation But Not Endothelial Growth
Authors: Smith Joshua D. , Bryant Shane R. , Couper Leslie L. , Vary Calvin P. H. , Gotwals Philip J. , Koteliansky Victor E. , Lindner Volkhard
Published: June 2012

Abstract
—Using the rat balloon catheter denudation model, we examined the role of transforming growth factor-β (TGF-β) isoforms in vascular repair processes. By en face in situ hybridization, proliferating and quiescent smooth muscle cells in denuded vessels expressed high levels of mRNA for TGF-β
1
, TGF-β
2
, TGF-β
3
, and lower levels of TGF-β receptor II (TGF-βRII) mRNA. Compared with normal endothelium, TGF-β
1
and TGF-β
2
, as well as TGF-βRII, mRNA were upregulated in endothelium at the wound edge. Injected recombinant soluble TGF-βRII (TGF-βR:Fc) localized preferentially to the adventitia and developing neointima in the injured carotid artery, causing a reduction in intimal lesion formation (up to 65%) and an increase in lumen area (up to 88%). The gain in lumen area was largely due to inhibition of negative remodeling, which coincided with reduced adventitial fibrosis and collagen deposition. Four days after injury, TGF-βR:Fc treatment almost completely inhibited the induction of smooth muscle α-actin expression in adventitial cells. In the vessel wall, TGF-βR:Fc caused a marked reduction in mRNA levels for collagens type I and III. TGF-βR:Fc had no effect on endothelial proliferation as determined by reendothelialization of the denuded rat aorta. Together, these findings identify the TGF-β isoforms as major factors mediating adventitial fibrosis and negative remodeling after vascular injury, a major cause of restenosis after angioplasty.
Replacement by Homologous Recombination of the
minK
Gene With
lacZ
Reveals Restriction of
minK
Expression to the Mouse Cardiac Conduction System
Authors: Kupershmidt Sabina , Yang Tao , Anderson Mark E. , Wessels Andy , Niswender Kevin D. , Magnuson Mark A. , Roden Dan M.
Published: June 2012

Abstract
—The
minK
gene encodes a 129-amino acid peptide the expression of which modulates function of cardiac delayed rectifier currents (
I
Kr
and
I
Ks
), and mutations in
minK
are now recognized as one cause of the congenital long-QT syndrome. We have generated
minK-
deficient mice in which the bacterial
lacZ
gene has been substituted for the
minK
coding region such that β-galactosidase expression is controlled by endogenous
minK
regulatory elements. In cardiac myocytes isolated from wild-type neonatal mice,
I
Ks
is rarely recorded, while
I
Kr
is common. In
minK
(–/–) myocytes,
I
Ks
is absent and
I
Kr
is significantly reduced and its deactivation slowed; these results further support a role for
minK
in modulating both
I
Ks
and
I
Kr
. Despite these changes, ECGs in (+/+) and (–/–) animals are no different at adult and at neonatal stages. ECG responses to isoproterenol are also similar in the 2 groups. β-Galactosidase staining in postnatal
minK
(–/–) hearts is highly restricted, to the sinus-node region, caudal atrial septum, and proximal conducting system. Moreover, as early as embryonal day 11, segmentally restricted β-galactosidase expression is observed in the portions of the sinoatrial and atrioventricular junctions that are thought to give rise to the conducting system, thereby implicating
minK
expression as an early event in conduction system development. More generally, the restricted nature of
minK
expression in the mouse heart suggests species-specific roles of this gene product in mediating the electrophysiological properties of the heart.
A Novel Mechanism of Anode-Break Stimulation Predicted by Bidomain Modeling
Authors: Ranjan Ravi , Tomaselli Gordon F. , Marbán Eduardo
Published: June 2012

Abstract
—Anodal stimulation by external pacemakers has been explained on the basis of bidomain models of cardiac tissue. Bidomain models predict that anodal stimuli will hyperpolarize the underlying tissue while adjacent regions become depolarized (virtual cathodes), initiating excitation. We investigated the contribution of active cellular properties to anode-break stimulation. A bidomain model was implemented in which each cell contained realistic ionic currents, including those recruited by hyperpolarization. Simulations reveal that anode-break excitation can originate at the site of stimulation itself and not only from adjacent regions of induced depolarization. The threshold for initiating excitation at the site of stimulation is lower than that for stimulation initiating from adjacent depolarized regions. Thus, incorporation of active cellular properties into a bidomain model predicts a novel mechanism for anode-break stimulation of the heart. The results will improve our understanding of anodal pacing and its risks and benefits in patients.
Proadrenomedullin N-Terminal 20 Peptide Hyperpolarizes the Membrane by Activating an Inwardly Rectifying K
+
Current in Differentiated PC12 Cells
Authors: Takano Koji , Fujita Toshiro
Published: June 2012

Abstract
—The mechanism of proadrenomedullin N-terminal 20 peptide (PAMP)–induced inhibition of catecholamine release from adrenergic nerve was investigated in nerve growth factor–treated PC12 cells that have differentiated characteristics somewhat similar to noradrenergic neurons. The effect of PAMP on the excitability of these cells was investigated with the use of perforated whole-cell clamp. PAMP hyperpolarized the membrane by increasing a K
+
conductance in a dose-dependent manner. The current-voltage relationship (
I
-V) relationship of the PAMP-induced K
+
conductance exhibited inward-going rectification. The activation was abolished by microinjecting GDPβS into the cells or pretreating the cells with pertussis toxin. These results indicate that a pertussis toxin–sensitive G protein is involved in the signal transduction. The PAMP-induced activation of the K
+
conductance was attenuated by microinjecting antibody against the carboxyl terminus of Gα
i3
, but it was not influenced by microinjecting antibody against the common carboxyl termini of Gα
i1
and Gα
i2
, which indicated that the G protein coupling the PAMP receptor to the inwardly rectifying K
+
current is Gα
i3.
The PAMP-induced hyperpolarization may inhibit the catecholamine release from the neurons by attenuating the action potential frequency.
Endothelial Injuries of Coronary Arteries Distal to Thrombotic Sites
Authors: Eguchi Hiroyuki , Ikeda Hisao , Murohara Toyoaki , Yasukawa Hideo , Haramaki Nobuya , Sakisaka Shotaro , Imaizumi Tsutomu
Published: June 2012

Abstract
—Intracoronary thrombus formation is associated with epicardial vasoconstriction distal to the thrombotic site. To investigate the mechanisms of abnormal vasomotor function of the artery distal to the thrombotic site, we studied coronary vessels in dogs with cyclic flow variations (CFVs) of the left anterior descending coronary artery (LAD) stenosis with endothelial injury. Coronary rings isolated from the LAD (proximal, stenotic, and distal sites) and control circumflex coronary arteries were tested for responsiveness to endothelium-dependent (acetylcholine and A23187) and endothelium-independent vasodilators (NaNO
2
). Endothelium-independent relaxation was intact in all 4 sites. Endothelium-dependent relaxation was intact in the control and proximal sites and impaired in the stenotic sites. Relaxations not only to acetylcholine and A23187 but also to serotonin, ADP, and thrombin were impaired in the distal sites after observing CFVs for 80 minutes. Electron microscopy revealed the loss of endothelial integrity with leukocyte adherence to the endothelium in the distal sites. Immunohistochemical expression of P-selectin on the endothelial cells was more upregulated in the distal site than in the proximal site, and P-selectin mRNA expression was significantly greater in the ischemic region distal to the thrombotic site than in the proximal nonischemic region. PB1.3, a neutralizing monoclonal antibody against P-selectin, and sialyl Lewis
X
(SLe
X
)–containing oligosaccharide SLe
X
, a carbohydrate analogue of selectin ligand, preserved endothelial function without affecting CFVs. SLe
X
-containing oligosaccharide preserved endothelial integrity of the distal site and inhibited P-selectin expression of the distal site. Thus, the adhesive interaction between endothelial P-selectin and leukocyte SLe
X
may play an important role in endothelial injuries of the coronary artery distal to the thrombotic site.
Mechanisms of Altered Excitation-Contraction Coupling in Canine Tachycardia-Induced Heart Failure, I
Authors: O’Rourke Brian , Kass David A. , Tomaselli Gordon F. , Kääb Stefan , Tunin Richard , Marbán Eduardo
Published: June 2012

Abstract
—Pacing-induced heart failure in the dog recapitulates many of the electrophysiological and hemodynamic abnormalities of the human disease; however, the mechanisms underlying altered Ca
2+
handling have not been investigated in this model. We now show that left ventricular midmyocardial myocytes isolated from dogs subjected to 3 to 4 weeks of rapid pacing have prolonged action potentials and Ca
2+
transients with reduced peaks, but durations ≈3-fold longer than controls. To discriminate between action potential effects on Ca
2+
kinetics and direct changes in Ca
2+
regulatory processes, voltage-clamp steps were used to examine the time constant for cytosolic Ca
2+
removal (τ
Ca
). τ
Ca
was prolonged by just 35% in myocytes from failing hearts after fixed voltage steps in physiological solutions (τ
Ca
control, 216±25 ms, n=17; τ
Ca
failing, 292±23 ms, n=22;
P
<0.05), but this difference was markedly accentuated when Na
+
/Ca
2+
exchange was eliminated (τ
Ca
control, 282±30 ms, n=13; τ
Ca
failing, 576±83 ms, n=11;
P
<0.005). Impaired sarcoplasmic reticular (SR) Ca
2+
uptake and a greater dependence on Na
+
/Ca
2+
exchange for cytosolic Ca
2+
removal was confirmed by inhibiting SR Ca
2+
ATPase with cyclopiazonic acid, which slowed Ca
2+
removal more in control than in failing myocytes. β-Adrenergic stimulation of SR Ca
2+
uptake in cells from failing hearts sufficed only to accelerate τ
Ca
to the range of unstimulated controls. Protein levels of SERCA2a, phospholamban, and Na
+
/Ca
2+
exchanger revealed a pattern of changes qualitatively similar to the functional measurements; SERCA2a and phospholamban were both reduced in failing hearts by 28%, and Na
+
/Ca
2+
exchange protein was increased 104% relative to controls. Thus, SR Ca
2+
uptake is markedly downregulated in failing hearts, but this defect is partially compensated by enhanced Na
+
/Ca
2+
exchange. The alterations are similar to those reported in human heart failure, which reinforces the utility of the pacing-induced dog model as a surrogate for the human disease.
Viral Myocarditis
Authors: Yang Decheng , Yu Jen , Luo Zongshu , Carthy Christopher M. , Wilson Janet E. , Liu Zhewei , McManus Bruce M.
Published: June 2012

Abstract
—Differences in host susceptibility to viral myocarditis caused by a given strain of coxsackievirus B3 (CVB3) are known to be largely related to host genetic factors. Little is known, however, about the key genes that encode determinants (mediators) of myocarditis development or the nature of injury. To identify these genes and further understand the molecular mechanisms of the disease process, we have used a murine model and the differential display technique to fingerprint mRNAs from CVB3-infected mouse hearts. Total RNA was extracted from hearts of 4- and 10-week-old A/J(H-2
a
) mice at day 4 after CVB3 infection, and mRNAs were detected by reverse transcriptase–polymerase chain reaction and subsequently analyzed on polyacrylamide DNA sequencing gels. The differentially displayed bands were confirmed by Northern hybridization using the bands as cDNA probes. Twenty-eight upregulated or downregulated bands were selected from the sequencing gels; among these, 2 upregulated and 3 downregulated cDNA fragments were confirmed by Northern hybridization. DNA sequence analysis and GenBank searching have determined that 4 of the 5 candidate genes are homologous to genes encoding
Mus musculus
inducible GTPase, mouse mitochondrial hydrophobic peptide (a subunit of NADH dehydrogenase), mouse β-globin, and
Homo sapiens
cAMP-regulated response element binding protein (CREB) binding protein (CBP), respectively. The remaining candidate gene matches an unpublished cDNA clone,
M musculus
Nip21 mRNA (GenBank accession number,
AF035207
), which is homologous to human Nip2, a Bcl-2 binding protein. Our data suggest preliminarily that both structural and nonstructural genes are involved in myocarditis development. For the structural gene, β-globin, we further confirmed its downregulation at the protein level by measuring the mean cell volume of red blood cells and found it was marginally reduced in the CVB3-infected group (
P
<0.06), with no change in hemoglobin concentration. Cardiac myoglobin concentration was also measured and found to be decreased (
P
<0.005), with a parallel decrease in total soluble protein in the CVB3-infected mouse myocardium (
P
<0.01). We also noted that the ratio of myoglobin to total protein was not significantly changed; this may be due to the downregulation of additional genes in the host heart, a number being observed on the differential display gels. The significant downregulation of β-globin major gene expression in the heart may be relevant to impaired cardiac function in both the early and late postinfection period. The other identified nonstructural genes are known to be involved in regulation of gene expression, signal transduction pathways, and apoptotic cell death. The altered expression of structural and nonstructural genes may play important roles in the mediation of myocarditis development and perhaps other pathological processes in the heart.
cdc25A Is Necessary but Not Sufficient for Optimal c-
myc–
Induced Apoptosis and Cell Proliferation of Vascular Smooth Muscle Cells
Authors: Macdonald Kirsty , Bennett Martin R.
Published: June 2012

Abstract
—Increasing evidence indicates that the control of cell proliferation and apoptosis are linked. The c-
myc
proto-oncogene is induced early after cell-cycle entry in vascular smooth muscle cells (VSMCs) in vitro and after arterial injury and regulates both cell proliferation and apoptosis. Although both proliferation and apoptosis are likely to be mediated via transcriptional activation of target genes, few c-
myc
targets have been identified. Therefore, the recent identification that cdc25A, a cell-cycle phosphatase involved in G
1
progression, is transcriptionally activated by c-
myc
and regulates c-
myc
–induced apoptosis has suggested that cdc25A may be the principal mediator of c-
myc
in VSMCs. We examined cdc25A regulation of c-
myc
–induced proliferation and apoptosis by expressing cdc25A or antisense cdc25A in primary rat VSMCs or in VSMCs expressing deregulated c-
myc
or adenovirus E1A. Ectopic c-
myc
increased cdc25A expression, but cdc25A was still responsive to serum components, which indicated that c-
myc
alone is not the main determinant of cdc25A expression. Antisense cdc25A inhibited c-
myc
–induced proliferation and apoptosis; however, drug and metabolic blocks indicated that this effect was limited to G
1
. Ectopic cdc25A augmented the proproliferative and proapoptotic action of c-
myc
but did not increase cell proliferation or apoptosis in the absence of ectopic c-
myc
. In contrast, E1A/E2F-induced apoptosis was independent of cdc25A. We conclude that cdc25A expression modulates the ability of c-
myc
to induce apoptosis in G
1
. However, cdc25A alone does not induce apoptosis and cannot substitute for c-
myc
in VSMCs. Additional targets of c-
myc
are therefore involved in apoptosis of both G
1
and post-G
1
VSMCs.
Acid-Evoked Currents in Cardiac Sensory Neurons
Authors: Benson Christopher J. , Eckert Stephani P. , McCleskey Edwin W.
Published: June 2012

Abstract
—Sensory neurons that innervate the heart sense ischemia and mediate angina. To use patch-clamp methods to study ion channels on these cells, we fluorescently labeled cardiac sensory neurons (CSNs) in rats so that they could later be identified in dissociated primary culture of either nodose or dorsal root ganglia (DRG). Currents evoked by a variety of different agonists imply the importance of lowered pH (≤7.0) in signaling ischemia. Acidic pH evoked extremely large depolarizing current in almost all cardiac afferent neurons from the DRG (CDRGNs). In contrast, only about half of the unlabeled DRG neurons responded to acid, and their current amplitudes were much less than that in CDRGNs. In all respects tested—kinetics, selectivity, and pharmacology—the acid-evoked current was similar to that of previously described native and cloned acid-sensing ion channels. Cardiac afferents from the nodose ganglia differed from CDRGNs in having smaller acid-evoked currents but clearly larger currents evoked by ATP. Serotonin, acetylcholine, bradykinin, and adenosine elicited currents in fewer CSNs than did ATP or lowered pH, and the currents were relatively small. Capsaicin, an activator of small nociceptive sensory neurons that innervate skin, evoked only small and rare currents in CDRGNs. The extremely large amplitude and prevalent expression of acid-evoked current in CSNs imply a critical role for acidity in sensation associated with myocardial ischemia.
Purification and Preliminary Characterization of a Cardiac Kv1.5 Repressor Element Binding Factor
Authors: Valverde Paloma , Koren Gideon
Published: June 2012

Abstract
—We have previously demonstrated that the cell-specific expression of Kv1.5 promoter is regulated by a silencer (Kv1.5 repressor element; KRE) containing a dinucleotide-repetitive element, (GT)
19
(GA)
1
(CA)
15
(GA)
16
. Electromobility gel shift assays (EMSAs) of KRE with GH3 nuclear extracts detected a unique DNA-protein complex, which was not detectable in Chinese hamster ovary or COS-7 cells. We further delineated KRE and determined that a 52-bp fragment that contained a (GT)
10
(GA)
1
(CA)
10
dinucleotide-repetitive element was sufficient for silencer activity. EMSAs using nuclear extracts isolated from the heart and from GH3 cells demonstrated that the 52-bp element formed specific and identical gel shift effects. These complexes were not detectable in EMSA experiments with liver nuclear extracts. Magnetic DNA affinity purification and UV cross-linking experiments identified a 27-kDa KRE binding factor (KBF) in GH3 cell nuclear extracts. Purified KBF reacted specifically with double-stranded KRE, abolishing the formation of multimeric KRE-DNA complexes. Thus, the interaction between KRE and KBF may play an important role in regulating the GH3- and cardiac-specific expression of Kv1.5.
Incidence, Evolution, and Spatial Distribution of Functional Reentry During Ventricular Fibrillation in Pigs
Authors: Rogers Jack M. , Huang Jian , Smith William M. , Ideker Raymond E.
Published: June 2012

Abstract
—Functional reentry has been hypothesized to be an underlying mechanism of ventricular fibrillation (VF); however, its contribution to activation patterns during fully developed VF is unclear. We applied new quantitative pattern analysis techniques to mapping data acquired from a 21×24 unipolar electrode array (2-mm spacing) located on the ventricular epicardium of 7 open-chest, unsupported pigs. Data epochs 4 seconds long beginning 1, 10, 20, 30, and 40 seconds after electrical induction were analyzed. Reentrant circuits were automatically identified and quantified. We found that 2.3% of activation pathways could unambiguously be classified as reentrant. From scaling analysis, an additional 28% of the pathways may also have been reentrant. Reentry was short-lived with 1.5±1.5 (mean±SD) complete cycles per circuit. The fraction of reentrant pathways, number of cycles per circuit, cycle duration, and area and perimeter of the cores all increased significantly as VF progressed. Core drift speed decreased significantly. Neither the orientation of the cores nor the direction of drift was well predicted by the epicardial fiber orientation (
r
2
=0.108 and 0.138, respectively, by linear regression). Reentrant circuits were clustered in regions of the epicardium. We conclude the following: (1) Epicardial reentry is relatively uncommon and short-lived during VF, suggesting either that sustained reentry is transmural or that mechanisms governing sustained reentry are relatively unimportant to the dynamics of VF. (2) Reentrant circuits become more common, larger, and longer-lived as VF progresses, which may explain a recently observed increase in VF organization during the first minute of VF. (3) The conditions necessary to induce and sustain reentry are distributed nonuniformly.
Manipulation of Cellular Excitability by Cell Fusion
Authors: Hoppe Uta C. , Johns David C. , Marbán Eduardo , O’Rourke Brian
Published: June 2012

Abstract
—To investigate the still-undetermined role of the Ca
2+
-independent transient outward current (
I
to1
) on repolarization of the cardiac action potential, we used cell fusion to introduce
I
to1
into guinea pig cardiomyocytes, which normally lack this current. This technique enables the rapid delivery of premade functional ion channels to cardiomyocytes within hours of isolation, thus eliminating the action potential alterations that complicate prolonged cell culture. Chinese hamster ovary (CHO) cells stably expressing Kv4.3 (CHO-Kv4.3) were loaded with a fluorescent dye and fused to guinea pig cardiomyocytes using polyethylene glycol. As controls, nontransfected CHO cells were fused using the same protocol. Myocytes fused with CHO-Kv4.3 cells exhibited a robust
I
to1
(16.5±2.6 pA/pF at +40 mV; 37°C; n=19), whereas controls had none.
I
to1
accelerated the early repolarization velocity (
r
=−0.68; 3 ms after the overshoot) and progressively suppressed the voltage of the plateau phase (
r
=−0.90) with increasing
I
to1
density. Reduction of the action potential duration to 50% repolarization (
r
=−0.76) and to 90% repolarization (
r
=−0.65) also correlated well with
I
to1
density. Thus,
I
to1
exerted a significant effect on the early repolarization phase and abbreviated action potential duration. Cell fusion is a valuable and generalizable technique to introduce preformed membrane proteins into native cells.
Opposing Effects of Reactive Oxygen Species and Cholesterol on Endothelial Nitric Oxide Synthase and Endothelial Cell Caveolae
Authors: Peterson Timothy E. , Poppa Veronica , Ueba Hiroto , Wu Albert , Yan Chen , Berk Bradford C.
Published: June 2012

Abstract
—Synthesis of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is critical for normal vascular homeostasis. eNOS function is rapidly regulated by agonists and blood flow and chronically by factors that regulate mRNA stability and gene transcription. Recently, localization of eNOS to specialized plasma membrane invaginations termed caveolae has been proposed to be required for maximal eNOS activity. Because caveolae are highly enriched in cholesterol, and hypercholesterolemia is associated with increased NO production, we first studied the effects of cholesterol loading on eNOS localization and NO production in cultured bovine aortic endothelial cells (BAECs). Caveolae-enriched fractions were prepared by OptiPrep gradient density centrifugation. Treatment of BAECs with 30 μg/mL cholesterol for 24 hours stimulated significant increases in total eNOS protein expression (1.50-fold), eNOS associated with caveolae-enriched membranes (2.23-fold), and calcium ionophore-stimulated NO production (1.56-fold). Because reactive oxygen species (ROS) contribute to endothelial dysfunction in hypercholesterolemia, we next studied the effects of ROS on eNOS localization and caveolae number. Treatment of BAECs for 24 hours with 1 μmol/L LY83583, a superoxide-generating napthoquinolinedione, decreased caveolae number measured by electron microscopy and prevented the cholesterol-mediated increases in eNOS expression. In vitro exposure of caveolae-enriched membranes to ROS (xanthine plus xanthine oxidase) dissociated caveolin more readily than eNOS from the membranes. These results show that cholesterol treatment increases eNOS expression, whereas ROS treatment decreases eNOS expression and the association of eNOS with caveolin in caveolae-enriched membranes. Our data suggest that oxidative stress modulates endothelial function by regulating caveolae formation, eNOS expression, and eNOS-caveolin interactions.
Ca
2+
Handling and Sarcoplasmic Reticulum Ca
2+
Content in Isolated Failing and Nonfailing Human Myocardium
Authors: Pieske Burkert , Maier Lars S. , Bers Donald M. , Hasenfuss Gerd
Published: June 2012

Abstract
—Disturbed sarcoplasmic reticulum (SR) Ca
2+
content may underlie the altered force-frequency and postrest contractile behavior in failing human myocardium. We used rapid cooling contractures (RCCs) to assess SR Ca
2+
content in ventricular muscle strips isolated from nonfailing and end-stage failing human hearts. With an increase in rest intervals (1 to 240 s; 37°C), nonfailing human myocardium (n=7) exhibited a parallel increase in postrest twitch force (at 240 s by 121±44%;
P
<0.05) and RCC amplitude (by 69±53%;
P
<0.05). In contrast, in failing myocardium (n=30), postrest twitch force decreased at long rest intervals and RCC amplitude declined monotonically with rest (by 25±9% and 53±9%, respectively;
P
<0.05). With an increase in stimulation frequencies (0.25 to 3 Hz), twitch force increased continuously in nonfailing human myocardium (n=7) by 71±17% (at 3 Hz;
P
<0.05) and RCC amplitude increased in parallel by 247±55% (
P
<0.05). In contrast, in failing myocardium (n=26), twitch force declined by 29±7% (
P
<0.05) and RCC amplitude increased only slightly by 36±14% (
P
<0.05). Paired RCCs were evoked to investigate the relative contribution of SR Ca
2+
uptake and Na
+
/Ca
2+
exchange to cytosolic Ca
2+
removal during relaxation. SR Ca
2+
uptake (relative to the Na
+
/Ca
2+
exchange) increased significantly in nonfailing but not in failing human myocardium as stimulation rates increased. We conclude that the negative force-frequency relation in failing human myocardium is due to an inability of SR Ca
2+
content to increase sufficiently at high frequencies and thus cannot overcome the frequency-dependent refractoriness of SR Ca
2+
release. The rest-dependent decay in twitch force in failing myocardium is due to rest-dependent decline in SR Ca
2+
content. These alterations could be secondary to depressed SR Ca
2+
-ATPase combined with enhanced cytosolic Ca
2+
extrusion via Na
+
/Ca
2+
exchange.
Two Distinct Mechanisms Mediate a Differential Regulation of Protein Kinase C Isozymes in Acute and Prolonged Myocardial Ischemia
Authors: Strasser Ruth H. , Simonis Gregor , Schön Steffen P. , Braun Martin U. , Ihl-Vahl Renate , Weinbrenner Christof , Marquetant Rainer , Kübler Wolfgang
Published: June 2012

Abstract
—An activation of protein kinase C (PKC) in acute myocardial ischemia has been shown previously using its translocation to the plasma membrane as an indirect parameter. However, whether PKC remains activated or whether other mechanisms such as altered gene expression may mediate an isozyme-specific regulation in prolonged ischemia have not been investigated. In isolated perfused rat hearts, PKC activity and the expression of PKC cardiac isozymes were determined on the protein level using enzyme activities and Western blot analyses and on the mRNA level using reverse transcriptase–polymerase chain reaction after various periods of global ischemia (1 to 60 minutes). As early as 1 minute after the onset of ischemia, PKC activity is translocated from the cytosol to the particulate fraction without change in total cardiac enzyme activity. This translocation involves all major cardiac isozymes of PKC (ie, PKCα, PKCδ, PKCε, and PKCζ). This rapid, nonselective activation of PKCs is only transient. In contrast, prolonged ischemia (≥15 minutes) leads to an increased cardiac PKC activity (119±7 versus 190±8 pmol/min per mg protein) residing in the cytosol. This is associated with an augmented, subtype-selective isozyme expression of PKCδ and PKCε (163% and 199%, respectively). The specific mRNAs for PKCδ (948±83 versus 1501±138 ag/ng total RNA, 30 minutes of ischemia) and PKCε (1597±166 versus 2611±252 ag/ng total RNA) are selectively increased. PKCα and PKCζ remain unaltered. In conclusion, two distinct activation and regulation processes of PKC are characterized in acute myocardial ischemia. The early, but transient, translocation involves all constitutively expressed cardiac isozymes of PKC, whereas in prolonged ischemia an increased total PKC activity is associated with an isozyme-selective induction of PKCε and PKCδ. Whether these fundamentally different activation processes interact remains to be elucidated.
Altered Structure, Regulation, and Function of the Gene Encoding the Atrial Natriuretic Peptide in the Stroke-Prone Spontaneously Hypertensive Rat
Authors: Rubattu Speranza , Lee-Kirsch Min Ae , DePaolis Paola , Giliberti Rosangela , Gigante Bruna , Lombardi Alessia , Volpe Massimo , Lindpaintner Klaus
Published: June 2012

Abstract
—Through the genotype/phenotype cosegregation analysis of an F
2
intercross, from the crossbreeding of stroke-prone spontaneously hypertensive rats (SHRSP) and stroke-resistant spontaneously hypertensive rats (SHR), we previously identified a quantitative trait locus for stroke on rat chromosome 5 (
STR2
) that colocalized with the genes encoding atrial and brain natriuretic peptides (
ANP
and
BNP
) and conferred a stroke-delaying effect. To further characterize
ANP
and
BNP
as candidates for stroke, we performed additional studies. Comparative sequence analysis revealed point mutations in both the coding and regulatory regions of
ANP
, whereas no interstrain differences were found for
BNP
. In in vitro studies in COS-7 and AtT-20 cells that were performed to test the relevance of a G→A substitution at position 1125, a Gly→Ser transposition in the SHRSP pro-ANP peptide resulted in different posttranslational processing of the SHRSP ANP gene product that was also associated with higher cGMP production (
P
<0.05). Furthermore, an analysis of a 5′ end mutation affecting a PEA2 regulatory binding site in the 5′ untranslated regulatory sequence of SHRSP
ANP
demonstrated a significantly lower
ANP
promoter activation in endothelial cells (P<0.05 versus the SHR
ANP
). In addition, the expression of
ANP
was significantly reduced in the brain, but not in the atria, of SHRSP compared with SHR (
P
<0.0001). No differences were detected with regard to
BNP
expression. The present results reveal substantial differences in
ANP
, but not
BNP
, structure and product among SHR and SHRSP, which supports a role of
ANP
in the pathogenesis of stroke in the SHRSP animal model.
Increase in Prostaglandin E
2
Production by Interleukin-1β in Arterial Smooth Muscle Cells Derived From Patients With Moyamoya Disease
Authors: Yamamoto Mari , Aoyagi Masaru , Fukai Naomi , Matsushima Yoshiharu , Yamamoto Kiyotaka
Published: June 2012

Abstract
—Moyamoya disease is a progressive cerebrovascular occlusive disease that primarily affects children. The cause is unknown. We examined the production of prostanoids and the expression of cyclooxygenase-2 (COX-2) in cultured arterial smooth muscle cells (SMCs) derived from patients with moyamoya disease. Twelve moyamoya and 8 control cell strains were examined. The steady-state levels of prostanoids in the culture medium did not differ between moyamoya and control SMCs. When the cells were stimulated with interleukin-1β (IL-1β), prostaglandin E
2
(PGE
2
) release into the medium was significantly greater from moyamoya SMCs than from control SMCs, whereas the amounts of prostacyclin and thromboxane B
2
did not differ. IL-1β–induced PGE
2
production by moyamoya SMCs was completely blocked by the addition of indomethacin or NS-398. IL-1β significantly stimulated cell migration and DNA synthesis in control SMCs but had an inhibitory effect on moyamoya SMCs. The inhibitory effects on the growth and migration of moyamoya SMCs were caused by excessive secretion of PGE
2
and was reversed with indomethacin treatment. Immunofluorescence studies and Western blot analysis showed greater amounts of COX-2 protein expression in IL-1β–stimulated moyamoya SMCs. These findings suggest that moyamoya SMCs respond to inflammatory stimuli to produce excess amounts of PGE
2
through the activation of COX-2, which increases vascular permeability and decreases vascular tone. This facilitates the exposure of vessels to blood constituents and promotes the development of intimal thickening in moyamoya disease.