bluuebunny/crossref_metadata_2025 huggingface dataset:
Impaired Modulation of Sympathetic Vasoconstriction in Contracting Skeletal Muscle of Rats With Chronic Myocardial Infarctions
Authors: Thomas Gail D. , Zhang Weiguo , Victor Ronald G.
Published: September 2007

Abstract
—Skeletal muscle perfusion during exercise is impaired in heart failure, but the underlying mechanisms are poorly understood. One possibility is that sympathetic vasoconstriction is enhanced in exercising muscle in heart failure as a result of impaired counterregulatory mechanisms that normally act to attenuate vasoconstrictor responses. In healthy animals, sympathetic vasoconstriction in contracting skeletal muscle is attenuated by endogenously produced nitric oxide (NO). Because the NO pathway may be dysfunctional in heart failure, we hypothesized that reduced NO in contracting muscle would result in enhanced sympathetic vasoconstriction. In sham rats and rats with chronic myocardial infarctions (MIs) produced by coronary artery ligation, we measured arterial pressure and femoral artery blood flow responses to sympathetic nerve stimulation (1, 2.5, and 5 Hz) in resting and contracting hindlimb. In resting hindlimb, sympathetic stimulation decreased femoral vascular conductance similarly in sham and MI rats. In contracting hindlimb, these vasoconstrictor responses were attenuated to a greater extent in sham than in MI rats. NO synthase inhibition enhanced sympathetic vasoconstriction in contracting hindlimb of sham, but not MI, rats. Conversely, infusion of
l
-arginine or a superoxide scavenger, tempol or tiron, attenuated sympathetic vasoconstriction in contracting hindlimb of MI rats. NO synthase expression was similar, but malondialdehyde (a marker of free radical damage) was greater in skeletal muscle from MI than from sham rats. These data suggest that impaired metabolic modulation of sympathetic vasoconstriction in contracting skeletal muscle of MI rats is a consequence of superoxide-mediated disruption of the NO pathway.
Nitric Oxide Insufficiency, Platelet Activation, and Arterial Thrombosis
Authors: Loscalzo Joseph
Published: September 2007

Abstract—Nitric oxide (NO) was originally discovered as a vasodilator product of the endothelium. Over the last 15 years, this vascular mediator has been shown to have important antiplatelet actions as well. By activating guanylyl cyclase, inhibiting phosphoinositide 3-kinase, impairing capacitative calcium influx, and inhibiting cyclooxygenase-1, endothelial NO limits platelet activation, adhesion, and aggregation. Platelets are also an important source of NO, and this platelet-derived NO pool limits recruitment of platelets to the platelet-rich thrombus. A deficiency of bioactive NO is associated with arterial thrombosis in animal models, individuals with endothelial dysfunction, and patients with a deficiency of the extracellular antioxidant enzyme glutathione peroxidase-3. This enzyme catalyzes the reduction of hydrogen and lipid peroxides, which limits the availability of these reactive oxygen species to react with and inactivate NO. The complex biochemical reactions that underlie the function and inactivation of NO in the vasculature represent an important set of targets for therapeutic intervention for the prevention and treatment of arterial thrombotic disorders.
Role of NADPH Oxidase in the Vascular Hypertrophic and Oxidative Stress Response to Angiotensin II in Mice
Authors: Wang Hui Di , Xu Shanqin , Johns Douglas G. , Du Yue , Quinn Mark T. , Cayatte Antonio J. , Cohen Richard A.
Published: September 2007

Abstract
—Oxygen-derived free radicals are involved in the vascular response to angiotensin II (Ang II), but the role of NADPH oxidase, its subunit proteins, and their vascular localization remain controversial. Our purpose was to address the role of NADPH oxidase in the blood pressure (BP), aortic hypertrophic, and oxidant responses to Ang II by taking advantage of knockout (KO) mice that are genetically deficient in gp91

phox

, an NADPH oxidase subunit protein. The baseline BP was significantly lower in KO mice than in wild-type (WT) (92±2 [KO] versus 101±1 [WT] mm Hg,
P
<0.01), but infusion of Ang II for 6 days caused similar increases in BP in the 2 strains (33±4 [KO] versus 38±2 [WT] mm Hg,
P
>0.4). Ang II increased aortic superoxide anion production 2-fold in the aorta of WT mice but did not do so in KO mice. Aortic medial area increased in WT (0.12±0.02 to 0.17±0.02 mm
2
,
P
<0.05), but did not do so in KO mice (0.10±0.01 to 0.11±0.01 mm
2
,
P
>0.05). Histochemistry and polymerase chain reaction demonstrated gp91

phox

localized in endothelium and adventitia of WT mice. Levels of reactive oxidant species as indicated by 3-nitrotyrosine immunoreactivity increased in these regions in WT but not in KO mouse aorta in response to Ang II. These results indicate an essential role in vivo of gp91

phox

and NADPH oxidase–derived superoxide anion in the regulation of basal BP and a pressure-independent vascular hypertrophic and oxidant stress response to Ang II.
Lysophosphatidic Acid Positively Regulates the Fluid Flow–Induced Local Ca
2+
Influx in Bovine Aortic Endothelial Cells
Authors: Ohata Hisayuki , Ikeuchi Tadahiro , Kamada Aya , Yamamoto Masayuki , Momose Kazutaka
Published: September 2007

Abstract
—Using real-time confocal microscopy, we have demonstrated that lysophosphatidic acid (LPA), a bioactive phospholipid existing in plasma, positively regulates fluid flow–induced [Ca
2+
]
i
response in fluo 4–loaded, cultured, bovine aortic endothelial cells. The initial increase in [Ca
2+
]
i
was localized to a circular area with a diameter of <4 μm and spread concentrically, resulting in a mean global increase in [Ca
2+
]
i
. The local increase often occurred in a stepwise manner or repetitively during constant flow. The percentage of cells that responded and the averaged level of increase in [Ca
2+
]
i
were dependent on both the concentration of LPA (0.1 to 10 μmol/L) and the flow rate (25 to 250 mm/s). The response was inhibited by removing extracellular Ca
2+
or by the application of Gd
3+
, an inhibitor of mechanosensitive (MS) channels, but not by thapsigargin, an inhibitor of the endoplasmic reticular Ca
2+
-ATPase. It was also inhibited by 8-bromo-cGMP, and the inhibition was completely reversed by KT5823, an inhibitor of protein kinase G (PKG). These results suggest that the [Ca
2+
]
i
response arises from Ca
2+
influx through Gd
3+
-sensitive MS channels, which are negatively regulated by the activation of PKG. The spatiotemporal properties of the [Ca
2+
]
i
response were completely different from those of a Ca
2+
wave induced by ATP, a Ca
2+
-mobilizing agonist. Therefore, we called the phenomenon Ca
2+
spots. We conclude that LPA positively regulates fluid flow–induced local and oscillatory [Ca
2+
]
i
increase, ie, the Ca
2+
spots, in endothelial cells via the activation of elementary Ca
2+
influx through PKG-regulating MS channels. This indicates an important role for LPA as an endogenous factor in fluid flow–induced endothelial function.
Defect in Insulin-Like Growth Factor-1 Survival Mechanism in Atherosclerotic Plaque–Derived Vascular Smooth Muscle Cells Is Mediated by Reduced Surface Binding and Signaling
Authors: Patel V. A. , Zhang Q.-J. , Siddle K. , Soos M. A. , Goddard M. , Weissberg P. L. , Bennett M. R.
Published: September 2007

Abstract
—Apoptosis of vascular smooth muscle cells (VSMCs) is increased in atherosclerosis compared with normal vessels, where it may contribute to plaque rupture. We have previously found that human plaque–derived VSMCs (pVSMCs) are intrinsically sensitive to apoptosis and not responsive to the protective effects of insulin-like growth factor-1 (IGF-1). We therefore examined the mechanism underlying this defect. Human pVSMCs showed <25%
125
I–IGF-1 surface binding, <20% IGF-1 receptor (IGF-1R) expression than that of normal medial VSMCs, and <40% Akt kinase activity in response to IGF-1. pVSMCs expressed and secreted high levels of IGF-1 binding proteins (IGFBPs), and the IGF-1 analogues, long R3 and Des 1,3 IGF-1, which do not bind to IGFBPs, were able to increase pVSMC survival to normal medial VSMC levels. The long R3 survival effect was phosphatidylinositol 3-kinase–mediated, but it was not dependent on Akt activity alone. Intimal pVSMCs in vivo showed reduced IGF-1R expression compared with medial VSMCs, in particular at the shoulder regions of plaques. We conclude that human pVSMCs show an intrinsic sensitivity to apoptosis caused in part by defective expression of IGF-1R, impaired IGF-1–mediated survival signaling and increased IGFBP secretion. This impaired IGF-1 protection against apoptosis may promote VSMC loss and plaque instability in atherosclerosis.
Current Perspective on the Role of Apoptosis in Atherothrombotic Disease
Authors: Mallat Ziad , Tedgui Alain
Published: September 2007

Abstract
—Thrombus formation on a disrupted atherosclerotic plaque is a threatening event that leads to vessel occlusion and acute ischemia. In this current perspective, we present evidence for apoptosis as a major determinant of the thrombogenicity of the plaque lipid core and a potential contributor to plaque erosion and associated thrombosis. Moreover, apoptosis may directly affect blood thrombogenicity through the release of apoptotic cells and microparticles into the bloodstream.
Diminished Cardioprotective Response to Inhibition of Angiotensin-Converting Enzyme and Angiotensin II Type 1 Receptor in B2Kinin Receptor Gene Knockout Mice
Authors: Yang Xiao-Ping , Liu Yun-He , Mehta Dharmesh , Cavasin Maria A. , Shesely Edward , Xu Jiang , Liu Fang , Carretero Oscar A.
Published: September 2007

Abstract—Using B2kinin receptor gene knockout mice (B2−/−), we tested the hypothesis that (l) lack of B2receptors may affect blood pressure and cardiac function and aggravate cardiac remodeling after myocardial infarction (MI), and (2) kinins partially mediate the cardiac beneficial effect of angiotensin-converting enzyme inhibitors (ACEi) or angiotensin II type 1 receptor antagonists (AT1-ant), whereas lack of B2receptors may diminish this cardioprotective effect. Chronic heart failure (HF) was induced by MI, which was caused by coronary artery ligation in both B2−/−and 129/SvEvTac mice (wild-type control, B2+/+). An ACEi (ramipril, 2.5 mg/kg/d) or AT1-ant (L-158809, 3 mg/kg/d) was given 1 week after MI and was continued for 12 weeks. Left ventricular (LV) ejection fraction, cardiac output (CO), diastolic LV dimension (LVDd), and LV mass were evaluated by echocardiography. Myocyte cross-sectional area and interstitial collagen fraction were studied histopathologically. We found that basal blood pressure and cardiac function were similar in B2+/+and B2−/−mice. After MI, development of HF and remodeling were also similar between the 2 strains. The ACEi improved cardiac function and remodeling in both strains; however, its effects were attenuated in B2−/−mice (respective values for B2+/+versus B2−/−mice: overall increase in ejection fraction, 64±10% versus 21±5% [P<0.01]; increase in CO, 69±17% versus 23±9% [P<0.01]; overall decrease in LVDd, −24±3% versus −7±4% [P<0.01]; and decrease in LV mass, −38±3% versus −6±6% [P<0.01]). AT1-ant had a beneficial cardiac effect similar to that produced by ACEi, and this effect was also diminished in B2−/−mice (respective values for B2+/+versus B2−/−mice: overall increase in ejection fraction, 46±10% versus 25±9% [P<0.01]; increase in CO, 44±14% versus 15±5% [P<0.01]; overall decrease in LVDd, −14±4% versus −6±3% [P<0.01]; and decrease in LV mass, −33±4 versus −16±7% [P<0.01]). The effect of ACEi or AT1-ant on myocyte cross-sectional area was similar between strains; however, their effect on the interstitial collagen fraction was diminished in B2−/−mice. We concluded that (1) lack of B2kinin receptors does not affect cardiac phenotype or function, either under normal physiological conditions or during the development of HF; and (2) kinins acting via the B2receptor play an important role in the cardioprotective effect of ACEi and AT1-ant.
Thrombin Regulates Insulin-Like Growth Factor-1 Receptor Transcription in Vascular Smooth Muscle
Authors: Du Jie , Brink Marijke , Peng Tao , Mottironi Bianca , Delafontaine Patrick
Published: September 2007

Abstract
—We have previously demonstrated that thrombin upregulation of insulin-like growth factor-1 receptor (IGF-1R) is essential for thrombin-induced mitogenic signaling. To characterize the mechanisms involved, we studied transcription of the IGF-1R gene in rat aortic smooth muscle cells. Thrombin markedly increased IGF-1R mRNA levels, peaking at 3 hours (112±7% above control). This effect was mimicked by the hexapeptide SFFLRN (that functions as a tethered ligand) and was blocked by the thrombin inhibitor hirudin. Nuclear run-on assays indicated that thrombin stimulated IGF-1R gene transcription by 2.1-fold, and this was confirmed with the use of actinomycin D. Thrombin-mediated upregulation of IGF-1R mRNA and protein levels was protein kinase C independent but was completely inhibited by the protein tyrosine kinase inhibitor genistein and by the antioxidants
N
-acetyl-
l
-cysteine and pyrrolidinedithiocarbamate, suggesting the involvement of reactive oxygen species. The thrombin-induced increase in IGF-1R mRNA was inhibitable by diphenyleneiodonium chloride but not by other inhibitors of cellular oxidase systems, suggesting that NAD(P)H oxidase was necessary for the increase. Furthermore, inhibitors of the epidermal growth factor receptor kinase, Janus kinase-2 kinase, and Src kinase did not block the effect. Thus, thrombin transcriptionally regulates the IGF-1R gene via a redox-sensitive protein tyrosine kinase–dependent pathway that does not require protein kinase C activation. In view of our prior data indicating that IGF-1R density is a critical determinant of vascular smooth muscle cell growth, our findings have particular relevance to understanding mechanisms whereby growth factors such as thrombin regulate vascular proliferation in vivo.
Hypoxia Alters the Sensitivity of the L-Type Ca
2+
Channel to α-Adrenergic Receptor Stimulation in the Presence of β-Adrenergic Receptor Stimulation
Authors: Hool Livia C.
Published: September 2007

Abstract
—The effects of α-adrenergic receptor (α-AR) stimulation alone and the effects in the presence of β-adrenergic receptor (β-AR) stimulation were examined on L-type Ca
2+
currents (
I
Ca-L
) in the absence and presence of hypoxia. The α-AR agonist methoxamine either had no effect or had a slight inhibitory effect on basal
I
Ca-L
in the absence and presence of hypoxia. Hypoxia significantly decreased the
K
0.5
for activation of
I
Ca-L
by norepinephrine from 79.8±6.6 to 13.3±0.7 nmol/L. To determine whether hypoxia specifically altered the sensitivity of the channel to α-AR stimulation, cells were exposed to increasing concentrations of methoxamine in the presence of 100 nmol/L isoproterenol (Iso). In the absence of hypoxia, methoxamine inhibited the Iso-activated
I
Ca-L
in a concentration-dependent manner with an EC
50
of 86.9±9.9 μmol/L. However, in the presence of hypoxia, the EC
50
for inhibition of
I
Ca-L
by methoxamine was significantly increased to 266.7±10.8 μmol/L. Methoxamine had little effect on
I
Ca-L
activated by forskolin or histamine in the absence or presence of hypoxia. In addition, inhibition of protein kinase C by bisindolylmaleimide 1 or protein kinase C β peptide inhibitor had no effect on the methoxamine-induced antagonism of
I
Ca-L
in the absence or presence of hypoxia. The tyrosine kinase inhibitor genistein attenuated the methoxamine response in nonhypoxic cells only. However, during hypoxia it was attenuated with the phospholipase A
2
inhibitors mepacrine and indomethacin. These findings represent a novel regulation of the L-type Ca
2+
channel by the phospholipase A
2
pathway and illustrate the complexity of regulation of the channel under hypoxic conditions.
Age-Associated Cardiac Dysfunction in
Drosophila melanogaster
Authors: Paternostro Giovanni , Vignola Carlo , Bartsch Dirk-Uwe , Omens Jeff H. , McCulloch Andrew D. , Reed John C.
Published: September 2007

Abstract
—The fruit fly,
Drosophila melanogaster
, has served as a valuable model/organism for the study of aging and was the first organism possessing a circulatory system to have its genome completely sequenced. However, little is known about the function of the heartlike organ of flies during the aging process. We have developed methods for studying cardiac function in vivo in adult flies. Using 2 different cardiovascular stress methods (elevated ambient temperature and external electrical pacing), we found that maximal heart rate is significantly and reproducibly reduced with aging in
Drosophila
, analogous to observations in elderly humans. We also describe for the first time several other aspects of the cardiac physiology of young adult and aging
Drosophila
, including an age-associated increase in rhythm disturbances. These observations suggest that the study of declining cardiac function in aging flies may serve as a genetically tractable model for genome-wide mutational screening for genes that participate in or protect against cardiac aging and disease.
ADAR1 Is Involved in the Development of Microvascular Lung Injury
Authors: Rabinovici Reuven , Kabir Koroush , Chen Meihong , Su Yingjun , Zhang Dexin , Luo Xiaoxing , Yang Jing-Hua
Published: September 2007

Abstract
—Deamination of adenosine on pre-mRNA to inosine is a recently discovered process of posttranscription modification of pre-mRNA, termed A-to-I RNA editing, which results in the production of proteins not inherent in the genome. The present study aimed to identify a role for A-to-I RNA editing in the development of microvascular lung injury. To that end, the pulmonary expression and activity of the RNA editase ADAR1 were evaluated in a mouse model of endotoxin (15 mg/kg IP)–induced microvascular lung injury (n=5) as well as in cultured alveolar macrophages stimulated with endotoxin, live bacteria, or interferon. ADAR1 expression and activity were identified in sham lungs that were upregulated in lungs from endotoxin-treated mice (at 2 hours). Expression was localized to polymorphonuclear and monocytic cells. These events preceded the development of pulmonary edema and leukocyte accumulation in lung tissue and followed the local production of interferon-γ, a known inducer of ADAR1 in other cell systems. ADAR1 was found to be upregulated in alveolar macrophages (MH-S cells) stimulated with endotoxin (1 to 100 μg/mL), live Escherichia coli (5×10
7
colony-forming units), or interferon-γ (1000 U/mL). Taken together, these data suggest that ADAR1 may play a role in the pathogenesis of microvascular lung injury possibly through induction by interferon.
Coupled Gating Between Cardiac Calcium Release Channels (Ryanodine Receptors)
Authors: Marx Steven O. , Gaburjakova Jana , Gaburjakova Marta , Henrikson Charles , Ondrias Karol , Marks Andrew R.
Published: September 2007

Abstract
—Excitation-contraction coupling in heart muscle requires the activation of Ca
2+
-release channels/type 2 ryanodine receptors (RyR2s) by Ca
2+
influx. RyR2s are arranged on the sarcoplasmic reticular membrane in closely packed arrays such that their large cytoplasmic domains contact one another. We now show that multiple RyR2s can be isolated under conditions such that they remain physically coupled to one another. When these coupled channels are examined in planar lipid bilayers, multiple channels exhibit simultaneous gating, termed “coupled gating.” Removal of the regulatory subunit, the FK506 binding protein (FKBP12.6), functionally but not physically uncouples multiple RyR2 channels. Coupled gating between RyR2 channels may be an important regulatory mechanism in excitation-contraction coupling as well as in other signaling pathways involving intracellular Ca
2+
release.
Recruitment of Serum Response Factor and Hyperacetylation of Histones at Smooth Muscle–Specific Regulatory Regions During Differentiation of a Novel P19-Derived In Vitro Smooth Muscle Differentiation System
Authors: Manabe Ichiro , Owens Gary K.
Published: September 2007

Abstract
—Little is known regarding transcriptional regulatory mechanisms that control the sequential and coordinate expression of genes during smooth muscle cell (SMC) differentiation. To facilitate mechanistic studies of SMC differentiation, we established a novel P19-derived clonal cell line (designated A404) harboring a smooth muscle (SM) α-actin promoter/intron-driven puromycin resistance gene. Retinoic acid plus puromycin treatment stimulated rapid differentiation of multipotential A404 cells into SMCs that expressed multiple SMC differentiation marker genes, including the definitive SM-lineage marker SM myosin heavy chain. Using this system, we demonstrated that various transcription factors were upregulated coincidentally with the expression of SMC differentiation marker genes. Of interest, the expression of serum response factor (SRF), whose function is critical for SMC-specific transcription, was high in undifferentiated A404 cells, and it did not increase over the course of differentiation. However, chromatin immunoprecipitation analyses showed that SRF did not bind the target sites of endogenous SMC marker genes in chromatin in undifferentiated cells, but it did in differentiated A404 cells, and it was associated with hyperacetylation of histones H3 and H4. The present studies define a novel cell system for studies of transcriptional regulation during the early stages of SMC differentiation, and using this system, we obtained evidence for the involvement of chromatin remodeling and selective recruitment of SRF to CArG elements in the induction of cell-selective marker genes during SMC differentiation.
Human Urotensin II–Induced Contraction and Arterial Smooth Muscle Cell Proliferation Are Mediated by RhoA and Rho-Kinase
Authors: Sauzeau Vincent , Le Mellionnec Erik , Bertoglio Jacques , Scalbert Elizabeth , Pacaud Pierre , Loirand Gervaise
Published: September 2007

The aim of this work was to investigate the coupling of human urotensin II (hU-II) to RhoA activation and regulation of RhoA-dependent functions. The use of the Rho-kinase inhibitor Y-27632 and the development of a membrane-permeant RhoA inhibitor (TAT-C3) allowed us to demonstrate that hU-II induced arterial smooth muscle contraction, actin stress fiber formation, and proliferation through the activation of the small GTPase RhoA and its downstream effector Rho-kinase.
Role of Intracellular Na
+
Kinetics in Preconditioned Rat Heart
Authors: Imahashi Kenichi , Nishimura Tsunehiko , Yoshioka Jun , Kusuoka Hideo
Published: September 2007

Abstract
—To elucidate the role of intracellular Na
+
kinetics in the mechanism for ischemic preconditioning (IPC), we measured intracellular Na
+
concentration ([Na
+
]
i
) using
23
Na–magnetic resonance spectroscopy in isolated rat hearts. IPC significantly delayed the initial [Na
+
]
i
increase (d[Na
+
]
i
/dt) compared with non-IPC control, resulting in attenuation of Na
+
accumulation (Δ[Na
+
]
i
) during 27 minutes of ischemia with better functional recovery. [Na
+
]
i
in IPC, but not in control, recovered to preischemic level during a 6-minute reperfusion. The Na
+
-H
+
exchange inhibitor further suppressed d[Na
+
]
i
/dt in both control and IPC hearts with concomitant improvement of functional recovery, suggesting little contribution to the mechanism of IPC. The mitochondrial ATP-sensitive K
+
(mito K
ATP
) channel activator diazoxide (30 μmol/L) completely mimicked both [Na
+
]
i
kinetics and functional recovery in IPC without any additive effects to IPC. The mito K
ATP
channel blocker 5-hydroxydecanoic acid (100 μmol/L) lost protective effect as well as the attenuation of d[Na
+
]
i
/dt and [Na
+
]
i
recovery induced by diazoxide. However, 5-hydroxydecanoic acid also lost IPC-induced protection, but incompletely abolished the alteration of d[Na
+
]
i
/dt and the [Na
+
]
i
recovery. The Na
+
/K
+
-ATPase inhibitor ouabain (200 μmol/L) did not change d[Na
+
]
i
/dt in non-IPC hearts, but it abolished the IPC- or diazoxide-induced reduction of d[Na
+
]
i
/dt and the [Na
+
]
i
recovery, whereas IPC followed by ouabain treatment showed partial functional recovery with smaller Δ[Na
+
]
i
than other ouabain groups. In conclusion, alteration of Na
+
kinetics by preserving Na
+
efflux via Na
+
/K
+
-ATPase mediated by mito K
ATP
channel activation mainly contributes to functional protection in IPC hearts. The contribution of mito K
ATP
channel–independent pathway relating to Na
+
kinetics including reduced Na
+
influx is limited in functional protection of IPC.
Contraction and Intracellular Ca
2+
Handling in Isolated Skeletal Muscle of Rats With Congestive Heart Failure
Authors: Lunde Per Kristian , Dahlstedt Anders J. , Bruton Joseph D. , Lännergren Jan , Thorén Peter , Sejersted Ole M. , Westerblad Håkan
Published: September 2007

Abstract
—A decreased exercise tolerance is a common symptom in patients with congestive heart failure (CHF). This decrease has been suggested to be partly due to altered skeletal muscle function. Therefore, we have studied contractile function and cytoplasmic free Ca
2+
concentration ([Ca
2+
]
i
, measured with the fluorescent dye indo 1) in isolated muscles from rats in which CHF was induced by ligation of the left coronary artery. The results show no major changes of the contractile function and [Ca
2+
]
i
handling in unfatigued intact fast-twitch fibers isolated from flexor digitorum brevis muscles of CHF rats, but these fibers were markedly more susceptible to damage during microdissection. Furthermore, CHF fibers displayed a marked increase of baseline [Ca
2+
]
i
during fatigue. Isolated slow-twitch soleus muscles of CHF rats displayed slower twitch contraction and tetanic relaxation than did muscles from sham-operated rats; the slowing of relaxation became more pronounced during fatigue in CHF muscles. Immunoblot analyses of sarcoplasmic reticulum proteins and sarcolemma Na
+
,K
+
-ATPase showed no difference in flexor digitorum brevis muscles of sham-operated versus CHF rats. In conclusion, functional impairments can be observed in limb muscle isolated from rats with CHF. These impairments seem to mainly involve structures surrounding the muscle cells and sarcoplasmic reticulum Ca
2+
pumps, the dysfunction of which becomes obvious during fatigue.
Mitochondrial ATP-Sensitive Potassium Channels Inhibit Apoptosis Induced by Oxidative Stress in Cardiac Cells
Authors: Akao Masaharu , Ohler Andreas , O’Rourke Brian , Marbán Eduardo
Published: September 2007

Abstract
—Mitochondria can either enhance or suppress cell death. Cytochrome
c
release from mitochondria and depolarization of the mitochondrial membrane potential (ΔΨ) are crucial events in triggering apoptosis. In contrast, activation of mitochondrial ATP-sensitive potassium (mitoK
ATP
) channels prevents lethal ischemic injury in vivo, implicating these channels as key players in the process of ischemic preconditioning. We probed the relationship between mitoK
ATP
channels and apoptosis in cultured neonatal rat cardiac ventricular myocytes. Incubation with 200 μmol/L hydrogen peroxide induced TUNEL positivity, cytochrome
c
translocation, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and dissipation of ΔΨ. Pharmacological opening of mitoK
ATP
channels by diazoxide (100 μmol/L) preserved mitochondrial integrity and suppressed all markers of apoptosis. Diazoxide prevented ΔΨ depolarization in a concentration-dependent manner (EC
50
≈40 μmol/L, with saturation by 100 μmol/L), as shown by both flow cytometry and quantitative image analysis of cells stained with fluorescent ΔΨ indicators. These cytoprotective effects of diazoxide were reproduced by pinacidil, another mitoK
ATP
agonist, and blocked by the mitoK
ATP
channel antagonist 5-hydroxydecanoate (500 μmol/L). Our findings identify a novel mitochondrial pathway that is protective against apoptosis. The results also pinpoint mitoK
ATP
channels as logical therapeutic targets in diseases of enhanced apoptosis and oxidative stress.
Novel Mechanism for Brugada Syndrome
Authors: Baroudi Ghayath , Pouliot Valerie , Denjoy Isabelle , Guicheney Pascale , Shrier Alvin , Chahine Mohamed
Published: September 2007

Abstract
—The
SCN5A
gene encodes the α subunit of the human heart sodium channel (hH1), which plays a critical role in cardiac excitability. Mutations of
SCN5A
underlie Brugada syndrome, an inherited disorder that leads to ventricular fibrillation and sudden death. This study describes changes in cellular localization and functional expression of hH1 in a naturally occurring
SCN5A
mutation (R1432G) reported for Brugada syndrome. Using patch-clamp experiments, we show that there is an abolition of functional hH1 expression in R1432G mutants expressed in human tsA201 cells but not in
Xenopus
oocytes. In tsA201 cells, a conservative positively charged mutant, R1432K, produced sodium currents with normal gating properties, whereas other mutations at this site abolished functional sodium channel expression. Immunofluorescent staining and confocal microscopy showed that the wild-type α subunit expressed in tsA201 cells was localized to the cell surface, whereas the R1432G mutant was colocalized with calnexin within the endoplasmic reticulum. The β
1
subunit was also localized to the cell surface in the presence of the α subunit; however, in its absence, the β
1
subunit was restricted to a perinuclear localization. These results demonstrate that the disruption of
SCN5A
cell-surface localization is one mechanism that can account for the loss of functional sodium channels in Brugada syndrome. The full text of this article is available at http://www.circresaha.org.
Adrenomedullin Induces Endothelium-Dependent Vasorelaxation via the Phosphatidylinositol 3-Kinase/Akt–Dependent Pathway in Rat Aorta
Authors: Nishimatsu Hiroaki , Suzuki Etsu , Nagata Daisuke , Moriyama Nobuo , Satonaka Hiroshi , Walsh Kenneth , Sata Masataka , Kangawa Kenji , Matsuo Hisayuki , Goto Atsuo , Kitamura Tadaichi , Hirata Yasunobu
Published: September 2007

Abstract
—To study the mechanisms by which adrenomedullin (AM) induces endothelium-dependent vasorelaxation, we examined whether AM-induced endothelium-dependent vasodilation was mediated by the phosphatidylinositol 3-kinase (PI3K)/Akt-dependent pathway in rat aorta, because it was recently reported that PI3K/Akt was implicated in the activation of endothelial NO synthase. AM-induced vasorelaxation in thoracic aorta with intact endothelium was inhibited by pretreatment with PI3K inhibitors to the same level as that in endothelium-denuded aorta. AM elicited Akt phosphorylation in a time- and dose-dependent manner. AM-induced Akt phosphorylation was inhibited by pretreatment with a calmodulin-dependent protein kinase inhibitor as well as with PI3K inhibitors. When an adenovirus construct expressing a dominant-negative Akt mutant (Ad/dnAkt) was injected into abdominal aortas so that the mutant was expressed predominantly in the endothelium layer, AM-induced vasodilation was diminished to the same level as that in endothelium-denuded aortas. Finally, AM-induced cGMP production, which was used as an indicator for NO production, was suppressed by PI3K inhibition or by Ad/dnAkt infection into the endothelium. These results suggested that AM induced Akt activation in the endothelium via the Ca
2+
/calmodulin-dependent pathway and that this was implicated in the production of NO, which in turn induced endothelium-dependent vasodilation in rat aorta.
Role for Tissue Factor Pathway in Murine Model of Vascular Remodeling
Authors: Singh Ripudamanjit , Pan Shuchong , Mueske Cheryl S. , Witt Tyra , Kleppe Laurel S. , Peterson Timothy E. , Slobodova Adriana , Chang Jen-Yea , Caplice Noel M. , Simari Robert D.
Published: September 2007

Abstract
—Tissue factor (TF) is a low-molecular-weight glycoprotein that initiates the extrinsic clotting cascade and is considered a major regulator of arterial thrombogenicity. TF pathway inhibitor (TFPI) is a major physiological inhibitor of TF-initiated coagulation. The aim of this study was to define the complex interplay between TF and TFPI and the regulation of vascular thrombogenicity in a model of vascular remodeling. To determine the levels and pattern of vascular expression of TF and TFPI associated with vascular remodeling, a murine model of flow cessation was studied. TF activity of the arteries increased after ligation (
P
<0.05). Quantitative analysis of homogenates of remodeled carotid arteries revealed increased TF expression but unchanged TFPI expression compared with normal carotid arteries, resulting in enhanced TF activity. To determine the potential therapeutic role of TFPI in this thrombogenic state, mice were treated with intravascular adenoviral delivery of either murine TFPI (Ad-mTFPImyc) or a control adenovirus (Ad-ΔE1). Overexpression of TFPI decreased vascular TF activity compared with viral control (
P
<0.01). Overexpression of TFPI inhibited neointimal formation (
P
=0.038), resulting in enhanced luminal area (
P
=0.001) 4 weeks after flow cessation. In this murine model of vascular remodeling, an imbalance between TF and TFPI expression is generated, resulting in increased TF activity. Overexpression of TFPI in this model inhibits vascular TF activity and results in attenuation of vascular remodeling associated with flow interruption.
Temporally Regulated and Tissue-Specific Gene Manipulations in the Adult and Embryonic Heart Using a Tamoxifen-Inducible Cre Protein
Authors: Sohal Dawinder S. , Nghiem Mai , Crackower Michael A. , Witt Sandra A. , Kimball Thomas R. , Tymitz Kevin M. , Penninger Josef M. , Molkentin Jeffery D.
Published: September 2007

Abstract
—The advent of conditional and tissue-specific recombination systems in gene-targeted or transgenic mice has permitted an assessment of single gene function in a temporally regulated and cell-specific manner. Here we generated transgenic mice expressing a tamoxifen-inducible Cre recombinase protein fused to two mutant estrogen-receptor ligand-binding domains (MerCreMer) under the control of the α-myosin heavy chain promoter. These transgenic mice were crossed with the
ROSA26 lacZ-flox
–targeted mice to examine Cre recombinase activity and the fidelity of the system. The data demonstrate essentially no Cre-mediated recombination in the embryonic, neonatal, or adult heart in the absence of inducing agent but >80% recombination after only four tamoxifen injections. Expression of the MerCreMer fusion protein within the adult heart did not affect cardiac performance, cellular architecture, or expression of hypertrophic marker genes, demonstrating that the transgene-encoded protein is relatively innocuous. In summary, MerCreMer transgenic mice represent a tool for temporally regulated inactivation of any loxP-targeted gene within the developing and adult heart or for specifically directing recombination and expression of a loxP-inactivated cardiac transgene in the heart.
Expression of R120G–αB-Crystallin Causes Aberrant Desmin and αB-Crystallin Aggregation and Cardiomyopathy in Mice
Authors: Wang Xuejun , Osinska Hanna , Klevitsky Raisa , Gerdes A. Martin , Nieman Michelle , Lorenz John , Hewett Timothy , Robbins Jeffrey
Published: September 2007

Abstract
—Upregulation of αB-crystallin (CryAB), a small heat shock protein, is associated with a variety of diseases, including the desmin-related myopathies. CryAB, which binds to both desmin and cytoplasmic actin, may participate as a chaperone in intermediate filament formation and maintenance, but the physiological consequences of CryAB upregulation are unknown. A mutation in CryAB, R120G, has been linked to a familial desminopathy. However, it is unclear whether the mutation is directly causative. We created multiple transgenic mouse lines that overexpressed either murine wild-type CryAB or the R120G mutation in cardiomyocytes. Overexpression of wild-type CryAB was relatively benign, with no increases in mortality and no induction of desmin-related cardiomyopathy even in a line in which CryAB mRNA expression was increased ≈104-fold and the protein level increased by 11-fold. In contrast, lines expressing the R120G mutation were compromised, with a high-expressing line exhibiting 100% mortality by early adulthood. Modest expression levels resulted in a phenotype that was strikingly similar to that observed for the desmin-related cardiomyopathies. The desmin filaments in the cardiomyocytes were overtly affected, myofibril alignment was significantly impaired, and a hypertrophic response occurred at both the molecular and cellular levels. The data show that the R120G mutation causes a desminopathy, is dominant negative, and results in cardiac hypertrophy.
Cyclic ADP-Ribose Is the Primary Trigger for Hypoxic Pulmonary Vasoconstriction in the Rat Lung In Situ
Authors: Dipp Michelle , Evans A. Mark
Published: September 2007

Abstract
—Hypoxic pulmonary vasoconstriction (HPV) is unique to pulmonary arteries, and it aids ventilation/perfusion matching. However, in diseases such as emphysema, HPV can promote hypoxic pulmonary hypertension. We recently showed that hypoxia constricts pulmonary arteries in part by increasing cyclic ADP-ribose (cADPR) accumulation in the smooth muscle and, thereby, Ca
2+
release by ryanodine receptors. We now report on the role of cADPR in HPV in isolated rat pulmonary arteries and in the rat lung in situ. In isolated pulmonary arteries, the membrane-permeant cADPR antagonist, 8-bromo-cADPR, blocked sustained HPV by blocking Ca
2+
release from smooth muscle ryanodine-sensitive stores in the sarcoplasmic reticulum. Most importantly, we showed that 8-bromo-cADPR blocks HPV induced by alveolar hypoxia in the ventilated rat lung in situ. Inhibition of HPV was achieved without affecting (1) constriction by membrane depolarization and voltage-gated Ca
2+
influx, (2) the release (by hypoxia) of an endothelium-derived vasoconstrictor, or (3) endothelium-dependent vasoconstriction. Our findings suggest that HPV is both triggered and maintained by cADPR in the rat lung in situ.
Tumor Necrosis Factor-α Induces Fibronectin Synthesis in Coronary Artery Smooth Muscle Cells by a Nitric Oxide–Dependent Posttranscriptional Mechanism
Authors: O’Blenes Catherine A. E. , Kinnear Caroline , Rabinovitch Marlene
Published: September 2007

Abstract
—Postcardiac transplant coronary arteriopathy is associated with tumor necrosis factor-α (TNF-α) induction of fibronectin-dependent smooth muscle cell (SMC) migration into the subendothelium, resulting in occlusive neointimal formation. Because expression of inducible nitric oxide synthase (iNOS) is elevated in neointimal formation after transplantation and upregulated in vascular SMCs by TNF-α, we investigated whether TNF-α induction of fibronectin synthesis in coronary artery (CA) SMCs is mediated by nitric oxide (NO). TNF-α caused a dose-dependent increase in reactive oxygen and nitrogen intermediates in CA SMCs (
P
<0.05). This correlated with increased NO production (
P
<0.05) and fibronectin synthesis (
P
<0.05). TNF-α induction of fibronectin synthesis was abrogated by the NOS inhibitor
N
G
-monomethyl-
l
-arginine (L-NMMA) (
P
<0.05) or the flavonoid-containing enzyme inhibitor diphenyleneiodonium (DPI) (
P
<0.05) and reproduced with the NO donor
S
-nitroso-
N
-acetyl-penicillamine (SNAP) (
P
<0.05). Northern blotting showed no effect of TNF-α on steady-state fibronectin mRNA levels. TNF-α increased expression of light chain 3 (LC-3), a protein shown previously to facilitate fibronectin mRNA translation through its interaction with an adenosine-uracil rich element (ARE) in the 3′-untranslated region of fibronectin mRNA. RNA gel mobility shift and UV cross-linking assays using CA SMC lysates revealed protein binding complexes with radiolabeled oligonucleotide containing the ARE, similar to those generated with recombinant LC-3. One of these complexes increased after TNF-α treatment, an effect inhibited with L-NMMA or DPI. These data demonstrate a novel paradigm whereby cytokines regulate mRNA translation of extracellular matrix proteins through NO-dependent modulation of RNA binding protein interaction with mRNA.
UT-A Urea Transporter Protein in Heart
Authors: Duchesne Rafael , Klein Janet D. , Velotta Jeffrey B. , Doran John J. , Rouillard Patricia , Roberts Brian R. , McDonough Alicia A. , Sands Jeff M.
Published: September 2007

Urea transporters have been cloned from kidney medulla (UT-A) and erythrocytes (UT-B). We determined whether UT-A proteins could be detected in heart and whether their abundance was altered by uremia or hypertension or in human heart failure. In normal rat heart, bands were detected at 56, 51, and 39 kDa. In uremic rats, the abundance of the 56-kDa protein increased 1.9-fold compared with pair-fed, sham-operated rats, whereas the 51- and 39-kDa proteins were unchanged. We also detected UT-A2 mRNA in hearts from control and uremic rats. Because uremia is accompanied by hypertension, the effects of hypertension per se were studied in uninephrectomized deoxycorticosterone acetate salt–treated rats, where the abundance of the 56-kDa protein increased 2-fold versus controls, and in angiotensin II–infused rats, where the abundance of the 56 kDa protein increased 1.8-fold versus controls. The 51- and 39-kDa proteins were unchanged in both hypertensive models. In human left ventricle myocardium, UT-A proteins were detected at 97, 56, and 51 kDa. In failing left ventricle (taken at transplant, New York Heart Association class IV), the abundance of the 56-kDa protein increased 1.4-fold, and the 51-kDa protein increased 4.3-fold versus nonfailing left ventricle (donor hearts). We conclude that (1) multiple UT-A proteins are detected in rat and human heart; (2) the 56-kDa protein is upregulated in rat heart in uremia or models of hypertension; and (3) the rat results can be extended to human heart, where 56- and 51-kDa proteins are increased during heart failure.
High Glucose Impairs Voltage-Gated K
+
Channel Current in Rat Small Coronary Arteries
Authors: Liu Yanping , Terata Ken , Rusch Nancy J. , Gutterman David D.
Published: September 2007

Hyperglycemia is associated with impaired endothelium-dependent dilation that is due to quenching of NO by superoxide (O
2
· −
). In small coronary arteries (CAs), dilation depends more on smooth muscle hyperpolarization, such as that mediated by voltage-gated K
+
(Kv) channels. We determined whether high glucose enhances O
2
·−
production and reduces microvascular Kv channel current and functional responses. CAs from Sprague-Dawley rats were incubated 24 hours in medium containing either normal glucose (NG, 5.5 mmol/L
d
-glucose), high glucose (HG, 23 mmol/L
d
-glucose), or
l
-glucose (LG, 5.5 mmol/L
d
-glucose and 17 mmol/L
l
-glucose). O
2
·−
production was increased in HG arteries. Whole-cell patch clamping showed a reduction of 4-aminopyridine (4-AP)–sensitive current (Kv current) from smooth muscle cells of HG CAs versus NG CAs or versus LG CAs (peak density was 9.95±5.3 pA/pF for HG versus 27.8±6.8 pA/pF for NG and 28.5±5.2 pA/pF for LG;
P
<0.05). O
2
·−
generation (xanthine+xanthine oxidase) decreased K
+
current density, with no further reduction by 4-AP. Partial restoration was observed with superoxide dismutase and catalase. Constriction to 3 mmol/L 4-AP was reduced in vessels exposed to HG (13±5%,
P
<0.05) versus NG (30±7%) or LG (34±4%). Responses to KCl and nifedipine were not different among groups. Superoxide dismutase and catalase increased contraction to 4-AP in HG CAs. This is the first direct evidence that exposure of CAs to HG impairs Kv channel activity. We speculate that this O
2
·−
-induced impairment may reduce vasodilator responsiveness in the coronary circulation of subjects with coronary disease or its risk factors.
Genetic Locus in Mice That Blocks Development of Atherosclerosis Despite Extreme Hyperlipidemia
Authors: Mehrabian Margarete , Wong Jack , Wang Xuping , Jiang Zhiming , Shi Weibin , Fogelman Alan M. , Lusis Aldons J.
Published: September 2007

The genes contributing to the common forms of atherosclerosis are largely unknown. One approach to dissecting complex traits such as atherosclerosis is to use animal models, such as the mouse, to map and characterize the genetic loci involved. We now report the identification of a locus for aortic lesion formation on mouse chromosome 6 that exhibits a highly significant lod score of 6.7 in a genetic cross between the susceptible strain, C57BL/6J, and the resistant strain, CAST/Ei. The locus was confirmed by constructing a congenic strain in which the chromosome 6 segment from CAST/Ei was transferred to a C57BL/6J background in a series of backcrosses. The congenic strain was almost completely resistant to diet-induced atherosclerosis. The chromosome 6 segment was also transferred onto the background of an LDL receptor–null mutation and resulted again in almost complete resistance to aortic lesion formation. This locus also influenced insulin levels but did not affect plasma lipoprotein levels, blood pressure, or body fat. The chromosome 6 gene, which we call
Artles
(for arterial lesions), did not affect endothelial cell responses to oxidized LDL, but lesion formation was partially reduced through bone marrow transplantation. The locus contains the candidate gene peroxisome proliferator–activated receptor-γ, and the congenic mice exhibited significantly reduced expression of peroxisome proliferator–activated receptor-γ.
Impaired Hyperpolarization in Regenerated Endothelium After Balloon Catheter Injury
Authors: Köhler Ralf , Brakemeier Susanne , Kühn Meike , Behrens Claudia , Real Robert , Degenhardt Christiane , Orzechowski Hans-Dieter , Pries Axel R. , Paul Martin , Hoyer Joachim
Published: September 2007

Ca
2+
-activated K
+
(K
Ca
) channels control endothelial Ca
2+
homeostasis and the formation of vasodilators. After angioplasty, dysfunction of the regenerated endothelium leads to abnormal vasoregulation. In this study, we tested the expression and function of K
Ca
channels in regenerated endothelium at 6 weeks after balloon catheter injury of rat carotid arteries (CAs) by using single-cell reverse transcription–polymerase chain reaction, patch-clamp techniques, and analysis of vasoreactivity. In single regenerated endothelial cells (ECs), the percentage of ECs expressing the K
Ca
genes, rSK3 (12±8%) and rIK1 (22±9%), was significantly lower compared with the percentage of native ECs expressing these genes (rSK3 58±8%, rIK1 64±10%). In patch-clamp experiments, K
Ca
currents and acetylcholine-induced hyperpolarization were markedly reduced in regenerated ECs (shift of membrane potential −6±3 mV) compared with those in native ECs (shift of membrane potential −21±5 mV). In pressure myograph experiments, acetylcholine-induced dilation was impaired in reendothelialized CAs compared with normal CAs. Intraluminal application of the K
Ca
blocker apamin and charybdotoxin inhibited dilation by 30% in normal CAs but was without effect in reendothelialized CAs. Intraluminal application of 1-ethyl-2-benzimidazolinone (100 μmol/L), an opener of K
Ca
channels, evoked dilation by 29% in normal CAs but had no effect in reendothelialized CAs. In conclusion, the impaired expression of K
Ca
channels in regenerated endothelium results in defective hyperpolarization and impaired dilation. Thus, the impaired K
Ca
channel function contributes to functional alterations of regenerated endothelium after angioplasty.