bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrin-Mediated Mechanotransduction in Vascular Smooth Muscle Cells
Authors: Goldschmidt Marc E. , McLeod Kenneth J. , Taylor W. Robert
Published: September 2007

Abstract
—Blood vessels are continuously exposed to mechanical forces that lead to adaptive remodeling and atherosclerosis. Although there have been many studies characterizing the responses of vascular cells to mechanical stimuli, the precise mechanical characteristics of the forces applied to cells to elicit these responses are not clear. We designed a magnetic exposure system capable of producing a defined normal force on ferromagnetic beads that are specifically bound to cultured cells coated with extracellular matrix proteins or integrin-specific antibodies. Rat aortic smooth muscle cells were incubated with engineered fibronectin–coated ferromagnetic beads and then exposed to a magnetic field. With activation of extracellular signal–regulated mitogen-activated protein kinase 1/2 (ERK 1/2
MAPK
) used as a prototypical marker for cell responsiveness to mechanical forces, Western blot analysis demonstrated an increase in phosphorylated ERK 1/2
MAPK
expression reaching a maximal response of a 3.5-fold increase at a total force of ≈2.5 pN per cell. The peak response occurred after 5 minutes of exposure and slowly decreased to baseline after 30 minutes. A cyclic, rather than static, force was required for this activation, and the frequency-response curve increased ≈2-fold between 0.5 and 2.0 Hz. Vitronectin- and β
3
antibody–coated beads showed a response nearly identical to those coated with engineered fibronectin, whereas forces applied to beads coated with α
2
and β
1
antibodies did not significantly activate ERK 1/2
MAPK
. Mechanical activation of the ERK 1/2
MAPK
system in rat aortic smooth muscle cells occurs through specific integrin receptors and requires a cyclic force with a magnitude estimated to be in the piconewton range.
Adaptive Mechanisms That Preserve Cardiac Function in Mice Without Myoglobin
Authors: Meeson Annette P. , Radford Nina , Shelton John M. , Mammen Pradeep P. A. , DiMaio J. Michael , Hutcheson Kelley , Kong Yanfeng , Elterman Joel , Williams R. Sanders , Garry Daniel J.
Published: September 2007

Abstract
—Mice lacking myoglobin survive to adulthood and meet the circulatory demands of exercise and pregnancy without cardiac decompensation. In the present study, we show that many myoglobin-deficient embryos die in utero at midgestation with signs of cardiac failure. Fetal mice that survive to gestational day 12.5, however, suffer no subsequent excess mortality. Survival in the absence of myoglobin is associated with increased vascularity and the induction of genes encoding the hypoxia-inducible transcription factors 1α and 2, stress proteins such as heat shock protein 27, and vascular endothelial growth factor. These adaptations are evident in late fetal life, persist into adulthood, and are sufficient to maintain normal myocardial oxygen consumption during stressed conditions. These data reveal that myoglobin is necessary to support cardiac function during development, but adaptive responses evoked in some animals can fully compensate for the defect in cellular oxygen transport resulting from the loss of myoglobin.
Microtubule Disruption by Colchicine Reversibly Enhances Calcium Signaling in Intact Rat Cardiac Myocytes
Authors: Kerfant B. G. , Vassort G. , Gómez A. M.
Published: September 2007

Abstract
—Using the whole-cell patch-clamp configuration in rat ventricular myocytes, we recently reported that microtubule disruption increases calcium current (
I
Ca
) and [Ca
2+
]
i
transient and accelerates their kinetics by adenylyl cyclase activation. In the present report, we further analyzed the effects of microtubule disruption by 1 μmol/L colchicine on Ca
2+
signaling in cardiac myocytes with intact sarcolemma. In quiescent intact cells, it is possible to investigate ryanodine receptor (RyR) activity by analyzing the characteristics of spontaneous Ca
2+
sparks. Colchicine treatment decreased Ca
2+
spark amplitude (F/F
0
: 1.78±0.01, n=983, versus 1.64±0.01, n=1660, recorded in control versus colchicine-treated cells;
P
<0.0001) without modifying the sarcoplasmic reticulum Ca
2+
load and enhanced their time to peak (in ms: 6.85±0.09, n=1185, versus 7.33±0.13, n=1647;
P
<0.0001). Microtubule disruption also induced the appearance of Ca
2+
sparks in doublets. These alterations may reflect RyR phosphorylation. To further investigate Ca
2+
signaling in cardiac myocytes with intact sarcolemma, we analyzed [Ca
2+
]
i
transient evoked by field stimulation. Cells were loaded with the fluorescence Ca
2+
indicator, Fluo-3 cell permeant, and stimulated at 1 Hz. [Ca
2+
]
i
transient amplitude was greater and its decay was accelerated in colchicine-treated, field-stimulated myocytes. This effect is reversible. When colchicine-treated myocytes were placed in a colchicine-free solution for 30 minutes, tubulin was repolymerized into microtubules, as shown by immunofluorescence, and the increase in [Ca
2+
]
i
transient was reversed. In summary, we demonstrate that microtubule disruption by colchicine reversibly modulates Ca
2+
signaling in cardiac cells with intact sarcolemma. The full text of this article is available at http://www.circresaha.org.
Macrophage p53 Deficiency Leads to Enhanced Atherosclerosis in APOE*3-Leiden Transgenic Mice
Authors: van Vlijmen Bart J.M. , Gerritsen Gery , Franken Arthur L. , Boesten Lianne S.M. , Kockx Mark M. , Gijbels Marion J. , Vierboom Michel P. , van Eck Miranda , van de Water Bob , van Berkel Theo J.C. , Havekes Louis M.
Published: September 2007

Abstract
—Cell proliferation and cell death (either necrosis or apoptosis) are key processes in the progression of atherosclerosis. The tumor suppressor gene p53 is an essential gene in cell proliferation and cell death and is upregulated in human atherosclerotic plaques, both in smooth muscle cells and in macrophages. In the present study, we investigated the importance of macrophage p53 in the progression of atherosclerosis using bone marrow transplantation in APOE*3-Leiden transgenic mice, an animal model for human-like atherosclerosis. APOE*3-Leiden mice were lethally irradiated and reconstituted with bone marrow derived from either p53-deficient (
p53
−/−
) or control (
p53
+/+
) donor mice. Reconstitution of mice with
p53
−/−
bone marrow did not result in any hemopoietic abnormalities as compared with
p53
+/+
transplanted mice. After 12 weeks on an atherogenic diet, APOE*3-Leiden mice reconstituted with
p53
−/−
bone marrow showed a significant (
P
=0.006) 2.3-fold increase in total atherosclerotic lesion area as compared with mice reconstituted with
p53
+/+
bone marrow. Although likely a secondary effect of the increased lesion area,
p53
−/−
transplanted mice also showed significantly more lesion necrosis (necrotic index, 1.1±1.3 versus 0.2±0.7;
P
=0.04) and lesion macrophages (macrophage area, 79.9±40.0 versus 39.7±27.3×10
3
μm
2
per section;
P
=0.02). These observations coincided with a tendency toward decreased apoptosis (terminal deoxynucleotidyl transferase end-labeling [TUNEL]–positive nuclei going from 0.42±0.39 to 0.14±0.15%,
P
=0.071), whereas the number of proliferating cells (5′-bromo-2′-deoxyuridine–positive nuclei) was not affected (3.75±0.98 versus 4.77±2.30%;
P
=0.59). These studies indicate that macrophage p53 is important in suppressing the progression of atherosclerosis and identify a novel therapeutic target for regulating plaque stability.
β-Adrenergic Stimulation Synchronizes Intracellular Ca
2+
Release During Excitation-Contraction Coupling in Cardiac Myocytes
Authors: Song Long-Sheng , Wang Shi-Qiang , Xiao Rui-Ping , Spurgeon Harold , Lakatta Edward G. , Cheng Heping
Published: September 2007

Abstract
—To elucidate microscopic mechanisms underlying the modulation of cardiac excitation-contraction (EC) coupling by β-adrenergic receptor (β-AR) stimulation, we examined local Ca
2+
release function, ie, Ca
2+
spikes at individual transverse tubule–sarcoplasmic reticulum (T-SR) junctions, using confocal microscopy and our recently developed technique for release flux measurement. β-AR stimulation by norepinephrine plus an α
1
-adrenergic blocker, prazosin, increased the amplitude of SR Ca
2+
release flux (J
SR
), its running integral (∫J
SR
), and L-type Ca
2+
channel current (
I
Ca
), and it shifted their bell-shaped voltage dependence leftward by ≈10 mV, with the relative effects ranking
I
Ca
> J
SR
>∫J
SR
. Confocal imaging revealed that the bell-shaped voltage dependence of SR Ca
2+
release is attributable to a graded recruitment of T-SR junctions as well as to changes in Ca
2+
spike amplitudes. β-AR stimulation increased the fractional T-SR junctions that fired Ca
2+
spikes and augmented Ca
2+
spike amplitudes, without altering the SR Ca
2+
load, suggesting that more release units were activated synchronously among and within T-SR junctions. Moreover, β-AR stimulation decreased the latency and temporal dispersion of Ca
2+
spike occurrence at a given voltage, delivering most of the Ca
2+
at the onset of depolarization rather than spreading it out throughout depolarization. Because the synchrony of Ca
2+
spikes affects Ca
2+
delivery per unit of time to contractile myofilaments, and because the myofilaments display a steep Ca
2+
dependence, our data suggest that synchronization of SR Ca
2+
release represents a heretofore unappreciated mechanism of β-AR modulation of cardiac inotropy.
Cardiac Na
+
-Ca
2+
Exchange
Authors: Shigekawa Munekazu , Iwamoto Takahiro
Published: September 2007

Abstract
—The Na
+
-Ca
2+
exchanger (NCX) is one of the essential regulators of Ca
2+
homeostasis in cardiomyocytes and thus an important modulator of the cardiac contractile function. The purpose of this review is to survey recent advances in cardiac NCX research, with particular emphasis on molecular and pharmacological aspects. The NCX function is thought to be regulated by a variety of cellular factors. However, data obtained by use of different experimental systems often appear to be in conflict. Where possible, we endeavor to provide a rational interpretation of such data. We also provide a summary of current work relating to the structure and function of the cardiac NCX. Recent molecular studies of the NCX protein are beginning to shed light on structural features of the ion translocation pathway in the NCX membrane domain, which seems likely to be formed, at least partly, by the phylogenetically conserved α-1 and α-2 repeat structures and their neighboring membrane-spanning segments. Finally, we discuss new classes of NCX inhibitors with improved selectivity. One of these, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), appears to exhibit unique selectivity for Ca
2+
-influx–mode NCX activity. Data obtained with these inhibitors should provide a basis for designing more selective and clinically useful drugs targeting NCX.
Exaggerated Left Ventricular Dilation and Reduced Collagen Deposition After Myocardial Infarction in Mice Lacking Osteopontin
Authors: Trueblood Nathan A. , Xie Zhonglin , Communal Catherine , Sam Flora , Ngoy Soeun , Liaw Lucy , Jenkins Alan W. , Wang Jing , Sawyer Douglas B. , Bing Oscar H. L. , Apstein Carl S. , Colucci Wilson S. , Singh Krishna
Published: September 2007

Abstract
—Osteopontin (OPN), an extracellular matrix protein, is expressed in the myocardium with hypertrophy and failure. We tested the hypothesis that OPN plays a role in left ventricular (LV) remodeling after myocardial infarction (MI). Accordingly, OPN expression and LV structural and functional remodeling were determined in wild-type (WT) and OPN knockout (KO) mice 4 weeks after MI. Northern analysis showed increased OPN expression in the infarcted region, peaking 3 days after MI and gradually decreasing over the next 28 days. In the remote LV, OPN expression was biphasic, with peaks at 3 and 28 days. In situ hybridization and immunohistochemical analyses showed increased OPN mRNA and protein primarily in the interstitium. Infarct size, heart weight, and survival were similar in KO and WT mice after MI (
P
=NS), whereas the lung wet weight/dry weight ratio was increased in the KO mice (
P
<0.005 versus sham-operated mice). Peak LV developed pressure was reduced to a similar degree after MI in the KO and WT mice. The number of terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL)-positive myocytes was similar in KO and WT mice after MI. In contrast, post-MI LV chamber dilation was approximately twice as great in KO versus WT mice (
P
<0.001). Myocyte length increased after MI in WT mice (
P
<0.001) but not in KO mice. Electron microscopy showed increased collagen content in WT mice after MI but not in KO mice after MI. Type I collagen content was increased ≈3-fold and ≈7-fold in remote and infarcted regions, respectively, of WT hearts after MI but not in KO hearts (
P
<0.01 versus WT hearts). Likewise, Northern analyses showed increased collagen I(α
1
) mRNA after MI in remote regions of WT hearts but not in KO hearts. Thus, increased OPN expression plays an important role in regulating post-MI LV remodeling, at least in part, by promoting collagen synthesis and accumulation.
Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor Differentially Modulate Early Postnatal Coronary Angiogenesis
Authors: Tomanek Robert J. , Sandra Alexander , Zheng Wei , Brock Tommy , Bjercke Robert J. , Holifield Jennifer S.
Published: September 2007

Abstract
—The roles of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF [FGF-2]) in early postnatal regulation of coronary angiogenesis were investigated by administering neutralizing antibodies to these growth factors between postnatal days 5 and 12. Immunohistochemistry and Western blotting both revealed decreases in VEGF protein in the hearts of rats treated with either antibody. In contrast, bFGF mRNA increased in both treated groups, whereas VEGF mRNA was unchanged. Using stereological assessment of perfusion-fixed hearts, we found that both anti-VEGF and anti-bFGF inhibited the rapid and marked capillary growth that occurs during this time period and that the effects of the two neutralizing antibodies are not additive. Arteriolar growth, as indicated by a lower length density, was inhibited by anti-bFGF, but not anti-VEGF. When both anti-VEGF and anti-bFGF were administered, arteriolar length density was not significantly lower, but the population of small arterioles (<15 μm) was markedly reduced, whereas the percentage of large arterioles (26 to 50 μm) more than doubled. Thus, inhibition of both growth factors negated or limited the formation of small arterioles and facilitated an expansion of the largest arterioles. These in vivo data are the first to document that during the early neonatal period, (1) both VEGF and bFGF modulate capillary growth, (2) bFGF facilitates arteriolar growth, and (3) the two growth factors interact to establish the normal hierarchy of the arteriolar tree.
Integrins and the Myocardium
Authors: Ross Robert S. , Borg Thomas K.
Published: September 2007

Abstract
—Extracellular matrix provides a structural, chemical, and mechanical substrate that is essential in cardiac development, growth, and responses to pathophysiological signals. Transmembrane receptors termed integrins provide a dynamic interaction of environmental cues and intracellular events. Integrins orchestrate multiple functions in the intact organism including organogenesis, regulation of gene expression, cell proliferation, differentiation, migration, and death. They are expressed in all cellular components of the cardiovascular system, including the vasculature, blood, cardiac myocytes and nonmuscle cardiac cells. The focus of this review will be on the role of integrins in the myocardium. We will provide background on integrin structure and function, discuss how the expression of integrins is critical to the form and function of the developing and postnatal myocardium, and review the known data on integrins as signaling molecules in the heart. Finally, we will offer insights to the future research directions into this important family of extracellular matrix receptors in the myocardium.
Circulation Research
Authors: Katz Arnold M.
Published: September 2007

Abstract
—
Circulation Research
, first published in 1953, was created by the American Heart Association as “the authoritative new journal for investigators of the basic sciences as they apply to the heart and circulation.” This review of the early years of the journal highlights the contributions of the first four Editors: Carl J. Wiggers, Carl F. Schmidt, Eugene M. Landis, and Julius H. Comroe, Jr. The success of
Circulation Research
is seen not only in the high quality of the articles published in its pages but also in the remarkable improvements in prevention and treatment of cardiovascular disease that have occurred over the past half century.
Endothelial Dysfunction and Elevation of
S
-Adenosylhomocysteine in Cystathionine β-Synthase–Deficient Mice
Authors: Dayal Sanjana , Bottiglieri Teodoro , Arning Erland , Maeda Nobuyo , Malinow M. René , Sigmund Curt D. , Heistad Donald D. , Faraci Frank M. , Lentz Steven R.
Published: September 2007

Abstract
—Hyperhomocysteinemia is associated with increased risk for cardiovascular events, but it is not certain whether it is a mediator of vascular dysfunction or a marker for another risk factor. Homocysteine levels are regulated by folate bioavailability and also by the methyl donor
S
-adenosylmethionine (SAM) and its metabolite
S
-adenosylhomocysteine (SAH). We tested the hypotheses that endothelial dysfunction occurs in hyperhomocysteinemic mice in the absence of folate deficiency and that levels of SAM and SAH are altered in mice with dysfunction. Heterozygous cystathionine β-synthase–deficient (CBS
+/–
) and wild-type (CBS
+/+
) mice were fed a folate-replete, methionine-enriched diet. Plasma levels of total homocysteine were elevated in CBS
+/–
mice compared with CBS
+/+
mice after 7 weeks (27.1±5.2 versus 8.8±1.1 μmol/L;
P
<0.001) and 15 weeks (23.9±3.0 versus 13.0±2.3 μmol/L;
P
<0.01). After 15 weeks, but not 7 weeks, relaxation of aortic rings to acetylcholine was selectively impaired by 35% (
P
<0.05) and thrombomodulin anticoagulant activity was decreased by 20% (
P
<0.05) in CBS
+/–
mice. Plasma levels of folate did not differ between groups. Levels of SAH were elevated ≈2-fold in liver and brain of CBS
+/–
mice, and correlations were observed between plasma total homocysteine and SAH in liver (
r
=0.54;
P
<0.001) and brain (
r
=0.67;
P
<0.001). These results indicate that endothelial dysfunction occurs in hyperhomocysteinemic mice even in the absence of folate deficiency. Endothelial dysfunction in CBS
+/–
mice was associated with increased tissue levels of SAH, which suggests that altered SAM-dependent methylation may contribute to vascular dysfunction in hyperhomocysteinemia.
Mechanisms Underlying the Reentrant Circuit of Atrioventricular Nodal Reentrant Tachycardia in Isolated Canine Atrioventricular Nodal Preparation Using Optical Mapping
Authors: Wu Jianyi , Wu Jiashin , Olgin Jeffrey , Miller John M. , Zipes Douglas P.
Published: September 2007

Abstract
—The reentrant pathways underlying different types of atrioventricular (AV) nodal reentrant tachycardia have not yet been elucidated. This study was performed to optically map Koch’s triangle and surrounding atrial tissue in an isolated canine AV nodal preparation. Multiple preferential AV nodal input pathways were observed in all preparations (n=22) with continuous (73%, n=16) and discontinuous (27%, n=6) AV nodal function curves (AVNFCs). AV nodal echo beats (EBs) were induced in 54% (12/22) of preparations. The reentrant circuit of the slow/fast EB (36%, n=8) started as a block in fast pathway (FP) and a delay in slow pathway (SP) conduction to the compact AV node, then exited from the AV node to the FP, and rapidly returned to the SP through the atrial tissue located at the base of Koch’s triangle. The reentrant circuit of the fast/slow EB (9%, n=2) was in an opposite direction. In the slow/slow EB (9%, n=2), anterograde conduction was over the intermediate pathway (IP) and retrograde conduction was over the SP. Unidirectional conduction block occurred at the junction between the AV node and its input pathways. Conduction over the IP smoothed the transition from the FP to the SP, resulting in a continuous AVNFC. A “jump” in AH interval resulted from shifting of anterograde conduction from the FP to the SP (n=4) or abrupt conduction delay within the AV node through the FP (n=2). These findings indicate that (1) multiple AV nodal anterograde pathways exist in all normal hearts; (2) atrial tissue is involved in reentrant circuits; (3) unidirectional block occurs at the interface between the AV node and its input pathways; and (4) the IP can mask the existence of FP and SP, producing continuous AVNFCs.
Investigation of Relationship Between Reduced, Oxidized, and Protein-Bound Homocysteine and Vascular Endothelial Function in Healthy Human Subjects
Authors: Chambers John C. , Ueland Per M. , Wright Melissa , Doré Caroline J. , Refsum Helga , Kooner Jaspal S.
Published: September 2007

Previous studies investigating homocysteine and vascular disease have relied on total plasma homocysteine as the sole index of homocysteine status. We examined the dynamic relationship between vascular endothelial function and concentrations of total, protein-bound oxidized, free oxidized, and reduced homocysteine to identify the homocysteine form associated with endothelial dysfunction in humans. We investigated 14 healthy volunteers (10 men, 4 women). Brachial artery flow–mediated dilatation was measured at baseline and at 30, 60, 120, 240, and 360 minutes after oral (1)
l
-methionine (50 mg/kg), (2)
l
-homocysteine (5 mg/kg), and (3) placebo. Plasma concentrations of total, protein-bound oxidized, free oxidized, and reduced homocysteine were measured at each time point, and nitroglycerin-induced dilatation at was assessed at 0, 120, and 360 minutes. Flow-mediated dilatation fell, and concentrations of total, protein-bound oxidized, free oxidized, and reduced homocysteine increased after oral homocysteine and oral methionine (all
P
<0.05 for difference in time course compared with placebo). Flow-mediated dilatation showed a reciprocal relationship with reduced homocysteine during both homocysteine and methionine loading. In both loading studies, peak reduction in flow-mediated dilatation coincided with maximal reduced homocysteine concentrations. In contrast, there was no consistent relationship between flow-mediated dilatation and free oxidized homocysteine, protein-bound oxidized homocysteine, or related species. Nitroglycerin-induced dilatation was unchanged by oral homocysteine and oral methionine (
P
>0.10 compared with placebo). Reduced homocysteine is closely associated with endothelial dysfunction during oral methionine and oral homocysteine loading. Our observations support the hypothesis that reduced homocysteine is the deleterious form of homocysteine for vascular function in vivo and suggest a less important role for other homocysteine species.
Sarcoplasmic Reticulum Ca
2+
ATPase (SERCA) 1a Structurally Substitutes for SERCA2a in the Cardiac Sarcoplasmic Reticulum and Increases Cardiac Ca
2+
Handling Capacity
Authors: Jane Lalli M. , Yong Ji , Prasad Vikram , Hashimoto Katsuji , Plank Dave , Babu Gopal J. , Kirkpatrick Darryl , Walsh Richard A. , Sussman Mark , Yatani Atsuko , Marbán Eduardo , Periasamy Muthu
Published: September 2007

Ectopic expression of the sarcoplasmic reticulum (SR) Ca
2+
ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca
2+
transients; however, the cellular mechanisms underlying altered Ca
2+
handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca
2+
load measurements revealed that TG myocytes have significantly enhanced SR Ca
2+
load. Confocal line-scan images of field-stimulated SR Ca
2+
release showed an increased rate of Ca
2+
removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by ≈30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca
2+
current density was reduced by ≈50%, whereas the time course of inactivation was unchanged in TG myocytes. These studies provide important evidence that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca
2+
transport and cardiac contractility.
Redox Signaling of the Arteriolar Myogenic Response
Authors: Nowicki P. T. , Flavahan S. , Hassanain H. , Mitra S. , Holland S. , Goldschmidt-Clermont P. J. , Flavahan N. A.
Published: September 2007

Arteriolar vascular smooth muscle cells (VSMCs) are mechanosensitive, constricting to elevations in transmural pressure (P
TM
). The goal of the present study was to determine using mouse isolated tail arterioles and arteries whether oxidant signaling regulates this myogenic response. In response to P
TM
elevation, VSMCs of arterioles but not arteries generated constriction and increased reactive oxygen species (ROS) activity (using the H
2
O
2
-sensitive probe dichlorodihydrofluorescein). Arterioles had increased expression of NADPH oxidase components compared with arteries. Inhibition of NADPH oxidase, using mice with targeted impairment of enzyme components (p47

phox

or rac1) or diphenyleneiodonium, prevented the pressure-induced generation of ROS. When ROS activity was inhibited, either by inhibiting NADPH oxidase or with
N
-acetylcysteine, the myogenic constriction was abolished. The myogenic constriction was also inhibited by catalase, which inactivates H
2
O
2
, but was unaffected by a cell-permeant mimic of superoxide dismutase (MnTMPyP). α
1
-Adrenergic constriction was not associated with altered ROS activity and was not affected by inhibition of NADPH oxidase or ROS. Exogenous H
2
O
2
constricted VSMCs of arterioles but not arteries. Thus, NADPH oxidase and ROS, in particular H
2
O
2
, contribute to the myogenic response of arteriolar VSMCs.
Nitric Oxide Synthase (NOS2) Mutation in Dahl/Rapp Rats Decreases Enzyme Stability
Authors: Ying Wei-Zhong , Xia Hongli , Sanders Paul W.
Published: September 2007

The pathogenesis of salt-sensitive hypertension remains poorly defined, but a role for nitric oxide (NO) has been suggested. The Dahl/Rapp salt-sensitive rat possesses a defect in NO synthesis that is overcome by supplementation with
l
-arginine, which increases NO and cGMP production and prevents salt-sensitive hypertension. An S714P mutation of inducible NO synthase (NOS2) was subsequently identified. The current report examined the functional significance of an S714P mutation in NOS2. COS-7 cells were transiently transfected with cDNA of wild-type NOS2 and S714P and S714A mutants of NOS2, and enzyme function was determined. Whereas steady-state mRNA levels did not differ, immunoblot analysis demonstrated decreased levels of NOS2 protein. Metabolic labeling experiments confirmed a reduced half-life of the S714P mutation. Nitrite production, which was dependent on the concentration of
l
-arginine in the medium, was diminished in cells transfected with the S714P mutant, compared with the wild type and the S714A mutant. These data provide a biochemical explanation of the physiological abnormalities of NOS2 in the Dahl/Rapp salt-sensitive rat and suggest that a posttranslational mechanism involving the proteasome may be responsible for the diminished NO production observed in response to increased dietary salt intake in these animals.
Mice Lacking Inducible Nitric Oxide Synthase Have Improved Left Ventricular Contractile Function and Reduced Apoptotic Cell Death Late After Myocardial Infarction
Authors: Sam Flora , Sawyer Douglas B. , Xie Zhonglin , Chang Donny L.F. , Ngoy Soeun , Brenner Daniel A. , Siwik Deborah A. , Singh Krishna , Apstein Carl S. , Colucci Wilson S.
Published: September 2007

Nitric oxide produced by inducible nitric oxide synthase (NOS2) has been implicated in the pathophysiology of chronic myocardial remodeling and failure. We tested the role of NOS2 in left ventricular (LV) remodeling early (1 month) and late (4 months) after myocardial infarction (MI) in mice lacking NOS2. MI size measured 7 days, 1 month, and 4 months after MI was the same in NOS2 knockout (KO) and wild-type (WT) mice. The LV end-diastolic pressure-volume relationship measured by the isovolumic Langendorff technique showed a progressive rightward shift from 1 to 4 months after MI in WT mice. LV developed pressure measured over a range of LV volumes was reduced at 1 and 4 months after MI in WT mice (
P
<0.05 and
P
<0.01 versus shams, respectively). In KO mice, the rightward shift was similar to that in WT mice at 1 and 4 months after MI, as was peak LV developed pressure at 1 month after MI. In contrast, at 4 months after MI, peak LV developed pressure in KO mice was higher than in WT mice (
P
<0.05 versus WT) and similar to that in sham-operated mice. At 1 month after MI, the frequency of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)–positive myocytes in the remote myocardium was increased to a similar extent in WT and KO mice. At 4 months after MI, the frequency of apoptotic myocytes was increased in WT mice but not in KO mice (
P
<0.05 versus WT). Improved contractile function and reduced apoptosis were associated with reduced mortality rate in KO mice at 4 months after MI. Thus, NOS2 does not play an important role in determining infarct size or early LV remodeling during the first month after MI. In contrast, during late (ie, 4 months after MI) remodeling, NOS2 in remote myocardium contributes to decreased contractile function, increased myocyte apoptosis in remote myocardium, and reduced survival.
Action Potential Characteristics and Arrhythmogenic Properties of the Cardiac Conduction System of the Murine Heart
Authors: Anumonwo Justus M.B. , Tallini Yvonne N. , Vetter Frederick J. , Jalife José
Published: September 2007

Studies have characterized conduction velocity in the right and left bundle branches (RBB, LBB) of normal and genetically engineered mice. However, no information is available on the action potential characteristics of the specialized conduction system (SCS). We have used microelectrode techniques to characterize action potential properties of the murine SCS, as well as epicardial and endocardial muscle preparations for comparison. In the RBB, action potential duration at 50%, 70%, and 90% repolarization (APD
50,70,90
) was 6±0.7, 35±6, and 90±7 ms, respectively. Maximum upstroke velocity (dV/dt
max
) was 153±14 V/s, and conduction velocity averaged 0.85±0.2 m/s. APD
90
was longer in the Purkinje network of fibers (web) than in the RBB (
P
<0.01). Web APD
50
was longer in the left than in the right ventricle (
P
<0.05). Yet, web APD
90
was longer in the right than in the left ventricle (
P
<0.001). APD
50,70
was significantly longer in the endocardial than in the epicardial (
P
<0.001;
P
<0.003). APD
90
in the epicardial and endocardial was shorter than in the RBB (≈36 ms versus ≈100 ms). Spontaneous electrical oscillations in phase 2 of the SCS occasionally resulted in early afterdepolarizations. These results demonstrate that APDs in the murine SCS are significantly (≈2-fold) longer than in the myocardium and implicate the role of the murine SCS in arrhythmias. The differences should have important implications in the use of the mouse heart to study excitation, propagation, and arrhythmias.
Effects of Cytosolic ATP on Ca
2+
Sparks and SR Ca
2+
Content in Permeabilized Cardiac Myocytes
Authors: Yang Zhaokang , Steele Derek S.
Published: September 2007

Abstract—

Confocal imaging was used to study the influence of cytosolic ATP on the properties of spontaneous Ca
2+
sparks in permeabilized ventricular myocytes. Cells were perfused with mock intracellular solutions containing fluo 3. Reducing [ATP] to <0.5 mmol/L decreased the frequency but increased the amplitude of spontaneous Ca
2+
sparks. In the presence of 20 μmol/L ATP, the amplitude increased by 48.7±10.9%, and the frequency decreased by 77.07±3.8%, relative to control responses obtained at 5 mmol/L ATP. After exposure to a solution containing zero ATP, the frequency of Ca
2+
sparks decreased progressively and approached zero within 90 seconds. As ATP washed out of the cell, the sarcoplasmic reticulum (SR) Ca
2+
content increased, until reaching a maximum after 3 minutes. Subsequent introduction of adenylyl imidodiphosphate precipitated a burst of large-amplitude Ca
2+
sparks. This was accompanied by a rapid decrease in SR Ca
2+
content to 80% to 90% of the steady-state value obtained in the presence of 5 mmol/L ATP. Thereafter, the SR Ca
2+
content declined much more slowly over 5 to 10 minutes. The effects of ATP withdrawal on Ca
2+
sparks may reflect reduced occupancy of the adenine nucleotide site on the SR Ca
2+
channel. These effects may contribute to previously reported changes in SR function during myocardial ischemia and reperfusion, in which ATP depletion and Ca
2+
overload occur.
Mechanistic Insights Into Very Slow Conduction in Branching Cardiac Tissue
Authors: Kucera Jan P. , Rudy Yoram
Published: September 2007

It is known that branching strands of cardiac tissue can form a substrate for very slow conduction. The branches slow conduction by acting as current loads drawing depolarizing current from the main strand (“pull” effect). It has been suggested that, upon depolarization of the branches, they become current sources reinjecting current back into the strand, thus enhancing propagation safety (“push” effect). It was the aim of this study to verify this hypothesis and to assess the contribution of the push effect to propagation velocity and safety. Conduction was investigated in strands of Luo-Rudy dynamic model cells that branch from either a single branch point or from multiple successive branch points. In single-branching strands, blocking the push effect by not allowing current to flow retrogradely from the branches into the strand did not significantly increase the branching-induced local propagation delay. However, in multiple branching strands, blocking the push effect resulted in a significant slowing of overall conduction velocity or even in conduction failure. Furthermore, for certain slow velocities, the safety factor for propagation was higher when slow conduction was caused by branching tissue geometry than by reduced excitability without branching. Therefore, these results confirm the proposed “pull and push” mechanism of slow, but nevertheless robust, conduction in branching structures. Slow conduction based on this mechanism could occur in the atrioventricular node, where multiple branching is structurally present. It could also support reentrant excitation in diseased myocardium where the substrate is structurally complex.
Store-Operated Channels Mediate Ca
2+
Influx and Contraction in Rat Pulmonary Artery
Authors: Ng Lih Chyuan , Gurney Alison M.
Published: September 2007

Cation channels activated by Ca
2+
store depletion have been proposed to mediate Ca
2+
influx in vascular smooth muscle cells. The aim of this study was to determine if store-operated channels have a functional role in pulmonary artery smooth muscle cells (PASMCs). In intact rat pulmonary artery rings, cyclopiazonic acid (CPA) produced a sustained contraction that was resistant to inhibition by nifedipine, but abolished in Ca
2+
-free solution and 50% blocked in the presence of 6 μmol/L Cd
2+
, 10 μmol/L Ni
2+
, 600 μmol/L La
3+
, and 7 μmol/L SKF96365. In freshly isolated PASMCs loaded with fura-2, CPA increased the intracellular Ca
2+
concentration by stimulating dihydropyridine-resistant Ca
2+
influx, which was ≈50% blocked by 10 μmol/L Ni
2+
and 7 μmol/L SKF96365. In perforated-patch recordings, CPA activated a sustained inward current at negative membrane potentials, which persisted in cells dialyzed with BAPTA, showed a near linear dependence on membrane potential when Cs
+
was the main intracellular cation, and was blocked by Ni
2+
, Cd
2+
, and SKF96365 at concentrations preventing contraction. The current showed a bimodal dependence on extracellular Ca
2+
, being enhanced 2-fold in the absence of Ca
2+
and around 10-fold on reducing Ca from 1.8 to 0.2 mmol/L. RT-PCR revealed the expression of Trp1, Trp3, Trp4, Trp5, and Trp6 mRNA, whereas immunostaining identified Trp1, Trp3, Trp4, and Trp6 channel proteins in isolated PASMCs. At least one of these subunits may contribute to cation channels in PASMCs, which are activated by store depletion to bring about Ca
2+
influx and contraction.
Hsp90 and Caveolin Are Key Targets for the Proangiogenic Nitric Oxide–Mediated Effects of Statins
Authors: Brouet Agnès , Sonveaux Pierre , Dessy Chantal , Moniotte Stéphane , Balligand Jean-Luc , Feron Olivier
Published: September 2007

3-Hydroxy-3-methylglutaryl (HMG)–coenzyme A reductase inhibitors or statins exert direct beneficial effects on the endothelium in part through an increase in nitric oxide (NO) production. Here, we examined whether posttranslational modifications of the endothelial NO synthase (eNOS) could account for the proangiogenic effects of statins. We used endothelial cells (ECs) isolated from cardiac microvasculature, aorta, and umbilical veins, as well as dissected microvessels and aortic rings, that were cultured on reconstituted basement membrane matrix (Matrigel). Tube or precapillary formation was evaluated after statin treatment, in parallel with immunoblotting and immunoprecipitation experiments. Atorvastatin stimulated NO-dependent angiogenesis from both isolated and outgrowing (vessel-derived) ECs, independently of changes in eNOS expression. We found that in macro- but not microvascular ECs, atorvastatin stabilized tube formation through a decrease in caveolin abundance and its inhibitory interaction with eNOS. We also identified the chaperone protein hsp90 as a key target for the proangiogenic effects of statins. Using geldanamycin, an inhibitor of hsp90 function, and overexpression of recombinant hsp90, we documented that the statin-induced phosphorylation of eNOS on Ser1177 was directly dependent on the ability of hsp90 to recruit Akt in the eNOS complex. Finally, we showed that statin promoted the tyrosine phosphorylation of hsp90 and the direct interaction of hsp90 with Akt, which further potentiated the NO-dependent angiogenic processes. Our study provides new mechanistic insights into the NO-mediated angiogenic effects of statins and underscores the potential of these drugs and other modulators of hsp90 and caveolin abundance to promote neovascularization in disease states associated or not with atherosclerosis.
Genetic Manipulation of Cardiac K
+
Channel Function in Mice
Authors: Nerbonne Jeanne M. , Nichols Colin G. , Schwarz Thomas L. , Escande Denis
Published: September 2007

In the mammalian myocardium, potassium (K
+
) channels control resting potentials, action potential waveforms, automaticity, and refractory periods and, in most cardiac cells, multiple types of K
+
channels that subserve these functions are expressed. Molecular cloning has revealed the presence of a large number of K
+
channel pore forming (α) and accessory (β) subunits in the heart, and considerable progress has been made recently in defining the relationships between expressed K
+
channel subunits and functional cardiac K
+
channels. To date, more than 20 mouse models with altered K
+
channel expression/functioning have been generated using dominant-negative transgenic and targeted gene deletion approaches. In several instances, the genetic manipulation of K
+
channel subunit expression has revealed the role of specific K
+
channel subunit subfamilies or individual K
+
channel subunit genes in the generation of myocardial K
+
channels. In other cases, however, the phenotypic consequences have been unexpected. This review summarizes what has been learned from the in situ genetic manipulation of cardiac K
+
channel functioning in the mouse, discusses the limitations of the models developed to date, and explores the likely directions of future research.
Nitric Oxide Modulates Cardiac Na
+
Channel via Protein Kinase A and Protein Kinase G
Authors: Ahmmed Gias U. , Xu Yanfang , Hong Dong Pei , Zhang Zhao , Eiserich Jason , Chiamvimonvat Nipavan
Published: September 2007

We directly tested the effects of nitric oxide (NO) on Na
+
channels in guinea pig and mouse ventricular myocytes using patch-clamp recordings. We have previously shown that NO donors have no observed effects on expressed Na
+
channels. In contrast, NO (half-blocking concentration of 523 nmol/L) significantly reduces peak whole-cell Na
+
current (
I
Na
) in isolated ventricular myocytes. The inhibitory effect of NO on
I
Na
was not associated with changes in activation, inactivation, or reactivation kinetics. At the single-channel level, the reduction in macroscopic current was mediated by a decrease in open probability and/or a decrease in the number of functional channels with no change in single-channel conductance. Application of cell permeable analogs of cGMP or cAMP mimics the inhibitory effects of NO. Furthermore, the effects of NO on
I
Na
can only be blocked by inhibition of both cGMP and cAMP pathways. Sulfhydryl-reducing agent does not reverse the effect of NO. In summary, although NO exerts its action via the known guanylyl cyclase (GC)/cGMP pathway, our findings provide evidence that NO can mediate its function via a GC/cGMP-independent mechanism involving the activation of adynylyl cyclase (AC) and cAMP-dependent protein kinase.
Inducible Activation of c-Myc in Adult Myocardium In Vivo Provokes Cardiac Myocyte Hypertrophy and Reactivation of DNA Synthesis
Authors: Xiao Guishan , Mao Songyan , Baumgarten Georg , Serrano Jennifer , Jordan Maria C. , Roos Kenneth P. , Fishbein Michael C. , MacLellan W. Robb
Published: September 2007

c-Myc, a protooncogene, mediates both proliferative and cellular growth in many cell types. Although not expressed in the adult heart under normal physiological conditions, Myc expression is rapidly upregulated in response to hypertrophic stimuli. Although Myc is capable of sustaining hyperplastic growth in fetal myocytes, the effects of its re-expression in adult postmitotic myocardium and its role in mediating cardiac hypertrophy are unknown. To determine the effects of de novo Myc activity in adult postmitotic myocardium in vivo, we created a novel transgenic model in which Myc is expressed and inducibly activated specifically in cardiac myocytes. Activation of Myc in adult myocardium was sufficient to reproduce the characteristic changes in myocyte size, protein synthesis, and cardiac-specific gene expression seen in cardiac hypertrophy. Despite the increased cardiac mass, left ventricular function remained normal. Activation of Myc also provoked cell cycle reentry in postmitotic myocytes, which led to increased nuclei per myocyte and DNA content per nuclei.
Contribution of Adventitial Fibroblasts to Neointima Formation and Vascular Remodeling
Authors: Sartore Saverio , Chiavegato Angela , Faggin Elisabetta , Franch Rafaella , Puato Massimo , Ausoni Simonetta , Pauletto Paolo
Published: September 2007

The adventitial layer surrounding the blood vessels has long been exclusively considered a supporting tissue the main function of which is to provide adequate nourishment to the muscle layers of tunica media. Although functionally interconnected, the adventitial and medial layers are structurally interfaced at the external elastic lamina level, clearly distinguishable at the maturational phase of vascular morphogenesis. Over the last few years the “passive” role that the adventitia seemed to play in experimental and spontaneous vascular pathologies involving proliferation, migration, differentiation, and apoptosis of vascular smooth muscle cells (VSMCs) has been questioned. It has been demonstrated that fibroblasts from the adventitia display an important partnership with the resident medial VSMCs in terms of phenotypic conversion, proliferation, apoptotic, and migratory properties the result of which is neointima formation and vascular remodeling. This article is an attempt at reviewing the major themes and more recent findings dealing with the phenotypic conversion process that leads adventitial “passive” (static) fibroblasts to become “activated” (mobile) myofibroblasts. This event shows some facets in common with vascular morphogenesis, ie, the process of recruitment, incorporation, and phenotypic conversion of cells surrounding the primitive endothelial tube in the definitive vessel wall. We hypothesize that during the response to vascular injuries in the adult, “activation” of adventitial fibroblasts is, at least in part, reminiscent of a developmental program that also invests, although with distinct spatiotemporal features, medial VSMCs.
Myocardial Protection by Insulin at Reperfusion Requires Early Administration and Is Mediated via Akt and p70s6 Kinase Cell-Survival Signaling
Authors: Jonassen Anne K. , Sack Michael N. , Mjøs Ole D. , Yellon Derek M.
Published: September 2007

The “metabolic cocktail” comprising glucose-insulin-potassium administrated at reperfusion reduces infarct size in the in vivo rat heart. We propose that insulin is the major component mediating this protection and acts via Akt prosurvival signaling. This hypothesis was studied in isolated perfused rat hearts (measuring infarct size to area of risk [%]) subjected to 35 minutes regional myocardial ischemia and 2 hours reperfusion. Insulin administered at the onset of reperfusion attenuated infarct size by ≥45% versus control hearts (
P
<0.001). Insulin-mediated cardioprotection was found to be independent of the presence of glucose at reperfusion. Moreover, the cell survival benefit of insulin is temporally dependent, in that insulin administration from the onset of reperfusion and maintained for either 15 minutes or for the duration of reperfusion reduced infarct size. In contrast, protection was abrogated if insulin administration was delayed until 15 minutes into reperfusion. Pharmacological inhibition of both upstream and downstream signals in the Akt prosurvival pathway abolished the cardioprotective effects of insulin. Here coadministration of insulin with the tyrosine kinase inhibitor lavendustin A, the phosphatidylinositol3-kinase (PI3-kinase) inhibitor wortmannin, and mTOR/p70s6 kinase inhibitor rapamycin abolished cardioprotection. Steady-state levels of activated/phosphorylated Akt correlated with insulin administration. Finally, downstream prosurvival targets of Akt including p70s6 kinase and BAD were modulated by insulin. In conclusion, insulin administration at reperfusion reduces myocardial infarction, is dependent on early administration during reperfusion, and is mediated via Akt and p70s6 kinase dependent signaling pathway. Moreover, BAD is maintained in its inert phosphorylated state in response to insulin therapy.