bluuebunny/crossref_metadata_2025 huggingface dataset:
Dystrophin Is Not a Specific Component of the Cardiac Costamere
Authors: Stevenson Shirley , Rothery Stephen , Cullen Michael J. , Severs Nicholas J.
Published: June 2012

Dystrophin is a key component of the subsarcolemmal skeleton of muscle cells, and lack of dystrophin is the direct cause of Duchenne muscular dystrophy. In skeletal muscle, dystrophin is reported to be localized specifically at costameres, transversely oriented riblike subsarcolemmal plaques that mechanically couple the contractile apparatus to the extracellular matrix. Costameres are characteristically rich in vinculin and are prominent in cardiac as well as skeletal muscle. To define the precise spatial relationship between dystrophin in relation to the costamere in cardiac muscle, we applied high-resolution single- and double-immunolabeling techniques, under a range of preparative conditions, with visualization of vinculin (as a costamere marker) and dystrophin by confocal microscopy and by the freeze-fracture cytochemical technique, fracture label. Immunoconfocal visualization revealed dystrophin as a continuous uniform layer at the cytoplasmic surface of the peripheral plasma membrane of the rat cardiac myocyte at both costameric and noncostameric regions. The pattern of labeling was reproducible with three different antibodies and was independent of time and antibody concentration. Platinum/carbon replicas and thin sections of fracture-label specimens permitted high-resolution visualization of the distribution of dystrophin in plan views of the freeze-fractured plasma membrane and in relation to the sarcomeric banding patterns of the underlying myofibrils. These results confirmed no preferential association of dystrophin with costameres or with any region of the sarcomeres of underlying myofibrils in rat cardiac tissue. We conclude that in contrast to skeletal muscle, dystrophin in cardiac muscle is not exclusively a component of the costamere.
The Giant Protein Titin
Authors: Labeit Siegfried , Kolmerer Bernhard , Linke Wolfgang A.
Published: June 2012

Titin is a giant protein of vertebrate striated muscles (

M
r

, ≥3000 kD). Its molecules are of filamentous shape and span from the Z disk to the M line, thereby forming a third filament system of the sarcomere. This filament system is important for both the structural integrity of the myofibril and the passive tension response of a stretched muscle fiber. The determination of the cDNA sequence of human cardiac titin has shown that the cardiac titin filament is formed by a single, giant, 27 000-residue-long polypeptide chain. The titin strand has a modular structure, and different modulular arrangements are expressed in different muscle tissue types by differential splicing. In the A band, the titin modules provide regular arrays of binding sites for other sarcomeric proteins, thereby contributing to a precise assembly of myofibrillar proteins in vivo. In the I band, two specific motif families, tandem-immunoglobulin domains and PEVK-rich sequences, confer extensibility to the titin filament. Expression of muscle tissue–specific length variants of the PEVK region by alternative splicing may explain the differences in the passive tension properties between various striated muscle types. Apart from the titin sequences with apparent functions for muscle structure and elasticity, the titin molecule contains a class of unique sequence insertions. Among these sequences are phosphorylation sites, a serine/threonine kinase domain, and binding sites for muscle-specific calpain proteases. Thus, it is likely that the titin filament also plays a role in myofibrillar signal transduction pathways.
Oxidative Stress-Induced Actin Reorganization Mediated by the p38 Mitogen-Activated Protein Kinase/Heat Shock Protein 27 Pathway in Vascular Endothelial Cells
Authors: Huot Jacques , Houle Francois , Marceau Francois , Landry Jacques
Published: June 2012

Vascular endothelial cells are constantly in contact with oxyradicals and must be especially well equipped to resist their toxic effects and generate appropriate physiological responses. Despite the importance of oxyradicals in the physiopathology of the vascular endothelium, the mechanisms regulating the oxidative response of endothelial cells are poorly understood. In the present study, we observed that H sub 2 O
2
in concentrations that induced severe fragmentation of F-actin in fibroblasts rather induced a reorganization of F-actin in primary cultures of human umbilical vein endothelial cells (HUVECs) that was characterized by the accumulation of stress fibers, the recruitment of vinculin to focal adhesions, and the loss of membrane ruffles. H
2
O
2
also induced in these cells a strong (10- to 14-fold) activation of the p38 mitogen-activated protein (MAP) kinase, which resulted in activation of MAP kinase-activated protein kinase-2/3 and phosphorylation of the F-actin polymerization modulator, heat shock protein 27 (HSP27). The MAP kinases extracellular-regulated kinase, and c-Jun N-terminal kinase/stress-activated protein kinase were only slightly increased by these treatments. Inhibiting p38 activity with the highly specific inhibitor SB203580 blocked the H
2
O
2
-induced endothelial microfilament responses. Moreover, fibroblasts acquired an endothelium-like SB203580-sensitive actin response when HSP27 concentration was increased by gene transfection to the same high level as found in HUVECs. The results indicate that activation of p38 MAP kinase in cells such as endothelial cells, which naturally express high level of HSP27, plays a central role in modulating microfilament responses to oxidative stress. Consequently, the p38 MAP kinase pathway may participate in the several oxyradical-activated functions of the endothelium that are associated with reorganization of microfilament network.
The Effect of Oligonucleotides to c
-myb
on Vascular Smooth Muscle Cell Proliferation and Neointima Formation After Porcine Coronary Angioplasty
Authors: Gunn Julian , Holt Cathy M. , Francis Sheila E. , Shepherd Lynda , Grohmann Malcolm , Newman Christopher M. H. , Crossman David C. , Cumberland David C.
Published: June 2012

Abstract
Proto-oncogenes, including c-
myb
, are expressed early after vascular injury. The application of antisense oligodeoxynucleotides (AS-ODNs) against these genes inhibits cell proliferation and neointima formation in small animals and in peripheral arteries. The aim of this study was to investigate the specificity of action of AS-ODN–c-
myb
in vitro and to assess its effect, when delivered locally, on neointima formation after percutaneous transluminal coronary angioplasty (PTCA) in porcine coronary arteries. AS-ODN–c-
myb
inhibited the proliferation of vascular smooth muscle cells (VSMCs) in vitro in a dose-dependent manner. There was a corresponding reduction in steady state levels of c-
myb
mRNA and protein. Expression of another early gene, c-
fos
, was unaffected. S1 nuclease analysis demonstrated intact full-length AS-ODN–c-
myb
retrieved from VSMCs in culture after 12 hours. A range of ODNs, related and unrelated to c-
myb
, with and without a GGGG sequence, inhibited VSMC proliferation. Phosphorothioated AS-ODN–c-
myb
was 30 times less potent than unphosphorothioated AS-ODN–c-
myb
. PTCA induced porcine coronary artery neointima formation. c
-myb
mRNA was maximally induced 18 hours after injury. Unmodified AS-ODN–c-
myb
, sense-ODN–c-
myb
, saline, or nothing was delivered immediately after balloon dilatation via a double-skinned porous balloon (Transport, SciMed). Fluorescence-labeled AS-ODN–c-
myb
was deposited throughout the vessel wall. Mean maximum intima/media cross-sectional area 4 weeks after PTCA was reduced with AS-ODN–c-
myb
by 79% compared with saline (
P
<.05), 82% compared with sense-ODN–c-
myb
, and 63% compared with nothing (
P
<.10). Conclusions are as follows: (1) c-
myb
is expressed in VSMCs after vascular injury. (2) AS-ODN–c-
myb
is retained intact in VSMCs, reducing their proliferation in vitro in dose-dependent fashion, with reduction in c-
myb
mRNA and protein, whereas sense-ODN–c-
myb
is not. (3) A range of ODNs can reduce VSMC proliferation by a non–sequence-specific mechanism. (4) Phosphorothioate protection of antisense molecules may reduce their efficacy. (5) Local delivery of unmodified AS-ODN
–
c-
myb
via the Transport catheter reduces neointima formation after porcine PTCA. (6) Local delivery of fluid may exacerbate neointimal thickening.
Conductances and Selective Permeability of Connexin43 Gap Junction Channels Examined in Neonatal Rat Heart Cells
Authors: Valiunas Virginijus , Bukauskas Feliksas F. , Weingart Robert
Published: June 2012

Abstract
Myocytes from neonatal rat hearts were used to assess the conductive properties of gap junction channels by means of the dual voltage-clamp method. The experiments were carried out on three types (groups) of preparations: (1) induced cell pairs, (2) preformed cell pairs with few gap junction channels (1 to 3 channels), and (3) preformed cell pairs with many channels (100 to 200 channels) after treatment with uncoupling agents such as SKF-525A (75 μmol/L), heptanol (3 mmol/L), and arachidonic acid (100 μmol/L). In group 1, the first opening of a newly formed channel was slow (20 to 65 ms) and occurred 7 to 25 minutes after physical cell contact. The rate of channel insertion was 1.3 channels/min. Associated with a junctional voltage gradient (V
j
), the channels revealed multiple conductances, a main open state [γ
j
(main state)], several substates [γ
j
(substates)], and a residual state [γ
j
(residual state)]. On rare occasions, the channels closed completely. The same phenomena were observed in groups 2 and 3. The existence of γ
j
(residual state) provides an explanation for the incomplete inactivation of the junctional current (I
j
) at large values of V
j
in cell pairs with many gap junction channels. The values of γ
j
(main state) and γ
j
(residual state) gained from groups 1, 2, and 3 turned out to be comparable and hence were pooled. The fit of the data to a Gaussian distribution revealed a narrow single peak for both conductances. The values of γ
j
were dependent on the composition of the pipette solution. Solutions were as follows: (1) KCl solution, γ
j
(main state)=96 pS and γ
j
(residual state)=23 pS; (2) Cs
+
aspartate
−
solution, γ
j
(main state)=61 pS and γ
j
(residual state)=12 pS; and (3) tetraethylammonium
+
aspartate
−
solution, γ
j
(main state)=19 pS and γ
j
(residual state)=3 pS. The respective γ
j
(main state)-to-γ
j
(residual state) ratios were 4.2, 5.1, and 6.3. This indicates that the residual state restricts ion permeation more efficiently than does the main state. Transitions of I
j
between open states (main open state, substates, and residual state) were fast (<2 ms), and transitions involving the closed state and an open state were slow (15 to 65 ms). This implies the existence of two gating mechanisms. The residual state may be regarded as the ground state of electrical gating controlled by V
j
; the closed state, as the ground state of chemical gating.
Epoxyeicosatrienoic Acids Activate K
+
Channels in Coronary Smooth Muscle Through a Guanine Nucleotide Binding Protein
Authors: Li Pin-Lan , Campbell William B.
Published: June 2012

Abstract
Epoxyeicosatrienoic acids (EETs) are endothelium-derived arachidonic acid metabolites of cytochrome P450. They dilate coronary arteries, open K
+
channels, and hyperpolarize vascular smooth muscles. However, the mechanisms of these smooth muscle actions remain unknown. This study examined the effects of EETs on the large-conductance Ca
2+
-activated K
+
channel (K
Ca
) in smooth muscle cells of small bovine coronary arteries. In cell-attached patch-clamp experiments, 11,12-EET produced a 0.5- to 10-fold increase in the activity of the K
Ca
channels when added in concentrations of 1, 10, and 100 nmol/L. In the inside-out excised membrane patch mode, 11,12-EET was without effect on the activity of the K
Ca
channel unless GTP (0.5 mmol/L) or GTP and ATP (1 mmol/L) were added to the bath solution. In the presence of GTP and ATP, the increase in the K
Ca
channel activity with 11,12-EET in inside-out patches was comparable to that in cell-attached patches. This effect of 11,12-EET in inside-out patches was blocked by the addition of GDP-β-S (100 μmol/L). In outside-out patches, 11,12-EET also increased the K
Ca
channel activity when GTP and ATP were added to the pipette solution. The addition of a specific anti-G
S
α antibody (100 nmol/L) in the pipette solution completely blocked the activation of the K
Ca
channels induced by 11,12-EET. An anti-Gβγ or anti-G
i
α antibody was without effect. We conclude that 11,12-EET activates the K
Ca
channels by a G
S
α-mediated mechanism. This mechanism contributes to the effects of EETs as endothelium-derived hyperpolarizing factors to hyperpolarize and relax arterial smooth muscle.
Induction of Vascular Endothelial Growth Factor in Balloon-Injured Baboon Arteries
Authors: Ruef Johannes , Hu Zhao Y. , Yin Li-Yan , Wu Yaxu , Hanson Stephen R. , Kelly Andrew B. , Harker Laurence A. , Rao Gadiparthi N. , Runge Marschall S. , Patterson Cam
Published: June 2012

Abstract
Neovascularization is a hallmark of neointimal formation in atherosclerotic plaques and restenotic lesions. Vascular endothelial growth factor (VEGF) promotes neovascular growth, whereas oxidative stress is a potent factor in vascular cell proliferation. To investigate the mechanisms of neovascular formation, we treated human and rat vascular smooth muscle cells (VSMCs) with H
2
O
2
. Northern blot analysis demonstrated a dose- and time-dependent increase in VEGF mRNA, with a maximum of 4-fold at 3 hours (200 μmol/L). As determined by immunoblotting and enzyme-linked immunosorbent assay, VEGF protein expression and secretion were similarly increased. Human umbilical vein endothelial cells were treated with conditioned medium from VSMCs incubated with 200 μmol/L H
2
O
2
. DNA synthesis, measured by thymidine incorporation, was increased 4-fold compared with control, an effect that was blocked by a neutralizing anti-VEGF antibody. The lipid peroxidation product 4-hydroxynonenal (1 μmol/L), an endogenous reactive oxygen species present in human atherosclerotic lesions, also increased VEGF secretion in VSMCs in a similar time-dependent fashion. Immunohistochemical staining and in situ hybridization of aortic sections from balloon-injured baboons demonstrated increased VEGF expression in discrete areas of the neointima and media compared with control sections, and expression correlated with the generation of 4-hydroxynonenal. Regulators of VEGF expression, such as reactive oxygen species, may enhance neovascularization of atherosclerotic and restenotic arteries.
Monocytes Harboring Cytomegalovirus: Interactions With Endothelial Cells, Smooth Muscle Cells, and Oxidized Low-Density Lipoprotein
Authors: Guetta Esther , Guetta Victor , Shibutani Tomoko , Epstein Stephen E.
Published: June 2012

Abstract
Cytomegalovirus (CMV) infection and its periodic reactivation from latency may contribute to atherogenesis and restenosis. It is unknown how CMV is delivered to the vessel wall and is reactivated. We examined the following hypothesis: CMV, present in monocytes recruited to sites of vascular injury, is activated by endothelial cell (EC) or smooth muscle cell (SMC) contact and by oxidized low-density lipoproteins (oxLDLs). The CMV major immediate-early promoter (MIEP) controls immediate-early (IE) gene expression, and thereby viral replication. To determine whether elements of the vessel wall can activate CMV present in monocytes, we transiently transfected the promonocytic cell line HL-60 with a chloramphenicol acetyltransferase reporter gene construct driven by MIEP. MIEP activity increased 1.7±0.5-fold (
P
=.02) when the transfected HL-60 cells were cocultured with ECs, 4.5±1.5-fold when cocultured with SMCs (
P
=.03), and 2.0±0.5-fold (
P
=.01) when exposed to oxLDL. The combination of oxLDL and EC coculture increased MIEP activity over 7-fold. We also found that freshly isolated human monocytes, infected with endothelium-passaged CMV, were capable of transmitting infectious virus to cocultured ECs or SMCs. CMV-related progression of atherosclerosis or restenosis may, at least in part, involve monocyte delivery of the virus to the site of vascular injury, where the vascular milieu, ie, contact with ECs, SMCs, and oxLDL, can contribute to viral reactivation and/or replication by enhancing CMV IE gene expression. The virus may then infect neighboring ECs or SMCs, initiating a cascade of events predisposing to the development of atherogenesis-related processes.
Death by Design
Authors: MacLellan W. Robb , Schneider Michael D.
Published: June 2012

Abstract
Programmed cell death (apoptosis) is recognized, increasingly, as a contributing cause of cardiac myocyte loss with ischemia/reperfusion injury, myocardial infarction, and long-standing heart failure. Although the exact mechanisms initiating apoptosis in these in vivo settings remain unproven, insights into the molecular circuitry controlling apoptosis more widely suggest the potential to protect mammalian ventricular muscle from apoptosis through one or more of these pathways, by pharmacological means or, conceivably, gene transfer.
Adenoviral Gene Transfer of Phospholamban in Isolated Rat Cardiomyocytes
Authors: Hajjar Roger J. , Schmidt Ulrich , Kang Jing X. , Matsui Takashi , Rosenzweig Anthony
Published: June 2012

Abstract
Phospholamban forms an integral part of the cardiac sarcoplasmic reticulum (SR) and regulates the activity of SR Ca
2+
-ATPase (SERCA2a). A number of studies have suggested a decrease in SERCA2a relative to phospholamban in heart failure. To test the hypothesis that changes in the relative abundance of phospholamban to SERCA2a could account for the pathophysiological abnormalities in Ca
2+
handling observed in failing myocardium, we created a recombinant adenovirus designed to overexpress phospholamban (Ad.RSV.PL). In neonatal rat cardiomyocytes, Ad.RSV.PL increased the expression of phospholamban in a concentration-dependent fashion, reaching 280±43% at a multiplicity of infection (MOI) of 10.0 plaque forming units (pfu)/cell at 48 hours. The relationship between Ca
2+
-ATPase activity and [Ca
2+
] was shifted rightward in membrane preparations from cardiomyocytes infected with Ad.RSV.PL. Intracellular Ca
2+
transients measured in the neonatal cells infected with Ad.RSV.PL (MOI, 10 pfu/cell) were characterized by (1) a significant prolongation of the relaxation phase (344±26 versus 710±56 milliseconds,
P
<.01), (2) a decrease in peak [Ca
2+
]
i
(967±43 versus 630±33 nmol/L,
P
<.01), and (3) an elevation in resting [Ca
2+
]
i
(143±14 versus 213±17 nmol/L,
P
<.05). Similarly, the time course of shortening was prolonged in myocytes infected with Ad.RSV.PL. These effects were partially restored by simultaneous transduction with an adeno-virus carrying SERCA2a. Cardiomyocytes infected with Ad.RSV.PL had an abnormal frequency response: a decrease in peak [Ca
2+
]
i
and an increase in resting [Ca
2+
]
i
with increasing frequency. These findings indicate that adenovirus-mediated gene transfer of phospholamban modifies intracellular Ca
2+
handling and the frequency response in cardiomyocytes. Our results suggest that alterations in the ratio of phospholamban to SERCA2a could account for the abnormalities in Ca
2+
handling observed in heart failure and that overexpression of SERCA2a can largely correct these abnormalities.
Rapamycin Inhibits α
1
-Adrenergic Receptor–Stimulated Cardiac Myocyte Hypertrophy but Not Activation of Hypertrophy-Associated Genes
Authors: Boluyt Marvin O. , Zheng Jing-Sheng , Younes Antoine , Long Xilin , O’Neill Lydia , Silverman Howard , Lakatta Edward G. , Crow Michael T.
Published: June 2012

Abstract
The 70-kD S6 kinase (p70
S6K
) has been implicated in the regulation of protein synthesis in many cell types and in the angiotensin II–stimulated hypertrophy of cardiac myocytes. Our purpose was to determine whether p70
S6K
plays a role in cardiomyocyte hypertrophy induced by the α
1
-adrenergic receptor (α
1
-AR) agonist phenylephrine (PE). PE stimulated the activity of p70
S6K
>3-fold, and this increase was blocked by rapamycin, an immunosuppressant macrolide that selectively inhibits p70
S6K
. When administered for 3 days, PE stimulated a 30% increase in total protein content, a 2-fold increase in the incorporation of [
14
C]phenylalanine (
14
C-Phe) into protein, and a 50% increase in two-dimensional myocyte area. Rapamycin pretreatment (≥500 pg/mL) significantly inhibited each of these PE-stimulated changes. Two days of PE treatment resulted in a 1.6-fold increase in total RNA yield per dish, a 2-fold increase in incorporation of [
14
C]uridine into myocyte RNA, and increases in relative mRNA levels of the hypertrophy-associated atrial natriuretic factor (ANF, 2.1-fold) and skeletal α-actin (SK, 2.2-fold) genes. Although rapamycin abolished the PE-stimulated increases in total RNA and incorporation of [
14
C]uridine, it had no effect on the induction of the ANF and SK genes. LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3-K) activity, inhibited PE-stimulated increases in p70
S6K
activity and the incorporation of labeled precursors into myocyte protein and RNA. These results demonstrate that p70
S6K
is activated by the hypertrophic agent PE and that a PI3-K or PI3-K–like activity is required for p70
S6K
activation and myocyte hypertrophy. The data suggest that p70
S6K
activation may be required for PE-stimulated hypertrophy of cardiac myocytes. Our results demonstrate that intracellular signaling pathways responsible for transcriptional and translational responses diverge early after α
1
-AR stimulation in cardiac myocytes.
Mechanism of Vascular Smooth Muscle Relaxation by Estrogen in Depolarized Rat and Mouse Aorta
Authors: Freay Ana D. , Curtis Sylvia W. , Korach Kenneth S. , Rubanyi Gabor M.
Published: June 2012

Abstract
17β-Estradiol induces vasodilation in vitro and in vivo, which has been suggested to contribute to the cardiovascular protection by this ovarian steroid hormone. However, the exact mechanism of vasorelaxation by estrogens remains to be elucidated. In this study, we analyzed the potential role of genomic mechanisms involving the nuclear estrogen receptor and inhibition of entry of extracellular Ca
2+
in 17β-estradiol–induced vasorelaxation in depolarized aortic rings, isolated from male and female rats and male mice. In both male and female rat aortic rings without endothelium and in intact male mouse aortic rings treated with
N
G
-nitro-
l
-arginine, 17β-estradiol caused dose-dependent (0.3 to 30 μmol/L) relaxation of contraction evoked by high-K
+
depolarization (30 and 45 mmol/L KCl, respectively). The estrogen receptor antagonist ICI 164384 had no effect on 17β-estradiol–induced relaxations.
125
I-17β-estradiol binding studies showed the presence of high-affinity cytosolic-nuclear estrogen receptors in control male mouse aortas. Comparable relaxations of aortic rings isolated from control and estrogen receptor–deficient transgenic mice provided direct evidence that the nuclear estrogen receptor is not involved in this response. 17β-Estradiol–induced relaxation of rat aortic rings could not be prevented by cycloheximide or actinomycin D, suggesting that the response was not mediated by de novo protein synthesis or gene transcription. In rat aortic rings, 17β-estradiol inhibited the increase of
45
Ca uptake by 30 mmol/L KCl at concentrations (10 and 30 μmol/L) that caused vasorelaxation in the same tissue, suggesting that inhibition of Ca
2+
entry contributes to the response. 17α-Estradiol was less effective, and estrone was devoid of vasorelaxing activity. Vasorelaxation by estrogens in female and male rat aortas was similar, indicating no gender difference in vascular responses under these conditions.
Angiotensin II Activates Phosphatidylinositol 3-Kinase in Vascular Smooth Muscle Cells
Authors: Saward Peter Zahradka Laura
Published: June 2012

Abstract
Phosphatidylinositol 3-kinase (PI3K) is an important component of the signal transduction systems activated by tyrosine kinase receptors. It has not been established, however, whether PI3K is also an essential mediator for G protein–coupled receptors. The potential involvement of PI3K in G protein–linked angiotensin II (Ang II)–dependent signaling was assessed in a primary cell culture system of porcine coronary artery smooth muscle cells (SMCs). Treatment of quiescent SMCs with Ang II (10
−5
to 10
−8
mol/L) resulted in a dose-dependent activation of PI3K when assayed in vivo and in vitro. The Ang II receptor antagonists losartan and PD123319 were used to establish that Ang II stimulates PI3K through the Ang II type-1 (AT
1
) receptor. Immunofluorescent microscopy revealed that Ang II (10
−6
mol/L) stimulated the translocation of p85, the regulatory subunit of PI3K, from the perinuclear region to distinct foci throughout the cell within 15 minutes. Western blot analysis of p85 subcellular distribution demonstrated that p85 concentrations were also increased within 15 minutes in the membrane fraction and concomitantly decreased in the cytoskeletal and nuclear fractions. These changes in PI3K location and activity were paralleled by increased tyrosine phosphorylation of p85. A potential correlation between angiotensin-mediated PI3K activation and SMC growth was found using LY294002, a specific inhibitor of PI3K, which blocked the increase in DNA and RNA synthesis as well as cellular hyperplasia generated by Ang II (10
−6
mol/L) stimulation of quiescent SMCs. These data indicate that PI3K may operate as a mediator of vascular SMC growth after stimulation with Ang II.
Flow-Induced Vascular Remodeling in the Rat Carotid Artery Diminishes With Age
Authors: Miyashiro Jody K. , Poppa Veronica , Berk Bradford C.
Published: June 2012

Abstract
Vascular remodeling is regulated by a combination of hemodynamic, environmental, and genetic factors and may be influenced by age. To evaluate age-dependent remodeling in rats, we developed and used a quantitative highly reproducible model of carotid flow alteration. Fourteen juvenile (99±3 g) and 9 adult (199±5 g) male inbred Fischer rats underwent ligation of the left internal and external carotid arteries under anesthesia. Left common carotid blood flow immediately decreased by ≈93%, whereas flow in the contralateral carotid increased by ≈46%. After 4 weeks, the left carotid outer diameter (OD) significantly decreased in both juvenile and adult rats (as measured in vivo and by histological morphometry) compared with sham-operated rats. Changes in shear stress acutely mirrored the changes in blood flow. OD increased and shear stress returned to initial values after chronic exposure to increased flow in juvenile but not adult rats. To develop a simple quantitative index of remodeling that would not require killing the animals, we measured the OD in vivo and compared the ratio of right to left OD (OD ratio [ODR]) between groups. The initial ODR for all groups was ≈1.0. After 4 weeks of altered flow, the ODR was significantly greater in juvenile than in adult rats (1.48±0.05 versus 1.29±0.04, respectively;
P
=.030), indicating that juvenile rats experienced more extensive remodeling than did the adult rats. We also found that unilateral carotid ligation caused a left versus right difference in endothelial NO synthase protein levels after 4 weeks that was not present in the sham-operated animals. Thus, the model described here shows that flow-induced vascular remodeling is dependent on age and supports the hypothesis that the driving force for remodeling involves shear stress and possibly NO. Because the model is quantitative, it allows dissection of the genetic factors that regulate remodeling in inbred rat strains.
Ca
2+
‘Sparks’ and Waves in Intact Ventricular Muscle Resolved by Confocal Imaging
Authors: Wier Withrow Gil , Keurs Henk E. D. J. ter , Marban Eduardo , Gao Wei Dong , Balke C. William
Published: June 2012

Abstract
The [Ca
2+
]
i
transient in heart is now thought to involve the recruitment and summation of discrete and independent “units” of Ca
2+
release (Ca
2+
“sparks”) from the sarcoplasmic reticulum, each of which is controlled locally by single coassociated L-type Ca
2+
channels (“local control theory of excitation-contraction coupling”). All prior studies on Ca
2+
sparks, however, have been performed in single enzymatically dissociated heart cells under nonphysiological conditions. In order to understand the possible significance of Ca
2+
sparks to normal working cardiac muscle, we used confocal microscopy to record Ca
2+
sparks, which are spatially averaged [Ca
2+
]
i
transients (and Ca
2+
waves), in individual cells of intact rat right ventricular trabeculae (composed of <15 cells in cross section) microinjected with the Ca
2+
indicator fluo 3 under physiological conditions ([Ca
2+
]
o
, 1 mmol/L; temperature, 33±1°C). Twitch force was recorded simultaneously. When stretched to optimal length (sarcomere length, 2.2 μm) and stimulated at 0.2 Hz, the trabeculae generated ≈700 μg of force per cell. Spatially averaged [Ca
2+
]
i
transients recorded from individual cells within a trabecula were similar to those recorded previously from single cells. The amplitude distribution of the peak ratio of Ca
2+
sparks was bimodal, with maxima at ratios of 1.8±0.3 and 2.7±0.2 (mean±SD), respectively. The amplitude of the peak of Ca
2+
sparks was ≈170 nmol/L. Ca
2+
sparks occurred at a frequency of 12.0±0.8/s (mean±SEM) in line scans covering 94 sarcomeres. Ca
2+
waves occurred randomly at a frequency of 0.57±0.08/s and propagated with a velocity of 29.5±1.7 μm/s. The extent of Ca
2+
wave propagation was 3.9±0.3 sarcomere lengths (sarcomere length, 2.2 μm). Ca
2+
sparks could be identified along the leading edge of the waves at intervals of 1.30±0.11 sarcomere length. Our observations suggest that (1) Ca
2+
sparks, similar to those recorded in single cells, occur in trabeculae under physiological conditions and (2) coupling of Ca
2+
spark generation between neighboring sites occurs and may lead to (3) the development of Ca
2+
waves, which propagate under physiological conditions at a low velocity over limited distances. The results suggest that concepts of excitation-contraction coupling recently derived from isolated myocytes are applicable to intact cardiac trabeculae.
Changing Activation Sequence in the Embryonic Chick Heart
Authors: Chuck Emil T. , Freeman David M. , Watanabe Michiko , Rosenbaum David S.
Published: June 2012

Abstract
In the mature heart, impulse propagation through the His-Purkinje system (HPS) is required for efficient ventricular contraction in an apex-to-base direction. However, the embryonic heart begins to contract as a myocardial tube without a specialized conduction system. To identify the developmental stage when the HPS begins to function, we mapped the ventricular depolarization sequence from microvolt-level electrograms recorded from embryonic myocardium using 50-μm extracellular electrodes, high-gain amplification, and signal-processing techniques. Analysis of left ventricular activation in 99 embryonic hearts revealed a transition in the activation sequence that was dependent on developmental stage. As the heart develops, a transition in the activation sequence occurred from the primitive base-to-apex pattern (in 20 of 33 hearts) at early stages (Hamburger-Hamilton stages 25 to 28) to the HPS-like apex-to-base pattern (12 of 17 hearts) late in development (stages 33 to 36). Immunohistological experiments (n=10) also confirm that the expression pattern of two biochemical HPS markers changes in parallel with the change to the mature ventricular activation pattern. These data indicate that the ventricular activation sequence in the chick heart develops to a mature pattern at stages 29 to 31, suggesting that preferential conduction through the HPS begins shortly after ventricular septation is complete.
Tie2 Expression and Phosphorylation in Angiogenic and Quiescent Adult Tissues
Authors: Wong Adrianne L. , Haroon Zishan A. , Werner Sabine , Dewhirst Mark W. , Greenberg Charles S. , Peters Kevin G.
Published: June 2012

Abstract
Angiogenesis, the process of new vessels sprouting from the existing vasculature, is a critical process during early development. However, angiogenesis rarely occurs in the adult, except in response to cyclic hormonal stimulation in the ovary and uterus, in response to injury, and in response to pathological conditions such as tumorigenesis and diabetes mellitus. Tie2 (also known as Tek) is a novel endothelium-specific receptor tyrosine kinase, which has been demonstrated to be essential for the development of the embryonic vasculature; Tie2 knockout mice die by embryonic day 10.5 with specific defects in the formation of microvessels. Tie2 is downregulated later in embryogenesis, and its function in the adult has been relatively unexplored. To gain insight into the potential functions of Tie2 in the adult vasculature, Tie2 expression was examined in adult tissues undergoing angiogenesis and in quiescent tissues. Tie2 expression was localized by immunohistochemistry to the endothelium of neovessels in rat tissues undergoing angiogenesis during hormonally stimulated follicular maturation and uterine development and in healing skin wounds. Immunoprecipitation and RNase protection assay demonstrated upregulation of Tie2 protein and mRNA in rat and mouse skin wounds, respectively. Moreover, Tie2 immunoprecipitated from skin wounds was tyrosine-phosphorylated, indicating active downstream signaling. Surprisingly, Tie2 was also expressed in the entire spectrum of the quiescent vasculature (arteries, veins, and capillaries) in a wide range of adult tissues, and Tie2 immunoprecipitated from quiescent adult tissues was also tyrosine-phosphorylated. Together, these results suggest a dual function for Tie2 in adult tissues involving both angiogenesis and vascular maintenance.
Evidence That Angiotensin II and Lipoxygenase Products Activate c-Jun NH
2
-Terminal Kinase
Authors: Wen Yeshao , Scott Stephen , Liu Yaxia , Gonzales Noe , Nadler Jerry L.
Published: June 2012

Abstract
The effect of angiotensin II (Ang II) to activate c-Jun amino-terminal kinase (JNK) was studied in a Chinese hamster ovary fibroblast cell line overexpressing the rat vascular type-1a Ang II receptor (CHO-AT
1a
). Ang II treatment induced a time-dependent activation of JNK. Ang II (10
−7
mol/L) activated JNK activity, with a peak at 30 minutes (9.39±2.52-fold, n=7,
P
<.02 versus control), which was maintained until 3 hours (2.7±0.65-fold, n=3,
P
<.02 versus control). Ang II–induced JNK activation at 30 minutes was inhibited by a specific lipoxygenase (LO) pathway inhibitor, cinnamyl-3,4-dihydroxy-α-cyanocinnamate (1 μmol/L) by 87.5% (n=4,
P
<.01 versus Ang II–induced JNK activity). The direct addition of 12-HETE also induced a time-dependent JNK activation. 12-HETE (10
−7
mol/L) activated JNK activity, with a peak at 10 minutes (3.43±0.87-fold, n=6,
P
<.02 versus control), which remained elevated until 1 hour. These results suggest that the LO pathway is a mediator of Ang II–induced JNK activation. 15-HETE can also activate JNK at 5 minutes, but this activity was reduced at 30 minutes and could not be seen at 1 hour, indicating that the time course was different from that seen with 12-HETE.
N
-Acetylcysteine (NAC), an antioxidant, was used to perturb intracellular reactive oxygen intermediate (ROI) levels to assess the role of endogenous ROIs in regulating JNK activity. Pretreatment of cells with 500 μmol/L NAC for 1 hour attenuated ≈50% of Ang II–induced JNK activation, suggesting that ROIs, at least partially, mediate Ang II–induced JNK activation. Furthermore, 12-HETE–induced JNK activation was reduced by ≈90% by NAC. Finally, pertussis toxin completely blocked 12-HETE–induced JNK activation, suggesting that G
i
-protein signaling participates in 12-HETE–induced effects. These results suggest that LO activation plays a role in mediating Ang II–induced JNK activation in part by altering the redox tone and G
i
-protein signaling of cells.
Ionic Mechanisms of Propagation in Cardiac Tissue
Authors: Shaw Robin M. , Rudy Yoram
Published: June 2012

Abstract
In cardiac tissue, reduced membrane excitability and reduced gap junction coupling both slow conduction velocity of the action potential. However, the ionic mechanisms of slow conduction for the two conditions are very different. We explored, using a multicellular theoretical fiber, the ionic mechanisms and functional role of the fast sodium current, I
Na
, and the L-type calcium current, I
Ca(L)
, during conduction slowing for the two fiber conditions. A safety factor for conduction (SF) was formulated and computed for each condition. Reduced excitability caused a lower SF as conduction velocity decreased. In contrast, reduced gap junction coupling caused a paradoxical increase in SF as conduction velocity decreased. The opposite effect of the two conditions on SF was reflected in the minimum attainable conduction velocity before failure: decreased excitability could reduce velocity to only one third of control (from 54 to 17 cm/s) before failure occurred, whereas decreased coupling could reduce velocity to as low as 0.26 cm/s before block. Under normal conditions and conditions of reduced excitability, I
Ca(L)
had a minimal effect on SF and on conduction. However, I
Ca(L)
played a major role in sustaining conduction when intercellular coupling was reduced. This phenomenon demonstrates that structural, nonmembrane factors can cause a switch of intrinsic membrane processes that support conduction. High intracellular calcium concentration, [Ca]
i
, lowered propagation safety and caused earlier block when intercellular coupling was reduced. [Ca]
i
affected conduction via calcium-dependent inactivation of I
Ca(L)
. The increase of safety factor during reduced coupling suggests a major involvement of uncoupling in stable slow conduction in infarcted myocardium, making microreentry possible. Reliance on I
Ca(L)
for this type of conduction suggests I
Ca(L)
as a possible target for antiarrhythmic drug therapy.
Pulsatile Stretch Stimulates Superoxide Production and Activates Nuclear Factor-κB in Human Coronary Smooth Muscle
Authors: Hishikawa Keiichi , Oemar Barry S. , Yang Zhihong , Lüscher Thomas F.
Published: June 2012

Abstract
There is increasing evidence that oxidative stress is of pathophysiological importance in cardiovascular disease. Mechanical forces such as pulsatility may also contribute. Using human coronary artery smooth muscle cells (HCAS), we tested the hypothesis that stretch-induced cell proliferation is associated with oxidative stress. Stretch induced DNA synthesis in HCAS, and this was prevented by the antioxidants
N
-acetylcysteine and pyrrolidinedithiocarbamate (PDTC). Pulsatile stretch also increased superoxide production from HCAS in a time- and stretch-dependent manner. Stretch-induced superoxide production was inhibited by diphenyleneiodoniumchloride, an NADPH oxidase inhibitor, and
p
-chloromercuriphenylsulfonic acid, an NADH oxidase inhibitor, but not by the xanthine oxidase inhibitor oxypurinol or the cyclooxygenase inhibitor indomethacin. In electrophoretic mobility shift assays, tumor necrosis factor-α activated nuclear factor-κB (NF-κB) with a peak at ≈3 hours, whereas pulsatile stretch showed sustained activation during stimulation for up to 24 hours. The sustained activation of NF-κB was abolished by cotreatment with
N
-acetylcysteine or PDTC. Furthermore, treatment of HCAS with antisense p65 and p50 oligodeoxynucleotides of NF-κB inhibited stretch-induced DNA synthesis. We propose that pulsatile stretch increases oxidative stress and, in turn, promotes DNA synthesis via NF-κB in cultured human coronary artery smooth muscle cells.
β-Arrestin1 Knockout Mice Appear Normal but Demonstrate Altered Cardiac Responses to β-Adrenergic Stimulation
Authors: Conner David. A. , Mathier Michael A. , Mortensen Richard M. , Christe Michael , Vatner Stephen F. , Seidman Christine E. , Seidman J. G.
Published: June 2012

Abstract
β-Arrestin1 knockout mice were studied to define the physiological role of β-arrestin1 in the regulation of G protein–coupled receptors. β-Arrestin1 is thought to be involved in the desensitization of many G protein–associated cell surface receptors, particularly β-adrenergic receptors. Homozygous knockout mice are overtly normal. Resting cardiovascular parameters modulated by β-adrenergic receptors such as heart rate, blood pressure, and left ventricular ejection fraction are not changed. However, homozygous mutants are more sensitive to β-receptor agonist–stimulated increases in ejection fraction, consistent with a role of β-arrestin1 in β-adrenergic receptor desensitization. We conclude that β-arrestin1 is important for in vivo G protein–coupled receptor desensitization and that this aspect of desensitization represents a mechanism for fine-tuning responses. However, β-arrestin1 does not appear to be required for development or for other essential biological functions.
Cardioprotective Effect of Diazoxide and Its Interaction With Mitochondrial ATP-Sensitive K
+
Channels
Authors: Garlid Keith D. , Paucek Petr , Yarov-Yarovoy Vladimir , Murray Holt N. , Darbenzio Raymond B. , D’Alonzo Albert J. , Lodge Nicholas J. , Smith Mark A. , Grover Gary J.
Published: June 2012

Abstract
Previous studies showed a poor correlation between sarcolemmal K
+
currents and cardioprotection for ATP-sensitive K
+
channel (K
ATP
) openers. Diazoxide is a weak cardiac sarcolemmal K
ATP
opener, but it is a potent opener of mitochondrial K
ATP
, making it a useful tool for determining the importance of this mitochondrial site. In reconstituted bovine heart K
ATP
, diazoxide opened mitochondrial K
ATP
with a K
1/2
of 0.8 μmol/L while being 1000-fold less potent at opening sarcolemmal K
ATP
. To compare cardioprotective potency, diazoxide or cromakalim was given to isolated rat hearts subjected to 25 minutes of global ischemia and 30 minutes of reperfusion. Diazoxide and cromakalim increased the time to onset of contracture with a similar potency (EC
25
, 11.0 and 8.8 μmol/L, respectively) and improved postischemic functional recovery in a glibenclamide (glyburide)-reversible manner. In addition, sodium 5-hydroxydecanoic acid completely abolished the protective effect of diazoxide. Whole-myocyte studies showed that diazoxide was significantly less potent than cromakalim in increasing sarcolemmal K
+
currents. Diazoxide shortened ischemic action potential duration significantly less than cromakalim at equicardioprotective concentrations. We also determined the effects of cromakalim and diazoxide on reconstituted rat mitochondrial cardiac K
ATP
activity. Cromakalim and diazoxide were both potent activators of K
+
flux in this preparation (K
1/2
values, 1.1±0.1 and 0.49±0.05 μmol/L, respectively). Both glibenclamide and sodium 5-hydroxydecanoic acid inhibited K
+
flux through the diazoxide-opened mitochondrial K
ATP
. The profile of activity of diazoxide (and perhaps K
ATP
openers in general) suggests that they protect ischemic hearts in a manner that is consistent with an interaction with mitochondrial K
ATP
.
The Protective Effect of Late Preconditioning Against Myocardial Stunning in Conscious Rabbits Is Mediated by Nitric Oxide Synthase
Authors: Bolli Roberto , Manchikalapudi Srinivas , Tang Xian-Liang , Takano Hitoshi , Qiu Yumin , Guo Yiru , Zhang Qin , Jadoon Asad K.
Published: June 2012

Abstract
Seventy-four conscious rabbits undergoing a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3) were assigned to nine groups. In group I (controls, n=8), the recovery of systolic wall thickening (WTh) after the sixth reperfusion was markedly improved on days 2 and 3 compared with day 1, indicating late preconditioning (PC) against myocardial stunning; the total deficit of WTh after the sixth reperfusion was reduced by 56% on day 2 and 50% on day 3 compared with day 1 (
P
<.01). Administration on day 2 of the nonselective NO synthase (NOS) inhibitor
N
ω
-nitro-
l
-arginine (L-NA) (group II, n=8) or of the selective inducible NOS inhibitors aminoguanidine (AG) (group IV, n=8) and
S
-methylisothiourea sulfate (SMT) (group VI, n=6) completely abrogated late PC against stunning on day 2. On day 3, the expected PC effect became manifest in all groups. Administration of L-NA, AG, or SMT on day 1 (groups III [n=7], V [n=6], and VII [n=5], respectively) had no discernible effect on the deficit of WTh on day 1, indicating that these agents do not augment the severity of myocardial stunning in nonpreconditioned myocardium. In group VIII (n=7), the abrogation of late PC by SMT on day 2 was completely reversed by the concomitant administration of
l
-arginine (595 mg/kg IV), indicating that it was not due to nonspecific NOS-unrelated actions. Administration of
l
-arginine alone on day 2 (group IX [n=5]) had no effect on the deficit of WTh. Furthermore, administration of L-NA on day 1 (group III) prevented the appearance of the PC effect on day 2, whereas AG (group V) and SMT (group VI) did not, suggesting that the development of late PC on day 1 is triggered by the endothelial (type III) isoform of NOS. This study demonstrates that three structurally different NOS inhibitors (L-NA, AG, and SMT), given 24 hours after the PC ischemia, consistently abrogate late PC against myocardial stunning in conscious rabbits, indicating that this cardioprotective effect is mediated by the activity of NOS. The results obtained with AG and SMT specifically implicate the inducible (type II) isoform as the mediator of the protection on day 2. Previous studies have shown that NO triggers the development of late PC. The present results indicate that NO plays a dual role in late PC against stunning, acting initially as the trigger and subsequently as the mediator of the protection.
Molecular Mechanisms of Anoxia/Reoxygenation-Induced Neutrophil Adherence to Cultured Endothelial Cells
Authors: Ichikawa Hiroshi , Flores Sonia , Kvietys Peter R. , Wolf Robert E. , Yoshikawa Toshikazu , Granger D. Neil , Aw Tak Yee
Published: June 2012

Abstract
The objectives of this study were to (1) determine the time course of neutrophil adhesion to monolayers of human umbilical vein endothelial cells (HUVECs) that were exposed to 60 minutes of anoxia followed by 30 to 600 minutes of reoxygenation and (2) define the mechanisms responsible for both the early (minutes) and late (hours) hyperadhesivity of postanoxic HUVECs to human neutrophils. The results clearly demonstrate that anoxia/reoxygenation (A/R) leads to a biphasic increase in neutrophil adhesion to HUVECs, with peak responses occurring at 30 minutes (phase 1) and 240 minutes (phase 2) after reoxygenation. Oxypurinol and catalase inhibited phase-1 adhesion, suggesting a role for xanthine oxidase and H
2
O
2
. In comparison, platelet activating factor (PAF) contributed to both phases of neutrophil adhesion. Anti–intercellular adhesion molecule-1 (ICAM-1) and anti–P-selectin antibodies (monoclonal antibodies [mAbs]) attenuated phase-1 neutrophil adhesion, consistent with roles for constitutively expressed ICAM-1 and enhanced surface expression of preformed P-selectin. Phase-2 neutrophil adhesion was attenuated by an anti–E-selectin mAb, indicating a dominant role of this adhesion molecule in the late phase response. Pretreatment with actinomycin D and cycloheximide or with competing
ds
-oligonucleotides containing the nuclear factor-κB or activator protein-1 cognate DNA sequences significantly attenuated phase-2 response, suggesting a role for de novo macromolecule synthesis. Surface expression of ICAM-1, P-selectin, and E-selectin on HUVECs correlated with the phase-1 and -2 neutrophil adhesion responses. Collectively, these findings indicate that A/R elicits a two-phase neutrophil–endothelial cell adhesion response that involves transcription-independent and transcription-dependent surface expression of different endothelial cell adhesion molecules.