bluuebunny/crossref_metadata_2025 huggingface dataset:
c-Myb–Dependent Cell Cycle Progression and Ca
2+
Storage in Cultured Vascular Smooth Muscle Cells
Authors: Husain Mansoor , Bein Kiflai , Jiang Lianwei , Alper Seth L. , Simons Michael , Rosenberg Robert D.
Published: June 2012

Abstract
Considerable controversy surrounds the role of the c-
myb
proto-oncogene in vascular smooth muscle cells (VSMCs). Previous investigations using antisense approaches have suggested a relationship between c-
myb
expression, cell cycle progression, and cytoplasmic Ca
2+
concentration ([Ca
2+
]
cyt
). However, the ability of certain antisense oligonucleotides to bind and inactivate growth factors allows alternative explanations. To define more specifically the role of c-Myb in cultured VSMCs (SVE and A10 cell lines), we have generated stable cell clones expressing a dominant-negative c-Myb lacking critical elements of the DNA binding domain (Δ5-SVE) and transiently transfected cell populations (GRE-M
En
-SVE and GRE-M
En
-A10) expressing a glucocorticoid-inducible chimeric protein that targets the Drosophila
Engrailed
repressor domain to c-Myb–responsive promoters. The Δ5-SVE clones and GRE-M
En
cell populations exhibit a 60% reduction in mean intracellular c-Myb activity, as measured by cotransfection assays with a c-Myb–responsive reporter, a 42% decrease in the mean S phase entry of growth-arrested (G
0
) cells after serum stimulation, and a 36% inhibition of mean cell proliferation over 4 days. These cells also display 28% (34-nmol/L) and 30% (42-nmol/L) reductions in mean [Ca
2+
]
cyt
at G
0
and at the G
1
/S interface, respectively, as well as significant reductions in the peak [Ca
2+
]
cyt
responses to thapsigargin (5 μmol/L) and caffeine (10 mmol/L). These latter reductions in operationally defined Ca
2+
pools were observed both at different stages of the cell cycle and after transient induction of the dominant-interfering construct, suggesting that c-Myb regulates these releasable Ca
2+
stores independent of its effects on cell cycle progression.
Inhibition of Myocardial Crossbridge Cycling by Hypoxic Endothelial Cells
Authors: Shah Ajay M. , Mebazaa Alexandre , Yang Zhao-Kang , Cuda Giovanni , Lankford Edward B. , Pepper Chris B. , Sollott Steven J. , Sellers James R. , Robotham James L. , Lakatta Edward G.
Published: June 2012

Abstract
Previous studies have shown that cardiac endothelial cells release substances that influence myocardial contraction. Since P
o
2
is an important stimulus that modulates endothelial function, we investigated the effects of acute moderate hypoxia and reoxygenation on the release of cardioactive factors by endothelial cells. Endothelial cells cultured from several vascular beds were superfused with normoxic (equilibrated with room air; P
o
2
, ≈160 mm Hg) or hypoxic (P
o
2
, 40 to 50 mm Hg) physiological buffer solution, and the superfusates were reequilibrated to a P
o
2
of ≈160 mm Hg and then tested for their effects on various myocardial assays. Endothelial cell viability and buffer ionic composition were unaltered after the superfusion procedures. The superfusates of hypoxic endothelial cells induced rapid, potent, reversible inhibition of isolated cardiac myocyte contraction without reducing cytosolic Ca
2+
transients. This activity was not lost after heating (95°C) and was present in low molecular weight (
M
r
, <500) superfusate fractions. Hypoxic endothelial superfusate reduced unloaded shortening velocity of human skinned soleus muscle fibers. It markedly depressed in vitro actin motility over cardiac myosin and reduced the rate of actin-activated cardiac myosin ATPase activity but had no effect on corresponding smooth muscle myosin assays. Reoxygenation of hypoxic endothelial cells resulted in loss of this inhibitory activity. These data indicate that cultured endothelial cells respond to acute moderate hypoxia by releasing an unidentified substance(s) that inhibits myocardial crossbridge cycling, independent of Ca
2+
or other second messenger signaling pathways. Such a mechanism could have important implications for the regulation of oxygen supply-demand balance in the heart and be relevant to conditions such as myocardial hibernation.
Rapid Inactivation Determines the Rectification and [K
+
]
o
Dependence of the Rapid Component of the Delayed Rectifier K
+
Current in Cardiac Cells
Authors: Yang Tao , Snyders Dirk J. , Roden Dan M.
Published: June 2012

Abstract
Two characteristic features of the rapid component of the cardiac delayed rectifier current (
I
Kr
) are prominent inward rectification and an unexpected reduction in activating current with decreased [K
+
]
o
. Similar features are observed with heterologous expression of
HERG
, the gene thought to encode the channel carrying
I
Kr
; moreover, recent studies indicate that the mechanism underlying rectification of
HERG
current is the inactivation that channels rapidly undergo during depolarizing pulses. The present studies were designed to determine the mechanism of
I
Kr
rectification and [K
+
]
o
sensitivity in the mouse atrial myocyte cell line, AT-1 cells. Reducing [Mg
2+
]
i
to 0, which reverses inward rectification of some K
+
channels, did not alter
I
Kr
current-voltage relationships, although it did decrease sensitivity to the
I
Kr
blockers dofetilide and quinidine 2- to 5-fold. To determine the presence and extent of fast inactivation of
I
Kr
in AT-1 cells, a brief hyperpolarizing pulse (20 ms to −120 mV) was applied during long depolarizations. Immediately after this pulse, a very large outward current that decayed rapidly to the previous activating current baseline was observed. This outward current component was blocked by the
I
Kr
-specific inhibitor dofetilide, indicating that it represented recovery from fast inactivation during the hyperpolarizing step, with fast reinactivation during the return to depolarized potential. With removal of inactivation using this approach, current-voltage relationships for
I
Kr
([K
+
]
o
, 1 to 20 mmol/L) were linear and reversed close to the predicted Nernst potential for K
+
. In addition, decreased [K
+
]
o
decreased the time constants for open→inactivated and inactivated→open transitions. Thus, in these cardiac myocytes, as with heterologously expressed
HERG
,
I
Kr
undergoes fast inactivation that determines its characteristic inward rectification. These studies demonstrate that the mechanism underlying decreased activating current observed at low [K
+
]
o
is more extensive fast inactivation.
Induction of P-Selectin by Oxidized Lipoproteins
Authors: Vora Devendra K. , Fang Zhuang-Ting , Liva Stephanie M. , Tyner Timothy R. , Parhami Farhad , Watson Andrew D. , Drake Thomas A. , Territo Mary C. , Berliner Judith A.
Published: June 2012

Abstract
Leukocyte binding to the endothelium is one of the earliest events in the occurrence of atherosclerosis. Leukocyte adhesion molecules involved in this process have not been definitely identified. We have found that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) for 24 hours caused a 2- to 3-fold increase of P-selectin protein, with little change in P-selectin surface expression. A 15-minute histamine treatment of cells exposed to MM-LDL caused a 50% to 100% increase in P-selectin surface expression compared with cells not treated with the lipoprotein. This increase resulted in a 2-fold increase in binding of leukocytes to the endothelium. Immunostaining of permeabilized HAECs after MM-LDL treatment also revealed a highly reproducible increase in intracellular P-selectin associated with rod-shaped structures, typical of Weibel-Palade bodies. Oxidized phospholipids were shown to be mainly responsible for the action of MM-LDL. This increased P-selectin expression was associated with MM-LDL–induced cAMP elevation. Like histamine, highly oxidized low-density lipoprotein, especially the oxidized fatty acids, caused immediate redistribution of P-selectin to the cell surface followed by reinternalization. Immunohistochemical staining showed that endothelial cells on human fatty streak lesions expressed increased levels of P-selectin compared with nonlesion areas. These studies suggest that P-selectin may play an important role in early recruitment of mononuclear cells to the subendothelium in human atherosclerosis and that oxidized lipoproteins may contribute to the increased expression of this molecule by increasing intracellular stores and causing redistribution to the cell surface.
Lipoprotein Lipase Increases Lipoprotein Binding to the Artery Wall and Increases Endothelial Layer Permeability by Formation of Lipolysis Products
Authors: Rutledge John C. , Woo Mable M. , Rezai Allen A. , Curtiss Linda K. , Goldberg Ira J.
Published: June 2012

Abstract
Mechanisms responsible for the accumulation of low-density lipoprotein (LDL) were investigated in a new model, the perfused hamster aorta. To do this, we developed a method to study LDL flux in real time in individually perfused arteries; each artery served as its own control. Using quantitative fluorescence microscopy, the rates of LDL accumulation and efflux were separately determined. Perfusion of arteries with buffer plus lipoprotein lipase (LpL) increased LDL accumulation 5-fold (0.1±0.03 mV/min [control] versus 0.5±0.05 mV/min [LpL]) by increasing LDL retention in the artery wall. This effect was blocked by heparin and monoclonal antibodies directed against the amino-terminal region of apolipoprotein B (apo B). This suggests that specific regions of apo B are involved in LDL accumulation within arteries. Also, the effect of hydrolysis of triglyceride-rich lipoproteins on endothelial barrier function was studied. We compared endothelial layer permeability using a water-soluble reference molecule, fluorescently labeled dextran. When LpL was added to hypertriglyceridemic plasma, dextran accumulation within the artery wall increased >4-fold (0.024±0.01 mV/min [control] versus 0.098±0.05 mV/min [LpL]). Under the same conditions, LpL increased LDL accumulation ≈3-fold (0.016±0.003 mV/min [control] versus 0.047±0.013 mV/min [LpL]). Rapid efflux of LDL from the artery wall indicated that increased endothelial layer permeability was the primary mechanism during periods of increased lipolysis. Our data demonstrate two LpL-mediated effects that may increase the amount of LDL in the artery wall. These findings may pertain to the observed relationship between increased postprandial lipemia and atherosclerosis.
Monocyte Chemotactic Protein-1 Increases Collateral and Peripheral Conductance After Femoral Artery Occlusion
Authors: Ito Wulf D. , Arras Margarete , Winkler Bernd , Scholz Dimitri , Schaper Jutta , Schaper Wolfgang
Published: June 2012

Abstract
Monocytes are activated during collateral artery growth in vivo, and monocyte chemotactic protein-1 (MCP-1) has been shown to be upregulated by shear stress in vitro. In order to investigate whether MCP-1 enhances collateral growth after femoral artery occlusion, 12 rabbits were randomly assigned to receive either MCP-1, PBS, or no local infusion via osmotic minipump. Seven days after occlusion, isolated hindlimbs were perfused with autologous blood at different pressures, measuring flows at maximal vasodilation via flow probe and radioactive microspheres, as well as peripheral pressures. This allowed the calculation of collateral (thigh) and peripheral (lower limb) conductances from pressure-flow tracings (slope of the curve). Collateral growth on postmortem angiograms was restricted to the thigh and was markedly enhanced with MCP-1 treatment. Both collateral and peripheral conductances were significantly elevated in animals with MCP-1 treatment compared with the control group, reaching values of nonoccluded hindlimbs after only 1 week of occlusion (collateral conductance, 70.6±19.23 versus 25.1±2.59 mL/min per 100 mm Hg;
P
<.01; peripheral conductance, 119.3±22.37 versus 45.4±6.80 mL/min per 100 mm Hg;
P
<.05). These results suggest that activation of monocytes plays an important role in collateral growth as well as in capillary sprouting.
Non–Voltage-Gated Ca
2+
Influx Through Mechanosensitive Ion Channels in Aortic Baroreceptor Neurons
Authors: Sullivan Margaret J. , Sharma Ram V. , Wachtel Ruth E. , Chapleau Mark W. , Waite Laurie J. , Bhalla Ramesh C. , Abboud Francois M.
Published: June 2012

Abstract
The mechanisms underlying mechanotransduction in baroreceptor neurons (BRNs) are undefined. In this study, we specifically identified aortic baroreceptor neurons in primary neuronal cell cultures from nodose ganglia of rats. Aortic baroreceptor neurons were identified by labeling their soma with the fluorescent dye 1,1′-dioleyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI) applied to the aortic arch. Using Ca
2+
imaging with fura 2, we examined these BRNs for evidence of Ca
2+
influx and determined its mechanosensitivity and voltage dependence. Mechanical stimuli were produced by ejecting buffer from a micropipette onto the cell surface with a pneumatic picopump, producing a shift in the center of mass of the cell that was related to intensity of stimulation. Ninety-three percent of DiI-labeled neurons responded to mechanical stimulation with an increase in [Ca
2+
]
i
. The magnitude of the increases in [Ca
2+
]
i
was directly related to the intensity of the stimulus and required the presence of external Ca
2+
. The trivalent cations Gd
3+
or La
3+
in equimolar concentrations (20 μmol/L) eliminated the K
+
-induced rises in [Ca
2+
]
i
, demonstrating that both trivalent cations are equally effective at blocking voltage-gated Ca
2+
channels in these baroreceptor neurons. In contrast, the mechanically induced increases in [Ca
2+
]
i
were blocked by Gd
3+
(20 μmol/L) only and not by La
3+
(20 μmol/L). Stretch-activated channels (SACs) have been shown in other preparations to be blocked by Gd
3+
specifically. Our data demonstrate that (1) BRNs, specifically identified as projecting to the aortic arch, have ion channels that are sensitive to mechanical stimuli; (2) mechanically induced Ca
2+
influx in these cells is mediated by a Gd
3+
-sensitive ion channel and not by voltage-gated Ca
2+
channels; (3) the magnitude of the Ca
2+
influx is dependent on the intensity of the stimulus and the degree and duration of deformation; and (4) repeated stimuli of the same intensity result in comparable increases in [Ca
2+
]
i
. We conclude that mechanical stimulation increases Ca
2+
influx into aortic BRNs independent of voltage-gated Ca
2+
channels. The results suggest that Gd
3+
-sensitive SACs are the mechanoelectrical transducers in baroreceptors.
Alterations of Na
+
Currents in Myocytes From Epicardial Border Zone of the Infarcted Heart
Authors: Pu Jielin , Boyden Penelope A.
Published: June 2012

Abstract
Previously, we have shown abnormalities in V̇
max
and in the recovery of V̇
max
in myocytes dispersed from the epicardial border zone (EBZ) of the 5-day infarcted canine heart (myocytes from the EBZ [IZs]). Thus, we sought to determine the characteristics of the whole-cell Na
+
current (
I
Na
) in IZs and compare them with the
I
Na
of cells from noninfarcted hearts (myocytes from noninfarcted epicardium [NZs]).
I
Na
was recorded using patch-clamp techniques under conditions that eliminated contaminating currents and controlled
I
Na
for measurement (19°C, 5 mmol/L [Na
+
]
o
). Peak
I
Na
density (at −25 mV) was significantly reduced in IZs (4.9±0.44 pA/pF, n=36) versus NZs (12.8±0.55 pA/pF, n=54;
P
<.001), yet the half-maximal activation voltage (V
0.5
), time course of decay, and time to peak
I
Na
were no different. However, in IZs, V
0.5
of the availability curve (
I
/
I
max
curve) was shifted significantly in the hyperpolarizing direction (−80.2±0.48 mV in NZs [n=45] versus −83.9±0.59 mV in IZs [n=27],
P
<.01). Inactivation of
I
Na
directly from a depolarized prepotential (−60 mV) was significantly accelerated in IZs versus NZs (fast and slow time constants [τ
1
and τ
2
, respectively] were as follows: NZs [n=28], τ
1
=71.5±5.6 ms and τ
2
=243.7±17.1 ms; IZs [n=21], τ
1
=36.3±2.4 ms and τ
2
=153±11.3 ms;
P
<.001). Recovery of
I
Na
from inactivation was dependent on the holding potential (V
H
) in both cell types but was significantly slower in IZs. At V
H
=−90 mV,
I
Na
recovery had a lag in 18 (82%) of 22 IZs (with a 17.6±1.5-ms lag) versus 2 (9%) of 22 NZs (with 5.9- and 8.7-ms lags); at V
H
=−100 mV, τ
1
=60.9±2.6 ms and τ
2
=352.8±28.1 ms in NZs (n=41) versus τ
1
=76.3±4.8 ms and τ
2
=464.4±47.2 ms in IZs (n=26) (
P
<.002 and
P
<.03, respectively); at V
H
=−110 mV, τ
1
=33.4±1.8 ms and τ
2
=293.5±33.6 ms in NZs (n=21) versus τ
1
=44.3±2.9 ms and τ
2
=388.4±38 ms in IZs (n=18) (
P
<.002 and
P
<.07, respectively). In sum,
I
Na
is reduced, and its kinetics are altered in IZs. These changes may underlie the altered excitability and postrepolarization refractoriness of the ventricular fibers of the EBZ, thus contributing to reentrant arrhythmias in the infarcted heart.
Emigrated Rat Neutrophils Adhere to Cardiac Myocytes via α
4
Integrin
Authors: Reinhardt Paul H. , Ward Christopher A. , Giles Wayne R. , Kubes Paul
Published: June 2012

Abstract
Previous work has shown that neutrophils isolated from whole blood adhere to cardiac myocytes via CD18 (β
2
integrin) to cause injury to the heart cells. In vitro, we have found that upon endothelial transmigration, neutrophils can also express α
4
β
1
; however, whether this contributes to neutrophil adhesion to parenchymal cells remains entirely unknown. Unstimulated and tumor necrosis factor-α–stimulated rat cardiac myocytes adherent to gelatin-coated coverslips supported
N
-formyl-Met-Leu-Phe (fMLP)–induced neutrophil (isolated from whole blood) adhesion entirely via CD18 (blocked with monoclonal antibody [mAb] WT-3). Emigrated neutrophils spontaneously adhered to cardiac myocytes also entirely via CD18. However, if fMLP was used to restimulate emigrated neutrophils, the adhesion to cardiac myocytes was entirely independent of CD18. Although an anti–α
4
integrin antibody (mAb TA-2) alone did not reduce the emigrated neutrophil-myocyte interaction, dual administration of TA-2 and WT-3 reduced adhesion by 81%. α
4
integrin was expressed in small amounts on the surface of circulating neutrophils, increased following transmigration, and then increased >5-fold after restimulation of these emigrated neutrophils. In the presence of the anti-CD18 antibody, a fibronectin fragment (FN-40) but not a vascular cell adhesion molecule-1 antibody (mAb 5F10) inhibited neutrophil-myocyte interactions by 80%. Similar results were seen when the rat chemokine CINC-gro was used instead of fMLP, suggesting that the α
4
-dependent adhesion was not specific to fMLP. These data demonstrate that α
4
integrin can be physiologically induced to increase in number and avidity after neutrophil emigration and that this adhesion molecule can cause firm adhesion to fibronectin on parenchymal cells, including rat cardiac myocytes.
Inhibition of L-Type Ca
2+
Channel Current in Rat Ventricular Myocytes by Terfenadine
Authors: Liu Shi , Melchert Russell B. , Kennedy Richard H.
Published: June 2012

Abstract
To elucidate possible mechanisms underlying the cardiotoxicity of terfenadine, the effect of this antihistamine on L-type Ca
2+
channel current (
I
Ca,L
) was studied in adult rat ventricular myocytes using the whole-cell patch-clamp technique. Myocytes were held at −70 mV and internally dialyzed and externally perfused with Na
+
- and K
+
-free solutions; exposure to terfenadine (10
−9
to 5×10
−6
mol/L) resulted in a concentration-dependent inhibition of peak
I
Ca,L
, with a half-maximum inhibition concentration (IC
50
) of 142 nmol/L. The terfenadine-induced inhibition of
I
Ca,L
was not mediated via effects on histamine H
1
receptors, because 1 μmol/L triprolidine, a more selective and potent H
1
antagonist, had no effect on I
Ca,L.
In this study, we found that terfenadine (1) increased both the fast and slow time constants of I
Ca,L
inactivation, (2) shifted the steady state inactivation of I
Ca,L
to more negative potentials, and (3) elicited a tonic block and a use-dependent block of I
Ca,L
. The terfenadine-induced tonic and use-dependent block and the steady state inhibition of I
Ca,L
were voltage dependent. Both tonic and use-dependent blocks of I
Ca,L
by terfenadine at −40 mV were greater than that at −70 mV, and blocks were partially released by applying a long hyperpolarizing prepulse to −90 mV. These results suggest that terfenadine binds to L-type Ca
2+
channels in inactivated and rested states and inhibits
I
Ca,L
predominantly by interacting with the inactivated state with an apparent dissociation constant of 60 nmol/L. Open-state block could be observed only at high concentrations of terfenadine. The high-affinity interaction of terfenadine with the inactivated state of L-type Ca
2+
channels may play an important role in its cardiotoxicity under pathophysiological conditions, such as ischemia.
Low-Density Lipoprotein Receptor Knockout Mice Exhibit Exaggerated Microvascular Responses to Inflammatory Stimuli
Authors: Henninger Dwight D. , Gerritsen Mary E. , Granger D. Neil
Published: June 2012

Abstract
The objective of this study was to determine whether genetically induced hypercholesterolemia affects leukocyte–endothelial cell interactions in postcapillary venules of the mouse cremaster muscle. Leukocyte adhesion, emigration, and other microvascular parameters were assessed in venules of normal (wild-type) and low-density lipoprotein receptor–deficient (LDLr
−/
−
) mice maintained on either normal rodent chow or on a high cholesterol diet (HCD). Measurements were obtained under control conditions and after administration of either leukotriene B
4
(LTB
4
), platelet-activating factor (PAF), or tumor necrosis factor-α (TNF-α). Elevated numbers of adherent and emigrated leukocytes were observed in venules of LDLr
−/
−
(compared with wild-type) mice on HCD, both under baseline conditions and after exposure to either LTB
4
, PAF, or TNF-α. Plasma TNF-α levels were also elevated in LDLr
−/
−
versus wild-type mice. Administration of blocking monoclonal antibodies demonstrated that intercellular adhesion molecule-1, but not vascular cell adhesion molecule-1, mediates the exaggerated leukocyte–endothelial cell adhesion observed in LDLr
−/
−
mice. The results of these studies indicate that chronic hypercholesterolemia predisposes the microvasculature to intense leukocyte–endothelial cell adhesion in response to different inflammatory stimuli.
FGF-1–Induced Platelet-Derived Growth Factor-A Chain Gene Expression in Endothelial Cells Involves Transcriptional Activation by Early Growth Response Factor-1
Authors: Delbridge Gabrielle J. , Khachigian Levon M.
Published: June 2012

Abstract
Fibroblast growth factor-1 (FGF-1), a prototype member of the heparin-binding growth factor family, is a potent mitogen for vascular endothelial cells and a variety of other cell types. FGF-1 can induce the expression of the platelet-derived growth factor-A chain (PDGF-A) gene in endothelial cells; however, the underlying transcriptional mechanisms are not known. We used serial 5′ deletion and transient transfection analysis of the human PDGF-A promoter to demonstrate that a 16-bp element, located 55 to 71 bp upstream of the transcriptional start site, is required for FGF-1–inducible promoter–dependent expression. This region contains nucleotide recognition elements for the early growth response gene product, early growth response factor-1 (Egr-1), and the related zinc-finger transcription factor, Sp1. Reverse-transcription polymerase chain reaction revealed that FGF-1 induced Egr-1 mRNA expression within 30 minutes. Electrophoretic mobility shift, supershift, and Western blot analysis demonstrated that Egr-1 protein accumulated in the nuclei of endothelial cells exposed to the growth factor, whereas levels of Sp1 did not change. Egr-1 bound to the FGF-1 response element in the proximal PDGF-A promoter in a specific and time-dependent manner. These findings indicate that Egr-1 plays a key regulatory role in FGF-1–inducible endothelial PDGF-A expression and implicate this transcription factor in pathological settings in which these mitogens are both expressed.
High Glucose Concentrations Increase Endothelial Cell Permeability via Activation of Protein Kinase Cα
Authors: Hempel Albrecht , Maasch Christian , Heintze Ute , Lindschau Carsten , Dietz Rainer , Luft Friedrich C. , Haller Hermann
Published: June 2012

Abstract
Endothelial cell permeability is impaired in diabetes mellitus and may be increased by high extracellular glucose concentrations. High glucose activates protein kinase C (PKC), a family of kinases vital to intracellular signaling. We tested the hypothesis that high glucose concentration activates PKC in endothelial cells and leads to an increase in endothelial cell permeability via distinct PKC isoforms. Porcine aortic endothelial cells were used, and the PKC isoforms α, δ, ε, ζ, and θ were identified in these cells. Glucose caused a rapid dose-dependent increase in endothelial cell permeability, with an EC
50
of 17.5 mmol/L. Phorbol 12-myristate 13-acetate (TPA) induced an increase in permeability very similar to that elicited by glucose. The effect of glucose and TPA was totally reversed by preincubating the cells with the PKC inhibitors staurosporine (10
−8
mol/L) and Goe 6976 (10
−8
mol/L). Downregulation of PKC by preincubation with TPA for 24 hours also abolished the effect of glucose and TPA on endothelial cell permeability. High glucose (20 mmol/L) caused an increase in PKC activity at 2, 10, and 30 minutes. Cell fractionation and Western blot analysis showed a glucose-induced translocation of PKCα and PKCε. Confocal microscopy confirmed the translocation and showed an association of PKCα and PKCε with nuclear structures and the cell membrane. Specific antisense oligodesoxynucleotides (ODNs) against PKCα reduced the expression of the isoform, abolished the effects of glucose on endothelial cell permeability completely, and reduced the TPA effect significantly. In contrast, specific antisense ODNs against PKCε had no effect on glucose-induced permeability and only a minor effect on the TPA-induced increase in permeability. We conclude that an increase in extracellular glucose leads to a rapid dose-dependent increase in endothelial cell permeability via the activiation of PKC and that this effect is mediated by the PKC isoform α.
Expression Pattern of Connexin Gene Products at the Early Developmental Stages of the Mouse Cardiovascular System
Authors: Delorme Bruno , Dahl Edgar , Jarry-Guichard Thérèse , Briand Jean-Paul , Willecke Klaus , Gros Daniel , Théveniau-Ruissy Magali
Published: June 2012

Abstract
The synchronized contraction of myocytes in cardiac muscle requires the structural and functional integrity of the gap junctions present between these cells. Gap junctions are clusters of intercellular channels formed by transmembrane proteins of the connexin (Cx) family. Products of several Cx genes have been identified in the mammalian heart (eg, Cx45, Cx43, Cx40, and Cx37), and their expression was shown to be regulated during the development of the myocardium. Cx43, Cx40, and Cx45 are components of myocyte gap junctions, and it has also been demonstrated that Cx40 was expressed in the endothelial cells of the blood vessels. The aim of the present work was to investigate the expression and regulation of Cx40, Cx43, and Cx37 during the early stages of mouse heart maturation, between 8.5 days post coitum (dpc), when the first rhythmic contractions appear, and 14.5 dpc, when the four-chambered heart is almost completed. At 8.5 dpc, only the reverse-transcriptase polymerase chain reaction technique has allowed identification of Cx43, Cx40, and Cx37 gene transcripts in mouse heart, suggesting a very low activity level of these genes. From 9.5 dpc, all three transcripts became detectable in whole-mount in situ–hybridized embryos, and the most obvious result was the labeling of the vascular system with Cx40 and Cx37 anti-sense riboprobes. Cx40 and Cx37 gene products (transcript and/or protein) were demonstrated to be expressed in the vascular endothelial cells at all stages examined. By contrast, only Cx37 gene products were found in the endothelial cells of the endocardium. In heart, Cx37 was expressed exclusively in these cells, which rules out any direct involvement of this Cx in the propagation of electrical activity between myocytes and the synchronization of contractions. Between 9.5 and 11.5 dpc, Cx40 gene activation in myocytes was demonstrated to proceed according to a caudorostral gradient involving first the primitive atrium and the common ventricular chamber (9.5 dpc) and then the right ventricle (11.5 dpc). During this period of heart morphogenesis, there is clearly a temporary and asymmetrical regionalization of the Cx40 gene expression that is superimposed on the functional regionalization. In addition, comparison of Cx40 and Cx43 distribution at the above developmental stages has shown that these Cxs have overlapping (left ventricle) or complementary (atrial tissue and right ventricle) expression patterns.
Interleukin-1β Inhibits Phospholamban Gene Expression in Cultured Cardiomyocytes
Authors: McTiernan Charles F. , Lemster Bonnie H. , Frye Carole , Brooks Steven , Combes Alain , Feldman Arthur M.
Published: June 2012

Abstract
Phospholamban is a key regulatory protein that defines diastolic function. Proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) can depress contractility and intracellular Ca
2+
currents and transients. An alteration in phospholamban expression is a possible pathway by which these cytokines modulate cardiac function. To test this hypothesis, primary cultures of neonatal rat cardiomyocytes were incubated with IL-1β, TNF-α, or both, and the level of phospholamban transcripts was examined by Northern blot analyses. Phospholamban transcript levels were decreased ≈50% (
P
<.0001) in cells exposed to 2 ng/mL IL-1β (20 hours), whereas TNF-α had no effect. Western blot analyses showed that IL-1β also reduced phospholamban protein levels (60% of control,
P
<.0001). The effects on transcript levels were gene specific; IL-1β induced transcripts for inducible NO synthase (iNOS), did not alter GAPDH transcripts, and reduced sarcoplasmic reticulum Ca
2+
-ATPase (65% of control,
P
<.001) transcripts. Cardiomyocytes treated with IL-1β showed no alterations in basal contractile parameters (maximum velocity of contraction and relaxation and maximal amplitude of contraction) but were unresponsive to β-adrenergic stimulation. Studies performed in the presence of second-messenger inhibitors showed that the effect of IL-1β on phospholamban transcript levels was blocked by dexamethasone, was insensitive to inhibitors of iNOS, cyclooxygenase, or tyrosine kinases, but was enhanced by the addition of the protein kinase inhibitor staurosporine. These data demonstrate that IL-1β alters the expression of phospholamban, a key regulator of cardiac contractility, at both the transcript and protein levels. The results suggest novel mechanisms by which IL-1β may modify cardiac function.
Angiotensin II Stimulates Tyrosine Phosphorylation of Phospholipase C-γ–Associated Proteins
Authors: Schmitz Udo , Ishida Mari , Berk Bradford C.
Published: June 2012

Abstract
Stimulation of phospholipase C-γ (PLC-γ) is a critical event in angiotensin II (Ang II) signal transduction. We have previously shown that in rat aortic smooth muscle (RASM) cells Ang II stimulates tyrosine phosphorylation of PLC-γ via activation of c-Src. Because we failed to demonstrate a direct association between c-Src and PLC-γ, we hypothesized that a linker protein mediates the interaction between these molecules. To identify PLC-γ–associated proteins, RASM cells were labeled with [
32
P]orthophosphate and stimulated with 100 nmol/L Ang II for 5 minutes. PLC-γ was immunoprecipitated, and associated proteins were characterized by autoradiography and Western blotting with anti-phosphotyrosine antibodies. Ang II stimulated the phosphorylation of 47-, 60-, 84-, and 97-kD PLC-γ–associated proteins. Because Ang II increased tyrosine phosphorylation of only the 97-kD protein, we characterized p97 further. An important role for Src in tyrosine phosphorylation of p97 was suggested by findings that p97 phosphorylation was inhibited by the selective Src-family kinase inhibitor CP-118,556, diminished in mouse aortic smooth muscle (MASM) cells from c-Src knockout mice compared with wild-type MASM cells, and increased in v-Src–transformed NIH-3T3 cells compared with wild-type NIH-3T3 cells. These studies are the first to define a PLC-γ–associated protein that may be required for Ang II–mediated signal transduction.
Left Ventricular Stretch Stimulates Angiotensin II– Mediated Phosphatidylinositol Hydrolysis and Protein Kinase C ε Isoform Translocation in Adult Guinea Pig Hearts
Authors: Paul Karamchand , Ball Nancy A. , Dorn Gerald W. , Walsh Richard A.
Published: June 2012

Abstract
Stretch of neonatal cardiomyocytes activates phospholipase C with production of inositol trisphosphate and diacylglycerol in part by formation of angiotensin II (Ang II). However, the response of this pathway to physical stimuli in the adult heart is poorly understood. Thus, in isovolumic perfused guinea pig hearts, we characterized stretch-mediated phosphatidylinositol (PI) hydrolysis and protein kinase C (PKC) isoform translocation using elevated diastolic pressure. Balloon dilatation (minimum diastolic pressure, 25 mm Hg) of the left ventricle (LV) stimulated PI hydrolysis. Pretreatment of stretched hearts with the specific angiotensin (AT
1
) receptor antagonist losartan abolished stretch-mediated accumulation of inositol phosphates. To examine PKC isoform expression and activation under these conditions, whole-heart extracts were examined by immunoblot analysis. Ang II translocated PKCε to the particulate fraction. 4β-Phorbol 12-myristate 13-acetate but not an inactive congener translocated PKCε to the particulate fraction and produced a decrease in myocardial contractile function. Mechanical stretch also translocated PKCε to the particulate fraction; however, this was attenuated but not abolished by losartan. We conclude that in the adult heart, LV dilatation produced stretch-mediated activation of phospholipase C, which resulted in PI hydrolysis and PKCε activation in part by stimulation of the local renin angiotensin system. In contrast to stretch-mediated inositol phosphate accumulation, PKCε translocation is not prevented by AT
1
receptor blockade, indicating that this PKC isoform can be activated in response to mechanical deformation by an Ang II–independent mechanism in the adult myocardium.
Activated
Rho
A Stimulates c-
fos
Gene Expression in Myocardial Cells
Authors: Ueyama Tomomi , Sakoda Tsuyoshi , Kawashima Seinosuke , Hiraoka Eiji , Hirata Ken-ichi , Akita Hozuka , Yokoyama Mitsuhiro
Published: June 2012

Abstract Rho
regulates various cell functions, including cell morphology and motility. However, the functional role of
Rho
on the signaling pathway in myocardial cells (MCs) is unknown. In the present study, we attempted to explore the mode of
Rho
action for c-
fos
gene expression in MCs. Expression of the c-
fos
promoter/enhancer linked to the luciferase reporter gene (c-
fos
luciferase) was stimulated by the wild type of
Rho
A and the point-mutated active form of
Rho
A (
Rho
A Val14) but not the biologically inactive effector domain mutant of
Rho
A.
Rho
GDP dissociation inhibitor inhibited the action of
Rho
A on c-
fos
luciferase expression. The deletion analysis revealed that the c-
fos
serum response element (SRE) and the 12-
O
-tetradecanoylphorbol-13-acetate response element (TRE) mainly account for c-
fos
luciferase expression by
Rho
A Val14. The c-
fos
SRE mutant, which contains an intact binding site for the serum response factor but lacks the ternary complex factor binding site, was activated by
Rho
A Val14. The action of
Rho
A Val14 on c-
fos
luciferase expression was not inhibited by downregulation of protein kinase C, protein kinase C inhibitors, or tyrosine kinase inhibitors. These results indicate that activated
Rho
A stimulates c-
fos
gene expression through the c-
fos
SRE and TRE and that the signaling pathway from activated
Rho
A to the c-
fos
promoter/enhancer is independent of these inhibitor-sensitive pathways in MCs.
Trophic Effect of Human Pericardial Fluid on Adult Cardiac Myocytes
Authors: Corda Stefano , Mebazaa Alexandre , Gandolfini Maria-Pia , Fitting Catherine , Marotte Françoise , Peynet Jacqueline , Charlemagne Danièle , Cavaillon Jean-Marc , Payen Didier , Rappaport Lydie , Samuel Jane-Lyse
Published: June 2012

Abstract
Pericardial fluid (PF) may contain myocardial growth factors that exert paracrine actions on cardiac myocytes. The aims of this study were (1) to investigate the effects of human PF and serum, collected from patients undergoing cardiac surgery, on the growth of cultured adult rat cardiac myocytes and (2) to relate the growth activity of both fluids to the adaptive changes in overloaded human hearts. Both PF and serum increased the rate of protein synthesis, measured by [
14
C]phenylalanine incorporation in adult rat cardiomyocytes (PF, +71.9±8.2% [n=17]; serum, +14.9±6.5% [n=13]; both
P
<.01 versus control medium). The effects of both PF and serum on cardiomyocyte growth correlated positively with the respective left ventricular (LV) mass. However, the magnitude of change with PF was 3-fold greater than with serum (
P
<.01). These trophic effects of PF were mimicked by exogenous basic fibroblast growth factor (FGF2) and inhibited by anti-FGF2 antibodies and transforming growth factor-β (TGF-β), suggesting a relationship to FGF2. In addition, FGF2 concentration in PF was 20 times greater than in serum. On the other hand, the LV mass–dependent trophic effect, present in both fluids, was independent of FGF2 concentration or other factors, such as angiotensin II, atrial natriuretic factor, and TGF-β. These data suggest that FGF2 in human PF is a major determining factor in normal myocyte growth, whereas unidentified LV mass–dependent factor(s), present in both PF and serum, participates in the development of ventricular hypertrophy.
Macrophage-Dependent Regulation of Syndecan Gene Expression
Authors: Li Jian , Brown Lawrence F. , Laham Roger J. , Volk Rudiger , Simons Michael
Published: June 2012

Abstract
Heparan sulfates in the extracellular matrix are required for a variety of biological processes, including cellular response to heparin-binding growth factors. However, little is known regarding the regulation of their expression and composition under pathophysiological conditions. In the present study, we have investigated the regulation of expression of two key heparan sulfate chain–carrying core proteins, syndecan-1 and syndecan-4, in a mouse/rat infarct model of tissue injury and repair. Induction of myocardial infarction was associated with a prompt increase in expression of both syndecan genes. Although infiltrating macrophages accounted for a substantial increase in syndecan expression, increased expression was noted in the levels of syndecan-1 mRNA in endothelial cells and syndecan-4 mRNA in cardiac myocytes. This increase in expression was limited to the immediate peri-infarct region and was absent from remote areas of the left or right ventricles. The influx of blood-derived macrophages in the heart correlated with the appearance of PR-39 peptide, which has previously been shown to increase syndecan expression in vitro. Studies in the op/op mice strain (which demonstrates sharply reduced levels of circulating monocytes) showed that myocardial infarction was associated with markedly reduced levels of macrophage influx and corresponding reduction in the expression of PR-39 and both syndecan genes. Pretreatment of op/op mice with granulocyte macrophage colony–stimulating factor restored myocardial macrophage content with corresponding restoration of PR-39/syndecan expression. In summary, myocardial infarction is associated with a distinct spatial and temporal pattern of syndecan-1 and -4 gene expression, which is induced by an influx of blood-derived macrophages.
Angiotensin II–Induced Leukocyte Adhesion on Human Coronary Endothelial Cells Is Mediated by E-Selectin
Authors: Gräfe Michael , Auch-Schwelk Wolfgang , Zakrzewicz Andreas , Regitz-Zagrosek Vera , Bartsch Petra , Graf Kristof , Loebe Matthias , Gaehtgens Peter , Fleck Eckart
Published: June 2012

Abstract
Clinical data suggest a link between the activation of the renin-angiotensin system and cardiovascular ischemic events. Leukocyte accumulation in the vessel wall is a hallmark of early atherosclerosis and plaque progression. E-Selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) are adhesion molecules participating in mediating interactions between leukocytes and endothelial cells and have been found to be expressed in atherosclerotic plaques. We investigated whether angiotensin II, the effector of the renin-angiotensin system, influences the endothelial expression of E-selectin, VCAM-1, and ICAM-1. In coronary endothelial cells derived from explanted human hearts, angiotensin II (10
−11
to 10
−5
mol/L) induced a concentration-dependent increase in E-selectin expression. The effect was measured by cell ELISA and duplex reverse-transcription polymerase chain reaction (RT-PCR) and reached its maximum at 10
−7
mol/L. Angiotensin II induced only a small increase in E-selectin expression in cardiac microvascular endothelial cells. VCAM-1 and ICAM-1 were not affected by angiotensin II stimulation. In addition, the effect of angiotensin II–induced E-selectin expression on leukocyte adhesion was quantified under flow conditions. Angiotensin II (10
−7
mol/L) increased leukocyte adhesion significantly to 67% of the maximal effect by tumor necrosis factor-α at a wall shear stress of 2 dyne/cm
2
. This adhesion was found to be E-selectin dependent, as demonstrated by blocking antibodies. The AT
1
-receptor antagonist DUP 753 significantly reduced E-selectin–dependent adhesion, whereas the AT
2
-receptor antagonist PD 123177 had no inhibitory effect. In addition, only AT
1
-receptor, but not AT
2
-receptor, mRNA could be detected by RT-PCR in coronary endothelial cells. Therefore, it is suggested that AT
1
receptors mediate the effects of angiotensin II on E-selectin expression and leukocyte adhesion on coronary endothelial cells.
Tachycardia-Induced Changes in Na
+
Current in a Chronic Dog Model of Atrial Fibrillation
Authors: Gaspo Rania , Bosch Ralph F. , Bou-Abboud Elias , Nattel Stanley
Published: June 2012

Abstract
We have previously shown that chronic rapid atrial activation (400 bpm) reduces atrial conduction velocity in dogs, contributing to the development of a substrate supporting sustained atrial fibrillation (AF). However, the cellular and ionic mechanisms underlying these functional changes have not been defined. We applied whole-cell patch-clamp techniques to atrial myocytes from dogs subjected to atrial pacing at 400 bpm for 7 days (P7, n=6) and 42 days (P42, n=5) and compared the results with those from sham-operated dogs similarly instrumented but without pacemaker activation (P0, n=6). Rapid atrial pacing allowed for the induction of sustained AF in 67% and 100% of dogs paced for 7 and 42 days, respectively, and significantly decreased conduction velocity under P7 and P42 conditions. In dogs paced for 7 days, Na
+
current (
I
Na
) density was reduced by 28% at −40 mV (
P
<.0001, n=59 cells).
I
Na
changes were even more decreased under P42 conditions, by ≈52% at −40 mV (
P
<.0001): from −78.7±4.6 pA/pF (P0, n=28 cells) to −37.7±3.0 pA/pF (P42, n=43 cells).
I
Na
was significantly reduced at all voltages ranging from −65 to −10 mV. Voltage-dependent activation and inactivation properties, activation kinetics, and recovery from inactivation were not altered by rapid atrial pacing; however, inactivation kinetics were slowed. AF duration was related to mean
I
Na
in each dog (
r
2
=.573,
P
<.001). We conclude that rapid atrial activation significantly reduces both conduction velocity and
I
Na
density. Since
I
Na
is a major determinant of conduction velocity, our data point to
I
Na
reduction as a potentially important mechanism contributing to the substrate for AF in this model.
Molecular Determinants of Stereoselective Bupivacaine Block of hKv1.5 Channels
Authors: Franqueza Laura , Longobardo Mónica , Vicente Javier , Delpón Eva , Tamkun Michael M. , Tamargo Juan , Snyders Dirk J. , Valenzuela Carmen
Published: June 2012

Abstract
Enantiomers of local anesthetics are useful probes of ion channel structure that can reveal three-dimensional relations for drug binding in the channel pore and may have important clinical consequences. Bupivacaine block of open hKv1.5 channels is stereoselective, with the
R
(+)-enantiomer being 7-fold more potent than the
S
(−)-enantiomer (
K
d
=4.1 μmol/L versus 27.3 μmol/L). Using whole-cell voltage clamp of hKv1.5 channels and site-directed mutants stably expressed in
Ltk
−
cells, we have identified a set of amino acids that determine the stereoselectivity of bupivacaine block. Replacement of threonine 505 by hydrophobic amino acids (isoleucine, valine, or alanine) abolished stereoselective block, whereas a serine substitution preserved it [
K
d
=60 μmol/L and 7.4 μmol/L for
S
(−)- and
R
(+)-bupivacaine, respectively]. A similar substitution at the internal tetraethylammonium binding site (T477S) reduced the affinity for both enantiomers similarly, thus preserving the stereoselectivity [
K
d
=45.5 μmol/L and 7.8 μmol/L for
S
(−)- and
R
(+)-bupivacaine, respectively]. Replacement of L508 or V512 by a methionine (L508M and V512M) abolished stereoselective block, whereas substitution of V512 by an alanine (V512A) preserved it. Block of Kv2.1 channels, which carry valine, leucine, and isoleucine residues at T505, L508, and V512 equivalent sites, respectively, was not stereoselective [
K
d
=8.3 μmol/L and 13 μmol/L for
S
(−)- and
R
(+)-bupivacaine, respectively]. These results suggest that (1) the bupivacaine binding site is located in the inner mouth of the pore, (2) stereoselective block displays subfamily selectivity, and (3) a polar interaction with T505 combined with hydrophobic interactions with L508 and V512 are required for stereoselective block.