bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological Basis for Potassium (
39
K) Magnetic Resonance Imaging of the Heart
Authors: Fieno David S. , Kim Raymond J. , Rehwald Wolfgang G. , Judd Robert M.
Published: June 2012

Abstract
—The potassium cation (K
+
) is fundamentally involved in myocyte metabolism. To explore the potential utility of direct MRI of the most abundant natural isotope of potassium,
39
K, we compared
39
K magnetic resonance (MR) image intensity with regional myocardial K
+
concentrations after irreversible injury. Rabbits were subjected either to 40 minutes of in situ coronary artery occlusion and 1 hour of reperfusion (n=26) or to 24 hours of permanent occlusion (n=4). The hearts were then isolated and imaged by
39
K MRI (n=10), or tissue samples were analyzed for regional
39
K content by MR spectroscopy (n=9), K
+
and Na
+
concentrations by atomic emission spectroscopy (inductively coupled plasma atomic emission spectroscopy; n=5), or intracellular K
+
content by electron probe x-ray microanalysis (n=6). Three-dimensional
39
K MR images of the isolated hearts were acquired in 44 minutes with 3×3×3–mm resolution.
39
K MR image intensity was reduced in infarcted regions (51.7±4.8% of remote;
P
<0.001). The circumferential extent and location of regions of reduced
39
K image intensity were correlated with those of infarcted regions defined histologically (
r
=0.97 and
r
=0.98, respectively). Compared with remote regions, tissue analysis revealed that infarcted regions had reduced
39
K concentration (by MR spectroscopy, 40.5±9.3% of remote;
P
<0.001), reduced potassium-to-sodium ratio (by inductively coupled plasma atomic emission spectroscopy, 20.7±2.1% of remote;
P
<0.01), and reduced intracellular potassium (by electron probe x-ray microanalysis, K
+
peak-to-background ratio 0.95±0.32 versus 2.86±1.10, respectively;
P
<0.01). We acquired the first
39
K MR images of hearts subjected to infarction. In the pathophysiologies examined, potassium (
39
K) MR image intensity primarily reflects regional intracellular K
+
concentrations.
Local Ca
2+
Entry Through L-Type Ca
2+
Channels Activates Ca
2+
-Dependent K
+
Channels in Rabbit Coronary Myocytes
Authors: Guia Antonio , Wan Xiaodong , Courtemanche Marc , Leblanc Normand
Published: June 2012

Abstract
—Large-conductance Ca
2+
-dependent K
+
channels (K
Ca
), which are abundant on the sarcolemma of vascular myocytes, provide negative feedback via membrane hyperpolarization that limits Ca
2+
entry through L-type Ca
2+
channels (
I
CaL
). We hypothesize that local accumulation of subsarcolemmal Ca
2+
during
I
CaL
openings amplifies this feedback. Our goal was to demonstrate that Ca
2+
entry through voltage-gated
I
CaL
channels can stimulate adjacent K
Ca
channels by a localized interaction in enzymatically isolated rabbit coronary arterial myocytes voltage clamped in whole-cell or in cell-attached patch clamp mode. During slow-voltage-ramp protocols, we identified an outward K
Ca
current that is activated by a subsarcolemmal Ca
2+
pool dissociated from bulk cytosolic Ca
2+
pool (measured with indo 1) and is dependent on L-type Ca
2+
channel activity. Transient activation of unitary K
Ca
channels in cell-attached patches could be detected during long step depolarizations to +40 mV (holding potential, −40 mV; 219 pS in near-symmetrical K
+
). This local interaction between the channels required the presence of Ca
2+
in the pipette solution, was enhanced by the
I
CaL
agonist Bay K 8644, and persisted after impairment of the sarcoplasmic reticulum by incubation with 10 μmol/L ryanodine and 30 μmol/L cyclopiazonic acid for at least 60 minutes. Furthermore, we provide the first direct evidence of simultaneous openings of single K
Ca
(67 pS) and
I
CaL
(3.9 pS) channels in near-physiological conditions, near resting membrane potential. Our data imply a novel sensitive mechanism for regulating resting membrane potential and tone in vascular smooth muscle.
Angiotensin II–Induced Transactivation of Epidermal Growth Factor Receptor Regulates Fibronectin and Transforming Growth Factor-β Synthesis via Transcriptional and Posttranscriptional Mechanisms
Authors: Moriguchi Yasutaka , Matsubara Hiroaki , Mori Yasukiyo , Murasawa Satoshi , Masaki Hiroya , Maruyama Katsuya , Tsutsumi Yoshiaki , Shibasaki Yasunobu , Tanaka Yoko , Nakajima Takuma , Oda Kinichiro , Iwasaka Toshiji
Published: June 2012

Abstract
—The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of a growth factor, and recent evidence indicates transactivation of epidermal growth factor receptor (EGF-R) by G protein–coupled receptors. Here, we report the involvement of EGF-R in Ang II–induced synthesis of fibronectin and transforming growth factor-β (TGF-β) in cardiac fibroblasts. Ang II stimulated fibronectin mRNA levels dose dependently, with a maximal increase (≈5-fold) observed after 12 hours of incubation. Fibronectin synthesis induced by Ang II or calcium ionophore was completely abolished by tyrosine kinase inhibitors and intracellular Ca
2+
chelating agents. Ang II–induced fibronectin mRNA was not affected by protein kinase C inhibitors or protein kinase C depletion, whereas specific inhibition of EGF-R function by a dominant negative EGF-R mutant and tyrphostin AG1478 abolished induction of fibronectin mRNA. We isolated the rat fibronectin gene, including the 5′-flanking region, and found that the activator protein-1 (AP-1) binding site present in the promoter region was responsible for the Ang II responsiveness of this gene. A gel retardation assay revealed the binding of nuclear protein to the AP-1 site, which was supershifted with anti–c
-fos
and anti–c
-jun
but not anti–activating transcription factor (ATF)-2 antibodies. Conditioned medium from Ang II–treated cells contained TGF-β bioactivity, and addition of neutralizing TGF-β antibody modestly (46%) inhibited induction of fibronectin. Ang II–induced synthesis of TGF-β was also abolished by inhibition of EGF-R function. The effect of TGF-β was exerted by stabilizing fibronectin mRNA without affecting the promoter activity and required de novo protein synthesis. We concluded that Ang II–induced expression of fibronectin and TGF-β is mediated by downstream signaling of EGF-R transactivated by Ca
2+
-dependent tyrosine kinase and that Ang II–induced fibronectin mRNA expression is regulated by 2 different mechanisms, which are transcriptional control by binding of the c-
fos
/c-
jun
complex to the AP-1 site and posttranscriptional control by mRNA stabilization due to autocrine or paracrine effects of TGF-β. Thus, this study suggests that the action of Ang II on extracellular matrix formation should be interpreted in association with the EGF-R signaling cascade.
Activated Protein Kinase C Isoforms Target to Cardiomyocyte Caveolae
Authors: Rybin Vitalyi O. , Xu Xiaohong , Steinberg Susan F.
Published: June 2012

Abstract
—Protein kinase C (PKC) isoforms constitute an important component of the signal transduction pathway used by cardiomyocytes to respond to a variety of extracellular stimuli. Translocation to distinct intracellular sites represents an essential step in the activation of PKC isoforms, presumably as a prerequisite for stable access to substrate. Caveolae are specialized subdomains of the plasma membrane that are reported to concentrate key signaling proteins and may represent a locus for PKC action, given that PKC activators have been reported to dramatically alter caveolae morphology. Accordingly, this study examines whether PKC isoforms initiate signaling in cardiomyocyte caveolae. Phorbol ester–sensitive PKC isoforms were detected at very low levels in caveolae fractions prepared from unstimulated cardiomyocytes; phorbol 12-myristate 13-acetate (PMA) (but not 4α-PMA, which does not activate PKC) recruited calcium-sensitive PKCα and novel PKCδ and PKCε to this compartment. The subcellular localization of the phorbol ester–insensitive PKCλ isoform was not influenced by PMA. Endothelin also induced the selective translocation of PKCα and PKCε (but not PKCδ or PKCλ) to caveolae. Multiple components of the extracellular signal–regulated protein kinase (ERK) cascade, including A-Raf, c-Raf-1, mitogen-activated protein kinase kinase, and ERK, were detected in caveolae under resting conditions. Although levels of these proteins were not altered by PMA, translocation of phorbol ester–sensitive PKC isoforms to caveolae was associated with the activation of a local ERK cascade as well as the phosphorylation of a ≈36-kDa substrate protein in this fraction. Finally, a minor fraction of a protein that has been designated as a receptor for activated protein kinase C resides in caveolae and (along with caveolin-3) could represent a mechanism to target PKC isoforms to cardiomyocyte caveolae. These studies identify cardiomyocyte caveolae as a meeting place for activated PKC isoforms and their downstream target substrates.
Mechanism of Block and Identification of the Verapamil Binding Domain to HERG Potassium Channels
Authors: Zhang Shetuan , Zhou Zhengfeng , Gong Qiuming , Makielski Jonathan C. , January Craig T.
Published: June 2012

Abstract
—Calcium channel antagonists have diverse effects on cardiac electrophysiology. We studied the effects of verapamil, diltiazem, and nifedipine on HERG K
+
channels that encode
I
Kr
in native heart cells. In our experiments, verapamil caused high-affinity block of HERG current (IC
50
=143.0 nmol/L), a value close to those reported for verapamil block of L-type Ca
2+
channels, whereas diltiazem weakly blocked HERG current (IC
50
=17.3 μmol/L), and nifedipine did not block HERG current. Verapamil block of HERG channels was use and frequency dependent, and verapamil unbound from HERG channels at voltages near the normal cardiac cell resting potential or with drug washout. Block of HERG current by verapamil was reduced by lowering pH
O
, which decreases the proportion of drug in the membrane-permeable neutral form.
N
-methyl-verapamil, a membrane-impermeable, permanently charged verapamil analogue, blocked HERG channels only when applied intracellularly. Verapamil antagonized dofetilide block of HERG channels, which suggests that they may share a common binding site. The C-type inactivation-deficient mutations, Ser620Thr and Ser631Ala, reduced verapamil block, which is consistent with a role for C-type inactivation in high-affinity drug block, although the Ser620Thr mutation decreased verapamil block 20-fold more than the Ser631Ala mutation. Our findings suggest that verapamil enters the cell membrane in the neutral form to act at a site within the pore accessible from the intracellular side of the cell membrane, possibly involving the serine at position 620. Thus, verapamil shares high-affinity HERG channel blocking properties with other class III antiarrhythmic drugs, and this may contribute to its antiarrhythmic mechanism.
Reduced Subendocardial Ryanodine Receptors and Consequent Effects on Cardiac Function in Conscious Dogs With Left Ventricular Hypertrophy
Authors: Hittinger Luc , Ghaleh Bijan , Chen Jie , Edwards John G. , Kudej Raymond K. , Iwase Mitsunori , Kim Song-Jung , Vatner Stephen F. , Vatner Dorothy E.
Published: June 2012

Abstract
—The goal of this study was to examine the transmural distribution of ryanodine receptors in left ventricular (LV) hypertrophy (LVH) and its in vivo consequences. Dogs were chronically instrumented with an LV pressure gauge, ultrasonic crystals for measurement of LV internal diameter and wall thickness, and a left circumflex coronary blood flow velocity transducer. Severe LVH was induced by chronic banding of the aorta (12±1 months), which resulted in a 78% increase in LV/body weight. When ryanodine was infused directly into the circumflex coronary artery, it did not affect LV global function or systemic hemodynamics; however, it reduced LV wall thickening and delayed relaxation in the posterior wall in control dogs but was relatively ineffective in dogs with LVH. In LV sarcolemmal preparations, [
3
H]ryanodine ligand binding revealed a subendocardial/subepicardial gradient in normal dogs. In LVH there was a 45% decrease in ryanodine receptor binding and a loss in the natural subendocardial/subepicardial gradient, which roughly correlated inversely with the extent of LVH and directly with regional wall motion. Both mRNA and Western analyses revealed similar findings, with a reduction of the transmural mRNA levels and a loss in the natural gradient between subendocardial and subepicardial layers in LVH. Thus, ryanodine receptor message and binding in LVH is reduced preferentially in the subendocardium with consequent attenuation of the action of ryanodine in vivo. The selectively altered ryanodine regulation subendocardially in LVH could reconcile some of the controversy in this field and may play a role in mediating decompensation from stable LVH.
Mechanotransduction of Rat Aortic Vascular Smooth Muscle Cells Requires RhoA and Intact Actin Filaments
Authors: Numaguchi Kotaro , Eguchi Satoru , Yamakawa Tadashi , Motley Evangeline D. , Inagami Tadashi
Published: June 2012

Abstract
—The growth-promoting effect of mechanical stress on vascular smooth muscle cells (VSMCs) has been implicated in the progress of vascular disease in hypertension. Extracellular signal–regulated kinases (ERKs) have been implicated in cellular responses, such as vascular remodeling, induced by mechanical stretch. However, it remains to be determined how mechanical stretch activates ERKs. The cytoskeleton seems the most likely candidate for force transmission into the interior of the cell. Therefore, we examined (1) whether the cytoskeleton involves mechanical stretch–induced signaling, (2) whether Rho is activated by stretch, and (3) whether Rho mediates the stretch-induced signaling in rat cultured VSMCs. Mechanical stretch activated ERKs, with a peak response observed at 20 minutes, followed by a significant increase in DNA synthesis. Treatment with the ERK kinase-1 inhibitor, PD98059, inhibited the stretch-induced increase in DNA synthesis. Cytochalasin D, which selectively disrupts the network of actin filaments, markedly inhibited stretch-induced ERK activation. In the control state, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to mechanical stretch. Botulinum C3 exoenzyme, which inactivates Rho p21 (known to participate in the reorganization of the actin cytoskeleton), attenuated stretch-induced ERK activation. Inhibition of Rho kinase (p160ROCK) also suppressed stretch-induced ERK activation dose dependently. Our results suggest that mechanotransduction in VSMCs is dependent on intact actin filaments, that Rho is activated by stretch, and that Rho/p160ROCK mediates stretch-induced ERK activation and vascular hyperplasia.
Cultured Porcine Coronary Artery Smooth Muscle Cells
Authors: Christen Thomas , Bochaton-Piallat Marie-Luce , Neuville Pascal , Rensen Sander , Redard Mireille , van Eys Guillaume , Gabbiani Giulio
Published: June 2012

Abstract
—Arterial intimal thickening after endothelial injury induced in rodents has proven to be a relatively unreliable model of restenosis for testing clinically useful compounds. The same has been found for cultured rat or rabbit vascular smooth muscle cells (SMCs). To test alternative possibilities, we have studied several differentiation features of porcine coronary artery SMCs, cultured up to the 5th passage after enzymatic digestion of the media. The effects of heparin, transforming growth factor (TGF)-β
1
or TGF-β
2
, and all-
trans
-retinoic acid (tRA) on proliferation, migration, and differentiation of these cells also were examined. Porcine arterial SMCs in culture not only express high levels of α-smooth muscle (SM) actin but, contrary to rodent SMCs, also maintain an appreciable expression of SM myosin heavy chain isoforms 1 and 2, desmin, and smoothelin, a recently described late differentiation marker of vascular SMCs. We demonstrate for the first time that smoothelin is colocalized with α-SM actin in these cells. Finally, we show that in the porcine model, heparin is more potent than TGF-β
1
or TGF-β
2
and tRA in terms of inhibition of proliferation and migration and of increasing the expression of differentiation markers. This model should be a useful complement to in vivo studies of SMC differentiation and of pathological situations such as restenosis and atheromatosis.
Characterization of Insulin-Like Growth Factor-1–Induced Activation of the JAK/STAT Pathway in Rat Cardiomyocytes
Authors: Takahashi Toshiyuki , Fukuda Keiichi , Pan Jing , Kodama Hiroaki , Sano Motoaki , Makino Shinji , Kato Takahiro , Manabe Tomohiro , Ogawa Satoshi
Published: June 2012

Abstract
—This study was designed to investigate whether insulin-like growth factor-1 (IGF-1) transduces signaling through the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in cardiomyocytes and to assess the upstream signals of serine and tyrosine phosphorylation of STAT family proteins. Primary cultured neonatal rat cardiomyocytes were stimulated with IGF-1 (10
−8
mol/L). JAK1, but not JAK2 or Tyk2, was phosphorylated by IGF-1 as early as 2 minutes and peaked at 5 minutes. IGF-1 induced both tyrosine and serine phosphorylation of STAT1 and STAT3. Tyrosine phosphorylation of STAT1 peaked at 15 minutes and correlated with that of JAK1, whereas that of STAT3 was sustained up to 120 minutes and was dissociated from the activation of JAK1. Tyrosine phosphorylation of STAT3 was unaffected by the preincubation with CV11974 (AT
1
blocker), TAK044 (endothelin-1 receptor blocker), RX435 (anti-gp130 blocking antibody), PD98058, wortmannin, EDTA, or KN62 but was significantly attenuated by BAPTA-AM and chelerythrine. The time course of a gel mobility shift of SIE (
sis
-inducing element) coincided with the phosphorylation of STAT3. Serine phosphorylation of STAT1 peaked at 30 minutes and that of STAT3 was observed from 5 to 60 minutes. These results indicated that (1) IGF-1 activated JAK1 but not JAK2 or Tyk2 in rat cardiomyocytes; (2) IGF-1 induced both tyrosine and serine phosphorylation of STAT1 and STAT3; and (3) the tyrosine phosphorylation of STAT3 was not caused by JAK1 alone, and protein kinase C and intracellular Ca
2+
were required for phosphorylation.
Human Activin-A Is Expressed in the Atherosclerotic Lesion and Promotes the Contractile Phenotype of Smooth Muscle Cells
Authors: Engelse Marten A. , Neele Jolanda M. , van Achterberg Tanja A. E. , van Aken Benien E. , van Schaik Ron H. N. , Pannekoek Hans , de Vries Carlie J. M.
Published: June 2012

Abstract
—Activin is a member of the transforming growth factor-β superfamily, and it modulates the proliferation and differentiation of various target cells. In this study, we investigated the role of activin in the initiation and progression of human atherosclerosis. The expression of activin, its physiological inhibitor follistatin, and activin receptors were assayed in human vascular tissue specimens that represented various stages of atherogenesis. In situ hybridization experiments revealed activin mRNA in endothelial cells and macrophages and a strong induction of activin expression in neointimal smooth muscle cells from the early onset of atherogenesis. We developed an “in situ free-activin binding assay” by using biotinylated follistatin, which allowed us to detect bioactive activin at specific sites in atherosclerotic lesions. The mRNAs encoding the activin receptors are expressed similarly in normal and atherosclerotic tissue, which indicates that activin-A signaling in atherogenesis is most likely dependent on changes in growth factor concentrations rather than on receptor levels. In vitro, activin induces the contractile, nonproliferative phenotype in cultured smooth muscle cells, as is reflected by increased expression of smooth muscle-specific markers (SMα-actin and SM22α). Our data provide evidence that activin induces redifferentiation of neointimal smooth muscle cells, and we hypothesize that activin is involved in plaque stabilization.
G Protein β3 Subunit 825T Allele and Enhanced Coronary Vasoconstriction on α
2
-Adrenoceptor Activation
Authors: Baumgart Dietrich , Naber Christoph , Haude Michael , Oldenburg Olaf , Erbel Raimund , Heusch Gerd , Siffert Winfried
Published: June 2012

Abstract
—Recently, α
2
-adrenoceptor activation was shown to play an important role in the vasoconstriction of normal coronary arteries, whereas in the presence of atherosclerosis, the activation of both α
1
- and α
2
-adrenoceptors reduces coronary blood flow in humans. α
2
-Adrenoceptors activate pertussis toxin (PTX)-sensitive G proteins, whereas α
1
-adrenoceptors couple to PTX-insensitive G proteins. Thus, the 825T allele of the β3 subunit of heterotrimeric G proteins, associated with enhanced PTX-sensitive G protein signaling, was expected to determine the α
2
-adrenoceptor–, but not the α
1
-adrenoceptor–, mediated reduction in coronary blood flow (CBF). Genotyping was performed on 48 individuals. Twelve of the 48 received the α
1
-adrenoceptor agonist methoxamine (MTX; 5 mg IC), and 12 received the α
2
-adrenoceptor agonist BHT 933 (BHT; 5 mg IC). Twenty-four additional individuals received both MTX and BHT during the same investigational procedure. CBF was calculated on the basis of coronary angiography and intracoronary Doppler flow velocity measurement. Drug-related ischemia was assessed on the basis of ST-segment changes and angina pectoris. In response to BHT, but not to MTX, CBF was reduced to a significantly greater extent in 825T allele carriers (58±4%, n=16) than in individuals homozygous for the C825 allele (28±4%, n=19,
P
=0.001). This finding was independent of cholesterol levels, mean arterial blood pressure, and the presence or absence of coronary artery disease. Ischemic events in response to BHT occurred more frequently in 825T allele carriers than in homozygous 825C allele carriers (
P
=0.01). α
2
-Adrenoceptor coronary vasoconstriction is genetically determined and significantly enhanced in
GNB3
825T allele carriers.
Estrogen Stimulates Neuronal Nitric Oxide Synthase Protein Expression in Human Neutrophils
Authors: García-Durán Margarita , de Frutos Trinidad , Díaz-Recasens Joaquín , García-Gálvez Gema , Jiménez Ana , Montón Mercedes , Farré Jerónimo , de Miguel Lourdes Sánchez , González-Fernández Fernando , Arriero María del Mar , Rico Luis , García Rosa , Casado Santos , López-Farré Antonio
Published: June 2012

Abstract
—Recent studies have postulated the contribution of nitric oxide (NO) released by the endothelium to the beneficial effects of estrogen. Despite a neuronal-type NO synthase (nNOS) described in neutrophils, less is known about the effect of estrogen in these cells. The aim of the present study was to analyze the expression of nNOS protein in human neutrophils under different estrogenic conditions. We first analyzed nNOS expression in neutrophils obtained from premenopausal women. During the first 2 days of the follicular phase (low circulating estrogen concentrations), nNOS expression in neutrophils was reduced with respect to that found in neutrophils obtained from the same donors during the ovulatory phase (high circulating estrogen concentrations). Moreover, the expression of nNOS protein in neutrophils obtained from postmenopausal women after transdermal estrogen therapy was markedly enhanced with respect to that observed before the treatment. In vitro incubation of neutrophils derived from men for 6 hours with 17β-estradiol (10
−10
to 10
−8
mol/L) upregulated the expression of nNOS protein. The 17β-estradiol receptor antagonists, tamoxifen (10
−8
mol/L) and ICI 182780 (10
−8
mol/L), inhibited the upregulation of nNOS protein induced by 17β-estradiol. The putative functional implication was denoted by a reduced expression of the CD18 antigen on the surface of 17β-estradiol–incubated neutrophils, which was accompanied by a decreased adhesive capacity. Both effects were prevented by an NO antagonist. In conclusion, the in vivo levels of circulating estrogen concentrations seem to be associated with the level of nNOS protein expression in neutrophils from women. Moreover, low doses of 17β-estradiol upregulate nNOS protein expression in neutrophils from men. The increased ability of 17β-estradiol–incubated neutrophils derived from men to produce NO reduced their adhesive properties.
Adenoviral
RB2/p130
Gene Transfer Inhibits Smooth Muscle Cell Proliferation and Prevents Restenosis After Angioplasty
Authors: Claudio Pier Paolo , Fratta Luigi , Farina Felicia , Howard Candace M. , Stassi Giorgio , Numata Shin-ichiro , Pacilio Carmen , Davis Alan , Lavitrano Marialuisa , Volpe Massimo , Wilson James M. , Trimarco Bruno , Giordano Antonio , Condorelli Gianluigi
Published: June 2012

Abstract
—Smooth muscle cell (SMC) proliferation that results in neointima formation is implicated in the pathogenesis of atherosclerotic plaques and accounts for the high rates of restenosis that occur after percutaneous transluminal coronary angioplasty, a widespread treatment for coronary artery disease. Endothelial lesions trigger intense proliferative signals to the SMCs of the subintima, stimulating their reentry into the cell cycle from a resting G
0
state, resulting in neointima formation and vascular occlusion. Cellular proliferation is negatively controlled by growth-regulatory or tumor-suppressor genes, or both, such as the retinoblastoma gene family members (
RB/p105, p107, RB2/p130
). In the present study, we show that
RB2/p130
inhibited SMC proliferation in vitro and in vivo. We used the rat carotid artery model of restenosis to demonstrate that adenovirus-mediated localized arterial transduction of
RB2/p130
at the time of angioplasty significantly reduced neointimal hyperplasia and prevented restenosis. Furthermore, the ability of pRb2/p130 to block proliferation correlated with its ability to bind and sequester the E2F family of transcription factors, which are important mediators of cell cycle progression. These results imply that
RB2/p130
could be an important target for vascular gene therapy.
Altered Expression of Cell Cycle Proteins and Prolonged Duration of Cardiac Myocyte Hyperplasia in p27
KIP1
Knockout Mice
Authors: Poolman Robert A. , Li Jian-Mei , Durand Beatrice , Brooks Gavin
Published: June 2012

Abstract
—The precise role of cell cycle–dependent molecules in controlling the switch from cardiac myocyte hyperplasia to hypertrophy remains to be determined. We report that loss of p27
KIP1
in the mouse results in a significant increase in heart size and in the total number of cardiac myocytes. In comparison to p27
KIP1
+/+ myocytes, the percentage of neonatal p27
KIP1
−/− myocytes in S phase was increased significantly, concomitant with a significant decrease in the percentage of G
0
/G
1
cells. The expressions of proliferating cell nuclear antigen, G
1
/S and G
2
/M phase–acting cyclins, and cyclin-dependent kinases (CDKs) were upregulated significantly in ventricular tissue obtained from early neonatal p27
KIP1
−/− mice, concomitant with a substantial decrease in the expressions of G
1
phase–acting cyclins and CDKs. Furthermore, mRNA expressions of the embryonic genes atrial natriuretic factor and α-skeletal actin were detectable at significant levels in neonatal and adult p27
KIP1
−/− mouse hearts but were undetectable in p27
KIP1
+/+ hearts. In addition, loss of p27
KIP1
was not compensated for by the upregulation of other CDK inhibitors. Thus, the loss of p27
KIP1
results in prolonged proliferation of the mouse cardiac myocyte and perturbation of myocyte hypertrophy.
Regulation of Vascular Smooth Muscle Cell Apoptosis
Authors: Bai Hong-zhi , Pollman Matthew J. , Inishi Yoji , Gibbons Gary H.
Published: June 2012

Abstract
—Our objective was to define the signaling mechanisms by which mitogens such as insulin-like growth factor-I (IGF-I) regulate vascular smooth muscle cell (VSMC) apoptosis. We confirmed that IGF-I inhibits serum withdrawal–induced apoptosis of cultured VSMCs in a dose-dependent and time-dependent fashion. To test the hypothesis that the phosphatidylinositol (PI) 3-kinase signaling pathway regulates VSMC survival, we examined the relationship between PI 3-kinase activity and cell fate. PI 3-kinase was elevated in viable VSMCs maintained in serum-containing medium, declined significantly in response to serum withdrawal, and increased in response to IGF-I–induced survival. Moreover, blockade of PI 3-kinase with 2 structurally dissimilar inhibitors (wortmannin or LY294002) abolished the capacity of IGF-I to maintain VSMC viability. Similarly, transient transfection of a dominant-negative Δp85 PI 3-kinase mutant construct abrogated the capacity of IGF-I to prevent VSMC death. Thus, PI 3-kinase is a critical antiapoptotic signal in VSMCs. To define the distal element of the antiapoptotic cascade, we tested the hypothesis that IGF-I inhibits the influence of the proapoptotic gene Bad. Indeed, IGF-I stimulates increased expression of the inactive, phosphorylated form of Bad by a PI 3-kinase–dependent pathway. Moreover, the proapoptotic effect of Bad was attenuated by the stimulation of IGF-I. Thus, growth factors appear to prevent VSMC death by activating signal transduction pathways linked to apoptotic regulatory genes.
Nitric Oxide Regulates Shear Stress–Induced Early Growth Response-1
Authors: Chiu J. J. , Wung B. S. , Hsieh H. J. , Lo L. W. , Wang D. L.
Published: June 2012

Abstract
—Endothelial cells (ECs) subjected to shear stress constantly release nitric oxide (NO). The effect of NO on shear stress–induced endothelial responses was examined. ECs subjected to shear stress induced a transient and shear force–dependent increase in early growth response-1 (Egr-1) mRNA levels. Treatment of ECs with an NO donor,
S
-nitroso-
N
-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1), inhibited this shear stress–induced Egr-1 expression. Conversely, an NO synthase inhibitor to ECs,
N
G
-monomethyl-
l
-arginine, augmented this Egr-1 expression. NO modulation of Egr-1 expression was demonstrated by functional analysis of Egr-1 promoter activity using a chimera containing the Egr-1 promoter region (–698 bp) and reporter gene luciferase. In contrast to the enhanced promoter activity after
N
G
-monomethyl-
l
-arginine treatment, shear stress–induced Egr-1 promoter activity was attenuated after ECs were treated with an NO donor. ECs cotransfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf301), or a catalytically inactive mutant of extracellular signal–regulated kinase (ERK)–2 (mERK) inhibited shear stress–induced Egr-1 promoter activity. NO modulation of the signaling pathway was shown by its inhibitory effect on shear stress–induced ERK1/ERK2 phosphorylation and activity. This inhibitory effect was further substantiated by the inhibition of NO on both the shear stress–induced transcriptional activity of Elk-1 (an ERK substrate) and the promoter activity of a reporter construct containing serum response element. NO-treated ECs resulted in a reduction of binding of nuclear proteins to the Egr-1 binding sequences in the platelet-derived growth factor-A promoter region. These results indicate that shear stress–induced Egr-1 expression is modulated by NO via the ERK signaling pathway in ECs. Our findings support the importance of NO as a negative regulator in endothelial responses to hemodynamic forces.
A Mammalian Myocardial Cell-Free System to Study Cell Cycle Reentry in Terminally Differentiated Cardiomyocytes
Authors: Engel Felix B. , Hauck Ludger , Cardoso M. Cristina , Leonhardt Heinrich , Dietz Rainer , von Harsdorf Rüdiger
Published: June 2012

Abstract
—Cardiomyocytes withdraw from the cell cycle in the early neonatal period, rendering the adult heart incapable to regenerate after injury. In the present study, we report the establishment of a cell-free system to investigate the control of cell cycle reentry in mammalian ventricular cardiomyocyte nuclei and to specifically address the question of whether nuclei from terminally differentiated cardiomyocytes can be stimulated to reenter S phase when incubated with extracts from S-phase cells. Immobilized cardiomyocyte nuclei were incubated with nuclei and cytoplasmic extract of synchronized H9c2 muscle cells or cardiac nonmyocytes. Ongoing DNA synthesis was monitored by biotin-16-dUTP incorporation as well as proliferating cell nuclear antigen expression and localization. Nuclei and cytoplasmic extract from S-phase H9c2 cells but not from H9c2 myotubes induced DNA synthesis in 92% of neonatal cardiomyocyte nuclei. Coincubation in the presence of cycloheximide indicated that de novo translation is required for the reinduction of S phase. Similar results were obtained with adult cardiomyocyte nuclei. When coincubated with both cytoplasmic extract and nuclei or nuclear extracts of S-phase cells, >70% of adult cardiomyocyte nuclei underwent DNA synthesis. In conclusion, these results demonstrate that postmitotic ventricular myocyte nuclei are responsive to stimuli derived from S-phase cells and can thus bypass the cell cycle block. This cell-free system now makes it feasible to analyze the molecular requirements for the release of the cell cycle block and will help to engineer strategies for regenerative growth in cardiac muscle.
c-Jun Triggers Apoptosis in Human Vascular Endothelial Cells
Authors: Wang Nanping , Verna Lynne , Hardy Stephen , Zhu Yi , Ma Kuo-Sheng , Birrer Michael J. , Stemerman Michael B.
Published: June 2012

Abstract
—In endothelial cells (ECs), the transcription factor c-Jun is induced by a variety of stimuli that perturb EC function. To extend our understanding of the role of c-Jun in EC physiology, we have directed overexpression of c-Jun in human umbilical vein ECs by using a tetracycline-regulated adenoviral expression system. In this study, we report a novel observation using this system. Specific expression of c-Jun is a sufficient trigger for ECs to undergo apoptosis, as demonstrated by a set of combined assays including an ELISA specific for histone-associated DNA fragmentation, DNA laddering, and TdT-mediated dUTP nick end labeling (TUNEL). Tetracycline can effectively shut off c-Jun overexpression and prevent EC apoptosis. Cleavage of poly(ADP-ribose) polymerase was also detected in ECs overexpressing c-Jun. Moreover, inhibitors of cysteine proteases blocked the apoptosis, suggesting a caspase-associated mechanism involved in proapoptotic effects of c-Jun. To gain further insight into the role of c-Jun as a pathophysiological regulator of EC death, TAM67, a dominant-negative mutant of c-Jun, was overexpressed in human umbilical vein ECs to abrogate endogenous c-Jun/activator protein-1 activation. H
2
O
2
-triggered apoptosis was largely attenuated in ECs overexpressing TAM67. Together, these results suggest that c-Jun, as a proapoptotic molecule, may play a role in mediating the cell death program in vascular endothelium.
Intravenous Allopurinol Decreases Myocardial Oxygen Consumption and Increases Mechanical Efficiency in Dogs With Pacing-Induced Heart Failure
Authors: Ekelund Ulf E. G. , Harrison Robert W. , Shokek Ori , Thakkar Rajiv N. , Tunin Richard S. , Senzaki Hideaki , Kass David A. , Marbán Eduardo , Hare Joshua M.
Published: June 2012

Abstract
—Allopurinol, an inhibitor of xanthine oxidase, increases myofilament calcium responsiveness and blunts calcium cycling in isolated cardiac muscle. We sought to extend these observations to conscious dogs with and without pacing-induced heart failure and tested the prediction that allopurinol would have a positive inotropic effect without increasing energy expenditure, thereby increasing mechanical efficiency. In control dogs (n=10), allopurinol (200 mg IV) caused a small positive inotropic effect; (dP/dt)
max
increased from 3103±162 to 3373±225 mm Hg/s (+8.3±3.2%;
P
=0.01), but preload-recruitable stroke work and ventricular elastance did not change. In heart failure (n=5), this effect was larger; (dP/dt)
max
rose from 1602±190 to 1988±251 mm Hg/s (+24.4±8.7%;
P
=0.03), preload-recruitable stroke work increased from 55.8±9.1 to 84.9±12.2 mm Hg (+28.1±5.3%;
P
=0.02), and ventricular elastance rose from 6.0±1.6 to 10.5±2.2 mm Hg/mm (
P
=0.03). Allopurinol did not affect myocardial lusitropic properties either in control or heart failure dogs. In heart failure dogs, but not controls, allopurinol decreased myocardial oxygen consumption (–49±4.6%;
P
=0.002) and substantially increased mechanical efficiency (stroke work/myocardial oxygen consumption; +122±42%;
P
=0.04). Moreover, xanthine oxidase activity was ≈4-fold increased in failing versus control dog hearts (387±125 versus 78±72 pmol/min · mg
–1
;
P
=0.04) but was not detectable in plasma. These data indicate that allopurinol possesses unique inotropic properties, increasing myocardial contractility while simultaneously reducing cardiac energy requirements. The resultant boost in myocardial contractile efficiency may prove beneficial in the treatment of congestive heart failure.
Oxygen Sensitivity of Cloned Voltage-Gated K
+
Channels Expressed in the Pulmonary Vasculature
Authors: Hulme Joanne T. , Coppock Elizabeth A. , Felipe Antonio , Martens Jeffery R. , Tamkun Michael M.
Published: June 2012

Abstract
—Hypoxic pulmonary vasoconstriction is initiated by inhibiting one or more voltage-gated potassium (Kv) channel in the vascular smooth muscle cells (VSMCs) of the small pulmonary resistance vessels. Although progress has been made in identifying which Kv channel proteins are expressed in pulmonary arterial (PA) VSMCs, there are conflicting reports regarding which channels contribute to the native O
2
-sensitive K
+
current. In this study, we examined the effects of hypoxia on the Kv1.2, Kv1.5, Kv2.1, and Kv9.3 α subunits expressed in mouse L cells using the whole-cell patch-clamp technique. Hypoxia (P
o
2
=≈30 mm Hg) reversibly inhibited Kv1.2 and Kv2.1 currents only at potentials more positive than 30 mV. In contrast, hypoxia did not alter Kv1.5 current. Currents generated by coexpression of Kv2.1 with Kv9.3 α subunits were reversibly inhibited by hypoxia in the voltage range of the resting membrane potential (E
M
) of PA VSMCs (≈28% at −40 mV). Coexpression of Kv1.2 and Kv1.5 α subunits produced currents that displayed kinetic and pharmacological properties distinct from Kv1.2 and Kv1.5 channels expressed alone. Moreover, hypoxia reversibly inhibited Kv1.2/Kv1.5 current activated at physiologically relevant membrane potentials (≈65% at −40 mV). These results indicate that (1) hypoxia reversibly inhibits Kv1.2 and Kv2.1 but not Kv1.5 homomeric channels, (2) Kv1.2 and 1.5 α subunits can assemble to form an O
2
-sensitive heteromeric channel, and (3) only Kv1.2/Kv1.5 and Kv2.1/Kv9.3 heteromeric channels are inhibited by hypoxia in the voltage range of the PA VSMC E
M
. Thus, these heteromeric channels are strong candidates for the K
+
channel isoforms initiating hypoxic pulmonary vasoconstriction.
Transient and Steady-State Effects of Shear Stress on Endothelial Cell Adherens Junctions
Authors: Noria Sabrena , Cowan Douglas B. , Gotlieb Avrum I. , Langille B. Lowell
Published: June 2012

Abstract
—Endothelial cells exhibit profound changes in cell shape in response to altered shear stress that may require disassembly/reassembly of adherens junction protein complexes that mediate cell-cell adhesion. To test this hypothesis, we exposed confluent porcine aortic endothelial cells to 15 dyne/cm
2
of shear stress for 0, 8.5, 24, or 48 hours, using a parallel plate flow chamber. Cells were fixed and stained with antibodies to vascular endothelial (VE) cadherin, α-catenin, β-catenin, or plakoglobin. Under static conditions, staining for all proteins was intense and peripheral, forming a nearly continuous band around the cells at cell-cell junctions. After 8.5 hours of shear stress, staining was punctate and occurred only at sites of continuous cell attachment. After 24 or 48 hours of shear, staining for VE-cadherin, α-catenin, and β-catenin was intense and peripheral, forming a band of “dashes” (adherens plaques) that colocalized with the ends of stress fibers that inserted along the lateral membranes of cells. Staining for plakoglobin was not observed after 24 hours of shear stress, but returned after 48 hours. Western blot analysis indicated that protein levels of VE-cadherin, α-catenin, and plakoglobin decreased, whereas β-catenin levels increased after 8.5 hours of shear stress. As cell shape change reached completion (24 to 48 hours), all protein levels were upregulated except for plakoglobin, which remained below control levels. The partial disassembly of adherens junctions we have observed during shear induced changes in endothelial cell shape may have important implications for control of the endothelial permeability barrier and other aspects of endothelial cell function.
Ca
2+
Influx Through Ca
2+
Channels in Rabbit Ventricular Myocytes During Action Potential Clamp
Authors: Puglisi José L. , Yuan Weilong , Bassani José W. M. , Bers Donald M.
Published: June 2012

Abstract
—Ca
2+
influx via Ca
2+
current (
I
Ca
) during the action potential (AP) was determined at 25°C and 35°C in isolated rabbit ventricular myocytes using AP clamp. Contaminating currents through Na
+
and K
+
channels were eliminated by using Na
+
- and K
+
-free solutions, respectively. DIDS (0.2 mmol/L) was used to block Ca
2+
-activated chloride current (
I
Cl(Ca)
). When the sarcoplasmic reticulum (SR) was depleted of Ca
2+
by preexposure to 10 mmol/L caffeine, total Ca
2+
entry via
I
Ca
during the AP was ≈12 μmol/L cytosol (at both 25°C and 35°C). Similar Ca
2+
influx at 35°C and 25°C resulted from a combination of higher and faster peak
I
Ca
, offset by more rapid
I
Ca
inactivation at 35°C. During repeated AP clamps, the SR gradually fills with Ca
2+
, and consequent SR Ca
2+
release accelerates
I
Ca
inactivation during the AP. During APs and contractions in steady state, total Ca
2+
influx via
I
Ca
was reduced by ≈50% but was again unaltered by temperature (5.6±0.2 μmol/L cytosol at 25°C, 6.0±0.2 μmol/L cytosol at 35°C). Thus, SR Ca
2+
release is responsible for sufficient
I
Ca
inactivation to cut total Ca
2+
influx in half. However, because of the kinetic differences in
I
Ca
, the amount of Ca
2+
influx during the first 10 ms, which presumably triggers SR Ca
2+
release, is much greater at 35°C.
I
Ca
during a first pulse, given just after the SR was emptied with caffeine, was subtracted from
I
Ca
during each of 9 subsequent pulses, which loaded the SR. These difference currents reflect
I
Ca
inactivation due to SR Ca
2+
release and thus indicate the time course of local [Ca
2+
] in the subsarcolemmal space near Ca
2+
channels produced by SR Ca
2+
release (eg, maximal at 20 ms after the AP activation at 35°C). Furthermore, the rate of change of this difference current may reflect the rate of SR Ca
2+
release as sensed by L-type Ca
2+
channels. These results suggest that peak SR Ca
2+
release occurs within 2.5 or 5 ms of AP upstroke at 35°C and 25°C, respectively.
I
Cl(Ca)
might also indicate local [Ca
2+
], and at 35°C in the absence of DIDS (when
I
Cl(Ca)
is prominent), peak
I
Cl(Ca)
also occurred at a time comparable to the peak
I
Ca
difference current. We conclude that SR Ca
2+
release decreases the Ca
2+
influx during the AP by ≈50% (at both 25°C and 35°C) and that changes in
I
Ca
(and
I
Cl(Ca)
), which depend on SR Ca
2+
release, provide information about local subsarcolemmal [Ca
2+
]. The full text of this article is available at http://www.circresaha.org.
Regulation of Ca
2+
Homeostasis by Atypical Na
+
Currents in Cultured Human Coronary Myocytes
Authors: Boccara Gilles , Choby Cécile , Frapier Jean Marc , Quignard Jean François , Nargeot Joël , Dayanithi Govindan , Richard Sylvain
Published: June 2012

Abstract
—Primary cultured human coronary myocytes (HCMs) derived from ischemic human hearts express an atypical voltage-gated tetrodotoxin (TTX)-sensitive sodium current (
I
Na
). The whole-cell patch-clamp technique was used to study the properties of
I
Na
in HCMs. The variations of intracellular calcium ([Ca
2+
]
i
) and sodium ([Na
+
]
i
) were monitored in non–voltage-clamped cells loaded with Fura-2 or benzofuran isophthalate, respectively, using microspectrofluorimetry. The activation and steady-state inactivation properties of
I
Na
determined a “window” current between −50 and −10 mV suggestive of a steady-state Na
+
influx at the cell resting membrane potential. Consistent with this hypothesis, the resting [Na
+
]
i
was decreased by TTX (1 μmol/L). In contrast, it was increased by Na
+
channel agonists that also promoted a large rise in [Ca
2+
]
i
. Veratridine (10 μmol/L), toxin V from
Anemonia sulcata
(0.1 μmol/L), and
N
-bromoacetamide (300 μmol/L) increased [Ca
2+
]
i
by 7- to 15-fold. This increase was prevented by prior application of TTX or lidocaine (10 μmol/L) and by the use of Na
+
-free or Ca
2+
-free external solutions. The Ca
2+
-channel antagonist nicardipine (5 μmol/L) blocked the effect of veratridine on [Ca
2+
]
i
only partially. The residual component disappeared when external Na
+
was replaced by Li
+
known to block the Na
+
/Ca
2+
exchanger. The resting [Ca
2+
]
i
was decreased by TTX in some cells. In conclusion,
I
Na
regulates [Ca
2+
]
i
in primary cultured HCMs. This regulation, effective at baseline, involves a tonic control of Ca
2+
influx via depolarization-gated Ca
2+
channels and, to a lesser extent, via a Na
+
/Ca
2+
exchanger working in the reverse mode.
Attenuation of the Slow Component of Delayed Rectification, Action Potential Prolongation, and Triggered Activity in Mice Expressing a Dominant-Negative Kv2 α Subunit
Authors: Xu Haodong , Barry Dianne M. , Li Huilin , Brunet Sylvain , Guo Weinong , Nerbonne Jeanne M.
Published: June 2012

Abstract
—An in vivo experimental strategy, involving cardiac-specific expression of a mutant Kv 2.1 subunit that functions as a dominant negative, was exploited in studies focused on exploring the role of members of the Kv2 subfamily of pore-forming (α) subunits in the generation of functional voltage-gated K
+
channels in the mammalian heart. A mutant Kv2.1 α subunit (Kv2.1N216) was designed to produce a truncated protein containing the intracellular N terminus, the S1 membrane–spanning domain, and a portion of the S1/S2 loop. The truncated Kv2.1N216 was epitope tagged at the C terminus with the 8–amino acid FLAG peptide to generate Kv2.1N216FLAG. No ionic currents are detected on expression of Kv2.1N216FLAG in HEK-293 cells, although coexpression of this construct with wild-type Kv2.1 markedly reduced the amplitudes of Kv2.1-induced currents. Using the α-myosin heavy chain promoter to direct cardiac specific expression of the transgene, 2 lines of Kv2.1N216FLAG-expressing transgenic mice were generated. Electrophysiological recordings from ventricular myocytes isolated from these animals revealed that
I
K, slow
is selectively reduced. The attenuation of
I
K, slow
is accompanied by marked action potential prolongation, and, occasionally, spontaneous triggered activity (apparently induced by early afterdepolarizations) is observed. The time constant of inactivation of
I
K, slow
in Kv2.1N216FLAG-expressing cells (mean±SEM=830±103 ms; n=17) is accelerated compared with the time constant of
I
K, slow
inactivation (mean±SEM=1147±57 ms; n=25) in nontransgenic cells. In addition, unlike
I
K, slow
in wild-type cells, the component of
I
K, slow
remaining in the Kv2.1N216FLAG-expressing cells is insensitive to 25 mmol/L tetraethylammonium. Taken together, these observations suggest that there are 2 distinct components of
I
K, slow
in mouse ventricular myocytes and that Kv2 α subunits underlie the more slowly inactivating, tetraethylammonium-sensitive component of
I
K, slow
. In vivo telemetric recordings also reveal marked QT prolongation, consistent with a defect in ventricular repolarization, in Kv2.1N216FLAG-expressing transgenic mice.