bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective Upregulation of Cardiac Endothelin System in Patients With Ischemic but Not Idiopathic Dilated Cardiomyopathy
Authors: Serneri Gian Gastone Neri , Cecioni Ilaria , Vanni Simone , Paniccia Rita , Bandinelli Brunella , Vetere Annamaria , Janming Xiao , Bertolozzi Iacopo , Boddi Maria , Lisi Gian Franco , Sani Guido , Modesti Pietro Amedeo
Published: June 2012

Abstract
—Only scarce information is available on the activity and modifications of the cardiac endothelin (ET)–1 system in heart failure due to ischemic (ICM) or idiopathic dilated (DCM) cardiomyopathy. The activity of the ET-1 system was investigated by measuring cardiac ET-1 and big ET-1 formation and quantifying cardiac mRNA for prepro–ET-1 (ppET-1), ET-converting enzyme-1, and ET
A
and ET
B
receptors both in myocardium and in isolated myocytes using Northern blot, reverse transcription–polymerase chain reaction, and in situ hybridization in 22 patients with DCM and 20 with ICM who underwent cardiac transplantation and in 7 potential heart transplant donors (nonfailing hearts). Notwithstanding a similar increase of plasma ET-1 in the 2 groups, cardiac ET formation, mRNA levels for ppET-1, and ET
A
and ET
B
receptors were higher on both the myocardium and isolated myocytes from ICM than on those from DCM hearts (
P
<0.001 for all). ppET-1 and ET-converting enzyme-1 mRNAs were expressed on myocytes and endothelial and interstitial cells in ICM, whereas in DCM and nonfailing hearts they were mainly expressed on nonmyocyte cells. In both ICM and DCM, the ET
A
mRNA signal was expressed on both myocytes and nonmyocyte cells, whereas ET
B
mRNA was almost exclusively localized on nonmyocyte cells. ET
A
- and ET
B
-specific receptor binding was increased on both myocytes and cardiac membranes, showing a positive correlation with left ventricular ejection fraction in ICM (
r
=0.78 and 0.70) but not in DCM patients. The present results show that human ventricular myocytes express all of the components of the ET-1 system, which is selectively upregulated in ICM patients and appears to be functionally important in the maintenance of cardiac function.
Novel Gain-of-Function Mechanism in K
+
Channel–Related Long-QT Syndrome:
Authors: Lees-Miller James P. , Duan Yanjun , Teng Guo Qi , Thorstad Kelly , Duff Henry J.
Published: June 2012

Abstract
—The N629D mutation, adjacent to the GFG signature sequence of the HERG1 A K
+
channel, causes long-QT syndrome (LQTS). Expression of N629D in
Xenopus
oocytes produces a rapidly activating, noninactivating current. N629D is nonselective among monovalent cations; permeation of K
+
was similar to that of Na
+
or Cs
+
. During repolarization to potentials between −30 and −70 mV, N629D manifested an inward tail current, which was abolished by replacement of extracellular Na
+
(Na
+
e
) with extracellular
N
-methyl-
d
-glucamine (NMG
e
). Because LQTS occurs in heterozygous patients, we coexpressed N629D and wild type (WT) at equimolar concentrations. Heteromultimer formation was demonstrated by analyzing the response to 0 [K
+
]
e
. The outward time-dependent current was nearly eliminated for WT at 0 [K
+
]
e
, whereas no reduction was observed for homomultimeric N629D or for the equimolar coexpressed current. To assess physiological significance, dofetilide-sensitive currents were recorded during application of simulated action potential clamps. During phase 3 repolarization, WT manifested outward currents, whereas homomultimeric N629D manifested inward depolarizing currents. During coexpression studies, variable phenotypes were observed ranging from a reduction in outward repolarizing current to net inward depolarizing current during phase 3. In summary, N629D replaces the WT outward repolarizing tail current with an inward depolarizing sodium current, which is expected to delay later stages of repolarization and contribute to arrhythmogenesis. Thus, the consequences of N629D resemble the pathophysiology seen in LQT3 Na
+
channel mutations and may be considered the first LQTS K
+
channel mutation that exhibits gain of function.
Potential Role for Kv3.1b Channels as Oxygen Sensors
Authors: Osipenko O. N. , Tate R. J. , Gurney A. M.
Published: June 2012

Abstract
—Hypoxia inhibits voltage-gated K channels in pulmonary artery smooth muscle (PASM). This is thought to contribute to hypoxic pulmonary vasoconstriction by promoting membrane depolarization, Ca
2+
influx, and contraction. Several of the K-channel subtypes identified in pulmonary artery have been implicated in the response to hypoxia, but contradictory evidence clouds the identity of the oxygen-sensing channels. Using patch-clamp techniques, this study investigated the effect of hypoxia on recombinant Kv1 channels previously identified in pulmonary artery (Kv1.1, Kv1.2, and Kv1.5) and Kv3.1b, which has similar kinetic and pharmacological properties to native oxygen-sensitive currents. Hypoxia failed to inhibit any Kv1 channel, but it inhibited Kv3.1b channels expressed in L929 cells, as shown by a reduction of whole-cell current and single-channel activity, without affecting unitary conductance. Inhibition was retained in excised membrane patches, suggesting a membrane-delimited mechanism. Using reverse transcription–polymerase chain reaction and immunocytochemistry, Kv3.1b expression was demonstrated in PASM cells. Moreover, hypoxia inhibited a K
+
current in rabbit PASM cells in the presence of charybdotoxin and capsaicin, which preserve Kv3.1b while blocking most other Kv channels, but not in the presence of millimolar tetraethylammonium ions, which abolish Kv3.1b current. Kv3.1b channels may therefore contribute to oxygen sensing in pulmonary artery.
Identification of a T-Type Ca
2+
Channel Isoform in Murine Atrial Myocytes (AT-1 Cells)
Authors: Satin Jonathan , Cribbs Leanne L.
Published: June 2012

Abstract
—Calcium channels are important targets for therapeutics, but their molecular diversity complicates characterization of these channels in native heart cells. In this study, we identify a new splice variant of a low-voltage activated, or T-type Ca
2+
, channel in murine atrial myocytes. To date, α1G and α1H are the only 2 T-type Ca
2+
channel isoforms found in cardiovascular tissue. We compared α1G and α1H channel current heterologously expressed in HEK 293 cells with T-type current from the murine atrial tumor cell, AT-1. AT-1 cell T-type current (
I
T
) has the same voltage dependence of activation and inactivation as α1G and α1H. The cloned T-type channels and AT-1 T-type current share similar kinetics of macroscopic inactivation and deactivation. The kinetics of recovery from inactivation of T-type currents serves as an electrophysiological signature for T-channel isoform. α1G and AT-1
I
T
have a similar recovery from inactivation time course that is faster than that for α1H. In all cases, T-type current recovers with a biexponential time course, and the relative amplitude of fast and slow time courses explains the slower α1H recovery kinetics, rather than differences in the time constants of the individual transitions. Thus, the T-type channels may be an important contributor to automaticity in heart cells, and molecular diversity is reflected in the pathway of recovery from inactivation.
Increased Microvascular Reactivity and Improved Mortality in Septic Mice Lacking Inducible Nitric Oxide Synthase
Authors: Hollenberg Steven M. , Broussard Marque , Osman Jailan , Parrillo Joseph E.
Published: June 2012

Abstract
—Persistent vasodilation characteristic of septic shock may result from overproduction of nitric oxide and can lead to pressor-refractory hypotension and death. To evaluate the significance of cytokine-inducible nitric oxide synthase (iNOS) in the pathogenesis of sepsis, we used a clinically relevant mouse model of sepsis and compared mortality and microvascular reactivity in wild-type (WT) mice and transgenic mice deficient in iNOS. WT C57BL/6 and iNOS-deficient mice were made septic by cecal ligation and puncture. Treated mice were given fluids and antibiotics every 6 hours. Microvascular vasoconstriction in response to topical norepinephrine was measured in cremasteric arterioles (15 to 30 μm) by videomicroscopy. Mortality at 48 hours was significantly lower in treated septic iNOS-deficient mice (45%) than in treated septic WT mice (76%), untreated septic iNOS-deficient mice (87%), or untreated WT mice (100%) (
P
<0.01). Norepinephrine-induced vasoconstriction was decreased in WT septic mice (EC
50
200±56 nmol/L) compared with WT and iNOS-deficient shams (16±4 and 13±6 nmol/L), and vasoconstriction was significantly improved in septic iNOS-deficient mice (35±13 nmol/L,
P
<0.01). Microvascular catecholamine responsiveness and survival were improved in iNOS-deficient mice in a clinically relevant model of sepsis, suggesting that iNOS plays an important, but not exclusive, role in refractory vasodilation in patients with septic shock.
Leukemia Inhibitory Factor Modulates Cardiogenesis in Embryoid Bodies in Opposite Fashions
Authors: Bader Alice , Al-Dubai Haifa , Weitzer Georg
Published: June 2012

Abstract
—Cardiogenesis is a multistep process regulated by a hierarchy of factors defining each developmental stage of the heart. One of these factors, leukemia inhibitory factor (LIF), a member of the interleukin-6 family of cytokines, is expressed in embryonic and neonatal cardiomyocytes and induces cardiomyocyte hypertrophy. Many aspects of embryogenesis are faithfully recapitulated during in vitro differentiation of embryonic stem cells in embryoid bodies. We exploited this model to study effects of growth factors on commitment and differentiation of cardiomyocytes and on maintenance of their phenotype. We identified LIF as a factor affecting commitment and differentiation of cardiomyocytes in an opposite manner. Diffusible LIF inhibited mesoderm formation and hampered commitment of cardiomyocytes. Lack of both the diffusible and matrix-bound isoforms of LIF in
lif
−/− embryoid bodies did not interfere with commitment, but it severely suppressed early differentiation of cardiomyocytes. Onset of differentiation was rescued by very low concentrations of diffusible LIF; however, consecutive differentiation was attenuated in a concentration-dependent manner by diffusible LIF both in wild-type and
lif
−/− cardiomyocytes. Differentiation of cardiomyocytes was severely hampered but not completely blocked in
lifr
−/− embryoid bodies, suggesting additional, LIF-receptor ligand independent pathways for commitment and differentiation of cardiomyocytes. At the fully differentiated state, both paracrine and autocrine LIF promoted proliferation and increased longevity of cardiomyocytes. These findings suggest that both paracrine and autocrine and both diffusible and matrix-bound isoforms of LIF contribute to the modulation of cardiogenesis in a subtle, opposite, and developmental stage–dependent manner and control proliferation and maintenance of the differentiated state of cardiomyocytes.
Effects of Hypoxia on Isometric Force, Intracellular Ca
2+
, pH, and Energetics in Porcine Coronary Artery
Authors: Shimizu Shunichi , Bowman Peggy Sue , Thorne George , Paul Richard J.
Published: June 2012

Abstract
—When exposed to hypoxic conditions, coronary arteries dilate, which is an important protective response. Although vessel sensitivity to oxygen is well documented, the mechanisms are not known with certainty. To further characterize the mechanisms of oxygen sensing in the coronary artery, we tested the major classes of hypotheses by measuring the effects of hypoxia on energetics, [Ca
2+
]
i
, K
+
channel function, and pH
i
. Hypoxia relaxes porcine coronary arteries stimulated with either KCl or U46619. The extent of relaxation is dependent on both the degree and kind of stimulation. [Ca
2+
]
i
was measured in endothelium-denuded arteries using fura 2-AM and ratiometric fluorescent techniques. At lower stimulus levels, hypoxia decreased both force and [Ca
2+
]
i
. Inhibitor studies suggest that K
Ca
and K
ATP
channels are not involved in the hypoxic relaxation, whereas K
V
channels may play a minor role, if any. Despite the hypoxia-mediated decrease in force, [Ca
2+
]
i
was unchanged or increased at high levels of stimulation. Despite a marked increase in lactate content, pH
i
(measured with the ratiometric fluorescent dye BCECF) was also little affected by hypoxia. Measurement of the phosphagen and metabolite profile of freeze-clamped arteries with analytical isotachophoresis indicated that hypoxia increased lactate content by 4-fold and decreased phosphocreatine to 60% of control. However, neither ATP nor P
i
was affected by hypoxia. Interestingly, additional stimulation under hypoxia increased force but not ATP utilization, as estimated from measurements of anaerobic lactate production. Thus, surprisingly, the economy of force maintenance is increased under hypoxia. In porcine coronary artery, both Ca
2+
-dependent and, importantly, Ca
2+
-independent mechanisms are involved in hypoxic vasodilatation. For the latter, mechanisms involving either ATP, [Ca
2+
]
i
, pH
i
, or P
i
cannot be invoked. This novel oxygen sensing mechanism involves a decreased Ca
2+
sensitivity.
Jumonji, a Nuclear Protein That Is Necessary for Normal Heart Development
Authors: Lee Youngsook , Song Alice J. , Baker Robert , Micales Bruce , Conway Simon J. , Lyons Gary E.
Published: June 2012

Abstract
—Jumonji (
jmj
) was cloned in a gene trap screen to identify and mutagenize genes important for heart development. To investigate the role of
jmj
in heart development, we generated mice homozygous for the
jmj
mutation. The
jmj
homozygous mouse embryos showed heart malformations, including ventricular septal defect, noncompaction of the ventricular wall, double-outlet right ventricle, and dilated atria. The
jmj
mutants died soon after birth, apparently as a result of respiratory insufficiency caused by rib and sternum defects in addition to the heart defects. In situ hybridization analyses suggested that cardiomyocytes were differentiated but developmental regulation of chamber-specific genes was defective in fetal hearts. Expression of
jmj
was detected in the myocardium, especially in the interventricular septum, ventricular wall, and outflow tract, which correlated well with the locations of defects observed in the hearts of mutant mice. Homozygous embryos failed to express the
jmj
transcript in all tissues except in the nervous system. Confocal microscopic examination using anti-JMJ antibodies indicated that the JMJ protein was localized in the nuclei of cells transfected with
jmj
. These data demonstrate that JMJ is a nuclear protein, which is essential for normal heart development and function.
Activation of Protein Kinase A Modulates Trafficking of the Human Cardiac Sodium Channel in
Xenopus
Oocytes
Authors: Zhou Jingsong , Yi Jianxun , Hu NingNing , George Alfred L. , Murray Katherine T.
Published: June 2012

Abstract
—Voltage-gated Na
+
channels are critical determinants of electrophysiological properties in the heart. Stimulation of β-adrenergic receptors, which activate cAMP-dependent protein kinase (protein kinase A [PKA]), can alter impulse conduction in normal tissue and promote development of cardiac arrhythmias in pathological states. Recent studies demonstrate that PKA activation increases cardiac Na
+
currents, although the mechanism of this effect is unknown. To explore the molecular basis of Na
+
channel modulation by β-adrenergic receptors, we have examined the effects of PKA activation on the recombinant human cardiac Na
+
channel, hH1. Both in the absence and the presence of hβ
1
subunit coexpression, activation of PKA caused a slow increase in Na
+
current that did not saturate despite kinase stimulation for 1 hour. In addition, there was a small shift in the voltage dependence of channel activation and inactivation to more negative voltages. Chloroquine and monensin, compounds that disrupt plasma membrane recycling, reduced hH1 current, suggesting rapid turnover of channels at the cell surface. Preincubation with these agents also prevented the PKA-mediated rise in Na
+
current, indicating that this effect likely resulted from an increased number of Na
+
channels in the plasma membrane. Experiments using chimeric constructs of hH1 and the skeletal muscle Na
+
channel, hSKM1, identified the I-II interdomain loop of hH1 as the region responsible for the PKA effect. These results demonstrate that activation of PKA modulates both trafficking and function of the hH1 channel, with changes in Na
+
current that could either speed or slow conduction, depending on the physiological circumstances. (

Circ Res. 2000;87:33-38.)
FK409, a Spontaneous Nitric Oxide Releaser, Attenuates Allograft Vasculopathy in a Rat Aortic Transplant Model
Authors: Fukada Johji , Schena Stefano , Tack Ivan , Ruiz Phillip , Kurimoto Yoshihiko , Pang Manhui , Aitouche Abdelouahab , Abe Tomio , Striker Liliane J. , Pham Si M.
Published: June 2012

Abstract
—Although systemic administration of NO donors has been shown to attenuate the development of neointimal hyperplasia in the balloon injury model, this strategy has not been tested in a model of allograft vasculopathy. In this study, we investigated the effect of FK409, a spontaneous NO releaser, on the development of allograft vasculopathy, using a rat aortic transplant model. Thoracic aortas from ACI rats were transplanted heterotopically into the abdominal aorta of Wistar-Furth rats. Postoperatively, recipients received FK409 orally every 8 hours from the day of transplantation to the time of euthanization. Morphometric and immunohistochemical analyses were performed on the aortic grafts 8 weeks after transplantation. Control allografts showed severe neointimal hyperplasia, which consists mainly of α-actin–containing vascular smooth muscle cells. The FK409-treated allografts showed a dose-dependent reduction (statistically significant compared with the control) in the neointimal thickness as the dose increased from 1 to 10 mg/kg (thrice per day). However, there was no significant difference in the neointimal thickness between groups treated with 10 and with 20 mg/kg. FK409 treatment (10 mg/kg) caused a significant decrease in DNA synthesis (5-bromo-2-deoxyuridine [BrdU] uptake), an increase in DNA fragmentation (terminal deoxynucleotidyltransferase–mediated uridine nick-end labeling [TUNEL]), and upregulation of Fas expression, in the neointimal vascular smooth muscle cells. These data suggest that FK409 attenuates the allograft vasculopathy in a rat aortic transplant model. (

Circ Res. 2000;87:66-72.)
C-Terminal Tails of Sulfonylurea Receptors Control ADP-Induced Activation and Diazoxide Modulation of ATP-Sensitive K
+
Channels
Authors: Matsuoka Tetsuro , Matsushita Kenji , Katayama Yusuke , Fujita Akikazu , Inageda Kiyoshi , Tanemoto Masayuki , Inanobe Atsushi , Yamashita Shizuya , Matsuzawa Yuji , Kurachi Yoshihisa
Published: June 2012

Abstract
—The ATP-sensitive K
+
(K
ATP
) channels are composed of the pore-forming K
+
channel Kir6.0 and different sulfonylurea receptors (SURs). SUR1, SUR2A, and SUR2B are sulfonylurea receptors that are characteristic for pancreatic, cardiac, and vascular smooth muscle–type K
ATP
channels, respectively. The structural elements of SURs that are responsible for their different characteristics have not been entirely determined. Here we report that the 42 amino acid segment at the C-terminal tail of SURs plays a critical role in the differential activation of different SUR-K
ATP
channels by ADP and diazoxide. In inside-out patches of human embryonic kidney 293T cells coexpressing distinct SURs and Kir6.2, much higher concentrations of ADP were needed to activate channels that contained SUR2A than SUR1 or SUR2B. In all types of K
ATP
channels, diazoxide increased potency but not efficacy of ADP to evoke channel activation. Replacement of the C-terminal segment of SUR1 with that of SUR2A inhibited ADP-mediated channel activation and reduced diazoxide modulation. Point mutations of the second nucleotide-binding domains (NBD2) of SUR1 and SUR2B, which would prevent ADP binding or ATP hydrolysis, showed similar effects. It is therefore suggested that the C-terminal segment of SUR2A possesses an inhibitory effect on NBD2-mediated ADP-induced channel activation, which underlies the differential effects of ADP and diazoxide on K
ATP
channels containing different SURs.
Vascular Smooth Muscle Cells Express the α
1A
Subunit of a P-/Q-Type Voltage-Dependent Ca
2+
Channel, and It Is Functionally Important in Renal Afferent Arterioles
Authors: Hansen Pernille B. , Jensen Boye L. , Andreasen Ditte , Friis Ulla G. , Skøtt Ole
Published: June 2012

Abstract
—In the present study, we tested whether the α
1A
subunit, which encodes a neuronal isoform of voltage-dependent Ca
2+
channels (VDCCs) (P-/Q-type), was present and functional in vascular smooth muscle and renal resistance vessels. By reverse transcription–polymerase chain reaction and Southern blotting analysis, mRNA encoding the α
1A
subunit was detected in microdissected rat preglomerular vessels and vasa recta, in cultures of rat preglomerular vascular smooth muscle cells (VSMCs), and in cultured rat mesangial cells. With immunoblots, α
1A
subunit protein was demonstrated in rat aorta, brain, aortic smooth muscle cells (A7r5), VSMCs, and mesangial cells. Immunolabeling with an anti-α
1A
antibody was positive in acid-macerated, microdissected preglomerular vessels and in A7r5 cells. Patch-clamp experiments on aortic A7r5 cells showed 22±4% (n=6) inhibition of inward Ca
2+
current by ω-Agatoxin IVA (10
–8
mol/L), which in this concentration is a specific inhibitor of P-type VDCCs. Measurements of intracellular Ca
2+
in afferent arterioles with fluorescence-imaging microscopy showed 32±9% (n=10) inhibition of the K
+
-induced rise in Ca
2+
in the presence of 10
–8
mol/L ω-Agatoxin IVA. In microperfused rabbit afferent arterioles, ω-Agatoxin IVA inhibited depolarization-mediated contraction with an EC
50
of 10
–17
mol/L and complete blockade at 10
–14
mol/L. We conclude that the α
1A
subunit is expressed in VSMCs from renal preglomerular resistance vessels and aorta, as well as mesangial cells, and that P-type VDCCs contribute to Ca
2+
influx in aortic and renal VSMCs and are involved in depolarization-mediated contraction in renal afferent arterioles.
Role of NAD(P)H Oxidase in Angiotensin II–Induced JAK/STAT Signaling and Cytokine Induction
Authors: Schieffer Bernhard , Luchtefeld Maren , Braun Sabine , Hilfiker Andres , Hilfiker-Kleiner Denise , Drexler Helmut
Published: June 2012

Abstract
—Inflammatory processes involve both synthesis of inflammatory cytokines, such as interleukin-6 (IL-6), and the activation of their distinct signaling pathways, eg, the janus kinases (JAKs) and signal transducers and activators of transcription (STAT). Superoxide (O
2
−
) anions activate this signaling cascade, and the vasoconstrictor angiotensin II (Ang II) enhances the formation of O
2
−
anions via the NAD(P)H oxidase system in rat aortic smooth muscle cells. Ang II activates the JAK/STAT cascade via its type 1 (AT
1
) receptor and induces synthesis and release of IL-6. Therefore, we investigated the role of O
2
−
anions generated by the NAD(P)H oxidase system on the Ang II activation of the JAK/STAT cascade and its impact on IL-6 synthesis. Ang II stimulation of rat aortic smooth muscle cells induced a rapid increase in O
2
−
anions determined by laser fluoroscopy, which can be abolished by DPI, a flavoprotein inhibitor. Ang II–induced phosphorylation of JAK2, STAT1α/β, STAT3, and IL-6-synthesis can be abolished by DPI, as determined by immunoprecipitations and Northern blot analysis. Electroporation of neutralizing antisera targeted against p47

phox

, a NAD(P)H oxidase subunit, abolished Ang II–induced JAK/STAT activation and IL-6 synthesis. Inhibition of JAK2 by its inhibitor AG490 (10 μmol/L) blocked not only JAK2 activation but also IL-6 synthesis. These results suggest that stimulation of the JAK/STAT cascade by Ang II requires O
2
−
anions generated by the NAD(P)H oxidase system, and O
2
−
anion–dependent activation of the JAK/STAT cascade seems to be additionally involved in Ang II–induced IL-6 synthesis. Thus, Ang II–induced inflammatory effects seem to require O
2
−
anions generated by the NAD(P)H oxidase system.
Transmission of Information From Cardiac Dihydropyridine Receptor to Ryanodine Receptor
Authors: Katoh Hideki , Schlotthauer Klaus , Bers Donald M.
Published: June 2012

Abstract
—Coupling between L-type Ca
2+
channels (dihydropyridine receptors, DHPRs) and ryanodine receptors (RyRs) plays a pivotal role in excitation-contraction (E-C) coupling in cardiac myocytes, and Ca
2+
influx is generally accepted as the trigger of sarcoplasmic reticulum (SR) Ca
2+
release. The L-type Ca
2+
channel agonist BayK 8644 (BayK) has also been reported to alter RyR gating via a functional linkage between DHPR and RyR, independent of Ca
2+
influx. Here, the effect of rapid BayK application on resting RyR gating in intact ferret ventricular myocytes was measured as Ca
2+
spark frequency (CaSpF) by confocal microscopy and fluo 3. BayK increased resting CaSpF by 401±15% within 10 seconds in Ca
2+
-free solution, and depolarization had no additional effect. The effect of BayK on CaSpF was dose-dependent, but even 50 nmol/L BayK induced a rapid 245±12% increase in CaSpF. Nifedipine (5 μmol/L) had no effect by itself on CaSpF, but it abolished the BayK effect (presumably by competitive inhibition at the DHPR). The nondihydropyridine Ca
2+
channel agonist FPL-64176 (1 μmol/L) did not alter CaSpF (despite rapid and potent enhancement of Ca
2+
current,
I
Ca
). In striking contrast to the very rapid and depolarization-independent effect of BayK on CaSpF, BayK increased
I
Ca
only slowly (τ=18 seconds), and the effect was greatly accelerated by depolarization. We conclude that in ferret ventricular myocytes, BayK effects on
I
Ca
and CaSpF both require drug binding to the DHPR, but postreceptor pathways may diverge in transmission to the gating of the L-type Ca
2+
channel and RyR. (Circ Res. 2000;87:106-111.)
Opioid Peptide Gene Expression Primes Cardiogenesis in Embryonal Pluripotent Stem Cells
Authors: Ventura Carlo , Maioli Margherita
Published: June 2012

Abstract
—Zinc finger–containing transcription factor GATA-4 and homeodomain Nkx-2.5 govern crucial developmental fates and have been found to promote cardiogenesis in embryonic cells exposed to the differentiating agent DMSO. Nevertheless, intracellular activators of these transcription factors are largely unknown. In this study, pluripotent P19 cells expressed the prodynorphin gene, an opioid gene encoding for the dynorphin family of opioid peptides. P19 cells were also able to synthesize and secrete dynorphin B, a biologically active end product of the prodynorphin gene. DMSO-primed GATA-4 and Nkx-2.5 gene expression was preceded by a marked increase in prodynorphin gene expression and dynorphin B synthesis and secretion. The DMSO effect occurred at the transcriptional level. In the absence of DMSO, dynorphin B triggered GATA-4 and Nkx-2.5 gene expression and led to the appearance of both α-myosin heavy chain and myosin light chain-2V transcripts, two markers of cardiac differentiation. Moreover, dynorphin B–exposed cells were positively stained in the presence of MF 20, a mouse monoclonal antibody raised against the α-myosin heavy chain. Opioid receptor antagonism and inhibition of opioid gene expression by a prodynorphin antisense phosphorothioate oligonucleotide blocked DMSO-induced cardiogenesis, suggesting an autocrine role of an opioid gene in developmental decisions.
Peroxynitrite Is a Major Contributor to Cytokine-Induced Myocardial Contractile Failure
Authors: Ferdinandy Péter , Danial Hajira , Ambrus Ildikó , Rothery Richard A. , Schulz Richard
Published: June 2012

Abstract
—Proinflammatory cytokines depress myocardial contractile function by enhancing the expression of inducible NO synthase (iNOS), yet the mechanism of iNOS-mediated myocardial injury is not clear. As the reaction of NO with superoxide to form peroxynitrite markedly enhances the toxicity of NO, we hypothesized that peroxynitrite itself is responsible for cytokine-induced cardiac depression. Isolated working rat hearts were perfused for 120 minutes with buffer containing interleukin-1β, interferon-γ, and tumor necrosis factor-α. Cardiac mechanical function and myocardial iNOS, xanthine oxidoreductase (XOR), and NAD(P)H oxidase activities (sources of superoxide) were measured during the perfusion. Cytokines induced a marked decline in myocardial contractile function accompanied by enhanced activity of myocardial XOR, NADH oxidase, and iNOS. Cardiac NO content, myocardial superoxide production, and perfusate nitrotyrosine and dityrosine levels, markers of peroxynitrite, were increased in cytokine-treated hearts. The peroxynitrite decomposition catalyst FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), the NO synthase inhibitor
N
G
-nitro-
l
-arginine, and the superoxide scavenger tiron each inhibited the decline in myocardial function and decreased perfusate nitrotyrosine levels. Proinflammatory cytokines stimulate the concerted enhancement in superoxide and NO-generating activities in the heart, thereby enhancing peroxynitrite generation, which causes myocardial contractile failure.
Accelerated Atherosclerosis by Placement of a Perivascular Cuff and a Cholesterol-Rich Diet in ApoE*3Leiden Transgenic Mice
Authors: Lardenoye J. H. P. , Delsing D. J. M. , de Vries M. R. , Deckers M. M. L. , Princen H. M. G. , Havekes L. M. , van Hinsbergh V. W. M. , van Bockel J. H. , Quax P. H. A.
Published: June 2012

Abstract
—Intimal hyperplasia after vascular injury is usually studied in animal models with healthy, normocholesterolemic animals. Here, we assess the effect of diet-induced hypercholesterolemia on the induction of intimal hyperplasia in ApoE*3Leiden mice. A nonconstrictive polyethylene cuff was placed around the femoral artery of ApoE3*Leiden mice fed a highly cholesterol-rich diet, a mildly cholesterol-rich diet, or a chow diet for 4 weeks. Diets were continued after cuff placement until euthanization. At several time points (1 to 14 days), mice were euthanized and the intimal hyperplasia in the cuffed arteries was analyzed. In mice fed a chow diet, a 2- to 4-cell-layer–thick intima, predominantly consisting of α smooth muscle cell actin–positive cells, was observed after 14 days. A mildly cholesterol-rich diet (mean plasma-cholesterol level, 10.5 mmol/L) resulted in a 2.7-fold increase of total intimal area, and a highly cholesterol-rich diet (mean plasma cholesterol level 28.6 mmol/L), in a 7.8-fold increase. In the high-cholesterol group, the intima consisted predominantly of lipid-loaded foam cells and α smooth muscle cell actin–positive cells. Foam cell accumulation could be observed by as early as 3 days, resulting in a near-total occlusion of the lumen after 14 days. Hypercholesterolemia resulted in a rapid, cholesterol-dependent induction of foam cell–rich intimal hyperplasia in cuffed femoral arteries of ApoE*3Leiden mice. In conclusion, the present data show that the combination of a local (cuff placement) and a systemic (hypercholesterolemic) risk factor of atherosclerosis results in a rapid induction (within 14 days) of atherosclerotic-like lesions in ApoE*3Leiden mice.
Fluid Shear Stress Activates Ca
2+
Influx Into Human Endothelial Cells via P2X4 Purinoceptors
Authors: Yamamoto Kimiko , Korenaga Risa , Kamiya Akira , Ando Joji
Published: June 2012

Abstract
—Ca
2+
signaling plays an important role in endothelial cell (EC) responses to shear stress generated by blood flow. Our previous studies demonstrated that bovine fetal aortic ECs showed a shear stress–dependent Ca
2+
influx when exposed to flow in the presence of extracellular ATP. However, the molecular mechanisms of this process, including the ion channels responsible for the Ca
2+
response, have not been clarified. Here, we demonstrate that P2X4 purinoceptors, a subtype of ATP-operated cation channels, are involved in the shear stress–mediated Ca
2+
influx. Human umbilical vein ECs loaded with the Ca
2+
indicator Indo-1/AM were exposed to laminar flow of Hanks’ balanced salt solution at various concentrations of ATP, and changes in [Ca
2+
]
i
were monitored with confocal laser scanning microscopy. A stepwise increase in shear stress elicited a corresponding stepwise increase in [Ca
2+
]
i
at 250 nmol/L ATP. The shear stress–dependent increase in [Ca
2+
]
i
was not affected by phospholipase C inhibitor (U-73122) but disappeared after the chelation of extracellular Ca
2+
with EGTA, indicating that the Ca
2+
increase was due to Ca
2+
influx. Antisense oligonucleotides designed to knockout P2X4 expression abolished the shear stress–dependent Ca
2+
influx seen at 250 nmol/L ATP in human umbilical vein ECs. Human embryonic kidney 293 cells showed no Ca
2+
response to flow at 2 μmol/L ATP, but when transfected with P2X4 cDNA, they began to express P2X4 purinoceptors and to show shear stress–dependent Ca
2+
influx. P2X4 purinoceptors may have a “shear-transducer” property through which shear stress is perceived directly or indirectly and transmitted into the cell interior via Ca
2+
signaling.
Synthesis of Extracellular Matrix and Adhesion Through β
1
Integrins Are Critical for Fetal Ventricular Myocyte Proliferation
Authors: Hornberger Lisa K. , Singhroy Sandra , Cavalle-Garrido Tiscar , Tsang Wendy , Keeley Fred , Rabinovitch Marlene
Published: June 2012

Abstract
—Extracellular matrix (ECM) regulates vascular smooth muscle cell proliferation. The role of ECM in myocardial growth is unexplored. We sought to determine whether human fetal ventricular myocytes (HFVMs) produce ECM and whether synthesis and attachment to ECM are necessary for their epidermal growth factor (EGF)–dependent and –independent proliferation. Cultured HFVMs proliferate in the presence but not absence of serum and EGF, as determined by increase in cell number and [
3
H]thymidine and [
14
C]leucine incorporation (measures of DNA and protein synthesis, respectively). Using a cyanogen bromide digestion technique to measure collagen and elastin and using affinity chromatography for fibronectin, we found that HFVMs synthesized collagen and fibronectin but not elastin. HFVMs grown on exogenous ECM (including fibronectin and type I collagen and laminin) demonstrated no change in proliferation or DNA and protein synthesis with or without EGF. However, inhibition of collagen synthesis using
cis
-4-hydroxyproline resulted in a decrease in EGF-related HFVM proliferation and DNA and protein synthesis, which was reversed by exposure to
l
-proline but not by growth on type I collagen. Use of β
1
but not β
3
integrin antibody to inhibit cell interaction with ECM resulted in a decrease in HFVM proliferation and DNA and protein synthesis in response to EGF. Furthermore, EGF-dependent proliferation was enhanced by α
1
β
1
and α
5
β
1
antibodies that act as functional ligands, but not α
3
β
1
, the only β
1
subtype expressed in adult myocytes. In conclusion, proliferating HFVMs synthesize collagen and fibronectin. The proliferative response of HFVMs to EGF requires the synthesis of collagen as well as attachment to specific α/β
1
integrin heterodimers.
Novel Embryonic Genes Are Preferentially Expressed by Autonomously Replicating Rat Embryonic and Neointimal Smooth Muscle Cells
Authors: Weiser-Evans Mary C. M. , Schwartz Philip E. , Grieshaber Nicole A. , Quinn Bryan E. , Grieshaber Scott S. , Belknap James K. , Mourani Peter M. , Majack Richard A. , Stenmark Kurt R.
Published: June 2012

Abstract
—We sought to identify and characterize the expression pattern of genes expressed by smooth muscle cells (SMCs) during periods of self-driven replication during vascular development and after vascular injury. Primary screening of a rat embryonic aortic SMC–specific cDNA library was accomplished with an autonomous embryonic SMC–enriched, nonautonomous adult SMC–subtracted cDNA probe. Positive clones were rescreened in parallel with embryonic SMC–specific and adult SMC–specific cDNA probes. We identified 14 clones that hybridized only with the embryonic cDNA (“emb” clones), 11 of which did not share significant homology with sequences in any of the databases. Five of these novel emb genes (emb7, emb8, emb20, emb37, and emb41) were selectively and only transiently reexpressed in vivo by neointimal SMCs during periods of rapid replication. The emb8:embryonic growth–associated protein (EGAP), which was studied the most extensively, was expressed at high levels by cultured, autonomously replicating embryonic and neointimal SMCs but was detected only at low levels even in mitogenically stimulated adult SMCs. Finally, the administration of antisense EGAP oligonucleotides markedly attenuated embryonic and neointimal SMC replication rates. We suggest that autonomous replication of SMCs may be essential for normal vascular morphogenesis and for the vascular response to injury and that these newly identified “embryonic” genes may be part of the molecular machinery that drives this unique growth phenotype.
Involvement of Rho GTPases in the Transcriptional Inhibition of Preproendothelin-1 Gene Expression by Simvastatin in Vascular Endothelial Cells
Authors: Hernández-Perera Octavio , Pérez-Sala Dolores , Soria Estrella , Lamas Santiago
Published: June 2012

Abstract
—Endothelial dysfunction is characterized by an impaired vasodilatory response to endothelial agonists as well as by alterations in adhesion and coagulation processes. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) have been shown to be useful in the reversal of endothelial dysfunction, an effect that may be independent of the reduction in cholesterol levels. Both the
l
-arginine–nitric oxide–cGMP and endothelin pathways are involved in the regulation of vascular tone. Here, we show that the basal transcription rate of the preproendothelin-1 gene was decreased by simvastatin (10 μmol/L) in bovine aortic endothelial cells. Transfection studies with the preproendothelin-1 gene promoter showed that mevalonate (100 μmol/L) was able to prevent the inhibitory effect mediated by simvastatin. Protein geranylgeranylation, but not farnesylation, proved to be crucial for a correct expression of the preproendothelin-1 gene. The C3 exotoxin from
Clostridium botulinum
that selectively inactivates Rho GTPases, the processing of which involves geranylgeranylation, reproduced the inhibitory effect of simvastatin on the expression of preproendothelin-1. Overexpression of dominant-negative mutants of RhoA and RhoB led to a significant reduction in the preproendothelin-1 promoter activity, whereas the expression of wild-type and constitutively active forms of these proteins resulted in an increase, in support that Rho proteins are required for the basal expression of the preproendothelin-1 gene. Finally, we show that the Rho-dependent activation of the preproendothelin-1 gene transcription was inhibited by simvastatin. Thus, the control of vascular tone and proliferative response mediated by endothelin-1 is regulated at multiple levels, among which the Rho proteins play an essential role.
iNOS Gene Expression Modulates Microvascular Responsiveness in Endotoxin-Challenged Mice
Authors: Boyle Walter A. , Parvathaneni Lakshmi S. , Bourlier Virginie , Sauter Craig , Laubach Victor E. , Cobb J. Perren
Published: June 2012

Abstract
—Septic shock is characterized by vasodilation and decreased responsiveness to vasoconstrictors. Recent studies suggest this results from nitric oxide (NO) overproduction after expression of the calcium-independent isoform of NO synthase (iNOS) in smooth muscle cells. However, direct evidence linking iNOS (NOS2) expression and decreased microvascular responsiveness after septic stimuli is lacking. In the present study, we determined the effect of bacterial lipopolysaccharide (LPS, 20 mg/kg, IP) on smooth muscle contraction and endothelial relaxation in mesenteric resistance arteries from wild-type and iNOS knockout mice. Four hours after challenge with LPS or saline in vivo, concentration-dependent responses to norepinephrine (NE) and acetylcholine (NE+ACh) were measured in cannulated, pressurized vessels ex vivo. In vessels from wild-type mice, NE-induced contraction was markedly impaired after LPS, and pretreatment with the iNOS inhibitor aminoguanidine (AG) partly restored the NE contraction. In contrast, NE contraction in microvessels from iNOS knockout mice was unaffected by LPS. ACh-induced relaxation was unaffected by LPS in vessels from either genotype. These data provide direct evidence that iNOS gene expression mediates the LPS-induced decrease in microvascular responsiveness to vasoconstrictors. Moreover, the observation that AG did not fully restore NE contraction after LPS, whereas iNOS gene deficiency did, suggests that iNOS expression plays a central role in the development of the NO-independent effect of LPS on microvascular responsiveness. Finally, our data indicate that LPS or iNOS expression has little effect on endothelium-dependent relaxation, and eNOS activity does not appear to play a role in the decreased smooth muscle responsiveness after LPS in this model. The full text of this article is available at http://www.circresaha.org.
Endothelial Cells of Hematopoietic Origin Make a Significant Contribution to Adult Blood Vessel Formation
Authors: Crosby Jeffrey R. , Kaminski Wolfgang E. , Schatteman Gina , Martin Paul J. , Raines Elaine W. , Seifert Ron A. , Bowen-Pope Daniel F.
Published: June 2012

Granulation tissue formation is an example of new tissue development in an adult. Its rich vascular network has been thought to derive via angiogenic sprouting and extension of preexisting vessels from the surrounding tissue. The possibility that circulating cells of hematopoietic origin can differentiate into vascular endothelial cells (ECs) in areas of vascular remodeling has recently gained credibility. However, no quantitative data have placed the magnitude of this contribution into a physiological perspective. We have used hematopoietic chimeras to determine that 0.2% to 1.4% of ECs in vessels in control tissues derived from hematopoietic progenitors during the 4 months after irradiation and hematopoietic recovery. By contrast, 8.3% to 11.2% of ECs in vessels that developed in sponge-induced granulation tissue during 1 month derived from circulating hematopoietic progenitors. This recruitment of circulating progenitors to newly forming vessels would be difficult to observe in standard histological studies, but it is large enough to be encouraging for attempts to manipulate this contribution for therapeutic gain.
Functional Reconstitution of the Angiotensin II Type 2 Receptor and G
i
Activation
Authors: Hansen Jakob Lerche , Servant Guy , Baranski Thomas J. , Fujita Toshiro , Iiri Taroh , Sheikh Søren P.
Published: June 2012

Abstract
—On the basis of the patterns of conserved amino acid sequence, the angiotensin II type 2 (AT
2
) receptor belongs to the family of serpentine receptors, which relay signals from extracellular stimuli to heterotrimeric G proteins. However, the AT
2
receptor signal transduction mechanisms are poorly understood. We have measured AT
2
-triggered activation of purified heterotrimeric proteins in urea-extracted membranes from cultured COS-7 cells expressing the recombinant receptor. This procedure removes contaminating GTP-binding proteins without inactivating the serpentine receptor. Binding studies using [
125
I] angiotensin (Ang) II revealed a single binding site with a
K
d
=0.45 and a capacity of 627 fmol/mg protein in the extracted membranes. The AT
2
receptor caused a rapid activation of α
i
and α
o
but not of α
q
and α
s
, as measured by radioactive guanosine 5′-3-
O
-(thio)triphosphate (GTPγS) binding. Activation required the presence of activated receptors, βγ, and α subunits. As a first step aimed at developing an in vitro assay to examine AT
2
receptor pharmacology, we tested a battery of Ang II–related ligands for their ability to promote AT
1
or AT
2
receptor–catalyzed G
i
activation. Two proteolytic fragments of Ang II, Ang III and Ang1–7, also promoted activation of α
i
through the AT
2
receptor. Furthermore, we found that [Sar
1
,Ala
8
]Ang II is an antagonist for both AT
1
and AT
2
receptors and that
CPG42112
behaves as a partial agonist for the AT
2
receptor. In combination with previous observations, these results show that the AT
2
receptor is fully capable of activating G
i
and provides a new tool for exploring AT
2
receptor pharmacology and interactions with G-protein trimers.
Crucial Role of Type 1, but Not Type 3, Inositol 1,4,5-Trisphosphate (IP
3
) Receptors in IP
3
-Induced Ca
2+
Release, Capacitative Ca
2+
Entry, and Proliferation of A7r5 Vascular Smooth Muscle Cells
Authors: Wang Yuepeng , Chen Jie , Wang Yue , Taylor Colin W. , Hirata Yasunobu , Hagiwara Hisashi , Mikoshiba Katsuhiko , Toyo-oka Teruhiko , Omata Masao , Sakaki Yoshiyuki
Published: June 2012

Abstract
—Stimulation of G protein– or tyrosine kinase–coupled receptors regulates cell proliferation through intracellular Ca
2+
([Ca
2+
]
i
) signaling. In A7r5 cells, we confirmed that inositol 1,4,5-trisphosphate (IP
3
) mediates vasopressin (VP)-evoked Ca
2+
release from intracellular stores and showed that types 1 (IP
3
R
1
) and 3 (IP
3
R
3
) IP
3
receptors were expressed. Using antisera selective for IP
3
R
1
or IP
3
R
3
and another that interacted equally well with both subtypes, together with membranes from
Sf
9 cells expressing only single IP
3
R subtypes to calibrate immunoblotting, we established that A7r5 cells express 81% IP
3
R
1
and 19% IP
3
R
3
. To elucidate the contributions of IP
3
R
1
and IP
3
R
3
to Ca
2+
signaling and proliferation, stable clones expressing promoter-inducible antisense cDNA fragments (−90 to +9) corresponding to the two IP
3
R subtypes were selected. Mild inhibition of IP
3
R
1
(71±8% of control level) slightly attenuated the IP
3
-evoked Ca
2+
release (IICR) induced by VP but significantly decreased the subsequent capacitative Ca
2+
entry (CCE) and proliferation. Moderate inhibition (34±6%) strongly decreased both IICR and CCE and further blocked proliferation. Complete inhibition almost abolished IICR and CCE and arrested proliferation entirely. Complete inhibition of IP
3
R
3
expression slightly attenuated IICR without affecting CCE or proliferation. In cells microinjected with a low dose of heparin, VP-induced CCE was more susceptible than IICR to mild inhibition of both IP
3
R
1
and IP
3
R
3
. A high dose of heparin had a similar effect to complete inhibition of IP
3
R
1
expression: it blocked VP-evoked IICR entirely and CCE by 90%. We conclude that IP
3
R
1
, but not IP
3
R
3
, is crucial for IICR, CCE, and proliferation of vascular smooth muscle cells.
Membrane-Bound Protein Kinase A Inhibits Smooth Muscle Cell Proliferation In Vitro and In Vivo by Amplifying cAMP–Protein Kinase A Signals
Authors: Indolfi Ciro , Stabile Eugenio , Coppola Carmela , Gallo Adriana , Perrino Cinzia , Allevato Giovanna , Cavuto Luigi , Torella Daniele , Di Lorenzo Emilio , Troncone Giancarlo , Feliciello Antonio , Avvedimento Enrico Vittorio , Chiariello Massimo
Published: June 2012

Abstract
—cAMP-dependent protein kinase is anchored to discrete cellular compartments by a family of proteins, the A-kinase anchor proteins (AKAPs). We have investigated in vivo and in vitro the biological effects of the expression of a prototypic member of the family, AKAP75, on smooth muscle cells. In vitro expression of AKAP75 in smooth muscle cells stimulated cAMP-induced transcription, increased the levels of the cyclin-dependent kinase-2 inhibitor p27
kip1
, and reduced cell proliferation. In vivo expression of exogenous AKAP75 in common carotid arteries, subjected to balloon injury, significantly increased the levels of p27
kip1
and inhibited neointimal hyperplasia. Both the effects in smooth muscle cells in vitro and in carotid arteries in vivo were specifically dependent on the amplification of cAMP-dependent protein kinase (PKA) signals by membrane-bound PKA, as indicated by selective loss of the AKAP75 biological effects in mutants defective in the PKA anchor domain or by suppression of AKAP effects by the PKA-specific protein kinase inhibitor. These data indicate that AKAP proteins selectively amplify cAMP-PKA signaling in vitro and in vivo and suggest a possible target for the inhibition of the neointimal hyperplasia after vascular injury.