bluuebunny/crossref_metadata_2025 huggingface dataset:
The Myocardial Na
+
-H
+
Exchange
Authors: Karmazyn Morris , Gan Xiaohong Tracey , Humphreys Rachael A , Yoshida Hiroyuki , Kusumoto Keiji
Published: June 2012

Abstract
—The Na
+
-H
+
exchange (NHE) is a major mechanism by which the heart adapts to intracellular acidosis during ischemia and recovers from the acidosis after reperfusion. There are at least 6 NHE isoforms thus far identified with the NHE1 subtype representing the major one found in the mammalian myocardium. This 110-kDa glycoprotein extrudes protons concomitantly with Na
+
influx in a 1:1 stoichiometric relationship rendering the process electroneutral, and its activity is regulated by numerous factors, including phosphorylation-dependent processes. There is convincing evidence that NHE mediates tissue injury during ischemia and reperfusion, which probably reflects the fact that under conditions of tissue stress, including ischemia, Na
+
-K
+
ATPase is inhibited, thereby limiting Na
+
extrusion, resulting in an elevation in [Na
+
]
i
. The latter effect, in turn, will increase [Ca
2+
]
i
via Na
+
-Ca
2+
exchange. In addition, NHE1 mRNA expression is elevated in response to injury, which may further contribute to the deleterious consequence of pathological insult. Extensive studies using NHE inhibitors have consistently shown protective effects against ischemic and reperfusion injury in a large variety of experimental models and has led to clinical evaluation of NHE inhibition in patients with coronary artery disease. Emerging evidence also implicates NHE1 in other cardiac disease states, and the exchanger may be particularly critical to postinfarction remodeling responses resulting in development of hypertrophy and heart failure.
In Vivo Modeling of Myosin Binding Protein C Familial Hypertrophic Cardiomyopathy
Authors: Yang Qinglin , Sanbe Atsushi , Osinska Hanna , Hewett Timothy E. , Klevitsky Raisa , Robbins Jeffrey
Published: June 2012

Abstract
—Myosin binding protein C (MyBP-C) is an integral part of the striated muscle sarcomere. As is the case for other sarcomeric genes in human populations, multiple mutations within the gene have been linked to familial hypertrophic cardiomyopathy. Although some MyBP-C lesions are the result of missense mutations, most show truncated polypeptides lacking either the myosin or myosin and titin binding sites. Previously, we generated transgenic (TG) mice with cardiac-specific expression of a MyBP-C mutant lacking the myosin and titin binding domains. Surprisingly, the mutant protein was stable and made up a majority of the MyBP-C species, with concomitant reductions in endogenous MyBP-C such that overall MyBP-C stoichiometry was conserved. In the present study, we created a second series of TG mice that express, in the heart, a mutant MyBP-C lacking only the myosin binding site. In contrast to the previous data for the MyBP-C lacking both titin and myosin binding sites, only very modest levels of protein were found, consistent with data obtained from human biopsies in which mutated MyBP-C could not be detected. Despite normal levels of wild-type MyBP-C, there were significant changes in the structure and ultrastructure of the heart. Fiber mechanics showed decreased unloading shortening velocity, maximum shortening velocity, and relative maximal power output.
Diminished Basal Phosphorylation Level of Phospholamban in the Postinfarction Remodeled Rat Ventricle
Authors: Huang Boyu , Wang Su , Qin Dayi , Boutjdir Mohamed , El-Sherif Nabil
Published: June 2012

Abstract
—Three weeks after myocardial infarction (MI) in the rat, remodeled hypertrophy of noninfarcted myocardium is at its maximum and the heart is in a compensated stage with no evidence of heart failure. Our hemodynamic measurements at this stage showed a slight but insignificant decrease of +dP/dt but a significantly higher left ventricular end-diastolic pressure. To investigate the basis of the diastolic dysfunction, we explored possible defects in the β-adrenergic receptor–G
s/i
protein–adenylyl cyclase–cAMP–protein kinase A–phosphatase pathway, as well as molecular or functional alterations of sarcoplasmic reticulum Ca
2+
-ATPase and phospholamban (PLB). We found no significant difference in both mRNA and protein levels of sarcoplasmic reticulum Ca
2+
-ATPase and PLB in post-MI left ventricle compared with control. However, the basal levels of both the protein kinase A–phosphorylated site (Ser16) of PLB (p16-PLB) and the calcium/calmodulin–dependent protein kinase-phosphorylated site (Thr17) of PLB (p17-PLB) were decreased by 76% and 51% in post-MI myocytes (
P
<0.05), respectively. No change was found in the β-adrenoceptor density, G
sα
protein level, or adenylyl cyclase activity. Inhibition of phosphodiesterase and G
i
protein by Ro-20-1724 and pertussis toxin, respectively, did not correct the decreased p16-PLB or p17-PLB levels. Stimulation of β-adrenoceptor or adenylyl cyclase increased both p16-PLB and p17-PLB in post-MI myocytes to the same levels as in sham myocytes, suggesting that decreased p16-PLB and p17-PLB in post-MI myocytes is not due to a decrease in the generation of p16-PLB or p17-PLB. We found that type 1 phosphatase activity was increased by 32% (
P
<0.05) with no change in phosphatase 2A activity. Okadaic acid, a protein phosphatase inhibitor, significantly increased p16-PLB and p17-PLB levels in post-MI myocytes and partially corrected the prolonged relaxation of the [Ca
2+
]
i
transient. In summary, prolonged relaxation of post-MI remodeled myocardium could be explained, in part, by altered basal levels of p16-PLB and p17-PLB caused by increased protein phosphatase 1 activity.
Molecular Cloning and Characterization of the Intermediate-Conductance Ca
2+
-Activated K
+
Channel in Vascular Smooth Muscle
Authors: Neylon Craig B. , Lang Richard J. , Fu Ying , Bobik Alex , Reinhart Peter H.
Published: June 2012

Abstract
—Recent evidence suggests that functional diversity of vascular smooth muscle is produced in part by a differential expression of ion channels. The aim of the present study was to examine the role of Ca
2+
-activated K
+
channels (K
Ca
channels) in the expression of smooth muscle cell functional phenotype. We found that smooth muscle cells exhibiting a contractile function express predominantly large-conductance (≈200 pS) K
Ca
(BK) channels. In contrast, proliferative smooth muscle cells express predominantly K
Ca
channels exhibiting a much smaller conductance (≈32 pS). These channels are blocked by low concentrations of charybdotoxin (10 nmol/L) but, unlike BK channels, are insensitive to iberiotoxin (100 nmol/L). To determine the molecular identity of this K
+
channel, we cloned a 1.9-kb cDNA from an immature-phenotype smooth muscle cell cDNA library. The cDNA contains an open reading frame for a 425 amino acid protein exhibiting sequence homology to other K
Ca
channels, in particular with mIK1 and hIK1. Expression in oocytes gives rise to a K
+
-selective channel exhibiting intermediate-conductance (37 pS at −60 mV) and potent activation by Ca
2+
(
K
d
120 nmol/L). Thus, we have cloned and characterized the vascular smooth muscle intermediate-conductance K
Ca
channel (SMIK), which is markedly upregulated in proliferating smooth muscle cells. The differential expression of these K
Ca
channels in functionally distinct smooth muscle cell types suggests that K
Ca
channels play a role in defining the physiological properties of vascular smooth muscle. The full text of this article is available at http://www.circresaha.org.
Phosphatidylinositol 3-Kinase Is Required for Insulin-Like Growth Factor-I–Induced Vascular Smooth Muscle Cell Proliferation and Migration
Authors: Duan Cunming , Bauchat Jeanette R. , Hsieh Tzefu
Published: June 2012

Abstract
—Insulin-like growth factor–I (IGF-I) plays an important role in regulating vascular smooth muscle cell (VSMC) proliferation and directed migration. The mitogenic and chemotactic actions of IGF-I are mediated through the IGF-I receptor, but how the activation of the IGF-I receptor leads to these biological responses is poorly understood. In this study, we examined the role of phosphatidylinositol 3-kinase (PI3 kinase) in mediating the mitogenic and chemotactic signals of IGF-I. IGF-I treatment resulted in a significant increase in phosphotyrosine-associated PI3 kinase activity in cultured primary VSMCs. To determine whether insulin receptor substrate (IRS)–1, -2, or both are involved in IGF-I signaling in VSMCs, cell lysates were immunoprecipitated with either an anti-IRS-1 or an anti-IRS-2 antibody, and the associated PI3 kinase activity was determined. IGF-I stimulation resulted in a significant increase in IRS-1– but not IRS-2–associated PI3 kinase activity, suggesting that IGF-I primarily utilizes IRS-1 to transmit its signal in VSMCs. The IGF-I–induced increase in IRS-I–associated PI3 kinase activity was concentration dependent. At the maximum concentration (50 ng/mL), IGF-I induced a 60-fold increase. This activation occurred within 5 minutes and was sustained at high levels for at least 6 hours. IGF-I also caused a concentration-dependent and long-lasting activation of protein kinase B (PKB/Akt). Inhibition of PI3 kinase activation by LY294002 or wortmannin abolished IGF-I–stimulated VSMC proliferation and reduced IGF-I–directed VSMC migration by ≈60%. These results indicate that activation of PI3 kinase is required for both IGF-I–induced VSMC proliferation and migration.
Microtubule Disruption Modulates Ca
2+
Signaling in Rat Cardiac Myocytes
Authors: Gómez A. M. , Kerfant B. G. , Vassort G.
Published: June 2012

Abstract
—Microtubules have been shown to alter contraction in cardiac myocytes through changes in cellular stiffness. However, an effect on excitation-contraction coupling has not been examined. Here we analyze the effects of microtubule disruption by 1 μmol/L colchicine on calcium currents (
I
Ca
) and [Ca
2+
]
i
transients in rat ventricular myocytes.
I
Ca
was studied using the whole-cell patch-clamp technique. Colchicine treatment increased
I
Ca
density (peak values, −4.6±0.4 and −9.1±1.3 pA/pF in 11 control and 12 colchicine-treated myocytes, respectively;
P
<0.05).
I
Ca
inactivation was well fitted by a biexponential function. The slow component of inactivation was unchanged, whereas the fast component was accelerated after colchicine treatment (at −10 mV, 11.8±1.0 versus 6.7±1.0 ms in control versus colchicine-treated cells;
P
<0.005). [Ca
2+
]
i
transients were analyzed by fluo-3 epifluorescence simultaneously with
I
Ca
. Peak [Ca
2+
]
i
transients were significantly increased in cardiac myocytes treated with colchicine. The values of F/F
0
at 0 mV were 1.1±0.02 in 9 control cells and 1.4±0.1 in 11 colchicine-treated cells (
P
<0.05). β-Adrenergic stimulation with 1 μmol/L isoproterenol increased both
I
Ca
and [Ca
2+
]
i
transient in control cells. However, no significant change was induced by isoproterenol on colchicine-treated cells. Colchicine and isoproterenol effects were similar and not additive. Inhibition of adenylyl cyclase by 200 μmol/L 2′-deoxyadenosine 3′-monophosphate blunted the colchicine effect. We suggest that β-adrenergic stimulation and microtubule disruption share a common pathway to enhance
I
Ca
and [Ca
2+
]
i
transient.
Hyperaldosteronemia in Rabbits Inhibits the Cardiac Sarcolemmal Na
+
-K
+
Pump
Authors: Mihailidou Anastasia S. , Bundgaard Henning , Mardini Mahidi , Hansen Peter S. , Kjeldsen Keld , Rasmussen Helge H.
Published: June 2012

Abstract
—Aldosterone upregulates the Na
+
-K
+
pump in kidney and colon, classical target organs for the hormone. An effect on pump function in the heart is not firmly established. Because the myocardium contains mineralocorticoid receptors, we examined whether aldosterone has an effect on Na
+
-K
+
pump function in cardiac myocytes. Myocytes were isolated from rabbits given aldosterone via osmotic minipumps and from controls. Electrogenic Na
+
-K
+
pump current, arising from the 3:2 Na
+
:K
+
exchange ratio, was measured in single myocytes using the whole-cell patch clamp technique. Treatment with aldosterone induced a decrease in pump current measured when myocytes were dialyzed with patch pipette solution containing Na
+
in a concentration of 10 mmol/L, whereas there was no effect measured when the solution contained 80 mmol/L Na
+
. Aldosterone had no effect on myocardial Na
+
-K
+
pump concentration evaluated by vanadate-facilitated [
3
H]ouabain binding or by K
+
-dependent paranitrophenylphosphatase activity in crude homogenates. Aldosterone induced an increase in intracellular Na
+
activity. The aldosterone-induced decrease in pump current and increased intracellular Na
+
were prevented by cotreatment with the mineralocorticoid receptor antagonist spironolactone. Our results indicate that hyperaldosteronemia decreases the apparent Na
+
affinity of the Na
+
-K
+
pump, whereas it has no effect on maximal pump capacity.
Myosin Binding Protein C, a Phosphorylation-Dependent Force Regulator in Muscle That Controls the Attachment of Myosin Heads by Its Interaction With Myosin S2
Authors: Kunst Gudrun , Kress Kai R. , Gruen Mathias , Uttenweiler Dietmar , Gautel Mathias , Fink Rainer H. A.
Published: June 2012

Abstract
—Myosin binding protein C (MyBP-C) is one of the major sarcomeric proteins involved in the pathophysiology of familial hypertrophic cardiomyopathy (FHC). The cardiac isoform is
tris
-phosphorylated by cAMP-dependent protein kinase (cAPK) on β-adrenergic stimulation at a conserved N-terminal domain (MyBP-C motif), suggesting a role in regulating positive inotropy mediated by cAPK. Recent data show that the MyBP-C motif binds to a conserved segment of sarcomeric myosin S2 in a phosphorylation-regulated way. Given that most MyBP-C mutations that cause FHC are predicted to result in N-terminal fragments of the protein, we investigated the specific effects of the MyBP-C motif on contractility and its modulation by cAPK phosphorylation. The diffusion of proteins into skinned fibers allows the investigation of effects of defined molecular regions of MyBP-C, because the endogenous MyBP-C is associated with few myosin heads. Furthermore, the effect of phosphorylation of cardiac MyBP-C can be studied in a defined unphosphorylated background in skeletal muscle fibers only. Triton skinned fibers were tested for maximal isometric force, Ca
2+
/force relation, rigor force, and stiffness in the absence and presence of the recombinant cardiac MyBP-C motif. The presence of unphosphorylated MyBP-C motif resulted in a significant (1) depression of Ca
2+
-activated maximal force with no effect on dynamic stiffness, (2) increase of the Ca
2+
sensitivity of active force (leftward shift of the Ca
2+
/force relation), (3) increase of maximal rigor force, and (4) an acceleration of rigor force and rigor stiffness development.
Tris
-phosphorylation of the MyBP-C motif by cAPK abolished these effects. This is the first demonstration that the S2 binding domain of MyBP-C is a modulator of contractility. The anchorage of the MyBP-C motif to the myosin filament is not needed for the observed effects, arguing that the mechanism of MyBP-C regulation is at least partly independent of a “tether,” in agreement with a modulation of the head-tail mobility. Soluble fragments occurring in FHC, lacking the spatial specificity, might therefore lead to altered contraction regulation without affecting sarcomere structure directly.
High-Frequency Periodic Sources Underlie Ventricular Fibrillation in the Isolated Rabbit Heart
Authors: Chen Jay , Mandapati Ravi , Berenfeld Omer , Skanes Allan C. , Jalife José
Published: June 2012

Abstract
—The mechanism(s) underlying ventricular fibrillation (VF) remain unclear. We hypothesized that at least some forms of VF are not random and that high-frequency periodic sources of activity manifest themselves as spatiotemporal periodicities, which drive VF. Twenty-four VF episodes from 8 Langendorff-perfused rabbit hearts were studied using high-resolution video imaging in conjunction with ECG recordings and spectral analysis. Sequential wavefronts that activated the ventricles in a spatially and temporally periodic fashion were identified. In addition, we analyzed the lifespan and dynamics of wavelets in VF, using a new method of phase mapping that enables identification of phase singularity points (PSs), which flank individual wavelets. Spatiotemporal periodicity was found in 21 of 24 episodes. Complete reentry on the epicardial surface was observed in 3 of 24 episodes. The cycle length of discrete regions of spatiotemporal periodicity correlated highly with the dominant frequency of the optical pseudo-ECG (
R
2
=0.75) and with the global bipolar electrogram (
R
2
=0.79). The lifespan of PSs was short (14.7±14.4 ms); 98% of PSs existed for <1 rotation. The mean number of waves entering (6.50±0.69) exceeded the mean number of waves that exited our mapping field (4.25±0.56;
P
<0.05). These results strongly suggest that ongoing stable sources are responsible for the majority of the frequency content of VF and therefore play a role in its maintenance. In this model, multiple wavelets resulting from wavebreaks do not appear to be responsible for the sustenance of this arrhythmia, but are rather the consequence of breakup of high-frequency activation from a dominant reentrant source.
Mechanisms for Regulation of Fluid Shear Stress Response in Circulating Leukocytes
Authors: Fukuda Shunichi , Yasu Takanori , Predescu Dan N. , Schmid-Schönbein Geert W.
Published: June 2012

Abstract
—We have shown that leukocytes retract their pseudopods and detach from substrates after exposure to physiological fluid shear stresses (≈1.5 dyn/cm
2
). In inflammation, however, pseudopod projection during spreading and firm adhesion on endothelium is observed even in microvessels with normal blood flow and fluid shear stresses. Thus, we examined mechanisms that may serve to regulate the shear stress response of circulating leukocytes. In the presence of inflammatory mediators (platelet-activating factor [PAF] f-met-leu-phe), a subgroup of cells ceases to respond to shear stress. cGMP analogs and nitric oxide (NO) donors enhance the shear stress response and reverse the inhibitory effect of inflammatory mediators on the shear stress response, whereas depletion of cGMP leads to cessation of the shear stress response even in unstimulated leukocytes. The ability of cGMP to enhance the shear stress response is not associated with CD18 expression, because cGMP has no effect on CD18 expression in response to shear stress. The shear stress response of leukocytes in endothelial nitric oxide synthase (−/−) mice, in which NO level in blood is decreased, is attenuated compared with that in wild-type mice. In rat mesentery venules stimulated by PAF under normal blood flow, a cGMP analog diminishes pseudopod projection of leukocytes, whereas inhibition of NO leads to enhanced pseudopod projection and spreading. The evidence suggests that inflammatory mediators suppress the shear stress response of leukocytes leading to spreading even under normal physiological shear stress, whereas cGMP may serve to maintain shear stress response even in inflammation. The full text of this article is available at http://www.circresaha.org.
Effects of Long-Term Nitroglycerin Treatment on Endothelial Nitric Oxide Synthase (NOS III) Gene Expression, NOS III–Mediated Superoxide Production, and Vascular NO Bioavailability
Authors: Münzel Thomas , Li Huige , Mollnau Hanke , Hink Ulrich , Matheis Edi , Hartmann Mark , Oelze Mathias , Skatchkov Mikhail , Warnholtz Ascan , Duncker Linda , Meinertz Thomas , Förstermann Ulrich
Published: June 2012

Abstract
—Long-term nitroglycerin (NTG) treatment has been shown to be associated with cross-tolerance to endothelium-dependent vasodilators. It may involve increased production of reactive oxygen species (such as superoxide, O
2
·−
) that rapidly inactivate the nitric oxide (NO) released from the endothelial cells. It remains to be elucidated, however, whether long-term treatment with NTG alters the activity and expression of the endothelial NO synthase (NOS III) and whether this enzyme can contribute to O
2
·−
formation. We studied the influence of long-term NTG treatment on the expression of NOS III as assessed by RNase protection assay and Western blot. Tolerance was measured ex vivo in organ chamber experiments with rat aortic rings. O
2
·−
and NO formation were quantified using lucigenin- and
Cypridina
luciferin analog–enhanced chemiluminescence as well as electron spin resonance (ESR) spectroscopy. Treatment of Wistar rats with NTG (Alzet osmotic minipumps, NTG concentration 10 μg · kg
−1
· min
−1
) for 3 days caused marked tolerance, cross-tolerance to the endothelium-dependent vasodilator acetylcholine, and a significant increase in O
2
·−
-induced chemiluminescence. Tolerance was associated with a significant increase in NOS III mRNA to 236±28% and NOS III protein to 239±17%. In control vessels, the NOS inhibitor
N
G
-nitro-
l
-arginine (L-NNA) increased the O
2
·−
-mediated chemiluminescence, indicating that basal production of endothelium-derived NO depresses the baseline chemiluminescence signal. In the setting of tolerance, however, L-NNA decreased steady-state O
2
·−
levels, indicating the involvement of NOS III in O
2
·−
formation. Likewise, A23187-induced, NOS III–mediated O
2
·−
production was more pronounced in tolerant than in control vessels. Vascular NO bioavailability as assessed with ESR spectroscopy using iron-thiocarbamate as a trap for NO was significantly reduced in tolerant vessels. Pretreatment of tolerant tissue in vitro with the protein kinase C (PKC) inhibitors reduced basal and stimulated NOS III–mediated O
2
·−
production and partially reversed vascular tolerance. These findings suggest that NTG treatment increases the expression of a dysfunctional NOS III gene, leading to increased formation of O
2
·−
and decreased vascular NO bioavailability. Normalization of NOS III–mediated O
2
·−
production and improvement of tolerance with PKC inhibition suggests an important role for PKC isoforms in mediating vascular dysfunction caused by long-term NTG treatment. The full text of this article is available at http://www.circresaha.org.
Folic Acid Reverts Dysfunction of Endothelial Nitric Oxide Synthase
Authors: Stroes E. S. G. , van Faassen E. E. , Yo M. , Martasek P. , Boer P. , Govers R. , Rabelink T. J.
Published: June 2012

Abstract
—5-Methyltetrahydrofolate (MTHF), the active form of folic acid, has been reported to restore NO status in hypercholesterolemic patients. The mechanism of this effect remains to be established. We assessed the effects of L- and D-MTHF on tetrahydrobiopterin (BH
4
)–free and partially BH
4
-repleted endothelial NO synthase (eNOS). Superoxide production of eNOS and the rate constants for trapping of superoxide by MTHF were determined with electron paramagnetic resonance using 5-diethoxyphosphoryl-5-methyl-1-pyrroline-
N
-oxide (DEPMPO) as spin trap for superoxide. NO production was measured with [
3
H]arginine–citrulline conversion or nitrite assay. The rate constants for scavenging of superoxide by L- and D-MTHF were similar, 1.4×10
4
ms
–1
. In BH
4
-free eNOS, L- and D-MTHF have no effect on enzymatic activity. In contrast, in partially BH
4
-repleted eNOS, we observe a 2-fold effect of MTHF on the enzymatic activity. First, superoxide production is reduced. Second, NO production is enhanced. In cultured endothelial cells, a similar enhancement of NO production is induced by MTHF. In the present study, we show direct effects of MTHF on the enzymatic activity of NO synthase both in recombinant eNOS as well as in cultured endothelial cells, which provides a plausible explanation for the previously reported positive effects of MTHF on NO status in vivo.
Inability to Induce Hypertension in Normotensive Rat Expressing AT
1
Receptor Antisense
Authors: Pachori Alok S. , Wang Hongwei , Gelband Craig H. , Ferrario Carlos M. , Katovich Michael J. , Raizada Mohan K.
Published: June 2012

Abstract
—Our previous studies have shown that neonatal delivery of angiotensin type 1 receptor antisense (AT
1
R-AS) in a retroviral vector prevents spontaneously hypertensive rats from developing hypertension for life but has no effect on blood pressure (BP) in normotensive animals. Based on these results, we hypothesized that AT
1
R-AS transduction in normotensive rats would protect them from developing experimental hypertension. The present study was designed to evaluate this hypothesis. A single intracardiac administration of AT
1
R-AS by a retroviral-mediated delivery system (LNSV-AT
1
R-AS) in 5-day-old normotensive Sprague-Dawley rats resulted in long-term expression of the AT
1
R-AS without an effect on basal BP. However, angiotensin II (Ang II)–induced BP, dipsogenic responses, and renovascular contractility were significantly attenuated in the LNSV–AT
1
R-AS–treated rats. Chronic infusion of low-dose Ang II (55 ng · kg
−
1
· min
−
1
) in LNSV-alone–treated rats caused a modest increase in BP, profound increase in cardiac hypertrophy, and increased vascular contractility. In contrast, the LNSV-AT
1
R-AS–treated rats were protected from developing these changes after Ang II infusion. These data establish that LNSV-AT
1
R-AS pretreatment protects healthy rats from developing Ang II–dependent hypertension.
Mechanism and Selectivity of the Effects of Halothane on Gap Junction Channel Function
Authors: He Ding Sheng , Burt Janis M.
Published: June 2012

Abstract
—Volatile anesthetics alter tissue excitability by decreasing the extent of gap junction–mediated cell-cell coupling and by altering the activity of the channels that underlie the action potential. In the present study, we demonstrate, using dual whole-cell voltage-clamp techniques, that coexpression of connexin (Cx) 40 and Cx43 rendered cells more sensitive to uncoupling by halothane than cells that express only Cx40 or only Cx43. The halothane-induced reduction in junctional conductance was caused by decreased channel mean open time and increased channel mean closed time. The magnitude of the effect of halothane on channel open time was least for Cx40-like channels and greatest for heteromeric channels. Thus, the data indicate that halothane gates gap junction channels to the closed state in a dose-dependent and connexin-specific manner. One consequence of the selectivity of halothane is that the profile of single-channel events observed in the presence of halothane may not be quantitatively representative of the population of channels contributing to macroscopic conductance in cells that express more than one connexin. In addition, in tissues that express multiple connexins, such as heart and blood vessels, the capacity of the gap junctions to transmit electrical and chemical signals in the presence of halothane could vary according to the pattern of connexin expression. The full text of this article is available at http://www.circresaha.org.
2-Deoxy-ATP Enhances Contractility of Rat Cardiac Muscle
Authors: Regnier M. , Rivera A. J. , Chen Y. , Chase P. B.
Published: June 2012

Abstract
—To investigate the kinetic parameters of the crossbridge cycle that regulate force and shortening in cardiac muscle, we compared the mechanical properties of cardiac trabeculae with either ATP or 2-deoxy-ATP (dATP) as the substrate for contraction. Comparisons were made in trabeculae from untreated rats (predominantly V1 myosin) and those treated with propylthiouracil (PTU; V3 myosin). Steady-state hydrolytic activity of cardiac heavy meromyosin (HMM) showed that PTU treatment resulted in >40% reduction of ATPase activity. dATPase activity was >50% elevated above ATPase activity in HMM from both untreated and PTU-treated rats.
V
max
of actin-activated hydrolytic activity was also >50% greater with dATP, whereas the
K
m
for dATP was similar to that for ATP. This indicates that dATP increased the rate of crossbridge cycling in cardiac muscle. Increases in hydrolytic activity were paralleled by increases of 30% to 80% in isometric force (F
max
), rate of tension redevelopment (
k
tr
), and unloaded shortening velocity (
V
u
) in trabeculae from both untreated and PTU-treated rats (at maximal Ca
2+
activation), and F-actin sliding speed in an in vitro motility assay (
V
f
). These results contrast with the effect of dATP in rabbit psoas and soleus fibers, where F
max
is unchanged even though
k
tr
,
V
u
, and
V
f
are increased. The substantial enhancement of mechanical performance with dATP in cardiac muscle suggests that it may be a better substrate for contractility than ATP and warrants exploration of ribonucleotide reductase as a target for therapy in heart failure.
The Insulin-Like Growth Factor Axis
Authors: Bayes-Genis Antoni , Conover Cheryl A. , Schwartz Robert S.
Published: June 2012

Abstract
—Insulin-like growth factors I and II (IGF-I and -II) and their regulatory proteins are secreted by cells of the cardiovascular system. They are growth promoters for arterial cells and mediators of cardiovascular disease. IGFs are bound to IGF binding proteins (IGFBPs), which modulate IGF ligand-receptor interaction and consequently to IGF action. IGFBPs are in turn posttranslationally modulated by specific proteases. This dynamic balance (IGFs, IGFBPs, and IGFBP proteases) constitutes the IGF axis and ultimately determines the extent of IGF-dependent cellular effects. Dysregulated actions of this axis influence coronary atherosclerosis through effects on vascular smooth muscle cell growth, migration, and extracellular matrix synthesis in the atherosclerotic plaque. IGF-I promotes macrophage chemotaxis, excess LDL cholesterol uptake, and release of proinflammatory cytokines. Endothelial cells also receive the effects of IGFs stimulating their migration and organization forming capillary networks. Neointimal hyperplasia of restenosis after coronary artery injury is also modulated by the IGF axis. IGFs stimulate vascular smooth muscle cell proliferation and migration to form the neointima and upregulate tropoelastin synthesis after disruption of the elastic layer. Understanding IGF axis regulation establishes a scientific basis for strategies directed to limit or reverse plaque growth and vulnerability in atherosclerosis and in the neointimal hyperplasia of restenosis.
Angiotensin-Converting Enzyme Inhibitors Downregulate Tissue Factor Synthesis in Monocytes
Authors: Napoleone Emanuela , Di Santo Angelomaria , Camera Marina , Tremoli Elena , Lorenzet Roberto
Published: June 2012

Abstract
—Angiotensin-converting enzyme (ACE) inhibitors reduce the risk of recurrent myocardial infarction in patients with left ventricular dysfunction. Tissue factor (TF), the initiator of blood coagulation, plays a pivotal role in arterial thrombosis that occurs after atherosclerotic plaque fissuring. Because monocytes synthesize TF and contain several components of the renin-angiotensin system, we investigated the possibility that ACE inhibitors could modulate monocyte TF expression. Mononuclear leukocytes from healthy volunteers were incubated with endotoxin in the presence or absence of different ACE inhibitors. Captopril reduced TF expression in endotoxin-stimulated mononuclear leukocytes, as measured by a 1-stage clotting assay and ELISA analysis, by ≈60%. The effect was dose-dependent and was attributable to ACE inhibition, given that other ACE inhibitors, such as idrapril or fosinopril, and losartan, an antagonist of the angiotensin II AT
1
receptor, caused a comparable reduction in TF activity. Reverse transcriptase–polymerase chain reaction indicated that endotoxin-mediated increased levels of TF mRNA were inhibited by ACE inhibitors. Moreover, endotoxin-induced nuclear factor-κB translocation to the promoter region of the gene encoding for TF was markedly inhibited by captopril. The finding that ACE inhibitors and angiotensin II AT
1
antagonists can potentially modulate TF expression by mononuclear cells has important biological and therapeutic implications for the evolution of thrombi. Our results suggest that the anti-ischemic effect of these drugs might be explained, at least in part, by their ability to reduce TF expression in monocytes.
Ischemic Preconditioning Activates MAPKAPK2 in the Isolated Rabbit Heart
Authors: Nakano Atsushi , Baines Christopher P. , Kim Sung O. , Pelech Steven L. , Downey James M. , Cohen Michael V. , Critz Stuart D.
Published: June 2012

Abstract
—Recent studies suggest that p38 mitogen-activated protein kinase (MAPK) may be involved in ischemic preconditioning (PC). To further test this possibility, the regulation of MAPK-activated protein kinase 2 (MAPKAPK2), a kinase immediately downstream from p38 MAPK, and the activity of c-Jun NH
2
-terminal kinase (JNK), a second MAPK, were examined in preconditioned hearts. Isolated, perfused rabbit hearts were subjected to 20 to 30 minutes of global ischemia. Ventricular biopsies before treatment and after 20 minutes of ischemia were homogenized, and the activities of MAPKAPK2 and JNK were evaluated. For the MAPKAPK2 experiments, 7 groups were studied, as follows: control hearts; preconditioned hearts; hearts treated with 500 nmol/L R(–)
N
6
-(2-phenylisopropyl) adenosine (PIA), an A
1
-adenosine receptor agonist; preconditioned hearts pretreated with 100 μmol/L 8-(
p
-sulfophenyl) theophylline (SPT), an adenosine receptor antagonist; preconditioned hearts also treated with SB 203580, a potent inhibitor of p38 MAPK activation; hearts treated with 50 ng/mL anisomycin (a p38 MAPK/JNK activator); and hearts treated with both anisomycin (50 ng/mL) and the tyrosine kinase inhibitor genistein (50 μmol/L). MAPKAPK2 activity was not altered in control hearts after 20 minutes of global ischemia. By contrast, there was a 3.8-fold increase in activity during ischemia in preconditioned hearts. Activation of MAPKAPK2 in preconditioned hearts was blocked by both SPT and SB 203580. MAPKAPK2 activity during ischemia increased 3.5-fold and 3.3-fold in hearts pretreated with PIA or anisomycin, respectively. MAPKAPK2 activation during ischemia in hearts pretreated with anisomycin was blocked by genistein. In separate hearts, anisomycin mimicked the anti-infarct effect of PC, and that protection was abolished by genistein. JNK activity was measured in control and preconditioned hearts. There was a comparable, modest decline in activity during 30 minutes of global ischemia in both groups. As a positive control, a third group of hearts was treated with anisomycin before global ischemia, and in these, JNK activity increased by 290% above baseline. These results confirm that the p38 MAPK/MAPKAPK2 pathway is activated during ischemia only if the heart is in a preconditioned state. These data further support p38 MAPK as an important signaling component in ischemic PC.
Antisense Intercellular Adhesion Molecule-1 (ICAM-1) Oligodeoxyribonucleotide Delivered During Organ Preservation Inhibits Posttransplant ICAM-1 Expression and Reduces Primary Lung Isograft Failure
Authors: Toda Koichi , Kayano Koichi , Karimova Ann , Naka Yoshifumi , Fujita Tomoyuki , Minamoto Kanji , Wang Catherine Y. , Pinsky David J.
Published: June 2012

Abstract
—Transiently increased expression of leukocyte adhesion receptors after lung preservation contributes to early graft demise by recruiting leukocytes, activating complement, and causing microcirculatory stasis. We hypothesized that inhibiting intercellular adhesion molecule-1 (ICAM-1) expression even briefly may significantly improve lung graft function and that the preservation period might provide a unique window to deliver a therapeutic pulse of antisense oligonucleotide ICAM-1 to inhibit ICAM-1 expression after transplantation. Interleukin-1β–treated rat pulmonary endothelial cells given a 20-mer phosphorothioate oligonucleotide comprising an antisense span targeted to the 3′-untranslated region of rat ICAM-1 demonstrated an oligonucleotide dose–dependent reduction in ICAM-1 expression. Using a cationic liposomal carrier, this same antisense oligonucleotide (but not the sense control) instilled into the pulmonary vasculature at the time of preservation reduced subsequent graft ICAM-1 expression and graft leukostasis and markedly improved oxygenation, pulmonary blood flow, and graft survival. These experiments demonstrate that the preservation period presents a window during which to target an anti–ICAM-1 expression strategy to inhibit early adhesion receptor expression and improve functional outcome after lung transplantation.
Interferon-γ Induces AT
2
Receptor Expression in Fibroblasts by Jak/STAT Pathway and Interferon Regulatory Factor-1
Authors: Horiuchi Masatsugu , Hayashida Wataru , Akishita Masahiro , Yamada Seiichiro , Lehtonen Jukka Y. A. , Tamura Kouichi , Daviet Laurent , Chen Yuquing E. , Hamai Meiko , Cui Tai-Xing , Iwai Masaru , Minokoshi Yasuhiko
Published: June 2012

Abstract
—The expression of angiotensin II type 2 (AT
2
) receptor is closely associated with cell growth, differentiation, and/or injury. We examined the effect of interferon (IFN)-γ on AT
2
receptor expression in mouse fibroblast R3T3 cells and demonstrated that IFN-γ treatment increased the expression of AT
2
receptor mRNA as well as its binding. Interferon regulatory factor (IRF)-1 was induced in mouse fibroblast R3T3 cells after IFN-γ stimulation, and electrophoretic mobility shift assay showed an increase in IRF-1 binding with the IRF-specific binding sequence in the AT
2
receptor gene promoter region after IFN-γ stimulation. The IRF-1 gene promoter contains an IFN-γ–activated sequence (GAS) motif for possible binding of signal transducer(s) and activator(s) of transcription (STAT). Indeed, in R3T3 cells, IFN-γ treatment resulted in rapid activation of Janus kinase (Jak) 1, Jak2, and STAT1 via tyrosine phosphorylation. Electrophoretic mobility shift assay with the GAS probe revealed increased STAT1 binding to the IRF-1 gene promoter in response to IFN-γ stimulation. Transfection of GAS-binding oligonucleotides inhibited the effect of IFN-γ on IRF-1 production, resulting in the AT
2
receptor
trans
-activation. Taken together, our data show that IFN-γ upregulates AT
2
receptor expression in R3T3 cells via the activation of the intracellular Jak/STAT pathway and production of IRF-1.
Tetrahydrobiopterin Improves Endothelium-Dependent Vasodilation in Chronic Smokers
Authors: Heitzer Thomas , Brockhoff Carsten , Mayer Bernd , Warnholtz Ascan , Mollnau Hanke , Henne Simone , Meinertz Thomas , Münzel Thomas
Published: June 2012

Abstract
—Conditions associated with impaired nitric oxide (NO) activity and accelerated atherosclerosis have been shown to be associated with a reduced bioavailability of tetrahydrobiopterin (BH4). We therefore hypothesized that BH4 supplementation may improve endothelial dysfunction of chronic smokers. Forearm blood flow (FBF) responses to the endothelium-dependent vasodilators acetylcholine (ACh; 0.75, 1.5, and 3.0 μg/100 mL tissue/min) or serotonin (5-HT; 0.7, 2.1, and 6.3 ng/100 mL tissue/min), to the inhibitor of endothelial nitric oxide synthase (NOS)
N
G
-monomethyl-
l
-arginine (L-NMMA; 2, 4, and 8 μmol/min), and to the endothelium-independent vasodilator sodium nitroprusside (SNP; 0.1, 0.3, and 1.0 μg/100 mL tissue/min) were measured by venous occlusion plethysmography in controls and chronic smokers. Drugs were infused into the brachial artery, and FBF was measured before and during concomitant intra-arterial infusion of BH4, tetrahydroneopterin (NH4; another reduced pteridine), or the antioxidant vitamin C (6 and 18 mg/min). In control subjects, BH4 had no effect on FBF in response to ACh, 5-HT, and SNP. In contrast, in chronic smokers, the attenuated FBF responses to ACh and 5-HT were markedly improved by concomitant administration of BH4, whereas the vasodilator responses to SNP were not affected. L-NMMA-induced vasoconstriction was significantly reduced in smokers compared with controls, suggesting impaired basal NO bioactivity. BH4 improved L-NMMA responses in smokers while having no effect on L-NMMA responses in controls. Pretreatment with vitamin C abolished BH4 effects on ACh-dependent vasodilation. In vitro, NH4 scavenged superoxide created by the xanthine/xanthine oxidase reaction equipotent like BH4 but failed to modify ACh-induced changes in FBF in chronic smokers in vivo. These data support the concept that in addition to the free radical burden of cigarette smoke, a dysfunctional NOS III due to BH4 depletion may contribute at least in part to endothelial dysfunction in chronic smokers. The full text of this article is available at http://www.circresaha.org.
Enhanced Dispersion of Repolarization and Refractoriness in Transgenic Mouse Hearts Promotes Reentrant Ventricular Tachycardia
Authors: Baker Linda C. , London Barry , Choi Bum-Rak , Koren Gideon , Salama Guy
Published: June 2012

Abstract
—The heterogeneous distribution of ion channels in ventricular muscle gives rise to spatial variations in action potential (AP) duration (APD) and contributes to the repolarization sequence in healthy hearts. It has been proposed that enhanced dispersion of repolarization may underlie arrhythmias in diseases with markedly different causes. We engineered dominant negative transgenic mice that have prolonged QT intervals and arrhythmias due to the loss of a slowly inactivating K
+
current. Optical techniques are now applied to map APs and investigate the mechanisms underlying these arrhythmias. Hearts from transgenic and control mice were isolated, perfused, stained with di-4-ANEPPS, and paced at multiple sites to optically map APs, activation, and repolarization sequences at baseline and during arrhythmias. Transgenic hearts exhibited a 2-fold prolongation of APD, less shortening (8% versus 40%) of APDs with decreasing cycle length, altered restitution kinetics, and greater gradients of refractoriness from apex to base compared with control hearts. A premature impulse applied at the apex of transgenic hearts produced sustained reentrant ventricular tachycardia (n=14 of 15 hearts) that did not occur with stimulation at the base (n=8) or at any location in control hearts (n=12). In transgenic hearts, premature impulses initiated reentry by encountering functional lines of conduction block caused by enhanced dispersion of refractoriness. Reentrant VT had stable (>30 minutes) alternating long/short APDs associated with long/short cycle lengths and T wave alternans. Thus, optical mapping of genetically engineered mice may help elucidate some electrophysiological mechanisms that underlie arrhythmias and sudden death in human cardiac disorders.
Lateral Zone of Cell-Cell Adhesion as the Major Fluid Shear Stress–Related Signal Transduction Site
Authors: Kano Yumiko , Katoh Kazuo , Fujiwara Keigi
Published: June 2012

Abstract
—It has been proposed previously that actin filaments and cell adhesion sites are involved in mechanosignal transduction. In this study, we present certain morphological evidence that supports this hypothesis. The 3D disposition of actin filaments and phosphotyrosine-containing proteins in endothelial cells in situ was analyzed by using confocal microscopy and image reconstruction techniques. Surgical coarctations were made in guinea pig aortas, and the same 3D studies were conducted on such areas 1 week later. Stress fibers (SFs) were present at both basal and apical regions of endothelial cells regardless of coarctation, and several phosphotyrosine-containing proteins were associated with SF ends. Apical SFs had one end attached to the apical cell membrane and the other attached to either the basal membrane or the lateral cell border. Within the coarctation area, the actin filament–containing and vinculin-containing structures became prominent, especially at the apical and the lateral regions. Substantially higher levels of anti-phosphotyrosine and anti-Src staining were detected in the constricted area, particularly at the cell-cell apposition, whereas the anti–focal adhesion kinase, anti–CT10-related kinase, anti–platelet endothelial cell adhesion molecule-l, anti-vinculin, and phalloidin staining intensities increased only slightly after coarctation. We propose that apical SFs directly transmit the mechanical force of flow from the cell apex to the lateral and/or basal SF anchoring sites and that the SF ends associated with signaling molecules are sites of signal transduction. Our results support the idea that the cell apposition area is the major fluid shear stress–dependent mechanosignal transduction site in endothelial cells.
Abnormal Cardiac Na
+
Channel Properties and QT Heart Rate Adaptation in Neonatal Ankyrin
B
Knockout Mice
Authors: Chauhan Vijay S. , Tuvia Shmuel , Buhusi Mona , Bennett Vann , Grant Augustus O.
Published: June 2012

Abstract
—The cytoskeleton of the cardiomyocyte has been shown to modulate ion channel function. Cytoskeletal disruption in vitro alters Na
+
channel kinetics, producing a late Na
+
current that can prolong repolarization. This study describes the properties of the cardiac Na
+
channel and cardiac repolarization in neonatal mice lacking ankyrin
B
, a cytoskeletal “adaptor” protein. Using whole-cell voltage clamp techniques,
I
Na
density was lower in ankyrin
B
(−/−) ventricular myocytes than in wild-type (WT) myocytes (−307±26 versus −444±39 pA/pF,
P
<0.01). Ankyrin
B
(−/−) myocytes exhibited a hyperpolarizing shift in activation and inactivation kinetics compared with WT. Slower recovery from inactivation contributed to the negative shift in steady-state inactivation in ankyrin
B
(−/−). Single Na
+
channel mean open time was longer in ankyrin
B
(−/−) versus WT at test potentials (
V
t
) of −40 mV (1.0±0.1 versus 0.61±0.04 ms,
P
<0.05) and −50 mV (0.8±0.1 versus 0.39±0.05 ms,
P
<0.05). Ankyrin
B
(−/−) exhibited late single-channel openings at
V
t
−40 and −50 mV, which were not seen in WT. Late
I
Na
contributed to longer action potential durations measured at 90% repolarization (APD
90
) at 1 Hz stimulation in ankyrin
B
(−/−) compared with WT (354±26 versus 274±22 ms,
P
<0.05). From ECG recordings of neonatal mice, heart rates were slower in ankyrin
B
(−/−) than in WT (380±14 versus 434±13 bpm,
P
<0.01). Although the QT interval was similar in ankyrin
B
(−/−) and WT at physiological heart rates, QT-interval prolongation in response to heart rate deceleration was greater in ankyrin
B
(−/−). In conclusion, Na
+
channels in ankyrin
B
(−/−) display reduced
I
Na
density and abnormal kinetics at the whole-cell and single-channel level that contribute to prolonged APD
90
and abnormal QT-rate adaptation.