bluuebunny/crossref_metadata_2025 huggingface dataset:
Multidimensional Rhythm Disturbances as a Precursor of Sustained Ventricular Tachyarrhythmias
Authors: Shusterman Vladimir , Aysin Benhur , Anderson Kelley P. , Beigel Anna
Published: September 2007

Abstract
—Cardiac cycle dynamics reflect underlying physiological changes that could predict imminent arrhythmias but are obscured by high complexity, nonstationarity, and large interindividual differences. To overcome these problems, we developed an adaptive technique, referred to as the modified Karhunen-Loeve transform (MKLT), that identifies an individual characteristic (“core”) pattern of cardiac cycles and then tracks the changes in the pattern by projecting the signal onto characteristic eigenvectors. We hypothesized that disturbances in the core pattern, indicating progressive destabilization of cardiac rhythm, would predict the onset of spontaneous sustained ventricular tachyarrhythmias (VTAs) better than previously reported methods. We analyzed serial ambulatory ECGs recorded in 57 patients at the time of VTA and non-VTA 24-hour periods. The disturbances in the pattern were found in 82% of the recordings before the onset of impending VTA, and their dimensionality, defined as the number of unstable orthogonal projections, increased gradually several hours before the onset. MKLT provided greater sensitivity and specificity (70% and 93%) compared with the best traditional method (68% and 67%, respectively). We present a theoretical analysis of MKLT and describe the effects of ectopy and slow changes in cardiac cycles on the disturbances in the pattern. We conclude that MKLT provides greater predictive accuracy than previously reported methods. The improvement is due to the use of individual patterns as a reference for tracking the changes. Because this approach is independent of the group reference values or the underlying clinical context, it should have substantial potential for predicting other forms of arrhythmic events in other populations.
The Carboxyl Terminal Domain Regulates the Unitary Conductance and Voltage Dependence of Connexin40 Gap Junction Channels
Authors: Anumonwo Justus M. B. , Taffet Steven M. , Gu Hong , Chanson Marc , Moreno Alonso P. , Delmar Mario
Published: September 2007

Abstract
—Chemical regulation of connexin (Cx) 40 and Cx43 follows a ball-and-chain model, in which the carboxyl terminal (CT) domain acts as a gating particle that binds to a receptor affiliated with the pore. Moreover, Cx40 channels can be closed by a heterodomain interaction with the CT domain of Cx43 and vice versa. Here, we report similar interactions in the establishment of the unitary conductance and voltage-dependent profile of Cx40 in N2A cells. Two mean unitary conductance values (“lower conductance” and “main”) were detected in wild-type Cx40. Truncation of the CT domain at amino acid 248 (Cx40tr248) caused the disappearance of the lower-conductance state. Coexpression of Cx40tr248 with the CT fragment of either Cx40 (homodomain interactions) or Cx43 (heterodomain interactions) rescued the unitary conductance profile of Cx40. In the N2A cells, the time course of macroscopic junctional current relaxation was best described by a biexponential function in the wild-type Cx40 channels, but it was reduced to a single-exponential function after truncation. However, macroscopic junctional currents recorded in the oocyte expression system were not significantly different between the wild-type and mutant channels. Concatenation of the CT domain of Cx43 to amino acids 1 to 248 of Cx40 yielded a chimeric channel with unitary conductance and voltage-gating profile indistinguishable from that of wild-type Cx40. We conclude that residence of Cx40 channels in the lower-conductance state involves a ball-and-chain type of interaction between the CT domain and the pore-forming region. This interaction can be either homologous (Cx40 truncation with Cx40CT) or heterologous (with the Cx43CT).
Gene Transfer of Heterologous G Protein–Coupled Receptors to Cardiomyocytes
Authors: Laugwitz Karl-Ludwig , Weig Hans-Jörg , Moretti Alessandra , Hoffmann Eva , Ueblacker Peter , Pragst Ingo , Rosport Kai , Schömig Albert , Ungerer Martin
Published: September 2007

Abstract
—In heart failure, reduced cardiac contractility is accompanied by blunted cAMP responses to β-adrenergic stimulation. Parathyroid hormone (PTH)–related peptide and arginine vasopressin are released from the myocardium in response to increased wall stress but do not stimulate contractility or adenylyl cyclase at physiological concentrations. To bypass the defective β-adrenergic signaling cascade, recombinant P1 PTH/PTH-related peptide receptors (rPTH1-Rs) and V
2
vasopressin receptors (rV
2
-Rs), which are normally not expressed in the myocardium and which are both strongly coupled to adenylyl cyclase, and recombinant β
2
-adrenergic receptors (rβ
2
-ARs) were overexpressed in cardiomyocytes by viral gene transfer. The capacity of endogenous hormones to increase contractility via the heterologous, recombinant receptors was compared. Whereas V
2
-Rs are uniquely coupled to Gs, PTH1-Rs and β
2
-ARs are also coupled to other G proteins. Gene transfer of rPTH1-Rs or rβ
2
-ARs to adult cardiomyocytes resulted in maximally increased basal contractility, which could not be further stimulated by adding receptor agonists. Agonists at rPTH1-Rs induced increased cAMP formation and phospholipase C activity. In contrast, healthy or failing rV
2
-R–expressing cardiomyocytes showed unaltered basal contractility. Their contractility and cAMP formation increased only at agonist exposure, which did not activate phospholipase C. In summary, we found that gene transfer of PTH1-Rs to cardiomyocytes results in constitutive activity of the transgene, as does that of β
2
-ARs. In the absence of receptor agonists, rPTH1-Rs and rβ
2
-ARs increase basal contractility, coupling to 2 G proteins simultaneously. In contrast, rV
2
-Rs are uniquely coupled to Gs and are not constitutively active, retaining their property to be activated exclusively on agonist stimulation. Therefore, gene transfer of V
2
-Rs might be more suited to test the effects of cAMP-stimulating receptors in heart failure than that of PTH1-Rs or β
2
-ARs.
Dynamics of Intramural and Transmural Reentry During Ventricular Fibrillation in Isolated Swine Ventricles
Authors: Valderrábano Miguel , Lee Moon-Hyoung , Ohara Toshihiko , Lai Angela C. , Fishbein Michael C. , Lin Shien-Fong , Karagueuzian Hrayr S. , Chen Peng-Sheng
Published: September 2007

Abstract
—The intramural dynamics of ventricular fibrillation (VF) remain poorly understood. Recent investigations have suggested that stable intramural reentry may underlie the mechanisms of VF. We performed optical mapping studies of VF in isolated swine right ventricles (RVs) and left ventricles (LVs). Nine RV walls were cut obliquely in their distal edge exposing the transmural surface. Six LV wedge preparations were also studied. Results showed that intramural reentry was present. In RV, 28 of 44 VF episodes showed reentry; 15% of the activation pathways were reentrant. Except for 4 episodes, reentry was transmural, involving subendocardial structures as the papillary muscle (PM) or trabeculae. In LV, reentry was observed in 27 of 27 VF episodes; 23% of the activations were part of reentrant pathways (
P
<0.05 compared with RV). All LV reentrant pathways were truly intramural (confined to the wall) and were frequently located at the PM insertion. In both ventricles, reentry was spatially and temporally unstable. Histological studies showed abrupt changes in fiber orientation at sites of reentry and wave splitting. Connexin 40 immunostaining demonstrated intramyocardial Purkinje fibers at sites of reentry in the PM root and around endocardial trabeculae. Our results confirm that reentry is frequent—but unstable—in the myocardial wall during VF. In RV, reentry is mostly transmural and requires participation of subendocardial structures. The LV has a greater incidence of reentry and is intramural. Anisotropic anatomic structures played key roles in the generation of wave splitting and in the maintenance of reentry.
Angiotensin Selectively Activates a Subpopulation of Postganglionic Sympathetic Neurons in Mice
Authors: Ma Xiuying , Chapleau Mark W. , Whiteis Carol A. , Abboud Francois M. , Bielefeldt Klaus
Published: September 2007

Abstract
—Angiotensin II (Ang II) increases renal sympathetic nerve activity in anesthetized mice before and after ganglionic blockade, suggesting that Ang II may directly activate postganglionic sympathetic neurons. The present study directly tested this hypothesis in vitro. Neurons were dissociated from aortic-renal and celiac ganglia of C57BL/6J mice. Cytosolic Ca
2+
concentration ([Ca
2+
]
i
) was measured with ratio imaging using fura 2. Ang II increased [Ca
2+
]
i
in a subpopulation of sympathetic neurons. At a concentration of 200 nmol/L, 14 (67%) of 21 neurons responded with a rise in [Ca
2+
]
i
. The Ang II type 1 (AT
1
) receptor blocker (losartan, 2 μmol/L) but not the Ang II type 2 (AT
2
) receptor blocker (PD123,319, 4 μmol/L) blocked this effect. The Ang II–induced [Ca
2+
]
i
increase was abolished by removal of extracellular Ca
2+
but not altered by depletion of intracellular Ca
2+
stores with thapsigargin. Ang II no longer elicited a [Ca
2+
]
i
increase in the presence of lanthanum (25 μmol/L). The specific N-type and L-type Ca
2+
channel blockers, ω-conotoxin GVIA and nifedipine, respectively, significantly inhibited the Ang II–induced [Ca
2+
]
i
increase. The protein kinase C inhibitor H7 but not the protein kinase A inhibitor H89 blocked the response to Ang II. These results demonstrate that Ang II selectively activates a subpopulation of postganglionic sympathetic neurons in aortic-renal and celiac ganglia, triggering Ca
2+
influx through voltage-gated Ca
2+
channels. This effect is mediated through AT
1
receptors and requires the activation of protein kinase C. The activation of a subgroup of sympathetic neurons by Ang II may exert unique effects on kidney function in pathological states associated with elevated Ang II.
Cardiac Angiotensin II Formation in the Clinical Course of Heart Failure and Its Relationship With Left Ventricular Function
Authors: Serneri Gian Gastone Neri , Boddi Maria , Cecioni Ilaria , Vanni Simone , Coppo Mirella , Papa Maria Letizia , Bandinelli Brunella , Bertolozzi Iacopo , Polidori Gianluca , Toscano Thomas , Maccherini Massimo , Modesti Pietro Amedeo
Published: September 2007

Abstract
—In 76 patients with heart failure (HF) (New York Heart Association [NYHA] classes I through IV) and in 15 control subjects, cardiac angiotensin II (Ang II) generation and its relationship with left ventricular function were investigated by measuring aorta–coronary sinus concentration gradients of endogenous angiotensins and in a part of patients by studying
125
I-labeled Ang I kinetics. Gene expression and cellular localization of the cardiac renin-angiotensin system components, the density of AT
1
and AT
2
on membranes and isolated myocytes, and the capacity of isolated myocytes for synthesizing the hypertrophying growth factors insulin-like growth factor-I (IGF-I) and endothelin (ET)-1 were also investigated on 22 HF explanted hearts (NYHA classes III and IV) and 7 nonfailing (NF) donor hearts. Ang II generation increased with progression of HF, and end-systolic wall stress was the only independent predictor of Ang II formation. Angiotensinogen and angiotensin-converting enzyme mRNA levels were elevated in HF hearts, whereas chymase levels were not, and mRNAs were almost exclusively expressed on nonmyocyte cells. Ang II was immunohistochemically detectable both on myocytes and interstitial cells. Binding studies showed that AT
1
density on failing myocytes did not differ from that of NF myocytes, with preserved AT
1
/AT
2
ratio. Conversely, AT
1
density was lower in failing membranes than in NF ones. Ang II induced IGF-I and ET-1 synthesis by isolated NF myocytes, whereas failing myocytes were unable to respond to Ang II stimulation. This study demonstrates that (1) the clinical course of HF is associated with progressive increase in cardiac Ang II formation, (2) AT
1
density does not change on failing myocytes, and (3) failing myocytes are unable to synthesize IGF-I and ET-1 in response to Ang II stimulation.>
Infection of Endothelium With E1
−
E4
+
, but Not E1
−
E4
−
, Adenovirus Gene Transfer Vectors Enhances Leukocyte Adhesion and Migration by Modulation of ICAM-1, VCAM-1, CD34, and Chemokine Expression
Authors: Rafii Shahin , Dias Sergio , Meeus Sarah , Hattori Koichi , Ramachandran Ramalingam , Feuerback Fred , Worgall Stefan , Hackett Neil R. , Crystal Ronald G.
Published: September 2007

Abstract
—Intravascular introduction of replication-deficient adenoviral vectors (Advectors) provides an ideal model of delivery of transgenes for the treatment of various vascular abnormalities. On the basis of the knowledge that Advectors can induce inflammatory responses after intravascular administration, we speculated that cellular activation by Advector infection could directly modulate the endothelial cell (EC) adhesion molecule/chemokine expression repertoire. Infection of human umbilical vein ECs or bone marrow microvascular ECs with an E1
−
E4
+
Advector resulted in the upregulation of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and CD34, but not E-selectin, P-selectin, CD36, CD13, CD44, HLA-DR or PECAM. Upregulation of ICAM-1, VCAM-1, and CD34 was apparent 12 hours after infection and persisted for weeks after infection. Selective induction of adhesion molecules was mediated by the presence of the E4 gene in the Advector, because infection of ECs with an E1
−
E4
−
Advector had no effect on adhesion molecule expression. ECs infected with E1
−
E4
+
Advector, but not those infected with E1
−
E4
−
Advector, supported the adhesion of leukocytes. Monoclonal antibodies to ICAM-1 and VCAM-1 inhibited adhesion of leukocytes to E1
−
E4
+
-infected ECs. Infection of the ECs with E1
−
E4
+
Advector, but not E1
−
E4
−
Advector, resulted in downregulation of expression of chemocytokines, including interleukin-8, MCP-1, RANTES, and GM-CSF. Nonetheless, a large number of leukocytes migrated through ECs infected with E1
−
E4
+
, but not those infected with E1
−
E4
l−
, in response to exogenous chemokines. These results demonstrate that infection of ECs with E1
−
E4
+
Advectors, but not E1
−
E4
−
Advectors, may directly augment inflammatory responses by upregulating expression of adhesion molecules and enhancing migration through Advector-infected ECs and suggest that E1
−
E4
−
Advectors may be a better choice for gene-transfer strategies directed to the ECs.
Novel gp91

phox

Homologues in Vascular Smooth Muscle Cells
Authors: Lassègue Bernard , Sorescu Dan , Szöcs Katalin , Yin QiQin , Akers Marjorie , Zhang Yong , Grant Sharon L. , Lambeth J. David , Griendling Kathy K.
Published: September 2007

Abstract
—Emerging evidence indicates that reactive oxygen species are important regulators of vascular function. Although NAD(P)H oxidases have been implicated as major sources of superoxide in the vessel wall, the molecular identity of these proteins remains unclear. We recently cloned nox1 (formerly mox-1), a member of a new family of gp91

phox

homologues, and showed that it is expressed in proliferating vascular smooth muscle cells (VSMCs). In this study, we examined the expression of three nox family members, nox1, nox4, and gp91

phox

, in VSMCs, their regulation by angiotensin II (Ang II), and their role in redox-sensitive signaling. We found that both nox1 and nox4 are expressed to a much higher degree than gp91
phox
in VSMCs. Although serum, platelet-derived growth factor (PDGF), and Ang II downregulated nox4, they markedly upregulated nox1, suggesting that this enzyme may account for the delayed phase of superoxide production in these cells. Furthermore, an adenovirus expressing antisense nox1 mRNA completely inhibited the early phase of superoxide production induced by Ang II or PDGF and significantly decreased activation of the redox-sensitive signaling molecules p38 mitogen-activated protein kinase and Akt by Ang II. In contrast, redox-independent pathways induced by PDGF or Ang II were unaffected. These data support a role for nox1 in redox signaling in VSMCs and provide insight into the molecular identity of the VSMC NAD(P)H oxidase and its potentially critical role in vascular disease.
Novel Polymorphisms in Promoter Region of ATP Binding Cassette Transporter Gene and Plasma Lipids, Severity, Progression, and Regression of Coronary Atherosclerosis and Response to Therapy
Authors: Lutucuta Silvia , Ballantyne Christie M. , Elghannam Hesham , Gotto Antonio M. , Marian A. J.
Published: September 2007

Abstract
—Identification of mutations in the ATP binding cassette transporter (
ABCA1
) gene in patients with Tangier disease, who exhibit reduced HDL cholesterol (HDL-C) and apolipoprotein A1 (apoA1) levels and premature coronary atherosclerosis, has led to the hypothesis that common polymorphisms in the
ABCA1
gene could determine HDL-C and apoA1 levels and the risk of coronary atherosclerosis in the general population. We sequenced a 660-bp 5′ fragment of the
ABCA1
gene in 24 subjects and identified 3 novel polymorphisms: −477C/T, −419A/C, and −320G/C. We developed assays, genotyped 372 participants in the prospective Lipoprotein Coronary Atherosclerosis Study (LCAS), and determined the association of the variants with fasting plasma lipids and indices of quantitative coronary angiograms obtained at baseline and 2.5 years after randomization to fluvastatin or placebo. Distribution of −477C/T and −320G/C genotypes were 127 CC, 171 CT, and 74 TT and 130 GG, 168 GC, and 75 CC, respectively, and were in complete linkage disequilibrium (
P
<0.0001). Data for −477C/T are presented. The −419A/C variant was uncommon (present in 1 of 63 subjects). Heterozygous subjects had a modest reduction in HDL-C (
P
=0.09) and apoA1 (
P
=0.05) levels and a lesser response of apoA1 to treatment with fluvastatin (
P
=0.04). The mean number of coronary lesions causing 30% to 75% diameter stenosis was greater in subjects with the TT genotype (3.1±2.1) or CT genotype (2.9±1.9) than in subjects with the CC genotype (2.2±1.8) (
P
=0.002). Similarly, compared with subjects with the CC genotype , greater numbers of subjects with the TT or CT genotype had ≥1 coronary lesion (
P
=0.001). No association between the genotypes and progression of coronary atherosclerosis or clinical events was detected. We conclude that
ABCA1
genotypes are potential risk factors for coronary atherosclerosis in the general population.
Targeted Replacement of Kv1.5 in the Mouse Leads to Loss of the 4-Aminopyridine–Sensitive Component of
I
K,slow
and Resistance to Drug-Induced QT Prolongation
Authors: London Barry , Guo Weinong , Pan Xiang-hua , Lee Joon S. , Shusterman Vladimir , Rocco Christopher J. , Logothetis Diomedes A. , Nerbonne Jeanne M. , Hill Joseph A.
Published: September 2007

Abstract
—The K
+
channel mKv1.5 is thought to encode a 4-aminopyridine (4-AP)–sensitive component of the current
I
K,slow
in the mouse heart. We used gene targeting to replace mKv1.5 with the 4-AP–insensitive channel rKv1.1 (SWAP mice) and directly test the role of Kv1.5 in the mouse ventricle. Kv1.5 RNA and protein were undetectable, rKv1.1 was expressed, and Kv2.1 protein was upregulated in homozygous SWAP hearts. The density of the K
+
current
I
K,slow
(depolarizations to +40 mV, pA/pF) was similar in left ventricular myocytes isolated from SWAP homozygotes (17±1, n=27) and littermate controls (16±2, n=19). The densities and properties of
I
peak
,
I
to,f
,
I
to,s
, and
I
ss
were also unchanged. In homozygous SWAP myocytes, the 50-μmol/L 4-AP–sensitive component of
IK,slow
was absent (n=6), the density of the 20-mmol/L tetraethylammonium-sensitive component of I
K,slow
was increased (9±1 versus 5±1,
P
<0.05), and no 100- to 200-nmol/L α-dendrotoxin–sensitive current was found (n=8). APD
90
in SWAP myocytes was similar to controls at baseline but did not prolong in response to 30 μmol/L 4-AP. Similarly, QTc (ms) was not prolonged in anesthetized SWAP mice (64±2, homozygotes, n=9; 62±2, controls, n=9), and injection with 4-AP prolonged QTc only in controls (63±1, homozygotes; 72±2, controls;
P
<0.05). SWAP mice had no increase in arrhythmias during ambulatory telemetry monitoring. Thus, Kv1.5 encodes the 4-AP–sensitive component of
I
K,slow
in the mouse ventricle and confers sensitivity to 4-AP–induced prolongation of APD and QTc. Compensatory upregulation of Kv2.1 may explain the phenotypic differences between SWAP mice and the previously described transgenic mice expressing a truncated dominant-negative Kv1.1 construct.
Leptin Enhances the Calcification of Vascular Cells
Authors: Parhami Farhad , Tintut Yin , Ballard Alex , Fogelman Alan M. , Demer Linda L.
Published: September 2007

Abstract
—Leptin, the product of the
ob
gene, regulates food intake, energy expenditure, and other physiological functions of the peripheral tissues. Leptin receptors have been identified in the hypothalamus and in extrahypothalamic tissues. Increased circulating leptin levels have been correlated with cardiovascular disease, obesity, aging, infection with bacterial lipopolysaccharide, and high-fat diets. All these conditions have also been correlated with increased vascular calcification, a hallmark of atherosclerotic and age-related vascular disease. In addition, the differentiation of marrow osteoprogenitor cells is regulated by leptin. Thus, we hypothesized that leptin may regulate the calcification of vascular cells. In this report, we tested the effects of leptin on a previously characterized subpopulation of vascular cells that undergo osteoblastic differentiation and calcification in vitro. When treated with leptin, these calcifying vascular cells had a significant 5- to 10-fold increase in alkaline phosphatase activity, a marker of osteogenic differentiation of osteoblastic cells. Prolonged treatment with leptin enhanced the calcification of these cells, further supporting the pro-osteogenic differentiation effects of leptin. Furthermore, the presence of the leptin receptor on calcifying vascular cells was demonstrated using reverse transcriptase polymerase chain reaction, immunocytochemistry, and Western blot analysis. We also identified the presence of leptin receptor in the mouse artery wall, localized to subpopulations of medial and adventitial cells, and the expression of leptin by artery wall cells and atherosclerotic lesions in mice. Taken together, these results suggest that leptin regulates the osteoblastic differentiation and calcification of vascular cells and that the artery wall may be an important peripheral tissue target of leptin action.
Titin-Based Modulation of Calcium Sensitivity of Active Tension in Mouse Skinned Cardiac Myocytes
Authors: Cazorla Olivier , Wu Yiming , Irving Thomas C. , Granzier Henk
Published: September 2007

Abstract
—We studied the effect of titin-based passive force on the length dependence of activation of cardiac myocytes to explore whether titin may play a role in the generation of systolic force. Force-pCa relations were measured at sarcomere lengths (SLs) of 2.0 and 2.3 μm. Passive tension at 2.3 μm SL was varied from ≈1 to ≈10 mN/mm
2
by adjusting the characteristics of the stretch imposed on the passive cell before activation. Relative to 2.0 μm SL, the force-pCa curve at 2.3 μm SL and low passive tension showed a leftward shift (ΔpCa
50
[change in pCa at half-maximal activation]) of 0.09±0.02 pCa units while at 2.3 μm SL and high passive tension the shift was increased to 0.25±0.03 pCa units. Passive tension also increased ΔpCa
50
at reduced interfilament lattice spacing achieved with dextran. We tested whether titin-based passive tension influences the interfilament lattice spacing by measuring the width of the myocyte and by using small-angle x-ray diffraction of mouse left ventricular wall muscle. Cell width and interfilament lattice spacing varied inversely with passive tension, in the presence and absence of dextran. The passive tension effect on length-dependent activation may therefore result from a radial titin-based force that modulates the interfilament lattice spacing.
New Tricks for Old Dogs
Authors: Patterson Cam , Stouffer George A. , Madamanchi Nageswara , Runge Marschall S.
Published: September 2007

Abstract
—Thrombin is a serine protease that potently activates platelets and catalyzes the conversion of fibrinogen to fibrin. Thrombin also exerts direct effects on vascular cells, such as smooth muscle cells, via interactions with members of the protease-activated receptor family. Evidence in several animal models implicates thrombin-mediated signaling events in the response to injury that typifies vascular lesion formation in atherosclerosis and restenosis. In this review, we examine the activation of protease-activated receptors by thrombin, the downstream signaling events mediated by these receptors, and the physiological role of thrombin in vascular cells and vascular disease.
Elevated Levels of
S
-Nitrosoalbumin in Preeclampsia Plasma
Authors: Tyurin Vladimir A. , Liu Shang-Xi , Tyurina Yulia Y. , Sussman Nancy B. , Hubel Carl A. , Roberts James M. , Taylor Robert N. , Kagan Valerian E.
Published: September 2007

Abstract
—The availability of nitric oxide (NO), which is required for the normal regulation of vascular tone, may be decreased in preeclampsia, thus contributing to the vascular pathogenesis of this pregnancy disorder. Because ascorbate is essential for the decomposition of
S
-nitrothiols and the release of NO, we speculated that the ascorbate deficiency typical of preeclampsia plasma might result in decreased rates of decomposition of
S
-nitrosothiols. We tested the hypothesis that total
S
-nitrosothiol and
S
-nitrosoalbumin concentrations are increased in preeclampsia plasma, reflecting a decreased release of NO from these major reservoirs of NO. Gestationally matched plasma samples were obtained (before labor or intravenous MgSO
4
) from 21 women with preeclampsia and 21 women with normal pregnancy, and plasma samples were also obtained from 12 nonpregnant women of similar age and body mass index during the follicular phase of the menstrual cycle. All were nonsmokers. The assay included ultraviolet-induced decomposition of
S
-nitrosothiols to liberate NO captured by a florigenic reagent, 4,5-diaminofluoresceine, to produce diaminofluoresceine-Triazole. Preeclampsia plasma contained significantly higher concentrations of total
S
-nitrosothiols (11.1±2.9 nmol/mL) than normal pregnancy samples (9.4±1.5 nmol/mL). Even greater differences were found between preeclampsia plasma and plasma samples from normal pregnancies and nonpregnant women (294±110, 186±25, and 151±25 pmol/mg protein, respectively) when
S
-nitrosothiol content was expressed per milligram protein. The albumin fraction contained 49.4% of total plasma
S
-nitrosothiols in the control samples and 53.7% and 56.8% of plasma
S
-nitrosothiols in normal pregnancy and preeclampsia, respectively. The level of
S
-nitrosoalbumin was significantly higher in preeclampsia than in normal pregnancy or nonpregnancy plasma (6.3±1.4, 5.1±0.7, and 4.2±1.0 nmol/mL, respectively). The increased concentration of
S
-nitrosoalbumin in preeclampsia almost completely accounted for the increased levels of
S
-nitrosothiols in total plasma. Due to combined increases in nitrosothiols and decreases in protein, the preeclampsia plasma concentration of
S
-nitrosoalbumin was greatly increased on a per milligram of protein basis (271% and 186% compared with normal nonpregnancy and normal pregnancy plasma, respectively). We conclude that
S
-nitrosoalbumin and total
S
-nitrosothiol concentrations are significantly increased in preeclampsia plasma and may reflect insufficient release of NO groups in this condition.
Developing a Strategy to Define the Effects of Insulin-Like Growth Factor-1 on Gene Expression Profile in Cardiomyocytes
Authors: Liu Tsun-jui , Lai Hui-chin , Wu Weihua , Chinn Steven , Wang Ping H.
Published: September 2007

Abstract
—Insulin-like growth factor (IGF)-1 activates intracellular signaling pathways and regulates myocardial structure and function. This study used DNA microarray to define the effects of IGF-1 on gene expression in cardiomyocytes. Despite DNA microarray becoming a popular tool for profiling gene expression, the specificity of DNA microarray results is rarely addressed. Our data showed that the specificity of a DNA microarray study can be increased by repetitive experiments and by excluding minimally expressed genes. In this study, the false-positive rates were reduced to <0.2%. Future DNA microarray studies should incorporate a proper strategy to minimize false-positive results. IGF-1 modulates the expression of genes in 17 functional categories, but most genes clustered around the regulation of intracellular signaling, cell cycle, transcription/translation, cellular respiration and mitochondrial function, cell survival, ion channels and calcium signaling, and humoral factors. To further explore whether extracellular signal–regulated kinase (ERK) and phosphatidylinositol (PI) 3 kinase specifically regulate different sets of genes, the effects of IGF-1 were inhibited with PD98059 or LY294002. The results showed that the majority of genes regulated by IGF-1 required activation of both ERK and PI 3 kinase. Thus, PI 3 kinase and ERK coordinately mediate the transcriptional regulatory effects of IGF-1 in cardiac muscle cells. These findings provide novel insight into how IGF-1 signaling modulates the programming of cardiac muscle gene expression.
Diabetes Mellitus Enhances Vascular Matrix Metalloproteinase Activity
Authors: Uemura Shiro , Matsushita Hidetsugu , Li Wei , Glassford Alexander J. , Asagami Tomoko , Lee Keun-Ho , Harrison David G. , Tsao Philip S.
Published: September 2007

Abstract
—Diabetes mellitus (DM) is a primary risk factor for cardiovascular disease. Although recent studies have demonstrated an important role for extracellular matrix metalloproteinases (MMPs) in atherosclerosis, little is known about the effects of hyperglycemia on MMP regulation in vascular cells. Gelatin zymography and Western blot analysis revealed that the activity and expression of 92-kDa (MMP-9) gelatinase, but not of 72 kDa (MMP-2) gelatinase, were significantly increased in vascular tissue and plasma of two distinct rodent models of DM. Bovine aortic endothelial cells (BAECs) grown in culture did not express MMP-9 constitutively; however, chronic (2-week) incubation with high glucose medium induced MMP-9 promoter activity, mRNA and protein expression, and gelatinase activity in BAECs. On the other hand, high glucose culture did not change MMP-9 activity from vascular smooth muscle cells or macrophages. Electron paramagnetic resonance studies indicate that BAECs chronically grown in high glucose conditions produce 70% more ROS than do control cells. Enhanced MMP-9 activity was significantly reduced by treatment with the antioxidants polyethylene glycol–superoxide dismutase and
N
-acetyl-
l
-cysteine but not by inhibitors of protein kinase C. In conclusion, vascular MMP-9 activity is increased in DM, in part because of enhanced elaboration from vascular endothelial cells, and oxidative stress plays an important role. This novel mechanism of redox-sensitive MMP-9 expression by hyperglycemia may provide a rationale for antioxidant therapy to modulate diabetic vascular complications.
Protein Kinase C ε–Src Modules Direct Signal Transduction in Nitric Oxide–Induced Cardioprotection
Authors: Vondriska Thomas M. , Zhang Jun , Song Changxu , Tang Xian-Liang , Cao Xinan , Baines Christopher P. , Pass Jason M. , Wang Shaoshan , Bolli Roberto , Ping Peipei
Published: September 2007

Abstract
—An essential role for protein kinase C ε (PKCε) has been shown in multiple forms of cardioprotection; however, there is a distinct paucity of information concerning the signaling architecture that is responsible for the manifestation of a protective phenotype. We and others have recently shown that signal transduction may proceed via the formation of signaling complexes (
Circ Res
. 2001;88:59–62). In order to understand if the assembly of multiprotein complexes is the manner by which signaling is conducted in cardioprotection, we designed a series of experiments to characterize the associations of Src tyrosine kinase with PKCε in a conscious rabbit model of nitric oxide (NO)-induced late preconditioning. Our data demonstrate that PKCε and Src can form functional signaling modules in vitro: PKCε interacts with Src; the association with PKCε activates Src; and adult cardiac cells receiving recombinant adenoviruses encoding PKCε exhibit increased Src activity. Furthermore, our results show that NO-induced late preconditioning involved PKCε-Src module formation and enhanced the enzymatic activity of PKCε-associated Src. Inhibition of PKC blocked cardioprotection, module formation, and PKCε-associated Src activity, providing direct evidence for a functional role of the PKCε-Src module in the orchestration of NO-induced cardioprotection in conscious rabbits.
MinK-Related Peptide 1
Authors: Yu H. , Wu J. , Potapova I. , Wymore R. T. , Holmes B. , Zuckerman J. , Pan Z. , Wang H. , Shi W. , Robinson R. B. , El-Maghrabi M. R. , Benjamin W. , Dixon J. , McKinnon D. , Cohen I. S. , Wymore R.
Published: September 2007

Abstract
—The HCN family of ion channel subunits underlies the currents
I
f
in heart and
I
h
and
I
q
in the nervous system. In the present study, we demonstrate that minK-related peptide 1 (MiRP1) is a β subunit for the HCN family. As such, it enhances protein and current expression as well as accelerating the kinetics of activation. Because MiRP1 also functions as a β subunit for the cardiac delayed rectifier
I
Kr
, these results suggest that this peptide may have the unique role of regulating both the inward and outward channels that underlie cardiac pacemaker activity. The full text of this article is available at http://www.circresaha.org.
Thrombin Activates the Hypoxia-Inducible Factor-1 Signaling Pathway in Vascular Smooth Muscle Cells
Authors: Görlach A. , Diebold I. , Schini-Kerth V. B. , Berchner-Pfannschmidt U. , Roth U. , Brandes R. P. , Kietzmann T. , Busse R.
Published: September 2007

Abstract
—The heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1) is activated under hypoxic conditions, resulting in the upregulation of its target genes plasminogen activator inhibitor-1 (PAI-1) and vascular endothelial growth factor (VEGF). PAI-1 and VEGF are also induced in response to vascular injury, which is characterized by the activation of platelets and the coagulation cascade as well as the generation of reactive oxygen species (ROS). However, it is not known whether HIF-1 is also stimulated by thrombotic factors. We investigated the role of thrombin, platelet-associated growth factors, and ROS derived from the p22

phox

-containing NADPH oxidase in the activation of HIF-1 and the induction of its target genes PAI-1 and VEGF in human vascular smooth muscle cells (VSMCs). Thrombin, platelet-derived growth factor-AB (PDGF-AB), and transforming growth factor-β
1
(TGF-β
1
) upregulated HIF-1α protein in cultured and native VSMCs. This response was accompanied by nuclear accumulation of HIF-1α as well as by increased HIF-1 DNA-binding and reporter gene activity. The thrombin-induced expression of HIF-1α, PAI-1, and VEGF was attenuated by antioxidant treatment as well as by transfection of p22

phox

antisense oligonucleotides. Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol-3-kinase significantly decreased thrombin-induced HIF-1α, PAI-1, and VEGF expression. These findings demonstrate that the HIF-1 signaling pathway can be stimulated by thrombin and platelet-associated growth factors and that a redox-sensitive cascade activated by ROS derived from the p22

phox

-containing NADPH oxidase is crucially involved in this response.
Redox Regulation of Vascular Smooth Muscle Cell Differentiation
Authors: Su B. , Mitra S. , Gregg H. , Flavahan S. , Chotani M. A. , Clark K. R. , Goldschmidt-Clermont P. J. , Flavahan N. A.
Published: September 2007

Abstract
—Experiments were performed to determine the role of reactive oxygen species (ROS) in regulating vascular smooth muscle cell (VSMC) phenotype. After quiescence, cultured human VSMCs increased their expression of differentiation proteins (α-actin, calponin, and SM1 and SM2 myosin), but not β-actin. ROS activity, determined using the H
2
O
2
-sensitive probe dichlorodihydrofluorescein (DCF), remained high in quiescent cells and was inhibited by catalase (3000 U/mL) or by N-acetylcysteine (NAC, 2 to 20 mmol/L). A superoxide dismutase mimic (SOD; MnTMPyP, 25 μmol/L) or SOD plus low concentrations of NAC (SODNAC2, 2 mmol/L) increased DCF fluorescence, which was inhibited by catalase or by NAC (10 to 20 mmol/L). Inhibition of ROS activity (by catalase or NAC) decreased the baseline expression of differentiation proteins, whereas elevation of ROS (by SOD or SODNAC2) increased expression of the differentiation markers. The latter effect was blocked by catalase or by NAC (10 to 20 mmol/L). None of the treatments altered β-actin expression. SODNAC2-treated cells demonstrated contractions to endothelin that were absent in proliferating cells. p38 Mitogen-activated protein kinase (MAPK) activity was decreased when ROS activity was reduced (NAC, 10 mmol/L) and was augmented when ROS activity was increased (SODNAC2). Inhibition of p38 MAPK with pyridyl imidazole compound (SB202190, 2 to 10 μmol/L) reduced expression of differentiation proteins occurring under basal conditions and in response to SODNAC2. Transduction of VSMCs with an adenovirus encoding constitutively active MKK6, an activator of p38 MAPK, increased expression of differentiation proteins, whereas transduction with an adenovirus encoding dominant-negative p38 MAPK decreased expression of the differentiation proteins. These findings demonstrate that ROS can increase VSMC differentiation through a p38 MAPK–dependent pathway.
Molecular Interactions Between Two Long-QT Syndrome Gene Products,
HERG
and
KCNE2
, Rationalized by In Vitro and In Silico Analysis
Authors: Mazhari Reza , Greenstein Joseph L. , Winslow Raimond L. , Marbán Eduardo , Nuss H. Bradley
Published: September 2007

Abstract
—The cardiac delayed rectifier potassium current mediates repolarization of the action potential and underlies the QT interval of the ECG. Mutations in either of the two molecular components of the rapid delayed rectifier (
I
K,r
), HERG and KCNE2, have been linked to heritable or acquired long-QT syndrome. Mechanisms whereby mutations of KCNE2 produce fatal cardiac arrhythmias characteristic of long-QT syndrome remain unclear. In this study, we characterize functional interactions between HERG and KCNE2 with a view to defining underlying mechanisms for action potential prolongation and long-QT syndrome. Whereas coexpression of hKCNE2 with HERG alters both kinetics and density of ionic current, incorporation of these effects into a quantitative model of the action potential predicts that only changes in current density significantly affect repolarization. Thus, the primary functional consequence of hKCNE2 on action potential morphology is through modulation of
I
K,r
density, as predicted by the model. Mutations associated with long-QT syndrome that result only in modest changes of gating kinetics may be epiphenomena or may modulate action potential repolarization via interaction with alternative pore-forming potassium channel α subunits.
HCN2 Overexpression in Newborn and Adult Ventricular Myocytes
Authors: Qu Jihong , Barbuti Andrea , Protas Lev , Santoro Bina , Cohen Ira S. , Robinson Richard B.
Published: September 2007

Abstract
—Ventricular pacemaker current (
I
f
) shows distinct voltage dependence as a function of age, activating outside the physiological range in normal adult ventricle, but less negatively in neonatal ventricle. However, heterologously expressed HCN2 and HCN4, the putative molecular correlates of ventricular
I
f
, exhibit only a modest difference in activation voltage. We therefore prepared an adenoviral construct (AdHCN2) of HCN2, the dominant ventricular isoform at either age, and used it to infect neonatal and adult rat ventricular myocytes to investigate the role of maturation on current gating. The expressed current exhibited an 18-mV difference in activation (V
1/2
−95.9±1.9 in adult; −77.6±1.6 mV in neonate), comparable to the 22-mV difference between native
I
f
in adult and neonatal cultures (V
1/2
−98.7 versus −77.0 mV). This did not result from developmental differences in basal cAMP, because saturating cAMP in the pipette caused an equivalent positive shift in both preparations. In the neonate, AdHCN2 caused a significant increase in spontaneous rate compared with control (88±5 versus 48±4 bpm). In adult, where HCN2 activates more negatively, the effect was evident only during anodal excitation, requiring significantly less stimulus energy than control (2149±266 versus 3140±279 mV · ms). Thus, ventricular maturational state influences the voltage dependence of expressed HCN2, resulting in distinct physiological impact of expressed channels in neonate and adult myocytes. The full text of this article is available at http://www.circresaha.org.
Chlamydia pneumoniae
and Chlamydial Heat Shock Protein 60 Stimulate Proliferation of Human Vascular Smooth Muscle Cells via Toll-Like Receptor 4 and p44/p42 Mitogen-Activated Protein Kinase Activation
Authors: Sasu Sebastian , LaVerda David , Qureshi Nilofer , Golenbock Douglas T. , Beasley Debbie
Published: September 2007

An early component of atherogenesis is abnormal vascular smooth muscle cell (VSMC) proliferation. The presence of
Chlamydia pneumoniae
in many atherosclerotic lesions raises the possibility that this organism plays a causal role in atherogenesis. In this study,
C pneumoniae
elementary bodies (EBs) rapidly activated p44/p42 mitogen-activated protein kinases (MAPKs) and stimulated proliferation of VSMCs in vitro. Exposure of VSMCs derived from human saphenous vein to
C pneumoniae
EBs (3×10
7
inclusion forming units/mL) enhanced bromodeoxyuridine (BrdU) incorporation 12±3-fold. UV- and heat-inactivated
C pneumoniae
EBs also stimulated VSMC proliferation, indicating a role of direct stimulation by chlamydial antigens. However, the mitogenic activity of
C pneumoniae
was heat-labile, thus excluding a role of lipopolysaccharide. Chlamydial hsp60 (25 μg/mL) replicated the effect of
C pneumoniae
, stimulating BrdU incorporation 7±3-fold. Exposure to
C pneumoniae
or chlamydial hsp60 rapidly activated p44/p42 MAPK, within 5 to 10 minutes of exposure. In addition, PD98059 and U0126, which are two distinct inhibitors of upstream MAPK kinase 1/2 (MEK1/2), abolished the mitogenic effect of
C pneumoniae
and chlamydial hsp60. Toll-like receptors (TLRs) act as sensors for microbial antigens and can signal via the p44/p42 MAPK pathway. Human VSMCs were shown to express TLR4 mRNA and protein, and a TLR4 antagonist abolished chlamydial hsp60–induced VSMC proliferation and attenuated
C pneumoniae
–induced MAPK activation and VSMC proliferation. Together these results indicate that
C pneumoniae
and chlamydial hsp60 are potent inducers of human VSMC proliferation and that these effects are mediated, at least in part, by rapid TLR4-mediated activation of p44/p42 MAPK.
Gene Expression and In Situ Localization of Diacylglycerol Kinase Isozymes in Normal and Infarcted Rat Hearts
Authors: Takeda Morihiko , Kagaya Yutaka , Takahashi Jun , Sugie Tadashi , Ohta Jun , Watanabe Jun , Shirato Kunio , Kondo Hisatake , Goto Kaoru
Published: September 2007

Diacylglycerol (DG) kinase (DGK) terminates signaling from DG, which serves as an activator of protein kinase C (PKC), by converting DG to phosphatidic acid. DGK is thus regarded as an attenuator of the PKC activity. In rats, five DGK isozymes have been cloned, but little is known about their role in the heart. In this study, the spatiotemporal expression of DGK isozymes was investigated in rat hearts under a normal condition and after myocardial infarction (MI) by in situ hybridization histochemistry and immunohistochemistry. In normal left ventricular myocardium, DGKα, DGKε, and DGKζ mRNAs were expressed evenly throughout the myocardium, although the DGKα expression was very low. In infarcted hearts, the expression of DGKζ was enhanced in the peripheral zone of the necrotic area and at the border zone 3 and 7 days after MI, and to a lesser extent in the middle layer of the granulation tissue 21 days after MI. The enhanced DGKζ expression in the infarcted and border areas could be attributed to granulocytes and macrophages. In contrast, the expression of DGKε in the infarcted and border areas was lower than that in the viable left ventricle (LV) throughout the postoperation period. Furthermore, DGKε expression in the viable myocardium 21 days after MI decreased significantly compared with left ventricular myocardium in the sham-operated rats and was completely restored by treatment with captopril. Our results demonstrate that three DGK isozymes are expressed in the heart and that each isozyme might have different functional characteristics in the healing and LV remodeling after MI.
Matrix Metalloproteinase Inhibition After Myocardial Infarction
Authors: Creemers Esther E.J.M. , Cleutjens Jack P.M. , Smits Jos F.M. , Daemen Mat J.A.P.
Published: September 2007

Increased activity of matrix metalloproteinases (MMPs) has been implicated in numerous disease processes, including tumor growth and metastasis, arthritis, and periodontal disease. It is now becoming increasingly clear that extracellular matrix degradation by MMPs is also involved in the pathogenesis of cardiovascular disease, including atherosclerosis, restenosis, dilated cardiomyopathy, and myocardial infarction. Administration of synthetic MMP inhibitors in experimental animal models of these cardiovascular diseases significantly inhibits the progression of, respectively, atherosclerotic lesion formation, neointima formation, left ventricular remodeling, pump dysfunction, and infarct healing. This review focuses on the role of MMPs in cardiovascular disease, in particular myocardial infarction and the subsequent progression to heart failure. MMPs, which are present in the myocardium and capable of degrading all the matrix components of the heart, are the driving force behind myocardial matrix remodeling. The recent finding that acute pharmacological inhibition of MMPs or deficiency in MMP-9 attenuates left ventricular dilatation in the infarcted mouse heart led to the proposal that MMP inhibitors could be used as a potential therapy for patients at risk for the development of heart failure after myocardial infarction. Although these promising results encourage the design of clinical trials with MMP inhibitors, there are still several unresolved issues. This review describes the biology of MMPs and discusses new insights into the role of MMPs in several cardiovascular diseases. Attention will be paid to the central role of the plasminogen system as an important activator of MMPs in the remodeling process after myocardial infarction. Finally, we speculate on the use of MMP inhibitors as potential therapy for heart failure.