bluuebunny/crossref_metadata_2025 huggingface dataset:
Chimera Analysis of Troponin I Domains That Influence Ca
2+
-Activated Myofilament Tension in Adult Cardiac Myocytes
Authors: Westfall Margaret V. , Albayya Faris P. , Turner Immanuel I. , Metzger Joseph M.
Published: June 2012

Abstract
—The goal of this study was to investigate isoform-specific functional domains of the inhibitory troponin subunit, troponin I (TnI), as it functions within the intact myofilaments of adult cardiac myocytes. Adenovirus-mediated gene transfer was used to deliver and express a TnI chimera composed of the amino terminus of cardiac TnI (cTnI) and the carboxy terminus of slow skeletal TnI (ssTnI) in adult rat cardiac myocytes. The TnI chimera, designated N-card/slow-C TnI, was expressed and incorporated into myofilaments after gene transfer, without detectable changes in contractile protein stoichiometry or sarcomere architecture. Interestingly, force at submaximal Ca
2+
levels was markedly elevated in single permeabilized myocytes expressing the N-card/slow-C TnI chimera relative to force generated in adult myocytes expressing ssTnI or cTnI. Based on these results, a hierarchy of myofilament Ca
2+
sensitivity is emerging by use of TnI chimera analysis, with the order of sensitivity being N-card/slow-C TnI≫ssTnI≫cTnI. These results also strongly suggest that independent isoform-specific domains in both the amino and carboxy portions of TnI influence myofilament Ca
2+
sensitivity. In additional studies carried out under pathophysiological ionic conditions (pH 6.2), the dramatic acidosis-induced decrease in myofilament Ca
2+
sensitivity observed in myocytes expressing cTnI was blunted in myocytes expressing N-card/slow-C TnI in a manner similar to that in ssTnI-expressing myocytes. These results demonstrate that there is a pH-sensitive domain residing in the carboxy-terminal portion of TnI. The dissection of isoform-specific functional domains under physiological and acidic pH conditions demonstrates the utility of TnI chimeras for analysis of TnI function and provides important insights into the overall function of TnI within the intact myofilament of adult cardiac myocytes.
Cardiac Dysfunction Caused by Myocardium-Specific Expression of a Mutant Thyroid Hormone Receptor
Authors: Pazos-Moura Carmen , Abel E. Dale , Boers Mary-Ellen , Moura Egberto , Hampton Thomas G. , Wang Jufeng , Morgan James P. , Wondisford Fredric E.
Published: June 2012

Abstract
—Thyroid hormone deficiency has profound effects on the cardiovascular system, resulting in decreased cardiac contractility, adrenergic responsiveness, and vascular volume and increased peripheral vascular resistance. To determine the importance of direct cardiac effects in the genesis of hypothyroid cardiac dysfunction, the cardiac myocyte was specifically targeted with a mutant thyroid hormone receptor (TR)-β (Δ337T-TR-β
1
) driven by the α-myosin heavy chain (α-MHC) gene promoter. As a control in these experiments, a wild-type (Wt) TR-β
1
was also targeted to the heart by using the same promoter. Transgenic mice expressing the mutant TR displayed an mRNA expression pattern consistent with cardiac hypothyroidism, even though their peripheral thyroid hormone levels were normal. When these animals were rendered hypothyroid or thyrotoxic, mRNA expression of MHC isoforms remained unchanged in the hearts of the Δ337T transgenic animals, in contrast to Wt controls or transgenic animals expressing Wt TR-β
1
, which exhibited the expected changes in steady-state MHC mRNA levels. Studies in Langendorff heart preparations from mutant TR-β
1
transgenic animals revealed evidence of heart failure with a significant reduction in +dP/dT, −dP/dT, and force-frequency responses compared with values in Wt controls and transgenic mice overexpressing the Wt TR-β
1
. In contrast, in vivo measures of cardiac performance were similar between Wt and mutant animals, indicating that the diminished performance of the mutant transgenic heart in vitro was compensated for by other mechanisms in vivo. This is the first demonstration indicating that isolated cardiac hypothyroidism causes cardiac dysfunction in the absence of changes in the adrenergic or peripheral vascular system.
Phenotypic and Functional Changes in Regenerated Porcine Coronary Endothelial Cells
Authors: Fournet-Bourguignon Marie-Pierre , Castedo-Delrieu Maria , Bidouard Jean-Pierre , Leonce Stephane , Saboureau Delphine , Delescluse Isabelle , Vilaine Jean-Paul , Vanhoutte Paul M.
Published: June 2012

Abstract
—Porcine coronary arteries with regenerated endothelium exhibit impaired endothelium-dependent relaxations. Experiments were designed to analyze the structural and functional changes occurring in regenerated endothelial cells. Primary cultures from regenerated endothelium contained giant endothelial cells, with an increased number of cells with diameter >14.5 μm, a reduced ability to proliferate, and signs of apoptosis. The uptake of fluorescent acetylated LDL was increased 2-fold in cultures from regenerated endothelium. The increased uptake of acetylated LDL was confirmed ex vivo in injured coronary arteries. In cultures from regenerated endothelium, cGMP production was decreased under basal conditions and during stimulation with serotonin, bradykinin, and
A23187.
Thus, during regeneration, there is accelerated senescence of endothelial cells accompanied by increased incorporation of modified LDL and reduction of NO production without decrease in endothelial NO synthase expression. These alterations help to explain the altered endothelium-dependent responses 28 days after balloon injury.
Remodeling of Gap Junctional Coupling in Hypertrophied Right Ventricles of Rats With Monocrotaline-Induced Pulmonary Hypertension
Authors: Uzzaman Mahmud , Honjo Haruo , Takagishi Yoshiko , Emdad Luni , Magee Anthony I. , Severs Nicholas J. , Kodama Itsuo
Published: June 2012

Abstract
—The present study investigates the remodeling of gap junctional organization in relation to changes in anisotropic conduction properties in hypertrophied right ventricles (RVs) of rats with monocrotaline (MCT)-induced pulmonary hypertension. In contrast to controls that showed immunolocalization of connexin43 (Cx43) labeling largely confined to the intercalated disks, RV myocytes from MCT-treated rats showed dispersion of Cx43 labeling over the entire cell surface. The disorganization of Cx43 labeling became more pronounced with the progression of hypertrophy. Desmoplakin remained localized to the intercalated disks, as in controls. In RV tissues, the proportion of Cx43 label at the intercalated disk progressively decreased. Quantitative analysis of en face views of intercalated disks revealed a significant decrease in the disk gap junctional density in RV tissues of MCT-treated rats (control, 0.18 versus MCT-treated, 0.14 at 2 weeks; control, 0.16 versus MCT-treated, 0.11 at 4 weeks). Conduction velocity in RVs parallel to the fiber orientation was significantly lower (30.2% [n=9]) in MCT-treated rats at 4 weeks than in control rats, whereas there was no significant difference observed in the conduction velocity across the fiber orientation between control and MCT-treated rats. The anisotropic ratio of MCT-treated rats (1.38±0.10) was significantly lower than that of control rats (1.98±0.12). These results suggest that RV hypertrophy induced by pressure overload is associated with both disorganization of gap junction distribution and alteration of anisotropic conduction properties.
Arterial Paclitaxel Distribution and Deposition
Authors: Creel Christopher J. , Lovich Mark A. , Edelman Elazer R.
Published: June 2012

Abstract
—Successful implementation of local arterial drug delivery requires transmural distribution of drug. The physicochemical properties of the applied compound, which govern its transport and tissue binding, become as important as the mode of delivery. Hydrophilic compounds distribute freely but are cleared rapidly. Hydrophobic drugs, insoluble in aqueous solutions, bind to fixed tissue elements, potentially prolonging tissue residence and biological effect. Paclitaxel is such a hydrophobic compound, with tremendous therapeutic potential against proliferative vascular disease. We hypothesized that the recent favorable preclinical data with this compound may derive in part from preferential tissue binding as a result of unique physicochemical properties. The arterial transport of paclitaxel was quantified through application ex vivo and measurement of the subsequent transmural distribution. Arterial paclitaxel deposition at equilibrium varied across the arterial wall and was everywhere greater in concentration than in the applied drug source. Permeation into the wall increased with time, from 15 minutes to 4 hours, and varied with the origin of delivery. In contrast to hydrophilic compounds, the concentration in tissue exceeds the applied concentration and the rate of transport was markedly slower. Furthermore, endovascular and perivascular paclitaxel application led to markedly differential deposition across the blood vessel wall. These data suggest that paclitaxel interacts with arterial tissue elements as it moves under the forces of diffusion and convection and can establish substantial partitioning and spatial gradients across the tissue. The complexity of paclitaxel pharmacokinetics requires in-depth investigation if this drug is to reach its full clinical potential in proliferative vascular diseases.
Expression, Release, and Biological Activity of Parathyroid Hormone–Related Peptide From Coronary Endothelial Cells
Authors: Schlüter K.-D. , Katzer C. , Frischkopf K. , Wenzel S. , Taimor G. , Piper H. M.
Published: June 2012

Abstract
—Ventricular cardiomyocytes have previously been identified as potential target cells for parathyroid hormone–related peptide (PTHrP). Synthetic PTHrP peptides exert a positive contractile effect. Because systemic PTHrP levels are normally negligible, this suggests that PTHrP is expressed in the ventricle and acts as a paracrine mediator. We investigated the ventricular expression of PTHrP and its expression in cultured cells isolated from the ventricle, studied the release of PTHrP from hearts and cultures, and investigated whether this authentic PTHrP mimics the biological effects previously described for synthetic PTHrP on ventricular cardiomyocytes. We found PTHrP expressed in ventricles of neonatal and adult rat hearts. In cells isolated from adult hearts, we found PTHrP expression exclusively in coronary endothelial cells but not in cardiomyocytes. The latter, however, are target cells for PTHrP. PTHrP was released from isolated perfused hearts during hypoxic perfusion and from cultured coronary endothelial cells under energy-depleting conditions. This PTHrP was biologically active; ie, it exerted a positive contractile and lusitropic effect on cardiomyocytes. Authentic PTHrP was glycosylated and showed a slightly higher potency than synthetic PTHrP. These results suggest that PTHrP is an endothelium-derived modulator of ventricular function.
Angiopoietin-1 Induces Endothelial Cell Sprouting Through the Activation of Focal Adhesion Kinase and Plasmin Secretion
Authors: Kim Injune , Kim Hwan Gyu , Moon Sang-Ok , Chae Soo Wan , So June-No , Koh Keum Nim , Ahn Byung Cook , Koh Gou Young
Published: June 2012

Abstract
—Angiopoietin-1 (Ang1) is a strong inducer of endothelial cell sprouting, which is a first step in both angiogenesis and neovascularization. We examined the mechanisms underlying Ang1-induced cell sprouting using porcine pulmonary artery endothelial cells. Ang1 induced the nondirectional and directional migration of endothelial cells mediated through the Tie2 but not the Tie1 receptor. Ang1 induced tyrosine phosphorylation of p125
FAK
, and this phosphorylation was dependent on phosphatidylinositol (PI) 3′-kinase activity. Ang1 induced the secretion of plasmin and matrix metalloproteinase-2 (MMP-2), which is inhibited by PI 3′-kinase inhibitors. Ang1 also induced the secretion of small amounts of proMMP-3 and proMMP-9 but not proMMP-1. Ang1 suppressed the secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), but not of TIMP-1. Addition of α
2
-antiplasmin, a combination of TIMP-1 and TIMP-2, or PI 3′-kinase inhibitors inhibited Ang1-induced sprouting activity. Therefore, Ang1-induced sprouting activity in endothelial cells may be accomplished by cytoskeletal changes and secretion of proteinases and may be largely mediated through intracellular PI 3′-kinase activation.
Hypoxia-Induced Endothelial Apoptosis Through Nuclear Factor-κB (NF-κB)–Mediated bcl-2 Suppression
Authors: Matsushita Hidetsugu , Morishita Ryuichi , Nata Toshie , Aoki Motokuni , Nakagami Hironori , Taniyama Yoshiaki , Yamamoto Kei , Higaki Jitsuo , Yasufumi Kaneda , Ogihara Toshio
Published: June 2012

Abstract
—The transcription factor nuclear factor-κB (NF-κB) plays a pivotal role in the coordinated transactivation of cytokine and adhesion molecule genes involved in endothelial activation. Although recent reports have documented the contribution of NF-κB to apoptosis, it is still controversial. Especially, the role of NF-κB in endothelial apoptosis is largely unknown. Hypoxia significantly induced human aortic endothelial cell death and apoptosis in a time-dependent manner (
P
<0.01), accompanied by NF-κB activation. Decrease in total cell number and increase in apoptotic cells induced by hypoxia were significantly attenuated by NF-κB decoy, but not by scrambled decoy, oligodeoxynucleotides (ODNs) (
P
<0.01). Increase in DNA fragmentation induced by hypoxia was also significantly inhibited by NF-κB decoy ODNs as compared with scrambled decoy ODNs (
P
<0.01). Moreover, transfection of NF-κB decoy ODNs resulted in a significant decrease in caspase-3–like activity, which is a common pathway for apoptosis, compared with scrambled decoy ODNs. Importantly, transfection of NF-κB decoy ODNs significantly increased protein of bcl-2, an inhibitor of apoptosis, and did not alter bax, a promoter of apoptosis, thereby resulting in a significant increase in the ratio of bcl-2 to bax (
P
<0.01). bcl-2 mRNA was also decreased by hypoxia, whereas transfection of NF-κB decoy ODNs significantly attenuated decrease in bcl-2 mRNA. These results demonstrate that activation of NF-κB by hypoxia induced endothelial apoptosis in a bcl-2–dependent manner. The importance of NF-κB in endothelial apoptosis was confirmed by the observation that pyrrolidine dithiocarbamate, a potent NF-κB inhibitor, prevented endothelial apoptosis, caspase 3–like activity, and bcl-2 downregulation induced by hypoxia. To test this hypothesis in vivo, we transfected NF-κB decoy ODNs into rat intact carotid artery after reperfusion injury. Reperfusion injury was associated with a significant increase in endothelial apoptosis at 24 hours, whereas NF-κB decoy ODN treatment markedly decreased terminal deoxynucleotidyltransferase–mediated dUTP nick end labeling (TUNEL)–positive endothelial cells at 24 hours after reperfusion (
P
<0.01). Here, using synthetic double-stranded DNA with high affinity for NF-κB as a decoy approach, we demonstrated that activation of NF-κB by hypoxia caused aortic endothelial cell death and apoptosis through the suppression of bcl-2. NF-κB–mediated endothelial apoptosis induced by hypoxia may be involved in the pathogenesis of endothelial dysfunction observed in cardiovascular ischemic diseases.
Allele-Specific Regulation of Matrix Metalloproteinase-12 Gene Activity Is Associated With Coronary Artery Luminal Dimensions in Diabetic Patients With Manifest Coronary Artery Disease
Authors: Jormsjö Sofia , Ye Shu , Moritz Joseffa , Walter Dirk H. , Dimmeler Stefanie , Zeiher Andreas M. , Henney Adriano , Hamsten Anders , Eriksson Per
Published: June 2012

Abstract
—Both the processes of atherosclerosis and plaque rupture are indicated to be influenced by matrix metalloproteinase (MMP) activity. We therefore searched for common functional variation in the matrix metalloelastase (MMP-12) gene locus that may be implicated in coronary artery disease. Single-strand conformation polymorphism analysis of DNA from healthy individuals detected a common polymorphism within the MMP-12 gene promoter (an A-to-G substitution at position –82). The frequency of the G allele was 0.19. The polymorphism influences the binding of the transcription factor activator protein-1 (AP-1) in electromobility shift assay. A higher binding affinity of AP-1 to the A allele was associated with higher MMP-12 promoter activity in vitro in transient transfection studies in U937 and murine lung macrophage (MALU) cells. Phorbol 12-myristate 13-acetate (PMA) and insulin, 2 known activators of AP-1, increased the binding of AP-1 to the MMP-12 promoter, with higher affinity for the A allele. In transfection experiments, both the A and the G alleles responded to insulin and PMA, the A allele showing higher promoter activity than the G allele. Furthermore, Western blot analysis demonstrated that insulin increased MMP-12 protein production. To analyze whether the –82 A/G polymorphism is associated with coronary artery disease, 367 consecutive patients who underwent percutaneous transluminal coronary angiography with stent implantation were genotyped. In patients (n=71) with diabetes, the A allele was associated with a smaller luminal diameter. In conclusion, a common functional polymorphism within the MMP-12 promoter influences coronary artery luminal dimensions in diabetic patients with manifest coronary artery disease.
Molecular Basis of Electrocardiographic ST-Segment Elevation
Authors: Li Ronald A. , Leppo Michelle , Miki Takashi , Seino Susumu , Marbán Eduardo
Published: June 2012

ST elevation is a classical hallmark of acute transmural myocardial ischemia. Indeed, ST elevation is the major clinical criterion for committing patients with chest pain to emergent coronary revascularization. Despite its clinical importance, the mechanism of ST elevation remains unclear. Various studies have suggested that activation of sarcolemmal ATP-sensitive potassium (K

ATP

) channels by ischemic ATP depletion may play a role, but little direct evidence is available. We studied mice with homozygous knockout (KO) of the Kir6.2 gene, which encodes the pore-forming subunit of cardiac surface K

ATP

channels. Patch-clamp studies in cardiomyocytes confirmed that surface K

ATP

current was indeed absent in KO, but robust in cells from wild-type mice (WT). We then measured continuous electrocardiograms in anesthetized adult mice before and after open-chest ligation of the left anterior descending artery (LAD). Whereas ST elevation was readily evident in WT after LAD ligation, it was markedly suppressed in KO. Such qualitative differences persisted for the rest of the 60-minute observation period of ischemia. In support of the concept that K

ATP

channels are responsible for ST elevation, the surface K

ATP

channel blocker HMR1098 (5 mg/kg IP) suppressed early ST elevation in WT. Thus, the opening of sarcolemmal K

ATP

channels underlies ST elevation during ischemia. These data are the first to link a specific gene product with a common electrocardiographic phenomenon.
Endothelial Dysfunction in Cardiovascular Diseases: The Role of Oxidant Stress
Authors: Cai Hua , Harrison David G.
Published: June 2012

Abstract
—Accumulating evidence suggests that oxidant stress alters many functions of the endothelium, including modulation of vasomotor tone. Inactivation of nitric oxide (NO
·
) by superoxide and other reactive oxygen species (ROS) seems to occur in conditions such as hypertension, hypercholesterolemia, diabetes, and cigarette smoking. Loss of NO
·
associated with these traditional risk factors may in part explain why they predispose to atherosclerosis. Among many enzymatic systems that are capable of producing ROS, xanthine oxidase, NADH/NADPH oxidase, and uncoupled endothelial nitric oxide synthase have been extensively studied in vascular cells. As the role of these various enzyme sources of ROS become clear, it will perhaps be possible to use more specific therapies to prevent their production and ultimately correct endothelial dysfunction.
Myocardial K
ATP
Channels in Preconditioning
Authors: O’Rourke Brian
Published: June 2012

Abstract
—We are on the brink of harnessing the cell’s natural defenses against ischemia and reperfusion injury after years of research into the destructive and protective mechanisms involved. Since the discovery of ischemic preconditioning, the surface receptors and signal transduction pathways underlying this phenomenon have been clarified, but many questions remain about the downstream targets that ultimately protect the cell. ATP-sensitive K
+
(K
ATP
) channels are thought to play a role in protection, but their mechanism of action has been unclear. Accumulating evidence now suggests that the location of the K
ATP
channels relevant to cytoprotection may be on the mitochondrial inner membrane instead of on the sarcolemma of the cardiac cell. This review discusses recent findings and unanswered questions about the role of K
ATP
channels in preconditioning and protection.
Apoptosis During Cardiovascular Development
Authors: Fisher Steven A. , Langille B. Lowell , Srivastava Deepak
Published: June 2012

Abstract
—Morphogenesis and developmental remodeling of cardiovascular tissues involve coordinated regulation of cell proliferation and apoptosis. In the heart, clear evidence points toward focal apoptosis as a contributor to development of the embryonic outflow tract, cardiac valves, conducting system, and the developing coronary vasculature. Apoptosis in the heart is likely regulated by survival and death signals that are also present in many other tissues. Cell type–specific regulation may be superimposed on general cell death/survival machinery through tissue-specific transcriptional pathways. In the vasculature, apoptosis almost certainly contributes to developmental vessel regression, and it is of proven importance in remodeling of arterial structure in response to local changes in hemodynamics. Physical forces, growth factors, and extracellular matrix drive vascular cell survival pathways, and considerable evidence points to local nitric oxide production as an important but complex regulator of vascular cell death. In both the heart and vasculature, progress has been impeded by inadequate information concerning the incidence of apoptosis, its relative importance compared with the diverse cell behaviors that remodel developing tissues, and by our primitive knowledge concerning regulation of cell death in these tissues. However, tools are now available to better understand apoptosis in normal and abnormal development of cardiovascular structures, and a framework has been established that should lead to considerable progress in the coming years.
The Role of Homeobox Genes in Vascular Remodeling and Angiogenesis
Authors: Gorski David H. , Walsh Kenneth
Published: June 2012

Abstract
—Homeodomain-containing transcription factors are critical in the regulation of cell proliferation, differentiation, and migration, and they play an important role in organogenesis and pattern formation during embryogenesis. There is evidence that some of them are oncogenes or tumor suppressors. The cardiovascular system undergoes extensive remodeling during embryogenesis and disease states such as atherosclerosis and tumor-induced angiogenesis, and homeobox genes may play an important role in regulating these processes. Recently, homeobox genes have been detected in both vascular smooth muscle and endothelial cells, and they are implicated in pathological processes such as arterial restenosis after balloon angioplasty and tumor-induced angiogenesis. The cellular function of some of these genes is beginning to be elucidated. Therefore, we briefly review what is currently known about the involvement of homeobox transcription factors in both physiological and pathological vascular remodeling and angiogenesis.
Gα
i2
but Not Gα
i3
Is Required for Muscarinic Inhibition of Contractility and Calcium Currents in Adult Cardiomyocytes
Authors: Nagata Kohzo , Ye Chianping , Jain Mohit , Milstone David S. , Liao Ronglih , Mortensen Richard M.
Published: June 2012

Abstract
—Parasympathetic stimulation of the heart acts through M
2
-muscarinic acetylcholine receptors to regulate ion channel activity and subsequent inotropic status. Although muscarinic signal transduction is mediated via pertussis toxin-sensitive G proteins Gα
i/o
, the specific signal transduction requirements of Gα
i2
and Gα
i3
in mediating muscarinic regulated L-type calcium currents (
I
Ca, L
), intracellular calcium, and cell contractility remain to be determined. Adult ventricular myocytes were isolated from Gα
i2
-null mice, Gα
i3
-null mice, and their wild-type littermates. Cell shortening, intracellular calcium levels, and
I
Ca, L
were all measured in response to isoproterenol, a β-adrenergic receptor agonist, and carbachol, a cholinergic receptor agonist. With isoproterenol stimulation, myocytes from all groups demonstrated a marked increase in calcium currents, correlating with augmented intracellular calcium transient amplitude and cell shortening. Carbachol significantly attenuated the isoproterenol response in wild-type and Gα
i3
-null cells but had no effect in Gα
i2
-null cells. This study demonstrates that Gα
i2
, but not Gα
i3
, is required for muscarinic inhibition of the β-adrenergic response in adult murine ventricular myocytes.
High-Resolution Optical Mapping of the Right Bundle Branch in Connexin40 Knockout Mice Reveals Slow Conduction in the Specialized Conduction System
Authors: Tamaddon Houman S. , Vaidya Dhananjay , Simon Alexander M. , Paul David L. , Jalife José , Morley Gregory E.
Published: June 2012

Abstract
—Connexin40 (Cx40) is a major gap junction protein that is expressed in the His-Purkinje system and thought to be a critical determinant of cell-to-cell communication and conduction of electrical impulses. Video maps of the ventricular epicardium and the proximal segment of the right bundle branch (RBB) were obtained using a high-speed CCD camera while simultaneously recording volume-conducted ECGs. In Cx40
–/–
mice, the PR interval was prolonged (47.4±1.4 in wild-type [WT] [n=6] and 57.5±2.8 in Cx40
–/–
[n=6];
P
<0.01). WT ventricular epicardial activation was characterized by focused breakthroughs that originated first on the right ventricle (RV) and then the left ventricle (LV). In Cx40
–/–
hearts, the RV breakthrough occurred after the LV breakthrough. Additionally, Cx40
–/–
mice showed RV breakthrough times that were significantly delayed with respect to QRS complex onset (3.7±0.7 ms in WT [n=6] and 6.5±0.7 ms in Cx40
–/–
[n=6];
P
<0.01), whereas LV breakthrough times did not change. Conduction velocity measurements from optical mapping of the RBB revealed slow conduction in Cx40
–/–
mice (74.5±3 cm/s in WT [n=7] and 43.7±6 cm/s in Cx40
–/–
[n=7];
P
<0.01). In addition, simultaneous ECG records demonstrated significant delays in Cx40
–/–
RBB activation time with respect to P time (P-RBB time; 41.6±1.9 ms in WT [n=7] and 55.1±1.3 ms in [n=7];
P
<0.01). These data represent the first direct demonstration of conduction defects in the specialized conduction system of Cx40
–/–
mice and provide new insight into the role of gap junctions in cardiac impulse propagation.
Calmodulin Kinases II and IV and Calcineurin Are Involved in Leukemia Inhibitory Factor–Induced Cardiac Hypertrophy in Rats
Authors: Kato Takahiro , Sano Motoaki , Miyoshi Shunichiro , Sato Toshihiko , Hakuno Daihiko , Ishida Hideyuki , Kinoshita-Nakazawa Hiroe , Fukuda Keiichi , Ogawa Satoshi
Published: June 2012

Abstract
—We recently reported that leukemia inhibitory factor (LIF) enhances Ca
2+
]
i
through an increase in L-type Ca
2+
current (
I
Ca,L
) in adult cardiomyocytes. The aim of this study was to investigate whether LIF activates Ca
2+
-dependent signaling molecules, such as calcineurin and calmodulin kinases II and IV (CaMKII and CaMKIV), and, if so, whether these Ca
2+
-mediated signaling events contribute to LIF-mediated cardiac hypertrophy. We first confirmed that LIF increased
I
Ca,L
and [Ca
2+
]
i
in primary cultured rat neonatal cardiomyocytes. Calcineurin, CaMKII, and CaMKIV activities increased at 2 minutes and peaked by 1.6-, 2.2-, and 2.2-fold, respectively, at 15 minutes. Nicardipine or verapamil fully inhibited these activities. Autophosphorylation of CaMKII was also observed to parallel the timing of CaMKII activity, and this phosphorylation was blocked by nicardipine, verapamil, or EGTA. LIF treatment led to a 3-fold increase in nuclear factor of activated T cell–luciferase activity. To confirm that inositol triphosphate (IP
3
)-induced Ca
2+
release from sarcoplasmic reticulum was not involved in this process, IP
3
content and phosphorylation of phospholipase Cγ were investigated. LIF did not increase IP
3
content or phosphorylate phospholipase Cγ. KN62 (an inhibitor of CaMKII and CaMKIV) attenuated c-fos, brain natriuretic peptide, α-skeletal actin, and atrial natriuretic peptide expression. KN62 suppressed the LIF-induced increase in [
3
H]phenylalanine uptake and cell size. Cyclosporin A and FK506 slightly attenuated brain natriuretic peptide but did not affect c-fos or atrial natriuretic peptide expression. Cyclosporin A significantly reduced the LIF-induced increase in [
3
H]phenylalanine uptake. These findings indicated that LIF activated CaMKII, CaMKIV, and calcineurin through an increase in
I
Ca,L
and [Ca
2+
]
i
and that CaMKII, CaMKIV, and calcineurin are critically involved in LIF-induced cardiac hypertrophy.
Probing the Interaction Between Inactivation Gating and
d
d-Sotalol Block of
HERG
Authors: Numaguchi Hirotaka , Mullins Franklin M. , Johnson J. P. , Johns David C. , Po Sunny S. , Yang Iris C.-H. , Tomaselli Gordon F. , Balser Jeffrey R.
Published: June 2012

Abstract
—Potassium channels encoded by HERG underlie
I
Kr
, a sensitive target for most class III antiarrhythmic drugs, including methanesulfonanilides such as
d
d-sotalol. Recently it was shown that these drugs are trapped in the channel as it closes during hyperpolarization. At the same time, HERG channels rapidly open and inactivate when depolarized, and methanesulfonanilide block is known to develop in a use-dependent manner, suggesting a potential role for inactivation in drug binding. However, the role of HERG inactivation in class III drug action is uncertain: pore mutations that remove inactivation reduce block, yet many of these mutations also modify the channel permeation properties and could alter drug affinity through gating-independent mechanisms. In the present study, we identify a definitive role for inactivation gating in
d
d-sotalol block of HERG, using interventions complementary to mutagenesis. These interventions (addition of extracellular Cd
2+
, removal of extracellular Na
+
) modify the voltage dependence of inactivation but not activation. In normal extracellular solutions, block of HERG current by 300 μmol/L
d
d-sotalol reached 80% after a 10-minute period of repetitive depolarization to +20 mV. Maneuvers that impeded steady-state inactivation also reduced
d
d-sotalol block of HERG: 100 μmol/L Cd
2+
reduced steady-state block to 55% at +20 mV (
P
<0.05); removing extracellular Na
+
reduced block to 44% (
P
<0.05). An inactivation-disabling mutation (G628C-S631C) reduced
d
d-sotalol block to only 11% (
P
<0.05 versus wild type). However, increasing the rate of channel inactivation by depolarizing to +60 mV reduced
d
d-sotalol block to 49% (
P
<0.05 versus +20 mV), suggesting that the drug does not primarily bind to the inactivated state. Coexpression of MiRP1 with HERG had no effect on inactivation gating and did not modify
d
d-sotalol block. We postulate that
d
d-sotalol accesses its receptor in the open pore, and the drug-receptor interaction is then stabilized by inactivation. Whereas deactivation traps the bound methanesulfonanilide during hyperpolarization, we propose that HERG inactivation stabilizes the drug-receptor interaction during membrane depolarization.
Apoptosis Regulates Human Vascular Calcification In Vitro
Authors: Proudfoot Diane , Skepper Jeremy N. , Hegyi Laszlo , Bennett Martin R. , Shanahan Catherine M. , Weissberg Peter L.
Published: June 2012

Abstract
—The mechanisms involved in the initiation of vascular calcification are not known, but matrix vesicles, the nucleation sites for calcium crystal formation in bone, are likely candidates, because similar structures have been found in calcified arteries. The regulation of matrix vesicle production is poorly understood but is thought to be associated with apoptotic cell death. In the present study, we investigated the role of apoptosis in vascular calcification. We report that apoptosis occurs in a human vascular calcification model in which postconfluent vascular smooth muscle cell (VSMC) cultures form nodules spontaneously and calcify after ≈28 days. Apoptosis occurred before the onset of calcification in VSMC nodules and was detected by several methods, including nuclear morphology, the TUNEL technique, and external display of phosphatidyl serine. Inhibition of apoptosis with the caspase inhibitor ZVAD.fmk reduced calcification in nodules by ≈40%, as measured by the cresolphthalein method and alizarin red staining. In addition, when apoptosis was stimulated in nodular cultures with anti-Fas IgM, there was a 10-fold increase in calcification. Furthermore, incubation of VSMC-derived apoptotic bodies with
45
Ca demonstrated that, like matrix vesicles, they can concentrate calcium. These observations provide evidence that apoptosis precedes VSMC calcification and that apoptotic bodies derived from VSMCs may act as nucleating structures for calcium crystal formation.
Mice With Disrupted BK Channel β1 Subunit Gene Feature Abnormal Ca
2+
Spark/STOC Coupling and Elevated Blood Pressure
Authors: Plüger Saskia , Faulhaber Jörg , Fürstenau Michael , Löhn Matthias , Waldschütz Ralph , Gollasch Maik , Haller Hermann , Luft Friedrich C. , Ehmke Heimo , Pongs Olaf
Published: June 2012

Abstract
—Large-conductance potassium (BK) channels in vascular smooth muscle cells (VSMCs) sense both changes in membrane potential and in intracellular Ca
2+
concentration. BK channels may serve as negative feedback regulators of vascular tone by linking membrane depolarization and local increases in intracellular Ca
2+
concentration (Ca
2+
sparks) to repolarizing spontaneous transient outward K
+
currents (STOCs). BK channels are composed of channel-forming BKα and auxiliary BKβ1 subunits, which confer to BK channels an increased sensitivity for changes in membrane potential and Ca
2+
. To assess the in vivo functions of this β subunit, mice with a disrupted BKβ1 gene were generated. Cerebral artery VSMCs from BKβ1 −/− mice generated Ca
2+
sparks of normal amplitude and frequency, but STOC frequencies were largely reduced at physiological membrane potentials. Our results indicate that BKβ1 −/− mice have an abnormal Ca
2+
spark/STOC coupling that is shifted to more depolarized potentials. Thoracic aortic rings from BKβ1 −/− mice responded to agonist and elevated KCl with an increased contractility. BKβ1 −/− mice had higher systemic blood pressure than BKβ1 +/+ mice but responded normally to α
1
-adrenergic vasoconstriction and nitric oxide–mediated vasodilation. We propose that the elevated blood pressure in BKβ1 −/− mice serves to normalize Ca
2+
spark/STOC coupling for regulating myogenic tone. The full text of this article is available at http://www.circresaha.org.
Role of Structural Barriers in the Mechanism of Alternans-Induced Reentry
Authors: Pastore Joseph M. , Rosenbaum David S.
Published: June 2012

Abstract
—Previously, using an animal model of T-wave alternans in structurally normal myocardium, we demonstrated that repolarization can alternate with opposite phase between neighboring myocytes (ie, discordant alternans), causing spatial dispersions of repolarization that form the substrate for functional block and reentrant ventricular fibrillation (VF). However, the mechanisms responsible for cellular discordant alternans and its electrocardiographic manifestation (ie, T-wave alternans) in patients with structural heart disease are unknown. We hypothesize that electrotonic uncoupling between neighboring regions of cells by a structural barrier (SB) is a mechanism for discordant alternans. Using voltage-sensitive dyes, ventricular action potentials were recorded from 26 Langendorff-perfused guinea pig hearts in the absence (ie, control) and presence of an insulating SB produced by an epicardial laser lesion. Quantitative analysis of magnitude and phase of cellular alternans revealed that in controls, action potential duration alternated in phase at all ventricular sites above a critical heart rate (269±17 bpm), ie, concordant alternans. Also, above a faster critical heart rate threshold (335±24 bpm), action potential duration alternated with opposite phase between sites, ie, discordant alternans. In contrast, only discordant but not concordant alternans was observed in 80% of hearts with the SB, and discordant alternans always occurred at a significantly slower heart rate (by 68±28 bpm) compared with controls. Therefore, the SB had a major effect on the alternans–heart rate relation, which served to facilitate the development of discordant alternans. Whether a SB was present or not, discordant alternans produced considerable increases (by ≈170%) in the maximum spatial gradient of repolarization, which in turn formed the substrate for unidirectional block and reentry. However, by providing a structural anchor for stable reentry, discordant alternans in the presence of a SB led most often to sustained monomorphic ventricular tachycardia rather than to VF, whereas in the absence of a SB discordant alternans caused VF. SBs facilitate development of discordant alternans between cells with different ionic properties by electrotonically uncoupling neighboring regions of myocardium. This may explain why arrhythmia-prone patients with structural heart disease exhibit T-wave alternans at lower heart rates. These data also suggest a singular mechanism by which T-wave alternans forms a substrate for initiation of both VF and sustained monomorphic ventricular tachycardia.
Independent Signals Control Expression of the Calcineurin Inhibitory Proteins MCIP1 and MCIP2 in Striated Muscles
Authors: Yang John , Rothermel Beverly , Vega Rick B. , Frey Norbert , McKinsey Timothy A. , Olson Eric N. , Bassel-Duby Rhonda , Williams R. Sanders
Published: June 2012

Abstract
—Calcineurin, a calcium/calmodulin-regulated protein phosphatase, modulates gene expression in cardiac and skeletal muscles during development and in remodeling responses such as cardiac hypertrophy that are evoked by environmental stresses or disease. Recently, we identified two genes encoding proteins (MCIP1 and MCIP2) that are enriched in striated muscles and that interact with calcineurin to inhibit its enzymatic activity. In the present study, we show that expression of MCIP1 is regulated by calcineurin activity in hearts of mice with cardiac hypertrophy, as well as in cultured skeletal myotubes. In contrast, expression of MCIP2 in the heart is not altered by activated calcineurin but responds to thyroid hormone, which has no effect on MCIP1. A ≈900-bp intragenic segment located between exons 3 and 4 of the MCIP1 gene functions as an alternative promoter that responds to calcineurin. This region includes a dense cluster of 15 consensus binding sites for NF-AT transcription factors. Because MCIP proteins can inhibit calcineurin, these results suggest that MCIP1 participates in a negative feedback circuit to diminish potentially deleterious effects of unrestrained calcineurin activity in cardiac and skeletal myocytes. Inhibitory effects of MCIP2 on calcineurin activity may be pertinent to gene switching events driven by thyroid hormone in striated muscles. The full text of this article is available at http://www.circresaha.org.
Endogenous Nitric Oxide and Myocardial Adaptation to Ischemia
Authors: Heusch Gerd , Post Heiner , Michel Martin C. , Kelm Malte , Schulz Rainer
Published: June 2012

Abstract
—Ischemic myocardium does not inevitably undergo necrosis but rather can survive through downregulation of contractile function, ie, “hibernate.” To study the role of endogenous NO in this adaptation, 41 enflurane-anesthetized swine were subjected to 90 minutes of moderate left anterior descending coronary artery hypoperfusion and assigned to placebo (P), to 30 mg/kg
N
G
-nitro-
l
-arginine (L-NNA) IV to inhibit NO synthase, or to aortic constriction (AO) to match the increased left ventricular pressure observed with L-NNA. During normoperfusion, a regional myocardial external work index (WI, mm Hg · mm, sonomicrometry and micromanometry) was reduced with L-NNA (from 326±27 [SEM] to 250±19,
P
<0.05) but increased with AO (from 321±16 to 363±19,
P
<0.05 versus L-NNA). At 10 minutes of ischemia, WI was lower with L-NNA (109±10,
P
<0.05) than P (180±22) and AO (170±11) and did not change further at 85 minutes of ischemia. Relationships between WI and transmural myocardial blood flow and oxygen consumption were shifted rightward by L-NNA versus P and AO at both 10 and 85 minutes of ischemia. The maximal increment in calcium-activated external work was not different during normoperfusion among groups but was decreased during ischemia with L-NNA. L-NNA transiently increased myocardial contractile calcium sensitivity along with systemic pressure but reduced it during ongoing ischemia. The free-energy change of ATP hydrolysis after an early ischemic decrease recovered toward baseline values in all groups, and necrosis was absent after 2 (triphenyltetrazolium chloride staining) or 8 (histology) hours of reperfusion. Thus, endogenous NO contributes to hibernation by reducing oxygen consumption and preserving calcium sensitivity and contractile function without an energy cost during ischemia. (Circ Res. 2000;87:146-152.)
Oxidant Signaling in Vascular Cell Growth, Death, and Survival
Authors: Irani Kaikobad
Published: June 2012

Abstract
—Reactive oxygen species (ROS) have been traditionally regarded as toxic byproducts of aerobic metabolism. However, ROS can also act as intracellular signaling molecules in vascular cells. ROS can mediate phenotypes in vascular endothelial and smooth muscle cells that may be considered both physiological and pathophysiological. Among these are growth, apoptosis, and survival. The specific response elicited by reactive oxygen intermediaries is determined by their specific intracellular target(s). This, in turn, is dependent on the species of oxidant(s) produced, the source and therefore subcellular localization of the oxidant(s), the kinetics of production, and the quantities produced. A fuller understanding of how ROS regulate mitogenesis and apoptosis in vascular smooth muscle and endothelial cells will permit the development of novel strategies to modify or prevent vascular diseases in which these phenotypes predominate.
Role for G
12
/G
13
in Agonist-Induced Vascular Smooth Muscle Cell Contraction
Authors: Gohla Antje , Schultz Günter , Offermanns Stefan
Published: June 2012

Abstract
—Receptor-induced vascular smooth muscle cell contraction is mediated by dual regulation of myosin light chain (MLC
20
) phosphorylation through Ca
2+
-dependent stimulation of myosin light chain kinase and Rho/Rho-kinase–mediated inhibition of myosin phosphatase. Although myosin light chain kinase regulation is initiated by the coupling of receptors to G proteins of the G
q
family, G
q
and G
11
, it is not known how receptors regulate the Rho/Rho-kinase–mediated pathway. In vascular smooth muscle cells, receptor-mediated MLC
20
phosphorylation and cell contraction was blocked by inhibitors of each of the pathways. Receptors of various vasocontractors were found to couple to G
q
/G
11
and G
12
/G
13
, and constitutively active forms of Gα
12
and Gα
13
induced a pronounced contraction of vascular smooth muscle cells that could be blocked by C3 exoenzyme, by inhibition of Rho-kinase, and by stable analogues of cGMP and cAMP. Receptor-mediated smooth muscle cell contraction was strongly inhibited by dominant-negative forms of Gα
12
and Gα
13
. These data indicate that a G
12
/G
13
-mediated Rho/Rho-kinase–dependent pathway operates in smooth muscle cells and that dual regulation of MLC
20
phosphorylation by vasocontractors is initiated by the dual coupling of their receptors to G proteins of the G
q
and G
12
families.
β
2
-Integrin Blockade Driven by E-Selectin Promoter Prevents Neutrophil Sequestration and Lung Injury in Mice
Authors: Xu Ning , Rahman Arshad , Minshall Richard D. , Tiruppathi Chinnaswamy , Malik Asrar B.
Published: June 2012

Abstract
—Interaction of CD11/CD18 β
2
integrins on polymorphonuclear leukocytes (PMNs) with their counterreceptor, intercellular adhesion molecule-1, on the surface of vascular endothelial cells is a critical event mediating stable PMN adhesion and migration across the pulmonary vascular endothelial barrier. Neutrophil inhibitory factor (NIF), a 41-kDa glycoprotein isolated from the canine hookworm (
Ancylostoma caninum
), binds to the I domain of CD11a and CD11b and inhibits β
2
integrin–dependent PMN adhesion. We describe a novel strategy using the endothelial cell–specific E-selectin promoter to induce NIF expression in an inflammation-specific manner in pulmonary vascular endothelial cells. A construct containing NIF cDNA driven by the inducible endothelial cell–specific E-selectin promoter (pESNIF) was transfected into human pulmonary artery endothelial cells (HPAECs). Lipopolysaccharide challenge (known to activate E-selectin) resulted in NIF mRNA and protein expression in transfected HPAECs. NIF expression induced by the E-selectin promoter prevented PMN adhesion to the activated HPAECs, whereas PMNs adhered avidly to activated HPAECs in the absence of NIF expression. To address the utility of this approach in conditionally preventing in vivo PMN sequestration, we injected mice intravenously with cationic liposomes containing the pESNIF construct. Analysis of lung tissue showed that intraperitoneal challenge of
Escherichia coli
resulted in NIF expression. Inflammation-specific NIF expression induced by the E-selectin promoter prevented lung PMN sequestration and vascular injury induced by
E coli
challenge. These studies suggest the feasibility of conditionally blocking β
2
integrin function at sites where the endothelium is activated and thereby of locally preventing PMN activation and migration responses that lead to tissue inflammation.
Ceramide-Coated Balloon Catheters Limit Neointimal Hyperplasia After Stretch Injury in Carotid Arteries
Authors: Charles Roger , Sandirasegarane Lakshman , Yun Jong , Bourbon Nicole , Wilson Ronald , Rothstein Raymond P. , Levison Steven W. , Kester Mark
Published: June 2012

Abstract
—Neointimal hyperplasia at the site of surgical intervention is a common and deleterious complication of surgery for cardiovascular diseases. We hypothesized that direct delivery of a cell-permeable growth-arresting lipid via the balloon tip of an embolectomy catheter would limit neointimal hyperplasia after stretch injury. We have previously demonstrated that sphingolipid-derived ceramide arrested the growth of smooth muscle cell pericytes in vitro. Here, we show that ceramide-coated balloon catheters significantly reduced neointimal hyperplasia induced by balloon angioplasty in rabbit carotid arteries in vivo. This ceramide treatment decreased the number of vascular smooth muscle cells entering the cell cycle without inducing apoptosis. In situ autoradiographic studies demonstrated that inflating the balloon catheter forced cell-permeable ceramide into the intimal and medial layers of the artery. Intercalation of ceramide into vascular smooth muscle cells correlated with rapid inhibition of trauma-associated phosphorylation of extracellular signal–regulated kinase and protein kinase B. These studies demonstrate the utility of cell-permeable ceramide as a novel therapy for reducing neointimal hyperplasia after balloon angioplasty.