bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Expression of Cytoskeletal, Linkage, and Extracellular Proteins in Failing Human Myocardium
Authors: Heling Annette , Zimmermann René , Kostin Sawa , Maeno Yoshi , Hein Stefan , Devaux Bruno , Bauer Erwin , Klövekorn Wolf-Peter , Schlepper Martin , Schaper Wolfgang , Schaper Jutta
Published: June 2012

Abstract
—Experimental studies have shown that in hypertrophy and heart failure, accumulation of microtubules occurs that impedes sarcomere motion and contributes to decreased ventricular compliance. We tested the hypothesis that these changes are present in the failing human heart and that an entire complex of structural components, including cytoskeletal, linkage, and extracellular proteins, are involved in causing functional deterioration. In explanted human hearts failing because of dilated cardiomyopathy (ejection fraction ≤20%), expression of α- and β-tubulin, desmin, vinculin, fibronectin, and vimentin was determined by Northern and Western blot analysis and compared with normal myocardium from explants not used for transplantation. The mRNA for α- and β-tubulin was increased to 2.4-fold (
P
<0.01) and 1.25-fold (NS), respectively; for desmin, 1.2-fold (
P
<0.05); for fibronectin, 5-fold (
P
<0.001); and for vimentin, 1.7-fold (
P
<0.05). Protein levels for α-tubulin increased 2.6-fold (
P
<0.02); for β-tubulin, 1.2-fold (
P
<0.005); for desmin, 2.1-fold (
P
<0.001); for vinculin, 1.2-fold (
P
<0.005); for fibronectin, 2.9-fold (
P
<0.001); and for vimentin, 1.5-fold (
P
<0.005). Confocal microscopy showed augmentation and disorganization of all proteins studied. In combination with the loss of myofilaments and sarcomeric skeleton previously reported, these changes suggest cardiomyocyte remodeling. Increased fibronectin and elevated interstitial cellularity (vimentin labeling) indicate progressive fibrosis. The present results suggest a causative role of cytoskeletal abnormalities and myofilament loss for intrinsic contractile and diastolic dysfunction in failing hearts.
Bone Sialoprotein Mediates Human Endothelial Cell Attachment and Migration and Promotes Angiogenesis
Authors: Bellahcène Akeila , Bonjean Karine , Fohr Berthold , Fedarko Neal S. , Robey Frank A. , Young Marian F. , Fisher Larry W. , Castronovo Vincent
Published: June 2012

Abstract
—Bone sialoprotein (BSP) is a secreted glycoprotein primarily found in sites of biomineralization. Recently, we demonstrated that BSP is strongly upregulated in osteotropic cancers and particularly those that exhibit microcalcifications. BSP contains an Arg-Gly-Asp (RGD) motif found in other adhesive molecules that interact with cellular integrins. In bone, BSP has been shown to mediate the attachment of osteoblasts and osteoclasts via α
v
β
3
integrin receptors. Ligands for α
v
β
3
integrin are considered to play a central role during angiogenesis. Therefore, we used human umbilical vein endothelial cells (HUVECs) to study the potential role of BSP in angiogenesis. We found that purified eukaryotic recombinant human BSP (rhBSP) is able to promote both adhesion and chemotactic migration of HUVECs in a dose-dependent manner. These interactions involve HUVEC α
v
β
3
integrin receptors and the RGD domain of BSP. Indeed, HUVECs attach to a recombinant BSP fragment containing the RGD domain, whereas this response is not observed with the same fragment in which RGD has been mutated to Lys-Ala-Glu (KAE). A cyclic RGD BSP peptide inhibits both adhesion and migration of HUVECs to rhBSP. Moreover, anti-α
v
β
3
but not anti-α
v
β
5
monoclonal antibodies also prevent BSP-mediated adhesion and migration of HUVECs. We observed that both rhBSP and the RGD BSP recombinant fragment stimulated ongoing angiogenesis on the chorioallantoic chick membrane assay. BSP angiogenic activity was inhibited by anti-α
v
β
3
antibody, and the KAE BSP fragment was inactive. Our findings represent the first report implicating BSP in angiogenesis. BSP could play a critical role in angiogenesis associated with bone formation and with tumor growth and metastatic dissemination.
Fluid Shear Stress Induces Lipocalin-Type Prostaglandin D
2
Synthase Expression in Vascular Endothelial Cells
Authors: Taba Yoji , Sasaguri Toshiyuki , Miyagi Megumi , Abumiya Takeo , Miwa Yoshikazu , Ikeda Toshiko , Mitsumata Masako
Published: June 2012

Abstract
—Ligands for peroxisome proliferator–activated receptor γ, such as the thiazolidinedione class of antidiabetic drugs and 15-deoxy-Δ
12,14
-prostaglandin J
2
(15d-PGJ
2
), modulate various processes in atherogenesis. In search of cells that generate prostaglandin D
2
(PGD
2
), the metabolic precursor of 15d-PGJ
2
, we identified PGD
2
from culture medium of endothelial cells. To study how PGD
2
production is regulated in endothelial cells, we investigated the role of fluid shear stress in the metabolism of PGD
2
. Endothelial cells expressed the mRNA for the lipocalin-type PGD
2
synthase (L-PGDS) both in vitro and in vivo. Loading laminar shear stress using a parallel-plate flow chamber markedly enhanced the gene expression of L-PGDS, with the maximal effect being obtained at 15 to 30 dyne/cm
2
. The expression began to increase within 6 hours after loading shear stress and reached the maximal level at 18 to 24 hours. In contrast, shear stress did not alter the expression levels of PGI
2
synthase and thromboxane A
2
synthase. In parallel with the increase in the expression level of L-PGDS, endothelial cells released PGD
2
and 15d-PGJ
2
into culture medium. These results demonstrate that shear stress promotes PGD
2
production by stimulating L-PGDS expression and suggest the possibility that a peroxisome proliferator–activated receptor γ ligand is produced in vascular wall in response to blood flow.
cAMP Pulse During Preservation Inhibits the Late Development of Cardiac Isograft and Allograft Vasculopathy
Authors: Wang Catherine Y. , Aronson Isaak , Takuma Shin , Homma Shunichi , Naka Yoshifumi , Alshafie Tarek , Brovkovych Viktor , Malinski Tadeusz , Oz Mehmet C. , Pinsky David J.
Published: June 2012

Abstract
—The causes of transplant-associated coronary artery disease remain obscure, and there is no known treatment. Preservation injury of murine heterotopic vascularized cardiac isografts caused a small, albeit significant, increase in neointimal formation; preservation injury of allografts markedly increased both the incidence and severity of transplant-associated coronary artery disease. As cAMP is an important vascular homeostatic mediator the levels of which decline during organ preservation, buttressing cAMP levels solely during initial preservation both improved acute allograft function and reduced the severity of transplant-associated coronary artery disease in grafts examined 2 months later. Inhibiting the cAMP-dependent protein kinase abrogated these beneficial effects. cAMP treatment was associated with an early reduction in leukocyte infiltration and a reciprocal decrease in superoxide and increase in NO levels. These data indicate that alloantigen-independent injury to the graft, which occurs at the time of cardiac preservation, can set in motion pathological vascular events that are manifest months later. Furthermore, a cAMP pulse during cardiac preservation reduces the incidence and severity of transplant-associated coronary artery disease.
Functional Consequences of Elimination of
I
to, f
and
I
to, s
Authors: Guo Weinong , Li Huilin , London Barry , Nerbonne Jeanne M.
Published: June 2012

Abstract
—It was recently reported that the slow transient outward K
+
current,
I
to, s
, that is evident in mouse left ventricular septal cells is eliminated in mice with a targeted deletion of the Kv1.4 gene (Kv1.4
−/−
). The rapidly inactivating transient outward K
+
current,
I
to, f
, in contrast, is selectively eliminated in ventricular myocytes isolated from transgenic mice expressing a dominant-negative Kv4 α subunit, Kv4.2W362F. Expression of Kv4.2W362F results in marked prolongation of action potentials and QT intervals. In addition, a slow transient outward K
+
current, that is similar to
I
to, s
in wild-type mouse left ventricular septal cells, is evident in all Kv4.2W362F-expressing (left and right) ventricular cells. To test directly the hypothesis that upregulation of Kv1.4 α subunit underlies the appearance of this slow transient outward K
+
current in Kv4.2W362F-expressing ventricular cells and to explore the functional consequences of elimination of
I
to, f
and
I
to, s
, mice expressing Kv4.2W362F in the Kv1.4
−/−
background (Kv4.2W362F×Kv1.4
−/−
) were generated. Histological and echocardiographic studies revealed no evidence of structural abnormalities or contractile dysfunction in Kv4.2W362F×Kv1.4
−/−
mouse hearts. Electrophysiological recordings from the majority (≈80%) of cells isolated from the right ventricle and left ventricular apex of Kv4.2W362F×Kv1.4
−/−
animals demonstrated that both
I
to, f
and
I
to, s
are eliminated; action potentials are prolonged significantly; and, in some cells, early afterdepolarizations were observed. In addition, in vivo telemetric ECG recordings from Kv4.2W362F×Kv1.4
−/−
animals revealed marked QT prolongation, atrioventricular block, and ventricular tachycardia. These observations demonstrate that upregulation of Kv1.4 contributes to the electrical remodeling evident in the ventricles of Kv4.2W362F-expressing mice and that elimination of both
I
to, f
and
I
to, s
has dramatic functional consequences. (

Circ Res. 2000;87:73-79.)
Calcium Modulation of Vascular Smooth Muscle ATP-Sensitive K
+
Channels
Authors: Wilson Andrew J. , Jabr Rita I. , Clapp Lucie H.
Published: June 2012

Abstract
—ATP-sensitive K
+
(K
ATP
) channels are broadly distributed in the vasculature and regulate arterial tone. These channels are inhibited by intracellular ATP ([ATP]
i
) and vasoconstrictor agents and can be activated by vasodilators. It is widely assumed that K
ATP
channels are insensitive to Ca
2+
, although regulation has not been examined in the intact cell where cytosolic regulatory processes may be important. Thus we investigated the effects of Ca
2+
on whole-cell K
ATP
current in rat aortic smooth muscle cells recorded in a physiological [ATP]
i
and K
+
gradient. Under control recording conditions, cells had a resting potential of ≈−40 mV when bathed in 1.8 mmol/L Ca
2+
. The K
ATP
channel inhibitor glibenclamide caused membrane depolarization (9 mV) and inhibited a small, time-independent background current. Reducing [ATP]
i
from 3 to 0.1 mmol/L hyperpolarized cells to ≈−60 mV and increased glibenclamide-sensitive current by 2- to 4-fold. Similar effects were observed when Ca
2+
levels were decreased either externally or internally by increasing EGTA from 1 to 10 mmol/L. Dialysis with solutions containing different free [Ca
2+
]
i
showed that K
ATP
current was maximally activated at 10 nmol/L [Ca
2+
]
i
and almost totally inhibited at 300 nmol/L. Moreover, under control conditions, when rat aortic smooth muscle cells were dialyzed with either cyclosporin A, FK-506, or calcineurin autoinhibitory peptide (structurally unrelated inhibitors of Ca
2+
-dependent protein phosphatase, type 2B), glibenclamide-sensitive currents were large and the resting potential was hyperpolarized by ≈20 to 25 mV. We report for the first time that K
ATP
channels can be modulated by Ca
2+
at physiological [ATP]
i
and conclude that modulation occurs via protein phosphatase type 2B.
Estrogen Receptor α and Endothelial Nitric Oxide Synthase Are Organized Into a Functional Signaling Module in Caveolae
Authors: Chambliss Ken L. , Yuhanna Ivan S. , Mineo Chieko , Liu Pingsheng , German Zohre , Sherman Todd S. , Mendelsohn Michael E. , Anderson Richard G. W. , Shaul Philip W.
Published: June 2012

Abstract
—Estrogen causes nitric oxide (NO)-dependent vasodilation due to estrogen receptor (ER) α-mediated, nongenomic activation of endothelial NO synthase (eNOS). The subcellular site of interaction between ERα and eNOS was determined in studies of isolated endothelial cell plasma membranes. Estradiol (E
2
, 10
–8
mol/L) caused an increase in eNOS activity in plasma membranes in the absence of added calcium, calmodulin, or eNOS cofactors, which was blocked by ICI 182,780 and ERα antibody. Immunoidentification studies detected the same 67-kDa protein in endothelial cell nucleus, cytosol, and plasma membrane. Plasma membranes from COS-7 cells expressing eNOS and ERα displayed ER-mediated eNOS stimulation, whereas membranes from cells expressing eNOS alone or ERα plus a myristoylation-deficient mutant eNOS were insensitive. Fractionation of endothelial cell plasma membranes revealed ERα protein in caveolae, and E
2
caused stimulation of eNOS in isolated caveolae that was ER-dependent; noncaveolae membranes were insensitive. Acetylcholine and bradykinin also activated eNOS in isolated caveolae. Furthermore, the effect of E
2
on eNOS in caveolae was prevented by calcium chelation. Thus, a subpopulation of ERα is localized to endothelial cell caveolae where they are coupled to eNOS in a functional signaling module that may regulate the local calcium environment. The full text of this article is available at http://www.circresaha.org.
Integrative Analysis of Calcium Cycling in Cardiac Muscle
Authors: Eisner D. A. , Choi H. S. , Díaz M. E. , O’Neill S. C. , Trafford A. W.
Published: June 2012

Abstract
—The control of intracellular calcium is central to regulation of contractile force in cardiac muscle. This review illustrates how analysis of the control of calcium requires an integrated approach in which several systems are considered. Thus, the calcium content of the sarcoplasmic reticulum (SR) is a major determinant of the amount of Ca
2+
released from the SR and the amplitude of the Ca
2+
transient. The amplitude of the transient, in turn, controls Ca
2+
fluxes across the sarcolemma and thence SR content. This control of SR content influences the response to maneuvers that modify, for example, the properties of the SR Ca
2+
release channel or ryanodine receptor. Specifically, modulation of the open probability of the ryanodine receptor produces only transient effects on the Ca
2+
transient as a result of changes of SR content. These interactions between various Ca
2+
fluxes are modified by the Ca
2+
buffering properties of the cell. Finally, we predict that, under some conditions, the above interactions can result in instability (such as alternans) rather than ordered control of contractility.
Hypoxia Increases the Sensitivity of the L-Type Ca
2+
Current to β-Adrenergic Receptor Stimulation via a C2 Region–Containing Protein Kinase C Isoform
Authors: Hool Livia C.
Published: June 2012

Abstract
—The effects of hypoxia on the L-type Ca
2+
current (
I
Ca-L
) in the absence and presence of the β-adrenergic receptor agonist isoproterenol (Iso) were examined. Exposing guinea pig ventricular myocytes to hypoxia alone resulted in a reversible inhibition of basal
I
Ca-L
. When cells were exposed to Iso in the presence of hypoxia, the
K
0.5
for activation of
I
Ca-L
by Iso was significantly decreased from 5.3±0.7 to 1.6±0.1 nmol/L. The membrane-impermeant thiol-specific oxidizing compound 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) attenuated the inhibition of basal
I
Ca-L
by hypoxia 81.3±9.4% but had no effect on the increase in sensitivity of
I
Ca-L
to Iso. In addition, DTT mimicked the effects of hypoxia on basal
I
Ca-L
and the increase in sensitivity to Iso. Neither the inhibitors of guanylate cyclase LY-83583 or methylene blue nor the NO synthase inhibitor
N
G
-monomethyl-
l
-arginine monoacetate had any effect on the basal inhibition of
I
Ca-L
or the decrease in
K
0.5
for activation of
I
Ca-L
by Iso during hypoxia. However, the protein kinase C (PKC) inhibitors bisindolylmaleimide I and Gö 7874 significantly attenuated the increase in sensitivity of
I
Ca-L
to Iso. More specifically, the response was attenuated when cells were dialyzed with a peptide inhibitor of the C2 region–containing classical PKC isoforms. The same effect was not observed with the PKCε peptide inhibitor. These results suggest that hypoxia regulates
I
Ca-L
through the following 2 distinct mechanisms: direct inhibition of basal
I
Ca-L
and an indirect effect on the sensitivity of the channel to β-adrenergic receptor stimulation that is mediated through a classical PKC isoform.
Involvement of Rho-Kinase–Mediated Phosphorylation of Myosin Light Chain in Enhancement of Cerebral Vasospasm
Authors: Sato Motohiko , Tani Eiichi , Fujikawa Hirokazu , Kaibuchi Kozo
Published: June 2012

Abstract
—Subarachnoid hemorrhage (SAH) often induces a long-term narrowing of the cerebral artery called cerebral vasospasm. Myosin light chain (MLC) in the spastic basilar artery was reported previously to be phosphorylated by Ca
2+
/calmodulin-dependent MLC kinase. Because Rho-kinase, which is activated by the small GTPase Rho, phosphorylates not only MLC but also myosin phosphatase at its myosin-binding subunit (MBS), thus inactivating myosin phosphatase, we examined whether Rho-kinase is involved in the development of vasospasm. Cerebral vasospasm was produced in the canine basilar artery by a 2-hemorrhage method, and vasocontractions were induced by topical application of 80 mmol/L KCl or 0.5 μmol/L serotonin to the canine basilar artery exposed transclivally. The phosphorylation of MLC in the basilar artery was increased concurrently with an enhancement in the intensity of vasospasm with the passage of time after SAH. In addition, Rho-kinase in the basilar artery was activated concurrently with an increase in the phosphorylation of MBS at Ser854 in vasospasm. The Rho-kinase activation levels in vasospasm on days 0 and 2 were comparable to those in KCl- and serotonin-induced sustained vasocontraction, respectively, and those in vasospasm on day 7 were markedly high. The topical application of Y-27632, a specific inhibitor of Rho-kinase, to the exposed spastic basilar artery on day 7 induced a dose-dependent dilation, and the intensities of vasospasm and the phosphorylation of MBS and MLC were simultaneously decreased by 10 μmol/L Y-27632, although the decrease in MBS phosphorylation was more marked than the decrease in MLC phosphorylation. These results indicate that the activation of Rho-kinase and the phosphorylation of MLC and MBS occur concomitantly during vasospasm induced by SAH and suggest that Rho-kinase is involved in the enhancement of cerebral vasospasm in addition to Ca
2+
/calmodulin-dependent MLC kinase by increasing the phosphorylation of MLC directly or indirectly as a result of the inhibition of myosin phosphatase by its phosphorylation.
Host Gene Regulation During Coxsackievirus B3 Infection in Mice
Authors: Taylor Lydia A. , Carthy Christopher M. , Yang Decheng , Saad Kareem , Wong Donald , Schreiner George , Stanton Lawrence W. , McManus Bruce M.
Published: June 2012

Abstract
—Host genetic responses that characterize enteroviral myocarditis have not yet been determined. The injurious and inflammatory process in heart muscle may reflect host responses of benefit to the virus and ultimately result in congestive heart failure and dilated cardiomyopathy. On the other hand, host responses within the myocardium may secure the host against acute or protracted damage. To investigate the nature of modified gene expression in comparison with normal tissue, mRNA species were assessed in myocardium using cDNA microarray technology at days 3, 9, and 30 after infection. Of 7000 clones initially screened, 169 known genes had a level of expression significantly different at 1 or more postinfection time points as compared with baseline. The known regulated genes were sorted according to their functional groups and normalized expression patterns and, subsequently, interpreted in the context of viremic, inflammatory, and healing phases of the myocarditic process.
Development of a Smooth Muscle–Targeted Cre Recombinase Mouse Reveals Novel Insights Regarding Smooth Muscle Myosin Heavy Chain Promoter Regulation
Authors: Regan Christopher P. , Manabe Ichiro , Owens Gary K.
Published: June 2012

Abstract
—The use of genetically modified mice has been an important model system to study gene function in cardiovascular development and under pathophysiological conditions. Although conventional gene knockout studies have provided important insights into gene function in the cardiovascular system, they may be limited by upregulation of compensatory pathways and the inability to differentiate direct versus indirect functions in vivo. As a first step in developing systems that can target gene activation or inactivation specifically to smooth muscle cells (SMCs), we coupled the smooth muscle myosin heavy chain (SMMHC) promoter to the cre recombinase gene and generated transgenic mice that express cre in SMCs. In addition, we used these mice to address whether the heterogeneous staining observed in SMMHC-LacZ mice was due to subsets of SMCs that required different regulatory cassettes of the promoter or if it reflected episodic expression of the transgene. To address both the feasibility of SMC targeting and the apparent heterogeneous expression, we bred SMMHC-cre mice to indicator mice containing a cre-activated LacZ gene. Results showed high-level expression in SMCs at various embryonic time points and in adult tissues. Because breeding of SMMHC-cre mice to an indicator line provided an integration of cre activity over time, results of this study revealed that expression of the SMMHC promoter fragment more closely resembled the expression of the endogenous gene, both with respect to the onset of activation during development and uniformity of staining among individual cells within tissues. Overall, these mice will provide a powerful tool to researchers to study gene function in vascular development/disease by using cre/lox technology to direct smooth muscle–specific gene activation or inactivation in vivo.
Abnormal Cardiac Conduction and Morphogenesis in Connexin40 and Connexin43 Double-Deficient Mice
Authors: Kirchhoff Susanne , Kim Jung-Sun , Hagendorff Andreas , Thönnissen Eva , Krüger Olaf , Lamers Wouter H. , Willecke Klaus
Published: June 2012

Abstract
—Connexin40-deficient (Cx40
−/−
/Cx43
+/+
) and connexin43-heterozygous knockout mice (Cx40
+/+
/Cx43
+/−
) are viable but show cardiac conduction abnormalities. The ECGs of adult double heterozygous animals (Cx40
+/−
/Cx43
+/−
) suggest additive effects of Cx40 and Cx43 haploinsufficiency on ventricular, but not on atrial, conduction. We also observed additive effects of both connexins on cardiac morphogenesis. Approximately half of the Cx40
−/−
/Cx43
+/+
embryos died during the septation period, and an additional 16% died after birth. The majority of the latter mice had cardiac hypertrophy in conjunction with common atrioventricular junction or a ventricular septal defect. All Cx40
−/−
/Cx43
+/−
progeny exhibited cardiac malformations and died neonatally. The most frequent defect was common atrioventricular junction with abnormal atrioventricular connection, which was more severe than that seen in Cx40
−/−
/Cx43
+/+
mice. Furthermore, muscular ventricular septal defects, premature closure of the ductus arteriosus, and subcutaneous edema were noticed in these embryos. Cx40
+/−
/Cx43
−/−
embryos showed the same phenotype (ie, obstructed right ventricular outflow tract) as reported for Cx40
+/+
/Cx43
−/−
mice. These findings demonstrate that Cx43 haploinsufficiency aggravates the abnormalities observed in the Cx40
−/−
phenotype, whereas Cx40 haploinsufficiency does not worsen the Cx43
−/−
phenotype. We conclude that the gap-junctional proteins Cx40 and Cx43 contribute to morphogenesis of the heart in an isotype-specific manner.
Nonlinear Changes of Transmembrane Potential During Defibrillation Shocks
Authors: Cheek Eric R. , Ideker Raymond E. , Fast Vladimir G.
Published: June 2012

Abstract
—Defibrillation shocks induce complex nonlinear changes of transmembrane potential (ΔV
m
). To elucidate the ionic mechanisms of nonlinear ΔV
m
, we studied the effects of ionic channel blockers on ΔV
m
in geometrically defined myocyte cultures. Experiments were carried out in cell strands with widths of 0.2 mm (narrow strands) and 0.8 mm (wide strands) produced using a technique of directed cell growth. Uniform-field shocks were applied across strands during the action potential (AP) plateau, and the distribution of shock-induced ΔV
m
was measured using an optical mapping technique. Nifedipine and 4-aminopyridine were applied to inhibit the L-type calcium current (
I
Ca
) and the transient outward current (
I
to
), respectively. In control conditions, the distribution of ΔV
m
across cell strands was highly asymmetrical with a large ratio of negative to positive ΔV
m
(ΔV
−
m
/ΔV
+
m
) measured at the opposite strand borders. Application of nifedipine caused a large increase of ΔV
+
m
and a decrease of ΔV
−
m
/ΔV
+
m
, indicating involvement of
I
Ca
in the asymmetrical ΔV
m
, likely as a result of the outward flow of
I
Ca
when V
m
exceeded the
I
Ca
reversal potential. ΔV
−
m
decreased in the narrow strands but remained unchanged in the wide strands, indicating that the changes of ΔV
−
m
were caused by electrotonic interaction with an area of depolarization. 4-Aminopyridine did not change ΔV
−
m
/ΔV
+
m
. These results provide evidence that (1) the asymmetry of shock-induced ΔV
m
during the AP plateau is due to outward flow of
I
Ca
in the depolarized portions of the strands, (2)
I
to
is not involved in the mechanism of ΔV
m
asymmetry, and (3) the effects of drugs on ΔV
m
are modulated by the tissue geometry.
Splice Variants Reveal the Region Involved in Oxygen Sensing by Recombinant Human L-Type Ca
2+
Channels
Authors: Fearon Ian M. , Varadi Gyula , Koch Sheryl , Isaacsohn Idit , Ball Stephen G. , Peers Chris
Published: June 2012

Regulation of vascular smooth muscle Ca

2+

channels by oxygen tension contributes importantly to hypoxic vasodilatation. We previously described the inhibitory effects of hypoxia on the recombinant human cardiac L-type Ca

2+

channel α

1C

subunit (hHT isoform) expressed in HEK 293 cells. We now demonstrate that hypoxia inhibits only one of the three naturally occurring splice variants of this channel that differ only in the C-terminal domain, permitting identification of a 71-amino acid insert in the C-terminal region of the channel that confers oxygen sensitivity. Selective restriction of the spliced insert allowed determination of a 39-amino acid region essential for oxygen sensing. This represents the first identification of the structural region of an ion channel required for sensing changes in oxygen tension.
A Comparison of Aorta and Vena Cava Medial Message Expression by cDNA Array Analysis Identifies a Set of 68 Consistently Differentially Expressed Genes, All in Aortic Media
Authors: Adams Lawrence D. , Geary Randolph L. , McManus Bruce , Schwartz Stephen M.
Published: June 2012

Abstract
—We performed a systematic analysis of gene expression in arteries and veins by comparing message profiles of macaque aorta and vena cava media using a cDNA array containing 4048 known human genes, ≈35% of currently named human genes (≈11 000). The data show extensive differences in RNA expression in artery versus vein media. Sixty-eight genes had consistent elevation in message expression by the aorta, but none were elevated in the vena cava. The most differentially expressed gene was regulator of G-protein signaling (RGS) 5, at an expression ratio of 46.5±12.6 (mean±SEM). The data set also contained 2 genes already known to be expressed in the aorta, elastin at 5.0±1.4, and the aortic preferentially expressed gene 1 (APEG-1) at 2.3±0.6. We chose to analyze RGS5 expression further because of its high level of differential expression in the aorta. Levels of RGS5 mRNA were confirmed by Northern analysis and in situ hybridization. A human tissue RNA dot blot showed that RGS5 message is highest in aorta, followed by small intestine, stomach, and then heart. Northern analysis confirmed that RGS5 expression in human aorta is higher than in any region of the heart. RGS5 is a G-protein signaling regulator of unknown specificity most homologous to RGS4, an inhibitory regulator of pressure-induced cardiac hypertrophy. The expression pattern of the 68 differential genes as a whole is a start toward identifying the molecular phenotypes of arteries and veins on a systematic basis.
Microvascular Integrity and the Time Course of Myocardial Sodium Accumulation After Acute Infarction
Authors: Rochitte Carlos E. , Kim Raymond J. , Hillenbrand Hanns B. , Chen Enn-ling , Lima João A. C.
Published: June 2012

Abstract
—Loss of membrane permeability caused by ischemia leads to cellular sodium accumulation and myocardial edema. This phenomenon has important implications to left ventricular structure and function in the first hours after myocardial infarction. We hypothesized that during this period of time, after prolonged coronary occlusion and complete reflow, the rate of myocardial sodium accumulation is governed by microvascular integrity. We used 3-dimensional
23
Na MRI to monitor myocardial sodium content changes over time in an in vivo closed-chest canine model (n=13) of myocardial infarction and reperfusion. Infarcts with microvascular obstruction (MO) defined by both radioactive microspheres and contrast-enhanced
1
H MRI showed a slower rate of sodium accumulation as well as lower blood flow at 20 minutes and 6 hours after reperfusion. Conversely, the absence of MO was associated with faster rates of sodium accumulation and greater blood flow restoration. In addition, infarct size by
23
Na MRI correlated best with infarct size by triphenyltetrazolium chloride and contrast-enhanced
1
H MRI at 9 hours after reperfusion. We conclude that in reperfused myocardial infarction, sodium accumulation is dependent on microvascular integrity and is slower in regions of MO compared with those with patent microvasculature. Finally,
23
Na MRI can be a useful tool for monitoring in vivo myocardial sodium content in acute myocardial infarction.
Enhanced Ca
2+
-Activated Na
+
-Ca
2+
Exchange Activity in Canine Pacing-Induced Heart Failure
Authors: Hobai Ion A. , O’Rourke Brian
Published: June 2012

Abstract
—Defective excitation-contraction coupling in heart failure is generally associated with both a reduction in sarcoplasmic reticulum (SR) Ca
2+
uptake and a greater dependence on transsarcolemmal Na
+
-Ca
2+
exchange (NCX) for Ca
2+
removal. Although a relative increase in NCX is expected when SR function is impaired, few and contradictory studies have addressed whether there is an absolute increase in NCX activity. The present study examines in detail NCX density and function in left ventricular midmyocardial myocytes isolated from normal or tachycardic pacing–induced failing canine hearts. No change of NCX current density was evident in myocytes from failing hearts when intracellular Ca
2+
([Ca
2+
]
i
) was buffered to 200 nmol/L. However, when [Ca
2+
]
i
was minimally buffered with 50 μmol/L indo-1, Ca
2+
extrusion via NCX during caffeine application was doubled in failing versus normal cells. In other voltage-clamp experiments in which SR uptake was blocked with thapsigargin, both reverse-mode and forward-mode NCX currents and Ca
2+
transport were increased >2-fold in failing cells. These results suggest that, in addition to a relative increase in NCX function as a consequence of defective SR Ca
2+
uptake, there is an absolute increase in NCX function that depends on [Ca
2+
]
i
in the failing heart.
Focal Adhesion Kinase Is Involved in Angiotensin II–Mediated Protein Synthesis in Cultured Vascular Smooth Muscle Cells
Authors: Govindarajan Geetha , Eble Diane M. , Lucchesi Pamela A. , Samarel Allen M.
Published: June 2012

Abstract
—The rate of vascular smooth muscle cell protein synthesis and cellular hypertrophy in response to angiotensin II (Ang II) is dependent on activation of protein tyrosine kinases (PTKs) and both the extracellular signal–regulated kinase (ERK) 1/2 and p70
S6K
pathways. One potential PTK that may regulate these signaling cascades is focal adhesion kinase (FAK), a nonreceptor PTK associated with focal adhesions. We used an actin depolymerizing agent, cytochalasin D (Cyt-D), and a replication-defective adenovirus encoding FAK-related nonkinase (FRNK), an inhibitor of FAK-dependent signaling, as tools to assess whether FAK was upstream of the ERK1/2 and/or the p70
S6K
pathways. Cyt-D reduced basal FAK phosphorylation and blocked Ang II–dependent FAK phosphorylation in a dose-dependent manner. Confocal microscopy indicated that Cyt-D induced actin filament disruption and FAK delocalization from focal adhesions. Cyt-D also reduced Ang II–induced ERK1/2 activation, but p70
S6K
activation was relatively unaffected. Cyt-D reduced basal protein synthetic rate and substantially reduced the Ang II–induced increase in protein synthesis. Similarly, FRNK overexpression blocked Ang II–induced FAK phosphorylation and ERK1/2 activation, but not p70
S6K
phosphorylation, and markedly inhibited protein synthesis. This is the first report to demonstrate that FAK is a critical component of the signal transduction pathways that mediate Ang II–induced ERK1/2 activation, c-
fos
induction, and enhanced protein synthesis in vascular smooth muscle cells.
Strongly Binding Myosin Crossbridges Regulate Loaded Shortening and Power Output in Cardiac Myocytes
Authors: McDonald Kerry S. , Moss Richard L.
Published: June 2012

Abstract
—This study investigated the possible roles of strongly binding myosin crossbridges in determining loaded shortening and power output in cardiac myocytes. Single skinned cardiac myocytes were attached between a force transducer and position motor, and shortening velocities were measured over a range of loads during varying levels of Ca
2+
activation. Lowering the [Ca
2+
] slowed shortening velocities, decreased relative power output, and increased the curvature of length traces. We tested the hypothesis that Ca
2+
activation dependence of loaded shortening is determined primarily by strongly binding crossbridges or by [Ca
2+
] per se, which was done by measuring loaded shortening before and after addition of
N
-ethylmaleimide–conjugated myosin subfragment-1 (NEM-S1), a strongly binding myosin analogue that cooperatively enhances thin filament activation. At fixed [Ca
2+
], NEM-S1 reduced the curvature of length traces and sped loaded shortening velocities. Even when [Ca
2+
] was adjusted so that force was equal with and without NEM-S1, myocyte shortening was faster and exhibited less curvature with NEM-S1. In the presence of NEM-S1, peak relative power output was also significantly greater during activations either at the same [Ca
2+
] or when [Ca
2+
] was adjusted to achieve the same force. Consequently, NEM-S1 eliminated any Ca
2+
dependence of relative power output that is normally observed in cardiac myocytes. These results indicate that strongly binding crossbridges play a significant role in determining loaded shortening and power output and suggest that previously observed Ca
2+
dependence of power output is mediated by alterations in numbers of crossbridges bound to the thin filament.
Soluble Guanylate Cyclase α
1
and β
1
Gene Transfer Increases NO Responsiveness and Reduces Neointima Formation After Balloon Injury in Rats via Antiproliferative and Antimigratory Effects
Authors: Sinnaeve Peter , Chiche Jean-Daniel , Nong Zengxuang , Varenne Olivier , Van Pelt Natascha , Gillijns Hilde , Collen Desire , Bloch Kenneth D. , Janssens Stefan
Published: June 2012

Abstract
—In vascular smooth muscle cells, NO stimulates the synthesis of cGMP by soluble guanylate cyclase (sGC), a heterodimer composed of α
1
and β
1
subunits. NO/cGMP signal transduction affects multiple cell functions that contribute to neointima formation after vascular injury. Balloon-induced vascular injury was found to decrease sGC subunit expression and enzyme activity in rat carotid arteries. The effect of restoring sGC enzyme activity on neointima formation was investigated using recombinant adenoviruses specifying sGC α
1
and β
1
subunits (Adα1 and Adβ1). Coinfection of cultured rat aortic smooth muscle cells with Adα1 and Adβ1 increased NO-stimulated intracellular cGMP levels 60-fold and decreased DNA synthesis and migration by 16% and 48%, respectively. Immunoreactivity for α
1
and β
1
subunits colocalized in carotid arteries infected with Adα1 and Adβ1. Molsidomine-stimulated carotid tissue cGMP levels were greater after coinfection with Adα1 and Adβ1 than after infection with a control virus, AdRR5 (0.53±0.09 pmol/mg protein, mean±SEM, versus 0.23±0.09,
P
<0.05). Mean intima/media ratio, 2 weeks after balloon injury and twice-daily administration of 5 mg/kg molsidomine, was less in rats coinfected with Adα1 and Adβ1 than in rats infected with AdRR5 or in uninfected rats (0.36±0.11 versus 0.81±0.13 and 0.75±0.25, respectively,
P
<0.05). Thus, Adα1 and Adβ1 gene transfer to balloon-injured rat carotid arteries increases NO responsiveness and attenuates neointima formation via a direct antiproliferative and antimigratory effect on vascular smooth muscle cells.
Microsatellite Instability of Endothelial Cell Growth and Apoptosis Genes Within Plexiform Lesions in Primary Pulmonary Hypertension
Authors: Yeager Michael E. , Halley George R. , Golpon Heiko A. , Voelkel Norbert F. , Tuder Rubin M.
Published: June 2012

Abstract
—Primary pulmonary hypertension (PPH) is a frequently fatal disease whose pathobiology is poorly understood. Monoclonal endothelial cell growth is present within plexiform lesions of patients with PPH but not secondary PH because of congenital heart malformations. We hypothesized that endothelial cells within PPH plexiform lesions harbor mutations permissive for clonal cell growth. We found that endothelial cells in PPH plexiform lesions demonstrated microsatellite instability within the human MutS Homolog 2 gene (10 of 20 lesions) and displayed microsatellite site mutations and reduced protein expression of transforming growth factor-β receptor type II (6 of 19 lesions) and Bax (4 of 19 lesions). These results suggest that, in PPH, proliferated endothelial cells have genetic alterations associated with microsatellite instability and concomitant perturbation of growth and apoptosis gene expression akin to neoplasia. The full text of this article is available at http://www.circresaha.org.
Heterogenous Nature of Flow-Mediated Dilatation in Human Conduit Arteries In Vivo
Authors: Mullen Michael J. , Kharbanda Rajesh K. , Cross Jenny , Donald Ann E. , Taylor Mia , Vallance Patrick , Deanfield John E. , MacAllister Raymond J.
Published: June 2012

Abstract
—Flow-mediated dilatation (FMD) of conduit arteries is dependent on an intact endothelium, although the mechanisms are not fully understood. Using high-resolution ultrasound, we examined the role of endothelial mediators in radial artery dilatation in response to transient (short period of reactive hyperemia) and sustained (prolonged period of reactive hyperemia, hand warming, or an incremental infusion of acetylcholine into the distal radial artery) hyperemia. After short episodes of reactive hyperemia, FMD was abolished by local infusion of the nitric oxide synthesis inhibitor
N
G
monomethyl-L-arginine (5.3±1.2% versus 0.7±0.7%,
P
<0.001). In contrast, basal vessel diameter and dilatation after prolonged episodes of reactive hyperemia, hand warming, and distal infusion of acetylcholine were not attenuated by nitric oxide synthesis inhibition. Inhibition of cyclooxygenase or local autonomic nervous system blockade also had no effect on FMD. Patients with hypercholesterolemia exhibited reduced FMD in response to transient hyperemia, but the response to sustained hyperemia was normal. These data suggest heterogeneity of endothelial responses to blood flow that are dependent on the characteristics of the flow stimulus. Dilatation after brief episodes of hyperemia is mediated by release of nitric oxide, whereas dilatation during sustained hyperemia is unaffected by NO synthesis inhibition. Hypercholesterolemia seems to differentially affect these pathways with impairment of the nitric oxide–dependent pathway and preservation of non nitric oxide–mediated dilatation to sustained flow stimuli.
Altered Cardiac Sarcoplasmic Reticulum Function of Intact Myocytes of Rat Ventricle During Metabolic Inhibition
Authors: Overend C. L. , Eisner D. A. , O’Neill S. C.
Published: June 2012

Abstract
—Changes in the behavior of the sarcoplasmic reticulum (SR) in rat ventricular myocytes were investigated under conditions of metabolic inhibition using laser-scanning confocal microscopy to measure intracellular Ca
2+
and the perforated patch-clamp technique to measure SR Ca
2+
content. Metabolic inhibition had several effects on SR function, including reduced frequency of spontaneous releases of Ca
2+
(sparks and waves of Ca
2+
-induced Ca
2+
release), increased SR Ca
2+
content (79.4±5.7 to 115.2±6.6 μmol/L cell volume [mean±SEM;
P
<0.001]), and, after a wave of Ca
2+
release, slower reuptake of Ca
2+
into the SR (rate constant of fall of Ca
2+
reduced from 8.5±1.1 s
−
1
in control to 5.2±0.4 s
−
1
in metabolic inhibition [
P
<0.01]). Inhibition of L-type Ca
2+
channels with Cd
2+
(100 μmol/L) did not reproduce the effects of metabolic inhibition on spontaneous Ca
2+
sparks. These results are evidence of inhibition of both Ca
2+
release and reuptake mechanisms. Reduced frequency of release could be attributable to either of these effects, but the increased SR Ca
2+
content at the time of reduced frequency of spontaneous release of Ca
2+
shows that the dominant effect of metabolic inhibition is to inhibit release of Ca
2+
from the SR, allowing the accumulation of greater than normal amounts of Ca
2+
. In the context of ischemia, this extra accumulation of Ca
2+
would present a risk of potentially arrhythmogenic, spontaneous release of Ca
2+
on reperfusion of the tissue.
Nitric Oxide Attenuates Signal Transduction
Authors: Li Hong , Brodsky Sergey , Basco Mary , Romanov Victor , De Angelis Dino A. , Goligorsky Michael S.
Published: June 2012

Abstract
—Caveolae harbor different serpentine receptors, intracellular components of signaling cascades, and certain enzymes, including endothelial nitric oxide synthase (eNOS). The regulation of eNOS activity by Ca
2+
/calmodulin and caveolin has been described. We have previously demonstrated that nitric oxide (NO) can modulate signaling initiated via receptors localized to caveolae. In the present study, we show that NO donors induced an increase in the monomeric form of this scaffolding protein in cultured endothelial cells, the effect mimicked by 8-bromo cGMP. Proximity imaging of endothelial cells transfected with the thermotolerant green fluorescent protein–caveolin-1 construct demonstrated that sodium nitroprusside resulted in the increased fluorescence ratio of 410:470 nm, consistent with the distancing of fluorescently tagged caveolin-1. Pulse labeling of endothelial cells with cholera toxin B subunit indicated that sodium nitroprusside reversibly decreased its binding. Signaling via G protein–coupled receptors resident to caveolae was inhibited by pretreatment with NO donor. The data demonstrate that NO modulation of cell signaling is accomplished in part by regulating the state of caveolin-1 oligomerization. NO-induced attenuation of signaling involves reversible dissociation of caveolin scaffold, thus providing both spatial and temporal modulation of signal transduction.
Development of the Myocardium of the Atrioventricular Canal and the Vestibular Spine in the Human Heart
Authors: Kim Jung-Sun , Virágh Szabolcs , Moorman Antoon F. M. , Anderson Robert H. , Lamers Wouter H.
Published: June 2012

Abstract
—To establish the morphogenetic mechanisms underlying formation and separation of the atrioventricular connections, we studied the remodeling of the myocardium of the atrioventricular canal and the extracardiac mesenchymal tissue of the vestibular spine in human embryonic hearts from 4.5 to 10 weeks of development. Septation of the atrioventricular junction is brought about by downgrowth of the primary atrial septum, fusion of the endocardial cushions, and forward expansion of the vestibular spine between atrial septum and cushions. The vestibular spine subsequently myocardializes to form the ventral rim of the oval fossa. The connection of the atrioventricular canal with the atria expands evenly. In contrast, the expression patterns of creatine kinase M and GlN2, markers for the atrioventricular and interventricular junctions, respectively, show that the junction of the canal with the right ventricle forms by local growth in the inner curvature of the heart. Growth of the caudal portion of the muscular ventricular septum to make contact with the inferior endocardial cushion occurs only after the canal has expanded rightward. The atrioventricular node develops from that part of the canal myocardium that retains its continuity with the ventricular myocardium.
Cardiac-Specific Overexpression of Cyclin-Dependent Kinase 2 Increases Smaller Mononuclear Cardiomyocytes
Authors: Liao Hai-Sun , Kang Peter M. , Nagashima Hirotaka , Yamasaki Naohito , Usheva Anny , Ding Bo , Lorell Beverly H. , Izumo Seigo
Published: June 2012

Abstract
—Cyclin-dependent kinase 2 (cdk2) plays a critical role in the G1- to S-phase checkpoint of the cell cycle. Adult cardiomyocytes are believed to withdraw from the cell cycle. To determine whether forced overexpression of cdk2 results in altered cell-cycle regulation in the adult heart, we generated transgenic mice specifically overexpressing cdk2 in hearts. Transgenic hearts expressed high levels of both cdk2 mRNA and catalytically active cdk2 proteins. Cdk2 overexpression significantly increased the levels of cdk4 and cyclins A, D3, and E. There was an increase in both DNA synthesis and proliferating cell nuclear antigen levels in the adult transgenic hearts. The ratio of heart weight to body weight in cdk2 transgenic mice was significantly increased in neonatal day 2 but not in adults compared with that of wild-type mice. Analysis of dispersed individual adult cardiomyocytes showed a 5.6-fold increase in the proportion of smaller mononuclear cardiomyocytes in the transgenic mice. Echocardiography revealed that transgenic heart was functionally normal. However, adult transgenic ventricles expressed β-myosin heavy chain and atrial natriuretic factor. Surgically induced pressure overload caused an exaggerated maladaptive hypertrophic response in transgenic mice but did not change the proportion of mononuclear cardiomyocytes. The data suggest that overexpression of cdk2 promotes smaller, less-differentiated mononuclear cardiomyocytes in adult hearts that respond in an exaggerated manner to pressure overload.
Chimeric Smooth Muscle–Specific Enhancer/Promoters
Authors: Ribault Sébastien , Neuville Pascal , Méchine-Neuville Agnès , Augé Fabrice , Parlakian Ara , Gabbiani Giulio , Paulin Denise , Calenda Valérie
Published: June 2012

Abstract
—Gene transfer with adenoviral vectors is an attractive approach for the treatment of atherosclerosis and restenosis. However, because expression of a therapeutic gene in nontarget tissues may have deleterious effects, artery-specific expression is desirable. Although expression vectors containing transcriptional regulatory elements of genes expressed solely in smooth muscle cells (SMCs) have proved efficient to restrict expression of the transgene, their use in the clinical setting can be limited by their reduced strength. In the present study, we show that low levels of transgene expression are obtained with the smooth muscle (SM)-specific SM22α promoter compared with the viral cytomegalovirus (CMV) enhancer/promoter. We have generated chimeric transcriptional cassettes containing either a SM (SM-myosin heavy chain) or a skeletal muscle (creatine kinase) enhancer combined with the SM22α promoter. With both constructs we observed significantly stronger expression that remains SM-specific. In vivo, reporter gene expression was restricted to arterial SMCs with no detectable signal at remote sites. Moreover, when interferon-γ expression was driven by one of these two chimeras, SMC growth was inhibited as efficiently as with the CMV promoter. Finally, we demonstrate that neointima formation in the rat carotid balloon injury model was reduced to the same extent by adenoviral gene transfer of interferon-γ driven either by the SM-myosin heavy chain enhancer/SM22α promoter or the CMV promoter. These results indicate that such vectors can be useful for the treatment of hyperproliferative vascular disorders.