bluuebunny/crossref_metadata_2025 huggingface dataset:
Epicardial ST Depression in Acute Myocardial Infarction
Authors: Li Danshi , Li Chuan Yong , Yong Ah Chot , Johnston Peter R. , Kilpatrick David
Published: June 2012

Abstract—
The presence of electrocardiographic ST depression in acute infarction remains controversial and poorly explained. A combined animal and modeling study was performed to evaluate the source of ST changes in acute infarction. In anaesthetized sheep, small infarcts showed uniform ST elevation over the infarction whereas larger infarcts showed marked ST depression over the normal myocardium in addition to the ST elevation. These findings were replicated by bidomain models of the heart. A hollow sphere was used to model a gradually increasing infarct, and this showed that there was a decrease in the ratio of ST elevation to ST depression as the infarct was increased. The current flowing out of the heart must be identical to the current flowing back into the heart. This means that any infarction will produce ST depression as well as ST elevation, the ratio between the two being related to the size of the infarction. Small infarction is associated with a small region of ST elevation and minor ST depression of the remaining myocardium, and as the infarct region increases, the amplitude of the epicardial ST elevation falls and the amplitude of the ST depression increases. Infarction size is proportional to both the height of the ST depression on the epicardium and the strength of the epicardial ST segment dipole.
Vascular Endothelial Growth Factor (VEGF) and VEGF-C Show Overlapping Binding Sites in Embryonic Endothelia and Distinct Sites in Differentiated Adult Endothelia
Authors: Lymboussaki Athina , Olofsson Birgitta , Eriksson Ulf , Alitalo Kari
Published: June 2012

Abstract
—Vascular endothelial growth factor (VEGF) is a key modulator of angiogenesis during development and in adult tissues, whereas the related VEGF-C has been shown to induce both lymphangiogenesis and angiogenesis. To better understand the specific functions of these growth factors, we have here analyzed their binding to sections of mouse embryonic and adult tissues and compared the distribution of the bound growth factors with the expression patterns of the 3 known members of the VEGF receptor family as well as with neuropilin-1, a coreceptor for VEGF
165
. Partially overlapping patterns of VEGF and VEGF-C binding were obtained in embryonic tissues, consistent with the expression of all known VEGF receptors by vascular endothelial cells. However, the most striking differences of binding were observed in the developing and adult heart, in which VEGF decorated all vessels, whereas strong VEGF-C signals were obtained only from epicardial vessels. In the lymph nodes, VEGF and VEGF-C showed distinct binding patterns in agreement with the differential location of their specific receptors. These results show that both VEGF-C and VEGF target embryonic blood vessels, whereas a more selective binding of VEGF-C occurs to its lymphatic vascular receptor in certain adult tissues. Our results suggest that VEGF and VEGF-C have both overlapping and distinct activities via their endothelial receptors.
Transcriptional Profile of Mechanically Induced Genes in Human Vascular Smooth Muscle Cells
Authors: Feng Yajun , Yang Jeong-Hee , Huang Hayden , Kennedy Scott P. , Turi Thomas G. , Thompson John F. , Libby Peter , Lee Richard T.
Published: June 2012

Abstract
—Vascular smooth muscle cells must monitor and respond to their mechanical environment; however, the molecular response of these cells to mechanical stimuli remains incompletely defined. By applying a highly uniform biaxial cyclic strain to cultured cells, we used DNA microarray technology to describe the transcriptional profile of mechanically induced genes in human aortic smooth muscle cells. We first identified vascular endothelial growth factor (VEGF) as a mechanically induced gene in these cells; VEGF served as a positive control for these experiments. We then used a DNA microarray with 5000 genes with putative functions to identify additional mechanically induced genes. Surprisingly, relatively few genes are mechanically induced in human aortic smooth muscle cells. Only 3 transcripts of 5000 were induced >2.5-fold: cyclooxygenase-1, tenascin-C, and plasminogen activator inhibitor-1. Downregulated transcripts included matrix metalloproteinase-1 and thrombomodulin. The transcriptional profile of mechanically induced genes in human aortic smooth muscle cells suggests a response of defense against excessive deformation. These data also demonstrate that in addition to identifying large clusters of genes that respond to a given stimulus, DNA microarray technology may be used to identify a small subset of genes that comprise a highly specific molecular response.
Glucocorticoid Regulation of Cardiac K
+
Currents and L-Type Ca
2+
Current in Neonatal Mice
Authors: Wang Li , Feng Zhong-Ping , Duff Henry J.
Published: June 2012

Abstract
—Previous studies have reported that dexamethasone (Dex) prolongs cardiac action potential repolarization in mice and rats. However, the cellular mechanisms of this effect have not been addressed. Because action potential duration is influenced by a complex interplay of both inward and outward currents, this study evaluated the role of K
+
currents and the L-type Ca
2+
current in response to chronic in vivo Dex treatment. Accordingly, neonatal mice were randomly allocated to treatment with Dex (1 mg/kg per day) or placebo (saline) given subcutaneously for 5 days. At 14 to 15 days of age, the L-type Ca
2+
current and K
+
currents were recorded in ventricular myocytes using whole-cell patch-clamp techniques. The density of peak outward K
+
currents was significantly decreased in the chronic Dex-treated group, but the current measured at the end of a 1-second depolarization pulse was similar in both groups. We further measured the magnitudes of the fast-inactivating (
I
to
) and the slowly inactivating (
I
slow
) currents that contribute to the peak outward K
+
currents.
I
to
was reduced from 17.5±3.0 pA/pF (control) to 10.6±2.5 pA/pF (Dex) at +50 mV (
P
<0.05), but
I
slow
was not significantly different. These data suggest that downregulation of
I
to
is responsible for the reduced peak outward current. Time courses of the onset and offset of in vivo Dex effects were also assessed. A period of 3 days of treatment was required to observe the Dex effect on peak outward K
+
currents, whereas a 7-day period after discontinuation of Dex was required to recover the baseline current density. Acute in vitro treatment with Dex (1 μmol/L) had no effect on K
+
current densities. In addition, chronic Dex treatment significantly increased the density of the L-type Ca
2+
current (
I
Ca-L
) from –7.2±0.5 pA/pF of control to –8.9±0.6 pA/pF of Dex at +10 mV,
P
<0.05. In conclusion, chronic in vivo Dex treatment decreases
I
to
and increases
I
Ca-L
in neonatal mouse ventricular myocytes, both of which contribute to the prolongation of cardiac action potential repolarization induced by glucocorticoids.
Downregulation of Soluble Guanylyl Cyclase in Young and Aging Spontaneously Hypertensive Rats
Authors: Ruetten Hartmut , Zabel Ulrike , Linz Wolfgang , Schmidt Harald H. H. W.
Published: June 2012

Abstract
—Endothelial dysfunction, as observed in hypertension and atherosclerosis, is associated with a reduction in the bioavailability of endothelium-derived nitric oxide (NO). We tested the hypothesis that alterations in the soluble guanylyl cyclase (sGC) pathway may also contribute to the pathogenesis of hypertension. Therefore, we investigated the expression and activity of sGC in young (6 weeks) and aging (17 months) spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto rats (WKY). Endothelium-independent relaxation of aortic rings in response to the sGC activator YC-1 was attenuated in SHR, and expression of both α
1
and β
1
subunits of heterodimeric sGC and the basal contents of cGMP were reduced specifically in SHR aorta. Moreover, mRNA expression of the cGMP receptor and effector protein cGMP-dependent protein kinase type Iα (cGKIα) was also reduced. Interestingly, downregulation of both sGC and cGKIα expression was observed in young, ie, normotensive SHR, whereas impairment of the endothelium-independent relaxation was found only in aging SHR. Accordingly, similar cGMP levels were reached in response to YC-1 in young SHR and young WKY, suggesting a compensatory increased sensitivity or effectiveness of the sGC pathway in young SHR. In aging SHR, however, increased sensitivity to YC-1 no longer compensated for the impairment of endothelium-independent relaxation, suggesting that other mechanisms were involved. In fact, endothelium-independent relaxations were partially restored by superoxide dismutase, suggesting a pathophysiological role of superoxide production, particularly at later disease stages. Thus, tissue-specific downregulation of components of the sGC/cGMP pathway is an early event in the pathogenesis of hypertension.
A Mouse Model of Arterial Gene Transfer
Authors: Vassalli Giuseppe , Agah Ramtin , Qiao Renli , Aguilar Christina , Dichek David A.
Published: June 2012

Abstract
—We developed a murine model of arterial gene transfer and used it to test the role of antigen-specific immunity in the loss of adenovirus-mediated transgene expression. Adenoviral vectors encoding either β-galactosidase (β-gal) or green fluorescent protein were infused to the lumen of normal common carotids of CD-1 and C57BL/6 mice and atherosclerotic carotids of
Apoe
−/−
mice. At 3 days after gene transfer, significant reporter gene expression was detected in all strains. Transgene expression was transient, with expression undetectable at 14 days. Next, a β-gal–expressing vector was infused into carotids of ROSA26 mice (transgenic for, and therefore tolerant of, β-gal) and RAG-2
−/−
mice (deficient in recombinase-activating gene [RAG]-2 and therefore lacking in antigen-specific immunity). β-Gal expression was again high at 3 days but declined substantially (>90%) by 14 days. In vivo labeling with bromodeoxyuridine revealed that carotid endothelial proliferation was increased dramatically by the gene-transfer procedure alone, likely leading to the loss of episomal adenoviral DNA. Gene transfer to normal and atherosclerotic mouse carotids can be accomplished; however, elimination of antigen-specific immune responses does not prevent the early loss of adenovirus-mediated transgene expression. Efforts to prolong adenovirus-mediated transgene expression in the artery wall must be redirected. These efforts will likely include strategies to avoid the consequences of increased cell turnover. Nevertheless, despite the brevity of expression, this mouse model of gene transfer to normal and severely atherosclerotic arteries will likely be useful for investigating the genetic basis of vascular disease and for developing gene therapies. The full text of this article is available at http://www.circresaha.org.
Extracellular Adenosine Induces Apoptosis of Human Arterial Smooth Muscle Cells via A
2b
-Purinoceptor
Authors: Peyot Marie-Line , Gadeau Alain-Pierre , Dandré Frédéric , Belloc Isabelle , Dupuch Françoise , Desgranges Claude
Published: June 2012

Abstract
—Apoptosis of arterial smooth muscle cells (ASMCs) could play an important role in the pathogenesis of atherosclerosis and restenosis. Recent studies have demonstrated that extracellular adenosine induces apoptosis in various cell types. Our aim was to delineate the capacity of this nucleoside to induce ASMC apoptosis in arterial diseases. We demonstrate that adenosine dose-dependently triggers apoptosis of cultured human ASMCs. Apoptotic cell death was quantified by analysis of nuclear chromatin morphology and characterized by DNA laddering. The involvement of adenosine receptors was suggested, because neither an adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, nor an inhibitor of cellular nucleoside transport, dipyridamole, was able to inhibit adenosine-induced ASMC apoptosis. In contrast, an A
1
/A
2
-adenosine receptor antagonist, xanthine amine congener, totally inhibited adenosine-induced apoptosis. Furthermore, among more selective inhibitors of P
1
purinoceptor subtypes, only alloxazine, an antagonist of A
1
- and A
2
-adenosine receptors, completely inhibited adenosine-induced ASMC apoptosis, suggesting that adenosine triggers ASMC apoptosis via either 1 or both of these receptors. However, 8-cyclopentyl-1,3-dipropylxanthine, 8-(3-chlorostyryl) caffeine, and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate, which are A
1
-, A
2a
-, and A
3
-adenosine receptor antagonists, did not inhibit adenosine-induced apoptosis, suggesting an involvement of the A
2b
-receptor in this process. Moreover, the cAMP increase followed by cAMP-dependent protein kinase activation appears essential to mediate adenosine-induced ASMC apoptosis, thus confirming the previous hypothesis. These results indicate that adenosine-induced apoptosis of ASMCs is essentially mediated via A
2b
-adenosine receptor and involves a cAMP-dependent pathway.
Sensitivity to Fas-Mediated Apoptosis Is Determined Below Receptor Level in Human Vascular Smooth Muscle Cells
Authors: Chan Shiu-Wan , Hegyi Laszlo , Scott Stephen , Cary Nathaniel R. B. , Weissberg Peter L. , Bennett Martin R.
Published: June 2012

Abstract
—Despite Fas expression, many cells resist Fas-induced apoptosis. Although differences in surface Fas expression can explain Fas resistance, multiple proteins below receptor level also inhibit Fas-induced apoptosis. To examine the mechanism of Fas resistance, we studied Fas-induced apoptosis in human medial vascular smooth muscle cells (VSMCs) from healthy coronary arteries. VSMCs showed marked heterogeneity to Fas-induced apoptosis, exhibiting both Fas-resistant (98.1±2.3% viable, n=4,
P
=NS) and Fas-sensitive (31.3±2.6% viable, n=3,
P
<0.01) cells. Fas-resistant VSMCs expressed surface Fas and could recruit RIP, indicating that functional receptor complexes were formed. However, Fas-resistant cells showed reduced expression of FADD, Fas ligand, and caspases 3, 7, and 8 and increased expression of FLIP and c-IAP-1. Fas-induced apoptosis was associated with cleavage of caspase 3 and blocked by inhibitors of caspase 3 or 8 but not caspase 1, 6, or 7. Selective inhibition of caspase 3 or 8 by antisense transfection inhibited Fas-induced apoptosis, but their reexpression could not rescue the Fas-resistant phenotype. In vivo, medial VSMCs showed marked heterogeneity of expression of caspase 3. We conclude that Fas sensitivity is determined not only by expression of surface Fas but by differential expression of Fas-signaling proteins below receptor level. Subpopulations of cells within the same tissue have different sensitivities to apoptosis, determined by expression of specific death-signaling proteins.
Effects of the Renin-Angiotensin System on the Current
I
to
in Epicardial and Endocardial Ventricular Myocytes From the Canine Heart
Authors: Yu Hangang , Gao Junyuan , Wang Hongsheng , Wymore Randy , Steinberg Susan , McKinnon David , Rosen Michael R. , Cohen Ira S.
Published: June 2012

Abstract
—The Ca
2+
-independent portion of transient outward K
+
current (
I
to
) exhibits a transmural gradient in ventricle. To investigate control mechanisms for this gradient, we studied canine epicardial and endocardial ventricular myocytes with use of the whole-cell patch-clamp technique.
I
to
was larger in amplitude, had a more negative voltage threshold for activation, and had a more negative midpoint of inactivation in epicardium. Recovery from inactivation was >10-fold slower in endocardium. Incubation of epicardial myocytes with angiotensin II for 2 to 52 hours altered
I
to
to resemble unincubated endocardium and reduced the amplitude of the phase 1 notch of the action potential. In contrast, incubation of endocardial myocytes with losartan for 2 to 52 hours altered
I
to
to resemble unincubated epicardium and induced a phase 1 notch in the action potential. With RNase protection assays, we determined that incubations with angiotensin II or losartan did not alter mRNA levels for either Kv4.3 or Kv1.4; thus, a change in the α subunit for
I
to
is unlikely to be responsible. To test whether posttranslational modification produced the effects of angiotensin II, we coexpressed Kv4.3 and the angiotensin II type 1a receptor in
Xenopus
oocytes. Incubation with angiotensin II increased the time constant for recovery from inactivation of the expressed current by 2-fold with an incubation time constant of 3.7 hours. No effect on activation or inactivation voltage dependence was observed. These results demonstrate that the properties of
I
to
in endocardium and epicardium are plastic and likely under the tonic-differing influence of the renin-angiotensin system.
Cardiotrophic Effects of Protein Kinase C ε
Authors: Mochly-Rosen Daria , Wu Guangyu , Hahn Harvey , Osinska Hanna , Liron Tamar , Lorenz John N. , Yatani Atsuko , Robbins Jeffrey , Dorn Gerald W.
Published: June 2012

Abstract
—Protein kinase C (PKC) is a key mediator of many diverse physiological and pathological responses. Although little is known about the specific in vivo roles of the various cardiac PKC isozymes, activation-induced translocation of PKC is believed to be the primary determinant of isozyme-specific functions. Recently, we have identified a catalytically inactive peptide translocation inhibitor (εV1) and translocation activator (ψεRACK [
r
eceptors for
a
ctivated
C
k
inase]) specifically targeting PKCε. Using cardiomyocyte-specific transgenic expression of these peptides, we combined loss- and gain-of-function approaches to elucidate the in vivo consequences of myocardial PKCε signaling. As expected for a PKCε RACK binding peptide, confocal microscopy showed that εV1 decorated cross-striated elements and intercalated disks of cardiac myocytes. Inhibition of cardiomyocyte PKCε by εV1 at lower expression levels upregulated α–skeletal actin gene expression, increased cardiomyocyte cell size, and modestly impaired left ventricular fractional shortening. At high expression levels, εV1 caused a lethal dilated cardiomyopathy. In contrast, enhancement of PKCε translocation with ψεRACK resulted in selectively increased β myosin heavy chain gene expression and normally functioning concentric ventricular remodeling with decreased cardiomyocyte size. These results identify for the first time a role for PKCε signaling in normal postnatal maturational myocardial development and suggest the potential for PKCε activators to stimulate “physiological” cardiomyocyte growth.
Angiotensin II Activates Nuclear Transcription Factor κB Through AT
1
and AT
2
in Vascular Smooth Muscle Cells
Authors: Ruiz-Ortega Marta , Lorenzo Oscar , Rupérez Mónica , König Sven , Wittig Burghardt , Egido Jesús
Published: June 2012

Abstract
—Nuclear factor-κB (NF-κB) regulates many genes involved in vascular physiopathology. We have previously observed in vivo NF-κB activation in injured vessels that diminished by angiotensin-converting enzyme inhibition. In the present work, we investigated the effect of angiotensin II (Ang II) on NF-κB activity in rat vascular smooth muscle cells, evaluating the molecular mechanisms and the specific receptor subtype involved. Ang II increased NF-κB DNA binding (5-fold, 10
−
9
mol/L at 1 hour; electrophoretic mobility shift assay), nuclear translocation of p50/p65 subunits, and cytosolic inhibitor κBα (IκBα) degradation. Ang II elicited NF-κB–mediated transcription (transfection of a reporter gene) and expression of NF-κB–related genes (monocyte chemoattractant protein-1 and angiotensinogen). AT
1
(DUP753) and AT
2
(PD123319 and
CGP42112
) receptor antagonists inhibited Ang II–induced NF-κB DNA binding in a dose-dependent manner (≈85% for each one; 10
−
5
mol/L at 1 hour). The AT
2
agonist
p
-aminophenylalanine
6
–Ang II augmented NF-κB binding (4.6-fold, 10
−
9
mol/L at 1 hour), p65 nuclear levels, and transcription of an NF-κB reporter gene. AT
1
antagonist markedly inhibited NF-κB–mediated transcription and gene expression. Some differences between AT
1
/AT
2
intracellular signals were found. Antioxidants and ceramide inhibitors, but not protein kinase C inhibitors, diminished NF-κB activation elicited by both Ang II and the AT
2
agonist, while tyrosine kinase inhibitors only decreased Ang II–induced NF-κB activity. Our results demonstrate that Ang II activates NF-κB via AT
1
and AT
2
, although NF-κB–mediated transcription occurred mainly through AT
1
. Both receptors share some signaling pathways (oxygen radicals and ceramide); however, tyrosine kinases only participate in AT
1
/NF-κB responses. These data provide novel insights into Ang II actions, suggesting a potential implication of the AT
2
in the pathobiology of vascular cells.
Nuclear Factor-κB and cAMP Response Element Binding Protein Mediate Opposite Transcriptional Effects on the Flk-1/KDR Gene Promoter
Authors: Illi Barbara , Puri PierLorenzo , Morgante Liliana , Capogrossi Maurizio C. , Gaetano Carlo
Published: June 2012

Abstract
—The vascular endothelial growth factor receptor Flk-1/KDR is highly expressed during development and almost disappears in adult tissues. Despite its biological relevance, little is known about the molecular mechanisms controlling its expression. In the present work, it is shown that cAMP response element binding protein (CREB) and nuclear factor-κB (NF-κB)–related antigens bind specific sequences in the Flk-1/KDR promoter. Functional studies demonstrate that cAMP represses whereas tumor necrosis factor-α, an activator of NF-κB, stimulates promoter activity. Histone acetyltransferases (HATs) P/CAF and CBP/p300 together with p65/RelA, the catalytic subunit of NF-κB, increase Flk-1/KDR promoter activity 10- to 20-fold. Consistently, inhibition by cAMP is reverted by increasing intracellular HATs and is completely abolished by site-specific mutagenesis of the cAMP response element. In contrast, specific mutations in the NF-κB response element abolish responsiveness to p65/RelA and HATs without affecting cAMP-dependent repression. These results suggest that opposing signaling pathways, activating NF-κB or CREB and requiring HAT molecules, control Flk-1/KDR promoter activity. The full text of this article is available at http://www.circresaha.org.
T-Lymphocyte–Derived Tumor Necrosis Factor Exacerbates Anoxia-Reoxygenation–Induced Neutrophil–Endothelial Cell Adhesion
Authors: Kokura Satoshi , Wolf Robert E. , Yoshikawa Toshikazu , Granger D. Neil , Aw Tak Yee
Published: June 2012

Abstract
—The overall objective of this study was to determine whether T lymphocytes can modulate the increased neutrophil adherence and upregulation of endothelial cell adhesion molecules in human umbilical vein endothelial cells (HUVECs) exposed to anoxia/reoxygenation (A/R). HUVEC monolayers were exposed to 60 minutes of anoxia, followed by 4 hours of reoxygenation in the absence or presence of human T lymphocytes. The A/R-induced neutrophil adhesion was significantly enhanced when T lymphocytes and HUVECs were cocultured for the first 45 minutes of reoxygenation. This was accompanied by a more pronounced increase in E-selectin expression. When T lymphocytes were cocultured with HUVECs by use of inserts that prevented direct cell-cell contact, a comparable A/R-induced enhancement of neutrophil adhesion and of E-selectin expression was observed, indicating that soluble factors produced by T lymphocytes mediate the exaggerated A/R-induced inflammatory responses. Treatment with either an anti–tumor necrosis factor-α antibody or catalase attenuated the T-cell–mediated responses in postanoxic HUVECs. Moreover, the T-cell–mediated neutrophil adhesion response was mimicked by exposure of naive HUVECs to H
2
O
2.
These findings indicate that H
2
O
2
produced by postanoxic endothelial cells stimulates T cells to produce tumor necrosis factor-α, which in turn elicits endothelial cell adhesion molecule expression and a corresponding increase in neutrophil adhesion.
Smooth Muscle α-Actin CArG Elements Coordinate Formation of a Smooth Muscle Cell–Selective, Serum Response Factor–Containing Activation Complex
Authors: Mack Christopher P. , Thompson Maria M. , Lawrenz-Smith Susan , Owens Gary K.
Published: June 2012

Abstract
—Previous studies have shown that multiple serum response factor (SRF)-binding CArG elements were required for smooth muscle cell (SMC)-specific regulation of smooth muscle (SM) α-actin expression. However, a critical question remains as to the mechanisms whereby a ubiquitously expressed transcription factor such as SRF might contribute to SMC-specific expression. The goal of the present study was to investigate the hypothesis that SMC-selective expression of SM α-actin is due at least in part to (1) unique CArG flanking sequences that distinguish the SM α-actin CArGs from other ubiquitously expressed CArG-dependent genes such as c-
fos
, (2) cooperative interactions between CArG elements, and (3) SRF-dependent binding of SMC-selective proteins to the CArG-containing regions of the promoter. Results demonstrated that specific sequences flanking CArG B were important for promoter activity in SMCs but not in bovine aortic endothelial cells. We also provided evidence indicating that the structural orientation between CArGs A and B was an important determinant of promoter function. Electrophoretic mobility shift assays and methylation interference footprinting demonstrated that a unique SRF-containing complex formed that was selective for SMCs and, furthermore, that this complex was probably stabilized by protein-protein interactions and not by specific interactions with CArG flanking sequences. Taken together, the results of these studies provide evidence that SM α-actin expression in SMCs is complex and may involve the formation of a unique multiprotein initiation complex that is coordinated by SRF complexes bound to multiple CArG elements.
Calcineurin-Mediated Hypertrophy Protects Cardiomyocytes From Apoptosis In Vitro and In Vivo
Authors: De Windt Leon J. , Lim Hae W. , Taigen Tyler , Wencker Detlef , Condorelli Gianluigi , Dorn Gerald W. , Kitsis Richard N. , Molkentin Jeffery D.
Published: June 2012

Abstract
—We have previously shown that the calcium-calmodulin–regulated phosphatase calcineurin (PP2B) is sufficient to induce cardiac hypertrophy that transitions to heart failure in transgenic mice. Given the rapid onset of heart failure in these mice, we hypothesized that calcineurin signaling would stimulate myocardial cell apoptosis. However, utilizing multiple approaches, we determined that calcineurin-mediated hypertrophy protected cardiac myocytes from apoptosis, suggesting a model of heart failure that is independent of apoptosis. Adenovirally mediated gene transfer of a constitutively active calcineurin cDNA (AdCnA) was performed in cultured neonatal rat cardiomyocytes to elucidate the mechanism whereby calcineurin affected myocardial cell viability. AdCnA infection, which induced myocyte hypertrophy and atrial natriuretic factor expression, protected against apoptosis induced by 2-deoxyglucose or staurosporine, as assessed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) labeling, caspase-3 activation, DNA laddering, and cellular morphology. The level of protection conferred by AdCnA was similar to that of adenoviral Bcl-x
L
gene transfer or hypertrophy induced by phenylephrine. In vivo, failing hearts from calcineurin-transgenic mice did not demonstrate increased TUNEL labeling and, in fact, demonstrated a resistance to ischemia/reperfusion–induced apoptosis. We determined that the mechanism whereby calcineurin afforded protection from apoptosis was partially mediated by nuclear factor of activated T cells (NFAT3) signaling and partially by Akt/protein kinase B (PKB) signaling. Although calcineurin activation protected myocytes from apoptosis, inhibition of calcineurin with cyclosporine was not sufficient to induce TUNEL labeling in Gqα-transgenic mice or in cultured cardiomyocytes. Collectively, these data identify a calcineurin-dependent mouse model of dilated heart failure that is independent of apoptosis.
Acidosis Inhibits Endothelial Cell Apoptosis and Function and Induces Basic Fibroblast Growth Factor and Vascular Endothelial Growth Factor Expression
Authors: D’Arcangelo Daniela , Facchiano Francesco , Barlucchi Laura Maria , Melillo Guido , Illi Barbara , Testolin Lucia , Gaetano Carlo , Capogrossi Maurizio C.
Published: June 2012

Abstract
—Endothelial cells are exposed to an acidotic environment in a variety of pathological and physiological conditions. However, the effect of acidosis on endothelial cell function is still largely unknown, and it was evaluated in the present study. Bovine aortic endothelial cells (BAECs) were grown in bicarbonate buffer equilibrated either with 20% CO
2
(pH 7.0, acidosis) or 5% CO
2
(pH 7.4, control). Acidosis inhibited BAEC proliferation in 10% FCS, whereas by day 7 in serum-free medium, cell number was 3-fold higher in acidotic cells than in control cells. Serum deprivation enhanced BAEC apoptosis, and apoptotic cell death was markedly inhibited by acidosis. Additionally, acidosis inhibited FCS-stimulated migration in a modified Boyden chamber assay and FCS-stimulated differentiation into capillary-like structures on reconstituted basement membrane proteins. Conditioned media from BAECs cultured for 48 hours either at pH 7.0 or pH 7.4 enhanced BAEC proliferation and migration at pH 7.4, and both effects were more marked with conditioned medium from BAECs grown in acidotic than in control conditions. Acidosis enhanced vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) mRNA expression as well as bFGF secretion, and a blocking bFGF antibody inhibited enhanced BAEC migration in response to conditioned medium from acidotic cells. These results show that acidosis protects endothelial cells from apoptosis and inhibits their proangiogenic behavior despite enhanced VEGF and bFGF mRNA expression and bFGF secretion.
Regulation of Endothelial Nitric Oxide Synthase Expression by Albumin-Derived Advanced Glycosylation End Products
Authors: Rojas Armando , Romay Sheyla , González Deyarina , Herrera Beatriz , Delgado Rene , Otero Karel
Published: June 2012

Abstract
—We examined whether albumin-derived advanced glycosylation end products (AGEs) downregulate the expression of endothelial nitric oxide synthase (NOS). Significant reductions in NOS activity and cGMP levels in bovine aortic endothelial cells were observed when exposed to different concentrations of albumin-derived AGEs. Western and Northern blot analyses showed significant decreases at the protein and transcript levels. Both reductions became evident after 24 hours of exposure. Nuclear run-on assays showed that AGE-BSA did not modify the transcription rate of the NOS III gene; however, AGE-BSA treatment markedly reduced the half-life of NOS III mRNA. In addition, AGE-treated endothelial cells displayed significant reduction on their antiplatelet properties. These results indicate that NOS expression is reduced by AGEs by increasing the rate of mRNA degradation and may be relevant to the impairment of some endothelial functions observed in diabetes and aging. The full text of this article is available at http://www.circresaha.org.
Possible Mechanism(s) of Arachidonic Acid–Induced Intracellular Acidosis in Rat Cardiac Myocytes
Authors: Wu Mei-Lin , Chan Chih-Chiang , Su Ming-Ja
Published: June 2012

Abstract
—Arachidonic acid (AA) and other nonesterified fatty acids (FAs) have been shown to exert harmful effects during cardiac ischemia. By continuously measuring intracellular pH (pH
i
) changes in neonatal and adult cardiac myocytes, we have found, for the first time, that 10 μmol/L AA induces a substantial intracellular acidosis (0.3 to 0.4 pH units). We have ruled out the possibilities that the AA-induced acidosis is caused by (1) inhibition or stimulation of the pH
i
regulators, (2) protein kinase C activation or the generation of AA metabolites or free radicals, or (3) activation of NADPH oxidase or an inward H
+
current. The AA-induced acidosis fits to a simple diffusion mechanism, as proposed by Kamp and Hamilton (flip-flop model) for artificial phospholipid bilayers. The important properties found in the cardiac myocyte are that (1) the initial rate of acid flux (J
H
) increases with the AA concentration (2 to 50 μmol/L), (2) FAs with a
−
COOH group (eg, AA, oleic acid, and linoleic acid) induce intracellular acidification, but FAs with a
−
COOCH
3
group (eg, AA methyl ester) have little effect on the pH
i
, (3) tetradecylamine (FA amine) induces intracellular alkalosis, and, most importantly, (4) both the AA- and tetradecylamine-induced pH
i
changes can be reversed by 0.3% BSA. Because a low concentration of AA (10 μmol/L) can induce a substantial acidosis, the possible involvement of the FA-evoked acidosis in the negative inotropic effect during cardiac ischemia is discussed. The full text of this article is available at http://www.circresaha.org.
Stimulation of Myocardial Na
+
-Independent Cl
−
-HCO
3
−
Exchanger by Angiotensin II Is Mediated by Endogenous Endothelin
Authors: de Hurtado Maria C. Camilión , Alvarez Bernardo V. , Ennis Irene L. , Cingolani Horacio E.
Published: June 2012

Abstract
—Experiments were performed in isolated cat papillary muscles loaded with the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in the esterified form to study the effect of endothelin-1 (ET-1) on the activity of the Na
+
-independent Cl
−
-HCO
3
−
exchanger. Exposure to ET-1 (10 nmol/L) raised pH
i
by 0.13±0.03 U (
P
<0.05) in papillary muscles superfused with nominally HCO
3
−
-free solution, whereas no significant change was detected under CO
2
/HCO
3
−
-buffered medium. However, if ET-1 was applied to muscles pretreated with the anion exchanger inhibitor 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid, pH
i
increased by 0.09±0.02 U (
P
<0.05) in the presence of CO
2
/HCO
3
−
buffer. The rate of pH
i
recovery from trimethylamine hydrochloride–induced intracellular alkaline load was enhanced so that net HCO
3
efflux increased about three times in the presence of ET-1 (2.74±0.25 versus 9.66±1.29 mmol · L
−1
· min
−1
at pH
i
7.55,
P
<0.05). This effect was canceled by previous exposure to either 50 nmol/L PD 142,893 (nonselective endothelin receptor blocker) or 300 nmol/L BQ 123 (selective blocker of ET
A
receptors). BQ 123 also abolished angiotensin II–induced activation of the Na
+
independent Cl
−
-HCO
3
−
exchanger. These results show that ET-1 increases the activity of the Na
+
-independent Cl
−
-HCO
3
−
exchanger in cardiac tissue through the ET
A
receptors. Furthermore, our data suggest that the previously described angiotensin II–induced stimulation of the anion exchanger activity is mediated by endogenous ET-1.
T-Type and Tetrodotoxin-Sensitive Ca
2+
Currents Coexist in Guinea Pig Ventricular Myocytes and Are Both Blocked by Mibefradil
Authors: Heubach Jürgen F. , Köhler Arndt , Wettwer Erich , Ravens Ursula
Published: June 2012

Abstract
—Under Na
+
-free conditions, low-voltage–activated Ca
2+
currents in cardiomyocytes from various species have been described either as Ni
2+
-sensitive T-type Ca
2+
current (
I
Ca(T)
) or as tetrodotoxin (TTX)-sensitive Ca
2+
current (
I
Ca(TTX)
). So far, coexistence of the 2 currents within the same type of myocyte has never been reported. We describe experimental conditions under which
I
Ca(T)
and
I
Ca(TTX)
can be separated and studied in the same cell. Rat and guinea pig ventricular myocytes were investigated with the whole-cell voltage-clamp technique in Na
+
-free solutions. Whereas rat myocytes lack
I
Ca(T)
and exhibit
I
Ca(TTX)
only, guinea pig myocytes possess both of these low-voltage–activated Ca
2+
currents, which are separated pharmacologically by superfusion with TTX or Ni
2+
.
I
Ca(T)
and
I
Ca(TTX)
were of similar amplitude but significantly differed in their electrophysiological properties:
I
Ca(TTX)
activated at more negative potentials than did
I
Ca(T)
, the potential for half-maximum steady-state inactivation was more negative, and current deactivation and recovery from inactivation were faster.
I
Ca(TTX)
but not
I
Ca(T)
increased after membrane rupture (“run-up”). Isolation of
I
Ca(TTX)
by application of the bivalent cation Ni
2+
is critical because of possible shifts in voltage dependence. Therefore, we investigated whether the T-type Ca
2+
channel blocker mibefradil (10 μmol/L) is a suitable tool for the study of
I
Ca(TTX)
. However, mibefradil not only blocked
I
Ca(T)
by 85±2% but also decreased
I
Ca(TTX)
by 48±8%. We conclude that under Na
+
-free conditions
I
Ca(T)
and
I
Ca(TTX)
coexist in guinea pig ventricular myocytes and that both currents are sensitive to mibefradil. Future investigations of
I
Ca(T)
will have to consider the TTX-sensitive current component to avoid possible interference.
Impaired Conduction of Vasodilation Along Arterioles in Connexin40-Deficient Mice
Authors: de Wit Cor , Roos Frederik , Bolz Steffen-Sebastian , Kirchhoff Susanne , Krüger Olaf , Willecke Klaus , Pohl Ulrich
Published: June 2012

Abstract
—Connexins have been hypothesized to play an important role in intercellular communication within the vascular wall and may provide a mechanistic explanation for conduction of vasomotor responses. To test this hypothesis, we studied the transmission of vasomotor responses in the intact skeletal muscle microcirculation of connexin40-deficient mice (Cx40
−/−
). Arterioles were locally stimulated with hyperpolarizing dilators (acetylcholine [ACh] as well as bradykinin [Bk]) or depolarizing K
+
solution, and the resulting changes in diameter were measured using a videomicroscopy technique at the site of application and up to 1.32 mm upstream. Arterial pressure was elevated 25% in Cx40
−/−
mice (94±5 versus 75±4 mm Hg). Vessels selected for study had equivalent basal diameter and vasomotor tone in both genotypes of mice. Vasomotion was present in small arterioles of both genotypes, but its intensity was exaggerated in Cx40
−/−
mice. ACh and Bk induced dilation (33% and 53%, respectively, of maximal response) at the site of application that was of similar magnitude in both genotypes. These dilations were observed to spread upstream within <1 second without significant attenuation in Cx40
+/+
mice. However, spreading was severely attenuated in Cx40
−/−
animals (11±4% versus 35±7% with ACh and 38±5% versus 60±7% with Bk in Cx40
−/−
and Cx40
+/+
, respectively;
P
<0.05). In contrast, conducted vasoconstrictions, induced by K
+
solution decreased equally with distance in both genotypes. These results support a significant role for Cx40 in vascular intercellular communication. Our observations indicate that Cx40 is required for normal transmission of endothelium-dependent vasodilator responses and may underlie altered vasomotion patterns.
Downregulation of Endothelial Nitric Oxide Synthase in Rat Aorta After Prolonged Hypoxia In Vivo
Authors: Toporsian Mourad , Govindaraju Karuthapillai , Nagi Mohammed , Eidelman David , Thibault Gaetan , Ward Michael E.
Published: June 2012

Abstract
—The goal of this study was to determine whether hypoxia alters expression of endothelial nitric oxide synthase (eNOS) in the systemic circulation. Rats breathed either air or 10% oxygen for 12 hours, 48 hours, or 7 days. Thoracic aortas were excised and either mounted in organ bath myographs or frozen in liquid nitrogen for later extraction of protein and RNA. eNOS protein (Western blotting) was decreased (20% of normoxic control) after 12 hours, 48 hours, and 7 days of hypoxia. eNOS mRNA (ribonuclease protection assay) was similarly reduced. Acetylcholine (10
−4
mol/L) reversed phenylephrine (10
−5
mol/L) preconstriction by 53.3±5.6% in aortic rings from normoxic rats and 26.1±4.8% in rings from rats exposed to hypoxia for 48 hours (
P
<0.05), with comparable impairment of relaxation by the calcium ionophore A23187 (10
−5
mol/L). Responses to diethylamine nitric oxide and 8-bromo-cGMP were unaffected. Aortic cGMP levels after incubation with acetylcholine (10
−6
mol/L) averaged 14.0±1.8 fmol/mg in rings from normoxic rats compared with 8.7±1.0 fmol/mg in rings from hypoxic rats (
P
<0.05). Similarly, nitrate concentration (by capillary electrophoresis) in the media in which the rings were incubated was reduced in the hypoxic group (5.6±0.23 μmol/L for hypoxic rats and 7.8±0.7 μmol/L for normoxic rats). Impaired endothelial NO release may handicap the vascular responses that defend vital organ function during hypoxia.
Local Expression of Bovine Decorin by Cell-Mediated Gene Transfer Reduces Neointimal Formation After Balloon Injury in Rats
Authors: Fischer Jens W. , Kinsella Michael G. , Clowes Monika M. , Lara Stephanie , Clowes Alexander W. , Wight Thomas N.
Published: June 2012

Abstract
—Decorin is an extracellular matrix (ECM) proteoglycan that may modify vascular smooth muscle cell (SMC) function by altering the response to growth factors and the accumulation of ECM proteins during vascular injury. To investigate these possibilities in vivo, decorin was overexpressed at the site of arterial injury by cell-mediated gene transfer. Fischer rat SMCs were transduced in vitro with a retroviral construct that contained the bovine decorin gene and were subsequently seeded into injured rat carotid arteries. A species-specific antibody to bovine decorin and polymerase chain reaction primers were used to detect bovine decorin and distinguish it from endogenous rat decorin. Immunohistochemical and Northern analyses of rat carotid arteries revealed only low levels of rat decorin expression up to 8 weeks after balloon injury. However, after cell-mediated transfer of bovine decorin, strong expression of bovine decorin was verified by immunohistochemistry and reverse transcriptase–polymerase chain reaction. Four weeks after injury, the intimal area in vessels seeded with bovine decorin–overexpressing SMCs was significantly reduced by 35±4% (mean±SEM, n=9;
P
<0.01). Decorin overexpression also induced a higher intimal nuclear density and decreased volume of ECM. Specifically, immunostaining for versican and fibronectin was markedly reduced. In contrast, immunostaining for collagen type I was increased, and electron microscopy confirmed that collagen accumulation was altered. Bromodeoxyuridine labeling indicated that intimal SMC proliferation was not affected by the expression of bovine decorin. In summary, we demonstrate that gene transfer of the ECM proteoglycan, decorin, into the injured arterial wall reduces intimal ECM volume and alters the composition of the ECM.
Transient Induction of Cytokine Production in Human Myocardial Fibroblasts by Coxsackievirus B3
Authors: Heim A. , Zeuke S. , Weiss S. , Ruschewski W. , Grumbach I. M.
Published: June 2012

Abstract
—Cytokine expression in enterovirus infections of the heart may trigger inflammation and have detrimental effects on myocytes. However, the induction of cytokines in human myocardial cells by cardiotropic enteroviruses, for example, Coxsackievirus B3 (CVB3), was not yet demonstrated. Fibroblasts are the predominant cell type of the myocardial interstitium before inflammatory infiltration develops. Hence, we investigated, by enzyme immunoassays, reverse transcription–quantitative polymerase chain reaction (RT-qPCR), and nucleic acid sequence–based amplification (NASBA), whether CVB3 induces cytokine expression in cultured human myocardial fibroblasts. As early as 3 hours after infection, RT-qPCR demonstrated a 2-fold increase of interleukin (IL)–6 and IL-8 mRNA compared with basal transcription, resulting in a significant increase of IL-6 and IL-8 to a median level of 1500 pg/mL (range, 1246 to 1858) and 529 pg/mL (range, 428 to 601) in culture supernatants, respectively. IL-6 and IL-8 expression returned to basal levels within 3 and 5 days, respectively, despite a persistent (carrier-state) CVB3 infection. For comparison, IL-6 and IL-8 were induced in dermal fibroblasts later than 3 days after CVB3 infection. Although the low-level IL-1α transcription of myocardial fibroblasts was not significantly increased, IL-1α was released from cells to culture supernatants 5 days after infection. Furthermore, a suppression of interferon-β transcription was demonstrated up to 24 hours after CVB3 infection of myocardial fibroblasts by highly sensitive NASBA. In conclusion, our results demonstrate a heart-specific pattern of a rapid and transient induction of proinflammatory cytokines after CVB3 infection, whereas the expression of protective interferon-β was suppressed by CVB3.
Altered Patterns of Gene Expression in Response to Myocardial Infarction
Authors: Stanton Lawrence W. , Garrard Lisa J. , Damm Deborah , Garrick Brett L. , Lam Andrew , Kapoun Ann M. , Zheng Qiang , Protter Andrew A. , Schreiner George F. , White R. Tyler
Published: June 2012

Abstract
—The use of cDNA microarrays has made it possible to simultaneously analyze gene expression for thousands of genes. Microarray technology was used to evaluate the expression of >4000 genes in a rat model of myocardial infarction. More than 200 genes were identified that showed differential expression in response to myocardial infarction. Gene expression changes were monitored from 2 to 16 weeks after infarction in 2 regions of the heart, the left ventricle free wall and interventricular septum. A novel clustering program was used to identify patterns of expression within this large set of data. Unique patterns were revealed within the transcriptional responses that illuminate changes in biological processes associated with myocardial infarction.
Two Distinct Congenital Arrhythmias Evoked by a Multidysfunctional Na
+
Channel
Authors: Veldkamp Marieke W. , Viswanathan Prakash C. , Bezzina Connie , Baartscheer Antonius , Wilde Arthur A. M. , Balser Jeffrey R.
Published: June 2012

Abstract
—The congenital long-QT syndrome (LQT3) and the Brugada syndrome are distinct, life-threatening rhythm disorders linked to autosomal dominant mutations in
SCN5A
, the gene encoding the human cardiac Na
+
channel. It is believed that these two syndromes result from opposite molecular effects: LQT3 mutations induce a gain of function, whereas Brugada syndrome mutations reduce Na
+
channel function. Paradoxically, an inherited C-terminal
SCN5A
mutation causes affected individuals to manifest electrocardiographic features of both syndromes: QT-interval prolongation (LQT3) at slow heart rates and distinctive ST-segment elevations (Brugada syndrome) with exercise. In the present study, we show that the insertion of the amino acid 1795insD has opposite effects on two distinct kinetic components of Na
+
channel gating (fast and slow inactivation) that render unique, simultaneous effects on cardiac excitability. The mutation disrupts fast inactivation, causing sustained Na
+
current throughout the action potential plateau and prolonging cardiac repolarization at slow heart rates. At the same time, 1795insD augments slow inactivation, delaying recovery of Na
+
channel availability between stimuli and reducing the Na
+
current at rapid heart rates. Our findings reveal a novel molecular mechanism for the Brugada syndrome and identify a new dual mechanism whereby single
SCN5A
mutations may evoke multiple cardiac arrhythmia syndromes by influencing diverse components of Na
+
channel gating function. The full text of this article is available at http://www.circresaha.org.