bluuebunny/crossref_metadata_2025 huggingface dataset:
Tissue-Destructive Macrophages in Giant Cell Arteritis
Authors: Rittner Heike L. , Kaiser Markus , Brack Alexander , Szweda Luke I. , Goronzy Jörg J. , Weyand Cornelia M.
Published: June 2012

Abstract
—Giant cell arteritis (GCA) is an inflammatory vasculopathy in which T cells and macrophages infiltrate the wall of medium and large arteries. Clinical consequences such as blindness and stroke are related to arterial occlusion. Formation of aortic aneurysms may result from necrosis of smooth muscle cells and fragmentation of elastic membranes. The molecular mechanisms of arterial wall injury in GCA are not understood. To identify mechanisms of arterial damage, gene expression in inflamed and unaffected temporal artery specimens was compared by differential display polymerase chain reaction. Genes differentially expressed in arterial lesions included 3 products encoded by the mitochondrial genome. Immunohistochemistry with antibodies specific for a 65-kDa mitochondrial antigen revealed that increased expression of mitochondrial products was characteristic of multinucleated giant cells and of CD68+ macrophages that cluster in the media and at the media-intima junction. 4-Hydroxy-2-nonenal adducts, products of lipid peroxidation, were detected on smooth muscle cells and on tissue infiltrating cells, in close proximity to multinucleated giant cells and CD68+ macrophages. Also, giant cells and macrophages with overexpression of mitochondrial products were able to synthesize metalloproteinase-2. Our data suggest that in the vascular lesions characteristic for GCA, a subset of macrophages has the potential to support several pathways of arterial injury, including the release of reactive oxygen species and the production of metalloproteinase-2. This macrophage subset is topographically defined and is also identified by overexpression of mitochondrial genes. Because these macrophages have a high potential to promote several mechanisms of arterial wall damage, they should be therapeutically targeted to prevent blood vessel destruction.
Subcellular Creatine Kinase Alterations
Authors: De Sousa E. , Veksler V. , Minajeva A. , Kaasik A. , Mateo P. , Mayoux E. , Hoerter J. , Bigard X. , Serrurier B. , Ventura-Clapier R.
Published: June 2012

Abstract
—We have tested the hypothesis that decreased functioning of creatine kinase (CK) at sites of energy production and utilization may contribute to alterations in energy fluxes and calcium homeostasis in congestive heart failure (CHF). Heart failure was induced by aortic banding in 3-week-old rats. Myofilaments, sarcoplasmic reticulum (SR), mitochondrial functions, and CK compartmentation were studied in situ using selective membrane permeabilization of left ventricular fibers with detergents (saponin for mitochondria and SR and Triton X-100 for myofibrils). Seven months after surgery, animals were in CHF. A decrease in total CK activity could be accounted for by a 4-fold decrease in activity and content (Western blots) of mitochondrial CK and a 30% decrease in M isoform of CK (MM-CK) activity. In myofibrils, maximal force, crossbridge kinetics, and α-myosin heavy-chain expression decreased, whereas calcium sensitivity of tension development remained unaltered. Myofibrillar CK efficacy was unchanged. Calcium uptake capacities of SR were estimated from the surface of caffeine-induced tension transient (S
Ca
) after loading with different substrates. In CHF, S
Ca
decreased by 23%, and phosphocreatine was 2 times less efficient in enhancing calcium uptake. Oxidative capacities of the failing myocardium measured as oxygen consumption per gram of fiber dry weight decreased by 28%. Moreover, the control of respiration by creatine, ADP, and AMP was severely impaired. Our observations provide evidence that alterations in CK compartmentation may contribute to alterations of energy fluxes and calcium homeostasis in CHF.
Regulation of Sarcolemmal Na
+
/H
+
Exchanger Activity by Angiotensin II in Adult Rat Ventricular Myocytes
Authors: Gunasegaram Suba , Haworth Robert S. , Hearse David J. , Avkiran Metin
Published: June 2012

Abstract
—Increased sarcolemmal Na
+
/H
+
exchanger activity has been implicated as a mediator of the cardiac actions of angiotensin II. We studied the receptor subtypes and signaling pathways involved in the regulation of sarcolemmal Na
+
/H
+
exchanger activity by angiotensin II in adult rat ventricular myocytes. Cells were loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1, and acid efflux rates estimated during recovery from intracellular acidosis were used to quantify exchanger activity. Sarcolemmal Na
+
/H
+
exchanger activity was not affected by angiotensin II alone but was increased by angiotensin II plus PD123319 (AT
2
antagonist). In contrast, angiotensin II plus losartan (AT
1
antagonist) or CGP42112A (AT
2
agonist) did not affect exchanger activity. The increase in Na
+
/H
+
exchanger activity induced by angiotensin II plus PD123319 was blocked by losartan, PD98059 (extracellular signal–regulated kinase inhibitor), GF109203X (protein kinase C inhibitor), and tyrphostin AG1478 (epidermal growth factor receptor kinase inhibitor). Extracellular signal–regulated kinase phosphorylation and activity, measured by immunoblot analysis and an immune-complex kinase assay, respectively, were increased significantly by angiotensin II plus PD123319; these increases were blocked by losartan and PD98059. The increase in extracellular signal–regulated kinase phosphorylation induced by angiotensin II plus PD123319 was blocked also by GF109203X and tyrphostin AG1478. These data show that AT
1
stimulation increases sarcolemmal Na
+
/H
+
exchanger activity in adult rat ventricular myocytes and that this response requires extracellular signal-regulated kinase activation through a protein kinase C– and epidermal growth factor receptor–mediated mechanism. The positive effect of AT
1
stimulation on Na
+
/H
+
exchanger activity is counteracted by simultaneous AT
2
stimulation through a mechanism that does not involve direct inhibition of the exchanger or attenuation of extracellular signal–regulated kinase activation.
Targeted Expression of a Dominant-Negative K
v
4.2 K
+
Channel Subunit in the Mouse Heart
Authors: Wickenden Alan D. , Lee Paul , Sah Rajan , Huang Qian , Fishman Glenn I. , Backx Peter H.
Published: June 2012

Abstract
—Action potential duration is prolonged in many forms of heart disease, often as a result of reductions in Ca
2+
-independent transient outward K
+
currents (ie,
I
to
). To examine the effects of a primary reduction in
I
to
current in the heart, transgenic mice were generated that express a dominant-negative N-terminal fragment of the K
v
4.2 pore-forming potassium channel subunit under the control of the mouse α-myosin heavy chain promoter. Two of 6 founders died suddenly, and only 1 mouse successfully transmitted the transgene in mendelian fashion. Electrophysiological analysis at 2 to 4 weeks of age demonstrated that
I
to
density was specifically reduced and action potential durations were prolonged in a subset of transgenic myocytes. The heterogeneous reduction in
I
to
was accompanied by significant prolongation of monophasic action potentials. In vivo hemodynamic studies at this age revealed significant elevations in the mean arterial pressure, peak systolic ventricular pressures, and ±dP/dt, indicative of enhanced contractility. Surprisingly, by 10 to 12 weeks of age, transgenic mice developed clinical and hemodynamic evidence of congestive heart failure. Failing transgenic hearts displayed molecular and cellular remodeling, with evidence of hypertrophy, chamber dilatation, and interstitial fibrosis, and individual myocytes showed sharp reductions in
I
to
and
I
K1
densities, action potential duration prolongation, and increased cell capacitance. Our results confirm that K
v
4.2 subunits contribute to
I
to
in the mouse and demonstrate that manipulation of cardiac excitability may secondarily influence contractile performance.
Distinct Role of cAMP and cGMP in the Cell Cycle Control of Vascular Smooth Muscle Cells
Authors: Fukumoto Shinya , Koyama Hidenori , Hosoi Masayuki , Yamakawa Kenjiro , Tanaka Shinji , Morii Hirotoshi , Nishizawa Yoshiki
Published: June 2012

Abstract
—cAMP and cGMP are known to suppress vascular smooth muscle cell (SMC) proliferation. In this study, our aim was to delineate the molecular mechanism underlying cAMP and cGMP suppression of cell cycle transition in human SMCs. cAMP inhibits both platelet-derived growth factor–stimulated cyclin-dependent kinase (cdk) 2 and cdk4 activation through upregulation of the cdk2 inhibitor p27
Kip1
and downregulation of cyclin D1 expression, which leads to a complete arrest of the cells in phase G
1
. In contrast, cGMP inhibits cyclin D1 expression, inhibits cdk4 activation, and delays platelet-derived growth factor–mediated cdk2 activation, resulting in a delay in G
1
/S transition. A transient increase in p27
Kip1
in cdk2 immunoprecipitates, without changes in total cellular p27
Kip1
levels, correlates with the delay in cdk2 activation caused by cGMP. Thus, cAMP and cGMP differentially affect cell cycle through distinct regulation of cell cycle molecules in human SMCs.
Aldosterone Upregulates Ca
2+
Current in Adult Rat Cardiomyocytes
Authors: Bénitah Jean-Pierre , Vassort Guy
Published: June 2012

Abstract
—Aldosterone is associated with the pathogenesis and progression of left ventricular hypertrophy and heart failure, independent of its relation with arterial blood pressure. However, little information exists about the possible influence of this mineralocorticoisteroid on cardiomyocyte electrical activity. The present study was designed to determine the role of aldosterone on whole-cell Ca
2+
current (
I
Ca
) in isolated adult rat ventricular myocytes using the patch-clamp technique. We found that incubation of cells with 1 μmol/L aldosterone for 24 hours increases the density of
I
Ca
significantly. This “long-term” aldosterone treatment had no significant effects on the kinetics and voltage dependence of
I
Ca
inactivation. Moreover, no demonstrable influence of aldosterone on
I
Ca
could be detected during short-term exposure (up to 6 hours), under our experimental conditions. The classical aldosterone intracellular receptor antagonist spironolactone (250-fold excess) was able to blunt the aldosterone-induced increase in
I
Ca
density. These effects were also observed with lower concentrations of aldosterone (10 and 100 nmol/L). Moreover, inhibitors of transcription (actinomycin D, 5 μg/mL) and protein synthesis (cycloheximide, 20 μg/mL) prevented the aldosterone-dependent increase in
I
Ca
. Therefore, the long latency
I
Ca
stimulation effect of aldosterone might result from an increased channel expression. We suggest that this genomic action contributes to the increased
I
Ca
observed during cardiac remodeling.
Tissue Inhibitor of Matrix Metalloproteinases-1 Impairs Arterial Neointima Formation After Vascular Injury in Mice
Authors: Lijnen H. R. , Soloway P. , Collen D.
Published: June 2012

Abstract
—The hypothesis that tissue inhibitor of metalloproteinases-1 (TIMP-1) plays a role in neointima formation was tested with the use of a vascular injury model in wild-type (TIMP-1
+/+
) and TIMP-1–deficient (TIMP-1
−/−
) mice. The neointimal area at 1 to 3 weeks after electric injury of the femoral artery was significantly higher in TIMP-1
−/−
as compared with TIMP-1
+/+
mice (0.012±0.0015 versus 0.0033±0.0008 mm
2
at 1 week,
P
<0.005). The medial areas were comparable, resulting in intima/media ratios that were significantly larger in TIMP-1
−/−
as compared with TIMP-1
+/+
arteries (1.2±0.22 versus 0.39±0.08 at 1 week,
P
<0.005). Nuclear cell counts in cross-sectional areas of the intima of the injured region were higher in TIMP-1
−/−
as compared with TIMP-1
+/+
arteries (138±15 versus 69±8 at 1 week,
P
<0.005). Immunocytochemical analysis revealed that α-actin–positive smooth muscle cells (SMCs) at 2 weeks after injury were more abundant in the intima of TIMP-1
−/−
arteries than in that of TIMP-1
+/+
arteries, whereas after 3 weeks the intimal cell population consisted mainly of SMCs in both genotypes. In in vitro scrape-wounding assays, SMCs of TIMP-1
−/−
mice migrated faster than those of TIMP-1
+/+
mice. Zymography of arterial extracts revealed a higher active matrix metalloproteinase (MMP)–2 level at 1 to 3 weeks after injury in TIMP-1
−/−
arteries, whereas active MMP-9 was only detected in TIMP-1
−/−
arteries at 1 week after injury. These data are compatible with a role of TIMP-1 in the impairment of SMC migration and neointima formation after vascular injury, as a result of inhibition of MMP activity.
BTEB2, a Krüppel-Like Transcription Factor, Regulates Expression of the SMemb/Nonmuscle Myosin Heavy Chain B (SMemb/NMHC-B) Gene
Authors: Watanabe Noboru , Kurabayashi Masahiko , Shimomura Yukio , Kawai-Kowase Keiko , Hoshino Yo-ichi , Manabe Ichiro , Watanabe Masafumi , Aikawa Masanori , Kuro-o Makoto , Suzuki Toru , Yazaki Yoshio , Nagai Ryozo
Published: June 2012

We have recently characterized the promoter region of the rabbit embryonic smooth muscle myosin heavy chain (SMemb/NMHC-B) gene and identified the 15-bp sequence, designated SE1, located at −105 from the transcriptional start site as an important regulatory element for its transcriptional activity in a smooth muscle cell (SMC) line. In this study, we attempted to isolate cDNA clones encoding for the transcription factors that control the expression of the SMemb gene through binding to this
cis
-regulatory element. We screened a λgt11 cDNA library prepared from C2/2 cells, a rabbit-derived SMC line, by using a radiolabeled concatenated oligonucleotide containing SE1 as a probe. Sequence analysis revealed that one of the cDNA clones corresponds to the rabbit homologue of basic transcriptional element binding protein-2 (BTEB2), which has previously been identified as one of the Krüppel-like transcription factor. Gel mobility shift assays and antibody supershift analyses with nuclear extracts from C2/2 cells indicate that BTEB2 is a major component of nuclear factor:SE1 complexes. Furthermore, a glutathione
S
-transferase-BTEB2 fusion protein binds to the SE1 in a sequence-specific manner. In support of the functionality of BTEB2 binding, basal promoter activity and BTEB2-induced transcriptional activation were markedly attenuated by the disruption of the SE1. In adult rabbit tissues, BTEB2 mRNA was most highly expressed in intestine, urinary bladder, and uterus. BTEB2 mRNA levels were downregulated in rabbit aorta during normal development. Moreover, immunohistochemical analysis indicated a marked induction of BTEB2 protein in the neointimal SMC after balloon injury in rat aorta. These results suggest that BTEB2 mediates the transcriptional regulation of the SMemb/NMHC-B gene and possibly plays a role in regulating gene expression during phenotypic modulation of vascular SMC.
Angiotensin II Stimulates Platelet-Derived Growth Factor-B Chain Expression in Newborn Rat Vascular Smooth Muscle Cells and Neointimal Cells Through Ras, Extracellular Signal–Regulated Protein Kinase, and c-Jun N-Terminal Protein Kinase Mechanisms
Authors: Deguchi Jun-o , Makuuchi Masatoshi , Nakaoka Takashi , Collins Tucker , Takuwa Yoh
Published: June 2012

Abstract
—Platelet-derived growth factors (PDGFs) have been implicated in the pathogenesis of vascular proliferative disorders. Vascular smooth muscle cells (VSMCs) are one of the cell types that produce PDGF-B chain in proliferative lesions, although the mechanism of regulation of PDGF-B chain production in these cells is not well understood. In the present study, we demonstrate that angiotensin II (Ang II), which is also implicated in vascular stenosis after angioplasty and atherosclerosis, markedly stimulates PDGF-B chain mRNA expression in cultured newborn rat medial VSMCs and neointimal VSMCs via an AT
1
, but not in adult rat VSMCs. In newborn rat VSMCs, Ang II activates extracellular signal–regulated protein kinase (ERK), c-Jun N-terminal protein kinase (JNK), and p38 mitogen–activated protein kinase. The mitogen-activated protein/ERK (MEK) inhibitor PD98059, but not the p38 inhibitor SB203580, abrogates Ang II–induced PDGF-B mRNA expression. Transient transfection analysis using a PDGF-B promoter-luciferase gene reporter construct reveals that Ang II induces transcriptional activation of PDGF-B chain gene, which is abolished by the expression of a dominant negative form of either ERK or JNK, but not of p38. The expression of a dominant negative form of Ras abolishes the stimulatory effects of Ang II on ERK activity and PDGF-B mRNA expression. In adult rat VSMCs, Ang II activates ERK and JNK, but weakly induces Egr-1, a transcription factor implicated in PDGF-B chain gene expression, compared with newborn VSMCs. These data indicate that Ang II activates PDGF-B chain gene expression in VSMCs through mechanisms involving Ras-ERK and JNK.
Role of Na
+
/H
+
Exchanger During Ischemia and Preconditioning in the Isolated Rat Heart
Authors: Xiao Xiao-Hui , Allen David G.
Published: June 2012

Abstract
—The role of the Na
+
/H
+
exchanger in ischemia, reperfusion, and preconditioning was investigated in isolated perfused rat hearts. Contractile function, [Na
+
]
i
, and pH
i
were measured; ischemic damage was assessed by the recovery of developed pressure (DP) on reperfusion. After 30 minutes of ischemia, DP recovered to only 14±4% of preischemic control. In contrast, after preconditioning (3×5-minute periods of ischemia) followed by 30 minutes of ischemia, DP recovered to 75±4%. Hearts treated with the Na
+
/H
+
exchange inhibitor 5-(
N
-methyl-
N
-isobutyl)amiloride (MIA) also showed an enhanced recovery after ischemia (DP 62±9%). Treatment with a low concentration of tetrodotoxin (TTX, 100 nmol/L), which blocks the persistent component of the Na
+
current, had a small beneficial effect on recovery (DP 37±8%). Thirty minutes of ischemia caused a small [Na
+
]
i
rise (3.2±0.9 mmol/L); reperfusion resulted in a further [Na
+
]
i
increase (+11.9±2.5 mmol/L), which partially recovered over 30 minutes. Preconditioning did not change the [Na
+
]
i
rise during ischemia but abolished the large [Na
+
]
i
rise on reperfusion, and [Na
+
]
i
instead fell (−3.6±1.3 mmol/L). In the presence of MIA, the [Na
+
]
i
rise was unchanged from ischemia only; on reperfusion, [Na
+
]
i
fell (−3.7±0.9 mmol/L), similar to the preconditioned hearts. TTX abolished the [Na
+
]
i
rise during ischemia (+0.3±0.7 mmol/L), and the increase on reperfusion was similar to ischemia only. We conclude that the rise of [Na
+
]
i
during ischemia is caused by Na
+
entry through persistent Na
+
channels. The rise of [Na
+
]
i
on reperfusion is caused by activation of the Na
+
/H
+
exchanger and is blocked by MIA and by preconditioning. It is known that the Na
+
/H
+
exchanger is inhibited during ischemia; the present result suggests that this inhibition is prolonged into the early part of reperfusion by preconditioning. To test this hypothesis, we measured the time course of pH
i
recovery after ischemia and preconditioning. Preconditioning slowed the rate of pH
i
recovery after ischemia, providing further support for the hypothesis that preconditioning inhibits the Na
+
/H
+
exchanger during early reperfusion. This inhibition of the Na
+
/H
+
exchanger during reperfusion prevents Na
+
entry, and therefore Ca
2+
loading, and is part of the protective pathway involved in preconditioning.
Shock-Induced Figure-of-Eight Reentry in the Isolated Rabbit Heart
Authors: Banville Isabelle , Gray Richard A. , Ideker Raymond E. , Smith William M.
Published: June 2012

Abstract
—The patterns of transmembrane potential on the whole heart during and immediately after fibrillation-inducing shocks are unknown. To study arrhythmia induction, we recorded transmembrane activity from the anterior and posterior epicardial surface of the isolated rabbit heart simultaneously using 2 charge-coupled device cameras (32,512 pixels, 480 frames/second). Isolated hearts were paced from the apex at a cycle length of 250 ms. Two shock coils positioned inside the right ventricle (−) and atop the left atrium (+) delivered shocks at 3 strengths (0.75, 1.5, and 2.25 A) and 6 coupling intervals (130 to 230 ms). The patterns of depolarization and repolarization were similar, as is evident in the uniformity of action potential duration at 75% repolarization (131.4±8.3 ms). At short coupling intervals (<180 ms), shocks hyperpolarized a large portion of the ventricles and produced a pair of counterrotating waves, one on each side of the heart. The first beat after the shock was reentrant in 90% of short coupling interval episodes. At long coupling intervals (>180 ms), increasingly stronger shocks depolarized an increasingly larger portion of the heart. The first beat after the shock was reentrant in 18% of long coupling interval episodes. Arrhythmias were most often induced at short coupling intervals (98%) than at long coupling intervals (35%). The effect and outcome of the shock were related to the refractory state of the heart at the time of the shock. Hyperpolarization occurred at short coupling intervals, whereas depolarization occurred at long coupling intervals. Consistent with the “critical point” hypothesis, increasing shock strength and coupling interval moved the location where reentry formed (away from the shock electrode and pacing electrode, respectively).
Identification and Characterization of a New Growth Hormone–Releasing Peptide Receptor in the Heart
Authors: Bodart V. , Bouchard J. F. , McNicoll N. , Escher E. , Carrière P. , Ghigo E. , Sejlitz T. , Sirois M. G. , Lamontagne D. , Ong H.
Published: June 2012

Abstract
—Hexarelin, a synthetic hexapeptide of the growth hormone–releasing peptide (GHRP) family with strong growth hormone (GH)–releasing activity, features protecting activity against postischemic ventricular dysfunction in hearts from GH-deficient and senescent rats. To document whether hexarelin action is mediated through specific cardiac receptors, perfusion of Langendorff rat hearts with hexarelin and binding studies were carried out. In the Langendorff rat heart system, hexarelin induced a dose-dependent increase in coronary perfusion pressure. Nifedipine, chelerythrine, and bisindolylmaleimide partially inhibited the vasoconstriction induced by hexarelin, suggesting that this effect was mediated at least in part by L-type Ca
2+
channels and protein kinase C. In contrast, diclofenac and 1-(7-carboxyheptyl)imidazole were without effect, suggesting that prostaglandins and thromboxanes were not involved in the coronary vasoconstriction induced by hexarelin. To characterize the hexarelin binding sites in the rat heart, [
125
I]Tyr-Bpa-Ala-hexarelin was used as photoactivatable radioligand in saturation and competitive binding studies. We specifically labeled a hexarelin receptor with an
M
r
of 84 000 in rat cardiac membranes. Saturation binding curves revealed a single class of binding sites with a
K
d
of 14.5 nmol/L and a density of 91 fmol/mg of protein. Competition binding studies gave an IC
50
of 2.9 μmol/L for hexarelin; MK-0677 and EP51389, both potent GH secretagogues, did not displace the binding of the photoactivatable derivative from rat cardiac membranes. Interestingly, both compounds were devoid of any vasoconstrictive activity. These results suggest the existence of a new class of hexarelin receptor in the heart, whose role in the regulation of the coronary vascular tone is yet to be determined.
Signaling Properties and Functions of Two Distinct Cardiomyocyte Protease-Activated Receptors
Authors: Sabri Abdelkarim , Muske Galina , Zhang HongLu , Pak Elena , Darrow Andrew , Andrade-Gordon Patricia , Steinberg Susan F.
Published: June 2012

Abstract
—Previous studies have established that cardiomyocytes express protease-activated receptor (PAR)-1, a high-affinity receptor for thrombin, which is also activated by the tethered-ligand domain sequence (SFLLRN) and which promotes inositol trisphosphate accumulation, stimulates extracellular signal–regulated protein kinase, and modulates contractile function. A single previous report identified PAR-1 as a hypertrophic stimulus, but there have been no subsequent investigations of the mechanism. This study reveals the coexpression of PAR-1 and PAR-2 (a second PAR, which is activated by trypsin/tryptase but not thrombin) by Northern blot analysis and compares their signaling properties in neonatal rat ventricular cardiomyocytes. SFLLRN and SLIGRL (an agonist peptide for PAR-2) promote inositol trisphosphate accumulation, stimulate mitogen-activated protein kinases (extracellular signal–regulated protein kinase and p38-mitogen-activated protein kinase), elevate calcium concentration, and increase spontaneous automaticity. SFLLRN (but not SLIGRL) also activates c-Jun NH
2
-terminal kinase and AKT. In keeping with their linkage to pathways that have been associated with growth and/or survival, SFLLRN and SLIGRL both induce hypertrophy. However, PAR agonists promote cell elongation, a morphology that is distinct from the uniform increase in cell dimension induced by α
1
-adrenergic receptor activation. These studies provide novel evidence that cardiomyocytes coexpress 2 functional PARs, which link to a common set of signals that culminate in changes in contractile function and hypertrophic growth. PAR actions may assume clinical importance in the border zone surrounding an infarction, where local proteolysis of PARs by serine proteases generated during inflammatory or thrombogenic pathways would elevate calcium concentration (setting the stage for arrhythmias), promote hypertrophic growth, and/or influence cardiomyocyte survival.
Determinants of Atherosclerosis Susceptibility in the C3H and C57BL/6 Mouse Model
Authors: Shi Weibin , Wang Nicholas J. , Shih Diana M. , Sun Victor Z. , Wang Xuping , Lusis Aldons J.
Published: June 2012

Abstract
—Lipids, monocytes, and arterial wall cells are primary components involved in atherogenesis. Using the inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H), which have been extensively studied as models of the genetic control of diet-induced atherosclerosis, we examined which of these components determine genetic susceptibility. To test whether dietary responsiveness is involved, a congenic strain of C3H carrying an apoE-null allele (apoE
−/−
) was constructed. Although C3H.apoE
−/−
mice had higher plasma cholesterol levels, they developed much smaller lesions than their B6.apoE
−/−
counterpart on either chow or Western diets. Reciprocal bone marrow transplantation between the strains, with congenics carrying the same H-2 haplotype, was performed to examine the role of monocytes. The atherosclerosis susceptibility was not altered in the recipient mice, indicating that variations in monocyte function were not involved. Endothelial cells isolated from the aorta of B6 mice exhibited a dramatic induction of monocyte chemotactic protein-1, macrophage colony–stimulating factor, vascular cell adhesion molecule-1, and heme oxygenase-1 in response to minimally modified LDL, whereas endothelial cells from C3H mice showed little or no induction. In a set of recombinant inbred strains derived from the B6 and C3H parental strains, endothelial responses to minimally modified LDL cosegregated with aortic lesion size. These data provide strong evidence that endothelial cells, but not monocytes or plasma lipid levels, account for the difference in susceptibility to atherosclerosis between the 2 mouse strains.
Requisite Role for Interleukin-4 in the Acceleration of Fatty Streaks Induced by Heat Shock Protein 65 or
Mycobacterium tuberculosis
Authors: George Jacob , Shoenfeld Yehuda , Gilburd Boris , Afek Arnon , Shaish Aviv , Harats Dror
Published: June 2012

Abstract
—Atherosclerotic lesions can be induced in rabbits and mice immunized with heat shock protein 65 (HSP65). In the current study, we investigated the role of interleukin (IL)-4 in the HSP65- and
Mycobacterium tuberculosis
(MT)–induced models that exhibit an inflammatory phenotype. Fatty streak formation in IL-4–knockout (IL-4 KO) mice immunized with HSP65 or MT was significantly reduced when compared with lesions in wild-type C57BL/6 mice. However, when injected with control (HSP-free) adjuvant, no differences were evident in the lesion size between wild-type and the IL-4 KO mice. Next, we studied comparatively the extent of humoral and cellular immune responses to HSP65 in the IL-4 KO and wild-type mice, as those are thought to be influential in murine atherosclerosis. Anti-HSP65 antibody levels were reduced in the HSP65-immunized IL-4 KO mice as compared with their wild-type littermates, whereas no differences were evident between the groups with respect to the primary cellular immune response to HSP65. Other than the absence of IL-4 in the knockout mice, the pattern of secreting cytokines interferon-γ and IL-10 in concanavalin A–primed splenocytes was similar between the groups. HSP65-primed inguinal lymphocytes from IL-4 KO mice immunized with HSP65 secreted higher levels of interferon-γ (previously shown to be proatherogenic in vivo) as compared with their wild-type controls. 12-/15-Lipoxygenase expression, known to be regulated by IL-4 and to contribute to murine atherosclerosis, in the lesions was not influenced by the immunization protocol used or by IL-4 disruption. Thus, IL-4 may prove a principal cytokine in the progression of early “inflammatory” atherosclerotic lesions and may serve as a target for immunomodulation.
Transgenic Overexpression of Constitutively Active Protein Kinase C ε Causes Concentric Cardiac Hypertrophy
Authors: Takeishi Yasuchika , Ping Peipei , Bolli Roberto , Kirkpatrick Darryl L. , Hoit Brian D. , Walsh Richard A.
Published: June 2012

Abstract
—To test the hypothesis that activation of the protein kinase C (PKC) ε isoform leads to cardiac hypertrophy without failure, we studied transgenic mice with cardiac-specific overexpression of a constitutively active mutant of the PKCε isoform driven by an α–myosin heavy chain promoter. In transgenic mice, the protein level of PKCε in heart tissue was increased 9-fold. There was a 6-fold increase of the membrane/cytosol ratio, and PKC activity in the membrane fraction was 4.2-fold compared with wild-type mice. The heart weight was increased by 28%, and upregulation of the mRNA for β-myosin heavy chain and α-skeletal actin was observed in transgenic mouse hearts. Echocardiography demonstrated increased anterior and posterior wall thickness with normal left ventricular function and dimensions, indicating concentric cardiac hypertrophy. Isolated cardiomyocyte mechanical function was slightly decreased, and Ca
2+
signals were markedly depressed in transgenic mice, suggesting that myofilament sensitivity to Ca
2+
was increased. No differences were observed in either the levels of cardiac Ca
2+
-handling proteins or the degree of cardiac regulatory protein phosphorylation between wild-type and transgenic mice. Unlike mice with PKCβ
2
overexpression, transgenic mice with cardiac-specific overexpression of the active PKCε mutant demonstrated concentric hypertrophy with normal in vivo cardiac function. Thus, PKC isoforms may play differential functional roles in cardiac hypertrophy and failure.
Influence of a Perfusing Bath on the Foot of the Cardiac Action Potential
Authors: Roth Bradley J.
Published: June 2012

Abstract
—Recently, Spach et al (
Circ Res
. 1998;83:1144–1164) measured the transmembrane action potential 150 to 200 μm below the tissue surface during longitudinal and transverse propagation. They found that “during longitudinal propagation there was initial slowing of V
m
[action potential] foot that resulted in deviations from a simple exponential… ” (p 1144). They attributed this behavior to the effects of capillaries on propagation. The purpose of this commentary is to show that the perfusing bath plays an important role in determining the time course of the action potential foot, even when the transmembrane potential is measured 150 μm below the tissue surface. Using numerical simulations based on the bidomain model, we find that the action potential foot for transverse propagation is nearly exponential (τ
foot
=314 μs). For longitudinal propagation, the action potential foot is not exponential because of an initial slowing (best-fit τ
foot
=483 μs). We conclude that the perfusing bath must be taken into account when interpreting data showing differences in the shape of the action potential foot with propagation direction, even if the transmembrane potential is measured 150 μm below the tissue surface. The full text of this article is available at http://www.circresaha.org.
Parallel Stimulation of Glucose and Mg
2+
Accumulation by Insulin in Rat Hearts and Cardiac Ventricular Myocytes
Authors: Romani Andrea M. P. , Matthews Veronica D. , Scarpa Antonio
Published: June 2012

Abstract
—The stimulation of β-adrenoceptors in cardiac cells results in a rapid loss of cellular Mg
2+
. Because insulin physiologically counteracts several of the cellular effects mediated by the activation of β-adrenoceptors and the elevation of cytosolic cAMP levels, we investigated whether insulin administration could prevent Mg
2+
mobilization from rat hearts and ventricular myocytes. Rat hearts were perfused in a retrograde Langendorff system, and the changes in extracellular Mg
2+
were measured by atomic absorbance spectrophotometry. Pretreatment of the hearts with 6 nmol/L insulin completely prevented the Mg
2+
extrusion induced by the β-adrenergic agonist isoproterenol. Furthermore, the administration of insulin per se induced an accumulation of Mg
2+
by the heart. This accumulation was small but detectable in the presence of 25 to 35 μmol/L [Mg
2+
]
o
and increased in proportion to [Mg
2+
]
o
. Insulin-mediated Mg
2+
accumulation was not observed in hearts perfused with a medium devoid of glucose or with a medium containing the inhibitors of glucose transport, cytochalasin B and phloretin. Insulin-stimulated [
3
H]2-deoxyglucose accumulation was measured in collagenase-dispersed cardiac ventricular myocytes in the presence of varying levels of [Mg
2+
]
o
. Glucose transport was not observed below 25 μmol/L [Mg
2+
]
o
, and it also increased in proportion to [Mg
2+
]
o
. Taken together, these results indicate the presence of a major uptake of Mg
2+
into cardiac cells that is stimulated by insulin and may require the insulin-induced operation of a glucose transporter. Hence, extracellular and/or intracellular Mg
2+
may modulate glucose transport and/or utilization.
Incidence of Myoendothelial Gap Junctions in the Proximal and Distal Mesenteric Arteries of the Rat Is Suggestive of a Role in Endothelium-Derived Hyperpolarizing Factor–Mediated Responses
Authors: Sandow Shaun L. , Hill Caryl E.
Published: June 2012

Abstract
—Although the chemical nature of endothelium-derived hyperpolarizing factor (EDHF) remains elusive, electrophysiological evidence exists for electrical communication between smooth muscle cells and endothelial cells suggesting that electrotonic propagation of hyperpolarization may explain the failure to identify a single chemical factor as EDHF. Anatomical evidence for myoendothelial gap junctions, or the sites of electrical coupling, is, however, rare. In the present study, serial-section electron microscopy and reconstruction techniques have been used to examine the incidence of myoendothelial gap junctions in the proximal and distal mesenteric arteries of the rat where EDHF responses have been reported to vary. Myoendothelial gap junctions were found to be very small in the mesenteric arteries, the majority being <100 nm in diameter. In addition, they were significantly more common in the distal compared with the proximal regions of this arterial bed. Pentalaminar gap junctions between adjacent endothelial cells were much larger and were common in both proximal and distal mesenteric arteries. These latter junctions were frequently found near the myoendothelial gap junctions. These results provide the first evidence for the presence of sites for electrical communication between endothelial cells and smooth muscle cells in the mesenteric vascular bed. Furthermore, the relative incidence of these sites suggests that there may be a relationship between the activity of EDHF and the presence of myoendothelial gap junctions.
Interactions Between Ca
2+
and H
+
and Functional Consequences in Vascular Smooth Muscle
Authors: Austin Clare , Wray Susan
Published: June 2012

Abstract
—Ca
2+
and H
+
ions can profoundly alter vascular tone. In many physiological and pathological processes, changes in the concentration of both ions occur. Thus, to understand the processes and mechanisms that modify force, it is necessary to understand what changes occur in these ions and, importantly, how they interact with each other. In this minireview, we highlight the quantitatively important mechanisms involved in the contractile responses of vascular tissues to pH change and discuss the cellular and molecular reasons underlying these responses.
Myosin Heavy Chain Isoform Expression in the Failing and Nonfailing Human Heart
Authors: Miyata Setsuya , Minobe Wayne , Bristow Michael R. , Leinwand Leslie A.
Published: June 2012

Abstract
—In the heart, the relative proportions of the 2 forms of the motor protein myosin heavy chain (MyHC) have been shown to be affected by a wide variety of pathological and physiological stimuli. Hearts that express the faster MyHC motor protein, α, produce more power than those expressing the slower MyHC motor protein, β, leading to the hypothesis that MyHC isoforms play a major role in the determination of cardiac contractility. We showed previously that a significant amount of αMyHC mRNA is expressed in nonfailing human ventricular myocardium and that αMyHC mRNA expression is decreased 15-fold in end-stage failing left ventricles. In the present study, we determined the MyHC protein isoform content of human heart samples of known MyHC mRNA composition. We demonstrate that αMyHC protein was easily detectable in 12 nonfailing hearts. αMyHC protein represented 7.2±3.2% of total MyHC protein (compared with ≈35% of the MyHC mRNA), suggesting that translational regulation may be operative; in contrast, there was effectively no detectable αMyHC protein in the left ventricles of 10 end-stage failing human hearts.
Distribution of Excitation Frequencies on the Epicardial and Endocardial Surfaces of Fibrillating Ventricular Wall of the Sheep Heart
Authors: Zaitsev Alexey V. , Berenfeld Omer , Mironov Sergey F. , Jalife José , Pertsov Arkady M.
Published: June 2012

Abstract
—Tissue heterogeneities may play an important role in the mechanism of ventricular tachycardia (VT) and fibrillation (VF) and can lead to a complex spatial distribution of excitation frequencies. Here we used optical mapping and Fourier analysis to determine the distribution of excitation frequencies in >20 000 sites of fibrillating ventricular tissue. Our objective was to use such a distribution as a tool to quantify the degree of organization during VF. Fourteen episodes of VT/VF were induced via rapid pacing in 9 isolated, coronary perfused, and superfused sheep ventricular slabs (3×3 cm
2
). A dual-camera video-imaging system was used for simultaneous optical recordings from the entire epi- and endocardial surfaces. The local frequencies of excitation were determined at each pixel and displayed as dominant frequency (DF) maps. A typical DF map consisted of several (8.2±3.6) discrete areas (domains) with a uniform DF within each domain. The DFs in adjacent domains were often in 1:2, 3:4, or 4:5 ratios, which was shown to be a result of an intermittent Wenckebach-like conduction block at the domain boundaries. The domain patterns were relatively stable and could persist from several seconds to several minutes. The complexity in the organization of the domains, the number of domains, and the dispersion of frequencies increased with the rate of the arrhythmia. Domain patterns on the epicardial and endocardial surfaces were not correlated. Sustained epicardial or endocardial reentry was observed in only 3 episodes. Observed frequency patterns during VT/VF suggest that the underlying mechanism may be a sustained intramural reentrant source interacting with tissue heterogeneities.
Reduced Neointima Hyperplasia of Vein Bypass Grafts in Intercellular Adhesion Molecule-1–Deficient Mice
Authors: Zou Yiping , Hu Yanhua , Mayr Manuel , Dietrich Hermann , Wick Georg , Xu Qingbo
Published: June 2012

Abstract
—Recently, we established a new mouse model of vein graft arteriosclerosis through the grafting of vena cava to carotid arteries. In many respects, the morphological features of this murine vascular graft model resemble those of human venous bypass graft disease. With this model, we studied the role of intercellular adhesion molecule-1 (ICAM-1) in the development of vein graft arteriosclerosis in ICAM-1–deficient mice. Neointimal hyperplasia of vein grafts in ICAM-1 −/− mice was reduced 30% to 50% compared with that of wild-type control animals. Immmunofluorescent analysis revealed that increased ICAM-1 expression was observed on the endothelium and smooth muscle cells (SMCs) of the grafted veins in wild-type, but not ICAM-1 −/−, mice. MAC-1 (CD11b/18)–positive cells that adhered to the surface of vein grafts in ICAM-1 −/− mice were significantly less as identified with en face immunofluorescence, and these positive cells were more abundant in the intimal lesions of vein grafts in wild-type mice. Furthermore, aortic SMCs cultivated from wild-type mice exhibited high ICAM-1 expression in response to tumor necrosis factor-α. When tumor necrosis factor-α–stimulated SMCs were incubated with mouse spleen leukocytes, the number of cells that adhered to ICAM-1 −/− SMCs was significantly lower than the number that adhered to ICAM-1 +/+ SMCs, which was markedly blocked through pretreatment of leukocytes with the anti–MAC-1 antibody. Taken together, our findings demonstrate that ICAM-1 is critical in the development of venous bypass graft arteriosclerosis, which provides essential information for therapeutic intervention for vein graft disease in patients undergoing bypass surgery.
Ca
2+
Transients and Ca
2+
Waves in Purkinje Cells
Authors: Boyden Penelope A. , Pu Jielin , Pinto Judith , Keurs Hendrik E. D. J. ter
Published: June 2012

Abstract
—Purkinje cells contain sarcoplasmic reticulum (SR) directly under the surface membrane, are devoid of t-tubuli, and are packed with myofibrils surrounded by central SR. Several studies have reported that electrical excitation induces a biphasic Ca
2+
transient in Purkinje fiber bundles. We determined the nature of the biphasic Ca
2+
transient in aggregates of Purkinje cells. Aggregates (n=12) were dispersed from the subendocardial Purkinje fiber network of normal canine left ventricle, loaded with Fluo-3/AM
,
and studied in normal Tyrode’s solution (24°C). Membrane action potentials were recorded with fine-tipped microelectrodes, and spatial and temporal changes in [Ca
2+
]
i
were obtained from fluorescent images with an epifluorescent microscope (×20; Nikon). Electrical stimulation elicited an action potential as well as a sudden increase in fluorescence (L
0
) compared with resting levels. This was followed by a further increase in fluorescence (L
1
) along the edges of the cells. Fluorescence then progressed toward the Purkinje cell core (velocity of propagation 180 to 313 μm/s). In 62% of the aggregates, initial fluorescent changes of L
0
were followed by focally arising Ca
2+
waves (L
2
), which propagated at 158±14 μm/s (n=13). Spontaneous Ca
2+
waves (L
2
*) propagated like L
2
(164±10 μm/s) occurred between stimuli and caused slow membrane depolarization; 28% of L
2
* elicited action potentials. Both spontaneous Ca
2+
wave propagation and resulting membrane depolarization were thapsigargin sensitive. Early afterdepolarizations were not accompanied by Ca
2+
waves. Action potentials in Purkinje aggregates induced a rapid rise of Ca
2+
through
I
CaL
and release from a subsarcolemmal compartment (L
0
). Ca
2+
release during L
0
either induced further Ca
2+
release, which propagated toward the cell core (L
1
), or initiated Ca
2+
release from small regions and caused L
2
Ca
2+
waves, which propagated throughout the aggregate. Spontaneous Ca
2+
waves (L
2
*) induce action potentials.
α
v
β
3
Integrin Induces Tyrosine Phosphorylation–Dependent Ca
2+
Influx in Pulmonary Endothelial Cells
Authors: Bhattacharya Sunita , Ying Xiaoyou , Fu Chenzhong , Patel Rashmi , Kuebler Wolfgang , Greenberg Steven , Bhattacharya Jahar
Published: June 2012

Abstract
—The endothelial α
v
β
3
integrin occurs luminally, where its ligation by soluble agents may induce inflammatory signaling. We tested this hypothesis in bovine pulmonary artery endothelial cell monolayers with the use of vitronectin and cross-linking antibodies to ligate and aggregate the integrin. We quantified the endothelial cytosolic Ca
2+
concentration ([Ca
2+
]
i
) according to the Fura 2 ratio imaging method in single cells of confluent monolayers. At baseline, endothelial [Ca
2+
]
i
levels remained steady at 86 nmol/L for >20 minutes. Cross-linking of the α
v
β
3
integrin through the sequential exposure of monolayers to anti-α
v
β
3
monoclonal antibody LM609 and secondary IgG resulted in a [Ca
2+
]
i
increase of 100% above baseline. This increase commenced in <0.5 minute, peaked in <2 minutes, and decayed to baseline in ≈5 minutes. Similar responses occurred after the addition of vitronectin (400 μg/mL). In contrast, external Ca
2+
depletion blunted the cross-linking–induced [Ca
2+
]
i
increase by 60%, a response that was completely inhibited when the monolayers were also pretreated with thapsigargin. Thus, the [Ca
2+
]
i
increase was attributable in part to the release of Ca
2+
from endosomal stores but mostly to Ca
2+
influx across the plasma membrane. Induced aggregation of the α
v
β
3
integrin enhanced tyrosine phosphorylation of phospholipase C-γ1 and increased the accumulation of inositol-1,4,5-trisphosphate. Genistein, a broad-spectrum tyrosine kinase inhibitor, abrogated both of these effects, as well as the α
v
β
3
-induced [Ca
2+
]
i
increases. We conclude that aggregation of the endothelial α
v
β
3
integrin induces a rapid tyrosine phosphorylation–dependent increase in [Ca
2+
]
i
. This response may subserve the inflammatory role of α
v
β
3
integrin in blood vessels.
Identification of
cis
Elements in the Cardiac Troponin T Gene Conferring Specific Expression in Cardiac Muscle of Transgenic Mice
Authors: Wang Q. , Sigmund C. D. , Lin J. J.-C.
Published: June 2012

Abstract
—To investigate the underlying mechanism regulating cardiac gene expression, transgenic mice carrying the rat cardiac troponin T proximal promoter (−497 bp from the transcriptional start site) fused to a
LacZ
or chloramphenicol acetyltransferase (
CAT
) reporter gene were analyzed. The
LacZ
expression pattern throughout development was very similar to that of the endogenous cardiac troponin T gene. Within this promoter, a high degree of sequence homology was found at 2 sites, modules D (−335 to −289 bp) and F (−249 to −209 bp). Both regions contain at least a TCTG(G/C) direct repeat and an A/T-rich site, whereas only the F module has a muscle enhancer factor 2 (MEF2)-like motif. No significant decrease in
CAT
transgene expression was observed when only the MEF2 core sequence was mutated. However, when the MEF2 core sequence and its flanking TCTGG site were mutated (Mut5),
CAT
transgene expression was significantly decreased in the heart, and ectopic expression of the transgene was also observed. When mutations were introduced into this promoter to destroy all upstream TCTG(G/C) direct repeats in the D module (MutD),
CAT
expression remained cardiac specific, but the expression level was dramatically decreased. Relaxation of cardiac-specific transgene expression became even more severe in transgenic mice carrying double mutations (Mut[D+5]). In addition,
CAT
activity in the heart was nearly abolished. These results suggest that D and F modules have an additive function in determining the level of expression in the heart and only the F module confers cardiac-specific expression.