bluuebunny/crossref_metadata_2025 huggingface dataset:
Cardiac Growth Factors in Human Hypertrophy
Authors: Serneri Gian Gastone Neri , Modesti Pietro Amedeo , Boddi Maria , Cecioni Ilaria , Paniccia Rita , Coppo Mirella , Galanti Giorgio , Simonetti Ignazio , Vanni Simone , Papa Letizia , Bandinelli Brunella , Migliorini Angela , Modesti Alessandra , Maccherini Massimo , Sani Guido , Toscano Michele
Published: June 2012

Abstract
—The aim of the present study was to investigate whether and which cardiac growth factors are involved in human hypertrophy, whether growth factor synthesis is influenced by overload type and/or by the adequacy of the hypertrophy, and the relationships between cardiac growth factor formation and ventricular function. Cardiac growth factor formation was assessed by measuring aorta-coronary sinus concentration gradient in patients with isolated aortic stenosis (n=26) or regurgitation (n=15) and controls (n=12). Gene expression and cellular localization was investigated in ventricular biopsies using reverse transcriptase–polymerase chain reaction and in situ hybridization. Cardiac hypertrophy with end-systolic wall stress <90 kdyne/cm
2
was associated with a selective increased formation of insulin-like growth factor (IGF)-I in aortic regurgitation and of IGF-I and endothelin (ET)-1 in aortic stenosis. mRNA levels for IGF-I and preproET-1 were elevated and mainly expressed in cardiomyocytes. At stepwise analysis, IGF-I formation was correlated to the mean velocity of circumferential fiber shortening (
r
=0.86,
P
<0.001) and ET-1 formation to relative wall thickness (
r
=0.82,
P
<0.001). When end-systolic wall stress was >90 kdyne/cm
2
, IGF-I and ET-1 synthesis by cardiomyocytes was no longer detectable, and only angiotensin (Ang) II was generated, regardless of the type of overload. The mRNA level for angiotensinogen was high, and the mRNA was exclusively expressed in the interstitial cells. Ang II formation was positively correlated to end-systolic stress (
r
=0.89,
P
<0.001) and end-diastolic stress (
r
=0.84,
P
<0.001). Multivariate stepwise analysis selected end-systolic stress as the most predictive variable and left ventricular end-diastolic pressure as the independent variable for Ang II formation (
r
=0.93,
P
<0.001). In conclusion, the present results indicate that the course of human left ventricular hypertrophy is characterized by the participation of different cardiac growth factors that are selectively related both to the type of hemodynamic overload and to ventricular function.
Local Anesthetic Anchoring to Cardiac Sodium Channels
Authors: Li Ronald A. , Tsushima Robert G. , Himmeldirk Klaus , Dime David S. , Backx Peter H.
Published: June 2012

Abstract
—Local anesthetics inhibit Na
+
channels in a variety of tissues, leading to potentially serious side effects when used clinically. We have created a series of novel local anesthetics by connecting benzocaine (BZ) to the sulfhydryl-reactive group methanethiosulfonate (MTS) via variable-length polyethylether linkers (L) (MTS-LX-BZ [X represents 0, 3, 6, or 9]). The application of MTS-LX-BZ agents modified native rat cardiac as well as heterologously expressed human heart (hH1) and rat skeletal muscle (rSkM1) Na
+
channels in a manner resembling that of free BZ. Like BZ, the effects of MTS-LX-BZ on rSkM1 channels were completely reversible. In contrast, MTS-LX-BZ modification of heart and mutant rSkM1 channels, containing a pore cysteine at the equivalent location as cardiac Na
+
channels (ie, Y401C), persisted after drug washout unless treated with DTT, which suggests anchoring to the pore via a disulfide bond. Anchored MTS-LX-BZ competitively reduced the affinity of cardiac Na
+
channels for lidocaine but had minimal effects on mutant channels with disrupted local anesthetic modification properties. These results establish that anchored MTS-LX-BZ compounds interact with the local anesthetic binding site (LABS). Variation in the linker length altered the potency of channel modification by the anchored drugs, thus providing information on the spatial relationship between the anchoring site and the LABS. Our observations demonstrate that local anesthetics can be anchored to the extracellular pore cysteine in cardiac Na
+
channels and dynamically interact with the intracellular LABS. These results suggest that nonselective agents, such as local anesthetics, might be made more selective by linking these agents to target-specific anchors.
Virtual Electrode–Induced Reexcitation
Authors: Cheng Yuanna , Mowrey Kent A. , Van Wagoner David R. , Tchou Patrick J. , Efimov Igor R.
Published: June 2012

Abstract
—Mechanisms of defibrillation remain poorly understood. Defibrillation success depends on the elimination of fibrillation without shock-induced arrhythmogenesis. We optically mapped selected epicardial regions of rabbit hearts (n=20) during shocks applied with the use of implantable defibrillator electrodes during the refractory period. Monophasic shocks resulted in virtual electrode polarization (VEP). Positive values of VEP resulted in a prolongation of the action potential duration, whereas negative polarization shortened the action potential duration, resulting in partial or complete recovery of the excitability. After a shock, new propagated wavefronts emerged at the boundary between the 2 regions and reexcited negatively polarized regions. Conduction velocity and maximum action potential upstroke rate of rise dV/dt
max
of shock-induced activation depended on the transmembrane potential at the end of the shock. Linear regression analysis showed that dV/dt
max
of postshock activation reached 50% of that of normal action potential at a
V
m
value of −56.7±0.6 mV postshock voltage (n=9257). Less negative potentials resulted in slow conduction and blocks, whereas more negative potentials resulted in faster conduction. Although wavebreaks were produced in either condition, they degenerated into arrhythmias only when conduction was slow. Shock-induced VEP is essential in extinguishing fibrillation but can reinduce arrhythmias by producing excitable gaps. Reexcitation of these gaps through progressive increase in shock strength may provide the basis for the lower and upper limits of vulnerability. The former may correspond to the origination of slow wavefronts of reexcitation and phase singularities. The latter corresponds to fast conduction during which wavebreaks no longer produce sustained arrhythmias.
Bifunctional Role of Protein Tyrosine Kinases in Late Preconditioning Against Myocardial Stunning in Conscious Rabbits
Authors: Dawn Buddhadeb , Xuan Yu-Ting , Qiu Yumin , Takano Hitoshi , Tang Xian-Liang , Ping Peipei , Banerjee Supratim , Hill Michael , Bolli Roberto
Published: June 2012

Abstract
—Although protein tyrosine kinases (PTKs) have been implicated in late preconditioning (PC) against infarction, their role in late PC against stunning is unknown. Furthermore, it is unknown whether PTK signaling is necessary only to trigger late PC on day 1 or also to mediate it on day 2. Thus, conscious rabbits underwent a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3). In the control group (group I, n=7), the recovery of systolic wall thickening after the 6 occlusion/reperfusion cycles was markedly improved on days 2 and 3 compared with day 1, indicating the development of late PC against stunning. Administration of the PTK inhibitor lavendustin-A (LD-A, 1 mg/kg IV) before the first occlusion on day 1 (group II, n=7) completely prevented the late PC effect against stunning on day 2. Late PC against stunning was also abrogated when LD-A was given before the first occlusion on day 2 (group III, n=7); however, in these rabbits, the late PC effect became apparent on day 3, indicating that LD-A itself did not have any delayed deleterious actions on myocardial stunning. In group V (n=5), the sequence of 6 occlusion/reperfusion cycles resulted in a robust increase in the activity of inducible NO synthase (iNOS [assessed as Ca
2+
-independent
l
-citrulline formation]) and nitrite+nitrate (NO
x
) tissue levels 24 hours later (on day 2), with no concomitant change in Ca
2+
-dependent NO synthase (endothelial NO synthase and/or neuronal NO synthase) activity. Similar results were obtained on day 3 (group VIII, n=6), indicating sustained upregulation of iNOS. Administration of LD-A either on day 1 (group VI, n=5) or on day 2 (group VII, n=6) abrogated the increase in iNOS activity and NO
x
levels on day 2. LD-A had no effect on iNOS activity or NO
x
levels in the absence of PC (group X, n=5). This study demonstrates that in conscious rabbits, PTK activity is necessary not only to trigger late PC against stunning on day 1 but also to mediate the protection on day 2. This investigation also provides the first direct evidence that cardiac iNOS activity is upregulated during the late phase of ischemic PC in rabbits. Furthermore, the data indicate that PTK signaling is essential for the augmentation of iNOS activity and that PTKs modulate this enzyme at two distinct levels: at an early stage on day 1 and at a late stage on day 2. This bifunctional role of PTKs in late PC has broad implications for the signaling mechanisms that underlie the response of the heart to ischemic stress and, possibly, other stresses.
ARC Inhibits Cytochrome
c
Release From Mitochondria and Protects Against Hypoxia-Induced Apoptosis in Heart-Derived H9c2 Cells
Authors: Ekhterae Daryoush , Lin Zhiwu , Lundberg Martha S. , Crow Michael T. , Brosius Frank C. , Núñez Gabriel
Published: June 2012

Abstract
—Ischemia induces apoptosis as well as necrosis of cardiac myocytes. We recently reported the cloning of a cDNA that encodes an apoptotic inhibitor, ARC, that is expressed predominantly in cardiac and skeletal muscle. In the present study, we examined the ability of ARC to protect rat embryonic heart–derived H9c2 cells from apoptosis induced by hypoxia, a component of ischemia. We found that H9c2 cells express ARC and that exposure to hypoxia substantially reduces ARC expression while inducing apoptosis. Transfected H9c2 cells in which cytosolic ARC protein levels remain elevated during hypoxia were significantly more resistant to hypoxia-induced apoptosis than parental H9c2 cells or H9c2 cells transfected with a control vector. Loss of endogenous ARC in the cytosol of H9c2 cells was associated with translocation of ARC from the cytosol to intracellular membranes, release of cytochrome
c
from the mitochondria, activation of caspase-3, poly(ADP-ribose)polymerase (PARP) cleavage, and DNA fragmentation. All of these events were inhibited in H9c2 cells overexpressing ARC when compared with control cells. In contrast, caspase inhibitors prevented PARP cleavage but not cytochrome
c
release, suggesting that exogenously expressed ARC acts upstream of caspase activation in this model of apoptosis. These results demonstrate that ARC can protect heart myogenic H9c2 cells from hypoxia-induced apoptosis, and that ARC prevents cytochrome
c
release by acting upstream of caspase activation, perhaps at the mitochondrial level. The full text of this article is available at http://www.circresaha.org.
Spatiotemporal Development and Distribution of Intercellular Junctions in Adult Rat Cardiomyocytes in Culture
Authors: Kostin Sawa , Hein Stefan , Bauer Erwin P. , Schaper Jutta
Published: June 2012

Abstract
—The mode of development of the intercalated disk (ID) is largely unknown, and the hypothesis was tested that the assembly of cell adhesion junctions may precede the formation of gap junctions (GJ) in developing ID in adult rat cardiomyocyte (ARC) in long-term culture. Immunostaining for connexin 43 (Cx43) and for cell adhesion junction proteins (N-cadherin, catenins, and desmoplakin) in single- and double-label techniques was analyzed and quantified by confocal and electron microscopy. All proteins investigated disappeared 48 hours after ARC isolation and reappeared parallel to redifferentiation of ARC. The newly formed ID, observed after 5 days, showed the presence of N-cadherin, catenins, and desmoplakin, low levels of Cx43, and absence of ultrastructurally discernible gap junctions. A progressive incorporation of Cx43 within ID was observed after 6 days, when cell adhesion junction proteins were already organized as zipper-like structures. Quantitative confocal analysis revealed a progressive augmentation of the fluorescence intensity of Cx43, associated with an increase in both the number and size of GJ, resulting in a substantial increase in the percentage of total GJ length per reassembled ID from 1.67% (day 6) to 15.58% (day 12). In the present study, we show that (1) the formation of the ID can be followed in ARC in culture and (2) the assembly of the adhering type of junction is the prerequisite for subsequent GJ formation within the ID. These findings may have clinical relevance in elaborating strategies for using myocardial grafts and for the potential restoration of GJ communication in cardiac diseases.
Bradycardia-Induced Coronary Angiogenesis Is Dependent on Vascular Endothelial Growth Factor
Authors: Zheng Wei , Brown Margaret D. , Brock Tommy A. , Bjercke Robert J. , Tomanek Robert J.
Published: June 2012

Abstract
—A marked coronary angiogenesis is known to occur with chronic bradycardia. We tested the hypothesis that vascular endothelial growth factor (VEGF), an endothelial cell mitogen and a major regulator of angiogenesis, is upregulated in response to low heart rate and consequential increased stroke volume. Bradycardia was induced in rats by administering the bradycardic drug alinidine (3 mg/kg body weight) twice daily. Heart rate decreased by 32% for 20 to 40 minutes after injection and was chronically reduced by 10%, 14%, and 18.5% after 1, 2, and 3 weeks of treatment, respectively. Arterial pressure and cardiac output were unchanged. Left ventricular capillary length density (mm/mm
3
) increased gradually with alinidine administration; a 15% increase after 2 weeks and a 40% increase after 3 weeks of alinidine treatment were documented. Left ventricular weight, body weight, and their ratio were not significantly altered by alinidine treatment. After 1 week of treatment, before an increase in capillary length density, VEGF mRNA increased >2-fold and then declined to control levels after 3 weeks of treatment. VEGF protein was higher in alinidine-treated rats than in controls after 2 weeks and increased further after 3 weeks of treatment. Injection of VEGF-neutralizing antibodies over a 2-week period completely blocked alinidine-stimulated angiogenesis. In contrast, bFGF mRNA was not altered by alinidine treatment. These data suggest that VEGF plays a key role in the angiogenic response that occurs with chronic bradycardia. The mechanism underlying this VEGF-associated angiogenesis may be an increase in stretch due to enhanced diastolic filling.
Responses of Cardiac Protein Kinase C Isoforms to Distinct Pathological Stimuli Are Differentially Regulated
Authors: Takeishi Yasuchika , Jalili Thunder , Ball Nancy A. , Walsh Richard A.
Published: June 2012

Abstract
—Currently at least 11 protein kinase C (PKC) isoforms have been identified and may play different roles in cell signaling pathways leading to changes in cardiac contractility, the hypertrophic response, and tolerance to myocardial ischemia. The purpose of the present study was to test the hypothesis that responses of individual PKC isoforms to distinct pathological stimuli were differentially regulated in the adult guinea pig heart. Isolated hearts were perfused by the Langendorff method and were exposed to ischemia, hypoxia, H
2
O
2
, or angiotensin II. Hypoxia and ischemia induced translocation of PKC isoforms α, β
2
, γ, and ζ, and H
2
O
2
translocated PKC isoforms α, β
2
, and ζ. Angiotensin II produced translocation of α, β
2
, ε, γ, and ζ isoforms. Inhibition of phospholipase C with tricyclodecan-9-yl-xanthogenate (D609) blocked hypoxia-induced (α, β
2
, and ζ) and angiotensin II–induced (α, β
2
, γ, and ζ) translocation of PKC isoforms. Inhibition of tyrosine kinase with genistein blocked translocation of PKC isoforms by hypoxia (β
2
and ζ) and by angiotensin II (β
2
). By contrast, neither D609 nor genistein blocked H
2
O
2
-induced translocation of any PKC isoform. We conclude that hypoxia-induced activation of PKC isoforms is mediated through pathways involving phospholipase C and tyrosine kinase, but oxidative stress may activate PKC isoforms independently of Gαq-phospholipase C coupling and tyrosine kinase signaling. Because oxidative stress may directly activate PKC, and PKC activation appears to be involved in human heart failure, selective inhibition of the PKC isoforms may provide a novel therapeutic strategy for the prevention and treatment of this pathological process.
Estradiol Inhibits Leukocyte Adhesion and Transendothelial Migration in Rabbits In Vivo
Authors: Nathan Lauren , Pervin Shehla , Singh Rajan , Rosenfeld Michael , Chaudhuri Gautam
Published: June 2012

Abstract
—The mechanism by which estrogens protect against atherosclerosis is not known. We evaluated in vivo whether there is a gender difference in monocyte adhesion and subendothelial migration in hypercholesterolemic rabbits and whether any gender differences observed are due to estradiol. Monocyte adhesion and subendothelial migration were assessed in a blinded fashion by analyzing a standardized segment of aorta using a scanning electron microscope. We also assessed whether estradiol modulates induction of vascular cell adhesion molecule-1 (VCAM-1) protein using Western blot and flow cytometric analyses. We observed that male rabbits develop more monocyte adhesion and subendothelial migration than do female rabbits during hypercholesterolemia. We also observed that oophorectomized rabbits given physiological estradiol supplementation demonstrate fewer adherent and subendothelial monocytes than do oophorectomized rabbits given placebo. VCAM-1 protein expression was increased in aortae obtained from hypercholesterolemic, oophorectomized animals supplemented with placebo, and this increase was attenuated by estradiol. Finally, in cultured rabbit aortic endothelial cells stimulated with lysophosphatidylcholine, we observed an increase in VCAM-1 protein that was inhibited in a concentration-dependent fashion by estradiol. We have demonstrated in vivo that there is a gender difference in monocyte adhesion to endothelial cells and transendothelial migration after hypercholesterolemia and that this gender difference is due in part to estradiol. Our results also suggest that estradiol inhibits monocyte adhesion by inhibiting expression of VCAM-1.
Heterogeneous Basal Expression of Nitric Oxide Synthase and Superoxide Dismutase Isoforms in Mammalian Heart
Authors: Brahmajothi Mulugu V. , Campbell Donald L.
Published: June 2012

Abstract
—The basal expression patterns of NO synthase (NOS; endothelial [eNOS], neuronal [nNOS], and cytokine-inducible [iNOS]) and superoxide dismutase (SOD; extracellular membrane bound [ECSOD], MnSOD, and CuZnSOD) isoforms in ferret heart (tissue sections and isolated myocytes) were determined by immunofluorescent localization. We demonstrate the following for the first time in the mammalian heart: (1) heterogeneous expression patterns of the 3 NOS and 3 SOD isoforms among different tissue and myocyte types; (2) colocalization of eNOS and ECSOD at both the tissue and myocyte levels; (3) a significant gradient of eNOS and ECSOD expression across the left ventricular (LV) wall, with both enzymes being highly expressed and colocalized in LV epicardial myocytes but markedly reduced in LV endocardial myocytes; and (4) specific subcellular localization patterns of eNOS and the 3 SOD isoforms. In particular, eNOS and ECSOD are demonstrated (electron and confocal microscopy) to be specifically localized to the sarcolemma of ventricular myocytes. Similar heterogeneous eNOS and ECSOD expression patterns were also obtained in human LV tissue sections, underscoring the general importance of these novel findings. Our data suggest a strong functional correlation between the activities of sarcolemmally localized myocyte eNOS and ECSOD in governing NO·/O
2
−
interactions and suggest that NO-related modulatory effects on cardiac myocyte protein and/or ion channel function may be significantly more complex than is presently believed.
Functional Role of c-Src in Gap Junctions of the Cardiomyopathic Heart
Authors: Toyofuku Toshihiko , Yabuki Masanori , Otsu Kinya , Kuzuya Tsunehiko , Tada Michihiko , Hori Masatsugu
Published: June 2012

Abstract
—Given the essential role played by gap junctions in the coordination of cardiac muscle contraction, it is plausible that down-regulation of gap junctional conduction is in part responsible for the contractile dysfunction observed in hypertrophied and failing hearts. In the present study, we analyzed the expression and function of the gap junction protein, connexin43, in the ventricular myocardium of hereditary cardiomyopathic, Syrian BIO 14.6 hamsters. Immunoprecipitation and immunoblot analyses revealed that levels of tyrosine phosphorylated connexin43 were increased in BIO 14.6 hamsters at the late stage of congestive heart failure. Furthermore, the increased tyrosine phosphorylation was correlated with increased c-Src activity. The functional consequences of tyrosine phosphorylation of connexin43 in gap junction were assessed using transfected cells expressing constitutively active c-Src. It was found that constitutively active c-Src diminished propagation of Ca
2+
waves in HEK293 cells and reduced gap junctional conductance between pairs of cardiac myocytes. We, therefore, conclude that during the progression of cardiac dysfunction in the cardiomyopathic heart, gap junctional communication is reduced via c-Src–mediated tyrosine phosphorylation of connexin43.
Simulated Ischemia in Flow-Adapted Endothelial Cells Leads to Generation of Reactive Oxygen Species and Cell Signaling
Authors: Wei Zhihua , Costa Karen , Al-Mehdi Abu B. , Dodia Chandra , Muzykantov Vladimir , Fisher Aron B.
Published: June 2012

Abstract
—We have previously shown that increased reactive oxygen species (ROS) generation occurs with ischemia in the oxygenated lung and have hypothesized that mechanotransduction is the initiating event. In the present study, we developed an in vitro model of oxygenated ischemia by interrupting medium flow to flow-adapted bovine pulmonary artery endothelial cells in an artificial capillary system. Cellular oxygenation during the “ischemic” period was maintained by perfusing medium over the abluminal surface of porous capillaries. Cells were assessed for ROS generation, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) binding activities, and DNA synthesis using dichlorofluorescein fluorescence by flow cytometry and spectrofluorometry, electrophoretic mobility shift assay of nuclear extracts with NF-κB–specific or AP-1–specific
32
P-labeled oligonucleotides, and
3
H-thymidine incorporation into DNA. Cells that were flow adapted for 2 to 7 days with 1 to 2 dyne/cm
2
shear stress exhibited a 1.6- to 1.9-fold increase in ROS generation during 1 hour of simulated ischemia compared with continuously perfused cells. This effect was abolished by diphenyleneiodonium chloride (DPI), indicating a role for a flavoprotein such as NADPH oxidase. The increase in ROS generation with ischemia was similar for cells from low and high passages. With ischemia, flow-adapted cells exhibited increases of 1.7-fold in nuclear NF-κB and 1.5-fold in nuclear AP-1; these changes were abolished by pretreatment with
N
-acetylcysteine or DPI. Ischemia for 24 hours resulted in a 1.8-fold increase of
3
H-thymidine incorporation into DNA and a significant increase of cells entering the cell cycle, as indicated by flow cytometry with propidium iodide. We conclude that flow-adapted endothelial cells generate ROS with ischemia that results in activation of NF-κB and AP-1 and an increase of DNA synthesis. This effect is not mediated by hypoxia, implicating a role for mechanotransduction in ischemia-mediated cell signaling.
Enhanced Cardiomyocyte DNA Synthesis During Myocardial Hypertrophy in Mice Expressing a Modified TSC2 Transgene
Authors: Pasumarthi Kishore B. S. , Nakajima Hidehiro , Nakajima Hisako O. , Jing Shaoliang , Field Loren J.
Published: June 2012

Abstract
—Tuberous sclerosis complex (TSC) is a rare genetic disorder characterized by the appearance of benign tumors in multiple organs, including the heart. Disease progression is accompanied by homozygous mutation at 1 of 2 loci (designated TSC1 or TSC2), leading to the suggestion that these genes function as tumor suppressors. In this study, we generated a series of TSC2 cDNAs in which one or more structural motifs were deleted, with the hope that expression of the modified gene product would override the growth-inhibitory activity of the endogenous TSC2 gene product. Several of the modified cDNAs enhanced growth rate, increased endocytosis, and promoted aberrant protein trafficking when expressed in NIH-3T3 cells, thereby mimicking phenotypes typical of TSC2-deficient cells. Surprisingly, targeted expression of the most potent TSC2 cDNA to the heart did not perturb cardiac development. However, the level of cardiomyocyte DNA synthesis in adult transgenic mice was elevated >35-fold during isoproterenol-induced hypertrophy compared with their nontransgenic siblings. These results suggest that alteration of TSC2 gene activity in combination with β-adrenergic stimulation can reactivate the cell cycle in a limited number of terminally differentiated adult cardiomyocytes.
Contribution of Caveolin Protein Abundance to Augmented Nitric Oxide Signaling in Conscious Dogs With Pacing-Induced Heart Failure
Authors: Hare Joshua M. , Lofthouse Robert A. , Juang George J. , Colman Laurence , Ricker Kelly M. , Kim Benjamin , Senzaki Hideaki , Cao Suyi , Tunin Richard S. , Kass David A.
Published: June 2012

Abstract
—Myocardial NO signaling appears elevated in heart failure (HF). Whether this results from increased NO production, induction of the high-output NO synthase (NOS)2 isoform, or changes in NOS regulatory pathways (such as caveolae) remains controversial. We tested the hypothesis that increased abundance of caveolin-3 and/or sarcolemmal caveolae contribute to increased NO signaling in pacing-induced HF. Abundance of caveolin-3 (0.59±0.08 versus 0.29±0.08 arbitrary units,
P
=0.01) but not caveolin-1 was increased in HF compared with control conditions, assessed by Western blot. Additionally, transmission electron microscopy revealed increased caveolae (2.7±0.4 versus 1.3±0.3 per micrometer myocyte membrane,
P
<0.005). The association between caveolin-3 and NOS3 at the sarcolemma and T tubules was unchanged in HF compared with control myocytes. The impact of NOS inhibition with
l
-
N
G
-methylarginine hydrochloride (L-NMMA) on β-adrenergic inotropy was assessed in conscious dogs before and after HF. In control dogs, dobutamine (5 μg · kg
−1
· min
−1
) increased +dP/dt by 36±7%, and this was augmented to 66±24% by 20 mg/kg L-NMMA (
P
=0.04 versus without L-NMMA, n=8) but not affected by 10 mg/kg L-NMMA (34±10%,
P
=NS; n=8). In HF, dobutamine +dP/dt response was depressed (
P
<0.001 versus control), and increased concentrations were required to match control inotropic responses (10 to 15 μg · kg
−1
· min
−1
, 48±7%). L-NMMA enhanced +dP/dt responses similarly at 10 mg/kg (61±17%,
P
=0.02; n=4) and 20 mg/kg (54±7%,
P
=0.04; n=7). Caveolin-3 abundance positively correlated with L-NMMA augmentation of dobutamine inotropic responses in HF (
r
=0.9,
P
=0.03; n=4). Thus, in canine pacing-induced HF, expression of caveolin-3 and of sarcolemmal caveolae is increased. This increase is associated with augmented agonist-stimulated NO signaling, likely via a compartmentation effect.
Series of Exon-Skipping Events in the Elastic Spring Region of Titin as the Structural Basis for Myofibrillar Elastic Diversity
Authors: Freiburg Alexandra , Trombitas Karoly , Hell Wolfgang , Cazorla Olivier , Fougerousse Francoise , Centner Thomas , Kolmerer Bernhard , Witt Christian , Beckmann Jaques S. , Gregorio Carol C. , Granzier Henk , Labeit Siegfried
Published: June 2012

Abstract
—Titins are megadalton-sized filamentous polypeptides of vertebrate striated muscle. The I-band region of titin underlies the myofibrillar passive tension response to stretch. Here, we show how titins with highly diverse I-band structures and elastic properties are expressed from a single gene. The differentially expressed tandem-Ig, PEVK, and N2B spring elements of titin are coded by 158 exons, which are contained within a 106-kb genomic segment and are all subject to tissue-specific skipping events. In ventricular heart muscle, exons 101 kb apart are joined, leading to the exclusion of 155 exons and the expression of a 2.97-MDa cardiac titin N2B isoform. The atria of mammalian hearts also express larger titins by the exclusion of 90 to 100 exons (cardiac N2BA titin with 3.3 MDa). In the soleus and psoas skeletal muscles, different exon-skipping pathways produce titin transcripts that code for 3.7- and 3.35-MDa titin isoforms, respectively. Mechanical and structural studies indicate that the exon-skipping pathways modulate the fractional extensions of the tandem Ig and PEVK segments, thereby influencing myofibrillar elasticity. Within the mammalian heart, expression of different levels of N2B and N2BA titins likely contributes to the elastic diversity of atrial and ventricular myofibrils.
Temporal Events Underlying Arterial Remodeling After Chronic Flow Reduction in Mice
Authors: Rudic Radu Daniel , Bucci Mariarosaria , Fulton David , Segal Steven S. , Sessa William C.
Published: June 2012

Abstract
—To define the cellular events of vascular remodeling in mice, we measured blood flow and analyzed the morphology of remodeled vessels at defined points after a flow-reducing remodeling stimulus for 3, 7, 14, and 35 days. Acute ligation of the left external carotid artery reduced blood flow in the left common carotid artery (LC) compared with sham and contralateral right common carotid arteries (RCs). In morphometric analyses, the decrease in diameter in LCs was reversible by vasodilator perfusion 3 days after ligation, whereas ligation for 7 days or greater resulted in a permanent diameter reduction. Coincident with structural remodeling at day 7 was an increase in cell death in remodeled LCs. Functionally, rings from remodeled LCs contracted to prostaglandin F
2α
and relaxed to acetylcholine in a manner identical to that of control arteries. However, remodeled LCs were hypersensitive to the nitrovasodilator sodium nitroprusside (at day 7) and exhibited a marked reduction in basal NO synthesis at 7 and 14 days after ligation. The impairment of endothelial NO synthase function was likely due to post-translational mechanisms, given that endothelial NO synthase mRNA and protein levels did not change in remodeled LCs. These data define the ontogeny of flow-triggered luminal remodeling in adult mice and suggest that endothelial dysfunction occurs during reorganization of the vessel wall.
The Stromal Cell–Derived Factor-1 Chemokine Is a Potent Platelet Agonist Highly Expressed in Atherosclerotic Plaques
Authors: Abi-Younes S. , Sauty A. , Mach F. , Sukhova G. K. , Libby P. , Luster A. D.
Published: June 2012

Abstract
—Chemokines are chemotactic cytokines that activate and direct the migration of leukocytes. However, their role in modulating platelet function has not been shown. We studied the direct effect of chemokines on human platelets and found that of the 16 tested only stromal cell–derived factor (SDF)-1 induced platelet aggregation, accompanied by a rise in intracellular calcium. Platelets expressed the SDF-1 receptor, CXCR4, and an antibody to CXCR4 and pertussis toxin inhibited SDF-1–induced platelet aggregation, confirming that this effect is mediated through CXCR4, a Gαi-coupled receptor. SDF-1–induced platelet aggregation was also inhibited by wortmannin, LY294002, and genistein, suggesting that phosphatidylinositol 3–kinase and tyrosine kinase are likely involved in SDF-1–induced platelet aggregation. Because chemokines are produced from multiple vascular cells and atherosclerotic vessels are prone to develop platelet-rich thrombi, we examined the expression of SDF-1 in human atheroma. SDF-1 protein was highly expressed in smooth muscle cells, endothelial cells, and macrophages in human atherosclerotic plaques but not in normal vessels. Our studies demonstrate a direct effect of a chemokine in inducing platelet activation and suggest a role for SDF-1 in the pathogenesis of atherosclerosis and thrombo-occlusive diseases.
Endothelin Receptor Antagonism Ameliorates Mast Cell Infiltration, Vascular Hypertrophy, and Epidermal Growth Factor Expression in Experimental Diabetes
Authors: Gilbert Richard E. , Rumble Jonathan R. , Cao Zemin , Cox Alison J. , van Eeden Pauline , Allen Terri J. , Kelly Darren J. , Cooper Mark E.
Published: June 2012

Abstract
—Vascular hypertrophy, a feature of experimental and human diabetes, has been implicated in the pathogenesis of the microvascular and macrovascular complications of the disease. In the present study, we sought to examine the role of endogenous endothelin and its relation to vascular growth factors in the mediation of vascular hypertrophy in experimental diabetes and to examine the contribution of mast cells to this process. Vessel morphology, endothelin, growth factor gene expression, and matrix deposition were studied in the mesenteric arteries of control and streptozotocin-induced diabetic Sprague-Dawley rats treated with or without the dual endothelin
A/B
receptor antagonist bosentan (100 mg · kg
−1
· d
−1
) during a 3-week period. Compared with control animals, diabetic animals had significant increases in vessel weight, wall-to-lumen ratio, mast cell infiltration, extracellular matrix deposition, and gene expression of epidermal growth factor (EGF) and transforming growth factor-β
1
. In diabetic, but not control, vessels, not only were EGF mRNA and endothelin present in endothelial cells, but also their expression was observed in adventitial mast cells. Immunoreactive endothelin was present in the media of mesenteric vessels of diabetic, but not control, animals. Bosentan treatment significantly reduced mesenteric weight, wall-to-lumen ratio, mast cell infiltration, matrix deposition, and EGF mRNA but did not prevent the overexpression of transforming growth factor-β
1
mRNA in diabetic rats. These findings suggest that endogenous endothelin and EGF may play a role in diabetes-induced vascular hypertrophy and that mast cells may be pathogenetically involved in this process.
Pharmacological and Immunohistochemical Characterization of Calmodulin-Stimulated (Ca
2+
+Mg
2+
)-ATPase in Cultured Porcine Aortic Endothelial Cells
Authors: McConnell Elizabeth J. , White Gary W. , Brokaw James J. , Raess Beat U.
Published: June 2012

Abstract
—Plasma membrane (Ca
2+
+Mg
2+
)-ATPase and Ca
2+
transport activities, best characterized in human erythrocytes, are stimulated by calmodulin and thought to play a crucial role in the termination of cellular Ca
2+
signaling in all cells. In plasma membranes isolated from cultured porcine aortic endothelial cells, the (Ca
2+
+Mg
2+
)-ATPase was not readily measured. This is in part because of an overabundance of nonspecific Ca
2+
- and/or Mg
2+
-activated ecto–5′-nucleotide phosphohydrolases. Moreover, addition of exogenous calmodulin (10
−9
to 10
−6
mol/L) produced no measurable stimulation of ATPase activities, suggesting a permanently activated state or, alternatively, a complete lack thereof. To establish and verify the presence of a calmodulin-regulated (Ca
2+
+Mg
2+
)-ATPase activity in these endothelial cells, immunohistochemical localization using a monoclonal mouse anti–(Ca
2+
+Mg
2+
)-ATPase antibody (clone 5F10) was applied to intact pig aorta endothelium, cultured endothelial monolayers, and isolated endothelial plasma membrane fractions. This approach clearly demonstrated Ca
2+
pump immunoreactivity in each of these preparations. To confirm functional calmodulin stimulation of the (Ca
2+
+Mg
2+
)-ATPase, 10
−5
mol/L calmidazolium (
R24571
) was added to the isolated plasma membrane preparation, which lowered the (Ca
2+
+Mg
2+
)-ATPase activity from 143.0 to 78.15 nmol P
i
/mg protein · min
–1
. This calmidazolium-reduced activity could then be stimulated 113.1±0.8% in a concentration-dependent manner by the addition of exogenous calmodulin (10
−7
to 2×10
−6
mol/L) with an EC
50
of 3.45±0.04×10
−7
mol/L (n=4). This represents a competitive lowering of the apparent calmodulin affinity by ≈100 compared with other unopposed calmodulin-stimulated processes. Together, these findings support evidence for the presence of a calmodulin-stimulated plasma membrane (Ca
2+
+Mg
2+
)-ATPase activity in cultured porcine aortic endothelial cells.
Effects of Cardiac Microstructure on Propagating Electrical Waveforms
Authors: Spach Madison S. , Barr Roger C.
Published: June 2012

Abstract—
Electrical waveforms measured during propagation at microscopic level are considerably affected by normal variations in cardiac microstructure as well as by the superfusing fluid. On the basis of evidence we present in this article, we argue that the anisotropic waveform variations discussed here are explained primarily by the associated variations in different microstructural components of myocardial architecture rather than by the effects of the perfusing bath. The results suggest that different components of myocardial architecture have preferential effects on V̇
max
and on the shape of the foot of the transmembrane action potential (V
m
foot). Resistive discontinuities primarily affect V̇
max
, and an additional capacitive component in the local circuit due to the capillaries in interstitial space primarily affects V
m
foot. Resistive discontinuities also have an important influence on cardiac conduction. These discontinuities include spatial variations in the size of interstitial space (interstitial resistive discontinuities) and the role of cellular scaling (effects of cell size) when changes occur in the cellular and multicellular distribution of gap junctions during remodeling of normal mature myocardium to proarrhythmic structural substrates. The full text of this article is available at http://www.circresaha.org.
Mice Lacking the Vascular Endothelial Growth Factor-B Gene (
Vegfb
) Have Smaller Hearts, Dysfunctional Coronary Vasculature, and Impaired Recovery From Cardiac Ischemia
Authors: Bellomo Daniela , Headrick John P. , Silins Ginters U. , Paterson Carol A. , Thomas Penny S. , Gartside Michael , Mould Arne , Cahill Marian M. , Tonks Ian D. , Grimmond Sean M. , Townson Steve , Wells Christine , Little Melissa , Cummings Margaret C. , Hayward Nicholas K. , Kay Graham F.
Published: June 2012

Abstract
—Vascular endothelial growth factor-B (VEGF-B) is closely related to VEGF-A, an effector of blood vessel growth during development and disease and a strong candidate for angiogenic therapies. To further study the in vivo function of VEGF-B, we have generated
Vegfb
knockout mice (
Vegfb
−/−
). Unlike
Vegfa
knockout mice, which die during embryogenesis,
Vegfb
−/−
mice are healthy and fertile. Despite appearing overtly normal,
Vegfb
−/−
hearts are reduced in size and display vascular dysfunction after coronary occlusion and impaired recovery from experimentally induced myocardial ischemia. These findings reveal a role for VEGF-B in the development or function of coronary vasculature and suggest potential clinical use in therapeutic angiogenesis. The full text of this article is available at http://www.circresaha.org.
Formation of Heterotypic Gap Junction Channels by Connexins 40 and 43
Authors: Valiunas Virginijus , Weingart Robert , Brink Peter R.
Published: June 2012

Abstract
—Gap junctions formed between transfected cells expressing connexin (Cx) 40 and Cx43 (Cx43-RIN, Cx40-HeLa, and Cx43-HeLa) revealed a relationship,
g
j
=
f
(
V
j
), at steady state, that is typified by a nonsymmetrical behavior similar to that previously reported for other heterotypic channels (gap junction conductance [
g
j
]; transjunctional voltage [
V
j
]). The unitary conductance of the channels was sensitive to the polarity of
V
j
. A main state conductance of 61 pS was found when the Cx43 cell was stepped positively or the Cx40 cell negatively (
V
j
=70 mV); the reverse polarities yielded a conductance of 100 pS. These heterotypic channels were permeable to carboxyfluorescein. In addition, two other heterotypic forms are illustrated to demonstrate that endogenous Cx45 expression cannot explain the results. The demonstration of heterotypic Cx40–Cx43 channels may have implications for the propagation of the electrical impulse in heart. For example, they may contribute to the slowing of the impulse propagation through the junctions between Purkinje fibers and ventricular muscle. The full text of this article is available at http://www.circresaha.org.
Electrophysiological Effects of Remodeling Cardiac Gap Junctions and Cell Size
Authors: Spach Madison S. , Heidlage J. Francis , Dolber Paul C. , Barr Roger C.
Published: June 2012

Abstract
—The increased incidence of arrhythmias in structural heart disease is accompanied by remodeling of the cellular distribution of gap junctions to a diffuse pattern like that of neonatal cardiomyocytes. Accordingly, it has become important to know how remodeling of gap junctions due to normal growth hypertrophy alters anisotropic propagation at a cellular level (V̇
max
) in relation to conduction velocities measured at a macroscopic level. To this end, morphological studies of gap junctions (connexin43) and in vitro electrical measurements were performed in neonatal and adult canine ventricular muscle. When cells enlarged, gap junctions shifted from the sides to the ends of ventricular myocytes. Electrically, normal growth produced different patterns of change at a macroscopic and microscopic level. Although the longitudinal and transverse conduction velocities were greater in adult than neonatal muscle, the anisotropic velocity ratios were the same. In the neonate, mean V̇
max
was not different during longitudinal (LP) and transverse (TP) propagation. However, growth hypertrophy produced a selective increase in mean TP V̇
max
(
P
<0.001), with no significant change in mean LP V̇
max
. Two-dimensional neonatal and adult cellular computational models show that the observed increases in cell size and changes in the distribution of gap junctions are sufficient to account for the experimental results. Unexpectedly, the results show that cellular scaling (cell size) is as important (or more so) as changes in gap junction distribution in determining TP properties. As the cells enlarged, both mean TP V̇
max
and lateral cell-to-cell delay increased. V̇
max
increased because increases in cell-to-cell delay reduced the electric current flowing downstream up to the time of V̇
max
, thus enhancing V̇
max
. The results suggest that in pathological substrates that are arrhythmogenic, maintaining cell size during remodeling of gap junctions is important in sustaining a maximum rate of depolarization.
Role of Kinins in the Control of Renal Papillary Blood Flow, Pressure Natriuresis, and Arterial Pressure
Authors: Tornel Jerónimo , Madrid María Isabel , García-Salom Miguel , Wirth Klaus J. , Fenoy Francisco J.
Published: June 2012

Abstract
—The present study evaluated the effects of blocking kinins with the bradykinin B
2
receptor antagonist Hoe140 on the relationship between renal perfusion pressure, papillary blood flow (PBF), and sodium excretion. To determine the relevance of renal kinins in the long-term control of arterial pressure, the effect of a chronic intrarenal infusion of Hoe140 on arterial pressure and sodium balance was also studied. PBF was not autoregulated in volume-expanded rats, and the administration of Hoe140 reduced PBF (−30%) and improved PBF autoregulation. The kinin antagonist also decreased sodium excretion (−35%) and blunted pressure natriuresis with no whole-kidney renal hemodynamic changes. These effects may be mediated through nitric oxide (NO), because in rats pretreated with
N
G
-nitro-
l
-arginine methyl ester, Hoe140 had no additional effects on PBF or pressure natriuresis. A role for NO in mediating the renal response to Hoe140 is also supported by the finding that Hoe140 reduced basal urinary NO
3
−
/NO
2
−
excretion (−33%), and it blunted the arterial pressure–induced increase in NO
3
−
/NO
2
−
excretion, which is compatible with the idea that the pressure-natriuresis response may be mediated through kinins and NO. The importance of kinins in long-term regulation of arterial pressure is demonstrated by the severe arterial hypertension (172±6 mm Hg) induced during the chronic intrarenal infusion of Hoe140 associated with sodium and volume retention. These data suggest that renal kinins and NO may be a part of the renal mechanism coupling changes in arterial pressure with modifications in PBF and sodium excretion, therefore contributing to the long-term control of arterial pressure.
Overexpressed A
1
Adenosine Receptors Reduce Activation of Acetylcholine-Sensitive K
+
Current by Native Muscarinic M
2
Receptors in Rat Atrial Myocytes
Authors: Wellner-Kienitz Marie-Cécile , Bender Kirsten , Meyer Thomas , Bünemann Moritz , Pott Lutz
Published: June 2012

Abstract
—In adult rat atrial myocytes, muscarinic acetylcholine (ACh)-sensitive K
+
current activated by a saturating concentration of adenosine (
I
K(ACh),(Ado)
) via A
1
receptors (A
1
Rs) amounts to only 30% of the current activated by a saturating concentration of ACh (
I
K(ACh),(ACh)
) via muscarinic M
2
receptors. The half-time of activation of
I
K(ACh),(Ado)
on a rapid exposure to agonist was ≈4-fold longer than that of
I
K(ACh),(ACh)
. Furthermore,
I
K(ACh),(Ado)
never showed fast desensitization. To study the importance of receptor density for A
1
R-
I
K(ACh),(Ado)
signaling, adult atrial myocytes in vitro were transfected with cDNA encoding for rat brain A
1
R and enhanced green fluorescent protein (EGFP) as a reporter. Whole-cell current was measured on days 3 and 4 after transfection. Time-matched cells transfected with only the EGFP vector served as controls. In ≈30% of EGFP-positive cells (group I), the density of
I
K(ACh),(Ado)
was increased by 72%, and its half-time of activation was reduced. Density and kinetic properties of
I
K(ACh),(ACh)
were not affected in this fraction. In ≈70% of transfection-positive myocytes (group II), the density of
I
K(ACh),(ACh)
was significantly reduced, its activation was slowed, and the fast desensitizing component was lost. Adenosine-induced currents were larger in group II than in group I, their activation rate was further increased, and a fast desensitizing component developed. These data indicate that in native myocytes the amplitude and activation kinetics of
I
K(ACh),(Ado)
are limited by the expression of A
1
R. Overexpression of A
1
R negatively interferes with signal transduction via the muscarinic M
2
receptor–linked pathway, which might reflect a competition of receptors with a common pool of G proteins. Negative interference of an overexpressed receptor with physiological regulation of a target protein by a different receptor should be considered in attempts to use receptor overexpression for gene therapy.