bluuebunny/crossref_metadata_2025 huggingface dataset:
Venous Myogenic Tone and Its Regulation Through K
+
Channels Depends on Chronic Intravascular Pressure
Authors: Szentiványi Mátyás , Bérczi Viktor , Hüttl Tivadar , Reneman Robert S. , Monos Emil
Published: June 2012

Abstract
In this study, we compared the level of myogenic tone and its negative-feedback control through specific K
+
channels in two types of human veins (saphenous [SV] and cephalic [CV] veins), which experience different ranges of pressure in vivo. We also investigated whether an experimental model of increased venous pressure in rats exposed to head-up tilt for 2 weeks produced changes similar to those observed in the human veins. Cylindrical vein segments were cannulated, their diameters were measured, and the intraluminal pressure was set at different levels (2 to 30 mm Hg) in vitro. Acetylcholine test showed that during the regular harvesting process 76% of the human SVs exposed for coronary bypass grafts had no functional endothelium. We found significant myogenic tone in the human SV, where the in vivo pressure is high, but it was not present in the human CV, where the in vivo pressure is low. The nonspecific K
+
channel antagonist, tetraethylammonium (TEA), decreased the diameter of the human SV but not the CV. Iberiotoxin and 4-aminopyridine, blockers of the Ca
2+
-sensitive (K
Ca
) and voltage-gated K
+
(K
V
) channels, also decreased the diameter of the human SV by 10.2±4.8% and 19.5±4.7%, respectively. In the rat SV, significant myogenic tone was found, but TEA had no effect, even after 2 weeks of in vivo pressure increase in the hindlimb by head-up tilt. We conclude that (1) an increased venous myogenic tone correlates with higher chronic intraluminal pressure loads, (2) K
Ca
and K
V
channels counterregulate the myogenic tone in human, but not in rat, saphenous vein, (3) the counterregulatory effect is more effective at high than at low intraluminal in vitro pressure levels, and (4) its development is probably a long-term process.
Altered Expression of Tropomodulin in Cardiomyocytes Disrupts the Sarcomeric Structure of Myofibrils
Authors: Sussman Mark A. , Baqué Susanna , Uhm Chang-Sub , Daniels Mathew P. , Price Robert L. , Simpson David , Terracio Louis , Kedes Larry
Published: June 2012

Abstract
—Tropomodulin is a tropomyosin-binding protein that terminates “pointed-end” actin filament polymerization. To test the hypothesis that regulation of tropomodulin:actin filament stoichiometry is critical for maintenance of actin filament length, tropomodulin levels were altered in cells by infection with recombinant adenoviral expression vectors, which produce either sense or antisense tropomodulin mRNA. Neonatal rat cardiomyocytes were infected, and sarcomeric actin filament organization was examined. Confocal microscopy indicated that overexpression of tropomodulin protein shortened actin filaments and caused myofibril degeneration. In contrast, decreased tropomodulin content resulted in the formation of abnormally long actin filament bundles. Despite changes in myofibril structure caused by altered tropomodulin expression, total protein turnover of the cardiomyocytes was unaffected. Biochemical analyses of infected cardiomyocytes indicated that changes in actin distribution, rather than altered actin content, accounted for myofibril reorganization. Ultrastructural analysis showed thin-filament disarray and revealed the presence of leptomeres after tropomodulin overexpression. Tropomodulin-mediated effects constitute a novel mechanism to control actin filaments, and our findings demonstrate that regulated tropomodulin expression is necessary to maintain stabilized actin filament structures in cardiac muscle cells.
Comparison of Unitary Displacements and Forces Between 2 Cardiac Myosin Isoforms by the Optical Trap Technique
Authors: Sugiura Seiryo , Kobayakawa Naoshi , Fujita Hideo , Yamashita Hiroshi , Momomura Shin-ichi , Chaen Shigeru , Omata Masao , Sugi Haruo
Published: June 2012

Abstract
—To provide information on the mechanism of cardiac adaptation at the molecular level, we compared the unitary displacements and forces between the 2 rat cardiac myosin isoforms, V
1
and V
3
. A fluorescently labeled actin filament, with a polystyrene bead attached, was caught by an optical trap and brought close to a glass surface sparsely coated with either of the 2 isoforms, so that the actin-myosin interaction took place in the presence of a low concentration of ATP (0.5 μmol/L). Discrete displacement events were recorded with a low trap stiffness (0.03 to 0.06 pN/nm). Frequency distribution of the amplitude of the displacements consisted of 2 gaussian curves with peaks at 9 to 10 and 18 to 20 nm for both V
1
and V
3
, suggesting that 9 to 10 nm is the unitary displacement for both isoforms. The duration of the displacement events was longer for V
3
than for V
1
. On the other hand, discrete force transients were recorded with a high trap stiffness (2.1 pN/nm), and their amplitude showed a broad distribution with mean values between 1 and 2 pN for V
1
and V
3
. The durations of the force transients were also longer for V
3
than for V
1
. These results indicate that both the unitary displacements and forces are similar in amplitude but different in duration between the 2 cardiac myosin isoforms, being consistent with the reports that the tension cost is higher in muscles consisting mainly of V
1
than those consisting mainly of V
3
.
β
1
Integrins Participate in the Hypertrophic Response of Rat Ventricular Myocytes
Authors: Ross Robert S. , Pham Can , Shai Shaw-Yung , Goldhaber Joshua I. , Fenczik Csilla , Glembotski Chris C. , Ginsberg Mark H. , Loftus Joseph C.
Published: June 2012

Abstract
—Multiple signaling pathways have been implicated in the hypertrophic response of ventricular myocytes, yet the importance of cell-matrix interactions has not been extensively examined. Integrins are cell-surface molecules that link the extracellular matrix to the cellular cytoskeleton. They can function as cell signaling molecules and transducers of mechanical information in noncardiac cells. Given these properties and their abundance in cardiac cells, we evaluated the hypothesis that β
1
integrin function is involved in the α
1
-adrenergic mediated hypertrophic response of neonatal rat ventricular myocytes. The hypertrophic response of this model required interaction with extracellular matrix proteins. Specificity of these results was confirmed by demonstrating that ventricular myocytes plated onto an anti–β
1
integrin antibody supported the hypertrophic gene response. Adenovirus-mediated overexpression of β
1
integrin augmented the myocyte hypertrophic response when assessed by protein synthesis and atrial natriuretic factor production, a marker gene of hypertrophic induction. DNA synthesis was not altered by integrin overexpression. Transfection of cultured cardiac myocytes with either the ubiquitously expressed β
1A
integrin or the cardiac/skeletal muscle–specific β
1
isoform (β
1D
) activated reporter expression from both the atrial natriuretic factor and myosin light chain-2 ventricular promoters, genetic markers of ventricular cell hypertrophy. Finally, suppression of integrin signaling by overexpression of free β
1
integrin cytoplasmic domains inhibited the adrenergically mediated atrial natriuretic factor response. These findings show that integrin ligation and signaling are involved in the cardiac hypertrophic response pathway.
Superoxide Production in Vascular Smooth Muscle Contributes to Oxidative Stress and Impaired Relaxation in Atherosclerosis
Authors: Miller Francis J. , Gutterman David D. , Rios C. David , Heistad Donald D. , Davidson Beverly L.
Published: June 2012

Abstract
—The endothelium is a source of reactive oxygen species in short-term models of hypercholesterolemia and atherosclerosis. We examined a chronic model of atherosclerosis for increased vascular production of superoxide (O
2
−
·) and determined whether endothelial overexpression of superoxide dismutase (SOD) would improve endothelium-dependent relaxation. Superoxide generation was 3 times higher in isolated aortas from Watanabe heritable hyperlipidemic (WHHL) rabbits (2 to 4 years old) compared with aortas from New Zealand White (NZ) rabbits (43±10 versus 14±2 relative light units · min
−1
· mm
−2
, n=9,
P
<0.05). After in vitro transduction with adenovirus containing the gene for CuZn-SOD (AdCMVCuZn-SOD) or extracellular SOD (AdCMVEC-SOD), endothelial O
2
−
· levels in WHHL aortas were significantly reduced. Gene transfer of SOD to WHHL aortas, however, failed to improve the impaired relaxation to acetylcholine or calcium ionophore. By use of the oxidative fluorescent dye hydroethidine, an in situ assay indicated markedly increased generation of O
2
−
· throughout the wall of WHHL aorta, especially within layers of smooth muscle. This finding was confirmed by demonstrating increased O
2
−
· levels in smooth muscle cells cultured from WHHL aorta. We conclude that elevated O
2
−
· levels in atherosclerotic vessels are not confined to the endothelium but occur throughout the vascular wall, including smooth muscle cells. Reduction in endothelial O
2
−
· levels is not sufficient to improve endothelium-dependent relaxation. Generation of reactive oxygen species within the media may contribute to vasomotor dysfunction in atherosclerosis.
Autonomic Control of Vasomotion in the Porcine Coronary Circulation During Treadmill Exercise
Authors: Duncker Dirk J. , Stubenitsky René , Verdouw Pieter D.
Published: June 2012

Abstract
—To date, no studies have investigated coronary vasomotor control of myocardial O
2
delivery (MD
o
2
) and its modulation by the autonomic nervous system in the porcine heart during treadmill exercise. We studied 8 chronically instrumented swine under resting conditions and during graded treadmill exercise. Exercise up to 85% to 90% of maximum heart rate produced an increase in myocardial O
2
consumption (MV̇
o
2
) from 163±16 μmol/min (mean±SE) at rest to 423±75 μmol/min (
P
≤0.05), which was paralleled by an increase in MD
o
2
, so that myocardial O
2
extraction (79±1% at rest) and coronary venous O
2
tension (cvP
o
2
, 23.7±1.0 mm Hg at rest) were maintained. β-Adrenoceptor blockade blunted the exercise-induced increase of MD
o
2
out of proportion compared with the attenuation of the exercise-induced increase in MV̇
o
2
, so that O
2
extraction rose from 78±1% at rest to 83±1% during exercise and cvP
o
2
fell from 23.5±0.9 to 19.6±1.1 mm Hg (both
P
≤0.05). In contrast, α-adrenoceptor blockade, either in the absence or presence of β-adrenoceptor blockade, had no effect on myocardial O
2
extraction or cvP
o
2
at rest or during exercise. Muscarinic receptor blockade resulted in a decreased O
2
extraction and an increase in cvP
o
2
at rest, an effect that waned during exercise. The vasodilation produced by muscarinic receptor blockade was likely due to an increased β-adrenoceptor activity, since combined muscarinic and β-adrenoceptor blockade produced similar changes in O
2
extraction and cvP
o
2
, as did β-adrenoceptor blockade alone. In conclusion, in swine myocardium, MV̇
o
2
and MD
o
2
are matched during exercise, which is the result of feed-forward β-adrenergic vasodilation in conjunction with minimal α-adrenergic vasoconstriction. β-Adrenergic vasodilation is due to an increase in sympathetic activity but may also be supported by withdrawal of muscarinic receptor–mediated inhibition of β-adrenergic coronary vasodilation. The observation that cvP
o
2
levels are maintained even during heavy exercise suggests that a decrease in cvP
o
2
is not essential for coronary vasodilation during exercise.
Coronary Hemodynamics in Endothelial NO Synthase Knockout Mice
Authors: Gödecke Axel , Decking Ulrich K. M. , Ding Zhaoping , Hirchenhain Jens , Bidmon Hans-Jürgen , Gödecke Stefanie , Schrader Jürgen
Published: June 2012

Abstract
—For the specific analysis of endothelial NO synthase (eNOS) function in the coronary vasculature, we generated a mouse homozygous for a defective eNOS gene (eNOS−/−). Western blot as well as immunohistochemical staining revealed the absence of eNOS protein in eNOS−/− mice. Aortic endothelial cells derived from eNOS−/− mice displayed only background levels of NO
x
formation compared with wild-type (WT) cells (88 versus 1990 pmol NO
x
· h
−1
/mg protein
−1
). eNOS−/− mice were hypertensive (mean arterial pressure, 135±15 versus 107±8 mm Hg in WT) without the development of cardiac hypertrophy. Coronary hemodynamics, analyzed in Langendorff-perfused hearts, showed no differences either in basal coronary flow or in maximal and repayment flow of reactive hyperemia. Acute NOS inhibition with
N
ω
-nitro-
l
-arginine methyl ester (L-NAME) in WT hearts substantially reduced basal flow and reactive hyperemia. The coronary response to acetylcholine (ACh) (500 nmol/L) was biphasic: An initial vasoconstriction (flow, −35%) in WT hearts was followed by sustained vasodilation (+190%). L-NAME significantly reduced vasodilation in WT hearts (+125%) but did not alter the initial vasoconstriction. In eNOS−/− hearts, the initial vasoconstriction was augmented (−70%), whereas the ACh-induced vasodilation was not affected. Inhibition of cyclooxygenase with diclofenac converted the ACh-induced vasodilation into vasoconstriction (−49% decrease of basal flow). This effect was even more pronounced in eNOS−/− hearts (−71%). Our results demonstrate that (1) acute inhibition of eNOS reveals a role for NO in setting the basal coronary vascular tone as well as participation in reactive hyperemia and the response to ACh; (2) chronic inhibition of NO formation in eNOS−/− mutant mice induces no changes in basal coronary flow and reactive hyperemia, suggesting the activation of important compensatory mechanisms; and (3) prostaglandins are the main mediators of the ACh-induced vasodilation in both WT and eNOS−/− mice.
Myogenic Activation and Calcium Sensitivity of Cannulated Rat Mesenteric Small Arteries
Authors: VanBavel Ed , Wesselman Jos P. M. , Spaan Jos A. E.
Published: June 2012

Abstract
—Pressure-induced activation of vascular smooth muscle may involve electromechanical as well as nonelectromechanical coupling mechanisms. We compared calcium-tone relations of cannulated rat mesenteric small arteries during pressure-induced activation, depolarization (16 to 46 mmol/L K
+
), and α
1
-adrenergic stimulation (1 μmol/L phenylephrine). The intracellular calcium concentration was expressed as the fura-2 ratio, normalized to the maximal and minimal ratios. In order to compare activation levels at various pressures, tone was expressed as the ratio of active wall tension to the maximal active tension. The passive and maximal active pressure-diameter relations needed for the calculation of tone were determined in a separate set of experiments, using isometric loading of cannulated vessels. Pressure steps from 20 to 60 and then to 100 mm Hg caused a modest rise of calcium. Nifedipine (1 μmol/L) blocked both the calcium rise and the resulting myogenic responses. Electromechanical coupling could not fully account for the myogenic response: the calcium sensitivity, defined as the slope of the calcium-tone relation, was five times higher during pressure-induced activation compared with potassium stimulation and twice as high as the sensitivity during α
1
-adrenergic stimulation. We therefore conclude that the myogenic response involves a small but necessary rise in calcium due to influx through L-type calcium channels, as well as a nonelectromechanical coupling mechanism that greatly enhances the calcium sensitivity of the contractile machinery.
Breakdown and Release of Myofilament Proteins During Ischemia and Ischemia/Reperfusion in Rat Hearts
Authors: Van Eyk Jennifer E. , Powers Francis , Law William , Larue Catherine , Hodges Robert S. , Solaro R. John
Published: June 2012

Abstract
—Our objective in experiments reported here was to identify myofilament proteins of rat hearts either lost or degraded by cardiac ischemia (15- or 60-minute duration) with and without 45 minutes of reperfusion. We correlated these changes with alterations in myofilament sensitivity to Ca
2+
and maximum force generation. Protein degradation and loss were assessed by high-performance liquid chromatography, SDS-PAGE, Western blotting analysis, and amino acid sequencing. Compared with nonischemic control hearts, bundles of skinned fibers from hearts subjected to ischemia alone demonstrated a decrease in maximum force generation and an increase in sensitivity to Ca
2+
. These changes in function were increased with the duration of the ischemia and with reperfusion. With increasing duration of ischemia, there was an increased loss and degradation of myofibrillar α-actinin and troponin I (TnI) at its C-terminus. α-Actinin and TnI were most susceptible to ischemia, but with 60 minutes of ischemia/reperfusion, there was also degradation of myosin light chain-1 (MLC1) involving a clip of residues 1 to 19. The MLC1 degradation product was detected in the reperfusion effluent (along with troponin T, tropomyosin, and α-actinin) but not in the tissue with 60 minutes of ischemia with no reperfusion. Moreover, with ischemia the following proteins became associated with the myofibrils: GAPDH and proteins of the mitochondrial ATP synthase complex. Our results provide new evidence regarding the mechanism by which ischemia/reperfusion causes myocardial injury and support the hypothesis that an important element in the injury is altered activity and structure of the myofilaments.
Inhibition of Nuclear Factor-κB–Mediated Adhesion Molecule Expression in Human Endothelial Cells
Authors: Lockyer Jean M. , Colladay John S. , Alperin-Lea Wendy L. , Hammond Timothy , Buda Andrew J.
Published: June 2012

Abstract
—The transcriptional regulatory protein nuclear factor-κB (NF-κB) participates in the control of gene expression of many modulators of the inflammatory and immune responses, including the adhesion molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1). NF-κB is found in the cytoplasm complexed with its inhibitory protein IκB. On activation, IκB is phosphorylated and degraded, thereby freeing NF-κB for translocation to the nucleus. We have generated populations of endothelial cells expressing wild-type and a proteolysis-resistant mutation of IκB that is lacking the 36 N-terminal amino acids (IκBΔN) in order to examine the effects of expression of the mutated IκB on tumor necrosis factor-α (TNF-α)–induced E-selectin and ICAM-1 expression. Wild-type and IκBΔN were introduced into primary endothelial cells using retrovirus infection followed by selection with G418. The IκBΔN protein remained at untreated control levels in endothelial cells treated with TNF-α and also remained complexed with the NF-κB family member p65. Furthermore, TNF-α–induced NF-κB DNA binding activity was inhibited in the population of endothelial cells expressing IκBΔN. That population of cells was also refractory to upregulation of E-selectin and ICAM-1 after treatment with TNF-α. The use of a truncated IκBα protein to prevent NF-κB–mediated gene expression provides a novel and specific approach for investigating the role of NF-κB in processes associated with adhesion molecule expression during inflammation.
Heart Defects in Connexin43-Deficient Mice
Authors: Ya Jing , Erdtsieck-Ernste Erna B. H. W. , de Boer Piet A. J. , van Kempen Marjan J. A. , Jongsma Habo , Gros Daniel , Moorman Antoon F. M. , Lamers Wouter H.
Published: June 2012

Abstract
—Cardiac malformation in connexin43 (CX43)-disrupted mice is restricted to the junction between right ventricle and outflow tract, even though CX43 is also expressed abundantly elsewhere. We analyzed cardiac morphogenesis in immunohistochemically and hybridohistochemically stained and three-dimensionally reconstructed serial sections of CX43-deficient embryos between embryonic day (ED) 10 and birth. The establishment of the
D
configuration in the ascending loop of CX43-deficient hearts is markedly retarded, so that the right ventricle retains a craniomedial position and is connected with the outflow tract by a more acute bend in ED10 and ED11 embryos. Because of the subsequent growth of the right ventricle, this condition usually evolves into a
D
loop, but when it persists, a “crisscross” configuration develops, with the atrioventricular cushions rotated 90°, a horizontal muscular ventricular septum, and a parallel course of the endocardial ridges of the outflow tract. After ED12, large intertrabecular pouches develop at the ventricular side of both shelflike myocardial structures that support the endocardial ridges of the outflow tract, ie, at the location that was earlier characterized by the acute bend between the right ventricle and the outflow tract and that subsequently develops into the anterosuperior leaflet of the tricuspid valve. Retarded development of the
D
configuration in the ascending loop of the embryonic heart predisposes the myocardium at the junction of the right ventricle and outflow tract to excessive development of intertrabecular pouches during subsequent development.
Molecular Determinants of Dofetilide Block of HERG K
+
Channels
Authors: Ficker Eckhard , Jarolimek Wolfgang , Kiehn Johann , Baumann Arnd , Brown Arthur M.
Published: June 2012

Abstract
—The human ether-a-go-go–related gene (HERG) encodes a K
+
channel with biophysical properties nearly identical to the rapid component of the cardiac delayed rectifier K
+
current (
I
Kr
). HERG/
I
Kr
channels are a prime target for the pharmacological management of arrhythmias and are selectively blocked by class III antiarrhythmic methanesulfonanilide drugs, such as dofetilide, E4031, and MK-499, at submicromolar concentrations. By contrast, the closely related bovine ether-a-go-go channel (BEAG) is 100-fold less sensitive to dofetilide. To identify the molecular determinants for dofetilide block, we first engineered chimeras between HERG and BEAG and then used site-directed mutagenesis to localize single amino acid residues responsible for block. Using constructs heterologously expressed in
Xenopus
oocytes, we found that transplantation of the S5-S6 linker from BEAG into HERG removed high-affinity block by dofetilide. A point mutation in the S5-S6 linker region, HERG S620T, abolished high-affinity block and interfered with C-type inactivation. Thus, our results indicate that important determinants of dofetilide binding are localized to the pore region of HERG. Since the loss of high-affinity drug binding was always correlated with a loss of C-type inactivation, it is possible that the changes observed in drug binding are due to indirect allosteric modifications in the structure of the channel protein and not to the direct interaction of dofetilide with the respective mutated site chains. However, the chimeric approach was not able to identify domains outside the S5-S6 linker region of the HERG channel as putative candidates involved in drug binding. Moreover, the reverse mutation BEAG T432S increased the affinity of BEAG K
+
channels for dofetilide, whereas C-type inactivation could not be recovered. Thus, the serine in position HERG 620 may participate directly in dofetilide binding; however, an intact C-type inactivation process seems to be crucial for high-affinity drug binding.
Time-Dependent Changes in Matrix Metalloproteinase Activity and Expression During the Progression of Congestive Heart Failure
Authors: Spinale Francis G. , Coker Mytsi L. , Thomas Chadwick V. , Walker Jennifer D. , Mukherjee Rupak , Hebbar Latha
Published: June 2012

Abstract
—The development of congestive heart failure (CHF) is associated with left ventricular (LV) dilation and myocardial remodeling. However, fundamental mechanisms that contribute to this remodeling process with the progression of CHF remain unclear. The matrix metalloproteinases (MMPs) have been demonstrated to play a significant role in tissue remodeling in a number of pathological processes. The present project tested the hypothesis that the LV dilation and remodeling during the progression of CHF is associated with early changes in MMP expression and zymographic activity. LV and myocyte function, collagen content, and MMP expression and zymographic activity were serially measured during the progression of CHF caused by pacing-induced supraventricular tachycardia (SVT) in pigs. After 7 days of SVT, LV end-diastolic dimension and myocyte length both increased by 15% from control values, and LV fractional shortening fell by 20%. At the level of the myocyte, percent shortening fell by 16% after 7 days of SVT, with no change in the steady-state velocity of shortening. Longer durations of SVT caused progressive LV dilation, LV pump failure, and myocyte contractile dysfunction. Specifically, 21 days of SVT resulted in a >50% increase in LV dimension, a 56% fall in LV fractional shortening, and a 33% decline in myocyte velocity of shortening. The decline in LV and myocyte function with 21 days of SVT was accompanied by signs and symptoms of CHF. Thus, SVT causes time-dependent changes in LV geometry and function and the subsequent development of CHF. LV myocardial collagen content and confluence fell by >25% after 7 days of SVT and were accompanied by an 80% increase in LV myocardial MMP zymographic activity against the substrate gelatin. After 14 days of SVT, total LV myocardial collagen content was reduced by 24%, and LV myocardial MMP zymographic activity increased by >100% from control values. Interstitial collagenase (MMP-1), stromelysin (MMP-3), and 72-kD gelatinase (MMP-2) were increased by ≈2-fold after 7 days of SVT. LV MMP zymographic activity and abundance remained elevated with longer durations of SVT. The results of the present study demonstrated that in this model of CHF, early changes in LV myocardial MMP zymographic activity and protein levels occurred with the initiation and progression of LV dilation and dysfunction. These findings suggest that an early contributory mechanism for the initiation of LV remodeling that occurred in this model of developing CHF is enhanced expression and potentially increased activity of LV myocardial MMPs.
Inhibition of Vascular Smooth Muscle Cell Growth by Inhibition of Fibronectin Matrix Assembly
Authors: Mercurius Kwesi O. , Morla Alex O.
Published: June 2012

Abstract
—The regulation of vascular smooth muscle cell (VSMC) proliferation by the fibronectin matrix was tested by treating human umbilical artery smooth muscle cells (HUASMCs) with a recombinant fragment of fibronectin (protein III
1
-C) that has previously been shown to modulate fibronectin matrix assembly. III
1
-C inhibited HUASMC proliferation by 75% to 90%. The inhibition of growth was time dependent; III
1
-C had no effect on DNA synthesis after 0 to 5 hours of treatment but did have an effect at 24 hours and beyond. III
1
-C did not stimulate apoptosis in these cells, indicating that the inhibition of proliferation was not due to an induction of programmed cell death. The effects of III
1
-C on cell growth were only specific for normal diploid smooth muscle cells. III
1
-C had no effect on the proliferation of IMR-90 fibroblasts, endothelial cells, NIH 3T3 cells, or the rat aortic smooth muscle cell line A7r5. However, III
1
-C did inhibit proliferation by primary rat aortic smooth muscle cells. An analysis of HUASMC fibronectin receptor (integrin α5β1) distribution revealed that III
1
-C did not inhibit α5β1 localization to focal contacts. Moreover, III
1
-C had no effect on the relative expression levels of seven different integrin subunits on HUASMCs. However, III
1
-C did inhibit fibronectin matrix assembly by rat aortic smooth muscle cells, HUASMCs, A7r5 cells, IMR-90 cells, and endothelial cells. An analysis of fibronectin synthesis indicated that the inhibition of fibronectin matrix assembly by III
1
-C was not due solely to a decrease in fibronectin synthesis. Finally, treatment of HUASMCs with anti-fibronectin monoclonal antibody L8 (which is known to inhibit fibronectin matrix assembly) also decreased the rate of HUASMC DNA synthesis. These results demonstrate that III
1
-C inhibits VSMC proliferation and suggest that this effect may be mediated by the inhibition of fibronectin matrix assembly.
Modulation of Ca
2+
Channels by Cyclic Nucleotide Cross Activation of Opposing Protein Kinases in Rabbit Portal Vein
Authors: Ruiz-Velasco Victor , Zhong Juming , Hume Joseph R. , Keef Kathleen D.
Published: June 2012

Abstract
—Cyclic nucleotides are known to modify voltage-gated (L-type) Ca
2+
channel activity in vascular smooth muscle cells, but the exact mechanism(s) underlying these effects is not well defined. The purpose of the present study was to investigate the modulatory role of the cAMP- and cGMP-dependent protein kinase (PKA and PKG, respectively) pathways in Ca
2+
channel function by using both conventional and perforated-patch–clamp techniques in rabbit portal vein myocytes. The membrane-permeable cAMP derivative, 8-bromo cAMP (0.1 to 10 μmol/L), significantly increased (14% to 16%) peak Ba
2+
currents, whereas higher concentrations (0.05 to 0.1 mmol/L) decreased Ba
2+
currents (23% to 31%). In contrast, 8-bromo cGMP inhibited Ba
2+
currents at all concentrations tested (0.01 to 1 mmol/L). Basal Ca
2+
channel currents were significantly inhibited by the PKA blocker 8-Bromo-2′-O-monobutyryladenosine-3′,5′-monophosphorothioate, Rp-isomer (Rp 8-Br-MP cAMPS, 30 μmol/L) and enhanced by the PKG inhibitor β-Phenyl-1,N
2
-etheno-8-bromoguanosine-3′,5′-monophosphorothioate, Rp-isomer (Rp-8-Br PET cGMPS, 10 nmol/L). In the presence of Rp 8-bromo PET cGMPS (10 to 100 nmol/L), both 8-bromo cAMP (0.1 mmol/L) and 8-bromo cGMP (0.1 mmol/L) enhanced Ba
2+
currents (13% to 39%). The excitatory effect of 8-bromo cGMP was blocked by Rp 8-bromo MB-cAMPS. Both 8-bromo cAMP (0.05 mmol/L) and forskolin (10 μmol/L) elicited time-dependent effects, including an initial enhancement followed by suppression of Ba
2+
currents. Ba
2+
currents were also enhanced when cells were dialyzed with the catalytic subunit of PKA. This effect was reversed by the PKA blocker KT 5720 (200 nmol/L). Our results suggest that cAMP/PKA stimulation enhances and cGMP/PKG stimulation inhibits L-type Ca
2+
channel activity in rabbit portal vein myocytes. Our results further suggest that both cAMP and cGMP have a primary action mediated by their own kinase as well as a secondary action mediated by the opposing kinase.
Formation of the Atrioventricular Septal Structures in the Normal Mouse
Authors: Webb Sandra , Brown Nigel A. , Anderson Robert H.
Published: June 2012

Abstract
—It is sometimes thought that formation of the atrioventricular septum is equated with fusion of the endocardial cushions and that failure of fusion can explain all deficiencies of atrioventricular septation. Clearly, this is simplistic, but the exact contribution of different primordia to atrioventricular septation is not well understood. To clarify this, we studied normal mouse embryos (days 10 to 15 of gestation), which were serially sectioned and examined by light microscopy. Another group of embryos was examined by scanning electron microscopy after microdissection. Our results show that development of the atrioventricular septal area is highly complex. Proper formation requires the following: remodeling of the inner heart curvature, rotation of the horns of the systemic venous sinus around the pulmonary portal, expansion of the right atrioventricular junction, formation of the muscular atrial and ventricular septa, bridging by the dextrodorsal outflow ridge and the superior endocardial cushion, fusion with the inferior margins of the venous valves, and formation of the mouth of the coronary sinus from the cranial muscular wall of the left sinus horn. Multiple primordia contribute to a central mesenchymal mass (the “septum intermedium”), including the mesenchyme on the leading edge of the primary atrial septum, the atrioventricular endocardial cushions, and the cap of mesenchyme on the spina vestibuli. Fusion of these components closes the ostium primum, completing atrial and atrioventricular septation. Additionally, the spina vestibuli has a mesodermal core, which contributes to the muscularization of the lower margin of the oval fossa. This contrasts with the formation of the upper rim, which occurs as a result of an infolding of the atrial wall itself.
Acute Glucose Overload Abolishes Ca
2+
Oscillation in Cultured Endothelial Cells From Bovine Aorta
Authors: Kimura Chiwaka , Oike Masahiro , Ito Yushi
Published: June 2012

Abstract
—Effects of acute glucose overload on [Ca
2+
]
i
were investigated in cultured endothelial cells from bovine aorta. Application of 0.1 μmol/L ATP elicited an oscillatory increase in [Ca
2+
]
i
(Ca
2+
oscillation) in Krebs solution containing 11.5 mmol/L glucose. The frequency of Ca
2+
oscillation induced by ATP increased in a concentration-dependent manner, ranging between 0.03 and 1 μmol/L. When cells were preincubated with 23 mmol/L glucose–containing Krebs solution (high glucose solution) for 3 hours, 0.1 μmol/L ATP failed to induce Ca
2+
oscillation but evoked only a phasic followed by sustained increase in [Ca
2+
]
i
. Application of a higher concentration of ATP (10 μmol/L) evoked a transient increase in [Ca
2+
]
i
both in control and high glucose–treated cells. However, the falling phase of [Ca
2+
]
i
was prolonged in high glucose–treated cells. Thapsigargin (1 μmol/L), an inhibitor of endoplasmic Ca
2+
-ATPase, induced a transient followed by a sustained increase in [Ca
2+
]
i
in control cells. Preincubation with high glucose solution increased the rate of rise of the thapsigargin-induced increase in [Ca
2+
]
i
and abolished the sustained increase, suggesting that glucose overload accelerates Ca
2+
leak from intracellular store sites and impairs Ca
2+
release–activated Ca
2+
entry. We found that all of the glucose overload–induced changes in Ca
2+
mobilization could be mimicked by xanthine with xanthine oxidase and abolished by superoxide dismutase. These results indicate that acute glucose overload accumulates superoxide anion in bovine aortic endothelial cells, thereby diminishing ATP-induced Ca
2+
oscillation through the impairment of Ca
2+
homeostasis.
Pulsatile Stretch in Coronary Arteries Elicits Release of Endothelium-Derived Hyperpolarizing Factor
Authors: Popp Rüdiger , Fleming Ingrid , Busse Rudi
Published: June 2012

Abstract
—To date, the release of the endothelium-derived hyperpolarizing factor (EDHF) has been demonstrated only in response to receptor-dependent Ca
2+
-elevating agonists. Since endothelial cells in situ are continuously subjected to rhythmic distension, we investigated the effect of rhythmic stretch on the release of EDHF from isolated porcine coronary arteries. In the combined presence of diclofenac and
N
G
-nitro-
l
-arginine (L-NNA), sinusoidal pressure oscillations (from 40 to 50 mm Hg, 4 minutes, 1.5 Hz) led to simultaneous oscillations in the external diameter of coronary artery segments, the amplitude of which were decreased by iberiotoxin and apamin and also by endothelial denudation. In order to directly demonstrate the release of EDHF, the intraluminal solution from endothelium-intact coronary segments exposed to pulsatile stretch was applied to detector rat aortic smooth muscle cells, the membrane potential of which was continuously measured using the patch-clamp technique. The hyperpolarization of detector cells induced by the intraluminal solution was proportional to the amplitude of the pressure oscillations applied to the donor artery and was attenuated by either preincubation of donor arteries with 17-octadecynoic acid or application of either tetrabutylammonium or iberiotoxin to detector cells. In contrast to the bradykinin-induced release of EDHF, the EDHF synthesized in response to pulsatile stretch did not exhibit any tachyphylaxis. These findings demonstrate for the first time that the synthesis of EDHF in coronary arteries can be mechanically stimulated by rhythmic vessel wall distension and suggest that the continuous release of EDHF may contribute to the adjustment of an adequate vascular compliance and to the control of coronary blood flow.
Protection of the Arterial Internal Elastic Lamina by Inhibition of the Renin-Angiotensin System in the Rat
Authors: Huang Wei , Gelas François Alhenc , Osborne-Pellegrin Mary J.
Published: June 2012

Abstract
—Spontaneous rupture of the internal elastic lamina (IEL) occurs in some arteries of the rat during growth and aging. Inbred, normotensive, Brown Norway (BN) rats are particularly susceptible to rupture of the IEL, especially in the abdominal aorta (AA). Preliminary experiments showed that different angiotensin-converting enzyme (ACE) inhibitors protect against rupture of the IEL in the BN rat to a greater extent than hydralazine, suggesting a role of the renin-angiotensin system (RAS) in this phenomenon. To explore this possibility, we have treated male BN rats from 4.5 to 14 weeks of age with either enalapril or losartan (both at 1, 3, and 10 mg · kg
−1
· d
−1
) or with the calcium antagonists mibefradil (at 3, 10, 30, and 45 mg · kg
−1
· d
−1
) and amlodipine (at 30 mg · kg
−1
· d
−1
). Systolic blood pressure (SBP) was measured weekly, and at the end of treatment we (1) recorded body and heart weights, (2) measured various parameters of the RAS in plasma, (3) quantified interruptions in the IEL on “en face” preparations of AA, and (4) quantified elastin, collagen, and cell proteins in the media of the thoracic aorta. Results showed that enalapril and losartan similarly decrease SBP and rupture of the IEL in the AA, suggesting that enalapril inhibits the latter via a decrease in the production of angiotensin II (Ang II) and not via another effect on ACE. The decrease in IEL rupture and in SBP, as well as the modifications in the parameters of the RAS, were all dose dependent. Mibefradil had little effect on the RAS and, at the highest doses, decreased SBP to an extent similar to that for enalapril at 3 mg · kg
−1
· d
−1
but did not significantly inhibit IEL rupture. Amlodipine decreased SBP, increased plasma renin concentration, and was without effect on IEL rupture. All treatments at the highest doses had a hypotrophic effect on the aortic media but differed in their effects on the heart, with enalapril and losartan decreasing and mibefradil and amlodipine increasing heart weight, suggesting that the inhibition of IEL rupture may be related to a cardiac hypotrophic effect. All these results, taken together, suggest that Ang II plays a role in the rupture of the IEL that is, in part, independent of SBP.
Impaired Cardiac Energetics in Mice Lacking Muscle-Specific Isoenzymes of Creatine Kinase
Authors: Saupe Kurt W. , Spindler Matthias , Tian Rong , Ingwall Joanne S.
Published: June 2012

Abstract—
Our purpose was to determine whether hearts from mice bioengineered to lack either the M isoform of creatine kinase (MCK
−/−
mice) or both the M and mitochondrial isoforms (M/MtCK
−/−
mice) have deficits in cardiac contractile function and energetics, which have previously been reported in skeletal muscle from these mice. The phenotype of hearts with deleted creatine kinase (CK) genes is of clinical interest, since heart failure is associated with decreased total CK activity and changes in the relative amounts of the CK isoforms in the heart. We measured isovolumic contractile performance in isolated perfused hearts from wild-type, MCK
−/−
, and M/MtCK
−/−
mice simultaneously with cardiac energetics (
31
P-nuclear magnetic resonance spectroscopy) at baseline, during increased cardiac work, and during recovery. Hearts from wild-type, MCK
−/−
, and M/MtCK
−/−
mice had comparable baseline function and responded to 10 minutes of increased heart rate and perfusate Ca
2+
with similar increases in rate-pressure product (48±5%, 42±6%, and 51±6%, respectively). Despite a similar contractile response, M/MtCK
−/−
hearts increased [ADP] by 95%, whereas wild-type and MCK
−/−
hearts maintained [ADP] at baseline levels. The free energy released from ATP hydrolysis decreased by 3.6 kJ/mol in M/MtCK
−/−
hearts during increased cardiac work but only slightly in wild-type (1.7 kJ/mol) and MCK
−/−
(1.5 kJ/mol) hearts. In contrast to what has been reported in skeletal muscle, M/MtCK
−/−
hearts were able to hydrolyze and resynthesize phosphocreatine. Taken together, our results demonstrate that when CK activity is lowered below a certain level, increases in cardiac work become more “energetically costly” in terms of high-energy phosphate use, accumulation of ADP, and decreases in free energy released from ATP hydrolysis, but not in terms of myocardial oxygen consumption.
Virtual Electrode–Induced Phase Singularity
Authors: Efimov Igor R. , Cheng Yuanna , Van Wagoner David R. , Mazgalev Todor , Tchou Patrick J.
Published: June 2012

Abstract
—Delivery of a strong electric shock to the heart remains the only effective therapy against ventricular fibrillation. Despite significant improvements in implantable cardioverter defibrillator (ICD) therapy, the fundamental mechanisms of defibrillation remain poorly understood. We have recently demonstrated that a monophasic defibrillation shock produces a highly nonuniform epicardial polarization pattern, referred to as a virtual electrode pattern (VEP). The VEP consists of large adjacent areas of strong positive and negative polarization. We sought to determine whether the VEP may be responsible for defibrillation failure by creating dispersion of postshock repolarization and reentry. Truncated exponential biphasic and monophasic shocks were delivered from a bipolar ICD lead in Langendorff-perfused rabbit hearts. Epicardial electrical activity was mapped during and after defibrillation shocks and shocks applied at the plateau phase of a normal action potential produced by ventricular pacing. A high-resolution fluorescence mapping system with 256 recording sites and a voltage-sensitive dye were used. Biphasic shocks with a weak second phase (<20% leading-edge voltage of the second phase with respect to the leading-edge voltage of the first phase) produced VEPs similar to monophasic shocks. Biphasic shocks with a strong second phase (>70%) produced VEPs of reversed polarity. Both of these waveforms resulted in extra beats and arrhythmias. However, biphasic waveforms with intermediate second-phase voltages (20% to 70% of first-phase voltage) produced no VEP, because of an asymmetric reversal of the first-phase polarization. Therefore, there was no substrate for postshock dispersion of repolarization. Shocks producing strong VEPs resulted in postshock reentrant arrhythmias via a mechanism of phase singularity. Points of phase singularity were created by the shock in the intersection of areas of positive, negative, and no polarization, which were set by the shock to excited, excitable, and refractory states, respectively. Shock-induced VEPs may reinduce arrhythmias via a phase-singularity mechanism. Strong shocks may overcome the preshock electrical activity and create phase singularities, regardless of the preshock phase distribution. Optimal defibrillation waveforms did not produce VEPs because of an asymmetric effect of phase reversal on membrane polarization.
Source of Electrocardiographic ST Changes in Subendocardial Ischemia
Authors: Li Danshi , Li Chuan Yong , Yong Ah Chot , Kilpatrick David
Published: June 2012

Abstract
—To clarify the source of electrocardiographic ST depression associated with ischemia, a sheep model of subendocardial ischemia was developed in which simultaneous epicardial and endocardial ST potentials were mapped, and a computer model using the bidomain technique was developed to explain the results. To produce ischemia in different territories of the myocardium in the same animal, the left anterior descending coronary artery and left circumflex coronary artery were partially constricted in sequence. Results from 36 sheep and the computer simulation are reported. The distributions of epicardial potentials from either ischemic source were very similar (
r
=0.77±0.14,
P
<0.0001), with both showing ST depression on the free wall of the left ventricle and no association between the ST depression and the ischemic region. However, endocardial potentials showed that ST elevation was directly associated with the region of reduced blood flow. Insulating the heart from the surrounding tissue with plastic increased the magnitude of epicardial ST potentials, which was consistent with an intramyocardial source. Increasing the percent stenosis of a coronary artery increased epicardial ST depression at the lateral boundary and resulted in ST elevation starting from the ischemic center as ischemia became transmural. Computer simulation using the bidomain model reproduced the epicardial ST patterns and suggested that the ST depression was generated at the lateral boundary between ischemic and normal territories. ST depression on the epicardium reflected the position of this lateral boundary. The boundaries of ischemic territories are shared, and only those appearing on the free wall contribute to external ST potential fields. These effects explain why body surface ST depression does not localize cardiac ischemia in humans.
Transcription Factor RTEF-1 Mediates α
1
-Adrenergic Reactivation of the Fetal Gene Program in Cardiac Myocytes
Authors: Stewart Alexandre F.R. , Suzow Joseph , Kubota Toru , Ueyama Takahisa , Chen Hsiao-Huei
Published: June 2012

Abstract
—α
1
-Adrenergic receptor stimulation induces cardiac myocytes to hypertrophy and reactivates many fetal genes, including β-myosin heavy chain (βMyHC) and skeletal α-actin (SKA), by signaling through myocyte-specific CAT (M-CAT)
cis
elements, binding sites of the transcriptional enhancer factor-1 (TEF-1) family of transcription factors. To examine functional differences between TEF-1 and related to TEF-1 (RTEF-1) in α
1
-adrenergic reactivation of the fetal program, expression constructs were cotransfected with βMyHC and SKA promoter/reporter constructs in neonatal rat cardiac myocytes. TEF-1 overexpression tended to transactivate a minimal βMyHC promoter but significantly interfered with a minimal SKA promoter. In contrast, RTEF-1 transactivated both the minimal βMyHC and SKA promoters. TEF-1 and RTEF-1 also affected the α
1
-adrenergic response of the βMyHC and SKA promoters differently. TEF-1 had no effect. In contrast, RTEF-1 potentiated the α
1
-adrenergic responses of the SKA promoter and of a −3.3-kb βMyHC promoter. To determine why the promoters responded differently to TEF-1 and RTEF-1, promoters with mutated M-CAT elements were tested in the same way. The βMyHC promoter required an intact M-CAT element to respond to TEF-1 and RTEF-1, whereas the SKA promoter M-CAT was required for the TEF-1 response but not for the RTEF-1 response, suggesting that SKA promoter–specific cofactors may be involved. By competition gel shift assay, the M-CAT of the minimal βMyHC promoter had a lower affinity than that of the SKA promoter, which partly explains the different responses of these promoters to TEF-1. These results highlight functional differences between TEF-1 and RTEF-1 and suggest a novel function of RTEF-1 in mediating the α
1
-adrenergic response in hypertrophic cardiac myocytes.