bluuebunny/crossref_metadata_2025 huggingface dataset:
Design Principles of Vascular Beds
Authors: Pries Axel R. , Secomb Timothy W. , Gaehtgens Peter
Published: June 2012

Abstract
Hemodynamic parameters were determined in each vessel segment of six complete microvascular networks in the rat mesentery by using a combination of experimental measurements and theoretical simulations. For a total number of 2592 segments, a strong unified dependence of wall shear stress on intravascular pressure for arterioles, capillaries, and venules was obtained. All three types of segments exhibit an essentially identical variation of shear stress from high to low values (from ≈100 to 10 dyne/cm
2
) as intravascular pressure falls from 70 to 15 mm Hg. On the basis of these observations, it is proposed that vascular beds grow and adapt so as to maintain the shear stress in each vessel at a level that depends on local transmural pressure. In contrast to Murray’s classic ‘minimum-cost’ hypothesis, which implies uniformity of wall shear rate throughout the vasculature, the proposed design principle provides an explanation for the functionally important arteriovenous asymmetry of wall shear rates and flow resistance in the circulation.
Cardiac Sarcoplasmic Reticulum Phosphorylation Increases Ca
2+
Release Induced by Flash Photolysis of Nitr-5
Authors: Patel Jitandrakumar R. , Coronado Roberto , Moss Richard L.
Published: June 2012

Abstract
Effects on Ca
2+
-induced Ca
2+
release due to phosphorylation of sarcoplasmic reticulum (SR) proteins were investigated in isoproterenol-treated saponin-permeabilized trabeculae from rat ventricles. In these experiments, Ca
2+
release from the SR was induced by a rapid change in concentration of free Ca
2+
(ie, trigger Ca
2+
) achieved by flash photolysis of nitr-5, and the amount of Ca
2+
released was assessed by measuring isometric tension. Ca
2+
uptake by the SR was more rapid, and the amount of Ca
2+
released by a given concentration of trigger Ca
2+
was greater in isoproterenol-treated trabeculae compared with control trabeculae. However, under the same conditions of Ca
2+
loading, the amplitudes of caffeine-elicited tension transients in control trabeculae were similar to those in isoproterenol-treated trabeculae, suggesting that the Ca
2+
available for release was similar in the two cases. Control experiments showed that there were no significant differences in Ca
2+
sensitivity of tension between isoproterenol-treated and control trabeculae. Also, application of alkaline phosphatase to trabeculae that had previously been treated with isoproterenol returned SR Ca
2+
release to control levels. We conclude that the greater release of Ca
2+
in isoproterenol-treated trabeculae in response to a given concentration of trigger Ca
2+
is due to phosphorylation of SR proteins, most likely the Ca
2+
release channel.
Isometric and Dynamic Contractile Properties of Porcine Skinned Cardiac Myocytes After Stunning
Authors: McDonald Kerry S. , Mammen Pradeep P.A. , Strang Kevin T. , Moss Richard L. , Miller William P.
Published: June 2012

Abstract
The purpose of this study was to investigate myofibrillar mechanisms of depressed contractile function associated with myocardial stunning. We first tested whether the degree of stunning was directly related to changes in myofilament Ca
2+
sensitivity. Variable degrees and durations of low-flow ischemia were followed by 30 minutes of reperfusion in an open-chest porcine model of regional myocardial stunning (n=27). Ca
2+
sensitivity of isometric tension was measured in skinned myocytes obtained from endocardial biopsies taken during control aerobic flow and after 30 minutes of reperfusion. The degree of stunning, as assessed by percent systolic wall thickening, ranged from −3% to 75% of control but did not correlate (
r
=.11) with changes in pCa
50
, ie, pCa for half-maximal tension. Only in the group (n=10) with the most severe level of ischemia was there a significant decrease in pCa
50
(from 5.97±0.06 in the control condition to 5.86±0.07 after ischemia,
P
<.05). Less severe levels of ischemia (n=17) resulted in significant stunning (percent systolic wall thickening, 38±4% of control) but no change in pCa
50
. To investigate the possibility that alterations in myofibrillar cross-bridge kinetics contribute to depressed function in stunning, maximum velocity of shortening (V
o
) was measured in postischemic myocytes. V
o
in postischemic myocytes was reduced to 56±4% of V
o
in control myocytes and was independent both of the degree of stunning (
r
=.26) and changes in Ca
2+
sensitivity. We conclude that the basis of stunning involves decreased cycling rates of myofibrillar cross-bridges and, after more severe ischemia, a reduction in myofilament Ca
2+
sensitivity.
Angiotensin II Activates pp60
c-
src
in Vascular Smooth Muscle Cells
Authors: Ishida Mari , Marrero Mario B. , Schieffer Bernhard , Ishida Takafumi , Bernstein Kenneth E. , Berk Bradford C.
Published: June 2012

Abstract
The angiotensin II type-1 (AT
1
) receptor, a G protein–coupled receptor, lacks intrinsic kinase activity. However, recent data show that angiotensin II (Ang II) stimulates tyrosine phosphorylation of phospholipase C-γ1 (PLC-γ1), Stat91 (one of the signal transducers and activators of transcription), and paxillin in vascular smooth muscle cells. The tyrosine kinases responsible for these phosphorylation events are unknown. Src family kinases have been shown to phosphorylate PLC-γ1 and to be activated by G protein–coupled receptors. We hypothesized that pp60
c-
src
associates with the AT
1
receptor and is activated after Ang II stimulation of smooth muscle cells. We immunoprecipitated pp60
c-
src
from Ang II–stimulated vascular smooth muscle cells and measured pp60
c-
src
activity by autophosphorylation and by phosphorylation of enolase. Both assays demonstrated an approximately threefold increase in pp60
c-
src
activity within 1 minute. A similar increase in Ang II–stimulated pp60
c-
src
activity was observed in Chinese hamster ovary cells transfected with the AT
1
receptor but not in untransfected cells. These data are the first to show that pp60
c-
src
is activated by Ang II. To determine if pp60
c-
src
associated with the AT
1
receptor, the AT
1
receptor was immunoprecipitated (with two different antibodies), and Western blots were performed with two different anti-pp60
c-
src
antibodies. No pp60
c-
src
was detected. In addition, direct interaction between the AT
1
receptor and pp60
c-
src
could not be demonstrated by using a glutathione S-transferase (GST)-AT
1
fusion protein to bind proteins from cell lysates stimulated by Ang II. In combination with recent findings that anti-pp60
c-
src
antibodies inhibit Ang II–mediated PLC-γ1 phosphorylation, our data suggest an important role for pp60
c-
src
in Ang II signal transduction.
Multiple Growth Factors Modulate mRNA Expression of Angiotensin II Type-2 Receptor in R3T3 Cells
Authors: Ichiki Toshihiro , Kambayashi Yoshikazu , Inagami Tadashi
Published: June 2012

Abstract
Previous studies showed that angiotensin II type-2 receptor (AT
2
) sites were increased when R3T3 cells were growth arrested and decreased when they were stimulated with fibroblast growth factor or serum. We examined the effects of several other growth factors on the expression of AT
2
mRNA to clarify the relation between the AT
2
receptor and growth factors. R3T3 cells were cultured in the medium containing 10% FCS until they were confluent, and then serum was removed. AT
2
mRNA was increased after serum was depleted, and the expression level reached a plateau after 2 days of serum depletion. The presence of serum (10%), fibroblast growth factor (10 ng/mL), or lysophosphatidic acid (1 μmol/L) reduced the AT
2
mRNA expression. Phorbol ester (1 to 100 nmol/L) also suppressed the AT
2
mRNA expression in a dose-dependent manner. Interleukin-1β (1 ng/mL) enhanced the AT
2
mRNA expression 1.6-fold and the AT
2
receptor number 1.4-fold. Insulin (100 nmol/L) enhanced AT
2
mRNA expression 1.4-fold and the AT
2
receptor number 1.6-fold. These results suggest that AT
2
mRNA expression is modulated by multiple growth factors in both positive and negative directions. The presence of potential
cis
DNA elements that respond to interleukin-1β (CCAAT enhancer binding protein site), insulin [insulin response sequence of phospho(enol)pyruvate carboxykinase gene], and phorbol ester (AP-1 site) in the promoter region of the mouse AT
2
gene suggests that the effects of these growth factors and phorbol ester may be mediated via these
cis
DNA elements.
Mechanisms of Transmural Heparin Transport in the Rat Abdominal Aorta After Local Vascular Delivery
Authors: Lovich Mark A. , Edelman Elazer R.
Published: June 2012

Abstract
Local vascular drug delivery systems provide elevated concentrations in target arterial tissues, while minimizing systemic side effects. Drug can now be released to isolated arterial segments from the endovascular or perivascular aspects of the blood vessel, yet the forces that determine drug distribution and deposition for these different modes of delivery have not been rigorously investigated. This study examines mechanisms of transmural transport of a model vasoactive drug, heparin, and compares estimates of the distribution after administration from either aspect of the artery. We showed that (1) heparin traversed the arterial wall rapidly; (2) diffusion far outweighed convection in the control of transmural heparin transport in the normal artery, but after endothelial injury, convective forces rose to one quarter the magnitude of diffusive forces; (3) the endothelium posed a minimal diffusive barrier to heparin; and (4) the diffusive barrier imposed by the adventitia depended on its thickness. These findings strongly suggest that vasoregulatory compounds can be administered to target tissue by either perivascular or endovascular means with equal efficacy, because the forces governing transport of heparin from either aspect of the blood vessel wall are not significantly different. Furthermore, the differences in arterial transport properties between heparin and other macromolecules suggest that distribution and the optimal aspect of delivery will depend just as much on the physicochemical properties of the drug as the state of the blood vessel wall.
Renal Effects of High-Dose Natriuretic Peptide Receptor Blockade in Rats With Congestive Heart Failure
Authors: Zhang Ping L. , Mackenzie Harald S. , Totsune Kazuhito , Troy Julia L. , Brenner Barry M.
Published: June 2012

Abstract
Previous studies suggest that elevated plasma atrial natriuretic peptide (ANP) levels participate in regulating renal excretory function in rats with congestive heart failure (CHF). To define the role of natriuretic peptides (NPs) in the regulation of renal function in CHF, the renal responses to HS-142-1 (HS), a potent NP receptor antagonist, were studied in anesthetized rats subjected to coronary ligation that developed left ventricular infarction and CHF or in sham-operated (SO) control rats. Plasma ANP levels averaged >14-fold higher in rats with CHF than in SO rats. In response to HS (20 mg/kg IV bolus), both mean arterial pressure and renal vascular resistance increased in rats with CHF but not in SO rats; glomerular filtration rate (GFR, 1.26±0.04 versus 0.76±0.11 mL/min) and renal plasma flow rate (RPF, 3.52±0.27 versus 2.70±0.32 mL/min) were significantly reduced in rats with CHF; and in SO rats, GFR (1.26±0.06 versus 1.20±0.07 mL/min) and RPF (3.98±0.21 versus 3.99±0.18 mL/min) were not significantly affected by HS. The sodium excretion rate (0.18±0.04 to 0.06±0.01 μEq/min) and fractional sodium excretion (0.01±0.02% to 0.04±0.01%) also fell markedly after HS administration in rats with CHF, but these parameters were unchanged in SO rats. These data indicate that NPs play a critical role in maintaining renal hemodynamic function and inhibiting tubule sodium reabsorption in rats with CHF, thus opposing sodium retention and preserving sodium balance in this model.
Electrical Activity Underlying Rhythmic Contraction in Human Pial Arteries
Authors: Gokina Natalia I. , Bevan Rosemary D. , Walters Carrie L. , Bevan John A.
Published: June 2012

Abstract
Human pial arteries obtained during surgery frequently exhibit spontaneous periodic contractions. Simultaneous measurements of membrane potential and vessel wall force were used to examine whether these contractions are associated with electrical activity of smooth muscle cells (SMCs). A total of 53 segments from 38 patients were studied, and of these, 26 showed spontaneous contractions related to periodic depolarization and generation of action potentials (APs). The resting membrane potential during the silent periods was −44.0±0.5 mV. APs without “overshoot” were observed when spontaneous depolarization reached levels of −40 to −35 mV. Just over half of the arterial segments failed to exhibit spontaneous activity; however, APs could be generated during K
+
-induced depolarization. The mean SMC resting membrane potential of these vessels was −53.5±1.5 mV, and this value differed significantly from that of SMCs in spontaneously active arteries. Application of tetrodotoxin did not change the amplitude and duration of APs. Removal of Ca
2+
from the bathing solution and addition of nifedipine completely inhibited the spontaneous APs and associated contractions. K
+
depolarization failed to induce APs and contraction in the presence of nifedipine. We conclude that periodic spontaneous depolarization and AP generation underlie the periodic spontaneous contractions of human pial arteries. Both the APs and associated contractions are related to the activation of dihydropyridine-sensitive voltage-dependent Ca
2+
channels. It is suggested that AP generation can be responsible for vasomotion of human pial arteries in vivo.
Acidosis-Induced Coronary Arteriolar Dilation Is Mediated by ATP-Sensitive Potassium Channels in Vascular Smooth Muscle
Authors: Ishizaka Hiroshi , Kuo Lih
Published: June 2012

Abstract
Although a decrease in extravascular pH has been suggested to be involved in coronary flow regulation during hypoxia, ischemia, and increased metabolic demand of the heart, its vasomotor control mechanism has not been elucidated. To examine the effect of acidosis on vasomotor tone, porcine coronary arterioles (40 to 110 μm) were isolated, cannulated, and pressurized to 60 cm H
2
O intraluminal pressure without flow for in vitro study. Acidosis (pH 7.4 to 7.0) was produced by adding HCl to the extravascular solution. The involvement of potassium channels in the vasomotor response to acidosis was evaluated by using BaCl
2
(100 μmol/L, nonspecific potassium channel inhibitor), glibenclamide (5 μmol/L, ATP-sensitive potassium channel inhibitor), and iberiotoxin (100 nmol/L, calcium-activated potassium channel inhibitor). To determine whether endothelial hyperpolarization contributes to the acidosis-induced dilation, the pH-diameter relation of the vessel was examined under a high intraluminal concentration of KCl (40 mmol/L). The involvement of nitric oxide and prostaglandins was assessed by using
N
G
-monomethyl-
l
-arginine (L-NMMA, 10 μmol/L) and indomethacin (10 μmol/L), respectively. To evaluate the role of endothelium in the acidosis-induced dilation, the pH-diameter relation was studied after endothelial removal. All vessels developed a similar level of spontaneous tone (internal diameter, 75±4 μm [≈69±1% of maximum diameter]) and dilated to HCl in a dose-dependent manner. Glibenclamide completely abolished vasodilation to a mild level of acidosis (pH 7.2 to 7.3) and attenuated the vasodilation by 70% at pH 7.0. Acidosis-induced dilation was also inhibited by BaCl
2
but not by iberiotoxin. L-NMMA, indomethacin, and intraluminal KCl did not alter the pH-diameter relation. Vasodilation to acidosis of the endothelium-denuded vessels was identical to that of the endothelium-intact vessels. In addition, glibenclamide attenuated the acidosis-induced arteriolar dilation of endothelium-denuded vessels in a manner similar to that of endothelium-intact vessels. These results suggest that the opening of ATP-sensitive potassium channels in vascular smooth muscle mediates the coronary arteriolar dilation during acidosis.
Frequency-Dependent Activation of a Constitutive Nitric Oxide Synthase and Regulation of Contractile Function in Adult Rat Ventricular Myocytes
Authors: Kaye David M. , Wiviott Stephen D. , Balligand Jean-Luc , Simmons William W. , Smith Thomas W. , Kelly Ralph A.
Published: June 2012

Abstract
Cardiac myocytes have recently been shown to express a constitutive Ca
2+
-sensitive isoform of NO synthase (NOS3), although the mechanism(s) responsible for activation of NOS3 and its physiological function remain to be determined. Since the activity of NOS3 is known to be regulated in part by the intracellular Ca
2+
activity ([Ca
2+
]
i
) in endothelial cells, we determined whether increasing myocyte [Ca
2+
]
i
by uniform electric field pacing was accompanied by an increase in NOS3 activity, detected as nitrite accumulation in the medium. A higher [Ca
2+
]
i
with increasing pacing frequencies was shown to be accompanied by a time-dependent accumulation of nitrite in medium that bathed adult rat ventricular myocytes stimulated at 3 Hz. Nitrite release by paced cells was significantly attenuated by treatment with either the NO synthase inhibitor nitro-
l-
arginine (L-NA, 1 mmol/L) or the intracellular Ca
2+
chelator BAPTA-AM (20 μmol/L). Paced myocytes also exhibited a frequency- and time-dependent increase in intracellular cGMP content that could be inhibited significantly by either L-NA or the soluble guanylate cyclase inhibitor LY83583 (5 μmol/L). To determine whether the increase in NOS3 activity with pacing affected contractile function, myocytes were sequentially paced at frequencies from 0.5 to 3 Hz. Methylene blue, L-NA, and LY83583 all increased the amplitude of shortening of myocytes paced at 3 Hz. Furthermore, a significantly greater positive inotropic response to high extracellular Ca
2+
(3 mmol/L) was demonstrated by myocytes pretreated with L-NA compared with control cells. These data indicate that myocyte NOS3 activity is regulated in part by [Ca
2+
]
i
, whether induced by changes in pacing frequency or [Ca
2+
]
o
, and depresses myocyte contractile responsiveness to higher stimulation frequencies.
Regulation of rDNA Transcription During Endothelin-1Induced Hypertrophy of Neonatal Cardiomyocytes
Authors: Luyken Joachim , Hannan Ross D. , Cheung Joseph Y. , Rothblum Lawrence I.
Published: June 2012

Abstract
Treatment of cultured neonatal cardiomyocytes with endothelin-1 and phorbol 12-myristate 13-acetate (PMA) results in cardiomyocyte hypertrophy. However, the signal transduction pathways involved in this process are poorly understood. Because increased ribosome biogenesis is a requisite for hypertrophy, we sought to (1) confirm the hypothesis that these two hypertrophic agents did indeed induce rRNA synthesis and (2) examine the mechanism through which this induction was accomplished. In this study, hypertrophy of contraction-arrested neonatal cardiomyocytes induced by treatment with either endothelin-1 or PMA was associated with increased rDNA transcription. Western blots demonstrated that the enhanced rates of rDNA transcription were not mediated by increased amounts of either RNA polymerase I or upstream binding factor (UBF), an rDNA transcription factor. However, immunoprecipitation of [
32
P]orthophosphate-labeled UBF from hypertrophying neonatal cardiomyocytes suggested that the increased rate of rDNA transcription may be due to the hyperphosphorylation of UBF, which would increase the activity of UBF. The increase in UBF phosphorylation occurred within 3 to 6 hours after exposure to either agent, was maximal at 12 hours, and was sustained for at least the first 24 hours of exposure. Phosphoamino acid analysis of UBF immunoprecipitated from control and treated cardiomyocytes demonstrated that UBF was phosphorylated exclusively on serine residues. Our previous studies have shown that the cellular UBF content increased in adrenergic- and contraction-induced models of cardiac hypertrophy. This study with endothelin-1 and PMA demonstrates that the modulation of UBF phosphorylation is an additional pathway by which ribosome biogenesis may be regulated in neonatal cardiomyocytes. These results support the hypothesis that UBF is an important regulatory factor during the initiation and maintenance of the accelerated rate of rDNA transcription observed during neonatal cardiomyocyte hypertrophy mediated by both phorbol esters and endothelin-1.
Identification of Epoxyeicosatrienoic Acids as Endothelium-Derived Hyperpolarizing Factors
Authors: Campbell William B. , Gebremedhin Debebe , Pratt Phillip F. , Harder David R.
Published: June 2012

Abstract
Endothelial cells release several compounds, including prostacyclin, NO, and endothelium-derived hyperpolarizing factor (EDHF), that mediate the vascular effects of vasoactive hormones. The identity of EDHF remains unknown. Since arachidonic acid causes endothelium-dependent relaxations of coronary arteries through its metabolism to epoxyeicosatrienoic acids (EETs) by cytochrome P
450
, we wondered if the EETs represent EDHFs. Precontracted bovine coronary arteries relaxed in an endothelium-dependent manner to methacholine. The cytochrome P
450
inhibitors, SKF 525A and miconazole, significantly attenuated these relaxations. They were also inhibited by tetraethylammonium (TEA), an inhibitor of Ca
2+
-activated K
+
channels, and by high [K
+
]
o
(20 mmol/L). Methacholine also caused hyperpolarization of coronary smooth muscle (−27±3.9 versus −40±5.1 mV), which was completely blocked by SKF 525A and miconazole. In vessels prelabeled with [
3
H]arachidonic acid, methacholine stimulated the release of 6-ketoprostaglandin F
1α
, 12-HETE, and the EETs. Arachidonic acid relaxed precontracted coronary arteries, which were also blocked by TEA, charybdotoxin, another Ca
2+
-activated K
+
channel inhibitor, and high [K
+
]
o
. 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET relaxed precontracted coronary vessels (EC
50
, 1×10
−6
mol/L). The four regioisomers were equally active. TEA, charybdotoxin, and high [K
+
]
o
attenuated the EET relaxations. 11,12-EET hyperpolarized coronary smooth muscle cells from −37±0.2 to −59±0.3 mV. In the cell-attached mode of patch clamp, both 14,15-EET and 11,12-EET increased the open-state probability of a Ca
2+
-activated K
+
channel in coronary smooth muscle cells. This effect was blocked by TEA and charybdotoxin. These data support the hypothesis that the EETs are EDHFs.
Transgenic Remodeling of the Contractile Apparatus in the Mammalian Heart
Authors: Palermo Joseph , Gulick James , Colbert Melissa , Fewell Jason , Robbins Jeffrey
Published: June 2012

Abstract
The structure-function relationships of the sarcomeric proteins in the mammalian cardiac compartment remain ill-defined because of the lack of a suitable model in which they can be readily manipulated or exchanged in vivo. To establish the validity of the transgenic paradigm for remodeling the mammalian heart, the murine α-cardiac myosin heavy chain gene promoter was used to express a ventricular myosin light chain-2 transgene (
MLC2v
) in both the atria and ventricles of the adult animal. Expression resulted in high levels of the transgene’s transcript in both compartments. In the ventricle, the transgene was expressed against the background expression of the normal isoform. In the atrium, the transgene’s expression would be ectopic, in that normally,
MLC2v
expression is restricted to the ventricle. Ectopic expression of the transgene in the atria resulted in a complete replacement of the atrial myosin light chain-2 protein isoform, although the endogenous isoform’s steady state transcript levels were unchanged. In contrast, ventricular expression of the transgene had no effect at the protein level, despite an eightfold increase in MLC2v transcript levels. The data show that sarcomeric protein stoichiometry is maintained rigorously via posttranscriptional regulation and that protein replacement can be achieved through a single transgenic manipulation.
Adverse Effects of Chronic Endogenous Sympathetic Drive Induced by Cardiac G
sα
Overexpression
Authors: Iwase Mitsunori , Bishop Sanford P. , Uechi Masami , Vatner Dorothy E. , Shannon Richard P. , Kudej Raymond K. , Wight David C. , Wagner Thomas E. , Ishikawa Yoshihiro , Homcy Charles J. , Vatner Stephen F.
Published: June 2012

Abstract
To study the physiological effect of the overexpression of myocardial G
sα
(protein levels increased by approximately threefold in transgenic mice), we examined the responsiveness to sympathomimetic amines by echocardiography (9 MHz) in five transgenic mice and five control mice (both 10.3±0.2 months old). Myocardial contractility in transgenic mice, as assessed by left ventricular (LV) fractional shortening (LVFS) and LV ejection fraction (LVEF), was not different from that of control mice at baseline (LVFS, 40±3% versus 36±2%; LVEF, 78±3% versus 74±3%). LVFS and LVEF values in transgenic mice during isoproterenol (ISO, 0.02 μg/kg per minute) infusion were higher than the values in control mice (LVFS, 68±4% versus 48±3%; LVEF, 96±1% versus 86±3%;
P
<.05). Norepinephrine (NE, 0.2 μg/kg per minute) infusion also increased LVFS and LVEF in transgenic mice more than in control mice (LVFS, 59±4% versus 47±3%; LVEF, 93±2% versus 85±3%;
P
<.05). Heart rates of transgenic mice were higher than those of control mice during ISO and NE infusion. In three transgenic mice with heart rates held constant, LV dP/dt rose by 33±2% with ISO (0.02 μg/kg per minute) and by only 13±2% in three wild-type control mice (
P
<.01). NE (0.1 μg/kg per minute) also induced a greater effect on LV dP/dt in the three transgenic mice with heart rates held constant compared with three wild-type control mice (65±8% versus 28±4%,
P
<.05). Pathological and histological analyses of older transgenic mouse hearts (16.0±0.8 months old) revealed hypertrophy, degeneration, atrophy of cells, and replacement fibrosis reflected by significant increases in collagen volume in the subendocardium (5.2±1.4% versus 1.2±0.3%,
P
<.05) and in the cross-sectional area of myocytes (298±29 versus 187±12 μm
2
,
P
<.05) compared with control mouse hearts. These results suggest that G
sα
overexpression enhances the efficacy of the β-adrenergic receptor–G
s
–adenylyl cyclase signaling pathway. This in turn leads to augmented inotropic and chronotropic responses to endogenous sympathetic stimulation. This action over the life of the animal results in myocardial damage characterized by cellular degeneration, necrosis, and replacement fibrosis, with the remaining cells undergoing compensatory hypertrophy. As a model, this transgenic mouse offers new insights into the mechanisms of cardiomyopathy and heart failure and provides a new tool for their study.
Thrombin Receptor Actions in Neonatal Rat Ventricular Myocytes
Authors: Jiang Tianrong , Kuznetsov Valery , Pak Elena , Zhang HongLu , Robinson Richard B. , Steinberg Susan F.
Published: June 2012

Abstract
Previous studies established that thrombin stimulates phosphoinositide hydrolysis and modulates contractile function in neonatal rat ventricular myocytes. The present study further defines the signaling pathways activated by the thrombin receptor and their role in thrombin’s actions in cardiac myocytes. The thrombin receptor–derived agonist peptide (TRAP, a portion of the tethered ligand created by thrombin’s proteolytic activity) stimulates the rapid and transient accumulation of inositol bis- and tris-phosphates (IP
2
and IP
3
, respectively), which is followed by the more gradual and sustained accumulation of inositol monophosphate (IP
1
). TRAP elicits a larger and more sustained accumulation of IP
1
than does thrombin. Thrombin and TRAP also activate mitogen-activated protein kinase (MAPK) in cultured neonatal rat ventricular myocytes. Differences in the kinetics and magnitude of thrombin- and TRAP-dependent inositol phosphate (IP) accumulation are paralleled by differences in the kinetics and magnitude of thrombin- and TRAP-dependent activation of MAPK. Pretreatment with phorbol 12-myristate 13-acetate (PMA) to downregulate protein kinase C (PKC) attenuates thrombin- and TRAP-dependent activation of MAPK, although small and equivalent effects of thrombin and TRAP to stimulate MAPK persist in PMA-pretreated cells. These results support the notion that the thrombin receptor activates MAPK through PKC-dependent and PKC-independent pathways and that the incremental activation of MAPK by TRAP over that induced by thrombin is the consequence of enhanced activation through the PKC limb of the phosphoinositide lipid pathway. TRAP also increases the beating rate of spontaneously contracting ventricular myocytes and elevates cytosolic calcium in myocytes electrically driven at a constant basic cycle length. The effects of TRAP to modulate contractile function and elevate intracellular calcium are not inhibited by tricyclodecan-9-yl-xanthogenate (D609, to block TRAP-dependent IP accumulation) or pretreatment with PMA (to downregulate PKC). The TRAP-dependent rise in intracellular calcium also is not inhibited by verapamil or removal of extracellular calcium but is markedly attenuated by depletion of sarcoplasmic reticular calcium stores by caffeine. Patch-clamp experiments demonstrate that TRAP elevates intracellular calcium in cells held at a membrane potential of −70 mV. Taken together, these results support the conclusion that the thrombin receptor modulates contractile function by mobilizing intracellular calcium through an IP
3
-independent mechanism and that this response does not require activation of voltage-gated ion channels.
Transient Ischemia Reduces Norepinephrine Release During Sustained Ischemia
Authors: Seyfarth Melchior , Richardt Gert , Mizsnyak Anna , Kurz Thomas , Schömig Albert
Published: June 2012

Abstract
Endogenous catecholamine release may play a role in ischemic preconditioning either as a trigger or as a target within the process of myocardial preconditioning. Therefore, we investigated the effect of transient ischemia (TI) on norepinephrine release during sustained ischemia in isolated rat hearts. TI was induced by multiple cycles of global ischemia followed by reperfusion with a duration of 5 minutes each, comparable to ischemic preconditioning protocols. After TI, norepinephrine release was evoked by either sustained global ischemia, anoxia, cyanide intoxication, tyramine, or electrical stimulation. During TI, no washout of norepinephrine was observed, and tissue concentrations of norepinephrine were not changed. TI, however, reduced norepinephrine overflow after 20 minutes of sustained ischemia from 239±26 pmol/g (control) to 79±8 pmol/g (67% reduction,
P
<.01). A similar reduction of ischemia-induced norepinephrine release from 192±22 pmol/g (control) to 90±15 pmol/g was observed when hearts underwent transient anoxia without glucose (
P
<.05). When reperfusion between TI and sustained ischemia was prolonged from 5 to 90 minutes, the inhibitory effect of TI on norepinephrine release was gradually lost. Susceptibility to TI was a unique feature of norepinephrine release induced by sustained ischemia, since release of norepinephrine evoked by anoxia, cyanide intoxication, tyramine, or electrical stimulation remained unaffected by TI. We propose a protective effect of TI on neural tissue, which may reduce norepinephrine-induced damage during prolonged myocardial ischemia.
Regulation of β
1
-Integrin Function in Cultured Human Vascular Smooth Muscle Cells
Authors: Seki Jiro , Koyama Noriyuki , Kovach Nicholas L. , Yednock Ted , Clowes Alexander W. , Harlan John M.
Published: June 2012

Abstract
Avidity modulation and function of β
1
-integrin receptors in cultured human vascular smooth muscle cells (SMCs) were investigated using monoclonal antibody (mAb) 8A2, which binds to the β
1
subunit of integrin heterodimers and induces a high avidity state. The adhesion of SMCs to extracellular matrix proteins, but not to poly-
l
-lysine, was enhanced by pretreatment with mAb 8A2. A qualitative alteration of β
1
integrin was assessed with mAb 15/7, which binds to an activation-dependent epitope on the β
1
subunit. Binding of mAb 15/7 was enhanced by mAb 8A2 in a dose-dependent manner. Arg-Gly-Asp peptide and soluble fibronectin also enhanced expression of the 15/7 epitope, suggesting that the 15/7 epitope is closely related to the ligand-occupied state of β
1
integrin. Platelet-derived growth factor (PDGF)-AA and -BB increased SMC adhesion to type I collagen but did not augment mAb 15/7 binding, suggesting that PDGFs increase binding avidity by a postreceptor mechanism. In addition, mAb 8A2 inhibited PDGF-BB–induced SMC migration through Matrigel-coated filters. These results suggest that avidity modulation of β
1
integrin may play an important role in the function of SMCs.
Adenosine-Induced Vasoconstriction In Vivo
Authors: Shepherd Rebecca K. , Linden Joel , Duling Brian R.
Published: June 2012

Abstract
Adenosine, a vasodilator metabolite, is often produced in tissues where the demand for oxygen exceeds the supply. We have recently demonstrated in isolated cannulated arterioles that adenosine and its metabolite, inosine, can also cause vasoconstriction by stimulation of mast cells. Secondary release of histamine and thromboxane is responsible for the inosine-induced constriction in vivo. In the present study, we explored the vasomotor effects of adenosine in vivo and investigated the role of the A
3
adenosine receptor in mediating vasoconstriction. In vivo, local application of adenosine (10
−6
to 10
−4
mol/L) to arterioles consistently caused dose-dependent vasodilation. A fraction of arterioles, however, exhibited a biphasic response, with constriction following dilation. This, too, was dose dependent; 37% of arterioles constricted by 12.7±4.3% of the initial diameter in response to 10
−4
mol/L adenosine. In the presence of 8-(
p
-sulfophenyl)theophylline (8-SPT), an antagonist of A
1
and A
2
adenosine receptors, dilation in response to the same dose of adenosine was reduced, and constriction was enhanced; 85% of the tested arterioles constricted by −44.3±6.0% of the initial diameter. The A
3
adenosine receptor has been shown to facilitate mediator release from mast cells, and its role was also examined.
N
6
-(3-Iodo-4-aminobenzyl)adenosine (I-ABA), an agonist of A
1
and A
3
adenosine receptors, produced dose-dependent vasoconstriction. 1,3-Dipropyl-8-(4-acrylate)phenylxanthine (BW-A1433), an antagonist of A
1
, A
2
, and A
3
receptors, significantly reduced the vasoconstrictor response to adenosine, which was unmasked during treatment with 8-SPT. In addition, both adenosine and I-ABA stimulated mast cell uptake of ruthenium red, indicating degranulation. The I-ABA–induced constriction was abolished by combined histamine and thromboxane receptor antagonists. We conclude that adenosine can cause vasoconstriction in vivo, which is often masked by A
2
receptor–mediated vasodilation. Mast cells are stimulated in the course of the response, and the A
3
adenosine receptor is involved in mediating constriction.
Lysophosphatidylcholine Promotes P-Selectin Expression in Platelets and Endothelial Cells
Authors: Murohara Toyoaki , Scalia Rosario , Lefer Allan M.
Published: June 2012

Abstract
Lysophosphatidylcholine (LysoPC), an atherogenic lysophospholipid contained in oxidized low-density lipoprotein (LDL), has been shown to stimulate protein kinase C (PKC). Since PKC activators are suggested to elicit rapid P-selectin expression in platelets and endothelial cells, we examined whether LysoPC promotes P-selectin expression in platelets and P-selectin–mediated leukocyte adherence to endothelial cells via a mechanism involving PKC activation. LysoPC, but not phosphatidylcholine (PC), which is a major phospholipid component in native LDL, significantly upregulated P-selectin on cat platelets by flow cytometric analysis. This P-selectin upregulation by LysoPC was significantly attenuated by two PKC inhibitors, 7-hydroxystaurosporine (UCN-01) and
N,N,N
-trimethylsphingosine, and by two NO donors, CAS1609 and sodium nitroprusside. Submicellar concentrations of LysoPC significantly activated PKC in platelets, and this was inhibited by either UCN-01 or CAS1609. LysoPC, but not PC, significantly increased adherence of autologous cat polymorphonuclear leukocytes to coronary vascular endothelium, which was also markedly attenuated by UCN-01 and by CAS1609. LysoPC induced P-selectin expression on the surface of cat coronary vascular endothelium as assessed by immunohistochemical analysis. These results suggest that LysoPC, an atherogenic lysophospholipid contained in oxidized LDL, rapidly induces P-selectin expression in both platelets and endothelial cells at least partially via PKC activation. Furthermore, NO-generating agents may inhibit P-selectin upregulation by LysoPC. Since P-selectin may play an important role in initiating atherosclerosis, our data provide further insight into the mechanism of early stages of atherogenesis and of NO-mediated inhibition of atherosclerosis.
Histamine H
3
-Receptor–Mediated Inhibition of Calcitonin Gene-Related Peptide Release From Cardiac C Fibers
Authors: Imamura Michiaki , Smith Neil C.E. , Garbarg Monique , Levi Roberto
Published: June 2012

Abstract
Antidromic stimulation of cardiac sensory C fibers releases calcitonin gene-related peptide (CGRP), which increases heart rate, contractility, and coronary flow. C-fiber endings are closely associated with mast cells, and CGRP may release mast-cell histamine. Because prejunctional histamine H
3
-receptors inhibit transmitter release from autonomic nerves, we tested the hypothesis that H
3
-receptors modulate CGRP release in the heart. CGRP released by bradykinin in the electrically paced guinea pig left atrium and by capsaicin in the spontaneously beating isolated heart caused marked positive inotropic and chronotropic effects, respectively. Capsaicin significantly enhanced the overflow of CGRP (fivefold) and histamine (twofold) into the coronary effluent. All of these effects were prevented by prior chemical destruction of C fibers in vivo. The H
3
-receptor agonist imetit attenuated the inotropic response to bradykinin by 50%. Imetit also decreased the capsaicin-induced tachycardia and the increase in CGRP overflow by 50%. Imetit, however, did not modify the response to exogenous CGRP. The effects of imetit were blocked by the H
3
-receptor antagonist thioperamide. Notably, thioperamide by itself potentiated the capsaicin-evoked increases in heart rate and CGRP overflow (by 25% and 50%, respectively). Thus, our findings identify a negative-feedback loop, whereby CGRP releases histamine from cardiac mast cells and histamine in turn inhibits CGRP release by activating H
3
-receptors on C-fiber terminals. Because CGRP release is augmented in pathophysiological conditions, such as septic shock, heart failure, and acute myocardial infarction, modulation of CGRP release may be clinically relevant.
Hepatocyte Growth Factor and Its Receptor Are Expressed in Cardiac Myocytes During Early Cardiogenesis
Authors: Rappolee Daniel A. , Iyer Anand , Patel Yogesh
Published: June 2012

Abstract
In the mouse, the heart primordium arises when mesoderm is set aside during gastrulation, is induced by pharyngeal endoderm, migrates ventrally to the midline of the embryo, forms a tube, and begins beating. Little is known of the molecular mechanisms that mediate the determination, mitosis, differentiation, and migration that lead to the beating heart. Transcripts for hepatocyte growth factor/scatter factor (HGF) and its receptor are coexpressed transiently and dynamically in the premyocardium but not in other heart progenitor cells. Transcripts for HGF ligand and receptor are first detected before cardiac function and looping and persist through the first looping stage, when heart morphology begins to elaborate. HGF ligand and receptor mRNA are detectable after the putative heart transcription factor, Csx/Nkx2-5, and concomitantly with the heart structural gene, cardiac actin. HGF receptor mRNA is detected in the mesoderm of the headfold stage and persists in myocardial precursors of the ventricles and atria (but not in the outflow-tract smooth muscle cells) through the 14-somite stage at ≈8.75 days after fertilization (day E8.75). At the headfold stage, between E7.5 and E8.0, HGF receptor mRNA was detected in myocardial cells before fusion at the ventral midline. HGF ligand and receptor mRNA transcripts are coexpressed in the embryo, except in the headfold stage (when only the HGF receptor can be detected) and in the heart at the 14- to 18-somite stage (when only HGF ligand can be detected). The dynamic pattern of coexpression suggests an autoregulatory role for HGF and its receptor in early heart development.
Upregulation of Cardiac Uptake
1
Carrier in Ischemic and Nonischemic Rat Heart
Authors: Ungerer Martin , Chlistalla Andreas , Richardt Gert
Published: June 2012

Abstract
Neuronal uptake
1
constitutes the main elimination process of cardiac norepinephrine under normoxic conditions. Uptake
1
may be subject to changes during myocardial ischemia. We therefore studied the regulation of the uptake
1
carrier in isolated perfused rat hearts, comparing ischemic and nonischemic conditions. Radioligand binding with [
3
H]mazindol was used to determine carrier densities and affinities, whereas cardiac clearance of [
3
H]norepinephrine served as a measure of the transport capacity of the uptake
1
carrier. When exocytotic norepinephrine release was induced in nonischemic rat hearts by electrical field stimulations, we observed an increase in the cardiac density of uptake
1
carriers (B
max
) to 210±5 fmol/mg protein (versus 134±3 fmol/mg in control hearts). Simultaneously, the cardiac clearance of [
3
H]norepinephrine increased to 41±4% versus 30±4% in control hearts. Both carrier density and norepinephrine clearance returned to baseline values within a period of 40 minutes after stimulation. Carrier affinities (
K
d
values) did not differ between the groups. Stop-flow ischemia induced a substantial overflow of norepinephrine by itself. Additionally, carrier density was increased to 144% after 40 minutes of stop-flow ischemia (
P
<.005 versus control hearts). When ischemia was followed by 20 minutes of reperfusion, the B
max
of the uptake
1
carrier remained significantly elevated. With a further extension of the reperfusion period to 40 minutes, however, carrier density declined to baseline values.
K
d
values were not influenced by any of these interventions. Clearance of [
3
H]norepinephrine was suppressed (to 5±2%) in the first minutes of reperfusion, which may reflect the inverse transport direction of the norepinephrine carrier known to occur in ischemia. After 20 minutes of reperfusion, clearance increased to 39±5% (
P
<.005 versus control hearts) and then fell to 29±5% after 40 minutes of reperfusion (NS). These results demonstrate that after both electrical field stimulation and myocardial ischemia, the density of uptake
1
carrier proteins temporarily increases, which may result in an increased transport capacity for norepinephrine.
Ca
2+
-Dependent Mitogen-Activated Protein Kinase Activation in Spontaneously Hypertensive Rat Vascular Smooth Muscle Defines a Hypertensive Signal Transduction Phenotype
Authors: Lucchesi Pamela A. , Bell Jeremy M. , Willis Laura S. , Byron Kenneth L. , Corson Marshall A. , Berk Bradford C.
Published: June 2012

Abstract
The mechanisms responsible for altered vascular smooth muscle cell (VSMC) function in hypertension remain unknown. In the spontaneously hypertensive rat (SHR) model of genetic hypertension, there are multiple abnormalities in VSMC function, including increased growth, Na
+
-H
+
exchange, and increased signal transduction by protein kinase C. The family of kinases termed mitogen-activated protein (MAP) kinases has recently been shown to be essential mediators of growth factor signal transduction. In the present study, alterations in MAP kinase function in the hypertensive phenotype were investigated using early-passage SHR and Wistar-Kyoto (WKY) VSMCs stimulated with angiotensin II (Ang II, 100 nmol/L) or platelet-derived growth factor-BB (PDGF-BB, 10 ng/mL). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Two differences between SHR and WKY rats were observed for Ang II–mediated MAP kinase activation: (1) Inactivation after Ang II stimulation was more rapid in SHR than WKY VSMCs. (2) Activity in SHR VSMCs showed a greater dependence on Ca
2+
mobilization, since chelation of intracellular Ca
2+
with BAPTA inhibited maximal activity by 95% in SHR VSMCs but by only 50% in WKY VSMCs. In contrast to the results with Ang II, no differences in PDGF-stimulated MAP kinase activity were observed. These findings establish activation of MAP kinase by Ang II as a feature that distinguishes SHR VSMCs from WKY VSMCs and suggest that differences in regulation of MAP kinase signaling may alter cellular events that are increased in the SHR genetic model of hypertension.
Protein Kinase C Isoform Expression in Normal and Failing Rabbit Hearts
Authors: Rouet-Benzineb Patricia , Mohammadi Kamiar , Pérennec Jeannine , Poyard Madeleine , El Houda Bouanani Nour , Crozatier Bertrand
Published: June 2012

Protein kinase C (PKC) is activated by α-adrenergic stimulation. Molecular analysis showed that PKC consists of a family of at least 12 isozymes. Studies of their distribution in the heart showed conflicting results. The first goal of our study was thus to characterize cardiac PKC in normal rabbits. PKC plays an important role in gene expression, cell growth, and differentiation and is involved in the hypertrophy phase of cardiac overload, but since its expression has never been evaluated in heart failure, the second goal of our study was to evaluate PKC activity and isoform expression in rabbits with heart failure induced by a double hemodynamic overload (aortic insufficiency followed by an aortic stenosis). In the first part of the study, PKC isoform expression analyzed in normal rabbits by immunoblotting showed that isoforms α, β, ε, and ζ were expressed along with PKCγ, which had never been detected in the heart. PKCγ expression was also identified by polymerase chain reaction, and immunofluorescence techniques showed a localization on intercalated disks associated with the membrane localization observed with the other isoforms. In the second part of the study, PKC activity, content, and isoform expression showed a decrease of 37% in the failing group. PKC immunodetection with a monoclonal antibody (Mab 1.9) recognizing the catalytic domain of all PKC isoforms revealed a 20% decrease in the failing ventricles compared with normal left ventricles. Expressed PKC isoforms quantified by Western blot showed, in the failing heart group compared with the control group, a decrease of 27%, 32%, 16%, and 9% of PKCα, PKCβ
1
, PKCγ, and PKCε, respectively, whereas PKCζ was not significantly modified. These results show that, in heart failure, PKC activity and expression of Ca
2+
-dependent PKC isoforms are decreased. This may lead to alterations of PKC-induced phosphorylations.