bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Effects of Pressure and Flow on DNA and Protein Synthesis and on Fibronectin Expression by Arteries in a Novel Organ Culture System
Authors: Bardy Nathalie , Karillon Gaëtan J. , Merval Régine , Samuel Jane-Lyse , Tedgui Alain
Published: June 2012

Abstract
Structural adaptation of the blood vessel wall occurs in response to mechanical factors related to blood pressure and flow. To elucidate the relative roles of pressure, flow, and medium composition, we have developed a novel organ culture system in which rabbit thoracic aorta, held at in vivo length, can be perfused and pressurized at independently varied flow and pressure for several days. Histology and histomorphometry, as well as scanning electron microscopy, revealed a well-preserved wall structure. In arteries perfused and pressurized at 80 mm Hg, endothelial injury led to a 2-fold increase in [
3
H]thymidine incorporation in the media, which peaked at 3 to 5 days and returned to baseline level at 6 to 8 days. In intact endothelialized vessels cultured for 3 days under no-flow conditions, pressure per se had no effect on DNA synthesis. In contrast, in the presence of serum, total protein synthesis, as assessed by [
35
S]methionine incorporation into the media, was enhanced 6-fold at 150 mm Hg compared with vessels pressurized at 0 or 80 mm Hg. In intact vessels perfused at a constant flow of 40 mL/min for 3 days, DNA synthesis was unchanged regardless of the pressure level when vessels were cultured in the presence of serum but increased 8-fold at both 80 and 150 mm Hg in the absence of serum. Unlike DNA synthesis, total protein synthesis was enhanced 12-fold by flow regardless of the presence or absence of serum. Expression of fibronectin was markedly enhanced at high transmural pressure, and serum potentiated its expression in the arterial wall. This novel organ culture system of perfused and pressurized vessels allowed identification of differential effects of pressure, flow, and serum on DNA and total protein synthesis, including cellular fibronectin expression.
cGMP-Dependent Protein Kinase Regulation of the L-Type Ca
2+
Current in Rat Ventricular Myocytes
Authors: Sumii Kotaro , Sperelakis Nicholas
Published: June 2012

Abstract
Regulation of L-type Ca
2+
channel current [I
Ca(L)
] by cGMP-dependent protein kinase (PK-G) was investigated in ventricular myocytes from 2- to 21-day-old rats using whole-cell voltage clamp with internal perfusion. I
Ca(L)
was elicited by a depolarizing pulse to +10 mV from a holding potential of −40 mV. Stimulated I
Ca(L)
(by 2 μmol/L isoproterenol) was inhibited to the basal level by internal perfusion with 50 nmol/L PK-G (activated by 8Br-cGMP, 0.1 μmol/L). When I
Ca(L)
was enhanced by Bay K 8644 (1 μmol/L), the enhanced basal I
Ca(L)
was also reduced by PK-G. Basal I
Ca(L)
(nonstimulated through the cAMP/cAMP-dependent protein kinase [PK-A] pathway) was also inhibited to various degrees (large, medium, or small) by internal application of PK-G (25 nmol/L). The average inhibition was 42.1% (n=36), and there were no differences in the inhibition during development. The inhibition by PK-G was blocked by the PK-G substrate peptide (cG-PKI, 300 μmol/L) and by heat inactivation of the PK-G. Relatively specific PK-G inhibitors (eg, cG-PKI and H-8) sometimes reversed the inhibition (5 of 25 cells), whereas isoproterenol stimulated I
Ca(L)
(7 of 8 cells). When a holding potential of −80 mV was used, the inhibition produced by PK-G was much less. The inhibitory effects of PK-G were not mediated by activating phosphodiesterase or protein phosphatase but most likely by a direct phosphorylation of the Ca
2+
channel or associated regulatory protein. The inhibitory effect of PK-G may be explained by a balance between activities of PK-A and PK-G in regulating the slow Ca
2+
channels at two separate sites.
Regional Capture of Fibrillating Ventricular Myocardium
Authors: KenKnight Bruce H. , Bayly Philip V. , Gerstle Ronald J. , Rollins Dennis L. , Wolf Patrick D. , Smith William M. , Ideker Raymond E.
Published: June 2012

Abstract
Previous investigations have suggested that during ventricular fibrillation (VF) pacing stimuli are incapable of evoking propagated ventricular activations. To determine whether regional myocardial capture could be achieved during rapid pacing in VF, extracellular unipolar potentials were sampled (2 kHz) and recorded from 506 Ag-AgCl electrodes arranged in a rectangular grid (22×23, 1.12-mm spacing) embedded in a plaque overlying two pacing electrodes in the epicardium of the anterobasal right ventricle in pentobarbital-anesthetized pigs (25 to 30 kg, n=6). During separate episodes of electrically induced VF, two bursts of 40 monophasic stimuli (10 mA, 2-millisecond duration) were asynchronously applied to the stimulating electrodes in either a bipolar, unipolar anodal, or unipolar cathodal mode. Evidence of regional capture was provided by (1) animating the first temporal derivative of the extracellular potentials, (2) analyzing interbeat interval patterns, and (3) employing the Karhunen-Loeve decomposition method to quantify the repetitiveness of spatiotemporal patterns of activation. Regional capture of ventricular myocardium during VF was observed when pacing stimuli fell late in the local myocardial activation interval and when the pacing cycle length was 80% to 115% of the mean subplaque activation cycle length. When myocardial activations became phase locked to the pacing stimuli, repeatable spatiotemporal patterns of activation followed each stimulus. Poincaré sections at the plaque border revealed that during VF prior to pacing, interbeat intervals were irregular but were driven by pacing to stable fixed values at times corresponding to our qualitative declaration of regional capture. A similar correspondence was demonstrated between the time of capture, defined by direct observation of the activation patterns, and a rise in the power contained in the first two spatial modes of a Karhunen-Loeve decomposition. These data demonstrate that appropriately timed stimuli produce regional capture of fibrillating right ventricular myocardium in the pig and support the existence of an excitable gap during VF in this model.
VEGF
165
Expressed by a Replication-Deficient Recombinant Adenovirus Vector Induces Angiogenesis In Vivo
Authors: Mühlhauser Judith , Merrill Marsha J. , Pili Roberto , Maeda Hiroyuki , Bacic Mima , Bewig Burkhard , Passaniti Antonino , Edwards Nancy A. , Crystal Ronald G. , Capogrossi Maurizio C.
Published: June 2012

Abstract
To evaluate the concept that localized delivery of angiogenic factors via virus-mediated gene transfer may be useful in the treatment of ischemic disorders, the replication-deficient adenovirus (Ad) vector AdCMV.VEGF
165
(where CMV is cytomegalovirus and VEGF is vascular endothelial growth factor) containing the cDNA for human VEGF
165
, a secreted endothelial cell–specific angiogenic growth factor, was constructed. Human umbilical vein endothelial cells (HUVECs) and rat aorta smooth muscle cells (RASMCs) infected with AdCMV.VEGF
165
(5 and 20 plaque-forming units [pfu] per cell) demonstrated VEGF mRNA expression and protein secretion into the supernatant. Furthermore, the conditioned medium from these cells enhanced vascular permeability in vivo. In contrast, neither VEGF mRNA nor secreted protein was found in uninfected HUVECs or RASMCs or in cells infected with the control vector AdCMV.βgal (where βgal is β-galactosidase). Assessment of starved HUVECs at 14 days demonstrated sixfold more cells for AdCMV.VEGF
165
-infected HUVECs (20 pfu per cell) than for either infected or uninfected control cells. RASMC proliferation was unaffected by infection with AdCMV.VEGF
165
. When plated in 2% serum on dishes precoated with reconstituted basement membrane (Matrigel), HUVECs infected with AdCMV.VEGF
165
(20 pfu per cell) differentiated into capillary-like structures. Under similar conditions, both uninfected HUVECs and HUVECs infected with AdCMV.βgal did not differentiate. To evaluate the ability of AdCMV.VEGF
165
to function in vivo, either AdCMV. VEGF
165
or AdCMV.βgal (2×10
10
pfu) was resuspended in 0.5 mL Matrigel and injected subcutaneously into mice. Immunohistochemical staining demonstrated VEGF in the tissues surrounding the Matrigel plugs containing AdCMV.VEGF
165
up to 3 weeks after injection, whereas no VEGF was found in the control plugs with AdCMV.βgal. Two weeks after injection, there was histological evidence of neovascularization in the tissues surrounding the Matrigel containing AdCMV.VEGF
165
, whereas no significant angiogenesis was observed in response to AdCMV.βgal. Furthermore, the Matrigel plugs with AdCMV.VEGF
165
demonstrated hemoglobin content fourfold higher than the plugs with AdCMV.βgal. Together, these in vitro and in vivo studies are consistent with the concept that Ad vectors may provide a useful strategy for efficient local delivery of VEGF
165
in the treatment of ischemic diseases.
T-Cell Lymphokines, Interleukin-4 and Gamma Interferon, Modulate the Induction of Vascular Smooth Muscle Cell Tissue Plasminogen Activator and Migration by Serum and Platelet-Derived Growth Factor
Authors: Wang Weizheng , Chen Hong Jun , Giedd Kenneth N. , Schwartz Allan , Cannon Paul J. , Rabbani LeRoy E.
Published: June 2012

Abstract
Platelet-derived growth factor (PDGF)–induced smooth muscle cell (SMC) fibrinolysis is necessary for SMC migration. In order to determine whether the T-cell lymphokines interleukin-4 (IL-4) and gamma interferon (γ-IFN) affect SMC fibrinolysis and migration, we examined the effects of human recombinant IL-4 and γ-IFN on human aortic SMC tissue-type plasminogen activator (TPA), urokinase-type plasminogen activator (UPA), and plasminogen activator inhibitor type-1 (PAI-1) antigen production, as determined by enzyme-linked immunosorbent assays. Although IL-4 had no direct effect on SMC TPA antigen, IL-4 potentiated SMC TPA antigen levels and activity in conditioned media and cellular lysates in media containing 2% fetal bovine serum but did not change UPA or PAI-1 production. γ-IFN attenuated IL-4 augmentation of SMC TPA antigen production in conditioned media, although γ-IFN itself had no direct effects on SMC TPA and PAI-1 antigen production. IL-4 augmented PDGF induction of SMC TPA antigen. γ-IFN inhibited PDGF induction of SMC TPA antigen and IL-4 potentiation of this process. γ-IFN diminished the promigratory effects of both IL-4 and PDGF on in vitro SMC migration. Tranexamic acid, a plasmin inhibitor, abrogated the stimulation of SMC migration by IL-4. Therefore, IL-4 and γ-IFN modulate the induction of SMC TPA and SMC migration by 2% fetal bovine serum and PDGF.
Effect of Ischemic Preconditioning of the Myocardium on cAMP
Authors: Sandhu Reena , Thomas Usha , Diaz Roberto J. , Wilson Gregory J.
Published: June 2012

Abstract
Reduction of cAMP has been implicated in the protection of ischemic preconditioning (IP), but until now, this possibility has not been directly addressed. In this study, we found that in the in vivo rabbit heart, 10 to 30 minutes of sustained regional ischemia was accompanied by a nearly twofold rise in cAMP levels. This increase in cAMP was attenuated when sustained ischemia was preceded by IP induced with a single cycle of transient ischemia and reperfusion (TI/R) and prevented when ischemia was preceded by three cycles of TI/R. The mechanism of cAMP reduction by IP does not involve activation of protein kinase C (PKC), since the PKC inhibitor polymyxin B (24 mg/kg) did not raise cAMP levels during sustained ischemia in IP hearts. Furthermore, this effect is also not mediated by reduced responsiveness of the β-adrenergic effector pathway, since both nonischemic hearts and hearts subjected to three cycles of TI/R exhibited similar increases in cAMP in response to 5 μg/kg isoproterenol. However, propranolol (0.75 mg/kg) abolished the rise in cAMP levels observed during sustained ischemia in control hearts but did not reduce cAMP levels further in IP hearts. These data indicate that the ischemia-induced rise in cAMP levels in control hearts was mediated by activation of the β-adrenergic receptor. Taken together with data demonstrating that β-adrenergic responsiveness was not affected by IP, these data support the conclusion that the lack of elevation in cAMP levels observed during sustained ischemia in IP hearts is mediated by an attenuation of norepinephrine release. To examine whether the protection of IP against necrosis was mediated by the lack of elevation in cAMP levels, we determined whether the infarct size–limiting effect of IP could be blocked by NKH477, an activator of adenylyl cyclase. Four groups of rabbits were subjected to 30 minutes of in vivo regional ischemia and 90 minutes of reperfusion. Control hearts (n=10) had 53.6±5.5% infarction of the area at risk. IP with three cycles of transient ischemia limited infarct size to 3.2±1.3% (n=13,
P
<.0001). NKH477 (45 μg/kg) increased average cAMP levels in IP hearts during sustained ischemia to levels similar to those in untreated control hearts. However, NKH477 did not block IP (50.2±7.7% of the area at risk was infarcted in the control+NKH477 group [n=10] versus 10.0±5.9% in the IP+NKH477 group [n=7],
P
<.05). Therefore, we conclude that although IP lowers cAMP levels during sustained ischemia, this effect is not necessary for its protection against necrosis, since raising cAMP does not block this protection of IP.
Hypoxia and Hypoxia/Reoxygenation Activate Raf-1, Mitogen-Activated Protein Kinase Kinase, Mitogen-Activated Protein Kinases, and S6 Kinase in Cultured Rat Cardiac Myocytes
Authors: Seko Yoshinori , Tobe Kazuyuki , Ueki Kohjiro , Kadowaki Takashi , Yazaki Yoshio
Published: June 2012

Abstract
In response to hypoxia and reoxygenation, mammalian cells are known to express a variety of genes to adapt to these external stresses or lead to further cell damage. We investigated the intracellular signaling cascades in cultured rat cardiac myocytes subjected to hypoxia or hypoxia followed by reoxygenation (hypoxia/reoxygenation). Here, we show that both hypoxia and hypoxia/reoxygenation caused rapid activation of the mitogen-activated protein kinase kinase kinase (MAPKKK) activity of Raf-1. This was followed by the sequential activation of mitogen-activated protein kinase kinase (MAPKK), mitogen-activated protein (MAP) kinases, and S6 kinase (p90

rsk

). Furthermore, hypoxia caused hyperphosphorylation of Raf-1. The maximal hyperphosphorylation of Raf-1 appeared to be accompanied by a significant decrease in MAPKKK activity. These results strongly suggest the following: (1) Intracellular signals initiated by both hypoxia and hypoxia/reoxygenation converge on Raf-1 and activate its MAPKKK activity. Then, Raf-1 activates downstream serine/threonine kinases including MAPKK, MAP kinases, and p90

rsk

. (2) Raf-1 is not only located upstream from MAPKK and MAP kinases but also may be phosphorylated by MAP kinases directly or indirectly, and at least Raf-1 kinase activity may be downregulated by this feedback mechanism.
Establishment of the Mesodermal Cell Line QCE-6
Authors: Eisenberg Carol A. , Bader David M.
Published: June 2012

Abstract
The QCE-6 cell line was derived from precardiac mesoderm of the Japanese quail. As previously reported, these cells are able to differentiate into two distinct cardiac cell types with myocardial or endocardial endothelial cell properties. This present communication describes in detail the derivation of this cell line and further characterizes the nontreated and induced myocardial and endothelial phenotypes of these cells. The QCE-6 cells exhibit an epithelial morphology, as well as the pattern of protein expression, that is characteristic of precardiac mesoderm. Treatment with retinoic acid, basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-β2, and TGF-β3 induces these cells to differentiate and produce mixed cultures of epithelial and mesenchymal cells. The epithelial cells express myosin, desmin, and cardiac troponin I in a punctate pattern throughout the cytoplasm. These sarcomeric proteins become organized in a premyofibrillar pattern when TGF-β1, platelet-derived growth factor (PDGF)-BB, and insulin-like growth factor (IGF) II are added in combination along with retinoic acid, bFGF, TGF-β2, and TGF-β3. Also, these treatments induce Na
+
,K
+
-ATPase expression. When the QCE-6 cells are cultured on collagen type I, the mesenchymal cells that are promoted by retinoic acid, bFGF, TGF-β2, and TGF-β3 will invade the gel. These mesenchymal cells are positive for QH1 and JB3, which are both markers for presumptive endocardial cells within the early cardiogenic mesoderm. The addition of both PDGF-BB and IGF II to QCE-6 cell cultures will inhibit the ability of retinoic acid, bFGF, TGF-β2, and TGF-β3 to induce both the mesenchymal morphology and QH1 and JB3 expression. Collectively, these results suggest that the process of cardiac cell differentiation is regulated by multiple signals and that early cardiogenic mesoderm contains a bipotential stem cell that can give rise to both the myocardial and endocardial lineages. More important, since the QCE-6 cells are representative of early cardiogenic cells, this cell line offers a unique model system to study cardiac cell differentiation.
Intestinal Absorption of Sodium and Nitric OxideDependent Vasodilation Interact to Dominate Resting Vascular Resistance
Authors: Bohlen H. Glenn , Lash Julia M.
Published: June 2012

Abstract
The villi of the small intestine maintain a hypertonic interstitium at all times, and the submucosal glands constantly secrete ions and accompanying water into the lumen. Generation of the 400- to 600-mOsm interstitial fluid in the villus and secretion by glands may require a large expenditure of energy and, consequently, have major effects on intestinal vascular regulation to supply oxygen and nutrients. Blood flow and oxygen consumption were measured in the ileum of anesthetized rats during natural resting conditions with physiological sodium chloride in the bathing fluid and during isosmotic replacement of sodium chloride with mannitol. Microvascular pressures and blood flow were used to determine the changes in resistance of the major arterioles and the terminal vasculature. When mannitol replaced sodium chloride in contact with the villi, intestinal blood flow decreased to 58.6±2.8% of control, and oxygen consumption was 54.2±3.4% of control. Resistance of the major arterioles increased 101.7±9.9%, and that of the terminal vasculature increased 40.4±6.2%. The increased resistance appeared to be caused by suppression of a nitric oxide mechanism. Local application of 10
−4
mol/L
N
G
-nitro-
l
-arginine methyl ester caused about the same reduction in flow and increases in regional vascular resistance as during replacement of sodium but did not alter the oxygen consumption. These data indicate that about half of the intestinal metabolic rate during natural resting conditions is devoted to sodium secretion/absorption. Large resistance vessels are dilated to maintain a high blood flow through release of nitric oxide. We propose that dilation of the terminal vasculature in the metabolically active tissues increased flow velocity sufficiently in the major resistance vessels to cause a flow-mediated release of nitric oxide.
Interaction of Cytotoxic T Lymphocytes and Guinea Pig Ventricular Myocytes
Authors: Felzen Bella , Lavy Revital , Garcia Maria , Berke Gideon , Gardner Phyllis , Binah Ofer
Published: June 2012

Abstract
Infiltrating cytotoxic T lymphocytes (CTLs) are important immune effectors that damage the myocardium during heart transplant rejection as well as in cardiomyopathy and Chagas’ heart disease. We have previously shown that in an in vitro model of murine-derived peritoneal exudate CTL (PEL)–guinea pig ventricular myocyte interaction, PEL induced in conjugated myocytes reduction of resting membrane potential and action potential (AP) amplitude, shortening of AP duration, delayed afterdepolarizations (DADs), and myocyte contracture and destruction. Since these findings indicated that cytotoxicity was largely caused by [Ca
2+
]
i
overload, in the present study we tested the hypothesis that blocking the L-type Ca
2+
current (I
Ca,L
) in the myocyte will eliminate the trigger for Ca
2+
release from intracellular stores and will reduce [Ca
2+
]
i
overload and subsequent myocyte deterioration. CoCl
2
(3 mmol/L) prevented PEL-induced AP changes, induction of DADs, and myocyte destruction. Since verapamil (2 μmol/L) was ineffective, indicating that the CoCl
2
protection was not due to block of I
Ca,L
, we tested whether the different action of these Ca
2+
channel blockers was due to their differential effect on the PEL’s K
+
current (I
K
), previously shown to participate in lymphocyte activation and cytotoxicity. In agreement with their protective efficacy, CoCl
2
but not verapamil blocked I
K
in PELs, suggesting that this is the mechanism for the protection provided by CoCl
2
. To support this notion, we tested the effect of the scorpion-derived peptide margatoxin (10 nmol/L), a specific K
+
channel blocker in lymphocytes, on PEL-myocyte interaction and on PEL’s I
K
; margatoxin prevented PEL-induced cytotoxicity and also blocked I
K
in PEL. Based on these findings, an alternative modality for attenuating CTL-induced lymphocytotoxicity is proposed.
In Vivo Survival and Function of Transplanted Rat Cardiomyocytes
Authors: Li Ren-Ke , Mickle Donald A.G. , Weisel Richard D. , Zhang Ji , Mohabeer Molly K.
Published: June 2012

Abstract
Recent studies have demonstrated the feasibility of transplanting fetal mouse cardiomyocytes into the hearts of adult syngeneic mice. However, the function of the transplanted cardiomyocytes and their capacity to survive in fibrous connective tissue were not assessed. In the present study, we evaluated the viability and contractility of transplanted fetal and neonatal rat cardiomyocytes in the connective tissue of the adult rat hindlimb. Purified fetal or neonatal rat cardiomyocytes were cultured. These cells contained sarcomeres, formed junctions composed of desmosomes and fascia adherens, and contracted regularly and spontaneously. A fetal or neonatal cardiomyocyte suspension was injected into the subcutaneous tissue of adult rat hindlimbs. Cyclosporin A (5 mg/kg) was administered subcutaneously daily for the 3-month duration of the study, at which time the animals were killed. The transplanted cardiomyocytes formed ‘tissue’ in vivo that increased in size for the first 2 weeks and remained the same size at the third week. The tissue derived from the transplanted fetal cardiomyocytes contracted spontaneously at a rate of 73±12 bpm, and that from the neonatal cardiomyocytes contracted at a rate of 43±21 bpm. The electrocardiogram was similar to that seen in myocardium with an idioventricular rhythm. Histologically, the tissue appeared to be cardiac muscle with sarcomeres. Angiogenesis occurred in the cardiomyocyte graft. In summary, a cell suspension of cultured fetal and neonatal rat cardiomyocytes transplanted into the adult rat hindlimb formed contractile cardiac tissue in the subcutaneous connective tissue.
DNA Synthesis in Adult Feline Ventricular Myocytes
Authors: Kozlovskis Patricia L. , Smets Marcel J.D. , Strauss William L. , Myerburg Robert J.
Published: June 2012

Abstract
Adult mammalian ventricular myocytes are terminally differentiated cells, and the prevailing perception has been that DNA synthesis and repair are not active. We tested the hypothesis that there is potential for DNA synthesis and repair by studying the ability of whole-cell extracts from adult myocytes to incorporate [α-
32
P]dCTP into damaged plasmids. Left ventricular myocytes were isolated from adult cat hearts by collagenase dissociation. Cells were maintained in room air (control extract, CE) or made ischemic (IE) with N
2
displacement of O
2
and extracted for total protein. The nicked form of the plasmid was produced by exposure to an Fe
3+
/ascorbic acid free radical generating system. Both IE and CE degraded the supercoiled form of the plasmid and incorporated [α-
32
P]dCTP into the nicked (
32
P/DNA mass; CE=2.2, IE=3.0) and linear forms (
32
P/DNA mass; CE=28.7, IE=25.2). Exposure of plasmids to UV light did not inhibit incorporation of label. Inhibition studies with the cell extracts suggested a participation of polymerase δ in myocyte DNA repair/synthesis. Myocyte extract was as active as extract from rapidly growing COS cells at incorporating labeled nucleotides into plasmid DNA. The ability of intact myocytes to incorporate [α-
32
P]dCTP into endogenous DNA was measured in isolated cells made permeable with saponin. Studies were done in room air or N
2
. Permeable cells incorporated [α-
32
P]dCTP into nuclear DNA, but maximal specific activity of DNA was observed at 15 minutes with ischemia and at 60 minutes with room air control cells (ischemia, 1.34±0.5, 0.86±0.33, 0.60±0.04; air, 1.0, 1.28±0.20, 1.87±0.38, at 15, 30, and 60 minutes, respectively). These data indicate that mammalian adult ventricular myocytes can actively repair and/or synthesize both exogenous and endogenous DNA. A DNA synthetic response to cellular damage may have important pathological and clinical implications.
Generation of New Intercellular Junctions Between Cardiocytes
Authors: Yamamoto Shoji , James Thomas N. , Sawada Ken-ichi , Okabe Makoto , Kawamura Keishiro
Published: June 2012

Abstract
Intercellular dehiscence is a common cardiocytic response to pathological conditions. However, little consideration has been given to the possibility of new intercellular junctions developing between cardiocytes within developed myocardium. To examine this possibility as it may relate to useful compensation for hemodynamic overloads, changes in cardiocytic connection were evaluated by scanning electron microscopy in hypertrophied myocardium of adult human hearts. Transmural myocardium of left ventricle was obtained at autopsy from five hearts with concentric hypertrophy, five hearts with eccentric hypertrophy, and five control hearts (noncardiac death). After formalin fixation, the number of cardiocytes connected to an individual cardiocyte was counted in tissues from the middle portion of the transmural samples by scanning electron microscopy. Cardiocytic diameter and connective tissue volume fraction were measured on the transmural sections by light microscopy. In concentrically hypertrophied hearts presenting both increased cardiocytic diameter and connective tissue volume fraction, the number of other cardiocytes connected to an individual cardiocyte (4.60±0.10 [mean±SE]) was significantly increased (
P
<.05) compared with control hearts (4.19±0.12) or eccentrically hypertrophied hearts (4.11±0.10). The increase in junctions per cardiocyte in concentrically hypertrophied hearts suggests that new connections had been generated. More junctions developing during hypertrophy could add another structural advantage to those of cardiocytic hypertrophy and connective tissue proliferation as compensatory adjustments to hemodynamic overload in concentrically hypertrophied hearts.
cGMP Formation in Rat Atrial Slices Is Ligand and Endothelin Receptor Subtype Specific
Authors: Shraga-Levine Zurit , Sokolovsky Mordechai
Published: June 2012

Abstract
Involvement of a cGMP pathway in signal transduction stimulated by endothelins (ETs) and sarafotoxins (SRTXs) was examined in rat atrial slices. These peptides activated different receptor-binding sites (ET-1 and SRTX-b reacted with the picomolar binding sites of the ET
A
receptor, and ET-3 and SRTX-c reacted with the nanomolar binding sites of the ET
B
receptor) to produce cGMP. ET-1 and SRTX-b stimulated an increase in cGMP levels via a Ca
2+
-dependent NO pathway involving a pertussis toxin–insensitive G protein, whereas ET-3 and SRTX-c elevated cGMP levels via a Ca
2+
-independent CO pathway involving a pertussis toxin–sensitive G protein. These results can best be explained in terms of formation of different ligand-receptor–G-protein complexes. The ligands had no effect on ventricular slices, indicating that these signal transduction mechanisms are unique to the atria.
Enhancement of the L-Type Ca
2+
Current by Mechanical Stimulation in Single Rabbit Cardiac Myocytes
Authors: Matsuda Naoki , Hagiwara Nobuhisa , Shoda Morio , Kasanuki Hiroshi , Hosoda Saichi
Published: June 2012

Abstract
Anion conductance is known to be activated by mechanical stimulation, such as osmotic cell swelling or cell inflation via the patch pipette, of canine or rabbit cardiac myocytes. The effects of mechanical stimulation on time-dependent currents, however, remain unsettled. Using the whole-cell voltage-clamp method, we have found that mechanical stimuli enhance the L-type Ca
2+
current (I
Ca,L
) in rabbit cardiac myocytes. At every membrane potential, I
Ca,L
was reversibly increased by osmotic cell swelling and by cell inflation caused by applying a positive pressure of 10 to 15 cm H
2
O via the patch pipette. I
Ca,L
was increased during cell inflation by 37±21% (mean±SD, n=17) in atrial cells and by 37±8% (n=7) in sinoatrial node cells in solution containing 2 mmol/L Ca
2+
. The current-voltage relationship, the inactivation time constant, the steady state inactivation curve, and the conductance properties of I
Ca,L
were all virtually unaffected by mechanical stimulation except for the open probability, which appears to increase. The increase in I
Ca,L
was not dependent on protein kinase A, since an inhibitor peptide of cAMP-dependent protein kinase failed to prevent the increase in I
Ca,L
during mechanical stimuli (n=5). The increase in I
Ca,L
caused by cell inflation was unaffected by the chelation of intracellular Ca
2+
by the addition of 10 mmol/L EGTA or 10 mmol/L BAPTA to the pipette solution, suggesting that the effect was not mediated by changes in intracellular Ca
2+
. Thus, mechanical stimulation due to cell swelling or inflation may itself directly increase I
Ca,L
in rabbit cardiac myocytes.
Inhibition of Inducible Nitric Oxide Synthase Prevents Myocardial and Systemic Vascular Barrier Dysfunction During Early Cardiac Allograft Rejection
Authors: Worrall Neil K. , Chang Kathy , Suau Gloria M. , Allison Wanda S. , Misko Thomas P. , Sullivan Patrick M. , Tilton Ronald G. , Williamson Joseph R. , Ferguson T. Bruce
Published: June 2012

Abstract
NO is produced during cardiac allograft rejection by expression of inducible NO synthase (iNOS) in the rejecting heart. Recent evidence indicates that NO modulates vascular permeability under both physiological and pathophysiological conditions. The present study explored the effects of early acute cardiac allograft rejection, and specifically the effects of NO, on myocardial and systemic vascular barrier function using a quantitative double-tracer permeation method in a rat cardiac transplant model. Early allograft rejection increased albumin permeation twofold to fivefold in the allograft heart and systemic vasculature (brain, lung, sciatic nerve, diaphragm, retina, muscle, kidney, and uvea) compared with isografts and controls. There were no detectable differences in regional blood flow or hemodynamics, suggesting that increased albumin permeation resulted from increased vascular permeability. iNOS mRNA was expressed in the allograft heart and native lung and was associated with increased serum nitrite/nitrate levels. iNOS inhibition with aminoguanidine prevented or attenuated allograft heart and systemic vascular barrier dysfunction and reduced allograft serum nitrite/nitrate levels to isograft values. Aminoguanidine did not affect the mild histological changes of rejection present in allografts. These data demonstrate the novel observations that (1) endothelial barrier function is compromised in the systemic vasculature, particularly in the brain, remote from the site of allograft rejection; (2) allograft vascular barrier dysfunction is associated with increased NO production and iNOS mRNA expression in the affected tissues (eg, native lung and grafted heart); and (3) inhibition of NO production by iNOS prevents vascular barrier dysfunction in the allograft heart and systemic vasculature.
Phospholamban Gene Dosage Effects in the Mammalian Heart
Authors: Luo Wusheng , Wolska Beata M. , Grupp Ingrid L. , Harrer Judy M. , Haghighi Kobra , Ferguson Donald G. , Slack Jay P. , Grupp Gunter , Doetschman Thomas , Solaro R. John , Kranias Evangelia G.
Published: June 2012

Abstract
Phospholamban ablation has been shown to result in significant increases in cardiac contractile parameters and loss of β-adrenergic stimulation. To determine whether partial reduction in phospholamban levels is also associated with enhancement of cardiac performance and to further examine the sensitivity of the contractile system to alterations in phospholamban levels, hearts from wild-type, phospholamban-heterozygous, and phospholamban-deficient mice were studied in parallel at the subcellular, cellular, and organ levels. The phospholamban-heterozygous mice expressed reduced cardiac phospholamban mRNA and protein levels (40±5%) compared with wild-type mice. The reduced phospholamban levels were associated with significant decreases in the EC
50
of the sarcoplasmic reticulum Ca
2+
pump for Ca
2+
and increases in the contractile parameters of isolated myocytes and beating hearts. The relative phospholamban levels among wild-type, phospholamban-heterozygous, and phospholamban-deficient mouse hearts correlated well with the (1) EC
50
of the Ca
2+
-ATPase for Ca
2+
in sarcoplasmic reticulum, (2) rates of relaxation and contraction in isolated cardiac myocytes, and (3) rates of relaxation and contraction in intact beating hearts. These findings suggest that physiological and pathological changes in the levels of phospholamban will result in parallel changes in sarcoplasmic reticulum function and cardiac contraction.
Variability of Spontaneous Ca
2+
Release Between Different Rat Ventricular Myocytes Is Correlated With Na
+
-Ca
2+
Exchange and [Na
+
]
i
Authors: Díaz M.E. , Cook S.J. , Chamunorwa J.P. , Trafford A.W. , Lancaster M.K. , O’Neill S.C. , Eisner D.A.
Published: June 2012

Abstract
We have studied the factors responsible for the variation of the frequency of “waves” caused by spontaneous Ca
2+
release in rat ventricular myocytes. The experiments were performed in isolated myocytes using the fluorescent indicators Indo-1 (to measure [Ca
2+
]
i
) and SBFI (to measure [Na
+
]
i
). After electrical stimulation (either with action potentials or voltage-clamp pulses), some cells showed spontaneous Ca
2+
release. The frequency of this release, where present, was variable. The Ca
2+
content of the sarcoplasmic reticulum (SR) was measured by applying caffeine (10 mmol/L). The resulting increase of [Ca
2+
]
i
activated the electrogenic Na
+
-Ca
2+
exchange, and the integral of this current was used to estimate the Ca
2+
content of the SR. The SR Ca
2+
content was significantly higher in cells that oscillated at high rates (>10·min
−1
) than in those that were quiescent. The rate of removal of Ca
2+
from the cytoplasm by non-SR mechanisms was measured by adding caffeine (10 mmol/L) and measuring the rate constant of decay of the resulting increase of [Ca
2+
]
i
. Cells that had a high rate constant of decay of [Ca
2+
]
i
had a low frequency of oscillations. Measurements of [Na
+
]
i
showed a positive correlation between the frequency of spontaneous SR Ca
2+
release and [Na
+
]
i
. After cessation of stimulation, there was a gradual decrease of [Na
+
]
i
, which was correlated with a parallel decrease of the frequency of oscillation rate. We conclude that the variability of frequency of spontaneous SR Ca
2+
release is due to variations of the rate of Ca
2+
removal from the cell, which are probably due to Na
+
-Ca
2+
exchange. The variability of Na
+
-Ca
2+
exchange rate, in turn, is likely to result from variations of [Na
+
]
i
.
Molecular Analysis of a Binding Site for Quinidine in a Human Cardiac Delayed Rectifier K
+
Channel
Authors: Yeola Sarita W. , Rich Tom C. , Uebele Vic N. , Tamkun Michael M. , Snyders Dirk J.
Published: June 2012

Abstract
The antiarrhythmic agent quinidine blocks the human cardiac hKv1.5 channel expressed in mammalian cells at therapeutically relevant concentrations (EC
50
, 6.2 μmol/L). Mechanistic analysis has suggested that quinidine acts as a cationic open-channel blocker at a site in the internal mouth of the ionic pore and that binding is stabilized by hydrophobic interactions. We tested these hypotheses using site-directed mutagenesis of residues proposed to line the internal mouth of the channel or of nearby residues. Amino acid substitutions in the midsection of S6 (T505I, T505V, T505S, and V512A) reduced the dissociation rate for quinidine, increased the affinity (0.7, 1.5, 3.4, and 1.4 μmol/L, respectively), and preserved both the voltage-dependent open channel–block mechanism and the electrical binding distance (0.19 to 0.22). In contrast, smaller or nonsignificant effects were observed for: deletion of the intracellular C-terminal domain, charge neutralizations in the region immediately C-terminal to S6, elimination of aromatic residues in S6, and mutations at the putative internal turn of the P loop, at the external entrance of the pore, and at sites in the S4S5 linker. The ≈10-fold increase in affinity with T505I and the reduction of the dissociation rate constant with the mutations that increased affinity are consistent with a hydrophobic stabilization of binding. Moreover, the T505 and V512 residues align on the same side of the putative α-helical S6 segment. Taken together, these results localize the hydrophobic binding site for this antiarrhythmic drug in the internal mouth of this human K
+
channel and provide molecular support for the open channel–block model and the role of S6 in contributing to the inner pore.
Novel Adenovirus Component System That Transfects Cultured Cardiac Cells With High Efficiency
Authors: Kohout Trudy A. , O’Brian Jeffrey J. , Gaa Shirley T. , Lederer W. Jonathan , Rogers Terry B.
Published: June 2012

Abstract
Although it is clear that gene transfection is a potentially valuable approach in the study of cardiac cell function and differentiation, classic transfection methods are limited by their poor efficiencies in cardiac cells. Recent studies show that recombinant replication-defective human adenovirus can transfect primary cardiac cultures with near 100% efficiency. Since such recombinants are time consuming to prepare, the goal of this study was to develop a plasmid/viral transfection system that would capitalize on the advantages of adenovirus. We have found that a “component system” formed by preincubation of Ad5dl312 adenovirus, poly-
l
-lysine, and an expression plasmid (
lac
Z reporter gene under control of the human cytomegalovirus (HCMV) major immediate early promoter) can transfect cultured cardiac cells. Optimal conditions were determined by quantifying β-galactosidase expression. Histochemical analysis of cultures revealed that the component system transfected 70% of the cells under these conditions.
Lac
Z-positive myocytes could be identified in intact myocytes with the fluorescent substrate C
12
-fluorescein di-β-galactopyranoside. Functional studies with such cells indicated that contractile behavior was maintained in transfected cardiocytes. Furthermore, the component system was used to transfect a DNA vector expressing a physiologically relevant protein, protein kinase Cδ. In summary, this powerful and simple approach can promote the expression of heterologous genes that can be studied at the biochemical and cellular level in cardiac cells.
Nitric Oxide Synthase (NOS3)–Mediated Cholinergic Modulation of Ca
2+
Current in Adult Rabbit Atrioventricular Nodal Cells
Authors: Han X. , Kobzik L. , Balligand J.-L. , Kelly R.A. , Smith T.W.
Published: June 2012

Abstract
We examined the role of endogenous NO in the autonomic regulation of atrioventricular (AV) nodal function by studying spontaneous action potentials (SAPs) and L-type Ca
2+
current (I
Ca-L
) in isolated single AV nodal cells from adult rabbit hearts. Both the perforated and the membrane-ruptured patch-clamp techniques in the whole-cell configuration were used under conditions known to alter NO production. Three NO donors, 3-morpholinosydnonimine (SIN-1, 0.1 mmol/L),
S
-nitroso-acetylcysteine (0.1 mmol/L), and sodium nitroprusside (0.1 mmol/L), suppressed the β-adrenergic agonist isoproterenol (ISO, 1 μmol/L)–stimulated increase in I
Ca-L
. SIN-1 also decreased the frequency and amplitude of SAPs. In cells in which I
Ca-L
had been previously attenuated by the muscarinic agonist carbamylcholine (CCh, 1 μmol/L), SIN-1 had no additive effect. CCh activated an acetylcholine-sensitive outward K
+
current (I
K(ACh)
) in AV nodal cells, in addition to the I
Ca-L
inhibition. Intracellular dialysis with the NO synthase inhibitor
N
-monomethyl-
l
-arginine (L-NMMA, 0.5 mmol/L) blocked CCh-induced, but not SIN-1–induced, I
Ca-L
attenuation. However, intracellular dialysis with methylene blue (20 μmol/L), which inhibits NO-mediated activation of guanylyl cyclase and cGMP production, blocked the effects of both CCh and SIN-1 on I
Ca-L
. In these cells, neither L-NMMA nor methylene blue affected the CCh-activated I
K(ACh)
. Direct application of cGMP (10 μmol/L) via internal dialysis significantly inhibited ISO-stimulated I
Ca-L
. In AV nodal cells internally perfused with either a nonhydrolyzable cAMP analogue, 8-Br-cAMP (0.5 mmol/L), or a high concentration of cAMP (0.5 mmol/L), CCh did not inhibit I
Ca-L
but still activated I
K(ACh)
. CCh-induced I
Ca-L
attenuation could be abolished or quickly reversed by the nonselective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (20 μmol/L). However, CCh still significantly suppressed ISO-stimulated I
Ca-L
after the cGMP-inhibited PDE isozyme (PDE3) had been selectively inhibited by milrinone (5 μmol/L). Immunohistochemical staining identified the presence of the endothelial constitutive NO synthase (ecNOS or NOS3) in both single AV nodal cells in vitro and in cryostat sections of AV nodal tissue in situ. These results demonstrate that endogenous NO is involved in the muscarinic cholinergic attenuation of I
Ca-L
in AV nodal cells; the mechanism likely involves the cGMP-stimulated PDE.
Modulatory Effects of Arachidonic Acid on the Delayed Rectifier K
+
Current in Rat Pulmonary Arterial Myocytes
Authors: Smirnov Sergey V. , Aaronson Philip I.
Published: June 2012

The effect of arachidonic acid (AA) on the delayed rectifier K
+
current (I
K
) was evaluated in rat pulmonary myocytes by using the whole-cell patch-clamp technique. Externally applied AA (50 μmol/L) caused a membrane depolarization, averaging 16 mV in six cells. AA (1 to 50 μmol/L) caused a dual effect on I
K
. First, AA accelerated the rate of I
K
activation, increasing current amplitude at the beginning of voltage step. Second, AA caused a marked acceleration of current decay, thereby reducing I
K
amplitude measured toward the end of the depolarizing steps. These effects were not prevented by indomethacin or nordihydroguaiaretic acid, blockers of cyclooxygenase and lipoxygenase, respectively. AA did not affect the voltage dependence of current activation or inactivation. The magnitude of the inhibitory effect on I
K
was correlated with the number of double bonds but was independent of tail length in fatty acids containing between 14 and 22 carbons. Linoleic acid (18:2,
cis
-9,12) inhibited I
K
much more than did its
trans
-stereoisomer, linolelaidic acid. Arachidonyl alcohol, which is uncharged, and arachidonyl coenzyme A, which does not ‘flip’ across the cell membrane, were less effective than AA in inhibiting I
K
; this effect of fatty acids may therefore require passage across the cell membrane. The enhancement of early I
K
was mimicked by the protein kinase C (PKC) stimulator 1-oleoyl-2-acetyl-
sn
-glycerol (10 μmol/L), was suppressed by ATP removal from the pipette solution, and was blocked by PKC inhibitors chelerythrine (10 μmol/L) and staurosporine (100 nmol/L). This effect may therefore require PKC-dependent phosphorylation.
In Vivo Assessment of Embryonic Cardiovascular Dimensions and Function in Day-10.5 to -14.5 Mouse Embryos
Authors: Keller Bradley B. , MacLennan Megan J. , Tinney Joseph P. , Yoshigi Masaaki
Published: June 2012

Embryonic cardiovascular function has been extensively studied in vivo in the chick embryo. However, the geometry of mammalian and avian hearts differs; the mammalian cardiovascular system is coupled to both yolk sac and placental circulations, and unique murine genetic models associated with structural and functional cardiovascular defects are now available. We therefore adapted techniques validated for the chick embryo to define cardiovascular dimensions and function in the mouse embryo. We bred C3HeB female and C57Bl/J6 male mice and ICR pairs for experiments on embryonic days (EDs) 10.5 to 14.5 (n=130 dams). After maternal anesthesia (pentobarbital, 60 mg/kg IP), laparotomy, and sequential regional hysterotomy, we exposed and then imaged individual embryos at 60 Hz (video) in the ventral and/or left anterior oblique views while maintaining uteroplacental continuity. We measured epicardial chamber dimensions and then calculated right and left ventricular elliptical volumes from areas. In addition, we measured pulsed-Doppler blood velocity across the atrioventricular cushions and ventricular outflow tract. We maintained embryonic temperature with a heated surgical platform, topical oxygenated and warmed buffer, and warming lamps. Embryonic heart rate increased from 124.7±5.2 to 194.3±13.2 bpm from EDs 10.5 to 14.5 (
P
<.01). Right and left ventricular end-diastolic and end-systolic dimensions increased (
P
<.05 by ANOVA for each). Maximal ventricular mean inflow and outflow velocities increased from 62.33±4.06 to 106.23±11.59 and from 55.79±6.11 to 91.61±6.93 mm/s, respectively (
P
<.05 by ANOVA for each). Thus, as has been done for chick and rat embryos, the maturation of murine embryonic cardiovascular function can be quantified in vivo, setting the stage for the investigation of structure-function relations in mouse models of cardiovascular development and disease.
No Prevention of Ischemic Preconditioning by the Protein Kinase C Inhibitor Staurosporine in Swine
Authors: Vahlhaus Christian , Schulz Rainer , Post Heiner , Onallah Raouf , Heusch Gerd
Published: June 2012

The delay of infarct size development by ischemic preconditioning involves the activation of protein kinase C in rats and rabbits. In dogs the role of protein kinase C in ischemic preconditioning is controversial. We investigated whether or not the activation of protein kinase C is a prerequisite for ischemic preconditioning in swine. Swine were used, since they are large mammals and since infarct development in this species, due to the lack of an innate collateral circulation, is similar to that in humans. In 20 enflurane-anesthetized swine, the proximal left anterior descending coronary artery was cannulated and perfused from an extracorporeal circuit. The impact of continuous intracoronary infusion of 10
−7
mol/L staurosporine, a potent protein kinase C inhibitor, on global and regional myocardial function (sonomicrometry), subendocardial blood flow (ENDO, microspheres), and infarct size (IS, triphenyltetrazolium chloride staining after 120 minutes of reperfusion) was analyzed. Staurosporine (10
−7
mol/L) abolished the 1.6-fold increase in coronary arterial resistance in response to 10
−6
mol/L IC 4β-phorbol 12-myristate 13-acetate, a potent protein kinase C activator. In the presence of staurosporine, 90 minutes of low-flow ischemia at an ENDO of 0.05±0.04 (mean±SD) mL·min
−1
·g
−1
resulted in an IS of 12.5±8.6% (n=10) of the area at risk. Also, in the presence of staurosporine, ischemic preconditioning by a cycle of 10 minutes of low-flow ischemia followed by 15 minutes reperfusion before the 90 minutes sustained ischemic period (ENDO, 0.05±0.03 mL·min
−1
·g
−1
) reduced IS to 3.3±3.4% (n=10,
P
<.05). The protein kinase C inhibitor staurosporine does not prevent ischemic preconditioning in swine.