bluuebunny/crossref_metadata_2025 huggingface dataset:
Medium perfusion enables engineering of compact and contractile cardiac tissue
Authors: Radisic Milica , Yang Liming , Boublik Jan , Cohen Richard J. , Langer Robert , Freed Lisa E. , Vunjak-Novakovic Gordana
Published: January 2004

We hypothesized that functional constructs with physiological cell densities can be engineered in vitro by mimicking convective-diffusive oxygen transport normally present in vivo. To test this hypothesis, we designed an in vitro culture system that maintains efficient oxygen supply to the cells at all times during cell seeding and construct cultivation and characterized in detail construct metabolism, structure, and function. Neonatal rat cardiomyocytes suspended in Matrigel were cultured on collagen sponges at a high initial density (1.35 × 108cells/cm3) for 7 days with interstitial flow of medium; constructs cultured in orbitally mixed dishes, neonatal rat ventricles, and freshly isolated cardiomyocytes served as controls. Constructs were assessed at timed intervals with respect to cell number, distribution, viability, metabolic activity, cell cycle, presence of contractile proteins (sarcomeric α-actin, troponin I, and tropomyosin), and contractile function in response to electrical stimulation [excitation threshold (ET), maximum capture rate (MCR), response to a gap junctional blocker]. Interstitial flow of culture medium through the central 5-mm-diameter × 1.5-mm-thick region resulted in a physiological density of viable and differentiated, aerobically metabolizing cells, whereas dish culture resulted in constructs with only a 100- to 200-μm-thick surface layer containing viable and differentiated but anaerobically metabolizing cells around an acellular interior. Perfusion resulted in significantly higher numbers of live cells, higher cell viability, and significantly more cells in the S phase compared with dish-grown constructs. In response to electrical stimulation, perfused constructs contracted synchronously, had lower ETs, and recovered their baseline function levels of ET and MCR after treatment with a gap junctional blocker; dish-grown constructs exhibited arrhythmic contractile patterns and failed to recover their baseline MCR levels.
Requirement of Rac activity for maintenance of capillary endothelial barrier properties
Authors: Waschke J. , Baumgartner W. , Adamson R. H. , Zeng M. , Aktories K. , Barth H. , Wilde C. , Curry F. E. , Drenckhahn D.
Published: January 2004

Our previous experiments indicated that GTPases, other than RhoA, are important for the maintenance of endothelial barrier integrity in both intact microvessels of rats and mice and cultured mouse myocardial endothelial (MyEnd) cell monolayers ( J Physiol 539: 295–308, 2002). In the present study, we inhibited the endothelial GTPase Rac by Clostridium sordellii lethal toxin (LT) and investigated the relation between the degree of inhibition of Rac by glucosylation and increased endothelial barrier permeability. In rat venular microvessels, LT (200 ng/ml) increased hydraulic conductivity from a control value of 2.5 ± 0.6 to 100.8 ± 18.7 × 10–7cm·s–1·cmH2O–1after 80 min. In cultured MyEnd cells exposed to LT (200 ng/ml), up to 60% of cellular Rac was glucosylated after 90 min, resulting in depolymerization of F-actin and interruptions of junctional distribution of vascular endothelial cadherin (VE-cadherin) and β-catenin as well as the formation of intercellular gaps. To understand the mechanism by which inhibition of Rac caused disassembly of adherens junctions, we used laser tweezers to quantify VE-cadherin-mediated adhesion. LT and cytochalasin D, an actin depolymerizing agent, both reduced adhesion of VE-cadherin-coated microbeads to the endothelial cell surface, whereas the inhibitor of Rho kinase Y-27632 did not. Stabilization of actin filaments by jasplakinolide completely blocked the effect of cytochalasin D but not of LT on bead adhesion. We conclude that Rac regulates endothelial barrier properties in vivo and in vitro by 1) modulation of actin filament polymerization and 2) acting directly on the tether between VE-cadherin and the cytoskeleton.
Na+ current through KATP channels: consequences for Na+ and K+ fluxes during early myocardial ischemia
Authors: Bollensdorff Christian , Knopp Andreas , Biskup Christoph , Zimmer Thomas , Benndorf Klaus
Published: January 2004

During early myocardial ischemia, the myocytes are loaded with Na+, which in turn leads to Ca2+ overload and cell death. The pathway of the Na+ influx has not been fully elucidated. The aim of the study was to quantify the Na+ inward current through sarcolemmal KATP channels ( IKATP,Na) in anoxic isolated cardiomyocytes at the actual reversal potential ( Vrev) and to estimate the contribution of this current to the Na+ influx in the ischemic myocardium. IKATP,Na was determined in excised single channel patches of mouse ventricular myocytes and macropatches of Xenopus laevis oocytes expressing SUR2A/Kir6.2 channels. In the presence of K+ ions, the respective permeability ratios for Na+ to K+ ions, PNa/ PK, were close to 0.01. Only in the presence of Na+ ions on both sides of the membrane was IKATP,Na similarly large to that calculated from the permeability ratio PNa/ PK, indicative of a Na+ influx that is largely independent of the K+ efflux at Vrev. With the use of a peak KATP channel conductance in anoxic cardiomyocytes of 410 nS, model simulations for a myocyte within the ischemic myocardium showed that the amplitude of the Na+ influx and K+ efflux is even larger than the respective fluxes by the Na+-K+ pump and all other background fluxes. These results suggest that during early ischemia the Na+ influx through KATP channels essentially contributes to the total Na+ influx and that it also balances the K+ efflux through KATP channels.
Effect of nitric oxide on capillary hemodynamics and cell injury in the pancreas duringPseudomonaspneumonia-induced sepsis
Authors: Tribl Barbara , Bateman Ryon M. , Milkovich Stephanie , Sibbald William J. , Ellis Christopher G.
Published: January 2004

Sepsis-induced nitric oxide (NO) overproduction has been implicated in a redistribution of flow from the pancreas making it vulnerable to ischemic injury in septic shock. To test this hypothesis in a remote injury model of normotensive sepsis, we induced Pseudomonas pneumonia in the rat and used intravital video microscopy (IVVM) of the pancreas to measure functional capillary density, capillary hemodynamics [red blood cell (RBC) velocity, lineal density, and supply rate], and lethal cellular damage (propidium iodine staining) at 6 and 24 h after the induction of pneumonia. With pneumonia, plasma nitrite/nitrate [[Formula: see text]] levels were doubled by 21 h ( P < 0.05). To assess the effect of NO overproduction on microvascular perfusion, N6-(1-iminoethyl)-l-lysine (l-NIL) was administered to maintain [Formula: see text] levels at baseline. Pneumonia did cause a decrease in RBC velocity of 23% by 6 h, but by 24 h RBC velocity and supply rate had increased relative to sham by 22 and 38%, respectively ( P < 0.05). l-NIL treatment demonstrated that this increase was due to NO overproduction. With pneumonia, there was no change in functional capillary density and only modest increases in cellular damage. We conclude that, in this normotensive pneumonia model of sepsis, NO overproduction was protective of microvascular perfusion in the pancreas.
Shear stress and VEGF activate IKK via the Flk-1/Cbl/Akt signaling pathway
Authors: Wang Yingxiao , Chang Joann , Li Yi-Chen , Li Yi-Shuan , Shyy John Y.-J. , Chien Shu
Published: January 2004

Vascular endothelial cells are continuously exposed to mechanical (e.g., shear stress) and chemical (e.g., growth factors) stimuli. It is important to elucidate the mechanisms by which cells perceive and integrate these different stimuli to regulate the downstream signaling pathways. We ( 50 ) have previously reported the shear-induced interplay between two membrane receptors, integrins and Flk-1. In the present study, we investigated the molecular mechanisms regulating the downstream IκB kinase (IKK) pathway in response to shear stress and VEGF. Both shear stress and VEGF induced a transient increase of IKK activity. These effects were inhibited by SU-1498, a specific Flk-1 inhibitor, and by a negative mutant of Casitas B-lineage lymphoma (Cbl) with tyrosine-to-phenylalanine mutations at sites 700, 731, and 774 (Cblnm). Because Flk-1 and Cbl form a complex upon shearing or VEGF applications ( 50 ), these results suggest that shear stress and VEGF activate IKK via the receptor Flk-1 and its recruitment of the adapter protein Cbl. The inhibition of the shear- and VEGF-induced IKK activities by a negative mutant of Akt indicates that Akt acts upstream to IKK in response to shear stress and VEGF. Furthermore, SU-1498 and Cbl-nmabolished the shear- and VEGF-induced Akt activity, indicating that Akt acts at a level downstream to Flk-1 and Cbl. Therefore, our results indicate that the signaling events induced by shear stress and VEGF converge at the membrane receptor Flk-1 and that these stimuli share the Flk-1/Cbl/Akt pathway in activating IKK activation.
Model of competitive binding of vascular endothelial growth factor and placental growth factor to VEGF receptors on endothelial cells
Authors: Mac Gabhann Feilim , Popel Aleksander S.
Published: January 2004

Placental growth factor (PlGF) competes with vascular endothelial growth factor (VEGF) for binding to VEGF receptor (VEGFR)-1 but does not bind VEGFR2. Experiments show that PlGF can augment the response to VEGF in pathological angiogenesis and in models of endothelial cell survival, migration, and proliferation. This synergy has been hypothesized to be due to a combination of the following: signaling by PlGF through VEGFR1 and displacement of VEGF from VEGFR1 to VEGFR2 by PlGF, causing increased signaling through VEGFR2. In this study, the relative contribution of PlGF-induced VEGF displacement to the synergy is quantified using a mathematical model of ligand-receptor binding to examine the effect on ligand-receptor complex formation of VEGF and PlGF acting together. Parameters specific to the VEGF-PlGF system are used based on existing data. The model is used to simulate in silico a specific in vitro experiment in which VEGF-PlGF synergy is observed. We show that, whereas a significant change in the formation of endothelial surface growth factor-VEGFR1 complexes is predicted in the presence of PlGF, the increase in the number of VEGFR2-containing signaling complexes is less significant; these results were shown to be robust to significant variation in the kinetic parameters of the model. Synergistic effects observed in that experiment thus appear unlikely to be due to VEGF displacement but to a shift from VEGF-VEGFR1 to PlGF-VEGFR1 complexes and an increase in total VEGFR1 complexes. These results suggest that VEGFR1 signaling can be functional in adult-derived endothelial cells.
Regulation of α2-adrenoceptors in human vascular smooth muscle cells
Authors: Chotani Maqsood A. , Mitra Srabani , Su Baogen Y. , Flavahan Sheila , Eid Ali H. , Clark K. Reed , Montague Christine R. , Paris Hervé , Handy Diane E. , Flavahan Nicholas A.
Published: January 2004

This study analyzed the regulation of α2-adrenoceptors (α2-ARs) in human vascular smooth muscle cells (VSMs). Saphenous veins and dermal arterioles or VSMs cultured from them expressed high levels of α2-ARs (α2C> α2A, via RNase protection assay) and responded to α2-AR stimulation [5-bromo- N-(4,5-dihydro-1 H-imidazol-2-yl)-6-quinoxalinamine (UK-14,304, 1 μM)] with constriction or calcium mobilization. In contrast, VSMs cultured from aorta did not express α2-ARs and neither cultured cells nor intact aorta responded to UK-14,304. Although α2-ARs (α2C>> α2A) were detected in aortas, α2C-ARs were localized by immunohistochemistry to VSMs of adventitial arterioles and not aortic media. In contrast with aortas, aortic arterioles constricted in response to α2-AR stimulation. Reporter constructs demonstrated higher activities for α2A- and α2C-AR gene promoters in arteriolar compared with aortic VSMs. In arteriolar VSMs, serum increased expression of α2C-AR mRNA and protein but decreased expression of α2A-ARs. Serum induction of α2C-ARs was reduced by inhibition of p38 mitogen-activated protein kinase (MAPK) with 2 μM SB-202190 or dominant-negative p38 MAPK. UK-14,304 (1 μM) caused calcium mobilization in control and serum-stimulated cells: in control VSMs, the response was inhibited by the α2A-AR antagonist BRL-44408 (100 nM) but not by the α2C-AR antagonist MK-912 (1 nM), whereas after serum stimulation, MK-912 (1 nM) but not BRL-44408 (100 nM) inhibited the response. These results demonstrate site-specific expression of α2-ARs in human VSMs that reflects differential activity of α2-AR gene promoters; namely, high expression and function in venous and arteriolar VSMs but no detectable expression or function in aortic VSMs. We found that α2C-ARs can be dramatically and selectively induced via a p38 MAPK-dependent pathway. Therefore, altered expression of α2C-ARs may contribute to pathological changes in vascular function.
Determination of cardiac contractility in awake unsedated mice with a fluid-filled catheter
Authors: Wang Qing , Brunner Hans R. , Burnier Michel
Published: January 2004

Today, cardiac contractility in mice is exclusively measured under anesthesia or in sedated animals because the catheters available are too rigid to be used in awake mice. We therefore developed a new catheter (Pebax 03) to measure cardiac contractility in conscious mice. In this study, we evaluated the accuracy and utility of this new catheter for assessment of cardiac contractility in anesthetized and conscious mice. With the use of a balloon-pop test, the Pebax catheter with an inner diameter of 0.3 mm was found to exhibit a high natural frequency, a low damping coefficient, and a flat frequency of up to 50.5 ± 0.6 Hz. Under anesthesia (0.5% or 1.0% halothane), no difference was found in heart rate (HR), left ventricular (LV) systolic pressure (LVSP), the maximum rates of LV pressure rise and fall (LV dP/d tmax and LV dP/d tmin, respectively), ejection time (ET), and isovolumic relaxation time constant (τ) when measured with either the 1.4-Fr Millar or Pebax 03 catheter. However, when HR, LVSP, LV dP/d tmax, and LV dP/d tmin were recorded with the Pebax catheter in awake mice, values were significantly higher, and ET and τ were lower, than under anesthesia, suggesting a major impact of anesthesia on these parameters. The Pebax catheter was also used in a normotensive one-renin gene mouse model of cardiac hypertrophy induced by DOCA and salt. In this model, DOCA-salt induced a severe decrease in cardiac contractility in the absence of changes in blood pressure. These data demonstrate that cardiac contractility can be measured very accurately in conscious mice. This new device can be of great help in the investigation of cardiac function in normal and genetically engineered mice.
Interstitial purine metabolites in hearts with LV remodeling
Authors: Gourine Andrey V. , Hu Qingsong , Sander Paul R. , Kuzmin Aleksandr I. , Hanafy Nadia , Davydova Svetlana A. , Zaretsky Dmitry V. , Zhang Jianyi
Published: January 2004

The myocardial ATP concentration is significantly decreased in failing hearts, which may be related to the progressive loss of the myocardial total adenine nucleotide pool. The total myocardial interstitial purine metabolites (IPM) in the dialysate of interstitial fluid could reflect the tissue ATP depletion. In rats, postmyocardial infarction (MI) left ventricular (LV) remodeling was induced by ligation of the coronary artery. Cardiac microdialysis was employed to assess changes of IPM in response to graded β-adrenergic stimulation with isoproterenol (Iso) in myocardium of hearts with post-MI LV remodeling (MI group) or hearts with sham operation (sham group). The dialysate samples were analyzed for adenosine, inosine, hypoxanthine, xanthine, and uric acid. LV volume was greater in the MI group (2.2 ± 0.2 ml/kg) compared with the sham group (1.3 ± 0.2 ml/kg, P < 0.05). Infarct size was 28 ± 4%. The baseline dialysate level of uric acid was higher in the MI group (18.9 ± 3.4 μmol) compared with the sham group (4.6 ± 0.7 μmol, P < 0.01). During and after Iso infusion, the dialysate levels of adenosine, xanthine, and uric acid were all significantly higher in the MI group. Thus the level of IPM is increased in hearts with postinfarction LV remodeling both at baseline and during Iso infusion. These results suggest that the decreased myocardial ATP level in hearts with post-MI LV remodeling may be caused by the chronic depletion of the total adenine nucleotide pool.
Effect of high-salt diet on NO release and superoxide production in rat aorta
Authors: Zhu Jiaxuan , Mori Takefumi , Huang Tianjian , Lombard Julian H.
Published: January 2004

Sprague-Dawley rats were fed either a high-salt (HS) diet (4.0% NaCl) or a low-salt (LS) diet (0.4% NaCl) for 3 days. Nitric oxide (NO) and superoxide production were assessed in the thoracic aorta by evaluating the fluorescence signal intensity from 4,5-diaminofluorescein (DAF-2DA) and dihydroethidine, respectively. Methacholine caused increased NO release in the aortas from rats on a LS but not HS diet. The SOD mimetic tempol restored methacholine-induced NO release in aortas from rats on a HS diet. Methacholine also caused superoxide production in the aortas of rats on a HS diet but not in the aortas of rats on a LS diet. Tempol and NG-monomethyl-l-arginine eliminated methacholine-induced superoxide production in the aortas of rats on a HS diet. Aortic rings from rats on the HS diet showed impaired methacholine-induced relaxation, which was improved by tempol. Tempol alone caused a NO-dependent relaxation of norepinephrine-precontracted aortas that was significantly greater in the aortas of rats on the HS diet than in vessels from rats on the LS diet. These data suggest that a HS diet impairs endothelium-dependent relaxation via reduced NO levels and increased superoxide production.
Hystereses in the force-length relation and regulation of cross-bridge recruitment in tetanized rat trabeculae
Authors: Levy Carmit , Landesberg Amir
Published: January 2004

Various mechanisms have been suggested to explain cardiac force-length Ca2+relations. The existence of a cooperativity mechanism, whereby cross-bridge (XB) recruitment is affected by the number of active XBs, suggests that the force response to length oscillations should lag length oscillations. Consequently, the oscillatory force response should be larger during shortening than during lengthening. To test this prediction, force responses to large-sarcomere length (SL) oscillations (36.7 ± 16.0 nm) at different SLs ( n = 6) and frequencies ( n = 7) were studied in intact tetanized trabeculae dissected from rat right ventricle ( n = 13). Stable tetani were obtained by utilizing 30 μM cyclopiazonic acid in Krebs-Henseleit solution containing 6 mM extracellular Ca2+at 25°C. SL was measured by laser diffraction techniques (Dalsa). Force was measured by silicone strain gauge. Instantaneous dynamic stiffness during large oscillations was measured by superimposing additional fast (50 or 200 Hz) and small-amplitude (2.25 ± 0.25 nm) oscillations. The force responses lagged the SL oscillations at slow frequencies (112 ± 41 ms at 1 Hz), and counterclockwise hystereses were obtained in the force-length plane: the force was higher during shortening than during lengthening. The delay in the force response decreased as the frequency of the SL oscillation was increased. Clockwise hysteresis, where the force preceded the SL, was obtained at frequencies >4 Hz. Similar hysteresis characteristics were obtained in the force-SL and stiffness-SL planes. Maximal lag was observed at the shortest SL, and the delay decreased with sarcomere elongation: 131.1 ± 31.7 ms at 1.78 ± 0.03 μm vs. 14.7 ± 18.5 ms at 1.99 ± 0.015 μm. The results establish the ability of cardiac fiber to adapt XB recruitment to changes in prevailing loading conditions. This study supports the stipulated existence of a cooperativity mechanism that regulates XB recruitment and highlights an additional method to characterize regulation of the force-length relation.
Variable efficacy ofN6-(1-iminoethyl)-L-lysine in acute cardiac transplant rejection
Authors: Pieper Galen M. , Nilakantan Vani , Hilton Gail , Zhou Xianghua , Khanna Ashwani K. , Halligan Nadine L. N. , Felix Christopher C. , Kampalath Bal , Griffith Owen W. , Hayward Mike A. , Roza Allan M. , Adams Mark B.
Published: January 2004

We examined the efficacy and mechanism of action of N6-(1-iminoethyl)-l-lysine (l-NIL), a highly selective inhibitor of inducible nitric oxide (NO) synthase (iNOS), on acute cardiac transplant rejection. l-NIL produced a concentration-dependent attenuation of plasma NO by-products and a decrease in nitrosylation of heme protein without altering protein levels of iNOS. At postoperative day 4, l-NIL did not alter the increased binding activities for transcription factors nuclear factor-κB and activator protein-1. Whereas l-NIL decreased inflammatory cell infiltration, graft survival was only prolonged at the dose of 1.0 μg/ml that incompletely blocked NO production. Higher l-NIL concentrations (30 and 60 μg/ml) ablated the increased NO production but failed to improve graft survival and even potentiated NF-κB binding activity examined at day 6. Alloimmune activation indicated by increased cytokine gene expression for interferon-γ, interleukin-6, and interleukin-10 was inhibited in grafts only by treatment with 1.0 μg/ml l-NIL. These findings suggest a complex role of NO in acute cardiac allograft rejection. Partial inhibition of iNOS is beneficial to graft survival, whereas total ablation may oppose any benefits to graft survival. These studies have important implications in understanding the dual role of NO in acute rejection and help to reconcile discrepancies in the literature.
Bradykinin induces mitochondrial ROS generation via NO, cGMP, PKG, and mitoKATP channel opening and leads to cardioprotection
Authors: Oldenburg Olaf , Qin Qining , Krieg Thomas , Yang Xi-Ming , Philipp Sebastian , Critz Stuart D. , Cohen Michael V. , Downey James M.
Published: January 2004

Bradykinin (BK) mimics ischemic preconditioning by generating reactive oxygen species (ROS). To identify intermediate steps that lead to ROS generation, rabbit cardiomyocytes were incubated in reduced MitoTracker Red stain, which becomes fluorescent after exposure to ROS. Fluorescence intensity in treated cells was expressed as a percentage of that in paired, untreated cells. BK (500 nM) caused a 51 ± 16% increase in ROS generation ( P < 0.001). Coincubation with either the BK B2-receptor blocker HOE-140 (5 μM) or the free radical scavenger N-(2-mercaptopropionyl)glycine (1 mM) prevented this increase, which confirms that the response was receptor mediated and ROS were actually being measured. Closing mitochondrial ATP-sensitive K+ (mitoKATP) channels with 5-hydroxydecanoate (5-HD, 1 mM) prevented increased ROS generation. BK-induced ROS generation was blocked by Nω-nitro-m-arginine methyl ester (m-NAME, 200 μM), which implicates nitric oxide as an intermediate. Blockade of guanylyl cyclase with 1- H-[1,2,4]oxadiazole[4,3- a]quinoxalin-1-one (ODQ, 10 μM) aborted BK-induced ROS generation but not that from diazoxide, a direct opener of mitoKATP channels. The protein kinase G (PKG) blocker 8-bromoguanosine-3′,5′-cyclic monophosphorothioate (25 μM) eliminated the effects of BK. Conversely, direct activation of PKG with 8-(4-chlorophenylthio)-guanosine-3′,5′-cyclic monophosphate (100 μM) increased ROS generation (39 ± 15%; P < 0.004) similar to BK. This increase was blocked by 5-HD. Finally, the nitric oxide donor S-nitroso- N-acetylpenicillamine (1 μM) increased ROS by 34 ± 6%. This increase was also blocked by 5-HD. In intact rabbit hearts, BK (400 nM) decreased infarction from 30.5 ± 3.0 of the risk zone in control hearts to 11.9 ± 1.4% ( P < 0.01). This protection was aborted by either 200 μM m-NAME or 2 μM ODQ (35.4 ± 5.7 and 30.4 ± 3.0% infarction, respectively; P = not significant vs. control). Hence, BK preconditions through receptor-mediated production of nitric oxide, which activates guanylyl cyclase. The resulting cGMP activates PKG, which opens mitoKATP. Subsequent release of ROS triggers cardioprotection.
Intracellular calcium handling heterogeneities in intact guinea pig hearts
Authors: Katra Rodolphe P. , Pruvot Etienne , Laurita Kenneth R.
Published: January 2004

Regional heterogeneities of ventricular repolarizing currents and their role in arrhythmogenesis have received much attention; however, relatively little is known regarding heterogeneities of intracellular calcium handling. Because repolarization properties and contractile function are heterogeneous from base to apex of the intact heart, we hypothesize that calcium handling is also heterogeneous from base to apex. To test this hypothesis, we developed a novel ratiometric optical mapping system capable of measuring calcium fluorescence of indo-1 at two separate wavelengths from 256 sites simultaneously. With the use of intact Langendorff-perfused guinea pig hearts, ratiometric calcium transients were recorded under normal conditions and during administration of known inotropic agents. Ratiometric calcium transients were insensitive to changes in excitation light intensity and fluorescence over time. Under control conditions, calcium transient amplitude near the apex was significantly larger (60%, P < 0.01) compared with the base. In contrast, calcium transient duration was significantly longer (7.5%, P < 0.03) near the base compared with the apex. During isoproterenol (0.05 μM) and verapamil (2.5 μM) administration, ratiometric calcium transients accurately reflected changes in contractile function, and, the direction of base-to-apex heterogeneities remained unchanged compared with control. Ratiometric optical mapping techniques can be used to accurately quantify heterogeneities of calcium handling in the intact heart. Significant heterogeneities of calcium release and sequestration exist from base to apex of the intact heart. These heterogeneities are consistent with base-to-apex heterogeneities of contraction observed in the intact heart and may play a role in arrhythmogenesis under abnormal conditions.
Increased focal Kv4.2 channel expression at the plasma membrane is the result of actin depolymerization
Authors: Wang Zhuren , Eldstrom Jodene R. , Jantzi Joshua , Moore Edwin D. , Fedida David
Published: January 2004

Voltage-dependent potassium channel trafficking and localization are regulated by proteins of the cytoskeleton, but the mechanisms by which these occur are still unclear. Using human embryonic kidney (HEK) cells as a heterologous expression system, we tested the role of the actin cytoskeleton in modulating the function of Kv4.2 channels. Pretreatment (≥1 h) of HEK cells with 5 μM cytochalasin D to disrupt the actin microfilaments greatly augmented whole cell Kv4.2 currents at potentials positive to –20 mV. However, no changes in the voltage dependence of activation and inactivation of macroscopic currents were observed to account for this increase. Similarly, single channel recordings failed to reveal any significant changes in the single channel conductance, open probability, and kinetics. However, the mean patch current was increased from 0.9 ± 0.2 pA in control to 6.7 ± 3.0 pA in the presence of cytochalasin D. Imaging experiments revealed a clear increase in the surface expression of the channels and the appearance of “bright spot” features, suggesting that large numbers of channels were being grouped at specific sites. Our data provide clear evidence that increased numbers and altered distribution of Kv4.2 channels at the cell surface are primarily the result of reorganization of the actin cytoskeleton.
A transmural pressure gradient induces mechanical and biological adaptive responses in endothelial cells
Authors: DeMaio Lucas , Tarbell John M. , Scaduto Russell C. , Gardner Thomas W. , Antonetti David A.
Published: January 2004

A sudden increase in the transmural pressure gradient across endothelial monolayers reduces hydraulic conductivity ( Lp), a phenomenon known as the sealing effect. To further characterize this endothelial adaptive response, we measured bovine aortic endothelial cell (BAEC) permeability to albumin and 70-kDa dextran, Lp, and the solvent-drag reflection coefficients (σ) during the sealing process. The diffusional permeability coefficients for albumin (1.33 ± 0.18 × 10–6cm/s) and dextran (0.60 ± 0.16 × 10–6cm/s) were measured before pressure application. The effective permeabilities (measured when solvent drag contributes to solute transport) of albumin and dextran ( Pealband Pedex) were measured after the application of a 10 cmH2O pressure gradient; during the first 2 h of pressure application, Pealb, Pedex, and Lpwere significantly reduced by 2.0 ± 0.3-, 2.1 ± 0.3-, and 3.7 ± 0.3-fold, respectively. Immunostaining of the tight junction (TJ) protein zonula occludens-1 (ZO-1) was significantly increased at cell-cell contacts after the application of transmural pressure. Cytochalasin D treatment significantly elevated transport but did not inhibit the adaptive response, whereas colchicine treatment had no effect on diffusive permeability but inhibited the adaptive response. Neither cytoskeletal inhibitor altered σ despite significantly elevating both Lpand effective permeability. Our data suggest that BAECs actively adapt to elevated transmural pressure by mobilizing ZO-1 to intercellular junctions via microtubules. A mechanical (passive) component of the sealing effect appears to reduce the size of a small pore system that allows the transport of water but not dextran or albumin. Furthermore, the structures of the TJ determine transport rates but do not define the selectivity of the monolayer to solutes (σ).
c-Jun is regulated by combination of enhanced expression and phosphorylation in acute-overloaded rat heart
Authors: Nadruz Wilson , Kobarg Claudia B. , Kobarg Jörg , Franchini Kleber G.
Published: January 2004

The transient increase in the expression of transcription factors encoded by immediate-early genes has been considered to play a critical role in the coordination of early gene expression during the hypertrophic growth of cardiac myocytes. Here, we investigated the regulation of c-Jun and its upstream activators JNKs in the myocardium of rats subjected to acute pressure overload induced by transverse aortic constriction. Western blotting and immunohistochemistry analysis demonstrated that both JNK1 and JNK2 were transiently activated by pressure overload, but only JNK1 was activated at the nuclei of cardiac myocytes. JNK1 activation was paralleled by phosphorylation of c-Jun at serine-63 in the myocardial nuclear fraction and by an increase in c-Jun expression in cardiac myocytes. A consistent increase in DNA binding of activator protein-1 (AP-1) complex was observed after 10 and 30 min of pressure overload and Supershift assays confirmed that c-Jun was a major component of activated AP-1 complex. Moreover, experiments performed with the specific JNK inhibitor SP-600125 abolished c-Jun phosphorylation and markedly attenuated its expression as well as the expression of the fetal gene β-myosin heavy chain. Overall, these findings demonstrate a molecular basis for load-induced activation of c-Jun in cardiac myocytes and its connection with the regulation of fetal gene, characteristic of the acute response to pressure overload.
Myocardial contractile function during postischemic low-flow reperfusion: critical thresholds of NADH and O2delivery
Authors: Stoner Jason D. , Angelos Mark G. , Clanton Thomas L.
Published: January 2004

The degree of myocardial oxygen delivery (Do2) that is necessary to reestablish functional contractile activity after short-term global ischemia in heart is not known. To determine the relationship between Do2and recovery of contractile and metabolic functions, we used tissue NADH fluorometric changes to characterize adequacy of reperfusion flow. Isolated perfused rat hearts were subjected to global ischemia and were reperfused at variable flow rates that ranged from 1 to 100% of baseline flow. Myocardial function and tissue NADH changes were continuously measured. NADH fluorescence rapidly increased and plateaued during ischemia. A strong inverse logarithmic correlation between NADH fluorescence and reperfusion Do2was demonstrated ( r = –0.952). Left ventricular function (rate-pressure product) was inversely related to NADH fluorescence at reperfusion flows from 25 to 100% of baseline ( r = –0.922) but not at lower reperfusion flow levels. An apparent reperfusion threshold of 25% of baseline Do2was necessary to resume contractile function. At very low reperfusion flows (1% of baseline), another threshold flow was identified at which NADH levels increased beyond that observed during global ischemia (3.4 ± 3.0%, means ± SE, n = 9), which suggests further reduction of the cellular redox state. This NADH increase at 1% of baseline reperfusion flow was blocked by removing glucose from the perfusate. NADH fluorescence is a sensitive indicator of myocardial cellular oxygen utilization over a wide range of reperfusion Do2values. Although oxygen is utilized at very low flow rates, as indicated by changes in NADH, a critical threshold of ∼25% of baseline Ḋo2is necessary to restore contractile function after short-term global ischemia.
Protective role of gap junctions in preconditioning against myocardial infarction
Authors: Miura Tetsuji , Ohnuma Yoshito , Kuno Atsushi , Tanno Masaya , Ichikawa Yoshihiko , Nakamura Yuichi , Yano Toshiyuki , Miki Takayuki , Sakamoto Jun , Shimamoto Kazuaki
Published: January 2004

The aim of the present study was to examine the hypothesis that acceleration of gap junction (GJ) closure during ischemia contributes to anti-infarct tolerance afforded by preconditioning (PC). First, the effects of PC on GJ communication during ischemia were assessed. Isolated buffer-perfused rabbit hearts were subjected to 5-min global ischemia with or without PC with two cycles of 5-min ischemia/5-min reperfusion or a GJ blocker (2 mM heptanol), and then the tissue excised from the ischemic region was incubated in anoxic buffer containing lucifer yellow (LY; 2.5 mg/ml), a tracer of GJ permeability, for 20 min at 37°C. PC and heptanol significantly reduced the area to which LY was transported in the ischemic myocardium by 39% and by 54%, respectively. In the second series of experiments, three GJ blockers (heptanol, 18β-glycyrrhetinic acid, and 2,3-butanedione monoxime) infused after the onset of ischemia reduced infarct size after 30-min ischemia/2-h reperfusion to an extent equivalent to that in the case of PC. In the third series of experiments, Western blotting for connexin43 (Cx43) showed that PC shortened the time to the onset of ischemia-induced Cx43 dephosphorylation but reduced the extent of Cx43 dephosphorylation during a 30-min period of ischemia. Calphostin C, a protein kinase C (PKC) inhibitor, abolished preservation of phosphorylated Cx43 but not the early onset of Cx43 dephosphorylation after ischemia in the preconditioned myocardium. These results suggest that PC-induced reduction of GJ permeability during ischemia, presumably by PKC-mediated Cx43 phosphorylation, contributes to infarct size limitation.
Increased susceptibility to ventricular arrhythmias in a rodent model of experimental depression
Authors: Grippo Angela J. , Santos Claudia M. , Johnson Ralph F. , Beltz Terry G. , Martins James B. , Felder Robert B. , Johnson Alan Kim
Published: January 2004

Depression is an important public health problem and is considered to be an independent risk factor for coronary artery disease. The pathophysiological mechanisms that link depression with adverse cardiovascular events (e.g., myocardial ischemia, myocardial infarction, and sudden death) are not well established. It is possible that an increased susceptibility to life-threatening cardiac arrhythmias in depressed patients influences the risk of morbidity and mortality in coronary artery disease. This idea was tested with the use of an experimental model of depression that was developed to induce anhedonia, the reduced responsiveness to pleasurable stimuli observed in human depressed patients. Rats exposed to 4 wk of chronic mild stress (e.g., paired housing, strobe light, and white noise) displayed anhedonia, which was operationally defined by the reduced intake of a palatable sucrose solution relative to an established baseline and to control animals. Furthermore, compared with control rats, the anhedonic rats showed increased basal heart rate and decreased heart rate variability. In response to an intravenously infused chemical challenge, aconitine, anhedonic rats exhibited an increased vulnerability to ventricular arrhythmias, as indicated by a reduced threshold for premature ventricular complexes, salvos, and ventricular tachycardia. These findings suggest that the presence of depressive symptoms is associated with a lower threshold for ventricular arrhythmias, which may contribute to the increased risk for adverse cardiovascular events in patients with depression.
Caveolin-1 regulates contractility in differentiated vascular smooth muscle
Authors: Je Hyun-Dong , Gallant Cynthia , Leavis Paul C. , Morgan Kathleen G.
Published: January 2004

Caveolin is a principal component of caveolar membranes. In the present study, we utilized a decoy peptide approach to define the degree of involvement of caveolin in PKC-dependent regulation of contractility of differentiated vascular smooth muscle. The primary isoform of caveolin in ferret aorta vascular smooth muscle is caveolin-1. Chemical loading of contractile vascular smooth muscle tissue with a synthetic caveolin-1 scaffolding domain peptide inhibited PKC-dependent increases in contractility induced by a phorbol ester or an α agonist. Peptide loading also resulted in a significant inhibition of phorbol ester-induced adducin Ser662phosphorylation, an intracellular monitor of PKC kinase activity, ERK1/2 activation, and Ser789phosphorylation of the actin binding protein caldesmon. α-Agonist-induced ERK1–1/2 activation was also inhibited by the caveolin-1 peptide. Scrambled peptide-loaded tissues or sham-loaded tissues were unaffected with respect to both contractility and signaling. Depolarization-induced activation of contraction was not affected by caveolin peptide loading. Similar results with respect to contractility and ERK1/2 activation during exposure to the phorbol ester or the α-agonist were obtained with the cholesterol-depleting agent methyl-β-cyclodextrin. These results are consistent with a role for caveolin-1 in the coordination of signaling leading to the regulation of contractility of smooth muscle.
Ventricular gradient and nondipolar repolarization components increase at higher heart rate
Authors: Smetana Peter , Batchvarov Velislav N. , Hnatkova Katerina , Camm A. John , Malik Marek
Published: January 2004

Differences in action potential duration reflect differences in ion channel properties. These properties also determine rate dependence of action potential duration, and transmural dispersion was confirmed experimentally to increase with cycle length. While several electrocardiographic indexes characterizing repolarization abnormalities have been proposed, studies of their heart rate dependence are missing. This study therefore investigated rate relationship of two repolarization descriptors, namely, the so-called total cosine of the QRS-T angle (TCRT), proposed to characterize global repolarization heterogeneity, and the so-called relative T wave residuum (TWR), linked to regional repolarization dispersion. During 24-h holter recordings in 60 healthy subjects (27 males), a 12-lead ECG was obtained every 30 s. RR intervals, QT intervals, and TCRT and TWR were calculated in each ECG and averaged over RR interval bins ranging from 550 to 1,150 ms in 10-ms steps. Women had uniformly greater TCRT and TWR values than men did over the entire range of investigated RR intervals. Whereas the TCRT in both sexes showed marked rate dependence with higher values at long RR intervals (550 vs. 1,150 ms: women, 0.46 ± 0.31 vs. 0.76 ± 0.18, P = 9 × 10–7; men, 0.08 ± 0.45 vs. 0.49 ± 0.35, P = 9 × 10–8), the rate dependence of TWR was more marked in women than in men, showing higher values at shorter RR intervals (550 ms vs. 1,150 ms: women: 0.29 ± 0.14% vs. 0.08 ± 0.06%, P = 2 × 10–8; men: 0.14 ± 0.12% vs. 0.04 ± 0.02%, P = 2 × 10–15). This suggests that both global and regional repolarization heterogeneity are increased at faster heart rates. Whereas in women at all heart rates the sequence of repolarization more closely replicates the sequence of depolarization, localized repolarization is more heterogeneous than in men especially at fast heart rates.
Atomic force microscopy measurement of leukocyte-endothelial interaction
Authors: Zhang Xiaohui , Chen Aileen , De Leon Dina , Li Hong , Noiri Eisei , Moy Vincent T. , Goligorsky Michael S.
Published: January 2004

Leukocyte adhesion to vascular endothelium is a key initiating step in the pathogenesis of many inflammatory diseases. In this study, we present real-time force measurements of the interaction between monocytic human promyelocytic leukemia cells (HL-60) cells and a monolayer of human umbilical vein endothelial cells (HUVECs) by using atomic force microscopy (AFM). The detachment of HL-60-HUVEC conjugates involved a series of rupture events with force transitions of 40–100 pN. The integrated force of these rupture events provided a quantitative measure of the adhesion strength on a whole cell level. The AFM measurements revealed that HL-60 adhesion is heightened in the borders formed by adjacent HUVECs. The average force and mechanical work required to detach a single HL-60 from the borders of a tumor necrosis factor-α-activated HUVEC layer were twice as high as those of the HUVEC bodies. HL-60 adhesion to the monolayer was significantly reduced by a monoclonal antibody against β1-integrins and partially inhibited by antibodies against selectins ICAM-1 and VCAM-1 but was not affected by anti-αVβ3. Interestingly, adhesion was also inhibited in a dose-dependent manner (IC50≈ 100 nM) by a cyclic arginine-glycine-aspartic acid (cRGD) peptide. This effect was mediated via interfering with the VLA-4-VCAM-1 binding. In parallel measurements, transmigration of HL-60 cells across a confluent HUVEC monolayer was inhibited by the cRGD peptide and by both anti-β1and anti-αVβ3antibodies. In conclusion, these data demonstrate the role played by β1-integrins in leukocyte-endothelial adhesion and transmigration and the role played by αVβ3in transmigration, thus underscoring the high efficacy of cRGD peptide in blocking both the adhesion and transmigration of monocytes.
Phosphatidylinositol 3-kinase modulates vascular smooth muscle contraction by calcium and myosin light chain phosphorylation-independent and -dependent pathways
Authors: Su Xiaoling , Smolock Elaine M. , Marcel Kristi N. , Moreland Robert S.
Published: January 2004

Regulation of smooth muscle contraction involves a number of signaling mechanisms that include both kinase and phosphatase reactions. The goal of the present study was to determine the role of one such kinase, phosphatidylinositol (PI)3-kinase, in vascular smooth muscle excitation-contraction coupling. Using intact medial strips of the swine carotid artery, we found that inhibition of PI3-kinase by LY-294002 resulted in a concentration-dependent decrease in the contractile response to both agonist stimulation and membrane depolarization-dependent contractions and a decrease in Ca2+-dependent myosin light chain (MLC) phosphorylation, the primary step in the initiation of smooth muscle contraction. Inhibition of PI3-kinase also depressed phorbol dibutyrate-induced contractions, which are not dependent on either Ca2+or MLC phosphorylation but are dependent on protein kinase C. To determine the Ca2+-dependent site of action of PI3-kinase, we determined the effect of several inhibitors of calcium metabolism on LY-294002-dependent inhibition of contraction. These inhibitors included nifedipine, SK&F-96365, and caffeine. Only SK&F-96365 blocked the LY-294002-dependent inhibition of contraction. Interestingly, all compounds blocked the LY-294002-dependent inhibition of MLC phosphorylation. Our results suggest that activation of PI3-kinase is involved in a Ca2+- and MLC phosphorylation-independent pathway for contraction likely to involve protein kinase C. In addition, our results also suggest that activation of PI3-kinase is involved in Ca2+-dependent signaling at the level of receptor-operated calcium channels.
Inhibition of endothelium-dependent vasorelaxation by extracellular K+: a novel controlling signal for vascular contractility
Authors: Seol Geun Hee , Ahn Seung Cheol , Kim Ji Aee , Nilius Bernd , Suh Suk Hyo
Published: January 2004

The effects of extracellular K+on endothelium-dependent relaxation (EDR) and on intracellular Ca2+concentration ([Ca2+]i) were examined in mouse aorta, mouse aorta endothelial cells (MAEC), and human umbilical vein endothelial cells (HUVEC). In mouse aortic rings precontracted with prostaglandin F2αor norepinephrine, an increase in extracellular K+concentration ([K+]o) from 6 to 12 mM inhibited EDR concentration dependently. In endothelial cells, an increase in [K+]oinhibited the agonist-induced [Ca2+]iincrease concentration dependently. Similar to K+, Cs+also inhibited EDR and the increase in [Ca2+]iconcentration dependently. In current-clamped HUVEC, increasing [K+]ofrom 6 to 12 mM depolarized membrane potential from –32.8 ± 2.7 to –8.6 ± 4.9 mV ( n = 8). In voltage-clamped HUVEC, depolarizing the holding potential from –50 to –25 mV decreased [Ca2+]isignificantly from 0.95 ± 0.03 to 0.88 ± 0.03 μM ( n = 11, P < 0.01) and further decreased [Ca2+]ito 0.47 ± 0.04 μM by depolarizing the holding potential from –25 to 0 mV ( n = 11, P < 0.001). Tetraethylammonium (1 mM) inhibited EDR and the ATP-induced [Ca2+]iincrease in voltage-clamped MAEC. The intermediate-conductance Ca2+-activated K+channel openers 1-ethyl-2-benzimidazolinone, chlorozoxazone, and zoxazolamine reversed the K+-induced inhibition of EDR and increase in [Ca2+]i. The K+-induced inhibition of EDR and increase in [Ca2+]iwas abolished by the Na+-K+pump inhibitor ouabain (10 μM). These results indicate that an increase of [K+]oin the physiological range (6–12 mM) inhibits [Ca2+]iincrease in endothelial cells and diminishes EDR by depolarizing the membrane potential, decreasing K+efflux, and activating the Na+-K+pump, thereby modulating the release of endothelium-derived vasoactive factors from endothelial cells and vasomotor tone.
Contribution of the respiratory rhythm to sinus arrhythmia in normal unanesthetized subjects during positive-pressure mechanical hyperventilation
Authors: Cooper H. E. , Clutton-Brock T. H. , Parkes M. J.
Published: January 2004

The precise contribution of the CO2-dependent respiratory rhythm to sinus arrhythmia in eupnea is unclear. The respiratory rhythm and sinus arrhythmia were measured in 12 normal, unanesthetized subjects in normocapnia and hypocapnia during mechanical hyperventilation with positive pressure. In normocapnia (41 ± 1 mmHg), the respiratory rhythm was always detectable from airway pressure and inspiratory electromyogram activity. The amplitude of sinus arrhythmia (138 ± 21 ms) during mechanical hyperventilation with positive pressure was not significantly different from that in eupnea. During the same mechanical hyperventilation pattern but in hypocapnia (24 ± 1 mmHg), the respiratory rhythm was undetectable and the amplitude of sinus arrhythmia was significantly reduced (to 40 ± 5 ms). These results show a greater contribution to sinus arrhythmia from the respiratory rhythm during hypocapnia caused by mechanical hyperventilation than previously indicated in normal subjects during hypocapnia caused by voluntary hyperventilation. We discuss whether the respiratory rhythm provides the principal contribution to sinus arrhythmia in eupnea.