bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracardiac Blood Flow Patterns Related to the Yolk Sac Circulation of the Chick Embryo
Authors: Hogers B. , DeRuiter M.C. , Baasten A.M.J. , Gittenberger-de Groot A.C. , Poelmann R.E.
Published: June 2012

Abstract
Intracardiac flow patterns during heart development were studied by injection of india ink into the yolk sac circulation of chick embryos at Hamburger-Hamilton stages 10 to 17. We injected india ink into a small venule or capillary, carefully preventing application of overpressure to the vascular system, and recorded the intracardiac route by video. From stage 12 onward, blood flow was laminar, and separate intracardiac currents were visualized. The yolk sac was divided into a left and a right half. Blood coursed through each half in concentric loops, ranging from the marginal sinus to the sinus venosus. This parallel array persisted within the heart. Bilateral to the embryo, two lateral regions arose that extended wedgelike within each half, resulting in six equally sized yolk sac regions at stage 16. The process of heart looping was not accompanied by a change in flow pattern. However, developmental changes of the yolk sac circulation were reflected in alteration of the intracardiac flow pattern. From stage 16 onward, the intracardiac flow pattern was no longer determined by the left- or right-hand side of the yolk sac but by bilateral anterior, lateral, and posterior regions of the yolk sac. Blood from the lateral regions of the yolk sac was preferentially distributed to the head. The results show that in preseptation stages a relatively stable flow pattern is present. We suggest that alterations in blood flow could influence the process of normal heart development.
Echocardiographic Assessment of Left Ventricular Mass and Systolic Function in Mice
Authors: Gardin Julius M. , Siri Francis M. , Kitsis Richard N. , Edwards John G. , Leinwand Leslie A.
Published: June 2012

Abstract
The increasing use of transgenic mouse models for investigating the mechanisms of cardiac growth and function has made it important to develop noninvasive methods for assessing murine cardiac anatomy, size, and function. At present, murine cardiac mass can be determined only at necropsy. Left ventricular (LV) function can be assessed by use of various catheterization techniques, but these approaches are usually terminal procedures and provide no information about chamber anatomy and dimensions. Although transthoracic echocardiography has been used to study the LVs of rats and larger animals, the considerably smaller LV masses and somewhat faster heart rates of mice pose significant challenges to obtaining good-quality echocardiograms. In this study we tested the hypothesis that transthoracic echocardiography can image the murine LV as well as provide assessments of LV mass and function. Our results in a series of 33 mice, including normal, transgenic, and aortic-banded subgroups, demonstrate the capability of transthoracic two-dimensionally directed M-mode echocardiography in mice to (1) obtain good-quality images, (2) produce estimates of LV mass having good correlations with directly determined LV mass in normal mice, (3) detect LV hypertrophy noninvasively in different experimental models, and (4) identify impaired LV systolic function. Thus, echocardiography appears to be a promising approach for noninvasively assessing LV mass and function in mice.
Local Continuity of Myocardial Blood Flow Studied by Monochromatic Synchrotron Radiation–Excited X-ray Fluorescence Spectrometry
Authors: Mori Hidezo , Chujo Mitsuaki , Haruyama Shuichi , Sakamoto Hiroyasu , Shinozaki Yoshiro , Uddin-Mohammed Minhaz , Iida Atsuo , Nakazawa Hiroe
Published: June 2012

Abstract
We have developed a monochromatic synchrotron radiation–excited system for two-dimensional mapping of x-ray fluorescence evoked from heavy element–loaded microspheres, which can evaluate myocardial blood flow in small contiguous regions with a small methodological error: 10.8±2.4% of the average of difference of the dual flow for 7- to 10-mg myocardial tissue (4 dogs). The fractal D value obtained from the slope of the log relative dispersion–log mass plot was 1.21±0.08 for a voxel size of 7 to 1260 mg (5 dogs) and that for a voxel size of 2.5 to 40 mg (1.12±0.06) was smaller than that for a voxel size of 40 to 1280 mg (1.25±0.14,
P
<.05, ANOVA, 4 dogs). The distance–correlation coefficient relation for paired myocardial regions was attenuated (correlation analysis), and the correlation coefficients between the original grouping and the two aggregates of the adjacent regions were dissociated (extended correlation analysis) under reduction of coronary perfusion pressure (6 dogs). Suppression of myocardial contraction with lidocaine (3 dogs) and vasodilation with adenosine partly improved the distance–correlation coefficient relation under reduced coronary perfusion pressure. Thus, an x-ray fluorescence system designed for precise flow measurement shows that the fractal nature of local flow distribution can be extended into regions smaller than previously reported, that in these regions the flow becomes more homogeneous, and that the self similarity and continuity of local flow are attenuated by the reduction of coronary perfusion pressure and improved by contractile suppression and coronary vasodilation.
Role of Atrial Contraction in Diastolic Pressure Elevation Induced by Rapid Pacing of Hypertrophied Canine Ventricle
Authors: Berger Ronald D. , Wolff Matthew R. , Anderson James H. , Kass David A.
Published: June 2012

Abstract
The mechanism of diastolic pressure elevation induced by acute rapid pacing in pressure-load hypertrophied left ventricles (LVs) remains incompletely understood. It has been ascribed to abnormalities of coronary flow, metabolism, and calcium cycling. However, rapid pacing also alters the timing of atrial and ventricular stimulation relative to the diastolic filling period, and this could also influence diastolic pressures. To test the role of such mechanical factors, LV pressure-volume hemodynamics were measured in closed-chested anesthetized dogs during and after abrupt cessation of rapid atrial pacing. Twenty-one dogs were studied: 6 dogs with LV hypertrophy (LVH) induced by perinephritic hypertension, 5 sham-operated normotensive dogs, and 10 acute normotensive control dogs. In LVH dogs, but not in sham-operated or control dogs, end-diastolic pressure rose progressively with increasing heart rate from 5.6±3.1 mm Hg at baseline to 22.6±8.1 mm Hg at 220 beats per minute. In all hearts, rapid pacing shifted the timing of left atrial contraction so that it occurred near the onset of LV filling rather than at end diastole. However, in LVH hearts, early LV diastolic pressure and peak atrial pressure were also markedly elevated. Most striking, immediately after terminating the pacing, diastolic pressure declined to near baseline. This rapid pressure decline occurred just when atrial systole would have ensued and before ventricular activation would have followed had pacing continued. Thus, diastolic pressure elevation resolved before a change in ventricular pacing rate. The role of atrial contraction was further explored by simultaneous atrioventricular pacing. This shifted the time of atrial systole so that it occurred during LV isovolumic contraction, while maintaining the identical LV pacing rate. This change eliminated the diastolic pressure elevation found previously. Further analysis revealed that the pressure increase during rapid pacing was not due simply to partial LV filling imposed on a relaxing ventricle or to hypertension or an intact pericardium. These data indicate that mechanical effects of atrioventricular interaction play an important role in tachycardia-induced diastolic dysfunction in this model of LVH and can be more causative than ischemia or metabolic factors in this setting.
Transverse Shear Along Myocardial Cleavage Planes Provides a Mechanism for Normal Systolic Wall Thickening
Authors: LeGrice I. J. , Takayama Y. , Covell J. W.
Published: June 2012

Abstract
Recent studies in humans and other species show that there is substantial transverse shear strain in the left ventricular myocardium, and others have shown transverse myocardial laminae separated by cleavage planes. We proposed that cellular rearrangement based on shearing along myocardial cleavage planes could account for >50% of normal systolic wall thickening, since <50% can be explained by increases in myocyte diameter. To test this hypothesis, we measured strains at two sites with different cleavage-plane anatomy in eight open-chest dogs. Columns of radiopaque markers were implanted in the left ventricular anterior free wall and septum. Markers were tracked with biplane cineradiography, and strains were quantified by using finite deformation techniques. Hearts were perfusion-fixed with glutaraldehyde, and cleavage-plane orientations at the bead sites were measured in three orthogonal planes. At subendocardial sites of the anterior left ventricular wall, where the cleavage planes approach the endocardium obliquely from the apical side of the surface normal in the longitudinal-radial plane (−67±11°), systolic longitudinal-radial transverse shear (E
23
) was positive (0.14±0.08). At the septal sites where the subendocardial cleavage planes approach the endocardium obliquely from above the surface normal (44±12°), E
23
was negative (−0.12±0.08). The differences in cleavage-plane angle and E
23
at the two sites were each highly significant (
P
<.0005). At both sites, the transverse shear strain accompanied substantial systolic wall thickening at the subendocardium (anterior, E
33
=0.44±0.16; septum, E
33
=0.22±0.14). These data are not representative of the behavior in midwall and outer wall sites, where cleavage-plane orientation was not consistently different between anterior left ventricle and septum. Our data indicate that rearrangement of myocytes by slippage along myocardial cleavage planes is in the correct direction and of sufficient magnitude in the subendocardium (inner third) to account for a substantial proportion (>50%) of systolic wall thickening. Furthermore, three-dimensional reconstruction of the myocardial laminae and local comparison with maximum strain vectors indicate that for the inner third of the ventricular wall the maximum shear deformation is a result of relative sliding between myocardial laminae.
Functional Identification of Histamine H
3
-Receptors in the Human Heart
Authors: Imamura Michiaki , Seyedi Nahid , Lander Harry M. , Levi Roberto
Published: June 2012

Abstract
Norepinephrine release contributes to ischemic cardiac dysfunction and arrhythmias. Because activation of histamine H
3
-receptors inhibits norepinephrine release, we searched for the presence of H
3
-receptors directly in sympathetic nerve endings (cardiac synaptosomes) isolated from surgical specimens of human atria. Norepinephrine was released by depolarization with K
+
. The presence of H
3
-receptors was ascertained because the selective H
3
-receptor agonists (
R
)α-methylhistamine and imetit reduced norepinephrine release, and the specific H
3
-receptor antagonist thioperamide blocked this effect. Norepinephrine release was exocytotic, since it was inhibited by the N-type Ca
2+
-channel blocker ω-conotoxin and the protein kinase C inhibitor Ro31-8220. Functional relevance of these H
3
-receptors was obtained by showing that transmural electrical stimulation of sympathetic nerve endings in human atrial tissue increased contractility, an effect blocked by propranolol and attenuated in a concentration-dependent manner by (
R
)α-methylhistamine. Also, thioperamide antagonized the effect of (
R
)α-methylhistamine. Our findings are the first demonstration that H
3
-receptors are present in sympathetic nerve endings in the human heart, where they modulate adrenergic responses by inhibiting norepinephrine release. Since myocardial ischemia causes intracardiac histamine release, H
3
-receptor–induced attenuation of sympathetic neurotransmission may be clinically relevant.
Inhibition of Protein Kinase C Prevents Rapid Desensitization of Type 1B Angiotensin II Receptor
Authors: Tang Hua , Shirai Heigoro , Inagami Tadashi
Published: June 2012

Abstract
The type 1B angiotensin II (AT
1B
) receptor cloned from rat kidney was stably expressed in Chinese hamster ovary cells. The stably expressed receptor was characterized by radioligand binding studies and functional coupling to inositol 1,4,5-triphosphate (IP
3
) formation. Exposure of cells expressing the AT
1B
receptor to angiotensin II (Ang II) resulted in a rapid and dose-dependent homologous desensitization of receptor-mediated production of IP
3
, with an essentially complete desensitization at an agonist concentration >10 nmol/L. Binding studies revealed no significant change in the number of AT
1B
receptors in transfected cells exposed to 1 nmol/L Ang II, whereas exposure to 100 nmol/L Ang II caused a rapid decrease of cell surface receptors, with a 75% loss of receptor number seen at 1 hour. Rapid desensitization occurred in the absence of receptor internalization. Blockade of receptor internalization with concanavalin A had at most only a slight effect on the agonist-induced desensitization. This indicates that factors other than internalization are chiefly responsible for the rapid agonist-induced desensitization. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, caused rapid desensitization of the receptor-mediated IP
3
response. Neither tyrosine kinase inhibitors nor a protein kinase A activator affected the receptor-mediated IP
3
response. The specific PKC inhibitor GF109203X or PKC depletion by prolonged treatment with 1 μmol/L PMA completely blocked the PMA-dependent desensitization. Desensitization evoked by a low Ang II agonist concentration (1 nmol/L) was reversed by the PKC-specific inhibitor GF109203X or PKC depletion, whereas the desensitizing effect at a high agonist concentration (100 nmol/L) is only partially prevented by PKC inhibitory treatment. These results demonstrate that PKC plays a crucial role in the desensitization of the AT
1B
receptor. They also suggest that receptor internalization and an additional PKC-independent pathway also contribute to desensitization of the AT
1B
receptor in transfected cells.
Shear Stress Induces ATP-Independent Transient Nitric Oxide Release From Vascular Endothelial Cells, Measured Directly With a Porphyrinic Microsensor
Authors: Kanai Anthony J. , Strauss Harold C. , Truskey George A. , Crews Anne L. , Grunfeld Saul , Malinski Tadeusz
Published: June 2012

Abstract
Shear stress causes the vascular endothelium to release nitric oxide (NO), which is an important regulator of vascular tone. However, direct measurement of NO release after the imposition of laminar flow has not been previously accomplished because of chemical (oxidative degradation) and physical (diffusion, convection, and washout) complications. Consequently, the mechanism, time course, kinetics, and Ca
2+
dependence of NO release due to shear stress remain incompletely understood. In this study, we characterized these parameters by using fura 2 fluorescence and a polymeric porphyrin/Nafion-coated carbon fiber microsensor (detection limit, 5 nmol/L; response time, 1 millisecond) to directly measure changes in [Ca
2+
]
i
and NO release due to shear stress or agonist (ATP or brominated Ca
2+
ionophore [Br-A23187]) from bovine aortic endothelial cells. The cells were grown to confluence on glass coverslips, loaded with fura 2-AM, and mounted in a parallel-plate flow chamber (volume, 25 μL). The microsensor was positioned ≈100 μm above the cells with its long axis parallel to the direction of flow. Laminar flow of perfusate was maintained from 0.04 to 1.90 mL/min, which produced shear stresses of 0.2 to 10 dyne/cm
2
. Shear stress caused transient NO release 3 to 5 seconds after the initiation of flow and 1 to 3 seconds after the rise in [Ca
2+
]
i
, which reached a plateau after 35 to 70 seconds. Although the amount (peak rate) of NO release increased as a function of the shear stress (0.08 to 3.80 pmol/s), because of the concomitant increase in the flow rate, the peak NO concentration (133±9 nmol/L) remained constant. Maintenance of flow resulted in additional transient NO release, with peak-to-peak intervals of 15.5±2.5 minutes. During this 13- to 18-minute period, when the cells were unresponsive to shear stress, exogenous ATP (10 μmol/L) or Br-A23187 (10 μmol/L) evoked NO release. Prior incubation of the cells with exogenous NO or the removal and EGTA (100 μmol/L) chelation of extracellular Ca
2+
blocked shear stress but not ATP-dependent NO release. The kinetics of shear stress–induced NO release (2.23±0.07 nmol/L per second) closely resembled the kinetics of Ca
2+
flux but differed markedly from the kinetics of ATP-induced NO release (5.64±0.32 nmol/L per second). These data argue that shear stress causes a Ca
2+
-mediated ATP-independent transient release of NO, where the peak rate of release but not the peak concentration depends on the level of shear stress. The transient nature of this response may be due to NO-induced inhibition of Ca
2+
influx via a mechanism yet to be determined.
Attenuation of the Exercise Pressor Reflex
Authors: Meintjes André F. , Nóbrega Antonio C. L. , Fuchs Ingbert E. , Ally Ahmmed , Wilson L. Britt
Published: June 2012

Abstract
Using α-chloralose–anesthetized cats, we studied blood pressure and heart rate responses to static contraction and passive stretch of the triceps surae muscle before and after microdialyzing the μ-opioid agonist [
d
-Ala
2
]-methionine enkephalinamide (DAME, 200 μmol/L) into the L-7 dorsal horn of the spinal cord. In addition, we measured contraction-induced substance P release in the dorsal horn before and after drug delivery. After 92±3 minutes of dialyzing the opioid agonist, contraction-induced increases in mean arterial pressure and heart rate were attenuated from control values of 58±7 mm Hg and 17±3 beats per minute to postdrug values of 27±7 mm Hg and 10±2 beats per minute, respectively. A similar attenuation was observed for the passive muscle stretches after 97±5 minutes of dialysis (control, 38±4 mm Hg and 8±2 beats per minute; after drug, 23±4 mm Hg and 5±1 beats per minute). Prior microdialysis of naloxone (300 μmol/L), a μ-antagonist, blocked this effect, suggesting that the opioid agonist has a specific receptor action. Naloxone alone had no effect on the pressor or tachycardiac responses. The contraction-induced increase in substance P–like immunoreactivity was reduced from a control value of 0.119±0.024 to 0.047±0.010 fmol/100 μL by DAME. Time-control experiments revealed no decrease in the release of substance P–like immunoreactivity. Thus, activation of opioid receptors modulates the transmission of group III and IV muscle afferent nerve activity through the L-7 dorsal horn. Presynaptic inhibition of substance P release from muscle afferents is a potential mechanism of action for DAME.
Effects of Rest Interval on the Release of Calcium From the Sarcoplasmic Reticulum in Isolated Guinea Pig Ventricular Myocytes
Authors: Terracciano Cesare M. N. , Naqvi Ruby U. , MacLeod Kenneth T.
Published: June 2012

Abstract
Guinea pig cardiac myocytes were loaded with the fluorescent dye indo 1, and cell contraction was measured by a video edge-detection system. Ca
2+
was released from the sarcoplasmic reticulum (SR) by rapidly cooling the myocytes or by rapid application of 10 mmol/L caffeine. Estimates of the amount of Ca
2+
released from the SR after different rest intervals (ie, under different loading conditions) were obtained by measuring the current evoked by rapid application of 10 mmol/L caffeine, which we call Na
+
/Ca
2+
exchange current. This current is completely inhibited by removal of extracellular Na
+
and Ca
2+
or by application of 5 mmol/L Ni
2+
. SR Ca
2+
release after rest intervals of 5 to 120 seconds (assuming cell volume to be 30×10
−12
L) was estimated to be 57.8±5.7 to 25.7±4.5 μmol/L accessible cell volume, respectively, equivalent to 23 to 10 μmol/kg wet wt, respectively. There was an exponential decline in Ca
2+
released from the SR after rest intervals of 2 to 120 seconds (rate constant, 0.029 s
−1
; t
1/2
, 24 seconds); thereafter, there remained a portion (56%) of Ca
2+
releasable to caffeine application. We found a similar exponential decay (rate constant, 0.020 s
−1
; t
1/2
, 35 seconds) of the size of rapid cooling contractures with increasing rest intervals. The time to peak of the Na
+
/Ca
2+
exchange current in the presence of caffeine slowed at long rest intervals, ie, at smaller SR loads. A decrease in SR load of 50% increased the time to peak of the exchange current by 213±37% (n=6). The rate of generation of a rapid cooling contracture was faster after short rest intervals and was associated with a faster rise in the corresponding increase in indo 1 fluorescence. A decrease in SR load of 50% decreased the rate of cell shortening to 34±5% and decreased the rate of change in fluorescence to 54±2% (n=5).
α-Adrenergic Control of Volume-Regulated Cl
−
Currents in Rabbit Atrial Myocytes
Authors: Duan Dayue , Fermini Bernard , Nattel Stanley
Published: June 2012

Abstract
α-Adrenergic stimulation is known to play a role in cardiac arrhythmogenesis and to modulate a variety of cardiac K
+
currents. The effects of α-adrenergic stimulation on Cl
−
currents are largely unknown. Many cardiac cell types show a volume-sensitive Cl
−
current induced by cell swelling (I
Cl.swell
). The present experiments were designed to assess the potential α-adrenergic modulation of I
Cl.swell
in rabbit atrial myocytes. I
Cl.swell
was induced with the use of a hypotonic superfusate, under conditions designed to prevent currents carried by K
+
, Na
+
, and Ca
2+
ions. A basal Cl
−
current (I
Cl.b
) was observed under isotonic conditions in 128 of 150 cells (85%), had the same dependency on [Cl
−
]
o
as I
Cl.swell
, and was reduced by cell shrinkage induced by hypertonic superfusion, suggesting that I
Cl.b
is carried by the same volume-sensitive Cl
−
conductance as I
Cl.swell
. Phenylephrine produced a concentration-dependent and near-complete inhibition of I
Cl.b
and I
Cl.swell
, with EC
50
values of 86±5 and 72±7 (mean±SEM) μmol/L, respectively, at +20 mV. Norepinephrine (administered in the presence of 1 μmol/L propranolol) also inhibited I
Cl.b
and I
Cl.swell
, with EC
50
values of 2.6±0.1 and 2.8±0.4 μmol/L, respectively. The concentration-response curve for phenylephrine was shifted significantly (
P
<.001) to the right by the α
1
-adrenoceptor antagonist prazosin and by the α
1A
-receptor antagonists (+)-niguldipine and 5-methylurapidil but was unaltered by the α
1B
-receptor antagonist chloroethylclonidine (100 μmol/L). Inhibition of protein kinase C (PKC) with staurosporine, H-7, or 18-hour preincubation with the phorbol ester 4β-phorbol 12-myristate 13-acetate (PMA, 500 nmol/L) blocked the effects of phenylephrine on I
Cl.swell
, and the highly selective PKC inhibitor bisindolylmaleimide blocked the effects of norepinephrine on I
Cl.swell
and I
Cl.b
. Both PMA and 1-oleoyl-2-acetylglycerol inhibited I
Cl.swell
in a concentration-dependent fashion. In blinded studies, the phorbol ester phorbol 12,13-didecanoate (PDD) reduced I
Cl.swell
by 91±3%; its inactive analogue 4α-PDD had no effect (mean change, 3±1%). Preincubation with pertussis toxin (PTX) prevented the actions of phenylephrine on I
Cl.swell
, indicating a role for a PTX-sensitive guanine nucleotide–binding (G) protein. We conclude that α-adrenergic agonists inhibit volume-sensitive Cl
−
currents in rabbit atrial cells by interacting with an α
1A
-adrenoceptor mechanism that is coupled to PKC via a PTX-sensitive G protein. These results suggest a potentially novel mechanism of α-adrenergic control of cardiac electrical activity, the inhibition of volume-sensitive Cl
−
currents, and indicate that PKC, well known to elicit phosphorylation-dependent Cl
−
currents in cat and guinea pig ventricular myocytes, is also capable of potently inhibiting other forms of cardiac Cl
−
current.
A Redox-Based Mechanism for Cardioprotection Induced by Ischemic Preconditioning in Perfused Rat Heart
Authors: Chen Weina , Gabel Scott , Steenbergen Charles , Murphy Elizabeth
Published: June 2012

Abstract
Recent studies have suggested that mild redox alterations can regulate cell function. Therefore, we tested the hypothesis that alteration in the thiol redox state might be responsible for the cardioprotective effects conferred by ischemic preconditioning in the perfused rat heart. We find that preconditioning with four 5-minute periods of ischemia, each separated by 5 minutes of reflow, is associated with a significant loss of glutathione (3.98±0.32 μmol/g dry wt, n=8) compared with no preconditioning (6.38±0.24 μmol/g dry wt, n=14). We further find that the addition of
N
-acetylcysteine (NAC, a glutathione precursor and antioxidant) during the preconditioning protocol not only blocks the loss of glutathione (5.60±0.31 μmol/g dry wt, n=9) but also blocks the protective effects of preconditioning. It is observed that after 20 minutes of ischemia followed by 20 minutes of reflow, untreated hearts recover 38±7% (n=5) of their initial preischemic contractile function, whereas preconditioned hearts recover 91±11% (n=7). Hearts preconditioned in the presence of NAC recover 24±3% (n=7) of their preischemic function. Similarly, the addition of NAC reverses the protective effect of preconditioning on creatine kinase release. On reflow after 60 minutes of ischemia, creatine kinase release from control hearts was 271±20 IU · 20 min
−1
· g dry wt
−1
(n=5), whereas preconditioned hearts release only 170±26 IU · 20 min
−1
· g dry wt
−1
(n=6), and hearts preconditioned in the presence of NAC release 361±30 IU · 20 min
−1
· g dry wt
−1
(n=5). We also find that hearts preconditioned in the presence of NAC have less attenuation of the decline in pH
i
than hearts preconditioned in the absence of drug. Thus, a redox-sensitive mechanism may be involved in the protection afforded by ischemic preconditioning.
Contractile Responsiveness of Ventricular Myocytes to Isoproterenol Is Regulated by Induction of Nitric Oxide Synthase Activityin Cardiac Microvascular EndothelialCells in Heterotypic Primary Culture
Authors: Ungureanu-Longrois Dan , Balligand Jean-Luc , Okada Ikutaro , Simmons William W. , Kobzik Lester , Lowenstein Charles J. , Kunkel Steven L. , Michel Thomas , Kelly Ralph A. , Smith Thomas W.
Published: June 2012

Abstract
Unlike large-vessel endothelial cells in cell culture, cardiac microvascular endothelial cells (CMEC) isolated from adult rat ventricular muscle exhibit little detectable constitutive nitric oxide (NO) synthase activity after isolation in vitro but respond to specific combinations of inflammatory mediators with an increase in inducible NO synthase (iNOS; type 2 NO synthase) activity. CMEC iNOS is induced by soluble inflammatory mediators in lipopolysaccharide-activated rat alveolar macrophage–conditioned medium at 24 hours, and this induction can be partially prevented by either interleukin-1 (IL-1) receptor antagonist or a polyclonal anti–rat tumor necrosis factor-α (TNF-α) antiserum. Interferon-γ (IFN-γ), which by itself does not induce iNOS in CMEC, potentiates and accelerates iNOS induction by IL-1β. Transforming growth factor-β (TGF-β) decreases iNOS activity, protein content, and mRNA abundance in IL-1β– and IFN-γ–pretreated CMEC. To determine whether NO released by CMEC would affect myocyte contractile function in vitro, freshly isolated ARVM were allowed to settle onto confluent, serum-starved CMEC that had been pretreated for 24 hours with IL-1β, a cytokine that alone does not affect myocyte contractile function in vitro. Baseline contractile amplitude, at 2 Hz and 37°C, of myocytes in heterotypic culture with IL-1β–pretreated CMEC was not different from that of myocytes in control, homotypic myocyte cultures. However, cocultured myocytes exhibited decreased contractile responsiveness to 2 nmol/L isoproterenol compared with control cells, and this could be reversed by the addition of 1 mmol/L
N
G
-monomethyl-
l
-arginine, an inhibitor of NOS. Thus, activation of endothelial cell iNOS by specific cytokines affects the contractile responsiveness to isoproterenol of adjacent cardiac myocytes in vitro. Reciprocal cell-cell signaling leading to TGF-β release and activation could act to limit the extent of endothelial cell iNOS induction in vivo.
Epidermal Growth Factor Receptor–Targeted Cytotoxin Inhibits Neointimal Hyperplasia In Vivo
Authors: Pastore Christopher J. , Isner Jeffrey M. , Bacha Patricia A. , Kearney Marianne , Pickering J. Geoffrey
Published: June 2012

Abstract
Smooth muscle cell accumulation is a key feature of restenosis that may be inhibited by the delivery of receptor-targeted cytotoxins. DAB
389
EGF is a recombinant fusion protein in which the receptor-binding domain of diphtheria toxin has been replaced by human epidermal growth factor (EGF). We investigated the effectiveness of DAB
389
EGF to inhibit neointimal hyperplasia in the balloon-injured rat carotid artery. Incubation of rat carotid arteries with
125
I-labeled EGF revealed extensive EGF binding sites in the neointima of balloon-injured arteries. Sixty rats subsequently received either saline or DAB
389
EGF (total dose, 0.15 mg) delivered immediately following balloon injury either systemically, via 14-day continuous osmotic pump infusion, or locally, via 30-minute intraluminal incubation. The effect of both treatment strategies was measured 2 weeks after injury by cross-sectional morphometric analysis of intimal area, the ratio of intimal/medial area (I/M), and the percent luminal narrowing (%LN). In addition, proliferative activity was assessed by immunostaining for the presence of the proliferating cell nuclear antigen (PCNA). Compared with controls, systemic delivery of fusion toxin significantly reduced intimal area, I/M, and %LN by 40%, 40%, and 29%, respectively. However, these rats exhibited 2% weight loss, indicating mild systemic toxicity. Local, intraluminal administration of DAB
389
EGF yielded a more pronounced reduction in intimal area, I/M, and %LN by 74%, 79%, and 72%, respectively. This inhibitory effect was preserved at 3 weeks postinjury, and PCNA immunostaining of locally treated arteries revealed a virtual absence of proliferative activity in the neointima and media at this timepoint. In contrast to systemically treated rats, rats receiving fusion toxin locally gained weight at a rate similar to controls, indicating avoidance of systemic toxicity. We conclude that DAB
389
EGF is a potent inhibitor of neointimal hyperplasia in vivo and that whereas an inhibitory effect may be achieved by systemic delivery, local delivery appears to be more potent, avoids systemic toxicity, and thus represents a feasible strategy to preempt restenosis.
Reperfusion of Rat Heart After Brief Ischemia Induces Proteolysis of Calspectin (Nonerythroid Spectrin or Fodrin) by Calpain
Authors: Yoshida Ken-ichi , Inui Makoto , Harada Kazuki , Saido Takaomi C. , Sorimachi Yoshihide , Ishihara Tokuhiro , Kawashima Sei-ichi , Sobue Kenji
Published: June 2012

Abstract
Rat myocardium expresses the 240- and 235-kD polypeptides antigenically related to α- and β-subunits of brain calspectin (nonerythroid spectrin or fodrin), respectively. In the subcellular fractions of the myocardium, α-calspectin was found in the 600
g
, 10 000
g
, and 100 000
g
pellets, whereas β-calspectin was localized to the 10 000
g
pellet. On the basis of the Na
+
,K
+
-ATPase activity and the contents of a gap junction protein, the sarcolemma was distributed to the 10 000
g
and 100 000
g
pellets, and the intercalated disks were enriched in the 10 000
g
pellet. Both α- and β-calspectin were proteolyzed by calpain in vitro. The two subunits were also proteolyzed in vivo, when the rat hearts underwent 10 to 60 minutes of global ischemia followed by 30 minutes of reperfusion. The reperfusion following the ischemia induced the proteolysis of α-calspectin in the 10 000
g
and 100 000
g
pellets, producing the 150-kD fragment. A synthetic calpain inhibitor, calpain inhibitor-1, suppressed the degradation of calspectin in vivo, which indicates that calpain is responsible for the reperfusion-induced proteolysis of calspectin. The inhibitor also improved myocardial stunning. Immunohistochemical study revealed that the proteolysis of α-calspectin occurs at the intercalated disks and the sarcolemma after postischemic reperfusion, in accord with the biochemical data. These results suggest that degradation of calspectin partly accounts for the contractile failure of the myocardium after postischemic reperfusion by disrupting the membrane skeleton and the intercalated disks.
Role of Bradykinin in Protection of Ischemic Preconditioning in Rabbit Hearts
Authors: Goto Mahiko , Liu Yongge , Yang Xi-Ming , Ardell Jeffrey L. , Cohen Michael V. , Downey James M.
Published: June 2012

Abstract
Bradykinin receptor activation has been proposed to be involved in ischemic preconditioning. In the present study, we further investigated the role of this agent in preconditioning in both isolated and in situ rabbit hearts. All hearts were subjected to 30 minutes of regional ischemia followed by reperfusion for 2 hours (in vitro hearts) and 3 hours (in situ hearts). Infarct size was measured by tetrazolium staining and expressed as a percentage of the size of the risk zone. Preconditioning in situ hearts with 5 minutes of ischemia and 10 minutes of reperfusion significantly reduced infarct size to 10.2±2.2% of the risk region (
P
<.0005 versus control infarct size of 36.7±2.6%). Pretreatment with HOE 140 (26 μg/kg), a bradykinin B
2
receptor blocker, did not alter infarct size in nonpreconditioned hearts (40.6±5.3% infarction) but abolished protection from ischemic preconditioning (34.1±1.6% infarction). However, when HOE 140 was administered during the initial reflow period following 5 minutes of ischemia, protection was no longer abolished (15.6±3.9% infarction versus 13.3±3.8% without HOE 140,
P
=NS). Bradykinin infusion in isolated hearts mimicked preconditioning, and protection was not affected by pretreatment with the nitric oxide synthase inhibitor
N
ω
-nitro-
l
-arginine methyl ester or the prostaglandin synthesis inhibitor indomethacin but could be completely abolished by the protein kinase C (PKC) inhibitors polymyxin B and staurosporine as well as by HOE 140. HOE 140 could not block the protection of ischemic preconditioning in isolated hearts. That failure was apparently due to the absence of blood-borne kininogens rather than autonomic nerves. When the preconditioning stimulus in the in situ model was amplified with four cycles of 5-minute ischemia/10-minute reperfusion, HOE 140 pretreatment could no longer block protection (infarct size was 10.7±3.5% versus 6.4±2.0% without HOE 140,
P
=NS). We propose that bradykinin receptors protect by coupling to PKC as do adenosine receptors, and blockade of either receptor will diminish the total stimulus of PKC below threshold and prevent protection. A more intense preconditioning ischemic stimulus can overcome bradykinin receptor blockade, however, by simply enhancing the amount of adenosine and possibly other agonists released.
Hypoxia Regulates Vascular Endothelial Growth Factor Gene Expression in Endothelial Cells
Authors: Liu Yuxiang , Cox Shanna R. , Morita Toshisuke , Kourembanas Stella
Published: June 2012

Abstract
Vascular endothelial growth factor (VEGF) is a potent mitogen specific for endothelial cells. Its expression is dramatically induced by low oxygen tension in a variety of cell types, and it has been suggested to be a key mediator of hypoxia-induced angiogenesis. Although VEGF action is targeted to endothelial cells, it is generally believed that these cells do not express VEGF. In addition, the mechanisms by which hypoxia regulates VEGF production remain unclear. We report in the present study that pulmonary artery endothelial cells do not express VEGF under basal conditions; however, significant VEGF mRNA levels accumulate when these cells are exposed to hypoxia. Using a DNA fragment containing human VEGF promoter sequence, we identified a 28-bp element that is necessary and sufficient to upregulate transcription in response to hypoxia. This element can act as a hypoxia-specific enhancer when placed upstream or downstream from a heterologous promoter. The enhancer includes, in addition to an octamer homologous to the hypoxia-inducible factor-1 (HIF-1) consensus, a sequence that resides 3′ to the consensus. Although this sequence may not be involved in the binding of HIF-1, it is absolutely required for the enhancer activity and may be the binding site for certain constitutive binding proteins. The expression of VEGF by endothelial cells in response to hypoxia may provide an important mechanism by which endothelial cell permeability and proliferation is regulated in an autocrine manner.
Ligation of Endothelial α
v
β
3
Integrin Increases Capillary Hydraulic Conductivity of Rat Lung
Authors: Tsukada Hiroshi , Ying Xiaoyou , Fu Chenzhong , Ishikawa Shigemi , McKeown-Longo Paula , Albelda Steven , Bhattacharya Sunita , Bray Bonnie Anderson , Bhattacharya Jahar
Published: June 2012

Abstract
Complement-mediated pulmonary edema results from increases in lung capillary hydraulic conductivity (Lp), possibly by receptor-mediated mechanisms. We considered the Lp effects of vitronectin and the vitronectin-containing complement complex SC5b-9, which ligate the integrin α
v
β
3
. Vitronectin, SC5b-9, and SC5b-9–enriched zymosan-activated serum all rapidly increased Lp, as determined by the split-drop technique in single lung capillaries of rat lung. The Lp increases were inhibited by a monospecific (LM609) and a polyclonal (R838) antibody against the α
v
β
3
integrin but not by an irrelevant monoclonal antibody isotype matched with LM609, by a monoclonal antibody against the α
v
β
5
integrin, or by preimmune rabbit serum. Vitronectin monomers failed to increase Lp. The tyrosine kinase blockers genistein and methyl 2,5-dihydroxycinnamate caused significant concentration-dependent inhibitions of Lp increases due to vitronectin and zymosan-activated serum. By contrast, the protein kinase C blocker calphostin C had no major effect. We conclude that (1) multivalent ligation of the luminally located α
v
β
3
integrin of lung capillary endothelium increases transcapillary liquid flux, and (2) the dominant signal transduction pathway for this effect occurs through tyrosine kinase activation.
Antisense to Thyrotropin Releasing Hormone Receptor Reduces Arterial Blood Pressure in Spontaneously Hypertensive Rats
Authors: Suzuki Satoshi , Pilowsky Paul , Minson Jane , Arnolda Leonard , Llewellyn-Smith Ida , Chalmers John
Published: June 2012

Abstract
We report in the present study the effect of intrathecal treatment with antisense oligonucleotides complementary to thyrotropin releasing hormone (TRH) receptor mRNA on the pressor response to intrathecal administration of TRH and on resting arterial blood pressure in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). In 16-week-old male WKY rats, 18-base phosphodiester antisense or mismatch oligonucleotides to TRH receptor mRNA (100 μg per day) were injected intrathecally for 3 days. Twenty-four hours after the last injection, the magnitude of the pressor response to intrathecal TRH (10 μg) was significantly smaller in the antisense-treated group (n=7) compared with mismatch-treated controls (n=7) (change in mean arterial pressure, +20.3±3.0 versus +32.6±2.5 mm Hg,
P
<.01). No differences were observed in the pressor responses to injection of
N
-methyl-
d
-aspartic acid. Resting arterial blood pressure was unaffected by antisense treatment in WKY rats. In separate experiments, 16-week-old male SHR were treated with antisense (n=7) or mismatch (n=6) oligonucleotides for 3 days. Mean resting arterial blood pressure was significantly reduced by treatment with antisense oligonucleotides (from 157±4.8 to 119±8.8 mm Hg,
P
<.01), but no significant changes were observed in mismatch-treated animals. Our results suggest that the expression of TRH receptors in spinal sympathetic preganglionic neurons can be selectively reduced by intrathecal treatment with antisense oligonucleotides and that TRH projections to sympathetic preganglionic neurons play an important role in the elevation of arterial blood pressure in SHR.
Nuclear Magnetic Resonance Studies of Cationic and Energetic Alterations With Oxidant Stress in the Perfused Heart
Authors: Yanagida S. , Luo C.S. , Doyle M. , Pohost G.M. , Pike M.M.
Published: June 2012

Abstract
The postischemic generation of oxygen-derived free radicals may contribute to myocardial reperfusion injury by affecting sarcolemmal ion transport. Recent evidence indicates that exposure to reactive oxygen intermediates induces rapid increases in myocardial cytosolic free Ca
2+
(Ca
2+
i
). The mechanism is undetermined but may involve disturbances in Na
+
homeostasis. We tested this hypothesis by interleaving
23
Na and
31
P nuclear magnetic resonance (NMR) measurements of Na
+
i
and high-energy phosphates in glucose-perfused rat hearts exposed to hydroxyl radicals generated from H
2
O
2
and Fe
3+
. In separate experiments, K
+
i
and Ca
2+
i
were measured with
39
K and
19
F NMR, respectively. The hearts rapidly exhibited contracture. Threefold Na
+
i
increases and substantial K
+
i
depletion were observed. Glycolytic inhibition was indicated by rapid sugar phosphate accumulation and cellular energy depletion. Notably, however, severe functional and energetic deterioration and substantial elevation of Ca
2+
i
occurred before substantial Na
+
i
accumulation or K
+
i
depletion was observed. Further experiments investigated the ability of pyruvate to scavenge H
2
O
2
and to protect the myocardium from oxidant stress. Pyruvate (1 or 2.5 mmol/L) dramatically attenuated functional and energetic alterations and alterations in Na
+
i
and K
+
i
, whereas acetate (2.5 mmol/L) offered no protection. Unlike pyruvate, lactate (5 mmol/L) has little or no capacity to scavenge H
2
O
2
but has similar protective effects. In conclusion, pyruvate effectively protects against H
2
O
2
/Fe
3+
, largely by direct H
2
O
2
scavenging. Protection with lactate may involve intracellular pyruvate augmentation. Without exogenous pyruvate or lactate, myocardial Na
+
homeostasis can be substantially altered by oxidant stress, possibly via cellular energy depletion. Excess Na
+
i
accumulation may, in turn, hasten metabolic and functional deterioration, but a causal link with the initial alterations in function or Ca
2+
i
was not supported.
Modulation of Cerebral Arteriolar Diameter by Intraluminal Flow and Pressure
Authors: Ngai Al C. , Winn H. Richard
Published: June 2012

Abstract
We determined whether cerebral arterioles in vitro adjust their diameters in response to changes in intraluminal flow rate and pressure. Intracerebral arterioles (38- to 55-μm diameter) were isolated from Sprague-Dawley rats and cannulated with a perfusion system that permitted separate control of intraluminal pressure and flow rates. Increasing pressure at 0 flow, in 20 mm Hg steps from 20 to 100 mm Hg, resulted in myogenic constriction, which was greatest at 60 mm Hg (≈20%). Increasing flow rate at a constant pressure of 60 mm Hg elicited a biphasic response. At flow rates of up to 10 μL/min, the arterioles dilated by up to 14.5±2.2% of their control diameter. At higher (>10 μL/min) flow rates, however, a progressive restoration of resting diameter was observed. Application of the nitric oxide synthase inhibitor
N
G
-monomethyl-
l
-arginine (L-NMMA, 0.1 mmol/L) caused a 15.4±1.7% decrease in control diameter (at 60 mm Hg, zero flow). Although L-NMMA did not affect the responses to increases in pressure or to vasodilators (adenosine and pH 6.8 buffer), it abolished the dilator responses to flow rate increases and to acetylcholine. In contrast, inhibition of prostaglandin synthesis by indomethacin (10 μmol/L) had no effect on flow-induced dilation. These results show that changes in intraluminal flow rates and pressure can independently influence cerebral arteriolar tone and suggest that the flow-induced dilator responses of cerebral arterioles are mediated by an arginine metabolite, such as nitric oxide.
Does Ischemic Preconditioning in the Human Involve Protein Kinase C and the ATP-Dependent K
+
Channel?
Authors: Speechly-Dick M. E. , Grover G. J. , Yellon D. M.
Published: June 2012

Abstract
Protein kinase C (PKC) and the ATP-dependent K
+
channel (K
ATP
channel) have been implicated in the mechanism of ischemic preconditioning in animal models. This study investigated the role of K
ATP
channels and PKC in preconditioning in human myocardium and whether K
ATP
channels are activated via a PKC-dependent pathway. Right atrial trabeculae were superfused with Tyrode’s solution and paced at 1 Hz. After stabilization, muscles underwent one of nine different protocols, followed by simulated ischemia (SI) consisting of 90 minutes of hypoxic substrate-free superfusion paced at 3 Hz and then by 120 minutes of reperfusion. Preconditioning consisted of 3 minutes of SI and 7 minutes of reperfusion. The experimental end point was recovery of contractile function after SI, presented here as percentage recovery (%Rec) of baseline function. %Rec was significantly improved by preconditioning by the K
ATP
channel opener cromakalim (CK), and by the PKC activator 1,2-dioctanoyl-
sn
-glycerol (DOG) compared with nonpreconditioned controls when these treatments were given before the SI insult (control group, 29.5±3.6%; preconditioned group, 63.5±5.4%, CK-treated group, 52.9±3.1%; and DOG-treated group, 48.0±3.5%;
P
<.01). The effects of CK could be blocked by the K
ATP
channel blocker glibenclamide (%Rec, 17.8±3.5%). Preconditioning could be blocked by the PKC antagonist chelerythrine (%Rec, 24.1±5.0%) and the K
ATP
blocker glibenclamide (%Rec, 24.8±3.1%). The effects of DOG could also be blocked by glibenclamide (%Rec, 23.1±2.3%). These findings show that protection against contractile dysfunction after SI can be induced by activation of PKC and by the opening of the K
ATP
channel and that the protection induced by PKC activation and preconditioning can be blocked by blocking the K
ATP
channel. This suggests that the mechanism of preconditioning in humans may act via PKC and rely on the action of the K
ATP
channel as the end effector.