bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of sinoaortic denervation on frequency-domain estimates of baroreflex sensitivity in conscious cats
Authors: Mancia Giuseppe , Parati Gianfranco , Castiglioni Paolo , di Rienzo Marco
Published: December 2017

In animals and humans, baroreceptor modulation of the sinus node in daily life can be studied by identification of the number of sequences in which systolic blood pressure (SBP) and pulse interval (PI) linearly decrease or increase for several beats. It is also studied by power spectral analysis of SBP and PI in regions where their powers are coherent, although, in contrast to the sequence method, whether this frequency-domain method specifically reflects the baroreceptor-heart rate reflex has not been adequately tested. We recorded intra-arterial BP for ∼3.5 h in eight conscious cats, first intact and then 7–10 days after sinoaortic denervation (SAD). Sensitivity of baroreceptor-heart rate reflex was assessed in 120-s segments by the square root of the ratio of PI and SBP spectral powers (α) in the regions around 0.1 (MF) and 0.3 (HF) Hz, and coherence between PI and SBP spectral powers in MF and HF regions was computed. SAD increased overall SBP variability and reduced PI variability throughout the frequency range examined. SAD markedly reduced ( P < 0.01) both α-MF (−65.6%) and α-HF (−79.9%) and consistently reduced the number of coherent segments [i.e., where coherence ( K 2) > 0.5] and average coherence values in the MF region. In the HF region, however, SAD did not alter the number of coherent segments, and although average coherence value throughout the HF band was reduced, in restricted portions of the band (different between animals), a high coherence value survived denervation. No significant changes were seen in any measured variables in five sham-operated cats. Thus the frequency-domain method specifically reflects baroreflex modulation of heart rate in the MF region only. In the HF region, in contrast, baroreflex and nonbaroreflex influences on the sinus node both contribute to a variable degree to determination of heart rate responses to BP oscillations. If used to study baroreflex function in daily life, this method should use the coefficient derived from MF data.
Ischemic preconditioning and myocardial hypothermia in rabbits with prolonged coronary artery occlusion
Authors: Hale Sharon L. , Kloner Robert A.
Published: December 2017

This study tests whether combining regional hypothermia and ischemic preconditioning (IP) provides greater myocardial protection during prolonged coronary artery occlusion (CAO) than either intervention alone, and whether increasing the duration of IP from 5 to 7 min extends the window of protection to include a 2-h CAO. Anesthetized rabbits were randomized to four groups ( n = 8 rabbits/group): control (C), hypothermia alone (H), IP alone for two 7-min episodes (IP7), and IP plus hypothermia (H + IP7). To compare differences in IP for 5 versus 7 min, additional rabbits ( n = 6) received one 5-min episode of ischemia (IP5). All rabbits got 2 h of CAO and 3 h of reperfusion. In comparison with the infarct size in the control group (72 ± 4% of the risk zone), infarct size was significantly reduced in H (50 ± 7%), IP7 (49 ± 5%), and H + IP7 (42 ± 6%) (all P < 0.05 vs. control group). IP5 failed to confer protection (67 ± 5% of the risk zone). Therefore, IP can protect against a 2-h CAO if the IP regimen is increased from 5 to 7 min. The combination therapy significantly improved regional myocardial blood flow in the previously ischemic region to a greater extent than either treatment alone.
NO modulates myocardial O2consumption in the nonhuman primate: an additional mechanism of action of amlodipine
Authors: Forfia Paul R. , Zhang Xiaoping , Knight Delvin R. , Smith Andrew H. , Doe Christopher P. A. , Wolfgang Eric A. , Flynn David M. , Wolin Michael S. , Hintze Thomas H.
Published: December 2017

Recent evidence from our laboratory and others suggests that nitric oxide (NO) is a modulator of in vivo and in vitro oxygen consumption in the murine and canine heart. Therefore, the goal of our study was twofold: to determine whether NO modulates myocardial oxygen consumption in the nonhuman primate heart in vitro and to evaluate whether the seemingly cardioprotective actions of amlodipine may involve an NO-mediated mechanism. Using a Clark-type O2electrode, we measured oxygen consumption in cynomologous monkey heart at baseline and after increasing doses of S-nitroso- N-acetylpenicillamine (SNAP; 10−7–10−4M), bradykinin (10−7–10−4M), ramiprilat (10−7–10−4M), and amlodipine (10−7–10−5M). SNAP (−38 ± 5.8%), bradykinin (−19 ± 3.9%), ramiprilat (−28 ± 2.3%), and amlodipine (−23 ± 4.5%) each caused significant ( P < 0.05) reductions in myocardial oxygen consumption at their highest dose. Preincubation of tissue with nitro-l-arginine methyl ester (10−4M) blunted the effects of bradykinin (−5.4 ± 3.2%), ramiprilat (−4.8 ± 5.0%), and amlodipine (−5.3 ± 5.0%) but had no effect on the tissue response to SNAP (−38 ± 5.8%). Our results indicate that NO can reduce oxygen consumption in the primate myocardium in vitro, and they support a role for the calcium-channel blocker amlodipine as a modulator of myocardial oxygen consumption via a kinin-NO mediated mechanism.
Differential regulation of cardiac expression of IL-6 and TNF-α by A2- and A3-adenosine receptors
Authors: Wagner Daniel R. , Kubota Toru , Sanders Virginia J. , McTiernan Charles F. , Feldman Arthur M.
Published: December 2017

The proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 have been implicated in the development of congestive heart failure. Adenosine inhibits the expression of TNF-α and IL-6 in macrophages. We determined the effect of adenosine on cytokine expression in rat cardiomyocytes and trabecular muscles obtained from patients with cardiomyopathy. In myocytes, adenosine suppressed TNF-α mRNA by 40% ( P < 0.05) and induced a 4.7-fold increase in IL-6 mRNA ( P < 0.05) with a twofold increase in IL-6 protein release ( P < 0.001). The effect on TNF-α could be replicated by A2agonist. The effect on IL-6 could be replicated by A3agonist, but not by A1and A2agonists, and was completely suppressed by A3antagonist. In human trabecular muscles, A2agonist suppressed TNF-α mRNA by 60% ( P < 0.05), but adenosine had no effect on IL-6. In the failing heart, IL-6 was immunolocalized to inflammatory cells. Thus A2and A3receptors differentially regulate cardiac expression of TNF-α and IL-6. Rat cardiomyocytes and the failing human heart respond differently to adenosine.
Plasma volume expansion with solutions of hemoglobin, albumin, and Ringer lactate in sheep
Authors: Fischer Stefanie R. , Burnet Michael , Traber Daniel L. , Prough Donald S. , Kramer George C.
Published: December 2017

We have measured plasma volume expansion (Evans blue and hematocrit changes) and hemodynamic responses in conscious hemorrhaged and normovolemic splenectomized sheep after a 30-min infusion of either 20 ml/kg of diaspirin cross-linked hemoglobin (DCLHb), 20 ml/kg of human albumin (Alb), or 60 ml/kg of a solution of Ringer lactate (RL). All regimens expanded blood volume and increased blood pressure and cardiac output after hemorrhage. However, only 15 ± 3% of the infused volume of RL was evident as intravascular expansion 10-min postinfusion, compared with 67 ± 16% and 139 ± 139% for Alb and DCLHb, respectively. DCLHb infusions were associated with higher blood pressures and lower cardiac outputs compared with RL and Alb infusions, but the increased oxygen content of blood with DCLHb resulted in systemic delivery of oxygen similar to that of the other infusions. These differences in hemodynamics and vascular volume continued for 6 h, and at 24 h vascular volume and all hemodynamics were similar in all three groups. The better volume expansion with DCLHb may be due to greater mobilization of endogenous interstitial protein or reduced transcapillary loss as total intravascular endogenous plasma protein increased after infusion of DCLHb, whereas there was an apparent loss of endogenous intravascular protein after infusions of Alb and RL. Vasoconstriction by DCLHb is one mechanism that could lower blood-to-tissue transport of fluid and protein. In addition to its oxygen-carrying capacity and vasoactivity, DCLHb is associated with volume expansion properties out of proportion to its colloid osmotic pressure.
Effect of amiloride analogs on DOCA-salt-induced hypertension in rats
Authors: Keep Richard F. , Si Xiaochen , Shakui Parvin , Ennis Steven R. , Betz A. Lorris
Published: December 2017

Intracerebroventricular infusions of an amiloride analog, benzamil, reduce blood pressure in several rat models of hypertension. This effect has been attributed to an inhibition of amiloride-sensitive Na+channels in the brain. This study examines whether intracerebroventricular benzamil would prevent the onset of deoxycorticosterone acetate (DOCA)-salt-induced hypertension in rats and whether this effect correlates with an inhibition of ion transport through the known amiloride-sensitive cation channels at the blood-brain barrier. We also examine whether the effects of benzamil on blood pressure are mediated by a Na+channel by comparing the effects of different amiloride analogs. Benzamil (0.15 and 0.5 μg/h icv) did significantly attenuate the increase in blood pressure induced by DOCA treatment. This antihypertensive effect, however, was not associated with an alteration in a blood-brain barrier ion transport as assessed by measurements of blood-to-brain22Na transport and cerebral spinal fluid Na+and K+concentrations. Indeed, intracerebroventricular infusion of dimethyl amiloride, an amiloride analog with low affinity for Na+channels, also attenuated the increase in blood pressure induced by DOCA-salt treatment. Comparisons of the effects of benzamil, dimethyl amiloride, and 3,4-dichlorobenzamil, another amiloride analog, suggest that these antihypertensive effects are mediated by an inhibition of Na+/Ca2+exchange in the brain.
Branching patterns of intramural coronary vessels determined by microangiography using synchrotron radiation
Authors: Tanaka Akira , Mori Hidezo , Tanaka Etsuro , Mohammed Minhaz Uddin , Tanaka Yutaka , Sekka Takafumi , Ito Kunihisa , Shinozaki Yoshiro , Hyodo Kazuyuki , Ando Masami , Umetani Keiji , Tanioka Kenkichi , Kubota Misao , Abe Sumihisa , Handa Shunnosuke , Nakazawa Hiroe
Published: December 2017

The intramural coronary artery (IMCA) with a diameter of 50–500 μm is critical for blood supply to the inner layers of heart muscle. We introduced digital measurement to microangiography using monochromatic synchrotron radiation and quantified branching patterns of the IMCA, the epicardial coronary artery (EPCA), and the distal ileal artery (DIA). The pre- and postbranching diameters were measured (95–1,275 μm) in seven dogs. A typical arterial segment divided into two nearly equivalent branches, and a regression line of daughter-to-mother diameter plots was almost identical among the EPCA ( y = 0.838 x − 16.7 in μm), IMCA ( y = 0.737 x− 2.18), and DIA ( y = 0.755 x + 8.63). However, a considerable difference was present at a segment where a proximal IMCA branched off from an EPCA ( y = 0.182 x + 90.2). Moreover, a proximal IMCA diameter had no relationship to the branching order from an EPCA. The precision of this method was confirmed by the good correlation of diameter measurements between two independent observers ( r = 0.999, y = 1.02 x − 1.07). In conclusion, using digital microangiography we demonstrated that the self-similar branching pattern of coronary arteries was discrete at the connection between the IMCA and EPCA.
Voltage-dependent K+ current in capillary endothelial cells isolated from guinea pig heart
Authors: Dittrich Michael , Daut Jürgen
Published: December 2017

Capillary fragments were isolated from guinea pig hearts, and their electrical properties were studied using the perforated-patch and cell-attached mode of the patch-clamp technique. A voltage-dependent K+ current was discovered that was activated at potentials positive to −20 mV and showed a sigmoid rising phase. For depolarizing voltage steps from −128 to +52 mV, the time to peak was 71 ± 5 ms (mean ± SE) and the amplitude of the current was 3.7 ± 0.5 pA/pF in the presence of 5 mM external K+. The time course of inactivation was exponential with a time constant of 7.2 ± 0.5 s at +52 mV. The current was blocked by tetraethylammonium (inhibitory constant ∼3 mM) but was not affected by charybdotoxin (1 μM) or apamin (1 μM). In the cell-attached mode, depolarization-activated single-channel currents were found that inactivated completely within 30 s; the single-channel conductance was 12.3 ± 2.4 pS. The depolarization-activated K+current described here may play a role in membrane potential oscillations of the endothelium.
Pathways of HERG inactivation
Authors: Kiehn Johann , Lacerda Antonio E. , Brown Arthur M.
Published: December 2017

The rapid, repolarizing K+ current in cardiomyocytes ( I Kr) has unique inwardly rectifying properties that contribute importantly to the downstroke of the cardiac action potential. The human ether-à-go-go-related gene ( HERG) expresses a macroscopic current virtually identical to I Kr, but a description of the single-channel properties that cause rectification is lacking. For this reason we measured single-channel and macropatch currents heterologously expressed by HERG in Xenopus oocytes. Our experiments had two main findings. First, the single-channel current-voltage relation showed inward rectification, and conductance was 9.7 pS at −100 mV and 3.9 pS at 100 mV when measured in symmetrical 100 mM K+ solutions. Second, single channels frequently showed no openings during depolarization but nevertheless revealed bursts of openings during repolarization. This type of gating may explain the inward rectification of HERG currents. To test this hypothesis, we used a three-closed state kinetics model and obtained rate constants from fits to macropatch data. Results from the model are consistent with rapid inactivation from closed states as a significant source of HERG rectification.
Two distinct HCO 3 − -dependent H+efflux pathways in human vascular endothelial cells
Authors: Sun Bing , Vaughan-Jones Richard D. , Kambayashi Jun-Ichi
Published: December 2017

Intracellular pH (pHi) regulation in human umbilical vein endothelial cells (HUVEC) was investigated. The pHiwas recorded using seminaphthorhodafluor-1 (SNARF-1). Cells were intracellularly acid loaded with NH4Cl prepulse. In HEPES-buffered Tyrode (nominally [Formula: see text]free), pHirecovery from acid load was inhibited by 1.5 mM amiloride or Na+-free solution. Additionally, in [Formula: see text]-buffered Tyrode, a[Formula: see text]-dependent pHirecovery from acidosis was evident in the presence of 1.5 mM amiloride, which mediated complete recovery of pHi(7.26). In Na+-free solution, the[Formula: see text]-dependent acid extruder mediated pHirecovery after an acid load but only back to 7.09. These results suggest that there are two[Formula: see text]-dependent acid extruders in the HUVEC. One is Na+dependent, and the other is Na+independent. The former was further shown to be completely inhibited by 0.5 mM DIDS, whereas the latter was only inhibited by 24.6%. In Cl−-free solution, both of the [Formula: see text]-dependent pathways were inhibited. In conclusion, one[Formula: see text]-dependent acid extruder in the HUVEC resembles the Na+-dependent Cl−/[Formula: see text]exchange found in other tissues, and the other is Cl−dependent but Na+independent.
NO and prostanoids: age dependence of hypercapniaand histamine-induced dilations of pig pial arterioles
Authors: Willis A. P. , Leffler C. W.
Published: December 2017

Responses to hypercapnia and acetylcholine by newborn piglet pial arterioles are prostanoid dependent but appear to require both prostanoids and nitric oxide in juvenile pigs. We hypothesized that cerebrovascular dilatory responses become less prostanoid dependent and more NO dependent with development. Pial arteriolar responses to hypercapnia and histamine were recorded from α-chloralose-anesthetized newborn and juvenile pigs with closed cranial windows. Responses were recorded during control, after indomethacin or N ω-nitro-l-arginine (l-NNA), and after inhibitor plus iloprost or sodium nitroprusside. Indomethacin blocked newborn hypercapnic responses and markedly attenuated histamine dilations, but only reduced the dilations to about half in juveniles. Iloprost at subdilator concentrations restored newborn responses to hypercapnia and histamine but did not alter either response in indomethacin-treated juveniles. l-NNA attenuated juvenile, but not newborn, hypercapnia-induced dilations. Sodium nitroprusside did not restore the response.l-NNA did not alter responses to histamine in either age group. Cerebrovascular dilations to hypercapnia and histamine are prostanoid dependent and nitric oxide independent in the newborn pig, whereas nitric oxide assumes an increasing role in hypercapnic, but not histamine, responses with development.
Separation of cardiomyocytes and coronary endothelial cells for cell-specific RT-PCR
Authors: Preisig-Müller Regina , y Schnitzler Michael Mederos , Derst Christian , Daut Jürgen
Published: December 2017

A simple method for analyzing the differential gene expression of coronary endothelial cells and cardiac muscle cells was developed. Cells were isolated from guinea pig hearts by collagenase digestion. In the diluted cell suspension, single cardiomyocytes and capillary fragments containing 6–15 endothelial cells could be identified morphologically. A simple “cell picker” was constructed using a polyethylene pipette with a tip diameter of ∼150 μm that was attached to a micromanipulator and connected to an electric miniature valve. Intermittent suction pulses (1- to 2-cm water column) were applied by opening the valve for 100–200 ms at 1-s intervals. Cardiomyocytes (800–1,000) or capillary fragments (150) were picked under visual control using an inverted microscope. The cells were transferred to a reaction tube for RNA extraction, reverse transcription (RT), and DNA amplification (RT-PCR) with gene-specific and intron-spanning primers. All PCR products were verified by sequencing. Troponin T and endothelin-1 were found to be specific markers for guinea-pig cardiac muscle cells and coronary endothelial cells, respectively.
Role of nitric oxide in regulation of renal sympathetic nerve activity during hemorrhage in conscious rats
Authors: Fujisawa Yoshihide , Mori Naoko , Yube Kouichi , Miyanaka Hiroshi , Miyatake Akira , Abe Youichi
Published: December 2017

The effect of inhibition of nitric oxide (NO) synthesis on the responses of blood pressure (BP), heart rate (HR), and renal sympathetic nerve activity (RSNA) during hemorrhaging was examined with the use of an NO synthase inhibitor, NG-nitro-l-arginine methyl ester (l-NAME), in conscious rats. In the 0.9% saline group, hemorrhage (10 ml/kg body wt) did not alter BP but significantly increased HR and RSNA by 88 ± 12 beats/min and 67 ± 12%, respectively. Intravenous infusion of l-NAME (50 μg ⋅ kg−1⋅ min−1) significantly attenuated these tachycardic and sympathoexcitatory responses to hemorrhage (14 ± 7 beats/min and 26 ± 12%, respectively). Pretreatment ofl-arginine (87 mg/kg) recovered the attenuation of HR and RSNA responses induced byl-NAME (92 ± 6 beats/min and 64 ± 10%, respectively).l-NAME by itself did not alter the baroreceptor reflex control of HR and RSNA. Hemorrhage increased the plasma vasopressin concentration, and its increment in thel-NAME-treated group was significantly higher than that in the 0.9% saline group. Pretreatment with the vascular arginine vasopressin V1-receptor antagonist OPC-21268 (5 mg/kg) recovered the attenuation of RSNA response induced byl-NAME (54 ± 7%). These results indicate that NO modulated HR and RSNA responses to hemorrhage but did not directly affect the baroreceptor reflex arch. It can be assumed that NO modulated the baroreflex function by altering the secretion of vasopressin induced by hemorrhage.
Cardiac muscle tissue engineering: toward an in vitro model for electrophysiological studies
Authors: Bursac N. , Papadaki M. , Cohen R. J. , Schoen F. J. , Eisenberg S. R. , Carrier R. , Vunjak-Novakovic G. , Freed L. E.
Published: December 2017

The objective of this study was to establish a three-dimensional (3-D) in vitro model system of cardiac muscle for electrophysiological studies. Primary neonatal rat ventricular cells containing lower or higher fractions of cardiac myocytes were cultured on polymeric scaffolds in bioreactors to form regular or enriched cardiac muscle constructs, respectively. After 1 wk, all constructs contained a peripheral tissue-like region (50–70 μm thick) in which differentiated cardiac myocytes were organized in multiple layers in a 3-D configuration. Indexes of cell size (protein/DNA) and metabolic activity (tetrazolium conversion/DNA) were similar for constructs and neonatal rat ventricles. Electrophysiological studies conducted using a linear array of extracellular electrodes showed that the peripheral region of constructs exhibited relatively homogeneous electrical properties and sustained macroscopically continuous impulse propagation on a centimeter-size scale. Electrophysiological properties of enriched constructs were superior to those of regular constructs but inferior to those of native ventricles. These results demonstrate that 3-D cardiac muscle constructs can be engineered with cardiac-specific structural and electrophysiological properties and used for in vitro impulse propagation studies.
Late systolic pressure augmentation: role of left ventricular outflow patterns
Authors: Karamanoglu Mustafa , Feneley Michael P.
Published: December 2017

Late systolic augmentation of the ascending aortic pressure waveform is believed to be caused by particular impedance patterns but also could be caused by particular left ventricular outflow patterns. Using a linear mathematical model of the entire human arterial tree, we derived realistic impedance patterns by altering 1) Young’s modulus of the arterial wall of the individual branches, 2) peripheral reflection coefficients, and 3) distal compliances at the terminations. These calculated impedance patterns were then coupled to realistic left ventricular outflow patterns determined by unique 1) end-diastolic and end-systolic pressure-volume relationships, 2) preload-recruitable stroke work relationships, and 3) shortening paths simulated by altered aortic flow contours. As determined by the ratio of the individual parameter coefficient of determination ( r 2) to the overall model r 2, late systolic pressure augmentation was more strongly determined by left ventricular outflow patterns than by arterial impedance parameters ( r 2 ratio: 53% vs. 33%). Thus left ventricular outflow patterns are at least as important as impedance parameters in determining late systolic pressure augmentation in this model.
Microembolization in pigs: effects on coronary blood flow and myocardial ischemic tolerance
Authors: Grund Frank , Sommerschild Hilchen T. , Lyberg Torstein , Kirkebøen Knut A. , Ilebekk Arnfinn
Published: December 2017

Coronary microembolization has been reported to increase coronary blood flow (CBF) through adenosine release. Because adenosine may increase ischemic tolerance against infarction, we tested the hypothesis that myocardial microembolization, a common finding in patients with ischemic heart disease, induces cardioprotection. Additionally, because the use of microspheres is a common tool to measure tissue perfusion, the effects of small amounts of microspheres on CBF were examined. Using anesthetized pigs, we measured CBF with a transit time flow probe on the left anterior descending coronary artery (LAD). In six pigs the relationship between the amount of injected microspheres (0–40 × 106, 15 μm in diameter, left atrial injections) and the effect on CBF was examined. Coronary hyperemia occurred, which was linearly related to the amount of microspheres injected: maximal increase in CBF (%) = 2.8 ± 1.5 (SE) + (5.8 ± 0.7 × 10−7× number of injected microspheres). Because injection of 40 × 106microspheres induced a long-lasting hyperemic response, which could be blocked by 8- p-sulfophenyl theophylline, ischemic tolerance was examined in five other pigs after two injections, each of 40 × 106microspheres, at a 30-min interval. Six control pigs had no injections. Ischemic tolerance was evaluated by measuring infarct size (tetrazolium stain) as the percentage of area at risk (fluorescent particles) after 45 min of LAD occlusion followed by 2 h of reperfusion. Pretreatment by microspheres increased infarct size from 60 ± 3% of area at risk in control animals to 84 ± 6% ( P< 0.05). The injection of microspheres induced a significant hyperemic flow response without causing necrosis by itself. We conclude that microembolization, evoking coronary hyperemia, does not improve but reduces myocardial ischemic tolerance against infarction in pigs.
Adenosine upregulates VEGF expression in cultured myocardial vascular smooth muscle cells
Authors: Gu Jian-Wei , Brady Ann L. , Anand Vivek , Moore Michael C. , Kelly Whitney C. , Adair Thomas H.
Published: December 2017

We tested whether adenosine has differential effects on vascular endothelial growth factor (VEGF) expression under normoxic and hypoxic conditions, and whether A1or A2receptors (A1R; A2R) mediate these effects. Myocardial vascular smooth muscle cells (MVSMCs) from dog coronary artery were exposed to hypoxia (1% O2) or normoxia (20% O2) in the absence and presence of adenosine agonists or antagonists for 18 h. VEGF protein levels were measured in media with ELISA. VEGF mRNA expression was determined with Northern blot analysis. Under normoxic conditions, the adenosine A1R agonists, N6-cyclopentyladenosine and R(-)- N6-(2-phenylisopropyl)adenosine did not increase VEGF protein levels at A1R stimulatory concentrations. However, adenosine (5 μM) and the adenosine A2R agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)]ethyl adenosine (DPMA; 100 nM) increased VEGF protein levels by 51 and 132% and increased VEGF mRNA expression by 44 and 90%, respectively, in cultured MVSMCs under normoxic conditions. Hypoxia caused an approximately fourfold increase in VEGF protein and mRNA expression, which could not be augmented with exogenous adenosine, A2R agonist (DPMA), or A1R agonist [1,3-diethyl-8-phenylxanthine (DPX)]. The A2R antagonist 8-(3-chlorostyryl)-caffeine completely blocked adenosine-induced VEGF protein and mRNA expression and decreased baseline VEGF protein levels by up to ∼60% under normoxic conditions but only by ∼25% under hypoxic conditions. The A1R antagonist DPX had no effect. These results are consistent with the hypothesis that 1) adenosine increases VEGF protein and mRNA expression by way of A2R. 2) Adenosine plays a major role as an autocrine factor regulating VEGF expression during normoxic conditions but has a relatively minor role during hypoxic conditions. 3) Endogenous adenosine can account for the majority of basal VEGF secretion by MVSMCs under normoxic conditions and could therefore be a maintenance factor for the vasculature.
Excitation-contraction coupling in rat ventricular myocytes after formamide-induced detubulation
Authors: Kawai Makoto , Hussain Munir , Orchard Clive H.
Published: December 2017

Formamide-induced osmotic shock has been used to detubulate isolated adult rat ventricular myocytes (i.e., disrupt the surface membrane-T tubule junction). Cell volume, calculated from cell length and width, rapidly decreased and increased upon application and removal of formamide, respectively. After treatment with formamide, membrane capacitance decreased by 26.4% (from 199.4 ± 18.7 pF in control cells to 146.7 ± 6.4 pF in formamide-treated cells; n = 13, P < 0.05). However, the amplitude of the L-type Ca2+ current ( I Ca) decreased by a greater extent (from 0.75 ± 0.14 to 0.18 ± 0.03 nA; n = 5, P < 0.05) so that the density of I Ca decreased by 74.5%. Simultaneous measurements of I Ca and Ca2+ transients (monitored using fura 2) showed that both decreased rapidly upon removal of formamide. However, the Ca2+ content of the sarcoplasmic reticulum showed little change. Cross-striations, visualized with the fluorescent dye di-8-aminonaphthylethenylpyridinium, were sparse or absent in cells that had been treated with formamide, suggesting that formamide can successfully detubulate cardiac cells and that I Ca is concentrated in the T tubules, which therefore play an important role in excitation-contraction coupling.
Blockade of AT1 receptors and Na+/H+exchanger and LV dysfunction after myocardial infarction in rats
Authors: Ruzicka Marcel , Yuan Baoxue , Leenen Frans H. H.
Published: December 2017

Mechanical stretch, ANG II, and α1-receptor stimulation may contribute to cardiac remodeling after myocardial infarction (MI). Each of these mechanisms involves different signaling pathways for the cellular hypertrophic response. All three also activate the Na+/H+exchanger. In the present study we evaluated the hypothesis that activation of the Na+/H+exchanger is involved in parallel with other signaling mechanisms for ANG II. Three days before coronary artery ligation, rats were randomly allocated to no treatment or treatment with amiloride, losartan, or amiloride and losartan in combination. Four weeks after coronary artery ligation, left ventricular (LV) function was assessed from in vivo resting cardiac pressures, hemodynamic responses to cardiac volume and pressure load, and cardiac remodeling by in vitro pressure-volume curves and LV and right ventricle (RV) weight. Amiloride and losartan given alone to a similar extent attenuated the shift of the pressure-volume curve to the right. This effect was significantly more pronounced with amiloride and losartan in combination. Each drug alone to a minor extent improved LV responses to pressure and volume load. However, with amiloride and losartan in combination, close-to-normal responses to pressure and volume load were observed. Losartan and amiloride alone had only a small effect on development of RV hypertrophy after MI but in combination completely prevented the RV hypertrophy. Amiloride and losartan appear to be complementary in prevention of cardiac remodeling and LV dysfunction after MI. This finding suggests that, besides ANG II, other mechanisms activating the Na+/H+exchanger contribute to cardiac remodeling after MI.
Myocardial infarction and remodeling in mice: effect of reperfusion
Authors: Michael Lloyd H. , Ballantyne Christie M. , Zachariah Justin P. , Gould Kenneth E. , Pocius Jennifer S. , Taffet George E. , Hartley Craig J. , Pham Thuy T. , Daniel Sherita L. , Funk Etai , Entman Mark L.
Published: December 2017

Anatomic and functional changes after either a permanent left anterior descending coronary artery occlusion (PO) or 2 h of occlusion followed by reperfusion (OR) in C57BL/6 mice were examined and compared with those in sham-operated mice. Both interventions generated infarcts comprising 30% of the left ventricle (LV) measured at 24 h and equivalent suppression of LV ejection velocity and filling velocity measured by Doppler ultrasound at 1 wk. Serial follow-up revealed that the ventricular ejection velocity and filling velocity returned to the levels of the sham-operated controls in the OR group at 2 wk and remained there; in contrast, PO animals continued to display suppression of both systolic and diastolic function. In contrast, ejection fractions of PO and OR animals were depressed equivalently (50% from sham-operated controls). Anatomic reconstruction of serial cross sections revealed that the percentage of the LV endocardial area overlying the ventricular scar (expansion ratio) was significantly larger in the PO group vs. the OR group (18 ± 1.7% vs. 12 ± 0.9%, P < 0.05). The septum that was never involved in the infarction had a significantly ( P < 0.002) increased mass in PO animals (22.5 ± 1.08 mg) vs. OR (17.8 ± 1.10 mg) or sham control (14.8 ± 0.99 mg) animals. Regression analysis demonstrated that the extent of septal hypertrophy correlated with LV expansion ratio. Thus late reperfusion appears to reduce the degree of infarct expansion even under circumstances in which it no longer can alter infarct size. We suggest that reperfusion promoted more effective ventricular repair, less infarct expansion, and significant recovery or preservation of ventricular function.
Reperfusion injury is not affected by blockade of P-selectin in the diabetic mouse heart
Authors: Jones Steven P. , Girod Wesley G. , Granger D. Neil , Palazzo Anthony J. , Lefer David J.
Published: December 2017

We examined the mechanisms responsible for myocardial ischemia-reperfusion (MI-R) injury in a well-characterized animal model of type II diabetes mellitus. Diabetic ( db/db) mice and their littermate nondiabetic controls were subjected to 30 min of left anterior descending coronary artery occlusion and 2 h of reperfusion. Diabetic and nondiabetic mice experienced similar-sized areas at risk per left ventricle: 50.4 ± 2.0 and 53.4 ± 4.1%, respectively. However, myocardial necrosis (percentage of area at risk) was significantly greater ( P < 0.001) in diabetic than in nondiabetic animals: 56.3 ± 2.8 and 27.2 ± 3.1%, respectively. Histological examination revealed significantly ( P < 0.05) more neutrophils (PMNs) in the diabetic than in the nondiabetic hearts. Coronary endothelial expression of P-selectin was determined using radiolabeled monoclonal antibodies (MAbs). MI-R elicited a more intense ( P < 0.05) upregulation of P-selectin in the ischemic zone of diabetic than of nondiabetic myocardium: 0.310 ± 0.034 and 0.161 ± 0.042 μg MAb/g tissue. Immunoneutralization of P-selectin (RB40.34) reduced PMN accumulation in the diabetic myocardium but failed to reduce the extent of myocardial necrosis. Conversely, administration of an MAb directed against CD18 (GAME46) reduced PMN infiltration and attenuated the infarct size in the diabetic hearts. These results suggest that the diabetic heart is more susceptible to ischemia-reperfusion injury than normal myocardium. Furthermore, the mechanism of this injury may not be critically dependent on P-selectin in diabetic hearts.
A model of low-flow ischemia and reperfusion in single, beating adult cardiomyocytes
Authors: Maddaford Thane G. , Hurtado Cecilia , Sobrattee Salisha , Czubryt Michael P. , Pierce Grant N.
Published: December 2017

The present study was undertaken to comprehensively characterize low-flow ischemia and reperfusion in single adult cardiomyocytes and to determine whether it is important to control contractile activity. The ischemia-mimetic solution was hypoxic, acidic (pH 6.0), and deficient in glucose but contained elevated KCl. Cardiomyocytes were stimulated to contract throughout ischemia and during reperfusion with control perfusate. After the ischemia-reperfusion insult, cells exhibited poor recovery of active cell shortening, a decrease in passive cell length, increased frequency of necrosis, lower ATP content, and evidence of the generation of oxygen-derived free radicals within the cells. Intracellular lactate concentration increased, pH decreased, and Ca2+transients were depressed during the ischemic insult, but the latter two parameters recovered partially on reperfusion. Basal intracellular Ca2+concentration was elevated during ischemia and early into reperfusion. Recovery was attenuated in cells that were electrically stimulated to contract throughout ischemia. The duration of ischemia, stimulation frequency, and composition of the ischemia-mimetic solution were important variables. The inclusion of 10 mM lactate in the ischemia-mimetic solution significantly aggravated all the parameters examined above. Our data demonstrate that 1) an ischemia-mimetic solution administered to single, isolated adult cardiomyocytes can reproduce many of the responses observed in whole hearts, 2) caution should be used in adding lactate to an ischemic solution, and 3) it is important to stimulate contractile activity throughout ischemia to reproduce the effects of ischemia in whole hearts.
Cardiac sympathetic afferent sensitivity is enhanced in heart failure
Authors: Wang Wei , Schultz Harold D. , Ma Rong
Published: December 2017

A previous study from this laboratory has shown that cardiac sympathetic afferent stimulation by epicardial application of bradykinin (BK) and capsaicin was significantly enhanced in the dog with experimental heart failure (HF). The present study determined whether activity from cardiac sympathetic chemosensitive afferent endings is enhanced in HF. Rapid ventricular pacing was induced in six dogs. Five sham dogs served as controls. At the time of the acute experiment, the dogs were anesthetized with pentobarbital sodium (30 mg/kg iv). A thoracotomy was performed in the second intercostal space, and single afferent fiber discharge from the left cardiac sympathetic nerve was recorded. Baseline cardiac sympathetic afferent discharge rate (spikes/s) and its responses to intra-atrial injection of BK were compared between sham and HF groups. Baseline cardiac sympathetic afferent discharge rate in the HF group was significantly elevated compared with the sham group (4.3 ± 0.5 vs. 2.2 ± 0.6 spikes/s, P < 0.05). In addition, cardiac sympathetic afferent responses to left intra-atrial injection of bradykinin (2 and 5 μg/kg) and capsaicin (5 and 10 μg/kg) were also significantly augmented. The sensitized cardiac sympathetic afferent responses to BK (2 and 5 μg/kg, left intra-atrial injection) in the HF group were significantly reduced by the cyclooxygenase inhibitor indomethacin (5 mg/kg iv). The sensitized cardiac sympathetic afferent response to capsaicin (5 and 10 μg/kg, left intra-atrial injection) in the HF group was preserved. It is suggested that the cardiac sympathetic chemosensitive afferent sensitivity is significantly enhanced in dogs with HF even though the baseline cardiac sympathetic afferent discharge is elevated.
ATP is involved in myocardial and vascular effects of exogenous bradykinin in ejecting guinea pig heart
Authors: Anning Peter B. , Prendergast Bernard D. , MacCarthy Philip A. , Shah Ajay M. , Buss Derek C. , Lewis Malcolm J.
Published: December 2017

It has recently been reported that bradykinin induces selective left ventricular (LV) relaxation in isolated guinea pig hearts via the release of nitric oxide. Exogenous bradykinin also induces vasodilation, which is only partly due to nitric oxide release. In the present study we investigated the role of adenyl purines on these bradykinin-induced effects. Isolated ejecting guinea pig hearts were studied. LV pressure was monitored by a 2-Fr micromanometer-tipped catheter. ATP concentrations were measured using a luciferin-luciferase assay. Bradykinin (1 and 100 nM) caused a progressive acceleration of LV relaxation together with a transient increase in coronary flow. These effects were inhibited by the nonselective P2 purinoceptor antagonist suramin (1 μM, n = 6) but were unaffected by the selective P2x purinoceptor antagonist pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid (1 μM, n = 6). These myocardial and vascular effects of bradykinin were associated with increased ATP levels in coronary effluent. These data suggest that the selective enhancement of LV relaxation and rise in coronary flow induced by exogenous bradykinin involve endogenous ATP and the subsequent stimulation of P2 purinoceptors.
Effects of estrogen on action potential and membrane currents in guinea pig ventricular myocytes
Authors: Tanabe Seiko , Hata Toshio , Hiraoka Masayasu
Published: December 2017

To explore a possible ionic basis for the prolonged Q-T interval in women compared with that in men, we investigated the electrophysiological effects of estrogen in isolated guinea pig ventricular myocytes. Action potentials and membrane currents were recorded using the whole cell configuration of the patch-clamp technique. Application of 17β-estradiol (10–30 μM) significantly prolonged the action potential duration (APD) at 20% (APD20) and 90% repolarization (APD90) at stimulation rates of 0.1–2.0 Hz. In the presence of 30 μM 17β-estradiol, APD20 and APD90 at 0.1 Hz were prolonged by 46.2 ± 17.1 and 63.4 ± 11.7% of the control ( n = 5), respectively. In the presence of 30 μM 17β-estradiol the peak inward Ca2+ current ( I CaL) was decreased to 80.1 ± 2.5% of the control ( n = 4) without a shift in its voltage dependence. Application of 30 μM 17β-estradiol decreased the rapidly activating component of the delayed outward K+ current ( I Kr) to 63.4 ± 8% and the slowly activating component ( I Ks) to 65.8 ± 8.7% with respect to the control; the inward rectifier K+ current was barely affected. The results suggest that 17β-estradiol prolonged APD mainly by inhibiting the I Kcomponents I Krand I Ks.
ANG II-induced Ca2+increase in smooth muscle cells from SHR is regulated by actin and microtubule networks
Authors: Samain Emmanuel , Bouillier Hèléne , Perret Claudine , Safar Michel , Dagher Georges
Published: December 2017

We hypothesized that the cytoskeletal network in vascular smooth muscle cells (VSMC) is critical to the signaling pathways from angiotensin (ANG) II-receptor subtype 1 (AT1) activation to intracellular Ca2+([Formula: see text]) release from internal stores and Ca2+influx. This was tested in spontaneously hypertensive rats (SHR), in which differences were reported in cultured aortic VSMC [Formula: see text]regulation and G protein function compared with those in normotensive Wistar-Kyoto (WKY) rats. In cultured aortic VSMC, disorganization of actin filaments with cytochalasin D (2 μmol/l) decreased the ANG II-induced [Formula: see text] release from internal stores and the ANG II-induced Ca2+influx in SHR in a reversible fashion, whereas it was without effect in WKY rats. On the other hand, blocking the dynamic state of the microtubule network significantly reduced ANG II-induced[Formula: see text] release from internal stores but was without effect on Ca2+influx in either SHR or WKY rats. This study demonstrates for the first time that, in the SHR, actin filaments play a major role in linking AT1-receptor activation to both[Formula: see text] release mechanisms and capacitative Ca2+influx. Furthermore, a functionally intact microtubule system is a necessary prerequisite for ANG II-induced [Formula: see text] release in both strains.
Increased Ca2+sensitivity as a key mechanism of PKC-induced constriction in pressurized cerebral arteries
Authors: Gokina Natalia I. , Knot Harm J. , Nelson Mark T. , Osol George
Published: December 2017

The effects of activating protein kinase C (PKC) with indolactam V (Indo-V) and 1,2-dioctanoyl- sn-glycerol (DOG) on smooth muscle intracellular Ca2+concentrations ([Ca2+]i) and arterial diameter were determined using ratiometric Ca2+imaging and video edge detection of pressurized rat posterior cerebral arteries. Elevation of intraluminal pressure from 10 to 60 mmHg resulted in an increase in [Ca2+]ifrom 74 ± 5 to 219 ± 8 nM and myogenic constriction. Application of Indo-V (0.01–3 μM) or DOG (0.1–30 μM) induced constriction and decreased [Ca2+]ito 140 ± 11 and 127 ± 12 nM, respectively, at the highest concentrations used. In the presence of Indo-V, the dihydropyridine Ca2+-channel-blocker nisoldipine produced nearly maximum dilation and decreased [Ca2+]ito 97 ± 7 nM. In α-toxin-permeabilized arteries, the constrictor effects of Indo-V and DOG were not observed in the absence of Ca2+. Both PKC activators significantly increased the degree of constriction of permeabilized arteries at different [Ca2+]i. We conclude that 1) Indo-V- or DOG-induced constriction of pressurized arteries requires Ca2+influx through voltage-dependent Ca2+channels, and 2) PKC-induced constriction of pressurized rat cerebral arteries is associated with a decrease in [Ca2+]i, suggesting an increase in the Ca2+sensitivity of the contractile process.
Relationship between baroreceptor reflex function and end-organ damage in spontaneously hypertensive rats
Authors: Shan Zheng-Zheng , Dai Sheng-Ming , Su Ding-Feng
Published: December 2017

The purpose of this study was to further illustrate the relationship between baroreceptor reflex sensitivity (BRS) and hypertensive end-organ damage (EOD) and to test the hypothesis that impairment of BRS aggravates EOD in hypertension. We studied baroreflex-mediated changes in heart rate [expressed as baroreceptor sensitivity to heart rate control (BRSHR)] and blood pressure [expressed as baroreceptor sensitivity to blood pressure control (BRSBP)] in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) that were used as controls, both at the age of 50–52 wk. Rats were also instrumented to record BP, HR, and BP variability (BPV) in the conscious, unrestrained state. In SHR compared with WKY, BP and BPV were significantly increased, whereas BRSHR and BRSBP were significantly decreased. SHR had remarkable EOD when compared with WKY (EOD score: 6.3 ± 2.5 vs. 2.9 ± 0.8, P < 0.01). Univariate regressive analysis demonstrated that EOD score was increased with BP and BPV and decreased with BRS. In multivariate analysis, EOD score was predicted by greater systolic BP and lower BRS and HR variability. These results indicate that BRS is negatively related to BPV and EOD score, and impaired BRS might be one of the major causes for hypertensive EOD.
Ischemia-reperfusion rapidly increases COX-2 expression in piglet cerebral arteries
Authors: Domoki Ferenc , Veltkamp Roland , Thrikawala Nishadi , Robins Greg , Bari Ferenc , Louis Thomas M. , Busija David W.
Published: December 2017

In the newborn, cyclooxygenase (COX)-derived products play an important role in the cerebrovascular dysfunction after ischemia-reperfusion (I/R). We examined effects of I/R on expression of COX-1 and COX-2 isoforms in large cerebral arteries of anesthetized piglets. The circle of Willis, the basilar, and the middle cerebral arteries were collected from piglets at 0.5–12 h after global ischemia (2.5–10 min, n = 50), hypoxia ( n = 3), or hypercapnia ( n = 2) and from time-control ( n = 19) or untreated animals ( n = 7). Tissues were analyzed for COX-1 and COX-2 mRNA and protein using RNase protection assay and immunoblot analysis, respectively. Ischemia increased COX-2 mRNA by 30 min, and maximal levels were reached at 2 h. Hypoxia or hypercapnia had minimal effects on COX-2 mRNA. COX-2 protein levels were also consistently elevated by 8 h after I/R. Increases in COX-2 mRNA or protein were not influenced by pretreatment with either indomethacin (5 mg/kg iv, n = 5) or nitro-l-arginine methyl ester (15 mg/kg iv, n = 7). COX-1 mRNA levels were low in time controls, and ischemic stress had no significant effect on COX-1 expression. Thus ischemic stress leads to relatively rapid, selective induction of COX-2 in cerebral arteries.