bluuebunny/crossref_metadata_2025 huggingface dataset:
Purinergic axis in cardiac blood vessels. Agonist-mediated release of ATP from cardiac endothelial cells.
Authors: Yang S , Cheek D J , Westfall D P , Buxton I L
Published: June 2012

Purified endothelial cells isolated from guinea pig hearts by enzymatic perfusion were grown in monolayer culture and used to test the ability of a variety of vasoactive agents to stimulate ATP release from these cells. Stimulation of endothelial cells with the peptide agonist bradykinin (1 nmol/L), acetylcholine (1 mumol/L), serotonin (1 mumol/L), or adenosine 5'-diphosphate (10 mumol/L) resulted in the rapid appearance of ATP in the incubation medium determined with the firefly luciferase assay for ATP. Addition of antagonists for muscarinic (atropine, 0.1 mumol/L) and purinergic (suramin, 100 mumol/L; reactive blue-2, 100 mumol/L) receptors suggested that ATP release from these cells was receptor-mediated. Bradykinin-induced release of ATP was rapid (peak < 30 seconds at 3 nmol/L bradykinin), dose-dependent (EC50, 0.18 nmol/L), and diminished with repeated administration of agonist. Desensitization to bradykinin also affected the ability of acetylcholine to induce release and was reversible when cells were returned to growth conditions for short periods. Measurement of released adenyl purines as their fluorescent N6-ethenopurine derivatives by high-performance liquid chromatography revealed the origin of the purine released to be ATP and confirmed its rapid dephosphorylation. Addition of the purine nucleotide analogues 2-methylthio-ATP (2-methyl-S-ATP), ADP, and beta gamma-methylene ATP to endothelial cell cultures resulted in a dose-dependent increase in the appearance of ATP measured in the medium bathing the cells at 30 seconds, suggesting the presence of ATP-induced ATP release.(ABSTRACT TRUNCATED AT 250 WORDS)
Impaired nitric oxide-mediated flow-induced dilation in arterioles of spontaneously hypertensive rats.
Authors: Koller A , Huang A
Published: June 2012

We tested the hypothesis that impairment of flow-dependent dilator mechanisms of skeletal muscle arterioles is one of the underlying reasons for the increased peripheral resistance in hypertension. Isolated, cannulated arterioles (approximately 55 microns) of gracilis muscle of 12-week-old spontaneously hypertensive (SH) and normotensive Wistar (NW) rats were investigated. At a constant perfusion pressure (80 mm Hg), the active diameters of NW and SH arterioles were 57.7 +/- 1.9 and 51.5 +/- 3.2 microns, whereas their passive diameters (Ca(2+)-free solution) were 113.6 +/- 2.9 and 101.7 +/- 2.9 microns, respectively. Flow-induced dilation was elicited by increases in flow of the perfusion solution from 0 to 25 microL/min in 5-microL/min steps. This response was significantly less in arterioles of SH compared with NW rats. For example, at 25-microL/min flow, the diameter of arterioles of SH rats was approximately 56% less (P < .05) than those of NW rats. Indomethacin, an inhibitor of prostaglandin synthesis, significantly attenuated the flow-diameter curve in both strains of rats. In contrast, N omega-nitro-L-arginine, a nitric oxide synthase inhibitor, significantly shifted the flow-diameter curve to the right in NW rats, but it did not affect the flow-diameter curve in SH rats. Thus, the present findings demonstrate that in gracilis muscle arterioles of normotensive rats in response to increases in flow (shear stress), prostaglandins and nitric oxide are co-released, resulting in a dilation. In early hypertension, however, there is a reduced arteriolar dilation to increases in flow that is due to the impairment of the nitric oxide-mediated portion of the flow-dependent arteriolar dilation.
Beta 2-adrenergic receptor and angiotensin II receptor modulation of sympathetic neurotransmission in human atria.
Authors: Rump L C , Schwertfeger E , Schaible U , Fraedrich G , Schollmeyer P
Published: June 2012

The aim of the present study was to investigate beta-adrenergic receptor and angiotensin II (Ang II) receptor modulation of norepinephrine release in human atria. Slices of human atrial appendages were incubated with [3H]norepinephrine, superfused with Krebs-Henseleit solution, and electrically stimulated in superfusion chambers. Pretreatment of the tissue with 6-hydroxydopamine (1.2 mmol/L) before the [3H]norepinephrine incubation to destroy sympathetic nerves reduced the uptake of radioactivity and abolished the stimulation-induced (S-I) outflow of radioactivity. Furthermore, S-I outflow of radioactivity was prevented by the addition of tetrodotoxin (1 mumol/L) to and omission of extracellular Ca2+ from the superfusion solution. Separation of [3H]norepinephrine from its metabolites revealed that the S-I outflow of radioactivity was mainly composed of intact [3H]norepinephrine. Thus, the S-I outflow of radioactivity was taken as an index of norepinephrine release. Isoproterenol (0.001 to 0.1 mumol/L) dose-dependently enhanced the S-I outflow of radioactivity. The concentration-response curve of isoproterenol was shifted to the right by the selective beta 2-adrenergic receptor antagonist ICI 118551 (0.01 and 0.1 mumol/L) but not by the beta 1-adrenergic receptor-selective antagonist atenolol (0.3 and 30 mumol/L). Ang II (0.001 to 1.0 mumol/L) also dose-dependently enhanced S-I outflow of radioactivity. The facilitatory effect of Ang II was blocked by either the peptide Ang II receptor antagonist saralasin (1.0 mumol/L) or EXP 3174 (0.1 mumol/L), the in vitro active form of the nonpeptide Ang II receptor antagonist losartan. The cell-permeable cAMP analogue 8-bromo-cAMP (30 to 300 mumol/L) dose-dependently enhanced S-I outflow of radioactivity.(ABSTRACT TRUNCATED AT 250 WORDS)
Beta-adrenergic receptor stimulation increases unloaded shortening velocity of skinned single ventricular myocytes from rats.
Authors: Strang K T , Sweitzer N K , Greaser M L , Moss R L
Published: June 2012

In vitro biochemical experiments have suggested that stimulation of beta-adrenergic receptor may increase the rate of crossbridge cycling in mammalian myocardium, but recent attempts to demonstrate a mechanical correlate have yielded conflicting results. To investigate this issue, we measured the effect of isoproterenol (ISO) and cAMP-dependent protein kinase (PKA) on unloaded shortening velocity (Vo). Vo is thought to be determined by the rate-limiting step of the crossbridge cycle, ie, the rate of crossbridge detachment from actin, and is therefore an index of the cycling rate. Single rat ventricular myocytes were enzymatically isolated, incubated in Ringer's solution without (control) or with 0.1 mumol/L ISO, and then rapidly skinned. Some control cells were subsequently treated with 3 micrograms/mL PKA for 40 minutes. Vo was then measured during maximal activation (pCa 4.5) in control, ISO-treated, and PKA-treated cells using the slack-test method. To test the efficacy of the agonist treatments, Ca2+ sensitivity of isometric tension was also assessed for each treatment by determining the [Ca2+] required for half-maximal tension (ie, pCa50). Both ISO and PKA treatment reduced the Ca2+ sensitivity of isometric tension compared with same-day control cells, in agreement with previous studies in intact and in skinned preparations. Vo was increased 38% by ISO treatment and 41% by PKA treatment compared with same-day control cells. 32P autoradiography showed that troponin I and C protein were the principal proteins phosphorylated by PKA treatment. We conclude that beta-adrenergic stimulation increases the rate of crossbridge release from actin, by a mechanism that most likely involves the phosphorylation of troponin I and/or C protein by PKA.
Relation between reverse sodium-calcium exchange and sarcoplasmic reticulum calcium release in guinea pig ventricular cells.
Authors: Kohomoto O , Levi A J , Bridge J H
Published: June 2012

Exchange-inhibitory peptide (XIP) can inhibit sodium-calcium exchange without inhibiting L-type calcium current (ICa). We therefore used this compound to test the hypothesis that reverse sodium-calcium exchange can trigger contraction in guinea pig ventricular myocytes. When cells were dialyzed with 20 mmol/L sodium, rapid blockade of ICa with nifedipine had little effect on cell shortening. However, if reverse exchange was inhibited by first dialyzing the cells with XIP, blockade of ICa largely inhibited cell shortening. In cells dialyzed with 10 mmol/L sodium, about 51% of the maximum cell shortening remained after ICa was blocked. When both ICa and reverse exchange were significantly inhibited with nifedipine and XIP, only 24% of the cell shortening remained; ie, 27% was XIP inhibitable. Cells dialyzed with solutions deficient in sodium exhibited contractions that were largely dependent on ICa (ie, not XIP inhibitable). If the sarcoplasmic reticulum (SR) was disabled with ryanodine and thapsigargin, reverse exchange could not cause contraction. We therefore conclude that with intact SR, reverse sodium-calcium exchange activates contraction by triggering calcium release from the SR in cells dialyzed with either 10 or 20 mmol/L sodium. A scrambled sequence of XIP, sXIP, caused no measurable effect on contraction.
Changes in type VI adenylyl cyclase isoform expression correlate with a decreased capacity for cAMP generation in the aging ventricle.
Authors: Tobise K , Ishikawa Y , Holmer S R , Im M J , Newell J B , Yoshie H , Fujita M , Susannie E E , Homcy C J
Published: June 2012

We investigated the developmental regulation of the beta-adrenergic receptor-Gs-adenylyl cyclase pathway in myocardial membranes from fetal, neonatal, adult, and mature adult rats by measuring the density of the beta-adrenergic receptor and the activities of the stimulatory guanine nucleotide-binding protein Gs and the adenylyl cyclase enzyme. Total beta-adrenergic receptor content (in femtomoles per milligram protein) was greatest in the fetal (124.4 +/- 20.5 fmol/mg) and neonatal (122.3 +/- 16.1 fmol/mg) stages and gradually decreased in the adult (90.9 +/- 8.0 fmol/mg) and mature adult (70.0 +/- 9.6 fmol/mg) stages. An equivalent pattern was seen for adenylyl cyclase activity: the basal activity of the effector enzyme or that measured in the presence of 0.1 mmol/L isoproterenol with 0.1 mmol/L Gpp(NH)p, 10 mmol/L NaF, or 0.05 mmol/L forskolin was greater in the fetus and the neonate than in the adult and the mature adult. These data suggested that decreased stimulation of the catalytic unit by Gs could be the underlying cause of diminished adenylyl cyclase activity with aging. However, quantification of Gs by reconstitution into S49 cyc- membranes (in picomoles cAMP per microgram for 10 minutes) demonstrated no significant decrease during development from fetus (1.55 +/- 0.1 pmol/microgram) to neonate (1.9 +/- 0.5 pmol/microgram) and subsequent aging to adult (2.6 +/- 0.2 pmol/micrograms) and mature adult (1.9 +/- 0.2 pmol/microgram). When Northern blot analysis was used to characterize the relative amounts of mRNA coding for Gs alpha, no significant differences were seen among the developmental stages studied.(ABSTRACT TRUNCATED AT 250 WORDS)
Inhibition of nitric oxide production aggravates myocardial hypoperfusion during exercise in the presence of a coronary artery stenosis.
Authors: Duncker D J , Bache R J
Published: June 2012

Regulation of coronary vasomotor tone during myocardial hypoperfusion is incompletely understood. The present study was performed to test the hypothesis that endogenous production of nitric oxide contributes to resistance vessel dilation distal to a coronary artery stenosis that results in myocardial ischemia during exercise. Seven dogs instrumented with a Doppler velocity probe, hydraulic occluder, and indwelling microcatheter in the left anterior descending coronary artery (LAD) were studied during treadmill exercise in the presence of a coronary artery stenosis before and after intracoronary infusion of NG-nitro-L-arginine (LNNA, 20 mg/kg). This dose of LNNA inhibited the maximal increase in LAD flow produced by intracoronary acetylcholine by 82 +/- 5% but did not alter the response to intracoronary nitroprusside. Coronary pressure distal to the stenosis was maintained constant during the control period and after administration of LNNA. LNNA increased aortic and left ventricular systolic and end-diastolic pressures at rest and during exercise. During control in the absence of a stenosis, LNNA had no effect on coronary blood flow. In the presence of a stenosis that decreased distal coronary pressure to 55 +/- 2 mm Hg, mean myocardial blood flow measured with microspheres was 1.09 +/- 0.13 mL.min-1.g-1 in the LAD-dependent and 2.57 +/- 0.50 mL.min-1.g-1 in the posterior control region, respectively. With no change in distal coronary pressure, LNNA decreased mean myocardial blood flow in the LAD region to 0.68 +/- 0.11 mL.min-1.g-1 (P < .01). To avoid systemic hemodynamic effects, LNNA was administered in a dose of 1.5 mg/kg IC to four additional dogs. This low dose inhibited the coronary blood flow increases produced by acetylcholine by 61 +/- 5% but was devoid of systemic hemodynamic effects. During exercise in the presence of a coronary stenosis that decreased coronary pressure to 52 +/- 1 mm Hg, this dose of LNNA decreased mean myocardial blood flow from 0.89 +/- 0.23 to 0.66 +/- 0.21 mL.min-1.g-1 (P < .02). These data demonstrate that nitric oxide contributes to the maintenance of myocardial perfusion distal to a flow-limiting coronary artery stenosis during exercise.
Ascorbyl free radical as a real-time marker of free radical generation in briefly ischemic and reperfused hearts. An electron paramagnetic resonance study.
Authors: Sharma M K , Buettner G R , Spencer K T , Kerber R E
Published: June 2012

The role of free radicals in myocardial reperfusion injury remains controversial. We have developed a new method using ascorbyl free radical (AFR) as a real-time, quantitative marker of free radical generation during myocardial reperfusion. A total of 35 dogs were studied. Twelve open-chest dogs underwent either 5 minutes (n = 5) or 20 minutes (n = 7) of coronary artery occlusion and 30 minutes of reperfusion. Seven additional animals undergoing 20 minutes of coronary occlusion also received the antioxidant enzymes superoxide dismutase and catalase, beginning 10 minutes before occlusion through the end of reperfusion. Exogenous ascorbate was infused intravenously, and the concentration of AFR in the great cardiac vein was continuously measured by electron paramagnetic resonance spectroscopy. Preocclusion AFR concentration was similar in the three groups. Upon reperfusion, AFR rose significantly in each animal group (P < .05). However, the AFR rise in the 20-minute-occlusion group, 38 +/- 17%, was significantly greater than in the 5-minute-occlusion group, 27 +/- 14% (P < .002). In addition, in the animals that received superoxide dismutase and catalase, the rise in the AFR was markedly attenuated, 13 +/- 6% (P < .002). Two dogs that received ascorbate but did not undergo coronary artery occlusion/reperfusion sequences showed no change in coronary venous AFR signal, indicating the stability of the signal over time. Five dogs received ascorbate while undergoing interventions to alter coronary venous flow: intravenous saline, dobutamine, dipyridamole, and nitroglycerin. Coronary venous AFR changes were minimal despite large coronary flow alterations, indicating that the AFR signal is independent of changes in coronary venous flow.(ABSTRACT TRUNCATED AT 250 WORDS)
ATP-sensitive K+ channels mediate dilatation of cerebral arterioles during hypoxia.
Authors: Taguchi H , Heistad D D , Kitazono T , Faraci F M
Published: June 2012

We tested the hypothesis that dilatation of cerebral arterioles during hypoxia is mediated by activation of ATP-sensitive K+ channels. The diameter of pial arterioles was measured through a closed cranial window in anesthetized rabbits. Topical application of aprikalim (10(-6) mol/L), a direct activator of ATP-sensitive K+ channels, dilated pial arterioles by 18 +/- 3% (mean +/- SEM). Glibenclamide (10(-6) mol/L), an inhibitor of ATP-sensitive K+ channels, virtually abolished aprikalim-induced vasodilatation. When arterial PO2 was reduced from 129 +/- 3 to 25 +/- 1 mm Hg, the diameter of cerebral arterioles increased by 66 +/- 9% (P < .05). Glibenclamide inhibited dilatation of pial arterioles during hypoxia by 46 +/- 5% (P < .05). In contrast, vasodilatation in response to sodium nitroprusside was not altered by glibenclamide. Topical application of adenosine (10(-4) mol/L) increased arteriolar diameter by 21 +/- 4%. Glibenclamide did not affect adenosine-induced vasodilatation. These findings suggest that dilatation of cerebral arterioles in response to hypoxia is mediated, in part, by activation of ATP-sensitive K+ channels.
Metabolic coupling of glutathione between mouse and quail cardiac myocytes and its protective role against oxidative stress.
Authors: Nakamura T Y , Yamamoto I , Kanno Y , Shiba Y , Goshima K
Published: June 2012

Cultured quail myocytes were much more resistant to H2O2 toxicity than cultured mouse myocytes. The intracellular concentration of glutathione ([GSH]i) and the activity of gamma-glutamylcysteine synthetase (gamma-GCS) in quail heart cells were about five and three times higher, respectively, than in mouse heart cells, although catalase and glutathione peroxidase (GSHpx) activity was similar in both. Preloading of gamma-glutamylcysteine monoethyl ester (gamma-GCE), a membrane-permeating GSH precursor, increased the H2O2 resistance of cultured mouse myocytes. These observations suggest that the high [GSH]i and the high activity of gamma-GCS in quail myocytes are responsible for their high resistance to H2O2. Both H2O2 sensitivity and [GSH]i of mosaic sheets composed of equal amounts of mouse and quail myocytes approximated those of sheets composed entirely of quail myocytes. From these observations, it is hypothesized that GSH was transferred from quail myocytes to mouse myocytes, probably through gap junctions between them, and that quail myocytes resynthesized GSH by a feedback mechanism, thus maintaining their intracellular GSH levels. When the fluorescent dye lucifer yellow was injected into a beating quail myocyte in a mosaic sheet, it spread to neighboring mouse myocytes but not to neighboring L cells (a cell line derived from mouse connective tissue). These observations indicate that existence of gap junctions in the region of cell contact between mouse and quail myocytes but not between quail myocytes and L cells. When quail myocytes preloaded with [3H]gamma-GCE were cocultured with mouse myocytes and L cells, the radioactivity was transmitted to neighboring mouse myocytes but not L cells. These observations show that GSH and/or its precursors can be transmitted from quail myocytes to mouse myocytes through gap junctions and that this can protect mouse myocytes from H2O2 toxicity. Mouse myocyte sheets composed of 10(4) cells or more showed higher resistance to H2O2 toxicity than single isolated mouse myocytes. Metabolic coupling of GSH between myocytes may contribute at least in part to this high resistance of the cell sheets.
Endothelial expression of thrombomodulin is reversibly regulated by fluid shear stress.
Authors: Malek A M , Jackman R , Rosenberg R D , Izumo S
Published: June 2012

The vascular endothelium, by virtue of its position at the interface between blood and the vessel wall, is known to play a critical role in the control of thrombosis and fibrinolysis. Thrombomodulin (TM) is a surface receptor that binds thrombin and is a potent activator of the protein C anticoagulant pathway. Although TM expression is known to be regulated by various cytokines, little is known about its response to ever-present biomechanical stimuli. We have explored the role of fluid shear stress, imparted on the luminal surface of the endothelial cell as a result of blood flow, on the expression of TM mRNA and protein in both bovine aortic endothelial (BAE) and bovine smooth muscle (BSM) cells in an in vitro system. We report in the present study that TM expression is regulated by flow. Subjecting BAE cells to fluid shear stress in the physiological range of magnitude of 15 (moderate shear stress) and 36 (elevated shear stress) dynes/cm2 resulted in a mild transient increase followed by a significant decrease in TM mRNA to 37% and 16% of its resting level, respectively, by 9 hours after the onset of flow. In contrast, shear stress at the low magnitude of 4 dynes/cm2 did not affect TM mRNA levels. The sensitivity of TM mRNA expression by flow was found to be specific to endothelium, since it was not observed in BSM cells exposed to steady laminar shear stress of 15 dynes/cm2. Furthermore, unlike BAE cells, BSM cells did not exhibit altered cell shape nor align in the direction of flow after 24 hours of shear stress at 15 dynes/cm2.(ABSTRACT TRUNCATED AT 250 WORDS)
Hypoxia inhibits myogenic reactivity of renal afferent arterioles by activating ATP-sensitive K+ channels.
Authors: Loutzenhiser R D , Parker M J
Published: June 2012

Recent findings implicate K+ channels as important modulators of myogenic tone and possible mediators of the vasodilatory effects of hypoxia. In the present report, we examined the effects of hypoxia on myogenic vasoconstriction of renal afferent arterioles. Using the in vitro perfused hydronephrotic rat kidney model, we observed precisely graded decreases in arteriolar diameter when renal perfusion pressure was increased. Normal myogenic reactivity was observed over PO2 levels of 150 to 80 mm Hg. Reducing PO2 to 60, 40, and 30 mm Hg resulted in a significant progressive inhibition of myogenic reactivity. At approximately 20 mm Hg, myogenic vasoconstriction was essentially abolished, whereas the vasoconstriction induced by 30 mmol/L KCl was unaffected. The addition of 1.0 mumol/L glibenclamide completely restored myogenic vasoconstriction during hypoxia. In contrast, 1.0 mmol/L tetraethylammonium did not alter the effects of hypoxia. To investigate the relation between hypoxia-induced vasodilation and smooth muscle oxidative phosphorylation, we monitored changes in arteriolar levels of reduced NADH during exposure to hypoxia. Arterioles preconstricted by elevated pressure were optically isolated for simultaneous monitoring of vessel diameter and NADH fluorescence (360-nm excitation, 450-nm emission). Reducing perfusate PO2 from 150 to 20 mm Hg resulted in progressive loss of myogenic tone with no change in arteriolar NADH. These findings indicate that lowering PO2 within a physiological range attenuates myogenic reactivity of the renal afferent arteriole by causing the activation of ATP-sensitive K+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)
Mapping the conversion of atrial flutter to atrial fibrillation and atrial fibrillation to atrial flutter. Insights into mechanisms.
Authors: Ortiz J , Niwano S , Abe H , Rudy Y , Johnson N J , Waldo A L
Published: June 2012

It is not generally believed that there is a relation between atrial flutter, thought to be due to a single reentrant circuit, and atrial fibrillation, thought to be due to simultaneously circulating multiple-reentrant wave fronts. However, there are many reasons to suggest that these rhythms are more closely related than previously thought. To test the hypothesis that the length of an area of functional block in the right atrial free wall is critical to the conversion of atrial flutter to atrial fibrillation and of atrial fibrillation to atrial flutter, we studied spontaneous and ATP-induced conversion of stable atrial flutter to sustained atrial fibrillation and spontaneous conversion of sustained atrial fibrillation to stable atrial flutter. We studied 13 episodes of the conversion of stable atrial flutter to sustained atrial fibrillation and sustained atrial fibrillation to stable atrial flutter in seven dogs with sterile pericarditis. Six episodes were spontaneous and seven were ATP related. All episodes were studied by using a multisite mapping system to record 190 unipolar electrograms (converted in the software to 95 bipolar electrograms) from the right atrial free wall along with ECG lead II. Atrial flutter induction was attempted by atrial stimulation (S1S2 or S1S2S3) or by rapid atrial pacing for > or = 20 beats from selected sites at selected rates. For both the spontaneous and the ATP-related episodes, stable atrial flutter was defined as any episode of > or = 5 minutes, and sustained atrial fibrillation was any episode of > or = 1 minute. During all the episodes of stable atrial flutter, a line of functional block with a mean length of 24 +/- 4 mm was localized on the right atrial free wall. When the previously stable line of functional block decreased to a mean of 16 +/- 3 mm (P < .05), either spontaneously or after ATP administration (40 mg i.v.), the new line of functional block was not long enough to maintain stable atrial flutter, and conversion to atrial fibrillation resulted. This shortened line of functional block continued to change and migrate over the right atrial free wall throughout sustained atrial fibrillation. These observations were similar for both spontaneous and ATP-induced conversions. When sustained atrial fibrillation evolved to stable atrial flutter, there was reformation of a long line of functional block, long enough (> or = prior length) to create a stable reentrant circuit, which then captured the right atrial free wall and subsequently both atria.(ABSTRACT TRUNCATED AT 400 WORDS)
Activation of cardiac vagal afferents in ischemia and reperfusion. Prostaglandins versus oxygen-derived free radicals.
Authors: Ustinova E E , Schultz H D
Published: June 2012

Myocardial ischemia and reperfusion can evoke excitation of cardiac vagal nerve endings and activation of a cardiogenic depressor reflex (Bezold-Jarisch effect). We postulate that oxygen-derived free radicals, which are known to be produced during prolonged ischemia and reperfusion, contribute to this afferent excitation. We recorded activity from 47 chemosensitive vagal afferent fibers in 31 rats; the endings of these fibers were located in the left ventricle. Chemosensitive endings were identified with topical applications of capsaicin (10 micrograms) to the surface of the heart. Reactivity of the endings to oxygen-derived free radicals was assessed by topical application of H2O2 (3 to 9 mumol). Activity of the vagal fibers was recorded during 30 minutes of occlusion of the left anterior descending coronary artery (LAD) and 10 minutes of subsequent reperfusion. The activity of chemosensitive endings within the ischemic zone increased in the first 2 minutes of LAD occlusion from 2.2 +/- 0.4 to 4.3 +/- 0.9 impulses per second (107 +/- 30% increase, P < .05). This increased activity waned after 3 to 5 minutes of occlusion. Endings outside the ischemic zone did not increase, their activity at the beginning of ischemia. Reperfusion caused a rapid elevation of activity only in chemosensitive fibers whose endings were found to respond to topical H2O2. The reperfusion-sensitive endings were located both within and outside the ischemic zone of the left ventricle. Indomethacin (5 mg/kg i.v., 20 minutes before occlusion) effectively prevented activation of chemosensitive afferent endings at the beginning of LAD occlusion regardless of their sensitivity to H2O2 but had no effect on the activation at reperfusion.
The failing human heart is unable to use the Frank-Starling mechanism.
Authors: Schwinger R H , Böhm M , Koch A , Schmidt U , Morano I , Eissner H J , Uberfuhr P , Reichart B , Erdmann E
Published: June 2012

There is evidence that the failing human left ventricle in vivo subjected to additional preload is unable to use the Frank-Starling mechanism. The present study compared the force-tension relation in human nonfailing and terminally failing (heart transplants required because of dilated cardiomyopathy) myocardium. Isometric force of contraction of electrically driven left ventricular papillary muscle strips was studied under various preload conditions (2 to 20 mN). To investigate the influence of inotropic stimulation, the force-tension relation was studied in the presence of the cardiac glycoside ouabain. In skinned-fiber preparations of the left ventricle, developed tension was measured after stretching the preparations to 150% of the resting length. To evaluate the length-dependent activation of cardiac myofibrils by Ca2+ in failing and nonfailing myocardium, the tension-Ca2+ relations were also measured. After an increase of preload, the force of contraction gradually increased in nonfailing myocardium but was unchanged in failing myocardium. There were no differences in resting tension, muscle length, or cross-sectional area of the muscles between both groups. Pretreatment with ouabain (0.02 mumol/L) restored the force-tension relation in failing myocardium and preserved the force-tension relation in nonfailing tissue. In skinned-fiber preparations of the same hearts, developed tension increased significantly after stretching only in preparations from nonfailing but not from failing myocardium. The Ca2+ sensitivity of skinned fibers was significantly higher in failing myocardium (EC50, 1.0; 95% confidence limit, 0.88 to 1.21 mumol/L) compared with nonfailing myocardium (EC50, 1.7; 95% confidence limit, 1.55 to 1.86 mumol/L). After increasing the fiber length by stretching, a significant increase in the sensitivity of the myofibrils to Ca2+ was observed in nonfailing but not in failing myocardium. These experiments provide evidence for an impaired force-tension relation in failing human myocardium. On the subcellular level, this phenomenon might be explained by a failure of the myofibrils to increase the Ca2+ sensitivity after an increase of the sarcomere length.
Conscious rabbits become tolerant to multiple episodes of ischemic preconditioning.
Authors: Cohen M V , Yang X M , Downey J M
Published: June 2012

Although ischemic preconditioning protects myocardium from infarction in isolated hearts and in anesthetized open-chest animals, its effects have not been examined in unanesthetized animals. Furthermore, it is unknown whether animals become tolerant to multiple episodes of ischemic preconditioning. Rabbits were chronically instrumented with a balloon occluder around a major branch of the left coronary artery for reversible coronary occlusion, a left atrial catheter for radioactive microsphere injections, ECG electrodes for monitoring of myocardial ischemia, and, in some cases, a carotid artery catheter for pressure measurements and timed withdrawal of reference arterial blood samples. Eight control rabbits underwent a 30-minute coronary occlusion and then 180 minutes of reperfusion. Five of the eight rabbits developed ventricular tachycardia or fibrillation during ischemia, and infarct size averaged 37.7 +/- 2.6% of the risk area. Eight rabbits experienced a 5-minute coronary occlusion and 10 minutes of reperfusion before the 30-minute occlusion. In these preconditioned animals, potentially fatal arrhythmias during ischemia were significantly reduced (one of eight, P < .05), and infarct size was much smaller (5.6 +/- 1.1%, P < .0001). The difference could not be explained by hemodynamics or collateral blood flow, which were nearly identical in the two groups. But when the 30-minute coronary occlusion was preceded by 40 to 65 five-minute occlusions during a 3- to 4-day period in seven animals, protection was markedly attenuated. Potentially lethal arrhythmias were very common, and infarct size averaged 26.5 +/- 2.9%, substantially larger than in rabbits with only one preconditioning occlusion (P < .0001).(ABSTRACT TRUNCATED AT 250 WORDS)
A dynamic model of the cardiac ventricular action potential. I. Simulations of ionic currents and concentration changes.
Authors: Luo C H , Rudy Y
Published: June 2012

A mathematical model of the cardiac ventricular action potential is presented. In our previous work, the membrane Na+ current and K+ currents were formulated. The present article focuses on processes that regulate intracellular Ca2+ and depend on its concentration. The model presented here for the mammalian ventricular action potential is based mostly on the guinea pig ventricular cell. However, it provides the framework for modeling other types of ventricular cells with appropriate modifications made to account for species differences. The following processes are formulated: Ca2+ current through the L-type channel (ICa), the Na(+)-Ca2+ exchanger, Ca2+ release and uptake by the sarcoplasmic reticulum (SR), buffering of Ca2+ in the SR and in the myoplasm, a Ca2+ pump in the sarcolemma, the Na(+)-K+ pump, and a nonspecific Ca(2+)-activated membrane current. Activation of ICa is an order of magnitude faster than in previous models. Inactivation of ICa depends on both the membrane voltage and [Ca2+]i. SR is divided into two subcompartments, a network SR (NSR) and a junctional SR (JSR). Functionally, Ca2+ enters the NSR and translocates to the JSR following a monoexponential function. Release of Ca2+ occurs at JSR and can be triggered by two different mechanisms, Ca(2+)-induced Ca2+ release and spontaneous release. The model provides the basis for the study of arrhythmogenic activity of the single myocyte including afterdepolarizations and triggered activity. It can simulate cellular responses under different degrees of Ca2+ overload. Such simulations are presented in our accompanying article in this issue of Circulation Research.
Taurine depletion and excitation-contraction coupling in rat myocardium.
Authors: Eley D W , Lake N , ter Keurs H E
Published: June 2012

Although the sulfur-containing amino acid taurine is found in high concentrations in mammalian myocardium, its involvement in function of the cardiac myocyte remains unclear. To examine the effects of taurine depletion on cardiac mechanical function, rats were treated in vivo with the taurine transport antagonist guanidinoethane sulfonate (GES). After 6 weeks of treatment, myocardial taurine concentrations were decreased to < 40% of control, with no change in tissue DNA content. Right ventricular trabeculas from taurine-depleted rats exhibited significant reductions (P < .05) in isometric twitch force (Ft) at all [Ca2+]o levels and systolic sarcomere lengths examined. Taurine-depleted trabeculas also exhibited increased passive compliance. A slight (P < .05) rightward shift in the Ft-[Ca2+]o relation suggested a decrease in the sensitivity of the taurine-depleted muscles to [Ca2+]o. No changes were observed in the force-interval relation, suggesting that the transsarcolemmal Ca(2+)-handling mechanisms remained unchanged. The fraction of Ca2+ recirculated through the sarcoplasmic reticulum, inferred from the decay of postextrasystolic potentiation, was also not different in the taurine-depleted muscles. When force was expressed relative to the rate of stimulation, length of rest periods, or postextrasystolic potentiation, virtually all curves were super-imposable for control and taurine-depleted muscles, suggesting that the deficit was not dependent on Ca2+ handling. Thus, we conclude that in taurine-depleted muscles the force-generating processes showed the same regulation as in control muscle. Furthermore, the substantial deficit in force development is consistent with a reduced population of force generators on the basis of three pieces of evidence.(ABSTRACT TRUNCATED AT 250 WORDS)
Wall shear stress temporal gradient and anastomotic intimal hyperplasia.
Authors: Ojha M
Published: June 2012

Unusual wall shear stress patterns on the bed of end-to-side arterial anastomoses appear to be the primary factor in the development of intimal hyperplasia that often leads to arterial bypass graft failure. In this in vitro study using the photochromic tracer technique, the shear stress variation on the bed of a 30 degrees anastomosis was examined before and after the development of hyperplasia. With the disease-free model, a rapid downstream shift in the stagnation point was seen on the bed during the systolic phase of the flow cycle, which led to sharp temporal changes in the shear stress from positive to negative values, ie, rapid changes in the direction of the shearing force. The resulting negative temporal gradients were roughly eight times larger than those seen in a straight tube, and it is suggested that this may lead to endothelial deformation or injury and eventually to intimal thickening. With the diseased model, the simulated tissue overgrowth on the bed appeared to act as a flow divider that restricted the motion of the stagnation point, and this drastically reduced the rapid changes in the direction of the shearing forces. Thus, it seems that the development of bed anastomotic intimal hyperplasia may be a response designed to reduce shear-induced endothelial deformation or injury.
Cytokine-stimulated human vascular smooth muscle cells synthesize a complement of enzymes required for extracellular matrix digestion.
Authors: Galis Z S , Muszynski M , Sukhova G K , Simon-Morrissey E , Unemori E N , Lark M W , Amento E , Libby P
Published: June 2012

Vascular matrix remodeling occurs during development, growth, and several pathological conditions that affect blood vessels. We investigated the capacity of human smooth muscle cells (SMCs) to express matrix metalloproteinases (MMPs), enzymes that selectively digest components of the extracellular matrix (ECM), in the basal state or after stimulation with certain cytokines implicated in vascular homeostasis and pathology. Enzymatic activity associated with various proteins secreted in the culture media was detected by gelatin or casein sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography. Proteins were identified by immunoprecipitation and mRNA by Northern blotting. SMCs constitutively secreted a 72-kD gelatinase and the tissue inhibitors of MMPs (TIMPs) types 1 and 2. SMCs stimulated with interleukin-1 or tumor necrosis factor-alpha synthesized de novo 92-kD gelatinase, interstitial collagenase, and stromelysin. Several lines of evidence suggest that when stimulated by cytokines, SMCs produce activated forms of MMPs. Together, the constitutive and the cytokine-induced enzymes can digest all the major components of the vascular ECM. Moreover, since these mediators augment the production of MMPs without appreciably affecting the synthesis of TIMPs, locally secreted cytokines may tip the regional balance of MMP activity in favor of vascular matrix degradation.
A role for endogenous endothelin-1 in neointimal formation after rat carotid artery balloon angioplasty. Protective effects of the novel nonpeptide endothelin receptor antagonist SB 209670.
Authors: Douglas S A , Louden C , Vickery-Clark L M , Storer B L , Hart T , Feuerstein G Z , Elliott J D , Ohlstein E H
Published: June 2012

The observation that levels of the mitogenic peptide endothelin-1 are elevated in the human coronary sinus after percutaneous transluminal coronary angioplasty (PTCA) has implicated endothelin-1 in the etiology of vascular restenosis. The present study examined this hypothesis in both an in vitro and an in vivo rat model of neointimal formation by using the novel nonpeptide endothelin receptor antagonist SB 209670. In vitro, endothelin-1 (1 nmol/L) induced a ninefold increase in rat aortic vascular smooth muscle [3H]thymidine incorporation. This endothelin A receptor-mediated effect was completely inhibited by SB 209670 (IC50, 6.2 +/- 2.2 nmol/L). In vivo, acute intra-arterial administration of exogenous endothelin-1 (5 to 500 pmol/kg over a 30-minute period immediately after angioplasty) dose-dependently augmented the degree of neointimal formation (by up to 150% when assessed 14 days after surgery). This response was evident as early as 7 days after angioplasty. Hemodynamic studies indicated that this action was unrelated to a systemic pressor action of the peptide. Administration of SB 209670 (2.5 mg/kg IP, twice a day for 3 days before and for 2 weeks after surgery) reduced neointimal formation by approximately 50% relative to control animals. Thus, the data indicate for the first time that (1) endothelin-1 promotes neointimal formation in vivo and (2) endogenous endothelin-1 is involved in the pathogenesis of angioplasty-induced lesion formation in the rat. Endothelin receptor antagonists such as SB 209670 may therefore serve as useful adjuncts to PTCA, attenuating the degree of vascular restenosis observed after vascular wall injury.
Migration of cultured vascular smooth muscle cells through a basement membrane barrier requires type IV collagenase activity and is inhibited by cellular differentiation.
Authors: Pauly R R , Passaniti A , Bilato C , Monticone R , Cheng L , Papadopoulos N , Gluzband Y A , Smith L , Weinstein C , Lakatta E G
Published: June 2012

The migration of vascular smooth muscle cells (VSMCs) from the tunica media to the neointima is a key event in the development and progression of many vascular diseases and a highly predictable consequence of mechanical injury to the blood vessel. In vivo, VSMCs are surrounded by and embedded in a variety of extracellular matrices (ECMs) that must be traversed during migration. One of the principal barriers to cell movement in the intact vessel is the basement membrane (BM) that surrounds each VSMC and separates the VSMC-containing medial cell layer from the endothelium. We have used a Boyden chamber to monitor the ability of VSMCs to degrade a BM barrier as they migrate toward a chemoattractant and to define the role of extracellular proteases in this process. We show that cultured VSMCs can migrate across a BM barrier and that this ability was dependent on the phenotypic state of the cell. VSMCs maintained in a proliferating or "synthetic" state readily migrated across a BM toward a chemoattractant, whereas the migration of serum-starved/differentiated VSMCs was suppressed by > 80% (P < .001). By use of a number of peptides that inhibit matrix metalloproteinase (MMP) activity, the migration of proliferating VSMCs across the BM barrier was inhibited by > 80% (P < .0001), whereas migration that occurred in the absence of the barrier was unaffected. Northern blotting and zymographic analyses indicated that 72-kD type IV collagenase (MMP2) was the principal MMP expressed and secreted by these cells. Accordingly, antisera capable of selectively neutralizing MMP2 activity also inhibited VSMC migration across the barrier without significantly affecting the migration of VSMCs in the absence of the barrier. Finally, MMP2 activity was also regulated by the phenotypic state of the cells in that MMP2 activity expressed by serum-starved/differentiated VSMCs was < 5% of that measured in proliferating VSMCs. Extrapolating to the in vivo situation in which VSMCs reside in an ECM composed of various BM barriers, these results suggest that VSMC migration in vivo may be dependent on MMP2 activity. That activity, in turn, could be regulated by the phenotypic state of VSMCs and increase as these cells undergo the transition from a quiescent and differentiated state to that of a dedifferentiated, proliferating, and motile phenotype after injury to the vessel.
Beneficial effect of SPM-5185, a cysteine-containing nitric oxide donor, in rat carotid artery intimal injury.
Authors: Guo J P , Milhoan K A , Tuan R S , Lefer A M
Published: June 2012

We studied the effects of an organic nitric oxide (NO) donor SPM-5185 in a rat carotid artery intimal injury model. Seven days after injury, the two end segments of the injured carotid arteries were studied for endothelial release of NO, and the middle segments were used for histological measurement of the intimal-to-medial (I/M) ratio and scanning electron microscopy of the luminal surface. The NO donor SPM-5185 or its non-NO-donating control compound SPM-5267 were infused intravenously at 30 micrograms/d. Full vasorelaxant responses of rat carotid arterial rings were obtained with the endothelium-dependent vasodilators acetylcholine (ACh), A23187, and the endothelium-independent vasodilator acidified NaNO2 in sham-operated control rings. Impaired relaxation occurred with 10 mumol/L ACh and 1 mumol/L A23187 in injured rings but not in rings infused with SPM-5185 for 7 days. Relaxation to 100 mumol/L acidified NaNO2 was not significantly different among any of the groups, indicating a normal vascular smooth muscle response after intimal injury. Morphometric analysis of injured carotid arteries given vehicle and SPM-5267 showed marked intimal thickening with an average I/M ratio of 0.78 +/- 0.03 and 0.74 +/- 0.05, respectively. SPM-5185 markedly attenuated intimal thickening, resulting in an I/M ratio of 0.13 +/- 0.03 (P < .01 from vehicle), representing an approximately 82% inhibition of intimal thickening. SPM-5185 infusion resulted in accelerated regeneration of endothelial cells on the intimal surface at 7 days.(ABSTRACT TRUNCATED AT 250 WORDS)
Thyroid hormone-induced alterations in phospholamban protein expression. Regulatory effects on sarcoplasmic reticulum Ca2+ transport and myocardial relaxation.
Authors: Kiss E , Jakab G , Kranias E G , Edes I
Published: June 2012

The aim of the present study was to determine the changes in phospholamban protein levels and their regulatory effect on sarcoplasmic reticulum (SR) Ca2+ uptake and left ventricular function in hypothyroid and hyperthyroid rat hearts. Hypothyroidism was associated with decreases in basal left ventricular function (+dP/dt and -dP/dt), whereas in hyperthyroidism these parameters were elevated compared with values for euthyroid hearts. The maximal SR Ca2+ uptake rates were 12.8 +/- 1.1, 15.5 +/- 1.2, and 21.4 +/- 1.4 nmol Ca2+ per milligram per minute, and the EC50 values for Ca2+ were 0.76 +/- 0.09, 0.41 +/- 0.07, and 0.30 +/- 0.05 mumol/L assayed in homogenates from hypothyroid, euthyroid, and hyperthyroid hearts, respectively. The relative tissue level of phospholamban was increased (135%) in hypothyroidism and decreased (75%) in hyperthyroidism compared with euthyroidism (100%). An opposite trend was observed for the SR Ca(2+)-ATPase, which was depressed (74%) in hypothyroid hearts but increased (134%) in hyperthyroid hearts. Consequently, the relative ratio of phospholamban to Ca(2+)-ATPase was highest in hypothyroid and lowest in hyperthyroid hearts, and these changes correlated with changes in the EC50 of the SR Ca2+ uptake for Ca2+. Stimulation of hearts with 0.1 mumol/L isoproterenol revealed that the relaxant effects were lower in hyperthyroid hearts and higher in hypothyroid hearts compared with euthyroid hearts, consistent with the alterations in the phospholamban levels. The maximal increases in the speed of relaxation, elicited by isoproterenol stimulation, correlated with the changes in the relative ratio of phospholamban to Ca(2+)-ATPase in these hearts.(ABSTRACT TRUNCATED AT 250 WORDS)