bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolic regulation of coronary vascular tone: role of endothelin-1
Authors: Merkus Daphne , Duncker Dirk J. , Chilian William M.
Published: March 2015

Coronary tone is determined by a balance between endogenously produced endothelin and metabolic dilators. We hypothesized that coronary vasodilation during augmented metabolism is the net result of decreased endothelin production and increased production of vasodilators. Isolated rat myocytes were stimulated at 0, 200, and 400 beats/min to modify metabolism. Supernatant from these preparations was added to isolated coronary arterioles with and without blocking vasoactive pathways (adenosine, bradykinin, and endothelin). Chronically instrumented swine were studied while resting and running on a treadmill before and after endothelin type A (ETA) receptor blockade. The vasodilatory properties of the supernatant increased with increased stimulation frequencies. Combined blockade of adenosine and bradykinin receptors abolished vasodilation in response to supernatant of stimulated myocytes. ETA blockade increased vasodilation to supernatant of unstimulated myocytes but did not affect dilation to supernatant of myocytes stimulated at 400 beats/min. In vivo, ETA blockade resulted in coronary vasodilation at rest, which waned during exercise. Thus endothelin has a tonic constrictor influence through the ETA receptor at low myocardial metabolic demand but its influence decreased during increased metabolism.
Pentose phosphate pathway coordinates multiple redox-controlled relaxing mechanisms in bovine coronary arteries
Authors: Gupte Sachin A. , Arshad Muhammad , Viola Steven , Kaminski Pawel M. , Ungvari Zoltan , Rabbani Golam , Koller Akos , Wolin Michael S.
Published: March 2015

Pentose phosphate pathway (PPP) inhibitors, 6-aminonicotinamide (6-AN) and epiandrosterone (Epi), were employed to examine whether changes in NADP(H) redox regulates contractile force in endothelium-removed bovine coronary arteries (BCAs). 6-AN (0.01–5 mM) or Epi (1–500 μM) elicited dose-dependent relaxation in BCAs contracted with 30 mM KCl, 0.1 μM U-44619, and endothelin-1 but not with phorbol 12,13-dibutyrate, a protein kinase C activator that causes Ca2+-independent contraction. Relaxation to PPP inhibition was associated with oxidation of NADPH and glutathione (GSH). Relaxation to 6-AN was not mediated by H2O2, because it was not altered by hypoxia or the peroxide scavenger ebselen (100 μM). The thiol reductant DTT (3 mM) attenuated the relaxation to 6-AN and Epi by 30–40%. Inhibition of glycolysis or mitochondrial electron transport did not elicit relaxation in BCAs contracted with 30 mM KCl, suggesting these pathways may not be involved in relaxation elicited by PPP inhibition. High doses of K+ channel blockers [e.g., TEA (10 mM) and 4-aminopyridine (10 mM)] only partially inhibited the relaxation to 6-AN. On the basis of changes in the fura-2 fluorescence ratio, 6-AN and Epi appeared to markedly reduce intracellular Ca2+. Thus PPP inhibition oxidizes NADPH and GSH and appears to activate a novel coordination of redox-controlled relaxing mechanisms in BCAs mediated primarily through decreasing intracellular Ca2+.
Theoretical analysis of rest and exercise hemodynamics in patients with total cavopulmonary connection
Authors: Magosso Elisa , Cavalcanti Silvio , Ursino Mauro
Published: March 2015

The objective of this study was to determine the impact of a total cavopulmonary connection on the main hemodynamic quantities, both at rest and during exercise, when compared with normal biventricular circulation. The analysis was performed by means of a mathematical model of the cardiovascular system. The model incorporates the main parameters of systemic and pulmonary circulation, the pulsating heart, and the action of arterial and cardiopulmonary baroreflex mechanisms. Furthermore, the effect of changes in intrathoracic pressure on venous return is also incorporated. Finally, the response to moderate dynamic exercise is simulated, including the effect of a central command, local metabolic vasodilation, and the “muscle pump” mechanism. Simulations of resting conditions indicate that the action of baroreflex regulatory mechanisms alone can only partially compensate for the absence of the right heart. Cardiac output and mean systemic arterial pressure at rest show a large decrease compared with the normal subject. More acceptable hemodynamic quantity values are obtained by combining the action of regulatory mechanisms with a chronic change in parameters affecting mean filling pressure. With such changes assumed, simulations of the response to moderate exercise show that univentricular circulation exhibits a poor capacity to increase cardiac output and to sustain aerobic metabolism, especially when the oxygen consumption rate is increased above 1.2–1.3 l/min. The model ascribes the poor response to exercise in these patients to the incapacity to sustain venous return caused by the high resistance to venous return and/or to exhaustion of volume compensation reserve.
α1-AR-induced positive inotropic response in heart is dependent on myosin light chain phosphorylation
Authors: Andersen Geir Øystein , Qvigstad Eirik , Schiander Iwona , Aass Halfdan , Osnes Jan-Bjørn , Skomedal Tor
Published: March 2015

The possible involvement of different kinases in the α1-adrenoreceptor (AR)-mediated positive inotropic effect (PIE) was investigated in rat papillary muscle and compared with β-AR-, endothelin receptor- and phorbol ester-induced changes in contractility. The α1-AR-induced PIE was not reduced by the inhibitors of protein kinase C (PKC), MAPK (ERK and p38), phosphatidyl inositol 3-kinase, or calmodulin kinase II. However, PKC inhibition attenuated the effect of phorbol 12-myristate 13-acetate (PMA) on contractility. α1-AR-induced PIE was reduced by ∼90% during inhibition of myosin light chain kinase (MLCK) by 1-(5-chloronaphthalene-1-sulfonyl)1 H-hexahydro-1,4-diazepine (ML-9). Endothelin-induced PIE was also reduced by ML-9, but ML-9 had no effect on β-AR-induced PIE. The Rho kinase inhibitor Y-27632 also reduced the α1-AR-induced PIE. The α1-AR-induced PIE in muscle strips from explanted failing human hearts was also sensitive to MLCK inhibition. α1-AR induced a modest increase in32P incorporation into myosin light chain in isolated rat cardiomyocytes. This effect was eliminated by ML-9. The PIE of α1-AR stimulation seems to be dependent on MLCK phosphorylation.
Role of intracellular antioxidant enzymes after in vivo myocardial ischemia and reperfusion
Authors: Jones Steven P. , Hoffmeyer Michaela R. , Sharp Brent R. , Ho Ye-Shih , Lefer David J.
Published: March 2015

Reactive oxygen species induce myocardial damage after ischemia and reperfusion in experimental animal models. Numerous studies have investigated the deleterious effects of ischemia-reperfusion (I/R)-induced oxidant production using various pharmacological interventions. More recently, in vitro studies have incorporated gene-targeted mice to decipher the role of antioxidant enzymes in myocardial reperfusion injury. We examined the role of cellular antioxidant enzymes in the pathogenesis of myocardial I/R (MI/R) injury in vivo in gene-targeted mice. Neither deficiency nor overexpression of Cu-Zn superoxide dismutase (SOD) altered the extent of myocardial necrosis. Overexpression of glutathione peroxidase did not affect the degree of myocardial injury. Conversely, overexpression of manganese (Mn)SOD significantly attenuated myocardial necrosis after MI/R. Transthoracic echocardiography was performed on MnSOD-overexpressing and wild-type mice that were subjected to a more prolonged period of reperfusion. Cardiac output was significantly depressed in the nontransgenic but not the transgenic MnSOD-treated mice. Anterior wall motion was significantly impaired in the nontransgenic mice. These findings demonstrate an important role for MnSOD but not Cu/ZnSOD or glutathione peroxidase in mice after in vivo MI/R.
Echocardiographic assessment of LV hypertrophy and function in aortic-banded mice: necropsy validation
Authors: Liao Yulin , Ishikura Fuminobu , Beppu Shintaro , Asakura Masanori , Takashima Seiji , Asanuma Hiroshi , Sanada Shoji , Kim Jiyoon , Ogita Hisakazu , Kuzuya Tsunehiko , Node Koichi , Kitakaze Masafumi , Hori Masatsugu
Published: March 2015

We characterized the time course of the left ventricular (LV) geometric and functional changes after aortic banding, validated them by necropsy, and investigated the sensitivity of echocardiographic findings on LV hypertrophy. C57BL/6 mice were subjected to transverse aortic constriction (TAC) or sham operation; echocardiographic assessments were performed before or at 2, 4, 6, and 11 wk after surgery; and some of the mice were euthanized at the corresponding time points. There was a progressive increase in diastolic posterior wall thickness and LV systolic dimension; the percentage of LV fractional shortening (LV%FS) decreased progressively at 4 wk, whereas these parameters remained stable in sham-operated mice. Echo LV mass and LV%FS correlated well with actual whole heart mass and ratio of lung weight to body weight, respectively ( r = 0.765 and −0.749, respectively; P < 0.0001). These results suggest that the development of myocardial hypertrophy and systolic dysfunction is a time-dependent process. Echocardiographic assessment of myocardial hypertrophy and functional changes correlate well with the actual heart mass and lung mass. Echocardiography is sensitive enough to assess myocardial hypertrophy and heart functional changes induced by pressure overload in mice.
Role of oxidative phosphorylation and ATP release in mediating birth-related pulmonary vasodilation in fetal lambs
Authors: Konduri Girija G. , Mattei Janine
Published: March 2015

We investigated the hypothesis that birth-related pulmonary vasodilation is mediated in part by an increase in oxidative phosphorylation and ATP release in response to oxygen exposure at birth. Studies were done in fetal lambs to evaluate the independent effects of oxygen, lung distension alone, or lung distension accompanied by oxygenation and shear stress on fetal pulmonary blood flow and resistance and plasma ATP levels in the pulmonary artery. The effect of each intervention was evaluated in lambs assigned to one of three groups: control or pretreatment with 2,4-dinitrophenol or antimycin-A, inhibitors of oxidative phosphorylation. Exposure to oxygen alone or with lung distension was associated with increases in plasma ATP levels and pulmonary blood flow and a decrease in pulmonary vascular resistance. Plasma ATP levels did not change during lung distension alone. 2,4-Dinitrophenol and antimycin-A attenuated the pulmonary vasodilator response to oxygen but did not attenuate the response to lung distension alone. An increase in oxidative phosphorylation and ATP release during oxygen exposure may contribute to birth-related pulmonary vasodilation in fetal lambs.
Collateral arteries grow from preexisting anastomoses in the rat hindlimb
Authors: Herzog Sandra , Sager Hendrik , Khmelevski Eugen , Deylig Andrea , Ito Wulf D.
Published: March 2015

Previous findings have suggested that collateral arteries grow from preexisting arteriolar anastomoses (“arteriogenesis”). To investigate whether collateral growth occurs without preceding angiogenesis, we obtained vascular casts and postmortem angiographies 3, 7, and 21 days after unilateral femoral artery occlusion in the rat. Proliferation kinetics were determined after 5′-bromo-2′-desoxyuridin infusion. A preexisting anastomosis was identified. Proliferation of this vessel began 24 h after femoral artery occlusion, increased maximally during the first 3 days, and reached 60% at day 7. Cell division was restricted to preexisting anastomoses and occurred neither in directly neighboring arterial vessels nor in capillaries. Collateral vessels doubled their diameter within 7 days and assumed a typical corkscrew appearance (increase of length: 21%). After 7 days of occlusion, we measured a further increase of length (14%) but no proliferation or increase of diameter. We conclude that arteriogenesis is a biphasic process involving rapid proliferation of preexisting arteriolar shunts followed by pronounced remodeling processes. Arteriogenesis occurs independently of angiogenesis and denotes a separate entity of vascular proliferation.
Upregulation of collagens detected by gene array in a model of flow-induced pulmonary vascular remodeling
Authors: Medhora Meetha , Bousamra Michael , Zhu Daling , Somberg Lewis , Jacobs Elizabeth R.
Published: March 2015

We recently reported localized increased pulmonary arterial resistance, neointimal lesions, and medial thickening induced by aortopulmonary anastomosis in young pigs. This model was used to investigate changes in expression of genes potentially involved in pulmonary vascular remodeling employing a high throughput Atlas Human Cardiovascular Array carrying ∼600 cardiovascular-related cDNA sequences. Data were confirmed by Northern analysis, Western blots, and histological examination. With the use of lower stringency conditions for hybridization, 56% of the 588 human genes on the array showed visible signal after autoradiography. Approximately 10% of the genes with visible hybridization were altered by shunt-induced high flow. Extracellular matrix and cell adhesion molecules were the most highly represented group of upregulated genes. To our knowledge, our data are the first to demonstrate flow-induced changes in gene expression using a combination of cross species cDNA arrays, homologous hybridization, immunospecific protein, and histology. Our observations expand the list of genes as putative candidates in pulmonary vascular remodeling and support the utility of cross-species microarray analysis in such applications.
Endothelin-1 increases calcium and attenuates renin gene expression in As4.1 cells
Authors: Ryan Michael J. , Black Thomas A. , Millard Susan L. , Gross Kenneth W. , Hajduczok George
Published: March 2015

Endothelin-1 (ET-1) is a potent vasoconstrictor and blood pressure modulator. Renin secretion from juxtaglomerular (JG) cells is crucial for blood pressure and electrolyte homeostasis and has been shown to be modulated by ET-1; however, the cellular and molecular mechanism of this regulation is not clear. The purpose of this study was to gain a better understanding of the cellular and molecular pathways activated by ET-1 by using a renin-producing cell line As4.1. ET-1 caused an increase in As4.1 cell intracelluar Ca2+concentration ([Ca2+]i) mediated by the ETAreceptor as its antagonist, BQ-123, abolished the response. The nitric oxide donor nitroprusside, but not 8-bromo-cGMP, reduced the time necessary for successive ET-1 responses. Endothelin-3 had no effect on [Ca2+]i. ET-1 dose dependently increased total inositol phosphates with an EC50of 2.1 nM. ET-1 reduced renin mRNA by 68% independently of changes in message decay. With the use of a renin-luciferase reporter system in As4.1 cells, ET-1 reduced luciferase activity by 51%, suggesting that renin gene transcription is directly modified by ET-1.
Effects of longitudinal stretch on VSM tone and distensibility of muscular conduit arteries
Authors: Zulliger Martin A. , Kwak Naomi T. M. R. , Tsapikouni Theodora , Stergiopulos Nikos
Published: March 2015

With progressing age, large arteries diminish their longitudinal stretch, which in extreme cases results in tortuosity. Increased age is also associated with loss of vessel distensibility. We measured pressure-diameter curves from muscular porcine carotid arteries ex vivo at different longitudinal stretch ratios (λ z = 1.4 and 1.8) and under different vascular smooth muscle (VSM) conditions (fully relaxed, normal VSM tone, and maximally contracted). Distensibility was found to be halved by decreasing longitudinal stretch from λ z = 1.8 to 1.4 at physiological pressures. This counterintuitive observation is possible because highly nonlinear elastic modulus of the artery and anisotropic properties. Furthermore, a significantly larger basal VSM contraction was observed at λ z = 1.8 than 1.4, although this was clearly not related to a myogenic response during inflation. This dependence of VSM tone to longitudinal stretch may have possible implications on the functional characteristics of the arterial wall.
Role of ANG II in coronary capillary angiogenesis at the insulin-resistant stage of a NIDDM rat model
Authors: Jesmin Subrina , Hattori Yuichi , Sakuma Ichiro , Mowa Chishimba N. , Kitabatake Akira
Published: March 2015

With the use of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of human non-insulin-dependent diabetes mellitus (NIDDM), we assessed whether ANG II is involved in coronary capillary angiogenesis at the insulin-resistant stage of NIDDM (20 wk of age). In OLETF rats, ANG II labeling and angiotensin type 1 (AT1) receptor expression in coronary vessels were increased more than in nondiabetic controls. A marked increase in vascular expression of vascular endothelial growth factor (VEGF) at both mRNA and protein levels was found in OLETF rats. The increased expression level of VEGF was associated with accumulation of hypoxia-inducible factor-1α (HIF-1α) activated by increased advanced glycation end products (AGEs). Morphometric analysis showed a significantly increased total coronary capillary density, which was a result of arterialization of the venular capillary portion in OLETF rats. Treatment of OLETF rats with candesartan, an AT1receptor blocker, inhibited vascular expressions of VEGF, HIF-1α, and AGEs, and ameliorated the morphometric changes. These results suggest a key role of ANG II in the pathogenesis of the coronary capillary remodeling in this NIDDM model.
Targeted O2delivery by low-P50hemoglobin: a new basis for O2therapeutics
Authors: Tsai Amy G. , Vandegriff Kim D. , Intaglietta Marcos , Winslow Robert M.
Published: March 2015

To assess O2delivery to tissue by a new surface-modified, polyethylene glycol-conjugated human hemoglobin [MP4; Po2at 50% saturation of hemoglobin (P50); 5.4 mmHg], we studied microcirculatory hemodynamics and O2release in golden Syrian hamsters hemodiluted with MP4 or polymerized bovine hemoglobin (PolyBvHb; P5054.2 mmHg). Comparisons were made with the animals' hemodiluted blood with a non-O2carrying plasma expander with similar solution properties (Dextran-70). Systemic hemodynamics (arterial blood pressure and heart rate) and acid-base parameters were not correlated with microhemodynamics (arteriolar and venular diameter, red blood cell velocity, and flow). Microscopic measurements of Po2and the O2equilibrium curves permitted analysis of O2release in precapillary and capillary vessels by red blood cells and plasma hemoglobin separately. No significant differences between the groups of animals with respect to arteriolar diameter, flow, or flow velocity were observed, but the functional capillary density was significantly higher in the MP4-treated animals (67%) compared with PolyBvHb-treated animals (37%; P < 0.05) or dextran-treated animals (53%). In the PolyBvHb-treated animals, predominant O2release (both red blood cells and plasma hemoglobin) occurred in precapillary vessels, whereas in MP4 animals most of the O2was released from both red blood cells and plasma hemoglobin in capillaries. Base excess correlated directly with capillary O2release but not systemic O2content or total O2release. Higher O2extraction of both red blood cell and plasma hemoglobin in capillaries represents a new mechanism of action of cell-free hemoglobin. High O2affinity appears to be an important property for cell-free hemoglobin solutions.
Defective intracellular Ca2+ signaling contributes to cardiomyopathy in Type 1 diabetic rats
Authors: Choi Kin M. , Zhong Yan , Hoit Brian D. , Grupp Ingrid L. , Hahn Harvey , Dilly Keith W. , Guatimosim Silvia , Lederer W. Jonathan , Matlib Mohammed A.
Published: March 2015

The goal of the study was to determine whether defects in intracellular Ca2+ signaling contribute to cardiomyopathy in streptozotocin (STZ)-induced diabetic rats. Depression in cardiac systolic and diastolic function was traced from live diabetic rats to isolated individual myocytes. The depression in contraction and relaxation in myocytes was found in parallel with depression in the rise and decline of intracellular free Ca2+ concentration ([Ca2+]i). The sarcoplasmic reticulum (SR) Ca2+ store and rates of Ca2+ release and resequestration into SR were depressed in diabetic rat myocytes. The rate of Ca2+ efflux via sarcolemmal Na+/Ca2+ exchanger was also depressed. However, there was no change in the voltage-dependent L-type Ca2+ channel current that triggers Ca2+ release from the SR. The depression in SR function was associated with decreased SR Ca2+-ATPase and ryanodine receptor proteins and increased total and nonphosphorylated phospholamban proteins. The depression of Na+/Ca2+ exchanger activity was associated with a decrease in its protein level. Thus it is concluded that defects in intracellular Ca2+ signaling caused by alteration of expression and function of the proteins that regulate [Ca2+]i contribute to cardiomyopathy in STZ-induced diabetic rats. The increase in phospholamban, decrease in Na+/Ca2+ exchanger, and unchanged L-type Ca2+ channel activity in this model of diabetic cardiomyopathy are distinct from other types of cardiomyopathy.
ANG II AT2receptor modulates AT1receptor-mediated descending vasa recta endothelial Ca2+signaling
Authors: Rhinehart Kristie , Handelsman Corey A. , Silldorff Erik P. , Pallone Thomas L.
Published: March 2015

We tested whether the respective angiotensin type 1 (AT1) and 2 (AT2) receptor subtype antagonists losartan and PD-123319 could block the descending vasa recta (DVR) endothelial intracellular calcium concentration ([Ca2+]i) suppression induced by ANG II. ANG II partially reversed the increase in [Ca2+]igenerated by cyclopiazonic acid (CPA; 10−5M), acetylcholine (ACh; 10−5M), or bradykinin (BK; 10−7M). Losartan (10−5M) blocked that effect. When vessels were treated with ANG II before stimulation with BK and ACh, concomitant AT2receptor blockade with PD-123319 (10−8M) augmented the suppression of endothelial [Ca2+]iresponses. Similarly, preactivation with the AT2receptor agonist CGP-42112A (10−8M) prevented AT1receptor stimulation with ANG II + PD-123319 from suppressing endothelial [Ca2+]i. In contrast to endothelial [Ca2+]isuppression by ANG II, pericyte [Ca2+]iexhibited typical peak and plateau [Ca2+]iresponses that were blocked by losartan but not PD-123319. DVR vasoconstriction by ANG II was augmented when AT2receptors were blocked with PD-123319. Similarly, AT2receptor stimulation with CGP-42112A delayed the onset of ANG II-induced constriction. PD-123319 alone (10−5M) showed no AT1-like action to constrict microperfused DVR or increase pericyte [Ca2+]i. We conclude that ANG II suppression of endothelial [Ca2+]iand stimulation of pericyte [Ca2+]iis mediated by AT1or AT1-like receptors. Furthermore, AT2receptor activation opposes ANG II-induced endothelial [Ca2+]isuppression and abrogates ANG II-induced DVR vasoconstriction.
Role of ceramide in TNF-α-induced impairment of endothelium-dependent vasorelaxation in coronary arteries
Authors: Zhang David X. , Yi Fu-Xian , Zou Ai-Ping , Li Pin-Lan
Published: March 2015

The present study tested the hypothesis that ceramide, a sphingomylinase metabolite, serves as an second messenger for tumor necrosis factor-α (TNF-α) to stimulate superoxide production, thereby decreasing endothelium-dependent vasorelaxation in coronary arteries. In isolated bovine small coronary arteries, TNF-α (1 ng/ml) markedly attenuated vasodilator responses to bradykinin and A-23187. In the presence of NG-nitro-l-arginine methyl ester, TNF-α produced no further inhibition on the vasorelaxation induced by these vasodilators. With the use of 4,5-diaminofluorescein diacetate fluorescence imaging analysis, bradykinin was found to increase nitric oxide (NO) concentrations in the endothelium of isolated bovine small coronary arteries, which was inhibited by TNF-α. Pretreatment of the arteries with desipramine (10 μM), an inhibitor of acidic sphingomyelinase, tiron (1 mM), a superoxide scavenger, and polyethylene glycol-superoxide dismutase (100 U/ml) largely restored the inhibitory effect of TNF-α on bradykinin- and A-23187-induced vasorelaxation. In addition, TNF-α activated acidic sphingomyelinase and increased ceramide levels in coronary endothelial cells. We conclude that TNF-α inhibits NO-mediated endothelium-dependent vasorelaxation in small coronary arteries via sphingomyelinase activation and consequent superoxide production in endothelial cells.
Postsystolic shortening of ischemic myocardium: a mechanism of abnormal intraventricular filling
Authors: Urheim Stig , Edvardsen Thor , Steine Kjetil , Skulstad Helge , Lyseggen Erik , Rodevand Olaf , Smiseth Otto A.
Published: March 2015

Acute myocardial ischemia has been associated with abnormal filling patterns in the left ventricular (LV) apex. We hypothesized that this may in part be due to postsystolic shortening of ischemic apical segments, which leads to reversal of early diastolic apical flow. Fourteen open-chest anesthetized dogs were instrumented with micromanometers in the LV apex and left atrium and myocardial sonomicrometers in the anterior apical LV wall. Intraventricular filling by color Doppler and wall motion by strain Doppler echocardiography (SDE) were assessed from an apical view. Measurements were taken before and after 5 min of left anterior descending coronary artery (LAD) occlusion. In four dogs, we measured the pressure difference between the LV apex and outflow tract. At baseline, peak early diastolic flow velocities in the distal one-third of the LV were directed toward apex (9.2 ± 1.6 cm/s). After LAD occlusion, the velocities reversed (−2.3 ± 0.4 cm/s, P < 0.01), indicating that blood was ejected from the apex toward the base during early filling. This interpretation was confirmed by wall motion analysis, which showed postsystolic shortening of apical myocardial segments. The postsystolic shortening represented 9.7 ± 1.7% ( P < 0.01) and 14.2 ± 2.4% ( P < 0.01) of end-diastolic segment length by SDE and sonomicrometry, respectively. Consistent with the velocity changes, we found reversal of the early diastolic pressure gradient from the LV apex to outflow tract. In the present model, acute LAD occlusion resulted in reversal of early diastolic apical flow, and this was attributed to postsystolic shortening of dyskinetic apical segments. The clinical diagnostic importance of this finding remains to be determined.
Oxygen tension distribution in postcapillary venules in resting skeletal muscle
Authors: Saltzman Darin J. , Toth Andras , Tsai Amy G. , Intaglietta Marcos , Johnson Paul C.
Published: March 2015

We tested the hypothesis that blood flow is distributed among capillary networks in resting skeletal muscle in such a manner as to maintain uniform end-capillary Po2. Oxygen tension in venules draining two to five capillaries was obtained by using the phosphorescence decay methodology in rat spinotrapezius muscle. For 64 postcapillary venules among 18 networks in 10 animals, the mean Po2was 30.1 Torr (range, 9.7–43.5 Torr) with a coefficient of variation (CV; standard deviation/mean) of 0.26. Oxygen levels of postcapillary venules within a single network or single animal, however, displayed a much smaller CV (0.064 and 0.094, respectively). By comparison, the CV of blood flow in 57 postcapillary venules of 17 networks in 9 animals was 1.27 with a mean flow of 0.011 ± 0.014 nl/s and a range of 3.7 × 10–4to 6.5 × 10–2nl/s. Blood flow of postcapillary venules within single networks displayed a lower CV (mean, 0.51), whereas that in individual animals was 0.78. Results indicate that among venular networks, heterogeneity of oxygen tension is less than that of blood flow and within venular networks the heterogeneity of oxygen tension is much less than that of blood flow. In addition, postcapillary Po2was independent of flow among venules in which both were measured. Results of this study may be attributable to three factors: 1) O2diffusion between adjacent capillaries and venules, 2) structural remodeling in regions of lower Po2, and 3) O2-dependent local control mechanisms.
Endothelium-dependent arterial wall tone elasticity modulated by blood viscosity
Authors: Cabrera Fischer Edmundo I. , Armentano Ricardo L. , Pessana Franco M. , Graf Sebastián , Romero Luis , Christen Alejandra I. , Simon Alain , Levenson Jaime
Published: March 2015

The role of blood viscosity on arterial wall elasticity before and after deendothelization (DE) was studied. Seven ovine brachiocephalic arteries were studied in vitro under physiological pulsatile flow conditions achieved by a mock circulation loop. Instantaneous pressure and diameter signals were assessed in each arterial segment. Incremental elastic modulus ( Einc) was calculated using the slope of the pure elastic stress-strain relationship. There was no significant difference between Eincvalues before and after DE (3.11 vs. 3.16 107dyn/cm2) at a blood viscosity of 2.00 mPa · s. Increases in blood viscosity (2.50, 3.00, 3.50, and 4.00 mPa · s) always resulted in decreases of Eincbefore DE; inversely, increases in blood viscosity resulted in increases of Eincafter DE. These values of Einc, for identical levels of blood viscosity, were always significantly lower ( P< 0.05) before DE than those obtained after DE. Arterial wall elasticity assessed through Eincwas strongly influenced by blood viscosity, probably due to presence or absence of endothelium relaxing factors or to direct shear smooth muscle activation when endothelial cells are removed.
Molecular basis of species-specific expression of repolarizing K+currents in the heart
Authors: Zicha Stephen , Moss Isaac , Allen Bruce , Varro Andras , Papp Julius , Dumaine Robert , Antzelevich Charles , Nattel Stanley
Published: March 2015

There are important species-specific differences in K+current profiles and arrhythmia susceptibility, but interspecies comparisons of K+channel subunit expression are lacking. We quantified voltage-gated K+channel (Kv) subunit mRNA and protein in rabbits, guinea pigs, and humans. Kv1.4, Kv4.2, and Kv4.3 mRNA was present in rabbits but undetectable in guinea pigs. MinK mRNA concentration in guinea pigs was almost threefold greater versus humans and 20-fold versus rabbits. MinK protein expression in guinea pigs was almost twofold that in humans and sixfold that in rabbits. KvLQT1 mRNA concentration was greatest in humans, and protein expression in humans was increased by ∼2- and ∼7-fold compared with values in rabbits and guinea pigs, respectively. The ether-a-go-go-related gene (ERG1) mRNA was more concentrated in humans, but ERG1 protein expression could not be compared across species because of epitope sequence differences. We conclude that important interspecies differences in cardiac K+channel subunit expression exist and may contribute to the following: 1) lack of a transient outward current in the guinea pig (α-subunit transcription absent in the guinea pig heart); 2) small slow delayed rectifier current and torsades de pointes susceptibility in the rabbit (low-level minK expression); and 3) large slow component of the delayed rectifier current in the guinea pig (strong minK expression).
Validation of formamide as a detubulation agent in isolated rat cardiac cells
Authors: Brette Fabien , Komukai Kimiaki , Orchard Clive H.
Published: March 2015

Kawai M, Hussain M, and Orchard CH. Am J Heart Circ Physiol 277: H603–H609, 1999 developed a technique to detubulate rat ventricular myocytes using formamide and showed that detubulation results in a decrease in cell capacitance, Ca2+ current density, and Ca2+ transient amplitude. We have investigated the mechanism of this detubulation and possible direct effects of formamide. Staining ventricular cells with di-8-ANEPPS showed that the t tubule membranes remain inside the cell after detubulation; trapping of FITC-labeled dextran within the t tubules showed that detubulation occurs during formamide washout and that the t tubules appear to reseal within the cell. Detubulation had no effect on the microtubule network but resulted in loss of synchronous Ca2+ release on electrical stimulation. In contrast, formamide treatment of atrial cells did not significantly change cell capacitance, Ca2+ current amplitude, action potential configuration, the Ca2+ transient or the response of the Ca2+ transient to isoprenaline. We conclude that formamide washout induces detubulation of single rat ventricular myocytes, leaving the t tubules within the cell, but without direct effects on cell proteins that might alter cell function.
Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression
Authors: Nunamaker Elizabeth A. , Zhang Hai-Ying , Shirasawa Yuichi , Benoit Joseph N. , Dean David A.
Published: March 2015

The development of inexpensive and effective approaches to transiently decrease gene expression in vivo would be useful for the study of physiological processes in living animals. DNAzymes are a novel class of DNA oligonucleotides that can catalytically cleave target mRNAs and thereby reduce protein production. However, current methods for their delivery in vivo are limited and inefficient. In this study, we show that electroporation can be used to deliver DNAzymes to the intact mesenteric vasculature of rats. With the use of PKC-ϵ as a target, a set of wild-type and mutant control DNAzymes was designed and shown to reduce both PKC-ϵ mRNA and protein levels in cultured smooth muscle cells in a specific manner. The wild-type DNAzyme reduced PKC-ϵ protein levels by 70% at 24 h in two different cell lines without decreasing the levels of the five other PKC isoforms tested. When delivered to the intact vasculature using electroporation, the DNAzyme reduced PKC-ϵ protein levels by >60% without affecting these other PKC isoforms. Electroporation was required for oligonucleotide transfer and was able to deliver the DNAzymes to multiple cell layers in the vessel wall. Protein levels were reduced maximally by 24 h postelectroporation and returned to normal by 48 h. These results suggest that electroporation can be used to deliver DNAzymes and other DNA oligonucleotides to the vasculature in vivo and can decrease gene expression for a window of time that can be used for experimental studies.
Adenoviral-mediated overexpression of catalase inhibits endothelial cell proliferation
Authors: Zanetti Michela , Katusic Zvonimir S. , O'Brien Timothy
Published: March 2015

Although hydrogen peroxide (H2O2) induces proliferation of vascular smooth muscle cells, its role in endothelial cell proliferation is unclear. Our aim was to study the role of hydrogen peroxide in endothelial cell proliferation by overexpressing catalase. Human aortic endothelial cells were transduced with adenoviral vectors encoding β-galactosidase (Adβgal) or catalase (AdCat) or were exposed to diluent alone (control). Transgene expression was demonstrated by β-galactosidase staining, Western analysis, and significantly increased enzyme activity in AdCat-transduced cells. Overexpression of catalase decreased DNA synthesis in AdCat compared with control and Adβgal-transduced cells (536.8 ± 31 vs. 1,875.1 ± 132.9 vs. 1,347.5 ± 93.7 dpm/well, respectively; P < 0.05 vs. control and Adβgal). Six days after transduction with AdCat (multiplicity of infection = 50), cell numbers were significantly reduced (AdCat: 38 ± 1.8% of cell counts in control, P < 0.05; and 45 ± 2% of cell count in Adβgal, P < 0.05). Incubation with aminotriazole 10 mmol/l, an inhibitor of catalase, prevented this effect. The number of apoptotic cells was increased one- and threefold 2 and 4 days, respectively, after transduction with AdCat. Exogenous administration of low concentrations of H2O2(50 μM) significantly increased cell proliferation, whereas it was inhibited by higher concentrations. These results suggest that H2O2is an important modulator of endothelial cell proliferation.
Inflammatory stress increases receptor for lysophosphatidylcholine in human microvascular endothelial cells
Authors: Lum Hazel , Qiao Jing , Walter Robert J. , Huang Fei , Subbaiah Papasani V. , Kim Kwang S. , Holian Oksana
Published: March 2015

The atherogenic serum lysophosphatidylcholine (LPC) is known to mediate vascular endothelial responses ranging from upregulation of adhesion molecules and growth factors to secretion of chemokines and superoxide anion. We investigated whether endothelial cells express receptors for LPC, which may account for their actions. Human brain microvascular (HBMEC) and dermal microvascular endothelial cells (HMEC) were prepared for RT-PCR analysis for possible expression of the G protein-coupled receptors, GPR4 and G2A, which are believed to be specific LPC receptors. Results indicated that HBMEC expressed low basal GPR4 mRNA, but stimulation with tumor necrosis factor-α (TNF-α) (100 U/ml) or H2O2 (50 μmol/l) for 2 h or overnight upregulated expression severalfold. In contrast, HMEC expressed high basal GPR4 mRNA, which was not further increased by either TNF-α or H2O2 stimulation. Another LPC receptor, G2A, was not detected in either endothelial cell type. Competition binding studies were made to evaluate specific binding of [3H]LPC to the intact endothelial cell monolayer. Basal specific [3H]LPC binding in HBMEC was approximately eight times lower than in HMEC; however, TNF-α or H2O2 stimulation increased [3H]LPC binding on HMBEC but not HMEC. The results indicated that GPR4 expression was consistent with specific [3H]LPC binding. Overall, we report that endothelial cells selectively expressed GPR4, a specific LPC receptor. Furthermore, GPR4 expression by HBMEC, but not HMEC, was increased by inflammatory stresses. We conclude that endogenous GPR4 in endothelial cells may be a potential G protein-coupled receptor by which LPC signals proinflammatory activities.
NOS inhibition accelerates atherogenesis: reversal by exercise
Authors: Niebauer Josef , Maxwell Andrew J. , Lin Patrick S. , Wang David , Tsao Philip S. , Cooke John P.
Published: March 2015

In this study, we assessed the effects of chronic exercise training (12 wk) on atherosclerotic lesion formation in hypercholesterolemic apolipoprotein E-deficient mice ( n = 31). At the age of 9 wk, mice were assigned to the following groups: sedentary (Sed; n = 9); exercise (Ex; n = 12); sedentary and oral NG-nitro-l-arginine (l-NNA, Sed-NA; n = 4), or exercise and oral l-NNA (Ex-NA; n = 6). Chronic exercise training was performed on a treadmill for 12 wk (6 times/wk and twice for 1 h/day) at a final speed of 22 m/min, and an 8° grade. l-NNA was discontinued 5 days before final treadmill testing. The farthest distance run to exhaustion was observed in Ex-NA mice (Sed: 306 ± 32 m; Ex: 640 ± 87; Sed-NA: 451 ± 109 m; Ex-NA: 820 ± 49 m; all P < 0.05). Lesion formation was assessed in the proximal ascending aorta by dissection microscopy after oil red O staining. The aortas of Sed-NA mice manifested a threefold increase in lesion formation compared with the other groups. This l-NNA-induced lesion formation was reduced by chronic exercise training (Sed, 786 ± 144; Ex, 780 ± 206; Sed-NA, 2,147 ± 522; Ex-NA, 851 ± 253; Sed-NA vs. all other groups: P < 0.001). In conclusion, treatment with oral l-NNA (an nitric oxide synthase antagonist) leads to accelerated atherogenesis in genetically determined hypercholesterolemic mice. This adverse effect can be overcome by chronic exercise training.
Preservation of ischemia and isoflurane-induced preconditioning after brain death in rabbit hearts
Authors: Chiari Pascal , Piriou Vincent , Hadour Guylaine , Rodriguez Claire , Loufouat Joseph , Lehot Jean-Jacques , Ovize Michel , Ferrera René
Published: March 2015

We sought to determine whether brain death-induced catecholamine release preconditions the heart, and if not, whether it precludes further protection by repetitive ischemia or isoflurane. Anesthetized rabbits underwent 30 min of coronary occlusion and 4 h of reperfusion. The effect on infarct size of either no intervention (controls), ischemic preconditioning (IPC), or isoflurane inhalation (Iso) was evaluated with or without previous brain death (BD) induced by subdural balloon inflation. Plasma catecholamine levels were measured at several time points. Although it dramatically increase plasma catecholamine levels, BD failed to reduce infarct size that averaged 0.49 ± 0.34 without BD versus 0.45 ± 0.27 g with BD. IPC and Iso, alone as well as after BD, significantly reduced infarct size that averaged 0.11 ± 0.04, 0.21 ± 0.15, 0.10 ± 0.09, and 0.22 ± 0.10 g in IPC, Iso, BD + IPC, and BD + Iso groups, respectively (means ± SD, P < 0.05 vs. controls). BD-induced catecholamines “storm” does not precondition the rabbit heart that however retains the ability to be protected by repetition of brief ischemia or isoflurane inhalation.
Synchronization of ventricular fibrillation with real-time feedback pacing: implication to low-energy defibrillation
Authors: Pak Hui-Nam , Liu Yen-Bin , Hayashi Hideki , Okuyama Yuji , Chen Peng-Sheng , Lin Shien-Fong
Published: March 2015

Wavefront synchronization is an important aspect preceding the termination of ventricular fibrillation (VF). We evaluated the defibrillation efficacy of a novel multisite pacing algorithm using optical recording-guided synchronized pacing (SyncP) in the excitable gaps. We compared the effects of SyncP with traditional overdrive pacing (ODP) at 90% of the VF cycle length (VFCL) and high-frequency pacing (HFP; 43–215 Hz) on spontaneous VF termination in isolated rabbit hearts. For SyncP, the pacing current was triggered by the activation of a reference site and was delivered when the optical potential of the pacing site was in an excitable gap. We measured VFCL and the spatial dispersion of VFCL (SDCL) from five points (3 points in the paced area and 2 points in the nonpaced area) and the distribution of phase singularities during the prepacing, pacing, and postpacing periods. The results showed that 1) the VF termination rate of SyncP (16.0%, n = 106) was higher than that of ODP (2.1%, n = 48, P < 0.01) or HFP (1.6%, n = 129, P < 0.0001); 2) energy consumption for SyncP (7.6 ± 9.3 mJ) was significantly lower than that of ODP (14.0 ± 14.8 mJ, P < 0.0001); and 3) SyncP, but not ODP or HFP, decreased SDCL in the paced area during the pacing ( P < 0.01) and postpacing ( P < 0.05) periods compared with the prepacing period. We conclude that SyncP is effective in inducing wavefront synchronization and is more effective at facilitating spontaneous VF termination than non-SyncP.
Peroxynitrite triggers a delayed resistance of coronary endothelial cells against ischemia-reperfusion injury
Authors: Laude Karine , Thuillez Christian , Richard Vincent
Published: March 2015

Experiments were designed to test whether nitric oxide (NO) and peroxynitrite trigger delayed coronary endothelial protection induced by preconditioning (PC) in rats. Prolonged ischemia reperfusion markedly reduced the response of isolated coronary arteries to acetylcholine, and this was prevented by PC performed 24 h earlier. The NO synthase (NOS) inhibitor N G-nitro-l-arginine methyl ester (l-NAME) administered during PC abolished its delayed endothelial protective effect, whereas the inducible NOS inhibitor N-(3(aminomethyl)benzyl)acetaminide had no effect. Delayed endothelial PC was also abolished by the peroxynitrite scavengers selenomethionine or uric acid given during PC. In parallel, the NO/peroxynitrite donor S-morpholinosydnonimine and authentic peroxynitrite, administered 24 h before prolonged ischemia-reperfusion mimicked endothelial PC, whereas the NO donor S-nitroso- N-acetylpencillamine had no effect. This suggests that peroxynitrite is an essential trigger of the delayed coronary endothelial protection induced by PC in rat hearts.
Downregulation of hypoxic vasoconstriction by chronic hypoxia in rabbits: effects of nitric oxide
Authors: Weissmann Norbert , Nollen Matthias , Gerigk Boris , Ghofrani Hossein Ardeschir , Schermuly Ralph Theo , Günther Andreas , Quanz Karin , Fink Ludger , Hänze Jörg , Rose Frank , Seeger Werner , Grimminger Friedrich
Published: March 2015

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation for optimizing pulmonary gas exchange. Chronic alveolar hypoxia results in vascular remodeling and pulmonary hypertension. Previous studies have reported conflicting results of the effect of chronic alveolar hypoxia on pulmonary vasoreactivity and the contribution of nitric oxide (NO), which may be related to species and strain differences as well as to the duration of chronic hypoxia. Therefore, we investigated the impact of chronic hypoxia on HPV in rabbits, with a focus on lung NO synthesis. After exposure of the animals to normobaric hypoxia (10% O2) for 1 day to 10 wk, vascular reactivity was investigated in ex vivo perfused normoxic ventilated lungs. Chronic hypoxia induced right heart hypertrophy and increased normoxic vascular tone within weeks. The vasoconstrictor response to an acute hypoxic challenge was strongly downregulated within 5 days, whereas the vasoconstrictor response to the thromboxane mimetic U-46619 was maintained. The rapid downregulation of HPV was apparently not linked to changes in the lung vascular NO system, detectable in the exhaled gas and by pharmacological blockage of NO synthesis. Treatment of the animals with long-term inhaled NO reduced right heart hypertrophy and partially maintained the reactivity to acute hypoxia, without any impact on the endogenous NO system being noted. We conclude that chronic hypoxia causes rapid downregulation of acute HPV as a specific event, preceding the development of major pulmonary hypertension and being independent of the lung vascular NO system. Long-term NO inhalation partially maintains the strength of the hypoxic vasoconstrictor response.
Mechanical force regulation of myofibroblast differentiation in cardiac fibroblasts
Authors: Wang J. , Chen H. , Seth A. , McCulloch C. A.
Published: March 2015

The myocardium responds to chronic pressure or volume overload by activation and proliferation of cardiac fibroblasts and their differentiation into myofibroblasts. Because α-smooth muscle actin (SMA) expression is the classical marker for myofibroblast differentiation, we examined force-induced SMA expression and regulation by specific MAPK pathways. Rat cardiac fibroblasts were separated from myocytes and smooth muscle cells, cultured, and phenotyped by using the presence of SMA, vimentin, and ED-A fibronectin and the absence of desmin as myofibroblast markers. Static tensile forces (0.65 pN/μm2) were applied to fibroblasts via collagen-coated magnetite beads. In neonatal cardiac fibroblasts cultured for 1 day, immunostaining and Western and Northern blotting showed very low basal levels of SMA. After the application of force, there were 1.5- to 2-fold increases of SMA protein and mRNA within 4 h. Force-induced SMA expression was dependent on ERK phosphorylation and on intact actin filaments. In contrast to cells cultured for 1 day, cells grown for 3 days on rigid substrates showed prominent stress fibers and high basal levels of SMA, which were reduced by 32% within 4 h after force application. ERK was not activated by force, but p38 phosphorylation was required for force-induced inhibition of SMA expression. These results indicate that mechanical force-induced regulation of SMA content is dependent on myofibroblast differentiation and by selective activation of MAPKs.