bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental changes of intracellular Ca2+transients in beating rat hearts
Authors: Escobar Ariel L. , Ribeiro-Costa Roberta , Villalba-Galea Carlos , Zoghbi María Elena , Pérez Claudia G. , Mejía-Alvarez Rafael
Published: February 2004

Postnatal maturation of the rat heart is characterized by major changes in the mechanism of excitation-contraction (E-C) coupling. In the neonate, the t tubules and sarcoplasmic reticulum (SR) are not fully developed yet. Consequently, Ca2+-induced Ca2+release (CICR) does not play a central role in E-C coupling. In the neonate, most of the Ca2+that triggers contraction comes through the sarcolemma. In this work, we defined the contribution of the sarcolemmal Ca2+entry and the Ca2+released from the SR to the Ca2+transient during the first 3 wk of postnatal development. To this end, intracellular Ca2+transients were measured in whole hearts from neonate rats by using the pulsed local field fluorescence technique. To estimate the contribution of each Ca2+flux to the global intracellular Ca2+transient, different pharmacological agents were used. Ryanodine was applied to evaluate ryanodine receptor-mediated Ca2+release from the SR, nifedipine for dihydropyridine-sensitive L-type Ca2+current, Ni2+for the current resulting from the reverse-mode Na+/Ca2+exchange, and mibefradil for the T-type Ca2+current. Our results showed that the relative contribution of each Ca2+flux changes considerably during the first 3 wk of postnatal development. Early after birth (1–5 days), the sarcolemmal Ca2+flux predominates, whereas at 3 wk of age, CICR from the SR is the most important. This transition may reflect the progressive development of the t tube-SR units characteristic of mature myocytes. We have hence directly defined in the whole beating heart the developmental changes of E-C coupling previously evaluated in single (acutely isolated or cultured) cells and multicellular preparations.
Role of cyclooxygenase in ventricular effects of adrenomedullin: is adrenomedullin a double-edged sword in sepsis?
Authors: Mittra Shivani , Hyvelin Jean-Marc , Shan Qixian , Tang Fai , Bourreau Jean-Pierre
Published: February 2004

Adrenomedullin (ADM) is upregulated in cardiac tissue under various pathophysiological conditions. However, the direct inotropic effect of ADM on normal and compromised cardiomyocytes is not clear. In rat ventricular myocytes, ADM produced an initial (<30 min) increase in cell shortening and Ca2+ transient and, on prolonged incubation (>1 h), a marked decrease in cell shortening and Ca2+ transient. Both effects were sensitive to inhibition by the ADM antagonist ADM-(22–52). The increase and decrease in cell shortening and Ca2+ transient were attenuated by pretreatment with indomethacin [a nonspecific cyclooxygenase (COX) inhibitor], nimesulide and SC-236 (specific COX-2 inhibitors), and tranylcypromine (a prostacyclin synthase inhibitor); SQ-29548 (a thromboxane receptor antagonist) was without effect. Cells isolated from LPS-treated rats that were in the late, hypodynamic phase of septic shock also showed a marked decrease in cell shortening and Ca2+ transient. Because ADM is overexpressed in sepsis, we repeated the above protocol in cells isolated from LPS-treated rats. At 4 h after LPS injection, ADM levels markedly increased in plasma, ventricles, and freshly isolated ventricular myocytes. Decreases in cell shortening and Ca2+ transient in LPS-treated cells were reversed by pretreatment with ADM-(22–52). Anti-ADM (rat) IgG also reversed the decrease in cell shortening and other parameters of cell kinetics. Indomethacin, SC-236, and tranylcypromine restored cell contractility and the decrease in Ca2+ transient, whereas SQ-29548 had no effect, implying that prostacyclin played a role in both effects. However, with regard to cell-shortening kinetics, indomethacin and SQ-29548 decreased the amount of time taken by the cells to return to baseline, whereas SC-236 and tranylcypromine did not, implying that not only prostacyclin, but also thromboxane, is involved. The results indicate that ADM interacts with COX to yield prostanoids, which mediate its negative inotropic effect in LPS-treated rat ventricular myocytes.
Urotensin II causes fatal circulatory collapse in anesthesized monkeys in vivo: a “vasoconstrictor” with a unique hemodynamic profile
Authors: Zhu Yi Zhun , Wang Zhong Jing , Zhu Yi Chun , Zhang Li , Oakley Reida M. E. , Chung Chin Wee , Lim Kiat Wee , Lee How Sung , Ozoux Marie L. , Linz Wolfgang , Böhm Michael , Kostenis Evi
Published: February 2004

Urotensin II (UII) is a vasoactive peptide that has recently emerged as a likely contributor to cardiovascular physiology and pathology. Acute infusion of UII into nonhuman primates results in circulatory collapse and death; however, the exact cause of death is not well understood. This study was undertaken to elucidate the mechanism underlying the fatal cardiovascular event on UII application in vivo in nonhuman primates. To this end, cynomolgus monkeys ( n = 4) were anesthetized and tracheal intubation was performed. One internal jugular vein was cannulated for administration of drugs, and one femoral artery for recording of blood pressure and heart rate using a transonic pressure transducer. Cardiac parameters were not significantly changed after administration of 0.003 nmol/kg human UII. A bolus of human UII (0.03 nmol/kg) caused a decrease of heart rate (HR) (13%), mean blood pressure (MBP) (18%), and first-order derivative of left ventricular pressure (dP/d t) (11%). Carotid and coronary blood flow were reduced by 9% and 7%, respectively; 0.3 nmol/kg of human UII resulted in a further reduction of HR (50.3%), MBP (65%), dP/d t (45%), carotid (38%), and coronary blood flow (30%), ultimately leading to cardiovascular breakdown and death. Pulmonary pressure, however, was increased by 30%. Plasma histamine levels were found to be unaffected by administration of UII. Our results indicate that systemic administration of human UII has negative inotropic and chronotropic effects and reduces total peripheral resistance ultimately leading to severe myocardial depression, pulmonary hypertension, and fatal circulation collapse in nonhuman primates. We suggest that successful design of UII antagonists might offer a new therapeutic principle in treating cardiovascular diseases.
Impact of acute hypoxic pulmonary hypertension on LV diastolic function in healthy mountaineers at high altitude
Authors: Allemann Yves , Rotter Martin , Hutter Damian , Lipp Ernst , Sartori Claudio , Scherrer Urs , Seiler Christian
Published: February 2004

In pulmonary hypertension right ventricular pressure overload leads to abnormal left ventricular (LV) diastolic function. Acute high-altitude exposure is associated with hypoxia-induced elevation of pulmonary artery pressure particularly in the setting of high-altitude pulmonary edema. Tissue Doppler imaging (TDI) allows assessment of LV diastolic function by direct measurements of myocardial velocities independently of cardiac preload. We hypothesized that in healthy mountaineers, hypoxia-induced pulmonary artery hypertension at high altitude is quantitatively related to LV diastolic function as assessed by conventional and TDI Doppler methods. Forty-one healthy subjects (30 men and 11 women; mean age 41 ± 12 yr) underwent transthoracic echocardiography at low altitude (550 m) and after a rapid ascent to high altitude (4,559 m). Measurements included the right ventricular to right atrial pressure gradient (ΔPRV-RA), transmitral early ( E) and late ( A) diastolic flow velocities and mitral annular early ( Em) and late ( Am) diastolic velocities obtained by TDI at four locations: septal, inferior, lateral, and anterior. At a high altitude, ΔPRV-RA increased from 16 ± 7to44 ± 15 mmHg ( P < 0.0001), whereas the transmitral E-to- A ratio ( E/ A ratio) was significantly lower (1.11 ± 0.27 vs. 1.41 ± 0.35; P < 0.0001) due to a significant increase of A from 52 ± 15 to 65 ± 16 cm/s ( P = 0.0001). ΔPRV-RA and transmitral E/ A ratio were inversely correlated ( r2 = 0.16; P = 0.0002) for the whole spectrum of measured values (low and high altitude). Diastolic mitral annular motion interrogation showed similar findings for spatially averaged (four locations) as well as for the inferior and septal locations: Am increased from low to high altitude (all P < 0.01); consequently, Em/ Am ratio was lower at high versus low altitude (all P < 0.01). These intraindividual changes were reflected interindividually by an inverse correlation between ΔPRV-RA and Em/ Am (all P < 0.006) and a positive association between ΔPRV-RA and Am (all P < 0.0009). In conclusion, high-altitude exposure led to a two- to threefold increase in pulmonary artery pressure in healthy mountaineers. This acute increase in pulmonary artery pressure led to a change in LV diastolic function that was directly correlated with the severity of pulmonary hypertension. However, in contrast to patients suffering from some form of cardiopulmonary disease and pulmonary hypertension, in these healthy subjects, overt LV diastolic dysfunction was not observed because it was prevented by augmented atrial contraction. We propose the new concept of compensated diastolic (dys)function.
Neuropeptide Y enhances permeability across a rat aortic endothelial cell monolayer
Authors: Nan Yong-Shan , Feng Guo-Gang , Hotta Yoshihiro , Nishiwaki Kimitoshi , Shimada Yasuhiro , Ishikawa Atsuko , Kurimoto Nakako , Shigei Tatsuro , Ishikawa Naohisa
Published: February 2004

Previously, in vivo studies showed that neuropeptide Y (NPY) elevates vascular permeability in isolated lung perfusion preparations, possibly through binding to the NPY Y3receptor. The present study used monolayers in a double-chamber culture method under conditions of normoxia (5% CO2-20% O2-75% N2) or hypoxia (5% CO2-5% O2-90% N2) to test the hypothesis that NPY directly affects rat aortic endothelial cells (RAECs). RAECs were cultured on the base of the upper chamber, into which FITC-labeled albumin was introduced, and permeation into the lower chamber was measured. The RAEC monolayer was treated with 10–8–3 × 10–7M NPY for 2 h in normoxia or hypoxia. In hypoxia, NPY concentration dependently increased the permeability of the RAEC monolayer, whereas in normoxia no significant change was observed. Peptide YY, NPY Y1, and NPY Y2receptor agonists and NPY Y1receptor antagonist exerted no significant effects under hypoxic conditions. NPY-(18–36), an NPY Y3receptor antagonist, elicited an inhibitory action on the NPY-induced increase in monolayer permeability. Furthermore, neither N-monomethyl-l-arginine, a nitric oxide synthase inhibitor, the bradykinin B2receptor antagonist FK-3657, nor the vascular endothelial growth factor receptor-coupled tyrosine kinase inhibitor tyrphostin SU-1498, injected into the medium of the upper chamber, affected the NPY-induced permeability changes under hypoxic conditions. The results suggest that the NPY-induced increase in permeability across the RAEC monolayer is closely related to low O2tension, possibly mediated by direct action on the NPY Y3receptor expressed on the endothelial cell membrane. Furthermore, this NPY-induced increase is not likely due to nitric oxide, bradykinin, or vascular endothelial growth factor.
Cardioprotective effects of ingliforib, a novel glycogen phosphorylase inhibitor
Authors: Tracey W. Ross , Treadway Judith L. , Magee William P. , Sutt Jill C. , McPherson R. Kirk , Levy Carolyn B. , Wilder Donald E. , Yu Li J. , Chen Yue , Shanker Ravi M. , Mutchler Alison K. , Smith Andrew H. , Flynn David M. , Knight Delvin R.
Published: February 2004

Interventions such as glycogen depletion, which limit myocardial anaerobic glycolysis and the associated proton production, can reduce myocardial ischemic injury; thus it follows that inhibition of glycogenolysis should also be cardioprotective. Therefore, we examined whether the novel glycogen phosphorylase inhibitor 5-Chloro- N-{(1 S,2 R)-3-[(3 R,4 S)-3,4-dihydroxy-1-pyrrolidinyl)]-2-hydroxy-3-oxo-1-(phenylmethyl)propyl}-1H-indole-2-carboxamide (ingliforib; CP-368,296) could reduce infarct size in both in vitro and in vivo rabbit models of ischemia-reperfusion injury (30 min of regional ischemia, followed by 120 min of reperfusion). In Langendorff-perfused hearts, constant perfusion of ingliforib started 30 min before regional ischemia and elicited a concentration-dependent reduction in infarct size; infarct size was reduced by 69% with 10 μM ingliforib. No significant drug-induced changes were observed in either cardiac function (heart rate, left ventricular developed pressure) or coronary flow. In open-chest anesthetized rabbits, a dose of ingliforib (15 mg/kg loading dose; 23 mg·kg–1·h–1infusion) selected to achieve a free plasma concentration equivalent to an estimated EC50in the isolated hearts (1.2 μM, 0.55 μg/ml) significantly reduced infarct size by 52%, and reduced plasma glucose and lactate concentrations. Furthermore, myocardial glycogen phosphorylase a and total glycogen phosphorylase activity were reduced by 65% and 40%, respectively, and glycogen stores were preserved in ingliforib-treated hearts. No significant change was observed in mean arterial pressure or rate-pressure product in the ingliforib group, although heart rate was modestly decreased postischemia. In conclusion, glycogen phosphorylase inhibition with ingliforib markedly reduces myocardial ischemic injury in vitro and in vivo; this may represent a viable approach for both achieving clinical cardioprotection and treating diabetic patients at increased risk of cardiovascular disease.
Evidence of myofibrillar protein oxidation induced by postischemic reperfusion in isolated rat hearts
Authors: Canton Marcella , Neverova Irina , Menabò Roberta , Van Eyk Jennifer , Di Lisa Fabio
Published: February 2004

Although the contribution of reactive oxygen species to myocardial ischemia is well recognized, the possible intracellular targets, especially at the level of myofibrillar proteins (MP), are not yet fully characterized. To assess the maximal extent of oxidative degradation of proteins, isolated rat hearts were perfused with 1 mM H2O2. Subsequently, the MP maximally oxidative damage was compared with the effects produced by 1) 30 min of no-flow ischemia (I) followed in other hearts by 3 min of reperfusion (I/R); and 2) I/R in the presence of a potent antioxidant N-(2-mercaptopropionyl)glycine (MPG). Samples from the H2O2group electrophoresed under nonreducing conditions and probed with actin, desmin, or tropomyosin monoclonal antibodies showed high-molecular mass complexes indicative of disulfide cross-bridges along with splitting and thickening of tropomyosin and actin bands, respectively. Only these latter changes could be detected in I/R samples and were prevented by MPG. Carbonyl groups generated by oxidative stress on MP were detected by Western blot analysis (oxyblot) under optimized conditions. The analyses showed one major band corresponding to oxidized actin, the density of which increased 1.2-, 2.8-, and 6.8-fold in I, I/R, and H2O2groups, respectively. The I/R-induced increase was significantly reduced by MPG. In conclusion, oxidative damage of MP occurs on reperfusion, although at a lower extent than in H2O2perfused hearts, whereas oxidative modifications could not be detected in ischemic hearts. Furthermore, the inhibition of MP oxidation by MPG might underlie the protective efficacy of antioxidants.
Roles for αB-crystallin and HSPB2 in protecting the myocardium from ischemia-reperfusion-induced damage in a KO mouse model
Authors: Morrison Lisa E. , Whittaker Ross J. , Klepper Robert E. , Wawrousek Eric F. , Glembotski Christopher C.
Published: February 2004

Overexpression studies have shown that the small heat shock proteins (sHSP) protect the myocardium from ischemia-reperfusion (I/R)-induced damage. However, gene deletion studies are necessary to demonstrate whether sHSPs are required for protection. The genes for αB-crystallin (αBC) and HSPB2, two sHSPs that are expressed in high levels in the heart, are in close proximity to one another; as a result, both genes were disrupted in a recently generated knockout (KO) mouse line. The αBC/HSPB2 KO mouse line is currently the only model that features disruption of sHSPs normally expressed in the heart. Accordingly, we examined the cardiac morphology, function, and response to I/R-induced stress in αBC-HSPB2 KO mice. Initial gross, light microscopic and echocardiographic characterization showed that the morphological and functional properties of hearts from adult KO mice were indistinguishable from age-matched wild-type (WT) mice. Electron microscopy showed that, compared with WT mouse hearts, KO mouse heart sarcomeres were relatively normal. Isolated perfused KO mouse hearts displayed normal contractility; however, when compared with WT, after I/R, KO mouse hearts exhibited a twofold reduction in contractile recovery, as well as increased necrosis and apoptosis. Additionally, when compared with WT, KO mouse hearts exhibited 43% less reduced glutathione, which is known to protect from I/R-induced damage. Thus, whereas neither αBC nor HSPB2 is essential for myocardial development and function under nonstressful conditions, one or both are required for maximal functional recovery and protection from I/R-induced necrosis and apoptosis.
Accelerated onset of heart failure in mice during pressure overload with chronically decreased SERCA2 calcium pump activity
Authors: Schultz Jo El J. , Glascock Betty J. , Witt Sandra A. , Nieman Michelle L. , Nattamai Kalpana J. , Liu Lynne H. , Lorenz John N. , Shull Gary E. , Kimball Thomas R. , Periasamy Muthu
Published: February 2004

We recently developed a mouse model with a single functional allele of Serca2 ( Serca2+/–) that shows impaired cardiac contractility and relaxation without overt heart disease. The goal of this study was to test the hypothesis that chronic reduction in sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2 levels in combination with an increased hemodynamic load will result in an accelerated pathway to heart failure. Age-matched wild-type and Serca2+/– mice were subjected to 10 wk of pressure overload via transverse aortic coarctation surgery. Cardiac hypertrophy and heart failure were assessed by echocardiography, gravimetry/histology, hemodynamics, and Western blotting analyses. Our results showed that ∼64% of coarcted Serca2+/– mice were in heart failure compared with 0% of coarcted wild-type mice ( P < 0.05). Overall, morbidity and mortality were greatly increased in Serca2+/– mice under pressure overload. Echocardiography assessment revealed a significant increase in left ventricular (LV) mass, and LV hypertrophy in coarcted Serca2+/– mice converted from a concentric to an eccentric pattern, similar to that seen in human heart failure. Coarcted Serca2+/– mice had decreased contractile/systolic and relaxation/diastolic performance and/or function compared with coarcted wild-type mice ( P < 0.05), despite a similar duration and degree of pressure overload. SERCA2a protein levels were significantly reduced (>50%) in coarcted Serca2+/– mice compared with noncoarcted and coarcted wild-type mice. Our findings suggest that reduction in SERCA2 levels in combination with an increased hemodynamic load results in an accelerated pathway to heart failure.
ROS are required for rapid reactivation of Na+/Ca2+exchanger in hypoxic reoxygenated guinea pig ventricular myocytes
Authors: Eigel B. N. , Gursahani H. , Hadley R. W.
Published: February 2004

The cardiac Na+/Ca2+exchanger (NCX) contributes to cellular injury during hypoxia, as its altered function is largely responsible for a rise in cytosolic Ca2+concentration ([Ca2+]i). In addition, the NCX in guinea pig ventricular myocytes undergoes profound inhibition during hypoxia and rapid reactivation during reoxygenation. The mechanisms underlying these changes in NCX activity are likely complex due to the participation of multiple inhibitory factors including altered cytosolic Na+concentration, pH, and ATP. Our main hypothesis is that oxidative stress is an essential trigger for rapid NCX reactivation in guinea pig ventricular myocytes and is thus a critical factor in determining the timing and magnitude of Ca2+overload. This hypothesis was evaluated in cardiac myocytes using fluorescent indicators to measure [Ca2+]iand oxidative stress. An NCX antisense oligonucleotide was used to decrease NCX protein expression in some experiments. Our results indicate that NCX activity is profoundly inhibited in hypoxic guinea pig ventricular myocytes but is reactivated within 1–2 min of reoxygenation at a time of rising oxidative stress. We also found that several interventions to decrease oxidative stress including antioxidants and diazoxide prevented NCX reactivation and Ca2+overload during reoxygenation. Furthermore, application of exogenous H2O2was sufficient by itself to reactivate the NCX during sustained hypoxia and could reverse the suppression of reoxygenation-mediated NCX reactivation by diazoxide. These data suggest that elevated oxidative stress in reoxygenated guinea pig ventricular myocytes is required for rapid NCX reactivation, and thus reactivation should be viewed as an active process rather than being due to the simple decline of NCX inhibition.
Stretch-induced regional mechanoelectric dispersion and arrhythmia in the right ventricle of anesthetized lambs
Authors: Chen Ruo L. , Penny Daniel J. , Greve Gottfried , Lab Max J.
Published: February 2004

Regional mechanical and electrophysiological changes accompany most ventricular arrhythmias and, it has been suggested, by mechanoelectric feedback. We hypothesized that an intervention producing regional mechanical dispersion was associated with regional, proarrhythmic electrical dispersion and studied the regional mechanoelectric feedback in the right ventricle (RV) of anesthetized lambs. Ten lambs were deeply anesthetized, and their hearts were exposed. Three tripodal devices, each incorporating three monophasic action potential electrodes and an integrated strain-gauge system, were placed on the RV apex outflow and inflow regions. Measurements were made before, during, and after 10-s pulmonary arterial occlusion. Pulmonary arterial occlusion increased RV pressure and overall regional segment length. Length excursion became out of phase with RV pressure beats immediately after occlusion, and the strain patterns were different in the three regions at the peak of occlusion. The occlusion resulted in different alterations in regional monophasic action potential morphology, including reduction in monophasic action potential amplitude and duration by different amounts and early afterdepolarizations that were unevenly distributed in the monophasic action potential recordings. This was associated with dispersion of repolarization and recovery time. The combination of electromechanical events precipitated a variety of arrhythmias. Acute RV distension is proarrhythmic, possibly through a causal relationship among mechanically induced afterdepolarizations, dispersion (heterogeneity) of mechanical strain, and dispersion of electrical recovery. The relationship among the different wall motions, the dispersion of repolarization, and arrhythmia underscored mechanoelectric feedback as an important part of arrhythmogenesis in pulmonary embolism and commotio cordis.
Pifithrin-α protects against doxorubicin-induced apoptosis and acute cardiotoxicity in mice
Authors: Liu Xuwan , Chua Chu C. , Gao Jinping , Chen Zhongyi , Landy Cathy L. C. , Hamdy Ronald , Chua Balvin H. L.
Published: February 2004

The present experiments were designed to evaluate the effects of pifithrin-α (PFT-α), which is a p53 inhibitor, on doxorubicin (DOX)-induced apoptosis and cardiac injury. Administration of DOX (22.5 mg/kg ip) in mice upregulated the mRNA levels of Bax and MDM2, whereas PFT-α attenuated those levels when administered at a total dose of 4.4 mg/kg at 30 min before and 3 h after DOX challenge. DOX treatment led to an upregulation of p53 protein levels, which was preceded by elevated levels of phosphorylated p53 at Ser15. PFT-α had no effect on the level of p53 or its phosphorylated form. The protein levels of Bax and MDM2 were elevated by DOX and attenuated by PFT-α. DOX gave rise to increased apoptosis-positive nuclei in cardiac cells, elevated serum creatine phosphokinase, ultrastructural alterations, and cardiac dysfunction. PFT-α offered protection against all of the aforementioned changes. Finally, PFT-α did not interfere with the antitumor potency of DOX. This study demonstrates that PFT-α effectively inhibits DOX-induced cardiomyocyte apoptosis, which suggests that PFT-α has the potential to protect cancer patients against DOX-induced cardiac injury.
Low-flow vascular remodeling in the metabolic syndrome X
Authors: Stepp David W. , Pollock David M. , Frisbee Jefferson C.
Published: February 2004

Peripheral microvascular dysfunction is a common affliction in patients with the metabolic syndrome X. Previous studies have described a number of vascular impairments in vasomotor control in both human patients and animal models of syndrome X, but the net effect of these impairments on microvascular structure has not been examined. The goal of the current study was to test the hypothesis that syndrome X reduces muscle perfusion and induces vascular remodeling. The obese Zucker rat was used as a model of syndrome X, and the microcirculation of the hindlimb and brain were examined. Obese Zucker rats were obese, hyperlipidemic, hyperinsulinemic, and hyperglycemic. Blood flow to the hindlimb was reduced by 59% in obese rats relative to lean rats. Skeletal muscle resistance arteries of the hindlimb microcirculation of obese rats had thinner walls, smaller lumens, and reduced distensibility. Hindlimb microvessels from obese rats also demonstrated reduced expression of vascular smooth muscle cell markers. Each of these traits is consistent with low-flow remodeling. In contrast, the cerebral microcirculation, where flow is vigorously autoregulated, showed no vascular remodeling nor were there changes in microvascular smooth muscle marker expression. Neither physical activity nor muscle mass were significantly different between lean and obese rats. Taken together, these findings suggest that syndrome X, by reducing hindlimb blood flow, induces a marked remodeling of microcirculation to favor smaller, less distensible vessels. This remodeling may result in an architectural limitation of maximum perfusion capacity and may be an important maladaption in the progression of peripheral microvascular disease.
Involvement of RhoA and Rho kinase in neutrophil-stimulated endothelial hyperpermeability
Authors: Breslin Jerome W. , Yuan Sarah Y.
Published: February 2004

Neutrophil-induced microvascular leakage is an early event in ischemic and inflammatory heart diseases. The specific signaling paradigm by which neutrophils increase microvascular permeability is not yet established. We investigated whether the small GTPase RhoA and its downstream effector Rho kinase mediate neutrophil-stimulated endothelial hyperpermeability. We assessed the effect of neutrophils on Rho activity in bovine coronary venular endothelial cells (CVEC) with a Rho-GTP pull-down assay. Permeability to FITC-albumin was evaluated using CVEC monolayers. We then tested the role of Rho kinase in the permeability response to neutrophils using two structurally distinct pharmacological inhibitors: Y-27632 and HA-1077. Furthermore, neutrophil-stimulated changes in endothelial F-actin organization were examined with fluorescence microscopy. The results show that C5a-activated neutrophils induced an increase in permeability coupled with RhoA activation in CVEC. Inhibition of Rho kinase with either Y-27632 or HA-1077 attenuated the hyperpermeability response. Rho kinase inhibition also attenuated increases in permeability stimulated by the neutrophil supernatant. In addition, activated neutrophils caused actin stress fiber formation in CVEC, which was diminished by either Y-27632 or HA-1077. These findings suggest that RhoA and Rho kinase are involved in the mediation of neutrophil-induced endothelial actin reorganization and barrier dysfunction.
Acute and specific collagen type I degradation increases diastolic and developed tension in perfused rat papillary muscle
Authors: Lamberts Regis R. , Willemsen Maurice J. J. M. F. , Pérez Néstor G. , Sipkema Pieter , Westerhof Nico
Published: February 2004

Collagen degradation is suggested to be responsible for long-term contractile dysfunction in different cardiomyopathies, but the effects of acute and specific collagen type I removal (main type in the heart muscle) on tension have not been studied. We determined the diastolic and developed tension length relations in isometric contracting perfused rat papillary muscles (perfusion pressure 60 cmH2O) before and after acute and specific removal of small collagen struts with the use of purified collagenase type I. At 95% of the maximal length (95% Lmax), diastolic tension increased 20.4 ± 8.1% ( P < 0.05, n = 6) and developed tension increased 15.0 ± 6.7% after collagenase treatment compared with time controls. Treatment increased the diastolic muscle diameter by 7.1 ± 3.4% at 95% Lmax, whereas the change in diameter due to contraction was not changed. Diastolic coronary flow and normalized coronary arterial flow impediment did not change after collagenase treatment. Electron microscopy revealed that the number of small collagen struts, interconnecting myocytes, and capillaries was reduced to ∼32% after treatment. We conclude that removal of the small collagen struts by acute and specific collagen type I degradation increases diastolic and developed tension in perfused papillary muscle. We suggest that diastolic tension is increased due to edema, whereas developed tension is increased because the removal of the struts poses a lower lateral load on the cardiac myocytes, allowing more myocyte thickening.
Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and function
Authors: White Steven M. , Constantin Phillip E. , Claycomb William C.
Published: February 2004

HL-1 cells are currently the only cardiomyocyte cell line available that continuously divides and spontaneously contracts while maintaining a differentiated cardiac phenotype. Extensive characterization using microscopic, genetic, immunohistochemical, electrophysiological, and pharmacological techniques has demonstrated how similar HL-1 cells are to primary cardiomyocytes. In the few years that HL-1 cells have been available, they have been used in a variety of model systems designed to answer important questions regarding cardiac biology at the cellular and molecular levels. Whereas HL-1 cells have been used to study normal cardiomyocyte function with regard to signaling, electrical, metabolic, and transcriptional regulation, they have also been used to address pathological conditions such as hypoxia, hyperglycemia-hyperinsulinemia, apoptosis, and ischemia-reperfusion. The availability of an immortalized, contractile cardiac cell line has provided investigators with a tool for probing the intricacies of cardiomyocyte function. In this review, we describe the culture and characterization of HL-1 cardiomyocytes as well as various model systems that have been developed using these cells to gain a better understanding of cardiac biology at the cellular and molecular levels.
Effect of changes in pH on wall tension in isolated rat pulmonary artery: role of the RhoA/Rho-kinase pathway
Authors: Hyvelin Jean-Marc , O’Connor Clare , McLoughlin Paul
Published: February 2004

Pulmonary arteries (PA) are resistant to the vasodilator effects of extracellular acidosis in systemic vessels; the mechanism underlying this difference between systemic and pulmonary circulations has not been elucidated. We hypothesized that RhoA/Rho-kinase-mediated Ca2+sensitization pathway played a greater role in tension development in pulmonary than in systemic vascular smooth muscle and that this pathway was insensitive to acidosis. In arterial rings contracted with the α1-agonist phenylephrine (PE), the Rho-kinase inhibitor Y-27632 (≤3 μM) induced greater relaxation in precontracted PA rings than in aortic rings. In PA rings stimulated by PE, the activation of RhoA was greater than in aorta. Normocapnic acidosis (NA) induced a smaller relaxation in precontracted PA than in aorta. However, in the presence of nifedipine and thapsigargin, when PE-induced contraction was predominantly mediated by Rho-kinase, the relaxant effect of NA was reduced and similar in both vessel types. Furthermore, in the presence of Y-27632, NA induced a greater relaxation in both PA and aorta, which was similar in both vessels. Finally, in α-toxin-permeabilized smooth muscle, PE-induced contraction at constant Ca2+activity was inhibited by Y-27632 and unaffected by acidosis. These results indicate that Ca2+sensitization induced by the RhoA/Rho-kinase pathway played a greater role in agonist-induced vascular smooth muscle contraction in PA than in aorta and that tension mediated by this pathway was insensitive to acidosis. The predominant role of the RhoA/Rho-kinase pathway in the pulmonary vasculature may account for the resistance of this circulation to the vasodilator effect of acidosis observed in the systemic circulation.
Identification of a novel region in the proximal promoter of the mouse renin gene critical for expression
Authors: Pan Li , Jones Craig A. , Glenn Sean T. , Gross Kenneth W.
Published: February 2004

An enhancer at −2.6 kb and a HOX·PBX-binding site at −60 bp have been demonstrated to be critical to expression of the mouse renin gene ( Ren-1c) in As4.1 cells. In this report, we show that a region (−197 to −70) immediately 5′ to the HOX·PBX-binding site is also critical for Ren-1cexpression. Deletion of this region in a construct containing 4.1 kb of the Ren-1c5′-flanking sequence resulted in a 99% reduction in Ren-1cpromoter activity in As4.1 cells, suggesting the pivotal role for the region in the regulation of the mouse renin gene. Electrophoretic mobility shift and supershift assays have identified two nuclear factor I-binding sites and a Sp1/Sp3-binding site within the distal portion of the region (−197 to −103). Mutation of these three sites caused a 90% decrease in Ren-1cpromoter activity. Mutational analysis and electrophoretic mobility shift assays have also identified three additional transcription factor-binding sites within the region from −103 to −69, each of which contributes to high-level expression of Ren-1cin As4.1 cells. Finally, we have shown that the Ren-1cenhancer is the target for endothelin-1 (ET-1)-induced inhibition of Ren-1cexpression and the transcription factor-binding sites in the proximal promoter are required for the maximal ET-1 inhibitory effect.
Nitric oxide-cGMP pathway is involved in endotoxin-induced contractile dysfunction in rat hearts
Authors: Tatsumi Tetsuya , Keira Natsuya , Akashi Kazuko , Kobara Miyuki , Matoba Satoaki , Shiraishi Jun , Yamanaka Satoshi , Mano Akiko , Takeda Mitsuo , Nishikawa Susumu , Asayama Jun , Fliss Henry , Nakagawa Masao
Published: February 2004

The mechanisms by which endotoxemia causes cardiac depression have not been fully elucidated. The present study examined the involvement of nitric oxide (NO) in this pathology. Rats were infused with lipopolysaccharide (LPS) or saline, and the plasma and myocardial [Formula: see text] and [Formula: see text] (NOx) concentrations were measured before or 3, 6, and 24 h after treatment. The hearts were then immediately isolated and mounted in a Langendorff apparatus, and left ventricular developed pressure (LVDP) was determined before biochemical analysis of the myocardium. LPS injection effected the expression of inducible NO synthase (iNOS) in the myocardium, a marked increase in plasma and myocardial NOx levels, and a significant decline in LVDP compared with saline controls. The LPS-induced NO production and concomitant cardiac depression were most pronounced 6 h after LPS injection and were accompanied by a significant increase in myocardial cGMP content. Myocardial ATP levels were not significantly altered after LPS injection. Significant negative correlation was observed between LVDP and myocardial cGMP content, as well as between LVDP and plasma NOx levels. Aminoguanidine, an inhibitor of iNOS, significantly attenuated the LPS-induced NOx production and contractile dysfunction. Furthermore, 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase, significantly decreased myocardial cGMP content and attenuated the contractile depression, although aminoguanidine or 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one was not able to completely reverse myocardial dysfunction. Our data suggest that endotoxin-induced contractile dysfunction in rat hearts is associated with NO production by myocardial iNOS and a concomitant increase in myocardial cGMP.
Influence of l-NAME on pulmonary O2 uptake kinetics during heavy-intensity cycle exercise
Authors: Jones Andrew M. , Wilkerson Daryl P. , Wilmshurst Sally , Campbell Iain T.
Published: February 2004

We hypothesized that inhibition of nitric oxide synthase (NOS) by NG-nitro-l-arginine methyl ester (l-NAME) would alleviate the inhibition of mitochondrial oxygen uptake (V̇o2) by nitric oxide and result in a speeding of phase II pulmonary V̇o2 kinetics at the onset of heavy-intensity exercise. Seven men performed square-wave transitions from unloaded cycling to a work rate requiring 40% of the difference between the gas exchange threshold and peak V̇o2 with and without prior intravenous infusion of l-NAME (4 mg/kg in 50 ml saline over 60 min). Pulmonary gas exchange was measured breath by breath, and V̇o2 kinetics were determined from the averaged response to two exercise bouts performed in each condition. There were no significant differences between the control (C) and l-NAME conditions (L) for baseline V̇o2, the duration of phase I, or the amplitude of the primary V̇o2 response. However, the time constant of the V̇o2 response in phase II was significantly smaller (mean ± SE: C: 25.1 ± 3.0 s; L: 21.8 ± 3.3 s; P < 0.05), and the amplitude of the V̇o2 slow component was significantly greater (C: 240 ± 47 ml/min; L: 363 ± 24 ml/min; P < 0.05) after l-NAME infusion. These data indicate that inhibition of NOS by l-NAME results in a significant (13%) speeding of V̇o2 kinetics and a significant increase in the amplitude of the V̇o2 slow component in the transition to heavy-intensity cycle exercise in men. The speeding of the primary component V̇o2 kinetics after l-NAME infusion indicates that at least part of the intrinsic inertia to oxidative metabolism at the onset of heavy-intensity exercise may result from inhibition of mitochondrial V̇o2 by nitric oxide. The cause of the larger V̇o2 slow-component amplitude with l-NAME requires further investigation but may be related to differences in muscle blood flow early in the rest-to-exercise transition.
Exercise prevents diabetes-induced impairment in superficial buffer barrier in porcine coronary smooth muscle
Authors: Witczak C. A. , Sturek M.
Published: February 2004

In healthy coronary smooth muscle cells, the superficial sarcoplasmic reticulum (SR) buffers rise in intracellular Ca2+ levels. In diabetic dyslipidemia, basal Ca2+ levels are increased, yet Ca2+ influx is decreased and SR Ca2+ uptake is increased. Exercise prevents diabetic dyslipidemia-induced increases in basal Ca2+ levels and decreases in Ca2+ influx. We tested the hypothesis that diabetic dyslipidemia impairs Ca2+ extrusion via a decrease in superficial SR and that exercise will prevent these losses. Male Yucatan swine were maintained in four treatment groups: control, hyperlipidemic, diabetic dyslipidemic, and diabetic dyslipidemic plus aerobically exercise trained. Intracellular Ca2+ levels were measured during depolarization-induced Ca2+ influx and caffeine-induced SR Ca2+ release. Na+/Ca2+ exchanger and plasmalemmal Ca2+-ATPase activity were assessed by inhibition with low extracellular Na+ and 5,6-carboxyeosin, respectively. Superficial SR was quantified using the internal membrane dye 3,3′-dihexyloxacarbocyanine iodide (DiOC6) and novel analysis techniques. We found that, in diabetic dyslipidemia, Ca2+ extrusion was impaired and superficial SR was decreased. Exercise prevented the diabetic dyslipidemia-induced decrease in superficial SR and restored plasmalemmal Ca2+ extrusion. On the basis of these results, we conclude exercise attenuates the diabetic dyslipidemia-induced impairment in intracellular Ca2+ regulation.
Effects of positive-pressure ventilation on the spontaneous baroreflex in healthy subjects
Authors: Fietze Ingo , Romberg Dietrich , Glos Martin , Endres Susanne , Theres Heinz , Witt Christian , Somers Virend K.
Published: February 2004

To determine the short-term effects of noninvasive positive-pressure ventilation (PPV) on spontaneous baroreflex sensitivity, we acquired time series of R-R interval and beat-to-beat blood pressure in 55 healthy volunteers (mean age 46.5 ± 10.5 yr) who performed breathing on four occasions at frequencies of 12 and 15 breaths/min without positive pressure (control) and also using PPV of 5 mbar. By using spectral and cross-spectral analysis, R-R interval variability and systolic blood pressure variability as well as the gain (α-index) of the baroreceptor reflex were estimated for the low-frequency and high-frequency (HF) bands. Compared with control breathing, PPV at 12 breaths/min and 15 breaths/min led to an increase in mean R-R ( P < 0.001) and blood pressure ( P < 0.05). The α-index of the HF band increased significantly for both respiratory frequencies ( P < 0.05) due to PPV. These results indicate that short-term administration of PPV in normal subjects elicits a significant enhancement in the HF index of the baroreflex gain. These findings may contribute to understanding the mechanisms, indications, and effectiveness of positive pressure breathing strategies in treating cardiorespiratory and other disease conditions.
Differential effects of sodium selenite in reducing tissue damage caused by three hemoglobin-based oxygen carriers
Authors: Baldwin Ann L. , Wiley Elizabeth B. , Alayash Abdu I.
Published: February 2004

Three “blood substitutes,” a diaspirin cross-linked human hemoglobin (DBBF-Hb), a bovine polymerized hemoglobin (PolyHbBv), and a human polymerized hemoglobin ( O-R-PolyHbA0), that have undergone clinical trials are used in this study. Previously, we showed in the rat that coadministration of sodium selenite (Na2SeO3) and DBBF-Hb significantly decreased mesenteric venular leakage and epithelial disruption produced by DBBF-Hb alone but did not reduce mast cell degranulation unless given orally. The purpose of this study was to determine whether Na2SeO3 produced similar beneficial responses when used with PolyHbBv and O-R-PolyHbA0. In anesthetized Sprague-Dawley rats, the mesenteric microvasculature was perfused with PolyHbBv or O-R-PolyHbA0, with and without Na2SeO3 in the perfusate and suffusate, for 10 min, followed by FITC-albumin for 3 min, and then fixed for microscopy. Na2SeO3 did not reduce leak number or area in preparations perfused with PolyHbBv and only reduced leak number (but not significantly) in preparations perfused with O-R-PolyHbA0. Na2SeO3 significantly increased mesenteric mast cell degranulation and impaired epithelial integrity in animals treated with PolyHbBv. In vitro, Na2SeO3 significantly reduced the oxidation rate of DBBF-Hb in the presence of oxidants, had little effect on PolyHbBv, and increased the oxidation rate of O-R-PolyHbA0. These results suggest that Na2SeO3 moderates hemoglobin-induced damage, at least partly, through its redox interactions with the heme sites in the hemoglobin molecules studied and that accessibility of the heme site to Na2SeO3 governs those interactions.
Electrical stimulation of human lower extremities enhances energy consumption, carbohydrate oxidation, and whole body glucose uptake
Authors: Hamada Taku , Hayashi Tatsuya , Kimura Tetsuya , Nakao Kazuwa , Moritani Toshio
Published: February 2004

Our laboratory has recently demonstrated that low-frequency electrical stimulation (ES) of quadriceps muscles alone significantly enhanced glucose disposal rate (GDR) during euglycemic clamp (Hamada T, Sasaki H, Hayashi T, Moritani T, and Nakao K. J Appl Physiol 94: 2107–2112, 2003). The present study is further follow-up to examine the acute metabolic effects of ES to lower extremities compared with voluntary cycle exercise (VE) at identical intensity. In eight male subjects lying in the supine position, both lower leg (tibialis anterior and triceps surae) and thigh (quadriceps and hamstrings) muscles were sequentially stimulated to cocontract in an isometric manner at 20 Hz with a 1-s on-off duty cycle for 20 min. Despite small elevation of oxygen uptake by 7.3 ± 0.3 ml·kg-1·min-1during ES, the blood lactate concentration was significantly increased by 3.2 ± 0.3 mmol/l in initial period (5 min) after the onset of the ES ( P < 0.01), whereas VE showed no such changes at identical oxygen uptake (7.5 ± 0.3 ml·kg-1·min-1). ES also induced enhanced whole body carbohydrate oxidation as shown by the significantly higher respiratory gas exchange ratio than with VE ( P < 0.01). These data indicated increased anaerobic glycolysis by ES. Furthermore, whole body glucose uptake determined by GDR during euglycemic clamp demonstrated a significant increase during and after the cessation of ES for at least 90 min ( P < 0.01). This post-ES effect was significantly greater than that of the post-VE period ( P < 0.01). These results suggest that ES can substantially enhance energy consumption, carbohydrate oxidation, and whole body glucose uptake at low intensity of exercise. Percutaneous ES may become a therapeutic utility to enhance glucose metabolism in humans.
Effect of resistance training on skeletal muscle-specific force in elderly humans
Authors: Reeves N. D. , Narici M. V. , Maganaris C. N.
Published: February 2004

This study assessed muscle-specific force in vivo following strength training in old age. Subjects were assigned to training ( n = 9, age 74.3 ± 3.5 yr; mean ± SD) and control ( n = 9, age 67.1 ± 2 yr) groups. Leg-extension and leg-press exercises (2 sets of 10 repetitions at 80% of the 5 repetition maximum) were performed three times/wk for 14 wk. Vastus lateralis (VL) muscle fascicle force was calculated from maximal isometric voluntary knee extensor torque with superimposed stimuli, accounting for the patella tendon moment arm length, ultrasound-based measurements of muscle architecture, and antagonist cocontraction estimated from electromyographic activity. Physiological cross-sectional area (PCSA) was calculated from the ratio of muscle volume to fascicle length. Specific force was calculated by dividing fascicle force by PCSA. Fascicle force increased by 11%, from 847.9 ± 365.3 N before to 939.3 ± 347.8 N after training ( P < 0.05). Due to a relatively greater increase in fascicle length (11%) than muscle volume (6%), PCSA remained unchanged (pretraining: 30.4 ± 8.9 cm2; posttraining: 29.1 ± 8.4 cm2; P > 0.05). Activation capacity and VL muscle root mean square electromyographic activity increased by 5 and 40%, respectively, after training ( P < 0.05), indicating increased agonist neural drive, whereas antagonist cocontraction remained unchanged ( P > 0.05). The VL muscle-specific force increased by 19%, from 27 ± 6.3 N/cm2 before to 32.1 ± 7.4 N/cm2 after training ( P < 0.01), highlighting the effectiveness of strength training for increasing the intrinsic force-producing capacity of skeletal muscle in old age.