bluuebunny/crossref_metadata_2025 huggingface dataset:
Microarray expression analysis of effects of exercise training: increase in atrial MLC-1 in rat ventricles
Authors: Diffee Gary M. , Seversen Eric A. , Stein Thor D. , Johnson Jeffrey A.
Published: March 2015

Previous studies have shown that endurance exercise training increases myocardial contractility. We have previously described training-induced alterations in myocardial contractile function at the cellular level, including an increase in the Ca2+ sensitivity of tension. To determine the molecular mechanism(s) of these changes, oligonucleotide microarrays were used to analyze the gene expression profile in ventricles from endurance-trained rats. We used an 11-wk treadmill training protocol that we have previously shown results in increased contractility in cardiac myocytes. After the training, the hearts were removed and RNA was isolated from the ventricles of nine trained and nine control rats. With the use of an Affymetrix Rat Genome U34A Array, we detected altered expression of 27 genes. Several genes previously found to have increased expression in hypertrophied myocardium, such as atrial natriuretic factor and skeletal α-actin, were decreased with training in this study. From the standpoint of altered contractile performance, the most significant finding was an increase in the expression of atrial myosin light chain 1 (aMLC-1) in the trained ventricular tissue. We confirmed microarray results for aMLC-1 using RT-PCR and also confirmed a training-induced increase in aMLC-1 protein using two-dimensional gel electrophoresis. aMLC-1 content has been previously shown to be increased in human cardiac hypertrophy and has been associated with increased Ca2+sensitivity of tension and increased power output. These results suggest that increased expression of aMLC-1 in response to training may be responsible, at least in part, for previously observed training-induced enhancement of contractile function.
Differential response of cardiac fibroblasts from young adult and senescent rats to ANG II
Authors: Shivakumar K. , Dostal David E. , Boheler Kenneth , Baker Kenneth M. , Lakatta Edward G.
Published: March 2015

The intracardiac ANG II-forming pathway is activated in the senescent myocardium, raising the possibility of enhanced ANG II effects on cardiac fibroblasts. This study established an in vitro model of cultured cardiac fibroblasts from aged rats to examine if the response of these cells to ANG II is modified in the aged heart. Levels of mRNA encoding renin, angiotensinogen, and the AT1 receptor subtype in cardiac fibroblasts from young adult and senescent rats were quantified by RT-PCR, net collagen production by a hydroxyproline-based assay, and transforming growth factor (TGF)-β levels using a commercial kit. In cardiac fibroblasts from young adult rats, ANG II significantly enhanced AT1mRNA levels, net collagen production, and TGF-β production. In fibroblasts from the aged myocardium, ANG II downregulated AT1 mRNA expression, had a less pronounced effect on net collagen production, and had no effect on TGF-β production. Such age-related modification of the response of cardiac fibroblasts to ANG II may counteract the effects of augmented intracardiac ANG II production in the senescent heart, limiting fibrogenesis.
Relationship between no reflow and infarct size as influenced by the duration of ischemia and reperfusion
Authors: Reffelmann Thorsten , Hale Sharon L. , Li Guohu , Kloner Robert A.
Published: March 2015

No reflow after acute myocardial infarction is an important predictor of infarct size and clinical outcome. However, the exact relationship between no reflow and infarct size remains to be determined, particularly because no reflow may progress during the time course of reperfusion. Control groups of five previous protocols using the anesthetized, open-chest rabbit model of coronary artery occlusion and reperfusion were retrospectively analyzed with respect to the correlation between regional myocardial blood flow (RMBF; radioactive microspheres) and infarct size (triphenyltetrazolium chloride) in the course of reperfusion. After 30 min of occlusion, reflow (defined as the ratio of RMBF in the risk area divided by the nonischemic area) declined from hyperemic values after 30 min of reperfusion (reflow ratio: 1.33 ± 0.81; RMBF in the risk area at the same time point: 2.25 ± 1.04 ml · g−1 · min−1) to 0.47 ± 0.22 after 120 min and 0.46 ± 0.13 after 180 min of reperfusion. After 120 min of ischemia, reflow at 30 min of reperfusion was 0.49 ± 0.24 and deteriorated by 120 min of reperfusion (0.26 ± 0.15). In every group, there was a strong correlation between infarct size and reflow (correlation coefficients: −0.62 to −0.82). The lines of regression for the groups with assessment of RMBF after 120 or 180 min of reperfusion were nearly identical regardless of the duration of ischemia. Thus microvascular reperfusion injury led to a striking decrease in RMBF within the first 2 h of reperfusion, with infarct size as the major determinant of reflow at a given time point of reperfusion.
Differing cardioprotective efficacy of the Na+/Ca2+exchanger inhibitors SEA0400 and KB-R7943
Authors: Magee William P. , Deshmukh Gayatri , Deninno Michael P. , Sutt Jill C. , Chapman Justin G. , Tracey W. Ross
Published: March 2015

KB-R7943 and SEA0400 are Na+/Ca2+exchanger (NCX) inhibitors with differing potency and selectivity. The cardioprotective efficacy of these NCX inhibitors was examined in isolated rabbit hearts (Langendorff perfused) subjected to regional ischemia (coronary artery ligation) and reperfusion. KB-R7943 and SEA0400 elicited concentration-dependent reductions in infarct size (SEA0400 EC50: 5.7 nM). SEA0400 was more efficacious than KB-R7943 (reduction in infarct size at 1 μM: SEA0400, 75%; KB-R7943, 40%). Treatment with either inhibitor yielded similar reductions in infarct size whether administered before or after regional ischemia. SEA0400 (1 μM) improved postischemic recovery of function (±dP/d t), whereas KB-R7943 impaired cardiac function at ≥1 μM. At 5–20 μM, KBR-7943 elicited rapid and profound depressions of heart rate, left ventricular developed pressure, and ±dP/d t. Thus the ability of KB-R7943 to provide cardioprotection is modest and limited by negative effects on cardiac function, whereas the more selective NCX inhibitor SEA0400 elicits marked reductions in myocardial ischemic injury and improved ±dP/d t. NCX inhibition represents an attractive approach for achieving clinical cardioprotection.
Protein tyrosine kinase signaling is necessary for NO donor-induced late preconditioning against myocardial stunning
Authors: Tang Xian-Liang , Kodani Eitaro , Takano Hitoshi , Hill Michael , Shinmura Ken , Vondriska Thomas M. , Ping Peipei , Bolli Roberto
Published: March 2015

Although protein tyrosine kinases (PTKs) signaling has been implicated in the late phase of ischemic preconditioning (PC), it is unknown whether PTK signaling is necessary for the development of nitric oxide (NO) donor-induced late PC. Thus conscious rabbits underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles followed by a 5-h recovery period of reperfusion for 3 consecutive days ( days 1, 2, and 3). On day 0 (24 h before the 6 O/R cycles on day 1), rabbits received no treatment (control), the NO donor diethylenetriamine (DETA)/NO (DETA/NO), the PTK inhibitor 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (PP2), or DETA/NO plus PP2 (DETA/NO + PP2). In control rabbits ( n = 6), the six O/R cycles on day 1resulted in delayed functional recovery, indicating severe myocardial stunning. In rabbits pretreated with DETA/NO ( n = 5) on day 1, myocardial stunning caused by the six O/R cycles on day 1 was markedly attenuated, with a significant reduction (∼60%) in the total deficit of wall thickening (WTh) compared with controls, indicating that DETA/NO induced a late PC effect against stunning. However, in rabbits pretreated with DETA/NO + PP2 ( n = 5), the total deficit of WTh was significantly greater than that in rabbits treated with DETA/NO alone and was similar to that in controls, indicating that PP2 prevented the development of DETA/NO-induced late PC. In rabbits pretreated with PP2 on day 0 ( n = 4), the total deficit of WTh was similar to that in controls, indicating that PP2 does not affect myocardial stunning in itself. We conclude that a PTK-dependent signaling mechanism is necessary for the development of NO donor-induced late PC against myocardial stunning in conscious rabbits.
Functional role, cellular source, and tissue distribution of rat elastase-2, an angiotensin II-forming enzyme
Authors: Santos Carlos F. , Caprio Marcos Antonio V. , Oliveira Eduardo B. , Salgado Maria Cristina O. , Schippers Daniela N. , Munzenmaier Diane H. , Greene Andrew S.
Published: March 2015

We recently described a chymostatin-sensitive elastase-2 as the major angiotensin (ANG) II-forming enzyme in the perfusate of the rat mesenteric arterial bed (MAB) with the same cDNA sequence as rat pancreatic elastase-2. The role of this enzyme in generating ANG II was examined in the rat isolated and perfused MAB. The vasoconstrictor effect elicited by ANG I and the renin substrate tetradecapeptide was only partially inhibited by captopril but abolished by the combination of captopril and chymostatin or N-acetyl-Ala-Ala-Pro-Leu-chloromethylketone (Ac-AAPL-CK; inhibitor originally developed for human elastase-2). The effect induced by [Pro11,d-Ala12]-ANG I, an ANG I-converting enzyme (ACE)-resistant biologically inactive precursor of ANG II, was blocked by chymostatin or Ac-AAPL-CK. It was also demonstrated that cultured rat mesenteric endothelial cells synthesize elastase-2 and that mRNA for this enzyme can be detected in different rat tissues such as the pancreas, MAB, lung, heart, kidney, liver, and spleen. In conclusion, the demonstration of a functional alternative pathway to ACE for ANG II generation in the rat MAB and the fact that cultured MAB endothelial cells are capable of producing and secreting elastase-2 represent strong evidence of a physiological role for this enzyme in the rat vasculature.
In vivo visualization of subendocardial arteriolar response in renovascular hypertensive hearts
Authors: Yada Toyotaka , Goto Masami , Hiramatsu Osamu , Tachibana Hiroyuki , Toyota Eiji , Nakamoto Hiroshi , Ogasawara Yasuo , Matsuda Hiroto , Arakawa Koki , Hayashi Koichi , Suzuki Hiromichi , Kajiya Fumihiko
Published: March 2015

Time-sequential responses to endothelium-dependent and -independent vasodilators and angiotensin-converting enzyme (ACE) inhibitors were studied in the subendocardial arterioles (Endo) of canine renovascular hypertension (HT) compared with subepicardial arterioles (Epi; both <120 μm) by charge-coupled device intravital microscope. Vascular responses to acetylcholine, papaverine, and cilazaprilat were compared between normotensive (NT) and HT dogs [4 wk and 12 wk of HT (4wHT and 12wHT)]. The acetylcholine-induced vasodilation of Endo in both 4wHT and 12wHT was smaller than that of NT (both P< 0.01 vs. 4wHT and 12wHT), and that of Epi was smaller than that of NT only in 12wHT ( P < 0.05). The papaverine-induced vasodilation of Endo, but not Epi, was impaired only in 12wHT (both P < 0.01 vs. NT and 4wHT). Vasodilation by cilazaprilat remained unchanged at 4wHT and 12wHT in both Epi and Endo. In conclusion, at the early stage, the endothelium-dependent response of Endo was impaired, whereas at the later stage, the endothelium-dependent and -independent responses of Endo and the endothelium-dependent response of Epi were impaired. However, the vasodilatory responses to the ACE inhibitor were maintained in both Endo and Epi of HT.
Normovolemic hemodilution with Hb vesicle solution attenuates hypoxia in ischemic hamster flap tissue
Authors: Erni Dominique , Wettstein Reto , Schramm Sören , Contaldo Claudio , Sakai Hiromi , Takeoka Shinji , Tsuchida Eishun , Leunig Michael , Banic Andrej
Published: March 2015

The aim of this study was to test whether oxygenation in acutely ischemic, collateralized tissue may be improved by normovolemic hemodilution with a solution containing liposome-encapsulated human Hb (HbV). A skin flap model in anesthetized hamsters was used, which consisted of two parts receiving either anatomic or collateral perfusion. Microhemodynamics were investigated with intravital microscopy. Partial tissue oxygen tension was measured with a Clark-type microprobe. Hemodilution was obtained by exchanging 50% of the total blood volume with HbV suspended in 8% human serum albumin (HSA8) or 6% Dextran 70 (Dx70). The size of the vesicles was 276 nm, the P50 was 22 mmHg, and the Hb concentration of the solutions was 7.5 g/dl. Colloid osmotic pressure and viscosity were 49.9 mmHg and 8.7 cP for HbV-Dx70 and 40.0 mmHg and 2.9 cP for HbV-HSA8, respectively. Hemodilution with HbV-Dx70 led to an increase in microvascular blood flow in the ischemic microvessels to maximally 158% (median, P < 0.01), whereas blood flow remained virtually unchanged after hemodilution with HbV-HSA8. In the ischemic tissue, oxygen tension was improved from 11.9 to 17.0 mmHg ( P < 0.01) after hemodilution with HbV-Dx70 but remained virtually unchanged after hemodilution with HbV-HSA8. Our study suggests that the oxygenation in acutely ischemic, collateralized tissue may be improved by normovolemic hemodilution with HbV suspended in Dx70. The effect was achieved by an increase in microcirculatory blood flow related to the rheological properties of the suspending medium.
Simvastatin restores endothelial NO-mediated vasorelaxation in large arteries after myocardial infarction
Authors: Bates Kathryn , Ruggeroli Charles E. , Goldman Steven , Gaballa Mohamed A.
Published: March 2015

Congestive heart failure (CHF) after myocardial infarction is associated with diminished endothelial nitric oxide (NO)-mediated vasorelaxation. The 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors have been shown to modulate vascular tone independent of the effects on lipid lowering. We hypothesized that simvastatin restores NO-dependent vasorelaxation with CHF. We found that incubation of the normal rat aorta with 0.1 mM simvastatin for 24 h enhanced ACh-mediated vasorelaxation ( P < 0.05). Moreover, simvastatin increased ( P < 0.05) endothelial NO synthase (eNOS) protein content by >200% (82.0 ± 14.0 vs. 21.6 ± 7.9% II/μg). In cultured endothelial cells, simvastatin (10 and 20 μM) increased eNOS levels by 114.7 ± 39.9 and 212.0 ± 75.0% II/μg protein, respectively (both P < 0.05; n = 8). In the rat coronary artery ligation model, oral gavage with 20 mg · kg−1 · day−1 simvastatin for 3 wk decreased ( P < 0.05) mean arterial pressure (121 ± 20 vs. 96.5 ± 10.8 mmHg) and left ventricular change in pressure with time (4,500 ± 700 vs. 4,091 ± 1,064 mmHg/s, n = 6). Simvastatin reduced ( P< 0.05) basal vasoconstriction and improved ACh-mediated vasorelaxation in CHF arterial rings. Inhibition of NO generation by N G-nitro-l-arginine methyl ester (100 μM) abolished the ACh-induced vasorelaxation in all rats. In conclusion, chronic treatment of CHF with simvastatin restores endothelial NO-dependent dysfunction and upregulates eNOS protein content in arterial tissue.
Endothelial NO formation does not control myocardial O2 consumption in mouse heart
Authors: Kojic Zvezdana Z. , Flögel Ulrich , Schrader Jürgen , Decking Ulrich K. M.
Published: March 2015

To test whether endothelium-derived nitric oxide (NO) regulates mitochondrial respiration, NO was pharmacologically modulated in isolated mouse hearts, which were perfused at constant flow to sensitively detect small changes in myocardial O2 consumption (MV̇O2). Stimulation of NO formation by 10 μM bradykinin (BK) increased coronary venous nitrite release fivefold to 58 ± 33 nM ( n = 17). Vasodilatation by BK, adenosine (1 μM), or papaverine (10 μM) decreased perfusion pressure, left ventricular developed pressure (LVDP), and MV̇O2. In the presence of adenosine-induced vasodilatation, stimulation of endothelial NO synthesis by BK had no effect on LVDP and MV̇O2. Also, inhibition of NO formation by NG-monomethyl-l-arginine (l-NMMA, 100 μM) did not significantly alter LVDP and MV̇O2. Similarly, intracoronary infusion of authentic NO ≤2 μM did not influence LVDP or MV̇O2 (-1 ± 1%). Only when NO was >2 μM were contractile dysfunction and MV̇O2 reduction observed. Because BK-induced stimulation of endothelial NO formation and basal NO are not sufficient to impair MV̇O2 in the saline-perfused mouse heart, a tonic control of the respiratory chain by endothelial NO is difficult to conceive.
AT1 and AT2 receptor expression and blockade after acute ischemia-reperfusion in isolated working rat hearts
Authors: Xu Yi , Kumar Dinender , Dyck Jason R. B. , Ford William R. , Clanachan Alexander S. , Lopaschuk Gary D. , Jugdutt Bodh I.
Published: March 2015

We assessed ANG II type 1 (AT1) and type 2 (AT2) receptor (R) expression and functional recovery after ischemia-reperfusion with or without AT1R/AT2R blockade in isolated working rat hearts. Groups of six hearts were subjected to global ischemia (30 min) followed by reperfusion (30 min) and exposed to no drug and no ischemia-reperfusion (control), ischemia-reperfusion and no drug, and ischemia-reperfusion with losartan (an AT1R antagonist; 1 μmol/l), PD-123319 (an AT2R antagonist; 0.3 μmol/l), N 6-cyclohexyladenosine (CHA, a cardioprotective adenosine A1 receptor agonist; 0.5 μmol/l as positive control), enalaprilat (an ANG-converting enzyme inhibitor; 1 μmol/l), PD-123319 + losartan, ANG II (1 nmol/l), or ANG II + losartan. Compared with controls, ischemia-reperfusion decreased AT2R protein (Western immunoblots) and mRNA (Northern immunoblots, RT-PCR) and impaired functional recovery. PD-123319 increased AT2R protein and mRNA and improved functional recovery. Losartan increased AT1R mRNA (but not AT1R/AT2R protein) and impaired recovery. Other groups (except CHA) did not improve recovery. The results suggest that, in isolated working hearts, AT2R plays a significant role in ischemia-reperfusion and AT2R blockade induces increased AT2R protein and cardioprotection.
Amelioration of ischemia-reperfusion injury with cyclic peptide blockade of ICAM-1
Authors: Merchant Shakil H. , Gurule Debbie M. , Larson Richard S.
Published: March 2015

Neutrophils are pivotal in the pathogenesis of ischemia-reperfusion (I/R) injury leading to muscle damage. Firm adhesion of neutrophils to the endothelium is initiated by an interaction between intercellular adhesion molecular-1 (ICAM-1) on the endothelium and β2-integrins on neutrophils. Inhibition of ICAM-1-dependent binding using monoclonal antibodies has been shown to be efficacious in ameliorating I/R injury by preventing the influx of neutrophils into the ischemic tissue. We recently described a cyclic peptide that is a potent and selective inhibitor of ICAM-1 (IP25) in vitro. In this study, we tested the hypothesis that IP25-mediated blockade of ICAM-1 would inhibit neutrophil influx during reperfusion of ischemic tissue and consequently attenuate muscle injury in a tourniquet hindlimb murine model of I/R injury. Varying amounts of peptide drug were injected at the beginning of the reperfusion period. The neutrophil influx and size of infarction at the end of 2 h of reperfusion were compared with those in untreated control mice and contralateral nonischemic limbs. Mice receiving IP25 immediately before reperfusion showed a 56% reduction in neutrophil infiltration in the ischemic muscle, accompanied by a 40% reduction in the infarct size. No effect on I/R injury was seen if IP25 administration was delayed for 60 min after reperfusion. We conclude that IP25 effectively inhibits ICAM-1-mediated adhesion of neutrophils to the endothelium in mice leading to a protective effect and suggests that synthetic peptide antagonists have a potential role as therapeutic tools.
Role of short-term cardiovascular regulation in heart period variability: a modeling study
Authors: Ursino Mauro , Magosso Elisa
Published: March 2015

A mathematical model of short-term cardiovascular regulation is used to investigate how heart period variability reflects the action of the autonomic regulatory mechanisms (vagal and sympathetic). The model includes the pulsating heart, the systemic (splanchnic and extrasplanchnic) and pulmonary circulation, the mechanical effect of respiration on venous return, two groups of receptors (arterial baroreceptors and lung stretch receptors), the sympathetic and vagal efferent branches, and a very low-frequency (LF) vasomotor noise. All model parameters were given on the basis of physiological data from the literature. We used data from humans whenever possible, whereas parameters for the regulation loops are derived from dog experiments. The model, with basal parameter values, produces a heart period power spectrum with two distinct peaks [a high frequency (HF) peak at the respiratory rate and a LF peak at ∼0.1 Hz]. Sensitivity analysis on the mechanism gains suggests that the HF peak is mainly affected by the vagal mechanism, whereas the LF peak is increased by a high sympathetic gain and reduced by a high vagal gain. Moreover, the LF peak depends significantly on the reactivity of resistance vessels and is affected by noise, amplified by the sympathetic control loop at its resonance frequency. The model may represent a new tool to study alterations in the heart period spectrum on the basis of quantitative physiological hypotheses.
24-h Langendorff-perfused neonatal rat heart used to study the impact of adenoviral gene transfer
Authors: Wiechert S. , El-Armouche A. , Rau T. , Zimmermann W. -H. , Eschenhagen T.
Published: March 2015

The human genome project has increased the demand for simple experimental systems that allow the impact of gene manipulations to be studied under controlled ex vivo conditions. We hypothesized that, in contrast to adult hearts, neonatal hearts allow long-term perfusion and efficient gene transfer ex vivo. A Langendorff perfusion system was modified to allow perfusion for >24 h with particular emphasis on uncompromised contractile activity, sterility, online measurement of force of contraction, inotropic response to β-adrenergic stimulation, and efficient gene transfer. The hearts were perfused with serum-free medium (DMEM + medium 199, 4 + 1) supplemented with hydrocortisone, triiodothyronine, ascorbic acid, insulin, pyruvate, l-carnitine, creatine, taurine, l-glutamine, mannitol, and antibiotics recirculating (500 ml/2 hearts) at 1 ml/min. Hearts from 2 day-old rats beat constantly at 135–155 beats/min and developed active force of 1–2 mN. During 24 h of perfusion, twitch tension increased to ∼165% of initial values ( P < 0.05), whereas the inotropic response to isoprenaline remained constant. A decrease in total protein content of 10% and histological examination indicated moderate edema, but actin and calsequestrin concentration remained unchanged and perfusion pressure remained constant at 7–11 mmHg. Perfusion with a LacZ-encoding adenovirus at 3 × 108 active virus particles yielded homogeneous transfection of ∼80% throughout the heart and did not affect heart rate, force of contraction, or response to isoprenaline compared with uninfected controls ( n = 7 each). Taken together, the 24-h Langendorff-perfused neonatal rat heart is a relatively simple, inexpensive, and robust new heart model that appears feasible as a test bed for functional genomics.
Attenuated sympathetic nerve responses after 24 hours of bed rest
Authors: Khan Mazhar H. , Kunselman Allen R. , Leuenberger Urs A. , Davidson William R. , Ray Chester A. , Gray Kristen S. , Hogeman Cynthia S. , Sinoway Lawrence I.
Published: March 2015

Bed rest reduces orthostatic tolerance. Despite decades of study, the cause of this phenomenon remains unclear. In this report we examined hemodynamic and sympathetic nerve responses to graded lower body negative pressure (LBNP) before and after 24 h of bed rest. LBNP allows for baroreceptor disengagement in a graded fashion. We measured heart rate (HR), cardiac output (HR × stroke volume obtained by echo Doppler), and muscle sympathetic nerve activity (MSNA) during a progressive and graded LBNP paradigm. Negative pressure was increased by 10 mmHg every 3 min until presyncope or completion of −60 mmHg. After bed rest, LBNP tolerance was reduced in 11 of 13 subjects ( P < .023), HR was greater ( P< .002), cardiac output was unchanged, and the ability to augment MSNA at high levels of LBNP was reduced (rate of rise for 30- to 60-mmHg LBNP before bed rest 0.073 bursts · min−1 · mmHg−1; after bed rest 0.035 bursts · min−1 · mmHg−1; P < 0.016). These findings suggest that 24 h of bed rest reduces sympathetic nerve responses to LBNP.
Preconditioning attenuates ischemia-reperfusion-induced remodeling of Na+-K+-ATPase in hearts
Authors: Elmoselhi Adel B. , Lukas Anton , Ostadal Petr , Dhalla Naranjan S.
Published: March 2015

The aim of this study was to determine whether changes in protein content and/or gene expression of Na+-K+-ATPase subunits underlie its decreased enzyme activity during ischemia and reperfusion. We measured protein and mRNA subunit levels in isolated rat hearts subjected to 30 min of ischemia and 30 min of reperfusion (I/R). The effect of ischemic preconditioning (IP), induced by three cycles of ischemia and reperfusion (10 min each), was also assessed on the molecular changes in Na+-K+-ATPase subunit composition due to I/R. I/R reduced the protein levels of the α2-, α3-, β1-, and β2-isoforms by 71%, 85%, 27%, and 65%, respectively, whereas the α1-isoform was decreased by <15%. A similar reduction in mRNA levels also occurred for the isoforms of Na+-K+-ATPase. IP attenuated the reduction in protein levels of Na+-K+-ATPase α2-, α3-, and β2-isoforms induced by I/R, without affecting the α1- and β1-isoforms. Furthermore, IP prevented the reduction in mRNA levels of Na+-K+-ATPase α2-, α3-, and β1-isoforms following I/R. Similar alterations in protein contents and mRNA levels for the Na+/Ca2+exchanger were seen due to I/R as well as IP. These findings indicate that remodeling of Na+-K+-ATPase may occur because of I/R injury, and this may partly explain the reduction in enzyme activity in ischemic heart disease. Furthermore, IP may produce beneficial effects by attenuating the remodeling of Na+-K+-ATPase and changes in Na+/Ca2+exchanger in hearts after I/R.
Effects of hyperoxia on local and remote microcirculatory inflammatory response after splanchnic ischemia and reperfusion
Authors: Waisman Dan , Brod Vera , Wolff Rafael , Sabo Edmond , Chernin Mark , Weintraub Zalman , Rotschild Avi , Bitterman Haim
Published: March 2015

Splanchnic ischemia-reperfusion (I/R) causes tissue hypoxia that triggers local and systemic microcirculatory inflammatory responses. We evaluated the effects of hyperoxia in I/R induced by 40-min superior mesenteric artery (SMA) occlusion and 120-min reperfusion in four groups of rats: 1) control (anesthesia only), 2) sham operated (all surgical procedures without vascular occlusion; air ventilation), 3) SMA I/R and air, 4) SMA I/R and 100% oxygen ventilation started 10 min before reperfusion. Leukocyte rolling and adhesion in mesenteric microvessels, pulmonary microvascular blood flow velocity (BFV), and macromolecular (FITC-albumin) flux into lungs were monitored by intravital videomicroscopy. We also determined pulmonary leukocyte infiltration. SMA I/R caused marked decreases in mean arterial blood pressure (MABP) and blood flow to the splanchnic and hindquarters vascular beds and pulmonary BFV and shear rates, followed by extensive increase in leukocyte rolling and adhesion and plugging of >50% of the mesenteric microvasculature. SMA I/R also caused marked increase in pulmonary sequestration of leukocytes and macromolecular leak with concomitant decrease in circulating leukocytes. Inhalation of 100% oxygen maintained MABP at significantly higher values ( P < 0.001) but did not change regional blood flows. Oxygen therapy attenuated the increase in mesenteric leukocyte rolling and adherence ( P < 0.0001) and maintained microvascular patency at values not significantly different from sham-operated animals. Hyperoxia also attenuated the decrease in pulmonary capillary BFV and shear rates, reduced leukocyte infiltration in the lungs ( P < 0.001), and prevented the increase in pulmonary macromolecular leak ( P < 0.001), maintaining it at values not different from sham-operated animals. The data suggest that beneficial effects of normobaric hyperoxia in splanchnic I/R are mediated by attenuation of both local and remote inflammatory microvascular responses.
B-type natriuretic peptide limits infarct size in rat isolated hearts via KATPchannel opening
Authors: D'Souza Savio P. , Yellon Derek M. , Martin Claus , Schulz Rainer , Heusch Gerd , Onody Annamaria , Ferdinandy Peter , Baxter Gary F.
Published: March 2015

B-type natriuretic peptide (BNP) has been reported to be released from the myocardium during ischemia. We hypothesized that BNP mediates cardioprotection during ischemia-reperfusion and examined whether exogenous BNP limits myocardial infarction and the potential role of ATP-sensitive potassium (KATP) channel opening. Langendorff-perfused rat hearts underwent 35 min of left coronary artery occlusion and 120 min of reperfusion. The control infarct-to-risk ratio was 44.8 ± 4.4% (means ± SE). BNP perfused 10 min before ischemia limited infarct size in a concentration-dependent manner, with maximal protection observed at 10−8M (infarct-to-risk ratio: 20.1 ± 5.2%, P < 0.01 vs. control), associated with a 2.5-fold elevation of myocardial cGMP above the control value. To examine the role of KATPchannel opening, glibenclamide (10−6M), 5-hydroxydecanoate (5-HD; 10−4M), or HMR-1098 (10−5M) was coperfused with BNP (10−8M). Protection afforded by BNP was abolished by glibenclamide or 5-HD but not by HMR-1098, suggesting the involvement of putative mitochondrial but not sarcolemmal KATPchannel opening. We conclude that natriuretic peptide/cGMP/KATPchannel signaling may constitute an important injury-limiting mechanism in myocardium.
Gap junction-dependent and -independent EDHF-type relaxations may involve smooth muscle cAMP accumulation
Authors: Chaytor Andrew T. , Taylor Hannah J. , Griffith Tudor M.
Published: March 2015

We have compared the mechanisms that contribute to endothelium-derived hyperpolarizing factor (EDHF)-type responses induced by ACh and the Ca2+ ionophore A-23187 in the rabbit iliac artery. Relaxations to both agents were associated with ∼1.5-fold elevations in smooth muscle cAMP levels and were attenuated by the adenylyl cyclase inhibitor 2′,5′-dideoxyadenosine (DDA) and potentiated by the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Mechanical responses were inhibited by coadministration of the Ca2+-activated K+channel blockers apamin and charybdotoxin, both in the absence and presence of IBMX, but were unaffected by blockade of ATP-sensitive K+ channels with the sulphonylurea glibenclamide. Relaxations and elevations in cAMP evoked by ACh were abolished by 18α-glycyrrhetinic acid, which disrupts gap junction plaques, whereas the corresponding responses to A-23187 were unaffected by this agent. Consistently, in “sandwich” bioassay experiments, A-23187, but not ACh, elicited extracellular release of a factor that evoked relaxations that were inhibited by DDA and potentiated by IBMX. These findings provide evidence that EDHF-type relaxations of rabbit iliac arteries evoked by ACh and A-23187 depend on cAMP accumulation in smooth muscle, but involve signaling via myoendothelial gap junctions and the extracellular space, respectively.
Glutamate release in midbrain periaqueductal gray by activation of skeletal muscle receptors and arterial baroreceptors
Authors: Li Jianhua , Mitchell Jere H.
Published: March 2015

We have previously reported that both skeletal muscle receptor and arterial baroreceptor afferent inputs activate neurons in the dorsolateral (DL) and lateral regions of the midbrain periaqueductal gray (PAG). In this study, we determined whether the excitatory amino acid glutamate (Glu) is released to mediate the increased activity in these regions. Static contraction of the triceps surae muscle for 4 min was evoked by electrical stimulation of the L7 and S1 ventral roots in cats. Activation of arterial baroreceptor was induced by intravenous injection of phenylephrine. The endogenous release of Glu from the PAG was recovered with the use of a microdialysis probe. Glu concentration was measured by the HPLC method. Muscle contraction increased mean arterial pressure (MAP) from 98 ± 10 to 149 ± 12 mmHg ( P < 0.05) and increased Glu release in the DL and lateral regions of the middle PAG from 0.39 ± 0.10 to 0.73 ± 0.12 μM (87%, P < 0.05) in intact cats. After sinoaortic denervation and vagotomy were performed, contraction increased MAP from 95 ± 12 to 158 ± 15 mmHg, and Glu from 0.34 ± 0.08 to 0.54 ± 0.10 μM (59%, P < 0.05). The increases in arterial pressure and Glu were abolished by muscle paralysis. Phenylephrine increased MAP from 100 ± 13 to 162 ± 22 mmHg and increased Glu from 0.36 ± 0.10 to 0.59 ± 0.18 μM (64%, P < 0.05) in intact animals. Denervation abolished this Glu increase. Summation of the changes in Glu evoked by muscle receptor and arterial baroreceptor afferent inputs was greater than the increase in Glu produced when both reflexes were activated simultaneously in intact state (123% vs. 87%). These data demonstrate that activation of skeletal muscle receptors evokes release of Glu in the DL and lateral regions of the middle PAG, and convergence of afferent inputs from muscle receptors and arterial baroreceptors in these regions inhibits the release of Glu. These results suggest that the PAG is a neural integrating site for the interaction between the exercise pressor reflex and the arterial baroreceptor reflex.
Role of carbon monoxide in glutamate receptor-induced dilation of newborn pig pial arterioles
Authors: Robinson James S. , Fedinec Alexander L. , Leffler Charles W.
Published: March 2015

The hypothesis that glutamate dilates pial arterioles of newborn pigs through the production of carbon monoxide (CO) was addressed. Anesthesized newborn pigs were equipped with cranial windows to measure pial arteriolar responses to stimuli. Heme oxygenase (HO) inhibitors added topically inhibited dilation to glutamate and to specific glutamate receptor agonists. The initial dilation to glutamate (10−5 M) was 22% from baseline without an inhibitor and decreased to 9% with the HO inhibitor chromium mesoporphyrin (CrMP). Inhibition of dilation upon HO inhibition was similar when specific glutamate receptor agonists were employed. RS-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid caused 24% dilation from the baseline without an inhibitor, and the dilation was decreased to 1% with tin protoporphyrin (SnPP). ( RS)-2-amino-3-(3-hydroxy-5- t-butylisoxazol-4-yl)propionic acid (kainate receptors) caused dilation of 18% from baseline without an inhibitor, but only 2% when tin mesoporphyrin was applied. 1-Aminocyclopropanecarboxylic acid ( N-methyl-d-aspartate receptors) dilated pial arterioles 33% from baseline in control, but only to 2% in the presence of SnPP. Neither copper mesoporphyrin, which does not inhibit HO, nor light-inactivated CrMP affected the dilations. Furthermore, cerebral microvessels removed from the brain produced CO (stable isotope dilution gas chromatography-mass spectrometry), and this production was dose dependently increased by glutamate and inhibited by metal porphyrin HO inhibitors. These data suggest that dilation of newborn pig pial arterioles to glutamate and specific glutamate receptor agonists involves vascular production of CO. Additional cerebral sources of CO also could be stimulated by glutamate and contribute to the dilation.
Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary hypertension in rats
Authors: Ikeda Yasuhiro , Yonemitsu Yoshikazu , Kataoka Chu , Kitamoto Shiro , Yamaoka Terutoshi , Nishida Ken-Ichi , Takeshita Akira , Egashira Kensuke , Sueishi Katsuo
Published: March 2015

Monocyte/macrophage chemoattractant protein-1 (MCP-1), a potent chemoattractant chemokine and an activator for mononuclear cells, may play a role in the initiation and/or progression of pulmonary hypertension (PH). To determine whether blockade of a systemic MCP-1 signal pathway in vivo may prevent PH, we intramuscularly transduced a naked plasmid encoding a 7-NH2terminus-deleted dominant negative inhibitor of the MCP-1 (7ND MCP-1) gene in monocrotaline-induced PH. We also simultaneously gave a duplicate transfection at 2-wk intervals or skeletal muscle-directed in vivo electroporation (EP) to evaluate whether a longer or higher expression might be more effective. The intramuscular reporter gene expression was enhanced 10 times over that by EP than by simple injection, and a significant 7ND MCP-1 protein in plasma was detected only in the EP group. 7ND MCP-1 gene transfer significantly inhibited the progression of MCT-induced PH as evaluated by right ventricular systolic pressure, right ventricular hypertrophy, medial hypertrophy of pulumonary arterioles, and mononuclear cell infiltration into the lung. Differential effects of longer or higher transgene expression were not apparent. Although the in vivo kinetics of 7ND MCP-1 gene therapy should be studied further, these encouraging results suggest that an anti-inflammatory strategy via blockade of the MCP-1 signal pathway may be an alternative approach to treat subjects with PH.
Ischemia and reperfusion of skeletal muscle lead to the appearance of a stable lipid free radical in the circulation
Authors: Pattwell David , Ashton Tony , McArdle Anne , Griffiths Richard D. , Jackson Malcolm J.
Published: March 2015

Both ischemia and reperfusion injury and contractile activity are associated with the generation of reactive oxygen species and free radicals by skeletal muscle. In addition, exercise has been reported to lead to the formation of a circulating free radical species that is detectable in the blood by spin trapping before analysis by electron-spin resonance (ESR) techniques. Previous analysis of the ESR signal indicated that the circulating species is either a carbon- or oxygen-centered lipid-derived free radical. The current data indicate that this species is present in the blood of anesthetized rats after 4-h ischemia and 1 h of reperfusion of a single hindlimb. During 4 h of ischemia, the species was also present in microdialysates from the tibialis anterior muscle but was unchanged in magnitude compared with control tissue. During 1 h of reperfusion, the signal intensity increased by a mean of 420% ( P < 0.05, n = 4). Hydroxyl radical activity in the interstitial fluid also significantly increased during ischemia and further increased by a mean of 210% ( P < 0.05, n = 4) during reperfusion. No changes in interstitial superoxide levels were seen, but interstitial PGE2 content also increased during reperfusion. A significant positive correlation was found between the magnitude of the ESR signal and both the hydroxyl radical activity and PGE2 content of microdialysis fluids. These data support the hypothesis that the circulating free radical species is formed in the interstitial fluid by hydroxyl radical interaction with a lipid that may be released from reperfused tissue with a similar pattern to prostanoids.
Differential distribution of Kir2.1 and Kir2.3 subunits in canine atrium and ventricle
Authors: Melnyk Peter , Zhang Liming , Shrier Alvin , Nattel Stanley
Published: March 2015

Ventricular inward rectifier K+current ( IK1) is substantially larger than atrial, producing functionally important action potential differences. To evaluate possible molecular mechanisms, we recorded IK1with patch-clamp techniques and studied Kir2.1 and Kir2.3 subunit expression. IK1density was >10-fold larger in the canine ventricle than atrium. Kir2.1 protein expression (Western blot) was 78% greater ( P < 0.01) in the ventricle, but Kir2.3 band density was 228% greater ( P < 0.01) in the atrium. Immunocytochemistry showed transverse tubular localization of Kir2.1 in 89% (17 of 19) of ventricular and 26% (5 of 19, P < 0.0001) of atrial cells. Both exhibited a weakly positive Kir2.1 signal at intercalated disks. Kir2.3 was strongly expressed at the intercalated disks in all cells and in the transverse tubular regions in 78% (14 of 18) of atrial and 22% (4 of 18, P < 0.001) of ventricular cells. Tissue immunohistochemical results qualitatively resembled isolated cell data. We conclude that the expression density and subcellular localization of Kir2.1 and Kir2.3 subunits differ in the canine atrium versus ventricle. Overall protein density differences are insufficient to explain IK1discrepancies, which may be related to differences in subcellular distribution.
Biphasic effects of cell volume on excitation-contraction coupling in rabbit ventricular myocytes
Authors: Li Gui-Rong , Zhang Min , Satin Leslie S. , Baumgarten Clive M.
Published: March 2015

We studied the effects of osmotic swelling on the components of excitation-contraction coupling in ventricular myocytes. Myocyte volume rapidly increased 30% in hyposmotic (0.6T) solution and was constant thereafter. Cell shortening transiently increased 31% after 4 min in 0.6T but then decreased to 68% of control after 20 min. In parallel, the L-type Ca2+ current ( I Ca-L) transiently increased 10% and then declined to 70% of control. Similar biphasic effects on shortening were observed under current clamp. In contrast, action potential duration was unchanged at 4 min but decreased to 72% of control after 20 min. Ca2+ transients were measured with fura 2-AM. The emission ratio with excitation at 340 and 380 nm (f340/f380) decreased by 12% after 3 min in 0.6T, whereas shortening and I Ca-L increased at the same time. After 8 min, shortening, I Ca-L, and the f340/f380 ratio decreased 28, 25, and 59%, respectively. The results suggest that osmotic swelling causes biphasic changes in I Ca-L that contribute to its biphasic effects on contraction. In addition, swelling initially appears to reduce the Ca2+ transient initiated by a given I Ca-L, and later, both I Ca-L and the Ca2+ transient are inhibited.
Ovariectomy upregulates expression of estrogen receptors, NOS, and HSPs in porcine platelets
Authors: Jayachandran Muthuvel , Miller Virginia M.
Published: March 2015

Platelets participate in normal and pathological thrombotic processes. Hormone replacement in postmenopausal women is associated with increase risk for thrombosis. However, little is known regarding how platelets are affected by hormonal status. Nitric oxide (NO) modulates platelet functions and is modulated by hormones. Therefore, the present study was designed to determine how loss of ovarian hormones changes expression of estrogen receptors and regulatory proteins for NO synthase (NOS) in platelets. Estrogen receptors (ERα and ERβ), NOS, heat shock proteins 70 and 90 (HSP70 and HSP90), caveolin-1, -2, and -3, calmodulin, NOS activity, and cGMP were analyzed in a lysate of platelets from gonadally intact and ovariectomized female pigs. Expression of ERβ and ERα receptors, endothelial NOS (eNOS), HSP70, and HSP90 increased with ovariectomy. NOS activity and cGMP also increased; calmodulin was unchanged. Caveolins were not detected. These results suggest that ovarian hormones influence expression of estrogen receptors and eNOS in platelets. Changes in estrogen receptors and NOS could affect platelet aggregation in response to hormone replacement.
Ischemic preconditioning inhibits mitochondrial respiration, increases H2O2release, and enhances K+transport
Authors: da Silva Mirian M. , Sartori Adriano , Belisle Eduardo , Kowaltowski Alicia J.
Published: March 2015

Ischemic preconditioning, or the protective effect of short ischemic episodes on a longer, potentially injurious, ischemic period, is prevented by antagonists of mitochondrial ATP-sensitive K+channels (mitoKATP) and involves changes in mitochondrial energy metabolism and reactive oxygen release after ischemia. However, the effects of ischemic preconditioning itself on mitochondria are still poorly understood. We determined the effects of ischemic preconditioning on isolated heart mitochondria and found that two brief (5 min) ischemic episodes are sufficient to induce a small but significant decrease (∼25%) in mitochondrial NADH-supported respiration. Preconditioning also increased mitochondrial H2O2release, an effect related to respiratory inhibition, because it is not observed in the presence of succinate plus rotenone and can be mimicked by chemically inhibiting complex I in the presence of NADH-linked substrates. In addition, preconditioned mitochondria presented more substantial ATP-sensitive K+transport, indicative of higher mitoKATPactivity. Thus we directly demonstrate that preconditioning leads to mitochondrial respiratory inhibition in the presence of NADH-linked substrates, increased reactive oxygen release, and activation of mitoKATP.
Characterization of the normal cardiac myofiber field in goat measured with MR-diffusion tensor imaging
Authors: Geerts L. , Bovendeerd P. , Nicolay K. , Arts T.
Published: March 2015

Cardiac myofiber orientation is a crucial determinant of the distribution of myocardial wall stress. Myofiber orientation is commonly quantified by helix and transverse angles. Accuracy of reported helix angles is limited. Reported transverse angle data are incomplete. We measured cardiac myofiber orientation postmortem in five healthy goat hearts using magnetic resonance-diffusion tensor imaging. A novel local wall-bound coordinate system was derived from the characteristics of the fiber field. The transmural course of the helix angle corresponded to data reported in literature. The mean midwall transverse angle ranged from −12 ± 4° near the apex to +9.0 ± 4° near the base of the left ventricle, which is in agreement with the course predicted by Rijcken et al. (18) using a uniform load hypothesis. The divergence of the myofiber field was computed, which is a measure for the extent to which wall stress is transmitted through the myofiber alone. It appeared to be <0.07 mm−1 throughout the myocardial walls except for the fusion sites between the left and right ventricles and the insertion sites of the papillary muscles.
TNF-α downregulates vascular endothelial Flk-1 expression in human melanoma xenograft model
Authors: Menon Chandrakala , Iyer Malini , Prabakaran Indira , Canter Robert J. , Lehr Shannon C. , Fraker Douglas L.
Published: March 2015

High-dose TNF with melphalan has significant antitumor activity in regional perfusion of the limbs and liver in human malignancies. TNF is believed to target tumor vasculature, but the precise molecular mechanism is unknown. The present study demonstrates that TNF downregulates the VEGF receptor, fetal liver kinase-1 (Flk-1), on tumor endothelium in a human melanoma xenograft model. NIH1286 human melanoma cells were transduced with a 720-bp fragment of the human VEGF121 gene to develop well-vascularized tumors that served as an amplified system for measuring Flk-1 expression changes. We injected 5 × 106 cells subcutaneously, each of two distinct single cell clones (NIH1286/3 and NIH1286/15), into athymic nude mice to produce tumors ∼10 mm in size. Each animal then received either BSA or TNF in BSA by tail vein. Tumors harvested at different time points post-TNF were analyzed for Flk-1 mRNA and protein expression. Data obtained showed that intravascular TNF downregulated Flk-1 expression in tumor endothelial cells. This effect could contribute to the antitumor activity of TNF known to target tumor vasculature.