bluuebunny/crossref_metadata_2025 huggingface dataset:
Losartan corrects abnormal frequency response of renal vasculature in congestive heart failure
Authors: DiBona Gerald F. , Sawin Linda L.
Published: March 2015

In congestive heart failure, renal blood flow is decreased and renal vascular resistance is increased in a setting of increased activity of both the sympathetic nervous and renin-angiotensin systems. The renal vasoconstrictor response to renal nerve stimulation is enhanced. This is associated with an abnormality in the low-pass filter function of the renal vasculature wherein higher frequencies (≥0.01 Hz) within renal sympathetic nerve activity are not normally attenuated and are passed into the renal blood flow signal. This study tested the hypothesis that excess angiotensin II action mediates the abnormal frequency response characteristics of the renal vasculature in congestive heart failure. In anesthetized rats, the renal vasoconstrictor response to graded frequency renal nerve stimulation was significantly greater in congestive heart failure than in control rats. Losartan attenuated the renal vasoconstrictor response to a significantly greater degree in congestive heart failure than in control rats. In control rats, the frequency response of the renal vasculature was that of a first order (–20 dB/frequency decade) low-pass filter with a corner frequency (–3 dB, 30% attenuation) of 0.002 Hz and 97% attenuation (–30 dB) at ≥0.1 Hz. In congestive heart failure rats, attenuation did not exceed 45% (–5 dB) over the frequency range of 0.001–0.6 Hz. The frequency response of the renal vasculature was not affected by losartan treatment in control rats but was completely restored to normal by losartan treatment in congestive heart failure rats. The enhanced renal vasoconstrictor response to renal nerve stimulation and the associated abnormality in the frequency response characteristics of the renal vasculature seen in congestive heart failure are mediated by the action of angiotensin II on renal angiotensin II AT1 receptors.
TNF-α antibodies are as effective as ischemic preconditioning in reducing infarct size in rabbits
Authors: Belosjorow Sergej , Bolle Ines , Duschin Alexej , Heusch Gerd , Schulz Rainer
Published: March 2015

Pretreatment with tumor necrosis factor-α (TNF-α) antibodies abolishes myocardial infarct size reduction by late ischemic preconditioning (IP). Whether or not TNF-α is also important for myocardial infarct size reduction by classic IP is unknown. Anesthetized rabbits were untreated ( group 1, n = 7), classically preconditioned by 5 min left coronary artery occlusion/10 min reperfusion ( group 2, n = 6), or pretreated with TNF-α antibodies without ( group 3, n = 6) or with IP ( group 4, n = 6) before undergoing 30 min of occlusion and 180 min of reperfusion. Infarct size in group 1 was 44 ± 11 (means ± SD)% of the area at risk. With a comparable area at risk, infarct size was reduced to 13 ± 7%, 23 ± 8%, and 19 ± 12% (all P < 0.05) in groups 2, 3, and 4, respectively. The circulating TNF-α concentration was increased during ischemia in group 1 from 752 ± 403 to 1,542 ± 482 U/ml ( P < 0.05) but remained unchanged in all other groups. Circulating TNF-α concentration during ischemia and infarct size correlated in all groups ( r = 0.76). IP, TNF-α antibodies, and the combined approach reduced infarct size to a comparable extent. Therefore, the question of whether or not TNF-α is causally involved in the infarct size reduction by IP in rabbits could not be answered.
Bradykinin stimulates ceramide production by activating specific BK-B1receptor in rat small artery
Authors: Kleine Leonard , Liu Gele , Leblanc Normand , Hébert Richard L.
Published: March 2015

Bradykinin (BK), a proinflammatory factor and vasodilator, causes functional change of the small artery. However, it is not clear whether any of these changes induced by BK are mediated by N-acetyl-d-sphingosine (ceramide). Therefore, we investigated whether BK affects the hydrolysis of sphingomyelin and generation of ceramide in the intact rat small artery. Our results suggest that BK induces sphingomyelin hydrolysis and increases ceramide production in a time- and dose-dependent manner. Relative to controls, BK causes a 50% decrease in sphingomyelin levels. Ceramide levels increase in response to BK with the highest level being obtained with 10−8M BK as well as similar amounts of ceramide are generated when exogenous sphingomyelinase (SMase) is added. We then determined which of the two BK receptors (BK-B1antagonist Lys-Des-Arg9-Leu8-BK or the BK-B2antagonist HOE-140) are implicated in the BK-induced generation of ceramide. The BK-B2antagonist did not alter the effect of BK on ceramide generation, whereas the BK-B1antagonist blocked the BK-induced production of ceramide. Although ceramide had no effect on KCl-induced constrictions, ceramide dilated preconstricted (phenylephrine) small pressurized rat mesenteric arteries by ∼40%. These results suggest that the activation of the BK-B1receptor mediates the BK-induced activation of SMase and of the production of ceramide. In conclusion, BK-mediated effects on vascular tone may be due, at least in part, to the increased production of ceramide.
Myocardial oxygenation in isolated hearts predicted by an anatomically realistic microvascular transport model
Authors: Beard Daniel A. , Schenkman Kenneth A. , Feigl Eric O.
Published: March 2015

An anatomically realistic model for oxygen transport in cardiac tissue is introduced for analyzing data measured from isolated perfused guinea pig hearts. The model is constructed to match the microvascular anatomy of cardiac tissue based on available morphometric data. Transport in the three-dimensional system (divided into distinct microvascular, interstitial, and parenchymal spaces) is simulated. The model is used to interpret experimental data on mean cardiac tissue myoglobin saturation and to reveal differences in tissue oxygenation between buffer-perfused and red blood cell-perfused isolated hearts. Interpretation of measured mean myoglobin saturation is strongly dependent on the oxygen content of the perfusate (e.g., red blood cell-containing vs. cell-free perfusate). Model calculations match experimental values of mean tissue myoglobin saturation, measured mean myoglobin, and venous oxygen tension and can be used to predict distributions of intracellular oxygen tension. Calculations reveal that ∼20% of the tissue is hypoxic with an oxygen tension of <0.5 mmHg when the buffer is equilibrated with 95% oxygen to give an arterial oxygen tension of over 600 mmHg. The addition of red blood cells to give a hematocrit of only 5% prevents tissue hypoxia. It is incorrect to assume that the usual buffer-perfused Langendorff heart preparation is adequately oxygenated for flows in the range of ≤10 ml · min–1· ml tissue–1.
LOX-1 inhibition in myocardial ischemia-reperfusion injury: modulation of MMP-1 and inflammation
Authors: Li Dayuan , Williams Victor , Liu Ling , Chen Hongjiang , Sawamura Tatsuya , Antakli Tamim , Mehta Jawahar L.
Published: March 2015

A recently identified lectin-like oxidized low-density lipoprotein receptor (LOX-1) mediates endothelial cell injury and facilitates inflammatory cell adhesion. We studied the role of LOX-1 in myocardial ischemia-reperfusion (I/R) injury. Anesthetized Sprague-Dawley rats were subjected to 60 min of left coronary artery (LCA) ligation, followed by 60 min of reperfusion. Rats were treated with saline, LOX-1 blocking antibody JXT21 (10 mg/kg), or nonspecific anti-goat IgG (10 mg/kg) before I/R. Ten other rats underwent surgery without LCA ligation and served as a sham control group. LOX-1 expression was markedly increased during I/R ( P < 0.01 vs. sham control group). Simultaneously, the expression of matrix metalloproteinase-1 (MMP-1) and adhesion molecules (P-selectin, VCAM-1, and ICAM-1) was also increased in the I/R area ( P < 0.01 vs. sham control group). There was intense leukocyte accumulation in the I/R area in the saline-treated group. Treatment of rats with the LOX-1 antibody prevented I/R-induced upregulation of LOX-1 and reduced MMP-1 and adhesion molecule expression as well as leukocyte recruitment. LOX-1 antibody, but not nonspecific IgG, also reduced myocardial infarct size ( P < 0.01 vs. saline-treated I/R group). To explore the link between LOX-1 and adhesion molecule expression, we measured expression of oxidative stress-sensitive p38 mitogen-activated protein kinase (p38 MAPK). The activity of p38 MAPK was increased during I/R ( P < 0.01 vs. sham control), and use of LOX-1 antibody inhibited p38 MAPK activation ( P < 0.01). These findings indicate that myocardial I/R upregulates LOX-1 expression, which through p38 MAPK activation increases the expression of MMP-1 and adhesion molecules. Inhibition of LOX-1 exerts an important protective effect against myocardial I/R injury.
Interaction between increased SERCA2a activity and β-adrenoceptor stimulation in adult rabbit myocytes
Authors: Chaudhri Babar , del Monte Federica , Hajjar Roger J. , Harding Sian E.
Published: March 2015

Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a overexpression and phospholamban depletion have been shown to have beneficial effects on contractility in heart failure. However, the high sympathetic tone during development of failure may interact with increases in SERCA2a activity in potentially deleterious ways. We used adenoviral vectors to overexpress SERCA2a or partially downregulate phospholamban in adult rabbit ventricular myocytes in culture and studied the responses of these cells to β-adrenoceptor stimulation. SERCA2a overexpression and phospholamban depletion had quantitatively similar effects on basal contraction amplitude and in accelerating relaxation. Increasing SERCA2a activity by either strategy had little effect on the increase in contraction amplitude or incidence of arrhythmias with increasing isoproterenol. Maximum acceleration of relaxation by β-adrenoceptor stimulation was similar to that produced by SERCA2a overexpression. Isoproterenol treatment of SERCA2a-overexpressing or phospholamban-deficient myocytes produced a further modest decrease in relaxation time, with similar final values in both groups. We find no evidence for Ca2+ overload induced by SERCA2a overexpression alone or in combination with catecholamines.
Estrogen increases iNOS expression in the ovine coronary artery
Authors: Mershon John L. , Baker R. Scott , Clark Kenneth E.
Published: March 2015

Estrogen is believed to protect postmenopausal women from coronary vascular disease, in part by increasing production of nitric oxide (NO). In this study, we investigated the possibility that transcriptional activation of inducible NO synthase (iNOS) is responsible for a component of the estrogen-induced increase in coronary blood flow. Twenty-two ewes were instrumented with Doppler flow probes on their left circumflex coronary and pulmonary arteries. Nine ewes received 17β-estradiol (1 μg/kg), and the coronary vascular response was followed for 16 h. Estradiol significantly increased coronary blood flow by 22 ± 4% over baseline and the peak response occurred at 2 h ( P < 0.01). To examine the effect of estrogen on NOS expression in the ovine coronary artery, 17 noninstrumented animals were killed 2 h after administration of estradiol or vehicle. Coronary arteries were analyzed for ovine iNOS and endothelial NOS (eNOS) expression by semiquantitative RT-PCR. PCR primers were based on partial cDNA clones for ovine eNOS and iNOS isolated as part of this study. The expression of iNOS was significantly increased (27-fold) by the administration of estradiol, whereas the expression of eNOS was much weaker (2-fold). To confirm these effects in vivo, additional instrumented animals received either the estrogen receptor (ER) antagonist ICI-182,780 ( n = 5), the iNOS antagonist dexamethasone ( n = 5), or pyrrolidine dithiocarbamic acid, an inhibitor of nuclear factor-κB ( n = 5). All three antagonists inhibited estrogen-induced increases in coronary blood flow and increases in cardiac output by over 85%. These results strongly support the hypothesis that 17β-estradiol increases coronary blood flow in the unanesthetized nonpregnant ewe via an ER-dependent mechanism that results in an increase in both eNOS and iNOS expression.
Troglitazone administration limits infarct size by reduced phosphorylation of canine myocardial connexin43 proteins
Authors: Lee Tsung-Ming , Chou Tsai-Fwu
Published: March 2015

Troglitazone, an antidiabetic thiazolidinedione, has been shown to have a scavenging effect on reactive oxygen species, which can modulate expression of connexin43. The study purpose was to evaluate whether troglitazone provides cardioprotection and to assess whether the cardioprotection is associated with an attenuated expression of connexin43 at the border of infarction in a canine model of acute myocardial infarction. Vehicle or troglitazone (1, 5, and 50 mg/kg; n = 14 for each group) was given intravenously 15 min before the coronary artery occlusion. Among the survivors, infarct size was significantly larger in the control than in the supplemented groups. There was a significantly lower infarct size in the high-dose group compared with that in the low-dose group (15 ± 7% vs. 23 ± 10% of the risk region in the low-dose group, P = 0.04). Reperfusion caused a significant elevation in superoxide anions as measured by lucigenin-derived chemiluminescence, which was significantly inhibited in animals treated with troglitazone. Connexin43 underwent dephosphorylation in response to ischemia-reperfusion measured by Western blot in control hearts at the border zone; these changes were significantly enhanced by troglitazone administration. Confocal microscopy confirmed the changes of junctional complexes. The magnitude of infarct size positively correlated with the magnitude of phosphorylated connexin43 expression assessed by Western blot analysis ( r = 0.73, P < 0.0001). This result demonstrated that the cardioprotective effect of troglitazone as an antioxidant may be associated with reduced phosphorylation of myocardial connexin43 protein.
Microgravity alters respiratory sinus arrhythmia and short-term heart rate variability in humans
Authors: Migeotte P-F. , Prisk G. Kim , Paiva M.
Published: March 2015

We studied heart rate (HR), heart rate variability (HRV), and respiratory sinus arrhythmia (RSA) in four male subjects before, during, and after 16 days of spaceflight. The electrocardiogram and respiration were recorded during two periods of 4 min controlled breathing at 7.5 and 15 breaths/min in standing and supine postures on the ground and in microgravity. Low (LF)- and high (HF)-frequency components of the short-term HRV (≤3 min) were computed through Fourier spectral analysis of the R-R intervals. Early in microgravity, HR was decreased compared with both standing and supine positions and had returned to the supine value by the end of the flight. In microgravity, overall variability, the LF-to-HF ratio, and RSA amplitude and phase were similar to preflight supine values. Immediately postflight, HR increased by ∼15% and remained elevated 15 days after landing. LF/HF was increased, suggesting an increased sympathetic control of HR standing. The overall variability and RSA amplitude in supine decreased postflight, suggesting that vagal tone decreased, which coupled with the decrease in RSA phase shift suggests that this was the result of an adaptation of autonomic control of HR to microgravity. In addition, these alterations persisted for at least 15 days after return to normal gravity (1G).
Inhibition of G protein-coupled receptor trafficking in neuroblastoma cells by MAP 4 decoration of microtubules
Authors: Cheng Guangmao , Iijima Yoshihiro , Ishibashi Yuji , Kuppuswamy Dhandapani , Cooper George
Published: March 2015

One mechanism for the reappearance of G protein-coupled receptors after agonist activation is microtubule-based transport. In pressure-overload cardiac hypertrophy, there is downregulation of G protein-coupled receptors and the appearance of a densified microtubule network extensively decorated by a microtubule-associated protein, MAP 4. Our hypothesis is that overdecoration of a dense microtubule network with this structural protein, as in hypertrophied myocardium, would impede receptor recovery. We tested this hypothesis by studying muscarinic acetylcholine receptor (mAChR) internalization and recovery after agonist stimulation in neuroblastoma cells. Exposure of cells to carbachol, a muscarinic receptor agonist, decreased membrane receptor binding activity. After carbachol withdrawal, receptor binding recovered toward the initial value. When microtubules were depolymerized before carbachol withdrawal, mAChR recovery was only 44% of that in intact cells. Cells were then infected with an adenovirus containing MAP 4 cDNA. MAP 4 protein decorated the microtubules extensively, and receptor recovery upon carbachol withdrawal was reduced to 54% of control. Thus muscarinic receptor recovery after agonist exposure is microtubule dependent, and MAP 4 decoration of microtubules inhibits receptor recovery.
11,12,15-Trihydroxyeicosatrienoic acid mediates ACh-induced relaxations in rabbit aorta
Authors: Campbell William B. , Spitzbarth Nancy , Gauthier Kathryn M. , Pfister Sandra L.
Published: March 2015

Rabbit aortic endothelium metabolizes arachidonic acid (AA) by the 15-lipoxygenase pathway to vasodilatory eicosanoids, hydroxyepoxyeicosatrienoic acids (HEETAs), and trihydroxyeicosatrienoic acids (THETAs). The present study determined the chemical identity of the vasoactive THETA and investigated its role in ACh-induced relaxation in the rabbit aorta. AA caused endothelium-dependent, concentration-related relaxations of the rabbit aorta. Increasing the extracellular KCl concentration from 4.8 to 20 mM inhibited the relaxations to AA by ∼60%, thereby implicating K+-channel activation in the relaxations. In addition, AA caused an endothelium-dependent hyperpolarization of aortic smooth muscle from –39.6 ± 2.7 to –56.1 ± 3.4 mV. In rabbit aortic rings, [14C]AA was metabolized to prostaglandins, HEETAs, THETAs, and 15-hydroxyeicosatetraenoic acid. Additional purification of the THETAs by HPLC resolved the mixture into its 14C-labeled products. Gas chromatography/mass spectrometry identified the metabolites as isomers of 11,12,15-THETA and 11,14,15-THETA. The 11,12,15-THETA relaxed and hyperpolarized the rabbit aorta, whereas 11,14,15-THETA had no vasoactive effect. The relaxations to 11,12,15-THETA were blocked by 20 mM KCl. In aortic rings pretreated with inhibitors of nitric oxide and prostaglandin synthesis, ACh caused a concentration-related relaxation that was completely blocked by 20 mM KCl. Pretreatment with the phospholipase A2 inhibitors mepacrine and 7,7-dimethyl-5,8-eicosadienoic acid, the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-α-cyanocinnamate, nordihydroguaiaretic acid, and ebselen, or the hydroperoxide isomerase inhibitors miconazole and clotrimazole also blocked ACh-induced relaxations. ACh caused a threefold increase in THETA release. These studies indicate that AA is metabolized by endothelial cells to 11,12,15-THETA, which activates K+ channels to hyperpolarize the aortic smooth muscle membrane and induce relaxation. Additionally, this lipoxygenase pathway mediates the nonnitric oxide, nonprostaglandin relaxations to ACh in the rabbit aorta by acting as a source of an endothelium-derived hyperpolarizing factor.
Nitric oxide-endothelin-1 interactions after acute ductal constriction in fetal lambs
Authors: Ovadia Boaz , Bekker Janine M. , Fitzgerald Robert K. , Kon Alexander , Thelitz Stephan , Johengen Michael J. , Hendricks-Munoz Karen , Gerrets Rene , Black Stephen M. , Fineman Jeffrey R.
Published: March 2015

Acute partial compression of the fetal ductus arteriosus (DA) results in an initial increase in pulmonary blood flow (PBF) that is followed by acute vasoconstriction. The objective of the present study was to determine the role of nitric oxide (NO)-endothelin-1 (ET-1) interactions in the acute changes in pulmonary vascular tone after in utero partial constriction of the DA. Twelve late-gestation fetal lambs (132–140 days) were instrumented to measure vascular pressures and left PBF. After a 24-h recovery period, acute constriction of the DA was performed by partially inflating a vascular occluder, and the hemodynamic variables were observed for 4 h. In control lambs ( n = 7), acute ductal constriction initially increased PBF by 627% ( P< 0.05). However, this was followed by active vasoconstriction, such that PBF was restored to preconstriction values by 4 h. This was associated with a 43% decrease in total NO synthase (NOS) activity ( P < 0.05) and a 106% increase in plasma ET-1 levels ( P < 0.05). Western blot analysis demonstrated no changes in lung tissue endothelial NOS, preproET-1, endothelin-converting enzyme-1, or ETB receptor protein levels. The infusion of PD-156707 (an ETA receptor antagonist, n = 5) completely blocked the vasoconstriction and preserved NOS activity. These data suggest that the fetal pulmonary vasoconstriction after acute constriction of the DA is mediated by NO-ET-1 interactions. These include an increase in ETA receptor-mediated vasoconstriction and an ETA receptor-mediated decrease in NOS activity. The mechanisms of these NO-ET-1 interactions, and their role in mediating acute changes in PBF, warrant further studies.
Early activation of cardiac and renal endothelin systems in experimental heart failure
Authors: Motte Sophie , van Beneden Ronald , Mottet José , Rondelet Benoit , Mathieu Myrielle , Havaux Xavier , Lause Pascale , Clercx Cécile , Ketelslegers Jean-Marie , Naeije Robert , McEntee Kathleen
Published: March 2015

We investigated the time course of the expression of cardiac and renal endothelin systems in tachycardia-induced heart failure in dogs. Eleven beagles underwent rapid pacing at a progressively increased rate over a period of 5 wk, with a weekly clinical examination, echocardiography, measurement of circulating and urinary endothelin-1 (ET-1), and myocardial and renal tissue biopsies. Real-time quantitative PCR was used for determinations of tissue prepro-ET-1 (ppET-1), ET-1-converting enzyme (ECE-1), and ETAand ETBreceptor mRNA. Cardiac and renal tissue ET-1 contents were evaluated by immunostaining and measured by radioimmunoassay at autopsy. Rapid pacing caused a progressive increase in end-systolic and end-diastolic ventricular volumes ( P < 0.05) from week 2 together with a decrease in ejection fraction and in mean velocity of circumferential shortening ( P < 0.05) from week 1. These changes were tightly correlated to myocardial ppET-1 and renal ETAreceptor mRNA and less so to myocardial ECE-1 mRNA, and they occurred before any increase in plasma and urinary ET-1 ( P < 0.05 from week 4) and clinical signs of heart failure. Renal ppET-1 did not change. Both cardiac and renal ET-1 peptide contents were increased at autopsy. We conclude that tachycardia-induced heart failure in dogs is characterized by an early activation of the cardiac and renal tissue endothelin systems, which occurs before any changes in circulating and urinary ET-1 and is closely related to altered ventricular function.
No ischemic preconditioning in heterozygous connexin43-deficient mice
Authors: Schwanke Uwe , Konietzka Ina , Duschin Alexej , Li Xiaokui , Schulz Rainer , Heusch Gerd
Published: March 2015

Protein kinase Cε (PKCε) plays a central role in ischemic preconditioning (IP) in mice and rabbits, and activated PKCε colocalizes with and phosphorylates connexin43 (Cx43) in rats and humans. Whether or not Cx43 contributes to the mechanism(s) of IP in vivo is yet unknown. Therefore, wild-type (n = 8) and heterozygous Cx43-deficient mice (n = 8) were subjected to 30 min occlusion and 120 min reperfusion of the left anterior descending coronary artery. IP was induced by one cycle of 5 min occlusion and 10 min reperfusion ( n = 8/8 mice) before the sustained occlusion. Infarct size was reduced by IP in wild-type mice [11.3 ± 3.4% vs. 23.7 ± 7.2% of the left ventricle (LV), P < 0.05] but not in Cx43-deficient mice (26.0 ± 6.0% vs. 25.1 ± 3.8% of LV). Also, three cycles of 5 min occlusion and 10 min reperfusion (n = 5) did not induce protection in Cx43-deficient mice (27.6 ± 5.5 % of LV). Thus Cx43 contributes to the protection of IP in mice in vivo.
Relative importance of malonyl CoA and carnitine in maturation of fatty acid oxidation in newborn rabbit heart
Authors: Besikci Arzu Onay , Campbell Fiona M. , Hopkins Teresa A. , Dyck Jason R. B. , Lopaschuk Gary D.
Published: March 2015

After birth, a dramatic increase in fatty acid oxidation occurs in the heart, which has been attributed to an increase in l-carnitine levels and a switch from the liver (L) to muscle (M) isoform of carnitine palmitoyltransferase (CPT)-1. However, because M-CPT-1 is more sensitive to inhibition by malonyl CoA, a potent endogenous regulator of fatty acid oxidation, a switch to the M-CPT-1 isoform should theoretically decrease fatty acid oxidation. Because of this discrepancy, we assessed the contributions of myocardiall-carnitine content and CPT-1 isoform expression and kinetics to the maturation of fatty acid oxidation in newborn rabbit hearts. Although fatty acid oxidation rates increased between 1 and 14 days after birth, myocardial l-carnitine concentrations did not increase. Changes in the expression of L-CPT-1 or M-CPT-1 mRNA after birth also did not parallel the increase in fatty acid oxidation. The Kmof CPT-1 for carnitine and the IC50for malonyl CoA remained unchanged between 1 and 10 days after birth. However, malonyl CoA levels dramatically decreased, due in part to an increase in malonyl CoA decarboxylase activity. Our data suggest that a decrease in malonyl CoA control of CPT-1 is primarily responsible for the increase in fatty acid oxidation seen in the newborn heart.
Acetoacetate augments β-adrenergic inotropism of stunned myocardium by an antioxidant mechanism
Authors: Squires Jeffrey E. , Sun Jie , Caffrey James L. , Yoshishige Darice , Mallet Robert T.
Published: March 2015

Blunted β-adrenergic inotropism in stunned myocardium is restored by pharmacological ( N-acetylcysteine) and metabolic (pyruvate) antioxidants. The ketone body acetoacetate is a natural myocardial fuel and antioxidant that improves contractile function of prooxidant-injured myocardium. The impact of acetoacetate on postischemic cardiac function and β-adrenergic signaling has never been reported. To test the hypothesis that acetoacetate restores contractile performance and β-adrenergic inotropism of stunned myocardium, postischemic Krebs-Henseleit-perfused guinea pig hearts were treated with 5 mM acetoacetate and/or 2 nM isoproterenol at 15–45 and 30–45 min of reperfusion, respectively, while cardiac power was monitored. The myocardium was snap frozen, and its energy state was assessed from phosphocreatine phosphorylation potential. Antioxidant defenses were assessed from GSH/GSSG and NADPH/NADP+redox potentials. Stunning lowered cardiac power and GSH redox potential by 90 and 70%, respectively. Given separately, acetoacetate and isoproterenol each increased power and GSH redox potential three- to fivefold. Phosphocreatine potential was 70% higher in acetoacetate- vs. isoproterenol-treated hearts ( P < 0.01). In combination, acetoacetate and isoproterenol synergistically increased power and GSH redox potential 16- and 7-fold, respectively, doubled NADPH redox potential, and increased cAMP content 30%. The combination increased cardiac power four- to sixfold vs. the individual treatments without a coincident increase in phosphorylation potential. Potentiation of isoproterenol's inotropic actions endured even after acetoacetate was discontinued and GSH potential waned, indicating that temporary enhancement of redox potential persistently restored β-adrenergic mechanisms. Thus acetoacetate increased contractile performance and potentiated β-adrenergic inotropism in stunned myocardium without increasing energy reserves, suggesting its antioxidant character is central to its beneficial actions.
Mechanisms of Arg-Pro-Pro-Gly-Phe inhibition of thrombin
Authors: Hasan Ahmed A. K. , Warnock Mark , Nieman Marvin , Srikanth Sujata , Mahdi Fakhri , Krishnan Raman , Tulinsky Alexander , Schmaier Alvin H.
Published: March 2015

Investigations determined the mechanism(s) by which Arg-Pro-Pro-Gly-Phe (RPPGF) inhibits thrombin-induced platelet activation. High concentrations of RPPGF inhibit thrombin-induced coagulant activity. RPPGF binds to the active site of thrombin by forming a parallel β-strand with Ser214-Gly216and interacts with His57, Asp189, and Ser195of the catalytic triad. RPPGF competitively inhibits α-thrombin from hydrolyzing Sar-Pro-Arg-paranitroanilide with a Ki= 1.75 ± 0.03 mM. Other mechanisms were sought to explain why RPPGF inhibits thrombin activation of platelets at concentrations below that which inhibits its active site. Soluble RPPGF blocks biotinylated NATLDPRSFLLR of the thrombin cleavage site on protease-activated receptor (PAR)1 from binding to the peptide RPPGC (IC50= 20 μM). The soluble recombinant extracellular domain of PAR1 (rPAR1EC) blocks biotinylated RPPGF binding to rPAR1EC(IC50= 50 μM) bound to microtiter plates, but rPAR1ECdeletion mutants missing the sequence LDPR or PRSF do not. RPPGF and related forms prevent the thrombin-like enzyme thrombocytin from proteolyzing rPAR1ECat concentrations that do not block thrombocytin's active site. These studies indicate that RPPGF is a bifunctional inhibitor of thrombin: it binds to PAR1 to prevent thrombin cleavage at Arg41and interacts with the active site of α-thrombin.
Load dependence of ventricular performance explained by model of calcium-myofilament interactions
Authors: Shimizu Juichiro , Todaka Koji , Burkhoff Daniel
Published: March 2015

Although a simple concept of load-independent behavior of the intact heart evolved from early studies of isolated, intact blood-perfused hearts, more recent studies showed that, as in isolated muscle, the mode of contraction (isovolumic vs. ejection) impacts on end-systolic elastance. The purpose of the present study was to test whether a four-state model of myofilament interactions with length-dependent rate constants could explain the complex contractile behavior of the intact, ejecting heart. Studies were performed in isolated, blood-perfused canine hearts with intracellular calcium transients measured by macroinjected aequorin. Measured calcium transients were used as the driving function for the model, and length-dependent rate constants yielding the highest concordance between measured and model-predicted midwall stress at different isovolumic volumes were determined. These length-dependent rate constants successfully predicted contractile behavior on ejecting contractions. This, along with additional model analysis, suggests that length-dependent changes in calcium binding affinity may not be an important factor contributing to load-dependent contractile performance in the intact heart under physiological conditions.
Increasing plasmalogen levels protects human endothelial cells during hypoxia
Authors: Zoeller Raphael A. , Grazia Todd J. , LaCamera Peter , Park James , Gaposchkin Daniel P. , Farber Harrison W.
Published: March 2015

Supplementation of cultured human pulmonary arterial endothelial cells (PAEC) with sn-1- O-hexadecylglycerol (HG) resulted in an approximately twofold increase in cellular levels of plasmalogens, a subclass of phospholipids known to have antioxidant properties; this was due, primarily, to a fourfold increase in the choline plasmalogens. Exposure of unsupplemented human PAEC to hypoxia (Po2= 20–25 mmHg) caused an increase in cellular reactive oxygen species (ROS) over a period of 5 days with a coincident decrease in viability. In contrast, HG-supplemented cells survived for at least 2 wk under these conditions with no evidence of increased ROS. Hypoxia resulted in a selective increase in the turnover of the plasmalogen plasmenylethanolamine. Human PAEC with elevated plasmalogen levels were also more resistant to H2O2, hyperoxia, and the superoxide generator plumbagin. This protection was seemingly specific to cellular stresses in which significant ROS were generated because the sensitivity to lethal heat shock or glucose deprivation was not altered in HG-treated human PAEC. HG, by itself, was not sufficient for protection; HG supplementation of bovine PAEC had no effect upon plasmalogen levels and did not rescue these cells from the cytotoxic effects of hypoxia. This is the initial demonstration that plasmalogen content can be substantially enhanced in a normal cell. These data also demonstrate that HG can protect cells during hypoxia and other ROS-mediated stress, likely due to the resulting increase in these antioxidant phospholipids.
Redox-regulated mechanisms of IL-4-induced MCP-1 expression in human vascular endothelial cells
Authors: Lee Yong Woo , Hennig Bernhard , Toborek Michal
Published: March 2015

The present study focused on the molecular signaling pathways of monocyte chemoattractant protein-1 (MCP-1) induction by interleukin-4 (IL-4) in human umbilical vein endothelial cells (HUVEC). RT-PCR showed that MCP-1 mRNA accumulation was markedly increased in IL-4-treated HUVEC in a time- and dose-dependent manner. Antioxidants, such as pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC), significantly inhibited IL-4-induced MCP-1 mRNA expression. These effects correlated well with the PDTC-mediated inhibition of MCP-1 promoter transcriptional activity observed in IL-4-treated HUVEC. IL-4-induced MCP-1 gene expression was paralleled by a concomitant production of MCP-1 protein. In agreement with MCP-1 gene expression, PDTC attenuated IL-4-mediated induction of MCP-1 protein expression. In addition, IL-4 dramatically increased the transcription factor signal transducers and activators of transcription 1 (STAT1) DNA binding activity, an effect that was attenuated by PDTC. The role of STAT1 in the regulation of the IL-4-induced MCP-1 gene expression was further confirmed in HUVEC transfected with a reporter construct of the MCP-1 promoter with a mutated STAT1 binding site. These results demonstrate that IL-4-dependent MCP-1 induction in HUVEC is mediated by redox-regulated STAT1 activation.
Recruitment of smooth muscle cells and arterial vasomotion
Authors: Lamboley Mathieu , Schuster Alexander , Bény Jean-Louis , Meister Jean-Jacques
Published: March 2015

Investigating the recruitment and synchronization of smooth muscle cells (SMCs) is the key to understanding the physical mechanisms leading to contraction and spontaneous diameter oscillations of arteries, called vasomotion. We improved a method that allows the correlation of calcium oscillations (flashing) of individual SMCs with mean calcium variations and arterial contraction using confocal microscopy. Endothelium-stripped rat mesenteric arteries were cut open, loaded with dual calcium fluorescence probes, and stimulated by increasing concentrations of the vasoconstrictors phenylephrine (PE) and KCl. We found that the number and synchronization of flashing cells depends on vasoconstrictor concentration. At low vasoconstrictor concentration, few cells flash asynchronously and no local contraction is detected. At medium concentration, recruitment of cells is complete and synchronous, leading to strip contraction after KCl stimulation and to vasomotion after PE stimulation. High concentration of PE leads to synchronous calcium oscillations and fully contracted vessels, whereas high concentration of KCl leads to a sustained nonoscillating increase of calcium and to fully contracted vessels. We conclude that the number of simultaneously recruited cells is an important factor in controlling rat mesenteric artery contraction and vasomotion.
Differential effects of heart rate reduction and β-blockade on left ventricular relaxation during exercise
Authors: Colin Patrice , Ghaleh Bijan , Hittinger Luc , Monnet Xavier , Slama Michel , Giudicelli Jean-François , Berdeaux Alain
Published: March 2015

Left ventricular (LV) relaxation is crucial for LV function, especially during exercise. We compared the effects of increasing doses of ivabradine, a selective inward hyperpolarization-activated current inhibitor, and atenolol on the rate and extent of LV relaxation (best fit method: time constant τBF, pressure asymptote PBF) at rest and during exercise. Eight dogs were chronically instrumented to measure LV pressure and LV wall stresses. During exercise under saline, heart rate increased from 108 ± 5 to 220 ± 6 beats/min and τBFwas significantly reduced from 22 ± 1 to 14 ± 2 ms. At rest, atenolol but not ivabradine increased τBF. For similar heart rate reductions during exercise, atenolol impeded the shortening of τBF(23 ± 2 ms) whereas ivabradine had no effect (15 ± 2 ms). The extent of the relaxation process (PBF) at peak exercise was increased by ivabradine, and to a greater extent by atenolol, compared with saline. Thus, for a similar reduction in heart rate at rest and during exercise, ivabradine, in contrast with atenolol, does not exert any negative lusitropic effect.
Gender differences in myosin heavy chain-β and phosphorylated phospholamban in diabetic rat hearts
Authors: Zhong Yan , Reiser Peter J , Matlib Mohammed A.
Published: March 2015

The objective of this study was to determine whether a gender difference exists in myosin heavy chain (MHC) isoform or sarcoplasmic reticulum protein levels in diabetic rat hearts. As is the case with normal rodent hearts, all four chambers of the control rat hearts expressed almost 100% MHC-α. In 6-wk diabetic rats, MHC-β expression in ventricles of males was significantly greater (78 ± 7%) than in females (50 ± 5%). The cardiac sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) protein level was decreased and the phospholamban (PLB) protein level was increased in the left ventricle of diabetic rats, but there was no difference between male and female diabetic rats. The phosphorylated PLB level was decreased more in male than in female diabetic rats. Insulin treatment completely normalized blood glucose level, cardiac SERCA2a and PLB protein levels, and the decrease in MHC-β levels in both male and female diabetic rats. Insulin treatment completely normalized serum insulin and almost completely normalized phosphorylation of PLB at serine 16 in male diabetic rats. Although insulin treatment completely normalized serum insulin levels in male diabetic rats, in females it only partially normalized serum insulin levels. Also, insulin treatment almost completely normalized phosphorylation of PLB at threonine 17 in female diabetic rats; however, the increase was significantly greater than that identified for insulin-treated male diabetic rats. We conclude that higher levels of MHC-β and dephosphorylated PLB may contribute to more contractile dysfunction in male than in female diabetic rat hearts, and that phosphorylation of PLB at threonine 17 is more responsive to insulin in female diabetic rat hearts.
Repeated inhalation of adrenomedullin ameliorates pulmonary hypertension and survival in monocrotaline rats
Authors: Nagaya Noritoshi , Okumura Hiroyuki , Uematsu Masaaki , Shimizu Wataru , Ono Fumiaki , Shirai Mikiyasu , Mori Hidezo , Miyatake Kunio , Kangawa Kenji
Published: March 2015

Adrenomedullin (AM) is a potent vasodilator peptide. We investigated whether inhalation of aerosolized AM ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats. Male Wistar rats given MCT (MCT rats) were assigned to receive repeated inhalation of AM ( n = 8) or 0.9% saline ( n = 8). AM (5 μg/kg) or saline was inhaled as an aerosol using an ultrasonic nebulizer for 30 min four times a day. After 3 wk of inhalation therapy, mean pulmonary arterial pressure and total pulmonary resistance were markedly lower in rats treated with AM than in those given saline [mean pulmonary arterial pressure: 22 ± 2 vs. 35 ± 1 mmHg (–37%); total pulmonary resistance: 0.048 ± 0.004 vs. 0.104 ± 0.006 mmHg · ml–1 · min–1 · kg–1 (–54%), both P < 0.01]. Neither systemic arterial pressure nor heart rate was altered. Inhalation of AM significantly attenuated the increase in medial wall thickness of peripheral pulmonary arteries in MCT rats. Kaplan-Meier survival curves demonstrated that MCT rats treated with aerosolized AM had a significantly higher survival rate than those given saline (70% vs. 10% 6-wk survival, log-rank test, P < 0.01). In conclusion, repeated inhalation of AM inhibited MCT-induced pulmonary hypertension without systemic hypotension and thereby improved survival in MCT rats.
Potassium channel openers protect cardiac mitochondria by attenuating oxidant stress at reoxygenation
Authors: Ozcan Cevher , Bienengraeber Martin , Dzeja Petras P. , Terzic Andre
Published: March 2015

K+ channel openers have been recently recognized for their ability to protect mitochondria from anoxic injury. Yet the mechanism responsible for mitochondrial preservation under oxidative stress is not fully understood. Here, mitochondria were isolated from rat hearts and subjected to 20-min anoxia, followed by reoxygenation. At reoxygenation, increased generation of reactive oxygen species (ROS) was associated with reduced ADP-stimulated oxygen consumption, blunted ATP production, and disrupted mitochondrial structural integrity coupled with cytochrome c release. The prototype K+ channel opener diazoxide markedly reduced mitochondrial ROS production at reoxygenation with a half-maximal effect of 29 μM. Diazoxide also preserved oxidative phosphorylation and mitochondrial membrane integrity, as indicated by electron microscopy and reduced cytochrome c release. The protective effect of diazoxide was reproduced by the structurally distinct K+ channel opener nicorandil and antagonized by 5-hydroxydecanoic acid, a short-chain fatty acid derivative and presumed blocker of mitochondrial ATP-sensitive K+ channels. Opener-mediated mitochondrial protection was simulated by the free radical scavenger system composed of superoxide dismutase and catalase. However, the effect of openers on ROS production was maintained in nominally K+-free medium in the presence or absence of the K+ ionophore valinomycin and was mimicked by malonate, a modulator of the mitochondrial redox state. This suggests the existence of a K+ conductance-independent pathway for mitochondrial protection targeted by K+ channel openers. Thus the cardioprotecive mechanism of K+ channel openers includes direct attenuation of mitochondrial oxidant stress at reoxygenation.
Reduced inotropic heart response in selenium-deficient mice relates with inducible nitric oxide synthase
Authors: Gomez Ricardo M. , Levander Orville A. , Sterin-Borda Leonor
Published: March 2015

Atria from mice fed a selenium-deficient (Se−) diet have a diminished β-adrenoceptor-inotropic cardiac response to isoproterenol or norepinephrine compared with atria from mice fed the same diet supplemented with 0.2 mg/kg Se as sodium selenite (Se+). This diminished response could be reversed by feeding Se− mice the Se+ diet for 1 wk or by pretreatment with nitric oxide synthase (NOS) inhibitors such as N G-monomethyl-l-arginine or aminopyridine. Elevated serum concentrations of nitrite/nitrate as well as a threefold increase in the atrial NOS activity were seen in the Se− versus Se+ mice. Western blotting and indirect immunofluorescence indicated an enhanced expression of inducible NOS in hearts from Se− mice. Increased expression and activity of NOS and increased nitrite/nitrate levels from Se− mice correlated with an impaired response to β-adrenoceptor inotropic cardiac stimulation. Elevated nitric oxide levels may account for some of the pathophysiological effects of Se deficiency on the heart.
Hypoxic vasorelaxation inhibition by organ culture correlates with loss of Kv channels but not Ca2+ channels
Authors: Thorne George D. , Conforti Laura , Paul Richard J.
Published: March 2015

We (Thorne GD, Shimizu S, and Paul RJ. Am J Physiol Cell Physiol 281: C24–C32, 2001) have recently shown that organ culture for 24 h specifically inhibits relaxation to acute hypoxia (95% N2-5% CO2) in the porcine coronary artery. Here we show similar results in the porcine carotid artery and the rat and mouse aorta. In the coronary artery, part of the inability to relax to hypoxia after organ culture is associated with a concomitant loss in ability to reduce intracellular Ca2+ concentration ([Ca2+]i) during hypoxia (Thorne GD, Shimizu S, and Paul RJ. Am J Physiol Cell Physiol 281: C24–C32, 2001). To elucidate the mechanisms responsible for the loss of relaxation to hypoxia, we investigated changes in K+ and Ca2+ channel activity and gene expression that play key roles in [Ca2+]iregulation in vascular smooth muscle (VSM). Reduced mRNA expression of O2-sensitive K+ channels (Kv1.5 and Kv2.1) was shown by reverse transcriptase-polymerase chain reaction in the rat aorta. In contrast, no change in other expressed voltage-gated K+ channels (Kv1.2 and Kv1.3) or Ca2+ channel subtypes was found. Modified K+ channel expression is supported by functional evidence indicating a reduced response to general K+ channel activation, by pinacidil, and to specific voltage-dependent K+ (Kv) channel blockade by 4-aminopyridine. In conclusion, organ culture decreases expression of specific Kv channels. These changes are consistent with altered mechanisms of VSM contractility that may be involved in Ca2+-dependent pathways of hypoxia-induced vasodilation.
Flow heterogeneity following global no-flow ischemia in isolated rabbit heart
Authors: Marshall Robert C. , Powers-Risius Patricia , Reutter Bryan W. , Schustz Amy M. , Kuo Chaincy , Huesman Michelle K. , Huesman Ronald H.
Published: March 2015

The purpose of this study was to evaluate flow heterogeneity and impaired reflow during reperfusion after 60-min global no-flow ischemia in the isolated rabbit heart. Radiolabeled microspheres were used to measure relative flow in small left ventricular (LV) segments in five ischemia + reperfused hearts and in five nonischemic controls. Relative flow heterogeneity was expressed as relative dispersion (RD) and computed as standard deviation/mean. In postischemic vs. preischemic hearts, RD was increased for the whole LV (0.92 ± 0.41 vs. 0.37 ± 0.07, P < 0.05) as well as the subendocardium (Endo) and subepicardium considered separately (1.28 ± 0.74 vs. 0.30 ± 0.09 and 0.69 ± 0.22 vs. 0.38 ± 0.08; P < 0.05 for both comparisons, respectively) during early reperfusion. During late reperfusion, the increased RD for the whole LV and Endo remained significant (0.70 ± 0.22 vs. 0.37 ± 0.07 and 1.06 ± 0.55 vs. 0.30 ± 0.09; P < 0.05 for both comparisons, respectively). In addition to the increase in postischemic flow heterogeneity, there were some regions demonstrating severely impaired reflow, indicating that regional ischemia can persist despite restoration of normal global flow. Also, the relationship between regional and global flow was altered by the increased postischemic flow heterogeneity, substantially reducing the significance of measured global LV reflow. These observations emphasize the need to quantify regional flow during reperfusion after sustained no-flow ischemia in the isolated rabbit heart.