bluuebunny/crossref_metadata_2025 huggingface dataset:
Stretch force on vascular smooth muscle cells enhances oxidation of LDL via superoxide production
Authors: Inoue Nobutaka , Kawashima Seinosuke , Hirata Ken-Ichi , Rikitake Yoshiyuki , Takeshita Saori , Yamochi Wataru , Akita Hozuka , Yokoyama Mitsuhiro
Published: December 2017

Hemodynamic forces on vasculature profoundly influence atherogenesis. We examined the effect of stretch force on the oxidation of low-density lipoprotein (LDL) by rat aortic smooth muscle cells (RASM) and superoxide production. Stretch force was imposed on RASM cultured on deformable dishes by stretching the dishes. Incubation of native LDL with static RASM for 24 h resulted in LDL oxidation as indicated by increases in thiobarbituric acid-reacting substances from 9.5 ± 2.3 to 24.5 ± 2.3 nmol malondialdehyde/mg. Stretch force on RASM augmented cell-mediated LDL oxidation to 149.3 ± 17.1% concomitantly with increase in superoxide production. LDL oxidation was inhibited by superoxide dismutase or depletion of the metal ion in the culture medium, indicating that it was a metal ion-dependent and superoxide-mediated process. The enhancement of LDL oxidation by stretch force was inhibited by diphenyliodonium, indicating the involvement of the NADH/NADPH oxidase system. Our findings suggest that the increased oxidant stress induced by stretch force is one of the potential mechanisms whereby hypertension facilitates atherosclerosis.
Disparate effects of biphasic and monophasic shocks on postshock refractory period dispersion
Authors: Sims J. Jason , Miller Allison Winecoff , Ujhelyi Michael R.
Published: December 2017

The magnitude by which a defibrillation shock extends the refractory period immediately postshock (refractory period extension, RPE) does not explain why biphasic shocks defibrillate with greater efficacy than monophasic shocks. It may be that spatial heterogeneity of RPE is a more important regulator of defibrillation efficacy. We measured RPE in 15 pentobarbital-anesthetized swine using 400-V biphasic and monophasic shocks of equal pulse duration at three discrete myocardial sites. Spatial heterogeneity of RPE was calculated as the difference between the maximum and minimum values of the three recording sites. Monophasic shocks produced greater magnitude of RPE than biphasic shocks at all sites tested (82 ± 6 to 99 ± 13 and 64 ± 6 to 68 ± 5 ms, respectively; P < 0.05). However, RPE dispersion was significantly less with biphasic shocks versus monophasic shocks (29 ± 4 and 48 ± 7 ms, respectively; P < 0.05). This suggests that one potential mechanism by which biphasic shocks defibrillate with greater efficacy is limiting postshock spatial heterogeneity of refractoriness. Thus these data support our hypothesis that RPE heterogeneity is a more likely predictor of defibrillation efficacy than magnitude of RPE.
Increased superoxide anion generation and altered vasoreactivity in rabbits on low-potassium diet
Authors: Yang B. C. , Li D. Y. , Weng Y. F. , Lynch J. , Wingo C. S. , Mehta J. L.
Published: December 2017

Potassium reduces blood pressure in populations at high risk of developing hypertension, which suggests that potassium depletion may increase vascular resistance. This study was designed to examine the effect of potassium depletion on thel-arginine-nitric oxide pathway in arterial tissues. New Zealand White rabbits were fed either a control diet, containing a normal amount of potassium, or a low-potassium diet for 1–3 wk. As expected, the low-potassium diet resulted in reduced serum and urinary potassium levels. Carotid arteries were excised, and their contractile and relaxant responses were determined in vitro. Carotid arterial ring contractile response to norepinephrine was enhanced, and relaxation in response to the endothelium-dependent vasodilators acetylcholine and calcium ionophore A-23187 was attenuated, in rabbits fed low-potassium diet (all P < 0.01 compared with responses in rabbits fed control diet). The vasomotor responses were similarly altered in rabbits fed low-potassium diet for 1 or 3 wk. Both the enhanced contraction and attenuated relaxation were abolished by treatment of arterial rings with superoxide dismutase but not by treatment with l-arginine or indomethacin. Carotid artery rings from rabbits fed the low-potassium diet showed ∼100% greater superoxide anion formation than those from rabbits fed control diet ( P < 0.01), whereas plasma and urinary nitrite levels were similar in both groups of rabbits. These observations indicate that low-potassium diet enhances the sensitivity of the carotid artery to vasoconstrictor stimuli and reduces the sensitivity to endothelium-dependent stimuli. Attenuation of endothelium-dependent relaxation appears to be secondary to increased free radical generation, which may degrade nitric oxide. Altered vasoreactivity may underlie the genesis of hypertension in populations consuming diets low in potassium.
Information scaling properties of heart rate variability
Authors: Roach Daniel E. , Sheldon Robert S.
Published: December 2017

Many chaos detection methods have proven inherently ambiguous in that they yield similar results for chaotic signals and correlated noise. The purpose of this work was to determine whether human resting heart period sequences have global properties characteristic of chaotic systems. We investigated the inherent global organization of heart period sequences by quantifying how the information content of the embedded sequences varied as a function of scale. We compared the information scaling characteristics of 60-min heart period sequences obtained from 10 healthy resting volunteers with those obtained from numerous periodic and chaotic control sequences. The information scaling properties of the heart period sequences were significantly different from those obtained for the controls, particularly at the coarsest scales ( P = 0.0003 vs. low-dimensional periodic controls; P = 0.0005 vs. low-dimensional chaotic controls; P = 0.0003 vs. low-dimensional periodic and chaotic controls). We also showed that nondeterministic components, such as large tachycardic (or bradycardic) events or aperiodic fluctuations, can lead to scaling characteristics similar to those observed for the resting heart period sequences. This, in addition to previous evidence from spectral, nonlinear predictability and lexical studies, favors an events-based approach to understanding heart rate variability.
A superfusion system to study border zones in confluent cultures of neonatal rat heart cells
Authors: Hyatt Christopher J. , Lemasters John J. , Muller-Borer Barbara J. , Johnson Timothy A. , Cascio Wayne E.
Published: December 2017

We present a new experimental method to study intracellular ion regulation in cultured cardiomyocytes at a border zone separating two different and distinct environments. Our system uses a dual-flow superfusion chamber to produce two different but adjacent environments over a monolayer of cardiomyocytes. Fluorescent microscopy of fluorescein showed that the transition between the two environments was nearly linear and was 220–320 μm wide depending on fluid viscosity and velocity. We superfused cultured monolayers on one side with a solution at pH 6.5 and on the other side with a solution at pH 7.4. We observed a sharply demarcated difference in intracellular pH (pHi) between the two halves of the cell monolayer as measured with the fluorescent pHi indicator carboxy-seminaphthorhodafluor-1. The demarcation of pHi corresponded well with the demarcation of the border measured with fluorescein. We conclude that our superfusion system will facilitate the study of intercellular communication and interactions across boundaries of cardiac tissue where different ionic or metabolic conditions are present, for example, between ischemic and nonischemic myocardium.
Intrinsic neural regulation of the heart in the chronic, conscious dog
Authors: Priola Donald V. , Cao Xiaoling , Anagnostelis Constantine , Bassenge Eberhard
Published: December 2017

The present experiments were performed to examine the capability of the intrinsic cardiac nerves (ICN) to modify cardiac performance in the resting chronic, conscious dog. Control and cardiac-denervated dogs were instrumented for recording of left atrial (LA) and ventricular (LV) contractility, heart rate, and atrioventricular (AV) conduction time. Acetylcholine (ACh) and nicotine (Nic) were administered via an indwelling coronary artery catheter. Limited distribution from the injection site only allowed access to the LA, LV, and AV node. Both β-blockade with timolol and cardiac denervation were used to separate direct effects of ICN stimulation from indirect (e.g., reflex) effects. ACh produced the expected negative inotropic and dromotropic changes. ICN stimulation with Nic caused large decreases in LA contractility along with depression of AV conduction but only trivial effects on the LV. We concluded that the ICN has limited effects on cardiac performance in the resting animal under minimal sympathetic drive. It is likely, however, that the ICN is capable of significantly depressing cardiac function under conditions of elevated sympathetic tone as would be encountered in exercise.
Postnatal changes in contractile time parameters, calcium regulatory proteins, and phosphatases
Authors: Gombosová Iva , Bokník Peter , Kirchhefer Uwe , Knapp Jörg , Lüss Hartmut , Müller Frank Ulrich , Müller Thorsten , Vahlensieck Ute , Schmitz Wilhelm , Bodor Geza S. , Neumann Joachim
Published: December 2017

Compared with isolated electrically driven neonatal ventricular preparations, the total time of contraction, the time to peak tension, and the time of relaxation were decreased to ∼50% in adult ventricular preparations. The expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) was increased to 133% at the protein level and to 154% at the mRNA level in adult vs. neonatal ventricular preparations, whereas phospholamban was unchanged at both the protein and mRNA levels. Moreover, Ca2+uptake was increased to 180% in adult vs. neonatal ventricular preparations. Phospholamban phosphorylation was enhanced in adult vs. neonatal ventricular preparations. In adult ventricular preparations, phosphatase activity was reduced to 53% of neonatal preparations, the protein levels of the immunologically detectable catalytic subunits of protein phosphatase types 1 and 2A were reduced to 28 and 61% of neonatal preparations, respectively, and the mRNA levels of type 1α, 1β, 1γ, 2Aα, and 2Aβ phosphatase isoforms were decreased to 69, 68, 54, 67, and 63%, respectively. We conclude that in the adult rat heart, the shortened time parameters of contraction can be explained by an elevated expression of SERCA. In addition, an increased phosphorylation state of phospholamban due to reduced phosphatase activity may be involved.
Right coronary autoregulation in conscious, chronically instrumented dogs
Authors: Bian Xiaoming , Williams Arthur G. , Gwirtz Patricia A. , Downey H. Fred
Published: December 2017

Right coronary (RC) autoregulation and right ventricular (RV) function were assessed in conscious dogs, chronically instrumented to measure RC flow and RC pressure (RCP) as a hydraulic occluder on the RC was inflated. Dogs were then anesthetized, and RC autoregulation and RV function were again assessed. In the conscious state, moderate RC autoregulation was present with closed loop gains (Gc) of 0.59–0.27 as RCP was reduced from 100 to 40 mmHg. In the anesthetized state, Gcwas not significantly less than in the conscious state at RCP >50 mmHg. The range and potency of RV autoregulation were greater in both groups than for previously reported findings in anesthetized dogs with RC perfused by an extracorporeal system. RV contractile function was well maintained in conscious and anesthetized dogs at RCP >45 mmHg. We conclude the following: 1) modest RC autoregulation is present in the conscious dog, 2) anesthesia limits the range but not the degree of RC autoregulation, 3) extracorporeal perfusion systems appear to depress RC autoregulation, and 4) RV contractile function remains constant in both conscious and anesthetized dogs until RCP falls below 50 mmHg.
Metabolic effects of aldose reductase inhibition during low-flow ischemia and reperfusion
Authors: Ramasamy Ravichandran , Trueblood Nathan , Schaefer Saul
Published: December 2017

Several studies have shown that maintenance of glycolysis limits the metabolic and functional consequences of low-flow ischemia. Because diabetic animals are known to have impaired glycolytic metabolism coupled with increased flux through the aldose reductase (AR) pathway, we hypothesized that inhibition of AR would enhance glycolysis and thereby improve metabolic and functional recovery during low-flow ischemia. Hearts ( n = 12) from nondiabetic control and diabetic rats were isolated and retrograde perfused using 11 mM glucose with or without the AR inhibitor zopolrestat (1 μM). Hearts were subjected to 30 min of low-flow ischemia (10% of baseline flow) and 30 min of reperfusion.31P NMR spectroscopy was used to monitor time-dependent changes in phosphocreatine (PCr), ATP, and intracellular pH. Changes in the cytosolic redox ratio of NADH to NAD+were obtained by measuring the ratio of tissue lactate to pyruvate. Effluent lactate concentrations and oxygen consumption were determined from the perfusate. AR inhibition improved functional recovery in both control and diabetic hearts, coupled with a lower cytosolic redox state and greater effluent lactate concentrations during ischemia. ATP levels during ischemia were significantly higher in AR-inhibited hearts, as was recovery of PCr. In diabetic hearts, AR inhibition also limited acidosis during ischemia and normalized pH recovery on reperfusion. These data demonstrate that AR inhibition maintains higher levels of high-energy phosphates and improves functional recovery upon reperfusion in hearts subjected to low-flow ischemia, consistent with an increase in glycolysis. Accordingly, this approach of inhibiting AR offers a novel method for protecting ischemic myocardium.
Aquaporin-1 and endothelial nitric oxide synthase expression in capillary endothelia of human peritoneum
Authors: Devuyst Olivier , Nielsen Soren , Cosyns Jean-Pierre , Smith Barbara L. , Agre Peter , Squifflet Jean-Paul , Pouthier Dominique , Goffin Eric
Published: December 2017

Water transport during peritoneal dialysis (PD) requires ultrasmall pores in the capillary endothelium of the peritoneum and is impaired in the case of peritoneal inflammation. The water channel aquaporin (AQP)-1 has been proposed to be the ultrasmall pore in animal models. To substantiate the role of AQP-1 in the human peritoneum, we investigated the expression of AQP-1, AQP-2, and endothelial nitric oxide synthase (eNOS) in 19 peritoneal samples from normal subjects ( n = 5), uremic patients treated by hemodialysis ( n = 7) or PD ( n = 4), and nonuremic patients ( n = 3), using Western blotting and immunostaining. AQP-1 is very specifically located in capillary and venule endothelium but not in small-size arteries. In contrast, eNOS is located in all types of endothelia. Immunoblot for AQP-1 in human peritoneum reveals a 28-kDa band (unglycosylated AQP-1) and diffuse bands of 35–50 kDa (glycosylated AQP-1). Although AQP-1 expression is remarkably stable in all samples whatever their origin, eNOS (135 kDa) is upregulated in the three patients with ascites and/or peritonitis (1 PD and 2 nonuremic patients). AQP-2, regulated by vasopressin, is not expressed at the protein level in human peritoneum. This study 1) supports AQP-1 as the molecular counterpart of the ultrasmall pore in the human peritoneum and 2) demonstrates that AQP-1 and eNOS are regulated independently of each other in clinical conditions characterized by peritoneal inflammation.
PTX-sensitive G proteins and permissive action of prostacyclin in newborn pig cerebral circulation
Authors: Zucker Boruch , Leffler Charles W.
Published: December 2017

The present study of newborn pig cerebral circulation investigated the role of pertussis toxin (PTX)-sensitive GTP binding proteins in the permissive action of prostacyclin in specific dilator responses. Pial arterioles of anesthetized piglets were observed through closed cranial windows. The piglets were treated topically with PTX and intravenously with indomethacin. The effects of hypercapnia (10% CO2ventilation) and topical 5,6-epoxyeicosatrienoic acid (5,6-EET) on pial arteriolar diameter were noted before and after the intervention. Samples of the artificial cerebrospinal fluid (aCSF) were collected from beneath the cranial windows for determination of the cAMP concentration. After administration of PTX, indomethacin still abolished pial arteriolar dilation to both hypercapnia and 5,6-EET and also inhibited the cAMP elevation caused by hypercapnia. The addition of phorbol 12-myristate 13-acetate (PMA), but not iloprost, restored the increase in cAMP and vascular responses to hypercapnia and 5,6-EET. Therefore, in the newborn pig cerebral microvasculature, PTX appears to inhibit a G protein involved in the permissive action of prostacyclin. However, the protein kinase C (PKC) activator PMA appears to act downstream from the block, and, therefore, the permissive action of PMA is not affected by PTX. We suggest that the prostacyclin IP receptor may be coupled to phospholipase C via a PTX-sensitive G protein that normally permits vasodilation to specific stimuli via activation of a PKC, resulting in phosphorylation of a component of the adenylyl cyclase pathway.
Intramitochondrial [Ca2+] and membrane potential in ventricular myocytes exposed to anoxia-reoxygenation
Authors: Delcamp T. J. , Dales C. , Ralenkotter L. , Cole P. S. , Hadley R. W.
Published: December 2017

The aim of this study was to investigate the role of mitochondrial ionic homeostasis in promoting reoxygenation-induced hypercontracture in cardiac muscle. Mitochondrial membrane potential and intramitochondrial Ca2+concentration ([Ca2+]) were measured using confocal imaging in guinea pig ventricular myocytes exposed to anoxia and reoxygenation. Anoxia produced a variable, but often profound, mitochondrial depolarization. Some cells mounted a recovery of their mitochondrial membrane potential during reoxygenation; the depolarization was sustained in other cells. Recovery of the mitochondrial membrane potential seemed essential to avoid reoxygenation-induced hypercontracture. Reoxygenation also caused a sizable elevation in intramitochondrial [Ca2+], the amplitude of which was correlated with the likelihood of a cell undergoing hypercontracture. A sustained Ca2+load analogous to that seen during reoxygenation was imposed on cardiac mitochondria through permeabilization of the plasma membrane. Elevation of intracellular [Ca2+] to 800 nM caused a substantial mitochondrial depolarization. We propose that the conditions seen in guinea pig ventricular myocytes during reoxygenation are well suited to produce Ca2+-dependent mitochondrial depolarization, which may play a significant role in promoting irreversible cell injury.
Accumulation of cAMP augments dynamic vagal control of heart rate
Authors: Nakahara Tsutomu , Kawada Toru , Sugimachi Masaru , Miyano Hiroshi , Sato Takayuki , Shishido Toshiaki , Yoshimura Ryoichi , Miyashita Hiroshi , Inagaki Masashi , Alexander Joe , Sunagawa Kenji
Published: December 2017

Recent investigations in our laboratory using a Gaussian white noise perturbation technique have shown that simultaneous sympathetic stimulation augmented the gain of the transfer function from vagal stimulation frequency to heart rate response. However, the mechanism of that augmentation remains to be elucidated. In this study, we examined in anesthetized rabbits how three pharmacological interventions known to cause intracellular accumulation of cAMP affected the transfer function. Isoproterenol (0.3 μg ⋅ kg−1 ⋅ min−1 iv) increased the dynamic gain of transfer function from 7.12 ± 0.67 to 12.4 ± 1.21 beats ⋅ min−1 ⋅ Hz−1( P < 0.05) without changing the corner frequency or the lag time. Similar augmentations were observed when forskolin (5 μg ⋅ kg−1 ⋅ min−1iv) or theophylline (20 mg/kg iv) was administered under conditions of β-adrenergic blockade. These results suggest that the accumulation of cAMP at postjunctional effector sites contributes, at least in part, to the sympathetic augmentation of the dynamic vagal control of heart rate.
Electrophysiological properties of neonatal rat ventricular myocytes with α1-adrenergic-induced hypertrophy
Authors: Gaughan John P. , Hefner Colleen A. , Houser Steven R.
Published: December 2017

The electrophysiology of neonatal rat ventricular myocytes with and without hypertrophy has not been characterized. The α1-adrenergic agonist phenylephrine induced hypertrophy in neonatal rat ventricular myocytes. After 48 h of exposure to 20 μM phenylephrine, cell surface area of hypertrophied myocytes was 44% larger than control. Action potential duration was significantly longer in hypertrophy than in control. There was an increase in L-type Ca2+current in control after 48 h in culture, but current density was significantly less in hypertrophy (−4.7 ± 0.8 hypertrophy vs. −10.7 ± 1.2 control pA/pF, n = 22, P < 0.05). T-type Ca2+current density was not different. The α-adrenergic antagonist prazosin blocked the hypertrophy and the chronic effect of phenylephrine on L-type Ca2+current. Transient outward K+current density was decreased 70% in hypertrophy and was blocked with 4-aminopyridine. No change in Na+current density was observed. Staurosporine, a protein kinase C inhibitor, eliminated the hypertrophy and the effect on L-type Ca2+current. These studies showed that phenylephrine-induced hypertrophy occurred via the α1-adrenergic pathway and caused electrophysiological changes and effects on ion channel expression.
Intracorporal injection ofhSlocDNA in rats produces physiologically relevant alterations in penile function
Authors: Christ G. J. , Rehman J. , Day N. , Salkoff L. , Valcic M. , Melman A. , Geliebter J.
Published: December 2017

The Ca2+-sensitive K+channel (maxi-K+) is an important modulator of corporal smooth muscle tone. The goal of these studies was twofold: 1) to determine the feasibility of transfecting corporal smooth muscle cells in vivo with the hSlo cDNA, which encodes for the human smooth muscle maxi-K+channel, and 2) to determine whether transfection of the maxi-K+channel would affect the physiological response to cavernous nerve stimulation in a rat model in vivo. Intracorporal microinjection of pCMVβ/Lac Z DNA in 10-wk-old rats resulted in significant incorporation and expression of β-galactosidase activity in 10 of 12 injected animals for up to 75 days postinjection. Moreover, electrical stimulation of the cavernous nerve revealed that, relative to the responses obtained in age-matched control animals ( N = 12), intracavernous injection of naked pcDNA/ hSlo DNA was associated with a statistically significant elevation in the mean amplitude of the intracavernous pressure response at all levels of current stimulation (range 0.5–10 mA) at both 1 mo ( N= 5) and 2 mo ( N = 8) postinjection. Furthermore, qualitatively similar observations were made at 3 mo ( N = 2) and 4 mo ( N = 2) postinjection. These data indicate that naked hSlo DNA is quite easily incorporated into corporal smooth muscle and, furthermore, that expression is sustained for at least 2 mo in corporal smooth muscle cells in vivo. Finally, after expression, hSlo is capable of measurably altering nerve-stimulated penile erection. Taken together, these data provide compelling evidence for the potential utility of gene therapy in the treatment of erectile dysfunction.
Acellular hemoglobin-mediated oxidative stress toward endothelium: a role for ferryl iron
Authors: Goldman Daniel W. , Breyer Richard J. , Yeh David , Brockner-Ryan Beth A. , Alayash Abdu I.
Published: December 2017

We tested the hypothesis that chemical modifications used to produce stable, oxygen-carrying, Hb-based blood substitutes can induce cytotoxicity in endothelial cells in culture because of altered redox activity. We examined the interaction of hydrogen peroxide with nonmodified hemoglobin (HbA0) and two chemically modified hemoglobins, α-cross-linked hemoglobin (α-DBBF) and its polymerized form (poly-α-DBBF). Hydrogen peroxide-induced cell death (as assessed by lactate dehydrogenase release) in bovine aortic endothelial cells (BAEC) was completely inhibited by all three hemoglobin preparations, consistent with their known pseudoperoxidase activity [hemoglobin consumes peroxide as it cycles between ferric (Fe3+) and ferryl (Fe4+) hemes]. However, reaction of the modified hemoglobins, but not HbA0, with hydrogen peroxide induced apoptotic cell death (as assessed by morphological changes and DNA fragmentation) that correlated with the formation of a long-lived ferrylhemoglobin. A preparation of ferryl-α-DBBF free of residual peroxide rapidly induced morphological changes and DNA fragmentation in BAEC, indicative of apoptotic cell death. Redox cycling of chemically modified hemoglobins by peroxide yielded a persistent ferryl iron that was cytotoxic to endothelial cells.
Increased renal endothelin formation is associated with sodium retention and increased free water clearance
Authors: Modesti Pietro Amedeo , Cecioni Ilaria , Migliorini Angela , Naldoni Alessandra , Costoli Alessandro , Vanni Simone , Serneri Gian Gastone Neri
Published: December 2017

To investigate whether renal endothelin (ET)-1 participates in water and sodium handling, we investigated the influence of different sodium intakes on renal production of ET-1 in eight healthy subjects. The functional relationship with the renin-angiotensin system was also studied. Renal ET-1 formation is affected by sodium intake, because 1 wk of high sodium decreased urinary ET-1 excretion (−34%, P< 0.05), whereas a low-sodium diet increased ET-1 excretion (66%, P < 0.05) and mRNA expression for preproendothelin-1 in epithelial cells of medullary collecting ducts and endothelial cells of the peritubular capillary network. Increased ET-1 renal synthesis was associated with sodium retention and increased free water clearance. Urinary ET-1 excretion changes from normal to low-sodium diet were negatively related to contemporary changes in sodium excretion ( r = 0.97, P < 0.05) and were positively correlated with free water clearance ( r = 0.97, P < 0.05). These correlations were maintained during angiotensin-converting enzyme inhibition, which only partially reduced ET-1 renal excretion. These results indicate that renal ET-1 production is indeed modulated by varying sodium intakes and may exert a role in sodium and water handling.
Effects of antisense oligonucleotide to iNOS on hemodynamic and vascular changes induced by LPS
Authors: Hoque Azizul M. , Papapetropoulos Andreas , Venema Richard C. , Catravas John D. , Fuchs Leslie C.
Published: December 2017

Lipopolysaccharide (LPS) causes impaired vascular contractility proposed to be mediated by induction of nitric oxide synthase (iNOS). Antisense (AS) oligonucleotide inhibits the translation of target mRNA into functional proteins. We hypothesize that in vivo pretreatment with AS oligonucleotide targeted to iNOS mRNA can prevent LPS-induced hyporeactivity to norepinephrine (NE). Three groups of conscious male Wistar rats received one of the following: saline, AS, or mismatch (MM) oligonucleotide at 0.4 mg/kg iv at 12 and 24 h before LPS (5 mg/kg iv). The fourth group received saline only. Mean arterial pressure (MAP) and heart rate (HR) were continuously recorded before and 6 h after LPS or saline administration. Aorta, lung lavage, and lung tissue were collected for determination of iNOS protein expression and NOS activity. Small mesenteric arteries (≈250 μm) were isolated, denuded of endothelium, and maintained at a constant intraluminal pressure of 40 mmHg for study in vitro. LPS produced significant tachycardia that was not altered by AS or MM oligonucleotide. AS, but not MM oligonucleotide, reduced the accumulation of cGMP, the increase in conversion ofl-[3H]arginine tol-[3H]citrulline, and iNOS protein expression in tissue from LPS-treated rats. Small mesenteric arterial contraction to NE was significantly impaired in vessels from LPS-treated rats and was restored by AS, but not MM, oligonucleotide. In a rat model of septic shock, AS oligonucleotide to iNOS mRNA inhibits NOS activity and iNOS protein expression and prevents the vascular hyporeactivity to NE, which may contribute to hypotension in shock.
Increased levels of myocardial IκB-α protein promote tolerance to endotoxin
Authors: Shames Brian D. , Meldrum Daniel R. , Selzman Craig H. , Pulido Edward J. , Cain Brian S. , Banerjee Anirban , Harken Alden H. , Meng Xianzhong
Published: December 2017

Endotoxin [lipopolysaccharide (LPS)] causes tumor necrosis factor-α (TNF-α)-mediated myocardial contractile depression. Tolerance to the cardiac toxicity of LPS can be induced by a prior exposure to LPS or by pretreatment with glucocorticoids. The mechanisms by which the myocardium acquires tolerance to LPS remain unknown. LPS causes phosphorylation and degradation of inhibitory κB-α (IκB-α), releasing nuclear factor-κB (NF-κB) to activate TNF-α gene transcription. We hypothesized that LPS induces supranormal synthesis of myocardial IκB-α protein and thus renders the myocardium tolerant to subsequent LPS. Rats were challenged with LPS after pretreatment with LPS, dexamethasone, or saline. In saline-pretreated rats, LPS caused a rapid decrease in myocardial IκB-α protein levels, activation of NF-κB, and increased TNF-α production. These events were followed by myocardial contractile depression. After the initial decrease in myocardial IκB-α, IκB-α protein levels rebounded to a level greater than control levels by 24 h. Dexamethasone pretreatment similarly increased myocardial IκB-α protein levels. In rats pretreated with either LPS or dexamethasone, myocardial IκB-α protein levels remained similar to control levels after LPS challenge. The preserved level of myocardial IκB-α protein was associated with diminished NF-κB activation, attenuated myocardial TNF-α production, and improved cardiac contractility. We conclude that LPS and dexamethasone upregulate myocardial IκB-α protein expression and that an increased level of myocardial IκB-α protein may promote cardiac tolerance to LPS by inhibition of NF-κB intranuclear translocation and myocardial TNF-α production.
Ca2+regulates the kinetics of tension development in intact cardiac muscle
Authors: Baker Anthony J. , Figueredo Vincent M. , Keung Edmund C. , Camacho S. Albert
Published: December 2017

The goal of this study was to determine whether Ca2+plays a role in regulating tension development kinetics in intact cardiac muscle. In cardiac muscle, this fundamental issue of Ca2+regulation has been controversial. The approach was to induce steady-state tetanic contractions of intact right ventricular trabeculae from rat hearts at varying external Ca2+concentrations ([Ca2+]) at 22°C. During tetani, cross bridges were mechanically disrupted and the kinetics of tension redevelopment were assessed from the rate constant of exponential tension redevelopment ( ktr). There was a relationship between ktrand external [Ca2+] that was similar in form to the relationship between tension and [Ca2+]. Thus a close relationship also existed between ktrand tension ( r = 0.88; P < 0.001); whereas at maximal tetanic tension (saturating cytosolic [Ca2+]), ktrwas 16.4 ± 2.2 s−1(mean ± SE, n = 7), at zero tension (low cytosolic [Ca2+]), ktrextrapolated to 20% of maximum (3.3 ± 0.7 s−1). Qualitatively similar results were obtained using different mechanical protocols to disrupt cross bridges. These data demonstrate that tension redevelopment kinetics in intact cardiac muscle are influenced by the level of Ca2+activation. These findings contrast with the findings of one previous study of intact cardiac muscle. Activation dependence of tension development kinetics may play an important role in determining the rate and extent of myocardial tension rise during the cardiac cycle in vivo.
Muscle chemoreflex-induced increases in right atrial pressure
Authors: Sheriff Don D. , Augustyniak Robert A. , O’Leary Donal S.
Published: December 2017

When oxygen delivery to active muscle is too low for the ongoing rate of metabolism, metabolites accumulate and stimulate sensory nerves within the muscle leading to sympathetic activation (muscle chemoreflex). To date, studies on this reflex have focused primarily on its ability to increase arterial pressure or on the activity of the nerves that mediate this response. Clearly, a rise in cardiac output (CO) constitutes an important adjustment, because it increases the total blood flow available to be distributed among organs competing for flow. However, increments in heart rate and contractility provide limited means of raising CO because of the inverse relationship that exists between CO and right atrial pressure (RAP) in the intact circulation. Our goal was to test whether muscle chemoreflex activation, achieved via graded reductions in hindlimb blood flow by partial vascular occlusion, elicits peripheral vascular adjustments that raise RAP. In four conscious dogs exercising on a treadmill at 3.2 km/h 0% grade, RAP was well maintained during reflex activation despite increases in CO and arterial pressure that are expected to reduce RAP. Thus peripheral vascular adjustments elicited by the reflex successfully defend RAP in a setting where it would otherwise fall. To isolate the effects of the reflex on RAP, CO was maintained constant by ventricular pacing in conjunction with β1-adrenergic blockade with atenolol. When the reflex was activated by reducing hindlimb blood flow from 0.6 to 0.3 l/min, RAP rose from 5.1 ± 0.8 to 7.4 ± 0.4 mmHg ( P < 0.05) despite continued large (40 mmHg) increases in arterial pressure. During heavier exercise (6.4 km/h 10% grade) in five dogs with normal ventricular function, the reflex raised RAP from 5.7 ± 0.9 to 6.6 ± 0.8 mmHg ( P < 0.05) despite increases in CO and arterial pressure. We conclude that the muscle chemoreflex is capable of eliciting substantial increases in RAP.
Altered inotropic response to IGF-I in diabetic rat heart: influence of intracellular Ca2+and NO
Authors: Ren Jun , Walsh Mary F. , Hamaty Marwan , Sowers James R. , Brown Ricardo A.
Published: December 2017

Normally, insulin-like growth factor I (IGF-I) exerts positive effects on cardiac growth and myocardial contractility, but resistance to its action has been reported in diabetes. This study was designed to determine whether IGF-I-induced myocardial contractile action is altered in diabetes as a result of an intrinsic alteration of contractile properties at the cellular level. Contractile responses to IGF-I were examined in left ventricular papillary muscles and ventricular myocytes from normal and short-term (5–7 days) streptozotocin-induced diabetic rats. Mechanical properties of muscles and myocytes were evaluated using a force transducer and an edge detector, respectively. Preparations were electrically stimulated at 0.5 Hz, and contractile properties analyzed include peak tension development (PTD) or peak twitch amplitude (PTA), time to peak contraction/shortening, and time to 90% relaxation/relengthening. Intracellular Ca2+transients were measured as fura 2 fluorescence intensity changes. IGF-I (1–500 ng/ml) caused a dose-dependent increase in PTD and PTA in preparations from normal but not diabetic animals. IGF-I did not alter time to peak contraction/shortening or time to 90% relaxation/relengthening. Pretreatment with the NO synthase inhibitor Nω-nitro-l-arginine methyl ester (100 μM) attenuated IGF-I-induced increases in PTD in normal myocardium but unmasked a positive inotropic action in diabetic animals. Pretreatment with Nω-nitro-l-arginine methyl ester blocked IGF-I-induced increases in PTA in single myocytes. Consistent with its inotropic actions on muscles and myocytes, IGF-I induced a dose-dependent increase in Ca2+transients in normal but not diabetic myocytes. These results suggest that the IGF-I-induced inotropic response is depressed in diabetes because of an intrinsic alteration at the myocyte level. Mechanisms underlying this alteration in IGF-I-induced myocardial response may be related to changes in intracellular Ca2+and/or NO production in diabetes.
Effect of pH, phosphate, and ADP on relaxation of myocardium after photolysis of diazo 2
Authors: Simnett S. J. , Johns E. C. , Lipscomb S. , Mulligan I. P. , Ashley C. C.
Published: December 2017

The aim of this study was to examine the effect of the metabolites H+, ADP, and Pion the rate of cardiac relaxation. We used guinea pig right ventricular trabeculae that had been chemically skinned, allowing the myofilaments to be studied in isolation. Laser-flash photolysis of the caged Ca2+chelator diazo 2, causing a rapid fall in intracellular Ca2+, enabled investigation of relaxation independently of the rate of Ca2+diffusion. On the photolysis of diazo 2, the trabeculae relaxed biphasically with exponential rate constants ( k1and k2) of 10.07 and 4.23 s−1, respectively, at 12°C and 18.35 and 2.52 s−1, respectively, at a nominal 20°C. Increasing the concentration of both protons (pH 7.2–6.8) and MgADP (0.5–3.4 mM) slowed the two phases of the relaxation transients. Raising the concentration of Pifrom the control level of 1.36 mM to 15.2 mM increased the rate of both phases, with relaxation becoming monoexponential at 19.4 mM Pi(with a k of 20.31 s−1at 12°C). Cardiac muscle was compared with skeletal muscle under identical conditions; in cardiac muscle 19.4 mM Piincreased the rate of relaxation, whereas in skeletal muscle this concentration of Pislowed relaxation. We conclude that the mechanism of relaxation differs between cardiac and skeletal muscle. This study is a direct demonstration of the effects of ATP metabolites on cardiac myofilament processes during relaxation.
Repetitive coronary artery occlusions induce release of growth factors into the myocardial interstitium
Authors: Weihrauch Dorothee , Tessmer John , Warltier David C. , Chilian William M.
Published: December 2017

Our objective was to delineate the temporal sequence of mitogenic activity in myocardial interstitial fluid (IF) during enhancement of collateral growth. Collateral development in chronically instrumented dogs was induced by eight 2-min coronary occlusions/day for 21 days. Collateralization was assessed by measurement of blood flow in the region distal to a total coronary occlusion. Myocardial IF was obtained periodically from an intramyocardial catheter, and mitogenic activity was assessed by proliferative response of cultured endothelial cells (EC) and vascular smooth muscle cells (VSMC) to the IF. Three experiments were conducted to test that the mitogenic activity is induced by protein growth factors: 1) protein digestion of the myocardial IF with Pronase-coupled latex beads; 2) heat inactivation (boiling) of the IF; and 3) neutralization of the mitogenic activity with antibodies for basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Blood flow was reconstituted to baseline levels during occlusion after 3 wk of repetitive coronary occlusions. After initiation of occlusion the mitogenic activity of the myocardial IF on VSMC and EC increased up to days 12–14 and was reduced on days 19–23. Pronase treatment and heat inactivation blocked the mitogenic effect. Treatment with antibodies for bFGF and VEGF neutralized the proliferative response to the myocardial IF at specific times. bFGF antibody inhibited the mitogenic effect significantly on days 12–14. VEGF antibody neutralized the mitogenicity of the myocardial IF on day 7, days 12 and 13, and days 19 and 20significantly. We conclude that myocardial IF harvested from ischemic myocardium is highly mitogenic up to 2 wk after initiation of repetitive coronary occlusions. After 3 wk of ischemia, the degree of mitogenic activity for VSMC and EC was decreased from peak levels. The antibodies could not neutralize the mitogenic effect of the myocardial IF during this time period. These results suggest that mitogens are expressed during various stages of collateral development in a time-dependent manner, that the mitogens are proteinaceous in nature, and that bFGF and VEGF are released into the myocardial IF.
Myocardial overexpression of GRK3 in transgenic mice: evidence for in vivo selectivity of GRKs
Authors: Iaccarino Guido , Rockman Howard A. , Shotwell Kyle F. , Tomhave Eric D. , Koch Walter J.
Published: December 2017

Transgenic mice were generated with cardiac-specific overexpression of the G protein-coupled receptor kinase 3 (GRK3) to explore the in vivo role of this GRK in cardiac function. GRK3 is expressed in the heart along with the β-adrenergic receptor kinase (β-ARK1) and GRK5. We have previously demonstrated that myocardial-targeted overexpression in transgenic mice of β-ARK1 (Koch, W.J., H. A. Rockman, P. Samama, R. A. Hamilton, R. A. Bond, C. A. Milano, and R. J. Lefkowitz. Science 268: 1350–1353, 1995) or GRK5 (Rockman, H.A., D.-J. Choi, N. U. Rahman, S. A. Akhter, R. J. Lefkowitz, and W. J. Koch. Proc. Natl. Acad. Sci. USA 93: 9954–9959, 1996) results in significant attenuation of β-adrenergic signaling and in vivo cardiac function and selective desensitization of angiotensin (ANG) II-mediated cardiac responses. Surprisingly, myocardial overexpression of GRK3 resulted in normal biochemical signaling through β-adrenergic receptors (β-ARs), and in vivo hemodynamic function in response to a β-AR agonist was indistinguishable from that in nontransgenic controls. Furthermore, in vivo signaling and functional responses to ANG II were unaltered. However, myocardial thrombin signaling, as assessed by p42/p44 mitogen-activated protein (MAP) kinase activation, was significantly attenuated in GRK3 transgenic mouse hearts, indicating a distinct in vivo substrate specificity for GRK3.
Functional evidence for alternative ANG II-forming pathways in hamster cardiovascular system
Authors: Nishimura Hikaru , Buikema Hendrik , Baltatu Ovidiu , Ganten Detlev , Urata Hidenori
Published: December 2017

Like human chymase, hamster chymase is an ANG II-forming enzyme, but pathophysiological roles of chymase are still unknown. We determined the functional conversion of ANG I and [Pro11, d-Ala12]ANG I, a chymase-selective substrate, to ANG II in the hamster cardiovascular system. ANG I and [Pro11, d-Ala12]ANG I produced similar dose-dependent pressor responses in conscious hamsters. Captopril and CV-11974, an ANG II type 1 (AT1)-receptor antagonist, inhibited the responses to ANG I; in contrast, the pressor responses to [Pro11,d-Ala12]ANG I were suppressed only by CV-11974. In the isolated aorta, captopril suppressed ANG I-induced contraction by 84%; administration of captopril with either chymostatin or aprotinin eliminated the contraction. [Pro11, d-Ala12]ANG I-induced contraction was not affected by captopril but was attenuated by chymostatin (71%) and aprotinin (57%). CV-11974 abolished the responses to both substrates, whereas PD-123319, an AT2-receptor antagonist, had no effect. In homogenates of the aorta and heart, soybean trypsin inhibitor-inhibitable ANG II formation predominated over captopril- or aprotinin-inhibitable ANG II formation. These data suggest that [Pro11,d-Ala12]ANG I and part of ANG I were functionally converted to ANG II by chymase and other serine protease(s) in hamster vessels, inducing AT1-receptor-mediated vasoconstriction. Biochemical data supported a role for chymase in the alternative pathway.
Intracellular acidosis differentially regulates KV channels in coronary and pulmonary vascular muscle
Authors: Berger Marcie G. , Vandier Christophe , Bonnet Pierre , Jackson William F. , Rusch Nancy J.
Published: December 2017

Decreases in intracellular pH (pHi) potently dilate coronary resistance arteries but constrict small pulmonary arteries. To define the ionic mechanisms of these responses, this study investigated whether acute decreases in pHi differentially regulate K+ currents in single vascular smooth muscle (VSM) cells isolated from rat coronary and pulmonary resistance arteries. In patch-clamp studies, whole cell K+ currents were elicited by 10-mV depolarizing steps between −60 and 0 mV in VSM cells obtained from 50- to 150-μm-OD arterial branches, and pHi was lowered by altering the NH4Cl gradient across the cell membrane. Progressively lowering pHi from calculated values of 7.0 to 6.7 and 6.4 increased the peak amplitude of K+ current in coronary VSM cells by 15 ± 5 and 23 ± 3% but reduced K+ current in pulmonary VSM cells by 18 ± 3 and 21 ± 3%, respectively. These changes were reversed by returning cells to the control pHi of 7.0 and were eliminated by dialyzing cells with pipette solution containing 50 mmol/l HEPES to buffer NH4Cl-induced changes in pHi. Pharmacological block of ATP-sensitive K+ channels and Ca2+-activated K+ channels by 1 μmol/l glibenclamide and 100 nmol/l iberiotoxin, respectively, did not prevent changes in K+ current levels induced by acidotic pHi. However, block of voltage-gated K+ channels by 3 mmol/l 4-aminopyridine abolished acidosis-induced changes in K+ current amplitudes in both VSM cell types. Interestingly, α-dendrotoxin (100 nmol/l), which blocks only select subtypes of voltage-gated K+ channels, abolished the acidosis-induced decrease in K+current in pulmonary VSM cells but did not affect the acidosis-induced increase in K+ current observed in coronary VSM cells. These findings suggest that opposing, tissue-specific effects of pHi on distinct subtypes of voltage-gated K+ channels in coronary and pulmonary VSM membranes may differentially regulate vascular reactivity in these two circulations under conditions of acidotic stress.
Stimulation of different phospholipase A2isoforms by TNF-α and IL-1β in adult rat ventricular myocytes
Authors: Liu Shi J. , McHowat Jane
Published: December 2017

We previously showed that in adult rat ventricular myocytes interleukin (IL)-1β activates a membrane-associated, Ca2+-independent phospholipase A2(iPLA2). In this study, we examined the possible existence of different PLA2isoforms and effects of tumor necrosis factor (TNF)-α on iPLA2activities. Western blot analysis identified iPLA2in both membrane (∼82 kDa) and cytosolic (∼40 kDa) fractions and identified Ca2+-dependent PLA2(cPLA2) only in cytosolic fractions. With plasmenylcholine or alkylacyl glycerophosphorylcholine as substrate, TNF-α elicited a twofold transient increase in cytosolic iPLA2activity accompanied by an increase in arachidonic acid release and decreased membrane-associated iPLA2activity with plasmenylcholine. With phosphatidylcholine as substrate, TNF-α decreased both cytosolic and membrane-associated iPLA2activities. TNF-α-induced increases in cytosolic iPLA2activity and arachidonic acid release were completely blocked by methyl arachidonyl fluorophosphonate (MAFP) but not by bromoenol lactone (BEL). TNF-α and IL-1β together enhanced synergistically cytosolic and membrane PLA2activities and arachidonic acid release that were blocked differentially by MAFP and BEL, respectively, and inhibited completely by MAFP plus BEL. These results suggest that TNF-α and IL-1β act on different PLA2isoforms in ventricular myocytes.