bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrical remodeling of the epicardial border zone in the canine infarcted heart: a computational analysis
Authors: Cabo Candido , Boyden Penelope A.
Published: March 2015

The density and kinetics of several ionic currents of cells isolated from the epicardial border zone of the infarcted heart (IZs) are markedly different from cells from the noninfarcted canine epicardium (NZs). To understand how these changes in channel function affect the action potential of the IZ cell as well as its response to antiarrhythmic agents, we developed a new ionic model of the action potential of a cell that survives in the infarct (IZ) and one of a normal epicardial cell (NZ) using formulations based on experimental measurements. The difference in action potential duration (APD) between NZ and IZ cells during steady-state stimulation (basic cycle length = 250 ms) was 6 ms (156 ms in NZ and 162 ms in IZ). However, because IZs exhibit postrepolarization refractoriness, the difference in the effective refractory period (ERP), calculated using a propagation model of a single fiber of 100 cells, was 43 ms (156 ms in NZ and 199 ms in IZ). Either an increase in L-type Ca2+ current (to simulate the effects of BAY Y5959) or a decrease of both or either delayed rectifier currents (e.g., to simulate the effects of azimilide, sotalol, and chromanol) had significant effects on NZ ERP. In contrast, the effects of these agents in IZs were minor, in agreement with measurements in the in situ canine infarcted heart. Therefore 1) because IZs exhibit postrepolarization refractoriness, conclusions drawn from APD measurements cannot be extrapolated directly to ERPs; 2) ionic currents that are the major determinants of APD and the ERP in NZs are less important in IZs; and 3) differential effects of either BAY Y5959 or azimilide in NZs versus IZs are predicted to decrease ERP dispersion and in so doing prevent initiation of arrhythmias in a substrate of inhomogeneous APD/ERPs.
Alterations in KATP and KCa channel function in cerebral arteries of insulin-resistant rats
Authors: Erdös Benedek , Miller Allison W. , Busija David W.
Published: March 2015

We examined whether insulin resistance alters the function of ATP-dependent and Ca2+-activated K+ channels (KATPand KCa channels, respectively) in pressurized isolated middle cerebral arteries (MCAs) from fructose-fed insulin-resistant (IR) and control rats. Blockade of KCa channels with tetraethylammonium chloride (TEA, 2.5 mM) or iberiotoxin (IBTX, 0.1 μM) increased the spontaneously developed tone in control MCAs by 10.5 ± 1.3% ( n = 10) and 13.3 ± 2.3% ( n = 6), respectively. In the IR arteries, TEA induced similar constrictions (8.0 ± 1.1%, n = 10), but IBTX constricted the IR arteries by only 3.1 ± 0.9% ( n = 8; P < 0.01). Bradykinin (BK)-induced endothelium-mediated relaxation was reduced in IR MCAs. Maximum relaxation to BK (10−6 M) was 42 ± 4% in control ( n = 9) and 19 ± 2% in IR ( n = 10; P < 0.01) arteries. Pretreatment with TEA, IBTX, or the KATP channel blocker glibenclamide (10 μM) inhibited relaxation to BK in control MCAs but did not alter dilation in IR arteries. Relaxation to the KATP channel opener cromakalim was also diminished in IR MCAs. Maximum relaxation to cromakalim (10−5 M) was 48 ± 3% in control ( n = 6) and 19 ± 2% in IR arteries ( n = 6; P < 0.01). These findings demonstrate that insulin resistance alters the function of KATP and KCa channels in isolated MCAs and affects the control of resting vascular tone and the mediation of dilator stimuli.
Myocardial infarction in rat eliminates regional heterogeneity of AP profiles,ItoK+currents, and [Ca2+]itransients
Authors: Kaprielian Roger , Sah Rajan , Nguyen Tin , Wickenden Alan D. , Backx Peter H.
Published: March 2015

Transient outward K+current density ( Ito) has been shown to vary between different regions of the normal myocardium and to be reduced in heart disease. In this study, we measured regional changes in action potential duration (APD), Ito, and intracellular Ca2+concentration ([Ca2+]i) transients of ventricular myocytes derived from the right ventricular free wall (RVW) and interventricular septum (SEP) 8 wk after myocardial infarction (MI). At +40 mV, Itodensity in sham-operated hearts was significantly higher ( P < 0.01) in the RVW (15.0 ± 0.8 pA/pF, n = 47) compared with the SEP (7.0 ± 1.1 pA/pF, n = 18). After MI, Itodensity was not reduced in SEP myocytes but was reduced ( P < 0.01) in RVW myocytes (8.7 ± 1.0 pA/pF, n = 26) to levels indistinguishable from post-MI SEP myocytes. These changes in Itodensity correlated with Kv4.2 (but not Kv4.3) protein expression. By contrast, Kv1.4 expression was significantly higher in the RVW compared with the SEP and increased significantly after MI in RVW. APD measured at 50% or 90% repolarization was prolonged, whereas peak [Ca2+]itransients amplitude was higher in the SEP compared with the RVW in sham myocytes. These regional differences in APD and [Ca2+]itransients were eliminated by MI. Our results demonstrate that the significant regional differences in Itodensity, APD, and [Ca2+]ibetween RVW and SEP are linked to a variation in Kv4.2 expression, which largely disappears after MI.
TNF-α and myocardial matrix metalloproteinases in heart failure: relationship to LV remodeling
Authors: Bradham William S. , Moe Gordon , Wendt Katherine A. , Scott Amelia A. , Konig Andrea , Romanova Marina , Naik George , Spinale Francis G.
Published: March 2015

The cytokine tumor necrosis factor (TNF)-α has been causally linked to left ventricular (LV) remodeling, but the molecular basis for this effect is unknown. Matrix metalloproteinases (MMPs) have been implicated in cardiac remodeling and can be regulated by TNF-α. This study tested the central hypothesis that administration of a TNF-α blocking protein would prevent the induction of MMPs and alter the course of myocardial remodeling in developing LV failure. Adult dogs were randomly assigned to the following groups: 1) chronic pacing (250 beats/min, 28 days, n = 12), 2) chronic pacing with concomitant administration of a TNF-α blocking protein (TNF block) using a soluble p75 TNF receptor fusion protein (TNFR:Fc; administered at 0.5 mg/kg twice a week subcutaneously, n = 7), and 3) normal controls ( n = 10). LV end-diastolic volume increased from control with chronic pacing (83 ± 12 vs. 118 ± 10 ml, P < 0.05) and was reduced with TNF block (97 ± 9 ml, P < 0.05). MMP zymographic levels (92 kDa, pixels) increased from control with chronic pacing (36,848 ± 9,593 vs. 87,247 ± 12,912, P < 0.05) and was normalized by TNF block. Myocardial MMP-9 and MMP-13 levels by immunoblot increased with chronic pacing relative to controls (130 ± 10% and 118 ± 6%, P < 0.05) and was normalized by TNF block. These results provide evidence to suggest that TNF-α contributes to the myocardial remodeling process in evolving heart failure through the local induction of specific MMPs.
A selective inducible NOS dimerization inhibitor prevents systemic, cardiac, and pulmonary hemodynamic dysfunction in endotoxemic mice
Authors: Ichinose Fumito , Hataishi Ryuji , Wu Justina C. , Kawai Noriko , Rodrigues Ana Clara Tude , Mallari Cornell , Post Joe M. , Parkinson John F. , Picard Michael H. , Bloch Kenneth D. , Zapol Warren M.
Published: March 2015

Increased nitric oxide (NO) production by inducible NO synthase (NOS2), an obligate homodimer, is implicated in the cardiovascular sequelae of sepsis. We tested the ability of a highly selective NOS2 dimerization inhibitor (BBS-2) to prevent endotoxin-induced systemic hypotension, myocardial dysfunction, and impaired hypoxic pulmonary vasoconstriction (HPV) in mice. Mice were challenged with Escherichia coli endotoxin before treatment with BBS-2 or vehicle. Systemic blood pressure was measured before and 4 and 7 h after endotoxin challenge, and echocardiographic parameters of myocardial function were measured before and 7 h after endotoxin challenge. The pulmonary vasoconstrictor response to left mainstem bronchus occlusion, which is a measure of HPV, was studied 22 h after endotoxin challenge. BBS-2 treatment alone did not alter baseline hemodynamics. BBS-2 treatment blocked NOS2 dimerization and completely inhibited the endotoxin-induced increase of plasma nitrate and nitrite levels. Treatment with BBS-2 after endotoxin administration prevented systemic hypotension and attenuated myocardial dysfunction. BBS-2 also prevented endotoxin-induced impairment of HPV. In contrast, treatment with NG-nitro-l-arginine methyl ester, which is an inhibitor of all three NOS isoforms, prevented the systemic hypotension but further aggravated the myocardial dysfunction associated with endotoxin challenge. Treatment with BBS-2 prevented endotoxin from causing key features of cardiovascular dysfunction in endotoxemic mice. Selective inhibition of NOS2 dimerization with BBS-2, while sparing the activities of other NOS isoforms, may prove to be a useful treatment strategy in sepsis.
Hypovolemia and neurovascular control during orthostatic stress
Authors: Kimmerly Derek S. , Shoemaker J. Kevin
Published: March 2015

Humans exposed to real or simulated microgravity experience decrements in blood pressure regulation during orthostatic stress that may be related to autonomic dysregulation and/or hypovolemia. We examined the hypothesis that hypovolemia, without the deconditioning effects of bed rest or spaceflight, would augment the sympathoneural and vasomotor response to graded orthostatic stress. Radial artery blood pressure (tonometry), stroke volume (SV), brachial blood flow (Doppler ultrasound), heart rate (electrocardiogram), peroneal muscle sympathetic nerve activity (MSNA; microneurography), and estimated central venous pressure (CVP) were recorded during five levels (−5, −10, −15, −20 and −40 mmHg) of randomly assigned lower body negative pressure (LBNP) ( n = 8). Forearm (FVR) and total peripheral vascular resistance (TPR) were calculated. The test was repeated under randomly assigned placebo (normovolemia) or diuretic (spironolactone: 100 mg/day, 3 days) (hypovolemia) conditions. The diuretic produced an ∼16% reduction in plasma volume. Compared with normovolemia, SV and cardiac output were reduced by ∼12% and ∼10% at baseline and during LBNP after the diuretic. During hypovolemia, there was an upward shift in the %ΔMSNA/ΔCVP, ΔFVR/ΔCVP, and ΔTPR/ΔCVP relationships during 0 to −20 mmHg LBNP. In contrast to normovolemia, blood pressure increased at −40 mmHg LBNP during hypovolemia due to larger gains in the %ΔMSNA/ΔCVP and ΔTPR/ΔCVP relationships. It was concluded that acute hypovolemia augmented the neurovascular component of blood pressure control during moderate orthostasis, effectively compensating for decrements in SV and cardiac output.
Effects of ischemia and myogenic activity on active and passive mechanical properties of rat cerebral arteries
Authors: Coulson Rebecca J. , Chesler Naomi C. , Vitullo Lisa , Cipolla Marilyn J.
Published: March 2015

Passive (papaverine induced) and active (spontaneous pressure induced) biomechanical properties of ischemic and nonischemic rat middle cerebral arteries (MCAs) were studied under pressurized conditions in vitro. Ischemic (1 h of occlusion), contralateral, and sham-operated control MCAs were isolated from male Wistar rats ( n = 22) and pressurized using an arteriograph system that allowed control of transmural pressure (TMP) and measurement of lumen diameter and wall thickness. Three mechanical stiffness parameters were computed: overall passive stiffness (β), pressure-dependent modulus changes ( E inc,p), and smooth muscle cell (SMC) activity-dependent changes ( E inc,a). The β-value for ischemic vessels was increased compared with sham vessels (13.9 ± 1.7 vs. 9.1 ± 1.4, P < 0.05), indicating possible short-term remodeling due to ischemia. E inc,p increased with pressure in the passive vessels ( P < 0.05) but remained relatively constant in the active vessels for all vessel types, indicating that pressure-induced SMC contractile activity (i.e., myogenic reactivity) in cerebral arteries leads to the maintenance of a constant elastic modulus within the autoregulatory pressure range. E inc,a increased with pressure for all conditions, signifying that changes in stiffness are influenced by SMC activity and vascular tone.
On the role of mechanosensitive mechanisms eliciting reactive hyperemia
Authors: Koller Akos , Bagi Zsolt
Published: March 2015

We hypothesized that changes in hemodynamic forces such as pressure (P) and flow (F) contribute importantly to the development of reactive hyperemia. To exclude the effects of vivo factors, isolated rat skeletal muscle arterioles (∼130 μm) were utilized. We found that changes in P or P + F following occlusions elicited reactive dilations (RD). The peak of RD (up to ∼45 μm), but not the duration of RD, increased to changes in P (80 to 10, then back to 80 mmHg) as a function of the length of occlusions (30, 60, and 120 s). However, changes in P + F (80–10 -80 mmHg + 25–0-25 μl/min) increased both the peak and duration of RD (from ∼25 to 90 s) with longer occlusions. When only P changed, inhibition of nitric oxide synthesis or endothelium removal (E−) reduced only the peak of RD, whereas when P + F were changed, both the peak and duration of RD became reduced. Inhibition of stretch-activated cation channels by gadolinium reduced the peak but enhanced the duration of RD (both to P or P + F) that was unaffected by N G-nitro-l-arginine methyl ester (l-NAME) or by E−. When only P changed, inhibition of tyrosine kinases by genistein reduced peak RD but did not affect the RD duration. However, when P + F changed, genistein reduced both the peak and the duration of RD, additional l-NAME reduced the peak RD, but did not affect the duration of RD. Thus in isolated arterioles an RD resembling the characteristics of reactive hyperemia can be generated that is elicited by deformation, stretch, pressure, and flow/shear stress-sensitive mechanisms and is, in part, mediated by nitric oxide.
Enhanced NO and superoxide generation in dysfunctional hearts from endotoxemic rats
Authors: Khadour Fadi H. , Panas Donna , Ferdinandy Péter , Schulze Costas , Csont Tamás , Lalu Manoj M. , Wildhirt Stephen M. , Schulz Richard
Published: March 2015

Free radicals have been implicated in the etiology of cardiac dysfunction during sepsis, but the actual species responsible remains unclear. We studied the alterations in myocardial nitric oxide (NO), superoxide, and peroxynitrite generation along with cardiac mechanical function and efficiency in hearts from lipopolysaccharide (LPS)-treated rats. Six hours after LPS (4 mg/kg ip) or saline (control) treatment, hearts were isolated and perfused for 1 h with recirculating Krebs-Henseleit buffer and paced at 300 beats/min. Cardiac work, O2consumption, and cardiac efficiency were markedly depressed in LPS hearts compared with controls. Plasma nitrate/nitrite level was elevated in LPS rats, and ventricular NO production was enhanced as measured by electron spin resonance spectroscopy, Ca2+-independent NO synthase (NOS) activity, and inducible NOS immunohistochemistry. Ventricular superoxide production was also enhanced in LPS-treated hearts as seen by lucigenin chemiluminescence and xanthine oxidase activity. Increased nitrotyrosine staining (immunohistochemistry) and higher lipid hydroperoxides levels were also detected in LPS-treated hearts, indicating oxygen radical-induced stress. Enhanced generation of both NO and superoxide, and thus peroxynitrite, occur in dysfunctional hearts from endotoxemic rats.
Gender differences in sarcoplasmic reticulum calcium loading after isoproterenol
Authors: Chen Jarvis , Petranka John , Yamamura Ken , London Robert E. , Steenbergen Charles , Murphy Elizabeth
Published: March 2015

Males exhibit enhanced myocardial ischemia-reperfusion injury versus females under hypercontractile conditions associated with increased sarcoplasmic reticulum (SR) Ca2+. We therefore examined whether there were gender differences in SR Ca2+. We used NMR Ca2+ indicator 1,2-bis(2-amino-5,6-difluorophenoxy)-ethane- N,N,N′, N′-tetraacetic acid to measure SR Ca2+ in perfused rabbit hearts. Isoproterenol increased SR Ca2+ in males from a baseline of 1.13 ± 0.07 to 1.52 ± 0.24 mM ( P < 0.05). Female hearts had basal SR Ca2+ that was not significantly different from males (1.04 ± 0.03 mM), and addition of isoproterenol to females resulted in a time-averaged SR Ca2+ (0.97 ± 0.07 mM) that was significantly less than in males. To confirm this difference, we measured caffeine-induced release of SR Ca2+ with fura-2 in isolated ventricular myocytes. Ca2+ release after caffeine in untreated male myocytes was 377 ± 41 nM and increased to 650 ± 55 nM in isoproterenol-treated myocytes ( P < 0.05). Ca2+ release after caffeine addition in untreated females was 376 ± 27 nM and increased to 503 ± 49 nM with isoproterenol, significantly less than in male myocytes treated with isoproterenol ( P < 0.05). Treatment of female myocytes with NG-nitro-l-arginine methyl ester, an inhibitor of nitric oxide synthase (NOS), resulted in higher SR Ca2+ release than that measured in females treated only with isoproterenol and was not significantly different from that measured in males with isoproterenol. Female myocytes also have significantly higher levels of neuronal NOS. This gender difference in SR Ca2+ handling may contribute to reduced ischemia-reperfusion injury observed in females.
Interaction of albumin with the endothelial cell surface
Authors: Osterloh Kurt , Ewert Uwe , Pries Axel R.
Published: March 2015

Endothelial cells (EC) are covered with cell-borne proteoglycans and glycoproteins. Blood plasma proteins (e.g., albumin) adsorb to this glycocalyx forming a complex endothelial surface layer (ESL). We determined the molecular mobility of albumin by electron spin resonance (ESR) in the presence and absence of ECs to analyze interactions with the ESL. Albumin was spin labeled with 5- or 12-4,4-dimethyloxazolidine- N-oxyl (DOXYL)-stearic acid yielding information on the mobility of the molecular surface (5-DOXYL) or the entire protein (12-DOXYL). EC cultures grown on glass coverslips were immersed in labeled albumin and placed in the temperature-regulated cavity of an ESR spectrometer. Alternatively, ECs were labeled and then exposed to native albumin. At 37°C, rotational correlation times determined by modified saturation transfer ESR (ST-ESR) were 26 and 48 ns for 5-DOXYL- and 12-DOXYL-labeled albumin, respectively. Presence of ECs increased rotational correlation time values for 5-DOXYL-stearic acid to 37 ns but not for 12-DOXYL-stearic acid. Albumin was able to completely take up the label from labeled EC within 2 min. The present study shows that modified ST-ESR can be used to determine the mobility of biological macromolecules interacting with cellular surfaces. Reduction in albumin surface mobility in the presence of EC at unchanged mobility of protein proper and fast removal of labeled fatty acids from EC membranes indicate rapid transient interactions between albumin surface and ESL but no rigid incorporation of albumin into a macromolecular network that would interfere with its transport function for poorly water-soluble substances.
Impaired parasympathetic heart rate control in mice with a reduction of functional G protein βγ-subunits
Authors: Gehrmann Josef , Meister Michael , Maguire Colin T. , Martins Donna C. , Hammer Peter E. , Neer Eva J. , Berul Charles I. , Mende Ulrike
Published: March 2015

Acetylcholine released on parasympathetic stimulation slows heart rate through activation of muscarinic receptors on the sinus nodal cells and subsequent opening of the atrial muscarinic potassium channel (KACh). KAChis directly activated by G protein βγ-subunits. To elucidate the physiological role of Gβγ for the regulation of heart rate and electrophysiological function in vivo, we created transgenic mice with a reduced amount of membrane-bound Gβ protein by overexpressing nonprenylated Gγ2-subunits in their hearts using the α-myosin heavy chain promoter. At baseline and after muscarinic stimulation with carbachol, heart rate and heart rate variability were determined with electrocardiogram telemetry in conscious mice and in vivo intracardiac electrophysiological studies in anesthetized mice. Reduction of the amount of functional Gβγ protein by >50% caused a pronounced blunting of the carbachol-induced bradycardia as well as the increases in time- and frequency-domain indexes of heart rate variability and baroreflex sensitivity that were observed in wild types. In addition, sinus node recovery time and inducibility of atrial arrhythmias were reduced in transgenic mice. Our data demonstrate in vivo that Gβγ plays a crucial role for parasympathetic heart rate control, sinus node automaticity, and atrial arrhythmia vulnerability.
Variation of mechanical properties along the length of the aorta in C57bl/6 mice
Authors: Guo Xiaomei , Kassab Ghassan S.
Published: March 2015

The objective of the present study is to obtain a systematic set of data on the mechanical properties along the entire length of the mouse aorta. The ascending aorta of seven mice was cannulated near the aortic valve, and the aorta was preconditioned with several cyclic changes in pressure. The perfusion pressure was then increased in 30-mmHg increments from 0 to 150 mmHg. Cab-O-Sil, colloidal silica, was mixed into the perfusate to prevent flow through the microvessels and hence attain zero-flow distensions. Our results show that the residual circumferential strain leads to a uniformity of transmural strain of the aorta in the loaded state along the entire length of the aorta. This uniformity is attained in the range of 60–120 mmHg. At pressures <60 mmHg, the outer strain is greater than the inner strain, whereas at pressures >120 mmHg, the converse is true. Furthermore, we found that the circumferential and longitudinal stress-strain relationships are linear in the pressure range of 30–120 mmHg. Finally, the circumferential modulus is greatest (most rigid) near the diaphragm, and the majority of volume compliance (85%) is in the thoracic compared with the abdominal aorta. These findings are important for an understanding of the hemodynamics of the cardiovascular system of the normal mouse and will serve as a reference state for the study of various diseases in knock-in and knock-out models of this species.
Effects of NADH and NADPH on superoxide levels and cerebral vascular tone
Authors: Didion Sean P. , Faraci Frank M.
Published: March 2015

Reactive oxygen species are important modulators of cerebral vascular tone. Recent evidence, mainly from the aorta, suggests that NAD(P)H oxidase is a major source of vascular superoxide. The goal of the present study was to examine the effects of NADH and NADPH that are commonly used to stimulate NAD(P)H oxidase activity, on superoxide levels and cerebral vascular tone. Basilar arteries and cerebral arterioles from normal rabbits were studied in vitro using isolated tissue baths and in vivo using a cranial window, respectively. In the basilar artery, NADH produced a biphasic response; low concentrations (0.1–10 μM NADH) produced marked relaxation, whereas higher concentrations (30–100 μM NADH) produced contraction. Responses to NADH were significantly ( P < 0.05) inhibited in the presence of 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron; a scavenger of superoxide, 10 mM). In contrast, NADPH (10–100 μM) produced moderate contraction of the basilar artery, which was inhibited in the presence of Tiron. In vivo, NADH produced Tiron-sensitive dilatation of cerebral arterioles. NADH and NADPH dose dependently increased superoxide levels in the basilar artery, as detected by lucigenin (5 μM)-enhanced chemiluminescence, but increases in superoxide were significantly greater for NADPH than NADH. These increases in superoxide were markedly reduced in the presence of polyethylene glycol-superoxide dismutase (300 U/ml) or diphenylene iodonium [0.1 mM, an inhibitor of flavin-containing enzymes, including NAD(P)H oxidase] but were not affected by indomethacin, N G-nitro-l-arginine, or allopurinol. These data suggest that NADH- and NADPH-induced changes in cerebral vascular tone are mediated by superoxide, produced by a flavin-containing enzyme, most likely NAD(P)H oxidase, but not xanthine oxidase or nitric oxide synthase.
Is neuroprotective efficacy of nNOS inhibitor 7-NI dependent on ischemic intracellular pH?
Authors: Coert Bernard A. , Anderson Robert E. , Meyer Fredric B.
Published: March 2015

The purpose of this study was to test the hypothesis that the efficacy of 7-nitroindazole (7-NI), a selective neuronal nitric oxide (NO) synthase (NOS) inhibitor, is pH dependent in vivo during focal cerebral ischemia. Wistar rats underwent 2 h of focal cerebral ischemia under 1% halothane anesthesia. 7-NI, 10 and 100 mg/kg in 0.1 ml/kg DMSO, was administered 30 min before occlusion. Ischemic brain acidosis was manipulated by altering serum glucose concentrations. Confirmation of the effects of these serum glucose manipulations on brain intracellular pH (pHi) was confirmed in a group of acute experiments utilizing umbelliferone fluorescence. The animals were euthanized at 72 h for histology. 7-NI significantly ( P < 0.05) reduced infarction volume in both the normoglycemic by 93.3% and hyperglycemic animals by 27.5%. In the moderate hypoglycemic animals, the reduction in infarction volume did not reach significance because moderate hypoglycemia in itself dramatically reduced infarction volume. We hypothesize that a mechanism to explain the published discrepancies on the effects of neuronal NOS inhibitors in vivo may be due to the effects by differences in ischemic brain acidosis on the production of NO.
Overexpression of tumor necrosis factor-α increases production of hydroxyl radical in murine myocardium
Authors: Machida Yoji , Kubota Toru , Kawamura Natsumi , Funakoshi Hajime , Ide Tomomi , Utsumi Hideo , Li Yun You , Feldman Arthur M. , Tsutsui Hiroyuki , Shimokawa Hiroaki , Takeshita Akira
Published: March 2015

Transgenic (TG) mice with cardiac-specific overexpression of tumor necrosis factor-α develop congestive heart failure with myocardial inflammation. The purpose of this study was to investigate the effects of tumor necrosis factor-α on reactive oxygen species (ROS) in this mouse model of cardiomyopathy. Myocardial production of hydroxyl radical detected by electron spin resonance spectroscopy was significantly increased in TG. Myocardial expression of Mn-SOD was significantly decreased in TG, whereas that of Cu,Zn-SOD was unaltered. Myocardial expression of catalase was unchanged, whereas that of glutathione peroxidase was significantly increased, in TG. Histological analysis revealed that macrophages and CD4-positive lymphocytes were increased in TG myocardium. To investigate whether these infiltrating inflammatory cells were the source of ROS, we treated TG mice with cyclophosphamide for 7 days. Although cyclophosphamide significantly suppressed the infiltration of inflammatory cells, it did not diminish the production of hydroxyl radical in TG myocardium. Damaged myocytes, but not infiltrating inflammatory cells, may be the source of ROS in TG.
Effect of NO on EDHF response in rat middle cerebral arteries
Authors: Schildmeyer Lisa A. , Bryan Robert M.
Published: March 2015

Whereas the actual identity of endothelium-derived hyperpolarizing factor (EDHF) is still not certain, it involves a process requiring the endothelium and eliciting hyperpolarization and relaxation of smooth muscle. It is neither nitric oxide (NO) nor prostacyclin, and its presence has been demonstrated in a variety of vessels. Recent studies in peripheral vessels report that EDHF-mediated dilations were either attenuated or blocked by NO. Studies presented here demonstrate that NO does not block EDHF-mediated dilations in cerebral vessels. Rat middle cerebral arteries were cannulated, pressurized, and luminally perfused. EDHF-mediated dilations were elicited by the luminal application of ATP in the presence of N G-nitro-l-arginine methyl ester (l-NAME) and indomethacin (inhibitors of NO synthase and cyclooxygenase, respectively). These dilations persisted when S-nitroso- N-acetylpenicillamine, an NO donor, was added exogenously in the presence of l-NAME, or when endogenous NO was present but its cGMP actions were blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of guanylate cyclase. These findings demonstrate that the EDHF response is not suppressed by NO in cerebral vessels and suggests a role for EDHF during normal physiological conditions.
l-Arginine protects human heart cells from low-volume anoxia and reoxygenation
Authors: Shiono Noritsugu , Rao Vivek , Weisel Richard D. , Kawasaki Muneyasu , Li Ren-Ke , Mickle Donald A. G. , Fedak Paul W. M. , Tumiati Laura C. , Ko Lawrence , Verma Subodh
Published: March 2015

Protective effects ofl-arginine were evaluated in a human ventricular heart cell model of low-volume anoxia and reoxygenation independent of alternate cell types. Cell cultures were subjected to 90 min of low-volume anoxia and 30 min of reoxygenation. l-Arginine (0–5.0 mM) was administered during the preanoxic period or the reoxygenation phase. Nitric oxide (NO) production, NO synthase (NOS) activity, cGMP levels, and cellular injury were assessed. To evaluate the effects of thel-arginine on cell signaling, the effects of the NOS antagonist N G-nitro-l-arginine methyl ester, NO donor S-nitroso- N-acetyl-penicillamine, guanylate cyclase inhibitor methylene blue, cGMP analog 8-bromo-cGMP, and ATP-sensitive K+ channel antagonist glibenclamide were examined. Our data indicate that low-volume anoxia and reoxygenation increased NOS activity and facilitated the conversion ofl-arginine to NO, which provided protection against cellular injury in a dose-dependent fashion. In addition,l-arginine cardioprotection was achieved by the activation of guanylate cyclase, leading to increased cGMP levels in human heart cells. This action involves a glibenclamide-sensitive, NO-cGMP-dependent pathway.
Activation of JNK in rat heart by exercise: effect of training
Authors: Boluyt M. O. , Loyd A. M. , Roth M. H. , Randall M. J. , Song E. Y. M.
Published: March 2015

The purpose of the study was to determine whether exercise would activate JNK in the heart and whether chronic exercise training would alter the response. Untrained rats were familiarized with the treadmill and assigned to one of four groups: low intensity (LI), 10 min, 0%, 15 m/min; medium intensity (MI), 10 min, 0%, 33 m/min; high intensity (HI), 10 min, 25%, 33 m/min; long duration (LD), 30 min, 0%, 15 m/min. Another cohort of rats was subjected to a progressive 6 wk high-intensity training protocol that produced a 12% increase in heart mass. In untrained rats, JNK activity was LI: 1.5 (fold nonrun control), MI: 2.0, HI: 2.5, LD: 1.25 immediately after a single bout of exercise. In trained rats, no activation of JNK above baseline was detected after either a 10-min or 1-h bout of exercise. We concluded that treadmill exercise activates JNK in the rat heart in an intensity-dependent manner and that chronic training abrogates the myocardial JNK response to a bout of exercise.
14,15-Dihydroxyeicosatrienoic acid relaxes bovine coronary arteries by activation of KCachannels
Authors: Campbell William B. , Deeter Christine , Gauthier Kathryn M. , Ingraham Richard H. , Falck J. R. , Li Pin-Lan
Published: March 2015

Epoxyeicosatrienoic acids (EETs) cause vascular relaxation by activating smooth muscle large conductance Ca2+-activated K+(KCa) channels. EETs are metabolized to dihydroxyeicosatrienoic acids (DHETs) by epoxide hydrolase. We examined the contribution of 14,15-DHET to 14,15-EET-induced relaxations and characterized its mechanism of action. 14,15-DHET relaxed U-46619-precontracted bovine coronary artery rings but was approximately fivefold less potent than 14,15-EET. The relaxations were inhibited by charybdotoxin, iberiotoxin, and increasing extracellular K+to 20 mM. In isolated smooth muscle cells, 14,15-DHET increased an iberiotoxin-sensitive, outward K+current and increased KCachannel activity in cell-attached patches and inside-out patches only when GTP was present. 14,15-[14C]EET methyl ester (Me) was converted to 14,15-[14C]DHET-Me, 14,15-[14C]DHET, and 14,15-[14C]EET by coronary arterial rings and endothelial cells but not by smooth muscle cells. The metabolism to 14,15-DHET was inhibited by the epoxide hydrolase inhibitors 4-phenylchalcone oxide (4-PCO) and BIRD-0826. Neither inhibitor altered relaxations to acetylcholine, whereas relaxations to 14,15-EET-Me were increased slightly by BIRD-0826 but not by 4-PCO. 14,15-DHET relaxes coronary arteries through activation of KCachannels. Endothelial cells, but not smooth muscle cells, convert EETs to DHETs, and this conversion results in a loss of vasodilator activity.
Cellular glutathione peroxidase deficiency and endothelial dysfunction
Authors: Forgione Marc A. , Weiss Norbert , Heydrick Stanley , Cap André , Klings Elizabeth S. , Bierl Charlene , Eberhardt Robert T. , Farber Harrison W. , Loscalzo Joseph
Published: March 2015

Cellular glutathione peroxidase (GPx-1) is the most abundant intracellular isoform of the GPx antioxidant enzyme family. In this study, we hypothesized that GPx-1 deficiency directly induces an increase in vascular oxidant stress, with resulting endothelial dysfunction. We studied vascular function in a murine model of homozygous deficiency of GPx-1 (GPx-1−/−). Mesenteric arterioles of GPx-1−/−mice demonstrated paradoxical vasoconstriction to β-methacholine and bradykinin, whereas wild-type (WT) mice showed dose-dependent vasodilation in response to both agonists. One week of treatment of GPx-1−/−mice withl-2-oxothiazolidine-4-carboxylic acid (OTC), which increases intracellular thiol pools, resulted in restoration of normal vascular reactivity in the mesenteric bed of GPx-1−/−mice. We observed an increase of the isoprostane iPF2α-III, a marker of oxidant stress, in the plasma and aortas of GPx-1−/−mice compared with WT mice, which returned toward normal after OTC treatment. Aortic sections from GPx-1−/−mice showed increased binding of an anti-3-nitrotyrosine antibody in the absence of frank vascular lesions. These findings demonstrate that homozygous deficiency of GPx-1 leads to impaired endothelium-dependent vasodilator function presumably due to a decrease in bioavailable nitric oxide and to increased vascular oxidant stress. These vascular abnormalities can be attenuated by increasing bioavailable intracellular thiol pools.
Tissue-specific distributions of alternatively spliced human PECAM-1 isoforms
Authors: Wang Yongji , Su Xiaojing , Sorenson Christine M. , Sheibani Nader
Published: March 2015

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion molecule that is highly expressed on the surface of endothelial cells and some hematopoietic cells. Its cytoplasmic domain is encoded by multiple exons, which undergo alternative splicing. Here, we demonstrate that the human PECAM-1 cytoplasmic domain undergoes alternative splicing, generating six different isoforms. RT-PCR cloning and DNA sequence analysis indicated that human tissue and endothelial cells express multiple isoforms of PECAM-1, including the full-length PECAM-1 and five other isoforms, which lack exon 12, 13, 14, or 15 or exons 14 and 15. The full-length PECAM-1 is the predominant isoform detected in human tissue and endothelial cells. This is in contrast to murine endothelium, in which the PECAM-1 isoform lacking exons 14 and 15 is the predominant isoform. The PECAM-1 isoform lacking exon 13 detected in human tissue and endothelial cells is absent in murine endothelium. The expression pattern of PECAM-1 isoforms changes during tube formation of endothelial cells on Matrigel, which may indicate specialized roles for specific isoforms of PECAM-1 during angiogenesis. The data presented here demonstrate that human PECAM-1 undergoes alternative splicing, generating multiple isoforms in vascular beds of various tissues. Therefore, the regulated expression of these isoforms may influence endothelial cell adhesive properties during angiogenesis and/or vasculogenesis.
Enhanced iNOS function in myocytes one day after brief ischemic episode
Authors: Kim Song-Jung , Kim Young-Kwon , Takagi Gen , Huang Cheng-Hsiung , Geng Yong-Jian , Vatner Stephen F.
Published: March 2015

There is increasing evidence that nitric oxide (NO) produced by inducible NO synthase (iNOS) plays a key role in cadioprotection during the “second window of protection” (SWOP). The goals of this study were to determine 1) whether a transient ischemic episode [10-min coronary artery occlusion (CAO), followed by full reperfusion] enhances NOS function in cardiac myocytes, 2) which specific NOS isoform is responsible for the enhanced NOS function in myocytes, and 3) to localize iNOS expression during SWOP. To address these questions, 10 dogs were instrumented to measure aortic and left ventricular pressures and wall thickness. At 1–2 wk after recovery, myocardial ischemia was induced regionally by a 10-min left circumflex CAO. After 24-h reperfusion, cardiac myocytes were isolated from the previously ischemic and nonischemic regions ( n = 6). Myocyte contractile function was assessed using a video motion detector at 1 Hz (35 ± 2°C). At baseline, myocyte contractile function (% contraction) was similar in the two regions (ischemic 7.8 ± 0.5% vs. nonischemic 7.8 ± 0.2%).l-Arginine (1 mM) significantly reduced ( P < 0.05) myocyte contraction in the ischemic (−34 ± 3%, P < 0.05) but not (−7 ± 4%) nonischemic regions; these responses were abolished by NG-nitro-l-arginine (1 mM), a nonspecific NOS inhibitor, as well as 2-amino-5,6-dihydro-6-methy-4H-1,3,thiazine (1 mM), a specific iNOS inhibitor. Immunohistochemistry also revealed enhanced iNOS expression in the myocardium and in particular the interstitial spaces in the ischemic zone. These results indicate that a brief ischemic episode upregulates iNOS function in myocytes as well as in the interstitial space between blood vessels and myocytes, strategically where it can regulate both vascular and myocyte function during the SWOP.
Inhaled NO inhibits platelet aggregation and elevates plasma but not intraplatelet cGMP in healthy human volunteers
Authors: Beghetti Maurice , Sparling Catherine , Cox Peter N. , Stephens Derek , Adatia Ian
Published: March 2015

Effects of inhaled nitric oxide (NO) on human platelet function are controversial. It is uncertain whether intraplatelet cGMP mediates the effect of inhaled NO on platelet function. We investigated the effect of 30 ppm inhaled NO on platelet aggregation and plasma and intraplatelet cGMP in 12 subjects. We performed platelet aggregation studies by using a photooptical aggregometer and five agonists (ADP, collagen, epinephrine, arachidonic acid, and ristocetin). During inhalation, the maximal extent of platelet aggregation decreased by 75% with epinephrine ( P < 0.005), 56% with collagen ( P < 0.005), and 20% with arachidonic acid ( P < 0.05). Responses to ADP (8% P > 0.05) and ristocetin (5% P > 0.05) were unaffected. Platelet aggregation velocity decreased by 64% with collagen ( P < 0.005), 60% with epinephrine ( P < 0.05), 33% with arachidonic acid ( P < 0.05), and 14% with ADP ( P > 0.05). Plasma cGMP levels increased from 2.58 ± 0.43 to 9.99 ± 5.57 pmol/ml ( P < 0.005), intraplatelet cGMP levels were unchanged (means ± SD: 1.96 ± 0.58 vs. 2.71 ± 1.67 pmol/109platelets; P > 0.05). Inhaled NO inhibits platelet aggregation via a cGMP independent mechanism.
Role of phospholipase C in development of myogenic tone in rat posterior cerebral arteries
Authors: Jarajapu Yagna P. R. , Knot Harm J.
Published: March 2015

Earlier studies have implicated phospholipase C (PLC) in the development of myogenic tone (MT) based on pharmacological studies in larger arteries. In the present study, we further investigated the cellular effects of PLC inhibition using pharmacological and electrophysiological approaches to provide more quantitative functional evidence for the involvement of PLC in the genesis of MT in small cerebral arteries. The phosphatidylinositol-selective PLC (PI-PLC) inhibitor U-73122 decreased MT by 87% in posterior cerebral arteries from Sprague-Dawley rats with pIC50 of 6.2 ± 0.09 ( n = 5). Similar potency (pIC50 of 6.2 ± 0.04, n = 5) was observed in arteries with MT that were further constricted with 30 nM serotonin. The phosphatidylcholine-specific (PC-PLC) inhibitor D609 had no effect on MT. U-73343, the inactive analog of U-73122, did not show any relaxant effect, but at higher concentrations (>1 μM) it reduced MT. In the presence of 125–500 nM U-73122, the pressure-diameter curves shifted toward that obtained in Ca-free conditions. U-73122-mediated decrease in MT was accompanied by a decrease in mean arterial wall calcium (maximum effect: 77 ± 3% of 16 mM KCl-mediated decrease, n = 4). This was due to a simultaneous membrane potential hyperpolarization of ∼9 mV or from −44 ± 1 to −53 ± 2 mV (10 μM, P < 0.001, n = 8). In summary, this study provides the first quantitative data suggesting a critical importance of PI-PLC in the genesis of pressure-induced MT in rat cerebral arteries via membrane potential depolarization and increased calcium influx.
Sequential opening of IP3-sensitive Ca2+channels and SOC during α-adrenergic activation of rabbit vena cava
Authors: Lee Cheng-Han , Rahimian Roshanak , Szado Tania , Sandhu Jasmin , Poburko Damon , Behra Tasniem , Chan Lally , van Breemen Cornelis
Published: March 2015

α1-Aderenoceptor-mediated constriction of rabbit inferior vena cava (IVC) is signaled by asynchronous wavelike Ca2+ oscillations in the in situ smooth muscle. We have shown previously that a putative nonselective cationic channel (NSCC) is required for these oscillations. In this report, we show that the application of 2-aminoethoxyphenyl borate (2-APB) to antagonize inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ release channels (IP3R channels) can prevent the initiation and abolish ongoing α1-aderenoceptor-mediated tonic constriction of the venous smooth muscle by inhibiting the generation of these intracellular Ca2+ concentration ([Ca2+]i) oscillations. The observed effects of 2-APB can only be attributed to its selective inhibition on the IP3R channels, not to its slight inhibition of the L-type voltage-gated Ca2+ channel and the sarco(endo)plasmic reticulum Ca2+ ATPase. Furthermore, 2-APB had no effect on the ryanodine-sensitive Ca2+ release channel and the store-operated channel (SOC) in the IVC. These results indicate that the putative NSCC involved in refilling the sarcoplasmic reticulum (SR) and maintaining the tonic contraction is most likely an SOC-type channel because it appears to be activated by IP3R-channel-mediated SR Ca2+ release or store depletion. This is in accordance with its sensitivity to Ni2+ and La3+ (SOC blockers). More interestingly, RT-PCR analysis indicates that transient receptor potential (Trp1) mRNA is strongly expressed in the rabbit IVC. The Trp1 gene is known to encode a component of the store-operated NSCC. These new data suggest that the activation of both the IP3R channels and the SOC are required for PE-mediated [Ca2+]i oscillations and constriction of the rabbit IVC.
Inferior vena caval hemodynamics quantified in vivo at rest and during cycling exercise using magnetic resonance imaging
Authors: Cheng Christopher P. , Herfkens Robert J. , Taylor Charles A.
Published: March 2015

Compared with the abdominal aorta, the hemodynamic environment in the inferior vena cava (IVC) is not well described. With the use of cine phase-contrast magnetic resonance imaging (MRI) and a custom MRI-compatible cycle in an open magnet, we quantified mean blood flow rate, wall shear stress, and cross-sectional lumen area in 11 young normal subjects at the supraceliac and infrarenal levels of the aorta and IVC at rest and during dynamic cycling exercise. Similar to the aorta, the IVC experienced significant increases in blood flow and wall shear stress as a result of exercise, with greater increases in the infrarenal level compared with the supraceliac level. At the infrarenal level during resting conditions, the IVC experienced higher mean flow rate than the aorta (1.2 ± 0.5 vs. 0.9 ± 0.4 l/min, P < 0.01) and higher mean wall shear stress than the aorta (2.0 ± 0.6 vs. 1.3 ± 0.6 dyn/cm2, P < 0.005). During exercise, wall shear stress remained higher in the IVC compared with the aorta, although not significantly. It was also observed that, whereas the aorta tapers inferiorly, the IVC tapers superiorly from the infrarenal to the supraceliac location. The hemodynamic and anatomic data of the IVC acquired in this study add to our understanding of the venous circulation and may be useful in a clinical setting.
Mouse heart Na+channels: primary structure and function of two isoforms and alternatively spliced variants
Authors: Zimmer Thomas , Bollensdorff Christian , Haufe Volker , Birch-Hirschfeld Eckhard , Benndorf Klaus
Published: March 2015

We isolated two full-length cDNA clones from the adult murine heart that encode two different voltage-gated Na+channels: mH1 and mH2. Sequence comparisons indicated that mH1 is highly homologous to rat SCN5A, whereas mH2 is highly homologous to SCN4A, expressed in rat skeletal muscle. Electrophysiological properties of mH1 channels strongly resembled the tetrodotoxin (TTX)-resistant Na+current of mouse ventricular cells, whereas mH2 channels activated at more positive potentials and were highly sensitive to TTX [50% inhibitory constant (IC50) = 11 nM]. We found that mH2 is not expressed in cardiac cells of neonatal mice, but appears to be upregulated during the development. Besides these Na+channel isoforms, we also detected two alternatively spliced mH1 variants that were characterized by deletions within the sequence coding for the intracellular loop between domains II and III. One of the shortened channels, mH1–2, developed Na+currents indistinguishable from those of mH1. The other splice variant (mH1–3) did not form functional channels. Quantitative reverse transcriptase-polymerase chain reaction indicated that RNA preparations of the adult mouse heart contain 54% mH1, 25% mH1–2, 16% mH2, and 5% mH1–3. Conclusively, mH1 generates the main portion of the mouse cardiac TTX-resistant Na+current and mH2 is a candidate for TTX-sensitive currents previously described in adult cardiomyocytes. Furthermore, the presence of mH1–2 and mH1–3 transcripts indicates that alternative splicing plays a role in the regulation of functional Na+channels in cardiomyocytes.
Cerebral artery responses to pressure and flow in uremic hypertensive and spontaneously hypertensive rats
Authors: New D. I. , Chesser A. M. S. , Thuraisingham R. C. , Yaqoob M. M.
Published: March 2015

Impaired cerebral blood flow autoregulation is seen in uremic hypertension, whereas in nonuremic hypertension autoregulation is shifted toward higher perfusion pressure. The cerebral artery constricts in response to a rise in either lumen pressure or flow; we examined these responses in isolated middle cerebral artery segments from uremic Wistar-Kyoto rats (WKYU), normotensive control rats (WKYC), and spontaneously hypertensive rats (SHR). Pressure-induced (myogenic) constriction developed at 100 mmHg; lumen flow was then increased in steps from 0 to 98 μl/min. Some vessels were studied after endothelium ablation. Myogenic constriction was significantly lower in WKYU (28 ± 2.9%) compared with both WKYC (39 ± 2.5%, P = 0.035) and SHR (40 ± 3.1%, P = 0.018). Flow caused constriction of arteries from all groups in an endothelium-independent manner. The response to flow was similar in WKYU and WKYC, whereas SHR displayed increased constriction compared with WKYU ( P < 0.001) and WKYC ( P < 0.001). We conclude that cerebral myogenic constriction is decreased in WKYU, whereas flow-induced constriction is enhanced in SHR.