bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhaled nitric oxide inhibits NOS activity in lambs: potential mechanism for rebound pulmonary hypertension
Authors: Black Stephen M. , Heidersbach R. Scott , McMullan D. Michael , Bekker Janine M. , Johengen Michael J. , Fineman Jeffrey R.
Published: December 2017

Life-threatening increases in pulmonary vascular resistance have been noted on acute withdrawal of inhaled nitric oxide (NO), although the mechanisms remain unknown. In vitro data suggest that exogenous NO exposure inhibits endothelial NO synthase (NOS) activity. Thus the objectives of this study were to determine the effects of inhaled NO therapy and its acute withdrawal on endogenous NOS activity and gene expression in vivo in the intact lamb. Six 1-mo-old lambs were mechanically ventilated and instrumented to measure vascular pressures and left pulmonary blood flow. Inhaled NO (40 ppm) acutely decreased left pulmonary vascular resistance by 27.5 ± 4.7% ( P < 0.05). This was associated with a 207% increase in plasma cGMP concentrations ( P < 0.05). After 6 h of inhaled NO, NOS activity was reduced to 44.3 ± 5.9% of pre-NO values ( P < 0.05). After acute withdrawal of NO, pulmonary vascular resistance increased by 52.1 ± 11.6% ( P < 0.05) and cGMP concentrations decreased. Both returned to pre-NO values within 60 min. One hour after NO withdrawal, NOS activity increased by 48.4 ± 19.1% to 70% of pre-NO values ( P < 0.05). Western blot analysis revealed that endothelial NOS protein levels remained unchanged throughout the study period. These data suggest a role for decreased endogenous NOS activity in the rebound pulmonary hypertension noted after acute withdrawal of inhaled NO.
Echocardiographic assessment of cardiac function in conscious and anesthetized mice
Authors: Yang Xiao-Ping , Liu Yun-He , Rhaleb Nour-Eddine , Kurihara Nobutaka , Kim Henry E. , Carretero Oscar A.
Published: December 2017

Using a high-frequency linear transducer (15L8), we studied 1) the feasibility of performing echocardiography in nonanesthetized mice compared with mice given pentobarbital sodium (Pento) or a mixture of ketamine and xylazine and 2) the feasibility of echocardiographic evaluation of left ventricular (LV) hypertrophy, dilatation, and function in mice with two-kidney, one-clip hypertension or myocardial infarction (MI). Heart rate (HR) in awake mice was 658 ± 9 beats/min; Pento and ketamine plus xylazine reduced HR to 377 ± 11 and 293 ± 19 beats/min, respectively, associated with a significant decrease in shortening fraction (SF), ejection fraction (EF), and cardiac output (CO) and an increase in LV end-diastolic (LVEDD) and end-systolic dimensions (LVESD). Mice with 4 wk of two-kidney, one-clip hypertension had increased LV mass (15.62 ± 0.62 vs. 22.17 ± 1.79 mg) without altered LV dimensions, SF, EF, or CO. Mice studied 4 wk post-MI exhibited obvious LV dilatation and systolic dysfunction, as evidenced by increased LVEDD and LVESD and decreased SF, EF, and CO. Our findings clearly show the adverse impact of anesthesia on basal cardiac function and the difficulty in interpreting data obtained from anesthetized mice. We believe this is the first study to demonstrate the feasibility of using echocardiography to assess cardiovascular function in the nonanesthetized mouse.
VEGF stimulates tyrosine phosphorylation of β-catenin and small-pore endothelial barrier dysfunction
Authors: Cohen Alex W. , Carbajal José M. , Schaeffer Richard C.
Published: December 2017

The purpose of this study was to test the hypothesis that tyrosine phosphorylation signaling events and protein kinase C (PKC) activation mediate vascular endothelial growth factor-A165(VEGF)-induced endothelial cell (EC) proliferation and barrier dysfunction in bovine pulmonary artery EC monolayers. A size-selective permeability assay showed that VEGF stimulated a delayed, prolonged (6–45 h), concentration-dependent (50–200 ng/ml, ∼1–4 nM) increase in the number of predominantly small-“pore” transport pathways (<60 Å) across EC monolayers. The tyrosine kinase inhibitor herbimycin A (HA) and the selective PKC inhibitor bisindolylmaleimide (BIM) prevented this phenomenon. After 6–24 h, VEGF-treated monolayers displayed an HA- and BIM-sensitive reorganization of β-catenin adherens junctions with fingerlike projections and the loss of β-catenin at sites of small paracellular hole formation. HA and BIM prevented the VEGF-induced increase in EC growth. HA blocked the VEGF-induced rapid and prolonged (10 min–45 h) increases in the phosphotyrosine (PY) contents of VEGF receptor 2, phospholipase C-γ1, paxillin, and β-catenin as well as ∼140- and 128- to 117-kDa proteins, whereas BIM inhibited only the tyrosine phosphorylation of β-catenin. These data suggest that VEGF initiates increased EC growth and chronic, small-pore endothelial barrier dysfunction by PY signaling through β-catenin that depends on PKC.
Mapping of capillary flow, cellular redox state, and resting membrane potential in hypoperfused rat myocardium
Authors: Brasch Frank , Neckel Marion , Volkmann Rolf , Schmidt Gerhard , Hellige Gerhard , Vetterlein Friedrich
Published: December 2017

The influence on myocyte viability of ischemia-induced changes in capillary perfusion was studied in the hearts of anesthetized rats subjected to partial occlusion of the left coronary artery for 45 min. Timed plasma labeling was applied to determine perfusion patterns. Changes in the fluorescence of preloaded potential-sensitive dyes [tetramethylrhodamine methyl ester (TMRM) and bis-oxonol], of trypan blue, and of endogeneous NADH were utilized in characterizing myocyte viability in histological sections of the heart. Within the hypoperfused zone, localized areas appeared vascularly nonlabeled for periods of at least 10 min. Within these areas a reduction in TMRM fluorescence occurred in 82.5% of the tissue, signaling a reduced resting membrane potential. In the same areas 37.7% of the myocytes revealed an NADH fluorescence lower than that regularly found in anoxic tissues. This correlated with an especially low level of TMRM, with increased fluorescence bis-oxonol and with an accumulation of trypan blue. In conclusion, in localized hypoperfusion-induced zones lacking capillary flow, an inhomogeneous pattern of reductions in myocyte viability develops, which appears to be relevant in ischemia-induced arrhythmias.
Capillary recruitment in response to tissue hypoxia and its dependence on red blood cell deformability
Authors: Parthasarathi Kaushik , Lipowsky Herbert H.
Published: December 2017

The effect of reduced red blood cell (RBC) deformability on microvessel recruitment attendant to a reduction in tissue[Formula: see text] was studied in rat cremaster muscle using indicator-dilution techniques. Transit times (TT) of fluorescently labeled RBCs (TTRBC) and plasma (TTPl) between functionally paired arterioles and venules were obtained from their dispersion throughout the microvascular network. Changes in[Formula: see text] were effected by superfusing the tissue with Ringer solution deoxygenated to different levels. Arteriolar blood flow (Q˙) was measured with the two-slit technique, and the vascular volume (V) occupied by RBCs and plasma was computed from the product of Q˙ × TT during bolus infusions of rat and less deformable human RBCs to obtain VRBCand fluorescently labeled albumin to obtain VPl. Measurements of TTRBCand TTPlpermitted computation of an average flow-weighted tissue (microvascular) hematocrit (HM) relative to systemic values (HS). During infusions of autologous rat RBCs, Q˙ and total V increased threefold in response to hypoxia, whereas normalized RBC TT (TTRBC/TTPl) and normalized tissue hematocrit (HM/HS) did not show a significant trend, indicating an increase in the number of pathways through which the RBCs can traverse the network because of spatial recruitment of capillaries. In contrast, during infusions of human RBCs, TTRBC/TTPland HM/HSdecreased significantly in response to hypoxia. AlthoughQ˙ exhibited an increase similar to that during rat RBC infusions, VRBCexhibited a smaller increase compared with VPl, suggesting that reduced RBC deformability leads to a redistribution of RBCs through larger-diameter pathways within the network and exclusion of these RBCs from pathways normally recruited during hypoxia. Hence, reduced RBC deformability may adversely affect capillary recruitment and physiological mechanisms that ensure adequate delivery of oxygen to tissue.
Caveolae require intact VAMP for targeted transport in vascular endothelium
Authors: McIntosh Deirdre P. , Schnitzer Jan E.
Published: December 2017

Caveolae appear to function in vesicular trafficking of specific molecular cargo into and across vascular endothelial and other cells. They contain the molecular machinery for docking and fusion, similar to other vesicular trafficking systems, yet the mechanisms mediating ligand internalization and targeted intracellular transport by caveolae remain unclear. Using immunoelectron microscopy, we show that caveolae in the microvascular endothelium of rat lung express vesicle-associated membrane protein (VAMP)-2 (also called synaptobrevin) on their cytoplasmic surface. Immunofluorescence studies of cholera toxin B (CTB)-FITC internalization in toxin-treated cells demonstrate that intact VAMP-2 is necessary for the efficient trafficking of caveolar ligands. The CTB subunit binds preferentially to GM1in caveolae, and N-ethylmaleimide treatment drastically inhibits the intracellular accumulation of CTB. The cleavage of caveolar VAMP-2 with VAMP-specific neurotoxins (botulinum D and F but not A) significantly inhibits CTB endocytosis and targeted intracellular accumulation in cultured endothelial cells. This impairment of caveolae-mediated trafficking provides evidence that caveolae require intact VAMP-2 for efficient targeted delivery via vesicle docking with target organelles.
Effect of aging on gender differences in neural control of heart rate
Authors: Kuo Terry B. J. , Lin Tsann , Yang Cheryl C. H. , Li Chia-Lin , Chen Chieh-Fu , Chou Pesus
Published: December 2017

To clarify the influence of gender on sympathetic and parasympathetic control of heart rate in middle-aged subjects and on the subsequent aging process, heart rate variability (HRV) was studied in normal populations of women ( n = 598) and men ( n = 472) ranging in age from 40 to 79 yr. These groups were divided into eight age strata at 5-yr intervals and were clinically diagnosed as having no hypertension, hypotension, diabetic neuropathy, or cardiac arrhythmia. Frequency-domain analysis of short-term, stationary R-R intervals was performed, which reveals very-low-frequency power (VLF; 0.003–0.04 Hz), low-frequency power (LF; 0.04–0.15 Hz), high-frequency power (HF; 0.15–0.40 Hz), the ratio of LF to HF (LF/HF), and LF and HF power in normalized units (LF% and HF%, respectively). The distribution of variance, VLF, LF, HF, and LF/HF exhibited acute skewness, which was adjusted by natural logarithmic transformation. Women had higher HF in the age strata from 40 to 49 yr, whereas men had higher LF% and LF/HF between 40 and 59 yr. No disparity in HRV measurements was found between the sexes in age strata ≥60 yr. Although absolute measurements of HRV (variance, VLF, LF, and HF) decreased linearly with age, no significant change in relative measurements (LF/HF, LF%, and HF%), especially in men, was detected until age 60 yr. We conclude that middle-aged women and men have a more dominant parasympathetic and sympathetic regulation of heart rate, respectively. The gender-related difference in parasympathetic regulation diminishes after age 50 yr, whereas a significant time delay for the disappearance of sympathetic dominance occurs in men.
Dietary fish oil promotes positive inotropy of ouabain in the rat heart
Authors: Maixent Jean-Michel , Gerbi Alain , Barbey Odile , Lan Carole , Jamme Isabelle , Burnet Henri , Nouvelot André , Lévy Samuel , Cozzone Patrick J. , Bernard Monique
Published: December 2017

We tested the hypothesis that a fish oil (FO) diet promotes positive inotropy of ouabain without increased toxicity. For 2 mo, two groups of adult male rats were fed 1) a regular food diet supplemented with dietary long-chain polyunsaturated fatty acid from FO or 2) a regular food diet (control). The responsiveness to ouabain was evaluated for the two groups in Langendorff-perfused hearts, by31P nuclear magnetic resonance spectroscopy, and on purified membrane-bound Na-K-ATPase. The maximum positive inotropy achieved with ouabain was nearly two times higher in the FO than in the control group and was not associated with significant changes in energetics. Alteration of function and energetic metabolism and inhibition of Na-K-ATPase in response to 3 × 10−4M ouabain were delayed in the FO group. This study demonstrates that dietary FO, by a cardiac membrane incorporation of n–3 polyunsaturated fatty acid, promotes positive inotropy of ouabain without toxicity and changes in cardiac metabolism.
Intravenous angiotensinogen antisense in AAV-based vector decreases hypertension
Authors: Tang Xiaoping , Mohuczy Dagmara , Zhang Y. Clare , Kimura Birgitta , Galli Sara M. , Phillips M. Ian
Published: December 2017

Angiotensinogen (AGT) has been linked to hypertension. Because there are no direct inhibitors of AGT, we have developed antisense (AS) inhibition of AGT mRNA delivered in an adeno-associated virus (AAV)-based plasmid vector. This plasmid, driven by the cytomegalovirus promoter, contains a green fluorescent protein reporter gene and AS cDNA for rat AGT. Transfection of the plasmid into rat hepatoma cells brought a strong expression of the transgenes and a significant reduction in the level of AGT. In the in vivo study, naked plasmid DNA was intravenously injected into adult spontaneously hypertensive rats at different doses (0.6, 1.5, and 3 mg/kg). Expression of AGT AS mRNA was present in liver and heart, and it lasted longer in the liver. All three doses produced a significant decrease in blood pressure (BP). BP decreased for 2, 4, and 6 days, respectively. The lowest dose decreased BP by 12 ± 3.0 mmHg, whereas the higher doses decreased BP by up to 22.5 ± 5.2 mmHg compared with the control rats injected with saline ( P < 0.01). The injection of the plasmid with liposomes produced a more profound and longer reduction (8 days) in BP. Consistent changes in plasma AGT level were observed. Sense plasmid had no effect. No liver toxicity was observed after injection of AS plasmid with or without liposomes. Our results suggest that the systemic delivery of AS against AGT mRNA by AAV-based plasmid vector, especially with liposomes, may have potential for gene therapy of hypertension and that further studies with the plasmid packaged into a recombinant AAV vector for a longer-lasting AS effect are warranted.
Novel method to estimate ventricular contractility using intraventricular pulse wave velocity
Authors: Shishido Toshiaki , Sugimachi Masaru , Kawaguchi Osamu , Miyano Hiroshi , Kawada Toru , Matsuura Wataru , Ikeda Yasuhiro , Sunagawa Kenji
Published: December 2017

We developed a novel technique for estimating ventricular contractility using intraventricular pulse wave velocity (PWV). In eight isolated, cross-circulated canine hearts, we used a fast servo pump to inject a volume pulse into the base of the left ventricular chamber at late diastole and at late systole. We measured the transit time of the volume pulse wave as it traversed the distance from base to apex and calculated the intraventricular PWV. The intraventricular PWV increased from diastole (2.3 ± 0.4 m/s) to systole (11.7 ± 2.4 m/s, P < 0.0001 vs. diastole). The square of the intraventricular PWV at late systole correlated linearly with the left ventricular end-systolic elastance ( r = 0.939, P < 0.0001) and with the end-systolic Young's modulus ( r = 0.901, P < 0.0001). Moreover, the intraventricular PWV was insensitive to preload. We conclude that the intraventricular PWV at late systole reflects left ventricular end-systolic elastance reasonably well. The fact that estimation of PWV does not require volume measurement or load manipulation makes this technique an attractive means of assessing ventricular contractility.
Voltage-dependent facilitation of cardiac L-type Ca channels expressed in HEK-293 cells requires β-subunit
Authors: Kamp Timothy J. , Hu Hai , Marban Eduardo
Published: December 2017

The activity of native L-type Ca channels can be facilitated by strong depolarizations. The cardiac Ca channel α1C-subunit was transiently expressed in human embryonic kidney (HEK-293) cells, but these channels did not exhibit voltage-dependent facilitation. Coexpression of the Ca channel β1a- or β2a-subunit with the α1C-subunit enabled voltage-dependent facilitation in 40% of cells tested. The onset of facilitation in α1C+ β1a-expressing HEK-293 cells was rapid after a depolarization to +100 mV (τ = 7.0 ms). The kinetic features of the facilitated currents were comparable to those observed for voltage-dependent relief of G protein inhibition demonstrated for many neuronal Ca channels; however, intracellular dialysis with guanosine 5′- O-(2-thiodiphosphate) and guanosine 5′- O-(3-thiotriphosphate) in the patch pipette had no effect on facilitation. Stimulation of G protein-coupled receptors, either endogenous (somatostatin receptors) or coexpressed (adenosine A1receptors), did not affect voltage-dependent facilitation. These results indicate that the cardiac Ca channel α1C-subunit can exhibit voltage-dependent facilitation in HEK-293 cells only when coexpressed with an auxiliary β-subunit and that this facilitation is independent of G protein pathways.
C-type natriuretic peptide inhibits ANP secretion and atrial dynamics in perfused atria: NPR-B-cGMP signaling
Authors: Lee Sook Jeong , Kim Sung Zoo , Cui Xun , Kim Suhn Hee , Lee Kyung Sun , Chung Yu Jeong , Cho Kyung Woo
Published: December 2017

The purpose of the present experiments was to define the role of C-type natriuretic peptide (CNP) in the regulation of atrial secretion of atrial natriuretic peptide (ANP) and atrial stroke volume. Experiments were performed in perfused beating and nonbeating quiescent atria, single atrial myocytes, and atrial membranes. CNP suppressed in a dose-related fashion the increase in atrial stroke volume and ANP secretion induced by atrial pacing. CNP caused a right shift in the positive relationships between changes in the secretion of ANP and atrial stroke volume or translocation of the extracellular fluid (ECF), which indicates the suppression of atrial myocytic release of ANP into the paracellular space. The effects of CNP on the secretion and contraction were mimicked by 8-bromoguanosine 3′,5′-cyclic monophosphate (8-BrcGMP). CNP increased cGMP production in the perfused atria, and the effects of CNP on the secretion of ANP and atrial dynamics were accentuated by pretreatment with an inhibitor of cGMP phosphodiesterase, zaprinast. An inhibitor of the biological natriuretic peptide receptor (NPR), HS-142-1, attenuated the effects of CNP. The suppression of ANP secretion by CNP and 8-BrcGMP was abolished by a depletion of extracellular Ca2+in nonbeating atria. Natriuretic peptides increased cGMP production in atrial membranes with a rank order of potency of CNP > BNP > ANP, and the effect was inhibited by HS-142-1. CNP and 8-BrcGMP increased intracellular Ca2+concentration transients in single atrial myocytes, and mRNAs for CNP and NPR-B were expressed in the rabbit atrium. From these results we conclude that atrial ANP release and stroke volume are controlled by CNP via NPR-B-cGMP mediated signaling, which may in turn act via regulation of intracellular Ca2+.
Endothelial cells potentiate phagocytic killing by macrophages via platelet-activating factor release
Authors: Owaki Tetsuhiro , Meneshian Avedis , Maemura Kosei , Takao Sonshin , Wang Dajie , Fuh Katherine C. , Bulkley Gregory B. , Klein Andrew S.
Published: December 2017

The immunomodulatory function of endothelial cells (EC) includes the initiation of leukocyte margination, diapedesis, and activation through the upregulation of various cell surface-associated molecules. However, the effect that EC have on the phagocytic function of neighboring monocytes and macrophages is less well described. To address this issue, microvascular EC were cocultured with murine peritoneal macrophages, first in direct contact, then in a noncontact coculture system, and macrophage phagocytosis and phagocytic killing were assessed. The presence of increasing concentrations of EC resulted in a dose-dependent increase in macrophage phagocytic killing. This stimulatory effect was inhibited in a dose-dependent manner by the pretreatment of macrophage/EC cocultures with WEB-2086 or CV-6209, specific platelet-activating factor (PAF)-receptor antagonists, but not by anti-tumor necrosis factor-α, anti-interleukin (IL)-1α, or anti-IL-1β. Furthermore, the effect was reproduced in the absence of EC by the exogenous administration of nanomolar concentrations of PAF. Microvascular EC potentiate macrophage phagocytic killing via the release of a soluble signal; PAF appears to be an important component of that signal.
Effects of natriuretic peptides on load and myocardial function in normal and heart failure dogs
Authors: Lainchbury John G. , Burnett John C. , Meyer Donna , Redfield Margaret M.
Published: December 2017

The effects on myocardial function and loading conditions of clinically relevant doses of the natriuretic peptides (NP) have not been established. The actions of single doses (100 ng ⋅ kg−1⋅ min−1iv over 30 min) of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) were studied in conscious normal dogs and in dogs with pacing-induced heart failure. All three NP reduced end-diastolic pressure in normal dogs, and ANP and BNP reduced end-diastolic volume. In heart failure ANP and BNP reduced EDP, and ANP reduced EDV. Arterial elastance was unchanged in normal dogs and in dogs with heart failure. ANP increased end-systolic elastance ( Ees) in normal dogs, whereas BNP tended to increase Ees( P = 0.06). In dogs with heart failure, no inotropic effect was seen. In normal dogs, all NP reduced the time constant of isovolumic relaxation (τ), and ANP and BNP reduced τ in dogs with heart failure. Increases in plasma cGMP in dogs with heart failure were blunted. The NP reduced preload and enhanced systolic and diastolic function in normal dogs. Effects of ANP and BNP on preload and diastolic function were maintained in heart failure. Lack of negative inotropic effects in heart failure supports the validity of the NP as therapeutic agents.
Dynamics of flow, resistance, and intramural vascular volume in canine coronary circulation
Authors: Spaan Jos A. E. , Cornelissen Annemiek J. M. , Chan Charles , Dankelman Jenny , Yin Frank C. P.
Published: December 2017

Varying coronary volume will vary vascular resistance and thereby have an effect on coronary hemodynamics. Six ventricular septa were isolated from anesthetized dogs, dispersed in a biaxial stretch apparatus at diastolic stress, and perfused artificially with an oxygenated perfluorochemical emulsion at maximal vasodilation. Flow and thickness were measured continuously by an electromagnetic flow probe and sonomicrometer. Pressure was varied sinusoidally around 30, 50, and 70 mmHg with an amplitude of 7.5 mmHg; frequencies ranged between 0.015 and 7 Hz. Bode plots of admittance (flow/pressure) and capacitance (scaled thickness/pressure) were constructed. A two-compartment model was used in which the resistances vary with volume. Realistic values of microvascular compliance (∼0.3 ml ⋅ mmHg−1 ⋅ 100 g−1) were found. Values 10 times higher were then found when resistances were forced to be constant. We concluded that volume dependence of resistances have to be taken into account when dynamic or static pressure-flow relations are studied and conceal the effect of a large intramyocardial compliance on arterial hemodynamics.
Electrical interactions between a real ventricular cell and an anisotropic two-dimensional sheet of model cells
Authors: Wang Yang-Gan , Kumar Rajiv , Wagner Mary B. , Wilders Ronald , Golod David A. , Goolsby William N. , Joyner Ronald W.
Published: December 2017

We have extended our “coupling clamp” technique, in which we couple a real cell to a real-time simulation of a model cell, to now incorporate a real cardiac cell as the central element of a two-dimensional sheet of model cells, in which the coupling conductances may be different in the x and y directions and a specific region of lack of coupling conductance may serve as a resistive barrier. We stimulated the real cell in the central location and determined the critical size of the real cell for successful activation of the entire sheet. We found that this critical size was decreased when anisotropy was present compared with the isotropic case and was further decreased when the central site of stimulation was close to the resistive barrier. The heart normally has some degree of anisotropy, and it has been shown that the remodeling that occurs in peri-infarction zones produces a particular loss of lateral connections compared with end-to-end connections among heart cells. We propose that the normal existence of anisotropy and enhancement of the degree of anisotropy both by loss of lateral gap junctions and the development of resistive barriers may play a facilitating role in the development of ectopic foci that may lead to cardiac arrhythmias.
Developmental differences in delayed rectifying outward current in feline ventricular myocytes
Authors: Barajas-Martínez Héctor , Elizalde Alejandro , Sánchez-Chapula José A.
Published: December 2017

In the present work, we found that the delayed rectifying outward potassium current ( I K) in adult and neonatal cat ventricular myocytes consists of both rapid and slow components, I Kr and I Ks, respectively, which can be isolated pharmacologically. Thus after complete blockade of I Kr with dofetilide, the remaining I Ks current is homogeneous, as shown by an envelope of tails test. I Kr maximum tail current density, measured at −40 mV, was similar in adult and neonatal myocytes. I Ks maximum tail current density in neonatal myocytes, measured at −40 mV, was significantly smaller than in adult myocytes. Activation kinetics of I Kr and I Ks was similar in both age groups. However, the I Kr deactivation time course was significantly faster in neonatal than in adult myocytes. Developmental differences in the subunit composition of I Kr that display distinctly different deactivation kinetics are suggested.
Effects of potassium channel modulators on myotropic responses of aortic rings of pregnant rats
Authors: Cadorette C. , Sicotte B. , Brochu M. , St-Louis J.
Published: December 2017

The contribution of potassium channels [ATP-sensitive potassium (KATP) and high-conductance calcium-activated potassium (BKCa) channels] in the resistance of aortic rings of term pregnant rats to phenylephrine (Phe), arginine vasopressin (AVP), and KCl was investigated. Concentration-response curves to tetraethylammonium (TEA), a nonselective K+ channel inhibitor, were obtained in the absence or presence of KCl. TEA induced by itself concentration-dependent responses only in aortic rings of nonpregnant rats. These responses to TEA could be modulated in both groups of rings by preincubation with different concentrations of KCl. Concentration-response curves to Phe, AVP, and KCl were obtained in the absence or presence of cromakalim or NS-1619 (KATP and BKCa openers, respectively) and glibenclamide or iberiotoxin (KATPand BKCa inhibitors, respectively). Cromakalim significantly inhibited the responses to the three agonists in a concentration-dependent manner in both groups of rats. Alternatively, in the pregnant group of rats, glibenclamide increased the sensitivity to all three agonists. NS-1619 also inhibited the response to all agonists. With AVP and KCl, its effect was greater in aortic rings of pregnant than nonpregnant rats. Finally, iberiotoxin increased the sensitivity to all three agents. This effect was more important in aortic rings of nonpregnant rats and was accompanied by an increase of the maximal response to Phe and AVP. These results suggest that potassium channels are implicated in the control of basal membrane potential and in the blunted responses to these agents during pregnancy.
Nonlinear changes of transmembrane potential caused by defibrillation shocks in strands of cultured myocytes
Authors: Fast Vladimir G. , Rohr Stephan , Ideker Raymond E.
Published: December 2017

Organization of cardiac tissue into cell strands and layers has been implicated in changes of transmembrane potential (Δ V m) during defibrillation. To determine the shock-induced Δ V m in such structures, cell strands of variable width [strand width (SW) = 0.15–2 mm] were grown in culture. Uniform-field shocks with variable strength [shock strength (SS) = 2–50 V/cm] were applied across strands during the action potential (AP) plateau, and Δ V m were measured optically. Three different types of Δ V m were observed. Small Δ V m [<40%AP amplitude (APA)] were linearly dependent on SS and SW and were symmetrically distributed about a strand centerline with maximal positive and negative Δ V m on opposite strand sides being equal. Intermediate Δ V m (<200%APA) were strongly asymmetric with negative Δ V m > positive Δ V m because of a negative time-dependent shift of V m at the depolarized side of the strands. For large Δ V m (>200%APA), a second time-dependent shift of V m to more positive levels was observed in the hyperpolarized portions of strands, causing reduction of the Δ V m asymmetry. We conclude that during application of shocks to cell strands during the AP plateau, passive changes of V m were followed by two voltage- and time-dependent shifts of V m, possibly reflecting changes of ionic currents or membrane electroporation.
Physiological cyclic stretch causes cell cycle arrest in cultured vascular smooth muscle cells
Authors: Chapman Gary B. , Durante William , Hellums J. David , Schafer Andrew I.
Published: December 2017

Smooth muscle cells (SMC) are the major cellular component of the blood vessel wall and are continuously exposed to cyclic stretch due to pulsatile blood flow. This study examined the effects of a physiologically relevant level of cyclic stretch on rat aortic vascular SMC proliferation. Treatment of static SMC with serum, platelet-derived growth factor, or thrombin stimulated SMC proliferation, whereas exposure of SMC to cyclic stretch blocked the proliferative effect of these growth factors. The stretch-mediated inhibition in SMC growth was not due to cell detachment or increased cell death. Flow cytometry analysis revealed that cyclic stretch increased the fraction of SMC in the G0/G1phase of the cell cycle. Stretch-inhibited G1/S phase transition was associated with a decrease in retinoblastoma protein phosphorylation and with a selective increase in the cyclin-dependent kinase inhibitor p21, but not p27. These results demonstrate that cyclic stretch inhibits SMC growth by blocking cell cycle progression and suggest that physiological levels of cyclic stretch contribute to vascular homeostasis by inhibiting the proliferative pathway of SMC.
Heart failure alters the strength and mechanisms of the muscle metaboreflex
Authors: Hammond Robert L. , Augustyniak Robert A. , Rossi Noreen F. , Churchill Paul C. , Lapanowski Karen , O'Leary Donal S.
Published: December 2017

We hypothesized that excessive sympathoactivation observed during strenuous exercise in subjects with heart failure (HF) may result from tonic activation of the muscle metaboreflex (MMR) via hypoperfusion of active skeletal muscle. We studied MMR responses in dogs during treadmill exercise by graded reduction of terminal aortic blood flow (TAQ) before and after induction of HF by rapid ventricular pacing. At a low workload, in both control and HF experiments, large decreases in TAQ were required to elicit the MMR pressor response. During control experiments, this pressor response resulted from increased cardiac output (CO), whereas in HF CO did not increase; thus the pressor response was solely due to peripheral vasoconstriction. In HF, MMR activation also induced higher plasma levels of vasopressin, norepinephrine (NE), and renin. At a higher workload, in control experiments any reduction of TAQ elicited MMR pressor responses. In HF, before any vascular occlusion, TAQ was already below MMR control threshold levels and reductions in TAQ again did not result in higher CO; thus SAP increased via peripheral vasoconstriction. NE rose markedly, indicating intense sympathetic activation. We conclude that in HF, the MMR is likely tonically active at moderate workloads and contributes to the tonic sympathoactivation.
Hydrogen peroxide induces LFA-1-dependent neutrophil adherence to cardiac myocytes
Authors: Lu Huifang , Youker Keith , Ballantyne Christie , Entman Mark , Smith C. Wayne
Published: December 2017

Adult cardiac myocytes express intercellular adhesion molecule (ICAM)-1 in response to cytokine stimulation. This allows stable adhesion of chemotactically stimulated but not unstimulated neutrophils. In the current study, we demonstrated that brief exposure of ICAM-1-expressing cardiac myocytes to H2O2promoted transient adhesive interactions between myocytes and neutrophils without added chemotactic factors. This transient adhesion differed in two ways from the stable adhesion promoted by exogenous chemotactic factors. It occurred more rapidly, peaking within 15 min, and it was dependent on leukocyte function-associated antigen (LFA)-1 (CD11a/CD18) on the neutrophil interacting with ICAM-1 on the myocyte. In contrast, chemotactic factor-induced adhesion peaked at 60 min and was dependent on Mac-1 (CD11b/CD18). The transient adhesion could be completely inhibited by platelet-activating factor (PAF)-receptor antagonists WEB-2086 and SDZ-64-412. These results indicate that canine neutrophils may utilize both LFA-1 and Mac-1 to adhere to adult cardiac myocytes, with LFA-1 triggered by a PAF-like activity induced in myocytes by H2O2.
Modulation of mouse cardiac function in vivo by eNOS and ANP
Authors: Gyurko Robert , Kuhlencordt Peter , Fishman Mark C. , Huang Paul L.
Published: December 2017

To study the role of endothelial nitric oxide synthase (eNOS) in cardiac function, we compared eNOS expression, contractility, and relaxation in the left ventricles of wild-type and eNOS-deficient mice. eNOS immunostaining is localized to the macro- and microvascular endothelium throughout the myocardium in wild-type mice and is absent in eNOS−/− mice. Whereas blood pressure is elevated in eNOS−/− mice, baseline cardiac contractility (dP/d tmax) is similar in wild-type and eNOS−/− mice (9,673 ± 2,447 and 9,928 ± 1,566 mmHg/s, respectively). The β-adrenergic agonist isoproterenol (Iso) at doses of ≥1 ng causes enhanced increases in dP/d tmaxin eNOS−/− mice compared with wild-type controls in vivo ( P < 0.01) as well as in Langendorff isolated heart preparations ( P < 0.02). β-Adrenergic receptor binding (Bmax) is not significantly different in the two groups of animals (Bmax= 41.4 ± 9.4 and 36.1 ± 5.1 fmol/mg for wild-type and eNOS−/−). Iso-stimulated ventricular relaxation is also enhanced in the eNOS−/− mice, as measured by dP/d tminin the isolated heart. However, baseline ventricular relaxation is normal in eNOS−/− mice (τ = 5.2 ± 1.0 and 5.6 ± 1.5 ms for wild-type and eNOS−/−, respectively), whereas it is impaired in wild-type mice after NOS inhibition (τ = 8.3 ± 2.4 ms). cGMP levels in the left ventricle are unaffected by eNOS gene deletion (wild-type: 3.1 ± 0.8 pmol/mg, eNOS−/−: 3.1 ± 0.6 pmol/mg), leading us to examine the level of another physiological regulator of cGMP. Atrial natriuretic peptide (ANP) expression is markedly upregulated in the eNOS−/− mice, and exogenous ANP restores ventricular relaxation in wild-type mice treated with NOS inhibitors. These results suggest that eNOS attenuates both inotropic and lusitropic responses to β-adrenergic stimulation, and it also appears to regulate baseline ventricular relaxation in conjunction with ANP.
Localization of dichlorofluorescin in cardiac myocytes: implications for assessment of oxidative stress
Authors: Swift Luther M. , Sarvazyan Narine
Published: December 2017

Localization and staining features of the oxidant-sensitive fluorescent probe 2′7′-dichlorofluorescin (DCFH) were evaluated in isolated cardiac muscle cells. Cardiomyocytes rapidly accumulated the probe and retained steady levels of DCFH and its highly fluorescent oxidized product dichlorofluorescein (DCF) in probe-free medium for 1.5 h. DCF was associated with mitochondria and was released by the proton ionophore carbonyl cyanide m-chlorophenylhydrazone but not by saponin, which permeabilizes the plasma membrane. A mitochondrial distribution of DCF was also suggested by experiments with the mitochondrial marker MitoTracker Red, in which quenching was observed between DCF and MitoTracker Red in live cells. Isolated cardiac mitochondria rapidly accumulated DCF, and high micromolar concentrations of the probe inhibited ADP-stimulated respiration rate. The study provides an information base essential for the interpretation and design of experiments with DCF as a marker of oxidative stress in cardiac muscle and reveals preferential localization of the probe in mitochondria.
Activation of mRNA translation in rat cardiac myocytes by insulin involves multiple rapamycin-sensitive steps
Authors: Wang Lijun , Wang Xuemin , Proud Christopher G.
Published: December 2017

Insulin acutely activates protein synthesis in ventricular cardiomyocytes from adult rats. In this study, we have established the methodology for studying the regulation of the signaling pathways and translation factors that may be involved in this response and have examined the effects of acute insulin treatment on them. Insulin rapidly activated the 70-kDa ribosomal S6 kinase (p70 S6k), and this effect was inhibited both by rapamycin and by inhibitors of phosphatidylinositol 3-kinase. The activation of p70 S6k is mediated by a signaling pathway involving the mammalian target of rapamycin (mTOR), which also modulates other translation factors. These include the eukaryotic initiation factor (eIF) 4E binding proteins (4E-BPs) and eukaryotic elongation factor 2 (eEF2). Insulin caused phosphorylation of 4E-BP1 and induced its dissociation from eIF4E, and these effects were also blocked by rapamycin. Concomitant with this, insulin increased the binding of eIF4E to eIF4G. Insulin also activated protein kinase B (PKB), which may lie upstream of p70 S6k and 4E-BP1, with the activation of the different isoforms being in the order α>β>γ. Insulin also caused inhibition of glycogen synthase kinase 3, which lies downstream of PKB, and of eEF2 kinase. The phosphorylation of eEF2 itself was also decreased by insulin, and this effect and the inactivation of eEF2 kinase were attenuated by rapamycin. The activation of overall protein synthesis by insulin in cardiomyocytes was substantially inhibited by rapamycin (but not by inhibitors of other specific signaling pathways, e.g., mitogen-activated protein kinase), showing that signaling events linked to mTOR play a major role in the control of translation by insulin in this cell type.
Silent α2C-adrenergic receptors enable cold-induced vasoconstriction in cutaneous arteries
Authors: Chotani Maqsood A. , Flavahan Sheila , Mitra Srabani , Daunt David , Flavahan Nicholas A.
Published: December 2017

Cold constricts cutaneous blood vessels by increasing the reactivity of smooth muscle α2-adrenergic receptors (α2-ARs). Experiments were performed to determine the role of α2-AR subtypes (α2A-, α2B-, α2C-ARs) in this response. Stimulation of α1-ARs by phenylephrine or α2-ARs by UK-14,304 caused constriction of isolated mouse tail arteries mounted in a pressurized myograph system. Compared with proximal arteries, distal arteries were more responsive to α2-AR activation but less responsive to activation of α1-ARs. Cold augmented constriction to α2-AR activation in distal arteries but did not affect the response to α1-AR stimulation or the level of myogenic tone. Western blot analysis demonstrated expression of α2A- and α2C-ARs in tail arteries: expression of α2C-ARs decreased in distal compared with proximal arteries, whereas expression of the glycosylated form of the α2A-AR increased in distal arteries. At 37°C, α2-AR-induced vasoconstriction in distal arteries was inhibited by selective blockade of α2A-ARs (BRL-44408) but not by selective inhibition of α2B-ARs (ARC-239) or α2C-ARs (MK-912). In contrast, during cold exposure (28°C), the augmented response to UK-14,304 was inhibited by the α2C-AR antagonist MK-912, which selectively abolished cold-induced amplification of the response. These experiments indicate that cold-induced amplification of α2-ARs is mediated by α2C-ARs that are normally silent in these cutaneous arteries. Blockade of α2C-ARs may prove an effective treatment for Raynaud's Phenomenon.
Pulsatile flow enhances endothelium-derived nitric oxide release in the peripheral vasculature
Authors: Nakano Toshihide , Tominaga Ryuji , Nagano Ichiro , Okabe Hayato , Yasui Hisataka
Published: December 2017

The effects of pulsatility in blood flow on endothelium-derived nitric oxide (EDNO) release in the peripheral vasculature were investigated. The basal and flow-stimulated EDNO release were compared between pulsatile and nonpulsatile systemic flows before and after the administration of NO synthase inhibitor NG-monomethyl-l-arginine (l-NMMA). Peripheral vascular resistance (PVR) was significantly lower in pulsatile flow than in nonpulsatile flow, but this difference disappeared after l-NMMA. The percent increase in PVR by l-NMMA was significantly larger in pulsatile flow. In reactive hyperemia in the hindlimb, the peak flow did not differ; however, both the repayment flow and the duration were significantly larger in pulsatile flow. Percent changes of these parameters by l-NMMA were significantly larger in pulsatile flow. These data indicated that pulsatility significantly enhances the basal and flow-stimulated EDNO release in the peripheral vasculature under in vivo conditions. We also studied the involvement of the Ca2+-dependent and Ca2+-independent pathways in flow-induced vasodilation using calmodulin inhibitor calmidazolium and tyrosine kinase inhibitor erbstatin A. PVR was significantly elevated by erbstatin A but not by calmidazolium, suggesting that flow-induced vasodilation was largely caused by tyrosine kinase inhibitor-sensitive activation of NO synthase.
Dominant role of cAMP in regulation of microvessel permeability
Authors: He P. , Zeng M. , Curry F. E.
Published: December 2017

We reported previously that increasing cAMP levels in endothelial cells attenuated ATP-induced increases in hydraulic conductivity ( Lp), and that the activation of cGMP-dependent pathways was a necessary step to increase Lpin response to inflammatory mediators. The aim of the present study was to evaluate the role of basal levels of cAMP in microvessel permeability under resting conditions and to evaluate the cross talk between cAMP- and cGMP-dependent signaling mechanisms in regulation of microvessel permeability under stimulated conditions, using individually perfused microvessels from frog and rat mesenteries. We found that reducing cAMP levels by inhibition of adenylate cyclase or inhibiting cAMP-dependent protein kinase through the use of H-89 increased basal Lpin both frog and rat mesenteric venular microvessels. We also found that 8-bromocAMP (8-BrcAMP, 0.2 and 2 mM) was sufficient to attenuate or abolish the increases in Lpdue to exposure of frog mesenteric venular microvessels to 8-BrcGMP (2 mM) and ATP (10 μM). Similarly, in rat mesenteric venular microvessels, application of 8-BrcAMP (2 mM) abolished the increases in Lpdue to exposure to 8-BrcGMP alone (2 mM) or with the combination of bradykinin (1 nM). In addition, application of erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of cGMP-stimulated phosphodiesterase, significantly attenuated both 8-BrcGMP- and bradykinin-induced increases in Lp. These results demonstrate that basal levels of cAMP are critical to maintaining normal permeability under resting conditions, and that increased levels of cAMP are capable of overcoming the activation of cGMP-dependent pathways, therefore preventing increases in microvessel permeability. The balance between endothelial concentrations of these two opposing cyclic nucleotides controls microvessel permeability, and cAMP levels play a dominant role.
Caveolin-1 regulates shear stress-dependent activation of extracellular signal-regulated kinase
Authors: Park Heonyong , Go Young-Mi , Darji Ritesh , Choi Jong-Whan , Lisanti Michael P. , Maland Matthew C. , Jo Hanjoong
Published: December 2017

Fluid shear stress activates a member of the mitogen-activated protein (MAP) kinase family, extracellular signal-regulated kinase (ERK), by mechanisms dependent on cholesterol in the plasma membrane in bovine aortic endothelial cells (BAEC). Caveolae are microdomains of the plasma membrane that are enriched with cholesterol, caveolin, and signaling molecules. We hypothesized that caveolin-1 regulates shear activation of ERK. Because caveolin-1 is not exposed to the outside, cells were minimally permeabilized by Triton X-100 (0.01%) to deliver a neutralizing, polyclonal caveolin-1 antibody (pCav-1) inside the cells. pCav-1 then bound to caveolin-1 and inhibited shear activation of ERK but not c-Jun NH2-terminal kinase. Epitope mapping studies showed that pCav-1 binds to caveolin-1 at two regions (residues 1–21 and 61–101). When the recombinant proteins containing the epitopes fused to glutathione- S-transferase (GST-Cav1–21 or GST-Cav61–101) were preincubated with pCav-1, only GST-Cav61–101 reversed the inhibitory effect of the antibody on shear activation of ERK. Other antibodies, including m2234, which binds to caveolin-1 residues 1–21, had no effect on shear activation of ERK. Caveolin-1 residues 61–101 contain the scaffolding and oligomerization domains, suggesting that binding of pCav-1 to these regions likely disrupts the clustering of caveolin-1 or its interaction with signaling molecules involved in the shear-sensitive ERK pathway. We suggest that caveolae-like domains play a critical role in the mechanosensing and/or mechanosignal transduction of the ERK pathway.
Focal extracellular potential: a means to monitor electrical activity in single cardiac myocytes
Authors: Riemer Tara L. , Tung Leslie
Published: December 2017

The focal extracellular potential (FEP) described in this study is an electrophysiological signal related to the transmembrane potential ( V m) of cardiac myocytes that avoids the mechanical fragility, interference with contraction, and intracellular contact associated with conventional whole cell recording. One end of a frog ventricular myocyte was secured into a glass holding pipette. The FEP was measured differentially between this pipette and a bath pipette while the cell was voltage- or current-clamped by a third whole cell pipette. The FEP appeared as an amplitude-truncated action potential, while FEP duration accurately reflected the action potential duration (APD) at 90% repolarization (APD90). FEP magnitude increased as the holding pipette K+ concentration ([K+]) was increased. The FEP-voltage relation was quasi-linear at negative V m with a slope that increased with elevated holding pipette [K+]. Increasing the membrane conductance inside the holding pipette by adding amphotericin B or cromakalim linearized the FEP-voltage relation across all V m. The FEP accurately reported electrical activation and APD90 during changes of stimulation frequency and episodes of cellular stretch.