bluuebunny/crossref_metadata_2025 huggingface dataset:
Factors inducing codistribution of marginal actin fibers and fibronectin in rat aortic endothelial cells
Authors: Sugimoto K. , Fujii S. , Takemasa T. , Yamashita K.
Published: December 2017

Rat endothelial cells have a unique arrangement of actin fibers running with subendothelial fibronectin along the cell margins. The present study was conducted to identify the factors that are associated with codistribution of these actin fibers and fibronectin in fetal rat aortic endothelial cells, mainly using rheological techniques. Fluorescence histochemistry revealed that the codistribution pattern was established between gestational days 14 and 15. The endothelial cells changed from polygonal to spindle shaped during this gestational period. Although the aortic length remained almost unchanged during this period of gestation, both the diameter and expansion ratio of the aorta increased, by 9 and 10%, respectively. On the other hand, the wall shear rate increased rapidly during gestational days 13-16. These results indicate that the codistribution of actin fibers and subendothelial fibronectin along the margins of endothelial cells in fetal rat aorta occurs in association with a rise in stretch and shear stress and that the endothelial cells may require this change to maintain structural integrity.
Effects of contraction, perfusion pressure, and length on intramyocardial pressure in rat papillary muscle
Authors: Heslinga J. W. , Allaart C. P. , Yin F. C. , Westerhof N.
Published: December 2017

If intramyocardial pressure (IMP) is the pressure that causes coronary flow to stop, i.e., “backpressure,” then it should be equal to the zero-flow perfusion pressure intercept (Pzf). Therefore we determined Pzf and IMP at zero flow (IMPzf) in papillary muscles suspended isometrically in a bath, superfused with a well-oxygenated Tyrode solution (27 degrees C), and perfused with Tyrode solution via the septal artery. For the IMP (servo-null) measurements, we used unbeveled glass micropipettes with a tip diameter of 3–4 microns. During diastolic arrest and systolic contracture (2 mM Ba21), perfusion pressure steps were applied, and the corresponding flow and IMP values were recorded. Fitting of the relationships, yielded Pzf and IMPzf. In the diastolically arrested muscle, perfusion pressure affected IMP. Pzf was much higher in systolically contracted muscle than in diastolically arrested muscle. The IMPzf in both conditions was significantly smaller than Pzf. Thus, even in this preparation with no ventricular pressure, IMP increases during contraction. We conclude that IMP arises from contraction per se but is not the pressure that causes the flow to stop.
Effect of nicotine on endothelium-dependent arteriolar dilatation in vivo
Authors: Mayhan W. G. , Patel K. P.
Published: December 2017

Smoking is a primary risk factor in coronary and peripheral vascular disease. However, the precise component of cigarette smoke that contributes to the pathogenesis of vascular disease remains unclear. The goal of this study was to determine the effect of nicotine on endothelium-dependent dilatation of peripheral resistance arterioles in vivo. We measured the diameter of resistance arterioles (approximately 50 microns in diameter) contained within the microcirculation of the hamster cheek pouch in response to endothelium-dependent (acetylcholine and ADP) and -independent (nitroglycerin) agonists before and after an intravenous infusion of vehicle or two concentrations of nicotine. Acetylcholine, ADP, and nitroglycerin produced a dose-related dilatation of the cheek pouch arterioles under control conditions. Endothelium-dependent, but not -independent, dilatation of arterioles was modestly impaired by an infusion of a low concentration of nicotine (1.0 microgram.kg-1.min-1). Infusion of a higher concentration of nicotine (2 micrograms.kg-1.min-1), which increased the plasma level of nicotine to 14 +/- 1.6 ng/ml, produced a profound selective impairment in endothelium-dependent vasodilatation. We suggest that elevations in plasma nicotine may contribute to the pathogenesis of the peripheral vascular disease observed in smokers.
Cardiac myocyte volume, Ca2+ fluxes, and sarcoplasmic reticulum loading in pressure-overload hypertrophy
Authors: Delbridge L. M. , Satoh H. , Yuan W. , Bassani J. W. , Qi M. , Ginsburg K. S. , Samarel A. M. , Bers D. M.
Published: December 2017

Alterations in cellular Ca2+ transport and excitation-contraction coupling may contribute to dysfunction in cardiac hypertrophy. Left ventricular myocytes were isolated from rat hearts after 15-18 wk of suprarenal abdominal aortic banding to evaluate the hypothesis that hypertrophy alters the relationship between Ca2+ current (ICa) and sarcoplasmic reticulum (SR) Ca2+ load during steady-state voltage-clamp depolarization. Mean arterial pressure (MAP) and heart weight-to-body weight ratio of banded (B) animals were significantly higher than in control or sham-operated animals (C). Isolated myocyte dimensions and volume increased in parallel with whole heart hypertrophy and elevation in MAP. However, the relationship between membrane surface area (measured by capacitance) and cell volume (measured by laser scanning confocal microscopy) was unaltered (C: 8.9 +/- 0.3; B: 8.5 +/- 0.4 pF/pl). No differences in the voltage dependence of ICa activation, steady-state inactivation, or recovery from inactivation were detected between C and B myocytes. Maximal ICa density for the two groups was also not different either under basal conditions (C: 4.28 +/- 0.98; B: 4.57 +/- 0.60 pA/pF) or in the presence of 1 microM isoproterenol (C: 16.6 +/- 2.3; B: 16.5 +/- 2.3 pA/pF). The fraction of Ca2+ released from the SR by a single twitch was 55.4 +/- 9.4% in C and 37.1 +/- 6.9% in B (not significantly different). Steady-state Ca2+ influx during a twitch was calculated in units of micromoles per liter of nonmitochondrial volume from the integral of ICa (C: 13.4 +/- 0.7 microM; B: 13.3 +/- 0.8 microM). The SR Ca2+ load was similarly calculated by integration of Na+/Ca2+ exchange current induced by rapid caffeine application (C: 140 +/- 9 microM; B: 169 +/- 18 microM). We conclude that significant cellular hypertrophy is associated with proportional increases in sarcolemmal ICa influx, SR Ca2+ loading, and the amount of SR Ca2+ released in this model of pressure overload.
Inhibition of NO synthesis minimally affects the dynamic baroreflex regulation of sympathetic nerve activity
Authors: Miyano H. , Kawada T. , Shishido T. , Sato T. , Sugimachi M. , Alexander J. , Sunagawa K.
Published: December 2017

The purpose of this investigation was to examine the role of nitric oxide (NO) in the dynamic baroreflex regulation of cardiac sympathetic nerve activity. In anesthetized rabbits, we imposed random pressure perturbations on the isolated carotid sinuses before and after the intravenous administration of NG-monomethyl-L-arginine. We characterized the dynamic properties relating carotid sinus pressure input to sympathetic nerve activity by means of a transfer function analysis. NG-monomethyl-L-arginine decreased the corner frequency of the transfer function (0.100 +/- 0.054 vs. 0.074 +/- 0.035 Hz; P < 0.05), whereas other parameters such as the steady-state gain and transmission lag time remained unchanged. Although cursory examination of these findings would suggest a possible contribution of NO in the dynamic baroreflex regulation of sympathetic nerve activity, quantitative assessment of the transfer function reveals only a minimal effect on the baroreflex regulation of arterial pressure, particularly under closed-loop conditions. We conclude that NO noticeably affects the dynamic baroreflex regulation of sympathetic nerve activity. However, it may not significantly affect arterial pressure regulation through central modulation of the carotid sinus baroreflex.
Do adult rat ventricular myocytes express protein kinase C-alpha?
Authors: Rybin V. , Steinberg S. F.
Published: December 2017

Although calcium-insensitive protein kinase C (PKC) isoforms (PKC-epsilon and PKC-delta) are consistently detected in adult ventricular myocytes, the evidence that adult ventricular myocytes also express calcium-sensitive PKC-alpha is inconsistent. The current study used four different anti-PKC-alpha-antibodies to resolve some of the uncertainties regarding the immunodetection of PKC-alpha in adult ventricular myocytes. Three of the antibodies used in this study barely (GIBCO-BRL) or rather faintly (Transduction Laboratories and Seikagaku America) recognize PKC-alpha in crude preparations from adult ventricular myocytes. Although each of these antibodies recognizes a prominent 80-kDa band, which is similar in size to PKC-alpha, this represents nonspecific immunoreactivity and should not be confused with PKC-alpha. This conclusion is based on peptide-blocking experiments (GIBCO-BRL), the absence of the requisite sensitivity to calcium- and phorbol 12-myristate 13-acetate-induced translocation (Seikagaku America and Transduction Laboratories), and/or the failure to copurify with PKC-alpha on DEAE-Sephacel chromatography. Nevertheless, an antibody from Upstate Biotechnology clearly recognizes PKC-alpha and not other unrelated nonspecific immunoreactive species in crude preparations from adult ventricular myocytes. Each of the antisera used in this study could detect PKC-alpha immunoreactivity following chromatographic purification of the samples to enrich for PKC-alpha and remove nonspecific immunoreactive proteins. These results suggest that PKC-alpha is expressed by adult ventricular myocytes and argue that differences in the sensitivity and/or specificity of available antisera contribute to at least some of the confusion regarding PKC-alpha expression in adult ventricular myocytes.
IGF-I regulates K(+)-channel expression of cultured neonatal rat ventricular myocytes
Authors: Guo W. , Kada K. , Kamiya K. , Toyama J.
Published: December 2017

Insulin-like growth factor (IGF) I has been known as an important peptide during heart development and myocardial hypertrophy. In the present study, the effects of IGF-I on cardiac K(+)-channel expression were investigated in cultured neonatal rat ventricular myocytes. Two distinct 4-aminopyridine (AP)-sensitive and rapidly activating outward K+ currents (IK) were observed. The predominant K(+)-channel current in cultured cells was a fast inactivating current similar to a 4-AP-sensitive transient outward current [I(to) half-maximal inhibitory concentration (IC60) = 0.87 mM]. Some cells lacking I(to) expressed an IK with little or no slow inactivation. IK exhibited higher sensitivity to inhibition by 4-AP (IC50 = 66.5 microM) and could be enhanced by isoproterenol but unaffected by tetraethylammonium. These characteristics indicate that IK might be the rat isoform of ultrarapid delayed rectifier K+ current IKur previously described in human atrial myocytes. Seventy-two-hour exposure to 60 ng/ml IGF-I induced myocyte hypertrophy and increased the percentage of cells expressing only IKur and the cells expressing both Ito and IKur. In some cultured myocytes, immunofluorescent staining with a polyclonal antibody specific to the COOH terminus of Kv1.5 K(+)-channel protein was performed in the same single cells after voltage-clamp recordings. The IGF-I-pretreated cells expressing larger IKur revealed a significantly intense antibody labeling. These observations indicate that the long-term administration of IGF-I can regulate the K(+)-channel expression of cultured neonatal rat ventricular myocytes. This is important for understanding the role of IGF-I in the modulation of cardiac excitability.
Regional differences in the role of the Ca2+ and Na+ currents in pacemaker activity in the sinoatrial node
Authors: Kodama I. , Nikmaram M. R. , Boyett M. R. , Suzuki R. , Honjo H. , Owen J. M.
Published: December 2017

The effect of block of the L-type Ca2+ current by 2 microM nifedipine and of the Na+ current by 20 microM tetrodotoxin on the center (normally the leading pacemaker site) and periphery (latent pacemaker tissue) of the rabbit sinoatrial node was investigated. Spontaneous action potentials were recorded with microelectrodes from either an isolated right atrium containing the whole node or small balls of tissue (approximately 0.3-0.4 mm in diameter) from different regions of the node. Nifedipine abolished the action potential in the center, but not usually in the periphery, in both the intact sinoatrial node and the small balls. Tetrodotoxin had no effect, on electrical activity in small balls from the center, but it decreased the takeoff potential and upstroke velocity and slowed the spontaneous activity (by 49 +/- 10%; n = 11) in small balls from the periphery. It is concluded that whereas the L-type Ca2- current plays an obligatory role in pacemaking in the center, the Na+ current plays a major role in pacemaking in the periphery.
An autocrine system for C-type natriuretic peptide within rat carotid neointima during arterial repair
Authors: Brown J. , Chen Q. , Hong G.
Published: December 2017

C-type natriuretic peptides (CNPs) are produced by endothelium and inhibit vascular smooth muscle cell (VSMC) proliferation. However, endothelial damage stimulates only transient VSMC proliferation in arteries. Here we report that a new source of CNP develops in rat carotid neointima 14 days after balloon angioplasty, when VSMC replication is subsiding despite continued absence of endothelium. CNP was detected immunohistochemically in neointimal but not medial VSMC. No other natriuretic peptides were detected immunohistochemically. CNP immunoreactivity (0.036 +/- 0.010 fmol/mg wet wt) was found in damaged arteries by radioimmunoassay, but none was detected in normal media. Reverse-phase chromatography suggested that this immunoreactivity consisted of CNP(1-53) and perhaps CNP(1-22). CNP transcript was identified by reverse transcription and polymerase chain reaction in carotid segments that had been stripped of endothelium but only once neointima had formed. Moreover, neointima expressed the NPR-C type of natriuretic peptide receptor at the same time as it synthesized CNP. Thus neointima develops an autocrine system for CNP that could regulate neointimal growth. Furthermore, the findings establish novel phenotypic differences between medial and neointimal VSMC in vivo.
A dynamic model of ventricular interaction and pericardial influence
Authors: Chung D. C. , Niranjan S. C. , Clark J. W. , Bidani A. , Johnston W. E. , Zwischenberger J. B. , Traber D. L.
Published: December 2017

A mathematical model describing the dynamic interaction between the left and the right ventricle over the complete cardiac cycle is presented. The pericardium-bound left and right ventricles are represented as two coupled chambers consisting of the left and right free walls and the interventricular septum. Time-varying pressure-volume relationships characterize the component compliances, and the interaction of these components produces the globally observed ventricular pump properties (total chamber pressure and volume). The model 1) permits the simulation of passive (diastolic) and active (systolic) ventricular interaction, 2) provides temporal profiles of hemodynamic variables (e.g., ventricular pressures, volumes, and flow) that agree well with reported observations, and 3) can be used to examine the effect of the pericardium on ventricular interaction and ventricular mechanics. It can be reduced to equivalency with models previously reported by invoking simplifying assumptions. Furthermore, model-generated "dynamic interaction gains" are employed to quantify the mode and degree of ventricular interaction. The model also yields qualitative predictions of septal and free wall displacements similar to those detected experimentally via M-mode echocardiography. Such analogies may be extended easily to the study of pathophysiological states via appropriate modifications to 1) the pressure-volume characteristics of the component walls (and/or pericardium) and/or 2) the specific time course of activation of the ventricular free wall or the septum. A limited number of examples are included to demonstrate the utility of the model, which may be used as an adjunct to new experimental investigations into ventricular interaction.
Coactivation of resistance vessels and muscle fibers with acetylcholine release from motor nerves
Authors: Welsh D. G. , Segal S. S.
Published: December 2017

Acetylcholine (ACh) released at the neuromuscular junction (NMJ) triggers muscle fiber contraction. We tested whether this source of ACh also triggers vasodilation. Arterioles [diameter: 4th order (4A), 18 +/- 3 microns; 2nd order (2A), 35 +/- 2 microns] and feed arteries (60 +/- 4 microns) were observed in retractor muscle of anesthetized hamsters. During stimulation [25% duty cycle (500-ms train, 1 per 2 s) at approximately 40% of maximum isometric tension], a nicotinic receptor antagonist (tubocurarine, 10 microM) prevented contraction, yet 2A and 4A arterioles and feed arteries rapidly (< or = 5 s) dilated (by 9 +/- 2, 11 +/- 3, and 8 +/- 1 microns, respectively; P < 0.05); neither cholinergic innervation of the vasculature nor ACh release from endothelium was apparent. Vasodilator responses doubled (P < 0.05) with cholinesterase inhibition (eserine, 1 microM) and were abolished with muscarinic receptor antagonism (atropine, 10 microM). Microiontophoresis of ACh onto arterioles triggered vasodilation that conducted into feed arteries, confirming functional continuity between intramuscular and extraparenchymal resistance vessels. To determine whether ACh served as a vasodilator during exercise, vascular responses to muscle contraction were measured in the presence or absence of atropine. With 2.5% duty cycle (50 ms, 1 per 2 s), atropine attenuated vasodilation by 35% in 2A and 51% in 4A arterioles and by 65% in feed arteries. With 25% duty cycle, arteriolar dilation was unaffected by atropine, yet feed artery dilation was attenuated by 60%; this was accompanied by a 50% reduction in functional hyperemia. Our findings indicate that ACh “spillover” from NMJs can coactivate muscarinic receptors, giving rise to a dilation that is conducted into feed arteries. This ascending vasodilation is integral to the full expression of functional hyperemia.
Contributions of the specialized conduction system to the activation sequence in the canine pulmonary conus
Authors: Pollard A. E. , Spitzer K. W. , Burgess M. J.
Published: December 2017

This study was designed to characterize the relative contributions of the specialized conduction system and the myocardial architecture to the ventricular activation sequence. In animal experiments, the activation sequence within a 14 x 14-mm region on each surface of the pulmonary conus from isolated canine hearts was determined from electrograms recorded during ventricular drives applied at the periphery of the measurement region. Recordings were obtained simultaneously from electrode arrays mounted on the endocardium and epicardium. Activation sequences were determined before and after the right ventricular cavity was bathed with a dilute Lugol-normal Tyrode (LNT) solution that selectively inhibited excitation of Purkinje cells. Simulations of action potential propagation in three-dimensional models (14.4 mm long x 7.2 mm wide x 3.6 mm thick) that included the major features of the midwall architecture were performed to aid in the interpretation of the experimental findings. During endocardial pacing (7 animals, 43 total drives), LNT application markedly prolonged the endocardial (13.7 +/- 1.3 ms) and epicardial (5.7 +/- 1.0 ms) activation sequences. However, epicardial isochrone maps constructed with electrograms recorded before LNT application showed no signs of multiple breakthrough sites and, with the exception of overall timing, closely resembled isochrone maps constructed with electrograms recorded after LNT application. During epicardial pacing (9 animals, 55 total drives), LNT application prolonged the endocardial (3.7 +/- 0.6 ms) and epicardial (1.9 +/- 0.6 ms) activation sequences much less dramatically than during endocardial pacing, suggesting a primary contribution of myocardial architecture. However, in those instances where nonuniform anisotropy slowed epicardial expansion of the depolarization wavefront, the specialized conduction system contributed to the activation sequence to a greater extent.
l-Citrulline conversion tol-arginine in sphenopalatine ganglia and cerebral perivascular nerves in the pig
Authors: Yu J. G. , Ishine T. , Kimura T. , O’Brien W. E. , Lee T. J. F.
Published: December 2017

The presence of nitric oxide synthase (NOS), argininosuccinate synthetase (ASS), and argininosuccinate lyase (ASL) and their coexistence with NADPH-diaphorase (NADPHd), a marker for NOS, in the porcine sphenopalatine ganglia (SPG), pial veins, and the anterior cerebral arteries was examined using immunohistochemical and histochemical staining techniques. NOS-immunoreactive (I), ASS-I, and ASL-I fibers were found in pial veins and the anterior cerebral arteries. NOS, ASS, and ASL immunoreactivities were also found in neuronal cell bodies in the SPG. Almost all neuronal cell bodies in the SPG and nerve fibers in pial veins and the anterior cerebral arteries that were reactive to ASS, ASL, and NOS were also stained positively with NADPHd, suggesting that ASS, ASL, and NOS were colocalized in the same neurons in the SPG and perivascular nerves. With the use of in vitro tissue bath techniques,l-citrulline but notd-citrulline reversed inhibition of neurogenic vasodilation in isolated porcine pial veins produced by NOS inhibitors such as NG-nitro-l-arginine methyl ester. In the presence of l-aspartate,l-arginine was synthesized froml-citrulline in homogenates of SPG and endothelium-denuded cerebral arteries and pial veins. These results provide evidence indicating that perivascular nerves in pial veins like cerebral arteries can convertl-citrulline tol-arginine for synthesizing nitric oxide. The conversion is most likely via an argininosuccinate pathway.
Decreased intracellular pH is not due to increased H+ extrusion in preconditioned rat hearts
Authors: Gabel Scott A. , Cross Heather R. , London Robert E. , Steenbergen Charles , Murphy Elizabeth
Published: December 2017

Ischemic preconditioning reduces intracellular acidification during a subsequent, prolonged period of ischemia. This may reflect decreased anaerobic glycolysis or increased H+ efflux. To distinguish between these hypotheses, we monitored intracellular and extracellular pH during a sustained period of ischemia to determine whether the preconditioned hearts had increased H+ efflux compared with nonpreconditioned hearts. At the end of 20 min of ischemia, intracellular pH in nonpreconditioned hearts was 5.90 ± 0.08 and extracellular pH was 5.51 ± 0.21, whereas in preconditioned hearts, intracellular pH was 6.50 ± 0.06 and extracellular pH was 6.62 ± 0.06. To investigate whether an Na+/H+exchange inhibitor would alter the reduced acidification during ischemia, we preconditioned hearts with and without dimethylamiloride (DMA). Intracellular pH during ischemia was similar in preconditioned hearts with and without DMA treatment (pH 6.42 ± 0.02 vs. 6.45 ± 0.03, respectively). These data do not support the hypothesis that enhanced proton efflux is responsible for the more alkaline intracellular pH during sustained ischemia in preconditioned hearts.
A novel phospholipase C- and cAMP-independent positive inotropic mechanism via a P2 purinoceptor
Authors: Podrasky Ernest , Xu David , Liang Bruce T.
Published: December 2017

Although ATP, acting through a P2 purinoceptor, can stimulate a pronounced positive inotropic effect in cardiac ventricular myocytes, the receptor-effector mechanism that underlies this stimulatory cardiac action is not well understood. The objectives of the present study were to develop the cultured chick embryo ventricular myocytes as a novel model for the cardiac P2 purinoceptor and to determine the mechanism underlying its positive inotropic effect. ATP caused an 89 ± 8.9% ( n = 14 cells) increase in the myocyte contractility, with an efficacy and potency order of ATP > ADP > AMP ≫ adenosine. 2-Methylthio-ATP (2-MeS-ATP) but not α,β-methylene-ATP was able to stimulate myocyte contractility, with a maximal increase of 54 ± 2.6% ( n = 11 cells). Although UTP potently stimulates phosphoinositide hydrolysis, it had an only modest positive inotropic effect (27 ± 7% maximal increase; n = 8 cells). In contrast to previous suggestions, the 2-MeS-ATP-stimulated positive inotropic response does not require the action of phospholipase C (PLC), such as that of the inositol phosphates; the UTP effect on contractility appears to be mediated via the 2-MeS-ATP-sensitive P2 receptor. The PLC inhibitor U-73122 had no effect on the 2-MeS-ATP-stimulated increase in contractility, providing further evidence against a role for PLC in the inotropic effect of 2-MeS-ATP. An adenosine 3′,5′-cyclic monophosphate-independent Ca2+entry-stimulating mechanism appears to underlie a direct coupling of the receptor to stimulation of the myocyte contractility. This new PLC- and adenosine 3′,5′-cyclic monophosphate-independent positive inotropic mechanism represents a target for developing novel positive inotropic therapeutics.
Contributions of acetylcholine and nitric oxide to forearm blood flow at exercise onset and recovery
Authors: Shoemaker J. K. , Halliwill J. R. , Hughson R. L. , Joyner M. J.
Published: December 2017

The contributions of acetylcholine and/or nitric oxide (NO) to the rapid changes in human forearm blood flow (FBF) at the onset and recovery from mild exercise were studied in eight subjects. Rhythmic handgrip contractions were performed during brachial artery infusions of saline (2 ml/min; control), atropine (0.2 mg over 3 min), to block acetylcholine binding to muscarinic receptors, or atropine + N G-monomethyl-l-arginine (l-NMMA; 4 mg/min for 4 min), to additionally inhibit NO synthase. Brachial artery mean blood velocity (MBV; pulsed Doppler ultrasound) and diameter (echo Doppler) were measured continuously, and FBF was calculated. Atropine reduced acetylcholine-induced increases in FBF by ∼71% ( P < 0.05). FBF at rest was reduced by atropine and further reduced with atropine +l-NMMA. Both drug conditions reduced FBF during exercise by ∼10% compared with control, with no difference between drug treatments. Brachial artery diameter was unchanged from rest by exercise, recovery, and drug treatments. Neither drug treatment altered the rate or magnitude of the increase in FBF above rest. Peak FBF after exercise was reduced by atropine and atropine + l-NMMA. Total FBF during 5 min of recovery was reduced with atropine +l-NMMA compared with control and atropine. The results suggest that 1) acetylcholine and NO mechanisms additively contribute to FBF levels at rest, 2) a cholinergic mechanism adjusts the absolute FBF levels during exercise, 3) neither acetylcholine nor NO is essential to observe the normal time course or magnitude of the exercise response, and 4) NO contributes to the FBF response during recovery from exercise.
Coronary constriction impairs cardiac function and induces myocardial damage and ventricular remodeling in mice
Authors: Li Baosheng , Li Qiong , Wang Xiaowei , Jana Kumar P. , Redaelli Giorgio , Kajstura Jan , Anversa Piero
Published: December 2017

To establish whether coronary artery narrowing (CAN) in mice was accompanied by depressed ventricular function, tissue injury, and modifications in cardiac anatomy, the left coronary artery was constricted in FVB/N mice and the animals were killed 7 days later. CAN consisted of a 53% reduction in luminal diameter, which resulted in a twofold increase in left ventricular end-diastolic pressure. Left ventricular systolic pressure and left ventricular + and −dP/d t decreased 15, 21, and 11%, respectively. Left ventricular weight-to-body weight ratio increased 33%. This hypertrophic adaptation was characterized by a 9 and 20% increase in the longitudinal and transverse cavitary diameters, which provoked a 1.5-fold expansion in chamber volume. In contrast, wall thickness decreased 15%. These anatomic and functional changes induced a threefold elevation in diastolic stress. Foci of reparative fibrosis were found in the endomyocardium and epimyocardium, involving 2–3% of the tissue. Finally, myocyte loss in the ventricle was 15%, and myocyte hypertrophy was 38%. Impaired ventricular function, diastolic Laplace overloading, myocyte loss, and decompensated eccentric hypertrophy in mice after CAN mimic the ischemic cardiomyopathic heart in humans.
Invasive hemodynamics and force-frequency relationships in open- versus closed-chest mice
Authors: Hoit Brian D. , Ball Nancy , Walsh Richard A.
Published: December 2017

We compared hemodynamics, ventricular function, and force-frequency relationships in six open-chest and six closed-chest anesthetized mice (FVB/N strain). Left ventricular (LV) pressure was measured with a 1.8- or 1.4-Fr Millar catheter placed via the right carotid artery and the LV apex in the closed- and open-chest state, respectively. Pacing was performed with electrodes placed either directly on atrial appendages (open chest) or with a 1-Fr bipolar catheter via the jugular vein (closed chest). Closed-chest animals had greater spontaneous heart rate (267 ± 106 vs. 147 ± 27 beats/min), LV systolic (81 ± 14 vs. 48 ± 9 mmHg) and diastolic pressures (11.2 ± 4.8 vs. 5.6 ± 2.4 mmHg), and maximal rise (+dP/d t max: 6,208 ± 2,519 vs. 3,682 ± 671 mmHg/s) and fall in pressure development (−dP/d t max: −6,094 ± 2,386 vs. −3,001 ± 399 mmHg/s). LV systolic pressure (98 ± 18 vs. 52 ± 11 mmHg), +dP/d t max (9,240 ± 2,459 vs. 5,777 ± 2,473 mmHg/s), and −dP/d t max(−8,375 ± 2,551 vs. −3,753 ± 1,170 mmHg/s) were significantly higher when animals were matched at a heart rate of 420 beats/min in closed-chest vs. open-chest animals. Biphasic force-frequency relationships were seen in all animals, but the critical heart rate was greater in the closed- than open-chest animals (432 ± 42 vs. 318 ± 42 beats/min). We conclude that 1) there are significant differences between invasive indexes of systolic and diastolic function between the closed- and open-chest preparations, 2) there is a biphasic force-frequency relationship in the anesthetized mouse, and 3) dP/d t max can be used to assess the cardiovascular phenotype.
Role of bradykinin-NO pathway in prevention of cardiac hypertrophy by ACE inhibitor in rat cardiomyocytes
Authors: Ishigai Yutaka , Mori Tomohiro , Ikeda Toshiko , Fukuzawa Akiko , Shibano Toshiro
Published: December 2017

To examine whether the bradykinin-nitric oxide (NO) pathway directly participates in the antihypertrophic property of angiotensin-converting enzyme (ACE) inhibitors in congestive heart failure, the effects of bradykinin were studied in rat cultured heart cells. Bradykinin (0.1, 1 nM) prevented the phenylephrine-induced increase in protein/DNA content, an index of hypertrophy of heart cells, and amplified the nitrite/nitrate content in the medium. Perindoprilat (1 μM), an ACE inhibitor, also restrained the progression of cardiac hypertrophy and augmented NO release. These effects of perindoprilat were abolished by HOE-140 (kinin B2antagonist), Nω-nitro-l-arginine (NO synthase inhibitor), and methylene blue (guanylate cyclase inhibitor). Furthermore, there was a significant correlation between protein/DNA content and nitrite/nitrate content. These results indicate that bradykinin inhibits the progression of cardiac hypertrophy due to the increase in NO release and that perindoprilat produces beneficial effects on cardiac hypertrophy by stimulating the bradykinin-NO pathway.
Estradiol induces C-type natriuretic peptide gene expression in mouse uterus
Authors: Acuff Cory G. , Huang Huaming , Steinhelper Mark E.
Published: December 2017

Previous experiments have demonstrated that C-type natriuretic peptide (CNP) expression in the uterus varies during the estrous cycle with maximal expression at proestrus. The present study was designed to determine whether exogenous steroid hormones regulate uterine CNP expression in ovariectomized mice. Estradiol increased significantly (3-fold) uterine immunoreactive CNP (irCNP) rapidly and dose dependently in ovariectomized mice as measured by radioimmunoassay. Other steroids produced either no significant change (testosterone, 1 mg; 2-methoxyestradiol, 1 μg) or weak induction (estriol, 1 μg) from vehicle controls. Progesterone (1 mg) significantly attenuated the estrogen-stimulated irCNP response by 50% when injected 30 min before estrogen (1 μg) in estrogen-primed ovariectomized mice. Estrogen-stimulated increases in uterine CNP transcripts detected by ribonuclease protection analyses were blocked by actinomycin D (160 μg) or ICI-164,384 (20 μg), a specific nuclear estrogen receptor antagonist. These results indicate that a nuclear estrogen receptor is required for estrogen to stimulate uterine CNP transcription and that progesterone negatively regulates estrogen-stimulated CNP expression.
Interstitial exclusion of macromolecules studied by graded centrifugation of rat tail tendon
Authors: Aukland Knut , Wiig Helge , Tenstad Olav , Renkin Eugene M.
Published: December 2017

Mechanical compression of cartilage and tendon has been shown to expel fluid both from collagen fibrils and from the extrafibrillar space. As reported previously, albumin (Alb) concentration and colloid osmotic pressure in tendon fluid (TF) expelled by repeated centrifugations fell progressively at increasing centrifugation force (G = 600, 2,400, and 13,100), suggesting either molecular sieving in compressed tendon or mobilization of protein-free (excluded) fluid. The present experiments, including analysis of 51Cr-EDTA, aprotinin (Ap), Alb, immunoglobulin G (IgG), and hyaluronan (hyaluronic acid; HA) with molecular weight (MW) ranging from 341 to 5 × 106, strongly favored the exclusion hypothesis; the fraction of Alb, IgG, and HA-free fluid (excluded) was already 0.23–0.36 in the first centrifugate, increasing to 0.73–0.82 in the third. The corresponding numbers were, respectively, 0.11 and 0.43 for Ap (MW 6,500), and 0 and 0.08 for51Cr-EDTA. These data, combined with calculated exclusion by collagen fibrils, proteoglycans, and HA, indicated that the first centrifugate was mainly derived from the extrafibrillar space, with increasing addition of macromolecular free intrafibrillar fluid in the second and third centrifugates, with each space contributing about equally to the total centrifugate volume. The calculations also indicated that Alb-, IgG-, and Ap-free fluid was mobilized from extrafibrillar space by increasing overlap of excluded territories. An excess of HA in tendon compared with that estimated from centrifugate concentrations suggests a large bound or immobilized HA fraction.
Regulatory effects of phospholamban on cardiac function in intact mice
Authors: Lorenz John N. , Kranias Evangelia G.
Published: December 2017

Phospholamban (PLB) regulates Ca2+- adenosinetriphosphatase activity in cardiac sarcoplasmic reticulum and participates in the regulation of myocardial performance. Animal models with altered levels of PLB permit in vivo evaluation of the physiological role of PLB. This study examined left ventricular (LV) performance in intact PLB heterozygous and homozygous mice under basal and stimulated conditions. A Millar Mikro-Tip transducer was inserted into the right carotid artery and advanced into the LV for direct measurement of ventricular pressure and the first derivative of intraventricular pressure (dP/d t). Baseline blood pressures were increased in PLB heterozygotes and even more so in PLB homozygotes compared with wild types (WT), and there were no differences in heart rate or LV end-diastolic pressure. The increase in pressure was primarily caused by an increase in systolic pressure. Baseline values for positive and negative dP/d t were linearly correlated with PLB levels. In PLB heterozygotes, contractile response to isoproterenol (Iso) was blunted compared with WT, but maximum rates of contraction were similar between the two groups. Contractile performance in PLB homozygous mice, which under baseline conditions was similar to maximum levels seen in WT, showed a blunted response to Iso, and maximum rates of contraction were significantly greater than in either of the other groups, indicating an essential but perhaps not exclusive role for PLB in mediating the inotropic effects of β-adrenergic agonists. The effects of Iso on negative dP/d t were also blunted in both PLB heterozygous and PLB homozygous animals. Our results demonstrate that myocardial function is highly dependent on PLB level and suggest that the cardiovascular effects of PLB perturbations are largely uncompensated for in the intact mouse.
Nitric oxide triggers late preconditioning against myocardial infarction in conscious rabbits
Authors: Qiu Yumin , Rizvi Ali , Tang Xian-Liang , Manchikalapudi Srinivas , Takano Hitoshi , Jadoon Asad K. , Wu Wen-Jian , Bolli Roberto
Published: December 2017

We tested the hypothesis that late preconditioning (PC) against myocardial infarction is triggered by the formation of nitric oxide (NO). Conscious rabbits underwent a 30-min coronary occlusion followed by 3 days of reperfusion. In group I (control group, n = 10), rabbits were not preconditioned, whereas in group II( n = 10), they were preconditioned 24 h earlier with a sequence of six 4-min occlusion/4-min reperfusion cycles. Myocardial infarct size (tetrazolium staining) was reduced by 50% by PC (28.6 ± 3.2% of the risk region in group II vs. 56.9 ± 5.9% in controls, P < 0.05). This reduction in cell death was associated with improved recovery of myocardial function [systolic thickening fraction (by sonomicrometry) at 3 days: 2.0 ± 11.0% of baseline in group II vs. −20.0 ± 2.8% in group I, P < 0.05]. Group III rabbits ( n = 11) underwent the same protocol as group II except that the rabbits received the NO synthase inhibitor Nω-nitro-l-arginine (l-NNA, 13 mg/kg) before the PC ischemia. In these animals, infarct size did not differ significantly from that observed in control rabbits, indicating thatl-NNA completely blocked the development of late PC against myocardial infarction. In group IV( n = 9), rabbits receivedl-NNA as in group III, but without the six occlusion-reperfusion cycles, and were subjected to the 30-min occlusion 24 h later. In this group, infarct size did not differ from that observed in controls, demonstrating that pretreatment withl-NNA, in itself, did not affect the extent of cell death. Taken together, these results indicate that, in the conscious rabbit, the development of late PC against myocardial infarction is triggered by the generation of NO during the PC ischemia. It is proposed that NO plays a key role in the delayed myocardial adaptation to ischemic stress.
Neuronal NOS-cGMP-dependent ACh-induced relaxation in pial arterioles of endothelial NOS knockout mice
Authors: Meng Wei , Ayata Cenk , Waeber Christian , Huang Paul L. , Moskowitz Michael A.
Published: December 2017

We evaluated the effects of superfusing 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), eNOS null ( B)an inhibitor of soluble guanylyl cyclase, and 7-nitroindazole sodium (7-NI), a selective neuronal nitric oxide synthase (nNOS) inhibitor, on the acetylcholine (ACh) response in endothelial NOS (eNOS) null mice. Pial arteriolar diameter was measured by intravital microscopy through a closed cranial window under α-chloralose anesthesia. NOS activity was measured by [3H]arginine-to-[3H]citrulline conversion in subjacent cortex in vitro. The density and distribution of muscarinic receptors in the brain were determined by quantitative [3H]quinuclidinyl benzilate autoradiography and did not differ between the eNOS mutants and wild-type mice. ACh superfusion (1 and 10 μM) dose dependently dilated pial arterioles in eNOS null and wild-type mice. ODQ (10 μM) attenuated ACh-induced dilation in both eNOS mutants (41% decrease at 10 μM ACh, P < 0.01, n = 6) and wild-type strains ( n = 5 per group). By contrast, topical superfusion of 7-NI (100 μM) attenuated the ACh response in eNOS mutants only (66%, P < 0.05, and 25% decrease, P < 0.05, at 1 and 10 μM ACh, respectively). Our findings suggest that nNOS-guanosine 3′,5′-cyclic monophosphate (cGMP)-dependent pathways dilate pial arterioles by compensatory mechanisms after eNOS gene disruption.
Electrophoretic separation and quantitation of cardiac myosin heavy chain isoforms in eight mammalian species
Authors: Reiser Peter J. , Kline William O.
Published: December 2017

A protocol for sample preparation and gel electrophoresis is described that reliably results in the separation of the α- and β-isoforms of cardiac myosin heavy chain (MHC-α and MHC-β) in eight mammalian species. The protocol is based on a simple, nongradient denaturing gel. The magnitude of separation of MHC-α and MHC-β achieved with this protocol is sufficient for quantitative determination of the relative amounts of these two isoforms in mouse, rat, guinea pig, rabbit, canine, pig, baboon, and human myocardial samples. The sensitivity of the protocol is sufficient for the detection of MHC isoforms in samples at least as small as 1 μg. The glycerol concentration in the separating gel is an important factor for successfully separating MHC-α and MHC-β in myocardial samples from different species. The effect of sample load on MHC-α and MHC-β band resolution is illustrated. The results also indicate that inclusion of a homogenization step during sample preparation increases the amount of MHC detected on the gel for cardiac samples to a much greater extent than for skeletal muscle samples. Although the protocol described in this study is excellent for analyzing cardiac samples, it should be noted that the same protocol is not optimal for separating MHC isoforms expressed in skeletal muscle, as is illustrated.
Measurement of heart rate and Q-T interval in the conscious mouse
Authors: Mitchell Gary F. , Jeron Andreas , Koren Gideon
Published: December 2017

Transgenic mouse models provided a powerful tool to evaluate the physiological significance of altered quantities or characteristics of specific gene products, such as cardiac ion channels. We have developed a system to record and analyze changes in the electrocardiogram in the mouse using an implantable telemetry system. The R-R and Q-T intervals were measured on individual beats and on signal-averaged complexes derived from 1, 2, or 4 s of contiguous data each hour during a 24-h period in three male and three female FVB mice. Duration of averaging had minimal effect on the measured Q-T. The Q-T interval was shown to be related to the square root of the R-R interval, and an appropriate formula for a rate-corrected Q-T interval (Q-Tc) was derived. Ketamine anesthesia was shown to markedly increase duration and variability in R-R, Q-T, and Q-Tc intervals. In conscious animals, variability in Q-T was low across animals and over time, suggesting that this should be a sensitive model for detection of changes in the Q-T interval in transgenic mice with ion channel defects.
Microsphere-induced bronchial artery vasodilation: role of adenosine, prostacyclin, and nitric oxide
Authors: Pearse David B. , Dahms Thomas E. , Wagner Elizabeth M.
Published: December 2017

We previously found that injection of 15-μm microspheres into the bronchial artery of sheep decreased bronchial artery resistance. This effect was inhibited partially by indomethacin or 8-phenyltheophylline, suggesting that microspheres caused release of a dilating prostaglandin and adenosine. To identify the prostaglandin and confirm adenosine release, we perfused the bronchial artery in anesthetized sheep. In 12 sheep, bronchial artery blood samples were obtained before and after the infusion of 1 × 106microspheres or microsphere diluent into the bronchial artery. Microspheres, but not diluent, decreased bronchial artery resistance by 40% and increased bronchial artery plasma 6-ketoprostaglandin F1α(194.7 ± 45.0 to 496.5 ± 101.3 pg/ml), the stable metabolite of prostacyclin, and prostaglandin (PG) F2α(28.1 ± 4.4 to 46.2 ± 9.7 pg/ml). There were no changes in PGD2, PGE2, thromboxane B2, adenosine, inosine, or hypoxanthine. Pretreatment with dipyridamole, an adenosine uptake inhibitor, did not affect bronchial artery nucleoside concentrations ( n = 7). Microsphere-induced vasodilation was not enhanced by dipyridamole ( n = 9) and was not inhibited by either the adenosine receptor antagonist xanthine amine congener ( n = 4) or the nitric oxide (NO) synthase inhibitor NG-monomethyl-l-arginine ( n = 8). These results do not support a role for either adenosine or NO and suggest that microspheres caused bronchial artery vasodilation through release of prostacylin and an unidentified vasodilator.
Hemostatic factors in rabbit limb lymph: relationship to mechanisms regulating extravascular coagulation
Authors: Le Dzung T. , Borgs Peter , Toneff Thomas W. , Witte Marlys H. , Rapaport Samuel I.
Published: December 2017

Mechanisms regulating extravascular coagulation in interstitial fluids of peripheral tissues are poorly understood, since measurements of hemostatic factors in these fluids are unavailable. Because lymph from a body region reflects the composition of its interstitial fluid, we measured hemostatic factors in limb lymph of rabbits both as activity and as antigen. Mean lymph-to-plasma activity ratios were the following: fibrinogen, 0.28; prothrombin, 0.26; factor X, 0.27; factor VII, 0.17; and factors V and VIII, 0.08. All lymph fibrinogen was clottable; fibrin degradation products were absent. Lymph von Willebrand factor antigen was <10% of plasma antigen and consisted primarily of lower molecular weight multimers. Mean lymph-to-plasma activity ratio for antithrombin was 0.38 and for tissue factor pathway inhibitor the ratio was 0.40. Low levels of antithrombin-factor Xa were measurable in lymph. The data are compatible with a basal factor VIIa-tissue factor-catalyzed extravascular activation of factor X that is prevented from progressing to generation of fibrin in limb interstitial fluid and lymph by low levels of factor VIII and factor V and by the inhibitory activity of antithrombin and tissue factor pathway inhibitor.
Induction and maintenance of increased VEGF protein by chronic motor nerve stimulation in skeletal muscle
Authors: Annex Brian H. , Torgan Carol E. , Lin Pengnian , Taylor Doris A. , Thompson Michael A. , Peters Kevin G. , Kraus William E.
Published: December 2017

Vascular endothelial growth factor (VEGF) causes endothelial cell proliferation in vitro and angiogenesis in vivo. Glycolytic skeletal muscles have a lower capillary density than oxidative muscles but can increase their capillary density and convert to a more oxidative phenotype when subject to chronic motor nerve stimulation (CMNS). We used Western analysis and immunohistochemical techniques to examine VEGF protein in a rabbit CMNS model of glycolytic skeletal muscle and in muscles with innate glycolytic versus oxidative phenotypes. VEGF protein per gram of total protein was increased in stimulated vs. control muscles 2.9 ± 1.0, 3.6 ± 1.3, 3.1 ± 0.5, 4.4 ± 1.6, and 2.7 ± 0.3 times after 3 ( n = 4), 5 ( n = 2), 10 ( n = 3), 21 ( n = 3), and 56 ( n = 2) days, respectively. VEGF protein was increased 3.1 ± 0.5 times ( P < 0.005) before (3, 5, and 10 days) and remained elevated 3.7 ± 1.0 times ( P < 0.05) after (21 and 56 days) the transition to an oxidative phenotype. By immunohistochemistry, VEGF protein was found primarily in the matrix between stimulated muscle fibers but not in the myocytes. In addition, VEGF protein was consistently lower in innate glycolytic compared with oxidative muscles. These findings suggest that VEGF plays a role in the alteration and maintenance of vascular density in mammalian skeletal muscles.