bluuebunny/crossref_metadata_2025 huggingface dataset:
Ischemic preconditioning inhibits glycolysis and proton production in isolated working rat hearts
Authors: Finegan B. A. , Lopaschuk G. D. , Gandhi M. , Clanachan A. S.
Published: December 2017

The effect of ischemic preconditioning (IPC) on glycolysis, glucose oxidation, adenine nucleotide and nucleoside levels, and mechanical function was studied in isolated paced working rat hearts under aerobic conditions or when reperfused following sustained ischemia. IPC inhibited glycolysis in aerobic hearts (4.48 +/- 0.66 vs. 3.18 +/- 0.39 mumol.min-1.g dry wt-1) and calculated proton production attributable to exogenous glucose (7.79 +/- 1.31 vs. 4.73 +/- 0.81 mumol.min-1.g dry wt-1). In hearts subjected to ischemia and reperfusion, IPC decreased, relative to controls, glycogen content before the onset of ischemia (116.6 +/- 4.3 vs. 158.0 +/- 8.4 mumol/dry wt) and decreased consumption of glycogen during ischemia (54 +/- 6 vs. 76 +/- 7 mumol/dry wt). During reperfusion, glycolysis was lower in IPC hearts (2.45 +/- 0.16 vs. 4.4 +/- 0.46 mumol.min-1.g dry wt-1), as was calculated proton production (3.57 +/- 0.30 vs. 8.38 +/- 0.93 mumol.min-1.g dry wt-1). Glucose oxidation was similar in control and IPC hearts. Adenosine and ATP content of IPC hearts, relative to controls, were higher at the end of ischemia, being 0.86 +/- 0.08 vs. 0.34 +/- 0.15 mumol/g dry wt and 11.3 +/- 0.8 vs. 5.0 +/- 1.6 mumol/g dry wt, respectively. IPC enhanced recovery of ventricular work during reperfusion of ischemic hearts from 37 to 68%. These results indicate that IPC is associated with a reduction in glycogen content, inhibition of glycolysis during ischemia and reperfusion, and a decrease in proton production from glucose. These changes may, in part, explain the enhanced recovery of mechanical function observed in IPC hearts.
Effect of chronic endothelin blockade in hyperinsulinemic hypertensive rats
Authors: Verma S. , Bhanot S. , McNeill J. H.
Published: December 2017

Evidence suggests that hyperinsulinemia may be causally related to the development of high blood pressure (BP) in fructose-hypertensive (FH) rats. Because plasma insulin has been shown to modulate endothelin (ET) release in vivo, we hypothesized that hyperinsulinemia may provide a continual stimulus for ET release, which could increase BP by altering plasma or blood vessel ET levels. To test this hypothesis, we studied the effect of chronic ET-receptor blockade (by using bosentan, a noncompetitive ET antagonist) on plasma insulin levels, plasma ET levels, blood vessel ET content, and BP in FH rats. Chronic oral bosentan treatment (100 mg.kg-1.day-1) was initiated in 6-wk-old Sprague-Dawley rats. One week after bosentan treatment was started, rats were fed either normal rat chow or a fructose-enriched diet. Plasma insulin, plasma glucose, and systolic BP were measured weekly. At termination (in 15-wk-old rats), plasma ET levels and total mesenteric ET content were determined. Bosentan treatment caused a sustained decrease in BP in the FH rats (treated 130 +/- 4 vs. untreated 149 +/- 2 mmHg, P < 0.001) but had no effect in the normotensive control group. FH rats had a higher total mesenteric ET content compared with the control group (21.5 +/- 3.2 vs. 14.1 +/- 2.1 fmol, P < 0.05). Bosentan treatment did not alter total mesenteric ET content (treated 18.8 +/- 5 fmol, P > 0.05 vs. untreated) nor did it affect plasma insulin or ET levels in any group. These data suggest that ET may be involved in the development of high BP in FH rats. Whether ET represents an intermediate, linking hyperinsulinemia to hypertension in rats, or is an independent hypertensinogenic mechanism remains to be determined.
Myocardial ischemia and reperfusion: a murine model
Authors: Michael L. H. , Entman M. L. , Hartley C. J. , Youker K. A. , Zhu J. , Hall S. R. , Hawkins H. K. , Berens K. , Ballantyne C. M.
Published: December 2017

Myocardial ischemia followed by reperfusion promotes a complex series of inflammatory reactions as noted in a variety of large animal studies. With development of genetically altered mice, there is intense interest in developing murine models to study mechanisms operative in cardiovascular disease. We developed a mouse model to study coronary artery occlusion and reperfusion effects and the method required to perform these studies both acutely and chronically. In mice, we applied a left anterior descending coronary artery occlusion either permanently or for 30 or 60 min followed by reperfusion allowing flow through the previously occluded coronary artery bed. Reperfusion was documented visually as well as by using Doppler ultrasound and histopathological techniques. The area at risk (AAR) and infarct size (IS) were assessed by EVans blue dye and triphenyltetrazolium chloride staining with computerized planimetry using an image analysis software program. The infarct as percentage of AAR and IS as percentage of the left ventricle in 13 mice with permanent occlusion was 68.6 +/- 4.4 and 28.0 +/- 2.8%, respectively. Reperfusion after occlusions of 60 and 30 min resulted in a significant decrease in IS as a percentage of the AAR compared with permanent occlusion. Histological examination of the ischemic and reperfused myocardium shows infiltration of leukocytes into the ischemic region as well as contraction bands classically associated with reperfusion. This new model allows assessment of AAR, IS, cardiac function, and pathophysiology in the mouse. With the current technology to develop genetically altered mice for overexpression or targeted mutations of various genes, this model is used to understand the complex pathophysiology of ischemia and reperfusion injury.
The erythrocyte as a regulator of vascular tone
Authors: Ellsworth M. L. , Forrester T. , Ellis C. G. , Dietrich H. H.
Published: December 2017

Local regulation of microvascular blood flow is a complex process in which the needs of the tissue must be communicated to the vasculature, enabling the appropriate matching of O2 supply to demand. We hypothesize that the red blood cell is not only the major O2 carrier but also serves as an O2 sensor and affecter of changes in O2 delivery via its release of ATP, which subsequently binds to P2y receptors on the vascular endothelium, altering vessel caliber. Using the hamster as a model, we determined that the efflux of ATP from red blood cells after exposure to low-PO2 (PO2 = 17 +/- 6 mmHg) and low-pH (pH = 7.06 +/- 0.07) solutions was significantly (P < 0.01) greater than that after exposure to normoxic, normal pH (PO2 = 87 +/- 4; pH = 7.38 +/- 0.04) solutions, indicating that two factors that are associated with an impaired O2 supply relative to demand increase the release of ATP from the red blood cell. To ascertain whether ATP alters vascular caliber, we applied 10(-6) M ATP intraluminally to arterioles of the retractor muscle, using a micropressure system. Vessel diameter increased 8 and 10%, 140 +/- 60 microns upstream of the site of infusion after 50- and 500-ms pulses, respectively. Application of ATP to arteriolar and venular capillaries induced a 31 and 81% increase in red blood cell supply rate, respectively. These results support our hypothesis that the red blood cell is more than just an O2 carrier and has a direct role in the regulation of vascular tone.
Inosine-induced vasoconstriction is mediated by histamine and thromboxane derived from mast cells
Authors: Shepherd R. K. , Duling B. R.
Published: December 2017

Mast cell degranulation has been shown to release products that cause arteriolar constriction. We previously reported that two nucleosides, adenosine and inosine, cause vasoconstriction of isolated hamster cheek pouch arterioles by stimulating degranulation of periarteriolar mast cells. The objectives of the present study were to characterize the nucleoside-dependent vasoconstriction in vivo and to determine the mediator or mediators responsible. We examined the vasomotor effect of inosine on arterioles in the cheek pouches of anesthetized hamsters (70 mg/kg pentobarbital sodium) in the control situation and in the presence of receptor antagonists for histamine (H1), thromboxane A2 (Tx), and leukotrienes (LT). Most experiments were carried out using inosine applied once locally via micropipette to arterioles and observing the subsequent response. Over a range of inosine concentrations from 10(-5) to 10(-3) M in the pipette, we observed a dose-dependent increase in the incidence and magnitude of constriction. In addition, mast cell staining with ruthenium red was observed after stimulation with inosine, an indication of mast cell degranulation. Neither the H1, Tx, nor LT antagonist alone had a significant effect on the vasomotor response to inosine. However, combined H1 and Tx blockade significantly reduced the incidence and magnitude of inosine-induced constriction. These data establish that inosine-induced constriction occurs in vivo and support the role of mast cells in this response. Furthermore they suggest that multiple mediators, primarily histamine and thromboxane, are responsible for the observed constriction.
Spatial characterization of contracting cardiac myocytes by computer-assisted, video-based image processing
Authors: Wang Z. , Mukherjee R. , Lam C. F. , Spinale F. G.
Published: December 2017

The goals of the present study were to develop and validate a computer-assisted, video-based image processing (CAVIP) system to measure time-dependent changes in isolated myocyte geometry during contraction and to use the CAVIP system to examine spatial characteristics of the myocyte during contraction in normal myocytes and in myocytes after development of dilated cardiomyopathy (DCM). Myocytes were isolated from the left ventricles of five control pigs and five pigs that developed chronic tachycardia (240 beats/min; 3 wk)-induced DCM. Isolated myocytes were stimulated and recorded using a high-speed camera interfaced with a standard video recording system. There was a significant linear relation between the indexes of time-dependent changes in myocyte length as measured by a conventional video edge-detector system and by the CAVIP system (r > 0.96; P < 0.01). After this validation procedure, dynamic changes in myocyte width and profile area with DCM were examined. Myocyte resting profile area was 33% larger in DCM myocytes compared with controls. However, there was no difference in the rate of area change with contraction between the two groups. Percent changes in myocyte width and profile area at peak contraction were significantly lower in the DCM group (43 and 46% respectively, P < 0.05). Therefore, the present study demonstrated that the CAVIP system provides unique information on time-dependent changes in myocyte geometry during contraction, particularly with the development of cardiomyopathic disease.
Sequential ischemia/reperfusion results in contralateral skeletal muscle salvage
Authors: Liauw S. K. , Rubin B. B. , Lindsay T. F. , Romaschin A. D. , Walker P. M.
Published: December 2017

Sequential ischemia/reperfusion in a paired canine gracilis muscle model resulted in significant muscle salvage. In this model, one randomly chosen gracilis muscle was subjected to 5 h of ischemia followed by 48 h of in vivo reperfusion. The contralateral (second) muscle was then made ischemic and reperfused using the same protocol. Muscle necrosis was determined at the end of 48 h of reperfusion. A mean 60% reduction in muscle necrosis was observed in the second group of muscles. Analysis of tissue adenine nucleotides indicated that significant sparing of ATP utilization occurred in the second muscle group during ischemia. Preliminary analysis of tissue heat shock proteins (HSP) showed that the second group of muscles had a different pattern of HSP expression before the onset of ischemia. The results suggest that reduced ATP utilization and altered HSP expression in the second muscle play a role in the tissue salvage observed in this sequential muscle ischemia model.
Left ventricular volumetric conductance catheter for rats
Authors: Ito H. , Takaki M. , Yamaguchi H. , Tachibana H. , Suga H.
Published: December 2017

Left ventricular (LV) volume (V) is an essential parameter for assessment of the cardiac pump function. Measurement of LVV in situ by a conductance catheter method has been widely used in dogs and humans but not yet in small experimental animals such as rats. We instituted a miniaturized six-electrode conductance catheter (3-F) for rat LVV measurement and its signal processing apparatus. We compared stroke volumes (SVs) simultaneously measured with this conductance catheter introduced into the LV through the apex and an electromagnetic flow probe placed on the ascending aorta during gradual decreases in LVV by an inferior vena caval occlusion. A high and linear correlation (r = 0.982) was obtained between these differently measured by SVs pooled from six rats. In another group of three rats, LV pressure was simultaneously measured with a 3-F catheter-tip micromanometer introduced into the LV through the apex. We obtained the slope of the end-systolic pressure-volume (P-V) relationship (Emax) by a gradual ascending aortic occlusion. After administration of propranolol, Emax obviously decreased with no change in volume intercept of the P-V relationship. The conductance volumetry proved to be useful in rats.
Properties of sodium and potassium currents of cultured adult human atrial myocytes
Authors: Feng J. , Li G. R. , Fermini B. , Nattel S.
Published: December 2017

Cultured cell systems are valuable for the study of regulation of phenotypic expression, but little is known about the electrophysiological properties of human cardiac tissues in culture. The present studies were designed to determine the feasibility of maintaining human atrial myocytes in primary culture and to assess changes in Na+ (INa) and K+ (Ito, transient outward, and Ikur, ultra-rapid delayed rectifier) currents. Within 24 h of culture, cells assumed an avoid shape, which they maintained for up to 7 days. The voltage dependence, kinetics, and density of INa were unchanged in culture. The activation properties of Ito (kinetics and voltage dependence) were not altered, but Ito density (current normalized to cell capacitance) was reduced and inactivation properties were altered (negative shift in voltage dependence and slowed kinetics) in cultured compared with fresh cells. The absolute current amplitude, kinetics, voltage dependence, and 4-aminopyridine sensitivity of IKur were unchanged, but current density was increased. All changes in ionic currents occurred within 24 h of culture and remained stable for the next 4 days. We conclude that human atrial myocytes can be maintained in primary culture, that the qualitative properties of INa, Ito, and IKur remain constant but that some quantitative changes occur, and that cultured human atrial myocytes may be valuable for studies of the molecular mechanisms and regulation of cardiac channel function in humans.
Endothelial vasodilator production by uterine and systemic arteries. I. Effects of ANG II on PGI2 and NO in pregnancy
Authors: Magness R. R. , Rosenfeld C. R. , Hassan A. , Shaul P. W.
Published: December 2017

Uterine vasculature is less responsive than systemic vasculature to angiotensin II (ANG II)-induced vasoconstriction. We hypothesized that pregnancy augments basal and ANG II-stimulated endothelial prostacyclin (PGI2) and/or nitric oxide (NO) production, which locally increase vascular smooth muscle (VSM) adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP), respectively. Uterine (UA) and systemic arteries (SA) from pregnant (P) and nonpregnant (NP) sheep were incubated with isobutylmethylxanthine. Basal PGI2, cAMP, and cGMP production was 2.4-, 1.6-, and 5.9-fold greater (P < 0.01) in UA from P vs. NP sheep; endothelium removal lowered (P < 0.05) values 69, 44, and 88%. Basal SA PGI2 and cAMP, but not cGMP, also were elevated by pregnancy. Indomethacin (Indo; 100 microM) decreased PGI2 and cAMP, but not cGMP production; N omega-nitro-L-arginine methyl ester (L-NAME; 10 microM) and methylene blue (MB, 10 microM) only decreased cGMP. Basal UA, but not SA, NO synthase activity (conversion of [3H]arginine to [3H]citrulline), was 1.8-fold higher in pregnancy and decreased (P < 0.01) after endothelium removal and with L-NAME. ANG II (50 nM) increased PGI2 (86%) and cAMP (56%) production only in UA from P sheep (P < 0.05); this was abolished by endothelium removal or Indo. ANG II also increased (P < 0.01) cGMP production by UA from both groups but only by SA from P ewes; this was absent in denuded, L-NAME-, or MB-treated vessels. Stimulation of VSM cGMP production with sodium nitroprusside (50 microM) was inhibited by MB, but not L-NAME or endothelium removal. In pregnancy, endothelial PGI2 and NO production are enhanced and may contribute to attenuated ANG II vasoconstriction via VSM cAMP and cGMP.
Enhanced vasodilation to acetylcholine in athletes is associated with lower plasma cholesterol
Authors: Kingwell B. A. , Tran B. , Cameron J. D. , Jennings G. L. , Dart A. M.
Published: December 2017

We investigated a change in vascular reactivity as a potential adaptive mechanism to chronic exercise. The study consisted of 2 separate protocols with 10 male athletes and 10 age-matched sedentary male control subjects participating in each. Protocol 1 investigated forearm blood flow responses to intra-arterial infusions of acetylcholine and sodium nitroprusside by use of venous occlusion plethysmography. Protocol 2 used identical techniques to study responses to norepinephrine, angiotensin II (ANG II), and NG-monomethyl-L-arginine (L-NMMA). The percent reduction in forearm vascular resistance to acetylcholine was significantly greater in the athletic compared with the sedentary group (multivariate analysis of variance for repeated measures, P = 0.03). Covariance analysis suggested that the lower total cholesterol level of the athletic group (P = 0.03) may contribute to their enhanced responsiveness to acetylcholine. There were no differences between athletic and sedentary groups in the forearm vascular resistance responses to norepinephrine, ANG II, sodium nitroprusside, or L-NMMA. These data support the hypothesis that long-term endurance training is associated with enhanced endothelium-dependent dilator reserve due to altered lipoprotein levels in athletes. This finding may have therapeutic application in conditions of elevated cholesterol and impaired vasodilator capacity including hypertension, hypercholesterolemia, atherosclerosis, and cardiac failure.
Two conformational states involved in the use-dependent TTX blockade of human cardiac Na+ channel
Authors: Dumaine R. , Hartmann H. A.
Published: December 2017

We used a fast inactivation-deficient mutant (QQQ) of the human heart Na+ channel alpha-subunit (hH1a) to assess the influence of the inactivation gate on tetrodotoxin (TTX) use-dependent block (UDB) and postrepolarization block (PRB). PRB had similar time courses in both channels, suggesting no direct interaction of the inactivation gate with the TTX binding site. The UDB saturated with high concentrations of TTX in hH1a but not in QQQ, revealing the modulatory action of fast inactivation on UDB. TTX did not stabilize the inactivated states of QQQ, and the extra block developing during long depolarizations suggests a higher-affinity site involved in the gating of the channel. These results cannot be solely explained by a slow recovery from the block in the inactivated states. They suggest a common use-dependent block mechanism for hH1a and QQQ involving a high-affinity site. We propose that an activated state is primarily responsible for UDB during short depolarization in the range of the action potential plateau and that fast inactivation modulates the accessibility of the toxin to this site.
Tissue specific expression of vascular smooth muscle angiotensin II receptor subtypes during ovine pregnancy
Authors: Cox B. E. , Rosenfeld C. R. , Kalinyak J. E. , Magness R. R. , Shaul P. W.
Published: December 2017

Uteroplacentral responses to infused angiotensin II (ANG II) are less than those elicited by systemic vasculature. This does not reflect ANG II receptor (AT) downregulation but may reflect differences in AT-receptor subtypes expressed. We examined AT-receptor subtypes in smooth muscle (SM) from uterine (UA), mesenteric, renal, and mammary arteries and aorta from nulliparous (n = 12), pregnant (n = 18; 105-140 days, term = 145 days), postpartum (n = 5; 6-9 days after delivery), and nonpregnant parous (n = 14) ewes by assessing displacement of 125I-labeled ANG II binding by [Sar1, Ile8]ANG II (AT1 and AT2), losartan (AT1) PD-123319 (AT2), and CGP-42112A (AT2). AT2 receptors accounted for 75-90% of total binding in UA. Except for mammary arteries, other arteries expressed only AT1 receptors. Receptor subtype expression was not altered by reproductive state in any artery studied. With the use of autoradiography, AT2 receptors appear to predominate in media of small intramyometrial arteries, whereas AT1 receptors predominate in the luminal portion. We therefore determined which subtype mediates endothelium-derived ANG II-induced increases in UA PGI2 synthesis during pregnancy. ANG II (0.05 microM) increased PGI2 synthesis 62%, from 214 +/- 13 to 346 +/- 23 pg.mg-1.h-1 (P < 0.05). Losartan (1.0 microM) inhibited the rise in PGI2 (257 +/- 24 vs. 238 +/- 25 pg.mg-1.h-1), whereas 1.0 microM PD-123319 had no effect (231 +/- 23 vs. 337 +/- 31 pg.mg-1.h-1; P < 0.05). AT2 receptors do not mediate ANG II-induced vasoconstriction, thus differences in uteroplacental and systemic sensitivity to ANG II may reflect predominance of AT2 receptors in UASM and ANG II-induced increases in UA prostacyclin synthesis by endothelial AT1 receptors.
Elevated heart rate variability in physically active postmenopausal women: a cardioprotective effect?
Authors: Davy K. P. , Miniclier N. L. , Taylor J. A. , Stevenson E. T. , Seals D. R.
Published: December 2017

Coronary heart disease (CHD) and cardiac sudden death (CSD) incidence accelerates after menopause, but the incidence is lower in physically active versus less active women. Low heart rate variability (HRV) is a risk factor for CHD and CSD. The purpose of the present investigation was to test the hypothesis that HRV at rest is greater in physically active compared with less active postmenopausal women. If true, we further hypothesized that the greater HRV in the physically active women would be closely associated with an elevated spontaneous cardiac baroreflex sensitivity (SBRS). HRV (both time and frequency domain measures) and SBRS (sequence method) were measured during 5-min periods of controlled frequency breathing (15 breaths/min) in the supine, sitting, and standing postures in 9 physically active postmenopausal women (age = 53 +/- 1 yr) and 11 age-matched controls (age = 56 +/- 2 yr). Body weight, body mass index, and body fat percentage were lower (P < 0.01) and maximal oxygen uptake was higher (P < 0.01) in the physically active group. The standard deviation of the R-R intervals (time domain measure) was higher in all postures in the active women (P < 0.05) as were the high-frequency, low-frequency, and total power of HRV. SBRS also was higher (P < 0.05) in the physically active women in all postures and accounted for approximately 70% of the variance in the high-frequency power of HRV (P < 0.05). The results of the present investigation indicate that physically active postmenopausal women demonstrate higher levels of HRV compared with age-matched, less active women. Furthermore, SBRS accounted for the majority of the variance in the high-frequency power of HRV, suggesting the possibility of a mechanistic link with cardiac vagal modulation of heart rate. Our findings may provide insight into a possible cardioprotective mechanism in physically active postmenopausal women.
I(to) and action potential notch are smaller in left vs. right canine ventricular epicardium
Authors: Di Diego J. M. , Sun Z. Q. , Antzelevitch C.
Published: December 2017

Transmural heterogeneities of repolarizing currents underlie prominent differences in the electrophysiology and pharmacology of ventricular epicardial, endocardial, and M cells in a number of species. The degree to which heterogeneities exist between the right and left ventricles is not well appreciated. The present study uses standard microelectrode and whole cell patch-clamp techniques to contrast the electrophysiological characteristics and pharmacological responsiveness of tissues and myocytes isolated from right (RVE) and left canine ventricular epicardium (LVE). RVE and LVE studied under nearly identical conditions displayed major differences in the early repolarizing phases of the action potential. The magnitude of phase 1 in RVE was nearly threefold that in LVE: 28.7 +/- 6.2 vs. 10.6 +/- 4.1 mV (basic cycle length = 2,000 ms). Phase 1 in RVE was also more sensitive to alterations of the stimulation rate and to 4-aminopyridine (4-AP), suggesting a much greater contribution of the transient outward current (I(to) 1) in RVE than in LVE. The combination of 4-AP plus ryanodine, low chloride, or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (chloride channel blocker) completely eliminated the notch and all rate dependence of the early phases of the action potential, making RVE and LVE indistinguishable. At +70 mV, RVE myocytes displayed peak I(to) 1 densities between 28 and 37 pA/pF. LVE myocytes included cells with similar I(to) 1 densities (thought to represent subsurface cells) but also cells with much smaller current levels (thought to represent surface cells). Average peak I(to) 1 density was significantly smaller in LVE than in RVE at voltages more than or equal to +10 mV. Our data point to prominent differences in the magnitude of the I(to) 1-mediated action potential notch in cells at the surface of RVE compared with the LVE and suggest that important distinctions may exist in the response of these two tissues to pharmacological agents and pathophysiological states, as previously demonstrated for epicardium and endocardium. Our findings also suggest that a calcium-activated outward current contributes to the early repolarization phase in RVE and LVE and that the influence of this current, although small, is more important in the left ventricle.
Fast sodium influx provides an initial step to trigger contractions in cat ventricle
Authors: Vites A. M. , Wasserstrom J. A.
Published: December 2017

We examined the possibility that Na+ current (INa) may play a role in excitation-contraction coupling in cat ventricular myocytes. A voltage step from -70 to -40 mV produced a fast INa, followed by a small transient inward current, a brief loss in voltage control to more positive potentials, and a transient contraction (reduction in cell length, delta L). We established that 10 microM nifedipine completely blocked Ca2+ current but did not prevent delta L; nifedipine reduced it by approximately 15%. This nifedipine-insensitive delta L was abolished by 1-10 microM ryanodine, 1-10 microM saxitoxin (STX), and 0.1-1.0 mM Cd2+. The size of delta L increased with more negative holding potential (VH; delta L-VH relation). Maximal delta L was achieved at a VH of approximately -70 mV. Anthopleura toxin A (APA, 3-10 nM), which selectively slows inactivation of INa, increased the size of the nifedipine-insensitive delta L at all VH, thus producing a +7-mV shift in the delta L-VH relation that was not affected by the state of the sarcoplasmic reticulum (SR). APA also produced an increase in maximal delta L, which was no longer observed once the SR was significantly loaded. These effects of APA were prevented by preexposure to STX. The state of the SR Ca2+ stores did not affect the presence of a nifedipine-insensitive delta L but determined its magnitude, suggesting that delta L was not associated with Ca2+ overload. In summary, cat and guinea pig ventricular myocytes are alike in that they both exhibit distinct INa-dependent contractions. Whether these contractions are due to a sudden increase in subsarcolemmal Na+ as a result of fast INa or the depolarization and thus reversal of the Na+/Ca2+ exchange remains undetermined.
Necrotic and apoptotic myocyte cell death in the aging heart of Fischer 344 rats
Authors: Kajstura J. , Cheng W. , Sarangarajan R. , Li P. , Li B. , Nitahara J. A. , Chapnick S. , Reiss K. , Olivetti G. , Anversa P.
Published: December 2017

To determine the effects of aging on myocyte cell death, Fischer 344 rats at 3, 7, 12, 16, and 24 mo of age were injected with myosin monoclonal antibody for the localization and quantification of necrotic myocyte cell death in the left ventricle, interventricular septum, and right ventricle. Conversely, the presence of DNA strand breaks in myocyte nuclei, indicative of programmed cell death, was evaluated by the terminal deoxynucleotidyl transferase assay and confirmed by DNA laddering. Myocyte necrosis, which involved nearly 1,000 myocytes in the left ventricular free wall at 3 mo, progressively increased with aging, reaching a value of 13,600 myocytes at 24 mo. Corre- sponding values in the interventricular septum were 300 and 9,400 myocytes. In the right ventricle, there were 270 necrotic myocytes at 3 mo and 9,000 at 24 mo. Programmed myocyte cell death was restricted to the left ventricular free wall and included 140 cells at 3 mo. This form of myocyte cell death increased at the subsequent age intervals, resulting in the involvement of 874 cells at 24 mo. The combination of necrosis and apoptosis in the left ventricular free wall was associated with 1,150 cells dying at 3 mo and 14,500 at 24 mo. In conclusion, myocyte cell death, apoptotic and necrotic in nature, constitutes an important determinant of the aging process, possibly mediating the occurrence of ventricular dysfunction and failure in the old heart.
Change in intramural strain distribution in rat aorta due to smooth muscle contraction and relaxation
Authors: Matsumoto T. , Tsuchida M. , Sato M.
Published: December 2017

Effect of smooth muscle contraction on intramural strain distribution in rat thoracic aorta was investigated with residual strain considered. Short segments sliced from the aortas were cut radially to release residual strain in an aerated Krebs-Ringer solution (37 degrees C). Each segment opened up immediately to form an arc. Opening angle was measured at this point and after smooth muscle contraction and relaxation. The angle increased from 97 +/- 11 degrees (means +/- SE, n = 14) to 153 +/- 14 degrees with the contraction and decreased to 68 +/- 9 degrees with the relaxation, indicating increase in residual strain with smooth muscle contraction. Intramural strain distribution at various pressures was calculated from the opening angle and pressure-diameter relations of the aortas. Strain distribution was almost uniform at 55 mmHg under smooth muscle relaxation, whereas this pressure exceeded 200 mmHg because of the increase in residual strain and decrease in vessel diameter under smooth muscle contraction. These results suggest aortic smooth muscle may change its contractile state through myogenic response to keep intramural strain distribution uniform against temporary change in blood pressure and thus maintain mechanical homeostasis in the aortic wall.
HSP induction inhibits iNOS mRNA expression and attenuates hypotension in endotoxin-challenged rats
Authors: Hauser G. J. , Dayao E. K. , Wasserloos K. , Pitt B. R. , Wong H. R.
Published: December 2017

Endotoxin (lipopolysaccharide, LPS)-induced hypotension is, in part, mediated via induction of nitric oxide synthase (iNOS), release of nitric oxide, and suppression of vascular reactivity (vasoplegia). Induction of heat shock proteins (HSP) or inhibition of iNOS expression improves survival in LPS-challenged rodents. We studied the effect of induction of HSP on LPS-mediated iNOS expression and on LPS-induced vasoplegia and hypotension. Rats were treated with the HSP inducer sodium arsenite (6 mg/kg iv) or saline control. Seventeen hours later, rats were challenged intravenously with 10 mg/kg of Escherichia coli LPS O127:B8 or saline control. Arsenite pretreatment resulted in expression of HSP 70 mRNA and of HSP 70 and heme oxygenase-1 proteins, inhibition of LPS-mediated iNOS mRNA induction, reversal of the LPS-induced hyporesponsiveness to norepinephrine ex vivo in isolated mesenteric arteries, and attenuation of LPS-induced hypotension in vivo. Our data suggest that induction of HSP expression protects rats from LPS by blocking LPS-induced iNOS expression, leading to inhibition of the overproduction of nitric oxide and thereby reversing LPS-induced vasoplegia and LPS-induced hypotension.
ATP: the red blood cell link to NO and local control of the pulmonary circulation
Authors: Sprague R. S. , Ellsworth M. L. , Stephenson A. H. , Lonigro A. J.
Published: December 2017

Recently, we reported that rabbit red blood cells (RBCs) were required for the expression of nitric oxide (NO) activity on pulmonary vascular resistance (PVR) in rabbit lungs. Here, we investigate the hypothesis that RBCs participate in the regulation of PVR via release of ATP in response to mechanical deformation that, in turn, evokes vascular NO synthesis. We found that rabbit and human RBCs, but not dog RBCs, release ATP in response to mechanical deformation. To determine the contribution of this ATP to NO synthesis and PVR, we compared the effects of human and dog RBCs on pressure-flow relationships in isolated rabbit lungs. In the presence of human RBCs, NG-nitro-L-arginine methyl ester (100 microM) produced a shift in the pressure-flow relationship consistent with a reduction in vascular caliber. NG-nitro-L-arginine methyl ester had no effect in lungs perfused with dog RBCs. These results suggest a unique mechanism for the control of PVR in rabbits and humans whereby release of ATP by RBCs in response to mechanical deformation leads to stimulation of NO synthesis that, in turn, modulates the PVR.
Velocity differences between L- and P-selectin-dependent neutrophil rolling in venules of mouse cremaster muscle in vivo
Authors: Jung U. , Bullard D. C. , Tedder T. F. , Ley K.
Published: December 2017

After surgical trauma, leukocyte rolling is initially normal in L-selectin-deficient mice and reduced at later times, whereas leukocyte rolling is initially absent in P-selectin-deficient mice but induced later. Here, we examined the possibility that P- and L-selectin support rolling at different characteristic velocities using intravital microscopy of venules of the exteriorized cremaster muscle venules of wild type (WT) and P- and L-selectin-deficient mice. At > 50 min after exteriorization, rolling in P-selectin-deficient mice occurred at significantly higher velocities (129 +/- 89 microns/s) than in WT mice (49 +/- 23 microns/s). Rolling velocity distribution in L-selectin-deficient mice was similar to WT mice immediately after exteriorization. Histological examination of Giemsa-stained whole-mount preparations in cremaster muscle venules revealed that the majority of rolling cells (approximately 90% in all genotypes) were granulocytes. We conclude that P-selectin mediates leukocyte rolling at velocities < 50 microns/s, whereas L-selectin sustains more rapid rolling. Under physiological conditions, P- and L-selectin synergize to support rolling at velocities between 20 and 70 microns/s as seen in WT mice.
Chemoreceptor dependence of very low frequency rhythms in advanced chronic heart failure
Authors: Ponikowski P. , Chua T. P. , Piepoli M. , Amadi A. A. , Harrington D. , Webb-Peploe K. , Volterrani M. , Colombo R. , Mazzuero G. , Giordano A. , Coats A. J.
Published: December 2017

Factors responsible for very low frequency oscillations (VLF; cycle > 30 s) in the cardiovascular system remain obscure. We tested the hypothesis that increased peripheral chemosensitivity is important in the pathogenesis of VLF oscillations in patients with chronic heart failure (CHF). Fourteen male patients with stable, moderate to severe CHF (age 60 +/- 1.1 yr, ejection fraction 23 +/- 11%) and reproducible VLF oscillations in heart rate underwent a protocol consisting of three consecutive 20-min phases during which they breathed air, hyperoxia (O2 via mask, 60% O2 concn), and air again. Autoregressive spectral analysis of R-R intervals, blood pressure, and respiration was used to quantify total oscillatory power (TP), VLF, low (0.04-0.15 Hz)- and high (0.15-0.40Hz)-frequency power, and the coherence between these signals. Peripheral chemosensitivity was studied by assessing the ventilatory response to hypoxia using transient inhalations of pure N2. Discrete VLF rhythms were seen in R-R intervals in all 14 patients, in blood pressure in 7 of 14, and in respiration in 8 of 14 patients. A significant coherence (> 0.5) between heart rate and systolic blood pressure within the VLF band with mean phase value of -140 degrees, suggesting an antibaroreflex relationship, was seen in six subjects. Transient hyperoxia abolished the VLF oscillations in most subjects (12 of 14 in R-R intervals) and decreased R-R variability power within the VLF band. This response significantly correlated with peripheral chemoreceptor sensitivity (r = 0.77, P = 0.014). This study suggests that in CHF, enhanced peripheral chemoreceptor activity may facilitate slow oscillations in the cardiorespiratory signals.
Plasmalemmal vesicles function as transcytotic carriers for small proteins in the continuous endothelium
Authors: Predescu S. A. , Predescu D. N. , Palade G. E.
Published: December 2017

We investigated the location and the structural identity of the small pore system, postulated by the pore theory of capillary permeability, using a murine heart perfusion system and small protein molecules as preferential probes for the small pores. Dinitrophenylated proteins were perfused in situ in the absence and in the presence of N-ethylmaleimide (NEM), a reagent known to interfere with membrane fusion of vesicular carriers with their target membranes. The exit pathways of the tracers from vascular lumina to the interstitia were followed by immunoelectron microscopy and by tissue fractionation biochemistry to quantitate their transport and to estimate the extent of transport inhibition by NEM. After 5 min of perfusion, all tracers used were found essentially restricted to plasmalemmal vesicles (PVs) within the endothelium and NEM inhibited their transport by 80-85%. The transport of [14C]inulin and [14C]sucrose, assumed to follow the paracellular pathway, was marginally affected by NEM. These findings indicate that PVs function as structural equivalents of the small pore system for molecules >2 nm in diameter.
Mechanical forces regulate focal adhesion and costamere assembly in cardiac myocytes
Authors: Sharp W. W. , Simpson D. G. , Borg T. K. , Samarel A. M. , Terracio L.
Published: December 2017

To determine whether the formation and maintenance of focal adhesions and costameres in cardiac myocytes are influenced by the mechanical forces that they transmit, we mechanically unloaded these cells by inhibiting their spontaneous contractile activity with the calcium-channel blocker nifedipine (12 microM). Interference-reflection and fluorescence microscopy revealed that within 24 h of arrest, beta 1-integrin- and vinculin-positive focal adhesions and costameres were disrupted. Loss of mature beta 1-integrin from the cell surface was observed in cell surface-labeling experiments and in Western blots. Subjecting nonbeating cells to a 5% static stretch for 24 h resulted in an increase of 21% for beta 1-integrin and 39% for vinculin. Stretching beating cells resulted in 71 and 9% increases, respectively. Intracellular concentrations of pre-beta 1 were not affected by contractile activity or by stretch. Our results indicate that mechanical forces stabilize the cellular levels of beta 1-integrin and vinculin by possibly regulating their association with the formation and maintenance of focal adhesions and costameres.
Hypoxia-induced expression of VEGF is reversible in myocardial vascular smooth muscle cells
Authors: Gu J. W. , Adair T. H.
Published: December 2017

We determined whether hypoxia-induced expression of vascular endothelial growth factor (VEGF) can be reversed by a normoxic environment. Dog myocardial vascular smooth muscle cells (MVSMCs) were exposed to hypoxia (1% O2) for 24 h and then returned to normoxia (20% O2). VEGF protein levels increased by more than fivefold after 24 h of hypoxia and returned to baseline within 24 h of the return of the cells to normoxia. Northern blot analysis showed that hypoxia caused a 5.5-fold increase in VEGF mRNA, and, again, the expression was reversed after reinstitution of normoxia. Additional measurements showed that basic fibroblast growth factor and platelet-derived growth factor protein levels were not induced by hypoxia and that hypoxia caused a fourfold decrease in transforming growth factor-beta 1 protein levels. Hypoxia conditioned media from MVSMCs caused human umbilical vein endothelial cells to increase [3H]thymidine incorporation by twofold, an effect that was blocked in a dose-dependent manner by anti-human VEGF antibody. The hypoxia conditioned media had no effect on MVSMC proliferation. These findings suggest that VEGF expression can be bidirectionally controlled by tissue oxygenation, and thus support the hypothesis that VEGF is a physiological regulator of angiogenesis.
Near-infrared monitoring of cerebral tissue oxygen saturation and blood volume in newborn piglets
Authors: Brun N. C. , Moen A. , Borch K. , Saugstad O. D. , Greisen G.
Published: December 2017

Near-infrared spectrophotometry (NIRS) potentially provides a tool for noninvasive tissue oxygenation and blood volume monitoring. Cerebral monitoring could be useful in the prevention of hypoxic ischemic brain injury in newborns. This study sought to validate such NIRS measurements in normoventilated, hypocapnic, and hypoxemic states in the brain of newborn piglets vs. arterial (SaO2) and sagittal sinus blood hemoglobin saturation (SssO2) and blood volume measurements with 99mTc-labeled erythrocytes. NIRS measurements of cerebral blood volume (CBV) were performed with both oxyhemoglobin and indocyanine green as tracers, and changes in CBV were monitored by following the change in the concentration of total hemoglobin (i.e., oxyhemoglobin + deoxyhemoglobin). NIRS CBV measurements did not correlate well with the radioactive measurements. NIRS measurements of oxygenation, however, correlated well with a weighted mean value of SaO2 and SssO2 (r = 0.90; P < 0.0001). Multiple linear regression of the oxygenation index (i.e., oxyhemoglobin - deoxyhemoglobin) on SaO2 and SssO2 suggested that NIRS sees hemoglobin in tissue in a venous-to-arterial ratio of 2:1. Therefore, in this study, NIRS reliably monitored changes in cerebral tissue oxygenation but not in CBV.
Contribution of chondroitin-dermatan sulfate-containing proteoglycans to the function of rat mesenteric arteries
Authors: Gandley R. E. , McLaughlin M. K. , Koob T. J. , Little S. A. , McGuffee L. J.
Published: December 2017

Proteoglycans are an important nonfibrous matrix component of the arterial wall. Direct evidence for their role in resistance-sized arteries is lacking, although they likely have an important role in coordinating and regulating vessel behavior, presumably via interactions of their glycosaminoglycan chains or core proteins with other matrix molecules and/or the smooth muscle cell surface. The purpose of this study was to determine whether the removal of specific glycosaminoglycan chains from proteoglycans in resistance-sized mesenteric arteries would change the mechanical properties of the arterial wall, thereby affecting their functional behavior. The major finding of the study was that 65% removal of chondroitin-dermatan sulfate-containing glycosaminoglycans from the arterial wall increased vascular wall stiffness and altered the myogenic behavior of the artery. The significant alterations in myogenic behavior associated with changes in passive mechanics following partial glycosaminoglycan chain removal support our hypothesis that chondroitin-dermatan sulfate-containing proteoglycans contribute significantly to the functional behavior of resistance arteries. We speculate that these alterations are the result of changes in stress transfer between collagen fibrils and/or stress transfer between cells and collagen fibrils under applied pressure.
Role of adenosine receptors in late preconditioning against myocardial stunning in conscious rabbits
Authors: Maldonado C. , Qiu Y. , Tang X. L. , Cohen M. V. , Auchampach J. , Bolli R.
Published: December 2017

Conscious rabbits underwent six 4-min coronary occlusions interspersed with 4-min periods of reperfusion for 2 consecutive days (days 1 and 2 of stage I); 2 wk later, they underwent the same protocol (days 1 and 2 of stage II) except that they received either 8-(p-sulfophenyl)theophylline (SPT) on day 1 (group I, n = 5) or 2-chloro-N6-cyclopentyl-adenosine (CCPA) on the day before day 1 (group II, n = 6). In both groups I and II, on day 1 of stage I, systolic wall thickening (WTh) remained significantly depressed for several hours, indicating myocardial stunning; on day 2, however, the total deficit of WTh was approximately 50% less than on day 1 (P < 0.01), indicating the development of late preconditioning (PC) against myocardial stunning. Despite administration of SPT, in group I the deficit of WTh during stage II was 55% less on day 2 than on day 1 (P < 0.05). Similar results were obtained in three other rabbits treated with PD-115199 on day 1. In group II, pretreatment with CCPA during stage II failed to decrease the deficit of WTh on day 1. This study presents a new conscious rabbit model for studying myocardial stunning that is relatively inexpensive and technically less demanding than larger animal models. In this model, the development of late PC against myocardial stunning is not blocked by nonselective blockade of adenosine receptors with either SPT or PD-115199, nor is it induced by activation of adenosine A1 receptors with CCPA, indicating that adenosine receptors are not involved in the pathogenesis of this phenomenon.
Myocardial ischemia decreases oxidative phosphorylation through cytochrome oxidase in subsarcolemmal mitochondria
Authors: Lesnefsky E. J. , Tandler B. , Ye J. , Slabe T. J. , Turkaly J. , Hoppel C. L.
Published: December 2017

The effect of myocardial ischemia on mitochondrial oxidative phosphorylation was investigated using isolated, buffer-perfused rabbit hearts. After 45 min of global ischemia, oxidative phosphorylation was decreased only in the subsarcolemmal population of mitochondria with all substrates tested. The oxidation of N,N,N',N' tetramethyl p-phenylenediamine-ascorbate, an electron donor to cytochrome oxidase via cytochrome c, was decreased in subsarcolemmal mitochondria [ischemia (n = 6): 76 +/- 3 vs. control (n = 5): 105 +/- 6 nanoatoms O.min-1.mg-1, P < 0.01] but not in interfibrillar mitochondria. Only minor morphological changes were observed by electron microscopy in the isolated mitochondria after ischemia. Neither cytochrome oxidase activity measured under conditions for maximal activity nor the apparent Michaelis constant and maximum velocity values of the two cytochrome c binding sites were different in subsarcolemmal mitochondria isolated from ischemic and control hearts. The cytochrome c content was decreased in subsarcolemmal mitochondria after ischemia (ischemia: 0.111 +/- 0.013 vs. control: 0.156 +/- 0.007 nmol/mg protein, P < 0.05). Thus ischemia decreased the rate of oxidative phosphorylation through cytochrome oxidase selectively in intact subsarcolemmal mitochondria. Ischemic damage to the terminal segment of the electron transport chain involves a decrease in the content of cytochrome c, whereas the expressible catalytic activity of cytochrome oxidase remains unchanged.