bluuebunny/crossref_metadata_2025 huggingface dataset:
Essential roles of IκB kinases α and β in serum- and IL-1-induced human VSMC proliferation
Authors: Sasu Sebastian , Beasley Debbie
Published: December 2017

Interleukin-1 (IL-1) is a potent vascular smooth muscle cell (VSMC) mitogen, which can stimulate cells via activation of nuclear factor-κB (NF-κB) following phosphorylation of its inhibitory subunit (IκB). Because the proliferative effect of IL-1 is additive with that of serum, the present studies assessed the role of IκB kinases (IKKs) and NF-κB in both IL-1- and serum-induced VSMC proliferation. IL-1β (1 ng/ml) induced marked and persistent NF-κB activation in VSMC that was maximal at 1 h and persisted for 3 days. There was a 3-fold increase in DNA synthesis after acute IL-1 exposure (24–96 h) and a 12-fold increase after chronic IL-1 exposure (>7 days). Electrophoretic mobility shift assay and supershift analysis indicated that IL-1-induced NF-κB complexes consisted of p65/p50 heterodimers and p50 homodimers. Human saphenous vein smooth muscle cells (HSVSMC) were transiently cotransfected with expression plasmids encoding a dominant negative mutant form of either IKKα or IKKβ, in which K44was mutated to A (K44A), and a green fluorescent protein expression plasmid that allows identification of transfected cells. IL-1 induced nuclear localization of p65 in 95% of cells transfected with vector alone but in only 69% and 26% of cells expressing IKKα (K44A) or IKKβ (K44A), respectively. Likewise, proliferation increased 3.2-fold in IL-1-treated HSVSMC which had been transfected with vector alone, but only 2.2- and 1.5-fold proliferation in HSVSMC expressing IKKα (K44A) or IKKβ (K44A), respectively. Although serum activated NF-κB transiently, serum-induced proliferation was markedly attenuated in HSVSMC expressing IKKα (K44A) and IKKβ (K44A) compared with HSVSMC transfected with vector alone. The results support an essential role of IKKs in the proliferative response of HSVSMC to IL-1 and to serum.
Chronic hypoxia, pregnancy, and endothelium-mediated relaxation in guinea pig uterine and thoracic arteries
Authors: White Margueritte Mabry , McCullough Robert E. , Dyckes Rebecca , Robertson Alastair D. , Moore Lorna Grindlay
Published: December 2017

Vasodilation that occurs during normal pregnancy is associated with enhanced relaxation and decreased contractile response to agonists, which are in part due to increased stimulated and basal nitric oxide (NO). In preeclampsia and/or pregnancies carried at high altitude (HA), this normal vascular adjustment is reversed or diminished. We previously reported that HA exposure did not inhibit the pregnancy-associated decrease in contractile response to agonist or basal NO in guinea pig uterine arteries (UA). We therefore sought to determine whether altitude interfered with effects of pregnancy on endothelium-dependent relaxation through a reduction in stimulated NO. We examined the relaxation response to ACh in UA and bradykinin in thoracic arteries (TA) and effects of NO inhibition with 200 μM N G-nitro-l-arginine (l-NNA) in arterial rings isolated from nonpregnant and pregnant guinea pigs exposed throughout gestation to low altitude (LA, 1,600 m, n = 26) or HA (3,962 m, n = 22). In pregnant UA, relaxation to ACh was enhanced ( P < 0.05) at both altitudes and NO inhibition diminished, but did not reverse, ACh relaxation. The effect of l-NNA on the relaxation response to ACh was less in HA than in LA animals ( P = 0.0021). In nonpregnant UA, relaxation to ACh was similar in LA and HA animals.l-NNA reversed the relaxation response to ACh at HA but not at LA. In TA, relaxation to bradykinin was unaltered by pregnancy or altitude and was completely reversed by NO inhibition. These data suggest that effects of NO inhibition are diminished in UA during pregnancy at HA. Additional studies are needed to confirm whether these effects are mediated through inhibition of stimulated NO. HA exposure did not inhibit relaxation to ACh, perhaps because of stimulation of other vasodilators.
Transmural distribution of FDG uptake in stunned myocardium
Authors: Fallavollita James A. , Trojan Christopher , Canty John M.
Published: December 2017

Fasting [18F]fluoro-2-deoxyglucose (FDG) uptake is increased in viable, chronically dysfunctional myocardium, but the relationship to acute episodes of ischemia remains undefined. To investigate FDG uptake in acute stunning, chronically instrumented pigs ( n = 9) and sham controls ( n = 8) were studied while in a fasted, closed-chest, anesthetized state. One-hour partial occlusion reduced subendocardial flow from 1.24 ± 0.14 to 0.35 ± 0.06 ml · min−1 · g−1 and wall thickening from 16.8 ± 2.1 to 3.7 ± 0.7%. Regional function remained depressed during reperfusion (8.3 ± 1.4%) despite the return of flow to resting levels. Triphenyl tetrazolium chloride staining showed no irreversible injury. FDG uptake in stunned myocardium was variably increased and averaged 1.5-fold higher than that of normal regions, with no consistent transmural variation. Subgroup analysis showed that variability in FDG uptake was related to alterations in insulin levels that varied directly with ischemic risk region.
Colocalization of dihydropyridine and ryanodine receptors in neonate rabbit heart using confocal microscopy
Authors: Sedarat Franklin , Xu Liqun , Moore Edwin D. W. , Tibbits Glen F.
Published: December 2017

Because of undeveloped T tubules and sparse sarcoplasmic reticulum, Ca2+-induced Ca2+release (CICR) may not be the major mechanism providing contractile Ca2+in the neonatal heart. Spatial association of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), a key factor for CICR, was examined in isolated neonatal rabbit ventricular myocytes aged 3–20 days by double-labeling immunofluorescence and confocal microscopy. We found a significant increase ( P < 0.0005) in the degree of colocalization of DHPR and RyR during development. The number of voxels containing DHPR that also contained RyR in the 3-day-old group (62 ± 1.8%) was significantly lower than in the other age groups (76 ± 1.3 in 6-day old, 75 ± 1.2 in 10-day old, and 79 ± 0.9% in 20-day old). The number of voxels containing RyR that also contained DHPR was significantly higher in the 20-day-old group (17 ± 0.5%) compared with the other age groups (10 ± 0.7 in 3-day old, 11 ± 0.6 in 6-day old, and 11 ± 0.5% in 10-day old). During this period, the pattern of colocalization changed from mostly peripheral to mostly internal couplings. Our results provide a structural basis for the diminished prominence of CICR in neonatal heart.
Isometric handgrip training reduces arterial pressure at rest without changes in sympathetic nerve activity
Authors: Ray Chester A. , Carrasco Dario I.
Published: December 2017

The purpose of this study was to determine whether isometric handgrip (IHG) training reduces arterial pressure and whether reductions in muscle sympathetic nerve activity (MSNA) mediate this drop in arterial pressure. Normotensive subjects were assigned to training ( n = 9), sham training ( n = 7), or control ( n = 8) groups. The training protocol consisted of four 3-min bouts of IHG exercise at 30% of maximal voluntary contraction (MVC) separated by 5-min rest periods. Training was performed four times per week for 5 wk. Subjects' resting arterial pressure and heart rate were measured three times on 3 consecutive days before and after training, with resting MSNA (peroneal nerve) recorded on the third day. Additionally, subjects performed IHG exercise at 30% of MVC to fatigue followed by muscle ischemia. In the trained group, resting diastolic (67 ± 1 to 62 ± 1 mmHg) and mean arterial pressure (86 ± 1 to 82 ± 1 mmHg) significantly decreased, whereas systolic arterial pressure (116 ± 3 to 113 ± 2 mmHg), heart rate (67 ± 4 to 66 ± 4 beats/min), and MSNA (14 ± 2 to 15 ± 2 bursts/min) did not significantly change following training. MSNA and cardiovascular responses to exercise and postexercise muscle ischemia were unchanged by training. There were no significant changes in any variables for the sham training and control groups. The results indicate that IHG training is an effective nonpharmacological intervention in lowering arterial pressure.
AGT M235T and ACE ID polymorphisms and exercise blood pressure in the HERITAGE Family Study
Authors: Rankinen Tuomo , Gagnon Jacques , Pérusse Louis , Chagnon Yvon C. , Rice Treva , Leon Arthur S. , Skinner James S. , Wilmore Jack H. , Rao D. C. , Bouchard Claude
Published: December 2017

We investigated the association between angiotensinogen (AGT) and angiotensin-converting enzyme (ACE) gene polymorphisms and exercise training responses of resting and exercise blood pressure (BP). BP at rest and during submaximal (50 watts) and maximal exercise tests was measured before and after 20 wk of endurance training in 476 sedentary normotensive Caucasian subjects from 99 families. AGT M235T and ACE insertion/deletion polymorphisms were typed with PCR-based methods. Men carrying the AGT MM and MT genotypes showed 3.7 ± 0.6 and 3.2 ± 0.5 (SE) mmHg reductions, respectively, in diastolic BP at 50 watts (DBP50), whereas, in the TT homozygotes, the decrease was 0.4 ± 1.0 mmHg ( P = 0.016 for trend, adjusted for age, body mass index, and baseline DBP50). Men with the ACE DD genotype showed a slightly greater decrease in DBP50 (4.4 ± 0.6 mmHg) than the II and ID genotypes (2.8 ± 0.7 and 2.4 ± 0.5 mmHg, respectively, P = 0.050). Furthermore, a significant ( P = 0.022) interaction effect between the AGT and ACE genes was noted for DBP50; the AGT TT homozygotes carrying the ACE D allele showed no response to training. Men with the AGT TT genotype had greater ( P = 0.007) diastolic BP (DBP) response to acute maximal exercise at baseline. However, the difference disappeared after the training period. No associations were found in women. These data suggest that, in men, the genetic variation in the AGT locus modifies the responsiveness of submaximal exercise DBP to endurance training, and interactions between the AGT and ACE loci can alter this response.
Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology
Authors: Zhou Ying-Ying , Wang Shi-Qiang , Zhu Wei-Zhong , Chruscinski Andrej , Kobilka Brian K. , Ziman Bruce , Wang Su , Lakatta Edward G. , Cheng Heping , Xiao Rui-Ping
Published: December 2017

Rapid development of transgenic and gene-targeted mice and acute genetic manipulation via gene transfer vector systems have provided powerful tools for cardiovascular research. To facilitate the phenotyping of genetically engineered murine models at the cellular and subcellular levels and to implement acute gene transfer techniques in single mouse cardiomyocytes, we have modified and improved current enzymatic methods to isolate a high yield of high-quality adult mouse myocytes (5.3 ± 0.5 × 105 cells/left ventricle, 83.8 ± 2.5% rod shaped). We have also developed a technique to culture these isolated myocytes while maintaining their morphological integrity for 2–3 days. The high percentage of viable myocytes after 1 day in culture (72.5 ± 2.3%) permitted both physiological and biochemical characterization. The major functional aspects of these cells, including excitation-contraction coupling and receptor-mediated signaling, remained intact, but the contraction kinetics were significantly slowed. Furthermore, gene delivery via recombinant adenoviral infection was highly efficient and reproducible. In adult β1/β2-adrenergic receptor (AR) double-knockout mouse myocytes, adenovirus-directed expression of either β1- or β2-AR, which occurred in 100% of cells, rescued the functional response to β-AR agonist stimulation. These techniques will permit novel experimental settings for cellular genetic physiology.
Construction of a very high-density extracellular electrode array
Authors: Malkin Robert A. , Pendley Bradford D.
Published: December 2017

Cellular activation mapping (specifying in time and space the electrical activation sequence of cells) is a well-established basic research tool in cardiac, neural, and gastric physiology. Much recent research in cardiac mapping has focused on large arrays (>200 electrodes) with small electrodes (<500 μm). Construction of such arrays using standard techniques is tedious and yields irregular electrode spacing. We present a novel construction technique that rapidly produces large arrays with regularly spaced small electrodes. For methods, fine-pitch copper ribbon cables, insulated with either polyvinylchloride (PVC) or polyimide (flexible printed circuit; FPC), were assembled together such that the active surface was the cut end of the cable. The cut end was sanded and polished, then coated with silver and sometimes silver chloride. Once completed, the alternating current (AC) root-mean-square (rms) potential was measured between two adjacent, individual electrodes. Polarization testing was conducted according to a previously reported protocol (Witkowski FX and Penkoske PA. J Electrocardiol 21: 273–282, 1988). Activation mapping was conducted in the open-chest guinea pig with both pacing- and defibrillation- strength stimuli. In terms of results, four PVC and three FPC arrays were constructed, ranging from 4 to 400 electrodes. Two hours of labor were needed to create a complete electrode array, independent of the number of electrodes, including connectors and silver/silver chloride coating. As expected, the addition of a silver/silver chloride coating significantly reduced (0.76–0.42 mV, P < 0.001) the AC rms potential difference between two electrodes. A nearly immediate recovery of the potential difference between adjacent pairs of silver/silver chloride electrodes was observed after defibrillation stimuli.
Nitric oxide exerts feedback inhibition on EDHF-induced coronary arteriolar dilation in vivo
Authors: Nishikawa Yasuhiro , Stepp David W. , Chilian William M.
Published: December 2017

We tested the hypothesis that nitric oxide (NO) inhibits endothelium-derived hyperpolarizing factor (EDHF)-induced vasodilation via a negative feedback pathway in the coronary microcirculation. Coronary microvascular diameters were measured using stroboscopic fluorescence microangiography. Bradykinin (BK)-induced dilation was mediated by EDHF, when NO and prostaglandin syntheses were inhibited, or by NO when EDHF and prostaglandin syntheses were blocked. Specifically, BK (20, 50, and 100 ng · kg−1 · min−1 ic) caused dose-dependent vasodilation similarly before and after administration of N G-monomethyl-l-arginine (l-NMMA) (3 μmol/min ic for 10 min) and indomethacin (Indo, 10 mg/kg iv). The residual dilation to BK withl-NMMA and Indo was completely abolished by suffusion of miconazole or an isosmotic buffer containing high KCl (60 mM), suggesting that this arteriolar vasodilation is mediated by the cytochrome P-450 derivative EDHF. BK-induced dilation was reduced by 39% after inhibition of EDHF and prostaglandin synthesis, and dilation was further inhibited by combined blockade withl-NMMA to a 74% reduction in the response. This suggests an involvement for NO in the vasodilation. After dilation to BK was assessed with l-NMMA and Indo, sodium nitroprusside (SNP, 1–3 μg · kg−1 · min−1ic), an exogenous NO donor, was administered in a dose to increase the diameter to the original control value. Dilation to BK was virtually abolished when administered concomitantly with SNP duringl-NMMA and Indo ( P < 0.01 vs. before SNP), suggesting that NO inhibits EDHF-induced dilation. SNP did not affect adenosine- or papaverine-induced arteriolar dilation in the presence ofl-NMMA and Indo, demonstrating that the effect of SNP was not nonspecific. In conclusion, our data are the first in vivo evidence to suggest that NO inhibits the production and/or action of EDHF in the coronary microcirculation.
Gonadal hormones affect diameter of male rat cerebral arteries through endothelium-dependent mechanisms
Authors: Geary Greg G. , Krause Diana N. , Duckles Sue P.
Published: December 2017

Gender is known to influence the incidence and severity of cerebrovascular disease. In the present study, luminal diameter was measured in vitro in pressurized middle cerebral artery segments from male rats that were either untreated, orchiectomized (ORX), ORX with testosterone treatment (ORX+TEST), or ORX with estrogen treatment (ORX+EST). The maximal passive diameters (0 Ca2+ + 3 mM EDTA) of arteries from all four groups were similar. In endothelium-intact arteries, myogenic tone was significantly greater in arteries from untreated and ORX+TEST compared with arteries from either ORX or ORX+EST. During exposure to N G-nitro-l-arginine-methyl ester (l-NAME), an NO synthase (NOS) inhibitor, myogenic tone significantly increased in all groups. The effect of l-NAME was significantly greater in arteries from untreated and ORX+EST compared with arteries from ORX and ORX+TEST rats. Differences in myogenic tone between ORX and ORX+TEST persisted after inhibition of NOS. After endothelium removal or inhibition of the cyclooxygenase pathway combined with K+ channel blockers, myogenic tone differences between ORX and ORX+TEST were abolished. Wall thickness and forced dilation were not significantly different between arteries from ORX and ORX+TEST. Our data show that gonadal hormones affect myogenic tone in male rat cerebral arteries through NOS- and/or endothelium-dependent mechanisms.
Ouabain augments Ca2+ transients in arterial smooth muscle without raising cytosolic Na+
Authors: Arnon Assaf , Hamlyn John M. , Blaustein Mordecai P.
Published: December 2017

Ouabain and other cardiotonic steroids (CTS) inhibit Na+ pumps and are widely believed to exert their cardiovascular effects by raising the cytosolic Na+ concentration ([Na+]cyt) and Ca2+. This view has not been rigorously reexamined despite evidence that low-dose CTS may act without elevating [Na+]cyt; also, it does not explain the presence of multiple, functionally distinct isoforms of the Na+ pump in many cells. We investigated the effects of Na+ pump inhibition on [Na+]cyt(with Na+ binding benzofuran isophthalate) and Ca2+ transients (with fura 2) in primary cultured arterial myocytes. Low concentrations of ouabain (3–100 nM) or human ouabain-like compound or reduced extracellular K+ augmented hormone-evoked mobilization of stored Ca2+ but did not increase bulk [Na+]cyt. Augmentation depended directly on external Na+, but not external Ca2+, and was inhibited by 10 mM Mg2+ or 10 μM La3+. Evoked Ca2+ transients in pressurized small resistance arteries were also augmented by nanomolar ouabain and inhibited by Mg2+. These results suggest that Na+ enters a tiny cytosolic space between the plasmalemma (PL) and the adjacent sarcoplasmic reticulum (SR) via an Mg2+- and La3+-blockable mechanism that is activated by SR store depletion. The Na+ and Ca2+ concentrations within this space may be controlled by clusters of high ouabain affinity (α3) Na+ pumps and Na/Ca exchangers located in PL microdomains overlying the SR. Inhibition of the α3 pumps by low-dose ouabain should raise the local concentrations of Na+ and Ca2+ and augment hormone-evoked release of Ca2+ from SR stores. Thus the clustering of small numbers of specific PL ion transporters adjacent to the SR can regulate global Ca2+ signaling. This mechanism may affect vascular tone and blood flow and may also influence Ca2+ signaling in many other types of cells.
p38 MAP kinase pathway regulates angiotensin II-induced contraction of rat vascular smooth muscle
Authors: Meloche Sylvain , Landry Jacques , Huot Jacques , Houle François , Marceau François , Giasson Edith
Published: December 2017

Angiotensin II (ANG II) is a multifunctional hormone that exerts potent vasoconstrictor and hypertrophic effects on vascular smooth muscle. Here, we demonstrate that the p38 mitogen-activated protein (MAP) kinase pathway is involved in ANG II-induced vascular contraction. Addition of ANG II to rat aortic smooth muscle cells (SMC) caused a rapid and transient increase of p38 activity through activation of the AT1receptor subtype. This response to ANG II was strongly attenuated by pretreating cells with antioxidants and diphenylene iodonium and was mimicked by exposure of cells to H2O2. Stimulation of p38 by ANG II resulted in the enzymatic activation of MAP kinase-activated protein (MAPKAP) kinase-2 and the phosphorylation of heat shock protein 27 (HSP27) in aortic SMC. Pretreatment of cells with the specific p38 MAP kinase inhibitor SB-203580 completely blocked the ANG II-dependent activation of MAPKAP kinase-2 and phosphorylation of HSP27. ANG II also caused a robust activation of MAPKAP kinase-2 in the intact rat aorta. Incubation with SB-203580 significantly decreased the potency of ANG II to induce contraction of rat aortic rings and depressed the maximal hormone response. These results suggest that the p38 MAP kinase pathway selectively modulates the vasoconstrictor action of ANG II in vascular smooth muscle.
The female reproductive cycle is an important variable in the response to trauma-hemorrhage
Authors: Jarrar Doraid , Wang Ping , Cioffi William G. , Bland Kirby I. , Chaudry Irshad H.
Published: December 2017

Although immune functions in proestrus females are maintained after hemorrhage as opposed to decreased responses in males, it remains unknown whether such a sexual dimorphism also exists with regard to cardiovascular and hepatocellular functions under those conditions. To study this, male and female (estrus and proestrus) rats underwent a 5-cm midline laparotomy and were bled to and maintained at a mean blood pressure of 40 mmHg until 40% of the maximal bleed-out volume was returned in the form of Ringer lactate (RL). Rats were then resuscitated with four times the shed blood volume with RL. At 24 h thereafter, cardiac index; heart performance; hepatocellular function; and plasma estradiol, testosterone, and prolactin levels were measured. Cardiovascular and hepatocellular functions were depressed in males and estrus females ( P < 0.05) but were not depressed in proestrus females after resuscitation. Plasma estradiol and prolactin levels were highest in proestrus females ( P < 0.05), whereas males had high testosterone and the lowest estradiol levels ( P < 0.05). Thus the female reproductive cycle is an important variable in the response to hemorrhage. Because low testosterone and high estradiol and prolactin levels appear to be beneficial for organ functions after trauma-hemorrhage, antagonism of testosterone receptors and/or increases in estradiol and prolactin levels in males and estrus females, respectively, may be novel approaches for improving organ functions under such conditions.
Virtual electrode polarization in the far field: implications for external defibrillation
Authors: Efimov Igor R. , Aguel Felipe , Cheng Yuanna , Wollenzier Brian , Trayanova Natalia
Published: December 2017

We recently suggested that failure of implantable defibrillation therapy may be explained by the virtual electrode-induced phase singularity mechanism. The goal of this study was to identify possible mechanisms of vulnerability and defibrillation by externally applied shocks in vitro. We used bidomain simulations of realistic rabbit heart fibrous geometry to predict the passive polarization throughout the heart induced by external shocks. We also used optical mapping to assess anterior epicardium electrical activity during shocks in Langendorff-perfused rabbit hearts ( n= 7). Monophasic shocks of either polarity (10–260 V, 8 ms, 150 μF) were applied during the T wave from a pair of mesh electrodes. Postshock epicardial virtual electrode polarization was observed after all 162 applied shocks, with positive polarization facing the cathode and negative polarization facing the anode, as predicted by the bidomain simulations. During arrhythmogenesis, a new wave front was induced at the boundary between the two regions near the apex but not at the base. It spread across the negatively polarized area toward the base of the heart and reentered on the other side while simultaneously spreading into the depth of the wall. Thus a scroll wave with a ribbon-shaped filament was formed during external shock-induced arrhythmia. Fluorescent imaging and passive bidomain simulations demonstrated that virtual electrode polarization-induced scroll waves underlie mechanisms of shock-induced vulnerability and failure of external defibrillation.
Less afterload sensitivity in short-term hibernating than in acutely ischemic and stunned myocardium
Authors: Schulz Rainer , Rose Jochen , Post Heiner , Skyschally Andreas , Heusch Gerd
Published: December 2017

Short-term hibernating myocardium is characterized by reduced contractile function during persistent moderate ischemia, the recovery of metabolic parameters, and the absence of necrosis. To study the afterload dependence of regional wall excursion in short-term hibernating myocardium, in 11 enflurane-anesthetized swine the left anterior descending coronary artery was cannulated and hypoperfused for 90 min to reduce anterior systolic wall thickening (WT, sonomicrometry) by 60%. Under control conditions, at 5 and 90 min ischemia the descending thoracic aorta was acutely constricted to increase left ventricular (LV) pressure by 30 mmHg. Under control conditions, increased LV pressure resulted in decreased WT [i.e., a negative slope of the relationship between WT and LV end-systolic pressure: −11.2 ± 4.2 (SD) μm/mmHg]. This slope was further significantly decreased at 5 min ischemia (−26.5 ± 8.8 μm/mmHg) but returned toward control values in short-term hibernating myocardium at 90 min ischemia (−17.2 ± 6.6 μm/mmHg). At 30 min reperfusion, the slope was once more significantly decreased (−27.8 ± 8.1 μm/mmHg). In conclusion, WT in short-term hibernating myocardium is less afterload dependent than in acutely ischemic and reperfused myocardium.
Inconsistent relation of MAPK activation to infarct size reduction by ischemic preconditioning in pigs
Authors: Behrends Matthias , Schulz Rainer , Post Heiner , Alexandrov Alexander , Belosjorow Sergej , Michel Martin C. , Heusch Gerd
Published: December 2017

The importance of the activation of mitogen-activated protein kinases (MAPK) for the cardioprotection achieved by ischemic preconditioning (IP) is still controversial. We therefore measured infarct size and p38, extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinase (JNK) MAPK phosphorylation (by biopsies) in enflurane-anesthetized pigs. After 90 min low-flow ischemia and 120 min reperfusion, infarct size averaged 18.3 ± 12.4 (SD)% ( group 1, n = 14). At similar subendocardial blood flows, IP by 10 min ischemia and 15 min reperfusion ( group 2, n = 14) reduced infarct size to 6.2 ± 5.1% ( P < 0.05). An inconsistent increase in p38, ERK, and p54 JNK phosphorylation (by Western blot) was found during IP; p46 JNK phosphorylation increased with the subsequent reperfusion. At 8 min of the sustained ischemia, p38, ERK, and p54 JNK phosphorylation were increased with no difference between groups (medians: p38: 207% of baseline in group 1vs. 153% in group 2; ERK: 142 vs. 144%; p54 JNK: 171 vs. 155%, respectively). MAPK phosphorylation and reduction of infarct size by IP were not correlated, thus not supporting the concept of a causal role of MAPK in mediating cardioprotection by IP.
MAPK and PKC activity are not required for H2O2-induced arterial muscle contraction
Authors: Pelaez N. J. , Osterhaus S. L. , Mak A. S. , Zhao Y. , Davis H. W. , Packer C. S.
Published: December 2017

H2O2-induced pulmonary arterial smooth muscle (PASM) contractions are independent of Ca2+and myosin light chain phosphorylation. The purpose of this study was to determine whether mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1 and ERK2, or protein kinase C (PKC) activation is required for H2O2-induced contraction. Porcine PASM strips were stimulated with 1 mM H2O2, 120 mM KCl, or 10 μM phorbol myristic acetate and freeze clamped at various times during the contractions. Changes in relative amounts of tyrosine/threonine phosphorylated MAPK compared with total MAPK were measured. MAPK tyrosine phosphorylation levels increased in correlation with tension development. However, 50 μM PD-98059, a MAPK/ERK kinase-MAPK kinase blocker, reduced MAPK phosphorylation below resting levels, even though the magnitude of the isometric tension development was unaltered. Freeze-clamped PASM strips were placed in a PKC activity assay buffer containing32P and CaCl2to measure the total myelin basic protein phosphorylation. The data show that: 1) the time courses of PKC activity and force produced in response to H2O2do not correlate, and 2) MAPK activation may be a concurrent event with, or a consequence of, tension development in response to a variety of agonists but is not responsible for contractions to H2O2, high K+, or phorbol esters.
Involvement of protein kinase C, tyrosine kinases, and Rho kinase in Ca2+handling of human small arteries
Authors: Martínez M. Carmen , Randriamboavonjy Voahanginirina , Ohlmann Patrick , Komas Narcisse , Duarte Juan , Schneider Francis , Stoclet Jean-Claude , Andriantsitohaina Ramaroson
Published: December 2017

The mechanisms of Ca2+handling and sensitization were investigated in human small omental arteries exposed to norepinephrine (NE) and to the thromboxane A2analog U-46619. Contractions elicited by NE and U-46619 were associated with an increase in intracellular Ca2+concentration ([Ca2+]i), an increase in Ca2+-independent signaling pathways, or an enhancement of the sensitivity of the myofilaments to Ca2+. The two latter pathways were abolished by protein kinase C (PKC), tyrosine kinase (TK), and Rho-associated protein kinase (ROK) inhibitors. In Ca2+-free medium, both NE and U-46619 elicited an increase in tension that was greatly reduced by PKC inhibitors and abolished by caffeine or ryanodine. After depletion of Ca2+stores with NE and U-46619 in Ca2+-free medium, addition of CaCl2in the continuous presence of the agonists produced increases in [Ca2+]iand contractions that were inhibited by nitrendipine and TK inhibitors but not affected by PKC inhibitors. NE and U-46619 induced tyrosine phosphorylation of a 42- or a 58-kDa protein, respectively. These results indicate that the mechanisms leading to contraction elicited by NE and U-46619 in human small omental arteries are composed of Ca2+release from ryanodine-sensitive stores, Ca2+influx through nitrendipine-sensitive channels, and Ca2+sensitization and/or Ca2+-independent pathways. They also show that the TK pathway is involved in the tonic contraction associated with Ca2+entry, whereas TK, PKC, and ROK mechanisms regulate Ca2+-independent signaling pathways or Ca2+sensitization.
A direct requirement of nuclear factor-κB for suppression of apoptosis in ventricular myocytes
Authors: Mustapha Shareef , Kirshner Alla , De Moissac Danielle , Kirshenbaum Lorrie A.
Published: December 2017

Nuclear factor-κB (NF-κB) is a ubiquitously expressed cellular factor regulated by the cytoplasmic factor inhibitor protein κBα (IκBα). Activation of NF-κB by cytokines, including tumor necrosis factor-α (TNF-α), requires the phosphorylation and degradation of IκBα. An anti-apoptotic role for NF-κB has recently been suggested. In the present study, we ascertained whether death-promoting signals and apoptosis mediated by TNF-α are suppressed by NF-κB in postnatal ventricular myocytes. Stimulation of myocytes with TNF-α resulted in a 12.1-fold increase ( P < 0.01) in NF-κB-dependent gene transcription and DNA binding compared with controls. This was accompanied by a corresponding increase in the NF-κB target protein A20 as determined by Western blot analysis. Vital staining revealed that TNF-α was not cytotoxic to myocytes and did not provoke apoptosis. Adenovirus-mediated delivery of a nonphosphorylatable form of IκBα to inactivate NF-κB prevented TNF-α-stimulated NF-κB-dependent gene transcription and nuclear NF-κB DNA binding. Importantly, myocytes stimulated with TNF-α and defective for NF-κB activation resulted in a 2.2-fold increase ( P < 0.001) in apoptosis. To our knowledge, the data provide the first indication that a functional NF-κB signaling pathway is crucial for suppressing death-promoting signals mediated by TNF-α in ventricular myocytes.
TNF-α induced bronchial vasoconstriction
Authors: Wagner Elizabeth M.
Published: December 2017

The pro-inflammatory characteristics of tumor necrosis factor-α (TNF-α) have been extensively characterized in in vitro systems. Furthermore, this cytokine has been shown to play a pivotal role in airways inflammation in asthma. Since the airway vasculature also performs an essential function in inflammatory cell transit to the airways, experiments were performed to determine the effects of TNF-α on bronchial vascular resistance (BVR). In anesthetized, ventilated sheep, the bronchial artery (BA) was cannulated and perfused with autologous blood. BVR was defined as inflow pressure/flow and averaged 6.3 ± 0.2 mmHg · ml−1 · min−1 (±SE) for the 25 sheep studied. Recombinant human TNF-α (10 μg for 20 or 40 min) infused directly into the BA resulted in a significant decrease in BVR to 87% of baseline ( P < 0.05). This vasodilation was followed by a reversal of tone by 120 min and a sustained increase in BVR to 126% of baseline ( P < 0.05). Since others have shown TNF-α caused coronary vasoconstriction through endothelial release of endothelin-1 (ET-1), an ET-1 antagonist was used to block bronchial vasoconstriction. BQ-123, a selective ETA receptor antagonist, was delivered to the bronchial vasculature prior to TNF-α challenge. Attenuation of bronchial vasoconstriction was observed at 120 min ( P < 0.03). Thus TNF-α causes bronchial vasoconstriction by the secondary release of ET-1. Although TNF-α exerts pro-inflammatory actions on most cells of the airways, vasoactive properties of this cytokine likely further contribute to the inflammatory status of the airways.
Right and left ventricular wall deformation patterns in normal and left heart hypoplasia chick embryos
Authors: Tobita Kimimasa , Keller Bradley B.
Published: December 2017

The vertebrate embryonic ventricle transforms from a smooth-walled single tube to trabeculated right ventricular (RV) and left ventricular (LV) chambers during cardiovascular morphogenesis. We hypothesized that ventricular contraction patterns change from globally isotropic to chamber-specific anisotropic patterns during normal morphogenesis and that these deformation patterns are influenced by experimentally altered mechanical load produced by chronic left atrial ligation (LAL). We measured epicardial RV and LV wall strains during normal development and left heart hypoplasia produced by LAL in Hamburger-Hamilton stage 21, 24, 27, and 31 chick embryos. Normal RV contracted isotropically until stage 24 and then contracted preferentially in the circumferential direction. Normal LV contracted isotropically at stage 21, preferentially in the longitudinal direction at stages 24 and 27, and then in the circumferential direction at stage 31. LAL altered both RV and LV strain patterns, accelerated the onset of preferential RV circumferential strain patterns, and abolished preferential LV longitudinal strain ( P < 0.05 vs. normal). Mature patterns of anisotropic RV and LV deformation develop coincidentally with morphogenesis, and changes in these deformation patterns reflect altered cardiovascular function and/or morphogenesis.
cADP ribose and [Ca2+]iregulation in rat cardiac myocytes
Authors: Prakash Y. S. , Kannan Mathur S. , Walseth Timothy F. , Sieck Gary C.
Published: December 2017

cADP ribose (cADPR)-induced intracellular Ca2+concentration ([Ca2+]i) responses were assessed in acutely dissociated adult rat ventricular myocytes using real-time confocal microscopy. In quiescent single myocytes, injection of cADPR (0.1–10 μM) induced sustained, concentration-dependent [Ca2+]iresponses ranging from 50 to 500 nM, which were completely inhibited by 20 μM 8-amino-cADPR, a specific blocker of the cADPR receptor. In myocytes displaying spontaneous [Ca2+]iwaves, increasing concentrations of cADPR increased wave frequency up to ∼250% of control. In electrically paced myocytes (0.5 Hz, 5-ms duration), cADPR increased the amplitude of [Ca2+]itransients in a concentration-dependent fashion, up to 150% of control. Administration of 8-amino-cADPR inhibited both spontaneous waves as well as [Ca2+]iresponses to electrical stimulation, even in the absence of exogenous cADPR. However, subsequent [Ca2+]iresponses to 5 mM caffeine were only partially inhibited by 8-amino-cADPR. In contrast, even under conditions where ryanodine receptor (RyR) channels were blocked with ryanodine, high cADPR concentrations still induced an [Ca2+]iresponse. These results indicate that in cardiac myocytes, cADPR induces Ca2+release from the sarcoplasmic reticulum through both RyR channels and via mechanisms independent of RyR channels.
Matrix metalloproteinase activity is required for activity-induced angiogenesis in rat skeletal muscle
Authors: Haas T. L. , Milkiewicz M. , Davis S. J. , Zhou A. L. , Egginton S. , Brown M. D. , Madri J. A. , Hudlicka O.
Published: December 2017

Proteolysis of the capillary basement membrane is a hallmark of inflammation-mediated angiogenesis, but it is undetermined whether proteolysis plays a critical role in the process of activity-induced angiogenesis. Matrix metalloproteinases (MMPs) constitute the major class of proteases responsible for degradation of basement membrane proteins. We observed significant elevations of mRNA and protein levels of both MMP-2 and membrane type 1 (MT1)-MMP (2.9 ± 0.7- and 1.5 ± 0.1-fold above control, respectively) after 3 days of chronic electrical stimulation of rat skeletal muscle. Inhibition of MMP activity via the inhibitor GM-6001 prevented the growth of new capillaries as assessed by the capillary-to-fiber ratio (1.34 ± 0.08 in GM-6001-treated muscles compared with 1.69 ± 0.03 in control 7-day-stimulated muscles). This inhibition correlated with a significant reduction in the number of capillaries with observable breaks in the basement membrane, as assessed by electron microscopy (0.27 ± 0.27% in GM-6001-treated muscles compared with 3.72 ± 0.65% in control stimulated muscles). Proliferation of capillary-associated cells was significantly elevated by 2 days and remained elevated throughout 14 days of stimulation. Capillary-associated cell proliferation during muscle stimulation was not affected by MMP inhibition (80.3 ± 9.3 nuclei in control and 63.5 ± 8.5 nuclei in GM-6001-treated animals). We conclude that MMP proteolysis of capillary basement membrane proteins is a critical component of physiological angiogenesis, and we postulate that capillary-associated proliferation precedes and occurs independently of endothelial cell sprout formation.
VEGF and ATP act by different mechanisms to increase microvascular permeability and endothelial [Ca2+]i
Authors: Pocock T. M. , Williams B. , Curry F. E. , Bates D. O.
Published: December 2017

Vascular endothelial growth factor (VEGF) increases hydraulic conductivity ( Lp) by stimulating Ca2+influx into endothelial cells. To determine whether VEGF-mediated Ca2+influx is stimulated by release of Ca2+from intracellular stores, we measured the effect of Ca2+store depletion on VEGF-mediated increased Lpand endothelial intracellular Ca2+concentration ([Ca2+]i) of frog mesenteric microvessels. Inhibition of Ca2+influx by perfusion with NiCl2significantly attenuated VEGF-mediated increased [Ca2+]i. Depletion of Ca2+stores by perfusion of vessels with thapsigargin did not affect the VEGF-mediated increased [Ca2+]ior the increase in Lp. In contrast, ATP-mediated increases in both [Ca2+]iand Lpwere inhibited by thapsigargin perfusion, demonstrating that ATP stimulated store-mediated Ca2+influx. VEGF also increased Mn2+influx after perfusion with thapsigargin, whereas ATP did not. These data showed that VEGF increased [Ca2+]iand Lpeven when Ca2+stores were depleted and under conditions that prevented ATP-mediated increases in [Ca2+]iand Lp. This suggests that VEGF acts through a Ca2+store-independent mechanism, whereas ATP acts through Ca2+store-mediated Ca2+influx.
Significance of ERK cascade compared with JAK/STAT and PI3-K pathway in gp130-mediated cardiac hypertrophy
Authors: Kodama Hiroaki , Fukuda Keiichi , Pan Jing , Sano Motoaki , Takahashi Toshiyuki , Kato Takahiro , Makino Shinji , Manabe Tomohiro , Murata Mitsushige , Ogawa Satoshi
Published: December 2017

We compared the role of the Raf-1/mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MEK)/extracellular signal-regulated protein kinase (ERK)/p90RSKcascade in gp130-mediated cardiac hypertrophy with the contribution of the Janus kinase (JAK)/signal transduction and activation of transcription (STAT) and phosphatidylinositide 3-kinase (PI3-K) pathways. Primary cultured neonatal rat cardiomyocytes were stimulated with leukemia inhibitory factor (LIF). LIF sequentially activated Raf-1, MEK1/2, ERK1/2, and p90RSK. We used PD-98059 (a specific MEK inhibitor), AG-490 (a JAK2 inhibitor), and wortmannin (a PI3-K inhibitor) to confirm that this cascade was independent of the JAK/STAT and PI3-K/p70 S6 kinase (S6K) pathways. PD-98059, AG-490, and wortmannin suppressed the LIF-induced increase in [3H]phenylalanine uptake by 54.7, 21.5, and 25.6%, respectively, and inhibited the increase in cell area by 61.2, 42.8, and 39.2%, respectively. Reorganization of myofilaments was predominantly suppressed by AG-490. LIF-induced expression of c- fos, brain natriuretic peptide, and skeletal α-actin mRNA was markedly suppressed by PD-98059 and moderately suppressed by wortmannin and AG-490. Atrial natriuretic peptide was significantly suppressed by AG-490. These findings indicate that this pathway is critically involved in protein synthesis, induction of c- fos, brain natriuretic peptide, and skeletal α-actin expression and is partially involved in myofilament reorganization and atrial natriuretic peptide induction in gp130-mediated cardiac hypertrophy.
Protein washdown as a defense mechanism against myocardial edema
Authors: Stewart Randolph H. , Geissler Hans J. , Allen Steven J. , Laine Glen A.
Published: December 2017

Myocardial edema occurs in many pathological conditions. We hypothesized that protein washdown at the myocardial microvascular exchange barrier would change the distribution of interstitial proteins from large to small molecules and diminish the effect of washdown on the colloid osmotic pressure (COP) of interstitial fluid and lymph. Dogs were instrumented with coronary sinus balloon-tipped catheters and myocardial lymphatic cannulas to manipulate myocardial lymph flow and to collect lymph. Myocardial venous pressure was elevated by balloon inflation to increase transmicrovascular fluid flux and myocardial lymph flow. COP of lymph was measured directly and was also calculated from protein concentration. Decreases occurred in both protein concentration and COP of lymph. The proportion of lymph protein accounted for by albumin increased significantly, whereas that accounted for by β-lipoprotein decreased significantly. The change in the calculated plasma-to-lymph COP gradient was significantly greater than the change in the measured COP gradient. We conclude that the change in the distribution of interstitial fluid protein species decreases the effect of protein washdown on interstitial fluid COP and limits its effectiveness as a defense mechanism against myocardial edema formation.
Diaspirin cross-linked Hb and norepinephrine prevent the sepsis-induced increase in critical O2delivery
Authors: Sielenkämper Andreas W. , Yu Pei , Eichelbrönner Otto , MacDonald Tammy , Martin Claudio M. , Chin-Yee Ian H. , Sibbald William J.
Published: December 2017

We hypothesized that support of arterial perfusion pressure with diaspirin cross-linked Hb (DCLHb) would prevent the sepsis-induced attenuation in the systemic O2delivery-O2uptake relationship. Awake septic rats were treated with a chronic infusion of DCLHb or a reference treatment [norepinephrine (NE)] to increase mean arterial pressure by 10–20% over 18 h. Septic and sham control groups received normal saline. Isovolemic hemodilution to create anemic hypoxia was then performed in a metabolic box during continuous measurement of systemic O2uptake. O2delivery was calculated from hemodynamic variables, and the critical point of O2delivery (D˙o2 crit) was determined using piecewise regression analysis of the O2delivery-O2uptake relationship. Sepsis increasedD˙o2 critfrom 4.99 ± 0.17 to 6.69 ± 0.42 ml · min−1· 100 g−1( P < 0.01), while O2extraction capacity was decreased ( P < 0.05). DCLHb and NE infusion prevented the sepsis-induced increase inD˙o2 crit[4.56 ± 0.42 ml · min−1· 100 g−1( P < 0.01) and 5.04 ± 0.56 ml · min−1· 100 g−1( P < 0.05), respectively]. This was explained by a 59% increase in O2extraction capacity in the DCLHb group compared with septic controls ( P < 0.05), whereas NE treatment decreased systemic O2uptake in anemic hypoxia (1.51 ± 0.08 vs. 1.87 ± 0.1 ml · min−1· 100 g−1in septic controls, P < 0.05). We conclude that DCLHb ameliorated O2extraction capacity in the septic microcirculation, whereas NE decreased the metabolic demands of the tissues.
Conservation of phosphorylation state of cardiac phosphofructokinase during in vitro hypothermic hypoxia
Authors: Pulis Randy P. , Wu Beatrice M. , Kneteman Norman M. , Churchill Thomas A.
Published: December 2017

We investigated the metabolic effects of buffering agents α-amino-4-imidazole-propionic acid (Histidine), N,N-bis(2-hydroxyethyl)glycine (bicine), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) on anaerobic energy production (via glycolysis) and conservation of key regulatory enzyme activity, and phosphofructokinase (PFK) throughout prolonged hypothermic hypoxia in porcine hearts. Hearts from 35 to 40 kg pigs were flushed with one of the following five solutions: St. Thomas' Hospital solution (STHS); modified University of Wisconsin (UW) solution; and three solutions containing modified UW plus 90 mM of histidine, bicine, or BES. The hearts were then stored at 4°C for 10 h. After 10 h of hypothermic hypoxia, lactate values were 6.7–12.9 μmol/g higher than control; this reflected an increase in anaerobic end product of 35–67%. The consequences of enhanced anaerobic metabolism were higher ATP, total adenylate, Energy Charge, and ATP/ADP ratios in most of the buffered groups after 4–10 h cold storage; effectiveness of the buffers employed correlated with buffering capacity (BES proved to be the most effective). PFK remained activated throughout most of the 10-h period in hearts stored with buffers and did not undergo the rapid inactivation experienced by hearts stored in STHS. Conservation of PFK integrity with buffering agents was not related to a pH-mediated event; changes in kinetic parameters suggested that this protection was due to an irreversible posttranslational modification, specifically a dephosphorylation event.
Expression and self-regulatory function of cardiac interleukin-6 during endotoxemia
Authors: Saito Hiroshi , Patterson Cam , Hu Zhaoyong , Runge Marschall S. , Tipnis Ulka , Sinha Mala , Papaconstantinou John
Published: December 2017

Interleukin (IL)-6 reportedly has negative inotropic and hypertrophic effects on the heart. Here, we describe endotoxin-induced IL-6 in the heart that has not previously been well characterized. An intraperitoneal injection of a bacterial lipopolysaccharide into C57BL/6 mice induced IL-6 mRNA in the heart more strongly than in any other tissue examined. Induction of mRNA for two proinflammatory cytokines, IL-1β and tumor necrosis factor (TNF)-α, occurred rapidly before the induction of IL-6 mRNA and protein. Although stimulation of isolated rat neonatal myocardial cells with IL-1β or TNF-α induced IL-6 mRNA in vitro, nonmyocardial heart cells produced higher levels of IL-6 mRNA upon stimulation with IL-1β. In situ hybridization and immunohistochemical analyses localized the IL-6 expression primarily in nonmyocardial cells in vivo. Endotoxin-induced expression of cardiac IL-1β, TNF-α, and intercellular adhesion molecule 1 was augmented in IL-6-deficient mice compared with control mice. Thus cardiac IL-6, expressed mainly by nonmyocardial cells via IL-1β action during endotoxemia, is likely to suppress expression of proinflammatory mediators and to regulate itself via a negative feedback mechanism.