bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein kinase C-ε-null mice have decreased hypoxic pulmonary vasoconstriction
Authors: Littler Cassana M. , Morris Kenneth G. , Fagan Karen A. , McMurtry Ivan F. , Messing Robert O. , Dempsey Edward C.
Published: March 2015

PKC contributes to regulation of pulmonary vascular reactivity in response to hypoxia. The role of individual PKC isozymes is less clear. We used a knockout (null, −/−) mouse to test the hypothesis that PKC-ε is important in acute hypoxic pulmonary vasoconstriction (HPV). We asked whether deletion of PKC-ε would decrease acute HPV in adult C57BL6×SV129 mice. In isolated, salt solution-perfused lung, reactivity to acute hypoxic challenges (0% and 3% O2) was compared with responses to angiotensin II (ANG II) and KCl. PKC-ε −/− mice had decreased HPV, whereas responses to ANG II and KCl were preserved. Inhibition of nitric oxide synthase (NOS) with nitro-l-arginine augmented HPV in PKC-ε +/+ but not −/− mice. Inhibition of Ca2+-gated K+ channels (KCa) with charybdotoxin and apamin did not enhance HPV in −/− mice relative to wild-type (+/+) controls. In contrast, the voltage-gated K+channel (KV) antagonist 4-aminopyridine increased the response of −/− mice beyond that of +/+ mice. This suggested that increased KV channel expression could contribute to blunted HPV in PKC-ε −/− mice. Therefore, expression of the O2-sensitive KV channel subunit Kv3.1b (100-kDa glycosylated form and 70-kDa core protein) was compared in whole lung and pulmonary artery smooth muscle cell (PASMC) lysates from +/+ and −/− mice. A subtle increase in Kv3.1b was detected in −/− vs. +/+ whole lung lysates. A much greater rise in Kv3.1b expression was found in −/− vs. +/+ PASMC. Thus deletion of PKC-ε blunts murine HPV. The decreased response could not be attributed to a general loss in vasoreactivity or derangements in NOS or KCa channel activity. Instead, the absence of PKC-ε allows increased expression of KV channels (like Kv3.1b) to occur in PASMC, which likely contributes to decreased HPV.
Vascular smooth muscle cell membrane depolarization after NOS inhibition hypertension
Authors: Bratz Ian N. , Falcon Ricardo , Partridge L. Donald , Kanagy Nancy L.
Published: March 2015

Nitric oxide (NO) synthase (NOS) inhibition with N ω-nitro-l-arginine (l-NNA) produces l-NNA hypertensive rats (LHR), which exhibit increased sensitivity to voltage-dependent Ca2+ channel-mediated vasoconstriction. We hypothesized that enhanced contractile responsiveness after NOS inhibition is mediated by depolarization of membrane potential ( E m) through attenuated K+ channel conductance. E m measurements demonstrated that LHR vascular smooth muscle cells (VSMCs) are depolarized in open, nonpressurized (−44.5 ± 1.0 mV in control vs. −36.8 ± 0.8 mV in LHR) and pressurized mesenteric artery segments (−41.8 ± 1.0 mV in control vs. −32.6 ± 1.4 mV in LHR). Endothelium removal or exogenous l-NNA depolarized control VSMCs but not LHR VSMCs. Superfused l-arginine hyperpolarized VSMCs from both the control and LHR groups and reversedl-NNA-induced depolarization (−44.5 ± 1.0 vs. −45.8 ± 2.1 mV). A Ca2+-activated K+channel agonist, NS-1619 (10 μM), hyperpolarized both groups of arteries to a similar extent (from −50.8 ± 1.0 to −62.5 ± 1.2 mV in control and from −43.7 ± 1.1 to −55.6 ± 1.2 mV in LHR), although E m was still different in the presence of NS-1619. In addition, superfused iberiotoxin (50 nM) depolarized both groups similarly. Increasing the extracellular K+ concentration from 1.2 to 45 mM depolarized E m, as predicted by the Goldman-Hodgkin-Katz equation. These data support the hypothesis that loss of NO activation of K+ channels contributes to VSMC depolarization inl-NNA-induced hypertension without a change in the number of functional large conductance Ca2+-activated K+ channels.
Decreased association of HSP90 impairs endothelial nitric oxide synthase in fetal lambs with persistent pulmonary hypertension
Authors: Konduri Girija G. , Ou Jingsong , Shi Yang , Pritchard Kirkwood A.
Published: March 2015

Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased nitric oxide (NO) release and impaired pulmonary vasodilation. We investigated the hypothesis that decreased association of heat shock protein 90 (HSP90) with endothelial NO synthase (eNOS) impairs NO release and vasodilation in PPHN. The responses to the NOS agonist ATP were investigated in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus, and in sham ligation controls. ATP caused dose-dependent vasodilation in control pulmonary resistance arteries, and this response was attenuated in PPHN vessels. The response of control pulmonary arteries to ATP was attenuated by NG-nitro-l-arginine methyl ester (l-NAME), a NOS antagonist, and geldanamycin, an inhibitor of HSP90-eNOS interaction. The attenuated response to ATP observed in PPHN was improved by pretreatment of vessels with l-NAME or 4,5-dihydroxy-1,3-benzene-disulfonate, a superoxide scavenger. Pulmonary arteries from PPHN lambs had decreased basal levels of HSP90 in association with eNOS. Association of HSP90 with eNOS and NO release increased in response to ATP in control pulmonary artery endothelial cells, but not in cells from PPHN lambs. Decreased HSP90-eNOS interactions may contribute to the impaired NO release and vasodilation observed in the ductal ligation model of PPHN.
Interaction between nitric oxide and prostanoids in arterioles of rat cremaster muscle in vivo
Authors: Laemmel E. , Bonnardel-Phu E. , Hou X. , Seror J. , Vicaut E.
Published: March 2015

We studied in vivo interactions of nitric oxide (NO), oxidative stress, and prostanoids derived from the cyclooxygenase pathway in the arterioles studied by intravital microscopy in peripheral muscle. Topical administration of NO synthase (NOS) inhibitor Nω-nitro-l-arginine (l-NNA) or cyclooxygenase inhibitor mefenamic acid (MA) alone leads to vasoconstriction. We found that l-NNA after MA induced an additional constriction, whereas MA after l-NNA induced a relative dilation. Therefore, an additional constriction was found when MA was administered after l-NNA in the presence of the thromboxane A2synthase-PGH2(TP) receptor antagonist SQ-29548. We also found a relative dilation when the TP receptor antagonist was administered after NOS inhibition by l-NNA. In the presence of superoxide dismutase and catalase, l-NNA-induced vasoconstriction is reduced, and the dilation observed after addition of MA in presence of the reactive oxygen species is no longer present. Taken together, these results showed that NO inhibition induced a shift in the synthesis or in the effects of cyclooxygenase products, in favor of constrictor prostanoids. This effect of NO inhibition disappears when reactive oxygen species are scavenged by superoxide dismutase and catalase.
Cortisol-mediated potentiation of uterine artery contractility: effect of pregnancy
Authors: Xiao Daliao , Huang Xiaohui , Bae Soochan , Ducsay Charles A. , Zhang Lubo
Published: March 2015

During pregnancy, maternal plasma cortisol concentrations approximately double. Given that cortisol plays an important role in the regulation of vascular reactivity, the present study investigated the potential role of cortisol in potentiation of uterine artery (UA) contractility and tested the hypothesis that pregnancy downregulated the cortisol-mediated potentiation. In vitro cortisol treatment (3, 10, or 30 ng/ml for 24 h) produced a dose-dependent increase in norepinephrine (NE)-induced contractions in both nonpregnant and pregnant (138–143 days gestation) sheep UA. However, this cortisol-mediated response was significantly attenuated by ∼50% in pregnant UA. The 11β-hydroxysteroid dehydrogenase (11-βHSD) inhibitor carbenoxolone did not change the effect of cortisol in nonpregnant UA but abolished its effect in pregnant UA by increasing the NE pD2 in control tissues from 6.20 ± 0.05 to 6.59 ± 0.11. The apparent dissociation constant value of NE α1-adrenoceptors was not changed by cortisol in pregnant UA but was decreased in nonpregnant UA. There was no difference in glucocorticoid receptor density between nonpregnant and pregnant UA. Cortisol significantly decreased endothelial nitric oxide (NO) synthase protein levels and NO release in both nonpregnant and pregnant UA, but the effect of cortisol was attenuated in pregnant UA by ∼50%. Carbenoxolone alone had no effects on NO release in nonpregnant UA but was decreased in pregnant UA. These results suggest that cortisol potentiates NE-mediated contractions by decreasing NO release and increasing NE-binding affinity to α1-adrenoceptors in nonpregnant UA. Pregnancy attenuates UA sensitivity to cortisol, which may be mediated by increasing type-2 11-βHSD activity in UA.
Targeting nucleotide-requiring enzymes: implications for diazoxide-induced cardioprotection
Authors: Dzeja Petras P. , Bast Peter , Ozcan Cevher , Valverde Arturo , Holmuhamedov Ekshon L. , Van Wylen David G. L. , Terzic Andre
Published: March 2015

Modulation of mitochondrial respiratory chain, dehydrogenase, and nucleotide-metabolizing enzyme activities is fundamental to cellular protection. Here, we demonstrate that the potassium channel opener diazoxide, within its cardioprotective concentration range, modulated the activity of flavin adenine dinucleotide-dependent succinate dehydrogenase with an IC50of 32 μM and reduced the rate of succinate-supported generation of reactive oxygen species (ROS) in heart mitochondria. 5-Hydroxydecanoic fatty acid circumvented diazoxide-inhibited succinate dehydrogenase-driven electron flow, indicating a metabolism-dependent supply of redox equivalents to the respiratory chain. In perfused rat hearts, diazoxide diminished the generation of malondialdehyde, a marker of oxidative stress, which, however, increased on diazoxide washout. This effect of diazoxide mimicked ischemic preconditioning and was associated with reduced oxidative damage on ischemia-reperfusion. Diazoxide reduced cellular and mitochondrial ATPase activities, along with nucleotide degradation, contributing to preservation of myocardial ATP levels during ischemia. Thus, by targeting nucleotide-requiring enzymes, particularly mitochondrial succinate dehydrogenase and cellular ATPases, diazoxide reduces ROS generation and nucleotide degradation, resulting in preservation of myocardial energetics under stress.
Endothelial contraction and monolayer hyperpermeability are regulated by Src kinase
Authors: Mucha David R. , Myers Carter L. , Schaeffer Richard C.
Published: March 2015

Endothelial monolayer hyperpermeability is regulated by a myosin light chain phosphorylation (MLCP)-dependent contractile mechanism. In this study, we tested the role of Src-dependent tyrosine phosphorylation to modulate endothelial contraction and monolayer barrier function with the use of the myosin phosphatase inhibitor calyculin A (CalA) to directly elevate MLCP with the Src family tyrosine kinase inhibitor herbimycin A (HA) in bovine pulmonary artery endothelial cells (EC). CalA stimulated an increase in MLCP, Src kinase activity, an increase in the tyrosine phosphorylation of paxillin and focal adhesion (FA) kinase (p125FAK), and monolayer hyperpermeability. Microscopic examination of CalA-treated EC revealed a contractile morphology characterized by peripheral contractile bands of actomyosin filaments and stress fibers linked to phosphotyrosine-containing FAs. These CalA-dependent events were HA sensitive. HA alone stimulated an improvement in monolayer barrier formation by reducing the levels of MLCP and phosphotyrosine-containing proteins and the number of large paracellular holes. These data show that Src kinase plays an important role in regulating monolayer hyperpermeability through adjustments in tyrosine phosphorylation, MLCP, and EC contraction.
Sodium restriction prevents cardiac hypertrophy and oxidative stress in angiotensin II hypertension
Authors: Rugale Caroline , Delbosc Sandrine , Cristol Jean-Paul , Mimran Albert , Jover Bernard
Published: March 2015

The influence of a low-sodium (LS) diet was assessed on the cardiac and renal alterations and pro-oxidant effect associated with a 10-day infusion of angiotensin II (200 or 400 ng · kg−1· min−1, osmotic pumps). Tail-cuff pressure (TCP), albuminuria, and renal blood flow were determined at the end of the experiments. Heart weight index (HWI) and production of superoxide anion (O[Formula: see text]·) by the left ventricle and H2O2by the aorta was measured with the use of bioluminescence. Although the final TCP was similar in LS and normal sodium (NS) rats infused with high and low doses of angiotensin II, respectively, the increase in HWI was prevented by the LS diet. Sodium restriction reduced the rise in albuminuria without a change in the renal effect of angiotensin II. The increased production of O[Formula: see text]· and H2O2observed in NS rats was abrogated in LS rats. The beneficial influence of dietary sodium restriction on target organ damage induced by angiotensin II is independent of arterial pressure reduction and possibly related to attenuation of the prooxidant effect of the peptide.
Spatial velocity profile in mouse embryonic aorta and Doppler-derived volumetric flow: a preliminary model
Authors: Phoon Colin K. L. , Aristizábal Orlando , Turnbull Daniel H.
Published: March 2015

Characterizing embryonic circulatory physiology requires accurate cardiac output and flow data. Despite recent applications of high-frequency ultrasound Doppler to the study of embryonic circulation, current Doppler analysis of volumetric flow is relatively crude. To improve Doppler derivation of volumetric flow, we sought a preliminary model of the spatial velocity profile in the mouse embryonic dorsal aorta using ultrasound biomicroscopy (UBM)-Doppler data. Embryonic hematocrit is 0.05–0.10 so rheologic properties must be insignificant. Low Reynolds numbers (<500) and Womersley parameters (<0.76) suggest laminar flow. UBM demonstrated a circular dorsal aortic cross section with no significant tapering. Low Dean numbers (<100) suggest the presence of minimal skewing of the spatial velocity profile. The inlet length allows for fully developed flow. There is no apparent aortic wall pulsatility. Extrapolation of prior studies to these vessel diameters (300–350 μm) and flow velocities (∼50–200 mm/s) suggests parabolic spatial velocity profiles. Therefore, mouse embryonic dorsal aortic blood flow may correspond to Poiseuille flow in a straight rigid tube with parabolic spatial velocity profiles. As a first approximation, these results are an important step toward precise in utero ultrasound characterization of blood flow within the developing mammalian circulation.
Relative contribution of vasodilator prostanoids, NO, and KATP channels to human forearm metabolic vasodilation
Authors: Farouque H. M. Omar , Meredith Ian T.
Published: March 2015

Isolated ATP-sensitive K+(KATP) channel inhibition with glibenclamide does not alter exercise-induced forearm metabolic vasodilation. Whether forearm metabolic vasodilation would be influenced by KATP channel inhibition in the setting of impaired nitric oxide (NO)- and prostanoid-mediated vasodilation is unknown. Thirty-seven healthy subjects were recruited. Forearm blood flow (FBF) was assessed using venous occlusion plethysmography, and functional hyperemic blood flow (FHBF) was induced by isotonic wrist exercise. Infusion of N G-monomethyl-l-arginine(l-NMMA), aspirin, or the combination reduced resting FBF compared with vehicle ( P < 0.05). Addition of glibenclamide to l-NMMA, aspirin, or the combination did not further reduce resting FBF. l-NMMA decreased peak FHBF by 26%, and volume was restored after 5 min ( P < 0.05). Aspirin reduced peak FHBF by 13%, and volume repaid after 5 min ( P < 0.05). Coinfusion of l-NMMA and aspirin reduced peak FHBF by 21% ( P < 0.01), and volume was restored after 5 min ( P < 0.05). Addition of glibenclamide to l-NMMA and aspirin did not further decrease FHBF. Vascular KATP channel blockade with glibenclamide does not affect resting FBF or FHBF in the setting of NO and vasodilator prostanoid inhibition.
Respiratory sinus arrhythmia is associated with efficiency of pulmonary gas exchange in healthy humans
Authors: Giardino Nicholas D. , Glenny Robb W. , Borson Soo , Chan Leighton
Published: March 2015

Respiratory sinus arrhythmia (RSA) may be associated with improved efficiency of pulmonary gas exchange by matching ventilation to perfusion within each respiratory cycle. Respiration rate, tidal volume, minute ventilation (V˙e), exhaled carbon dioxide (V˙co 2), oxygen consumption (V˙o 2), and heart rate were measured in 10 healthy human volunteers during paced breathing to test the hypothesis that RSA contributes to pulmonary gas exchange efficiency. Cross-spectral analysis of heart rate and respiration was computed to calculate RSA and the coherence and phase between these variables. Pulmonary gas exchange efficiency was measured as the average ventilatory equivalent of CO2(V˙e/V˙co 2) and O2(V˙e/V˙o 2). Across subjects and paced breathing periods, RSA was significantly associated with CO2 (partial r = −0.53, P = 0.002) and O2 (partial r = −0.49, P = 0.005) exchange efficiency after controlling for the effects of age, respiration rate, tidal volume, and average heart rate. Phase between heart rate and respiration was significantly associated with CO2 exchange efficiency (partial r = 0.40, P = 0.03). These results are consistent with previous studies and further support the theory that RSA may improve the efficiency of pulmonary gas exchange.
Dobutamine responsiveness, PET mismatch, and lack of necrosis in low-flow ischemia: is this hibernation in the isolated rat heart?
Authors: Southworth Richard , Garlick Pamela B.
Published: March 2015

The clinical hallmarks of hibernating myocardium include hypocontractility while retaining an inotropic reserve (using dobutamine echocardiography), having normal or increased [18F]fluoro-2-deoxyglucose-6-phosphate (18FDG6P) accumulation associated with decreased coronary flow [flow-metabolism mismatch by positron emission tomography (PET)], and recovering completely postrevascularization. In this study, we investigated an isolated rat heart model of hibernation using experimental equivalents of these clinical techniques. Rat hearts ( n = 5 hearts/group) were perfused with Krebs-Henseleit buffer for 40 min at 100% flow and 3 h at 10% flow and reperfused at 100% flow for 30 min (paced at 300 beats/min throughout). Left ventricular developed pressure fell to 30 ± 8% during 10% flow and recovered to 90 ± 7% after reperfusion. In an additional group, this recovery of function was found to be preserved over 2 h of reperfusion. Electron microscopic examination of hearts fixed at the end of the hibernation period demonstrated a lack of ischemic injury and an accumulation of glycogen granules, a phenomenon observed clinically. In a further group, hearts were challenged with dobutamine during the low-flow period. Hearts demonstrated an inotropic reserve at the expense of increased lactate leakage, with no appreciable creatine kinase release. PET studies used the same basic protocol in both dual- and globally perfused hearts (with 250MBq18FDG in Krebs buffer ± 0.4 mmol/l oleate). PET data showed flow-metabolism “mismatch;” whether regional or global,18FDG6P accumulation in ischemic tissue was the same as (glucose only) or significantly higher than (glucose + oleate) control tissue (0.023 ± 0.002 vs. 0.011 ± 0.002 normalized counts · s-1· g-1· min-1, P < 0.05) despite receiving 10% of the flow. This isolated rat heart model of acute hibernation exhibits many of the same characteristics demonstrated clinically in hibernating myocardium.
Role of c-Src in regulation of endothelial nitric oxide synthase expression during exercise training
Authors: Davis Michael E. , Cai Hua , McCann Louise , Fukai Tohru , Harrison David G.
Published: March 2015

We have shown that c-Src plays a role in shear stress stimulation of endothelial nitric oxide synthase (eNOS) expression in cultured cells. To examine the role of c-Src in vivo, we exercised C57Blk/6 and c-Src heterozygous (c-Src+/−) mice on a treadmill for 3 wk. Western analysis demonstrated that c-Src+/− mice express less than one-half the normal amount of c-Src. Exercise increased heart rate and blood pressure to identical levels in both strains as determined using radiotelemetry. Exercise training increased eNOS protein >2-fold in the aorta and 1.7-fold in the heart in C57Blk/6 mice but had no effect on eNOS protein levels in c-Src+/− mice. In contrast to exercise, treatment of mice with mevastatin, which stimulates expression of eNOS posttranscriptionally, increased eNOS protein in both strains. Training also increased aortic extracellular superoxide dismutase protein expression, which is regulated by nitric oxide, in C57Blk/6 mice but not in c-Src+/−mice. These data indicate that c-Src has an important role in modulating vascular adaptations to exercise training, in particular increasing eNOS and extracellular superoxide dismutase protein expression.
Del1 mediates VSMC adhesion, migration, and proliferation through interaction with integrin αvβ3
Authors: Rezaee Mehrdad , Penta Kalyani , Quertermous Thomas
Published: March 2015

Del1 is a matrix protein transiently expressed by embryonic endothelial cells. It was recently demonstrated that vascular endothelial cells adhere and interact with Del1 through αvβ3- integrins, providing an autocrine angiogenic signaling pathway in this cell type. To determine whether Del1 might signal to other cell types in the vessel wall in a paracrine fashion, studies were conducted with vascular smooth muscle cells (VSMC). Del1 promoted adhesion and migration of VSMC in a dose-dependent fashion. These functions were mediated through αvβ3-integrins, as the vitronectin receptor inhibitory peptide containing penacillamine (PCN) arginine-glycine-aspartic acid (PCN-RGD) and an antibody specific for the αvβ3-integrin specifically blocked both adhesion and migration. Adhesion of VSMC to Del1 was associated with organization of actin filaments and formation of focal contacts enriched in vinculin and αvβ3. Furthermore, Del1 supported VSMC proliferation at least in part by inhibiting these cells from undergoing apoptosis. These data, in conjunction with evidence that Del1 expression is reactivated in vascular injury, suggest that Del1 may have a paracrine role in vessel wall development and remodeling.
Mechanosensitive release of parathyroid hormone-related peptide from coronary endothelial cells
Authors: Degenhardt Heike , Jansen Johanna , Schulz Rainer , Sedding Daniel , Braun-Dullaeus Ruediger , Schlüter Klaus-Dieter
Published: March 2015

10.1152/ajpheart.00925. 2001.—Parathyroid hormone-related peptide (PTHrP) is expressed throughout the cardiovascular system and is able to dilate vessels. This study investigated whether mechanical forces generated by changes in regional perfusion influence PTHrP release from the coronary vascular bed. Experiments were performed in vitro on saline-perfused rat hearts or isolated coronary endothelial cells exposed to cyclic strain and in vivo in anesthetized pigs. In vitro, PTHrP release from saline-perfused rat hearts was strongly correlated with coronary flow ( r = 0.84). Increasing coronary flow from 5 to 10 ml/min increased PTHrP release from 442 ± 42 to 1,563 ± 167 pg/min. Increasing the viscosity of the perfusate did not change basal PTHrP release. Increasing flow without a concomitant increase in pressure did not lead to an increase in release rate, but reduction in pressure under flow-constant conditions reduced PTHrP release rate. Cyclic strain induced a strain-dependent release of PTHrP from endothelial cells that was inhibited by the addition of a calcium-chelating agent. In vivo, there was a net release of PTHrP in the coronary circulation and decreases in coronary flow and pressure decreased the PTHrP release rate. Bradykinin in the presence of constant pressure increased PTHrP release, probably by increasing the intracellular calcium concentration in coronary endothelial cells. In summary, mechanical forces evoked by blood flow can trigger a constant PTHrP release.
Cardioprotective effect of ischemic preconditioning is preserved in food-restricted senescent rats
Authors: Abete Pasquale , Testa Gianluca , Ferrara Nicola , De Santis Domenico , Capaccio Pina , Viati Luisa , Calabrese Claudio , Cacciatore Francesco , Longobardi Giancarlo , Condorelli Mario , Napoli Claudio , Rengo Franco
Published: March 2015

Ischemic preconditioning (PC) has been proposed as an endogenous form of protection against-ischemia reperfusion injury. We have shown that PC does not prevent postischemic dysfunction in the aging heart. This phenomenon could be due to the reduction of cardiac norepinephrine release, and it has also been previously demonstrated that age-related decrease of norepinephrine release from cardiac adrenergic nerves may be restored by caloric restriction. We investigated the effects on mechanical parameters of PC against 20 min of global ischemia followed by 40 min of reperfusion in isolated hearts from adult (6 mo) and “ad libitum”-fed and food-restricted senescent (24 mo) rats. Norepinephrine release in coronary effluent was determined by high-performance liquid chromatography. Final recovery of percent developed pressure was significantly improved after PC in adult hearts versus unconditioned controls (85.2 ± 19% vs. 51.5 ± 10%, P < 0.01). The effect of PC on developed pressure recovery was absent in ad libitum-fed rats, but it was restored in food-restricted senescent hearts (66.6 ± 13% vs. 38.3 ± 11%, P < 0.05). Accordingly, norepinephrine release significantly increased after PC in both adult and in food-restricted senescent hearts, and depletion of myocardial norepinephrine stores by reserpine abolished the PC effect in both adult and in food-restricted senescent hearts. We conclude that PC reduces postischemic dysfunction in the hearts from adult and food-restricted but not in ad libitum-fed senescent rats. Despite the possibility of multiple age-related mechanisms, the protection afforded by PC was correlated with increased norepinephrine release, and it was blocked by reserpine in both adult and food-restricted senescent hearts. Thus caloric restriction may restore PC in the aging heart probably via increased norepinephrine release.
Altered function in atrium of transgenic mice overexpressing triadin 1
Authors: Kirchhefer Uwe , Baba Hideo A. , Kobayashi Yvonne M. , Jones Larry R. , Schmitz Wilhelm , Neumann Joachim
Published: March 2015

Triadin 1 is a protein in the cardiac junctional sarcoplasmic reticulum (SR) that interacts with the ryanodine receptor, junctin, and calsequestrin, proteins that are important for Ca2+release. To better understand the role of triadin 1 in SR-Ca2+release, we studied the time-dependent expression of SR proteins and contractility in atria of 3-, 6-, and 18-wk-old transgenic mice overexpressing canine cardiac triadin 1 under control of the α-myosin heavy chain (MHC) promoter. Three-week-old transgenic atria exhibited mild hypertrophy. Finally, atrial weight was increased by 110% in 18-wk-old transgenic mice. Triadin 1 overexpression was accompanied by time-dependent changes in the protein expression of the ryanodine receptor, junctin, and cardiac/slow-twitch muscle SR Ca2+-ATPase isoform. Force of contraction was already decreased in 3-wk-old transgenic atria. The application of caffeine led to a positive inotropic effect in transgenic atria of 3-wk-old mice. Rest pauses resulted in an increased potentiation of force of contraction after restimulation in 3- and 6-wk-old mice and a reduced potentiation of force of contraction in 18-wk-old transgenic mice. Hence, triadin 1 overexpression triggered time-dependent alterations in SR protein expression, Ca2+homeostasis, and contractility, indicating for the first time an inhibitory function of triadin 1 on SR-Ca2+release in vivo.
Vitamin C prevents hyperoxia-mediated vasoconstriction and impairment of endothelium-dependent vasodilation
Authors: Mak Susanna , Egri Zoltan , Tanna Gemini , Colman Rebecca , Newton Gary E.
Published: March 2015

High arterial blood oxygen tension increases vascular resistance, possibly related to an interaction between reactive oxygen species and endothelium-derived vasoactive factors. Vitamin C is a potent antioxidant capable of reversing endothelial dysfunction due to increased oxidant stress. We tested the hypotheses that hyperoxic vasoconstriction would be prevented by vitamin C, and that acetylcholine-mediated vasodilation would be blunted by hyperoxia and restored by vitamin C. Venous occlusion strain gauge plethysmography was used to measure forearm blood flow (FBF) in 11 healthy subjects and 15 congestive heart failure (CHF) patients, a population characterized by endothelial dysfunction and oxidative stress. The effect of hyperoxia on FBF and derived forearm vascular resistance (FVR) at rest and in response to intra-arterial acetylcholine was recorded. In both healthy subjects and CHF patients, hyperoxia-mediated increases in basal FVR were prevented by the coinfusion of vitamin C. In healthy subjects, hyperoxia impaired the acetylcholine-mediated increase in FBF, an effect also prevented by vitamin C. In contrast, hyperoxia had no effect on verapamil-mediated increases in FBF. In CHF patients, hyperoxia did not affect FBF responses to acetylcholine or verapamil. The addition of vitamin C during hyperoxia augmented FBF responses to acetylcholine. These results suggest that hyperoxic vasoconstriction is mediated by oxidative stress. Moreover, hyperoxia impairs acetylcholine-mediated vasodilation in the setting of intact endothelial function. These effects of hyperoxia are prevented by vitamin C, providing evidence that hyperoxia-derived free radicals impair the activity of endothelium-derived vasoactive factors.
Exercise improves postischemic function in aging hearts
Authors: Starnes Joseph W. , Taylor Ryan P. , Park Yoonjung
Published: March 2015

Exercise improves cardioprotection against ischemia-reperfusion in young animals but has not been investigated in older animals, which represent the population most likely to suffer an ischemic event. Therefore, we sought to determine the effects of aging on exercise-induced cardioprotection. Young, middle-aged, and old (4, 12, and 21 mo old) male Fischer 344 rats ran 60 min at 70–75% of maximum oxygen consumption. Twenty-four hours postexercise, isolated perfused working hearts underwent 22.5 min of global ischemia and then 30 min of recovery (reperfusion). Compared with sedentary rats ( n = 8–9 rats/group), recovery of function (cardiac output × systolic pressure) improved after exercise ( n = 9 rats/group) by 40% at 4 mo, 78% at 12 mo, and 59% at 21 mo. Exercise increased inducible heat shock protein 70 expression 105% at 4 mo but only 27% at 12 mo and 24% at 21 mo. Catalase activity progressively increased with age ( P < 0.05) and was increased by exercise at 4 mo (26%) and 21 mo (19%). Manganese superoxide dismutase activity was increased by exercise only at 21 mo (45%). No exercise-related change in any antioxidant enzyme was observed at 12 mo. We conclude that exercise can enhance cardioprotection regardless of age, but the cardioprotective protein phenotype changes with age.
Alterations in EDHF-type relaxation and phosphodiesterase activity in mesenteric arteries from diabetic rats
Authors: Matsumoto Takayuki , Kobayashi Tsuneo , Kamata Katsuo
Published: March 2015

In isolated superior mesenteric artery rings from age-matched control rats and streptozotocin (STZ)-induced diabetic rats, we investigated the role of cAMP in endothelium-derived hyperpolarizing factor (EDHF)-type relaxation. The ACh-induced EDHF-type relaxation was significantly weaker in STZ-induced diabetic rats than in control rats, and in both groups of rats it was attenuated by 18α-glycyrrhetinic acid (18α-GA), an inhibitor of gap junctions, and enhanced by IBMX, a cAMP-phosphodiesterase (PDE) inhibitor. These enhanced EDHF-type responses were very similar in magnitude between diabetic and age-matched control rats. The EDHF-type relaxation was enhanced by cilostamide, a PDE3-selective inhibitor, but not by Ro 20–1724, a PDE4-selective inhibitor. The expression levels of the mRNAs and proteins for two cAMP PDEs (PDE3A, PDE3B) were significantly increased in STZ-induced diabetic rats, but those for PDE4D were not. We conclude that the impairment of EDHF-type relaxations in STZ-induced diabetic rats may be attributed to a reduction in the action of cAMP via increased PDE activity.
Expression of TASK-1, a pH-sensitive twin-pore domain K+ channel, in rat myocytes
Authors: Jones Sandra A. , Morton Michael J. , Hunter Malcolm , Boyett Mark R.
Published: March 2015

We have investigated the expression of TASK-1, a pH-sensitive, twin-pore domain K+channel in the rat heart. A mammalian cell line of Chinese hamster ovary cells (CHO), transfected with a plasmid containing mouse TASK-1, demonstrated the specificity of the anti-TASK-1 antibody. TASK-1 expression in cardiac tissue was initially demonstrated by Western blot and then localized by immunofluorescence. In single rat ventricular myocytes, strong staining of the TASK-1 protein was located at the intercalated disks and across the cell in a striated pattern, corresponding to the transverse axial tubular network (T tubules). In contrast, single rat atrial myocytes were stained at the intercalated disks with a weak punctate, striated pattern corresponding to underdeveloped T tubules. Also, formamide was used to induce the detubulation of ventricular myocytes, which enabled confirmation that TASK-1 protein expression occurs in T tubules. Consistent with this, RT-PCR revealed the expression of TASK-1 mRNA in total RNA from both the ventricles and atria. In this study, we conclusively demonstrated that TASK-1 protein and mRNA were expressed in rat atrial and ventricular tissue. The extensive distribution of TASK-1 shown to exist within myocyte membranes may provide a potential future target for antiarrhythmic drugs.
Structural and functional changes in left ventricle during normotensive and preeclamptic pregnancy
Authors: Simmons Lisa A. , Gillin Adrian G. , Jeremy Richmond W.
Published: March 2015

Increased cardiac output in pregnancy is associated with cardiac remodeling and possible reduction in contractility, which may worsen in preeclampsia. Left ventricular (LV) geometry and function were compared between nonpregnant controls ( n = 12) and normotensive ( n = 44) and preeclamptic ( n = 15) pregnant women using echocardiography. Load-independent comparisons of LV systolic function compared end-systolic stress (ESS) and rate-corrected velocity of circumferential fiber shortening ( VCFC). Mean arterial pressures were 101 ± 14 mmHg in preeclampsia, 76 ± 6 mmHg in normotensive pregnancy, and 78 ± 6 mmHg in controls ( P < 0.005 vs. preeclampsia). LV mass increased during normotensive pregnancy (66 ± 13 to 76 ± 16 g/m2; P < 0.05; controls, 65 ± 10 g/m2; P < 0.05) and was greater in preeclampsia (90 ± 18 g/m2; P < 0.05). In normotensive pregnancy, ESS decreased (59 ± 9 to 52 ± 11 g/cm2; P < 0.05; controls, 66 ± 14 g/cm2; P < 0.005). ESS was greater in preeclampsia (60 ± 14 g/cm2; P < 0.05). In controls, there was an inverse relationship between ESS and VCFC( r = −0.78). The ESS- VCFCrelationships in normotensive and preeclamptic pregnancy were unchanged from controls. We conclude that LV hypertrophy in normotensive and preeclamptic pregnancy matches changes in cardiac work, and LV contractility is preserved.
In vivo gene transfer of Kv1.5 normalizes action potential duration and shortens QT interval in mice with long QT phenotype
Authors: Brunner Michael , Kodirov Sodikdjon A. , Mitchell Gary F. , Buckett Peter D. , Shibata Katsushi , Folco Eduardo J. , Baker Linda , Salama Guy , Chan Danny P. , Zhou Jun , Koren Gideon
Published: March 2015

Mutations in cardiac voltage-gated K+channels cause long QT syndrome (LQTS) and sudden death. We created a transgenic mouse with a long QT phenotype (Kv1DN) by overexpression of a truncated K+channel in the heart and investigated whether the dominant negative effect of the transgene would be overcome by the direct injection of adenoviral vectors expressing wild-type Kv1.5 (AV-Kv1.5) into the myocardium. End points at 3–10 days included electrophysiology in isolated cardiomyocytes, surface ECG, programmed stimulation of the right ventricle, and in vivo optical mapping of action potentials and repolarization gradients in Langendorff-perfused hearts. Overexpression of Kv1.5 reconstituted a 4-aminopyridine-sensitive outward K+current, shortened the action potential duration, eliminated early afterdepolarizations, shortened the QT interval, decreased dispersion of repolarization, and increased the heart rate. Each of these changes is consistent with a physiologically significant primary effect of adenoviral expression of Kv1.5 on ventricular repolarization of Kv1DN mice.
Effect of AT1 receptor blockade on cardiac apoptosis in angiotensin II-induced hypertension
Authors: Diep Quy N. , El Mabrouk Mohammed , Yue Ping , Schiffrin Ernesto L.
Published: March 2015

Angiotensin II (ANG II) via AT1 receptors induces apoptosis in cardiomyocytes in vitro. We tested the hypothesis that in vivo AT1 receptor stimulation is accompanied by cardiac apoptosis and attempted to elucidate the molecular mechanisms involved in the death signaling pathway. Male Sprague-Dawley rats received ANG II (120 ng · kg−1 · min−1 sc) for 7 days with or without the AT1 receptor antagonist losartan (10 mg · kg−1 · day−1orally). Cardiac function was assessed by echocardiography. Apoptosis in the heart was detected and quantified by in situ TdT-mediated dUTP nick-end labeling (TUNEL) and radiolabeled DNA laddering. Expression of bax, bcl-2, caspase 3, and AT1 and AT2receptors was examined by Western blot analysis. Activity of caspase 3 was also measured by a fluorometric immunosorbent enzyme assay. Tail cuff systolic blood pressure was elevated ( P < 0.01, n = 6) in ANG II-infused rats (173 ± 3 mmHg) versus controls (111 ± 2 mmHg) and reduced by losartan (134 ± 4 mmHg). Cardiac function was essentially unchanged in ANG II-infused rats. Increased internucleosomal DNA cleavage by TUNEL assay and radiolabeled DNA laddering showed results compatible with enhanced cardiomyocyte apoptosis in the hearts of ANG-II infused rats. The bax-to-bcl-2 ratio, expression of the active form of caspase 3 (17 kDa), and activity of caspase 3 in the hearts of the ANG II group increased more than twofold above controls. Protein expression of AT1 and AT2 receptors was significantly increased in ANG II-infused rats compared with control rats. Losartan-treated ANG II-infused rats exhibited normalized apoptosis, bax, caspase 3 activity, and AT1 receptors. ANG II stimulation of AT1 receptors in the heart in vivo is associated with an increased rate of apoptosis without major hemodynamic consequences. Bax and caspase 3 are involved in the apoptotic signaling pathway in this experimental paradigm.
Delayed arteriolar relaxation after prolonged agonist exposure: functional remodeling involving tyrosine phosphorylation
Authors: Hill Michael A. , Potocnik Simon J. , Martinez-Lemus Luis A. , Meininger Gerald A.
Published: March 2015

Although arteriolar contraction is dependent on Ca2+-induced myosin phosphorylation, other mechanisms including Ca2+sensitization and time-dependent phenomena such as cytoskeletal and cellular reorganization may contribute to contractile events. We hypothesized that if arteriolar smooth muscle exhibits time-dependent behavior this may be manifested in differences in relaxation after short- and long-term exposure to contractile agonists. Studies were conducted in isolated arterioles pressurized to 70 mmHg. In initial experiments ( n = 10), rate of relaxation was measured after acute (5 min) or prolonged (4 h) exposure to 5 μM norepinephrine (NE). Prolonged exposure to NE resulted in significantly ( P < 0.05) increased time for relaxation in physiological salt solution. Rapid relaxation of vessels exposed to NE for 4 h was observed after superfusion with 0 mM Ca2+buffer, indicating that the alteration in relaxation was reversible and Ca2+dependent. A similarly impaired dilation was not observed with 4-h exposure to KCl (75 mM). To determine mechanisms contributing to the effects of prolonged NE exposure, studies were performed in the presence of the microtubule depolymerizing agent demecolcine (10 μM) or a series of tyrosine phosphorylation inhibitors. Although demecolcine caused significant vasoconstriction ( P < 0.05) and potentiated NE vasoconstriction, it did not prevent the effect of long-term NE exposure on relaxation. Genistein, although having no effect on acute NE-induced contraction, concentration-dependently inhibited prolonged NE constriction. Similarly, Src (PP1) and p42/44 MAP kinase (PD-98059) inhibitors prevented maintenance of long-term NE contraction. The data indicate that prolonged exposure to NE induces biochemical alterations that impair relaxation after removal of the agonist. The contractile effects are Ca2+dependent and involve tyrosine phosphorylation but do not appear to involve the polymerization state of the microtubule network.
Nonlinear effects in subthreshold virtual electrode polarization
Authors: Sambelashvili Aleksandre T. , Nikolski Vladimir P. , Efimov Igor R.
Published: March 2015

Introduction of the virtual electrode polarization (VEP) theory suggested solutions to several century-old puzzles of heart electrophysiology including explanation of the mechanisms of stimulation and defibrillation. Bidomain theory predicts that VEPs should exist at any stimulus strength. Although the presence of VEPs for strong suprathreshold pulses has been well documented, their existence at subthreshold strengths during diastole remains controversial. We studied cardiac membrane polarization produced by subthreshold stimuli in 1) rabbit ventricular muscle using high-resolution fluorescent imaging with the voltage-sensitive dye pyridinium 4-{2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl}-1-(3-sulfopropyl)hydroxide (di-4-ANEPPS) and 2) an active bidomain model with Luo-Rudy ion channel kinetics. Both in vitro and in numero models show that the common dog-bone-shaped VEP is present at any stimulus strength during both systole and diastole. Diastolic subthreshold VEPs exhibited nonlinear properties that were expressed in time-dependent asymmetric reversal of membrane polarization with respect to stimulus polarity. The bidomain model reveals that this asymmetry is due to nonlinear properties of the inward rectifier potassium current. Our results suggest that active ion channel kinetics modulate the transmembrane polarization pattern that is predicted by the linear bidomain model of cardiac syncytium.
TGF-β1attenuates myocardial ischemia-reperfusion injury via inhibition of upregulation of MMP-1
Authors: Chen Hongjiang , Li Dayuan , Saldeen Tom , Mehta Jawahar L.
Published: March 2015

Ischemia-reperfusion (I/R) is thought to upregulate the expression and activity of matrix metalloproteinases (MMPs), which regulate myocardial and vascular remodeling. Previous studies have shown that transforming growth factor-β1(TGF-β1) can attenuate myocardial injury induced by I/R. TGF-β1is also reported to suppress the release of MMPs. To study the modulation of MMP-1 by TGF-β1in I/R myocardium, Sprague-Dawley rats were given saline and subjected to 1 h of myocardial ischemia [total left coronary artery (LCA) ligation] followed by 1 h of reperfusion ( n = 9). Parallel groups of rats were pretreated with recombinant TGF-β1(rTGF-β1, 1 mg/rat, n = 9) before reperfusion or exposure to sham I/R (control group). I/R caused myocardial necrosis and dysfunction, indicated by decreased first derivative of left ventricular pressure, mean arterial blood pressure, and heart rate (all P < 0.01 vs. sham-operated control group). Simultaneously, I/R upregulated MMP-1 ( P < 0.01). Treatment of rats with rTGF-β1reduced the extent of myocardial necrosis and dysfunction despite I/R (all P < 0.01). rTGF-β1treatment also inhibited the upregulation of MMP-1 in the I/R myocardium ( P < 0.05). To determine the direct effect of MMP-1 on the myocardium, isolated adult rat myocytes were treated with active MMP-1, which caused injury and death of cultured myocytes, measured as lactate dehydrogenase release and trypan blue staining, in a dose- and time-dependent manner ( P < 0.05). Pretreatment with PD-166793, a specific MMP inhibitor, attenuated myocardial injury and death induced by active MMP-1. The present study for the first time shows that MMP-1 can directly cause myocyte injury or death and that attenuation of myocardial I/R injury by TGF-β1may, at least partly, be mediated by the inhibition of upregulation of MMP-1.
Fibroblast alignment under interstitial fluid flow using a novel 3-D tissue culture model
Authors: Ng Chee Ping , Swartz Melody A.
Published: March 2015

Interstitial flow is an important component of the microcirculation and interstitial environment, yet its effects on cell organization and tissue architecture are poorly understood, in part due to the lack of in vitro models. To examine the effects of interstitial flow on cell morphology and matrix remodeling, we developed a tissue culture model that physically supports soft tissue cultures and allows microscopic visualization of cells within the three-dimensional matrix. In addition, pressure-flow relationships can be continuously monitored to evaluate the bulk hydraulic resistance as an indicator of changes in the overall matrix integrity. We observed that cells such as human dermal fibroblasts aligned perpendicular to the direction of interstitial flow. In contrast, fibroblasts in static three-dimensional controls remained randomly oriented, whereas cells subjected to fluid shear as a two-dimensional monolayer regressed. Also, the dynamic measurements of hydraulic conductivity suggest reorganization toward a steady state. These primary findings help establish the importance of interstitial flow on the biology of tissue organization and interstitial fluid balance.
Exacerbation of myocardial injury in transgenic mice overexpressing FGF-2 is T cell dependent
Authors: Meij Johanna T. A. , Sheikh Farah , Jimenez Sarah K. , Nickerson Peter W. , Kardami Elissavet , Cattini Peter A.
Published: March 2015

Fibroblast growth factor-2 (FGF-2) is cardioprotective when added exogenously, stimulates cardiac myocyte proliferation, and is a mediator of tissue repair after injury. Furthermore, transgenic (TG) mice overexpressing FGF-2 in cardiac muscle demonstrate increased resistance to injury in an isolated heart model of ischemia-reperfusion. We investigated how increasing the endogenous FGF-2 levels in the heart affects the extent of myocardial damage induced by isoproterenol in vivo. Histopathological evaluation of hearts after intraperitoneal injection of isoproterenol yielded significantly higher scores for myocardial damage in FGF-2 TG lines compared with non-TG mice. After 1 day, FGF-2 TG mouse hearts displayed more cellular infiltration correlating with increased tissue damage. Immunostaining of non-TG and FGF-2 TG mouse hearts showed the presence of leukocytes in the infiltrate, including T cells expressing FGF receptor-1. Treatment of mice with T cell suppressors cyclosporin A and anti-CD3ε significantly decreased the level of myocardial injury observed after isoproterenol and equalized the histopathology scores in FGF-2 TG and non-TG hearts. These data demonstrate a direct T cell involvement in the response to isoproterenol-induced injury in vivo. Moreover, the findings indicate that the exacerbation of myocardial damage in FGF-2 TG mice was dependent on T cell infiltration, implicating FGF-2 in the inflammatory response seen in cardiac tissue after injury in vivo.