bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in [Na+]i, compartmental [Ca2+], and NADH with dysfunction after global ischemia in intact hearts
Authors: Varadarajan Srinivasan G. , An Jianzhong , Novalija Enis , Smart Steven C. , Stowe David F.
Published: December 2017

We measured the effects of global ischemia and reperfusion on intracellular Na+, NADH, cytosolic and mitochondrial (subscript mito) Ca2+, relaxation, metabolism, contractility, and Ca2+sensitivity in the intact heart. Langendorff-prepared guinea pig hearts were crystalloid perfused, and the left ventricular (LV) pressure (LVP), first derivative of LVP (LV dP/d t), coronary flow, and O2extraction and consumption were measured before, during, and after 30-min global ischemia and 60-min reperfusion. Ca2+, Na+, and NADH were measured by luminescence spectrophotometry at the LV free wall using indo 1 and sodium benzofuran isophthalate, respectively, after subtracting changes in tissue autofluorescence (NADH). Mitochondrial Ca2+was assessed by quenching cytosolic indo 1 with MnCl2. Mechanical responses to changes in cytosolic-systolic (subscript sys), diastolic (subscript dia), and mitochondrial Ca2+were tested over a range of extracellular [Ca2+] before and after ischemia-reperfusion. Both [Ca2+]sysand [Ca2+]diadoubled at 1-min reperfusion but returned to preischemia values within 10 min, whereas [Ca2+]mitowas elevated over 60-min reperfusion. Reperfusion dissociated [Ca2+]diaand [Ca2+]sysfrom contractile function as LVPsys-diaand the rise in LV dP/d t (LV dP/d tmax) were depressed by one-third and the fall in LV dP/d t (LV dP/d tmin) was depressed by one-half at 30-min reperfusion, whereas LVPdiaremained markedly elevated. [Ca2+]sys-diasensitivity at 100% LV dP/d tmaxwas not altered after reperfusion, but [Ca2+]diaat 100% LV dP/dtminand [Ca2+]mitoat 100% LV dP/d tmaxwere markedly shifted right on reperfusion (ED50+36 and +125 nM [Ca2+], respectively) with no change in slope. NADH doubled during ischemia but returned to normal on initial reperfusion. The intracellular [Na+] ([Na+]i) increased minimally during ischemia but doubled on reperfusion and remained elevated at 60-min reperfusion. Thus Na+and Ca2+temporally accumulate during initial reperfusion, and cytosolic Ca2+returns toward normal, whereas [Na+]iand [Ca2+]mitoremain elevated on later reperfusion. Na+loading likely contributes to Ca2+overload and contractile dysfunction during reperfusion.
Cardiac expression of a gain-of-function α5-integrin results in perinatal lethality
Authors: Valencik Maria L. , McDonald John A.
Published: December 2017

Communication between the extracellular matrix and the intracellular signal transduction and cytoskeletal system is mediated by integrin receptors. α5β1-Integrin and its cognate ligand fibronectin are essential in development of mesodermal structures, myocyte differentiation, and normal cardiac development. To begin to explore the potential roles of α5β1-integrin specifically in cardiomyocytes, we used a transgenic expression strategy. We overexpressed two forms of the human α5-integrin in cardiomyocytes: the full-length wild-type α5-integrin and a putative gain-of-function mutation created by truncating the cytoplasmic domain, designated α5-1-integrin. Overexpression of the wild-type α5-integrin has no detectable adverse effects in the mouse, whereas expression of α5-1-integrin caused electrocardiographic abnormalities, fibrotic changes in the ventricle, and perinatal lethality. Thus physiological regulation of integrin function appears essential for maintenance of normal cardiomyocyte structure and function. This strengthens the role of inside-out signaling in regulation of integrins in vivo and suggests that integrins and associated signaling molecules are important in cardiomyocyte function.
Real-time measurements of endogenous CO production from vascular cells using an ultrasensitive laser sensor
Authors: Morimoto Yuji , Durante William , Lancaster David G. , Klattenhoff Jens , Tittel Frank K.
Published: December 2017

Carbon monoxide (CO) has been implicated as a biological messenger molecule analogous to nitric oxide. A compact gas sensor based on a midinfrared laser absorption spectroscopy was developed for direct and real-time measurement of trace levels (in approximate pmol) of CO release by vascular cells. The midinfrared light is generated by difference frequency mixing of two nearinfrared lasers in a nonlinear optical crystal. A strong infrared absorption line of CO (4.61 μm) is chosen for convenient CO detection without interference from other gas species. The generation of CO from cultured vascular smooth muscle cells was detected every 20 s without any chemical modification to the CO. The sensitivity of the sensor reached 6.9 pmol CO. CO synthesis was measured from untreated control cells (0.25 nmol per 107cells/h), sodium nitroprusside-treated cells (0.29 nmol per 107cells/h), and hemin-treated cells (0.49 nmol per 107cells/h). The sensor also detected decreases in CO production after the addition of the heme oxygenase (HO) inhibitor tin protoporphyrin-IX (from 0.49 to 0.02 nmol per 107cells/h) and increases after the administration of the HO substrate hemin (from 0.27 to 0.64 nmol per 107cells/h). These results demonstrate that midinfrared laser absorption spectroscopy is a useful technique for the noninvasive and real-time detection of trace levels of CO from biological tissues.
Elevated interstitial adenosine concentrations do not activate the muscle reflex
Authors: MacLean David A. , Vickery Lisa M. , Sinoway Lawrence I.
Published: December 2017

The purpose of the present study was to examine the effects of adenosine perfusion of the isolated triceps surae muscle group in the decerebrate cat on interstitial adenosine concentrations as well as heart rate and blood pressure responses. In six male cats (6.0 ± 0.21 kg), the triceps surae muscle group of both legs was perfused with an artificial blood solution containing no additives (control) and then with blood containing 20 mM or 100 μM adenosine for 10 min. An intact muscle reflex was confirmed by bolus injections of 50 mM phosphate and/or saturated KCl administered into the triceps surae muscle via the cannulated popliteal artery before and after adenosine blood perfusion. Microdialysis of the triceps surae muscle group during muscle perfusion revealed that interstitial adenosine was elevated ( P < 0.05) from 0.9 ± 0.3 μM during control blood perfusion to 2,421 ± 547 μM during 20 mM adenosine perfusion. In addition, interstitial adenosine levels were increased ( P < 0.05) from 1.1 ± 0.3 μM during control blood perfusion to 4.1 ± 1.2 μM during perfusion with 100 μM adenosine. Despite the large increases in interstitial adenosine levels, perfusion of the triceps surae muscle group with the two blood adenosine solutions resulted in no significant increases in heart rate or blood pressure. These data strongly suggest that elevated interstitial adenosine concentrations do not play a role in activating the muscle reflex and confirm our previous in vivo human findings ( J Appl Physiol 83: 1045–1053, 1997).
Regional nonuniformity of normal adult human left ventricle
Authors: Bogaert Jan , Rademakers Frank E.
Published: December 2017

Regional nonuniformity is a feature of both diseased and normal left ventricles (LV). With the use of magnetic resonance (MR) myocardial tagging, we performed three-dimensional strain analysis on 87 healthy adults in local cardiac and fiber coordinate systems (radial, circumferential, longitudinal, and fiber strains) to characterize normal nonuniformities and to test the validity of wall thickening as a parameter of regional function. Regional morphology included wall thickness and radii of curvature measurements. With respect to transmural nonuniformity, subendocardial strains exceeded subepicardial strains. Going from base to apex, wall thickness and circumferential radii of curvature decreased, whereas longitudinal radii of curvature increased. All of the strains increased from LV base to apex, resulting in a higher ejection fraction (EF) at the apex than at the base (70.9 ± 0.4 vs. 62.4 ± 0.4%; means ± SE, P < 0.0001). When we looked around the circumference of the ventricle, the anterior part of the LV was the flattest and thinnest and showed the largest wall thickening (46.6 ± 1.2%) but the lowest EF (64.7 ± 0.5%). The posterior LV wall was thicker, more curved, and showed a lower wall thickening (32.8 ± 1.0%) but a higher EF (71.3 ± 0.5%). The regional contribution of the LV wall to the ejection of blood is thus highly variable and is not fully characterized by wall thickening alone. Differences in regional LV architecture and probably local stress are possible explanations for this marked functional nonuniformity.
Nitric oxide-mediated arteriolar dilation after endothelial deformation
Authors: Sun Dong , Huang An , Recchia Fabio A. , Cui Yanning , Messina Edward J. , Koller Akos , Kaley Gabor
Published: December 2017

Previously, we frequently observed dilation of arterioles after agonist-induced constrictions. We hypothesized that deformation of the endothelium during decreases in diameter of isolated arterioles elicits the release of nitric oxide (NO). In isolated arterioles of rat mesentery, phenylephrine (PE, 10−7 M)-, U-46619 (10−7 M)-, and KCl (50 mM)-induced constrictions were followed by potent dilations. Inhibition of NO synthase with N ω-nitro-l-arginine (l-NNA, 2 × 10−4 M) or removal of the endothelium significantly enhanced constriction and reduced the postconstriction dilation. In the presence of 80 mmHg of intraluminal pressure, an increase in extraluminal pressure (Pe) to 75 mmHg for 20 s and 1 and 2 min decreased vessel diameter. After release of Pe, arterioles dilated as a function of the duration of diameter reduction by Pe. Removal of the endothelium or administration of l-NNA significantly diminished the post-Pe dilations. In cultured mesenteric arteriolar endothelial cells (EC), PE, U-46619, or KCl did not increase, whereas ACh did increase, the production of NO, as measured by a fluorometric assay for nitrite. Furthermore, when EC, cultured on a stretched silicone membrane, were subjected to deformation by shortening the membrane to 50% of its original length, NO release increased significantly. Based on all of the above, we propose that deformation of EC per se elicits release of NO, a mechanism that modulates arteriolar constriction.
Gadolinium prevents stretch-mediated contractile dysfunction in isolated papillary muscles
Authors: Nicolosi Alfred C. , Kwok Chiaki S. , Contney Stephen J. , Olinger Gordon N. , Bosnjak Zeljko J.
Published: December 2017

We tested the hypothesis that overstretching the myocardium could induce and/or exacerbate contractile dysfunction via stretch-activated (SA) ion channels. Maximum developed tension (Tmax), normalized to a control value, was compared in guinea pig papillary muscles held at one of three resting lengths (physiological stretch, overstretch, and unloaded) for 85 min. Overstretched muscles exhibited decreased contractile force (Tmax= 0.77 ± 0.03) compared with physiological and unloaded muscles (Tmax= 0.93 ± 0.05 and 1.03 ± 0.07, respectively). Gd3+, an SA channel antagonist, eliminated the adverse effect of overstretching (Tmax= 0.98 ± 0.06), but nifedipine, a dihydropyridine (DHP) antagonist of L-type calcium channels, did not (Tmax= 0.82 ± 0.04). Exposure to modified hypoxia-reoxygenation (MHR) during physiological stretch resulted in decreased contractility (Tmax= 0.63 ± 0.07), an effect that was exacerbated by overstretching (Tmax= 0.44 ± 0.04). Gd3+mitigated the effects of overstretch during MHR (Tmax= 0.64 ± 0.05), but DHP did not (Tmax= 0.48 ± 0.04). These data suggest that overstretching of the myocardium contributes to contractile abnormalities via SA channels that are distinct from L-type calcium channels.
Vascular β-adrenergic receptor system is dysfunctional after myocardial infarction
Authors: Gaballa Mohamed A. , Eckhart Andrea , Koch Walter J. , Goldman Steven
Published: December 2017

We identified abnormalities in the vascular β-adrenergic receptor (β-AR) signaling pathway in heart failure after myocardial infarction (MI). To examine these abnormalities, we measured β-AR-mediated hemodynamics, vascular reactivity, and the vascular β-AR molecular signaling components in rats with heart failure after MI. Six weeks after MI, these rats had an increased left ventricular (LV) end-diastolic pressure, decreased LV systolic pressure, and decreased rate of LV pressure change (dP/d t). LV dP/d t responses to isoproterenol were shifted downward, although the responses for systemic vascular resistance were shifted upward in heart failure rats ( P < 0.05). Isoproterenol- and IBMX-induced vasorelaxations were blunted in heart failure rats ( P< 0.05) with no change in the forskolin-mediated vasorelaxation. These changes were associated with the following alterations in β-AR signaling ( P < 0.05): decreases in β-AR density (aorta: 58.7 ± 6.0 vs. 35.7 ± 1.9 fmol/mg membrane protein; carotid: 29.6 ± 5.6 vs. 18.0 ± 3.9 fmol/mg membrane protein, n = 5), increases in G protein-coupled receptor kinase activity levels (relative phosphorimage counts of 191 ± 39 vs. 259 ± 26 in the aorta and 115 ± 30 vs. 202 ± 7 in the carotid artery, n = 5), and decreases in cGMP and cAMP in the carotid artery (0.85 ± 0.10 vs. 0.31 ± 0.06 pmol/mg protein and 2.3 ± 0.3 vs. 1.2 ± 0.1 pmol/mg protein, n = 5) with no change in Gαsor Gαiin the aorta. Thus in heart failure there are abnormalities in the vascular β-AR system that are similar to those seen in the myocardium. This suggests a common neurohormonal mechanism and raises the possibility that treatment in heart failure focused on the myocardium may also affect the vasculature.
Reciprocal regulation of cGMP-mediated vasorelaxation by soluble and particulate guanylate cyclases
Authors: Hussain Monira B. , MacAllister Raymond J. , Hobbs Adrian J.
Published: December 2017

Nitric oxide (NO) and atrial natriuretic peptides (ANP) activate soluble (sGC) and particulate guanylate cyclase (pGC), respectively, and play important roles in the maintenance of cardiovascular homeostasis. However, little is known about potential interactions between these two cGMP-generating pathways. Here we demonstrate that sGC and pGC cooperatively regulate cGMP-mediated relaxation in human and murine vascular tissue. In human vessels, the potency of spermine-NONOate (SPER-NO) and ANP was increased after inhibition of endogenous NO synthesis and decreased by prior exposure to glyceryl trinitrate (GTN). Aortas from endothelial NO synthase (eNOS) knockout (KO) mice were more sensitive to ANP than tissues from wild-type (WT) animals. However, in aortas from WT mice, the potency of ANP was increased after pretreatment with NOS or sGC inhibitor. Vessels from eNOS KO animals were less sensitive to ANP after GTN pretreatment, an effect that was reversed in the presence of an sGC inhibitor. cGMP production in response to SPER-NO and ANP was significantly greater in vessels from eNOS KO animals compared with WT animals. This cooperative interaction between NO and ANP may have important implications for human pathophysiologies involving deficiency in either mediator and the clinical use of nitrovasodilators.
Influence of permeating ions on Kv1.5 channel block by nifedipine
Authors: Lin S. , Wang Z. , Fedida D.
Published: December 2017

Nifedipine can block K+ currents through Kv1.5 channels in an open-channel manner (32). Replacement of internal and external K+ with equimolar Rb+ or Cs+ reduced the potency of nifedipine block of Kv1.5 from an IC50 of 7.3 μM (K+) to 16.0 μM (Rb+) and 26.9 μM (Cs+). The voltage dependence of block was unaffected, and a single binding site block model was used to describe block for all three ions. By varying ion species at the intra- and extracellular mouth of the channel and by using a nonconducting W472F-Kv1.5 mutant, we demonstrated that block was conditioned by the ion permeating the pore and, to a lesser extent, by the extracellular ion species alone. In Kv1.5, the outer pore mutations R487V and R487Y reduced nifedipine potency close to that of Kv4.2 and other Kv channels with an equivalent valine. Although changing this residue can affect C-type inactivation of Kv channels, the normalized reduction and time course of currents blocked by nifedipine in 5, 135, and 300 mM extracellular K+concentration was the same. Similarly, a mean recovery time constant from nifedipine block of 316 ms was unchanged (332 ms) after 5-s prepulses to allow C-type inactivation. This is consistent with the conclusion that nifedipine block and C-type inactivation in the Kv1.5 channel can coexist but are mediated by distinct mechanisms coordinated by outer pore conformation.
IGF-I differentially regulates Bcl-xL and Bax and confers myocardial protection in the rat heart
Authors: Yamamura Tadashi , Otani Hajime , Nakao Yoshihisa , Hattori Reiji , Osako Motohiko , Imamura Hiroji
Published: December 2017

Bcl-2 family proteins play a crucial role in the cytoprotective action of insulin-like growth factor-I (IGF-I) by regulating cell death signaling at the mitochondrial level. The present study examined the effect of IGF-I on the expression of Bcl-2 family proteins in the rat heart mitochondria in relation to myocardial protection against ischemia-reperfusion injury. Systemic IGF-I (1 mg) treatment in the rat increased Bcl-xL and attenuated Bax 12–24 h later in the heart mitochondria fraction. Permeability transition and cytochrome c release occurred in a Ca2+concentration-dependent manner in the vehicle-treated mitochondria. This was significantly inhibited by the IGF-I-pretreatment. Moreover, ATP synthesis was significantly greater in the IGF-I-pretreated mitochondria. IGF-I pretreatment 24 h before 25 min of global ischemia in the isolated rat heart model significantly improved recovery of isovolumic left ventricular function and inhibited creatine kinase release during reperfusion. This was associated with a significantly less number of terminal transferase labeling-positive myocytes and nonmyocytes 2 h after reperfusion. These results suggest that IGF-1 differentially regulates Bcl-xL and Bax in heart mitochondria, which may be causally related to myocardial protection against ischemia-reperfusion injury.
Evidence against oxidative stress as mechanism of endothelial dysfunction in methionine loading model
Authors: Nightingale Angus K. , James Philip P. , Morris-Thurgood Jayne , Harrold Fraser , Tong Richard , Jackson Simon K. , Cockcroft John R. , Frenneaux Michael P.
Published: December 2017

Endothelial dysfunction reflects reduced nitric oxide (NO) bioavailability due to either reduced production, inactivation of NO, or reduced smooth muscle responsiveness. Oral methionine loading causes acute endothelial dysfunction in healthy subjects and provides a model in which to study mechanisms. Endothelial function was assessed using flow-mediated dilatation (FMD) of the brachial artery in humans. Three markers of oxidative stress were measured ex vivo in venous blood. NO responsiveness was assessed in vascular smooth muscle and platelets. Oral methionine loading induced endothelial dysfunction (FMD decreased from 2.8 ± 0.8 to 0.3 ± 0.3% with methionine and from 2.8 ± 0.8 to 1.3 ± 0.3% with placebo; P < 0.05). No significant changes in measures of plasma oxidative stress or in vascular or platelet sensitivity to submaximal doses of NO donors were detected. These data suggest that oxidative stress is not the mechanism of endothelial dysfunction after oral methionine loading. Furthermore, the preservation of vascular and platelet NO sensitivity makes a signal transduction abnormality unlikely.
In vivo regulation of Na/Ca exchanger expression by adrenergic effectors
Authors: Golden Kish L. , Ren Jun , O'Connor Jessica , Dean Andrea , DiCarlo Stephen E. , Marsh James D.
Published: December 2017

The Na/Ca exchanger encoded by the NCX1gene plays an important role in calcium homeostasis in cardiac muscle. We previously identified three in vitro signaling pathways that are of major importance in the regulation of Na/Ca exchanger gene expression in neonatal cardiac myocytes, the protein kinase A (PKA) and protein kinase C (PKC) pathways, and intracellular Ca2+. To determine whether these pathways are important in vivo, we stimulated the PKA and PKC pathways and examined functional expression of the Na/Ca exchanger in adult rat heart. After a 3- and 7-day treatment, norepinephrine (200 μg · kg−1· h−1), isoproterenol (150 μg · kg−1· h−1), and phenylephrine (200 μg · kg−1· h−1) each stimulated a significant increase in NCX1 mRNA levels (35–85%, P < 0.05). Norepinephrine also stimulated a 35% increase in protein abundance ( P < 0.05), a 20% decrease in relaxation duration ( P < 0.05), and a 25% reduction in the fluorescence decay constant ( P < 0.05) after a 7-day treatment. We conclude that a 7-day treatment of α- and β-adrenergic agonists increases the expression of functional Na/Ca exchangers in adult rat heart.
PKCε activation induces dichotomous cardiac phenotypes and modulates PKCε-RACK interactions and RACK expression
Authors: Pass Jason M. , Zheng Yuting , Wead William B. , Zhang Jun , Li Richard C. X. , Bolli Roberto , Ping Peipei
Published: December 2017

Receptors for activated C kinase (RACKs) have been shown to facilitate activation of protein kinase C (PKC). However, it is unknown whether PKC activation modulates RACK protein expression and PKC-RACK interactions. This issue was studied in two PKCε transgenic lines exhibiting dichotomous cardiac phenotypes: one exhibits increased resistance to myocardial ischemia (cardioprotected phenotype) induced by a modest increase in PKCε activity (228 ± 23% of control), whereas the other exhibits cardiac hypertrophy and failure (hypertrophied phenotype) induced by a marked increase in PKCε activity (452 ± 28% of control). Our data demonstrate that activation of PKC modulates the expression of RACK isotypes and PKC-RACK interactions in a PKCε activity- and dosage-dependent fashion. We found that, in mice displaying the cardioprotected phenotype, activation of PKCε enhanced RACK2 expression (178 ± 13% of control) and particulate PKCε-RACK2 protein-protein interactions (178 ± 18% of control). In contrast, in mice displaying the hypertrophied phenotype, there was not only an increase in RACK2 expression (330 ± 33% of control) and particulate PKCε-RACK2 interactions (154 ± 14% of control) but also in RACK1 protein expression (174 ± 10% of control). Most notably, PKCε-RACK1 interactions were identified in this line. With the use of transgenic mice expressing a dominant negative PKCε, we found that the changes in RACK expression as well as the attending cardiac phenotypes were dependent on PKCε activity. Our observations demonstrate that RACK expression is dynamically regulated by PKCε and suggest that differential patterns of PKCε-RACK interactions may be important determinants of PKCε-dependent cardiac phenotypes.
A common mechanism for concurrent changes of diastolic muscle length and systolic function in intact hearts
Authors: Lu Li , Xu Ya , Zhu Peili , Greyson Clifford , Schwartz Gregory G.
Published: December 2017

Mechanical properties of the myocardium at end diastole have been thought to be dominated by passive material properties rather than by active sarcomere cross-bridge interactions. This study tested the hypothesis that residual cross-bridges significantly contribute to end-diastolic mechanics in vivo and that changes in end-diastolic cross-bridge interaction parallel concurrent changes in systolic cross-bridge interaction. Open-chest anesthetized pigs were treated with intracoronary verapamil ( n = 7) or 2,3-butanedione monoxime (BDM; n = 8). Regional left ventricular external work and end-diastolic pressure (EDP) versus end-diastolic segment length (EDL) relations were determined in the treated and untreated regions of each heart. Both agents reduced external work of treated regions to 31–38% of baseline and concurrently shifted EDP versus EDL relations to the right (i.e., greater EDL at a given EDP) by an average of 5% ( P < 0.05). During washout of the drugs, EDP versus EDL returned to baseline in parallel with recovery of external work. Sarcomere length, measured by transmission electron microscopy in BDM-treated and untreated regions of the same hearts after diastolic arrest and perfusion fixation, was 8% greater in BDM-treated regions ( P < 0.01). We concluded that residual diastolic cross-bridges significantly and reversibly influence end-diastolic mechanics in vivo. Alterations of end-diastolic and systolic cross-bridge interactions occur in parallel.
Juxtacrine effects of IL-1α precursor promote iNOS expression in vascular smooth muscle cells
Authors: Sasu Sebastian , Cooper Angela L. , Beasley Debbie
Published: December 2017

After injury to the blood vessel wall, vascular smooth muscle cells (SMC) synthesize interleukin (IL)-1 and inducible nitric oxide (NO) synthase (iNOS). The present study tested whether endogenous production of IL-1α stimulates iNOS expression in vascular SMC, and assessed whether IL-1α exerts autocrine effects on the cells producing IL-1α or juxtacrine effects on cells that contact the IL-1α producing cells. Rat aortic SMC were transiently transfected with expression plasmids encoding either IL-1α precursor, which localizes to the plasma membrane, or mature IL-1α, which remains cytosolic. iNOS mRNA levels, determined by RT-PCR, and production of nitrite, a stable oxidation product of NO, were markedly elevated in SMC overexpressing IL-1α precursor, and modestly elevated in SMC overexpressing mature IL-1α, relative to SMC transfected with vector alone. Exposure to exogenous IL-1β or TNF-α further stimulated iNOS gene expression in SMC producing IL-1α; low levels of IL-1β (20 pg/ml) were effective in SMC transfected with IL-1α precursor plasmid, whereas SMC transfected with mature IL-1α plasmid or vector alone required higher concentrations of IL-1β (200 and 2,000 pg/ml, respectively). The increases in iNOS mRNA levels and NO production in SMC overexpressing IL-1α precursor were prevented by exogenous IL-1 receptor antagonist, suggesting that these effects were mediated by the type I IL-1 receptor. Immunostaining studies indicated that IL-1α precursor stimulates iNOS gene expression via cell-cell contact. Expression of iNOS was enhanced in cells that were in contact with a cell overexpressing IL-1α precursor (identified by coexpression of green fluorescent protein), and in cells that were overexpressing IL-1α themselves, but only when the cell contacted another cell. Together these results indicate that IL-1α precursor acts by cell-cell contact as an autocrine and juxtacrine enhancer of iNOS gene expression, inducing moderate iNOS expression on its own, and markedly augmenting the responsiveness of rat aortic SMC to exogenous cytokines.
Endothelial vasodilator production by uterine and systemic arteries. VII. Estrogen and progesterone effects on eNOS
Authors: Rupnow Heidi L. , Phernetton Terrance M. , Shaw Cynthia E. , Modrick Mary L. , Bird Ian M. , Magness Ronald R.
Published: December 2017

Uterine blood flow (UBF) and uterine artery endothelial nitric oxide synthase (eNOS) expression are greatest during the follicular vs. luteal phase. 17β-Estradiol (E2β) increases UBF and elevates eNOS in ovine uterine but not systemic arteries; progesterone (P4) effects on E2β changes of eNOS remain unclear. Nonpregnant ovariectomized sheep received either vehicle ( n = 10), P4 (0.9 g Controlled Internal Drug Release vaginal implants; n = 13), E2β (5 μg/kg bolus + 6 μg · kg−1 · day−1; n = 10), or P4 + E2β ( n = 12). Reproductive (uterine/mammary) and nonreproductive (omental/renal) artery endothelial proteins were procured on day 10, and eNOS was measured by Western analysis. P4 and E2β alone and in combination increased ( P < 0.05) eNOS expression in uterine artery endothelium (vehicle = 100 ± 16%, P4 = 251 ± 59%, E2β = 566 ± 147%, P4 + E2β = 772 ± 211% of vehicle). Neither omental, renal, nor mammary artery eNOS was altered, demonstrating the local nature of steroid-induced maintenance of uterine arterial eNOS. In the myometrial microvasculature, eNOS was increased slightly ( P = 0.06) with E2β and significantly with P4 + E2β. Systemic NOx was increased with P4 and P4 + E2β, but not E2β, suggesting differential regulation of eNOS expression and activity, since P4 increased eNOS in uterine artery endothelium while E2β and the combination further increased eNOS protein.
Characterization of stretch-activated cation current in coronary smooth muscle cells
Authors: Wu Xin , Davis Michael J.
Published: December 2017

Stretch-activated ion currents were recorded from vascular smooth muscle (VSM) after enzymatic isolation of single cells from porcine coronary arterioles. Patch pipettes were used to record whole cell current and control cell length. Under voltage clamp in physiological saline solution, an inward cation current ( ICAT) was activated by 105–135% longitudinal stretch. ICATcoincided with an increase in intracellular Ca2+concentration. Under current clamp, membrane depolarization was induced by stretch. The magnitude of ICATvaried from −0.8 to −6.9 pA/pF at a holding potential of −60 mV. ICATwas graded with stretch, inactivated on release, and could be repeatedly induced. A potassium current ( IK) activated in unstretched cells by depolarization was also enhanced by stretch. In Ca2+-free bath solution, stretch-induced enhancement of IKwas blocked, but ICATwas still present. Hexamethyleneamiloride (50 μM), a reputed inhibitor of mechanosensitive channels, blocked ICATand the stretch-induced increase in IKbut not basal IK. Grammostolla spatulata venom (1:100,000) blocked basal IK, blocked stretch-induced increases in IK, and blocked ICAT. Iberiotoxin, a specific Ca2+-activated K+channel blocker, did not alter ICATbut blocked the stretch-induced increase in IKand increased the magnitude of stretch-induced depolarization. We concluded that longitudinal stretch directly activates a cation current and secondarily activates a Ca2+-activated K+current in isolated coronary myocytes. Although these two currents would partially counteract each other, the predominance of ICATat physiological potentials is likely to explain the depolarization and contraction observed in intact coronary VSM during pressure elevation.
Modeling of the Norwood circulation: effects of shunt size, vascular resistances, and heart rate
Authors: Migliavacca Francesco , Pennati Giancarlo , Dubini Gabriele , Fumero Roberto , Pietrabissa Riccardo , Urcelay Gonzalo , Bove Edward L. , Hsia Tain-Yen , de Leval Marc R.
Published: December 2017

Hypoplastic left heart syndrome is the most common lethal cardiac malformation of the newborn. Its treatment , apart from heart transplantation , is the Norwood operation. The initial procedure for this staged repair consists of reconstructing a circulation where a single outlet from the heart provides systemic perfusion and an interpositioning shunt contributes blood flow to the lungs. To better understand this unique physiology, a computational model of the Norwood circulation was constructed on the basis of compartmental analysis. Influences of shunt diameter, systemic and pulmonary vascular resistance, and heart rate on the cardiovascular dynamics and oxygenation were studied. Simulations showed that 1) larger shunts diverted an increased proportion of cardiac output to the lungs, away from systemic perfusion, resulting in poorer O2 delivery, 2) systemic vascular resistance exerted more effect on hemodynamics than pulmonary vascular resistance, 3) systemic arterial oxygenation was minimally influenced by heart rate changes, 4) there was a better correlation between venous O2 saturation and O2 delivery than between arterial O2 saturation and O2delivery, and 5) a pulmonary-to-systemic blood flow ratio of 1 resulted in optimal O2 delivery in all physiological states and shunt sizes.
Calorie restriction attenuates inflammatory responses to myocardial ischemia-reperfusion injury
Authors: Chandrasekar B. , Nelson J. F. , Colston J. T. , Freeman G. L.
Published: December 2017

The life-prolonging effects of calorie restriction (CR) may be due to reduced damage from cumulative oxidative stress. Our goal was to determine the long-term effects of moderate dietary CR on the myocardial response to reperfusion after a single episode of sublethal ischemia. Male Fisher 344 rats were fed either an ad libitum (AL) or CR (40% less calories) diet. At age 12 mo the animals were anaesthetized and subjected to thoracotomy and a 15-min left-anterior descending coronary artery occlusion. The hearts were reperfused for various periods. GSH and GSSG levels, nuclear factor-κB (NF-κB) DNA binding activity, cytokine, and antioxidant enzyme expression were assessed in the ischemic zones. Sham-operated animals served as controls. Compared with the AL diet, chronic CR limited oxidative stress as seen by rapid recovery in GSH levels in previously ischemic myocardium. CR reduced DNA binding activity of NF-κB. The κB-responsive cytokines interleukin-1β and tumor necrosis factor-α were transiently expressed in the CR group but persisted longer in the AL group. Furthermore, expression of manganese superoxide dismutase, a key antioxidant enzyme, was significantly delayed in the AL group. Collectively these data indicate that CR significantly attenuates myocardial oxidative stress and the postischemic inflammatory response.
Regulation of muscle sympathetic nerve activity after bed rest deconditioning
Authors: Pawelczyk James A. , Zuckerman Julie H. , Blomqvist C. Gunnar , Levine Benjamin D.
Published: December 2017

Cardiovascular deconditioning reduces orthostatic tolerance. To determine whether changes in autonomic function might produce this effect, we developed stimulus-response curves relating limb vascular resistance, muscle sympathetic nerve activity (MSNA), and pulmonary capillary wedge pressure (PCWP) with seven subjects before and after 18 days of −6° head-down bed rest. Both lower body negative pressure (LBNP; −15 and −30 mmHg) and rapid saline infusion (15 and 30 ml/kg body wt) were used to produce a wide variation in PCWP. Orthostatic tolerance was assessed with graded LBNP to presyncope. Bed rest reduced LBNP tolerance from 23.9 ± 2.1 to 21.2 ± 1.5 min, respectively (means ± SE, P= 0.02). The MSNA-PCWP relationship was unchanged after bed rest, though at any stage of the LBNP protocol PCWP was lower, and MSNA was greater. Thus bed rest deconditioning produced hypovolemia, causing a shift in operating point on the stimulus-response curve. The relationship between limb vascular resistance and MSNA was not significantly altered after bed rest. We conclude that bed rest deconditioning does not alter reflex control of MSNA, but may produce orthostatic intolerance through a combination of hypovolemia and cardiac atrophy.
Nitric oxide modulates arterial baroreflex control of heart rate in conscious lambs in an age-dependent manner
Authors: Sener Alp , Smith Francine G.
Published: December 2017

Experiments were carried out in conscious chronically instrumented lambs aged 1 ( n = 6) and 6 wk ( n = 5) to evaluate the arterial baroreflex control of heart rate (HR) during postnatal maturation and to investigate any modulatory role of endogenously produced nitric oxide (NO). Before and after intravenous administration of 20 mg/kg of the l-arginine analog NG-nitro-l-arginine methyl ester (l-NAME), the arterial baroreflex was assessed by measuring HR responses to increases and decreases in systolic arterial pressure achieved by intravenous administration of phenylephrine and sodium nitroprusside. The HR range over which the baroreflex operates and minimum HR as well as maximum gain were greater at 1 than at 6 wk of age. These age differences were abolished in the presence ofl-NAME, which decreased the HR range and gain of the arterial baroreflex control of HR at 1 but not at 6 wk of age. These data provide new information that age-dependent effects of the arterial baroreflex appear to result from effects of endogenously produced NO.
Effects of soluble TNF receptor treatment on lipopolysaccharide-induced myocardial cytokine expression
Authors: Kadokami Toshiaki , McTiernan Charles F. , Kubota Toru , Frye Carole S. , Bounoutas George S. , Robbins Paul D. , Watkins Simon C. , Feldman Arthur M.
Published: December 2017

Tumor necrosis factor (TNF)-α plays a key role in the pathogenesis of septic shock syndrome, and myocardial TNF-α expression may contribute to this pathophysiology. We examined the myocardial expression of TNF-α-related cytokines and chemokines in mice exposed to lipopolysaccharide (LPS) and tested the effects of anti-TNF therapy on myocardial cytokine expression. Cytokine mRNA levels were measured by RNase protection assay, and protein levels in the plasma and myocardium were assessed by enzyme-linked immunosorbent assays. LPS (4 μg/g body wt ip) induced marked cytokine expression, including TNF-α, interleukin (IL)-1β, IL-6, and monocyte chemotactic protein (MCP)-1, in both the plasma and myocardium. Pretreatment with adenovirus-mediated TNF receptor fusion protein (AdTNFR1; 109plaque-forming units iv) decreased plasma cytokine levels. In contrast, whereas myocardial IL-1β expression was also suppressed, expression of IL-6 and MCP-1 was not inhibited by AdTNFR1. In summary, anti-TNF treatment differentially altered the cytokine expression in the plasma and myocardium during endotoxemia. Inability to block myocardial expression of IL-6 and MCP-1 suggests a possible mechanism for the failure of anti-TNF therapies in the treatment of endotoxin shock.
Inhibition of hypoxia/reoxygenation-induced apoptosis in metallothionein-overexpressing cardiomyocytes
Authors: Wang Guang-Wu , Zhou Zhanxiang , Klein Jon B. , Kang Y. James
Published: December 2017

To study possible mechanisms for metallothionein (MT) inhibition of ischemia-reperfusion-induced myocardial injury, cardiomyocytes isolated from MT-overexpressing transgenic neonatal mouse hearts and nontransgenic controls were subjected to 4 h of hypoxia (5% CO2-95% N2, glucose-free modified Tyrode's solution) followed by 1 h of reoxygenation in MEM + 20% fetal bovine serum (FBS) (5% CO2-95% air), and cytochrome c-mediated caspase-3 activation apoptotic pathway was determined. Hypoxia/reoxygenation-induced apoptosis was significantly suppressed in MT-overexpressing cardiomyocytes, as measured by both terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling and annexin V-FITC binding. In association with apoptosis, mitochondrial cytochrome c release, as determined by Western blot, was observed to occur in nontransgenic cardiomyocytes. Correspondingly, caspase-3 was activated as determined by laser confocal microscopic examination with the use of FITC-conjugated antibody against active caspase-3 and by enzymatic assay. The activation of this apoptotic pathway was significantly inhibited in MT-overexpressing cells, as evidenced by both suppression of cytochrome c release and inhibition of caspase-3 activation. The results demonstrate that MT suppresses hypoxia/reoxygenation-induced cardiomyocyte apoptosis through, at least in part, inhibition of cytochrome c-mediated caspase-3 activation.
Preconditioning limits mitochondrial Ca2+during ischemia in rat hearts: role of KATPchannels
Authors: Wang Lianguo , Cherednichenko Gennady , Hernandez Lisa , Halow Jessica , Camacho S. Albert , Figueredo Vincent , Schaefer Saul
Published: December 2017

Prolonged myocardial ischemia results in an increase in intracellular calcium concentration ([Ca2+]i), which is thought to play a critical role in ischemia-reperfusion injury. Ischemic preconditioning (PC) improves myocardial function during ischemia-reperfusion, a process that may involve opening mitochondrial ATP-sensitive potassium (KATP) channels. Because pharmacological limitation of mitochondrial calcium concentration ([Ca2+]m) overload during ischemia-reperfusion has been shown to improve myocardial function, we hypothesized that PC would reduce [Ca2+]mduring ischemia-reperfusion and that this effect was mediated by opening mitochondrial KATPchannels. Isolated rat hearts were subjected to 25 min of global ischemia and 30 min of reperfusion with or without PC in the presence of mitochondrial KATPchannel opening (diazoxide, 100 μM) and blockade [5-hydroxydecanoic acid (5-HD), 100 μM]. Contracture during ischemia (end-diastolic pressure) and functional recovery on reperfusion (developed pressure) were assessed. Total [Ca2+]iand [Ca2+]mwere measured using indo 1 fluorescence. Both PC and diazoxide limited the increase in end-diastolic pressure and resulted in greater functional recovery after 30 min of reperfusion, functional effects that were partially or completely abolished by 5-HD. PC and diazoxide also significantly limited the increase in [Ca2+]mduring ischemia-reperfusion. In addition, PC lowered [Ca2+]iduring reperfusion, whereas diazoxide paradoxically resulted in increased [Ca2+]iduring reperfusion. There was an inverse linear relationship between [Ca2+]mand developed pressure during reperfusion. PC limits the ischemia-induced increase in mitochondrial, but not total, [Ca2+]i, an effect mediated by opening mitochondrial KATPchannels. These data suggest that the lowering of mitochondrial calcium overload is a mechanism of cardioprotection in PC.
Characterization of PGE2receptors in fetal and newborn lamb ductus arteriosus
Authors: Bouayad Asmàa , Kajino Hiroki , Waleh Nahid , Fouron Jean-Claude , Andelfinger Gregor , Varma Daya R. , Skoll Amanda , Vazquez Alejandro , Gobeil Fernand , Clyman Ronald I. , Chemtob Sylvain
Published: December 2017

Although the role of PGE2in maintaining ductus arteriosus (DA) patency is well established, the specific PGE2receptor subtype(s) (EP) involved have not been clearly identified. We used late gestation fetal and neonatal lambs to study developmental regulation of EP receptors. In the fetal DA, radioligand binding and RT-PCR assays virtually failed to detect EP1but detected EP2, EP3D, and EP4receptors in equivalent proportions. In the newborn lamb, DA total density was one-third of that found in the fetus and only EP2was detected. Stimulation of EP2and EP4increased cAMP formation and was associated with DA relaxation. Though stimulation of EP3inhibited cAMP formation, it surprisingly relaxed the fetal DA both in vitro and in vivo. This EP3-induced relaxation was specifically diminished by the ATP-sensitive K+(KATP) channel blocker glibenclamide. In conclusion, PGE2dilates the late gestation fetal DA through pathways that involve either cAMP (EP2and EP4) or KATPchannels (EP3). The loss of EP3and EP4receptors in the newborn DA is consistent with its decreased responsiveness to PGE2.
Adrenergic stimulation of rat resistance arteries affects Ca2+ sparks, Ca2+ waves, and Ca2+oscillations
Authors: Mauban Joseph R. H. , Lamont Christine , Balke C. William , Wier W. Gil
Published: December 2017

Confocal laser scanning microscopy and fluo 4 were used to visualize local and whole cell Ca2+ transients within individual smooth muscle cells (SMC) of intact, pressurized rat mesenteric small arteries during activation of α1-adrenoceptors. A method was developed to record the Ca2+ transients within individual SMC during the changes in arterial diameter. Three distinct types of “Ca2+signals” were influenced by adrenergic activation (agonist: phenylephrine). First, asynchronous Ca2+ transients were elicited by low levels of adrenergic stimulation. These propagated from a point of origin and then filled the cell. Second, synchronous, spatially uniform Ca2+ transients, not reported previously, occurred at higher levels of adrenergic stimulation and continued for long periods during oscillatory vasomotion. Finally, Ca2+sparks slowly decreased in frequency of occurrence during exposure to adrenergic agonists. Thus adrenergic activation causes a decrease in the frequency of Ca2+ sparks and an increase in the frequency of asynchronous wavelike Ca2+ transients, both of which should tend to decrease arterial diameter. Oscillatory vasomotion is associated with spatially uniform synchronous oscillations of cellular [Ca2+] and may have a different mechanism than the asynchronous, propagating Ca2+ transients.
EET homologs potently dilate coronary microvessels and activate BKCachannels
Authors: Zhang Yongde , Oltman Christine L. , Lu Tong , Lee Hon-Chi , Dellsperger Kevin C. , VanRollins Mike
Published: December 2017

Epoxyeicosatrienoic acids (EETs) are released from endothelial cells and potently dilate small arteries by hyperpolarizing vascular myocytes. In the present study, we investigated the structural specificity of EETs in dilating canine and porcine coronary microvessels (50–140 μm ID) and activating large-conductance Ca2+-activated K+(BKCa) channels. The potencies and efficacies of EET regioisomers and enantiomers were compared with those of two EET homologs: epoxyeicosaquatraenoic acids (EEQs), which are made from eicosapentaenoic acid by the same cytochrome P-450 epoxygenase that generates EETs from arachidonic acid, and epoxydocosatetraenoic acids (EDTs), which are EETs that are two carbons longer. With EC50values of 3–120 pM but without regio- or stereoselectivity, EETs potently dilated canine and porcine microvessels. Surprisingly, the EEQs and EDTs had comparable potencies and efficacies in dilating microvessels. Moreover, 50 nM 13,14-EDT activated the BKCachannels with the same efficacy as either 11,12-EET enantiomer at 50 nM. We conclude that coronary microvessels and BKCachannels possess low structural specificity for EETs and suggest that EEQs and EDTs may thereby also be endothelium-derived hyperpolarizing factors.
Extending fluorescent microsphere methods for regional organ blood flow to 13 simultaneous colors
Authors: Schimmel Carmel , Frazer David , Glenny Robb W.
Published: December 2017

Seven fluorescent microsphere colors can be used in a single experiment to estimate regional blood flow without correcting for spillover of emitted fluorescence. To extend the method to 13 colors, we compared the accuracy of three methods for spillover correction. Fixed wavelength intensities were corrected by matrix inversion, and synchronous scan spectra were corrected by least squares fit of an overdetermined system of linear equations and by least squares fit of a sum of Gaussian and Lorentzian functions. Correction methods were validated in pigs and sheep by simultaneous injections of radioactive microspheres and fluorescent microspheres of 7, 10, and 13 different colors. We induced extreme changes in flow to create regions with low fluorescent signals bound on either side by high fluorescent signals. Blood flow was determined by radioactivity and by fluorescence using both fixed excitation and emission wavelength pairs and synchronous scanning and then corrected for spillover. Correlation between fluorescent intensity and radioactivity were excellent for all three correction methods [ R 2 = 0.98 ± 0.02 (mean ± SD)]. Low-flow regions requiring large spillover correction had systematic errors for some color combinations in all methods. We conclude that for 13 fluorescent colors spillover error can be minimized so that all three correction methods provide accurate estimates of regional blood flow.