bluuebunny/crossref_metadata_2025 huggingface dataset:
Multitrack system for superfusing isolated cardiac myocytes
Authors: Heller Lois Jane , Mohrman David E. , Smith Juline A. , Wallace Kendall B.
Published: March 2015

A new system for studying mechanical activity of freshly isolated cardiac myocytes from up to four experimental groups simultaneously is described. Suspensions of cardiac myocytes isolated from adult rat hearts were drawn into microhematocrit capillary tubes, which were then mounted in parallel fashion between two four-channel tubing manifolds placed on the movable stage of an inverted microscope. Within a few minutes, cells settled and attached to the bottom of the tubes and then could be superfused with various test solutions. The system allowed for electrical field stimulation, rapid changes in bathing solutions, control of temperature, and simulation of ischemia and reperfusion with measurements of the effects of such interventions on both populations of cells (low power survey) and individual myocytes (high power). Myocyte responses to these various interventions are described. The primary advantage of this system is the ability to conduct experiments on cardiac myocytes isolated concurrently from multiple experimental groups at the same time and under identical conditions.
Strain-specific patterns of autonomic nervous system activity and heart failure susceptibility in mice
Authors: Shusterman Vladimir , Usiene Irmute , Harrigal Chivonne , Lee Joon Sup , Kubota Toru , Feldman Arthur M. , London Barry
Published: March 2015

Transgenic mice are widely used to study cardiac function, but strain-dependent differences in autonomic nervous system activity (ANSA) have not been explored. We compared 1) short-term pharmacological responses of cardiac rhythm in FVB vs. C57Black6/SV129 wild-type mice and 2) long-term physiological dynamics of cardiac rhythm and survival in tumor necrosis factor (TNF)-α transgenic mice with heart failure (TNF-α mice) on defined backgrounds. Ambulatory telemetry electrocardiographic recordings and response to saline, adrenergic, and cholinergic agents were examined in FVB and C57Black6/SV129 mice. In FVB mice, baseline heart rate (HR) was higher and did not change after injection of isoproterenol or atropine but decreased with propranolol. In C57Black6/SV129 mice, HR did not change with propranolol but increased with isoproterenol or atropine. Mean HR, but not indexes of HR variability, was an excellent predictor of response to autonomic agents. The proportion of surviving animals was higher in TNF-α mice on an FVB background than on a mixed FVB/C57Black6 background. The homeostatic states of ANSA are strain specific, which can explain the interstrain differences in mean HR, pharmacological responses, and survival of animals with congestive heart failure. Strain-specific differences should be considered in selecting the strains of mice used for transgenic and gene targeting experiments.
Evidence that NOS2 acts as a trigger and mediator of late preconditioning induced by acute systemic hypoxia
Authors: Xi Lei , Tekin Demet , Gursoy Erdal , Salloum Fadi , Levasseur Joseph E. , Kukreja Rakesh C.
Published: March 2015

Chronic systemic hypoxia (SH) enhances myocardial ischemic tolerance in mammals. We studied the delayed cardioprotection caused by acute SH and associated signaling mechanism. Conscious adult male mice were exposed to one or two cycles of hypoxia (H; 10% O2) or normoxia (21% O2) for various durations (30 min, 2 h, 4 h) followed by 24 h of reoxygenation. Hearts were isolated 24 h later and subjected to ischemia-reperfusion in a Langendorff model. Infarct size was reduced in mice pretreated with one (H4h) or two cycles (H4hx2) of 4 h SH compared with normoxia mice ( P < 0.05), which was abolished by an inducible nitric oxide synthase (NOS2) inhibitor ( S-methylisothiourea, 3 mg/kg) given before SH or ischemia. H4hx2 also failed to reduce infarct size in NOS2 knockout mice. Cyclooxygenase-2 (COX-2) inhibitor (NS-398, 10 mg/kg) did not block the protection given either before H4hx2 or ischemia. A two- to three fold increase in myocardial NOS2 expression was observed in H4h, H2hx2, and H4hx2 ( P < 0.05), whereas endothelial NOS (NOS3) or COX-2 remained unchanged. We conclude that acute SH induces delayed cardioprotection, which is triggered and mediated by NOS2, but not by NOS3 or COX-2.
Involvement of Ca2+/calmodulin-dependent protein kinase II in endothelial NO production and endothelium-dependent relaxation
Authors: Schneider Jean-Christophe , El Kebir Driss , Chéreau Christiane , Lanone Sophie , Huang Xiao-Lin , De Buys Roessingh Anthony S. , Mercier Jean-Christophe , Dall'Ava-Santucci Josette , Dinh-Xuan A. Tuan
Published: March 2015

Nitric oxide (NO) is synthesized froml-arginine by the Ca2+/calmodulin-sensitive endothelial NO synthase (NOS) isoform (eNOS). The present study assesses the role of Ca2+/calmodulin-dependent protein kinase II (CaMK II) in endothelium-dependent relaxation and NO synthesis. The effects of three CaMK II inhibitors were investigated in endothelium-intact aortic rings of normotensive rats. NO synthesis was assessed by a NO sensor and chemiluminescence in culture medium of cultured porcine aortic endothelial cells stimulated with the Ca2+ionophore A23187 and thapsigargin. Rat aortic endothelial NOS activity was measured by the conversion ofl-[3H]arginine tol-[3H]citrulline. Three CaMK II inhibitors, polypeptide 281–302, KN-93, and lavendustin C, attenuated the endothelium-dependent relaxation of endothelium-intact rat aortic rings in response to acetylcholine, A23187, and thapsigargin. None of the CaMK II inhibitors affected the relaxation induced by NO donors. In a porcine aortic endothelial cell line, KN-93 decreased NO synthesis and caused a rightward shift of the concentration-response curves to A23187 and thapsigargin. In rat aortic endothelial cells, KN-93 significantly decreased bradykinin-induced eNOS activity. These results suggest that CaMK II was involved in NO synthesis as a result of Ca2+-dependent activation of eNOS.
Channels involved in transient currents unmasked by removal of extracellular calcium in cardiac cells
Authors: Macianskiene Regina , Moccia Francesco , Sipido Karin R. , Flameng Willem , Mubagwa Kanigula
Published: March 2015

In cardiac cells that lack macroscopic transient outward K+ currents ( I to), the removal of extracellular Ca2+ can unmask “ I to-like” currents. With the use of pig ventricular myocytes and the whole cell patch-clamp technique, we examined the possibility that cation efflux via L-type Ca2+channels underlies these currents. Removal of extracellular Ca2+ and extracellular Mg2+ induced time-independent currents at all potentials and time-dependent currents at potentials greater than −50 mV. Either K+ or Cs+ could carry the time-dependent currents, with reversal potential of +8 mV with internal K+ and +34 mV with Cs+. Activation and inactivation were voltage dependent [Boltzmann distributions with potential of half-maximal value ( V 1/2) = −24 mV and slope = −9 mV for activation; V 1/2 = −58 mV and slope = 13 mV for inactivation]. The time-dependent currents were resistant to 4-aminopyridine and to DIDS but blocked by nifedipine at high concentrations (IC50 = 2 μM) as well as by verapamil and diltiazem. They could be increased by BAY K-8644 or by isoproterenol. We conclude that the I to-like currents are due to monovalent cation flow through L-type Ca2+ channels, which in pig myocytes show low sensitivity to nifedipine.
Targeted myocardial transgenic expression of HIV Tat causes cardiomyopathy and mitochondrial damage
Authors: Raidel Scott M. , Haase Chad , Jansen Natalie R. , Russ Rodney B. , Sutliff Roy L. , Velsor Leonard W. , Day Brian J. , Hoit Brian D. , Samarel Allen M. , Lewis William
Published: March 2015

Cardiac effects of human immunodeficiency virus (HIV) transactivator (Tat) are unclear, but Tat decreases liver glutathione (an important mitochondrial antioxidant) when ubiquitously expressed in transgenic mice (TG). With an α-myosin heavy chain promoter, Tat was selectively targeted to murine cardiac myocytes. One high-expression hemizygous (+/−Tathigh; 12 copies) and two low-expression (+/−TatlowA,B; 2–5 copies) TG lines were created. Cardiomyopathy was documented with increased left ventricle (LV) mass, ventricular expression of atrial natriuretic factor (ANF) mRNA, mitochondrial ultrastructural defects, and myocardial depletion of glutathione. In+/−TathighTGs, normalized LV mass (determined echocardiographically) increased 46% (90 days), 134% (240 days), and 96% (365 days) compared with wild-type littermates (WT). LV fractional shortening was decreased to 28% (90 days), 27% (240 days), and 19% (365 days).+/−TatlowLV mass was unchanged (≤365 days). ANF in+/−Tathighventricles (180 days) was twofold WT values. Glutathione was selectively decreased in+/−Tathighhearts (120 days).+/−Tathighhearts contained damaged mitochondria (≥210 days); however, profound mitochondrial destruction occurred in homozygous+/+Tathighhearts (10 days) and the pups died (14 days). Tat caused cardiac dysfunction in this TG and may impact on cardiomyopathy in acquired immunodeficiency syndrome.
Modulation of protein phosphatase 2a by adenosine A1 receptors in cardiomyocytes: role for p38 MAPK
Authors: Liu Qinghang , Hofmann Polly A.
Published: March 2015

Adenosine A1 receptor activation causes protein phosphatase 2a (PP2a) activation in ventricular myocytes. This attenuates β-adrenergic functional effects in the heart (Liu Q and Hofmann PA. Am J Physiol Heart Circ Physiol 283: H1314–H1321, 2002). The purpose of the present study was to identify the signaling pathway involved in the translocation/activation of PP2a by adenosine A1 receptors in ventricular myocytes. We found that N6-cyclopentyladenosine (CPA; an adenosine A1 receptor agonist)-induced PP2a translocation was blocked by p38 MAPK inhibition but not by JNK inhibition. CPA increased phosphorylation of p38 MAPK, and this effect was abolished by pertussis toxin and inhibitors of the cGMP pathway. Moreover, CPA-induced PP2a translocation was blocked by inhibition of the cGMP pathway. Guanylyl cyclase activation mimicked the effects of CPA and caused p38 MAPK phosphorylation and PP2a translocation. Finally, CPA-induced dephosphorylations of troponin I and phospholamban were blocked by pertussis toxin and attenuated by p38 MAPK inhibition. These results suggest that adenosine A1 receptor-mediated PP2a activation uses a pertussis toxin-sensitive Gi protein-guanylyl cyclase-p38 MAPK pathway. This proposed, novel pathway may play a role in acute modulation of cardiac function.
Increased nonoxidative glycolysis despite continued fatty acid uptake during demand-induced myocardial ischemia
Authors: Chandler Margaret P. , Huang Hazel , McElfresh Tracy A. , Stanley William C.
Published: March 2015

During stress, patients with coronary artery disease frequently fail to increase coronary flow and myocardial oxygen consumption (MV˙o 2) in response to a greater demand for oxygen, resulting in “demand-induced” ischemia. We tested the hypothesis that dobutamine infusion with flow restriction stimulates nonoxidative glycolysis without a change in MV˙o 2 or fatty acid uptake. Measurements were made in the anterior wall of anesthetized open-chest swine hearts ( n = 7). The left anterior descending (LAD) coronary artery flow was controlled via an extracorporeal perfusion circuit, and substrate uptake and oxidation were measured with radiotracers. Demand-induced ischemia was produced with intravenous dobutamine (15 μg · kg−1 · min−1) and 20% reduction in LAD flow for 20 min. Despite no change in MV˙o 2, there was a switch from lactate uptake (5.9 ± 3.1) to production (74.5 ± 16.3 μmol/min), glycogen depletion (66%), and increased glucose uptake (105%), but no change in anterior wall power or the index of anterior wall energy efficiency. There was no change in the rate of tracer-measured fatty acid uptake; however, exogenous fatty acid oxidation decreased by 71%. Thus demand-induced ischemia stimulated nonoxidative glycolysis and lactate production, but did not effect fatty acid uptake despite a fall in exogenous fatty acid oxidation.
Effect of endurance training on coronary artery size and function in healthy men: an invasive followup study
Authors: Windecker Stephan , Allemann Yves , Billinger Michael , Pohl Tilmann , Hutter Damian , Orsucci Thomas , Blaga Laurent , Meier Bernhard , Seiler Christian
Published: March 2015

In eight healthy male volunteers (cardiologists; age 36 ± 5 yr), bicycle spiroergometry, Doppler echocardiography, and quantitative coronary angiography with intracoronary Doppler measurements before and after completion of a physical endurance exercise program of >5 mo duration were performed. Maximum oxygen uptake increased from 46 ± 6 to 54 ± 5 ml · kg−1 · min−1( P = 0.04), maximum ergometric workload changed from 3.8 ± 0.3 to 4.4 ± 0.3 W/kg ( P = 0.001), and left ventricular mass index increased from 82 ± 18 to 108 ± 29 g/m2 ( P = 0.001). The right, left main, and left anterior descending coronary artery cross-sectional area increased significantly in repsonse to exercise. Before versus at the end of the exercise program, flow-induced left anterior descending coronary artery cross-sectional area was 10.1 ± 3.5 and 11.0 ± 3.9 mm2, respectively ( P = 0.03), nitroglycerin-induced left coronary calibers increased significantly, and coronary flow velocity reserve changed from 3.8 ± 0.8 to 4.5 ± 0.7 ( P = 0.001). Left coronary artery correlated significantly with ventricular mass and maximum oxygen uptake, and coronary flow velocity reserve was significantly associated with maximum workload.
Serial noninvasive assessment of progressive pulmonary hypertension in a rat model
Authors: Jones John E. , Mendes Lisa , Rudd M. Audrey , Russo Giulia , Loscalzo Joseph , Zhang Ying-Yi
Published: March 2015

Current methods used to investigate pulmonary hypertension in rat models of the disease allow for only one to two measurements of pulmonary artery (PA) pressure in the life of a rat. We investigated whether transthoracic echocardiography can be used to assess the progression of pulmonary hypertension in rats at multiple time points. Serial echocardiographic measurements were performed over a 6-wk period on rats injected with monocrotaline (MCT) or placebo. Development of a midsystolic notch in the PA waveform, a decrease in the PA flow acceleration time (PAAT), an increase in right ventricular (RV) free-wall thickness, and the development of tricuspid regurgitation (TR) were observed as pulmonary hypertension developed. Changes in the PA waveform and PAAT began in week 3 of disease development as the PA systolic pressure (PASP) reached 25–30 mmHg according to right heart catheterization. The RV free-wall thickness increased significantly by week 5 (PASPs 40–50 mmHg). Development of quantifiable TR occurred in week 6 or at PASPs > 65 mmHg. A linear correlation was found between the PAAT and PASP in the range of 30–65 mmHg and between the RV-right atrial pressure gradient (derived from TR velocity) and PASP at pressures >65 mmHg, which enabled a noninvasive estimate of the PASP over a wide range of pressures based on these parameters. These data indicate that transthoracic echocardiography can be used for monitoring the progress of pulmonary hypertension in a rat model.
Age and attenuation of exercise-induced myocardial HSP72 accumulation
Authors: Demirel Haydar A. , Hamilton Karyn L. , Shanely R. Andrew , Tümer Nihal , Koroly Mary Jo , Powers Scott K.
Published: March 2015

Overexpression of heat shock protein (HSP)72 is associated with cardioprotection. Hyperthermia-induced HSP72 overexpression is attenuated with senescence. While exercise also increases myocardial HSP72 in young animals, it is unknown whether this effect is attenuated with aging. Therefore, we investigated the effect of aging on exercise-induced myocardial heat shock factor (HSF)-1 activation and HSP72 expression. Male Fischer-344 rats (6 or 24 mo) were randomized to control, exercise, and hyperthermic groups. Exercise consisted of 2 days of treadmill running (60 min/day, ∼75% maximal oxygen consumption). Hyperthermia, 15 min at ∼41°C (colonic temperature), was achieved using a temperature-controlled heating blanket. Analyses included Western blotting for myocardial HSP72 and HSF-1, electromobility shift assays for HSF-1 activation, and Northern blotting for HSP72 mRNA. Exercise and hyperthermia increased ( P < 0.05) myocardial HSP72 in both young (>3.5- and 2.5-fold, respectively) and aged (>3- and 1.5-fold, respectively) animals. Both exercise and hyperthermic induction of HSP72 was attenuated with age. Myocardial HSF-1 protein, HSF-1 activation, and HSP72 mRNA did not differ with age. These data demonstrate that aging is associated with diminished exercise-induced myocardial HSP72 expression. Mechanisms other than HSF-1 activation and transcription of HSP72 mRNA are responsible for this age-related impairment.
Expression of genes involved in vascular development and angiogenesis in endothelial cells of adult lung
Authors: Favre Cecile J. , Mancuso Michael , Maas Kevin , McLean John W. , Baluk Peter , McDonald Donald M.
Published: March 2015

Profiling gene expression in endothelial cells advances the understanding of normal vascular physiology and disease processes involving angiogenesis. However, endothelial cell purification has been challenging because of the difficulty of isolating cells and their low abundance. Here we examine gene expression in endothelial cells freshly isolated from lung capillaries after in vivo labeling with fluorescent cationic liposomes and purification by fluorescence-activated cell sorting (FACS). Of the 39,000 genes and expressed sequence tags evaluated on custom oligonucleotide arrays, 555 were enriched in endothelial cell fraction. These included familiar endothelial cell-associated genes such as VEGF, VEGF receptor (VEGFR)-1, VEGFR-2, angiopoietin-2, Tie1, Tie2, Edg1 receptor, VE-cadherin, claudin 5, connexin37, CD31, and CD34. Also enriched were genes in semaphorin/neuropilin (Sema3c and Nrp1), ephrin/Eph (ephrin A1, B1, B2, and EphB4), delta/notch (Hey1, Jagged 2, Notch 1, Notch 4, Numb, and Siah1b), and Wingless (Frizzled-4 and Tle1) signaling pathways involved in vascular development and angiogenesis. Expression of representative genes in alveolar capillary endothelial cells was verified by immunohistochemistry. Such expression reflects features that endothelial cells of normal lung capillaries have in common with embryonic and growing blood vessels. About half of the enriched genes, including exostosin 2, lipocalin 7, phospholipid scramblase 2, pleckstrin 2, protocadherin 1, Ryk, scube 1, serpinh1, SNF-related kinase, and several tetraspanins, had little or no previous association with endothelial cells. This approach can readily be used to profile genes expressed in blood vessels in tumors, chronic inflammation, and other sites in which endothelial cells avidly take up cationic liposomes.
Interleukin-11 attenuates human vascular smooth muscle cell proliferation
Authors: Zimmerman Michael A. , Selzman Craig H. , Reznikov Leonid L. , Raeburn Christopher D. , Barsness Katherine , McIntyre Robert C. , Hamiel Christine R. , Harken Alden H.
Published: March 2015

Interleukin (IL)-11 is a growth factor for megakaryocytes, osteoclasts, and intestinal mucosa. IL-11 is also an anti-inflammatory agent, mediating many of its effects by inhibition of the transcriptional activator nuclear factor (NF)-κB. The purposes of this study were to examine the effects of IL-11 on human vascular smooth muscle cell (VSMC) proliferation and NF-κB activity. VSMC were cultured from human transplant donor aortas, stimulated with basic fibroblastic growth factor (bFGF), and treated with IL-11. VSMC stimulated with bFGF demonstrated an increase in cell number by direct cell counting and mitochondrial activity. IL-11 caused a concentration-dependent decrease in bFGF-induced VSMC proliferation. Furthermore, IL-11 attenuated bFGF-induced increases in cytoplasmic and intranuclear unbound NF-κB p65. Similarly, IL-11 attenuated VSMC expression of two NF-κB-dependent cytokines, IL-8 and IL-6. Stimulated VSMC did not secrete IL-11, suggesting that endogenous IL-11 did not account for our observations. In conclusion, IL-11 inhibits human VSMC proliferation in vitro and is associated with suppression of NF-κB.
UDP acts as a growth factor for vascular smooth muscle cells by activation of P2Y6receptors
Authors: Hou Mingyan , Harden T. Kendall , Kuhn Cynthia M. , Baldetorp Bo , Lazarowski Eduardo , Pendergast William , Möller Sebastian , Edvinsson Lars , Erlinge David
Published: March 2015

Mitogenic effects of the extracellular nucleotides ATP and UTP are mediated by P2Y1, P2Y2, and P2Y4receptors. However, it has not been possible to examine the highly expressed UDP-sensitive P2Y6receptor because of the lack of stable, selective agonists. In rat aorta smooth muscle cells (vascular smooth muscle cells; VSMC), UDP and UTP stimulated3H-labeled thymidine incorporation with similar pEC50values (5.96 and 5.69). Addition of hexokinase did not reduce the mitogenic effect of UDP. In cells transfected with P2Y receptors the stable pyrimidine agonist uridine 5'- O-(2-thiodiphosphate) (UDPβS) was specific for P2Y6with no effect on P2Y1, P2Y2, or P2Y4receptors. UDPβS stimulated [3H]thymidine and [3H]leucine incorporation and increased cell number in VSMC. Flow cytometry demonstrated that UDP stimulated cell cycle progression to both the S and G2phases. The intracellular signal pathways were dependent on phospholipase C, possibly protein kinase C-δ, and a tyrosine kinase pathway but independent of Giproteins, eicosanoids, and protein kinase A. The half-life of P2Y6receptor mRNA was <1 h by competitive RT-PCR. The mitogen-activated protein kinase kinase inhibitor PD-098059 significantly suppressed, whereas ATP and interleukin-1β upregulated, expression of P2Y6receptor mRNA. The results demonstrate that UDP stimulates mitogenesis through activation of P2Y6receptors and that the receptor is regulated by factors important in the development of vascular disease.
Gender differences in the expression of heat shock proteins: the effect of estrogen
Authors: Voss M. R. , Stallone J. N. , Li Min , Cornelussen R. N. M. , Knuefermann P. , Knowlton A. A.
Published: March 2015

The heat shock proteins (HSPs) are an important family of endogenous, protective proteins that are found in all tissues. In the heart, HSP72, the inducible form of HSP70, has been the most intensely studied. It is well established that HSP72 is induced with ischemia and is cardioprotective. Overexpression of other HSPs also is protective against cardiac injury. Recently, we observed that 17β-estradiol increases levels of HSPs in male rat cardiac myocytes. We hypothesized that there were gender differences in HSP72 expression in the heart secondary to estrogen. To test this hypothesis, we examined cardiac levels of HSP72 by ELISA in male and female Sprague-Dawley rats. In addition, three other HSPs were assessed by Western blot (HSP27, HSP60, and HSP90). To determine whether estrogen status affected HSP72 expression in other muscles or tissues, two other muscle tissues, slow twitch muscle (soleus muscle) and fast twitch muscle (gastrocnemius muscle), were studied as well as two other organs, the kidney and liver. Because HSP72 is cardioprotective, and females are known to have less cardiovascular disease premenopause, the effects of ovariectomy were examined. We report that female Sprague-Dawley rat hearts have twice as much HSP72 as male hearts. Ovariectomy reduced the level of HSP72 in female hearts, and this could be prevented by estrogen replacement therapy. These data show that the expression of cardiac HSP72 is greater in female rats than in male rats, due to upregulation by estrogen.
Copper-induced vascular endothelial growth factor expression and wound healing
Authors: Sen Chandan K. , Khanna Savita , Venojarvi Mika , Trikha Prashant , Ellison E. Christopher , Hunt Thomas K. , Roy Sashwati
Published: March 2015

Angiogenesis plays a central role in wound healing. Among many known growth factors, vascular endothelial growth factor (VEGF) is believed to be the most prevalent, efficacious, and long-term signal that is known to stimulate angiogenesis in wounds. Whereas a direct role of copper to facilitate angiogenesis has been evident two decades ago, the specific targets of copper action remained unclear. This report presents first evidence showing that inducible VEGF expression is sensitive to copper and that the angiogenic potential of copper may be harnessed to accelerate dermal wound contraction and closure. At physiologically relevant concentrations, copper sulfate induced VEGF expression in primary as well as transformed human keratinocytes. Copper shared some of the pathways utilized by hypoxia to regulate VEGF expression. Topical copper sulfate accelerated closure of excisional murine dermal wound allowed to heal by secondary intention. Copper-sensitive pathways regulate key mediators of wound healing such as angiogenesis and extracellular matrix remodeling. Copper-based therapeutics represents a feasible approach to promote dermal wound healing.
KCa channel blockers reveal hyperpolarization and relaxation to K+ in rat isolated mesenteric artery
Authors: Dora Kim A. , Ings Nicola T. , Garland Christopher J.
Published: March 2015

Raising extracellular K+concentration ([K+]o) around mesenteric resistance arteries reverses depolarization and contraction to phenylephrine. As smooth muscle depolarizes and intracellular Ca2+ and tension increase, this effect of K+ is suppressed, whereas efflux of cellular K+ through Ca2+-activated K+ (KCa) channels is increased. We investigated whether K+ efflux through KCa suppresses the action of exogenous K+ and whether it prestimulates smooth muscle Na+-K+-ATPase. Under isometric conditions, 10.8 mM [K+]o had no effect on arteries contracted >10 mN, unless 100 nM iberiotoxin (IbTX), 100 nM charybdotoxin (ChTX), and/or 50 nM apamin were present. Simultaneous measurements of membrane potential and tension showed that phenylephrine depolarized and contracted arteries to −32.2 ± 2.3 mV and 13.8 ± 1.6 mN ( n = 5) after blockade of KCa, but 10.8 mM K+ reversed fully the responses (107.6 ± 8.6 and 98.8 ± 0.6%, respectively). Under isobaric conditions and preconstriction with phenylephrine, 10.7 mM [K+]o reversed contraction at both 50 mmHg (77.0 ± 8.5%, n = 9) and 80 mmHg (83.7 ± 5.5%, n = 5). However, in four additional vessels at 80 mmHg, raising K+ failed to reverse contraction unless ChTX was present. Increases in isometric and decreases in isobaric tension with phenylephrine were augmented by either ChTX or ouabain (100 μM), whereas neither inhibitor altered tension under resting conditions. Inhibition of cellular K+ efflux facilitates hyperpolarization and relaxation to exogenous K+, possibly by indirectly reducing the background activation of Na+-K+-ATPase.
Single-beat estimation of right ventricular end-systolic pressure-volume relationship
Authors: Brimioulle Serge , Wauthy Pierre , Ewalenko Patricia , Rondelet Benoı̂t , Vermeulen Françoise , Kerbaul François , Naeije Robert
Published: March 2015

Assessement of right ventricular (RV) contractility from end-systolic pressure-volume relationships (ESPVR) is difficult due to problems in measuring RV instantaneous volume and to effects of changes in RV preload or afterload. We therefore investigated in anesthetized dogs whether RV ESPVR and contractility can be determined without measuring RV volume and without changing RV preload or afterload. The maximal RV pressure of isovolumic beats (Pmax) was predicted from isovolumic portions of RV pressure during ejecting beats and compared with Pmaxmeasured during the first beat after pulmonary artery clamping. In RV pressure-volume loops obtained from RV pressure and integrated pulmonary arterial flow, end-systolic elastance ( E es) was assessed as the slope of Pmax-derived ESPVR, pulmonary artery effective elastance ( E a) as the slope of end-diastolic to end-systolic relation, and coupling efficiency as the E es-to- E a ratio ( E es/ E a). Predicted Pmax correlated with observed Pmax( r = 0.98 ± 0.02). Dobutamine increased E es from 1.07 to 2.00 mmHg/ml and E es/ E a from 1.64 to 2.49, and propranolol decreased E es/ E a from 1.64 to 0.91 (all P < 0.05). After adrenergic blockade, preload reduction did not affect E es, whereas hypoxia and arterial constriction markedly increased E aand somewhat increased E es due to the Anrep effect. Low preload did not affect E es/ E a and high afterload decreased E es/ E a. In conclusion, in the right ventricle 1) Pmax can be calculated from normal beats, 2) Pmax can be used to determine ESPVR without change in load, and 3) Pmax-derived ESPVR can be used to assess ventricular contractility and ventricular-arterial coupling efficiency.
Extracellular ATP signaling in the rabbit lung: erythrocytes as determinants of vascular resistance
Authors: Sprague Randy S. , Olearczyk Jeffrey J. , Spence Dana M. , Stephenson Alan H. , Sprung Robert W. , Lonigro Andrew J.
Published: March 2015

Previously, it was reported that red blood cells (RBCs) are required to demonstrate participation of nitric oxide (NO) in the regulation of rabbit pulmonary vascular resistance (PVR). RBCs do not synthesize NO; hence, we postulated that ATP, present in millimolar amounts in RBCs, was the mediator, which evoked NO synthesis in the vascular endothelium. First, we found that deformation of RBCs, as occurs on passage across the pulmonary circulation with increasing flow rate, evoked increments in ATP release. Here, ATP (300 nM), administered to isolated, salt solution-perfused (PSS) rabbit lungs, decreased total and upstream (arterial) PVR, a response inhibited by NG-nitro-l-arginine methyl ester (l-NAME, 100 μM). In lungs perfused with PSS containing RBCs, l-NAME increased total and upstream PVR. In lungs perfused with PSS containing glibenclamide-treated RBCs, which inhibits ATP release, l-NAME was without effect. Apyrase grade VII (8 U/ml), which degrades ATP to AMP, was without effect on PVR in PSS-perfused lungs. These results are consistent with the hypothesis that ATP, released from RBCs as they traverse the pulmonary circulation, evokes endogenous NO synthesis.
Renal denervation chronically lowers arterial pressure independent of dietary sodium intake in normal rats
Authors: Jacob Frédéric , Ariza Pilar , Osborn John W.
Published: March 2015

The present study was designed to test the hypothesis that renal nerves chronically modulate arterial pressure (AP) under basal conditions and during changes in dietary salt intake. To test this hypothesis, continuous telemetric recording of AP in intact (sham) and renal denervated (RDNX) Sprague-Dawley rats was performed and the effect of increasing and decreasing dietary salt intake on AP was determined. In protocol 1, 24-h AP, sodium, and water balances were measured in RDNX ( n = 11) and sham ( n = 9) rats during 5 days of normal (0.4% NaCl) and 10 days of high (4.0% NaCl) salt intake, followed by a 3-day recovery period (0.4% NaCl). Protocol 2 was similar with the exception that salt intake was decreased to 0.04% NaCl for 10 days after the 5-day period of normal salt (0.04% NaCl) intake (RDNX; n = 6, sham; n = 5). In protocol 1, AP was lower in RDNX (91 ± 1 mmHg) compared with sham (101 ± 2 mmHg) rats during the 5-day 0.4% NaCl control period. During the 10 days of high salt intake, AP increased <5 mmHg in both groups so that the difference between sham and RDNX rats remained constant. In protocol 2, AP was also lower in RDNX (93 ± 2 mmHg) compared with sham (105 ± 4 mmHg) rats during the 5-day 0.4% NaCl control period, and AP did not change in response to 10 days of a low-salt diet in either group. Overall, there were no between-group differences in sodium or water balance in either protocol. We conclude that renal nerves support basal levels of AP, irrespective of dietary sodium intake in normal rats.
Gender differences on the effects of aging on cardiac and peripheral adrenergic stimulation in old conscious monkeys
Authors: Takagi Gen , Asai Kuniya , Vatner Stephen F. , Kudej Raymond K. , Rossi Franco , Peppas Athanasios , Takagi Ikuyo , Resuello Ranillo R. G. , Natividad Filipinas , Shen You-Tang , Vatner Dorothy E.
Published: March 2015

We examined the effects of gender and aging on cardiac and peripheral hemodynamic responses to β-adrenergic receptor (β-AR) stimulation in young (male = 5.9 ± 0.4 yr old and female = 6.5 ± 0.7 yr old) and old (male = 19.8 ± 0.7 yr old and female = 21.2 ± 0.2 yr old) conscious monkeys ( Macaca fascicularis), chronically instrumented for measurements of left ventricular (LV) and arterial pressures as well as cardiac output. Baseline LV pressure, the first derivative of LV pressure (LV dP/d t), cardiac index, mean arterial pressure, total peripheral resistance (TPR), and heart rate in conscious monkeys were not different among the four groups. Increases in LV dP/d t in response to 0.1 μg/kg isoproterenol (Iso) were diminished ( P < 0.05) in old males (+99 ± 11%) compared with young males (+194 ± 18%). In addition, the inotropic responses to norepinephrine (NE) and forskolin (FSK) were significantly depressed ( P < 0.05) in old males. Iso-induced reductions of TPR were less ( P < 0.05) in old males (–28 ± 2%) than in young males (–49 ± 2%). The changes of TPR in response to NE and FSK were also significantly attenuated ( P < 0.05) in old males. However, the LV dP/d t responses to BAY y 5959 (15 μg · kg–1 · min–1), a Ca2+ channel promotor independent of β-AR signaling, were not significantly different between old and young males. In contrast to results in male monkeys, LV dP/d t and TPR responses to Iso, NE, and FSK in old females were similar to those observed in young females. Thus both cardiac contractile and peripheral vascular dynamic responses to β-AR stimulation are preserved in old female but not old male monkeys. This may explain, in part, the reduced cardiovascular risk in the older female population.
Role of δ-opioid receptor agonists on infarct size reduction in swine
Authors: Sigg Daniel C. , Coles James A. , Oeltgen Peter R. , Iaizzo Paul A.
Published: March 2015

Opioids are involved in cardiac ischemic preconditioning. Important species differences in cellular signaling mechanisms, antiarrhythmic, and antistunning effects have been described. The role of the δ-opioid receptor activation in swine remains unknown. Forty minutes before a 45-min occlusion and 180-min reperfusion of the left anterior descending coronary artery, open-chest, pentobarbital-anesthetized swine received either 1) saline (controls); 2) [d-Ala2,d-Leu5]enkephalin (DADLE); 3) [d-Pen2,5]enkephalin (DPDPE); 4) deltorphin-D, a novel δ2-opioid agonist; or 5) ischemic preconditioning (IP). Assessed were 1) infarct size to area at risk (IS, triphenyltetrazolium staining), 2) regional and global myocardial function (sonomicrometry, ventricular pressure catheters), and 3) arrhythmias (electrocardiogram analyses). It was found that DPDPE and deltorphin-D pretreatment reduced IS from 64.7 ± 5 to 36.5 ± 6% and 27.4 ± 11% ( P < 0.01), respectively, whereas DADLE had no effect (66.8 ± 3%). Both IP and DADLE had a proarrhythmic effect ( P < 0.01). However, no differences in global or regional myocardial function or arrhythmia scores were observed between groups. This suggests that δ-receptor-specific opioids provide cardioprotection in swine.
Cytochrome P-450 epoxygenase products contribute to attenuated vasoconstriction after chronic hypoxia
Authors: Earley Scott , Pastuszyn Andrzej , Walker Benjimen R.
Published: March 2015

The systemic vasculature exhibits attenuated vasoconstriction following chronic hypoxia (CH) that is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization. We hypothesized that increased production of arachidonic acid metabolites such as the cyclooxygenase product prostacyclin or cytochrome P-450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) contributes to VSM cell hyperpolarization following CH. VSM cell resting membrane potential ( Em) was measured in superior mesenteric artery strips isolated from rats with control barometric pressure (Pb, ≅630 Torr) and CH (Pb, 380 Torr for 48 h). VSM cell Em was normalized between groups following administration of the CYP inhibitors 17-octadecynoic acid and SKF-525A. VSM cell hyperpolarization after CH was not altered by cyclooxygenase inhibition, whereas the selective CYP2C9 inhibitor sulfaphenazole normalized VSM cell Em between groups. Iberiotoxin also normalized VSM cell Em, which suggests that large-conductance, Ca2+-activated K+ (BKCa) channel activity is increased after CH. Sulfaphenazole administration restored phenylephrine-induced and myogenic vasoconstriction and Ca2+ responses of mesenteric resistance arteries isolated from CH rats to control levels. Western blot experiments demonstrated that CYP2C9 protein levels were greater in mesenteric arteries from CH rats. In addition, 11,12-EET levels were elevated in endothelial cells from CH rats compared with controls. We conclude that enhanced CYP2C9 expression and 11,12-EET production following CH contributes to BKCa channel-dependent VSM cell hyperpolarization and attenuated vasoreactivity.
Gender differences in cardiac ACE expression are normalized in androgen-deprived male mice
Authors: Freshour Judy R. , Chase Sharon E. , Vikstrom Karen L.
Published: March 2015

Gender differences have been described in the response of the cardiovascular system to a number of stimuli, including ventricular remodeling in response to pressure overload, but the molecular basis for these differences remains unclear. Because gender differences in the cardiac expression of angiotensin-converting enzyme (ACE) could contribute to differences in myocardial remodeling, we examined myocardial ACE expression in age-matched male and female mice. Ventricular ACE was more abundant in male than female mice at both mRNA and protein levels. These differences became apparent once the mice reached sexual maturity and became more pronounced with increasing age. The influence of mouse gonadal status on ventricular ACE expression was also examined. Oophorectomy slightly increased ACE levels in female mice, whereas ventricular ACE levels were substantially decreased in androgen-deprived males. The antithetical changes in ventricular ACE abundance seen in agonadal male and female mice suggest that testosterone as well as estrogen may play a role in regulating ACE expression in the heart.
Diazoxide triggers cardioprotection against apoptosis induced by oxidative stress
Authors: Ichinose Masashi , Yonemochi Hidetoshi , Sato Toshiaki , Saikawa Tetsunori
Published: March 2015

Although mitochondrial ATP-sensitive potassium (mitoKATP) channels have been reported to reduce the extent of apoptosis, the critical timing of mitoKATP channel opening required to protect myocytes against apoptosis remains unclear. In the present study, we examined whether the mitoKATP channel serves as a trigger of cardioprotection against apoptosis induced by oxidative stress. Apoptosis of cultured neonatal rat cardiomyocytes was determined by flow cytometry (light scatter and propidium iodide/annexin V-FITC fluorescence) and by nuclear staining with Hoechst 33342. Mitochondrial membrane potential (ΔΨ) was measured by flow cytometry of cells stained with rhodamine-123 (Rh-123). Exposure to H2O2 (500 μM) induced apoptosis, and the percentage of apoptotic cells increased progressively and peaked at 2 h. This H2O2-induced apoptosis was associated with the loss of ΔΨ, and the time course of decrease in Rh-123 fluorescence paralleled that of apoptosis. Pretreatment of cardiomyocytes with diazoxide (100 μM), a putative mitoKATP channel opener, for 30 min before exposure to H2O2 elicited transient and mild depolarization of ΔΨ and consequently suppressed both apoptosis and ΔΨ loss after 2-h exposure to H2O2. These protective effects of diazoxide were abrogated by the mitoKATP channel blocker 5-hydroxydecanoate (500 μM) but not by the sarcolemmal KATP channel blocker HMR-1098 (30 μM). Our results suggest for the first time that diazoxide-induced opening of mitoKATP channels triggers cardioprotection against apoptosis induced by oxidative stress in rat cardiomyocytes.
Regional gap junction inhibition increases defibrillation thresholds
Authors: Sims J. Jason , Schoff Kell L. , Loeb Jennifer M. , Wiegert Nicholas A.
Published: March 2015

It is clear that ischemia inhibits successful defibrillation by altering regional electro-physiology. However, the exact mechanisms are unclear. This study investigated whether regional gap junction inhibition increases biphasic shock defibrillation thresholds (DFT). Sixteen swine were instrumented with a mid-left anterior descending (LAD) perfusion catheter for regional infusion of 0.5 mM/h heptanol ( n = 8) or saline ( n = 8). DFT values and effective refractory periods (ERP) at five myocardial sites were determined. Regional conduction velocity (CV) was determined in an LAD drug-perfused and nondrug-perfused region in an additional seven swine. Regional heptanol infusion increased 50% DFT values by 33% ( P = 0.01) and slowed CV by 42–59% ( P < 0.01) but did not affect ERP. Regional heptanol also increased CV dispersion by ∼270% ( P < 0.05) but did not change ERP dispersion. Regional placebo did not alter any of these parameters. Furthermore, regional heptanol infusion induced spontaneous ventricular fibrillation in eight of eight animals. Increasing spatial conduction velocity dispersion by impairing regional gap junction conductance increased DFT values. Dispersion in conduction velocity slowing during regional ischemia may be an important determinant of defibrillation efficacy.
Effects of aging on capillary geometry and hemodynamics in rat spinotrapezius muscle
Authors: Russell John A. , Kindig Casey A. , Behnke Brad J. , Poole David C. , Musch Timothy I.
Published: March 2015

The effects of aging on muscle microvascular structure and function may play a key role in performance deficits and impairment of O2 exchange within skeletal muscle of senescent individuals. To determine the effects of aging on capillary geometry, red blood cell (RBC) hemodynamics, and hematocrit in a muscle of mixed fiber type, spinotrapezius muscles from Fischer 344 × Brown Norway hybrid rats aged 6–8 mo [young (Y); body mass 421 ± 10 g, n = 6] and 26–28 mo [old (O); 561 ± 12 g, n = 6] were observed by high-resolution transmission light microscopy under resting conditions. The percentage of RBC-perfused capillaries (Y: 78 ± 3%; O: 75 ± 2%) and degree of tortuosity and branching (Y: 13 ± 2%; O: 13 ± 2%, additional capillary length) were not different in O vs. Y muscles. Lineal density of RBC-perfused capillaries in O was significantly reduced (Y: 30.7 ± 1.8, O: 22.8 ± 3.1 capillaries/mm; P < 0.05). However, RBC-perfused capillaries from O rats ( n = 78) exhibited increased RBC velocity ( VRBC) (Y: 219 ± 12, O: 310 ± 14 μm/s; P < 0.05) and RBC flux ( FRBC) (Y: 27 ± 2, O: 41 ± 2 RBC/s; P < 0.05) vs. Y rats ( n = 66). Thus O2 delivery per unit of muscle was not different between groups (Y: 894 ± 111, O: 887 ± 118 RBC · s-1 · mm muscle-1). Capillary hematocrit was not different in Y vs. O rats (Y: 26 ± 1%, O: 28 ± 1%: P > 0.05). These data indicate that in resting spinotrapezius muscle, aging decreases the lineal density of RBC-perfused capillaries while increasing mean VRBC and FRBC within those capillaries. Whereas muscle conductive O2 delivery and capillary hematocrit were unchanged, elevated VRBC reduces capillary RBC transit time and may impair the diffusive transport of O2 from blood to myocyte particularly under exercise conditions.
Correlation of repolarization of ventricular monophasic action potential with ECG in the murine heart
Authors: Danik Stephan , Cabo Candido , Chiello Christine , Kang Sacha , Wit Andrew L. , Coromilas James
Published: March 2015

Transgenic mice have become important experimental models in the investigation of mechanisms causing cardiac arrhythmias because of the ability to create strains with alterations in repolarizing membrane currents. It is important to relate alterations in membrane currents in cells to their phenotypic expression on the electrocardiogram (ECG). The murine ECG, however, has unusual characteristics that make interpretation of the phenotypic expression of changes in ventricular repolarization uncertain. The major deflection representing the QRS (referred to as “ a”) is often followed by a secondary slower deflection (“ b”) and sometimes a subtle third deflection (“ c”). To determine whether the second or third deflections or both represent ventricular repolarization, we recorded the ventricular monophasic action potential (MAP) in open-chest mice and correlated repolarization with the ECG. There was no significant correlation by linear regression, between action potential duration to 50% or 90% repolarization (APD50or APD90), respectively, of the MAP and either the interval from onset of Q to onset of b (Qb interval) or onset of c (Qc interval). Administration of 4-aminopyridine (4-AP) significantly prolonged APD50and APD90and the Qb interval, indicating that this deflection on the ECG represents part of ventricular repolarization. After 4-AP, the c wave disappeared, also suggesting that it represents a component of ventricular repolarization. Although it appears that both the b and c waves that follow the Q wave on the ECG represent ventricular repolarization, neither correlates exactly with APD90of the MAP. Therefore, an accurate measurement of complete repolarization of the murine ventricle cannot be obtained from the surface ECG.
Neuronal control of heart rate in isolated mouse atria
Authors: Choate J. K. , Feldman R.
Published: March 2015

A novel mouse isolated atrial preparation with intact postganglionic autonomic innervation was used to investigate the neuronal control of heart rate. To establish whether autonomic activation was likely to alter heart rate by modulating the hyperpolarization-activated current ( If), the L-type Ca2+ current ( ICa,L), or the ACh-activated K+ current ( IK,ACh), the effects of nerve stimulation (right stellate ganglion or right vagus, 1–30 Hz) and autonomic agonists (0.1 μM norepinephrine or 0.3 μM carbachol) on heart rate were investigated in the presence of inhibitors of these currents, cesium chloride (Cs+, 1 mM), nifedipine (200 nM), and barium chloride (Ba2+, 0.1 mM), respectively. The positive chronotropic response to stellate ganglion stimulation was reduced by ∼20% with Cs+ and nifedipine ( P < 0.05), whereas the heart rate response to norepinephrine was only reduced with Cs+ ( P < 0.05). Ba2+ attenuated the decrease in heart rate with vagal stimulation and carbachol by ∼60% ( P < 0.05). These results are consistent with the idea that sympathetic nerve stimulation modulates If to increase heart rate in the mouse. Activation of ICa,L also appears to contribute to the sympathetic heart rate response. However, the decrease in heart rate with vagal stimulation or carbachol is likely to result primarily from the activation of IK,ACh.