bluuebunny/crossref_metadata_2025 huggingface dataset:
MRI/MRS assessment of in vivo murine cardiac metabolism, morphology, and function at physiological heart rates
Authors: Chacko V. P. , Aresta Francesca , Chacko Sonia M. , Weiss Robert G.
Published: December 2017

Transgenic mice are increasingly used to probe genetic aspects of cardiovascular pathophysiology. However, the small size and rapid rates of murine hearts make noninvasive, physiological in vivo studies of cardiac bioenergetics and contractility difficult. The aim of this report was to develop an integrated, noninvasive means of studying in vivo murine cardiac metabolism, morphology, and function under physiological conditions by adapting and modifying noninvasive cardiac magnetic resonance imaging (MRI) with image-guided 31P magnetic resonance spectroscopy techniques used in humans to mice. Using spatially localized, noninvasive 31P nuclear magnetic resonance spectroscopy and MRI at 4.7 T, we observe mean murine in vivo myocardial phosphocreatine-to-ATP ratios of 2.0 ± 0.2 and left ventricular ejection fractions of 65 ± 7% at physiological heart rates (∼600 beats/min). These values in the smallest species studied to date are similar to those reported in normal humans. Although these observations do not confirm a degree of metabolic scaling with body size proposed by prior predictions, they do suggest that mice can serve, at least at this level, as a model for human cardiovascular physiology. Thus it is now possible to noninvasively study in vivo myocardial bioenergetics, morphology, and contractile function in mice under physiological conditions.
Simvastatin upregulates coronary vascular endothelial nitric oxide production in conscious dogs
Authors: Mital Seema , Zhang Xiaoping , Zhao Gong , Bernstein Robert D. , Smith Carolyn J. , Fulton David L. , Sessa William C. , Liao James K. , Hintze Thomas H.
Published: December 2017

Statin drugs can upregulate endothelial nitric oxide (NO) synthase (eNOS) in isolated endothelial cells independent of lipid-lowering effects. We investigated the effect of short-term simvastatin administration on coronary vascular eNOS and NO production in conscious dogs and canine tissues. Mongrel dogs were instrumented under general anesthesia to measure coronary blood flow (CBF). Simvastatin (20 mg · kg−1 · day−1) was administered orally for 2 wk; afterward, resting CBF was found to be higher compared with control ( P < 0.05) and veratrine- (activator of reflex cholinergic NO-dependent coronary vasodilation) and ACh-mediated coronary vasodilation were enhanced ( P < 0.05). Response to endothelium-independent vasodilators, adenosine and nitroglycerin, was not potentiated. After simvastatin administration, plasma nitrate and nitrite (NOx) levels increased from 5.22 ± 1.2 to 7.79 ± 1.3 μM ( P < 0.05); baseline and agonist-stimulated NO production in isolated coronary microvessels were augmented ( P < 0.05); resting in vivo myocardial oxygen consumption (MV˙o 2) decreased from 6.8 ± 0.6 to 5.9 ± 0.4 ml/min ( P < 0.05); NO-dependent regulation of MV˙o 2 in response to NO agonists was augmented in isolated myocardial segments ( P < 0.05); and eNOS protein increased 29% and eNOS mRNA decreased 50% in aortas and coronary vascular endothelium. Short-term administration of simvastatin in dogs increases coronary endothelial NO production to enhance NO-dependent coronary vasodilation and NO-mediated regulation of MV˙o 2.
Dobutamine enhances both contractile function and energy reserves in hypoperfused canine right ventricle
Authors: Yi Kun Don , Downey H. Fred , Bian Xiaoming , Fu Min , Mallet Robert T.
Published: December 2017

Although the β1-adrenergic agent dobutamine is used clinically to provide inotropic support to the failing myocardium, it could jeopardize the myocardium by depleting energy reserves. This investigation delineated the contractile and energetic effects of low versus high dobutamine doses in the hypoperfused right ventricular (RV) myocardium. The right coronary artery (RCA) of anesthetized dogs was cannulated for controlled perfusion with arterial blood, and regional RV contractile function was measured. RCA perfusion pressure was lowered from 100 mmHg baseline to 40 mmHg, and flow fell by 54%. At 15-min hypoperfusion, dobutamine was infused into the RCA at either 0.01 (low-dose dobutamine) or 0.06 μg · kg−1· min−1(high-dose dobutamine) for 15 min. Regional power (systolic segment shortening × isometric developed force × heart rate) stabilized at 63% of baseline during hypoperfusion. Low-dose dobutamine restored power to baseline but did not increase RV myocardial O2consumption (MV˙o2) and thus increased myocardial O2utilization efficiency (O2UE:power/MV˙o2). At 5 min, high-dose dobutamine enhancement of power was similar to that of low-dose dobutamine, but by 15 min, power and O2UE fell to untreated levels. Remarkably, low-dose dobutamine tripled cytosolic phosphorylation potential; in contrast, high-dose dobutamine lowered phosphorylation potential to 45% of the untreated value. Analyses of glucose uptake and glycolytic intermediates revealed sustained enhancement of glycolysis by low-dose dobutamine, but glycolysis became limited at glyceraldehyde 3-phosphate dehydrogenase during high-dose dobutamine treatment. In summary, low-dose dobutamine improved mechanical performance and efficiency of the hypoperfused RV myocardium while increasing myocardial energy reserves, but high-dose dobutamine failed to sustain improved function and depleted energy reserves. Dobutamine is capable of improving both contractile function and cellular energetics in the hypoperfused RV myocardium, but dosage should be carefully selected.
Heat-shock factor-1, steroid hormones, and regulation of heat-shock protein expression in the heart
Authors: Knowlton A. A. , Sun Limin
Published: December 2017

Heat-shock proteins (HSPs) are an important family of endogenous, protective proteins. Overexpression of HSPs is protective against cardiac injury. Previously, we observed that dexamethasone activated heat-shock factor-1 (HSF-1) and induced a 60% increase in HSP72 in adult cardiac myocytes. The mechanism responsible for this effect of dexamethasone is unknown. Because HSP90 is known to bind the intracellular hormone receptors, we postulated that the interaction between HSP90, the receptors, and HSF was an important element in activation of HSF-1 by hormones. We hypothesized that there is an equilibrium between HSP90 and the various receptors/enzymes that it binds and that alteration in levels of certain hormones will alter the intracellular distribution of HSP90 and activate HSF-1. We report that, in adult cardiac myocytes, HSF-1 coimmunoprecipitates with HSP90. HSP90 redistributes in cardiac myocytes after treatment with 17β-estradiol or progesterone. Estrogen and progesterone activate HSF-1 in adult male isolated cardiac myocytes, and this is followed by an increase in HSP72 protein. Testosterone had no effect on HSP levels; however, no androgen receptor was found in cardiac myocytes; therefore, testosterone would not be expected to effect binding of HSP90 to HSF. Geldanamycin, which inactivates HSP90 and prevents it from binding to receptors, activates HSF-1 and stimulates HSP72 synthesis. Activation of HSF-1 by steroid hormones, resulting from a change in the interaction of HSP90 and HSF-1, represents a novel pathway for regulating expression of HSPs. These findings may explain some of the gender differences in cardiovascular disease.
Selectin-independent leukocyte rolling and adhesion in mice deficient in E-, P-, and L-selectin and ICAM-1
Authors: Forlow S. Bradley , Ley Klaus
Published: December 2017

To study selectin-independent leukocyte recruitment and the role of intercellular adhesion molecule-1 (ICAM-1), we generated mice lacking all three selectins and ICAM-1 (E/P/L/I−/−) by bone marrow transplantation. These mice were viable and appeared healthy under vivarium conditions, although they showed a 97% reduction in leukocyte rolling, a 63% reduction in leukocyte firm adhesion, and a 99% reduction of neutrophil recruitment in a thioglycollate-induced model of peritonitis at 4 and 24 h. Mononuclear cell recruitment was almost unaffected. All residual leukocyte rolling and most leukocyte adhesion in these mice depended on α4-integrins, but a small number of leukocytes (6% of wild-type control) still became adherent in the absence of all known rolling mechanisms (E-, P-, L-selectin and α4-integrins). A striking similarity of leukocyte adhesion efficiency in E/P/L−/− and E/P/I−/− mice suggests a pathway in which leukocyte rolling through L-selectin requires ICAM-1 for adhesion and recruitment. Comparison of our data with mice lacking individual or other combinations of adhesion molecules reveal that elimination of more adhesion molecules further reduces leukocyte recruitment but the effect is less than additive.
Inhaled nitric oxide-induced rebound pulmonary hypertension: role for endothelin-1
Authors: McMullan D. Michael , Bekker Janine M. , Johengen Michael J. , Hendricks-Munoz Karen , Gerrets Rene , Black Stephen M. , Fineman Jeffrey R.
Published: December 2017

Clinically significant increases in pulmonary vascular resistance have been noted on acute withdrawal of inhaled nitric oxide (NO). Endothelin (ET)-1 is a vasoactive peptide produced by the vascular endothelium that may participate in the pathophysiology of pulmonary hypertension. The objectives of this study were to determine the effects of inhaled NO on endogenous ET-1 production in vivo in the intact lamb and to determine the potential role of ET-1 in the rebound pulmonary hypertension associated with the withdrawal of inhaled NO. Seven 1-mo-old vehicle-treated control lambs and six PD-156707 (an ETA receptor antagonist)-treated lambs were mechanically ventilated. Inhaled NO (40 parts per million) was administered for 24 h and then acutely withdrawn. After 24 h of inhaled NO, plasma ET-1 levels increased by 119.5 ± 42.2% ( P < 0.05). Western blot analysis revealed that protein levels of preproET-1, endothelin-converting enzyme-1α, and ETA and ETB receptors were unchanged. On acute withdrawal of NO, pulmonary vascular resistance (PVR) increased by 77.8% ( P < 0.05) in control lambs but was unchanged (−5.5%) in PD-156707-treated lambs. Inhaled NO increased plasma ET-1 concentrations but not gene expression in the intact lamb, and ETA receptor blockade prevented the increase in PVR after NO withdrawal. These data suggest a role for ET-1 in the rebound pulmonary hypertension noted on acute withdrawal of inhaled NO.
Effects of endotoxin on neutrophil-mediated I/R injury in isolated perfused rat hearts
Authors: Lipton Brian P. , Bautista Abraham P. , Delcarpio Joseph B. , McDonough Kathleen H.
Published: December 2017

With the use of a syngeneic model, we demonstrate that rat polymorphonuclear neutrophils (PMNs) exacerbate ischemia-reperfusion injury in the isolated rat heart. However, PMNs (19 × 106cells) from lipopolysaccharide (LPS)-treated rats (LPS-PMNs; 100 mg/kg administered 7 h before exsanguination) induce less reperfusion injury in the isolated heart. Average recovery of left ventricular developed pressure after 20 min of ischemia and 60 min of reperfusion was 51 ± 4% in hearts receiving PMNs from saline-treated control rats (saline-PMNs) versus 78 ± 2% in hearts receiving LPS-PMNs. Ischemic hearts reperfused with LPS-PMNs recovered to the same extent as did hearts reperfused with Krebs buffer only. LPS-PMNs and saline-PMNs showed no difference in basal or phorbol ester-induced superoxide production. Whereas twice the number of LPS-PMNs was positive for nitroblue tetrazolium, the percent positive for L-selectin, a receptor integral in PMN-adhesion to endothelium, was 50% less in LPS-PMNs than in controls. After reperfusion, three-fourths of the saline-PMNs remained within the hearts, whereas only one-fourth of LPS-PMNs were trapped. These data suggest that PMNs from LPS-treated rats do not exacerbate ischemia-reperfusion injury as do control PMNs, possibly, due to impaired PMN adhesion to endothelium as a result of decreased L-selectin receptors.
Regional differences in mechanisms of cerebral circulatory response to neuronal activation
Authors: Gotoh Jun , Kuang Tang-Yong , Nakao Yasuaki , Cohen David M. , Melzer Peter , Itoh Yoshiaki , Pak Hazel , Pettigrew Karen , Sokoloff Louis
Published: December 2017

Vibrissal stimulation raises cerebral blood flow (CBF) in the ipsilateral spinal and principal sensory trigeminal nuclei and contralateral ventroposteromedial (VPM) thalamic nucleus and barrel cortex. To investigate possible roles of adenosine and nitric oxide (NO) in these increases, local CBF was determined during unilateral vibrissal stimulation in unanesthetized rats after adenosine receptor blockade with caffeine or NO synthase inhibition with N G-nitro-l-arginine methyl ester (l-NAME) or 7-nitroindazole (7-NI). Caffeine lowered baseline CBF in all structures but reduced the percent increase during stimulation only in the two trigeminal nuclei. l-NAME and 7-NI lowered baseline CBF but reduced the percent increase during stimulation only in the higher stations of this sensory pathway, i.e.,l-NAME in the VPM nucleus and 7-NI in both the VPM nucleus and barrel cortex. Combinations of caffeine with 7-NI orl-NAME did not have additive effects, and none alone or in combination completely eliminated functional activation of CBF. These results suggest that caffeine-sensitive and NO-dependent mechanisms are involved but with different regional distributions, and neither fully accounts for the functional activation of CBF.
Inhibitory effect of valves on endothelium-dependent relaxations to calcium ionophore in canine saphenous vein
Authors: Eguchi Daihiko , Katusic Zvonimir S.
Published: December 2017

The present study was designed to evaluate endothelium-dependent relaxation to the calcium ionophore A-23187 in isolated canine saphenous veins. Isometric force recordings and cGMP measurements using isolated veins with and without valves were performed. During contractions to U-46619 (3 × 10−7 M), endothelium-dependent relaxations to A-23187 (10−9–10−6 M) were significantly reduced in rings with valves compared with rings without valves. Endothelial removal abolished A-23187-induced relaxation. Relaxations to forskolin (FK; 10−8–10−5 M) and diethylaminodiazen-1-ium-1,2-dionate; DEA-NONOate, 10−9–10−5 M) were identical in rings with and without valves. In rings without valves, a nitric oxide synthase inhibitor, N G-nitro-l-arginine methyl ester (l-NAME; 3 × 10−4 M), and a cyclooxygenase inhibitor, indomethacin (10−5 M), partially reduced A-23187-induced relaxation. However, in rings with valves,l-NAME had no effect, whereas indomethacin abolished the relaxation to A-23187. A selective soluble guanylate cyclase inhibitor, 1 H-[1,2,4]-oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 3×10−6 M), had no effect on the relaxation to A-23187 in either group. In contrast, ODQ abolished the A-23187-induced increase in cGMP levels, suggesting that relaxation to nitric oxide released by A-23187 is independent of increases in cGMP. These results demonstrate that endothelium-dependent relaxation to A-23187 is reduced in regions of veins with valves compared with relaxation in the nonvalvular venous wall. Lower production of nitric oxide in endothelial cells of valvular segments appears to be a mechanism responsible for reduced reactivity to A-23187.
Effect of changing vascular volume on measurement of protein reflection coefficient in ischemic lungs
Authors: Pearse David B. , Becker Patrice M. , Permutt Solbert
Published: December 2017

In ischemic organs, the protein reflection coefficient (ς) can be estimated by measuring blood hematocrit (Hct) and protein after increasing static vascular pressure (Pv). Our original equation for ς ( J Appl Physiol 73: 2616–2622, 1992) assumed a constant vascular volume during convective fluid flux ( J˙). In this study, we 1) quantified the rate of vascular volume change (dV/d t) still present in ischemic single ferret lungs after 20 min of Pv = 30 Torr and 2) developed an equation for ς that allowed a finite dV/d t. In 25 lungs, we estimated the dV/d t after 20 min at Pv = 30 Torr by subtracting J˙ from the rate of lung weight gain (W˙L). The relationship between J˙ (0.15 ± 0.02 ml/min) andW˙L (0.24 ± 0.02 g/min) was significant ( R = 0.66, P < 0.001), but the slope was <1 (0.41 ± 0.10, P < 0.05). dV/d t(0.10 ± 0.02 ml/min) was similar in magnitude to J˙ at 20 min. The modified equation for ς revealed that a finite dV/d t caused the original ς measurement to underestimate true ς. A low ς, high J˙, high baseline Hct, and long filtration time enhanced the error. The error was small, however, and could be minimized by adjusting experimental parameters.
Elevated capillary tube hematocrit reflects degradation of endothelial cell glycocalyx by oxidized LDL
Authors: Constantinescu Alina A. , Vink Hans , Spaan Jos A. E.
Published: December 2017

Proteoglycans and plasma proteins bound to the endothelial cell glycocalyx are essential for vascular function, but at the same time, they lower capillary tube hematocrit by reducing capillary volume available to flowing blood. Because oxidized low-density lipoproteins (oxLDL) reduce the effective thickness of the glycocalyx (Vink H, Constantinescu AA, and Spaan JAE. Circulation 101: 1500–1502, 2000), we designed the present study to determine whether this is caused by pathological degradation of glycocalyx constituents or increased glycocalyx deformation by elevated shear forces of flowing blood. Capillaries from the right cremaster muscle of 24 hamsters were examined by using intravital microscopy after systemic administration of normal LDL ( n = 4), moderate oxLDL (6-h oxidation with CuSO4, n = 7), severe oxLDL (18-h oxidation, n = 5), and moderate oxLDL plus superoxide dismutase (SOD) and catalase ( n = 8). Capillary tube hematocrit increased from 0.16 ± 0.03 to 0.37 ± 0.05 and from 0.15 ± 0.01 to 0.31 ± 0.03 after moderate oxLDL and severe oxLDL, respectively. These changes were paralleled by increases in red blood cell flux from 8.7 ± 1.9 to 13.8 ± 3 and from 10.7 ± 2.1 to 16.3 ± 3.2 cells/s after moderate oxLDL and severe oxLDL, respectively, in the absence of changes in anatomic capillary diameter. Red blood cell velocity, as a measure for the shear forces on the glycocalyx, was not affected by oxLDL, whereas tissue pretreatment with SOD and catalase completely abolished the effects of oxLDL on glycocalyx thickness, capillary hematocrit, and red blood cell flux. We conclude that elevation of capillary tube hematocrit by oxLDL reflects degradation of the endothelial glycocalyx by oxygen-derived free radicals.
Gender and aging in a transgenic mouse model of hypertrophic cardiomyopathy
Authors: Olsson M. Charlotte , Palmer Bradley M. , Leinwand Leslie A. , Moore Russell L.
Published: December 2017

Mutations in the cardiac myosin heavy chain (MHC) can cause familial hypertrophic cardiomyopathy (FHC). A transgenic mouse model has been developed in which a missense (R403Q) allele and an actin-binding deletion in the α-MHC are expressed in the heart. We used an isovolumic left heart preparation to study the contractile characteristics of hearts from transgenic (TG) mice and their wild-type (WT) littermates. Both male and female TG mice developed left ventricular (LV) hypertrophy at 4 mo of age. LV hypertrophy was accompanied by LV diastolic dysfunction, but LV systolic function was normal and supranormal in the young TG females and males, respectively. At 10 mo of age, the females continued to present with LV concentric hypertrophy, whereas the males began to display LV dilation. In female TG mice at 10 mo of age, impaired LV diastolic function persisted without evidence of systolic dysfunction. In contrast, in 10-mo-old male TG mice, LV diastolic function worsened and systolic performance was impaired. Diminished coronary flow was observed in both 10-mo-old TG groups. These types of changes may contribute to the functional decompensation typically seen in hypertrophic cardiomyopathy. Collectively, these results further underscore the potential utility of this transgenic mouse model in elucidating pathogenesis of FHC.
In vivo assessment of microvascular nitric oxide production and its relation with blood flow
Authors: Figueroa X. F. , Martínez A. D. , González D. R. , Jara P. I. , Ayala S. , Boric M. P.
Published: December 2017

To assess the hypothesis that microvascular nitric oxide (NO) is critical to maintain blood flow and solute exchange, we quantified NO production in the hamster cheek pouch in vivo, correlating it with vascular dynamics. Hamsters (100–120 g) were anesthetized and prepared for measurement of microvessel diameters by intravital microscopy, of plasma flow by isotopic sodium clearance, and of NO production by chemiluminescence. Analysis of endothelial NO synthase (eNOS) location by immunocytochemistry and subcellular fractionation revealed that eNOS was present in arterioles and venules and was 67 ± 7% membrane bound. Basal NO release was 60.1 ± 5.1 pM/min ( n = 35), and plasma flow was 2.95 ± 0.27 μl/min ( n = 29). Local NO synthase inhibition with 30 μM N ω-nitro-l-arginine reduced NO production to 8.6 ± 2.6 pmol/min (−83 ± 5%, n = 9) and plasma flow to 1.95 ± 0.15 μl/min (−28 ± 12%, n = 17) within 30–45 min, in parallel with constriction of arterioles (9–14%) and venules (19–25%). The effects of N ω-nitro-l-arginine (10–30 μM) were proportional to basal microvascular conductance ( r = 0.7, P < 0.05) and fully prevented by 1 mM l-arginine. We conclude that in this tissue, NO production contributes to 35–50% of resting microvascular conductance and plasma-tissue exchange.
Endothelial cell protein kinase G inhibits release of EDHF through a PKG-sensitive cation channel
Authors: Dora K. A. , Garland C. J. , Kwan H. Y. , Yao X.
Published: December 2017

The release of dilator agents from vascular endothelial cells is modulated by changes in cytosolic Ca2+ concentration ([Ca2+]i). In this study, we demonstrate the presence of a Ca2+-permeable cation channel in inside-out membrane patches of endothelial cells isolated from small mesenteric arteries. The activity of the channel is increased by KT-5823, a highly selective inhibitor of protein kinase G (PKG), while it is decreased by direct application of active PKG. Application of KT-5823 induces Ca2+ influx in the endothelial cells isolated from small mesenteric arteries, and it also causes endothelium-dependent relaxations in isolated small mesenteric arteries. KT-5823-induced relaxations in small mesenteric arteries are greatly reduced by 35 mM K+ or 50 nM charybdotoxin + 50 nM apamin, suggesting that endothelium-derived hyperpolarizing factor (EDHF) is the participating dilator. The involvement of EDHF is further supported by experiments in which the relaxations of small mesenteric arteries are shown to be accompanied by membrane repolarization. These data strongly argue for a major role of a PKG-sensitive cation channel in modulating the release of EDHF from endothelial cells in rat small mesenteric arteries.
Stenosis differentially affects subendocardial and subepicardial arterioles in vivo
Authors: Merkus D. , Vergroesen I. , Hiramatsu O. , Tachibana H. , Nakamoto H. , Toyota E. , Goto M. , Ogasawara Y. , Spaan J. A. E. , Kajiya F.
Published: December 2017

The presence of a coronary stenosis results primarily in subendocardial ischemia. Apart from the decrease in coronary perfusion pressure, a stenosis also decreases coronary flow pulsations. Applying a coronary perfusion system, we compared the autoregulatory response of subendocardial ( n = 10) and subepicardial ( n = 12) arterioles (<120 μm) after stepwise decreases in coronary arterial pressure from 100 to 70, 50, and 30 mmHg in vivo in dogs ( n = 9). Pressure steps were performed with and without stenosis on the perfusion line. Maximal arteriolar diameter during the cardiac cycle was determined and normalized to its value at 100 mmHg. The initial decrease in diameter during reductions in pressure was significantly larger at the subendocardium. Diameters of subendocardial and subepicardial arterioles were similar 10–15 s after the decrease in pressure without stenosis. However, stenosis decreased the dilatory response of the subendocardial arterioles significantly. This decreased dilatory response was also evidenced by a lower coronary inflow at similar average pressure in the presence of a stenosis. Inhibition of nitric oxide production with N G-monomethyl-l-arginine abrogated the effect of the stenosis on flow. We conclude that the decrease in pressure caused by a stenosis in vivo results in a larger decrease in diameter of the subendocardial arterioles than in the subepicardial arterioles, and furthermore stenosis selectively decreases the dilatory response of subendocardial arterioles. These two findings expand our understanding of subendocardial vulnerability to ischemia.
Heat pretreatment differentially affects cardiac fatty acid accumulation during ischemia and postischemic reperfusion
Authors: Cornelussen Richard N. M. , Van Der Vusse Ger J. , Roemen Theo H. M. , Snoeckx Luc H. E. H.
Published: December 2017

We investigated whether the cardioprotection induced by heat stress (HS) pretreatment is associated with mitigation of phospholipid degradation during the ischemic and/or postischemic period. The hearts, isolated from control rats and from heat-pretreated rats (42°C for 15 min) either 30 min (HS0.5-h) or 24 h (HS24-h) earlier, were subjected to 45 min of no-flow ischemia, followed by 45 min of reperfusion. Unesterified arachidonic acid (AA) accumulation was taken as a measure for phospholipid degradation. Significantly improved postischemic ventricular functional recovery was only found in the HS24-h group. During ischemia, AA accumulated comparably in control and both HS groups. During reperfusion in control and HS0.5-h hearts, AA further accumulated (control hearts from 82 ± 33 to 109 ± 51 nmol/g dry wt, not significant; HS-0.5h hearts from 52 ± 22 to 120 ± 53 nmol/g dry wt; P < 0.05). In contrast, AA was lower at the end of the reperfusion phase in HS24-h hearts than at the end of the preceding ischemic period (74 ± 18 vs. 46 ± 23 nmol/g dry wt; P < 0.05). Thus accelerated reperfusion-induced degradation of phospholipids in control hearts is completely absent in HS24-h hearts. Furthermore, the lack of functional improvement in HS0.5-h hearts is also associated with a lack of beneficial effect on lipid homeostasis. Therefore, it is proposed that enhanced membrane stability during reperfusion is a key mediator in the heat-induced cardioprotection.
Dichloroacetate improves cardiac efficiency after ischemia independent of changes in mitochondrial proton leak
Authors: Taniguchi Masayuki , Wilson Craig , Hunter Charlene A. , Pehowich Daniel J. , Clanachan Alexander S. , Lopaschuk Gary D.
Published: December 2017

Dichloroacetate (DCA) is a pyruvate dehydrogenase activator that increases cardiac efficiency during reperfusion of ischemic hearts. We determined whether DCA increases efficiency of mitochondrial ATP production by measuring proton leak in mitochondria from isolated working rat hearts subjected to 30 min of ischemia and 60 min of reperfusion. In untreated hearts, cardiac work and efficiency decreased during reperfusion to 26% and 40% of preischemic values, respectively. Membrane potential was significantly lower in mitochondria from reperfused (175.6 ± 2.2 mV) versus aerobic (185.8 ± 3.1 mV) hearts. DCA (1 mM added at reperfusion) improved recovery of cardiac work (1.9-fold) and efficiency (1.5-fold) but had no effect on mitochondrial membrane potential (170.6 ± 2.9 mV). At the maximal attainable membrane potential, O2consumption (nmol O2· mg−1· min−1) did not differ between untreated or DCA-treated hearts (128.3 ± 7.5 and 120.6 ± 7.6, respectively) but was significantly greater than aerobic hearts (76.6 ± 7.6). During reperfusion, DCA increased glucose oxidation 2.5-fold and decreased H+production from glucose metabolism to 53% of untreated hearts. Because H+production decreases cardiac efficiency, we suggest that DCA increases cardiac efficiency during reperfusion of ischemic hearts by increasing the efficiency of ATP use and not by increasing the efficiency of ATP production.
Cardiomyopathy in transgenic mice with cardiac-specific overexpression of serum response factor
Authors: Zhang Xiaomin , Azhar Gohar , Chai Jianyuan , Sheridan Pamela , Nagano Koichiro , Brown Thomas , Yang Jihong , Khrapko Konstantin , Borras Ana M. , Lawitts Joel , Misra Ravi P. , Wei Jeanne Y.
Published: December 2017

Serum response factor (SRF), a member of the MCM1, agamous, deficiens, SRF (MADS) family of transcriptional activators, has been implicated in the transcriptional control of a number of cardiac muscle genes, including cardiac α-actin, skeletal α-actin, α-myosin heavy chain (α-MHC), and β-MHC. To better understand the in vivo role of SRF in regulating genes responsible for maintenance of cardiac function, we sought to test the hypothesis that increased cardiac-specific SRF expression might be associated with altered cardiac morphology and function. We generated transgenic mice with cardiac-specific overexpression of the human SRF gene. The transgenic mice developed cardiomyopathy and exhibited increased heart weight-to-body weight ratio, increased heart weight, and four-chamber dilation. Histological examination revealed cardiomyocyte hypertrophy, collagen deposition, and interstitial fibrosis. SRF overexpression altered the expression of SRF-regulated genes and resulted in cardiac muscle dysfunction. Our results demonstrate that sustained overexpression of SRF, in the absence of other stimuli, is sufficient to induce cardiac change and suggest that SRF is likely to be one of the downstream effectors of the signaling pathways involved in mediating cardiac hypertrophy.
Human cardiac myosin heavy chain isoforms in fetal and failing adult atria and ventricles
Authors: Reiser Peter J. , Portman Michael A. , Ning Xue-Han , Moravec Christine Schomisch
Published: December 2017

The goal of this study was to test the hypothesis that the relative amounts of the cardiac myosin heavy chain (MHC) isoforms MHC-α and MHC-β change during development and transition to heart failure in the human myocardium. The relative amounts of MHC-α and MHC-β in ventricular and atrial samples from fetal (gestational days 47–110) and nonfailing and failing adult hearts were determined. The majority of the fetal right and left ventricular samples contained small relative amounts of MHC-α (mean < 5% of total MHC). There was a small significant decrease in the level of MHC-α in the ventricles between 7 and 12 wk of gestation. Fetal atria expressed predominantly MHC-α (mean > 95%), with MHC-β being detected in most samples. The majority of adult nonfailing right and left ventricular samples had detectable levels of MHC-α ranging from 1 to 10%. Failing right and left ventricles expressed a significantly lower level of MHC-α. MHC-α comprised ∼90% of the total MHC in adult nonfailing left atria, whereas the relative amount of MHC-α in the left atria of individuals with dilated or ischemic cardiomyopathy was ∼50%. The differences in MHC isoform composition between fetal and nonfailing adult atria and between fetal and nonfailing adult ventricles were not statistically significant. We concluded that the MHC isoform compositions of fetal human atria are the same as those of nonfailing adult atria and that the ventricular MHC isoform composition is different between adult nonfailing and failing hearts. Furthermore, the marked alteration in atrial MHC isoform composition, associated with cardiomyopathy, does not represent a regression to a pattern that is uniquely characteristic of the fetal stage.
Influence of timing and magnitude of arterial wave reflection on left ventricular relaxation
Authors: Yano Masafumi , Kohno Michihiro , Kobayashi Shigeki , Obayashi Masakazu , Seki Kohzaburo , Ohkusa Tomoko , Miura Toshiro , Fujii Takashi , Matsuzaki Masunori
Published: December 2017

The influence of timing and magnitude of arterial wave reflection (WR) on afterload-dependent relaxation was evaluated in patients with a variety of heart diseases ( group 1, age < 30 yr; group 2, age > 40 yr) and in dogs. While both femoral arteries were compressed (FC), WR returned just after the dicrotic notch (early diastole) in group 1but before the dicrotic notch (late systole) in group 2. The time constant of the left ventricular pressure decay (τ) was shortened during FC in group 1, whereas it was prolonged in group 2. In dogs, a constriction of the thoracic aorta induced a late systolic augmentation of WR with a prolongation of τ (cf. group 2), whereas constriction of the lower abdominal aorta induced an early diastolic augmentation of WR with a shortening of τ (cf. group 1). With aortic constriction, coronary flow increased, and there was a close correlation between the peak change in backward aortic pressure and that in coronary flow regardless of the timing of WR. Thus the time at which WR returns during the cardiac cycle may have an important effect on left ventricular relaxation and coronary flow.
Pregnancy enhances G protein activation and nitric oxide release from uterine arteries
Authors: Thompson L. P. , Weiner C. P.
Published: December 2017

We hypothesized that pregnancy modulates receptor-mediated responses of the uterine artery (UA) by altering G protein activation or coupling. Relaxation and contraction to NaF (0.5–11.5 mM), acetylcholine (10−9–10−5M), and bradykinin (10−12–3 × 10−5M) were measured in isolated UA of pregnant and nonpregnant guinea pigs. Responses were measured in the presence and absence of either cholera toxin (2 μg/ml) or pertussis toxin (Gαsand Gαiinhibitors, respectively). NaF relaxation was endothelium dependent and nitro-l-arginine sensitive (a nitric oxide synthase inhibitor). Relaxation to NaF, acetylcholine, and bradykinin were potentiated by pregnancy. Cholera but not pertussis toxin increased relaxation to acetylcholine and bradykinin in UA from nonpregnant animals, had no effect in UA from pregnant animals, and abolished the pregnancy-induced differences in acetylcholine relaxation. Cholera toxin potentiated the bradykinin-induced contraction of UA of both pregnant and nonpregnant animals, whereas pertussis toxin inhibited contraction of UA from pregnant animals only. Therefore, pregnancy may enhance agonist-stimulated endothelium-dependent relaxation and bradykinin-induced contraction of UA by inhibiting GTPase activity or enhancing Gαsbut not Gαiactivation in pregnant animals. Thus the diverse effects of pregnancy on UA responsiveness may result from hormonal modulation of G proteins coupled to their specific receptors.
Gap junctions in the rabbit sinoatrial node
Authors: Verheule Sander , van Kempen Marjan J. A. , Postma Sjoerd , Rook Martin B. , Jongsma Habo J.
Published: December 2017

In comparison to the cellular basis of pacemaking, the electrical interactions mediating synchronization and conduction in the sinoatrial node are poorly understood. Therefore, we have taken a combined immunohistochemical and electrophysiological approach to characterize gap junctions in the nodal area. We report that the pacemaker myocytes in the center of the rabbit sinoatrial node express the gap junction proteins connexin (Cx)40 and Cx46. In the periphery of the node, strands of pacemaker myocytes expressing Cx43 intermingle with strands expressing Cx40 and Cx46. Biophysical properties of gap junctions in isolated pairs of pacemaker myocytes were recorded under dual voltage clamp with the use of the perforated-patch method. Macroscopic junctional conductance ranged between 0.6 and 25 nS with a mean value of 7.5 nS. The junctional conductance did not show a pronounced sensitivity to the transjunctional potential difference. Single-channel recordings from pairs of pacemaker myocytes revealed populations of single-channel conductances at 133, 202, and 241 pS. With these single-channel conductances, the observed average macroscopic junctional conductance, 7.5 nS, would require only 30–60 open gap junction channels.
Sp1-mediated downregulation of P2X4receptor gene transcription in endothelial cells exposed to shear stress
Authors: Korenaga Risa , Yamamoto Kimiko , Ohura Norihiko , Sokabe Takaaki , Kamiya Akira , Ando Joji
Published: December 2017

Endothelial purinoceptors play an important role in vascular responses to extracellular adenine nucleotides and hemodynamic forces. Here we report that P2X4purinoceptor expression in human umbilical vein endothelial cells is transcriptionally downregulated by fluid shear stress. When human umbilical vein endothelial cells were subjected to a laminar shear stress of 15 dyn/cm2, P2X4mRNA levels began to decrease within 1 h and further decreased with time, reaching 60% at 24 h. Functional analysis of the 1.9-kb P2X45′-promoter indicated that a 131-bp segment (−112 to +19 bp relative to the transcription start site) containing a consensus binding site for the Sp1 transcription factor was critical for the shear stress responsiveness. Mutations of the Sp1 site decreased the basal level of transcription and abolished the response of the P2X4promoter to shear stress. Electrophoretic mobility shift assays showed a marked decrease in binding of Sp1 to the Sp1 consensus element in shear-stressed cells, suggesting that Sp1 mediates the shear stress-induced downregulation of P2X4gene transcription.
Hydrogen peroxide stimulates macrophage vascular endothelial growth factor release
Authors: Cho Michael , Hunt Thomas K. , Hussain M. Zamirul
Published: December 2017

Neutrophils gather at the wound site shortly after trauma and release bactericidal reactive oxygen species (ROS) and H2O2to kill bacteria and prevent infection. Macrophages arrive at the wound in response to environmental stimuli, phagocytose foreign particles, and release vascular endothelial growth factor (VEGF), an angiogenic factor crucial for wound healing. Because oxidants are released early in inflammation and have been found to regulate transcription factors, we investigated a possible role of H2O2in VEGF stimulation. Human U937 macrophages exposed to H2O2and allowed to recover in H2O2-free medium rapidly showed an increase in VEGF mRNA. The H2O2-mediated mRNA increase was dose dependent, blocked by catalase, and associated with elevated VEGF in conditioned media. The increase in VEGF was also found in primary rat peritoneal macrophages and the RAW 264.7 murine macrophage cell line. Transcriptional inhibition with actinomycin D revealed no significant difference in mRNA half-life. Transient transfections with a 1.6-kb VEGF promoter-luciferase construct (Shima DT, Kuroki M, Deutsch U, Ng YS, Adamis AP, and D'Amore PA. J Biol Chem 271: 3877–3883, 1996) showed a ninefold stimulation of VEGF gene promoter activity. We concluded that H2O2increases macrophage VEGF through an oxidant induction of VEGF promoter. This oxidant stimulation can be mediated by activated neutrophils.
Caveolin-1 peptide exerts cardioprotective effects in myocardial ischemia-reperfusion via nitric oxide mechanism
Authors: Young Lindon H. , Ikeda Yasuhiko , Lefer Allan M.
Published: December 2017

Caveolin-1 is a protein constituent of cell membranes. The caveolin-1 scaffolding region (residues 82–101) is a known inhibitor of protein kinase C. Inhibition of protein kinase C results in maintained nitric oxide (NO) release from the endothelium, which attenuates cardiac dysfunction after ischemia-reperfusion (I/R). Therefore, we hypothesized that the caveolin-1 scaffolding region of the molecule, termed caveolin-1 peptide, might attenuate postischemia polymorphonuclear neutrophil (PMN)-induced cardiac dysfunction. We examined the effects of caveolin-1 peptide in isolated ischemic (20 min) and reperfused (45 min) rat hearts reperfused with PMNs. Caveolin-1 peptide (165 or 330 μg) given intravenously 1 h before I/R significantly attenuated postischemic PMN-induced cardiac dysfunction, as exemplified by left ventricular developed pressure (LVDP) ( P < 0.01) and the maximal rate of develped pressure (+dP/d t max) ( P < 0.01), compared with I/R hearts obtained from rats given 0.9% NaCl. In addition, caveolin-1 peptide significantly reduced cardiac PMN infiltration from 195 ± 5 PMNs/mm2 in untreated hearts to 103 ± 5 and 60 ± 5 PMNs/mm2 in hearts from 165 and 330 μg caveolin-1 peptide-treated rats, respectively ( P < 0.01). PMN adherence to the rat coronary vasculature was also significantly reduced in rats given either 165 or 330 μg caveolin-1 peptide compared with rats given 0.9% NaCl ( P < 0.01). Moreover, caveolin-1 peptide-treated rat aortas exhibited a 2.2-fold greater basal release of NO than vehicle-treated aortas ( P < 0.01), and this was inhibited by N G-nitro-l-arginine methyl ester. These results provide evidence that caveolin-1 peptide significantly attenuated PMN-induced post-I/R cardiac contractile dysfunction in the isolated perfused rat heart, probably via enhanced release of endothelium-derived NO.
PKA accelerates rate of force development in murine skinned myocardium expressing α- or β-tropomyosin
Authors: Patel Jitandrakumar R. , Fitzsimons Daniel P. , Buck Scott H. , Muthuchamy Mariappan , Wieczorek David F. , Moss Richard L.
Published: December 2017

In myocardium, protein kinase A (PKA) is known to phosphorylate troponin I (TnI) and myosin-binding protein-C (MyBP-C). Here, we used skinned myocardial preparations from nontransgenic (NTG) mouse hearts expressing 100% α-tropomyosin (α-Tm) to examine the effects of phosphorylated TnI and MyBP-C on Ca2+sensitivity of force and the rate constant of force redevelopment ( ktr). Experiments were also done using transgenic (TG) myocardium expressing ∼60% β-Tm to test the idea that the α-Tm isoform is required to observe the mechanical effects of PKA phosphorylation. Compared with NTG myocardium, TG myocardium exhibited greater Ca2+sensitivity of force and developed submaximal forces at faster rates. Treatment with PKA reduced Ca2+sensitivity of force in NTG and TG myocardium, had no effect on maximum ktrin either NTG or TG myocardium, and increased the rates of submaximal force development in both kinds of myocardium. These results show that PKA-mediated phosphorylation of myofibrillar proteins significantly alters the static and dynamic mechanical properties of myocardium, and these effects occur regardless of the type of Tm expressed.
Abnormal calcium and protein kinase C-ε signaling in hypertrophied atrial tumor myocytes (AT-1 cells)
Authors: Kline Richard , Jiang Tianrong , Xu Xiaohong , Rybin Vitalyi O. , Steinberg Susan F.
Published: December 2017

Cardiac hypertrophy leads to contractile dysfunction and altered hormone responsiveness through incompletely understood mechanisms. Atrial tumor (AT-1) myocytes (AT-1 cells) are a cardiomyocyte lineage that proliferates but hypertrophies when proliferation is prevented with mitomycin C. Because both states maintain a highly differentiated phenotype, AT-1 cells were used to explore the signaling pathways that accompany and/or contribute to hypertrophic cardiomyocyte growth. Mitomycin C-induced AT-1 cell enlargement is associated with a pronounced increase in the amplitude and the duration of both electrically stimulated calcium transients and endothelin receptor-dependent calcium responses. Studies with caffeine indicate that the intracellular pool of releasable calcium is similar in control and hypertrophied AT-1 cells. This agrees with the results of Northern analyses that show similar steady-state levels of transcripts encoding the sarcoplasmic reticulum Ca-ATPase (and higher levels of transcripts encoding the Na+/Ca2+exchanger) in hypertrophied AT-1 cells, relative to proliferating control cultures. However, immunoblot analyses reveal a marked increase in the expression of protein kinase C (PKC)-ε (a critical intermediate in the signaling pathway for endothelin receptor-dependent modulation of intracellular calcium) during AT-1 cell hypertrophy; the abundance of other PKC isoforms is not changed. Collectively, these results identify reciprocal regulation between calcium/PKC signaling and hypertrophic growth. The evidence that AT-1 cell hypertrophy leads to abnormalities in calcium regulation and specific changes in PKC-ε expression that alter endothelin receptor responsiveness supports the notion that pathophysiological changes in PKC-ε abundance lead to functionally important changes in hormonal modulation of cardiomyocyte function.
Sarcoplasmic reticulum Ca2+uptake is impaired in coronary smooth muscle distal to coronary occlusion
Authors: Heaps Cristine L. , Sturek Michael , Price Elmer M. , Laughlin M. Harold , Parker Janet L.
Published: December 2017

After chronic occlusion, collateral-dependent coronary arteries exhibit alterations in both vasomotor reactivity and associated myoplasmic free Ca2+levels that are prevented by chronic exercise training. We tested the hypotheses that coronary occlusion diminishes Ca2+uptake by the sarcoplasmic reticulum (SR) and that exercise training would prevent impaired SR Ca2+uptake. Ameroid constrictors were surgically placed around the proximal left circumflex (LCx) artery of female swine 8 wk before initiating 16-wk sedentary (pen confined) or exercise-training (treadmill run) protocols. Twenty-four weeks after Ameroid placement, smooth muscles cells were enzymatically dissociated from both the LCx and nonoccluded left anterior descending (LAD) arteries of sedentary and exercise-trained pigs, and myoplasmic free Ca2+was studied using fura 2 microfluorometry. After the SR Ca2+store was partially depleted with caffeine (5 mM), KCl-induced membrane depolarization produced a significant decrease in the time to half-maximal ( t½) myoplasmic free Ca2+accumulation in LCx versus LAD cells of sedentary pigs. Furthermore, inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA; 10 μM cyclopiazonic acid) significantly reduced t½in cells isolated from the LAD but not from the LCx. Exercise training did not prevent the differences in t½myoplasmic free Ca2+accumulation observed between LCx and LAD cells. Occlusion or exercise training did not alter SERCA protein levels. These results support our hypothesis of impaired SR Ca2+uptake in coronary smooth muscle cells isolated distal to chronic occlusion. Impaired SR Ca2+uptake was independent of SERCA protein levels and was not prevented by exercise training.
Preconditioning attenuates apoptosis and necrosis: role of protein kinase Cε and -δ isoforms
Authors: Liu Huiping , McPherson Bradley C. , Yao Zhenhai
Published: December 2017

Preconditioning reduces cardiomyocyte necrosis in vivo and in vitro, but it is unknown whether preconditioning blocks apoptosis. We wanted to compare the effects of preconditioning on necrosis and apoptosis in cardiomyocytes. Necrosis was detected with propidium iodide, and apoptosis was quantified by three complementary techniques: flow cytometry, TdT-mediated dUTP nick-end labeling assay, and DNA-laddering electrophoresis. Apoptosis increased with simulated ischemia time (6 h, 19 ± 1%; 12 h, 27 ± 2%; 18 h, 40 ± 4%; 24 h, 54 ± 4%; and 36 h, 83 ± 4%; n = 6 for each group). Simulated ischemia and reoxygenation contributed equally to apoptosis (12-h ischemia, 27 ± 2%, n = 6; 12-h ischemia and 12-h reoxygenation, 51 ± 4%, n = 6; and 24-h ischemia, 54 ± 5%, n = 8). Necrosis occurred primarily during reoxygenation; none was detected during simulated ischemia. Preconditioning with 10 min of simulated ischemia reduced necrosis (18 ± 6%, n = 8) but had no effect on apoptosis. However, three 1-min cycles of simulated ischemia separated by 5 min of reoxygenation reduced necrosis and apoptosis similarly. The protein kinase C (PKC) inhibitors Go6976 (0.1 μM) or chelerythrene (4 μM) abolished the effect of preconditioning. Preconditioning selectively activated PKCε but had no effect on PKCδ and on total PKC enzyme activity. Preconditioning protected against necrosis and apoptosis, but the preconditioning ischemia required for blocking apoptosis was less than that for reducing necrosis. Activation of PKCε isoform is important in mediating the protection.