bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of Heart-Infiltrating T-Cell Clonotypes in Experimental Autoimmune Myocarditis in Rats
Authors: Hanawa Haruo , Inomata Takayuki , Sekikawa Hiroho , Abo Toru , Kodama Makoto , Izumi Tohru , Shibata Akira
Published: June 2012

Abstract
Experimental autoimmune myocarditis (EAM) resembles the lethal giant cell myocarditis seen in humans, and the recurrent forms lead to dilated cardiomyopathy (DCM). EAM in rats induced by a subcutaneous injection of cardiac myosin has been shown to be a T cell–mediated autoimmune disease. αβ T cells have proved to be important by the observation that antibodies to αβ T-cell receptor (TCR) prevent disease progression. αβ T cells recognize antigenic peptides bound to major histocompatibility (MHC) molecules by αβ TCR, and complementarity determining region 3 (CDR3) is considered the most important region for antigen recognition. To elucidate the nature of this T cell–mediated myocarditis, we analyzed TCR Vβ chains of heart-infiltrating T cells. In the early stage of EAM, none of 22 TCR Vβ chain transcripts seemed to be dominant by reverse transcription–polymerase chain reaction analysis of total RNA and flow cytometric analysis. On the other hand, single-strand conformation polymorphism analysis of TCR Vβ8.2, Vβ8.5, Vβ10, and Vβ16 cDNA amplified by polymerase chain reaction encompassing the CDR3 revealed oligoclonal expansion in heart-infiltrating T cells isolated from animals at various disease stages. cDNA encoding Vβ CDR3 from heart-infiltrating and pericardial effusion T cells in rats with EAM revealed more restricted sequences than did cells from rats with normal spleens. Clones from distinct lesions of the same animal were identical, and clones from heart-infiltrating and pericardial effusion T cells from the same animal showed overlap. Thus, CDR3 of the TCR β chain may be important in rat EAM, and heart-infiltrating T cells are considered to recognize the specific antigen.
Tumor Necrosis Factor-α Induces a Biphasic Effect on Myocardial Contractility in Conscious Dogs
Authors: Murray David R. , Freeman Gregory L.
Published: June 2012

Abstract
Tumor necrosis factor-α (TNF-α) likely plays a role in the pathophysiology of myocardial depression observed in septic shock. To evaluate the hemodynamic effects of TNF-α in vivo while eliminating the influence of altered sympathetic tone, eight conscious chronically instrumented dogs were studied after pretreatment with propranolol (2 mg/kg) and atropine (2 mg). Using three sets of piezoelectric crystals to measure left ventricular (LV) volume and LV manometers to measure pressure, we determined load-independent parameters of LV systolic performance before, during, and after infusion of recombinant human TNF-α (rhTNF-α, 40 μg/kg for 1 hour). Plasma was analyzed for epinephrine and norepinephrine. Between 1 and 7 hours of exposure, rhTNF-α induced significant increases in circulating catecholamines. Norepinephrine rose from 268.6±47.2 to 426.2±87.0 pg/mL (
P
<.05) at 1 hour and peaked at 921.2±156.8 pg/mL (
P
<.001) at 4 hours after initiating rhTNF-α treatment. Similarly, epinephrine increased from 130.2±30.9 to 884.5±210.2 pg/mL (
P
<.05) at 1 hour and peaked at 3195.3±476 pg/mL (
P
<.001) at 4 hours. Before the surge of circulating catecholamines and despite complete β-adrenergic blockade, rhTNF-α induced a 7% to 40% increase in LV contractile performance during the 60-minute infusion. After this initial positive inotropic effect, rhTNF-α treatment led to precipitous systolic dysfunction between 2 and 7 hours of exposure; this myocardial depressant effect persisted at 25 hours. LV systolic performance declined to 19% to 35% of baseline values, depending on the specific contractile parameter evaluated. We conclude that rhTNF-α affects LV systolic function in a time-dependent biphasic manner. Increases in circulating catecholamines after rhTNF-α infusion cannot account for the early improvement in LV systolic performance.
Myocyte Enhancer Binding Factor-2 Expression and Activity in Vascular Smooth Muscle Cells
Authors: Firulli Anthony B. , Miano Joseph M. , Bi Weizhen , Johnson A. Daniel , Casscells Ward , Olson Eric N. , Schwarz John J.
Published: June 2012

Abstract
Proliferation and phenotypic modulation of smooth muscle cells (SMCs) are major components of the vessel’s response to injury in experimental models of restenosis. Some of the growth factors involved in restenosis have been identified, but to date little is known about the transcription factors that ultimately regulate this process. We examined the expression of the four members of the myocyte enhancer binding factor-2 (MEF2) family of transcription factors in cultured rat aortic SMCs (RASMCs) and a rat model of restenosis because of their known importance in regulating the differentiated phenotype of skeletal and cardiac muscle. In skeletal and cardiac muscle, the MEF2s are believed to be important for activating the expression of contractile protein and other muscle-specific genes. Therefore, we anticipated that the MEF2s would be expressed at high levels in medial SMCs that are producing contractile proteins and that they would be downregulated along with the contractile protein genes in neointimal SMCs. On the contrary, we observe that MEF2A, MEF2B, and MEF2D mRNAs are upregulated in the neointima, with the highest levels in the layer of cells nearest to the lumen, whereas MEF2C mRNA levels do not appreciably increase. Moreover, few cells in the media are making MEF2 proteins detectable by immunohistochemistry, whereas large numbers of neointimal cells are positive for all four MEF2s. These data suggest that the MEF2s are involved in the activated smooth muscle phenotype and not in the maintenance of contractile protein gene expression.
Angiotensin II Decreases a Resting K
+
Conductance in Rat Bulbospinal Neurons of the C1 Area
Authors: Li Yu-Wen , Guyenet Patrice G.
Published: June 2012

Abstract
In the rostral ventrolateral medulla (RVLM), angiotensin II (Ang II) receptors are concentrated in the region that contains neurons innervating sympathetic preganglionic neurons. We sought to determine whether these bulbospinal cells are sensitive to Ang II. Retrogradely labeled bulbospinal RVLM neurons (N=125) were recorded in thin slices from neonatal rats. Most (33 of 46) histologically recovered bulbospinal neurons were C1 cells (immunoreactive for tyrosine hydroxylase [TH-ir] or phenylethanolamine
N
-methyltransferase [PNMT-ir]). Bulbospinal RVLM neurons were spontaneously active (2.7±0.2 spikes per second, n=69) with ‘resting’ potential of −54±0.4 mV (n=77) and input resistance of 879±53 MΩ (n=47). Ang II (0.3 to 1 μmol/L) increased the spontaneous firing rate of most bulbospinal neurons (+250%, 28 of 39). In current-clamp mode, Ang II (1 μmol/L) produced depolarization (+6.8±0.6 mV, n=59 neurons) and increased input resistance (+21±2%, n=36 neurons). In voltage-clamp mode, Ang II elicited an inward current (9.7±0.9 pA; holding potential, −40 to −55 mV; n=25 neurons) that reversed polarity at the K
+
equilibrium potential

(n=8 neurons) and was barium sensitive (n=4 neurons). Ang II–evoked conductance change was voltage independent (−40 to −140 mV, n=8 neurons). The effects of Ang II were blocked by losartan (9 of 9 neurons) but persisted in low Ca
2+
/high Mg
2+
(7 of 7 neurons). Ang II–sensitive cells were inhibited by α
2
-adrenergic receptor agonists (12 of 15 neurons). Ang II excited 91% (30 of 33) of TH-ir or PNMT-ir cells but 23% (3 of 13) of non–TH-ir neurons. In conclusion, RVLM bulbospinal cells express Ang II type-1 receptors whose activation leads to a reduction in resting K
+
conductance.
Mouse Mutants and Cardiac Development
Authors: Rossant J.
Published: June 2012

Abstract
Understanding the genetic control of cardiac development has been greatly assisted by the newly acquired ability to generate targeted mutations in the mouse. A number of mutations in genes that directly or indirectly affect cardiac development have now been reported, and the phenotypes of these mutations have suggested that cardiac development is under complex genetic control. Further analysis of the mouse model system should help elucidate the etiology of human congenital cardiovascular anomalies.
Cardiomyopathy in Transgenic
myf5
Mice
Authors: Edwards J.G. , Lyons G.E. , Micales B.K. , Malhotra A. , Factor S. , Leinwand L.A.
Published: June 2012

Abstract
To explore the compatibility of skeletal and cardiac programs of gene expression, transgenic mice that express a skeletal muscle myogenic regulator,
bmyf5,
in the heart were analyzed. These mice develop a severe cardiomyopathy and exhibit a significantly shorter life span than do their nontransgenic littermates. The transgene was expressed from day 7.5 post coitum forward, resulting in activation of skeletal muscle genes not normally seen in the myocardium. Cardiac pathology was not apparent at midgestation but was evident by day 2 of postnatal life, and by 42 days, hearts exhibited multifocal interstitial inflammation, fibrosis, cellular hypertrophy, and occasional myocyte degeneration. All four chambers of the heart were enlarged to varying degrees, with the atria demonstrating the most significant hypertrophy (>100% in 42-day-old mice). The transgene and several skeletal muscle–specific genes were expressed only in patchy areas of the heart in heterozygous mice. However, molecular markers of hypertrophy (such as α-skeletal actin and atrial myosin light chain-1) were expressed with a wider distribution, suggesting that their induction was secondary to the expression of the transgene. In older (28-week-old) mice, lung weights were also significantly increased, consistent with congestive heart failure. The life span of
bmyf5
mice was significantly shortened, with an average life span of 109 days, compared with at least a twofold longer life expectancy for nontransgenic littermates. Expression of the transgene was associated with an increase in Ca
2+
-stimulated myofibrillar ATPase in myofibrils obtained from the left ventricles of 42-day-old
bmyf5
mice. Myocardial
bmyf5
expression therefore induces a program of skeletal muscle gene expression that results in progressive cardiomyopathy that may be due to incompatibility of heart and skeletal muscle structural proteins.
Vital Staining of Cardiac Myocytes During Embryonic Stem Cell Cardiogenesis In Vitro
Authors: Metzger Joseph M. , Lin Wan-In , Samuelson Linda C.
Published: June 2012

Abstract
Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types, including spontaneously contracting cardiac myocytes. The primary aim of this work was to use vital stain techniques for real-time detection of developing cardiac myocytes in ES cell differentiation cultures. The −440 to +6 human cardiac α-actin promoter was used to direct expression of the
Escherichia coli
reporter gene
lacZ
(pHCActlacZ) into ES cell–derived cardiac myocytes during cardiogenesis in vitro. Undifferentiated ES cells were electroporated with HCActlacZ together with a plasmid containing the neomycin gene under the direction of the phosphoglycerate kinase promoter, and stable transformants were selected in G418. Individual clones were screened for activation of
lacZ
gene expression in cardiac myocytes developing in vitro. Results showed that expression of the HCActlacZ reporter construct was activated very early during the ES cell differentiation program, at a time point before the appearance of spontaneous contractile activity. The earliest detection was at day 6 of differentiation, when ≈25% of the differentiation cultures expressed the reporter construct, with expression increasing to ≈70% at day 9 and continuing throughout the duration of spontaneous contractile activity exhibited by the ES cell–derived cardiac myocytes. Indirect immunofluorescence assays provide evidence that expression was restricted to the cardiac myocytes in culture. In the present study, we show vital staining of transgene expression in living cardiac myocytes using lipophilic fluorogenic β-galactopyranoside substrates for real-time detection of the reporter gene during continuous contraction of the ES cell myocytes in vitro. The vital stain approach used in the present study will permit the identification of differentiating ES cells that are committed to the cardiac lineage for analysis of gene expression at early time points of ES cell cardiogenesis and, in addition, will aid in selecting genetically modified ES cell cardiac myocytes for use in functional studies.
Evidence for Two Components of Delayed Rectifier K
+
Current in Human Ventricular Myocytes
Authors: Li Gui-Rong , Feng Jianlin , Yue Lixia , Carrier Michel , Nattel Stanley
Published: June 2012

Abstract
Previous voltage-clamp studies have suggested that the delayed rectifier current (I
K
) is small or absent in the human ventricle and, when present, consists only of the rapid component (I
Kr
); however, molecular studies suggest the presence of functionally important I
K
in the human heart, specific I
Kr
blockers are known to delay ventricular repolarization and cause the long QT syndrome in humans, and we have shown that the expression of I
K
is strongly influenced by cell isolation techniques. The present experiments were designed to assess the expression of I
K
in myocytes obtained by arterial perfusion of right ventricular tissue from explanted human hearts. Of 35 cells from three hearts, 33 (94%) showed time-dependent currents typical of I
K
. The envelope-of-tails test was not satisfied under control conditions but became satisfied in the presence of the benzenesulfonamide E-4031 (5 μmol/L). E-4031 suppressed a portion of I
K
in 32 of 33 cells, with properties of the drug-sensitive and -resistant components consistent with previous descriptions of I
Kr
and the slow component (I
Ks
), respectively. Action potential duration to 95% repolarization at 1 Hz was prolonged by E-4031 from 336±16 (mean±SEM) to 421±19 ms (n=5,
P
<.01), indicating a functional role for I
K
. Indapamide, a diuretic agent previously shown to inhibit I
Ks
selectively, suppressed E-4031–resistant current. The presence of a third type of delayed rectifier, the ultrarapid delayed rectifier current (I
Kur
), was evaluated with the use of depolarizing prepulses and low concentrations (50 μmol/L) of 4-aminopyridine. Although these techniques revealed clear I
Kur
in five of five human atrial cells, no corresponding component was observed in any of five human ventricular myocytes. We conclude that a functionally significant I
K
, with components corresponding to I
Kr
and I
Ks
, is present in human ventricular cells, whereas I
Kur
appears to be absent. These findings are important for understanding the molecular, physiological, and pharmacological determinants of human ventricular repolarization and arrhythmias.
Ca
2+
-Induced Current Oscillations in Rabbit Ventricular Myocytes
Authors: Laflamme Michael A. , Becker Peter L.
Published: June 2012

Abstract
Transient currents are activated by spontaneous Ca
2+
oscillations in rabbit ventricular myocytes. We investigated the ionic basis for these transient currents under conditions in which K
+
currents would be expected to be blocked. Holding cells under voltage clamp at positive potentials leads to a rise in intracellular Ca
2+
via reversal of the Na
+
-Ca
2+
exchanger and subsequently to the initiation of spontaneous Ca
2+
transients, presumably from a Ca
2+
-overloaded sarcoplasmic reticulum. The current transients associated with these Ca
2+
transients reversed at about +10 to +15 mV under conditions of approximately symmetrical Cl
−
. In the absence of Cl
−
, this current was inward at all potentials examined over the range from −88 to +72 mV, consistent with a Na
+
-Ca
2+
exchanger current. In the absence of Na
+
, the repetitive spontaneous Ca
2+
transients could be initiated by a brief train of depolarizations to activate the inward Ca
2+
current. Under such conditions, the current was found to reverse at −3 mV when the equilibrium potential of Cl
−
(E
Cl
) was −2 mV, and the reversal potential shifted to −32 mV when internal Cl
−
was lowered, to make E
Cl
−33 mV. Thus, in the absence of Na
+
, it appears that the current is exclusively a Ca
2+
-activated Cl
−
current. There is no evidence to indicate the presence of a Ca
2+
-activated cationic conductance. Further, our results demonstrate that the Ca
2+
-activated Cl
−
conductance can carry inward current at potentials more negative to E
Cl
in rabbit ventricular myocytes and is therefore likely to contribute to the arrhythmogenic delayed afterdepolarizations that occur in Ca
2+
-overloaded cells.
Nitric Oxide Synthase Activity in Guinea Pig Ventricular Myocytes Is Not Involved in Muscarinic Inhibition of cAMP-Regulated Ion Channels
Authors: Zakharov Sergey I. , Pieramici Sean , Kumar Ganesh K. , Prabhakar Nanduri R. , Harvey Robert D.
Published: June 2012

Abstract
It has recently been demonstrated that NO plays an obligatory role in muscarinic inhibition of β-adrenergically stimulated ion channels in cardiac sinoatrial node cells (
J Gen Physiol.
1995;106:45-65). We looked for evidence that NO might play a similar role in ventricular cells by using histochemical staining for NO synthase (NOS) activity and whole-cell patch-clamp recording of cAMP-regulated Cl
−
currents. Myocytes isolated from guinea pig hearts stained positively for NADPH-diaphorase activity, suggesting that these cells do express NOS. Acetylcholine (ACh) inhibition of the
R
(−)-isoproterenol bitartrate (Iso)–activated Cl
−
current was also reversed by the cGMP-lowering agents LY-83583 and methylene blue, consistent with the idea that NO activation of guanylate cyclase may contribute to muscarinic responses. However, LY-83583 and methylene blue activated the Cl
−
current in the presence of subthreshold concentrations of Iso alone, suggesting that their effects may not be due to antagonism of an NO/cGMP-dependent response. Furthermore, ACh inhibition of Iso-activated Cl
−
currents could not be mimicked by the NO donors sodium nitroprusside, 3-morpholinosydnonimine, and spermine-NO. Similarly, ACh inhibition of the Iso-activated Cl
−
current could not be blocked by the NOS inhibitor
N

G

-monomethyl-
l
-arginine. These results indicate that even though ventricular myocytes possess NOS activity, NO production does not play an important role in muscarinic inhibition of β-adrenergically regulated Cl
−
channels in these cells.
Morphine Mimics the Cardioprotective Effect of Ischemic Preconditioning via a Glibenclamide-Sensitive Mechanism in the Rat Heart
Authors: Schultz Jo El J. , Hsu Anna K. , Gross Garrett J.
Published: June 2012

Abstract
Previous results from our laboratory have suggested that opioid receptors are involved in ischemic preconditioning (PC) in rat heart. Furthermore, other investigators have suggested that μ- and δ-opioid receptors mediate analgesia and hypoxic cerebral vasodilatation via opening of ATP-sensitive K
+
(K
ATP
) channels. Thus, the purpose of the present study was to test the hypothesis that activation of opioid receptors mimics the cardioprotective effect of ischemic PC and that this effect is produced by activation of K
ATP
channels in the rat heart. Anesthetized open-chest Wistar rats were subjected to six different protocols. All groups were subjected to 30 minutes of occlusion and 2 hours of reperfusion. Ischemic PC was elicited by three 5-minute occlusion periods interspersed with 5 minutes of reperfusion. Similarly, morphine-induced PC was elicited by three 5-minute drug infusions (100 μg/kg IV) interspersed with 5-minute drug-free periods before the prolonged 30-minute occlusion. Infarct size (IS) as a percentage of the area at risk (AAR) was determined by triphenyltetrazolium staining. Ischemic PC and morphine infusions resulted in similar reductions in IS/AAR from 56±5% to 11±3% and 12±5%, respectively (
P
<.05). Administration of glibenclamide (0.3 mg/kg IV), a K
ATP
channel antagonist, or naloxone (3 mg/kg IV), a nonselective opioid receptor antagonist, both blocked the cardioprotective effects of morphine. These results indicate that opioid receptor stimulation results in a reduction in infarct size similar to that produced by ischemic PC. The effect of morphine is most likely mediated via an opioid receptor-K
ATP
channel–linked mechanism in the rat heart, since glibenclamide abolished its protection.
Depletion of Mitogen-Activated Protein Kinase Using an Antisense Oligodeoxynucleotide Approach Downregulates the Phenylephrine-Induced Hypertrophic Response in Rat Cardiac Myocytes
Authors: Glennon Peter E. , Kaddoura Samer , Sale Elizabeth M. , Sale Graham J. , Fuller Stephen J. , Sugden Peter H.
Published: June 2012

Abstract
An antisense oligodeoxynucleotide (ODN) approach was used to investigate whether mitogen-activated protein kinase (MAPK) is necessary for the hypertrophic response in cardiac myocytes. A phosphorothioate-protected 17-mer directed against the initiation of translation sites of the p42 and p44 MAPK isoform mRNAs was introduced into cultured cardiac myocytes by liposomal transfection. At an antisense ODN concentration of 0.2 μmol/L, p42 MAPK protein was reduced by 82% (immunoblot) after 48 hours, and p42 and p44 MAPK activities were reduced by 44% and 60%, respectively. The same concentration of anti-MAPK ODN inhibited development of the morphological features of hypertrophy (sarcomerogenesis, increased cell size) in myocytes exposed to phenylephrine. Phenylephrine-induced activation of the atrial natriuretic factor (ANF) promoter (measured by the activity of a transfected ANF promoter/luciferase reporter gene) and induction of ANF mRNA (measured by RNase protection assay) were also attenuated. We conclude that MAPK is important for the development of the hypertrophic phenotype in this model of hypertrophy.
A Cellular Mechanism Contributing to Postvagal Tachycardia Studied in Isolated Pacemaker Cells From Cat Right Atrium
Authors: Wang Yong Gao , Lipsius Stephen L.
Published: June 2012

Vagal nerve-induced inhibition of the heartbeat is followed by a postvagal increase in heart rate above control levels, postvagal tachycardia. In the present study, we used a perforated-patch/whole-cell recording method to determine the role of L-type Ca
2+
current (I
Ca,L
) and the hyperpolarization-activated inward current (I
f
) in the positive chronotropic response elicited by withdrawal of acetylcholine (ACh). Experiments were performed on sinoatrial node (SAN) and latent atrial pacemaker (LAP) cells isolated from cat right atrium. Withdrawal of a 2-minute exposure to 1 μmol/L ACh elicited a rebound stimulation of I
Ca,L
in both SAN (33±4%) and LAP (50±6%) cells above control. Similarly, withdrawal of ACh (1 μmol/L) elicited a rebound stimulation of I
f
in both SAN (21±4%) and LAP (20±6%) cells. During the rebound stimulation of I
Ca,L
, peak amplitude was increased throughout the voltage range, and the voltage dependence of activation was shifted to more negative voltages. Action potential recordings from both SAN and LAP cells showed that following ACh-induced inhibition, withdrawal of ACh elicited a concomitant rebound increase in action potential amplitude (+21±2% and +21±3%, respectively) and decrease in pacemaker cycle length (30±5% and 44±5%, respectively) compared with control. H-89 (2 μmol/L), an inhibitor of cAMP-dependent protein kinase A, abolished the rebound increase of I
Ca,L
, I
f
, action potential amplitude, and decrease in pacemaker cycle length elicited by withdrawal of ACh. In the presence of 2 mmol/L cesium, a blocker of I
f
, the rebound decrease in pacemaker cycle length elicited by withdrawal of ACh was unchanged. We conclude that in SAN and LAP cells, withdrawal of ACh elicits a positive chronotropic response primarily through a cAMP-mediated rebound stimulation of I
Ca,L
. These findings are the first demonstration of an intrinsic cellular mechanism that may contribute directly to the nonadrenergic component of postvagal tachycardia.
Heart Failure in Rats Causes Changes in Skeletal Muscle Morphology and Gene Expression That Are Not Explained by Reduced Activity
Authors: Simonini Americo , Long Carlin S. , Dudley Gary A. , Yue Ping , McElhinny Jill , Massie Barry M.
Published: June 2012

In patients with congestive heart failure, skeletal muscle is characterized by a smaller proportion of slow-twitch oxidative fibers and reduced oxidative enzyme activity. However, whether these changes result from disuse or occur as a direct consequence of heart failure is unresolved. To address this issue, 18 rats with heart failure 8 weeks after left coronary artery ligation and 13 sham-operated control rats underwent quantification of locomotor activity by a photocell activation technique, measurements of hemodynamics and infarct size, histochemical and morphological analyses of the soleus and plantaris muscles, and Northern analyses of muscle contractile protein and oxidative enzyme mRNA expression. Although the rats with heart failure had elevated left ventricular end-diastolic pressures (24.1±2.6 mm Hg) and a mean infarct size of 35.1±4.1%, activity levels were similar to those found in the sham-operated rats (3849±304 versus 3526±130 counts per hour). With heart failure, there was a significant reduction of type I fibers in the soleus muscle and type IIa fibers in the plantaris muscle, with corresponding increases in intermediate staining of type IIab fibers in both muscles. This was associated with a 17% decrease in citrate synthase activity in both the soleus and plantaris muscles (26.2±1.6 versus 30.7±3.4 and 29.1±2.4 versus 35.7±3.4 μmol/L per minute per gram, respectively [
P
<.05]). In the soleus muscle, mRNA for both β-myosin heavy chains and cytochrome C oxidase III (normalized to 18S RNA) was reduced (0.27±0.02 versus 0.65±0.02 and 0.23±0.04 versus 0.64±0.02 U), whereas the messages for IIx and IIb myosin heavy chains were increased. A similar decrease in messages for cytochrome oxidase and the primary myosin isoform was observed in the plantaris muscle. Both soleus β-myosin heavy chain and cytochrome C oxidase expression show significant inverse relationships to left ventricular end-diastolic pressure and infarct size. In contrast, there was no relationship between either β-myosin heavy chain or cytochrome C oxidase expression and locomotor activity. These results indicate that in rats heart failure produces changes in skeletal muscle gene expression at the pretranslational level that cannot be explained by inactivity.
Acute Reductions in Blood Flow and Shear Stress Induce Platelet-Derived Growth Factor-A Expression in Baboon Prosthetic Grafts
Authors: Kraiss Larry W. , Geary Randolph L. , Mattsson Erney J.R. , Vergel Selina , Au Y.P. Tina , Clowes Alexander W.
Published: June 2012

Abrupt reductions in fluid shear stress induce subendothelial smooth muscle cells (SMCs) to proliferate in experimental prosthetic grafts. Platelet-derived growth factor (PDGF), an important SMC mitogen, is expressed by cultured endothelial cells and modulated by shear stress. We hypothesized that this growth factor would be modulated by changes in shear stress in vivo. Bilateral aortoiliac prosthetic grafts were implanted into five baboons. High flow was generated by construction of femoral arteriovenous fistulas on both sides. Two months later, one of the fistulas was ligated, reducing shear stress in the upstream graft by 78±6%. Four days after fistula ligation, all grafts were removed and analyzed. As previously reported, SMC proliferation in low-flow grafts exceeded that in high-flow grafts, although the neointimal area was similar. mRNA levels for PDGF-A were significantly increased in low-flow grafts compared with high-flow grafts. In situ hybridization and immunohistochemical studies localized the increased PDGF-A mRNA and protein to the luminal endothelium and subjacent SMCs. Abrupt reductions in blood flow and fluid shear stress may induce accelerated neointimal thickening by a PDGF-A–mediated mechanism, since endothelial expression of this gene is temporally and anatomically associated with neointimal SMC proliferation.
Exogenously Administered Growth Hormone and Insulin-like Growth Factor-I Alter Intracellular Ca
2+
Handling and Enhance Cardiac Performance
Authors: Strömer Hinrik , Cittadini Antonio , Douglas Pamela S. , Morgan James P.
Published: June 2012

It has been proposed that chronic treatment with growth hormone (GH) or insulin-like growth factor-I (IGF-I) in the rat may enhance cardiac function in vivo. To confirm these findings and elucidate the mechanisms by which cardiac function is modulated, we studied isolated buffer-perfused rat hearts after 4 weeks of treatment with high doses of GH and IGF-I alone or in combination. Mechanical parameters were measured at 50% of the intracardiac balloon volume at which maximal developed pressure (DevP) occurred. EC
50
of the force-Ca
2+
relationship and maximal Ca
2+
-activated systolic wall stress (max σ
s
) were assessed by increasing Ca
2+
in the perfusate in a stepwise fashion and plotting systolic wall stress (σ
s
) versus intracellular peak systolic Ca
2+
, measured by the aequorin bioluminescence method. We found a marked increase of systolic pressure (P
s
), DevP, and (+dP/dt)/DevP in the treated groups compared with the control group. The combination group showed a blunted effect. σ
s
was increased in all treated groups for a perfusate Ca
2+
concentration of >1.5 mmol/L. The enhanced systolic performance can be explained by an increase of the overall Ca
2+
responsiveness due to an increased maximal response to Ca
2+
even though the EC
50
of the Ca
2+
-dose response was also slightly increased. P
s
was further enhanced by an increase of the relative wall thickness induced by the treatment. Diastolic pressure, diastolic Ca
2+
, and the amplitude and time course of the Ca
2+
transient were not influenced by any treatment protocol. All treatments caused increases of body and heart weight. These data support the hypothesis that both IGF-I and GH directly affect cardiac performance by altering cardiac geometry as well as by enhancing max σ
s
.
Prostacyclin Formation Elicited by Endothelin-1 in Rat Aorta Is Mediated via Phospholipase D Activation and Not Phospholipase C or A
2
Authors: Wright Harold M. , Malik Kafait U.
Published: June 2012

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that also stimulates production of prostacyclin (PGI
2
) from arachidonic acid. The purpose of this study was to determine the contribution of phospholipases (PLs) A
2
, C, and/or D in ET-1–induced PGI
2
formation in the rat aorta, measured as immunoreactive 6-ketoprostaglandin (PG) F
1α
. ET-1 increased 6-keto-PGF
1α
formation, which was not affected by a PLA
2
inhibitor, 7,7-dimethyl eicosadienoic acid (DEDA). Furthermore, ET-1 failed to stimulate PLA
2
activity measured in the cytosol (cPLA
2
), using phosphatidylcholine,
l
-a-1-palmitoyl-2-arachidonyl[
14
C] as a substrate. However, the adrenergic agonist norepinephrine increased 6-keto-PGF
1α
formation, which was attenuated by DEDA, and enhanced PLA
2
activity. ET-1 enhanced PLC activity, as indicated by increased inositol phosphate production, which was prevented by a PLC inhibitor, U-73122. However, ET-1–induced 6-keto-PGF
1α
production was not altered by U-73122. An inhibitor of PLD activation, C
2
-ceramide, attenuated ET-1–induced PLD activity, as indicated by the production of phosphatidylethanol. Furthermore, ET-1–induced 6-keto-PGF
1α
formation was inhibited by C
2
-ceramide as well as by ethanol treatment. Moreover, inhibitors of phosphatidate phosphohydrolase (propranolol) and diacylglycerol lipase (RHC-80267), attenuated ET-1–induced 6-keto-PGF
1α
formation. Finally, ET-1–induced activation of PLD was not attenuated by a selective PKC inhibitor, bisindolylmaleimide I. These data suggest a novel pathway for ET-1–induced PGI
2
formation in the rat aorta involving activation of PLD but not cPLA
2
and independent of PLC or PKC activation.
Alterations in Rat Interlobar Artery Membrane Potential and K
+
Channels in Genetic and Nongenetic Hypertension
Authors: Martens Jeffrey R. , Gelband Craig H.
Published: June 2012

The renal vasculature plays an important role in the control of blood pressure. K
+
channels have been demonstrated to regulate smooth muscle membrane potential and thereby control smooth muscle tone. However, few data are available on K
+
channel function in the renal vasculature of hypertensive animals. This study details changes in K
+
currents and membrane potential in genetic and nongenetic models of hypertension. The patch-clamp technique and Ca
2+
-imaging fluorescence were used to examine the differences in Wistar-Kyoto (WKY), Sprague-Dawley (SD), spontaneously hypertensive (SHR), and deoxycorticosterone acetate (DOCA) hypertensive single cells of rat kidney interlobar arteries. In current-clamp experiments, SHR and DOCA hypertensive cells were ≈20 mV more depolarized than the control cells. In voltage-clamp experiments with 4-aminopyridine and niflumic acid present to inhibit voltage-dependent K
+
(K
(v)
) and Ca
2+
-activated Cl
−
(Cl
(Ca)
) currents, SHR and DOCA hypertensive Ca
2+
-activated K
+
(K
(Ca)
) currents were significantly larger and activated at more negative potentials than the control. Conversely, with charybdotoxin and niflumic acid present to inhibit K
(Ca)
and Cl
(Ca)
currents, SHR and DOCA hypertensive K
(v)
current was significantly smaller than the control. Finally, basal and angiotensin II–stimulated peak intracellular free [Ca
2+
] was greater in the SHR and DOCA hypertensive cells compared with control cells. These results suggest that membrane potential and the activity of K
(Ca)
and K
(v)
channels are altered in hypertensive rat renal interlobar arteries and may play a role in the regulation of renal blood flow under physiological and pathophysiological conditions.
Role of Endothelium-Derived Nitric Oxide in Coronary Vasodilatation Induced by Pacing Tachycardia in Humans
Authors: Egashira Kensuke , Katsuda Yousuke , Mohri Masahiro , Kuga Takeshi , Tagawa Tatuya , Kubota Toru , Hirakawa Youji , Takeshita Akira
Published: June 2012

Endothelium-derived NO contributes to the control of coronary perfusion. We investigated the roles of NO in the metabolic coronary vasodilatation induced by rapid pacing in humans. We evaluated the dilatation of large epicardial and resistance coronary arteries during rapid atrial pacing before and after intracoronary infusion of
N
G
-monomethyl-
l
-arginine (L-NMMA), an inhibitor of NO synthesis, in 19 patients without significant coronary artery disease. The diameter of the large epicardial coronary artery and coronary blood flow (CBF) were assessed by quantitative coronary arteriography and by a Doppler flow velocity measurement. An increase in the heart rate increased CBF (
P
<.01) and the coronary artery diameter (
P
<.05). L-NMMA at a total dose of 200 μmol reduced basal CBF but did not significantly affect basal coronary artery diameter, arterial pressure, or heart rate. L-NMMA inhibited the pacing-induced dilatation of the large coronary arteries (
P
<.05) but did not affect pacing-induced increases in CBF. L-NMMA inhibited the acetylcholine-induced increase in CBF (
P
<.01) and acetylcholine-induced dilatation of the large epicardial coronary artery (
P
<.05). These results show that the contribution of NO to the metabolic vasodilatation during rapid pacing may differ between large epicardial and resistance coronary arteries in patients without significant coronary artery disease.
Effect of Tetrahydrobiopterin on Endothelial Function in Canine Middle Cerebral Arteries
Authors: Tsutsui Masato , Milstien Sheldon , Katusic Zvonimir S.
Published: June 2012

Tetrahydrobiopterin is an essential cofactor required for activation of NO synthase. However, in intact arteries, the exact role of tetrahydrobiopterin in the regulation of NO synthase activity is not fully understood. The present study was designed to determine the effect of increasing intracellular tetrahydrobiopterin levels on endothelial function in isolated canine middle cerebral arteries. The arterial segments were incubated in MEM for 24 hours at 37°C in the presence or absence of a tetrahydrobiopterin precursor, sepiapterin (10
−4
mol/L), and/or superoxide dismutase (150 U/mL). The rings were suspended for isometric tension recording. Tetrahydrobiopterin levels were assayed by high-performance liquid chromatography. Production of cGMP was measured by radioimmunoassay. Incubation with sepiapterin markedly increased intracellular tetrahydrobiopterin levels. In sepiapterin-treated arteries, endothelium-dependent relaxations to calcium ionophore A23187 and intracellular cGMP levels were significantly reduced. Superoxide dismutase alone did not affect either relaxation to A23187 or production of cGMP. However, when arteries were incubated with superoxide dismutase plus sepiapterin, endothelium-dependent relaxations to A23187, as well as cGMP production, were significantly augmented. The augmentation of cGMP was observed in rings with (but not without) endothelium. Incubation of arteries in calcium-free medium almost abolished the synergistic effect of tetrahydrobiopterin and superoxide dismutase on cGMP production. These results demonstrate that increased availability of tetrahydrobiopterin may activate endothelial NO synthase. This effect appears to be critically dependent on the presence of superoxide dismutase.
Cellular and Ionic Basis of Arrhythmias in Postinfarction Remodeled Ventricular Myocardium
Authors: Qin Dayi , Zhang Zhi-Hao , Caref Edward B. , Boutjdir Mohamed , Jain Praveer , El-Sherif Nabil
Published: June 2012

After myocardial infarction (MI), the noninfarcted myocardium undergoes significant hypertrophy as part of the post-MI structural remodeling. Electrophysiological changes associated with the hypertrophied remodeled myocardium may play a key role in arrhythmia generation in the post-MI heart. We investigated the cellular and ionic basis of arrhythmias in remodeled left ventricular (LV) myocardium 3 to 4 weeks after MI in the rat. We analyzed (1) the incidence of induced ventricular tachyarrhythmias (VTs) in the in vivo heart, (2) action potential characteristics and arrhythmia mechanisms in multicellular preparations and isolated remodeled LV myocytes, and (3) the density and kinetics of the L-type Ca
2+
current (I
Ca-L
) and the fast and slow components of transient outward K
+
currents (I
to-f
and I
to-s
, respectively). The results were compared with those from sham-operated rats. In vivo, programmed stimulation induced sustained VT in 80% of post-MI rats but not in sham-operated rats. The capacitance of post-MI hypertrophied myocytes was significantly increased compared with myocytes from sham-operated rats. Post-MI myocytes had prolonged action potential duration (APD) with marked heterogeneity of the time course of repolarization. The prolongation of APD could be explained by the significant decrease of the density of both I
to-f
and I
to-s
. There was no change in the kinetics of both currents compared with control. Both the density and kinetics of I
Ca-L
were not significantly different in post-MI remodeled myocytes compared with control. The cellular studies showed that reentrant excitation secondary to dispersion of repolarization and triggered activity from both early and delayed afterdepolarizations are potential mechanisms for VT in the post-MI remodeled heart.
Modulation of Ventricular Repolarization by a Premature Stimulus
Authors: Laurita Kenneth R. , Girouard Steven D. , Rosenbaum David S.
Published: June 2012

Recent evidence suggests that ion channels governing the response of action potential duration (APD) to a premature stimulus (ie, APD restitution) are heterogeneously dispersed throughout the heart. However, because of limitations of conventional electrophysiological recording techniques, the effects of restitution in single cells on ventricular repolarization at the level of the intact heart are poorly understood. Using high-resolution optical mapping with voltage-sensitive dyes, we measured APD restitution kinetics at 128 simultaneous sites on the epicardial surface (1 cm
2
) of intact guinea pig hearts (n=15). During steady state baseline pacing, APD gradients that produced a spatial dispersion of repolarization were observed. Mean APD was shortened monotonically from 186±19 ms during baseline pacing (S
1
-S
1
cycle length, 393±19 ms) to 120±4 ms as single premature stimuli were introduced at progressively shorter coupling intervals (shortest S
1
-S
2
, 190±15 ms). In contrast, premature stimuli caused biphasic modulation of APD dispersion (defined as the variance of APD measured throughout the mapping field). Over a broad range of increasingly premature coupling intervals, APD dispersion decreased from 70±29 ms
2
to a minimum of 10±7 ms
2
at a critical S
1
-S
2
interval (216±18 ms), and then, at shorter premature coupling intervals, APD dispersion increased sharply to 66±25 ms
2
. Modulation of APD dispersion by premature stimuli was attributed to coupling interval–dependent changes in the magnitude and direction of ventricular APD gradients, which, in turn, were explained by systematic heterogeneities of APD restitution across the epicardial surface. There was a characteristic pattern in the spatial distribution of cellular restitution such that faster restitution kinetics were closely associated with longer baseline APD. This relationship explained the reversal of APD between single cells, inversion of APD gradients across the heart, and ECG T-wave inversion during closely coupled premature stimulation. Therefore, because of the heterogeneous distribution of cellular restitution kinetics across the epicardial surface, a single premature stimulus profoundly altered the pattern and synchronization of ventricular repolarization in the intact ventricle. This response has important mechanistic implications in the initiation of arrhythmias that are dependent on dispersion of repolarization.
Quantitative Relation Between Interstitial Adenosine Concentration and Coronary Blood Flow
Authors: Stepp David W. , Van Bibber Richard , Kroll Keith , Feigl Eric O.
Published: June 2012

The effect of exogenous and endogenous adenosine in controlling coronary flow was determined using an axially distributed mathematical model of the myocardium to estimate interstitial adenosine concentration from coronary arterial and venous adenosine values. The left main coronary artery was perfused at constant pressure in closed-chest, anesthetized dogs, and exogenous adenosine was infused intracoronary to increase coronary flow. Basal interstitial adenosine was 92 nmol/L, just at the threshold for increasing coronary flow. An increase in interstitial adenosine concentration of only 62% was sufficient to increase coronary flow from 5% to 50% of maximal flow. The possible contribution of an endothelial dilator secondary to activation of adenosine receptors on endothelial cells was tested by comparing the response to exogenous intracoronary adenosine infusion with increases in endogenous adenosine produced by inhibition of adenosine kinase and adenosine deaminase. If adenosine increases coronary flow by an endothelial mechanism, then the interstitial ED
50
of exogenous adenosine would be lower than that for endogenous adenosine due to the postulated additional endothelial dilator. The interstitial ED
50
for exogenous adenosine was 156 nmol/L, not different from the endogenous ED
50
of 150 nmol/L. In conclusion, basal interstitial adenosine concentration is at the threshold of a remarkably steep dose-response curve for increasing coronary blood flow. No evidence was found for an endothelium-mediated vasodilator mechanism secondary to adenosine receptor activation of endothelial cells in vivo. The steep adenosine dose-response curve indicates that measurements of adenosine concentration should be interpreted with caution, because small changes in adenosine concentration cause large changes in coronary flow.
Fibroblast Growth Factor-2 Decreases Metabolic Coupling and Stimulates Phosphorylation as Well as Masking of Connexin43 Epitopes in Cardiac Myocytes
Authors: Doble Bradley W. , Chen Yijing , Bosc Denis G. , Litchfield David W. , Kardami Elissavet
Published: June 2012

Cardiac gap junction (GJ) channels, composed of connexins, allow electrical and metabolic couplings between cardiomyocytes, properties important for coordinated action of the heart as well as tissue homeostasis and control of growth and differentiation. Fibroblast growth factor-2 (FGF-2) is an endogenous growth-promoting protein, believed to participate in the short- and long-term responses of the heart to injury. We have examined short-term effects of FGF-2 on cardiac myocyte GJ–mediated metabolic coupling, using cultures of neonatal rat cardiomyocytes. FGF-2 decreased coupling between cardiomyocytes assessed by scrape dye loading as well as microinjection and dye transfer within 30 minutes of administration. Genistein blocked the effects of FGF-2. To determine the mechanism, we next assessed the effect of FGF-2 on expression, distribution, and phosphorylation of connexin43 (Cx43), which is a major cardiomyocyte connexin. FGF-2 did not affect Cx43 mRNA or protein accumulation and synthesis, and it did not change Cx43 localization at sites of intercellular contact as assessed by immunostaining with a polyclonal anti-Cx43 antibody raised against a synthetic peptide containing residues 346 to 363 of Cx43. FGF-2, however, decreased staining intensity at sites of intermyocyte contact when a monoclonal anti-Cx43 antibody was used, suggesting a localized masking of epitope(s) recognized by the monoclonal but not the polyclonal antibody. These epitopes appear to reside within residues 261 to 270 of Cx43, as indicated by full quenching of monoclonal antibody staining with synthetic peptides. In addition, FGF-2 induced a more than twofold increase in Cx43 phosphorylation. Phosphoamino acid analysis indicated increased phosphorylation of Cx43 on serine residues. Although tyrosine phosphorylation of Cx43 was not detected in either treated or control cells, a fraction of Cx43 was immunoprecipitated with anti–phosphotyrosine-specific antibodies in FGF-2–treated myocytes, suggesting interaction (and hence coprecipitation) with phosphotyrosine-containing protein(s). In conclusion, we have identified Cx43 and intercellular communication as targets of FGF-2–triggered and tyrosine phosphorylation–dependent signal transduction in cardiac myocytes. It is suggested that phosphorylation of Cx43 on serine induced by FGF-2 contributes to decreased metabolic coupling between cardiomyocytes.
The Fumagillin Analogue TNP-470 Inhibits DNA Synthesis of Vascular Smooth Muscle Cells Stimulated by Platelet-Derived Growth Factor and Insulin-like Growth Factor-I
Authors: Koyama Hidenori , Nishizawa Yoshiki , Hosoi Masayuki , Fukumoto Shinya , Kogawa Kyoko , Shioi Atsushi , Morii Hirotoshi
Published: June 2012

The effect of an angiogenesis inhibitor, TNP-470, on DNA synthesis and its underlying signaling cascades stimulated by platelet-derived growth factor (PDGF)-BB and insulin-like growth factor (IGF)-I were examined in bovine vascular smooth muscle cells (SMCs). PDGF-BB (10 ng/mL)– and IGF-I (100 ng/mL)–stimulated increase in DNA synthesis was completely abolished by simultaneous treatment with TNP-470 (1.0 ng/mL). TNP-470 had no effects on PDGF receptor autophosphorylation or early signal transduction, such as activation of mitogen-activated protein kinase and immediate early gene expression. PDGF-BB induced an increase in mRNA levels of cyclin D1, cyclin-dependent kinase (cdk) 4, and cdk2, as well as the activity of cdk2, which preceded the G1/S boundary, as estimated by the kinetics of DNA synthesis. The PDGF-BB–induced activation of cdk2 was inhibited by TNP-470, which was correlated with decreased cdk2 mRNA levels. In contrast, TNP-470 had no or less marked effect on cyclin D1 and cdk4 mRNA levels induced by PDGF-BB. TNP-470 also inhibited a much smaller increase in cdk2 mRNA levels and activation stimulated by IGF-I. In conclusion, TNP-470 potently inhibits DNA synthesis of SMCs, and this inhibition is associated with decreased levels of cdk2 mRNA and activity.