bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Inducible Nitric Oxide Synthase in Rat Experimental Autoimmune Myocarditis With Special Reference to Changes in Cardiac Hemodynamics
Authors: Hirono Satoru , Islam M. Omedul , Nakazawa Mikio , Yoshida Yutaka , Kodama Makoto , Shibata Akira , Izumi Tohru , Imai Shoichi
Published: June 2012

Excessive NO produced by an inducible NO synthase (iNOS) has been implicated in many types of immune-associated disorders of the cardiovascular system, but it remains to be determined whether NO plays a role in myocarditis. Thus, the significance of iNOS expression in the development of experimental autoimmune myocarditis (EAM), an animal model of human giant cell myocarditis, was investigated. Lewis rats were immunized with cardiac myosin and were killed 7, 14, 21, 28, and 49 days after immunization. The development of severe myocarditis was observed on days 14, 21, and 28 in association with significant deterioration of hemodynamics determined by cardiac catheterization, which peaked on day 21. In parallel with histological severity of myocarditis and deterioration of cardiac performance, iNOS activity in the heart measured by [
14
C]
l
-citrulline formation was markedly increased on days 14, 21, and 28. The expression of iNOS was confirmed by immunoblotting and was localized to the infiltrating inflammatory cells found in the vicinity of necrotic myocytes by immunohistochemical analysis. Aminoguanidine, a selective inhibitor of iNOS, significantly decreased the iNOS activity (1.04±0.37 compared with 29.1±8.62 pmol·min
−1
·mg protein
−1
in untreated myosin-immunized rats,
P
<.01) and effectively attenuated histopathological changes of EAM on day 21. Hemodynamic parameters were also improved from 64±3 to 89±3 mm Hg for mean blood pressure, from 80±2 to 113±4 mm Hg for left ventricular systolic pressure, from 7.8±0.3 to 3.2±0.3 mm Hg for left ventricular end-diastolic pressure, from 2867±137 to 4180±102 mm Hg/s for +dP/dt, and from 2717±132 to 4180±184 mm Hg/s for −dP/dt (
P
<.01). The values after aminoguanidine treatment were not significantly different from the control values. These results suggest an important role for NO in mediating pathophysiological changes in myocarditis of autoimmune origin.
p22phox mRNA Expression and NADPH Oxidase Activity Are Increased in Aortas From Hypertensive Rats
Authors: Fukui Toshiki , Ishizaka Nobukazu , Rajagopalan Sanjay , Laursen Jørn Bech , Capers Quinn , Taylor W. Robert , Harrison David G. , de Leon Hector , Wilcox Josiah N. , Griendling Kathy K.
Published: June 2012

Recent studies suggest that superoxide production by the NADPH/NADH oxidase may be involved in smooth muscle cell growth and the pathogenesis of hypertension. We previously showed that angiotensin II (Ang II) activates a p22phox-based NADPH/NADH oxidase in cultured rat vascular smooth muscle cells and in animals made hypertensive by infusion of Ang II. To investigate the mechanism responsible for this increased oxidase activity, we examined p22phox mRNA expression in rats made hypertensive by implanting an osmotic minipump that delivered Ang II (0.7 mg/kg per day). Blood pressure began to increase 3 days after the start of Ang II infusion and remained elevated for up to 14 days. Expression of p22phox mRNA in aorta was also increased after 3 days and reached a maximum increase of 338±41% by 5 days after pump implantation compared with the value after sham operation. This increase in mRNA expression was accompanied by an increase in the content of the corresponding cytochrome (twofold) and NADPH oxidase activity (179±11% of that in sham-operated rats 5 days after pump implantation). Treatment with the antihypertensive agents losartan (25 mg/kg per day) or hydralazine (15 mg/kg per day) inhibited this upregulation of mRNA levels and activity. Furthermore, infusion of recombinant heparin-binding superoxide dismutase decreased both blood pressure and p22phox mRNA expression. In situ hybridization of aortic tissue showed that p22phox mRNA was expressed in medial smooth muscle as well as in the adventitia. These findings suggest that Ang II–induced hypertension activates the NADPH/NADH oxidase system by upregulating mRNA levels of one or several components of this oxidase system, including the p22phox, and that the NADPH/NADH oxidase system is associated with the pathology of hypertension in vivo.
Modulation of Renal Cortical Blood Flow During Static Exercise in Humans
Authors: Middlekauff Holly R. , Nitzsche Egbert U. , Nguyen Alison H. , Hoh Carl K. , Gibbs G. Gary
Published: June 2012

During static exercise, several reflex systems that increase sympathetic nerve activity, heart rate, arterial pressure, and cardiac output are activated. At rest, the renal circulation receives the most blood flow per tissue weight of any organ in the body. However, the renal circulatory response to static exercise has not been studied in humans because of technical limitations in methods for measuring rapid changes in renal blood flow. The aim of this study was to determine the renal blood flow response to static exercise in healthy humans and, specifically, to clarify the reflex mechanisms underlying this response. Renal cortical blood flow was measured using dynamic positron emission tomography and the blood flow agent oxygen-15 water. Graded handgrip exercise, posthandgrip circulatory arrest, and administration of intra-arterial adenosine were performed to clarify the mechanisms controlling renal blood flow during static exercise. The major new findings in this study are that in healthy humans (1) renal cortical blood flow decreases (basal versus handgrip, 4.4±0.1 versus 3.5±0.1 mL·min
−1
·g
−1
;
P
=.008) and renal cortical vascular resistance increases (basal versus handgrip, 17±1 versus 26±2 U;
P
=.01) in response to static handgrip exercise; (2) central command and/or the mechanoreflex contributes importantly to the early decrease in renal blood flow (basal versus handgrip, 4.2±0.2 versus 3.5±0.3 mL·min
−1
·g
−1
;
P
=.04) and to the increase in renal cortical vascular resistance (basal versus handgrip, 20±1 versus 25±2 U;
P
=.04); (3) the muscle metaboreflex contributes to further decreases in renal blood flow (basal versus posthandgrip circulatory arrest, 4.3±0.1 versus 3.5±0.2 mL·min
−1
·g
−1
;
P
=.002) and increases in renal cortical vascular resistance (basal versus handgrip, 18±1 versus 25±3 U;
P
=.002); and (4) exogenous adenosine activates the muscle metaboreflex producing reflex renal vasoconstriction and decreased renal blood flow, which may implicate endogenous adenosine generated during ischemic exercise as a potential activator of the muscle metaboreflex during ischemic handgrip exercise.
The Integrin Very Late Antigen-4 Is Expressed in Human Smooth Muscle Cell
Authors: Dupla`a Ce´cile , Couffinhal Thierry , Dufourcq Pascale , Llanas Brigitte , Moreau Catherine , Bonnet Jacques
Published: June 2012

Vascular cell adhesion molecule-1 (VCAM-1) and its counterreceptor, the integrin very late antigen-4 (VLA-4), have recently been identified in smooth muscle cells during intimal thickening in humans and in newly forming vessels during ontogeny in mice, respectively. We examined the coexpression of VCAM-1 and the α
4
integrin subunit in human smooth muscle cells. The expression of VCAM-1 and α
4
subunit were studied during development of the aorta. In the 10-week-old human fetal aorta, VCAM-1 and α
4
were strongly expressed in smooth muscle cells. Their expression was dramatically reduced within the 24th week of gestation and disappeared in the adult aortic media. However, smooth muscle cells from intimal atherosclerotic thickening of adult aorta reexpressed both VCAM-1 and α
4
. In a culture model mimicking smooth muscle differentiation, VCAM-1 mRNA and protein and α
4
integrin protein were coexpressed with smooth muscle–specific variants of cytoskeletal and contractile proteins, smooth muscle myosin heavy chain, caldesmon heavy chain, and desmin. Treatment with antibodies against VCAM-1 or α
4
integrin subunit interfered with the mRNA induction of smooth muscle–specific markers of differentiation. These results in vitro, associated with the transitory expression of VCAM-1 and VLA-4 during vascular ontogeny and the atherosclerosis process, point to a possible role of VCAM-1 and VLA-4 in the induction of smooth muscle differentiation.
Modulation by pH
o
and Intracellular Ca
2+
of Na
+
-H
+
Exchange in Diabetic Rat Isolated Ventricular Myocytes
Authors: Le Prigent Karine , Lagadic-Gossmann Dominique , Feuvray Danielle
Published: June 2012

We have previously shown that diabetes is associated with a decrease in Na
+
-H
+
exchange activity in rat cardiac papillary muscle. The present work has been carried out in order to elucidate the factors responsible for such an alteration. Thus, we have studied the effects of pH
o
and intracellular Ca
2+
on Na
+
-H
+
exchange in ventricular myocytes isolated from streptozotocin-induced diabetic rat hearts. pH
i
was recorded using carboxy-seminaphthorhodafluor (SNARF-1). The NH
4
+
(10 mmol/L) prepulse method was used to induce an acid load in order to activate Na
+
-H
+
exchange in HEPES-buffered Tyrode's solution. Whereas diabetes did not change intracellular buffering power, it significantly decreased acid efflux through Na
+
-H
+
exchange (acid efflux, 4.32±0.4 [n=32, normal cells] versus 2.5±0.2 [n=43, diabetic cells] meq/L per minute at pH
i
6.9;
P
<.02). Upon changes of pH
o
(at a range of 8.0 to 6.8), acid efflux similarly varied in normal and diabetic cells, thus pointing to an unchanged pH
o
sensitivity of Na
+
-H
+
exchange. Buffering of intracellular Ca
2+
by pretreatment of the cells with BAPTA-AM (25 μmol/L, Ca
2+
-chelator) resulted in a decrease by ≈58% of acid efflux in the diabetic group. This decrease was even more marked in normal cells (by ≈74%). Interestingly, the pH
i
dependence of the acid efflux carried by Na
+
-H
+
exchange then became identical in both groups of cells, thus pointing to a role for intracellular Ca
2+
in the diabetes-related alterations of the exchange. Inhibition of calmodulin (by 1.5 μmol/L calmidazolium) and of Ca
2+
/calmodulin-dependent protein kinase II (by 2 μmol/L 1-[
N
,
O
-bis(5-isoquinolinesulfonyl)-
N
-methyl-
l
-tyrosyl]-4-phenylpiperazine [KN-62]) significantly slowed down pH
i
recovery in both normal and diabetic cells. However, the effect of KN-62 was significantly lower in diabetic cells (efflux decreased by ≈17%) compared with normal cells (decrease by 45%). In conclusion, these data, in light of recent observations showing a decreased [Ca
2+
]
i
associated with diabetes in isolated ventricular myocytes, suggest that changes in intracellular Ca
2+
may play an important role in altering Na
+
-H
+
exchange activity in diabetic ventricular myocytes. They also point to diabetes-related alterations in the Ca
2+
/calmodulin protein kinase II–dependent phosphorylation of Na
+
-H
+
exchange.
Tissue and Species Distribution of mRNA for the i
kr
-like K
+
Channel, ERG
Authors: Wymore Randy S. , Gintant Gary A. , Wymore Rigel T. , Dixon Jane E. , McKinnon David , Cohen Ira S.
Published: June 2012

The human K
+
channel gene, HERG, has been linked to the type 2 form of the autosomal dominant long-QT syndrome and has been suggested to encode the fast component of the delayed rectifier K
+
current (I
Kr
) found in heart. To date, the published electrophysiological and pharmacological data on the
Xenopus
-expressed HERG are very similar but are not identical to those of the endogenous I
Kr
. In an effort to provide a different type of correlative data on the relationship between erg and I
Kr
, cDNA fragments of erg homologues from guinea pig, rabbit, human, dog, and rat were cloned and used to test for the presence of erg mRNA in cardiac tissue. RNase protection assays reveal that erg message is found in the hearts of all five species and that it is expressed uniformly throughout the heart. The erg transcript is expressed at relatively high levels, being ≈50% more abundant than the most prevalent Kv-class K
+
channel transcript in canine ventricle (Kv4.3). Erg transcripts were found to have a wide tissue distribution in rat and are abundant in the brain, retina, thymus, and adrenal gland and are also found in skeletal muscle, lung, and cornea. Since there were no published reports of an I
Kr
-like current in the rat heart, electrophysiological studies were performed to test whether the significant level of erg message in rat heart was correlated with the presence of an I
Kr
-like current in rat. In isolated rat ventricular myocytes, an E-4031–sensitive current was observed, which is consistent with the presence of I
Kr
. These results strengthen the link between erg and the native I
Kr
in heart and suggest that erg may play an important role in other noncardiac tissues.
Cytoskeletal Mechanics in Pressure-Overload Cardiac Hypertrophy
Authors: Tagawa Hirofumi , Wang Ning , Narishige Takahiro , Ingber Donald E. , Zile Michael R. , Cooper George
Published: June 2012

We have shown that the cellular contractile dysfunction characteristic of pressure-overload cardiac hypertrophy results not from an abnormality intrinsic to the myofilament portion of the cardiocyte cytoskeleton but rather from an increased density of the microtubule component of the extramyofilament portion of the cardiocyte cytoskeleton. To determine how, in physical terms, this increased microtubule density mechanically overloads the contractile apparatus at the cellular level, we measured cytoskeletal stiffness and apparent viscosity in isolated cardiocytes via magnetic twisting cytometry, a technique by which magnetically induced force is applied directly to the cytoskeleton through integrin-coupled ferromagnetic beads coated with Arg-Gly-Asp (RGD) peptide. Measurements were made in two groups of cardiocytes from cats with right ventricular (RV) hypertrophy induced by pulmonary artery banding: (1) those from the pressure-overloaded RV and (2) those from the normally loaded same-animal control left ventricle (LV). Cytoskeletal stiffness increased almost twofold, from 8.53±0.77 dyne/cm
2
in the normally loaded LV cardiocytes to 16.46±1.32 dyne/cm
2
in the hypertrophied RV cardiocytes. Cytoskeletal apparent viscosity increased almost fourfold, from 20.97±1.92 poise in the normally loaded LV cardiocytes to 87.85±6.95 poise in the hypertrophied RV cardiocytes. In addition to these baseline data showing differing stiffness and, especially, apparent viscosity in the two groups of cardiocytes, microtubule depolymerization by colchicine was found to return both the stiffness and the apparent viscosity of the pressure overload–hypertrophied RV cells fully to normal. Conversely, microtubule hyperpolymerization by taxol increased the stiffness and apparent viscosity values of normally loaded LV cardiocytes to the abnormal values given above for pressure-hypertrophied RV cardiocytes. Thus, increased microtubule density constitutes primarily a viscous load on the cardiocyte contractile apparatus in pressure-overload cardiac hypertrophy.
Effects of Tityus Serrulatus Scorpion Toxin gamma on Voltage-Gated Na sup + Channels
Authors: Marcotte P. , Chen L.-Q. , Kallen R.G. , Chahine M.
Published: June 2012

The effects of Brazilian scorpion Tityus serrulatus toxin gamma (TiTx gamma) were studied on voltage-gated Na sup + channels from human heart (hH1) and rat skeletal muscle (rSkM1). The Na sup + channels were expressed in Xenopus laevis oocytes, and Na sup + currents were recorded using two-microelectrode voltageclamp techniques. In control experiments, the threshold of activation of hH1 is more negative than that of rSkM1 by [nearly =]20 mV. The toxin induces a shift of the voltage dependence of activation toward more negative potential values and reduces the amplitude of the current when administered to rSkM1. In contrast, TiTx gamma has little discernible effect on the current-voltage curve for hH1 at 100 nmol/L. Chimeric channels formed from these two isoforms were constructed to localize the binding site of TiTx gamma on rSkM1. TiTx gamma shifts the activation of a chimera (SSHH) in which domains 1 (D1) and 2 (D2) derive from rSkM1 and domains 3 (D3) and 4 (D4) derive from hH1. This finding suggests that the toxin acts on the activation of rSkM1 by binding either to D1 and/or D2. TiTx gamma shifted the activation of another chimera with D2-D3-D4 from rSkM1 (HSSS) toward more hyperpolarizing potentials and had no effect on the activation of other chimeras with only D1-D3-D4 from rSkM1 (SHSS) or only D3 from rSkM1 (HHSH). Finally, a chimera in which D2 is from rSkM1 and all others domains are from hH1 (HSHH) provides further compelling support for our hypothesis. TiTx gamma shifts the activation of this chimera toward more negative potential values. Thus, TiTx gamma action on chimeras segregates with the source of D2: when D2 is from rSkM1, the toxin affects activation. We infer that D2 plays an important role in the activation process of voltage-gated Na sup + channels.
Upregulation of 5′-AMP–Activated Protein Kinase Is Responsible for the Increase in Myocardial Fatty Acid Oxidation Rates Following Birth in the Newborn Rabbit
Authors: Makinde A-Olufemi , Gamble James , Lopaschuk Gary D.
Published: June 2012

Abstract
In newborn rabbits, fatty acid oxidation rates in the heart significantly increase between 1 and 7 days after birth. This is due in part to a decrease in malonyl coenzyme A (CoA) production by acetyl CoA carboxylase (ACC). In other tissues, 5′-AMP–activated protein kinase (AMPK) can phosphorylate and inhibit ACC activity. In this study, we show that 1- and 7-day-old rabbit hearts have a high AMPK activity, with AMPK expression and activity being greatest in 7-day-old hearts. Hearts were also perfused in the Langendorff mode with Krebs-Henseleit buffer containing 0.4 mmol/L [
14
C]palmitate and 11 mmol/L glucose±100 μU/mL insulin. In the absence of insulin, fatty acid oxidation rates were significantly higher in 7-day-old hearts compared with 1-day-old hearts. AMPK activity was also greater in 7-day-old hearts compared with 1-day-old hearts (909±60 and 585±75 pmol·min
−1
·mg protein
−1
, respectively;
P
<.05). In 1-day-old hearts, the presence of insulin resulted in a significant decrease in AMPK activity, an increase in ACC activity, and a decrease in fatty acid oxidation rates. In 7-day-old hearts, AMPK activity was also decreased by insulin, although ACC activity remained low and fatty acid oxidation rates remained high. Stimulation of AMPK in 7-day-old hearts with 200 μmol/L 5-amino 4-imidazolecarboxamide ribotide resulted in a further decrease in ACC activity and an increase in fatty acid oxidation rates. These data suggest that AMPK, ACC, and fatty acid oxidation are sensitive to insulin in 1-day-old rabbit hearts and that the decrease in circulating insulin levels seen after birth leads to an increased activity of AMPK. This can then lead to a phosphorylation and inhibition of ACC activity, with a resultant increase in fatty acid oxidation rates.
Targeted Ablation of the Phospholamban Gene Is Associated With a Marked Decrease in Sensitivity in Aortic Smooth Muscle
Authors: Lalli Jane , Harrer Judy M. , Luo Wusheng , Kranias Evangelia G. , Paul Richard J.
Published: June 2012

Abstract
Phospholamban (PLB) is a protein associated with the Ca
2+
-ATPase of the sarcoplasmic reticulum (SR) in cardiac, slow-twitch skeletal, and smooth muscle. PLB inhibits the SR Ca
2+
-ATPase in cardiac muscle; this inhibition is relieved on phosphorylation. The role of PLB in smooth muscle contractility is less clear. To elucidate the role of PLB in vascular smooth muscle contractility in vivo, we used a model in which the PLB gene was targeted in murine embryonic stem cells, generating mice deficient in PLB (PLB
−
). The PLB
−
mice exhibited no gross developmental abnormalities, but marked changes in aortic contractility were observed. The time course of force development with phenylephrine stimulation was faster in the PLB
−
aorta, suggesting changes in SR Ca
2+
release. No differences were observed for KCl contractures between tissue types for either maximum forces observed or time course of force production; relaxation was faster in 7 of 11 arteries, but this trend did not attain statistical significance. The cumulative concentration–isometric force relations for the PLB
−
aorta were to the right of the wild-type for both KCl and phenylephrine stimulation, indicating a less sensitive tissue. To investigate whether the observed changes were related to SR function, we inhibited the SR Ca
2+
-ATPase with cyclopiazonic acid (CPA). CPA treatment resulted in a leftward shift of the concentration–isometric force relations for both aorta types, as expected after removal of a major Ca
2+
uptake system. Most interestingly, the differences between PLB and wild-type aorta were abolished by SR inhibition. Our results suggest that PLB is a regulator of the SR Ca
2+
pump in mouse aorta and plays a regulatory role in both KCl-induced and receptor-mediated contractility in vascular smooth muscle.
Antisense Oligodeoxynucleotides Directed Against Kv1.5 mRNA Specifically Inhibit Ultrarapid Delayed Rectifier K
+
Current in Cultured Adult Human Atrial Myocytes
Authors: Feng Jianlin , Wible Barbara , Li Gui-Rong , Wang Zhiguo , Nattel Stanley
Published: June 2012

Abstract
Several cloned K
+
channel subunits are candidates to underlie macroscopic currents in the human heart, but direct evidence bearing on their role is lacking. The Kv1.5 K
+
channel subunit has been suggested to play a potential role in human cardiac ultrarapid delayed rectifier (I
Kur
) and transient outward (I
to
) currents. To evaluate the role of proteins encoded by the Kv1.5 gene, we incubated cultured human atrial myocytes for 48 hours in medium containing antisense phosphorothioate oligodeoxynucleotides directed against octodecameric segments of the Kv1.5 mRNA coding sequence, the same concentration of homologous oligodeoxynucleotides with four mismatch mutations, or vehicle (control group). Cells exposed to antisense showed a highly significant (≈50%) reduction in I
Kur
, whether measured by step current at the end of a 400-millisecond depolarizing pulse, tail current at −20 mV, or current sensitive to a concentration of 4-aminopyridine (50 μmol/L) that is highly selective for I
Kur
, compared with control cells or cells exposed to mismatch oligodeoxynucleo-tides. In contrast, I
to
was not different among the three experimental groups. When cultured human ventricular myocytes were exposed to Kv1.5 antisense oligodeoxynucleotides with the same controls, no changes occurred in either I
to
or the sustained current at the end of a depolarizing pulse. We conclude that Kv1.5 channel subunits are essential to the expression of I
Kur
and do not play a role in I
to
in cultured human atrial myocytes. These studies provide the first direct evidence with an antisense approach for the equivalence between a macroscopic cardiac K
+
current and a cloned K
+
channel subunit and offer insights into the molecular electrophysiology of the human heart.
Angiotensin II Signal Transduction in Vascular Smooth Muscle
Authors: Berk Bradford C. , Corson Marshall A.
Published: June 2012

Abstract
In this review, the role of tyrosine kinases in angiotensin II–mediated signal transduction pathways in vascular smooth muscle is discussed. Angiotensin II was isolated by virtue of its vasoconstrictor abilities and has long been thought to play a critical role in hypertension. However, recent studies indicate important roles for angiotensin II in inflammation, atherosclerosis, and congestive heart failure. The expanding role of angiotensin II indicates that multiple signal transduction pathways are likely to be activated in a tissue-specific manner. Exciting recent data show that angiotensin II directly stimulates tyrosine kinases, including pp60
c-src
kinase (c-Src), focal adhesion kinase (FAK), and Janus kinases (JAK2 and TYK2). Angiotensin II may activate receptor tyrosine kinases, such as Axl and platelet-derived growth factor, by as-yet-undefined autocrine mechanisms. Finally, unknown tyrosine kinases may mediate tyrosine phosphorylation of Shc, Raf, and phospholipase C-γ after angiotensin II stimulation. These angiotensin II–regulated tyrosine kinases appear to be required for angiotensin II effects, such as vasoconstriction, proto-oncogene expression, and protein synthesis, on the basis of studies with tyrosine kinase inhibitors. Thus, understanding angiotensin II–stimulated signaling events, especially those related to tyrosine kinase activity, may form the basis for the development of new therapies for cardiovascular diseases.
Fibroblast Growth Factor-2 Potentiates Vascular Smooth Muscle Cell Migration to Platelet-Derived Growth Factor
Authors: Pickering J. Geoffrey , Uniyal Shashi , Ford Carol M. , Chau Thu , Laurin Mary Ann , Chow Lawrence H. , Ellis Christopher G. , Fish Jonathan , Chan Bosco M. C.
Published: June 2012

Abstract
Fibroblast growth factor-2 (FGF-2) has been implicated in vascular smooth muscle cell (SMC) migration, a key process in vascular disease. We demonstrate here that FGF-2 promotes SMC motility by altering β
1
integrin–mediated interactions with the extracellular matrix (ECM). FGF-2 significantly increased surface expression of α
2
β
1
, α
3
β
1
, and α
5
β
1
integrins on human SMCs, as assessed by flow cytometry. The greatest increase was for the collagen-binding α
2
β
1
integrin. Despite this, FGF-2 did not increase SMC adhesion to type I collagen but instead promoted SMC elongation and SMC motility. The latter was evaluated by using a microchemotaxis chamber and by digital time-lapse video microscopy. Although FGF-2 was not chemotactic for human SMCs, cells preincubated with FGF-2 displayed a 3.1-fold increase in migration to the undersurface of porous type I collagen–coated membranes and a 2.1-fold increase in migration speed on collagen. Furthermore, chemotaxis to platelet-derived growth factor-BB on collagen was significantly greater in SMCs exposed to FGF-2. FGF-2–induced elongation and migration on collagen were inhibited by a blocking anti-α
2
β
1
antibody; however, SMC adhesion to collagen was unaffected. SMC migration on fibronectin was also enhanced by FGF-2, although less prominently: migration through porous membranes increased 1.8-fold, and migration speed increased 1.3-fold. Also, FGF-2 completely disassembled the smooth muscle α-actin–containing stress fiber network contemporaneously with the change in integrin expression and cell shape. We conclude that (1) exogenous FGF-2 promotes SMC migration and potentiates chemotaxis to PDGF-BB; (2) the promigratory effect of FGF-2 is especially prominent on type I collagen and is mediated by upregulation of α
2
β
1
integrin; and (3) FGF-2 disassembles actin stress fibers, which may promote differential utilization of α
2
β
1
integrin for motility but not adhesion. This dynamic SMC-ECM interplay may be an important mechanism by which FGF-2 facilitates SMC motility in vivo.
Changes in Cell-to-Cell Electrical Coupling Associated With Left Ventricular Hypertrophy
Authors: Cooklin M. , Wallis W. R. J. , Sheridan D. J. , Fry C. H.
Published: June 2012

Abstract
The impedance to current flow in the intracellular compartment of guinea pig left ventricular myocardium was measured at 20°C and 37°C using tissue from hypertrophied hearts subjected to aortic constriction. Alternating current of varying frequency was passed longitudinally along myocardial preparations, which revealed two time constants: one attributed to the surface membrane at the ends of the preparation and a second lying in the intracellular pathway. The longitudinal impedance was quantitatively analyzed in terms of a parallel intracellular and extracellular pathway; the former had two series components, one attributable to the sarcoplasm and the other to the low-resistance junctions between adjacent cells. This interpretation was consistent (1) with control experiments using
n
-heptanol, which increased the component attributed to intercellular junctions but not sarcoplasmic resistivity, and (2) with suspensions of isolated myocytes, which yielded a similar value for the sarcoplasmic resistivity. Aortic constriction increased the heart weight–to–body weight ratio of experimental animals from a mean value of 3.10±0.28 to 5.05±0.83 g/kg after 50 days of constriction and 5.60±0.95 g/kg after 150 days of constriction. An increase of heart weight–to–body weight ratio at 150 days of constriction was associated with an increased intracellular resistivity, which could be attributed solely to an increase of the junctional resistance between adjacent cells by ≈44% at 20°C and 140% at 37°C; the sarcoplasmic resistivity was unchanged. The results are discussed in terms of altered conduction in hypertrophied myocardium as a possible basis for arrhythmias in this tissue.
Outward K
+
Current Densities and Kv1.5 Expression Are Reduced in Chronic Human Atrial Fibrillation
Authors: Van Wagoner David R. , Pond Amber L. , McCarthy Patrick M. , Trimmer James S. , Nerbonne Jeanne M.
Published: June 2012

Abstract
Chronic atrial fibrillation is associated with a shortening of the atrial action potential duration and atrial refractory period. To test the hypothesis that these changes are mediated by changes in the density of specific atrial K
+
currents, we compared the density of K
+
currents in left and right atrial myocytes and the density of delayed rectifier K
+
channel α-subunit proteins (Kv1.5 and Kv2.1) in left and right atrial appendages from patients (n=28) in normal sinus rhythm with those from patients (n=15) in chronic atrial fibrillation (AF). Contrary to our expectations, nystatin–perforated patch recordings of whole-cell K
+
currents revealed significant reductions in both the inactivating (
I
TO
) and sustained (
I
Ksus
) outward K
+
current densities in left and right atrial myocytes isolated from patients in chronic AF, relative to the
I
TO
and
I
Ksus
densities in myocytes isolated from patients in normal sinus rhythm. Quantitative Western blot analysis revealed that although there was no change in the expression of the Kv2.1 protein, the expression of Kv1.5 protein was reduced by >50% in both the left and the right atrial appendages of AF patients. The finding that Kv1.5 expression is reduced in parallel with the reduction in delayed rectifier K
+
current density is consistent with recent suggestions that Kv1.5 underlies the major component of the delayed rectifier K
+
current in human atrial myocytes, the ultrarapid delayed rectifier K
+
current, I
Kur
. The unexpected finding of reduced voltage-gated outward K
+
current densities in atrial myocytes from AF patients demonstrates the need to further examine the details of the electrophysiological remodeling that occurs during AF to enable more effective and safer therapeutic strategies to be developed.
Ca
2+
as a Mediator of Ischemic Preconditioning
Authors: Miyawaki Hiroshi , Ashraf Muhammad
Published: June 2012

Abstract
We tested the hypothesis that elevation of [Ca
2+
]
i
during ischemic preconditioning (IPC) stimulates protein kinase C (PKC), which confers the protection against the ischemic injury. Langendorff-perfused rat hearts were subjected to 40-minute global ischemia followed by 30-minute reperfusion (I/R). In preconditioned groups, hearts were subjected to either IPC, consisting of 5-minute global ischemia and 10-minute reperfusion, or high-Ca
2+
preconditioning (HCPC), ie, the 5-minute perfusion of higher Ca
2+
perfusate (2.3 mmol/L Ca
2+
) followed by 10-minute perfusion of normal perfusate (1.8 mmol/L Ca
2+
), and then were subjected to I/R. A significant functional recovery and decreased lactate dehydrogenase release were observed in HCPC and IPC hearts compared with ischemic control hearts. ATP contents of preconditioned hearts were significantly higher than those of the ischemic control hearts. The cell structure in preconditioned hearts was preserved better than that in the ischemic control hearts. Furthermore, the activation and translocation of PKC from cytoplasm to sarcolemma were observed in the preconditioned hearts. Verapamil administered during IPC significantly attenuated the salutary effects of IPC. Administration of chelerythrine, a specific PKC inhibitor, completely abolished the HCPC- and IPC-induced cardioprotection. The translocation of PKC by IPC was blocked by verapamil or chelerythrine. Immunohistochemical study using rabbit polyclonal antibody against PKC isoforms indicated that stress induced by IPC or HCPC evoked the translocation of PKCα and PKCδ to the cell membrane. Translocation of PKC isoforms was attenuated by the treatment with verapamil or chelerythrine. These results demonstrate that (1) a transient increase in [Ca
2+
]
i
during IPC is an important trigger for the activation of PKC, which is responsible for cardioprotection; (2) the elevation of [Ca
2+
]
i
during IPC, at least partly, resulted from Ca
2+
entry via voltage-dependent Ca
2+
channel; and (3) activation and translocation of PKCα and PKCδ occur during IPC and HCPC and may be important in preconditioning.
Selective Activation of A
3
Adenosine Receptors With
N
6
-(3-Iodobenzyl)Adenosine-5′-
N
-Methyluronamide Protects Against Myocardial Stunning and Infarction Without Hemodynamic Changes in Conscious Rabbits
Authors: Auchampach John A. , Rizvi Ali , Qiu Yumin , Tang Xian-Liang , Maldonado Claudio , Teschner Steffi , Bolli Roberto
Published: June 2012

Abstract
To examine the cardioprotective role of A
3
adenosine receptors during myocardial ischemia/reperfusion injury, we tested the effect of
N
6
-(3-iodobenzyl)adenosine-5′-
N
-methyluronamide (IB-MECA), a potent and selective A
3
adenosine receptor agonist, in models of myocardial stunning and infarction in chronically instrumented conscious rabbits. In phase I (studies of myocardial stunning), rabbits were subjected to six 4-minute coronary occlusions, each separated by 4-minute reperfusion periods, after which the recovery of systolic wall thickening was measured (ultrasonic crystals). In phase II (studies of myocardial infarction), rabbits were subjected to a 30-minute coronary occlusion followed by 3 days of reperfusion. In both phases, IB-MECA was administered as an intravenous bolus (100 μg/kg) 10 minutes before the first coronary occlusion. This dose of IB-MECA was determined in pilot studies to have no effect on heart rate, arterial blood pressure, or plasma histamine concentration in rabbits. In phase I, IB-MECA markedly improved the recovery of wall thickening after the six occlusion/reperfusion cycles, and this effect was sustained throughout the 5-hour observation period; the total deficit of wall thickening (a measure of the overall severity of myocardial stunning) was reduced by 68% (control, 129±16 arbitrary units, n=7; IB-MECA, 41±6 arbitrary units, n=6;
P
<.01). The protective effects of IB-MECA against stunning were completely blocked by pretreatment with the nonselective adenosine receptor antagonist 8-
p
-sulfophenyl theophylline or the specific protein kinase C inhibitor chelerythrine. In phase II, IB-MECA reduced myocardial infarct size by 61%; infarct size (tetrazolium staining) was 41±4% of the risk region in control animals (n=8) and 16±6% in IB-MECA–treated animals (n=8,
P
<.01). These results demonstrate that in conscious rabbits the A
3
adenosine receptor agonist IB-MECA confers a powerful protection against both reversible (stunning) and irreversible (infarction) injury during acute myocardial ischemia and reperfusion by a protein kinase C–mediated pathway, suggesting that selective activation of A
3
receptors is an effective means of protecting the ischemic myocardium without hemodynamic changes.
Endothelin-1 Alters the Contractile Phenotype of Cultured Embryonic Smooth Muscle Cells
Authors: Fisher Steven A. , Ikebe Mitsuo , Brozovich Frank
Published: June 2012

Abstract
Smooth muscle tissues may be classified into phasic (fast) or tonic (slow) contractile phenotypes. This study was initiated to examine the specification of these phenotypes during development and the role of growth factors in this process. We used myosin light chain 17 (MLC
17
) and myosin heavy chain transcript splice variants as markers of the tonic (aortic) and phasic (intestinal) smooth muscle phenotypes in chick embryos. By reverse transcription–polymerase chain reaction, we determined embryonic days 6 to 16 to be a critical period for the establishment of these phenotypes. During this period, endothelin-1 is present at 40-fold-higher levels in aortic compared with intestinal tissues. To test the hypothesis that endothelin-1 may be involved in establishing the aortic (tonic) phenotype, we developed a system in which embryonic smooth muscle cells exhibit phasic and tonic contractile properties in vitro. Single-cell force measurements showed that cultured embryonic gizzard (phasic) cells developed force more rapidly (8±2 seconds) and achieved greater force (3.0±0.7 μN) than did cultured embryonic aortic (tonic) cells (20±0.7 seconds, 0.76±0.01 μN;
P
<.05) in response to depolarization. Chronic exposure of the phasic (gizzard) cells to endothelin-1 prolonged the time to peak force (24±3 seconds) and reduced the peak force (1.0±0.1 μN), so that the contraction resembled the tonic type. This effect, mediated by the endothelin-A receptor, was associated with a shift in MLC
17
splicing to the tonic pattern. These results demonstrate that endothelin-1 is highly enriched in developing aortic compared with intestinal tissues and can convert phasic smooth muscle cells to the tonic type in vitro, suggesting a role for this growth factor during development in determining the contractile phenotype of smooth muscle cells.
Developmental Changes in Transient Outward Current in Mouse Ventricle
Authors: Wang Li , Duff Henry J.
Published: June 2012

Abstract
Developmental changes in the transient outward K
+
current (
I
to
) in mouse ventricular myocytes were assessed by the whole-cell patch-clamp technique. The density of
I
to
in mouse ventricular myocytes was significantly increased from the day-1 neonate to the adult. At +50 mV, the density of
I
to
was 3±1 pA/pF in the day-1 neonate, 15±3 pA/pF in the day-14 neonate, and 19±4 pA/pF in the adult (
P
<.01). Unlike other species, the rate of
I
to
inactivation significantly slowed in mouse ventricular cells during development. Moreover, the time courses of inactivation and recovery from inactivation of
I
to
were well described by a monoexponential function in day-1 neonatal cells, whereas they were best fitted by a biexponential function in day-14 neonatal and adult cells. The characteristics of steady state inactivation were also significantly different in day-1 neonatal cells (half-inactivation potential [V
h
]=−66±4 mV, slope factor [k]=12±2 mV), in day-14 neonatal cells (V
h
=−40±3 mV, k=13±1 mV), and in adult cells (V
h
=−34±4 mV, k=6±1 mV). Microelectrode studies revealed that action potential duration progressively decreased in mouse ventricles during normal postnatal development. In addition, 4-aminopyridine (1 mmol/L) prolonged action potential duration more in adult than in neonatal mouse ventricles, suggesting that the developmental increase in the density of
I
to
contributes to the age-related shortening of action potential duration in mouse ventricles. In conclusion,
I
to
in adult mouse ventricular myocytes exhibits a higher density, slower inactivation kinetics, and a relatively more positive half-inactivation potential. All these characteristics result in
I
to
being a physiologically more important repolarizing K
+
current in adult than in neonatal mouse hearts.
Pulmonary Vasoconstriction and Hypertension in Mice With Targeted Disruption of the Endothelial Nitric Oxide Synthase (NOS 3) Gene
Authors: Steudel Wolfgang , Ichinose Fumito , Huang Paul L. , Hurford William E. , Jones Rosemary C. , Bevan John A. , Fishman Mark C. , Zapol Warren M.
Published: June 2012

Abstract
NO, synthesized in endothelial cells by endothelial NO synthase (NOS 3), is believed to be an important endogenous pulmonary vasodilator substance that contributes to the normal low pulmonary vascular resistance. To selectively investigate the role of NOS 3 in the pulmonary circulation, mice with targeted disruption of the NOS 3 gene were studied. Pulmonary hemodynamics were studied by measuring pulmonary artery pressure, left ventricular end-diastolic pressure, and lower thoracic aortic flow by using a novel open-chest technique. Transient partial occlusion of the inferior vena cava was used to assess the pulmonary artery pressure-flow relationship. Tension developed by isolated pulmonary artery segments after acetylcholine stimulation was measured in vitro. The histological appearance of NOS 3–deficient and wild-type murine lungs was compared. NOS 3–deficient mice (n=27), when compared with wild-type mice (n=32), had pulmonary hypertension (pulmonary artery pressure, 19.0±0.8 versus 16.4±0.6 mm Hg [mean±SE];
P
<.05) that was due to an increased total pulmonary resistance (62±6 versus 33±2 mm Hg·min·g·mL
−1
;
P
<.001). In vitro, acetylcholine induced vasodilation in the main pulmonary arteries of wild-type but not NOS 3–deficient mice. The morphology of the lungs of NOS 3–deficient mice did not differ from that of wild-type mice. We conclude that NOS 3 is a key enzyme responsible for providing basal pulmonary NO release. Congenital NOS 3 deficiency produces mild pulmonary hypertension in mice.
Mitochondrial Response to Heart Rate Steps in Isolated Rabbit Heart Is Slowed After Myocardial Stunning
Authors: Zuurbier C.J. , van Beek J.H.G.M.
Published: June 2012

Abstract
The oxidative capacity of mitochondria isolated from myocardium is undiminished after myocardial stunning, which is remarkable because stunning affects many other cellular functions. The aim of the present study was to assess the mitochondrial oxidative response in intact rather than isolated myocardium. The mean response time of mitochondrial O
2
consumption to heart rate steps (t
mito
) was measured before and after 15-minute ischemia or high-flow hypoxia in isolated rabbit hearts. The t
mito
was calculated from the time course of venous O
2
tension to steps in heart rate, with corrections made for diffusion and vascular transport delay. Isovolumic hearts were perfused with Tyrode’s solution at 37°C. Developed left ventricular pressure at 35 minutes of reperfusion was decreased significantly to 67±3% after ischemia (mean±SEM, n=8) and to 79±6% after hypoxia (n=8) relative to the control condition (n=8), without increased cellular creatine kinase release. Before ischemia or hypoxia, t
mito
was 4.3±0.3 seconds. During reperfusion after ischemia or hypoxia, the increase in t
mito
(by 62±10% and 64±18%, respectively) was significantly larger than that in time controls (24±12% increase). The major determinant of decreased contractility and slower mitochondrial response appeared to be O
2
deprivation and/or reintroduction rather than other consequences of stopped flow. O
2
consumption at a given rate-pressure product was not increased after ischemia or hypoxia, indicating undiminished cardiac contractile economy. Brief ischemia or hypoxia, resulting in stunning, was associated with a slowing of the in vivo mitochondrial oxidative response, indicating that energy transfer and/or signaling between energy-consuming sites and mitochondria is affected in stunned myocardium.
Endothelial Cells of the Human Microvasculature Express Epidermal Fatty Acid–Binding Protein
Authors: Masouyé Isabelle , Hagens Gerry , Van Kuppevelt Toin H. , Madsen Peder , Saurat Jean-Hilaire , Veerkamp Jacques H. , Pepper Michael S. , Siegenthaler Georges
Published: June 2012

Abstract
Epidermal fatty acid–binding protein (E-FABP), previously characterized in human keratinocytes, is a cytoplasmic protein of 15 kD that specifically binds fatty acids (FAs). Previous PAGE-immunoblotting studies indicated that several human tissues display an immunoreactive band with an electrophoretic mobility identical to that of E-FABP. The aim of this study was to determine in which cells, other than keratinocytes, E-FABP might be expressed. By immunohistochemistry, we show that E-FABP is expressed in endothelial cells of the microvasculature of the placenta, heart, skeletal muscle, small intestine, lung, and renal medulla. Interestingly, in lung, a tissue of endodermal origin, E-FABP staining was also localized to secretory cells, ie, Clara cells, goblet cells, and probably a subpopulation of pneumocytes. RNA isolated from cultured human umbilical vein and normal human dermal microvascular endothelial cells was analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR). Southern blotting and sequencing of the cloned RT-PCR products demonstrate that endothelial E-FABP is identical to keratinocyte E-FABP. These data suggest that E-FABP–mediated FA transport occurs at the level of the microvasculature in several FA target organs.
Exacerbation of Cerebral Injury in Mice That Express the P-Selectin Gene
Authors: Connolly E. S. , Winfree C. J. , Prestigiacomo C. J. , Kim S. C. , Choudhri T. F. , Hoh B. L. , Naka Y. , Solomon R. A. , Pinsky D. J.
Published: June 2012

Abstract
There is currently a stark therapeutic void in the treatment of evolving stroke. Although P-selectin is rapidly expressed by hypoxic endothelial cells in vitro, the functional significance of P-selectin expression in stroke remains unexplored. In order to identify the pathophysiological consequences of P-selectin expression and to identify P-selectin blockade as a potential new approach for the treatment of stroke, experiments were performed using a murine model of focal cerebral ischemia and reperfusion. Early P-selectin expression in the postischemic cerebral cortex was demonstrated by the specific accumulation of radiolabeled anti-murine P-selectin IgG, with the increased P-selectin expression localized to the ipsilateral cerebral microvascular endothelial cells by immunohistochemistry. In experiments designed to test the functional significance of increased P-selectin expression in stroke, neutrophil accumulation in the ischemic cortex of mice expressing the P-selectin gene (PS +/+) was demonstrated to be significantly greater than that in homozygous P-selectin–null mice (PS −/−). Reduced neutrophil influx was accompanied by greater postischemic cerebral reflow (measured by laser Doppler) in the PS −/− mice. In addition, PS −/− mice demonstrated smaller infarct volumes (5-fold reduction,
P
<.05) and improved survival compared with PS +/+ mice (88% versus 44%,
P
<.05). Functional blockade of P-selectin in PS +/+ mice using a monoclonal antibody directed against murine P-selectin also improved early reflow and stroke outcome compared with control mice, with reduced cerebral infarction volumes noted even when the blocking antibody was administered after occlusion of the middle cerebral artery. These data are the first to demonstrate a pathophysiological role for P-selectin in stroke and suggest that P-selectin blockade may represent a new therapeutic target in the treatment of stroke.