bluuebunny/crossref_metadata_2025 huggingface dataset:
Tracer Oxygen Distribution Is Barrier-Limited in the Cerebral Microcirculation
Authors: Kassissia Ibrahim G. , Goresky Carl A. , Rose Colin P. , Schwab Andreas J. , Simard André , Huet Pierre-Michel , Bach Glen G.
Published: June 2012

Abstract
The kinetics of tracer oxygen distribution in the brain microcirculation of the awake dog were investigated with the multiple indicator dilution technique. A bolus containing
51
Cr-labeled red blood cells, previously totally desaturated and then resaturated with [
18
O]
2
(oxygen),
125
I-albumin,
22
Na, and [
3
H]water, was injected into the carotid artery, and serial anaerobic blood samples were collected from the sagittal sinus over the next 30 seconds. The outflow recovery curves were analyzed with a distributed-in-space two-barrier model for water and a one-barrier model for oxygen. The analysis provided an estimate of flow per gram brain weight as well as estimates for the tracer water and oxygen rate constants for blood-to-brain exchange and tracer oxygen parenchymal sequestration. Flow to tissue was found to vary between different animals, in concert with parallel changes in oxygen consumption. The
18
O
2
outflow curves showed an early peak, coincident with and more than half the magnitude of its vascular reference curve (labeled red blood cells), whereas the [
3
H]water curve increased abruptly to a low-in-magnitude curve at low flow values and to a small early peak at high flow values. Analysis indicates that the transfers of both
18
O
2
and [
3
H]water indicators from blood to brain are barrier-limited, with the former highly so because of the large red blood cell capacity for oxygen, and that the proportion of the tracer oxygen returning to the circulation from tissue is a small fraction of the total tracer emerging at the outflow.
Cutaneous Active Vasodilation in Humans Is Mediated by Cholinergic Nerve Cotransmission
Authors: Kellogg Dean L. , Pérgola Pablo E. , Piest Kenneth L. , Kosiba Wojciech A. , Crandall Craig G. , Grossmann Mattias , Johnson John M.
Published: June 2012

Abstract
During heat stress, increases in blood flow in nonglabrous skin in humans are mediated through active vasodilation by an unknown neurotransmitter mechanism. To investigate this mechanism, a three-part study was performed to determine the following: (1) Is muscarinic receptor activation necessary for active cutaneous vasodilation? We iontophoretically applied atropine to a small area of forearm skin. At that site and an untreated control site, we measured the vasomotor (laser-Doppler blood flow [LDF]) and sudomotor (relative humidity) responses to whole-body heat stress. Blood pressure was monitored. Cutaneous vascular conductance (CVC) was calculated (LDF÷mean arterial pressure). Sweating was blocked at treated sites only. CVC rose at both sites (
P
<.05 at each site); thus, cutaneous active vasodilation is not effected through muscarinic receptors. (2) Are nonmuscarinic cholinergic receptors present on cutaneous arterioles? Acetylcholine (ACh) was iontophoretically applied to forearm skin at sites pretreated by atropine iontophoresis and at untreated sites. ACh increased CVC at untreated sites (
P
<.05) but not at atropinized sites. Thus, the only functional cholinergic receptors on cutaneous vessels are muscarinic. (3) Does cutaneous active vasodilation involve cholinergic nerve cotransmission? Botulinum toxin was injected intradermally in the forearm to block release of ACh and any coreleased neurotransmitters. Heat stress was performed as in part 1 of the study. At treated sites, CVC and relative humidity remained at baseline levels during heat stress (
P
>.05). Active vasodilator and sudomotor responses to heat stress were abolished by botulinum toxin. We conclude that cholinergic nerve activation mediates cutaneous active vasodilation through release of an unknown cotransmitter, not through ACh.
Inward Rectification and Implications for Cardiac Excitability
Authors: Nichols C.G. , Makhina E.N. , Pearson W.L. , Sha Q. , Lopatin A.N.
Published: June 2012

Abstract
Since the cloning of the first inwardly rectifying K
+
channel in 1993, a family of related clones has been isolated, with many members being expressed in the heart. Exogenous expression of different clones has demonstrated that between them they encode channels with the essential functional properties of classic inward rectifier channels, ATP-sensitive K
+
channels, and muscarinic receptor–activated inward rectifier channels. High-level expression of cloned channels has led to the discovery that classic strong inward, or anomalous, rectification is caused by very steeply voltage-dependent block of the channel by polyamines, with an additional contribution by Mg
2+
ions. Knowledge of the primary structures of inward rectifying channels and the ability to mutate them have led to the determination of many of the structural requirements of inward rectification. The implications of these advances for basic understanding and pharmacological manipulation of cardiac excitability may be significant. For example, cellular concentrations of polyamines are altered under different conditions and can be manipulated pharmacologically. Simulations predict that changes in polyamine concentrations or changes in the relative proportions of each polyamine species could have profound effects on cardiac excitability.
Inhibition of Matrix Metalloproteinase Activity Inhibits Smooth Muscle Cell Migration but Not Neointimal Thickening After Arterial Injury
Authors: Bendeck Michelle P. , Irvin Colleen , Reidy Michael A.
Published: June 2012

Abstract
Smooth muscle cell (SMC) migration and replication are important for neointimal formation after arterial injury. Migration of SMCs requires degradation of basement membrane and extracellular matrix surrounding the cell, and our previous work has shown a correlation between expression of two matrix-degrading metalloproteinases (MMPs), MMP-2 and MMP-9, and smooth muscle migration into the intima in the balloon catheter–injured rat carotid artery. In the present study, an MMP inhibitor, GM 6001, was administered to rats for various times after balloon injury of the carotid artery. Inhibition of MMP activity resulted in a 97% decrease in the number of SMCs that migrated into the intima by 4 days after injury, and lesion growth was retarded by continuous treatment with GM 6001 for up to 10 days after injury. At 10 days, intimal area in GM 6001–treated rats was 0.035±0.008 mm
2
compared with 0.095±0.01 mm
2
in the control group. Neither intimal nor medial SMC replication rates were decreased by GM 6001 treatment, supporting our hypothesis that the decrease in lesion size was due to inhibition of MMP-mediated migration and not inhibition of replication. By 14 days after injury, however, intimal area and SMC number were the same in control and inhibitor-treated rats. An increased rate of SMC replication in the GM 6001 rats (replication rates at 10 days were 56.7±10.0% in the GM 6001 group and 16.97±1.73% in the control group) contributed to “catch-up” growth of the neointima. Thus, it appears that inhibiting SMC migration with MMP inhibitors is not sufficient to inhibit lesion growth, and lesion size eventually catches up to control via increased SMC replication.
Regulation of Matrix Metalloproteinases and Plasminogen Activator Inhibitor-1 Synthesis by Plasminogen in Cultured Human Vascular Smooth Muscle Cells
Authors: Lee Elaine , Vaughan Douglas E. , Parikh Smruti H. , Grodzinsky Alan J. , Libby Peter , Lark Michael W. , Lee Richard T.
Published: June 2012

Abstract
Plasmin and matrix metalloproteinases (MMPs) both participate in extracellular matrix remodeling. This study examined the effects of tumor necrosis factor-α (TNF-α) and plasminogen on collagenase, stromelysin, and plasminogen activator inhibitor-1 (PAI-1) synthesis by cultured human vascular smooth muscle cells (SMCs). TNF-α induced the concentration-dependent synthesis of collagenase and stromelysin, which remained predominantly in proenzyme forms, as determined by Western analysis of culture media. In contrast, plasminogen and plasmin not only increased secretion of MMPs but also induced cleavage to their active forms. The serine protease inhibitor aprotinin inhibited this activation of MMPs by plasminogen and plasmin. TNF-α reduced plasminogen-induced activation of MMPs, suggesting induction of an inhibitor of plasmin generation, such as PAI-1. Enzyme-linked immunosorbent assay of culture media showed that TNF-α (10 ng/mL) increased PAI-1 secretion by 4.2-fold compared with control (105.5±9.6 versus 24.9±1.7 ng/mL, n=3). Surprisingly, plasminogen also increased PAI-1 secretion by vascular SMCs (3.6-fold over control). These results demonstrate coordination of cytokines and serine proteases in regulating MMP secretion and activation. In addition, the induction of PAI-1 by TNF-α and plasminogen suggests a negative-feedback mechanism to limit both plasmin-mediated and MMP-mediated matrix degradation.
Targeting of Transgene Expression to the Vascular Endothelium of Mice by Homologous Recombination at the Thrombomodulin Locus
Authors: Weiler-Guettler Hartmut , Aird William C. , Husain Mansoor , Rayburn Helen , Rosenberg Robert D.
Published: June 2012

Abstract
We describe a straightforward gene-targeting technique to achieve uniform, stable, and genetically invariant expression of a transgene in the vascular endothelium of mice. To demonstrate the feasibility of this approach, the reporter gene bacterial β-galactosidase was inserted via homologous recombination into the intronless thrombomodulin locus of murine embryonic stem cells. In this fashion, the lacZ gene is placed under the regulatory control of the endogenous thrombomodulin promoter. The expression of the transgene in adult mice recapitulated the widespread, stable, and high-level expression of the thrombomodulin gene in vascular endothelium. These data indicate that targeting of cDNAs into the thrombomodulin locus serves as a viable strategy to express transgenes in endothelial cells. Analysis of reporter gene expression revealed a heterogeneous pattern of thrombomodulin gene activity in the endothelium of the aorta and its tributaries. We also show that embryonic stem cells with a targeted thrombomodulin locus contribute in a mosaic fashion to the vascular endothelium of chimeric mice. This method for generating animals with a functionally heterogeneous cardiovascular system should provide an experimental technique for studying how localized genetic abnormalities in endothelial cell function lead to the development of vascular diseases.
SM22α, a Marker of Adult Smooth Muscle, Is Expressed in Multiple Myogenic Lineages During Embryogenesis
Authors: Li Li , Miano Joseph M. , Cserjesi Peter , Olson Eric N.
Published: June 2012

Abstract
SM22α is a calponin-related protein that is expressed specifically in adult smooth muscle. To begin to define the mechanisms that regulate the establishment of the smooth muscle lineage, we analyzed the expression pattern of the SM22α gene during mouse embryogenesis. In situ hybridization demonstrated that SM22α transcripts were first expressed in vascular smooth muscle cells at about embryonic day (E) 9.5 and thereafter continued to be expressed in all smooth muscle cells into adulthood. In contrast to its smooth muscle specificity in adult tissues, SM22α was expressed transiently in the heart between E8.0 and E12.5 and in skeletal muscle cells in the myotomal compartment of the somites between E9.5 and E12.5. The expression of SM22α in smooth muscle cells, as well as early cardiac and skeletal muscle cells, suggests that there may be commonalities between the regulatory programs that direct muscle-specific gene expression in these three myogenic cell types.
Ionic Mechanism of Action Potential Prolongation in Ventricular Myocytes From Dogs With Pacing-Induced Heart Failure
Authors: Kääb Stefan , Nuss H. Bradley , Chiamvimonvat Nipavan , O’Rourke Brian , Pak Peter H. , Kass David A. , Marban Eduardo , Tomaselli Gordon F.
Published: June 2012

Abstract
Membrane current abnormalities have been described in human heart failure. To determine whether similar current changes are observed in a large animal model of heart failure, we studied dogs with pacing-induced cardiomyopathy. Myocytes isolated from the midmyocardium of 13 dogs with heart failure induced by 3 to 4 weeks of rapid ventricular pacing and from 16 nonpaced control dogs did not differ in cell surface area or resting membrane potential. Nevertheless, action potential duration (APD) was significantly prolonged in myocytes isolated from failing ventricles (APD at 90% repolarization, 1097±73 milliseconds [failing hearts, n=30] versus 842±56 milliseconds [control hearts, n=25];
P
<.05), and the prominent repolarizing notch in phase 1 was dramatically attenuated. Basal L-type Ca
2+
current and whole-cell Na
+
current did not differ in cells from failing and from control hearts, but significant differences in K
+
currents were observed. The density of the inward rectifier K
+
current (I
K1
) was reduced in cells from failing hearts at test potentials below −90 mV (at −150 mV, −19.1±2.2 pA/pF [failing hearts, n=18] versus −32.2±5.1 pA/pF [control hearts, n=15];
P
<.05). The small outward current component of I
K1
was also reduced in cells from failing hearts (at −60 mV, 1.7±0.2 pA/pF [failing hearts] versus 2.5±0.2 pA/pF [control hearts];
P
<.05). The peak of the Ca
2+
-independent transient outward current (I
to
) was dramatically reduced in myocytes isolated from failing hearts compared with nonfailing control hearts (at +80 mV, 7.0±0.9 pA/pF [failing hearts, n=20] versus 20.4±3.2 pA/pF [control hearts, n=15];
P
<.001), while the steady state component was unchanged. There were no significant differences in I
to
kinetics or single-channel conductance. A reduction in the number of functional I
to
channels was demonstrated by nonstationary fluctuation analysis (0.4±0.03 channels per square micrometer [failing hearts, n=5] versus 1.2±0.1 channels per square micrometer [control hearts, n=3];
P
<.001). Pharmacological reduction of I
to
by 4-aminopyridine in control myocytes decreased the notch amplitude and prolonged the APD. Current clamp–release experiments in which current was injected for 8 milliseconds to reproduce the notch sufficed to shorten the APD significantly in cells from failing hearts. These data support the hypothesis that downregulation of I
to
in pacing-induced heart failure is at least partially responsible for the action potential prolongation. Because the repolarization abnormalities mimic those in cells isolated from failing human ventricular myocardium, canine pacing-induced cardiomyopathy may provide insights into the development of repolarization abnormalities and the mechanisms of sudden death in patients with heart failure.
Endothelin-1 and Angiotensin II Receptors in Cells From Rat Hypertrophied Heart
Authors: Fareh Jeannette , Touyz Rhian M. , Schiffrin Ernesto L. , Thibault Gaetan
Published: June 2012

Abstract
This study investigates the cellular localization and regulation of endothelin-1 (ET-1) and angiotensin II (Ang II) receptors and the effects of ET-1 and Ang II on [Ca
2+
]
i
in cardiac hypertrophy due to volume overload in the rat. Radioligand binding assays and [Ca
2+
]
i
measurements by fura 2 methodology were performed on isolated ventricular cardiomyocytes and fibroblasts from the heart of rats with a 4-week aortocaval shunt. In the hypertrophied myocardium, ET-1 and Ang II concentrations were unchanged in ventricles. Ventricular ET-1 receptors had a cell-specific distribution: >90% of ET receptors in cardiomyocytes are of the ET
A
subtype, whereas fibroblasts had a nearly equal proportion of the ET
A
and ET
B
subtypes. ET-1 receptor densities, affinities, and ET-1–induced [Ca
2+
]
i
were not significantly different from control in both ventricular cell types from hypertrophied myocardium. Ang II specific binding was very low on isolated ventricular cardiomyocytes, suggesting few receptors in control conditions. However, [Ca
2+
]
i
responses induced by Ang II at concentrations >10
−8
mol/L were detectable and were significantly higher in hypertrophied cardiomyocytes. Ang II receptor density (exclusively AT
1
) on fibroblasts was significantly reduced (42 970±3330 versus 73 870±7940 sites per cell for control cells,
P
<.01), but AT
1
receptor affinity was unchanged after volume overload. The maximum increase in [Ca
2+
]
i
evoked by 10
−6
to 10
−4
mol/L Ang II was significantly lower in fibroblasts from overloaded hearts. In conclusion, ET-1 receptor proportion is cell specific, with cardiomyocytes possessing predominantly the ET
A
subtype and fibroblasts possessing both ET
A
and ET
B
receptors. Plasma and cardiac ET-1 concentrations and ET-1 receptor regulation on both ventricular cell types are not altered in cardiac volume overload, suggesting that cardiac ET-1 may not play a significant role in this model. Cardiac hypertrophy induced a significant downregulation of AT
1
receptors on fibroblasts, whereas total binding and [Ca
2+
]
i
sensitivity to Ang II were significantly enhanced in hypertrophied cardiomyocytes. This suggests that cardiac Ang II may be involved in the pathophysiology of the cardiac hypertrophy of volume overload.
Tropoelastin Gene Expression in Individual Vascular Smooth Muscle Cells
Authors: Belknap James K. , Grieshaber Nicole A. , Schwartz Phillip E. , Orton E. Christopher , Reidy Michael A. , Majack Richard A.
Published: June 2012

Abstract
After vascular injury, quiescent adult smooth muscle cells (SMCs) revert to a more immature synthetic-state phenotype concomitant with the onset of cell replication. The relationship between SMC proliferation and the reexpression of genes characteristic of immature SMCs (eg, tropoelastin [TE]), on an individual cell basis, has not been determined. Using a combined bromodeoxyuridine (BrdU) immunocytochemistry–TE in situ hybridization technique, we determined the relationship between DNA synthesis and TE gene expression in the rat vascular wall during development and after experimental injury. During the early development of the aortic media (embryonic days 13 to 18), low but detectable levels of TE expression occurred equally in both replicating and nonreplicating SMCs. TE message levels dramatically increased in the late fetal and early postnatal periods (fetal day 19 to 1 month postpartum), after a precipitous drop in SMC replication, and then decreased to undetectable levels by postpartum day 60. After balloon catheter injury in the adult, a developmental sequence of SMC replication followed by TE gene expression was reiterated in both the media and in the developing neointima. On an individual cell basis, adult SMCs replicating after injury expressed little or no TE message; detectable TE gene expression occurred only in nonreplicating SMCs. The most important implications of these data are that (1) adult SMCs replicating after injury appear to revert to a preelastogenic embryonic phenotype; (2) maximal TE expression occurs in SMCs only after the cessation of cell replication; and (3) in both the media and the neointima, adult SMCs responding to injury undergo temporally sequential changes in phenotype reflective of SMC development.
Differential Distribution of Electrophysiologically Distinct Myocytes in Conduit and Resistance Arteries Determines Their Response to Nitric Oxide and Hypoxia
Authors: Archer Stephen L. , Huang James M.C. , Reeve Helen L. , Hampl Václav , Tolarová Simona , Michelakis Evangelos , Weir E. Kenneth
Published: June 2012

Abstract
The cellular mechanisms that determine differences in reactivity of arteries of varying size and origin are unknown. We evaluated the hypothesis that there is diversity in the distribution of K
+
channels between vascular smooth muscle (VSM) cells within a single segment of the pulmonary arteries (PAs) and that there are differences in the prevalence of these cell types between conduit and resistance arteries, which contribute to segmental differences in the vascular response to NO and hypoxia. Three types of VSM cells can be identified in rat PAs on the basis of their whole-cell electrophysiological properties—current density and the pharmacological dissection of whole-cell K
+
current (I
K
)—and morphology. Cells are referred to as “K
Ca
, K
DR
, or mixed,” acknowledging the type of K
+
channel that dominates the I
K
: the Ca
2+
-sensitive (K
Ca
) channel, delayed rectifier (K
DR
) channel, or a mixture of both. The three cell types were identified by light and electron microscopy. K
Ca
cells are large and elongated, and they have low current density and currents that are inhibited by tetraethylammonium (5 mmol/L) or charybdotoxin (100 nmol/L). K
DR
cells are smaller, with a perinuclear bulge, but have high current density and currents that are inhibited by 4-aminopyridine (5 mmol/L). Conduit arteries contain significant numbers of K
Ca
cells, whereas resistance arteries have a majority of K
DR
cells and few K
Ca
cells. NO rapidly and reversibly increases I
K
and hyperpolarizes K
Ca
cells because of an increase in open probability of a 170-pS K
Ca
channel. Hypoxia depolarizes K
DR
cells by rapidly and reversibly inhibiting one or more of the tonically active K
DR
channels (including a 37-pS channel) that control resting membrane potential. The effects of both hypoxia and NO on K
+
channels are evident at negative membrane potentials, supporting their physiological relevance. The functional correlate of this electrophysiological diversity is that K
DR
-enriched resistance vessels constrict to hypoxia, whereas conduit arteries have a biphasic response predominated by relaxation. Although effective in both segments, NO relaxes conduit more than resistance rings, in both cases by a cGMP-dependent mechanism. We conclude that regional electrophysiological diversity among smooth muscle cells is a major determinant of segmental differences in vascular reactivity.
Class III Antiarrhythmic Drugs Block HERG, a Human Cardiac Delayed Rectifier K
+
Channel
Authors: Spector Peter S. , Curran Mark E. , Keating Mark T. , Sanguinetti Michael C.
Published: June 2012

Abstract
We recently reported that mutations in HERG, a potassium channel gene, cause long QT syndrome. Heterologous expression of HERG in
Xenopus
oocytes revealed that this channel had biophysical properties nearly identical to a cardiac delayed rectifier K
+
current, I
Kr
, but had dissimilar pharmacological properties. Class III antiarrhythmic drugs such as E-4031 and MK-499 are potent and specific blockers of I
Kr
in cardiac myocytes. Our initial studies indicated that these compounds did not block HERG at a concentration of 1 μmol/L. In the present study, we used standard two-microelectrode voltage-clamp techniques to further characterize the effects of these drugs on HERG channels expressed in oocytes. Consistent with initial findings, 1 μmol/L MK-499 and E-4031 had no effect on HERG when oocytes were voltage clamped at a negative potential and not pulsed during equilibration with the drug. However, MK-499 did block HERG current if oocytes were repetitively pulsed, or clamped at a voltage positive to the threshold potential for channel activation. This finding is in contrast to previous studies that showed significant block of I
Kr
in isolated myocytes by similar drugs, even in the absence of pulsing. This apparent discrepancy may be due to differences in channel characteristics (HERG versus guinea pig and mouse I
Kr
), tissue (oocytes versus myocytes), or specific drugs. Under steady state conditions, block of HERG by MK-499 was half maximal at 123±12 nmol/L at a test potential of −20 mV. MK-499 (150 nmol/L) did not affect the voltage dependence of activation and rectification nor the kinetics of activation and deactivation of HERG. These data indicate that MK-499 preferentially blocks open HERG channels and further support the conclusion that HERG subunits form I
Kr
channels in cardiac myocytes.
Mechanism of Hydrogen Peroxide and Hydroxyl Free Radical–Induced Intracellular Acidification in Cultured Rat Cardiac Myoblasts
Authors: Wu Mei-Lin , Tsai Ke-Li , Wang Seu-Mei , Wu Jiahn-Chun , Wang Bor-Sen , Lee Yuan-Teh
Published: June 2012

Abstract
After a transient ischemic attack of the cardiac vascular system, reactive oxygen-derived free radicals, including the superoxide (O
2
−•
) and hydroxyl (
•
OH) radicals can be easily produced during reperfusion. These free radicals have been suggested to be responsible for reperfusion-induced cardiac stunning and reperfusion-induced arrhythmia. Hydrogen peroxide (H
2
O
2
) is often used as an experimental source of oxygen-derived free radicals. Using freshly dissociated single rat cardiac myocytes and the rat cardiac myoblast cell line, H9c2, we have shown, for the first time, that an intriguing pH
i
acidification (≈0.24 pH unit) is induced by the addition of 100 μmol/L H
2
O
2
and that this dose is without effect on the intracellular free Ca
2+
levels or viability of the cells. Using H9c2 as a model cardiac cell, we have shown that it is the intracellular production of
•
OH, and not O
2
−•
or H
2
O
2
, that results in this acidification. We have excluded any involvement of (1) the three known cardiac pH
i
regulators (the Na
+
-H
+
exchanger, the Cl
−
-HCO
3
exchanger, and the Na
+
-HCO
3
cotransporter), (2) a rise in intracellular Ca
2+
levels, and (3) inhibition of oxidative phosphorylation. However, we have found that H
2
O
2
-induced acidosis is due to inhibition of the glycolytic pathway, with hydrolysis of intracellular ATP and the resultant intracellular acidification. In cardiac muscle and in skinned cardiac muscle fiber, it has been shown that a small intracellular acidification may severely inhibit contractility. Therefore, the sustained pH
i
decrease caused by hydroxyl radicals may contribute, in some part, to the well-documented impairment of cardiac mechanical function (ie, reperfusion cardiac stunning) seen during reperfusion ischemia.
K
+
Currents in Human Coronary Artery Vascular Smooth Muscle Cells
Authors: Gollasch Maik , Ried Christian , Bychkov Rostislav , Luft Friedrich C. , Haller Hermann
Published: June 2012

Abstract
K
+
channels and their currents are important in vascular tone regulation and are potential therapeutic targets; however, K
+
channels in human coronary artery vascular smooth muscle cells (VSMCs) have received little attention. We examined K
+
currents in freshly isolated VSMCs from human coronary arteries (n=368 from 32 human hearts) with conventional patch-clamp or perforated-patch techniques with nystatin. We detected four different K
+
currents: (1) the delayed rectifier K
+
current, I
K(dr)
; (2) the Ca
2+
-activated K
+
current, I
K(Ca)
; (3) the nonrectifying noninactivating outward ATP-dependent K
+
current, I
K(ATP)
; and (4) the spontaneous transient outward K
+
current, I
K(STOC)
. K
+
channels underlying spontaneous transient outward currents probably represent a single clustered population of Ca
2+
-activated K
+
channels functionally associated with Ca
2+
release channels in the sarcoplasmic reticulum. Inwardly rectifying K
+
currents were not observed. K
+
currents were unevenly distributed in that they were not uniformly exhibited by all cells. The most prominent K
+
currents were I
K(Ca)
(100%) and I
K(dr)
(46%). I
K(STOC)
s, which have not been previously described in humans, were present in 67% of VSMCs. I
K(ATP)
was small under physiological conditions; however, I
K(ATP)
increased markedly after cell stimulation with exogenous or endogenous coronary vasodilators. Thus, I
K(ATP)
may be particularly relevant in ischemia and could be of special importance as a therapeutic target. We conclude that human coronary VSMCs have unique K
+
currents that differ sufficiently from those of other species, thus making the investigation of human material clinically relevant. The findings suggest potential avenues for further therapeutic research.
Cellular Basis for the Negative Dromotropic Effect of Adenosine on Rabbit Single Atrioventricular Nodal Cells
Authors: Wang Desuo , Shryock John C. , Belardinelli Luiz
Published: June 2012

Abstract
The effects of adenosine on action potentials, rate-dependent activation failure (the cellular basis for second-degree atrioventricular [AV] block), and the recovery of excitability in rabbit isolated single AV nodal cells were studied using the whole-cell patch-clamp technique. Adenosine (1 μmol/L) shortened the duration, depressed the amplitude, and reduced the rate of rise of the AV nodal cell action potential. Adenosine (10 μmol/L) caused a significant hyperpolarization (7±1 mV) of AV nodal cells. Adenosine increased the occurrence and the rate dependence of activation failure (Wenckebach periodicity) of AV nodal cells; this effect was concentration dependent and mediated by A
1
adenosine receptors. The rate-dependent activation failure caused by adenosine was associated with a prolongation of the effective refractory period by 18±2 ms (
P
<.05), an increase in the duration of activation delay, and an elevation (from 0.22±0.04 to 0.30±0.03 nA,
P
<.05) of the threshold current amplitude required to activate AV nodal cells. The results suggest that the slowed recovery of excitability of AV nodal cells caused by adenosine forms the cellular basis for adenosine-induced second-degree AV block. Adenosine decreased I
Ca,L
and activated I
K,ADO
of AV nodal cells. These actions of adenosine on ion currents may contribute to the effect of this nucleoside to depress excitability of AV nodal cells. The enhancement by adenosine of rate-dependent activation failure of AV nodal cells implies that the negative dromotropic effect of adenosine should be more pronounced during an episode of supraventricular tachycardia than during normal rhythm.
Endogenous Retinoic Acid Signaling Colocalizes With Advanced Expression of the Adult Smooth Muscle Myosin Heavy Chain Isoform During Development of the Ductus Arteriosus
Authors: Colbert Melissa C. , Kirby Margaret L. , Robbins Jeffrey
Published: June 2012

Abstract
During fetal development, a specialized vessel, the ductus arteriosus, shunts blood from the pulmonary artery to the aorta, thus bypassing the lungs. The ductus differs primarily from the great vessels in that it is a muscular rather than an elastic artery, and the etiology of this differential development remains controversial. We present evidence that retinoic acid (RA) may contribute to the unique muscle phenotype of the ductus arteriosus. Using a transgenic mouse carrying an RA response element–
lacZ
transgene that expresses β-galactosidase (β-gal) in response to endogenous RA signals during embryonic and fetal development, we observe a strong β-gal signal in the ductus arteriosus. By immunofluorescence, this signal colocalizes with the expression of the adult-specific smooth muscle myosin heavy chain isoform, SM2. The β-gal signal is present throughout fetal development and persists in the neonate until the ductus arteriosus is completely closed. β-Gal–positive cells are first detected by immunofluorescence at 13.5 days postcoitum (dpc) in the mesenchyme surrounding the ductus. By 15.5 dpc, very intense β-gal staining localizes to the ductus arteriosus but is absent or minimal in the pulmonary trunk and aortic arch; by 17.5 dpc, the smooth muscle layers of the tunica media in the ductus arteriosus exhibit positive β-gal staining. Immunostaining with antibodies against smooth muscle myosins shows that, while SM1 is expressed in all embryonic vessels, SM2 is precociously expressed in the ductus arteriosus. Furthermore, SM2 expression can be detected in the ductus as early as 15.5 dpc. In the neonate, the β-gal signal persists in the smooth muscle layer of the ductus and immunostaining colocalizes with SM2 expression. These data suggest that RA may play a role in inducing and maintaining smooth muscle differentiation in the developing ductus arteriosus and may promote precocious expression of the adult vascular phenotype.
Modification of Viral Myocarditis in Mice by Interleukin-6
Authors: Kanda Tsugiyasu , Wilson McManus Janet E. , Nagai Ryozo , Imai Susumu , Suzuki Tadashi , Yang DeCheng , McManus Bruce M. , Kobayashi Isao
Published: June 2012

Abstract
Inflammatory cytokines play a key role in the myocardial injury produced by viral myocarditis. Although interleukin-6 (IL-6) reportedly possesses antiviral properties, its effect in viral myocarditis is unclear. To investigate the role of IL-6 in viral myocarditis induced by encephalomyocarditis virus (EMCV) in mice, we evaluated (1) the survival rate following IL-6 administration, (2) the viral titer in the heart, (3) viral replication in the heart by in situ hybridization, (4) histopathological changes using immunohistochemical staining, (5) neutralizing antibody against EMCV, (6) circulating interferon and tumor necrosis factor-α (TNF-α), (7) viral suppression in vitro by IL-6, and (8) natural killer (NK)–cell activity. Eight-week-old C3H/HeJ mice were injected intraperitoneally with EMCV (day 0) and were also injected subcutaneously twice daily for 4 consecutive days with 10 μg/0.1 mL of human IL-6 on day −4 (group A), day 0 (group B), or day +4 (group D) for 4 days. As a control, 0.1 mL PBS instead of IL-6 was injected on day 0 for 4 days (group C). Certain mice were killed on day 4. The myocardial virus titers, viral replication in situ, and NK-cell activity in the spleen were determined. Decreased viral titer and viral replication in the heart reduced the titer of circulating TNF-α, and lower NK-cell activity was observed in group B versus group C (control group). The titer of neutralizing antibodies against EMCV was significantly (
P
<.05) increased in group B compared with group C. The remaining mice were killed on days 10 and 30 after infection. The ratio of heart weight (HW) to body weight (BW) and myocardial injury in group B were reduced versus group C on days 10 and 30. The HW of group B on day 30 did not differ from the normal control group. The ratio of splenic weight to BW and the ratio of thymic weight to BW of group B increased on day 10, with expanded follicles observed in the spleen and enlargement of the medulla observed in the thymus. Immunohistochemical study revealed an increased percentage of macrophages in the heart and spleen of group B. In summary, IL-6 reduces myocardial damage in mice with viral myocarditis. Modification of immune responses together with reduction in viral replication appears to be the mechanism of the IL-6 effect. Although IL-6 is likely important in the process of viral antigen presentation, early activation of immune responses and attenuation of viral replication appear most significant, as reflected in the limited time window during which IL-6 is effective in myocarditis.
Altered Ventricular and Myocyte Response to Angiotensin II in Pacing-Induced Heart Failure
Authors: Cheng Che-Ping , Suzuki Makoto , Ohte Nobuyuki , Ohno Michiya , Wang Zhong-Min , Little William C.
Published: June 2012

Abstract
Alterations in the cardiac response to angiotensin II (Ang II) may contribute to the functional impairment in tachycardia-induced heart failure (congestive heart failure [CHF]). Accordingly, we studied the response to Ang II in eight conscious instrumented dogs before and after inducing CHF. Left ventricular (LV) performance was assessed by measuring LV pressure and LV volume. Isolated myocyte function was evaluated using computer-assessed videomicroscopy. In conscious animals before CHF, Ang II produced a load-dependent slowing of the time constant of LV relaxation (τ) and did not depress intact LV contractile function. After CHF, although Ang II produced a similar increase in LV systolic pressure, the increases in LV diastolic pressure and time constant τ were much greater, and contractile performance was depressed. These changes persisted when the elevation of end-systolic pressure was prevented by nitroprusside. Similar changes were also present after autonomic blockade. In isolated myocytes, before CHF, Ang II (10
−6
mol/L) produced a slight positive inotropic effect. In contrast, after CHF, Ang II produced a negative inotropic effect and slowed the rate of relengthening. The effects in the intact LV and myocytes were reversed by an Ang II AT
1
receptor blocker (losartan). We conclude that pacing-induced CHF alters the LV and myocyte response to Ang II, so that Ang II produces direct depressions in intact LV contraction, relaxation, and filling and exacerbates myocyte contractile dysfunction. These effects are mediated through the activation of AT
1
receptors.
Decreased Energy Reserve in an Animal Model of Dilated Cardiomyopathy
Authors: Liao Ronglih , Nascimben Luigino , Friedrich Jan , Gwathmey Judith K. , Ingwall Joanne S.
Published: June 2012

Abstract
An animal model was used to test the hypothesis that in heart failure the decrease in the ability to resynthesize ATP through the creatine kinase (CK) reaction (which we call energy reserve) contributes to the inability of the heart to maintain its normal function and contractile reserve. One-week-old turkey poults were fed furazolidone for 14 days to induce dilated cardiomyopathy. Isolated Langendorff-perfused hearts from these myopathic animals showed a 73% decrease in baseline isovolumic contractile performance. Neither increasing [Ca
2+
]
o
nor electrical pacing rate increased isovolumic contractile performance. Measured by
31
P nuclear magnetic resonance magnetization transfer and chemical assay, ATP concentration was decreased by 23%, phosphocreatine concentration by 42%, CK enzyme activity by 34%, and the pseudo first-order rate constant for the CK reaction by 50%. Measured CK reaction velocity decreased by 71%. The reduced ability to increase cardiac performance in response to increasing [Ca
2+
]
o
in hearts with lower CK reaction velocity was reproduced in part by feeding a separate group of turkey poults β-guanidinopropionic acid to specifically reduce CK reaction velocity by decreasing guanidino substrate concentration. These hearts had normal baseline performance but blunted contractile reserve. These observations provide further support for the hypothesis that a decrease in energy reserve via the CK system contributes to reduced cardiac function in the failing heart.
Role of Poly-ADP Ribosyltransferase Activation in the Vascular Contractile and Energetic Failure Elicited by Exogenous and Endogenous Nitric Oxide and Peroxynitrite
Authors: Szabó Csaba , Zingarelli Basilia , Salzman Andrew L.
Published: June 2012

Abstract
Stimulation of vascular smooth muscle with bacterial lipopolysaccharide (LPS) and proinflammatory cytokines induces the expression of a distinct isoform of NO synthase (inducible NOS [iNOS]) contributing to the suppression of vascular contractility. We have obtained evidence of the involvement of an indirect pathway triggered by NO and its reaction product peroxynitrite (ONOO
−
) through the activation of the nuclear enzyme poly-ADP ribosyltransferase (PARS) in the pathogenesis of cellular energetic and contractile failure in vascular smooth muscle. Exposure of vascular smooth muscle cells caused DNA strand breaks, activation of PARS, depletion of NAD
+
, and inhibition of mitochondrial respiration. The NAD
+
depletion and inhibition of mitochondrial respiration were reduced by pharmacological inhibition of PARS. Stimulation of vascular smooth muscle cells with LPS and interferon gamma (IFN-γ) triggered the production of superoxide anion over 3 to 48 hours and NO and ONOO
−
over 24 to 48 hours and resulted in significant DNA strand breakage. The decrease in mitochondrial respiration in response to LPS and IFN-γ stimulation was inhibited by the ONOO
−
scavenger uric acid (100 μmol/L) and by inhibitors of iNOS. The PARS inhibitors 3-aminobenzamide (1 mmol/L), nicotinamide (1 mmol/L), and PD 128763 (100 μmol/L) inhibited the reduction in cellular NAD
+
and ATP and the suppression of mitochondrial respiration in response to LPS and IFN-γ stimulation. Administration of 3-aminobenzamide also reduced PARS activation and vascular hyporeactivity of rat thoracic aortas exposed to ONOO
−
(300 μmol/L to 1.5 mmol/L) in vitro. 3-Aminobenzamide (10 mg/kg IP) preserved the ex vivo contractility of aortas obtained from endotoxic rats and improved survival in lethal murine endotoxic shock. These data suggest that PARS activation due to iNOS induction (1) is involved in the energetic depletion of vascular smooth muscle cells that express iNOS and (2) contributes to the pathogenesis of vascular energetic and contractile failure in endotoxic shock. Inhibition of PARS may be a novel concept of therapeutic potential in shock.
In Situ Hybridization Reveals Extensive Diversity of K
+
Channel mRNA in Isolated Ferret Cardiac Myocytes
Authors: Brahmajothi Mulugu V. , Morales Michael J. , Liu Shuguang , Rasmusson Randall L. , Campbell Donald L. , Strauss Harold C.
Published: June 2012

Abstract
The molecular basis of K
+
currents that generate repolarization in the heart is uncertain. In part, this reflects the similar functional properties different K
+
channel clones display when heterologously expressed, in addition to the molecular diversity of the voltage-gated K
+
channel family. To determine the identity, regional distribution, and cellular distribution of voltage-sensitive K
+
channel mRNA subunits expressed in ferret heart, we used fluorescent labeled oligonucleotide probes to perform in situ hybridization studies on enzymatically isolated myocytes from the sinoatrial (SA) node, right and left atria, right and left ventricles, and interatrial and interventricular septa. The most widely distributed K
+
channel transcripts in the ferret heart were Kv1.5 (present in 69.3% to 85.6% of myocytes tested, depending on the anatomic region from which myocytes were isolated) and Kv1.4 (46.1% to 93.7%), followed by Kv1.2, Kv2.1, and Kv4.2. Surprisingly, many myocytes contain transcripts for Kv1.3, Kv2.2, Kv4.1, Kv5.1, and members of the Kv3 family. Kv1.1, Kv1.6, and Kv6.1, which were rarely expressed in working myocytes, were more commonly expressed in SA nodal cells. IRK was expressed in ventricular (84.3% to 92.8%) and atrial (52.4% to 64.0%) cells but was nearly absent (6.6%) in SA nodal cells; minK was most frequently expressed in SA nodal cells (33.7%) as opposed to working myocytes (10.3% to 29.3%). Two gene products implicated in long-QT syndrome,
ERG
and KvLQT1, were common in all anatomic regions (41.1% to 58.2% and 52.1% to 71.8%, respectively). These results show that the diversity of K
+
channel mRNA in heart is greater than previously suspected and that the molecular basis of K
+
channels may vary from cell to cell within distinct regions of the heart and also between major anatomic regions.
Cell Cycle-Dependent Expression of L- and T-Type Ca
2+
Currents in Rat Aortic Smooth Muscle Cells in Primary Culture
Authors: Kuga Takeshi , Kobayashi Sei , Hirakawa Yoji , Kanaide Hideo , Takeshita Akira
Published: June 2012

The expression of L- and T-type Ca
2+
channels has been reported to change during various biological events, including cellular differentiation and proliferation. The present study aimed to examine whether or not the expression of L- and T-type Ca
2+
channels depends on the cell cycle in rat aortic smooth muscle cells in primary culture. Both the phase of the cell cycle and the functional expression of Ca
2+
channels were determined in the same single cell, using an immunocytochemical analysis of cell cycle-specific nuclear antigens and a whole-cell voltage-clamp method, respectively. In the G
0
(n=130) and M (n=75) phases, all cells showed only L-type Ca
2+
currents. The cells showing a T-type Ca
2+
current appeared in the G
1
phase (37%, n=85) and increased in the S phase (90%, n=21). For L-type Ca
2+
channels, the current density was significantly greater in the G
1
phase than in the G
0
and M phases. However, either the voltage-dependent properties or the dose-response relationships of Bay K 8644- and second messenger-induced modulations of L-type Ca
2+
current did not differ in the four phases of the cell cycle. These findings thus indicate that the expression of L- and T-type Ca
2+
channels depends on the cell cycle, whereas the characteristics of L-type Ca
2+
channels do not differ between the phases of the cell cycle.
Developmental Changes in the Delayed Rectifier K
+
Channels in Mouse Heart
Authors: Wang Li , Feng Zhong-Ping , Kondo Colleen S. , Sheldon Robert S. , Duff Henry J.
Published: June 2012

Expression of cardiac transient outward current and inwardly rectifying K
+
current is age dependent. However, little is known about age-related changes in cardiac delayed rectifier K
+
current (I
K
, with rapidly and slowly activating components, I
Kr
and I
Ks
, respectively). Accordingly, the purpose of the present study was to assess developmental changes in I
K
channels in fetal, neonatal, and adult mouse ventricles. Three techniques were used: conventional microelectrode to measure the action potential, voltage clamp to record macroscopic currents of I
K
, and radioligand assay to examine [
3
H]dofetilide binding sites. The extent of prolongation of action potential duration at 95% repolarization (APD
95
) by a selective I
Kr
blocker, dofetilide (1 μmol/L), dramatically decreased from fetal (137%±18%) to day-1 (75%±29%) and day-3 (20%±15%) neonatal mouse ventricular tissues (
P
<.01). Dofetilide did not prolong APD
95
in adult myocardium. I
Kr
is the sole component of I
K
in day-18 fetal mouse ventricular myocytes. However, both I
Kr
and I
Ks
were observed in day-1 neonatal ventricular myocytes. With further development, I
Ks
became the dominant component of I
K
in day-3 neonates. In adult mouse ventricular myocytes, neither I
Kr
nor I
Ks
was observed. Correspondingly, a high-affinity binding site for [
3
H]dofetilide was present in fetal mouse ventricles but was absent in adult ventricles. The complementary data from microelectrode, voltage-clamp, and [
3
H]dofetilide binding studies demonstrate that expression of the I
K
channel is developmentally regulated in the mouse heart.
Molecular and Functional Identification of m
1
Muscarinic Acetylcholine Receptors in Rat Ventricular Myocytes
Authors: Sharma Virendra K. , Colecraft Henry M. , Wang David X. , Levey Allan I. , Grigorenko Elena V. , Yeh Hermes H. , Sheu Shey-Shing
Published: June 2012

The expression of muscarinic acetylcholine receptor (mAChR) subtypes in freshly isolated adult rat ventricular myocytes was investigated by reverse transcription of cellular mRNA followed by amplification of cDNA using the polymerase chain reaction (PCR). After reverse-transcriptase PCR, bands were obtained corresponding to the expected sizes for the m
1
and m
2
but not for the m
3
to m
5
mAChRs. The identity of the m
1
and m
2
bands was confirmed by single-cell PCR, restriction digest mapping, and Southern blot analysis. The presence of m
1
and m
2
, but not m
3
, mAChR protein in these cells was shown by indirect immunofluorescence studies using subtype-specific antibodies. It was further investigated whether the identified m
1
mAChR was responsible for the stimulatory effects on Ca
2+
transients by high concentrations of carbachol (>10 μmol/L) known to occur in these cells. In pertussis toxin–treated ventricular myocytes electrically stimulated at 1 Hz, carbachol (300 μmol/L) increased the basal Ca
2+
level from 96±7 to 118±8 nmol/L and the peak Ca
2+
transient level from 519±32 to 640±36 nmol/L (mean±SEM,
P
<.05 for both, n=8). These effects of carbachol on Ca
2+
transients were antagonized by 10 nmol/L pirenzepine, an m
1
mAChR–selective antagonist. In contrast, the m
2
mAChR–selective antagonist methoctramine (up to 100 nmol/L) did not inhibit the response. These results are the first to use single-cell PCR to probe cardiomyocyte-specific gene expression and indicate that m
1
mAChRs are expressed on adult rat ventricular myocytes in addition to m
2
mAChRs. The results further suggest that m
1
mAChRs mediate the stimulatory responses on Ca
2+
transients to high concentrations of cholinergic agonists seen in these cells.