bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipid Lowering Promotes Accumulation of Mature Smooth Muscle Cells Expressing Smooth Muscle Myosin Heavy Chain Isoforms in Rabbit Atheroma
Authors: Aikawa Masanori , Rabkin Elena , Voglic Sami J. , Shing Helen , Nagai Ryozo , Schoen Frederick J. , Libby Peter
Published: June 2012

Abstract
—Smooth muscle cells (SMCs) in the atherosclerotic intima characteristically differ from those in the arterial media, for example, by reduced expression of SMC differentiation/maturation markers such as smooth muscle myosin heavy chain isoforms (SM1 and SM2). This study tested the hypothesis that lipid lowering promotes maturation of intimal SMCs in 33 rabbits subjected to balloon injury and cholesterol feeding (0.3%) for 4 months (Baseline group, n=15); some of which then were switched to a low-cholesterol diet for 8 months (Low group at 8 months, n=3) or 16 months (Low group at 16 months, n=10). The remaining rabbits continued to consume a high-cholesterol diet for 16 months (High group, n=5). We monitored SMC phenotype by expression of immunoreactive α-smooth muscle actin, SM1, and SM2. α-Actin is an early marker, and SM1 and SM2 are late markers for SMC differentiation/maturation. Only fully differentiated or mature SMCs express SM2. Data are reported as the percentage of the α-actin-positive intimal area occupied by smooth muscle myosin–positive SMCs determined by color image analysis of immunostained sections. Levels of SM1 and SM2, highly expressed by SMCs in the normal aortic media (n=5) decreased in the aortic intima of the Baseline and High groups, indicating a less mature phenotype. In contrast, SM1 and SM2 increased in the Low (16 months) group, indicating that intimal SMCs exhibit a more mature phenotype after lipid lowering. Electron microscopy also showed the presence of mature intimal SMCs with abundant myofilaments. Furthermore, lipid lowering reduced levels of platelet-derived growth factor-B in the arterial intima, a factor known to suppress smooth muscle myosin expression. These data demonstrate that lipid lowering favors accumulation of mature SMCs in the atherosclerotic intima in association with reduced levels of platelet-derived growth factor-B expression. Intimal SMCs in the Low group also displayed reduced expression of matrix metalloproteinases-3 and -9 compared with the Baseline and High groups. These findings shed new light on the effects of lipid lowering at the level of the vascular wall, which may influence the biology of the atheroma.
Embryonic Expression Suggests an Important Role for CRP2/SmLIM in the Developing Cardiovascular System
Authors: Jain Mukesh K. , Kashiki Saori , Hsieh Chung-Ming , Layne Matthew D. , Yet Shaw-Fang , Sibinga Nicholas E. S. , Chin Michael T. , Feinberg Mark W. , Woo Ian , Maas Richard L. , Haber Edgar , Lee Mu-En
Published: June 2012

Abstract
—Proteins of the LIM family are critical regulators of development and differentiation in various cell types. We have described the cloning of cysteine-rich protein 2/smooth muscle LIM protein (CRP2/SmLIM), a LIM-only protein expressed in differentiated vascular smooth muscle cells. As a first step toward understanding the potential functions of CRP2/SmLIM, we analyzed its expression after gastrulation in developing mice and compared the expression of CRP2/SmLIM with that of the other 2 members of the CRP subclass, CRP1 and CRP3/MLP. In situ hybridization in whole-mount and sectioned embryos showed that CRP2/SmLIM was expressed in the sinus venosus and the 2 cardiac chambers at embryonic day 9. Vascular expression of CRP2/SmLIM was first seen at embryonic day 10. At subsequent time points, CRP2/SmLIM expression decreased in the heart but remained high in the vasculature. CRP1 was expressed both in vascular and nonvascular tissues containing smooth muscle cells, whereas CRP3/MLP was expressed only in tissues containing striated muscle. These patterns of expression were maintained in the adult animal and suggest an important role for this gene family in the development of smooth and striated muscle.
Ontogeny of Local Sarcoplasmic Reticulum Ca
2+
Signals in Cerebral Arteries
Authors: Gollasch Maik , Wellman George C. , Knot Harm J. , Jaggar Jonathan H. , Damon Deborah H. , Bonev Adrian D. , Nelson Mark T.
Published: June 2012

Abstract
—Ca
2+
release through ryanodine receptors (RyRs) in the sarcoplasmic reticulum is a key element of excitation-contraction coupling in muscle. In arterial smooth muscle, Ca
2+
release through RyRs activates Ca
2+
-sensitive K
+
(K
Ca
) channels to oppose vasoconstriction. Local Ca
2+
transients (“Ca
2+
sparks”), apparently caused by opening of clustered RyRs, have been observed in smooth and striated muscle. We explored the fundamental issue of whether RyRs generate Ca
2+
sparks to regulate arterial smooth muscle tone by examining the function of RyRs during ontogeny of arteries in the brain. In the present study, Ca
2+
sparks were measured using the fluorescent Ca
2+
indicator fluo-3 combined with laser scanning confocal microscopy. Diameter and arterial wall [Ca
2+
] measurements obtained from isolated pressurized arteries were also used in this study to provide functional insights. Neonatal arteries (<1 day postnatal), although still proliferative, have the molecular components for excitation-contraction coupling, including functional voltage-dependent Ca
2+
channels, RyRs, and K
Ca
channels and also constrict to elevations in intravascular pressure. Despite having functional RyRs, Ca
2+
spark frequency in intact neonatal arteries was ≈1/100 of adult arteries. In marked contrast to adult arteries, neonatal arteries did not respond to inhibitors of RyRs and K
Ca
channels. These results support the hypothesis that RyRs organize during postnatal development to cause Ca
2+
sparks, and RyRs must generate Ca
2+
sparks to regulate the function of the intact tissue.
Extracellular Discontinuities in Cardiac Muscle
Authors: Spach Madison S. , Heidlage J. Francis , Dolber Paul C. , Barr Roger C.
Published: June 2012

Abstract
—It has become of fundamental importance to understand variations in the shape of the upstroke of the action potential in order to identify structural loading effects. One component of this goal is a detailed experimental analysis of the time course of the foot of the cardiac action potential (V
m
foot) during propagation in different directions in anisotropic cardiac muscle. To this end, we performed phase-plane analysis of transmembrane action potentials during anisotropic propagation in adult working myocardium. The results showed that during longitudinal propagation there was initial slowing of V
m
foot that resulted in deviations from a simple exponential; corollary changes occurred at numerous sites during transverse propagation. We hypothesized that the effect on V
m
foot observed in the experimental data was created by the microscopic structure, especially the capillaries. This hypothesis predicts that the phase-plane trajectory of V
m
foot will deviate from linearity in the presence of a high density of capillaries, and that a linear trajectory will occur in the absence of capillaries. Comparison of the results of Fast and Kléber (
Circ Res
. 1993;73:914–925) in a monolayer of neonatal cardiac myocytes, which is devoid of capillaries, and our results in newborn ventricular muscle, which is rich in capillaries, showed drastic differences in V
m
foot as predicted. Because this comparison provided experimental support for the capillary hypothesis, we explored the underlying biophysical mechanisms due to interstitial electrical field effects, using a “2-domain” model of myocytes and capillaries separated by interstitial space. The model results show that a propagating interstitial electrical field induces an inward capacitive current in the inactive capillaries that causes a feedback effect on the active membrane (source) that slows the initial rise of its action potential. The results show unexpected mechanisms related to extracellular structural loading that may play a role in selected conduction disturbances, such as in a reperfused ischemic region surrounded by normal myocardium.
Bay K 8644 Increases Resting Ca
2+
Spark Frequency in Ferret Ventricular Myocytes Independent of Ca Influx
Authors: Satoh Hiroshi , Katoh Hideki , Velez Patricio , Fill Michael , Bers Donald M.
Published: June 2012

Abstract
—Bay K 8644, an L-type Ca
2+
channel agonist, was shown previously to increase resting sarcoplasmic reticulum (SR) Ca
2+
loss and convert post-rest potentiation to decay in dog and ferret ventricular muscle. Here, the effects of Bay K 8644 on local SR Ca
2+
release events (Ca
2+
sparks) were measured in isolated ferret ventricular myocytes, using laser scanning confocal microscopy and the fluorescent Ca
2+
indicator fluo-3. The spark frequency under control conditions was fairly constant during 20 s of rest after interruption of electrical stimulation. Bay K 8644 (100 nmol/L) increased the spark frequency by 466±90% of control at constant SR Ca
2+
load but did not change the spatial and temporal characteristics of individual sparks. The increase in spark frequency was maintained throughout the period of rest. The increase in Ca
2+
spark frequency induced by Bay K 8644 was not affected by superfusion with Ca
2+
-free solution (with 10 mmol/L EGTA) but was suppressed by the addition of 10 μmol/L nifedipine (which by itself did not alter resting Ca
2+
spark frequency). This suggests that the effect of Bay K 8644 on Ca
2+
sparks is mediated by the sarcolemmal dihydropyridine receptor but is also independent of Ca
2+
influx. Low concentrations of caffeine (0.5 mmol/L) increased both the average frequency and duration of sparks. Ryanodine (50 nmol/L) increased the spark frequency and also induced long-lasting Ca
2+
signals. This may indicate long-lasting openings of SR Ca
2+
release channels and a lack of local SR Ca
2+
depletion. In lipid bilayers, Bay K 8644 had no effect on either single-channel current amplitude or open probability of the cardiac ryanodine receptor. It is concluded that Bay K 8644 activates SR Ca
2+
release at rest, independent of Ca
2+
influx and perhaps through a functional linkage between the sarcolemmal dihydropyridine receptor and the SR ryanodine receptor. In contrast, caffeine and ryanodine modulate Ca
2+
sparks by a direct action on the SR Ca
2+
release channels.
Thrombin Stimulates Vascular Smooth Muscle Cell Polyamine Synthesis by Inducing Cationic Amino Acid Transporter and Ornithine Decarboxylase Gene Expression
Authors: Durante William , Liao Lan , Peyton Kelly J. , Schafer Andrew I.
Published: June 2012

Abstract
—Thrombin, a serine protease, is a potent mitogen for vascular smooth muscle cells (SMCs), but its mechanism of action is not known. Since
l
-ornithine is metabolized to growth-stimulatory polyamines, we examined whether thrombin regulates the transcellular transport and metabolism of
l
-ornithine by vascular SMCs. Treatment of SMCs with thrombin initially (0 to 2 hours) decreased
l
-ornithine uptake, whereas longer exposures (6 to 24 hours) progressively increased transport. Kinetic studies indicated that thrombin-induced inhibition was associated with a decrease in affinity for
l
-ornithine, whereas stimulation was mediated by an increase in transport capacity. Thrombin induced the expression of both cationic amino acid transporter (CAT)-1 and CAT-2 mRNA. Furthermore, thrombin stimulated
l
-ornithine metabolism by inducing ornithine decarboxylase (ODC) mRNA expression and activity. The stimulatory effect of thrombin on both
l
-ornithine transport and ODC activity was reversed by hirudin, a thrombin inhibitor, and was mimicked by a 14–amino acid thrombin receptor–activating peptide. Thrombin also markedly increased the capacity of SMCs to generate putrescine, a polyamine, from extracellular
l
-ornithine. The thrombin-mediated increase in putrescine production was reversed by
N
G
-methyl-
l
-arginine, a competitive inhibitor of cationic amino acid transport, or by α-difluoromethylornithine (DFMO), an ODC inhibitor. DFMO also inhibited thrombin-induced SMC proliferation. These results demonstrate that thrombin stimulates polyamine synthesis by inducing CAT and ODC gene expression and that thrombin-stimulated SMC proliferation is dependent on polyamine formation. The ability of thrombin to upregulate
l
-ornithine transport and direct its metabolism to growth-stimulatory polyamines may contribute to postangioplasty restenosis and atherosclerotic lesion formation.
Modulation of Cardiac Na
+
Current Phenotype by β
1
-Subunit Expression
Authors: Kupershmidt Sabina , Yang Tao , Roden Dan M.
Published: June 2012

Abstract
—Na
+
current (
I
Na
) is smaller, activates and inactivates more slowly, and displays less negative voltage dependence of inactivation in the neonatal rat than in the adult rat. We have observed very similar changes when
I
Na
is recorded as a function of time in culture in mouse atrial tumor (AT-1) cells. The differences between mature and immature
I
Na
are reminiscent of those observed when skeletal muscle Na
+
channel α subunits are expressed alone (immature) or with the β
1
subunit (mature). In the present experiments, we tested the hypothesis that suppression of β
1
-subunit expression by antisense oligonucleotides would prevent the development of a mature
I
Na
. The mouse β
1
subunit was cloned from an AT-1 cDNA library and found to be identical to that in the rat at 216/218 amino acids. AT-1 cells exposed to anti-β
1
antisense oligonucleotides displayed an immature
I
Na
at day 8 in culture, whereas untreated cells or cells exposed to sense oligonucleotides displayed a mature
I
Na
. This result was observed with 2 different oligonucleotides, and neither affected the rapidly activating component of the delayed rectifier K
+
current, another current recorded in AT-1 cells. These findings indicate that in these cells, the gating of
I
Na
is modulated by β
1
expression and that α-β
1
coexpression is required for the development of a mature cardiac
I
Na
phenotype.
3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase and Isoprenylation Inhibitors Induce Apoptosis of Vascular Smooth Muscle Cells in Culture
Authors: Guijarro Carlos , Blanco-Colio Luis Miguel , Ortego Mónica , Alonso Covadonga , Ortiz Alberto , Plaza Juan José , Díaz Cristina , Hernández Gonzalo , Egido Jesús
Published: June 2012

Abstract
—Recent evidence suggests that apoptosis may be involved in the control of vascular smooth muscle cell (VSMC) number in atherosclerotic lesions. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been reported to induce apoptosis in a variety of tumor cell lines. To evaluate whether these agents also induce apoptosis of VSMCs, cultured rat VSMCs were treated with increasing doses of atorvastatin in the presence of FBS as a survival factor. The presence of apoptosis was evaluated by morphological criteria, annexin V binding, and DNA fragmentation and quantified as the proportion of hypodiploid cells by flow cytometry. Atorvastatin induced apoptosis in a dose-dependent manner, an effect also seen with simvastatin and lovastatin, but not with the hydrophilic drug pravastatin. The proapoptotic effect of statins was seen only when the inhibition of acetate incorporation into sterols was >95% and was fully reversed by mevalonate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate but not by isopentenyl adenosine, ubiquinone, or squalene, suggesting a role for prenylated proteins in the regulation of VSMC apoptosis. To further assess the role of protein prenylation, VSMCs were exposed to the prenyl transferase inhibitors perillic acid and manumycin A. Both agents induced VSMC apoptosis as evaluated by the above-mentioned criteria. Finally, VSMC treatment with lipophilic statins was associated with decreased prenylation of p21-Rho B, further supporting the role of protein prenylation inhibition in statin-induced VSMC apoptosis. The present data suggest that interference with protein prenylation by HMG-CoA reductase inhibitors or other agents may provide new strategies for the prevention of neointimal thickening.
Ionic Mechanisms of Regional Action Potential Heterogeneity in the Canine Right Atrium
Authors: Feng Jianlin , Yue Lixia , Wang Zhiguo , Nattel Stanley
Published: June 2012

Abstract
—Atrial action potential heterogeneity is a major determinant of atrial reentrant arrhythmias, but the underlying ionic mechanisms are poorly understood. To evaluate the basis of spatial heterogeneity in canine right atrial repolarization, we isolated cells from 4 regions: the crista terminalis (CT), appendage (APG), atrioventricular ring (AVR) area, and pectinate muscles. Systematic action potential (AP) differences were noted: CT cells had a “spike-and-dome” morphology and the longest AP duration (APD; value to 95% repolarization at 1 Hz, 270±10 ms [mean±SEM]); APG and pectinate muscle cells had intermediate APDs (180±3 and 190±3 ms, respectively;
P
<0.001 versus CT for each), with APG cells having a small phase 1; and AVR cells had the shortest APD (160±4 ms,
P
<0.001 versus other regions). The inward rectifier and the slow and ultrarapid delayed rectifier currents were similar in all regions. The transient outward K
+
current was significantly smaller in APG cells, explaining their small phase 1 and high plateau. L-type Ca
2+
current was greatest in CT cells and least in AVR cells, contributing to their longer and shorter APD, respectively. The E-4031–sensitive rapid delayed rectifier K
+
current was larger in AVR cells compared with other regions. Voltage- and time-dependent current properties were constant across regions. We conclude that myocytes from different right atrial regions of the dog show systematic variations in AP properties and ionic currents and that the spatial variation in ionic current density may explain AP differences. Regional variation in atrial ionic currents may play an important role in atrial arrhythmia generation and may present opportunities for improving antiarrhythmic drug therapy.
Slow Conduction in Cardiac Tissue, I
Authors: Rohr Stephan , Kucera Jan P. , Kléber André G.
Published: June 2012

Abstract
—It was the aim of this study to characterize the spread of activation at the cellular level in cardiac tissue during conduction slowing, a key element of reentrant arrhythmias; therefore, activation patterns were assessed at high spatiotemporal resolution in narrow (70 to 80 μm) and wide (230 to 270 μm) linear strands of cultured neonatal rat ventricular myocytes, using multiple site optical recording of transmembrane voltage. Slow conduction was induced by graded elevation of [K
+
]
o
, by applying tetrodotoxin, or by exposing the preparations to the gap junctional uncouplers palmitoleic acid or 1-octanol. The main findings of the study are 4-fold: (1) gap junctional uncoupling reduced conduction velocity (range, 37 to 47 cm/s under control conditions) to a substantially larger extent before block (≤1 cm/s; ultra-slow conduction) than did a reduction of excitability (range, ≈10 to 15 cm/s); (2) activation wavefronts during uncoupling meandered within the boundaries of the preparations, resulting in a pronounced additional slowing of conduction in wide cell strands; (3) at the cellular level, propagation during uncoupling-induced ultra-slow conduction was sustained by sequentially activated tissue patches, each of which consisted of a few cells being activated simultaneously; and (4) depending on the uncoupler used, maximal action potential upstroke velocities during ultra-slow conduction were either slightly (palmitoleic acid) or highly (1-octanol) depressed. Thus, depolarizing inward currents, the spatial pattern and degree of gap junctional coupling, and geometrical factors all contribute in a concerted manner to conduction slowing, which, at its extreme (0.25 cm/s measured over 1 mm), can reach values low enough to permit, theoretically, reentrant excitation to occur in minuscule areas of cardiac tissue (≪1 mm
2
).
Epoxyeicosatrienoic Acids and Dihydroxyeicosatrienoic Acids Are Potent Vasodilators in the Canine Coronary Microcirculation
Authors: Oltman Christine L. , Weintraub Neal L. , VanRollins Mike , Dellsperger Kevin C.
Published: June 2012

Abstract
—Cytochrome P
450
epoxygenases convert arachidonic acid into 4 epoxyeicosatrienoic acid (EET) regioisomers, which were recently identified as endothelium-derived hyperpolarizing factors in coronary blood vessels. Both EETs and their dihydroxyeicosatrienoic acid (DHET) metabolites have been shown to relax conduit coronary arteries at micromolar concentrations, whereas the plasma concentrations of EETs are in the nanomolar range. However, the effects of EETs and DHETs on coronary resistance arterioles have not been examined. We administered EETs and DHETs to isolated canine coronary arterioles (diameter, 90.0±3.4 μm; distending pressure, 20 mm Hg) preconstricted by 30% to 60% of the resting diameter with endothelin. All 4 EET regioisomers produced potent, concentration-dependent vasodilation (EC
50
values ranging from −12.7 to −10.1 log [M]) and were approximately 1000 times more potent than reported in conduit coronary arteries. The vasodilation produced by 14,15-EET was not attenuated by removal of the endothelium and indicated a direct action of 14,15-EET on microvascular smooth muscle. Likewise, 14,15-DHET, 11,12-DHET, 8,9-DHET, and the δ-lactone of 5,6-EET produced extremely potent vasodilation (EC
50
values ranging from −15.8 to −13.1 log [M]). The vasodilation produced by these eicosanoids was highly potent in comparison to that produced by other vasodilators, including arachidonic acid (EC
50
=−7.5 log [M]). The epoxide hydrolase inhibitor, 4-phenylchalone oxide, which blocked the conversion of [
3
H]14,15-EET to [
3
H]14,15-DHET by canine coronary arteries, did not alter arteriolar dilation to 11,12-EET; thus, the potent vasodilation induced by EETs does not require formation of DHETs. In contrast, charybdotoxin (a K
Ca
channel inhibitor) and KCl (a depolarizing agent) blocked vasodilation by 11,12-EET and 11,12-DHET. We conclude that EETs and DHETs potently dilate canine coronary arterioles via activation of K
Ca
channels. The preferential ability of these compounds to dilate resistance blood vessels suggests that they may be important regulators of coronary circulation.
Upregulation of Estrogen Receptor-α Expression in Rabbit Cardiac Allograft
Authors: Lou Hong , Martin Mary Beth , Stoica Adriana , Ramwell Peter W. , Foegh Marie L.
Published: June 2012

Abstract
—Estrogen receptor (ER) expression has been detected in different tissues, and estradiol-17β treatment protects against experimental transplant arteriosclerosis. In this study, ER-α expression in the rabbit hearts and attached aortas before and after cardiac-aorta allograft transplantation was examined. Ten male New Zealand White rabbits were transplanted with cardiac-aorta allografts from male Dutch Belted rabbits. This transplant arteriosclerosis model uses a 0.5% cholesterol diet and immunosuppression with cyclosporin A (10 mg · kg
−1
· d
−1
) until euthanatization 42 days later. The cardiac grafts with the attached aorta were harvested. Strong staining of ER-α protein was shown in the coronary arteries of the cardiac allografts by immunohistochemistry with the use of a mouse anti-human ER-α monoclonal antibody (ID5). In contrast, both the nongrafted hearts of the recipients and donor hearts expressed only weak staining. RNase protection assay with the use of a
32
P–labeled ER-α antisense riboprobe (pOR 300) proved that the basal expression of ER-α mRNA is similar in the nongrafted aorta of both recipients and donors. A marked increase of ER-α mRNA was observed in the allograft aorta compared with the nongrafted aorta (289±69%,
P
<0.02) by reverse transcription and polymerase chain reaction. The DNA sequence analysis confirmed that the polymerase chain reaction–amplified fragment corresponded to ER-α. This is the first observation of ER-α upregulation in the allograft vasculature and may relate to the allograft cardiovascular protective effects of estrogen.
Nitric Oxide–Independent Relaxations to Acetylcholine and A23187 Involve Different Routes of Heterocellular Communication
Authors: Hutcheson Iain R. , Chaytor Andrew T. , Evans W. Howard , Griffith Tudor M.
Published: June 2012

Abstract
—NO- and prostanoid-independent relaxations are generally assumed to be mediated by an endothelium-derived hyperpolarizing factor (EDHF) that has been postulated to be an arachidonic acid metabolite. Recent evidence also suggests that direct heterocellular gap junctional communication (GJC) between endothelium and smooth muscle contributes to NO-independent relaxations. In the present study we have investigated the contribution of phospholipase A
2
(PLA
2
)-linked metabolites and GJC to EDHF-type relaxations in rabbit mesenteric artery. In isolated rings preconstricted with 10 μmol/L phenylephrine in the presence of
N
G
-nitro-
l
-arginine methyl ester (L-NAME) and indomethacin, acetylcholine (ACh) and the Ca
2+
ionophore A23187 evoked relaxations that were markedly attenuated by the Ca
2+
-dependent PLA
2
inhibitors 2-(
p
-amylcinnamoyl)amino-4-chlorobenzoic acid (3 μmol/L) and arachidonyl trifluoromethyl ketone (3 μmol/L), but were potentiated by the sulfhydryl agent thimerosal (300 nmol/L). In intact rings, relaxations to ACh were attenuated synergistically by L-NAME and Gap 27 peptide, an inhibitor of GJC, whereas ACh-evoked relaxations of “sandwich” preparations were unaffected by the peptide but were abolished by L-NAME. In both ring and sandwich preparations A23187-induced relaxations were attenuated by inhibition of PLA
2
but were insensitive to L-NAME and Gap 27 peptide. We conclude that EDHF-type relaxations of rabbit mesenteric artery to ACh and A23187 depend on a common pathway that involves activation of PLA
2
. In the case of ACh, relaxation requires transfer of a factor or factors from the endothelium to smooth muscle via gap junctions, whereas A23187 permits release directly into the extracellular space.
Troponin I Degradation and Covalent Complex Formation Accompanies Myocardial Ischemia/Reperfusion Injury
Authors: McDonough Jason L. , Arrell D. Kent , Van Eyk Jennifer E.
Published: June 2012

Abstract
—Selective troponin I (TnI) modification has been demonstrated to be in part responsible for the contractile dysfunction observed with myocardial ischemia/reperfusion injury. We have isolated and characterized modified TnI products in isolated rat hearts after 0, 15, or 60 minutes of ischemia followed by 45 minutes of reperfusion using affinity chromatography with cardiac troponin C (TnC) and an anti-TnI antibody, immunological mapping, reversed-phase high-performance liquid chromatography, and mass spectrometry. Rat cardiac TnI becomes progressively degraded from 210 amino acid residues to residues 1–193, 63–193, and 73–193 with increased severity of injury. Degradation is accompanied by formation of covalent complexes between TnI 1–193 and, respectively, TnC residues 1–94 and troponin T (TnT) residues 191–298. The covalent complexes are likely a result of isopeptide bond formation between lysine 193 of TnI and glutamine 191 of TnT by the cross-linking enzyme transglutaminase. With severe ischemia, cellular necrosis results in specific release of TnI 1–193 into the reperfusion effluent and TnT degradation in the myocardium (25-, 27-, and 33-kDa products). Two-dimensional electrophoresis demonstrated that phosphorylation of TnI prevents ischemia-induced degradation. This study characterized the modified TnI products in isolated rat hearts reperfused after a brief or severe period of ischemia, revealing the progressive nature of TnI degradation, changes in phosphorylation, and covalent complexes with ischemia/reperfusion injury. Finally, we propose a model for ischemia/reperfusion injury in which the extent of proteolytic and transglutaminase activities ultimately determines whether apoptosis or necrosis is achieved.
Strain Differences in Neointimal Hyperplasia in the Rat
Authors: Assadnia Shahin , Rapp John P. , Nestor Andrea L. , Pringle Timothy , Cerilli Greg J. , Gunning William T. , Webb Thomas H. , Kligman Mark , Allison David C.
Published: June 2012

Abstract
—We performed an initial screen of 11 rat strains by use of a standard balloon injury to the left iliac artery to observe whether genetically determined differences existed in the development of neointimal hyperplasia. Neointimal hyperplasia was assayed 8 weeks after the vascular injury on coded microscopic sections. Statistically significant differences in the percentages of the vascular wall cross-sectional areas composed of intima (percentage intima) secondary to neointimal hyperplasia were noted among the different rat strains (
P
<0.02), with the Brown-Norway (BN), Dark Agouti, and Milan normotensive strain rats having the highest and the spontaneously hypertensive rats (SHR) having the lowest percentages of intima. In a separate experiment, F
1
hybrids of SHR×BN strains and parental BN and SHR underwent the vascular injury, and the parental strains again showed a statistically significant difference from one another in the mean percentage of intima (
P
<0.0001). The F
1
hybrids showed an average percentage of intima intermediate between those of the parental strains. The average lumen size of the injured BN vessels were significantly smaller than that of the noninjured control vessels (
P
=0.044), but this significance disappeared when the circular areas of these vessels were calculated without taking neointimal growth into consideration (
P
=0.649). These results provide the groundwork for a genetic linkage analysis to identify the genes that influence the development of neointimal hyperplasia after vascular injury.
Swelling-Activated Chloride Current Is Persistently Activated in Ventricular Myocytes From Dogs With Tachycardia-Induced Congestive Heart Failure
Authors: Clemo Henry F. , Stambler Bruce S. , Baumgarten Clive M.
Published: June 2012

Abstract
—The hypothesis that cellular hypertrophy in congestive heart failure (CHF) modulates mechanosensitive (ie, swelling- or stretch-activated) anion channels was tested. Digital video microscopy and amphotericin-perforated-patch voltage clamp were used to measure cell volume and ion currents in ventricular myocytes isolated from normal dogs and dogs with rapid ventricular pacing-induced CHF. In normal myocytes, osmotic swelling in 0.9T to 0.6T solution (T, relative osmolarity; isosmotic solution, 296 mOsmol/L) was required to elicit
I
Cl,swell
, an outwardly rectifying swelling-activated Cl
–
current that reversed near –33 mV and was inhibited by 1 mmol/L 9-anthracene carboxylic acid (9AC), an anion channel blocker. Block of
I
Cl,swell
by 9AC simultaneously increased the volume of normal cells in hyposmotic solutions by up to 7%, but 9AC had no effect on volume in isosmotic or hyperosmotic solutions. In contrast,
I
Cl,swell
was persistently activated under isosmotic conditions in CHF myocytes, and 9AC increased cell volume by 9%. Osmotic shrinkage in 1.1T to 1.5T solution inhibited both
I
Cl,swell
and 9AC-induced cell swelling in CHF cells, whereas osmotic swelling only slightly increased
I
Cl,swell
. The current density for fully activated 9AC-sensitive
I
Cl,swell
was 40% greater in CHF than normal myocytes. In both groups, 9AC-sensitive current and 9AC-induced cell swelling were proportional with changes in osmolarity and 9AC concentration, and the effects of 9AC on current and volume were blocked by replacing bath Cl
–
with methanesulfonate. CHF thus altered the set point and magnitude of
I
Cl,swell
and resulted in its persistent activation. We previously observed analogous regulation of mechanosensitive cation channels in the same CHF model. Mechanosensitive anion and cation channels may contribute to the electrophysiological and contractile derangements in CHF and may be novel targets for therapy.
Daunorubicin-Induced Apoptosis in Rat Cardiac Myocytes Is Inhibited by Dexrazoxane
Authors: Sawyer Douglas B. , Fukazawa Ryuji , Arstall Margaret A. , Kelly Ralph A.
Published: June 2012

Abstract
—The clinical efficacy of anthracycline antineoplastic agents is limited by a high incidence of severe and usually irreversible cardiac toxicity, the cause of which remains controversial. In primary cultures of neonatal and adult rat ventricular myocytes, we found that daunorubicin, at concentrations ≤1 μmol/L, induced myocyte programmed cell death within 24 hours, as defined by several complementary techniques. In contrast, daunorubicin concentrations ≥10 μmol/L induced necrotic cell death within 24 hours, with no changes characteristic of apoptosis. To determine whether reactive oxygen species play a role in daunorubicin-mediated apoptosis, we monitored the generation of hydrogen peroxide with dichlorofluorescein (DCF). However, daunorubicin (1 μmol/L) did not increase DCF fluorescence, nor were the antioxidants
N
-acetylcysteine or the combination of α-tocopherol and ascorbic acid able to prevent apoptosis. In contrast, dexrazoxane (10 μmol/L), known clinically to limit anthracycline cardiac toxicity, prevented daunorubicin-induced myocyte apoptosis, but not necrosis induced by higher anthracycline concentrations (≥10 μmol/L). The antiapoptotic action of dexrazoxane was mimicked by the superoxide-dismutase mimetic porphyrin manganese(II/III)tetrakis(1-methyl-4-peridyl)porphyrin (50 μmol/L). The recognition that anthracycline-induced cardiac myocyte apoptosis, perhaps mediated by superoxide anion generation, occurs at concentrations well below those that result in myocyte necrosis, may aid in the design of new therapeutic strategies to limit the toxicity of these drugs.
Phospholamban Is Present in Endothelial Cells and Modulates Endothelium-Dependent Relaxation
Authors: Sutliff Roy L. , Hoying James B. , Kadambi Vivek J. , Kranias Evangelia G. , Paul Richard J.
Published: June 2012

Abstract
—Vascular endothelial cells regulate vascular smooth muscle tone through Ca
2+
-dependent production and release of vasoactive molecules. Phospholamban (PLB) is a 24- to 27-kDa phosphoprotein that modulates activity of the sarco(endo)plasmic reticulum Ca
2+
ATPase (SERCA). Expression of PLB is reportedly limited to cardiac, slow-twitch skeletal and smooth muscle in which PLB is an important regulator of [Ca
2+
]
i
and contractility in these muscles. In the present study, we report the existence of PLB in the vascular endothelium, a nonmuscle tissue, and provide functional data on PLB regulation of vascular contractility through its actions in the endothelium. Endothelium-dependent relaxation to acetylcholine was attenuated in aorta of PLB-deficient (PLB-KO) mice compared with wild-type (WT) controls. This effect was not due to actions of nitric oxide on the smooth muscle, because sodium nitroprusside–mediated relaxation in either denuded or endothelium-intact aortas was unaffected by PLB ablation. Relative to denuded vessels, relaxation to forskolin was enhanced in WT endothelium-intact aortas. The endothelium-dependent component of this relaxation was attenuated in PLB-KO aortas. To investigate whether these changes were due to PLB, WT mouse aorta endothelial cells were isolated. Both reverse transcriptase–polymerase chain reaction and Western blot analyses revealed the presence of PLB in endothelial cells, which were shown to be >98% pure by diI-acetylated LDL uptake and nuclear counterstaining. These data indicate that PLB is present and modulates vascular function as a result of its actions in endothelial cells. The presence of PLB in endothelial cells opens new fields for investigation of Ca
2+
regulatory pathways in nonmuscle cells and for modulation of endothelial-vascular interactions.
Potential Molecular Basis of Different Physiological Properties of the Transient Outward K
+
Current in Rabbit and Human Atrial Myocytes
Authors: Wang Zhiguo , Feng Jianlin , Shi Hong , Pond Amber , Nerbonne Jeanne M. , Nattel Stanley
Published: June 2012

Abstract
—The properties of the transient outward current (
I
to
) differ between rabbit and human atrial myocytes. In particular, rabbit
I
to
is known to recover more slowly than its human counterpart and to show much more frequency dependence. To assess the possibility that these physiological differences may reflect differing expression of K
+
channel subunit gene products, we used a combination of whole-cell voltage-clamp, heterologous expression, pharmacological, antisense, and Western blot techniques. The inactivation of
I
to
in rabbit atrial myocytes was significantly slowed by hydrogen peroxide, with human
I
to
being unaffected. Use-dependent unblocking with 4-aminopyridine was not seen for rabbit
I
to
nor for Kv1.4 currents in
Xenopus
oocytes, whereas human
I
to
showed strong use-dependent unblock (as did Kv4 currents). Western blots indicated the presence of Kv4 proteins in both human and rabbit atrial membranes, but Kv1.4 was only detected in the rabbit. Antisense oligodeoxynucleotides directed against Kv4.3, Kv4.2, or Kv1.4 subunit sequences significantly inhibited
I
to
current density in cultured rabbit atrial myocytes, whereas only Kv4.3 antisense significantly inhibited
I
to
in human cells. Neither mismatch oligodeoxynucleotides nor vehicle altered currents in either species. We conclude that, unlike human atrial myocytes, rabbit atrial myocytes express Kv1.4 channel subunits, which likely contribute to a number of important physiological differences in
I
to
properties between the species. To our knowledge, these studies constitute the first demonstration of a functional role for Kv1.4 channels in cardiac membranes and provide insights into the molecular mechanisms of an important cardiac repolarizing current.
Overexpression of Insulin-Like Growth Factor-1 Attenuates the Myocyte Renin-Angiotensin System in Transgenic Mice
Authors: Leri Annarosa , Liu Yu , Wang Xiaowei , Kajstura Jan , Malhotra Ashwani , Meggs Leonard G. , Anversa Piero
Published: June 2012

Abstract
—Constitutive overexpression of insulin-like growth factor-1 (IGF-1) in myocytes protects them from apoptosis and interferes with myocyte hypertrophy in the normal and pathological heart. Conversely, angiotensin II (Ang II) triggers cell death and promotes myocyte hypertrophy. Moreover, activation of p53 upregulates the cellular renin-angiotensin system (RAS). Therefore, IGF-1 overexpression in FVB.
Igf
+/− mice may downregulate the local RAS through the attenuation of p53 and p53-inducible genes. On this basis, p53 DNA binding activity to angiotensinogen (Aogen), bax, and the AT
1
receptor was determined in left ventricular myocytes from FVB.
Igf
−/− and FVB.
Igf
+/− mice. The quantity of Bax, Bcl-2, Aogen, and AT
1
receptor in these cells was evaluated. The presence of Mdm2-p53 complexes was also established. Finally, Ang II levels in myocytes were measured. Upregulation of IGF-1 in myocytes was associated with a protein-to-protein interaction between Mdm2 and p53, which attenuated p53 transcriptional activity for bax, Aogen, and AT
1
receptor. Similarly, the amount of Bax, Aogen, and AT
1
receptor proteins in these cells decreased. In contrast, the expression of Bcl-2 remained constant. The downregulation of Aogen in myocytes from FVB.
Igf
+/− mice was characterized by a reduction in Ang II. In conclusion, IGF-1 negatively influences the myocyte RAS through the upregulation of Mdm2 and its binding to p53. This may represent the molecular mechanism responsible for the effects of IGF-1 on cell viability and myocyte hypertrophy in the nonpathological and pathological heart in vivo.
Analysis of the Presence and Physiological Relevance of Subconducting States of Connexin43-Derived Gap Junction Channels in Cultured Human Corporal Vascular Smooth Muscle Cells
Authors: Christ George J. , Brink Peter R.
Published: June 2012

Abstract
—Subconductance states are a commonly observed feature of gap junction channels. Their overt frequency and consistent appearance in both single and multichannel records have led to speculation that they might be of physiological importance in terms of altering the rate of small solute transfer from cell to cell. Among the connexin gene family, connexin43 (Cx43) is the most ubiquitous connexin that has been shown to generate subconductive states. Therefore, it was the explicit aim of this investigation to more fully evaluate the potential contribution of human Cx43-derived subconducting states to intercellular communication in cultured human corporal vascular smooth muscle cells. To determine the weight of subconductive states in our records, we analyzed amplitude histograms of multichannel and single-channel data during the application of transjunctional voltages larger than expected for physiological conditions but still smaller than transjunctional voltages known to induce lower conductive states (V
o
>V
j
). The data clearly indicated that the subconducting states occupy only a small fraction of the total channel open time. This was reflected by the fact that the average open probability for the subconductive state(s) determined from the 9 records analyzed was ≈2%. Closer inspection of the data revealed that the frequency of subconductive states was actually higher than the frequency of the main state conductance. In summary, recording conditions sufficient for evaluation of the intrinsic gating characteristics of human Cx43-derived gap junction channels have been used. Under these conditions, our data clearly indicate that despite their greater frequency, the duration of subconductance events is so short relative to the main state duration as to render them physiologically insignificant.
Liposomal Delivery of Purified Inhibitory-κBα Inhibits Tumor Necrosis Factor-α–Induced Human Vascular Smooth Muscle Proliferation
Authors: Selzman Craig H. , Shames Brian D. , Reznikov Leonid L. , Miller Stephanie A. , Meng Xianzhong , Barton Hazel A. , Werman Ariel , Harken Alden H. , Dinarello Charles A. , Banerjee Anirban
Published: June 2012

Abstract
—Vessel injury results in the elaboration of various cytokines, including tumor necrosis factor-α (TNF-α), which may influence vascular smooth muscle cell (VSMC) function and contribute to atherogenesis. We tested the hypothesis that TNF-α–induced VSMC proliferation requires activation of the transcription factor nuclear factor-κB (NF-κB), which could be prevented by delivery of the NF-κB inhibitory peptide, IκBα. TNF-α induced concentration-dependent human VSMC proliferation, and neutralizing antibody to interleukin-6 reduced TNF-α–induced VSMC proliferation by 65%. In TNF-α–stimulated VSMCs, there was a 3-fold increase in NF-κB–dependent luciferase reporter activity that was associated with degradation of ΙκBα. To determine an essential role for NF-κB in TNF-α–induced VSMC proliferation, recombinant ΙκBα was introduced into VSMCs via liposomal delivery. Under these conditions, TNF-α–induced NF-κB nuclear translocation and DNA binding were inhibited, NF-κB–dependent luciferase activity was reduced by 50%, there was no degradation of native ΙκBα detected, interleukin-6 production was reduced by 54%, and VSMC proliferation was decreased by 60%. In conclusion, the mitogenic effect of TNF-α on human arterial VSMCs is dependent on NF-κB activation and may be prevented by exogenously delivered ΙκBα. Furthermore, liposomal delivery of endogenous inhibitory proteins may represent a novel, therapeutically accessible method for selective transcriptional suppression in the response to vascular injury.
Electrical Restitution and Spatiotemporal Organization During Ventricular Fibrillation
Authors: Riccio Mark L. , Koller Marcus L. , Gilmour Robert F.
Published: June 2012

Abstract
—Despite recent advances in our understanding of the mechanism for ventricular fibrillation (VF), important electrophysiological aspects of the development of VF still are poorly defined. It has been suggested that the onset of VF involves the disintegration of a single spiral wave into many self-perpetuating waves. It has been further suggested that such a process requires that the slope of the electrical restitution relation be ≥1. The same theory anticipates that a single spiral wave will be stable (not disintegrate) if the maximum slope of the restitution relation is <1. We have shown previously that the slope of the restitution relation during rapid pacing and during VF is ≥1 in canine ventricle. We now show that drugs that reduce the slope of the restitution relation (diacetyl monoxime and verapamil) prevent the induction of VF and convert existing VF into a periodic rhythm. In contrast, a drug that does not reduce the slope of the restitution relation (procainamide) does not prevent the induction of VF, nor does it regularize VF. These results indicate that the kinetics of electrical restitution is a key determinant of VF. Moreover, they suggest novel approaches to preventing the induction or maintenance of VF.
Modulation of Extracellular Superoxide Dismutase Expression by Angiotensin II and Hypertension
Authors: Fukai Tohru , Siegfried Martin R. , Ushio-Fukai Masuko , Griendling Kathy K. , Harrison David G.
Published: June 2012

Abstract
—Angiotensin II and hypertension increase vascular oxidant stress. We examined how these might affect expression of the extracellular superoxide dismutase (ecSOD), a major form of vascular SOD. In mice, angiotensin II infusion (1.1 mg/kg for 7 days) increased systolic blood pressure from 107±3 to 152±9 mm Hg and caused a 3-fold increase in ecSOD, but there was no change in the cytosolic Cu/Zn SOD protein, as determined by Western blot analysis. This was associated with a similar increase in ecSOD mRNA as assessed by RNase protection assay and was prevented by losartan. Induction of ecSOD by angiotensin II was not due to hypertension alone, because hypertension caused by norepinephrine (5.6 mg · kg
−1
· d
−1
) had no effect on ecSOD. Similarly, exposure of mouse aortas to angiotensin II (100 nmol/L) in organoid culture increased ecSOD by ≈2-fold. In the organoid culture, angiotensin II–induced upregulation of ecSOD was prevented by losartan (10 μmol/L) and PD985059 (30 μmol/L), a specific inhibitor of p42/44 MAP kinase kinase. Angiotensin II activates the NADH/NADPH oxidase; however, diphenyleneiodonium chloride (10 μmol/L), an inhibitor of this oxidase, did not prevent p42/44 MAP kinase phosphorylation or ecSOD induction by angiotensin II. Finally, in human aortic smooth muscle cells, angiotensin II moderately increased transcriptional rate (as assessed by nuclear run-on analysis) but markedly increased ecSOD mRNA stability. Thus, angiotensin II increases ecSOD expression independent of hypertension, and this increase involves both an increase in ecSOD transcription and stabilization of ecSOD mRNA. This effect of angiotensin II on ecSOD expression may modulate the oxidative state of the vessel wall in pathological processes in which the renin-angiotensin system is activated.