bluuebunny/crossref_metadata_2025 huggingface dataset:
The Cardiac Renin-Angiotensin System
Authors: Dostal David E. , Baker Kenneth M.
Published: June 2012

Abstract
—Angiotensin II, the effector peptide of the renin-angiotensin system, regulates cellular growth in response to developmental, physiological, and pathological processes. The identification of renin-angiotensin system components and angiotensin II receptors in cardiac tissue suggests the existence of an autocrine/paracrine system that has effects independent of angiotensin II derived from the circulatory system. To be functional, a local renin-angiotensin system should produce sufficient amounts of the autocrine and/or paracrine factor to elicit biological responses, contain the final effector (angiotensin II receptor), and respond to humoral, neural, and/or mechanical stimuli. In this review, we discuss evidence for a functional cardiac renin-angiotensin system.
A Novel K
ATP
Current in Cultured Neonatal Rat Atrial Appendage Cardiomyocytes
Authors: Baron Anne , van Bever Laurianne , Monnier Dominique , Roatti Angela , Baertschi Alex J.
Published: June 2012

Abstract
—The functional and pharmacological properties of ATP-sensitive K
+
(K
ATP
) channels were studied in primary cultured neonatal rat atrial appendage cardiomyocytes. Activation of a whole-cell inward rectifying K
+
current depended on the pipette ATP concentration and correlated with a membrane hyperpolarization close to the K
+
equilibrium potential. The K
ATP
current could be activated either spontaneously or by a hypotonic stretch of the membrane induced by lowering the osmolality of the bathing solution from 290 to 260 mOsm/kg H
2
O or by the K
+
channel openers diazoxide and cromakalim with EC
50
≈1 and 10 nmol/L, respectively. The activated atrial K
ATP
current was highly sensitive to glyburide, with an IC
50
of 1.22±0.15 nmol/L. Recorded in inside-out patches, the neonatal atrial K
ATP
channel displayed a conductance of 58.0±2.2 pS and opened in bursts of 133.8±20.4 ms duration, with an open time duration of 1.40±0.10 ms and a close time duration of 0.66±0.04 ms for negative potentials. The channel had a half-maximal open probability at 0.1 mmol/L ATP, was activated by 100 μmol/L diazoxide, and was inhibited by glyburide, with an IC
50
in the nanomolar range. Thus, pending further tests at low concentrations of K
ATP
channel openers, the single-channel data confirm the results obtained with whole-cell recordings. The neonatal atrial appendage K
ATP
channel thus shows a unique functional and pharmacological profile resembling the pancreatic β-cell channel for its high affinity for glyburide and diazoxide and for its conductance, but also resembling the ventricular channel subtype for its high affinity for cromakalim, its burst duration, and its sensitivity to ATP. Reverse transcriptase–polymerase chain reaction experiments showed the expression of Kir6.1, Kir6.2, SUR1A, SUR1B, SUR2A, and SUR2B subunits, a finding supporting the hypothesis that the neonatal atrial K
ATP
channel corresponds to a novel heteromultimeric association of K
ATP
channel subunits.
Mechanisms Underlying the Increase in Force and Ca
2+
Transient That Follow Stretch of Cardiac Muscle
Authors: Alvarez Bernardo V. , Pérez Néstor G. , Ennis Irene L. , Camilión de Hurtado María C. , Cingolani Horacio E.
Published: June 2012

Abstract
—Myocardial stretch produces an increase in developed force (DF) that occurs in two phases: the first (rapidly occurring) is generally attributed to an increase in myofilament calcium responsiveness and the second (gradually developing) to an increase in [Ca
2+
]
i
. Rat ventricular trabeculae were stretched from ≈88% to ≈98% of L
max
, and the second force phase was analyzed. Intracellular pH, [Na
+
]
i
, and Ca
2+
transients were measured by epifluorescence with BCECF-AM, SBFI-AM, and fura-2, respectively. After stretch, DF increased by 1.94±0.2 g/mm
2
(
P
<0.01, n=4), with the second phase accounting for 28±2% of the total increase (
P
<0.001, n=4). During this phase, SBFI
340/380
ratio increased from 0.73±0.01 to 0.76±0.01 (
P
<0.05, n=5) with an estimated [Na
+
]
i
rise of ≈6 mmol/L. [Ca
2+
]
i
transient, expressed as fura-2
340/380
ratio, increased by 9.2±3.6% (
P
<0.05, n=5). The increase in [Na
+
]
i
was blocked by 5-(
N
-ethyl-
N
-isopropyl)-amiloride (EIPA). The second phase in force and the increases in [Na
+
]
i
and [Ca
2+
]
i
transient were blunted by AT
1
or ET
A
blockade. Our data indicate that the second force phase and the increase in [Ca
2+
]
i
transient after stretch result from activation of the Na
+
/H
+
exchanger (NHE) increasing [Na
+
]
i
and leading to a secondary increase in [Ca
2+
]
i
transient. This reflects an autocrine-paracrine mechanism whereby stretch triggers the release of angiotensin II, which in turn releases endothelin and activates the NHE through ET
A
receptors.
Protective Role of Interleukin-10 in Atherosclerosis
Authors: Mallat Ziad , Besnard Sandrine , Duriez Micheline , Deleuze Virginie , Emmanuel Florence , Bureau Michel F. , Soubrier Fabienne , Esposito Bruno , Duez Hélène , Fievet Catherine , Staels Bart , Duverger Nicolas , Scherman Daniel , Tedgui Alain
Published: June 2012

Abstract
—The potential role of anti-inflammatory cytokines in the modulation of the atherosclerotic process remains unknown. Interleukin (IL)-10 has potent deactivating properties in macrophages and T cells and modulates many cellular processes that may interfere with the development and stability of the atherosclerotic plaque. IL-10 is expressed in human atherosclerosis and is associated with decreased signs of inflammation. In the present study, we show that IL-10–deficient C57BL/6J mice fed an atherogenic diet and raised under specific pathogen-free conditions exhibit a significant 3-fold increase in lipid accumulation compared with wild-type mice. Interestingly, the susceptibility of IL-10–deficient mice to atherosclerosis was exceedingly high (30-fold increase) when the mice were housed under conventional conditions. Atherosclerotic lesions of IL-10–deficient mice showed increased T-cell infiltration, abundant interferon-γ expression, and decreased collagen content. In vivo, transfer of murine IL-10 achieved 60% reduction in lesion size. These results underscore the critical roles of IL-10 in both atherosclerotic lesion formation and stability. Moreover, IL-10 appears to be crucial as a protective factor against the effect of environmental pathogens on atherosclerosis. The full text of this article is available at http://www.circresaha.org.
Ionic Mechanisms Responsible for the Electrocardiographic Phenotype of the Brugada Syndrome Are Temperature Dependent
Authors: Dumaine Robert , Towbin Jeffrey A. , Brugada Pedro , Vatta Matteo , Nesterenko Dmitri V. , Nesterenko Vladislav V. , Brugada Josep , Brugada Ramon , Antzelevitch Charles
Published: June 2012

Abstract
—The Brugada syndrome is a major cause of sudden death, particularly among young men of Southeast Asian and Japanese origin. The syndrome is characterized electrocardiographically by an ST-segment elevation in V1 through V3 and a rapid polymorphic ventricular tachycardia that can degenerate into ventricular fibrillation. Our group recently linked the disease to mutations in
SCN5A
, the gene encoding for the α subunit of the cardiac sodium channel. When heterologously expressed in frog oocytes, electrophysiological data recorded from the Thr1620Met missense mutant failed to adequately explain the electrocardiographic phenotype. Therefore, we sought to further characterize the electrophysiology of this mutant. We hypothesized that at more physiological temperatures, the missense mutation may change the gating of the sodium channel such that the net outward current is dramatically augmented during the early phases of the right ventricular action potential. In the present study, we test this hypothesis by expressing Thr1620Met in a mammalian cell line, using the patch-clamp technique to study the currents at 32°C. Our results indicate that Thr1620Met current decay kinetics are faster when compared with the wild type at 32°C. Recovery from inactivation was slower for Thr1620Met at 32°C, and steady-state activation was significantly shifted. Our findings explain the features of the ECG of Brugada patients, illustrate for the first time a cardiac sodium channel mutation of which the arrhythmogenicity is revealed only at temperatures approaching the physiological range, and suggest that some patients may be more at risk during febrile states.
Regulation of the Transient Outward K
+
Current by Ca
2+
/Calmodulin-Dependent Protein Kinases II in Human Atrial Myocytes
Authors: Tessier Sophie , Karczewski Peter , Krause Ernst-Georg , Pansard Yves , Acar Christophe , Lang-Lazdunski Michel , Mercadier Jean-Jacques , Hatem Stéphane N.
Published: June 2012

Abstract
—Ca
2+
/calmodulin-dependent protein kinases II (CaMKII) have important functions in regulating cardiac excitability and contractility. In the present study, we examined whether CaMKII regulated the transient outward K
+
current (
I
to
) in whole-cell patch-clamped human atrial myocytes. We found that a specific CaMKII inhibitor, KN-93 (20 μmol/L), but not its inactive analog, KN-92, accelerated the inactivation of
I
to
(τ
fast
: 66.9±4.4 versus 43.0±4.4 ms, n=35;
P
<0.0001) and inhibited its maintained component (at +60 mV, 4.9±0.4 versus 2.8±0.4 pA/pF, n=35;
P
<0.0001), leading to an increase in the extent of its inactivation. Similar effects were observed by dialyzing cells with a peptide corresponding to CaMKII residues 281 to 309 or with autocamtide-2–related inhibitory peptide and by external application of the calmodulin inhibitor calmidazolium, which also suppressed the effects of KN-93. Furthermore, the phosphatase inhibitor okadaic acid (500 nmol/L) slowed
I
to
inactivation, increased
I
sus
, and inhibited the effects of KN-93. Changes in [Ca
2+
]
i
by dialyzing cells with ≈30 nmol/L Ca
2+
or by using the fast Ca
2+
buffer BAPTA had opposite effects on
I
to
. In BAPTA-loaded myocytes,
I
to
was less sensitive to KN-93. In myocytes from patients in chronic atrial fibrillation, characterized by a prominent
I
sus
, KN-93 still increased the extent of inactivation of
I
to
. Western blot analysis of atrial samples showed that δ-CaMKII expression was enhanced during chronic atrial fibrillation. In conclusion, CaMKII control the extent of inactivation of
I
to
in human atrial myocytes, a process that could contribute to
I
to
alterations observed during chronic atrial fibrillation.
Hybrid Transgenic Mice Reveal In Vivo Specificity of G Protein–Coupled Receptor Kinases in the Heart
Authors: Eckhart Andrea D. , Duncan Sandra J. , Penn Raymond B. , Benovic Jeffrey L. , Lefkowitz Robert J. , Koch Walter J.
Published: June 2012

Abstract
—G protein–coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including α
1B
-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the β-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type α
1B
AR. Transgenic mice with cardiac CAMα
1B
AR overexpression had enhanced myocardial α
1
AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMα
1B
ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMα
1B
AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type α
1B
AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type α
1B
AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial α
1
AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo α
1
AR mitogen–activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with α
1B
ARs in vivo such that GRK3 desensitizes all α
1B
AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo α
1B
AR signaling in the heart.
Differential Expression of Cardiac Titin Isoforms and Modulation of Cellular Stiffness
Authors: Cazorla O. , Freiburg A. , Helmes M. , Centner T. , McNabb M. , Wu Y. , Trombitás K. , Labeit S. , Granzier H.
Published: June 2012

Abstract
—Extension of the I-band segment of titin gives rise to part of the diastolic force of cardiac muscle. Previous studies of human cardiac titin transcripts suggested a series of differential splicing events in the I-band segment of titin leading to the so-called N2A and N2B isoform transcripts. Here we investigated titin expression at the protein level in a wide range of mammalian species. Results indicate that the myocardium coexpresses 2 distinct titin isoforms: a smaller isoform containing the N2B element only (N2B titin) and a larger isoform with both the N2B and N2A elements (N2BA titin). The expression ratio of large N2BA to small N2B titin isoforms was found to vary greatly in different species; eg, in the left ventricle the ratio is ≈0.05 in mouse and ≈1.5 in pig. Differences in the expression ratio were also found between atria and ventricles and between different layers of the ventricular wall. Immunofluorescence experiments with isoform-specific antibodies suggest that coexpression of these isoforms takes place at the single-myocyte level. The diastolic properties of single cardiac myocytes isolated from various species expressing high levels of the small (rat and mouse) or large (pig) titin isoform were studied. On average, pig myocytes are significantly less stiff than mouse and rat myocytes. Gel analysis indicates that this result cannot be explained by varying amounts of titin in mouse and pig myocardium. Rather, low stiffness of pig myocytes can be explained by its high expression level of the large isoform: the longer extensible region of this isoform results in a lower fractional extension for a given sarcomere length and hence a lower force. Implications of our findings to cardiac function are discussed.
Differentiation-Inducing Factor-1, a Morphogen of
Dictyostelium
, Induces G
1
Arrest and Differentiation of Vascular Smooth Muscle Cells
Authors: Miwa Yoshikazu , Sasaguri Toshiyuki , Kosaka Chiya , Taba Yoji , Ishida Akio , Abumiya Takeo , Kubohara Yuzuru
Published: June 2012

Abstract
—Differentiation-inducing factor-1 (DIF-1) is a morphogen that induces differentiation of
Dictyostelium
. Recently, DIF-1 has been shown to inhibit proliferation and induce differentiation in tumor cells, although the underlying mechanisms remain unknown. In this study, we examined the effects of DIF-1 on the proliferation and differentiation of vascular smooth muscle cells, to explore novel therapeutic strategies for atherosclerosis. DIF-1 nearly completely inhibited DNA synthesis and cell division in mitogen-stimulated cells. DIF-1 inhibited the phosphorylation of the retinoblastoma protein and the activities of cyclin-dependent kinase (Cdk) 4, Cdk6, and Cdk2, which phosphorylate the retinoblastoma protein. DIF-1 strongly suppressed the expression of cyclins D1, D2, and D3, as well as those of cyclins E and A, which normally began after that of the D-type cyclins. The mRNAs for the smooth muscle myosin heavy chains SM1 and SM2 were expressed in quiescent cells in primary culture, and these expression levels decreased after mitogenic stimulation. In the presence of DIF-1, the rate of the reduction was significantly decelerated. Moreover, the addition of DIF-1 to dedifferentiated cells induced the expressions of SM1 and SM2, accompanied by a reduction in the level of SMemb, a nonmuscle-type myosin heavy chain. Therefore, DIF-1 seemed to interrupt a very early stage of G
1
probably by suppressing the expressions of the D-type cyclins. Furthermore, this compound may prevent phenotypic modulation and induce differentiation of vascular smooth muscle cells.
Endothelial Cell Pathway for Conduction of Hyperpolarization and Vasodilation Along Hamster Feed Artery
Authors: Emerson Geoffrey G. , Segal Steven S.
Published: June 2012

Abstract
—Acetylcholine (ACh) evokes the conduction of vasodilation along resistance microvessels. However, it is not known which cell layer (endothelium or smooth muscle) serves as the conduction pathway. In isolated, cannulated feed arteries (≈70 μm in diameter at 75 mm Hg; length ≈4 mm) of the hamster retractor muscle, we tested the hypothesis that endothelial cells provide the pathway for conduction. Microiontophoresis of ACh (500 ms, 500 nA) onto the distal end of a feed artery evoked hyperpolarization (−13±2 mV) of both cell layers with vasodilation (15±1 μm) along the entire vessel. To selectively damage endothelial cells (confirmed by loss of vasodilation to ACh and labeling of disrupted cells with propidium iodide), an air bubble was perfused through a portion of the vessel lumen, or a 70-kDa fluorescein-conjugated dextran (FCD) was illuminated within a segment (300 μm) of the lumen. After endothelial cell damage, hyperpolarization and vasodilation conducted up to, but not through, the treated segment. To selectively damage smooth muscle cells (confirmed by loss of vasoconstriction to phenylephrine and labeling with propidium iodide), FCD was perifused around the vessel and illuminated. Vasodilation and hyperpolarization conducted past the disrupted smooth muscle cells without attenuation. We conclude that endothelial cells provide the pathway for conducting hyperpolarization and vasodilation along feed arteries in response to ACh.
Effects of MgADP on Length Dependence of Tension Generation in Skinned Rat Cardiac Muscle
Authors: Fukuda Norio , Kajiwara Hidetoshi , Ishiwata Shin’ichi , Kurihara Satoshi
Published: June 2012

Abstract
—The effect of MgADP on the sarcomere length (SL) dependence of tension generation was investigated using skinned rat ventricular trabeculae. Increasing SL from 1.9 to 2.3 μm decreased the muscle width by ≈11% and shifted the midpoint of the pCa-tension relationship (pCa
50
) leftward by about 0.2 pCa units. MgADP (0.1, 1, and 5 mmol/L) augmented maximal and submaximal Ca
2+
-activated tension and concomitantly diminished the SL-dependent shift of pCa
50
in a concentration-dependent manner. In contrast, pimobendan, a Ca
2+
sensitizer, which promotes Ca
2+
binding to troponin C (TnC), exhibited no effect on the SL-dependent shift of pCa
50
, suggesting that TnC does not participate in the modulation of SL-dependent tension generation by MgADP. At a SL of 1.9 μm, osmotic compression, produced by 5% wt/vol dextran (molecular weight ≈464 000), reduced the muscle width by ≈13% and shifted pCa
50
leftward to a similar degree as that observed when increasing SL to 2.3 μm. This favors the idea that a decrease in the interfilament lattice spacing is the primary mechanism for SL-dependent tension generation. MgADP (5 mmol/L) markedly attenuated the dextran-induced shift of pCa
50
, and the degree of attenuation was similar to that observed in a study of varying SL. The actomyosin-ADP complex (AM.ADP) induced by exogenous MgADP has been reported to cooperatively promote myosin attachment to the thin filament. We hereby conclude that the increase in the number of force-generating crossbridges on a decrease in the lattice spacing is masked by the cooperative effect of AM.ADP, resulting in depressed SL-dependent tension generation. The full text of this article is available at http://www.circresaha.org.
Immunoneutralization of Glycoprotein Ibα Attenuates Endotoxin-Induced Interactions of Platelets and Leukocytes With Rat Venular Endothelium In Vivo
Authors: Katayama Tomihiro , Ikeda Yasuo , Handa Makoto , Tamatani Takuya , Sakamoto Shinji , Ito Masaharu , Ishimura Yuzuru , Suematsu Makoto
Published: June 2012

Abstract
—This study aimed to examine molecular mechanisms for endotoxin-induced adhesive changes in platelets in vivo. Platelets labeled with carboxyfluorescein diacetate succinimidyl ester were visualized in rat mesenteric venules through intravital microscopy assisted by a high-speed fluorescence video imager at 1000 frames per second or by a normal-speed intensifier under monitoring of erythrocyte velocity. Leukocyte rolling was examined by normal-speed transmission video images. The velocity of platelets traveling along the centerline of venules followed that of erythrocytes, whereas that measured at the periendothelial space was significantly smaller than the erythrocyte velocity; a majority of these cells exhibited transient but notable rolling with endothelium. Administration of endotoxin increased the density of periendothelial platelets and reduced the rolling velocities of platelets and leukocytes in venules: All events were attenuated by anti–rat P-selectin monoclonal antibody s789G or by anti–human glycoprotein (GP) Ibα monoclonal antibody GUR83/35, which blocks ristocetin-induced aggregation of rat platelets. Isolated rat platelets injected into endotoxin-pretreated rats were able to roll on the venules. This event was attenuated by pretreatment of platelets in vitro with GUR83/35 but not with s789G, suggesting involvement of endothelial P-selectin and platelet GP Ibα in the endotoxin-induced responses. Furthermore, isolated human platelets showed similar rolling interactions with endotoxin-preexposed rat venules, and pretreatment of the platelets with GUR83/35, but not with s789G, significantly reduced such interactions. Our results provide the first evidence for involvement of GP Ibα in endotoxin-induced microvascular rolling of platelets and leukocytes, and this system serves as a potentially useful tool to examine GP Ibα–associated function of human platelets in vivo.
Investigation of a Truncated Cardiac Troponin T That Causes Familial Hypertrophic Cardiomyopathy
Authors: Redwood Charles , Lohmann Karin , Bing Wu , Esposito Giovanna M. , Elliott Kathryn , Abdulrazzak Hassan , Knott Adam , Purcell Ian , Marston Steven , Watkins Hugh
Published: June 2012

Abstract
—Familial hypertrophic cardiomyopathy (HCM) is caused by mutations in at least 8 contractile protein genes, most commonly β myosin heavy chain, myosin binding protein C, and cardiac troponin T. Affected individuals are heterozygous for a particular mutation, and most evidence suggests that the mutant protein acts in a dominant-negative fashion. To investigate the functional properties of a truncated troponin T shown to cause HCM, both wild-type and mutant human cardiac troponin T were overexpressed in
Escherichia coli
, purified, and combined with human cardiac troponins I and C to reconstitute human cardiac troponin. Significant differences were found between the regulatory properties of wild-type and mutant troponin in vitro, as follows. (1) In actin-tropomyosin–activated myosin ATPase assays at pCa 9, wild-type troponin caused 80% inhibition of ATPase, whereas the mutant complex gave negligible inhibition. (2) Similarly, in the in vitro motility assay, mutant troponin failed to decrease both the proportion of actin-tropomyosin filaments motile and the velocity of motile filaments at pCa 9. (3) At pCa 5, the addition of mutant complex caused a greater increase (21.7%) in velocity of actin-tropomyosin filaments than wild-type troponin (12.3%). These data suggest that the truncated troponin T prevents switching off of the thin filament at low Ca
2+
. However, the study of thin filaments containing varying ratios of wild-type and mutant troponin T at low Ca
2+
indicated an opposite effect of mutant troponin, causing enhancement of the inhibitory effect of wild-type complex, when it is present in a low ratio (10% to 50%). These multiple effects need to be taken into account to explain the physiological consequences of this mutation in HCM. Further, these findings underscore the importance of studying mixed mutant:wild-type preparations to faithfully model this autosomal-dominant disease.
Gap Junctions in Cardiovascular Disease
Authors: Jongsma Habo J. , Wilders Ronald
Published: June 2012

Abstract
—Connexins, the protein molecules forming gap junction channels, are reduced in number or redistributed from intercalated disks to lateral cell borders in a variety of cardiac diseases. This “gap junction remodeling” is considered to be arrhythmogenic. Using a simple model of human ventricular myocardium, we found that quantitative remodeling data extracted from the literature gave rise to only small to moderate changes in conduction velocity and the anisotropy ratio. Especially for longitudinal conduction, cytoplasmic resistivity (and thus cellular geometry) is much more important than commonly realized. None of the remodeling data gave rise to slow conduction on the order of a few centimeters per second.
Interleukin-1β and Tumor Necrosis Factor-α Decrease Collagen Synthesis and Increase Matrix Metalloproteinase Activity in Cardiac Fibroblasts In Vitro
Authors: Siwik Deborah A. , Chang Donny L.-F. , Colucci Wilson S.
Published: June 2012

Abstract
—We tested the hypothesis that the inflammatory cytokines can regulate fibroblast extracellular matrix metabolism. Neonatal and adult rat cardiac fibroblasts cultures in vitro were exposed to interleukin (IL)–1β (4 ng/mL), tumor necrosis factor-α (TNF-α; 100 ng/mL), IL-6 (10 ng/mL), or interferon-γ (IFN-γ; 500 U/mL) for 24 hours. IL-1β, and to a lesser extent TNF-α, decreased collagen synthesis, which was measured as collagenase-sensitive [
3
H]proline incorporation, but had no effect on cell number or total protein synthesis. IL-1β decreased the expression of procollagen α
1
(I), α
2
(I), and α1(III) mRNA, but increased the expression of procollagen α
1
(IV), α
2
(IV), and fibronectin mRNA, indicating a selective transcriptional downregulation of fibrillar collagen synthesis. IL-1β and TNF-α each increased total matrix metalloproteinase (MMP) activity as measured by in-gel zymography, causing specific increases in the bands corresponding to MMP-13, MMP-2, and MMP-9. IL-1β increased the expression of proMMP-2 and proMMP-3 mRNA, suggesting that increased metalloproteinase activity is due, at least in part, to increased transcription. The effects of IL-1β were not dependent on NO production. Thus, IL-1β and TNF-α decrease collagen synthesis and activate MMPs that degrade collagen. These observations suggest that IL-1β and TNF-α may contribute to ventricular dilation and myocardial failure by promoting the remodeling of interstitial collagen.
Ca
2+
Channel Modulation by Recombinant Auxiliary β Subunits Expressed in Young Adult Heart Cells
Authors: Wei Shao-kui , Colecraft Henry M. , DeMaria Carla D. , Peterson Blaise Z. , Zhang Rui , Kohout Trudy A. , Rogers Terry B. , Yue David T.
Published: June 2012

Abstract
—L-type Ca
2+
channels contribute importantly to the normal excitation-contraction coupling of physiological hearts, and to the functional derangement seen in heart failure. Although Ca
2+
channel auxiliary β
1–4
subunits are among the strongest modulators of channel properties, little is known about their role in regulating channel behavior in actual heart cells. Current understanding draws almost exclusively from heterologous expression of recombinant subunits in model systems, which may differ from cardiocytes. To study β-subunit effects in the cardiac setting, we here used an adenoviral-component gene-delivery strategy to express recombinant β subunits in young adult ventricular myocytes cultured from 4- to 6-week-old rats. The main results were the following. (1) A component system of replication-deficient adenovirus, poly-
l
-lysine, and expression plasmids encoding β subunits could be optimized to transfect young adult myocytes with 1% to 10% efficiency. (2) A reporter gene strategy based on green fluorescent protein (GFP) could be used to identify successfully transfected cells. Because fusion of GFP to β subunits altered intrinsic β-subunit properties, we favored the use of a bicistronic expression plasmid encoding both GFP and a β subunit. (3) Despite the heteromultimeric composition of L-type channels (composed of α
1C
, β, and α
2
δ), expression of recombinant β subunits alone enhanced Ca
2+
channel current density up to 3- to 4-fold, which argues that β subunits are “rate limiting” for expression of current in heart. (4) Overexpression of the putative “cardiac” β
2a
subunit more than halved the rate of voltage-dependent inactivation at +10 mV. This result demonstrates that β subunits can tune inactivation in the myocardium and suggests that other β subunits may be functionally dominant in the heart. Overall, this study points to the possible therapeutic potential of β subunits to ameliorate contractile dysfunction and excitability in heart failure.
Targeted Disruption of the Mouse
Sod I
Gene Makes the Hearts Vulnerable to Ischemic Reperfusion Injury
Authors: Yoshida Tetsuya , Maulik Nilanjana , Engelman Richard M. , Ho Ye-Shih , Das Dipak K.
Published: June 2012

Abstract
—The role of Cu/Zn–superoxide dismutase (SOD) in myocardial ischemic reperfusion injury was studied by using a mouse model with targeted disruption of the mouse
Sod I
gene. Inactivation of the functional mouse
Sod I
gene in hearts by gene targeting (
Sod I
+/−
) resulted in a 50% reduction of Cu/Zn-SOD mRNA and significant reduction of Cu/Zn-SOD enzyme activity compared with that of wild-type
Sod I
+/+
mice. Cu/Zn-SOD mRNA could not be detected in
Sod I
−/−
heart. The isolated buffer-perfused hearts from the knockout mice devoid of any functional copy of the Sod I (
Sod I
−/−
) and matched nontransgenic control mice were subjected to 30 minutes of global ischemia followed by 2 hours of reperfusion. For both groups of mice, the postischemic functional recovery for the hearts was lower than the baseline, but the recovery for the
Sod I
−/−
was less compared with the wild-type mice. Thus, the postischemic recovery of the developed force and the maximum first derivative of the developed force were consistently lower for the
Sod I
−/−
mouse hearts compared with wild-type control hearts. The coronary flow was lower compared with the baseline levels for both groups of hearts, but there was no significant difference between the groups. The myocardial infarction determined from the ratio of infarct size/area of risk was higher for the
Sod I
−/−
mice compared with the control mice. The amount of creatine kinase release from the wild-type mouse hearts was less compared with the
Sod I
−/−
mouse hearts. In concert, a reduced amount of oxidative stress was found in the hearts of wild-type mice compared with
Sod I
−/−
mouse hearts. These results documented that
Sod I
−/−
mouse hearts were more susceptible to ischemic reperfusion injury compared with corresponding wild-type mouse hearts, suggesting that the
Sod I
gene constitutes an important defense element for the hearts.
Expression of Coxsackievirus and Adenovirus Receptor in Hearts of Rats With Experimental Autoimmune Myocarditis
Authors: Ito Masahiro , Kodama Makoto , Masuko Masayoshi , Yamaura Masayuki , Fuse Koichi , Uesugi Yumiko , Hirono Satoru , Okura Yuji , Kato Kiminori , Hotta Yuko , Honda Takao , Kuwano Ryozo , Aizawa Yoshifusa
Published: June 2012

Abstract
—The expression of coxsackievirus and adenovirus receptor (CAR) was dominant in the brains and hearts of mice until the newborn phase. There is no detailed information concerning the relation between the expression of CAR and development of hearts. It is also uncertain whether CAR is able to be induced in adult hearts after cardiac injury. We demonstrated that CAR was abundant in the hearts of newborn rats but was barely detectable in the hearts of adult rats. The expression of CAR in rat hearts with experimental autoimmune myocarditis, which was induced by immunization of purified cardiac myosin, was serially investigated. Active myocarditis was observed from day 15 after immunization. By immunohistochemistry, cardiomyocytes were strongly stained for CAR antibody from days 24 to 42. CAR mRNA was also detected from days 18 to 30 by using reverse transcription–polymerase chain reaction. In the next experiment, the induction of CAR on isolated cardiomyocytes was investigated. CAR was barely detectable in cultured cardiomyocytes by Western blot analysis after isolation. This molecule gradually appeared along with the creation of clusters and beating of cardiomyocytes. Furthermore, the induction of CAR in cultured cardiomyocytes increased after supplement with conditioned medium of rat splenocytes activated by concanavalin A. In conclusion, rat CAR is expressed strongly in the hearts of newborn rats and is suppressed in those of adult rats. The expression of CAR is enhanced during the active phase of experimental autoimmune myocarditis and is induced by inflammatory mediators. CAR may play a role in cell-to-cell contact and adhesion of cardiomyocytes.
Hypoxic Regulation of Inducible Nitric Oxide Synthase via Hypoxia Inducible Factor-1 in Cardiac Myocytes
Authors: Jung Frank , Palmer Lisa A. , Zhou Nan , Johns Roger A.
Published: June 2012

Abstract
—The relationship between hypoxia and regulation of nitric oxide synthase (NOS) in myocardial tissue is not well understood. We investigated the role of hypoxia inducible factor-1 (HIF-1) on expression of the inducible NOS (iNOS) in myocardial cells in vivo and in vitro. In situ hybridization in myocardial tissue from rats exposed to hypoxia for 3 weeks demonstrated increased iNOS mRNA expression. Northern analysis of RNA from hearts of those animals and from cells exposed to hypoxia for 12 hours in vitro demonstrated an increase of HIF-1 RNA expression. Electrophoretic mobility shift assays using oligonucleotides containing the iNOS HIF-1 DNA binding site and nuclear extracts from cardiac myocytes showed induction of specific DNA binding in cells subjected to hypoxia. Transient transfection of cardiac myocytes using the murine iNOS promoter resulted in a 3.43-fold increase in promoter activity under hypoxia compared with normoxia. Mutation or deletion of the HIF-1 site eliminated the hypoxic response. As cytokines have been shown to regulate iNOS expression in myocardial cells, cultured neonatal cardiac myocytes were stimulated with interleukin-1β causing a dramatic induction of iNOS protein expression under normoxia, with further augmentation under hypoxia. Transient transfection of cells stimulated with interleukin-1β showed an increased iNOS promoter activity under normoxic conditions compared with unstimulated cells, with a further increase in response to hypoxia, which was dependent on HIF-1. These results demonstrate that hypoxia causes an increase in iNOS expression in cardiac myocytes and that HIF-1 is essential for the hypoxic regulation of iNOS gene expression.
Photodynamic Therapy Generates a Matrix Barrier to Invasive Vascular Cell Migration
Authors: Overhaus Marcus , Heckenkamp Joerg , Kossodo Sylvie , Leszczynski Dariusz , LaMuraglia Glenn M.
Published: June 2012

Abstract
—Photodynamic therapy (PDT) inhibits experimental intimal hyperplasia. PDT results in complete vascular wall cell eradication with subsequent adventitia but minimal media repopulation. This study was designed to test the hypothesis that PDT alters the vascular wall matrix thereby inhibiting invasive cell migration, and as such, provides an important barrier mechanism to favorably alter the vascular injury response. Untreated smooth muscle cells (SMCs) and fibroblasts were seeded on control and PDT-treated (100 J/cm
2
; photosensitizer was chloroaluminum-sulfonated phthalocyanine, 5 μg/mL) 3-dimensional collagen matrix gels. Invasive cell migration was temporally quantified by calibrated microscopy. Zymography and ELISA assessed SMC matrix metalloproteinase levels. Molecular changes of gel proteins and their susceptibility to collagenase were analyzed by SDS-PAGE and Western blot. Limited pepsin digestion and histology were used to assess the in vivo relevance of the model, using an established rat carotid artery model at 1 and 4 weeks after balloon injury and PDT. PDT of 3-dimensional matrix of gels led to a 52% reduction of invasive SMCs and to a 59% reduction of fibroblast migration (
P
<0.001) but did not significantly affect secretion of matrix metalloproteinases. PDT induced collagen matrix changes, including cross-linking, which resulted in resistance to protease digestion. PDT led to a durable 45% reduction in pepsin digestion susceptibility of treated arteries (
P
<0.001) and inhibition of periadventitial cell migration into the media. These data suggest that PDT of matrix gels generates a barrier to invasive cellular migration. This newly identified effect on matrix proteins underscores its pleiotropic actions on the vessel wall, and as such, PDT may be of considerable potential therapeutic value to inhibit restenosis.
A Variant of p22

phox

, Involved in Generation of Reactive Oxygen Species in the Vessel Wall, Is Associated With Progression of Coronary Atherosclerosis
Authors: Cahilly Carolyn , Ballantyne Christie M. , Lim Do-Sun , Gotto Antonio , Marian Ali J.
Published: June 2012

Abstract
—A series of pro-oxidant and antioxidant enzymes, such as the NADPH oxidase system, maintain the redox state in the vessel wall. A major component of NADPH oxidase is p22

phox

, which is implicated in atherosclerosis. We prospectively studied the association of the histidine (H)
72
→tyrosine (Y) mutation in p22

phox

with the severity and progression/regression of coronary artery disease (CAD), plasma lipid levels, clinical events, and response to treatment with fluvastatin in a well-characterized population. Genotypes were determined by polymerase chain reaction and restriction digestion with
Rsa
I enzyme in 368 subjects in the Lipoprotein and Coronary Atherosclerosis Study (LCAS). Fasting plasma lipids and quantitative coronary angiograms were obtained at baseline and 2.5 years after randomization to fluvastatin or placebo. Subjects with CC genotype (n=157) were identified by the presence of 396-bp and 113-bp products on gel electrophoresis. Those with TT (n=39) were identified by the presence of 316-bp, 113-bp, and 80-bp products, and those with CT (n=172) by the presence of 396-bp, 316-bp, 113-bp, and 80-bp products. Baseline and final plasma levels of lipids and the baseline severity of CAD were not significantly different among the genotypes. In the placebo group, subjects with the mutation had a 3- to 5-fold greater loss in mean minimum lumen diameter (MLD) (TT: −0.15±0.15; CT: −0.17±0.26; and CC: −0.03±0.22 mm;
P
=0.006) and lesion-specific MLD (TT: −0.15±0.06; CT: −0.18±0.03; and CC: −0.06±0.03 mm;
P
=0.038) than those without. Progression was also more (TT: 8/17 [47%]; CT: 35/73 [48%]; and CC: 17/62 [27%]) and regression less (TT: 0/17 [0%]; CT: 1/73 [1%]; and CC: 11/72 [18%]) common in those with the mutation (
P
=0.002). The C
242
T mutation in p22

phox

, involved in maintaining the redox state in the vessel wall, is associated with progression of coronary atherosclerosis in the LCAS population.
Asynchronous Ca
2+
Waves in Intact Venous Smooth Muscle
Authors: Ruehlmann Dietrich O. , Lee Cheng-Han , Poburko Damon , van Breemen Cornelis
Published: June 2012

Abstract
—The rabbit inferior vena cava (IVC) is a large-capacitance vessel that displays typical contractile dose-response curves for caffeine and phenylephrine (PE). Using confocal microscopy on the endothelium-denuded IVC, we undertook experiments to correlate these whole-tissue contractile dose-response curves with changes in subcellular [Ca
2+
]
i
signals in the in situ vascular smooth muscle cells (VSMCs). We observed that both caffeine and PE initially elicited Ca
2+
waves in individual VSMCs. The [Ca
2+
]
i
in cells challenged with caffeine subsequently returned to baseline whereas the [Ca
2+
]
i
in cells challenged with PE exhibited repetitive asynchronous Ca
2+
waves. These [Ca
2+
]
i
oscillations were related to Ca
2+
release from the sarcoplasmic reticulum as they were inhibited by ryanodine and caffeine. The lack of synchronicity of the [Ca
2+
]
i
oscillations between VSMCs can explain the observed tonic contraction at the whole-tissue level. The nature of these Ca
2+
waves was further characterized. For caffeine, the amplitude was all-or-none in nature, with individual cells differing in sensitivity, leading to their recruitment at different concentrations of the agonist. This concentration dependency of recruitment appears to form the basis for the concentration dependency of caffeine-induced contraction. Furthermore, the speed of the Ca
2+
waves correlated positively with the concentration of caffeine. In the case of PE, we observed the same characteristics with respect to wave speed, amplitude, and recruitment. Increasing concentrations of PE also enhance the frequency of the [Ca
2+
]
i
oscillations. We therefore conclude that PE stimulates whole-tissue contractility through differential recruitment of VSMCs and enhancement of the frequency of asynchronous [Ca
2+
]
i
oscillations once the cells are recruited. The full text of this article is available at http://www.circresaha.org.
NAD(P)H Oxidase
Authors: Griendling Kathy K. , Sorescu Dan , Ushio-Fukai Masuko
Published: June 2012

Abstract
—Reactive oxygen species have emerged as important molecules in cardiovascular function. Recent work has shown that NAD(P)H oxidases are major sources of superoxide in vascular cells and myocytes. The biochemical characterization, activation paradigms, structure, and function of this enzyme are now partly understood. Vascular NAD(P)H oxidases share some, but not all, characteristics of the neutrophil enzyme. In response to growth factors and cytokines, they produce superoxide, which is metabolized to hydrogen peroxide, and both of these reactive oxygen species serve as second messengers to activate multiple intracellular signaling pathways. The vascular NAD(P)H oxidases have been found to be essential in the physiological response of vascular cells, including growth, migration, and modification of the extracellular matrix. They have also been linked to hypertension and to pathological states associated with uncontrolled growth and inflammation, such as atherosclerosis.
Loss of Expression of the β Subunit of Soluble Guanylyl Cyclase Prevents Nitric Oxide–Mediated Inhibition of DNA Synthesis in Smooth Muscle Cells of Old Rats
Authors: Chen Lihua , Daum Günter , Fischer Jens W. , Hawkins Suzanne , Bochaton-Piallat Marie-Luce , Gabbiani Giulio , Clowes Alexander W.
Published: June 2012

Abstract
—We compared the effects of NO donors and cGMP analogues on the growth of aortic smooth muscle cells (SMCs) derived from newborn, adult (aged 3 months), and old (aged 2 years) rats. We found that the NO donor
S
-nitroso-
N
-acetylpenicillamine failed to block DNA synthesis in SMCs from old rats but was effective in SMCs from newborn and adult rats. However, cGMP analogues were inhibitory in all 3 SMC types. We demonstrated that in SMCs from old rats, NO was unable to increase the concentration of intracellular cGMP, suggesting that either cGMP synthesis was defective or cGMP degradation was enhanced. Western blot analysis revealed that SMCs from old rats do not express the β subunit of soluble guanylyl cyclase. To confirm the importance of this observation in vivo, we balloon-injured the carotid arteries of adult and old rats. Whereas soluble guanylyl cyclase was expressed at the same level in the media of injured vessels and uninjured vessels of both groups, its expression in the intimas of old rats was reduced by 70% compared with intimas from adult animals. Furthermore,
N
ω
-nitro-
l
-arginine, an inhibitor of NO synthesis, enhanced the intimal thickening in injured vessels in adult rats but not in old rats. We conclude that the loss of NO responsiveness in aged rats is due to the lack of the β subunit of soluble guanylyl cyclase, and we speculate that this defect contributes to the enhanced intimal thickening in response to injury in old animals.
Preconditioning in Cardiomyocytes Protects by Attenuating Oxidant Stress at Reperfusion
Authors: Hoek Terry L. Vanden , Becker Lance B. , Shao Zuo-Hui , Li Chang-Qing , Schumacker Paul T.
Published: June 2012

Abstract
—Cardiomyocyte death after ischemia/reperfusion correlates with oxidant stress, and antioxidants confer protection in that model. Preconditioning (PC) with hypoxia or adenosine also confers protection, leading us to hypothesize that PC protects by attenuating oxidant generation during subsequent ischemia/reperfusion. Chick cardiomyocytes were preconditioned with 10 minutes of hypoxia or adenosine (100 μmol/L), followed by 1 hour of simulated ischemia and 3 hours of reperfusion. Adenosine PC decreased cell death from 50±3% to 18±4% and enhanced the return of contractions during reperfusion, as observed previously with hypoxic PC. A transient burst of dichlorofluorescein (sensitive to H
2
O
2
) oxidation that was significantly attenuated by PC initiated by hypoxia or adenosine was seen at reperfusion. The protein kinase C (PKC) inhibitor Go-6976 and the mitochondrial ATP-sensitive K
+
(K
ATP
) channel inhibitor 5-hydroxydecanoate each abolished protection and abrogated the PC-induced attenuation of reperfusion oxidant stress. By contrast, when given only at reperfusion, the K
+
channel opener pinacidil or the antioxidants 2-mercaptopropionylglycine and 1,10-phenanthroline decreased oxidant stress at reperfusion and improved survival and return of contractions. Thus, PC protection is associated with an attenuation of the oxidant burst at reperfusion, regardless of the method by which PC is triggered. Loss of PC protection associated with PKC inhibition or K
ATP
channel inhibitors is associated with a restoration of that oxidant stress. These results suggest a mechanism for PC protection and reveal a functional link between PKC activation and K
ATP
channel activation in that pathway.
Redox Changes of Cultured Endothelial Cells and Actin Dynamics
Authors: Moldovan Leni , Moldovan Nicanor I. , Sohn Richard H. , Parikh Sahil A. , Goldschmidt-Clermont Pascal J.
Published: June 2012

Abstract
—We studied the association between the production of reactive oxygen species, actin organization, and cellular motility. We have used an endothelial cell monolayer–wounding assay to demonstrate that the cells at the margin of the wound thus created produced significantly more free radicals than did cells in distant rows. The rate of incorporation of actin monomers into filaments was fastest at the wound margin, where heightened production of free radicals was detected. We have tested the effect of decreasing reactive oxygen species production on the migration of endothelial cells and on actin polymerization. The NADPH inhibitor diphenylene iodonium and the superoxide dismutase mimetic manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) virtually abolished cytochalasin D–inhibitable actin monomer incorporation at the fast-growing barbed ends of filaments. Moreover, endothelial cell migration within the wound was significantly retarded in the presence of both diphenylene iodonium and MnTMPyP. We conclude that migration of endothelial cells in response to loss of confluence includes the intracellular production of reactive oxygen species, which contribute to the actin cytoskeleton reorganization required for the migratory behavior of endothelial cells.