bluuebunny/crossref_metadata_2025 huggingface dataset:
Elevated Blood Pressure in Transgenic Mice With Brain-Specific Expression of Human Angiotensinogen Driven by the Glial Fibrillary Acidic Protein Promoter
Authors: Morimoto Satoshi , Cassell Martin D. , Beltz Terry G. , Johnson Alan Kim , Davisson Robin L. , Sigmund Curt D.
Published: September 2007

In addition to the circulatory renin (REN)–angiotensin system (RAS), a tissue RAS having an important role in cardiovascular function also exists in the central nervous system. In the brain, angiotensinogen (AGT) is expressed in astrocytes and in some neurons important to cardiovascular control, but its functional role remains undefined. We generated a transgenic mouse encoding the human AGT (hAGT) gene under the control of the human glial fibrillary acidic protein (GFAP) promoter to experimentally dissect the role of brain versus systemically derived AGT. This promoter targets expression of transgene products to astrocytes, the most abundant cell type expressing AGT in brain. All transgenic lines exhibited hAGT mRNA expression in brain, with variable expression in other tissues. In one line examined in detail, transgene expression was high in brain and low in tissues outside the central nervous system, and the level of plasma hAGT was not elevated over baseline. In the brain, hAGT protein was mainly localized in astrocytes, but was present in neurons in the subfornical organ. Intracerebroventricular (ICV) injection of human REN (hREN) in conscious unrestrained mice elicited a pressor response, which was abolished by ICV preinjection of losartan. Double-transgenic mice expressing the hREN gene and the GFAP-hAGT transgene exhibited a 15–mm Hg increase in blood pressure and an increased preference for salt. Blood pressure in the hREN/GFAP-hAGT mice was lowered after ICV, but not intravenous losartan. These studies suggest that AGT synthesis in the brain has an important role in the regulation of blood pressure and electrolyte balance.
Targeted α1A-Adrenergic Receptor Overexpression Induces Enhanced Cardiac Contractility but not Hypertrophy
Authors: Lin Fang , Owens W. Andrew , Chen Songhai , Stevens Mary E. , Kesteven Scott , Arthur Jane F. , Woodcock Elizabeth A. , Feneley Michael P. , Graham Robert M.
Published: September 2007

Activation of the α1A-adrenergic receptor (α1A-AR)/Gq pathway has been implicated as a critical trigger for the development of cardiac hypertrophy. However, direct evidence from in vivo studies is still lacking. To address this issue, transgenic mice with cardiac-targeted overexpression of the α1A-AR (4- to 170-fold) were generated, using the rodent α-myosin heavy chain promoter. Heterozygous animals displayed marked enhancement of cardiac contractility, evident from increases in dP/dtmax(80%,P<0.0001), dP/dtmax/LVPinst(76%,P<0.001), dP/dtmax:dP/dtmin(104%,P<0.0001), and fractional shortening (33%,P<0.05). Moreover, changes in the dP/dtmax–end-diastolic volume relationship provided load-independent evidence of a primary increase in contractility. Blood pressure and heart rate were largely unchanged, and there was a small increase in (−)norepinephrine-stimulated, but not basal, phospholipase C activity. Increased contractility was directly related to the level of receptor overexpression and could be completely reversed by acute α1A- but not β-AR blockade. Despite the robust changes in contractility, transgenic animals displayed no morphological, histological, or echocardiographic evidence of left ventricular hypertrophy. In addition, apart from an increase in atrial natriuretic factor mRNA, expression of other hypertrophy-associated genes was unchanged. To our knowledge, these data provide the first in vivo evidence for an inotropic action of the α1A-AR.
Transgenic Expression of Sarcoplasmic Reticulum Ca
2+
ATPase Modifies the Transition From Hypertrophy to Early Heart Failure
Authors: Ito Kenta , Yan Xinhua , Feng Xin , Manning Warren J. , Dillmann Wolfgang H. , Lorell Beverly H.
Published: September 2007

To examine the contribution of sarcoplasmic reticulum Ca
2+
ATPase (SERCA2a) to early heart failure, we subjected transgenic (TG) mice expressing SERCA2a gene and wild-type (WT) mice to aortic stenosis (AS) for 7 weeks. At an early stage of hypertrophy (4-week AS), in vivo hemodynamic and echocardiographic indices were similar in TG and WT mice. By 7 weeks of AS, which is the stage of early failure in this model, TG mice with AS had lower mortality than WT mice with AS (6.7% versus 29%). The magnitude of left ventricular (LV) hypertrophy was similar in WT and TG 7-week AS mice. In vivo LV systolic function was higher in TG than in WT 7-week AS mice. In LV myocytes loaded with fluo-3, fractional cell shortening and the amplitude of the [Ca
2+
]
i
transients were higher in TG than in WT 7-week AS mice under baseline conditions (0.5 Hz, 1.5 mmol/L [Ca
2+
]
o
, 25°C). The rates of relengthening and decay in [Ca
2+
]
i
were faster in TG than in WT 7-week AS myocytes. In myocytes from WT 7-week AS compared with sham-operated WT mice, contractile reserve in response to rapid pacing was depressed with impaired augmentation of both peak-systolic [Ca
2+
]
i
and the SR Ca
2+
load. In contrast, contractile reserve and the capacity to augment SR Ca
2+
load were maintained in TG 7-week AS mice. SERCA2a protein levels were depressed in WT 7-week AS mice, but were preserved in TG 7-week AS mice. These data suggest that defective SR Ca
2+
loading contributes to the onset of contractile failure in animals with chronic pressure overload.
Transcriptional Regulation of Vascular Development
Authors: Oettgen Peter
Published: September 2007

Vascular development is a highly organized sequence of events that requires the correct spatial and temporal expression of specific sets of genes leading to the development of a primary vascular network. The first step in this process is the differentiation of pluripotent stem cells into endothelial cells. This is followed by endothelial proliferation, migration, and eventual formation of endothelial tubes. Maturation of these primitive tubes into fully developed blood vessels requires the recruitment of surrounding pericytes and their differentiation into vascular smooth muscle cells. Many of the events that occur during vasculogenesis are recapitulated during angiogenesis. Transcription factors have been shown to serve as master switches for regulating a number of developmental processes. Using a candidate gene approach, the genomic regulatory regions required to direct vascular-specific gene expression of several receptor tyrosine kinases that are critical for vasculogenesis have been characterized and some of the transcription factors that are involved in the regulation of these genes have recently been identified. Many of these factors are also involved in the regulation of hematopoiesis and may have overlapping functions in determining hematopoietic and endothelial differentiation. Targeted disruption of other transcription factors that were not previously thought to be involved in vascular development have also been recently shown to play a role in blood vessel development. The purpose of this review is to provide an update on the progress that has been made in our understanding of the transcriptional regulation of vascular development over the past few years.
Role of the Sphingosine 1-Phosphate Receptor EDG-1 in Vascular Smooth Muscle Cell Proliferation and Migration
Authors: Kluk Michael J. , Hla Timothy
Published: September 2007

Abstract—

Sphingosine 1-phosphate (S1P), a platelet-derived ligand for the EDG-1 family of G protein–coupled receptors (GPCRs), has recently emerged as a regulator of vascular development. Although S1P has potent effects on endothelial cells and vascular smooth muscle cells (VSMCs), the functions of the specific S1P receptors in the latter cell type are not known. Here we show that pup-intimal VSMCs express higher levels of EDG-1 mRNA than adult-medial VSMCs. Stable transfection of EDG-1 into adult-medial VSMCs enhanced their proliferative response to S1P, concomitant with induction of p70 S6 kinase activity and expression of cyclin D1. Pertussis toxin treatment inhibited S1P-induced p70 S6 kinase activation, cyclin D1 expression and proliferation, suggesting that EDG-1–coupling to the G
i
pathway is critical. Furthermore, blocking p70 S6 kinase phosphorylation with rapamycin inhibited cyclin D1 expression and proliferation, suggesting that activation of p70 S6 kinase is critical in EDG-1/G
i
–mediated cell proliferation. EDG-1 expression also profoundly enhanced the migratory response of adult-medial VSMCs to S1P. S1P-induced migration of adult-medial VSMCs expressing exogenous EDG-1 required G
i
activation but not p70 S6 kinase. These results suggest that enhanced expression of EDG-1 in VSMCs dramatically stimulates both the proliferative and migratory responses to S1P. Since EDG-1 is expressed in the pup-intimal phenotype of VSMCs, S1P signaling via EDG-1 may play a role in vascular diseases in which the proliferation and migration of VSMCs are dysregulated.
Innate Diversity of Adult Human Arterial Smooth Muscle Cells
Authors: Li Shaohua , Fan Yao-Shan , Chow Lawrence H. , Van Den Diepstraten Caroline , van der Veer Eric , Sims Stephen M. , Pickering J. Geoffrey
Published: September 2007

Abstract—

Vascular smooth muscle cells (SMCs) perform diverse functions and this functional heterogeneity could be based on differential recruitment of distinct SMC subsets. In humans, however, there is little support for such a paradigm, partly because isolation of pure human SMC subsets has proven difficult. We report the cloning of 12 SMC lines from a single fragment of human internal thoracic artery and the elucidation of 2 distinct cellular profiles. Epithelioid clones (n=9) were polygonal at confluence, 105±9 μm in length, and had a doubling time of 39±2 hours. Spindle-shaped clones (n=3) were larger (267±18 μm long,
P
<0.01) and grew slower (doubling time 65±4 hours,
P
<0.01). Both types of clones expressed smooth muscle (SM) α-actin, SM-myosin heavy chains, h-caldesmon, and calponin, but only spindle-shaped clones expressed metavinculin. Epithelioid clones displayed greater proliferation in response to platelet-derived growth factor-BB and fibroblast growth factor-2 and were more responsive to the migratory effect of platelet-derived growth factor-BB. Spindle-shaped clones showed more robust Ca
2+
transients in response to angiotensin II, histamine, and norepinephrine, crawled more quickly, and expressed more type I collagen. On serum withdrawal, spindle-shaped clones differentiated into a contraction-competent cell. A regional basis for diversity among SMCs was suggested by stepwise arterial digestion, which liberated small, SM α-actin–positive cells from the abluminal medial layers and larger SMCs from all layers. These results identify inherent SMC diversity in the media of the adult internal thoracic artery and suggest differential participation of SMC subsets in the regulation of human arterial behavior.
Effects of Estrogen on the Vascular Injury Response in Estrogen Receptor α,β (Double) Knockout Mice
Authors: Karas Richard H. , Schulten Henny , Pare Gary , Aronovitz Mark J. , Ohlsson Claes , Gustafsson Jan-Ake , Mendelsohn Michael E.
Published: September 2007

Abstract—

The two known estrogen receptors, ERα and ERβ, mediate the effects of estrogen in all target tissues, including blood vessels. We have shown previously that estrogen inhibits vascular injury response to the same extent in female wild-type (WT), ERα knockout (ERαKO
CH
), and ERβ knockout (ERβKO
CH
) mice. We generated mice harboring disruptions of both ERα and ERβ genes (ERα,βKO
CH
) by breeding and studied the effect of 17β-estradiol (E2) on vascular injury responses in ovariectomized female ERα,βKO
CH
mice and WT littermates. E2 inhibited increases in vascular medial area following injury in the WT mice but not in the ERα,βKO
CH
mice, demonstrating for the first time that the two known estrogen receptors are necessary and sufficient to mediate estrogen inhibition of a component of the vascular injury response. Surprisingly, as in WT littermates, E2 still significantly increased uterine weight and inhibited vascular smooth muscle cell (VSMC) proliferation following injury in the ERα,βKO
CH
mice. These data support that the role of estrogen receptors differs for specific components of the vascular injury response in the ERα,βKO
CH
mice. The results leave unresolved whether E2 inhibition of VSMC proliferation in ERα,βKO
CH
mice is caused by a receptor-independent mechanism, an unidentified receptor responsive to estrogen, or residual activity of the ERα splice variant reported previously in the parental ERαKO
CH
mice. These possibilities may be resolved by studies of mice in which ERα has been fully disrupted (ERαKO
St
), which are in progress.
Proteomic Analysis of Pharmacologically Preconditioned Cardiomyocytes Reveals Novel Phosphorylation of Myosin Light Chain 1
Authors: Arrell D. Kent , Neverova Irina , Fraser Heather , Marbán Eduardo , Van Eyk Jennifer E.
Published: September 2007

Abstract—

Proteomic analysis of rabbit ventricular myocytes revealed a novel posttranslational modification to myosin light chain 1 (MLC1), consisting of phosphorylation at two sites. Subproteomic extraction to isolate myofilament-enriched fractions enabled determination of the extent of phosphorylation, which increased from 25.7±1.6% to 34.0±2.7% (mean±SE, n=4;
P
<0.05) after adenosine treatment at levels sufficient to pharmacologically precondition the myocytes (100 μmol/L). Mass spectrometry of MLC1 tryptic digests identified two peptide fragments modified by phosphorylation. These two phosphopeptides were characterized by peptide mass fingerprinting to determine the phosphorylation sites within rabbit ventricular MLC1, which correspond to Thr69 and Ser200 of rat MLC1, and to Thr64 and Ser194 or 195 of human MLC1. This proteomic analysis of preconditioned myocardium has revealed a previously unsuspected in vivo posttranslational modification to MLC1.
Mechanisms of L-Type Ca
2+
Current Downregulation in Rat Atrial Myocytes During Heart Failure
Authors: Boixel Christophe , Gonzalez Walter , Louedec Liliane , N. Hatem Stéphane
Published: September 2007

Abstract—

Downregulation of the L-type Ca
2+
current (
I
Ca
) is an important determinant of the electrical remodeling of diseased atria. Using a rat model of heart failure (HF) due to ischemic cardiopathy, we studied
I
Ca
in isolated left atrial myocytes with the whole-cell patch-clamp technique and biochemical assays.
I
Ca
density was markedly reduced (1.7±0.1 pA/pF) compared with sham-operated rats (S) (4.1±0.2 pA/pF), but its gating properties were unchanged. Calcium channel α
1C
-subunit quantities were not significantly different between S and HF. The β-adrenergic agonist isoproterenol (1 μmol/L) had far greater stimulatory effects on
I
Ca
in HF than in S (2.5- versus 1-fold), thereby suppressing the difference in current density. Dialyzing cells with 100 μmol/L cAMP or pretreating them with the phosphatase inhibitor okadaic acid also increased
I
Ca
and suppressed the difference in density between S and HF. Intracellular cAMP content was reduced more in HF than in S. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine had a greater effect on
I
Ca
in HF than in S (76.0±11.2% versus 15.8±21.2%), whereas the inhibitory effect of atrial natriuretic peptide on
I
Ca
was more important in S than in HF (54.1±4.8% versus 24.3±8.8%). Cyclic GMP extruded from HF myocytes was enhanced compared with S (55.8±8.0 versus 6.2±4.0 pmol · mL
−1
). Thus,
I
Ca
downregulation in atrial myocytes from rats with heart failure is caused by changes in basal cAMP-dependent regulation of the current and is associated with increased response to catecholamines.
Regulation of Thyroid Hormone Receptor Isoforms in Physiological and Pathological Cardiac Hypertrophy
Authors: Kinugawa Koichiro , Yonekura Katsunori , Ribeiro Ralff C.J. , Eto Yoko , Aoyagi Teruhiko , Baxter John D. , Camacho S. Albert , Bristow Michael R. , Long Carlin S. , Simpson Paul C.
Published: September 2007

Abstract—

Physiological and pathological cardiac hypertrophy have directionally opposite changes in transcription of thyroid hormone (TH)-responsive genes, including α- and β-myosin heavy chain (MyHC) and sarcoplasmic reticulum Ca
2+
-ATPase (SERCA), and TH treatment can reverse molecular and functional abnormalities in pathological hypertrophy, such as pressure overload. These findings suggest relative hypothyroidism in pathological hypertrophy, but serum levels of TH are usually normal. We studied the regulation of TH receptors (TRs) β1, α1, and α2 in pathological and physiological rat cardiac hypertrophy models with hypothyroid- and hyperthyroid-like changes in the TH target genes, α- and β-MyHC and SERCA. All 3 TR subtypes in myocytes were downregulated in 2 hypertrophy models with a hypothyroid-like mRNA phenotype, phenylephrine in culture and pressure overload in vivo. Myocyte TRβ1 was upregulated in models with a hyperthyroid-like phenotype, TH (triiodothyronine, T3), in culture and exercise in vivo. In myocyte culture, TR overexpression, or excess T3, reversed the effects of phenylephrine on TH-responsive mRNAs and promoters. In addition, TR cotransfection and treatment with the TRβ1-selective agonist GC-1 suggested different functional coupling of the TR isoforms, TRβ1 to transcription of β-MyHC, SERCA, and TRβ1, and TRα1 to α-MyHC transcription and increased myocyte size. We conclude that TR isoforms have distinct regulation and function in rat cardiac myocytes. Changes in myocyte TR levels can explain in part the characteristic molecular phenotypes in physiological and pathological cardiac hypertrophy.
Aging Enhances the Sensitivity of Endothelial Cells Toward Apoptotic Stimuli
Authors: Hoffmann Jörg , Haendeler Judith , Aicher Alexandra , Rössig Lothar , Vasa Mariuca , Zeiher Andreas M. , Dimmeler Stefanie
Published: September 2007

Abstract—

Advanced aging leads to impaired endothelial NO synthesis and enhanced endothelial cell apoptosis; therefore, we investigated the sensitivity of aged endothelial cells toward apoptotic stimuli and determined the role of NO. Human umbilical vein endothelial cells (HUVECs) were cultured until 14th passage. In aged cells, oxLDL and tumor necrosis factor-α–induced apoptosis and caspase-3–like activity were significantly enhanced more than 3-fold compared with young cells (passage 3). Because NO contributes to protection against endothelial cell death via S-nitrosylation of caspases, we determined endothelial NO synthase (eNOS) protein expression and the content of S-nitrosylated proteins. Aged HUVECs showed significantly reduced eNOS expression (35±10%) and a decrease in the overall S-NO content (33±3%), suggesting that eNOS downregulation may be involved in age-dependent increase of apoptosis sensitivity. Indeed, eNOS knockout endothelial cells showed a significantly enhanced apoptosis induction. Exogenous NO donors abolished increased apoptosis and caspase-3–like activity. In contrast, the application of shear stress, which exerts a profound apoptosis inhibitory effect via upregulation of NO synthesis in young cells, failed to inhibit apoptosis in aged cells. Moreover, no upregulation of eNOS protein expression and S-NO content in response to shear stress was detected in aged cells. Overexpression of wild-type eNOS completely restored the antiapoptotic effect of shear stress, whereas only a partial inhibitory effect was detected under steady conditions. Strikingly, transfection of constitutively active phosphomimetic eNOS (S1177D) further abrogated apoptosis in aged HUVECs. Thus, aging of endothelial cells is associated with decreased NO synthesis and concomitantly increased sensitivity of apoptosis, which may contribute to functional impairment of the endothelial monolayer.
Phosphoinositide 3-Kinase Isoforms Selectively Couple Receptors to Vascular L-Type Ca
2+
Channels
Authors: Macrez Nathalie , Mironneau Chantal , Carricaburu Valérie , Quignard Jean-François , Babich Aleksei , Czupalla Cornelia , Nürnberg Bernd , Mironneau Jean
Published: September 2007

Heterodimeric class I phosphoinositide 3-kinase (PI3K) has been shown to be involved in the stimulation of voltage-gated Ca
2+
channels by various mediators. In this study, we bring evidences that vascular L-type Ca
2+
channels can be modulated by both tyrosine kinase–regulated class Ia and G protein–regulated class Ib PI3Ks. Purified recombinant PI3Ks increased the peak Ca
2+
channel current density when applied intracellularly. Furthermore, PI3Kα-, β-, and δ-mediated stimulations of Ca
2+
channel currents were increased by preactivation by a phosphotyrosyl peptide, whereas PI3Kγ- and β-mediated effects were increased by Gβγ. In freshly isolated and cultured vascular myocytes, angiotensin II and Gβγ stimulated L-type Ca
2+
channel current. In contrast, platelet-derived growth factor (PDGF)-BB and the phosphotyrosyl peptide did not stimulate Ca
2+
channel current in freshly isolated cells despite the presence of endogenous PDGF receptors and PI3Kα and PI3Kγ. Interestingly, when endogenous PI3Kβ expression arose in cultured myocytes, both PDGF and phosphotyrosyl peptide stimulated Ca
2+
channels through PI3Kβ, as revealed by the inhibitory effect of an anti-PI3Kβ antibody. These results suggest that endogenous PI3Kβ but not PI3Kα is specifically involved in PDGF receptor–induced stimulation of Ca
2+
channels and that different isoforms of PI3K regulate physiological increases of Ca
2+
influx in vascular myocytes stimulated by vasoconstrictor or growth factor.
cis
Element Decoy Against Nuclear Factor-κB Attenuates Development of Experimental Autoimmune Myocarditis in Rats
Authors: Yokoseki Osamu , Suzuki Jun-ichi , Kitabayashi Hiroshi , Watanabe Noboru , Wada Yuko , Aoki Motokuni , Morishita Ryuichi , Kaneda Yasufumi , Ogihara Toshio , Futamatsu Hideki , Kobayashi Yasushi , Isobe Mitsuaki
Published: September 2007

Nuclear factor-κB (NFκB) plays a significant role in the coordinated transactivation of cytokine, inducible NO synthase (iNOS), and adhesion molecule genes. Although inflammation is an essential pathological feature of myocarditis, the role of NFκB in this process remains obscure. We examined the role of NFκB in the progression of rat experimental autoimmune myocarditis (EAM) and tested the hypothesis that NFκB blockade with a decoy against the
cis
element of NFκB can prevent the progression of EAM. Lewis rats were immunized with purified porcine cardiac myosin to establish EAM on day 0. NFκB decoy was infused into the rat coronary artery on day 0 (group NF0), 7 (group NF7), or 14 (group NF14) and harvested on day 21. Scrambled decoy was infused on day 0 (group SD0), 7 (group SD7), or 14 (group SD14) and served for control groups. The ratios of myocarditis-affected areas to the ventricular cross-sectional area of all treatment groups were significantly lower than those of the control groups (group NF0, 33±18% versus SD0, 53±14%; group NF7, 19±15% versus SD7, 50±16%; and group NF14, 34±10% versus SD14, 52±14%). Immunohistochemical and immunoblot analyses showed expression of ICAM-1, iNOS, IL-2, and TNFα in myocardium of scrambled decoy groups, and this expression was effectively suppressed by NFκB decoy treatment. Thus, we found that NFκB is a key regulator in the progression of EAM and that in vivo transfection of NFκB decoy reduces the severity of EAM.
Interaction Between PEVK-Titin and Actin Filaments
Authors: Kulke M. , Fujita-Becker S. , Rostkova E. , Neagoe C. , Labeit D. , Manstein D.J. , Gautel M. , Linke W.A.
Published: September 2007

The giant muscle protein titin contains a unique sequence, the PEVK domain, the elastic properties of which contribute to the mechanical behavior of relaxed cardiomyocytes. Here, human N2-B–cardiac PEVK was expressed in
Escherichia coli
and tested—along with recombinant cardiac titin constructs containing immunoglobulin-like or fibronectin-like domains—for a possible interaction with actin filaments. In the actomyosin in vitro motility assay, only the PEVK construct inhibited actin filament sliding over myosin. The slowdown occurred in a concentration-dependent manner and was accompanied by an increase in the number of stationary actin filaments. High [Ca
2+
] reversed the PEVK effect. PEVK concentrations ≥10 μg/mL caused actin bundling. Actin-PEVK association was found also in actin fluorescence binding assays without myosin at physiological ionic strength. In cosedimentation assays, PEVK-titin interacted weakly with actin at 0°C, but more strongly at 30°C, suggesting involvement of hydrophobic interactions. To probe the interaction in a more physiological environment, nonactivated cardiac myofibrils were stretched quickly, and force was measured during the subsequent hold period. The observed force decline could be fit with a three-order exponential-decay function, which revealed an initial rapid-decay component (time constant, 4 to 5 ms) making up 30% to 50% of the whole decay amplitude. The rapid, viscous decay component, but not the slower decay components, decreased greatly and immediately on actin extraction with Ca
2+
-independent gelsolin fragment, both at physiological sarcomere lengths and beyond actin-myosin overlap. Steady-state passive force dropped only after longer exposure to gelsolin. We conclude that interaction between PEVK-titin and actin occurs in the sarcomere and may cause viscous drag during diastolic stretch of cardiac myofibrils. The interaction could also oppose shortening during contraction.
Opening of Mitochondrial K
ATP
Channels Attenuates the Ouabain-Induced Calcium Overload in Mitochondria
Authors: Ishida Hideyuki , Hirota Yuki , Genka Chokoh , Nakazawa Hiroe , Nakaya Haruaki , Sato Toshiaki
Published: September 2007

We tested whether opening of mitochondrial ATP-sensitive K
+
(mitoK
ATP
) channels depolarizes mitochondrial membrane potential (ΔΨ
m
) and thereby prevents the mitochondrial Ca
2+
overload. With the use of a Nipkow disk confocal system, the mitochondrial Ca
2+
concentration ([Ca
2+
]
m
) and ΔΨ
m
in rat ventricular myocytes were measured by loading cells with Rhod-2 and JC-1, respectively. Exposure to ouabain (1 mmol/L) for 30 minutes produced mitochondrial Ca
2+
overload, and the intensity of Rhod-2 fluorescence significantly increased to 173±16% of baseline (
P
<0.001). Treatment of myocytes with the mitoK
ATP
channel opener diazoxide (100 μmol/L) blunted the ouabain-induced mitochondrial Ca
2+
overload (131±10% of baseline;
P
<0.001 versus ouabain). Moreover, diazoxide significantly depolarized the ΔΨ
m
and reduced the intensity of JC-1 fluorescence during application of ouabain to 89±2% of baseline (
P
<0.05). These effects of diazoxide were blocked by the mitoK
ATP
channel blocker 5-hydroxydecanoate (500 μmol/L). These results indicate that opening of mitoK
ATP
channels prevents a mitochondrial Ca
2+
overload in association with ΔΨ
m
depolarization and thereby protects myocardium against ischemic damage.
Dilated Cardiomyopathy and Sudden Death Resulting From Constitutive Activation of Protein Kinase A
Authors: Antos Christopher L. , Frey Norbert , Marx Steven O. , Reiken Steven , Gaburjakova Marta , Richardson James A. , Marks Andrew R. , Olson Eric N.
Published: September 2007

β-Adrenergic receptor (βAR) signaling, which elevates intracellular cAMP and enhances cardiac contractility, is severely impaired in the failing heart. Protein kinase A (PKA) is activated by cAMP, but the long-term physiological effect of PKA activation on cardiac function is unclear. To investigate the consequences of chronic cardiac PKA activation in the absence of upstream events associated with βAR signaling, we generated transgenic mice that expressed the catalytic subunit of PKA in the heart. These mice developed dilated cardiomyopathy with reduced cardiac contractility, arrhythmias, and susceptibility to sudden death. As seen in human heart failure, these abnormalities correlated with PKA-mediated hyperphosphorylation of the cardiac ryanodine receptor/Ca
2+
-release channel, which enhances Ca
2+
release from the sarcoplasmic reticulum, and phospholamban, which regulates the sarcoplasmic reticulum Ca
2+
-ATPase. These findings demonstrate a specific role for PKA in the pathogenesis of heart failure, independent of more proximal events in βAR signaling, and support the notion that PKA activity is involved in the adverse effects of chronic βAR signaling.
Tolerance for ATP-Insensitive KATPChannels in Transgenic Mice
Authors: Koster Joseph C. , Knopp Andreas , Flagg Thomas P. , Markova Kamelia P. , Sha Qun , Enkvetchakul Decha , Betsuyaku Tetsuo , Yamada Kathryn A. , Nichols Colin G.
Published: September 2007

To examine the role of sarcolemmal KATPchannels in cardiac function, we generated transgenic mice expressing GFP-tagged Kir6.2 subunits with reduced ATP sensitivity under control of the cardiac α-myosin heavy chain promoter. Four founder mice were isolated, and both founders and progeny were all apparently normal and fertile. Electrocardiograms from conscious animals also appeared normal, although mean 24-hour heart rate was approximately 10% lower in transgenic animals compared with littermate controls. In excised membrane patches, KATPchannels were very insensitive to inhibitory ATP: mean K1/2([ATP] causing half-maximal inhibition) was 2.7 mmol/L in high-expressing line 4 myocytes, compared with 51 μmol/L in littermate control myocytes. Counterintuitively, KATPchannel density was ≈4-fold lower in transgenic membrane patches than in control. This reduction of total KATPconductance was confirmed in whole-cell voltage-clamp conditions, in which KATPwas activated by metabolic inhibition. KATPconductance was not obvious after break-in of either control or transgenic myocytes, and there was no action potential shortening in transgenic myocytes. In marked contrast to the effects of expression of similar transgenes in pancreatic β-cells, these experiments demonstrate a profound tolerance for reduced ATP sensitivity of cardiac KATPchannels and highlight differential effects of channel activity in the electrical activity of the 2 tissues.
Sphingolipid Mediators in Cardiovascular Cell Biology and Pathology
Authors: Levade Thierry , Augé Nathalie , Veldman Robert Jan , Cuvillier Olivier , Nègre-Salvayre Anne , Salvayre Robert
Published: September 2007

Sphingolipids have emerged as a new class of lipid mediators. In response to various extracellular stimuli, sphingolipid turnover can be stimulated in vascular cells and cardiac myocytes. Subsequent generation of sphingolipid molecules such as ceramide, sphingosine, and sphingosine-1-phosphate, is followed by regulation of ion fluxes and activation of various signaling pathways leading to smooth muscle cell proliferation, endothelial cell differentiation or apoptotic cell death, cell contraction, retraction, or migration. The importance of sphingolipids in cardiovascular signaling is illustrated by recent observations implicating them in physiological processes such as vasculogenesis as well as in frequent pathological conditions, including atherosclerosis and its complications.
Heteromultimeric Kv1.2-Kv1.5 Channels Underlie 4-Aminopyridine-Sensitive Delayed Rectifier K+Current of Rabbit Vascular Myocytes
Authors: Kerr Paul M. , Clément-Chomienne Odile , Thorneloe Kevin S. , Chen Tim T. , Ishii Kuniaki , Sontag David P. , Walsh Michael P. , Cole William C.
Published: September 2007

The molecular identity of vascular delayed rectifier K+channels (KDR) is poorly characterized. Inhibition by 4-aminopyridine (4-AP) of KDRof rabbit portal vein (RPV) myocytes was studied by patch clamp and compared with that of channels composed of Kv1.5 and/or Kv1.2 subunits cloned from the RPV and expressed in mammalian cells. 4-AP block of KDRwas pulse-frequency dependent, required channel activation, and was associated with a positive shift in voltage dependence of activation. 4-AP caused a voltage-dependent reduction in mean open time of KDR. Relief of 4-AP block of whole cell currents during washout required channel activation and was unaffected by voltage. Homotetrameric Kv1.5 channels did not exhibit the shift in voltage dependence of activation exhibited by the native channels. In contrast, Kv1.2 channels displayed a shift in voltage dependence of activation, and this characteristic was also evident during 4-AP treatment when Kv1.2 was coexpressed with Kv1.5 or coupled to Kv1.5 in a tandem construct to produce heterotetrameric [Kv1.5/Kv1.2]2channels. KDRcurrents were not sensitive to charybdotoxin, which blocks homotetrameric Kv1.2 channels. The findings of this study (1) indicate that vascular KDRare inhibited by 4-AP via an open-state block mechanism and trapping of the drug within the pore on channel closure and (2) provide novel evidence based on a comparison of functional characteristics that indicate the dominant form of vascular KDRchannel complex in RPV involves the heteromultimeric association of Kv1.2 and Kv1.5 subunits.
Increased Cardiac Sympathetic Activity and Insulin-Like Growth Factor-I Formation Are Associated With Physiological Hypertrophy in Athletes
Authors: Neri Serneri Gian Gastone , Boddi Maria , Modesti Pietro Amedeo , Cecioni Ilaria , Coppo Mirella , Padeletti Luigi , Michelucci Antonio , Colella Andrea , Galanti Giorgio
Published: September 2007

Physiological hypertrophy represents the adaptive changes of the heart required for supporting the increased hemodynamic load in regularly trained healthy subjects. Mechanisms responsible for the athlete’s hypertrophy still remain unknown. In 15 trained competitive soccer players and in 15 healthy men not engaged in sporting activities (sedentary control subjects) of equivalent age, we investigated the relationship among cardiac growth factor formation, cardiac sympathetic activity, and left ventricular morphology and function. Cardiac formation of insulin-like growth factor (IGF)-I, endothelin (ET)-1, big ET-1, and angiotensin (Ang) II was investigated at rest by measuring artery-coronary sinus concentration gradients. Cardiac sympathetic activity was studied by [
3
H]norepinephrine (NE) kinetics. Cardiac IGF-I, but not ET-1, big ET-1, and Ang II, formation was higher in athletes than in control subjects (
P
<0.01). NE levels in arterial and peripheral venous blood did not differ between groups. In contrast, coronary sinus NE concentration was higher in athletes than in control subjects (
P
<0.01). Cardiac, but not total systemic, NE spillover was also increased in athletes (
P
<0.01), whereas cardiac [
3
H]NE reuptake and clearance were not different. Echocardiographic modifications indicated a volume overload-induced hypertrophy associated with increased myocardial contractility. Multivariate stepwise analysis selected left ventricular mass index as the most predictive independent variable for cardiac IGF-I formation and velocity of circumferential fiber shortening for cardiac NE spillover. In conclusion, increased cardiac IGF-I formation and enhanced sympathetic activity selectively confined to the heart appear to be responsible for the physiological hypertrophy in athletes performing predominantly isotonic exercise.
Increased Myocardial Rab GTPase Expression
Authors: Wu Guangyu , Yussman Martin G. , Barrett Thomas J. , Hahn Harvey S. , Osinska Hanna , Hilliard George M. , Wang Xuejun , Toyokawa Tsuyoshi , Yatani Atsuko , Lynch Roy A. , Robbins Jeffrey , Dorn Gerald W.
Published: September 2007

The Ras-like Rab GTPases regulate vesicle transport in endocytosis and exocytosis. We found that cardiac Rabs1, 4, and 6 are upregulated in a dilated cardiomyopathy model overexpressing β2-adrenergic receptors. To determine if increased Rab GTPase expression can contribute to cardiomyopathy, we transgenically overexpressed in mouse hearts prototypical Rab1a, the small G protein that regulates vesicle transport from endoplasmic reticulum to and through Golgi. In multiple independent mouse lines, Rab1a overexpression caused cardiac hypertrophy that progressed in a time- and transgene dose–dependent manner to heart failure. Isolated cardiac myocytes were hypertrophied and exhibited contractile depression with impaired calcium reuptake. Ultrastructural analysis revealed enlarged Golgi stacks and increased transitional vesicles in ventricular myocytes, with increased secretory atrial natriuretic peptide granules and degenerative myelin figures in atrial myocytes; immunogold studies localized Rab1a to these abnormal vesicular structures. A survey of hypertrophy signaling molecules revealed increased protein kinase C (PKC) α and δ, and confocal microscopy showed abnormal subcellular distribution of PKCα in Rab1a transgenics. These results indicate that increased expression of Rab1 GTPase in myocardium distorts subcellular localization of proteins and is sufficient to cause cardiac hypertrophy and failure.
Rectification of the Background Potassium Current
Authors: Samie Faramarz H. , Berenfeld Omer , Anumonwo Justus , Mironov Sergey F. , Udassi Sharda , Beaumont Jacques , Taffet Steven , Pertsov Arkady M. , Jalife José
Published: September 2007

Ventricular fibrillation (VF) is the leading cause of sudden cardiac death. Yet, the mechanisms of VF remain elusive. Pixel-by-pixel spectral analysis of optical signals was carried out in video imaging experiments using a potentiometric dye in the Langendorff-perfused guinea pig heart. Dominant frequencies (peak with maximal power) were distributed throughout the ventricles in clearly demarcated domains. The fastest domain (25 to 32 Hz) was always on the anterior left ventricular (LV) wall and was shown to result from persistent rotor activity. Intermittent block and breakage of wavefronts at specific locations in the periphery of such rotors were responsible for the domain organization. Patch-clamping of ventricular myocytes from the LV and the right ventricle (RV) demonstrated an LV-to-RV drop in the amplitude of the outward component of the background rectifier current (
I
B
). Computer simulations suggested that rotor stability in LV resulted from relatively small rectification of
I
B
(presumably
I
K1
), whereas instability, termination, and wavebreaks in RV were a consequence of strong rectification. This study provides new evidence in the isolated guinea pig heart that a persistent high-frequency rotor in the LV maintains VF, and that spatially distributed gradients in
I
K1
density represent a robust ionic mechanism for rotor stabilization and wavefront fragmentation.
Integrin and Growth Factor Receptor Crosstalk
Authors: Eliceiri Brian P.
Published: September 2007

Crosstalk between integrins and growth factor receptors are an important signaling mechanism to provide specificity during normal development and pathological processes in vascular biology. Evidence from several model systems demonstrates the physiological importance of the coordination of signals from growth factors and the extracellular matrix to support cell proliferation, migration, and invasion in vivo. Several examples of crosstalk between these two important classes of receptors indicate that integrin ligation is required for growth factor–induced biological processes. Furthermore, integrins can directly associate with growth factor receptors, thereby regulating the capacity of integrin/growth factor receptor complexes to propagate downstream signaling. Recent data suggest that antagonists of αvintegrins can provide a therapeutic benefit in human cancer patients, whereas knockout mice lacking specific integrins can provide an interesting insight into the role of integrins during development. This review will focus on the biological importance of integrin and growth factor receptor crosstalk that occurs during cell growth, migration, and invasion as well as in endothelial cells during angiogenesis.
HERG K
+
Channel Activity Is Regulated by Changes in Phosphatidyl Inositol 4,5-Bisphosphate
Authors: Bian Jinsong , Cui Jie , McDonald Thomas V.
Published: September 2007

Autonomic stimulation controls heart rate and myocardial excitability and may underlie the precipitation of both acquired and hereditary arrhythmias. Changes in phosphatidyl inositol bisphosphate (PIP2) concentration results from activation of several muscarinic and adrenergic receptors. We sought to investigate whether PIP2 changes could alter HERG K
+
channel activity in a manner similar to that seen with inward rectifier channels. PIP2 (10 μmol/L) internally dialyzed increased the K
+
current amplitude and shifted the voltage-dependence of activation in a hyperpolarizing direction. Elevated PIP2 accelerated activation and slowed inactivation kinetics. When 10 μmol/L PIP2 was applied to excised patches, no significant change in single channel conductance occurred, indicating that PIP2-dependent effects were primarily due to altered channel gating. PIP2 significantly attenuated the run-down of HERG channel activity that we normally observe after patch excision, suggesting that channel run-down is due, in part, to membrane depletion of PIP2. Inclusion of a neutralizing anti-PIP2 monoclonal antibody in whole cell pipette solution produced the opposite effects of PIP2. The physiological relevance of PIP2–HERG interactions is supported by our finding that phenylephrine reduced the K
+
current density in cells coexpressing α1A-receptor and HERG. The effects of α-adrenergic stimulation, however, were prevented by excess PIP2 in internal solutions but not by internal Ca
2+
buffering nor PKC inhibition, suggesting that the mechanism is due to G-protein–coupled receptor stimulation of PLC resulting in the consumption of endogenous PIP2. Thus, dynamic regulation of HERG K
+
channels may be achieved via receptor-mediated changes in PIP2 concentrations.
Power Output Is Increased After Phosphorylation of Myofibrillar Proteins in Rat Skinned Cardiac Myocytes
Authors: Herron Todd J. , Korte F. Steven , McDonald Kerry S.
Published: September 2007

β-Adrenergic stimulation increases stroke volume in mammalian hearts as a result of protein kinase A (PKA)-induced phosphorylation of several myocyte proteins. This study investigated whether PKA-induced phosphorylation of myofibrillar proteins directly affects myocyte contractility. To test this possibility, we compared isometric force, loaded shortening velocity, and power output in skinned rat cardiac myocytes before and after treatment with the catalytic subunit of PKA. Consistent with previous studies, PKA increased phosphorylation levels of myosin binding protein C and troponin I, and reduced Ca
2+
sensitivity of force. PKA also significantly increased both maximal force (25.4±8.3 versus 31.6±11.3 μN [
P
<0.001, n=12]) and peak absolute power output (2.48±1.33 versus 3.38±1.52 μW/mg [
P
<0.05, n=5]) during maximal Ca
2+
activations. Furthermore, PKA elevated power output at nearly all loads even after normalizing for the increase in force. After PKA treatment, peak normalized power output increased ≈20% during maximal Ca
2+
activations (n=5) and ≈33% during half-maximal Ca
2+
activations (n=9). These results indicate that PKA-induced phosphorylation of myofibrillar proteins increases the power output–generating capacity of skinned cardiac myocytes, in part, by speeding the step(s) in the crossbridge cycle that limit loaded shortening rates, and these changes likely contribute to greater contractility in hearts after β-adrenergic stimulation.
Changes in Expression of Antioxidant Enzymes Affect Cell-Mediated LDL Oxidation and Oxidized LDL–Induced Apoptosis in Mouse Aortic Cells
Authors: Guo ZhongMao , Van Remmen Holly , Yang Hong , Chen XinLian , Mele James , Vijg Jan , Epstein Charles J. , Ho Ye-Shih , Richardson Arlan
Published: September 2007

Abstract
—Transgenic mice overexpressing Cu/Zn superoxide dismutase (
hSod1Tg
+/0
) or catalase (
hCatTg
+/0
) and knockout mice underexpressing manganese superoxide dismutase (
Sod2
+/
−
) or glutathione peroxidase-1 (
Gpx1
−/−
) were used to study the effect of antioxidant enzymes on cell-mediated low density lipoprotein (LDL) oxidation and oxidized LDL (oxLDL)-induced apoptosis. Incubation of LDL with mouse aortic segments or smooth muscle cells (SMCs) resulted in a significant increase in LDL oxidation. However, LDL oxidation was significantly reduced when LDL was incubated with aortic segments and SMCs obtained from
hSod1Tg
+/0
and
hCatTg
+/0
mice compared with those obtained from wild-type mice. In contrast, LDL oxidation was significantly increased when LDL was incubated with aortic segments and SMCs obtained from
Sod2
+/
−
and
Gpx1
−/−
mice. CuSO
4
-oxidized LDL increased DNA fragmentation and caspase activities in the primary cultures of mouse aortic SMCs. However, oxLDL-induced DNA fragmentation and caspase activities were reduced 50% in SMCs obtained from
hSod1Tg
+/0
and
hCatTg
+/0
mice compared with wild-type control mice. In contrast, oxLDL-induced DNA fragmentation and caspase activities were significantly increased in SMCs obtained from
Sod2
+/
−
and
Gpx1
−/−
mice. These findings suggest that overexpression of Cu/Zn superoxide dismutase or catalase reduces cell-mediated LDL oxidation and oxLDL-induced apoptosis, whereas underexpression of manganese superoxide dismutase or glutathione peroxidase-1 increases cell-mediated LDL oxidation and oxLDL-induced apoptosis.
4-Hydroxynonenal Prevents NO Production in Vascular Smooth Muscle Cells by Inhibiting Nuclear Factor-κB–Dependent Transcriptional Activation of Inducible NO Synthase
Authors: Hattori Yoshiyuki , Hattori Sachiko , Kasai Kikuo
Published: September 2007

Abstract
—The role of lipid peroxidation products in atherogenesis was studied. We investigated whether 4-hydroxy-2-nonenal (HNE) modulates activation of the nuclear factor (NF)-κB system or alters expression of the NF-κB target gene product, inducible NO synthase (iNOS), in vascular smooth muscle cells (VSMCs) stimulated by lipopolysaccharide (LPS) in combination with interferon (IFN)-γ (LPS/IFN). NO production induced by LPS/IFN was dose-dependently inhibited by HNE. NF-κB activation by LPS/IFN was inhibited by HNE in a dose-dependent manner. HNE significantly decreased LPS/IFN-stimulated proteolysis of IκB-α. iNOS promoter activity stimulated by LPS/IFN was also decreased by HNE dose-dependently. The treatment of VSMCs with LPS/IFN strongly stimulated iNOS mRNA and protein expression. The LPS/IFN-induced increases in iNOS mRNA and protein levels were dose-dependently decreased by HNE. Our data suggest that treatment with HNE blocks signaling events required for IκB-α degradation, thereby preventing NF-κB activation. Inhibition of NF-κB–regulated gene expression, especially modulation of NO production, may contribute to atherogenesis.