bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of Na
+
Current Inactivation by Stimulation of Protein Kinase C in Cardiac Cells
Authors: Watson Cheryl L. , Gold Michael R.
Published: June 2012

Abstract
Modulation of the inward Na
+
current (
I
Na
) by protein kinase C (PKC) was investigated by intracellular perfusion of a peptide corresponding to the catalytic subunit of PKC (PKCP). The effects of PKC activation independent of membrane-receptor pathways were studied in neonatal rat ventricular myocytes using whole-cell patch-clamp techniques. Perfusion with 2 nmol/L PKCP caused a depolarizing shift in steady state half-inactivation relative to control (−83.2±1.3 versus −74.9±1.6 mV for control versus PKCP, respectively) without a change in current-voltage relationships or peak
I
Na
. The development of resting inactivation was slowed by PKCP (τ, 69.1±7.6 [control] versus 100.4±5.1 ms). Open-channel inactivation, estimated by measuring
I
Na
decay from peak current at test voltages between −10 and +30 mV was significantly slowed by PKCP. Recovery from inactivation was more rapid during PKCP perfusion, with a shortening of both the fast (τ
f
) and slow (τ
s
) components of τ (τ
f
, 38.5±7.0 [control] versus 14.2±4.7 ms; τ
s
, 163.4±47.9 [control] versus 51.3±9.2 ms). All of the effects of PKCP on
I
Na
were antagonized by the PKC inhibitors chelerythrine chloride or staurosporine or by downregulation of PKC using phorbol ester preincubation. We conclude that the actions of PKC on the Na
+
channel result in slowing the development of inactivation and accelerating reactivation, resulting in less resting inactivation.
Ischemic Preconditioning Prevents the Impairment of Hypoxic Coronary Vasodilatation Caused by Ischemia/Reperfusion
Authors: Giannella Eliana , Mochmann Hans-Christian , Levi Roberto
Published: June 2012

Abstract
We previously reported that hypoxic coronary vasodilatation (HCVD) is initiated by endothelial NO and sustained by adenosine. Prolonged ischemia/reperfusion impairs endothelium-dependent coronary vasodilatation, whereas transient ischemia (ie, preconditioning) protects the myocardium from subsequent ischemic events. Accordingly, we assessed whether prolonged ischemia/reperfusion impairs HCVD and whether preconditioning prevents this dysfunction. HCVD, elicited in isolated guinea pig hearts by a 1-minute exposure to 100% N
2
, consisted of an ≈70% increase in coronary flow associated with enhanced nitrite/nitrate and adenosine overflow (+40% and 5-fold, respectively). After 30-minute global ischemia and 20-minute reperfusion, HCVD was decreased by ≈60%, and the increases in nitrite/nitrate and adenosine overflow were abolished. Preconditioning (ie, three cycles of 5-minute global ischemia+5-minute reperfusion) prevented the impairment of HCVD and fully restored the increase in nitrite/nitrate overflow, but not that of adenosine. The protective effect of preconditioning was mimicked by perfusion with the adenosine A
1
receptor agonist
N
6
-cyclopentyladenosine and prevented by the A
1
receptor antagonist N-0861. In addition, the A
3
receptor agonist
N
6
-(3-iodobenzyl)adenosine-5′-
N
-methyl-carboxamide had a similar protective effect. The bradykinin B
2
receptor antagonist HOE 140 abolished the protective effect of preconditioning, whereas the NO synthase inhibitor
N
ω
-methyl-
l
-arginine and the cycloxygenase inhibitor indomethacin did not. Our data indicate that preconditioning restores HCVD by a process that is triggered by activation of adenosine A
1
/A
3
and bradykinin B
2
receptors. The action of bradykinin is independent of NO and prostacyclin production. Once restored by preconditioning, HCVD is mediated by NO but no longer sustained by adenosine.
Conserved Enhancer Elements in Human and Mouse Renin Genes Have Different Transcriptional Effects in As4.1 Cells
Authors: Yan Yan , Jones Craig A. , Sigmund Curt D. , Gross Kenneth W. , Catanzaro Daniel F.
Published: June 2012

Abstract
Despite the strong conservation of proximal 5′-flanking DNA sequences, cell transfection and transgenic animal studies have failed to provide a unifying hypothesis to explain the expression of both mouse and human renin genes. Recently, sequences contained in the mouse
Ren
-1
C
gene 5′-flanking DNA (–2866 to –2625) were shown to contain an enhancer-like element that stimulates
Ren
-1
C
promoter activity in renin-expressing As4.1 cells ≈80-fold. Earlier studies using transgenic mice had suggested that this same region is required for the cell-specific expression of mouse renin genes. Since existing human renin genomic clones lack sequences homologous to the mouse renin enhancer, we isolated several human P1 and P1 artificial chromosome genomic clones that contain >80 kb spanning the human renin gene. Analysis of these clones by Southern blot hybridization and long-range polymerase chain reaction showed that they contain sequences homologous to the mouse enhancer at ≈12 kb upstream of the transcription start site. Mouse and human sequences were 59% identical over a 650-bp region that contained the minimal enhancer from the mouse
Ren
-1
C
gene. However, a 1-kb fragment containing the entire human enhancer homology failed to stimulate human renin promoter activity in transiently transfected As4.1 cells. Further deletional analysis showed that a 220-bp region of the human sequence highly conserved in the mouse
Ren
-1
C
gene exhibited up to 47-fold transcriptional stimulation, although this was lower than the maximal effect exhibited by the minimal mouse enhancer (223-fold). Taken together, these observations suggest that sequences surrounding the conserved enhancer core stimulate enhancer activity in the mouse gene but suppress activity in the human gene. The high transcriptional activity of the mouse enhancer may have evolved to support the exceptionally high plasma renin concentrations found in mice. However, the enhancer core and surrounding conserved sequences may play an additional role in directing cell specificity.
Interaction of the Heavy and Light Chains of Cardiac Myosin Subfragment-1 With F-Actin
Authors: Andreev O.A. , Borejdo J.
Published: June 2012

Abstract
The interaction of the heavy chain (HC) and the light chain (cdLC1) of cardiac S1 (cdS1) with F-actin was studied by cross-linking, Western blotting, and fluorescence polarization methods. Incorporation of cdLC1 in cross-linked products was examined by Western blots using the primary antibody against 71-74 residues of cdLC1. Cross-linking with zero-length, water-soluble reagent yielded three products with apparent molecular masses of 150, 160, and 210 kD. Like in the case of cross-linking of skeletal S1 with actin, these complexes included only HC of S1 and actin. The composition of the products were as follows: 150 kD, one HC of S1 cross-linked through a primary site (on the C-terminal of the 20-kD fragment) to the N-terminus of actin; 160 kD, one HC of S1 cross-linked through a secondary site (on the 50 kD fragment) to the N-terminus of actin; and 210 kD, one HC of S1 cross-linked through primary and secondary sites to two actins. Four additional products with apparent molecular masses of 66, 120, 185, and 235 kD contained cdLC1 and were identified as cdLC1+actin, cdLC1+HC
S1
, cdLC1+actin+HC
S1
, and cdLC1+two actins+HC
S1
, respectively. The same products were observed when cross-linking was performed in cardiac myofibrils incubated with cdS1. The production of cross-linked complexes of the heavy and light chain with actin decreased with an increase in the molar ratio of cdS1:actin. To test whether the orientation of myosin heads depended on a degree of occupation of thin filaments, myofibrils were irrigated with varying concentrations of cdS1. Fluorescence polarization measurements of cdS1 bound to individual I-bands revealed that the orientation depended on the concentration.
Bradykinin B
2
-Receptor Activation Augments Norepinephrine Exocytosis From Cardiac Sympathetic Nerve Endings
Authors: Seyedi Nahid , Win Terrance , Lander Harry M. , Levi Roberto
Published: June 2012

Abstract
We determined whether local bradykinin production modulates cardiac adrenergic activity. Depolarization of guinea pig heart sympathetic nerve endings (synaptosomes) with 1 to 100 mmol/L K
+
caused the release of endogenous norepinephrine (10% to 50% above basal level). This release was exocytotic, because it depended on extracellular Ca
2+
, was inhibited by the N-type Ca
2+
-channel blocker ω-conotoxin and the protein kinase C inhibitor Ro31-8220, and was potentiated by the neuronal uptake-1 inhibitor desipramine. Typical of adrenergic terminals, norepinephrine exocytosis was enhanced by activation of prejunctional angiotensin AT
1
-receptors and attenuated by adrenergic α
2
-receptors, adenosine A
1
-receptors, and histamine H
3
-receptors. Exogenous bradykinin enhanced norepinephrine exocytosis by 7% to 35% (EC
50
, 17 nmol/L), without inhibiting uptake 1. B
2
-receptor, but not B
1
-receptor, blockade antagonized this effect. The kininase II/angiotensin-converting enzyme inhibitor enalaprilat and the addition of kininogen or kallikrein enhanced norepinephrine exocytosis by ≈6% to 40% (EC
50
, 20 nmol/L) and ≈25% to 60%, respectively. This potentiation was prevented by serine protease inhibitors and was antagonized by B
2
-receptor blockade. Therefore, norepinephrine exocytosis is augmented when bradykinin synthesis is increased or when its breakdown is inhibited. This is the first report of a local kallikrein-kinin system in adrenergic nerve endings capable of generating enough bradykinin to activate B
2
-receptors in an autocrine/paracrine fashion and thus enhance norepinephrine exocytosis. This amplification process may operate in disease states, such as myocardial ischemia, associated with severalfold increases in local kinin concentrations.
Targeted Ablation of the Murine α-Tropomyosin Gene
Authors: Blanchard Edward M. , Iizuka Kenji , Christe Michael , Conner David A. , Geisterfer-Lowrance Anja , Schoen Frederick J. , Maughan David W. , Seidman Christine E. , Seidman J.G.
Published: June 2012

Abstract
We created a mouse that lacks a functional α-tropomyosin gene using gene targeting in embryonic stem cells and blastocyst-mediated transgenesis. Homozygous α-tropomyosin “knockout” mice die between embryonic day 9.5 and 13.5 and lack α-tropomyosin mRNA. Heterozygous α-tropomyosin knockout mice have ≈50% as much cardiac α-tropomyosin mRNA as wild-type littermates but similar α-tropomyosin protein levels. Cardiac gross morphology, histology, and function (assessed by working heart preparations) of heterozygous α-tropomyosin knockout and wild-type mice were indistinguishable. Mechanical performance of skinned papillary muscle strips derived from mutant and wild-type hearts also revealed no differences. We conclude that haploinsufficiency of the α-tropomyosin gene produces little or no change in cardiac function or structure, whereas total α-tropomyosin deficiency is incompatible with life. These findings imply that in heterozygotes there is a regulatory mechanism that maintains the level of myofibrillar tropomyosin despite the reduction in α-tropomyosin mRNA.
Vascular Endothelial Growth Factor Increases the Mitogenic Response to Fibroblast Growth Factor-2 in Vascular Smooth Muscle Cells In Vivo via Expression of
fms-
Like Tyrosine Kinase-1
Authors: Couper Leslie L. , Bryant Shane R. , Eldrup-Jørgensen Jens , Bredenberg Carl E. , Lindner Volkhard
Published: June 2012

Abstract
Vascular endothelial growth factor (VEGF) has traditionally been considered an endothelial cell–specific factor inducing angiogenesis and vascular permeability in vivo. In the present study, expression of VEGF and its receptors, fetal liver kinase-1 (flk-1) and
fms
-like tyrosine kinase-1 (flt-1), was examined in rat carotid arteries after balloon injury. Although VEGF and flk-1 were not detectable, high levels of flt-1 mRNA and protein were expressed by smooth muscle cells (SMCs) in the neointima, as demonstrated by en face in situ hybridization and Western blotting. Intimal SMC proliferation in chronically denuded rat carotid arteries was unaffected by intraluminal infusion of VEGF, whereas fibroblast growth factor (FGF)-2 increased the number of replicating SMCs 4-fold. Pretreatment with VEGF doubled the mitogenic response to infused FGF-2 by increasing SMC replication in deeper layers of the intima. VEGF increased the permeability of chronically denuded vessels to plasma proteins but had no effect on the uptake of locally infused biotinylated FGF-2. These findings demonstrate that vascular SMCs express functional flt-1 receptors after arterial injury and that VEGF has synergistic effects with FGF-2 on SMC proliferation. These effects are likely to be mediated by a VEGF-mediated increase in permeability as well as a direct interaction between the VEGF and FGF signaling pathways.
Mechanisms of Action of Troglitazone in the Prevention of High Glucose–Induced Migration and Proliferation of Cultured Coronary Smooth Muscle Cells
Authors: Yasunari Kenichi , Kohno Masakazu , Kano Hiroaki , Yokokawa Koji , Minami Mieko , Yoshikawa Junichi
Published: June 2012

Abstract
Recent findings suggest that high glucose levels may promote atherosclerosis in coronary vascular smooth muscle cells (VSMCs). To explore the intracellular mechanisms of action by which troglitazone affects this process, we examined the effect of troglitazone on the migration and growth characteristics of cultured rabbit coronary VSMCs. Treatment with chronic high glucose medium (22.2 mmol/L) for 5 days increased VSMC migration by 92%, [
3
H]thymidine incorporation by 135%, and cell number by 32% compared with VSMCs treated with normal glucose (5.5 mmol/L glucose+16.6 mmol/L mannose) medium. Troglitazone at 100 nmol/L and 1 μmol/L significantly suppressed high glucose–induced VSMC migration by 34% and 42%, respectively, the proliferative effect (as measured by cell number) by 17% and 27%, and [
3
H]thymidine incorporation by 45% and 60% (n=6,
P
<.05). The high glucose–induced impairment of insulin-mediated [
3
H]deoxyglucose uptake was blocked by a protein kinase C (PKC) inhibitor (calphostin C, 1 μmol/L) and was also improved by troglitazone without any change in insulin receptor number and affinity. The high glucose–induced insulin-mediated increase in cell number and in [
3
H]thymidine incorporation was suppressed by troglitazone. Troglitazone (1 μmol/L) also suppressed high glucose–induced phospholipase D activation, elevation of the cytosolic NADH/NAD
+
ratio (as measured by the cytosolic ratio of lactate/pyruvate), and membrane-bound PKC activation. Flow cytometric DNA histogram analysis of cell cycle stage showed that high glucose–induced increase in the percentage of cells in the S phase was suppressed by 1 μmol/L troglitazone. These findings suggest that PKC may be a link between impairment of insulin-mediated glucose uptake and the increase in migration and proliferation induced by high glucose levels and that troglitazone may be clinically useful for the treatment of high glucose–induced coronary atherosclerosis.
Molecular and Physiological Effects of α-Tropomyosin Ablation in the Mouse
Authors: Rethinasamy Prabhakar , Muthuchamy Mariappan , Hewett Tim , Boivin Greg , Wolska Beata M. , Evans Christian , Solaro R. John , Wieczorek David F.
Published: June 2012

Abstract
—Tropomyosin (TM) is an integral component of the thin filament in muscle fibers and is involved in regulating actin-myosin interactions. TM is encoded by a family of four alternatively spliced genes that display highly conserved nucleotide and amino acid sequences. To assess the functional and developmental significance of α-TM, the murine α-TM gene was disrupted by homologous recombination. Homozygous α-TM null mice are embryonic lethal, dying between 8 and 11.5 days post coitum. Mice that are heterozygous for α-TM are viable and reproduce normally. Heterozygous knockout mouse hearts show a 50% reduction in cardiac muscle α-TM mRNA, with no compensatory increase in transcript levels by striated muscle β-TM or TM-30 isoforms. Surprisingly, this reduction in α-TM mRNA levels in heterozygous mice is not reflected at the protein level, where normal amounts of striated muscle α-TM protein are produced and integrated in the myofibril. Quantification of α-TM mRNA bound in polysomal fractions reveals that both wild-type and heterozygous knockout animals have similar levels. These data suggest that a change in steady-state level of α-TM mRNA does not affect the relative amount of mRNA translated and amount of protein synthesized. Physiological analyses of myocardial and myofilament function show no differences between heterozygous α-TM mice and control mice. The present study suggests that translational regulation plays a major role in the control of TM expression.
Overexpression of Inducible Nitric Oxide Synthase by Neointimal Smooth Muscle Cells
Authors: Yan Zhong-qun , Hansson Göran K.
Published: June 2012

Abstract
—The formation of a neointima represents an important repair mechanism in response to vascular injury. It is associated with the expression of a specific set of genes by the intimal smooth muscle cells. Recently, expression of the inducible isoform of NO synthase (iNOS) has been identified in injured arteries during neointimal formation, suggesting that intimal SMCs have a unique mechanism for regulating NO production. Therefore, we have analyzed the expression of iNOS in intimal SMCs. Although first expressed in the media within 1 day after injury, iNOS was confined to neointimal smooth muscle cells at 1 to 2 weeks after injury. Isolated intimal SMCs were found to consistently reexpress iNOS in reaction to proinflammatory mediators. This was associated with a 5- to 8-fold higher output of NO in comparison with SMCs derived from the media of uninjured arteries. Western blot and Northern blot analyses likewise revealed that the high production of NO by intimal SMCs was due to overexpression of iNOS. Moreover, the same stimuli induced a higher transcriptional activity in intimal than in medial SMCs, as detected by transfection of a reporter gene under the iNOS promoter. Induction of iNOS led to a reduced proliferation in both medial and intimal SMCs. This inhibitory effect was, however, less pronounced in intimal than in medial SMCs. Similarly, intimal cells were less sensitive to NO-induced inhibition of mitochondrial respiration. When SMC clones were analyzed, there was no correlation between iNOS expression and growth pattern, suggesting that iNOS expression is independent of the morphological phenotype of SMCs. Together, our data show that the intimal SMC is the main iNOS-expressing cell type in the injured artery, that it responds more vividly to iNOS-inducing cytokines because of a more efficient activation of the iNOS promoter, and that it is more resistant to the actions of NO compared with medial SMCs. Intimal production of NO via the inducible pathway may be important for the restoration of vascular homeostasis after injury.
Antithrombotic Effects of Recombinant Human, Active Site–Blocked Factor VIIa in a Rabbit Model of Recurrent Arterial Thrombosis
Authors: Golino Paolo , Ragni Massimo , Cirillo Plinio , D’Andrea Davide , Scognamiglio Annalisa , Ravera Amelia , Buono Chiara , Ezban Mirella , Corcione Nicola , Vigorito Francesco , Condorelli Mario , Chiariello Massimo
Published: June 2012

Abstract
—The extrinsic coagulation pathway is activated when circulating factor VII (FVII) gains access to tissue factor (TF) exposed as a consequence of vascular injury. Increasing evidence indicates that this TF-dependent activation of the coagulation plays an important role in the pathophysiology of intravascular thrombus formation. In the present study, we tested the effects of recombinant human, active site–blocked activated FVII (FVIIai) in a rabbit model of carotid artery thrombosis. Cyclic flow variations (CFVs), due to recurrent thrombus formation, were obtained in stenotic rabbit carotid arteries with endothelial injury. Carotid blood flow velocity was measured by a Doppler flow probe. After 30 minutes of CFVs, the animals received FVIIai (100 μg · kg
−1
· min
−1
intracarotid infusion for 10 minutes, n=9). If CFVs were abolished, animals were followed for 30 additional minutes, after which recombinant human activated FVII (FVIIa) was infused into the carotid artery (100 μg · kg
−1
· min
−1
for 10 minutes) to determine whether FVIIai could be displaced from TF by FVIIa, thus restoring CFVs. To establish the duration of action of FVIIai, an additional group of animals received FVIIai at the same dose as above, and after CFVs were inhibited, they were followed until CFVs were restored or for up to 6 hours. To determine whether CFVs could be restored by epinephrine after their abolition with FVIIai, increasing doses of epinephrine were administered to a third group of 6 animals. FVIIai abolished CFVs in 8 of 9 rabbits (
P
<.01). This effect was reversible, as FVIIa administration restored CFVs in all animals. Prothrombin times and activated partial thromboplastin times did not change significantly throughout the study. One single 10-minute infusion exerted complete antithrombotic effects for at least 6 hours, despite the fact that at this time point, plasma FVIIai levels were well below threshold concentrations. Epinephrine restored CFVs in 3 of 6 animals in which CFVs were inhibited by FVIIai. FVIIai exerts potent antithrombotic effects in this model; these effects were prolonged even after FVIIai was almost completely cleared from the circulation, probably as a result of the tight binding of FVIIai to TF. Thus, FVIIai might represent an antithrombotic substance of potential interest.
Myocardial Presynaptic and Postsynaptic Autonomic Dysfunction in Hypertrophic Cardiomyopathy
Authors: Schäfers Michael , Dutka David , Rhodes Christopher G. , Lammertsma Adriaan A. , Hermansen Flemming , Schober Otmar , Camici Paolo G.
Published: June 2012

Abstract
—Although hypertrophic cardiomyopathy (HCM) is genetically determined, several other factors, including autonomic dysfunction, may play a role in the phenotypic expression. A recent study using positron emission tomography with [
11
C]CGP 12177 ([
11
C]CGP) demonstrated that β-adrenoceptor (βAR) density is reduced in HCM and is correlated with disease progression. This present study tested the hypothesis that this downregulation is associated with reduced catecholamine reuptake (uptake 1) by myocardial sympathetic nerve terminals leading to increased local norepinephrine concentration. Myocardial presynaptic catecholamine reuptake was assessed by measuring the volume of distribution (V
d
) of the catecholamine analogue [
11
C]hydroxyephedrine ([
11
C]HED) in 9 unrelated HCM patients aged 45±15 years. The maximum number of binding sites (B
max
) for myocardial βAR density was measured in 13 unrelated HCM patients aged 40±12 years using the nonselective β blocker [
11
C]CGP. Six patients were studied with both [
11
C]HED and [
11
C]CGP. Comparison was made with two groups of healthy control subjects for each ligand ([
11
C]HED, n=10, aged 35±8 years; [
11
C]CGP, n=19, aged 44±16 years). Myocardial V
d
of [
11
C]HED (33.4±4.3 mL/g tissue) and βAR density (7.3±2.6 pmol/g tissue) were significantly reduced in HCM patients compared with control subjects (71.0±18.8 mL/g tissue,
P
<.001, and 10.2±2.9 pmol/g tissue,
P
=.008, respectively). These results are consistent with our hypothesis that myocardial βAR downregulation in HCM is associated with an impaired uptake-1 mechanism and hence increased local catecholamine levels.
Activation of Extracellular Signal–Regulated Kinases (ERK1/2) by Angiotensin II Is Dependent on c-Src in Vascular Smooth Muscle Cells
Authors: Ishida Mari , Ishida Takafumi , Thomas Sheila M. , Berk Bradford C.
Published: June 2012

Abstract
—Among the angiotensin II (Ang II)–mediated signal events likely to be important in vascular smooth muscle cells (VSMCs) is activation of extracellular signal–regulated kinases 1 and 2 (ERK1/2). The upstream mediators by which Ang II activates ERK1/2 remain poorly defined. Recently, we showed that Ang II activated c-Src, a nonreceptor kinase, which is a candidate to mediate Ang II signal events. To determine whether c-Src is required for ERK1/2 activation by Ang II, we studied the effects of Src family–selective tyrosine kinase inhibitors on ERK1/2 activation and also studied Ang II–mediated signal events in Src-deficient and Src-overexpressing VSMCs. The tyrosine kinase inhibitors, genistein and CP-188,556, blocked Ang II–mediated ERK1/2 activation in rat VSMCs (rVSMCs). We derived Src-deficient VSMCs from the aortas of c-Src knockout mice (Src
−/−
mVSMCs). Basal ERK1/2 activity was lower, and activation of ERK1/2 by Ang II was significantly decreased in Src
−/−
mVSMCs compared with wild-type mVSMCs, whereas ERK1/2 protein expression and ERK1/2 activation by phorbol 12-myristate 13-acetate were similar. To examine the role of c-Src further, we overexpressed wild-type or dominant-negative c-Src in rVSMCs using retroviral vectors. ERK1/2 activation by Ang II was significantly increased in rVSMCs that overexpressed c-Src, whereas ERK1/2 activation by Ang II was significantly inhibited in rVSMCs that overexpressed dominant-negative c-Src compared with control rVSMCs. These findings demonstrate that c-Src activation is required for Ang II stimulation of ERK1/2 in VSMCs and suggest an important role for c-Src in Ang II–mediated signal transduction.
Ineffective Perfusion-Contraction Matching in Conscious, Chronically Instrumented Pigs With an Extended Period of Coronary Stenosis
Authors: Kudej Raymond K. , Ghaleh Bijan , Sato Naoki , Shen You-Tang , Bishop Sanford P. , Vatner Stephen F.
Published: June 2012

Abstract
—Several models purported to represent hibernating myocardium involve a coronary stenosis (CS) to reduce blood flow (BF) and function without eliciting necrosis in anesthetized pigs. The goal of the present study was to determine whether sustained moderate reduction in coronary BF in conscious pigs induced hibernating myocardium, ie, perfusion-contraction matching with no necrosis. These experiments were conducted in conscious pigs chronically instrumented with a coronary artery BF probe and hydraulic occluder, left ventricular (LV) pressure gauge, and wall thickening (WT) crystals in the potentially ischemic and nonischemic zones. The hydraulic occluder was inflated to induce a stable 41±4% reduction in BF for 24 hours. Ischemic zone systolic WT fell initially with CS and then continued to decline during the period of CS even though blood flow did not change further, suggesting the induction of myocardial stunning. At 2 days after release of CS, WT was still depressed by 48±15%. Assessment of necrosis by histology or triphenyltetrazolium chloride showed 40±5% multifocal patchy necrosis interspersed with normal tissue involving the inner one third to one half of the ventricular wall. Regional myocardial BF (radioactive microsphere technique) was assessed by dividing the entire LV into an average of 488±59 pieces and examining the spatial distribution of BF within the area at risk (AAR). BF in the samples in the area of patchy necrosis was reduced (−66±4% from a baseline of 1.55±0.27 mL · min
−1
· g
−1
), whereas BF was maintained in samples in the AAR without necrosis (−2±7% from a baseline of 1.25±0.22 mL · min
−1
· g
−1
). These findings indicate that when hypoperfusion induced by CS in conscious pigs is sustained, the result is necrosis rather than hibernating myocardium. The remainder of the AAR, which lacked necrosis, might have been mistaken for hibernating myocardium had only histology been evaluated and BF not been measured and found to be at normal levels.
Gene Transfer Into Normal and Atherosclerotic Human Blood Vessels
Authors: Rekhter Mark D. , Simari Robert D. , Work Carolyn W. , Nabel Gary J. , Nabel Elizabeth G. , Gordon David
Published: June 2012

Abstract
—Gene transfer to blood vessels is a promising new approach to the treatment of the vascular diseases, but the feasibility of gene transfer to adult human vessels has not been explored. We introduced an adenovirus vector encoding a marker gene human placental alkaline phosphatase into normal and atherosclerotic human vessels in organ culture. In the normal vessels, recombinant gene was expressed preferentially in the endothelial cells (≈100%), intimal smooth muscle cells (1.3±0.4%, 1.4±1.0%, and 3.8±0.8% in the internal mammary arteries, saphenous veins, and normal coronary arteries, respectively), and various adventitial cells. Advanced, complicated atherosclerotic plaques demonstrated a similar efficiency of recombinant gene expression (3.1±0.5% and 3.8±0.3% of nonendothelial intimal cells in the coronary artery and carotid artery plaques, respectively). Of these intimal cells, macrophages and smooth muscle cells expressed a transgene, identifying them as targets for gene transfer. Areas of plaque rupture and thrombus are sites of predilection for expression of recombinant genes. Collagenase and elastase treatment increased the percentage of transgenic alkaline phosphatase–positive cells 7 times (
P
<0.001), suggesting that the pattern of gene expression was affected by the amount of surrounding extracellular matrix. These studies demonstrate the feasibility of gene transfer to human blood vessels. However, these studies also highlight important barriers to adenoviral gene delivery to the actual normal and atherosclerotic human vessels of clinical interest.
Cyclic GMP–Dependent Protein Kinase Inhibits Osteopontin and Thrombospondin Production in Rat Aortic Smooth Muscle Cells
Authors: Dey Nupur B. , Boerth Nancy J. , Murphy-Ullrich Joanne E. , Chang Pi-Ling , Prince Charles W. , Lincoln Thomas M.
Published: June 2012

Abstract
—Vascular lesions resulting from injury are characterized by a thickening of the intima brought about in part through the production of increased amounts of extracellular matrix proteins by the vascular smooth muscle cells (VSMCs). In this study, we tested the hypothesis that cGMP-dependent protein kinase (PKG), an important mediator of NO and cGMP signaling in VSMCs, inhibits the production of two extracellular matrix proteins, osteopontin and thrombospondin, which are involved in the formation of the neointima. VSMCs deficient in PKG were stably transfected with cDNAs encoding either the holoenzyme PKG-Iα or the constitutively active catalytic domain of PKG-I in order to directly examine the effects of PKG on osteopontin and thrombospondin production. Cells expressing either of the PKG constructs had dramatically reduced levels of osteopontin and thrombospondin-1 protein compared with control-transfected PKG-deficient cells. PKG transfection also altered the morphology of the VSMCs. These results indicate that PKG may be involved in suppressing extracellular matrix protein expression, which is one important characteristic of synthetic secretory VSMCs. Suppression of these matrix proteins may underlie the effects of NO-cGMP signaling to inhibit VSMC migration and phenotypic modulation.
Integrin-Induced Protein Kinase Cα and Cε Translocation to Focal Adhesions Mediates Vascular Smooth Muscle Cell Spreading
Authors: Haller Hermann , Lindschau Carsten , Maasch Christian , Olthoff Heike , Kurscheid Doris , Luft Friedrich C.
Published: June 2012

Abstract
—The extracellular matrix influences the cellular spreading of vascular smooth muscle cells (VSMCs) via integrin receptors. However, the intracellular signaling mechanisms are still incompletely understood. We investigated the hypothesis that VSMCs binding to fibronectin activates the protein kinase C (PKC) pathway, causes differential intracellular PKC isoform translocation, and mediates cell spreading. VSMCs binding to poly-
l
-lysine or preincubated with Arg-Gly-Asp (RGD) peptides were used as controls. Diacylglycerol (DAG) and phospholipase D (PLD) activity were measured by thin-layer chromatography. Intracellular distribution of PKC isoforms was assessed by confocal microscopy. VSMCs binding to fibronectin induced focal adhesions and cell spreading within 30 minutes. Fibronectin induced a rapid increase in DAG content, peaking at 10 minutes with a sustained response for <1 hour. In contrast, PLD activity was not influenced by specific binding to fibronectin. PKC isoforms α, δ, ε, and ζ were assessed by confocal microscopy. Fibronectin induced a PKC isoform translocation to the cell nucleus and to focal adhesions within minutes. The nuclear PKCα immunoreactivity was transiently increased. PKC isoforms α and ε were both translocated to focal adhesions. The intracellular distributions of other PKC isoforms were not influenced by fibronectin. The effects of fibronectin on DAG generation, the translocation of PKCα and PKCε, and cell spreading were all abolished by the incubation with RGD peptides. Downregulation of PKC isoforms α and ε with specific antisense oligodinucleotides resulted in a significant inhibition of cell spreading. Our results show that integrins induce intracellular signaling in VSMCs via DAG and PKC. PKC isoform α is translocated to the nucleus, whereas PKC isoforms α and ε are translocated to focal adhesions. Both isoforms seem to play a role in inside-out integrin signaling and cell spreading.
Cell Type–Specific Angiotensin II–Evoked Signal Transduction Pathways
Authors: Zou Yunzeng , Komuro Issei , Yamazaki Tsutomu , Kudoh Sumiyo , Aikawa Ryuichi , Zhu Weidong , Shiojima Ichiro , Hiroi Yukio , Tobe Kazuyuki , Kadowaki Takashi , Yazaki Yoshio
Published: June 2012

Abstract
—Angiotensin II (Ang II) induces hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts. To determine the molecular mechanism by which Ang II displayed different effects on cardiac myocytes and fibroblasts, we examined signal transduction pathways leading to activation of extracellular signal–regulated kinases (ERKs). Ang II–induced ERK activation was abolished by pretreatment with pertussis toxin and by overexpression of the G
βγ
subunit–binding domain of the β-adrenergic receptor kinase 1 in cardiac fibroblasts but not in cardiac myocytes. Inhibition of protein kinase C strongly inhibited activation of ERKs by Ang II in cardiac myocytes, whereas inhibitors of tyrosine kinases but not of protein kinase C abolished Ang II–induced ERK activation in cardiac fibroblasts. Overexpression of C-terminal Src kinase (Csk), which inactivates Src family tyrosine kinases, suppressed the activation of transfected ERK in cardiac fibroblasts. Ang II rapidly induced phosphorylation of Shc and association of Shc with Grb2. Cotransfection of the dominant-negative mutant of Ras or Raf-1 kinase abolished Ang II–induced ERK activation in cardiac fibroblasts. Overexpression of Csk or the dominant-negative mutant of Ras had no effects on Ang II–induced ERK activation in cardiac myocytes. These findings suggest that Ang II–evoked signal transduction pathways differ among cell types. In cardiac fibroblasts, Ang II activates ERKs through a pathway including the G
βγ
subunit of G
i
protein, tyrosine kinases including Src family tyrosine kinases, Shc, Grb2, Ras, and Raf-1 kinase, whereas G
q
and protein kinase C are important in cardiac myocytes.
Role of Ion Channels and Exchangers in Mechanical Stretch–Induced Cardiomyocyte Hypertrophy
Authors: Yamazaki Tsutomu , Komuro Issei , Kudoh Sumiyo , Zou Yunzeng , Nagai Ryozo , Aikawa Ryuichi , Uozumi Hiroki , Yazaki Yoshio
Published: June 2012

Abstract
—We have previously reported that stretching of cardiomyocytes activates the phosphorylation cascade of protein kinases, including Raf-1 kinase and mitogen-activated protein (MAP) kinases, followed by an increase in protein synthesis partly through enhanced secretion of angiotensin II and endothelin-1. Membrane proteins, such as ion channels and exchangers, have been postulated to first receive extracellular stimuli and evoke intracellular signals. The present study was performed to determine whether mechanosensitive ion channels and exchangers are involved in stretch-induced hypertrophic responses. Neonatal rat cardiomyocytes cultured on expandable silicone dishes were stretched after pretreatment with a specific inhibitor of stretch-sensitive cation channels (gadolinium and streptomycin), of ATP-sensitive K
+
channels (glibenclamide), of hyperpolarization-activated inward channels (CsCl), or of the Na
+
-H
+
exchanger (HOE 694). Pretreatment with gadolinium, streptomycin, glibenclamide, and CsCl did not show any inhibitory effects on MAP kinase activation by mechanical stretch. HOE 694, however, markedly attenuated stretch-induced activation of Raf-1 kinase and MAP kinases by ≈50% and 60%, respectively, and attenuated stretch-induced increase in phenylalanine incorporation into proteins. In contrast, HOE 694 did not inhibit angiotensin II–and endothelin-1–induced Raf-1 kinase and MAP kinase activation. These results suggest that among many mechanosensitive ion channels and exchangers, the Na
+
-H
+
exchanger plays a critical role in mechanical stress–induced cardiomyocyte hypertrophy.
Fas (CD95/Apo-1)–Mediated Damage to Ventricular Myocytes Induced by Cytotoxic T Lymphocytes From Perforin-Deficient Mice
Authors: Felzen Bella , Shilkrut Mark , Less Hadar , Sarapov Israel , Maor Gila , Coleman Raymond , Robinson Richard B. , Berke Gideon , Binah Ofer
Published: June 2012

Abstract
—Cytotoxic T lymphocytes (CTLs) that infiltrate the heart are important immune effectors implicated in heart transplant rejection, myocarditis, and other cardiomyopathies. To investigate the mechanism(s) underlying CTL damage to the myocardium through activation of the Fas receptor (Fas/CD95/Apo-1) by the Fas ligand, we explored the interaction between peritoneal exudate CTLs (PELs), derived from perforin gene–knockout (P−/−) mice, and murine ventricular myocytes. Fas expression on isolated ventricular myocytes was demonstrated immunohistochemically. Action potentials, [Ca
2+
]
i
transients, and contractions of myocytes conjugated to P−/− PELs or treated with the apoptosis-inducing anti-Fas monoclonal antibody Jo2 were recorded. Action potential characteristics of nonconjugated myocytes and myocytes conjugated with P−/− PELs were, respectively, as follows: V
m
, −73.2±1.5 and −53.6±6.4 mV (mean±SEM); action potential amplitude, 117.9±3.9 and 74.3±21.2 mV; and action potential duration at 80% repolarization, 17±6 and 42±13 milliseconds (all
P
<.05). P−/− PELs also induced early and delayed afterdepolarizations as well as arrhythmogenic activity. Diastolic [Ca
2+
]
i
increased during the cytocidal interaction with P−/− PELs, from a fluorescence ratio of 0.82±0.05 (n=7) to 1.98±0.09 (n=13) (
P
<.05). All of the effects caused by P−/− PELs were reproduced by incubating the myocytes with Jo2. Heparin (50 μg/mL), an antagonist of inositol trisphosphate (IP
3
)–operated sarcoplasmic reticulum Ca
2+
channels, or U-73122 (2 μmol/L), a phospholipase C inhibitor, but not the inactive agonist U-73343, prevented Fas-mediated myocyte dysfunction. Additionally, intracellular application (through the patch pipette) of the active IP
3
analogue, inositol 1,4,5-trisphosphate, but not the inactive analogue, inositol 1,3,4-trisphosphate, caused electrophysiological changes resembling those resulting from P−/− PELs and Jo2, suggesting that CTL-induced Fas-based myocyte dysfunction is mediated by IP
3
. We conclude that a Fas-based perforin-independent mechanism of CTL action can account for the immunopathology seen in the allotransplanted heart, myocarditis, and dilated cardiomyopathy.
Protection of Rat Cardiomyocytes Against Simulated Ischemia and Reoxygenation by Treatment With Protein Kinase C Activator
Authors: Ladilov Y. V. , Balser C. , Piper H. M.
Published: June 2012

Abstract
—The aim of this study was to investigate whether treatment with the protein kinase C (PKC) agonist 1,2-dioctanoyl-
sn
-glycerol (1,2DOG
)
can protect isolated adult Wistar rat cardiomyocytes against simulated ischemia and reoxygenation. Cytosolic Ca
2+
(assessed by fura 2 fluorescence), pH
i
(assessed by BCECF fluorescence), and cell length were measured during 80 minutes of simulated ischemia (anoxia, pH
o
6.4) and 20 minutes of reoxygenation (pH
o
7.4) and compared between control cells and cells treated with 20 μmol/L 1,2DOG before anoxia (10-minute treatment and 10-minute washout), before and during anoxia (two-step treatment), or only during anoxia. Treatment before anoxia attenuated rigor contracture but did not influence anoxic Ca
2+
overload. In contrast, two-step treatment before and during anoxia accelerated rigor contracture but reduced the rate of anoxic Ca
2+
accumulation. During reoxygenation, control cells developed irreversible hypercontracture (reduction of cell length to 43±2% of the initial cell length, n=62), which was accompanied by spontaneous oscillations of cytosolic Ca
2+
(19.6±1.6 per minute). Two-step treatment with 1,2DOG before and during anoxia significantly reduced hypercontracture (reduction of cell length to 60±2%,
P
<.01 versus control, n=41) and suppressed spontaneous Ca
2+
oscillations (2.8±0.9 per minute,
P
<.01 versus control). These effects could not be reproduced by treatment with 1,2DOG before anoxia or during anoxia or by a two-step treatment with the PKC-inactive 1,3-dioctanoyl-
sn
-glycerol and were fully abolished with 1 μmol/L bisindolylmaleimide (PKC inhibitor). We conclude that a two-step activation of PKC before and during anoxia is required for effective protection of cardiomyocytes against anoxic Ca
2+
overload and reoxygenation-induced hypercontracture.
Experimental Autoimmune Myocarditis Produced by Adoptive Transfer of Splenocytes After Myocardial Infarction
Authors: Maisel Alan , Cesario David , Baird Stephen , Rehman Jalees , Haghighi Parviz , Carter Steve
Published: June 2012

Abstract
—One possible mechanism for neurohumoral activation after myocardial infarction may be the generation of an immune response against cardiac self-antigens. We hypothesize that if there is a T cell–mediated reaction to self-antigens, the transfer of splenic lymphocytes from postinfarct rats into syngeneic rats with normal hearts should result in a T cell–mediated autoimmune myocarditis in the healthy syngeneic rats. Rats were killed 6 weeks after coronary ligation. Splenocytes from animals with large and small infarcts were purified from spleens, activated with concanavalin A, and injected in varying doses into normal syngeneic rats. These recipient rats were killed 6 weeks later, and histopathological studies were performed. Our results demonstrate in vivo evidence of lymphocyte-mediated myocardial injury by adoptive transfer of sensitized lymphocytes from rats who developed congestive heart failure after acute myocardial infarction. The amount of infiltrate and necrosis in the recipient rats appeared directly related to the size of the infarct from the donor rats. This suggests that larger infarcts lead to a greater inflammatory response as well as a greater propensity for alteration of cardiac surface antigens or the emergence of previously sequestered antigens. None of the other organs (kidney, liver, lung, or brain) had evidence of infiltrates. Two-dimensional echocardiography did not reveal systolic dysfunction. This study provides direct evidence of autoimmune myocardial injury produced by adoptive transfer of concanavalin A–activated splenocytes after myocardial infarction. We propose that neurohumoral activation early in the postinfarction period triggers a series of specific inflammatory and immunological events that lead to formation of specific clones of T cells. When these are activated and transferred into normal rats, cardiac-specific cellular infiltration occurs, occasionally accompanied by myocardial necrosis. This model should help to further explore the link between neurohumoral activation after myocardial infarction and the subsequent immune alterations that might be associated with the development and/or progression of congestive heart failure. Additionally, this might be a useful model in which to study other immune-mediated cardiomyopathies.
Angiotensin II Activates RhoA in Cardiac Myocytes
Authors: Aoki Hiroki , Izumo Seigo , Sadoshima Junichi
Published: June 2012

Abstract
—The organization of actin into striated fibers (myofibrils) is one of the major features of cardiac hypertrophy. However, its signal transduction mechanism is not well understood. Although Rho-family small G proteins have been implicated in actin organization in many cell types, it is not fully elucidated whether Rho mediates the organization of actin fibers by hypertrophic stimuli in cardiac myocytes. Therefore, we examined (1) whether Rho is activated by the hypertrophic stimulus, angiotensin II (Ang II), and (2) whether Rho mediates the Ang II–induced organization of actin fibers in cultured neonatal rat cardiac myocytes. Treatment of myocytes with Ang II caused a rapid formation of both striated (mature myofibrils) and nonstriated (premyofibrils) actin fibers within 30 minutes, as determined by phalloidin stainings of the polymerized actin and troponin T stainings. Immunoblot analyses and immunostainings have indicated that cardiac myocytes express RhoA, but RhoB is undetectable. In the control state, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to Ang II, consistent with activation of RhoA by Ang II. Incubation of myocytes with exoenzyme C3 for 48 hours completely ADP-ribosylated Rho in vivo. The C3 treatment abolished formation of premyofibrils induced by Ang II, suggesting that Ang II causes premyofibril formation via a Rho-dependent mechanism. The Ang II–induced mature myofibril formation was only partly abolished by C3. Expression of constitutively active RhoA (V14RhoA) caused the formation of premyofibrils but not mature myofibrils. The C3 treatment inhibited Ang II–induced atrial natriuretic factor induction, whereas it had no effect on c-
fos
induction. These results indicate that RhoA is activated by Ang II and mediates the Ang II–induced formation of premyofibrils and induction of a subset of genes. Distinct signaling mechanisms seem to be responsible for striated mature myofibril formation by Ang II.
Hypertrophy, Pathology, and Molecular Markers of Cardiac Pathogenesis
Authors: Vikstrom Karen L. , Bohlmeyer Teri , Factor Stephen M. , Leinwand Leslie A.
Published: June 2012

Abstract
—Increased ventricular expression of several genes, including atrial natriuretic factor (ANF), has been documented in experimental models of cardiac hypertrophy. It remains to be clarified whether altered expression of these genes is a consistent marker of the hypertrophy itself or a marker of some parallel pathogenetic process. Using a transgenic mouse model of hypertrophic cardiomyopathy as a tool, we assessed the relationship between the amount of ventricular ANF gene expression and the degree of hypertrophy as well as the relationship between the cells expressing ANF and tissue pathology. We determined that hypertrophy is not always associated with increased ventricular expression of ANF and that cells expressing ANF are found in regions of tissue pathology. We propose that alteration in the ventricular expression of this gene is a sensitive indicator of cardiac pathogenesis and may result from a number of different stimuli that include, among others, abnormal tissue architecture and hemodynamic load.