bluuebunny/crossref_metadata_2025 huggingface dataset:
Myocyte Adaptation to Chronic Hypoxia and Development of Tolerance to Subsequent Acute Severe Hypoxia
Authors: Silverman Howard S. , Wei Shao-kui , Haigney Mark C. P. , Ocampo Christopher J. , Stern Michael D.
Published: June 2012

Abstract
Studies in animal models and humans suggest that myocardium may adapt to chronic or intermittent prolonged episodes of reduced coronary perfusion. Stable maintenance of partial flow reduction is difficult to achieve in experimental models; thus, in vitro cellular models may be useful for establishing the mechanisms of adaptation. Since moderate hypoxia is likely to be an important component of the low-flow state, isolated adult rat cardiac myocytes were exposed to 1% O
2
for 48 hours to study chronic hypoxic adaptation. Hypoxic culture did not reduce cell viability relative to normoxic controls but did enhance glucose utilization and lactate production, which is consistent with an anaerobic pattern of metabolism. Lactate production remained transiently increased after restoration of normal O
2
tension. Myocyte contractility was reduced (video-edge analysis), as was the amplitude of the intracellular Ca
2+
transient (indo 1 fluorescence) in hypoxic cells. Relaxation was slowed and was accompanied by a slowed decay of the Ca
2+
transient. These changes were not due to alterations in the action potential. Tolerance to subsequent acute severe hypoxia occurred in cells cultured in 1% O
2
and was manifested as a delay in the time to full ATP-depletion rigor contracture during severe hypoxia and enhanced morphological recovery of myocytes at reoxygenation. The latter was still seen after normalization of the data for the prolonged time to rigor, suggesting a multifactorial basis for tolerance. An intervening period of normoxic exposure before subsequent acute severe hypoxia did not result in loss of tolerance but rather increased the delay to subsequent ATP depletion rigor. Cellular glycogen was preserved during chronic hypoxic exposure and increased after the restoration of normal O
2
tension. As mitochondrial cytochromes should be fully oxygenated at levels well below 1% O
2
, hypoxic adaptation may be mediated by a low-affinity O
2
-sensing process. Thus, adaptations that occur during prolonged periods of moderate hypoxia are proposed to poise the myocyte in a better position to tolerate impending episodes of severe O
2
deprivation.
Nitric Oxide Promotes Proliferation and Plasminogen Activator Production by Coronary Venular Endothelium Through Endogenous bFGF
Authors: Ziche Marina , Parenti Astrid , Ledda Fabrizio , Dell’Era Patrizia , Granger Harris J. , Maggi Carlo A. , Presta Marco
Published: June 2012

Abstract
We reported previously that NO is responsible for the angiogenesis produced by endothelium-dependent vasodilating peptides. To investigate the mechanisms by which NO controls angiogenesis, NO was assessed for the ability to affect cell proliferation and upregulation of urokinase-type plasminogen activator (uPA) induced by basic fibroblast growth factor (bFGF) when added exogenously to or when produced endogenously by coronary venular endothelial cells (CVECs). The treatment of the cells with the NO donor sodium nitroprusside (NaNp) induced uPA upregulation and cell proliferation, which were prevented by anti-bFGF antibodies. Similarly, the NO-dependent mitogenic activity of the vasodilating peptide substance P (SP) was blocked by anti-bFGF antibodies, thus implicating endogenous bFGF in the NO-induced response. NaNp and SP induced bFGF expression as measured by Western blot analysis of CVEC extracts and by differential reverse transcriptase–polymerase chain reaction of bFGF mRNA. SP-induced upregulation of bFGF was prevented by the NO synthase inhibitor
N
ω
-monomethyl-
l
-arginine. We conclude that NO promotes cell proliferation and uPA upregulation in CVECs by inducing endogenous bFGF and that this pathway mediates the angiogenetic response to the vasoactive neuropeptide SP. This signaling paradigm may provide an important link between shear rate, NO, bFGF, and coronary angiogenesis.
Short-term Exercise Training Enhances Reflex Cholinergic Nitric Oxide–Dependent Coronary Vasodilation in Conscious Dogs
Authors: Zhao Gong , Zhang Xiaoping , Xu Xiaobin , Ochoa Manuel , Hintze Thomas H.
Published: June 2012

Abstract
The effects of exercise training on the coronary vasodilation following activation of the Bezold-Jarisch reflex were examined in conscious dogs. Mongrel dogs were chronically instrumented using sterile techniques for measurements of systemic hemodynamics and left circumflex coronary blood flow (CBF). With the heart rate controlled (150 bpm), veratrine (0.5 to 20 μg/kg) caused dose-dependent increases in CBF; eg, 5 μg/kg of veratrine increased CBF by 61±6% from 31±1.3 mL/min (
P
<.05). After exercise training, the dose-response curve of CBF in response to veratrine was shifted to the left; eg, 5 μg/kg of veratrine increased CBF by 101±12% (
P
<.05 compared with control) from 34±2.3 mL/min. The enhanced coronary vasodilation was blunted by nitro-
l
-arginine (NLA, 35 mg/kg). In anesthetized dogs after exercise training, electrical stimulation of the left vagus nerve caused greater increases in CBF, and NLA inhibited increases in CBF. Acetylcholine, norepinephrine, angiotensin II, and bradykinin caused greater increases in NO
2
−
production in coronary microvessels from exercise-trained dogs compared with those from normal dogs. Our results indicate that the coronary vasodilation following activation of the Bezold-Jarisch reflex is enhanced in conscious dogs after exercise training. Since electrical stimulation of the vagus nerve caused greater coronary vasodilation and since the agonists resulted in greater increases in NO production in coronary microvessels from exercise-trained dogs, the mechanism responsible for the enhanced coronary vasodilation following activation of the Bezold-Jarisch reflex is most likely due to the increased release of NO from the endothelial cells.
Diverse Effects of Heparin on Mitogen-Activated Protein Kinase–Dependent Signal Transduction in Vascular Smooth Muscle Cells
Authors: Daum Günter , Hedin Ulf , Wang Yunxia , Wang Trevina , Clowes Alexander W.
Published: June 2012

Abstract
Proliferation of vascular smooth muscle cells (SMCs) is implicated in pathological events, including atherosclerosis and intimal hyperplasia following angioplasty. The glycosaminoglycan heparin is a growth inhibitor of SMCs in vitro and in vivo. The underlying mechanism, however, is still poorly understood. In the present study, we report that heparin inhibited the activation of the mitogen-activated protein kinase (MAPK) in baboon SMCs by serum but not by platelet-derived growth factor (PDGF). When fibroblast growth factor was used, heparin had a stimulatory effect on MAPK. The only MAPK-activating kinase found in SMCs was MAPK kinase (MAPKK)-1, although MAPKK-2 was present in comparable amounts. Activation of MAPKK-1 and DNA synthesis were affected by heparin in a similar fashion. Heparin does not appear to exert its effects through members of the protein kinase C family, which are downregulated by phorbol esters, because it was still capable of inhibiting MAPK/MAPKK-1 stimulation by FCS in phorbol ester–pretreated cells. The present findings support the conclusions that the effects of heparin depend on the nature of the mitogen and that heparin inhibits SMC proliferation by preventing activation of MAPKK-1.
Nitric Oxide Can Increase Heart Rate by Stimulating the Hyperpolarization-Activated Inward Current, I
f
Authors: Musialek Piotr , Lei Ming , Brown Hilary F. , Paterson David J. , Casadei Barbara
Published: June 2012

Abstract
We investigated the chronotropic effect of increasing concentrations of sodium nitroprusside (SNP, n=8) or 3-morpholinosydnonimine (SIN-1, n=6) in isolated guinea pig spontaneously beating sinoatrial node/atrial preparations. Low concentrations of NO donors (nanomolar to micromolar) gradually increased the beating rate, whereas high (millimolar) concentrations decreased it. The increase in rate was (1) enhanced by superoxide dismutase (50 to 100 U/mL, n=6), (2) prevented by the guanylyl cyclase inhibitors 6-anilino-5,8-quinolinedione (5 μmol/L, n=6) or 1
H
-(1,2,4)oxadiazolo(4,3-
a
)quinoxalin-1-one (10 μmol/L, n=6), and (3) mimicked by 8-bromo-cGMP (n=6) with no additional positive chronotropic effect of SIN-1 (n=5). The response to 10 μmol/L SNP (n=28) or 50 μmol/L SIN-1 (n=16) was unaffected by I
Ca-L
antagonism with nifedipine (0.2 μmol/L) but was abolished after blockade of the hyperpolarization-activated inward current (I
f
) by Cs
+
(2 mmol/L) or 4-(
N
-ethyl-
N
-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium chloride (1 μmol/L). The effect on I
f
was further evaluated in rabbit isolated patch-clamped sinoatrial node cells (n=21), where we found that 5 μmol/L SNP or SIN-1 caused a reversible Cs
+
-sensitive increase in this current (+130% at −70 mV and +250% at −100 mV). In conclusion, NO donors can affect pacemaker activity in a concentration-dependent biphasic fashion. Our results indicate that the increase in beating rate is due to stimulation of I
f
via the NO-cGMP pathway. This may contribute to the sinus tachycardia in pathological conditions associated with an increase in myocardial production of NO.
Altered Metabolite Exchange Between Subcellular Compartments in Intact Postischemic Rabbit Hearts
Authors: Lewandowski E. Douglas , Yu Xin , LaNoue Kathryn F. , White Lawrence T. , Doumen Chris , O’Donnell J. Michael
Published: June 2012

Abstract
To examine metabolic regulation in postischemic hearts, we examined oxidative recycling of
13
C within the glutamate pool (GLU) of intact rabbit hearts. Isolated hearts oxidized 2.5 mmol/L [2-
13
C]acetate during normal conditions (n=6) or during reperfusion after 10 minutes of ischemia (n=5).
13
C-Nuclear magnetic resonance spectra were acquired every 1 minute. Kinetic analysis of
13
C incorporation into GLU provided both tricarboxylic acid (TCA) cycle flux and the interconversion rate (F
1
) between the TCA cycle intermediate, α-ketoglutarate (α-KG), and the largely cytosolic GLU. The rate-pressure product in postischemic hearts was 46% of normal (
P
<.05). No difference in substrate utilization occurred between groups, with acetate accounting for 92% of the carbon units entering the TCA cycle at the citrate synthase step. TCA cycle flux in postischemic hearts was normal (normal hearts, 10.7 μmol·min
−1
·g
−1
; postischemic hearts, 9.4 μmol·min
−1
·g
−1
), whereas F
1
was 72% lower at 2.9±0.4 versus 10.2±2.5 μmol·min
−1
·g
−1
(mean±SE) in normal hearts (
P
<.05). From additional hearts perfused with 2.5 mmol/L [2-
13
C]acetate plus supplemental 5 mmol/L glucose, any potential differences in endogenous carbohydrate availability were proved not to account for the reduced rate α-KG and GLU exchange, which remained depressed in postischemic hearts. However, specific activities of the transaminase enzyme, catalyzing chemical exchange of α-KG and GLU, were the same, and transaminase flux was 100 μmol·min
−1
·g
−1
in postischemic hearts versus 68 μmol·min
−1
·g
−1
in normal hearts. Normal transaminase activity and the increased flux in postischemic hearts are contrary to the reduced F
1
. The findings indicate reduced metabolite transport rates across the mitochondrial membranes of stunned myocardium, particularly through the reversible α-KG–malate carrier.
Angiotensin II Stimulates p90
rsk
in Vascular Smooth Muscle Cells
Authors: Takahashi Eiichi , Abe Jun-ichi , Berk Bradford C.
Published: June 2012

Abstract
Angiotensin II is a multifunctional agonist for vascular smooth muscle cells (VSMCs), stimulating increases in signal events, cell growth, and ion flux. We previously defined protein kinase C (PKC)–dependent and –independent mechanisms by which angiotensin II stimulated activity of the Na
+
-H
+
exchanger isoform-1 (NHE-1) and identified a 90-kD kinase that exhibited increased activity in VSMCs isolated from genetically hypertensive rats. To determine whether this 90-kD kinase was p90
rsk
(RSK), VSMCs were stimulated with 100 nmol/L angiotensin II, and NHE-1 kinase activity was measured by phosphorylation of recombinant NHE-1 (a glutathione S-transferase fusion protein containing amino acids 516 to 815 of the cytoplasmic carboxyl tail) in vitro. NHE-1 kinase (90 kD) activity was markedly decreased by immunodepletion of RSK. Characterization of RSK activation by angiotensin II revealed many similarities to the 90-kD NHE-1 kinase, including time course and NHE-1 domain phosphorylation, as well as regulation by extracellular signal–regulated kinases (ERK1/2), intracellular Ca
2+
, and PKC. Specifically, angiotensin II stimulated a rapid and transient (peak, 5 minutes) increase in RSK activity. Analysis of several NHE-1 fusion proteins revealed that only proteins containing amino acids 670 to 714 were phosphorylated by RSK. Inhibiting ERK1/2 (30 μmol/L PD098059 for 30 minutes) or chelating intracellular Ca
2+
prevented RSK activation. In contrast, downregulating PKC (1 μmol/L phorbol dibutyrate for 24 hours) had little effect. These findings establish RSK as a putative NHE-1 kinase and potential mediator of increased Na
+
-H
+
exchange in hypertension.
Effects of Changes in Blood Flow Rate on Cell Death and Cell Proliferation in Carotid Arteries of Immature Rabbits
Authors: Cho Aesim , Mitchell Lylieth , Koopmans Donna , Langille B. Lowell
Published: June 2012

Abstract
Spontaneous and experimental changes in arterial blood flow rates affect tissue accumulation in developing arteries. To examine whether cell proliferation and/or cell death are affected by alterations in blood flow, we ligated the left external carotid artery of 3-week-old rabbits, which reduces left common carotid blood flow by 71%. In control arteries and after 2 days of flow reduction, agarose gel electrophoresis of DNA extracted from all carotid arteries resolved multiple low molecular weight bands characteristic of apoptosis; however, DNA fragmentation in arteries carrying reduced blood flow was 2.5-fold higher than that of control arteries. The effect of reduced blood flow on cell death subsequently waned but remained significant at 7 days. Cell death in carotid arteries was also detected by in vivo uptake of propidium iodide, a DNA-binding fluorescent dye that labels the nuclei of nonviable cells. Both smooth muscle and endothelial cells exhibited large and statistically significant increases in labeling index in the flow-reduced artery. Propidium iodide–labeled cells were cleared from the vessel wall within 1 to 4 hours of labeling, and nuclear staining displayed condensation (clumping) of chromatin in all labeled cells at later time points. This time course and nuclear morphology and the rapid clearance of labeled cells are consistent with death via apoptosis. Many propidium iodide–positive cells did not display chromatin condensation immediately after labeling; however, this was also true of cultured endothelial cells that were driven into apoptosis with sphingomyelinase treatment and then double-labeled with propidium iodide and the apoptosis marker annexin V. We infer that propidium iodide can label apoptotic vascular cells before these cells display chromatin condensation that is detectable with fluorescence labeling of DNA. Replication rates of smooth muscle and endothelial cells, determined by 5-bromo-2′-deoxyuridine uptake, were inhibited by >75% with decreased blood flow. The inhibition of proliferation was unabated after 7 days of reduced flow. These findings indicate that the coordinated regulation of cell death and cell proliferation, in response to changes in arterial blood flow rates, contributes to arterial remodeling during development.
Comparison of Aorta and Pulmonary Artery
Authors: Schwenke Dawn C.
Published: June 2012

Abstract
In rabbits, the pulmonary artery and the aorta are susceptible to atherosclerosis. However, susceptibility of the pulmonary artery, compared with the aortic arch, to atherosclerosis and the relationship between the accumulation of cholesterol during the early stages of atherogenesis and the development of atheromatous lesions for these arterial regions remain to be clarified. Cholesterol concentrations for the pulmonary artery and aorta were measured in normal rabbits and in rabbits fed a 0.5% cholesterol diet for 8, 12, and 16 days and 17 weeks. In normal rabbits, the rank order of arterial cholesterol concentrations was pulmonary artery>aortic arch>descending thoracic aorta, with concentrations of total and nonesterified cholesterol 17% and 25% (both
P
<.05) greater, respectively, for the pulmonary artery than for the descending thoracic aorta. Rank order remained the same during 16 days of cholesterol feeding, but differences between arterial regions were exaggerated. After rabbits were fed cholesterol for 16 days, total and esterified cholesterol concentrations were 57% and 920% (both
P
<.01) greater, respectively, for the pulmonary artery than for the descending thoracic aorta, with much smaller differences between the aortic regions. In contrast, after rabbits were fed cholesterol for 17 weeks, concentrations of total, esterified, and nonesterified cholesterol were similar for the pulmonary artery and aortic arch, but these forms of cholesterol were increased 100%, 130%, and 53% (all
P
<.03), respectively, for the aortic arch compared with the descending thoracic aorta. Cholesterol concentrations for the pulmonary artery were positively associated with those for the aortic regions during the first 16 days of cholesterol feeding, but for rabbits fed cholesterol for 17 weeks the associations were either negative or absent. These results indicate that relative rates of cholesterol accumulation in the pulmonary artery and aorta differ at different stages of atherogenesis and suggest that the balance between processes that deliver cholesterol to, and remove cholesterol from, the artery may change in different ways in these arterial regions during atherogenesis.
Comparison of Aorta and Pulmonary Artery
Authors: Schwenke Dawn C.
Published: June 2012

Abstract
The pulmonary artery and the aorta are similarly susceptible to atherosclerosis in rabbits. However, the mechanism(s) that accounts for this is not yet known. This study investigated the hypothesis that one or more aspects of arterial low-density lipoprotein (LDL) transport and metabolism might explain the similar susceptibility of the aortic arch and pulmonary artery to atherosclerosis and the increased susceptibility of these arterial regions compared with the descending thoracic aorta. We determined permeability to LDL, rates of LDL degradation, and concentrations of undegraded LDL for the intima-media of normal rabbits and those fed cholesterol for ≈8 days. Intima-media permeability did not differ between corresponding arterial regions of normal rabbits and rabbits fed cholesterol for 8 days and was similar for the aortic arch and pulmonary artery. Rates of LDL degradation and concentrations of undegraded LDL for the intima-media were influenced by cholesterol feeding. These measures were reduced in fractional terms but increased in absolute terms as a result of hypercholesterolemia, without differences between corresponding parameters for the pulmonary artery and aortic arch. However, permeability to LDL, rates of LDL degradation, and concentrations of undegraded LDL were increased for the intima-media of the aortic arch compared with the descending thoracic aorta. Similar, although not always significant, trends were evident for the comparison of the pulmonary artery and descending thoracic aorta. Differences in LDL transport and metabolism and changes after feeding cholesterol for 8 days parallel the relative susceptibility to atherosclerosis for the three arterial regions studied. These results support the role of arterial LDL transport and metabolism in atherogenesis and potentially provide a mechanistic explanation for the differences in susceptibility to atherosclerosis for these three arterial regions.
Lysophosphatidylcholine Modulates Cardiac
I
Na
via Multiple Protein Kinase Pathways
Authors: Watson Cheryl L. , Gold Michael R.
Published: June 2012

Abstract
Lysophosphatidylcholine (LPC) is a naturally occurring intracellular phospholipid metabolite that has been implicated in arrhythmogenesis during ischemia. LPC has been shown to affect the cardiac Na
+
current (
I
Na
), but the mechanism of modulation remains undescribed. Recently, low concentrations of LPC have been shown to activate protein kinase C (PKC) independent of the receptor-delineated pathway. The purposes of this study were to describe the effects of intracellularly introduced LPC on
I
Na
and to determine if these effects were mediated by kinases. Modulation of
I
Na
was studied in ventricular cells with LPC (1 nmol/L to 1 μmol/L) internally applied using whole-cell patch-clamp techniques. Intracellular LPC caused a dose-dependent depolarizing shift of steady state inactivation that was accompanied by a change in slope factor. The development of resting inactivation from closed states was delayed 40%, whereas the recovery from inactivation was significantly accelerated. These results were mimicked by another bioactive lipid, lysophosphatidylethanolamine, or by a peptide analogue of PKC, which is a potent stimulator of endogenous PKC activity. Maximal recruitable current was significantly increased by LPC but not by PKC activation. Some of the effects of LPC on
I
Na
could be partially inhibited by the specific PKC inhibitor chelerythrine chloride or by downregulation of PKC with phorbol ester pretreatment. However, genistein, a specific tyrosine kinase inhibitor, completely inhibited all the modulation of
I
Na
caused by LPC. These data suggest that LPC modulates
I
Na
in cardiac myocytes by a pathway that involves both PKC-dependent and tyrosine kinase dependent phosphorylation.
Cross Talk Between Angiotensin AT
1
and α
1
-Adrenergic Receptors
Authors: Li Hong-Tai , Long Carlin S. , Gray Mary O. , Rokosh D. Gregg , Honbo Norman Y. , Karliner Joel S.
Published: June 2012

Abstract
Signaling mediated by the angiotensin (Ang) II and α
1
-adrenergic receptor (α
1
-AR) pathways is important for cardiovascular homeostasis. However, it is unknown whether Ang II has any direct effect on α
1
-AR expression and signaling in cardiac myocytes. In the present study, we determined α
1
-AR subtype mRNA levels by RNase protection; receptor density by competition binding with 5-methylurapidil; and α
1
-AR mediated c-
fos
expression by Northern blot analysis. We found that Ang II had no effect on α
1b
- and α
1d
-AR mRNA levels but decreased the α
1a
-AR mRNA level in a time- and dose-dependent manner. The maximal effect occurred at 6 hours with 100 nmol/L Ang II (40.0±8.2% reduction, n=4,
P
<.01). The decrease in α
1a
-AR mRNA level induced by Ang II is mediated by the Ang II AT
1
receptor subtype and is associated with decreased stability of α
1a
-AR mRNA. Corresponding to the changes in the α
1a
-AR mRNA level, Ang II (100 nmol/L, 24 hours) reduced the density of high-affinity sites for 5-methylurapidil (α
1A
-AR) by 29% (56.5±6.4 versus 79.0±11.6 fmol/mg protein, n=4,
P
<.05). α
1
-AR stimulated c-
fos
induction, which could be blocked by 5-methylurapidil but not by chloroethylclonidine, was attenuated by Ang II preincubation (100 nmol/L, 24 hours). We conclude that there is previously undescribed cross talk between AT
1
receptors and α
1
-ARs. Ang II selectively downregulates α
1a
-AR subtype mRNA and its corresponding receptor as well as α
1a
-AR mediated expression of the immediate-early gene c-
fos
in cardiac myocytes.
Left Ventricular Norepinephrine and Epinephrine Kinetics at Birth in Lambs
Authors: Smolich Joseph J. , Cox Helen S. , Berger Philip J. , Walker Adrian M. , Eisenhofer Graeme , Esler Murray D.
Published: June 2012

Abstract
Little is known about the changes in the left ventricular (LV) kinetics of the catecholamines norepinephrine and epinephrine occurring at birth and their relationship to perinatal alterations in LV function and whole-body catecholamine kinetics. To address this issue, whole-body and LV catecholamine kinetics (radiotracer dilution methodology) and fetal LV output and myocardial blood flow (radioactive microspheres) were measured in chronically instrumented near-term fetuses and in the same animals 1 and 4 hours after birth. Between fetal and 1-hour lambs, LV external work increased 115% (
P
<.005); carotid arterial plasma norepinephrine concentration, 148% (
P
<.01); carotid arterial plasma epinephrine concentration, 546% (
P
<.005); LV norepinephrine spillover, a measure of LV sympathetic activity, 4.1-fold (
P
<.005); LV epinephrine spillover, 3-fold (
P
<.05); total-body spillover of norepinephrine, 52% (
P
<.025); and total-body spillover of epinephrine, 460% (
P
<.005). Arterial catecholamine concentrations and total-body catecholamine spillovers were unchanged between 1- and 4-hour lambs, but LV external work fell (
P
<.05) to a level still 77% greater than in fetal lambs (
P
<.005); LV norepinephrine spillover returned to near-fetal levels, and LV epinephrine spillover became undetectable. These results suggest that (1) a transient increase in LV sympathetic activity occurs at birth and may contribute to the immediate postnatal augmentation of LV performance, (2) organ differences in the pattern of sympathetic activation occur at birth, and (3) birth-related increases in LV sympathetic activity are accompanied by release of epinephrine from the heart.
Decreased Expression of Kv4.2 and Novel Kv4.3 K
+
Channel Subunit mRNAs in Ventricles of Renovascular Hypertensive Rats
Authors: Takimoto Koichi , Li Danqing , Hershman Kenneth M. , Li Ping , Jackson Edwin K. , Levitan Edwin S.
Published: June 2012

Abstract
Hypertension-induced cardiac hypertrophy is associated with alterations in ventricular action potentials. To understand molecular mechanisms underlying this electrical abnormality, expression of cardiac voltage-gated K
+
channel subunit genes was examined in ventricles of renovascular hypertensive rats. While generating a rat Kv4.3 probe, we discovered a previously unreported 19–amino acid insertion in the C-terminal intracellular region of the channel subunit. RNase protection assays indicated that this novel isoform is predominant in rat lung and heart. Effects of renovascular hypertension were then determined by using renal artery clipping models: two-kidney, one clip (2K-1C) rats, a model of high-renin hypertension with a normal plasma volume, and one-kidney, one clip (1K-1C) rats, a model of normal renin with a raised plasma volume. Expression of Kv4.2 and Kv4.3 mRNAs was diminished by >50% in ventricles of 2K-1C rats; however, no changes in the expression of Kv1.2, Kv1.4, Kv1.5, Kv2.1, or KvLQT1 mRNAs were detected. Similar downregulation of Kv4.2 and Kv4.3 mRNAs was detected in 1K-1C rats. Chronic administration of captopril, an angiotensin-converting enzyme inhibitor, blocked the development of hypertension and the suppression of Kv4 subfamily channel mRNA expression in 2K-1C rats. Furthermore, captopril administration to sham-operated rats significantly increased Kv4.2 mRNA. These results indicate that renovascular hypertension causes specific reductions in Kv4 subfamily channel mRNA expression and that this effect is likely to be mediated primarily by an increase in cardiac afterload.
Effect of Natriuretic Peptide Family on the Oxidized LDL–Induced Migration of Human Coronary Artery Smooth Muscle Cells
Authors: Kohno Masakazu , Yokokawa Koji , Yasunari Kenichi , Kano Hiroaki , Minami Mieko , Ueda Makiko , Yoshikawa Junichi
Published: June 2012

Abstract
The migration of medial smooth muscle cells (SMCs) into the intima is proposed to be an important process of intimal thickening in atherosclerotic lesions. The present study examined the possible effect of a novel endothelium-derived relaxing peptide, C-type natriuretic peptide (CNP), on oxidized low-density lipoprotein (LDL)–induced migration of cultured human coronary artery SMCs by the Boyden’s chamber method. The effect of CNP was compared with that of atrial and brain natriuretic peptides (ANP and BNP, respectively). Oxidized LDL stimulates SMC migration in a concentration-dependent manner between 20 and 200 μg/mL. This stimulation was chemotactic in nature but was not chemokinetic. By contrast, native LDL was without significant activity. CNP-22 clearly inhibited SMC migration stimulated with 200 μg/mL oxidized LDL in a concentration-dependent manner between 10
−9
and 10
−6
mol/L. ANP-(1-28) and BNP-32 also inhibited oxidized LDL–induced SMC migration at concentrations of 10
−7
and 10
−6
mol/L, but these effects were weaker than the effect of CNP-22. Such inhibition by these natriuretic peptides was paralleled by an increase in the cellular level of cGMP. Oxidized LDL–induced migration was significantly inhibited by a stable analogue of cGMP, 8-bromo-cGMP, or an activator of the cytosolic guanylate cyclase, sodium nitroprusside. These natriuretic peptides did not suppress the cell adhesion either in the absence or presence of oxidized LDL. These data indicate that oxidized LDL stimulates migration of human coronary artery SMCs and that natriuretic peptides, especially CNP, inhibit this stimulated SMC migration, at least in part, through a cGMP-dependent process. Taken together with the finding that oxidized LDL is present in the intima, CNP may play a role as a local antimigration factor during the process of intimal thickening in hypercholesterolemia-induced coronary atherosclerosis.
Increased Sensitivity of Human Vascular Smooth Muscle Cells From Atherosclerotic Plaques to p53-Mediated Apoptosis
Authors: Bennett Martin R. , Littlewood Trevor D. , Schwartz Stephen M. , Weissberg Peter L.
Published: June 2012

Abstract
The recent demonstration that apoptosis of vascular smooth muscle cells (VSMCs) occurs in human atherosclerotic plaques suggests that VSMC apoptosis may promote plaque rupture and subsequent myocardial infarction. In culture, human plaque VSMCs show higher rates of apoptosis than VSMCs from normal vessels, although the mechanism of this effect is unknown. In earlier studies, we have shown that the tumor suppressor gene p53 regulates apoptosis of rat VSMCs after deregulated cell cycle control. We therefore analyzed p53 function in cultured VSMCs derived from human coronary plaques or the media of normal coronary arteries. VSMCs with reduced or increased p53 activity were created by infecting VSMCs with retroviruses containing a dominant-negative p53 minigene or a chimeric p53 protein (p53TMER), which could be activated pharmacologically. Basal p53 protein expression and transcriptional activity were similar in plaque and normal VSMCs, and suppression of p53 activity blocked growth arrest in response to DNA damage in both VSMC types. In contrast, suppression of p53 activity failed to block apoptosis of plaque or normal VSMCs in low- or high-serum conditions or after DNA damage. Furthermore, in plaque VSMCs, p53 overexpression induced apoptosis in all conditions tested and also induced growth arrest. p53-mediated apoptosis was independent of new gene transcription or protein synthesis but was suppressed by prior growth arrest of cells, indicating that growth status can regulate sensitivity to p53-mediated apoptosis. No effect of increased p53 activity was seen in normal VSMCs. We conclude that VSMCs from human plaques have an increased sensitivity to p53-mediated apoptosis compared with normal VSMCs. Our data also suggest that the mechanism of p53-mediated apoptosis of plaque VSMCs may be distinct from that inducing growth arrest.
Involvement of Transcriptional and Posttranscriptional Mechanisms in Cardiac Overload–Induced Increase of B-Type Natriuretic Peptide Gene Expression
Authors: Magga Jarkko , Vuolteenaho Olli , Tokola Heikki , Marttila Minna , Ruskoaho Heikki
Published: June 2012

Abstract
The induction of atrial and ventricular B-type natriuretic peptide (BNP) gene expression is one of the earliest events occurring during hemodynamic overload. To examine the molecular mechanisms for increased BNP gene expression during cardiac overload, we studied the induction of the BNP gene expression compared with that of atrial natriuretic peptide (ANP) in a modified perfused rat heart preparation. An increase in right atrial pressure of 5 mm Hg resulted in a 1.4-fold (
P
<.05) and 2.2-fold (
P
<.01) increase in BNP mRNA levels after 1 and 2 hours, respectively, whereas ANP mRNA levels remained unchanged. Stretching for up to 2 hours also significantly increased right atrial immunoreactive BNP (ir-BNP) levels (from 15.8±2.2 to 20.1±1.2 ng/mg,
P
<.05). Actinomycin D (10 μg/mL), a transcriptional inhibitor, completely inhibited the stretch-induced increase in atrial BNP mRNA levels at 1 hour (
P
<.05) and 2 hours (
P
<.001), whereas a protein synthesis inhibitor, cycloheximide (90 μg/mL), had no effect on basal or direct mechanical stretch-induced increase in right atrial BNP mRNA levels. Furthermore, we examined the role of tyrosine kinase and protein kinase C activities in acute mechanical stretch–induced increase in BNP synthesis. Tyrosine kinase inhibitors lavendustin A (1 μmol/L) and tyrphostin A25 (3 μmol/L) and protein kinase C inhibitors staurosporine (30 nmol/L) and chelerythrine (1 μmol/L) prevented the stretch-induced increase in right atrial ir-BNP concentrations at 2 hours. In addition, chelerythrine inhibited the increase of right atrial BNP mRNA levels stimulated by cardiac overload. These results demonstrate that the early increase of BNP mRNA levels by mechanical stretch results from increased transcriptional activation and is independent of protein synthesis. Our results also suggest that protein kinase C and tyrosine kinases activities may be involved in coupling cardiac overload to alterations in atrial BNP synthesis.
Coagulation Factor Xa Induces Endothelium-Dependent Relaxations in Rat Aorta
Authors: Schaeffer Paul , Mares Anne-Marie , Dol Frédérique , Bono Françoise , Herbert Jean-Marc
Published: June 2012

Abstract
The relaxing effect of coagulation factor Xa on phenylephrine-contracted rat aortic rings was compared with the effect of thrombin and trypsin. All three proteases induced a dose-dependent relaxation in the presence of an intact endothelium. EC
50
values were 3±1, 24±9, and 16±1 nmol/L for thrombin, trypsin, and factor Xa, respectively. Whereas thrombin induced rapid relaxations followed by partial recontraction, trypsin and factor Xa induced slower sustained effects. Factor Xa–induced relaxations were not affected by hirudin at high concentrations (1 μmol/L) but were abolished by DX9065A, a specific inhibitor of the catalytic activity of factor Xa. Furthermore, no relaxations to factor Xa could be elicited in the presence of the NO synthase inhibitor
N
ω
-nitro-
l
-arginine methyl ester (100 μmol/L), whereas relaxations were not altered in the presence of the inactive enantiomer
N
ω
-nitro-
d
-arginine methyl ester (100 μmol/L). Addition of factor Xa together with thrombin induced relaxations that were larger than those induced by thrombin alone, whereas factor Xa had no additional effects on trypsin-induced relaxations. Furthermore, factor Xa relaxed thrombin-desensitized aortic rings but was ineffective in trypsin-desensitized tissues. These data suggest that factor Xa acts on a cleavable endothelial receptor that induces NO release, resulting in the relaxation of precontracted rat aortic rings. Factor Xa does not act through endothelial thrombin receptors but may activate another cleavable trypsin-sensitive receptor.
Nitric Oxide–Independent Dilation of Conductance Coronary Arteries to Acetylcholine in Conscious Dogs
Authors: Ming Zhi , Parent Robert , Lavallée Michel
Published: June 2012

Abstract
NO and prostacyclin formation cannot entirely account for receptor-operated endothelium-dependent dilation of coronary vessels, since vasodilator responses are not completely suppressed by inhibitors of these agents. Therefore, we considered that another factor, such as an endothelium-derived hyperpolarizing factor described in vitro, may participate in NO- and prostacyclin-independent coronary dilator responses. In conscious instrumented dogs, intracoronary acetylcholine (ACh, 30.0 ng · kg
−1
· min
−1
) increased the external epicardial coronary diameter (CD) by 0.18±0.03 mm (from 3.44±0.11 mm) when increases in coronary blood flow (CBF) were prevented and increased the CD by 0.20±0.05 when CBF was allowed to increase. After the administration of intracoronary
N
ω
-nitro-
l
-arginine methyl ester (L-NAME), CBF responses to ACh were abolished, but CD responses (0.23±0.05 from 3.22±0.09 mm) were maintained. Blockade of NO formation was confirmed by reduced CD baselines and blunted flow-dependent CD responses caused by adenosine and transient coronary artery occlusions after L-NAME administration. ACh-induced CD increases resistant to L-NAME and indomethacin were reduced after the administration of intracoronary quinacrine, an inhibitor of phospholipase A
2
, or proadifen, an inhibitor of cytochrome P-450. Quinacrine or proadifen alone (without L-NAME) did not alter CD responses to ACh, but L-NAME given after proadifen blunted ACh-induced increases in CD. The increases in CD caused by arachidonic acid given after L-NAME+indomethacin were antagonized by proadifen but not altered by quinacrine. Thus, a cytochrome P-450 metabolite of arachidonic acid accounts for L-NAME–resistant and indomethacin-resistant dilation of large epicardial coronary arteries to ACh. Conversely, NO formation is the dominant mechanism of ACh-induced dilation after blockade of the cytochrome P-450 pathway.
Isoform Transitions of the Myosin Binding Protein C Family in Developing Human and Mouse Muscles
Authors: Gautel Mathias , Fürst Dieter O. , Cocco Alessandra , Schiaffino Stefano
Published: June 2012

Abstract
—Mutations in the gene for the cardiac isoform of myosin binding protein C (MyBP-C) have been identified as the cause of chromosome 11–associated autosomal-dominant familial hypertrophic cardiomyopathy (FHC). Most mutations produce a truncated polypeptide that lacks the sarcomeric binding region. We have now investigated the expression pattern of the cardiac and skeletal isoforms of cMyBP-C in mice and humans by in situ hybridization and immunofluorescence microscopy using specific antibodies and probes. We demonstrate that the cardiac isoform is expressed only in cardiac muscle throughout development. The slow and fast isoforms of MyBP-C remain specific for skeletal muscle, where they can be coexpressed. Immunological evidence also suggests that an embryonic isoform of MyBP-C precedes the expression of slow MyBP-C in developing skeletal muscle. This suggests that transcomplementation of MyBP-C isoforms is possible in skeletal but not cardiac muscle.
Spatial Relationship of the C-Terminal Domains of Dystrophin and β-Dystroglycan in Cardiac Muscle Support a Direct Molecular Interaction at the Plasma Membrane Interface
Authors: Stevenson Shirley , Rothery Stephen , Cullen Michael J. , Severs Nicholas J.
Published: June 2012

Abstract
—Dystrophin and β-dystroglycan are components of a complex of at least nine proteins (the dystrophin-glycoprotein complex) that physically link the membrane cytoskeleton in skeletal and cardiac muscle, through the plasma membrane, to the extracellular matrix. Mutations in the dystrophin gene, which result in an absence or a quantitative or qualitative alteration of dystrophin, cause a subset of familial dilated cardiomyopathies as well as Duchenne and Becker muscular dystrophy. Biochemical studies on isolated skeletal muscle molecules indicate that dystrophin is bound to the glycoprotein complex via β-dystroglycan, with the C-terminus of β-dystroglycan binding to the cysteine-rich domain and first half of the C-terminal domain of dystrophin. Ultrastructural labeling has demonstrated a close spatial relationship between dystrophin and β-dystroglycan in intact skeletal muscle, but no previous ultrastructural labeling studies have examined the dystrophin/β-dystroglycan interaction in cardiac muscle. In the present study, we have applied complementary immunoconfocal microscopy and double immunogold fracture-label, a freeze-fracture cytochemical technique that allows high-resolution visualization of labeled membrane components in thin section and in platinum-carbon replicas, to investigate the spatial relationship between dystrophin and β-dystroglycan in rat cardiac muscle. When immunogold probes of two different sizes for the two proteins were used, “doublets” representing side-by-side antibody labeling were demonstrated in en face views at the level of the plasma membrane. The results support the conclusions that dystrophin and β-dystroglycan directly interact at the cytoplasmic face of the rat cardiac muscle plasma membrane.
Epicardium-Derived Cells Contribute a Novel Population to the Myocardial Wall and the Atrioventricular Cushions
Authors: Gittenberger-de Groot Adriana C. , Vrancken Peeters Mark-Paul F.M. , Mentink Monica M.T. , Gourdie Robert G. , Poelmann Robert E.
Published: June 2012

Abstract
—The epicardium and dorsal mesocardium are known to be the source of structures that form the wall of the coronary vessels. Because mouse knockout studies have shown that proper epicardial formation is also essential for myocardial development, we have studied in detail the migration and differentiation of epicardium-derived cells (EPDCs) within the developing heart. We constructed chicken-quail chimeras by grafting the quail epicardial organ, including a piece of primordial liver, at essentially stages 16 and 17. The embryos were studied at stages 25 to 43. To detect quail-derived EPDCs, an anti-quail nucleus antibody was used in combination with several differentiation markers, eg, for muscle actin, for vascular smooth muscle cells, for procollagen-I, for quail endothelium, and for Purkinje fibers. At stages 25 to 31, EPDCs are encountered in the myocardial wall and the subendocardial region. The latter deposition is spatially facilitated as the endocardium protrudes through transient discontinuities in the myocardium to contact the subepicardial layer. Later on, at stages 32 to 43, EPDCs invaded, by way of the atrioventricular sulcus, the atrioventricular cushion tissue. The localization is apparent at the interface with the myocardium, as well as subendocardially, but never within the endocardial lining. The origin of endothelium, smooth muscle cells, and fibroblasts of the coronary vessel wall from the epicardial graft were confirmed in accordance with already published data. The functional role of the novel EPDCs in the subendocardium, myocardium, and atrioventricular cushions remains to be investigated. A close positional relationship is found with the differentiating Purkinje fibers. Furthermore, a regulatory role is postulated in the process of endocardial-mesenchymal transformation. The ultimate fate of EPDCs seems to be a cardiac fibroblast cell line involved in the formation of the fibrous heart skeleton.
Purkinje-Muscle Reentry as a Mechanism of Polymorphic Ventricular Arrhythmias in a 3-Dimensional Model of the Ventricles
Authors: Berenfeld Omer , Jalife José
Published: June 2012

Abstract
—Multiple electrode mapping of the ventricles during complex tachyarrhythmias has revealed focal subendocardial activation whose mechanism remains unexplained. We hypothesized that reentry involving the Purkinje-muscle junctions (PMJs) may be a mechanism for such focal excitations. We have constructed an anatomically appropriate computerized 3-dimensional model of the mammalian ventricles that includes the Purkinje conduction system and 214 PMJs distributed throughout the endocardium. Isochronal maps during normal excitation, as well as during right or left bundle branch block, resembled experimental measurements and compared well with isochronal maps of propagation in the human heart. Activity observed at both sides of a PMJ in the model showed that propagation from Purkinje fibers to muscle was slower than in the opposite direction. Under these realistic and normal conditions, the evolution of reentrant activity involving muscle and the Purkinje network was simulated. The reentry pattern was independent of the initiation site. It evolved with drifting epicardial breakthroughs and transformed on the endocardium from focal activity to figure-of-8 reentry. In addition, the ECG amplitude undulated during the evolution, and decrease in the cycle period, apparent wavelength, and propagation velocity were observed. Finally, the reentry was terminated if the Purkinje system was disconnected from the muscle before it reached a relative steady state. The simulation results suggest the following: (1) Epicardial breakthroughs and endocardial focal activity may originate at the PMJs. (2) The ECG amplitude may decrease as the reentry stabilizes and the excitation wavelength decreases. (3) The Purkinje system may have a double role in the evolution of reentry: first, it is essential to the reentry at the initial stage; second, it may lead to the establishment of intramyocardial reentry, at which time the Purkinje system becomes irrelevant.
Plasminogen Activator Expression in Rat Arterial Smooth Muscle Cells Depends on Their Phenotype and Is Modulated by Cytokines
Authors: Bochaton-Piallat Marie-Luce , Gabbiani Giulio , Pepper Michael S.
Published: June 2012

Abstract
—Cultured rat aortic smooth muscle cells (SMCs) exhibit at least 2 phenotypic variants: (1) a spindle-shaped phenotype, obtained from normal adult media, and (2) an epithelioid phenotype, obtained from intimal thickening 15 days after endothelial injury. Both phenotypes can be cloned from each location, with normal media yielding a majority of spindle-shaped clones and intimal thickening yielding a majority of epithelioid clones. These findings suggest that intimal thickening develops essentially from a subpopulation of medial SMCs exhibiting epithelioid features in vitro. Using zymographic and Northern blot analyses, we have studied plasminogen activator (PA) expression by these SMCs. Our results show that epithelioid SMCs, cultured as whole SMC populations or as clones, display higher PA activity than do spindle-shaped SMCs, irrespective of their origin. This is mainly due to differences in the expression of tissue PA and, to a lesser extent, urokinase PA and is accompanied by a decrease in PA inhibitor 1. Tissue PA activity is increased by basic fibroblast growth factor and platelet-derived growth factor-BB, particularly in epithelioid SMCs. Taken together, these results indicate that SMCs are heterogeneous with respect to their proteolytic profile, at least as far as the PA system is concerned. Proteolytic activity of the different SMC populations is modulated by cytokines that play a role in intimal thickening. Our results are in agreement with the suggestion that epithelioid SMCs are mainly responsible for intimal thickening.