bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin-like Growth Factor-1 but Not Growth Hormone Augments Mammalian Myocardial Contractility by Sensitizing the Myofilament to Ca
2+
Through a Wortmannin-Sensitive Pathway
Authors: Cittadini Antonio , Ishiguro Yoshiki , Strömer Hinrik , Spindler Matthias , Moses Alan C. , Clark Ross , Douglas Pamela S. , Ingwall Joanne S. , Morgan James P.
Published: June 2012

Abstract
—A growing body of evidence has been accumulated recently suggesting that growth hormone (GH) and insulin-like growth factor-1 (IGF-1) affect cardiac function, but their mechanism(s) of action is unclear. In the present study, GH and IGF-1 were administered to isolated isovolumic aequorin-loaded rat whole hearts and ferret papillary muscles. Although GH had no effect on the indices of cardiac function, IGF-1 increased isovolumic developed pressure by 24% above baseline. The aequorin transients were abbreviated and demonstrated decreased amplitude. The positive inotropic effects of IGF-1 were not associated with increased intracellular Ca
2+
availability to the contractile machinery but to a significant increase of myofilament Ca
2+
sensitivity. Accordingly, the Ca
2+
-force relationship obtained under steady-state conditions in tetanized muscle was shifted significantly to the left (EC
50
, 0.44±0.02 versus 0.52±0.03 μmol/L with and without IGF-1 in the perfusate, respectively;
P
<0.05); maximal Ca
2+
-activated tetanic pressure was increased significantly by 12% (211±3 versus 235±2 mm Hg in controls and IGF-1–treated hearts, respectively;
P
<0.01). The positive inotropic actions of IGF-1 were not associated with changes in either pH
i
or high-energy phosphate content, as assessed by
31
P nuclear magnetic resonance spectroscopy, and were blocked by the phosphatidylinositol 3-kinase inhibitor wortmannin. Concomitant administration of IGF binding protein-3 blocked IGF-1–positive inotropic action in ferret papillary muscles. In conclusion, IGF-1 is an endogenous peptide that through a wortmannin-sensitive pathway displays distinct positive inotropic properties by sensitizing the myofilaments to Ca
2+
without increasing myocyte [Ca
2+
]
i
.
Relation Between Crossbridge Structure and Actomyosin ATPase Activity in Rat Heart
Authors: Weisberg Andrea , Winegrad Saul
Published: June 2012

Abstract
—Cardiac myofilaments contain proteins that regulate the interaction between actin and myosin. In the thick filament, there are several proteins that may contribute to the regulation of the contraction. The myosin binding protein C, or C protein, has 4 sites that can be phosphorylated by a Ca
2+
-calmodulin–controlled kinase, protein kinase A or protein kinase C. Using electron microscopy and optical diffraction, we examined the structure of thick filaments isolated from rat ventricles with either the α or β isoform of myosin heavy chain (MHC) and the effect of specific phosphorylation of C protein on the structure. In thick filaments with α-MHC, crossbridges were clearly visible. Phosphorylation of C protein by protein kinase A extended the crossbridges from the backbone of the filament, changed their orientation, increased the degree of order of the crossbridges, and decreased the flexibility of the crossbridges. Crossbridges in filaments with β-MHC were less ordered and apparently more flexible. Phosphorylation of C protein in β-MHC–containing filaments did not extend the crossbridges and did not alter degree of order or flexibility. The relative flexibility of the crossbridges inferred from the optical diffraction pattern correlated well with the rate of ATP hydrolysis by actomyosin. These results suggest that (1) crossbridge flexibility is an important parameter in setting the rate of crossbridge cycling, and (2) C protein–mediated control of the position and flexibility of crossbridges may regulate actomyosin ATPase activity by modifying the kinetics of crossbridge cycling.
Sox4-Deficiency Syndrome in Mice Is an Animal Model for Common Trunk
Authors: Ya Jing , Schilham Marco W. , de Boer Piet A. J. , Moorman Antoon F. M. , Clevers Hans , Lamers Wouter H.
Published: June 2012

Abstract
—Embryonic mice lacking functional Sox4 transcription factor die from cardiac failure at embryonic day (ED) 14. Heart morphogenesis in these embryos was analyzed in hematoxylin-azophlochsin or immunohistochemically stained, 3-dimensionally reconstructed serial sections between ED12 and ED14. Although Sox4 is expressed in the endocardially derived tissue of both the outflow tract and atrioventricular canal, Sox4-deficient hearts only suffer from defective transformation of the endocardial ridges into semilunar valves and from lack of fusion of these ridges, usually resulting in common trunk, although the least affected hearts should be classified as having a large infundibular septal defect. The more serious cases are, in addition, characterized by an abnormal number and position of the semilunar valve-leaflet anlagen, a configuration of the ridges typical for transposition of the great arteries (with linear rather than spiral course of both ridges and posterior position of the pulmonary trunk at the level of the valve), and variable size of the aorta relative to the pulmonary trunk. The coronary arteries always originated from the aorta, irrespective of its position relative to the pulmonary trunk. The restriction of the malformations to the arterial pole implies that the interaction between the endocardially derived tissue of the outflow tract and the neural crest–derived myofibroblasts determines proper development of the arterial pole.
Peroxisome Proliferator-Activated Receptor Gamma Activators Inhibit Gene Expression and Migration in Human Vascular Smooth Muscle Cells
Authors: Marx Nikolaus , Schönbeck Uwe , Lazar Mitchell A. , Libby Peter , Plutzky Jorge
Published: June 2012

Abstract
—Migration of vascular smooth muscle cells (VSMCs) plays an important role in atherogenesis and restenosis after arterial interventions. The expression of matrix metalloproteinases (MMPs), particularly MMP-9, contributes to VSMC migration. This process requires degradation of basal laminae and other components of the arterial extracellular matrix. Peroxisome proliferator-activated receptors (PPARs), members of the nuclear receptor family, regulate gene expression after activation by various ligands. Recent studies have suggested opposing effects of PPAR gamma (PPARγ) activation on atherogenesis. The present study tested the hypotheses that human VSMCs express PPAR alpha (PPARα) and PPARγ and that PPAR agonists in VSMCs modulate MMP-9 expression and activity, as well as VSMC migration. Human VSMCs expressed PPARα and PPARγ mRNA and protein. Treatment of VSMCs with the PPARγ ligands troglitazone and the naturally occurring 15-deoxy-
Δ12,14
-prostaglandin J
2
(15d-PGJ
2
) decreased phorbol 12-myristate 13-acetate–induced MMP-9 mRNA and protein levels, as well as MMP-9 gelatinolytic activity in the supernatants in a concentration-dependent manner. Six different PPARα activators lacked such effects. Addition of prostaglandin F
2α
, known to limit PPARγ activity, diminished the MMP-9 inhibition seen with either troglitazone or 15d-PGJ
2
, further implicating PPARγ in these effects. Finally, troglitazone and 15d-PGJ
2
inhibited the platelet-derived growth factor-BB–induced migration of VSMCs in vitro in a concentration-dependent manner. PPARγ activation may regulate VSMC migration and expression and activity of MMP-9. Thus, PPARγ activation in VSMCs, via the antidiabetic agent troglitazone or naturally occurring ligands, may act to counterbalance other potentially proatherosclerotic PPARγ effects.
Reconstituted Human Cardiac K
ATP
Channels
Authors: Babenko Andrey P. , Gonzalez Gabriela , Aguilar-Bryan Lydia , Bryan Joseph
Published: June 2012

Abstract
—ATP-sensitive potassium (K
ATP
) channels in striated myocytes are heteromultimers of K
IR
6.2, a weak potassium inward rectifier, plus SUR2A, a low-affinity sulfonylurea receptor. We have cloned human K
IR
6.2 (huK
IR
6.2) and a huSUR2A that corresponds to the major, full-length splice variant identified by polymerase chain reaction analysis of human cardiac poly A
+
mRNA. ATP- and glibenclamide-sensitive K
+
channels were produced when both subunits were coexpressed in COSm6 and Chinese hamster ovary cells lacking endogenous K
ATP
channels, but not when huSUR2A or huK
IR
6.2 were transfected alone. Recombinant channels activated by metabolic inhibition in cell-attached configuration or in inside-out patches with ATP-free internal solution were compared with sarcolemmal K
ATP
channels in human ventricular cells. The single-channel conductance of ≈80 pS measured at −40 mV in quasi-symmetrical ≈150 mmol/L K
+
solutions, the intraburst kinetics that were dependent on K
+
driving force, and the weak inward rectification were indistinguishable for both channels. Similar to the native channels, huSUR2A/huK
IR
6.2 recombinant channels were inhibited by ATP at quasi-physiological free Mg
2+
(≈0.7 mmol/L) or in the absence of Mg
2+
, with an apparent IC
50
of ≈20 μmol/L and a pseudo-Hill coefficient of ≈1. They were “refreshed” by MgATP and stimulated by ADP in the presence of Mg
2+
when inhibited by ATP. The huSUR2A/huK
IR
6.2 channels were stimulated by cromakalim and pinacidil in the presence of ATP and Mg
2+
but were insensitive to diazoxide. The results suggest that reconstituted huSUR2A/huK
IR
6.2 channels represent K
ATP
channels in sarcolemma of human cardiomyocytes and are an adequate experimental model with which to examine structure-function relationships, molecular physiology, and pharmacology of these channels from human heart.
Reduced Reperfusion–Induced Ins(1,4,5)P
3
Generation and Arrhythmias in Hearts Expressing Constitutively Active α
1B
-Adrenergic Receptors
Authors: Harrison Sharon N. , Autelitano Dominic J. , Wang Bing Hui , Milano Carmelo , Du Xiao-Jun , Woodcock Elizabeth A.
Published: June 2012

Abstract
—Reperfusion of globally ischemic rat hearts causes the generation of inositol(1,4,5)
tris
phosphate [Ins(1,4,5)P
3
] and the initiation of arrhythmias. These responses are mediated by α
1
-adrenergic receptors (ARs), but the subtype of receptor involved has not been identified. Under normoxic conditions, hearts from transgenic animals expressing constitutively active α
1B
-ARs in heart (α
1B
-constitutively active mutant [CAM]) showed higher [
3
H] inositol phosphate responses to norepinephrine (2.3-fold) than hearts from nontransgenic animals (α
1B
-WT) (1.6-fold). α
1B
-WT hearts responded to 2 minutes of reperfusion after 20 minutes of global ischemia by generation of Ins(1,4,5)P
3
(5301±1310 to 11 413±1597 CPM/g tissue; mean±SEM; n=6;
P
<0.01 in [
3
H] labeling studies and 3.8±0.2 to 6.3±0.6 nmol/g by mass analysis, n=6;
P
<0.05). In contrast to findings in normoxia, hearts from α
1B
-CAM animals showed no Ins(1,4,5)P
3
response in early reperfusion. In parallel studies, α
1B
-WT hearts developed ventricular tachycardia and ventricular premature beats (VPB) during 5 minutes of reperfusion after 20 minutes of ischemia. The incidence of these arrhythmias was reduced in the α
1B
-CAM hearts (95% to 62% for VPB and 47% to 12% for ventricular tachycardia; both
P
<0.05). The resistance of the α
1B
-CAM hearts was not due to α
1B
-AR–mediated preconditioning, as the Ins(1,4,5)P
3
response to thrombin receptor activation during reperfusion was not different between the 2 groups. To investigate the possibility of reduced α
1A
-receptor activity in the α
1B
-CAM hearts, expression of the mRNA for α
1A
- and α
1B
-receptors was measured. α
1B
-WT hearts contained mRNA for both receptor subtypes, but the levels of α
1B
-receptor mRNA were 5-fold higher than α
1A
-receptor mRNA. α
1B
-CAM hearts contained very high levels of α
1B
-receptor mRNA (26-fold increase), but the expression of mRNA for the α
1A
-receptors (0.141±0.035 amol/μg RNA; mean±SEM; n=6) was reduced by 50% relative to α
1B
-WT controls (0.276±0.046 amol/μg RNA; n=6;
P
<0.01). The reduction in arrhythmogenic and Ins(1,4,5)P
3
responses in α
1B
-CAM hearts provides evidence that these response are not mediated by α
1B
-receptors.
Nicotine Enhances Presynaptic and Postsynaptic Glutamatergic Neurotransmission to Activate Cardiac Parasympathetic Neurons
Authors: Neff Robert A. , Humphrey John , Mihalevich Mark , Mendelowitz David
Published: June 2012

Abstract
—Although peripheral cholinergic neurotransmission has long been known to play a pivotal role in the control of heart rate and blood pressure, recent evidence has suggested that central cholinergic mechanisms may be involved in the genesis of hypertension, anxiety, cardiorespiratory control, and, in particular, the respiratory modulation of heart rate. Yet, the sites, mechanisms, and receptor subtypes involved in the action of nicotine within the central nervous system are controversial. The present study demonstrates that nicotine has at least 3 sites of action to increase the activity of vagal cardiac neurons. Nicotine, but not muscarinic agonists, activates postsynaptic receptors and a depolarizing inward current in vagal cardiac neurons studied with the perforated patch-clamp technique in a visualized brain stem slice. In addition, nicotine acts at different presynaptic and postsynaptic sites to facilitate glutamatergic neurotransmission. Presynaptic nicotinic receptors increase the frequency of transmitter release and are sensitive to block by α-bungarotoxin. Nicotine also elicits a previously undescribed augmentation of postsynaptic non-NMDA currents. The presynaptic and postsynaptic receptors may prove to be future targets in the search for agonists to increase vagal cardiac activity and reduce the fatality associated with cardiac hyperexcitability and for antagonists to reduce cardiac vagal activity in pathological conditions associated with abnormally low heart rates and cardiac function such as sudden infant death syndrome.
Neuronal Nitric Oxide Synthase Is Expressed in Rat Vascular Smooth Muscle Cells
Authors: Boulanger Chantal M. , Heymes Christophe , Benessiano Joëlle , Geske Robert S. , Lévy Bernard I. , Vanhoutte Paul M.
Published: June 2012

Abstract
—The nitric oxide synthase (NOS) inhibitor nitro-
l
-arginine augmented the contractions to angiotensin (Ang) II in carotid artery rings without endothelium from spontaneously hypertensive rats (SHR) but not normotensive Wistar-Kyoto rats, suggesting the possibility of nonendothelial NOS activity in SHR arteries. In SHR artery without endothelium, the potentiation of Ang II contraction by nitro-
l
-arginine was prevented by
l-
arginine, but not by
d-
arginine, and was observed also in the presence of oxyhemoglobin, monomethyl
-l
-arginine, and 7-nitroindazole, but not in the presence of aminoguanidine. In further support of NOS activation by Ang II in nonendothelial cells, Ang II but not acetylcholine stimulated cGMP levels by 2-fold in SHR arteries without endothelium; nitro-
l
-arginine decreased both basal and Ang II–stimulated cGMP levels. When NOS activity in SHR arteries was measured, no calcium-independent
l-
citrulline formation was detectable, while up to 47% of the total calcium-dependent NOS activity was present in nonendothelial cells. Expression of neuronal NOS was revealed in the media of SHR arteries by immunohistochemistry, Western blot, and reverse transcriptase–polymerase chain reaction. Expression of this NOS isoform was greater in SHR than in Wistar-Kyoto rat preparations. Finally, endothelial NOS was observed in the endothelium, but no detectable levels of inducible NOS were found in these tissues. These results demonstrate the expression of neuronal NOS in rat vascular smooth muscle cells and its activation on stimulation by Ang II in spontaneously hypertensive, but not normotensive, animals.
Stress (Heat Shock) Proteins
Authors: Benjamin Ivor J. , McMillan D. Randy
Published: June 2012

Abstract
—How a cell responds to stress is a central problem in cardiovascular biology. Diverse physiological stresses (eg, heat, hemodynamics, mutant proteins, and oxidative injury) produce multiple changes in a cell that ultimately affect protein structures and function. Cells from different phyla initiate a cascade of events that engage essential proteins, the molecular chaperones, in decisions to repair or degrade damaged proteins as a defense strategy to ensure survival. Accumulative evidence indicates that molecular chaperones such as the heat shock family of stress proteins (HSPs) actively participate in an array of cellular processes, including cytoprotection. The versatility of the ubiquitous HSP family is further enhanced by stress-inducible regulatory networks, both at the transcriptional and posttranscriptional levels. In the present review, we discuss the regulation and function of HSP chaperones and their clinical significance in conditions such as cardiac hypertrophy, vascular wall injury, cardiac surgery, ischemic preconditioning, aging, and, conceivably, mutations in genes encoding contractile proteins and ion channels.
Novel LQT-3 Mutation Affects Na
+
Channel Activity Through Interactions Between α- and β
1
-Subunits
Authors: An R. H. , Wang X. L. , Kerem B. , Benhorin J. , Medina A. , Goldmit M. , Kass R. S.
Published: June 2012

Abstract
—The congenital long-QT syndrome (LQT), an inherited cardiac arrhythmia characterized in part by prolonged ventricular repolarization, has been linked to 5 loci, 4 of which have been shown to harbor genes that encode ion channels. Previously studied LQT-3 mutations of SCN5A (or hH1), the gene that encodes the human Na
+
channel α-subunit, have been shown to encode voltage-gated Na
+
channels that reopen during prolonged depolarization and hence directly contribute to the disease phenotype: delayed repolarization. Here, we report the functional consequences of a novel SCN5A mutation discovered in an extended LQT family. The mutation, a single A→G base substitution at nucleotide 5519 of the SCN5A cDNA, is expected to cause a nonconservative change from an aspartate to a glycine at position 1790 (D1790G) of the SCN5A gene product. We investigated ion channel activity in human embryonic kidney (HEK 293) cells transiently transfected with wild-type (hH1) or mutant (D1790G) cDNA alone or in combination with cDNA encoding the human Na
+
channel β
1
-subunit (hβ
1
) using whole-cell patch-clamp procedures. Heteromeric channels formed by coexpression of α- and β
1
-subunits are affected: steady-state inactivation is shifted by –16 mV, but there is no D1790G-induced sustained inward current. This effect is independent of the β
1
-subunit isoform. We find no significant effect of D1790G on the biophysical properties of monomeric α- (hH1) channels. We conclude that the effects of the novel LQT-3 mutation on inactivation of heteromeric channels are due to D1790G-induced changes in α- and β
1
-interactions.
Neuropeptide Y
Authors: Zukowska-Grojec Zofia , Karwatowska-Prokopczuk Ewa , Rose Wesley , Rone Janice , Movafagh Sharareh , Ji Hong , Yeh Yunyun , Chen Wen-Tien , Kleinman Hynda K. , Grouzmann Eric , Grant Derrick S.
Published: June 2012

Abstract
—Sympathetic nerves have long been suspected of trophic activity, but the nature of their angiogenic factor has not been determined. Neuropeptide Y (NPY), a sympathetic cotransmitter, is the most abundant peptide in the heart and the brain. It is released during nerve activation and ischemia and causes vasoconstriction and smooth muscle cell proliferation. Here we report the first evidence that NPY is angiogenic. At low physiological concentrations, in vitro, it promotes vessel sprouting and adhesion, migration, proliferation, and capillary tube formation by human endothelial cells. In vivo, in a murine angiogenic assay, NPY is angiogenic and is as potent as a basic fibroblast growth factor. The NPY action is specific and is mediated by Y1 and Y2 receptors. The expression of both receptors is upregulated during cell growth; however, Y2 appears to be the main NPY angiogenic receptor. Its upregulation parallels the NPY-induced capillary tube formation on reconstituted basement membrane (Matrigel); the Y2 agonist mimics the tube-forming activity of NPY, whereas the Y2 antagonist blocks it. Endothelium contains not only NPY receptors but also peptide itself, its mRNA, and the “NPY-converting enzyme” dipeptidyl peptidase IV (both protein and mRNA), which terminates the Y1 activity of NPY and cleaves the Tyr
1
-Pro
2
from NPY to form an angiogenic Y2 agonist, NPY
3–36
. Endothelium is thus not only the site of action of NPY but also the origin of the autocrine NPY system, which, together with the sympathetic nerves, may be important in angiogenesis during tissue development and repair.
Increased Expression of Estrogen Receptor-β mRNA in Male Blood Vessels After Vascular Injury
Authors: Lindner Volkhard , Kim Sung K. , Karas Richard H. , Kuiper George G. J. M. , Gustafsson Jan-Åke , Mendelsohn Michael E.
Published: June 2012

Abstract
—Estrogen exerts direct effects on vascular endothelial and smooth muscle cells that are important for vascular protection. Estrogen receptor-α (ERα) is expressed in vascular cells from males and females and may mediate some of the effects of estrogen on vascular tissue. However, we recently found that estrogen is able to protect against vascular injury in ovariectomized female ERα knockout mice. These mice express the newly described estrogen receptor-β (ERβ) in their aortas, suggesting that ERβ may also mediate some of the direct effects of estrogen on the vasculature. In this study, the level of expression of ERα and ERβ mRNA in male rat aortas was examined before and after vascular injury using en face (Häutchen) preparations and in situ hybridization. Little or no change in ERα expression was observed after vascular injury in either vascular endothelial or smooth muscle cells at any time point. In contrast, ERβ mRNA was found to be expressed markedly after balloon injury. In endothelial cells, ERβ was increased by 2 days after injury, and high levels of expression were maintained at 8 and 14 days. Furthermore, ERβ expression was high in luminal smooth muscle cells at 8 and 14 days after injury and had decreased to low levels by 28 days after injury. These data demonstrate the presence of ERβ in male vascular tissues and the induction of ERβ mRNA expression after vascular injury, supporting a role for ERβ in the direct vascular effects of estrogen.
Fluid Shear Stress Transcriptionally Induces Lectin-like Oxidized LDL Receptor-1 in Vascular Endothelial Cells
Authors: Murase Takatoshi , Kume Noriaki , Korenaga Risa , Ando Joji , Sawamura Tatsuya , Masaki Tomoh , Kita Toru
Published: June 2012

Abstract
—Fluid shear stress has been shown to modulate various endothelial functions, including gene expression. In this study, we examined the effect of fluid shear stress on the expression of lectin-like oxidized LDL receptor-1 (LOX-1), a novel receptor for atherogenic oxidized LDL in cultured bovine aortic endothelial cells (BAECs). Exposure of BAECs to the physiological range of shear stress (1 to 15 dyne/cm
2
) upregulated LOX-1 protein and mRNA in a time-dependent fashion. LOX-1 mRNA levels peaked at 4 hours, and LOX-1 protein levels peaked at 8 hours. Inhibition of de novo RNA synthesis by actinomycin D totally abolished shear stress–induced LOX-1 mRNA expression. Furthermore, nuclear runoff assay showed that shear stress directly stimulates transcription of the LOX-1 gene. Chelation of intracellular Ca
2+
with quin 2-AM completely reduced shear stress–induced LOX-1 mRNA expression; furthermore, the treatment of BAECs with ionomycin upregulated LOX-1 mRNA levels in a dose-dependent manner. Taken together, physiological levels of fluid shear stress can regulate LOX-1 expression by a mechanism dependent on intracellular Ca
2+
mobilization. Inducible expression of LOX-1 by fluid mechanics may play a role in localized expression of LOX-1 and atherosclerotic lesion formation in vivo.
Troponin and Tropomyosin
Authors: Solaro R. John , Rarick Helen M.
Published: June 2012

Abstract
—We present a current perception of the regulation of activation of cardiac myofilaments with emphasis on troponin (Tn) and tropomyosin (Tm). Activation involves both a Ca
2+
-regulated molecular switch and a potentiated state, dependent on feedback effects of force-generating crossbridges. Recent developments in the elucidation of the structure and arrangement of the myofilament proteins offer insights into the molecular interactions that constitute the switching and potentiating mechanisms. Transgenic mice overexpressing myofilament proteins, in vitro studies of mutant myofilament proteins, multidimensional multinuclear nuclear magnetic resonance, and fluorescence resonance energy transfer offer important approaches to understanding the molecular signaling processes. These studies reveal special features of the cardiac myofilament proteins that appear specialized for the unique functions of the heart. An important aspect of these special features is their role in mechanical, chemical, and neurohumoral coupling processes that tune myofilament activation to hemodynamics and beating frequency. Understanding these processes has become essential to understanding cardiac pathologies such as heart failure, ischemia and reperfusion injury, stunning, and familial hypertrophic cardiac myopathies.
Regulation of Cardiomyocyte Apoptotic Signaling by Insulin-like Growth Factor I
Authors: Wang Lei , Ma Weiqiong , Markovich Rachelle , Chen Jaw-Wen , Wang Ping H.
Published: June 2012

Abstract
—Apoptosis is regulated by specific intracellular signaling pathways. The development of cardiomyopathy involves the apoptosis of cardiomyocytes; however, the details of their apoptotic signaling are not yet known. Insulin-like growth factor I (IGF I) is an important survival growth factor for myocardium and other tissues, but the effects of IGF I on apoptotic signaling remain largely unknown. To study apoptotic signaling pathways in cardiomyocytes and to understand IGF I actions on the apoptotic signaling of cardiac muscle cells, we have defined the effects of IGF I on Bcl-2, Bax, caspase 3, DNA fragmentation, and cell survival in primary cardiomyocytes. Compared with Bax levels, the levels of Bcl-2 were found to be quite low in these cells. Serum withdrawal and doxorubicin reduced cell viability, increased fragmentation of DNA, increased cellular contents of Bax, and activated caspase 3. IGF I enhanced cell viability, suppressed DNA fragmentation, attenuated Bax induction, and suppressed caspase 3 activation. The levels of Bcl-2–associated Bax were increased after serum withdrawal and incubation with doxorubicin and were reduced by IGF I. Thus, cardiomyocyte apoptosis induced by serum withdrawal and doxorubicin likely results, in part, from the induction of Bax and activation of caspase 3, but IGF I may inhibit cardiomyocyte apoptosis by attenuating Bax induction and caspase 3 activation. These findings provide new insight into the mechanisms of cardiomyocytes apoptosis and may help elucidate how IGF I modulates apoptotic signaling in cardiac muscle.
Critical Role of AT
1
Receptor Expression After Ischemia/Reperfusion in Isolated Rat Hearts
Authors: Yang B. C. , Phillips M. I. , Zhang Y. C. , Kimura B. , Shen L. P. , Mehta P. , Mehta J. L.
Published: June 2012

Abstract
—To examine the relevance of angiotensin II type 1 receptor (AT
1
R) expression in the determination of myocardial function after ischemia/reperfusion, Sprague-Dawley rats were treated intravenously with antisense oligodeoxynucleotides (AS-ODNs) directed at AT
1
R mRNA (100 μg/rat, n=9) or scrambled antisense oligodeoxynucleotides (Scr-ODNs, 100 μg/rat, n=6). Both AS-ODNs and Scr-ODNs were given along with 300 μg/rat of liposome DOTAP/DOPE, a positive electron carrier (wt:wt=1:1). The hearts from AS-ODN– or Scr-ODN–treated rats were excised 24 hours later, perfused in vitro, and subjected to 25 minutes of global ischemia followed by 30 minutes of reperfusion. Parallel groups of rats were given the specific AT
1
R antagonist losartan (10 mg/kg IV, n=6) or saline (n=7) 4 to 6 hours before excising the hearts. Ischemia/reperfusion resulted in a significant increase in myocardial AT
1
R expression (autoradiography and binding assay) and myocardial dysfunction, indicated by increases in coronary perfusion pressure and left ventricular end-diastolic pressure and a decrease in developed left ventricular pressure (all
P
<0.01 versus baseline) in the saline-treated group. AT
1
R protein and mRNA levels also increased in ischemic/reperfused myocardial tissues. Administration of AS-ODNs or losartan, but not Scr-ODNs, preserved myocardial function and blocked the increased AT
1
R binding after ischemia/reperfusion (both
P
<0.01). Myocardial AT
1
R mRNA levels were not affected by either AS-ODNs or losartan, and the AT
1
R protein levels were significantly reduced by AS-ODN, but not losartan, treatment. Plasma angiotensin II levels increased after administration of losartan but not after administration of AS-ODNs. These observations imply a critical role of AT
1
R upregulation in determining myocardial function immediately after ischemia/reperfusion. AS-ODNs to AT
1
R mRNA may be more beneficial than losartan, because losartan does not affect the plasma angiotensin II level. The sustained increase in AT
1
R mRNA, but diminished protein expression, in rat hearts treated with AS-ODNs suggests that AS-ODNs block AT
1
R at the translational level.
Thyroid Hormone–Induced Alterations in Phospholamban-Deficient Mouse Hearts
Authors: Kiss Eva , Brittsan Angie G. , Edes Istvan , Grupp Ingrid L. , Grupp Gunter , Kranias Evangelia G.
Published: June 2012

Abstract
—Alterations in the expression levels of the sarcoplasmic reticulum (SR) Ca
2+
-ATPase and its regulator, phospholamban, have been implicated in the effects of thyroxine hormone on cardiac function. To determine the role of phospholamban in these effects, hypothyroidism and hyperthyroidism were induced in phospholamban-deficient mice and their isogenic wild types. Hypothyroidism resulted in significant decreases of left ventricular contractility, which could be moderately stimulated by increases in preload or afterload, in both phospholamban-deficient and wild-type mice. However, the basal contractile parameters in hypothyroid phospholamban-deficient hearts were at least as high as those exhibited by hyperthyroid wild-type hearts. In hyperthyroidism, there was no further enhancement of the hyperdynamic contractile parameters in phospholamban-deficient hearts, although the wild-type hearts exhibited significantly increased contractile function compared with their respective euthyroid groups. Furthermore, increases in preload or afterload did not enhance contractility in either phospholamban-deficient or wild-type hyperthyroid hearts. Examination of the relative tissue levels of cardiac SR Ca
2+
-ATPase revealed increases in hyperthyroidism and decreases in hypothyroidism compared with euthyroidism, and these changes were similar between phospholamban-deficient and wild-type hearts. An opposite trend was observed for phospholamban expression levels in the wild-type group, which were depressed in hyperthyroid hearts but increased in hypothyroid hearts. These findings indicate that (1) thyroid hormones induce similar changes in the cardiac SR Ca
2+
-ATPase levels in either the presence or absence of phospholamban, (2) the thyroxine-induced increases in SR Ca
2+
-ATPase levels are not associated with any further stimulation of the hyperdynamic cardiac function in phospholamban-deficient mice, and (3) the decreased contractile parameters in hypothyroid phospholamban-deficient hearts associated with decreases in SR Ca
2+
-ATPase levels and myosin heavy chain isoform switches are at least as high as those of the stimulated hyperthyroid wild-type hearts. Thus, alterations in the phospholamban level or its activity may be a critical determinant of the contractile responses to altered thyroid states in the mammalian heart.
Rapid Turnover of Connexin43 in the Adult Rat Heart
Authors: Beardslee Michael A. , Laing James G. , Beyer Eric C. , Saffitz Jeffrey E.
Published: June 2012

Abstract
—Remodeling of the distribution of gap junctions is an important feature of anatomic substrates of arrhythmias in patients with healed myocardial infarcts. Mechanisms underlying this process are poorly understood but probably involve changes in gap junction protein (connexin) synthesis, assembly into channels, and degradation. The half-life of the principal cardiac gap junction protein, connexin43 (Cx43), is only 1.5 to 2 hours in primary cultures of neonatal myocytes, but it is unknown whether rapid turnover of Cx43 occurs in the adult heart or is unique to disaggregated neonatal myocytes that are actively reestablishing connections in vitro. To characterize connexin turnover dynamics in the adult heart and to elucidate its potential role in remodeling of gap junctions, we measured Cx43 turnover kinetics and characterized the proteolytic pathways involved in Cx43 degradation in isolated perfused adult rat hearts. Hearts were labeled for 40 minutes with Krebs-Henseleit buffer containing [
35
S]methionine, and then chase perfusions were performed with nonradioactive buffer for 0, 60, 120, and 240 minutes. Quantitative immunoprecipitation assays of Cx43 radioactivity in 4 hearts at each time point yielded a monoexponential decay curve indicating a Cx43 half-life of 1.3 hours. Proteolytic pathways responsible for Cx43 degradation were elucidated by perfusing isolated rat hearts for 4 hours with specific inhibitors of either lysosomal or proteasomal proteolysis. Immunoblot analysis demonstrated significant increases (≈30%) in Cx43 content in hearts perfused with either lysosomal or proteasomal pathway inhibitors. Most of the Cx43 in hearts perfused with lysosomal inhibitors consisted of phosphorylated isoforms, whereas nonphosphorylated Cx43 accumulated selectively in hearts perfused with a specific proteasomal inhibitor. These results indicate that Cx43 turns over rapidly in the adult heart and is degraded by multiple proteolytic pathways. Regulation of Cx43 degradation could play an important role in gap junction remodeling in response to cardiac injury.
Increased Expression of Axl Tyrosine Kinase After Vascular Injury and Regulation by G Protein–Coupled Receptor Agonists in Rats
Authors: Melaragno Matthew G. , Wuthrich Daniel A. , Poppa Veronica , Gill Denzil , Lindner Volkhard , Berk Bradford C. , Corson Marshall A.
Published: June 2012

Abstract
—Axl is a receptor tyrosine kinase originally identified as a transforming gene product in human myeloid leukemia cells. Cultured rat vascular smooth muscle cells also express Axl, where it has been proposed that Axl may play a role in cell proliferation. In the current study, we tested the hypotheses that Axl expression would parallel neointima formation in balloon-injured rat carotid, and that Axl expression would be regulated by growth factors present at sites of vascular injury. Ribonuclease protection assay showed dynamic increases in Axl mRNA in vessels, with peak expression 7 and 14 days after injury. Immunohistochemical analysis confirmed these results and demonstrated that Axl protein expression was localized primarily to cells of the neointima after injury. Northern blot analysis indicated increased mRNA expression for the secreted Axl ligand, Gas6, in injured carotids, with a time course paralleling that of Axl upregulation. Axl and Gas6 expression were temporally correlated with neointima formation, suggesting a role for Axl signaling in this process. Other studies, performed in cultured rat vascular smooth muscle cells, revealed positive regulation of Axl mRNA expression by thrombin or angiotensin II but not by basic fibroblast growth factor, platelet-derived growth factor-BB, or transforming growth factor-β
1
. Western blot analysis confirmed these results, showing that Axl protein expression was specifically increased by thrombin or angiotensin II. Our results implicate Axl as a potential mediator of vascular smooth muscle migration and proliferation caused by vascular injury and G protein–coupled receptor agonists.
Cardiac Angiotensin II Receptors Are Upregulated by Long-Term Inhibition of Nitric Oxide Synthesis in Rats
Authors: Katoh Makoto , Egashira Kensuke , Usui Makoto , Ichiki Toshihiro , Tomita Hideharu , Shimokawa Hiroaki , Rakugi Hiromi , Takeshita Akira
Published: June 2012

Abstract
—It has been shown that nitric oxide (NO) may regulate angiotensin II (Ang II) receptors in vitro. To determine whether the chronic inhibition of NO synthesis upregulates cardiac Ang II receptors in a rat model, we evaluated the in vivo effect of N
ω
-nitro-
l
-arginine methyl ester (L-NAME) on several Ang II receptors and on the expression of AT1 receptor mRNA in heart tissue. The chronic administration of L-NAME to normal rats increased the arterial blood pressure. The number of AT1 and AT2 receptors was increased, with no change in affinity, during the first week of L-NAME administration but returned to control levels after 4 weeks of treatment. The AT1 receptor mRNA was changed parallel to AT1 receptor number. Inflammatory changes (monocyte infiltration and myofibroblast formation) in perivascular areas surrounding coronary vessels and myocardial interstitial spaces were observed during the first week. The immunohistochemistry revealed that myofibroblasts expressed AT1 receptor. AT1 receptor blockade or cotreatment with
l
-arginine, but not cotreatment with hydralazine, prevented the L-NAME–induced increase in Ang II receptors and inflammatory changes. In conclusion, rat cardiac Ang II receptors are upregulated at an early phase of chronic inhibition of NO synthesis. This may contribute to cardiovascular inflammatory changes in an early phase and to remodeling at the later phase, which occurs after inhibition of NO synthesis.
p38 Kinase Is a Negative Regulator of Angiotensin II Signal Transduction in Vascular Smooth Muscle Cells
Authors: Kusuhara Masatoshi , Takahashi Eiichi , Peterson Timothy E. , Abe Jun-ichi , Ishida Mari , Han Jiahuai , Ulevitch Richard , Berk Bradford C.
Published: June 2012

Abstract
—Activation of the Na
+
/H
+
exchanger isoform-1 (NHE-1) by angiotensin II is an early signal transduction event that may regulate vascular smooth muscle cell (VSMC) growth and migration. Many signal transduction events stimulated by angiotensin II are mediated by the mitogen-activated protein (MAP) kinases. To define their roles in angiotensin II–mediated NHE-1 activity, VSMCs were treated with angiotensin II and the activities of p38, c-Jun
N
-terminal kinase (JNK), and extracellular signal–regulated kinases 1 and 2 (ERK1/2) were measured. Angiotensin II rapidly (peak, 5 minutes) activated p38 and ERK1/2, whereas JNK was activated more slowly (peak, 30 minutes). Because angiotensin II stimulated Na
+
/H
+
exchange within 5 minutes, the effects of p38 and ERK1/2 antagonists on Na
+
/H
+
exchange were studied. The MEK-1 inhibitor PD98059 decreased ERK1/2 activity and Na
+
/H
+
exchange stimulated by angiotensin II. In contrast, the specific p38 antagonist SKF-86002 increased Na
+
/H
+
exchange. Two mechanisms were identified that may mediate the effects of p38 and SKF-86002 on angiotensin II–stimulated Na
+
/H
+
exchange. First, angiotensin II activation of ERK1/2 was increased 1.5- to 2.5-fold (depending on assay technique) in the presence of SKF-86002, demonstrating that p38 negatively regulates ERK1/2. Second, the ability of angiotensin II–stimulated MAP kinases to phosphorylate a glutathione
S
-transferase fusion protein containing amino acids 625 to 747 of NHE-1 in vitro was analyzed. The relative activities of endogenous immunoprecipitated p38, ERK1/2, and JNK were 1.0, 2.0, and 0.05 versus control, respectively suggesting that p38 and ERK1/2, but not JNK, may phosphorylate NHE-1 in VSMC. These data indicate important roles for p38 and ERK1/2 in angiotensin II–mediated regulation of the Na
+
/H
+
exchanger in VSMC.
Enhanced Myocardial Contractility and Increased Ca
2+
Transport Function in Transgenic Hearts Expressing the Fast-Twitch Skeletal Muscle Sarcoplasmic Reticulum Ca
2+
-ATPase
Authors: Loukianov Evgeny , Ji Yong , Grupp Ingrid L. , Kirkpatrick Darryl L. , Baker Debra L. , Loukianova Tanya , Grupp Gunter , Lytton Jonathan , Walsh Richard A. , Periasamy Muthu
Published: June 2012

Abstract
—In this study, we investigated whether the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca
2+
transport pump (SERCA1a) can functionally substitute the cardiac SERCA2a isoform and how its overexpression affects cardiac contractility. For this purpose, we generated transgenic (TG) mice that specifically overexpress SERCA1a in the heart, using the cardiac-specific α-myosin heavy chain promoter. Ectopic expression of SERCA1a resulted in a 2.5-fold increase in the amount of total SERCA protein. At the same time, the level of the endogenous SERCA2a protein was decreased by 50%, whereas the level of other muscle proteins, including calsequestrin, phospholamban, actin, and tropomyosin, remained unchanged. The steady-state level of SERCA phosphoenzyme intermediate was increased 2.5-fold, and the maximal velocity of Ca
2+
uptake was increased 1.7-fold in TG hearts, demonstrating that the overexpressed protein is functional. Although the basal cytosolic calcium signal was decreased by 38% in TG cardiomyocytes, the amplitude of cytosolic calcium signal was increased by 71.8%. The rate of calcium resequestration was also increased in TG myocytes, which was reflected by a 51.6% decrease in the normalized time to 80% decay of calcium signal. This resulted in considerably increased peak rates of myocyte shortening and relengthening (50.0% and 66.6%, respectively). Cardiac functional analysis using isolated work-performing heart preparations revealed significantly faster rates of contraction and relaxation in TG hearts (41.9% and 39.5%, respectively). The time to peak pressure and the time to half-relaxation were shorter (29.1% and 32.7%, respectively). In conclusion, our study demonstrates that the SERCA1a pump can functionally substitute endogenous SERCA2a, and its overexpression significantly enhances Ca
2+
transport and contractile function of the myocardium. These results also demonstrate that the SERCA pump level is a critical determinant of cardiac contractility.
Sulfhydryl Redox State Affects Susceptibility to Ischemia and Sarcoplasmic Reticulum Ca
2+
Release in Rat Heart
Authors: Zucchi Riccardo , Yu Gongyuan , Galbani Paola , Mariani Mario , Ronca Giovanni , Ronca-Testoni Simonetta
Published: June 2012

Abstract
—We investigated the effect of sulfhydryl and disulfide reagents on ischemic preconditioning and on sarcoplasmic reticulum Ca
2+
release. Isolated working rat hearts were subjected to ischemic preconditioning (three 3-minute periods of global ischemia) or to control aerobic perfusion, which was followed by 30 minutes of global ischemia and 120 minutes of retrograde reperfusion. Necrosis was evaluated on the basis of lactate dehydrogenase release and triphenyltetrazolium chloride staining. In parallel experiments, sarcoplasmic reticulum Ca
2+
release and [
3
H]-ryanodine binding were determined before the sustained ischemia. Ischemic preconditioning was associated with protection versus ischemic injury, decreased Ca
2+
release and reduced [
3
H]-ryanodine binding. The disulfide reducing agent dithiothreitol (1 mmol/L) removed the protection provided by ischemic preconditioning, if added to the perfusion buffer either before or after the preconditioning procedure. In preconditioned hearts, dithiothreitol increased sarcoplasmic reticulum Ca
2+
release and ryanodine binding, whereas in control hearts it had no effect on either tissue injury or sarcoplasmic reticulum function. Perfusion of control hearts with the sulfhydryl blocking agents 4,4′-dithiodipyridine (25 μmol/L) and N-ethylmaleimide (16 μmol/L) increased the resistance to ischemia and reduced sarcoplasmic reticulum Ca
2+
release and [
3
H]-ryanodine binding. These effects were not additive with those induced by preconditioning. Sulfhydryl and disulfide reagents produced similar effects on Ca
2+
release and [
3
H]-ryanodine binding if added in vitro to preparations obtained from control and preconditioned hearts. We conclude that ischemic preconditioning is associated with the oxidation of sulfhydryl groups involved in the modulation of sarcoplasmic reticulum Ca
2+
release.
Ascorbate Prevents the Interaction of Superoxide and Nitric Oxide Only at Very High Physiological Concentrations
Authors: Jackson Terence S. , Xu Aiming , Vita Joseph A. , Keaney John F.
Published: June 2012

Abstract
—The bioactivity of nitric oxide (
•
NO) depends, in part, on its interaction with superoxide. Usually, superoxide dismutase (SOD) preserves
•
NO bioactivity by limiting the availability of superoxide. Ascorbic acid also effectively scavenges superoxide, but the extent to which this interaction is necessary for intact
•
NO bioactivity is not known. Therefore, the present study examined the effect of ascorbic acid on
•
NO bioactivity with isolated rabbit arterial segments. A steady flux of superoxide (1.15 to 2.3 μmol · L
−1
· min
−1
) produced either by pyrogallol autoxidation or a hypoxanthine/xanthine oxidase system inhibited endothelium-derived
•
NO-mediated arterial relaxation elicited by acetylcholine. This effect of superoxide was completely blocked by SOD (300 IU/mL) and the manganese SOD mimic EUK-8 (300 μmol/L) and partially inhibited by ascorbic acid (10 mmol/L). Lower concentrations of ascorbic acid were ineffective despite scavenging >90% of superoxide. We increased the endogenous flux of superoxide (3.2±0.3-fold) by inhibiting vascular copper-zinc SOD with diethyldithiocarbamate. This increased endogenous flux of superoxide produced an impairment of
•
NO-mediated arterial relaxation that was reversed by EUK-8 (300 μmol/L) but not ascorbic acid (10 mmol/L) despite equivalent scavenging of the endogenous superoxide flux. We used 3-nitrotyrosine formation (from peroxynitrite) as an indicator of
•
NO interaction with superoxide and found that SOD and EUK-8 compete more effectively with
•
NO for superoxide than does ascorbic acid. These data indicate that preservation of
•
NO bioactivity by superoxide scavengers depends not only on superoxide scavenging activity, but also on the rate of superoxide scavenging. Normal extracellular concentrations of ascorbic acid (30 to 150 μmol/L) are not likely to prevent the interaction of
•
NO with superoxide under physiological conditions.