bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin-Like Growth Factor-1 Attenuates the Detrimental Impact of Nonocclusive Coronary Artery Constriction on the Heart
Authors: Li Baosheng , Setoguchi Manabu , Wang Xiaowei , Andreoli Anna Maria , Leri Annarosa , Malhotra Ashwani , Kajstura Jan , Anversa Piero
Published: June 2012

Abstract
—Coronary artery narrowing (CAN) induces tissue injury, which may involve myocyte necrosis and apoptosis. Insulin-like growth factor (IGF)–1 may counteract cell death, modifying the detrimental effects of myocardial ischemia. On this basis, CAN was produced in female FVB.
Igf
+/– mice and nontransgenic littermates, and the animals were euthanized 7 days later. CAN consisted of an 82% reduction in the vessel luminal cross-sectional area in both groups of mice. Severe left ventricular dysfunction was present in CAN nontransgenic and transgenic mice, but heart and left ventricular weights increased more in littermates than in FVB.
Igf
+/– mice. Similarly, the changes in chamber volume and diastolic wall stress were greater in nontransgenic mice. Subacute tissue injury, represented by foci of replacement fibrosis, was 2.6-fold higher in CAN littermates than in FVB.
Igf
+/– mice. Ongoing myocyte necrosis was 5-fold greater in nontransgenic mice, whereas apoptosis was low and did not differ in the 2 groups of mice. In CAN nontransgenic mice, myocyte necrosis was 12-fold more frequent than apoptosis but, in CAN transgenic mice, these 2 types of cell death were comparable. α-Myosin and β-myosin isoform mRNAs were affected by CAN, but α-myosin mRNA was reduced more in nontransgenic mice. In conclusion, myocyte necrosis and replacement fibrosis are the prevailing forms of myocardial damage induced by CAN. Constitutive overexpression of IGF-1 attenuates myocyte necrosis and tissue injury, having no effect on cell apoptosis. These factors limit ventricular dilation, myocardial loading, cardiac hypertrophy, and alterations in α- and β-myosin isoform expression.
Activation of Distinct cAMP-Dependent and cGMP-Dependent Pathways by Nitric Oxide in Cardiac Myocytes
Authors: Vila-Petroff Martin G. , Younes Antoine , Egan Josephine , Lakatta Edward G. , Sollott Steven J.
Published: June 2012

Abstract
—Nitric oxide (NO) donors were recently shown to produce biphasic contractile effects in cardiac tissue, with augmentation at low NO levels and depression at high NO levels. We examined the subcellular mechanisms involved in the opposing effects of NO on cardiac contraction and investigated whether NO modulates contraction exclusively via guanylyl cyclase (GC) activation or whether some contribution occurs via cGMP/PKG-independent mechanisms, in indo 1–loaded adult cardiac myocytes. Whereas a high concentration of the NO donor
S
-nitroso-
N
-acetylpenicillamine (SNAP, 100 μmol/L) significantly attenuated contraction amplitude by 24.4±4.5% (without changing the Ca
2+
transient or total cAMP), a low concentration of SNAP (1 μmol/L) significantly increased contraction amplitude (38±10%), Ca
2+
transient (26±10%), and cAMP levels (from 6.2 to 8.5 pmol/mg of protein). The negative contractile response of 100 μmol/L SNAP was completely abolished in the presence of the specific blocker of PKG KT 5823 (1 μmol/L); the positive contractile response of 1 μmol/L SNAP persisted, despite the presence of the selective inhibitor of GC 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μmol/L) alone, but was completely abolished in the presence of ODQ plus the specific inhibitory cAMP analog Rp-8-CPT-cAMPS (100 μmol/L), as well as by the NO scavenger oxyhemoglobin. Parallel experiments in cell suspensions showed significant increases in adenylyl cyclase (AC) activity at low concentrations (0.1 to 1 μmol/L) of SNAP (AC, 18% to 20% above basal activity). We conclude that NO can regulate both AC and GC in cardiac myocytes. High levels of NO induce large increases in cGMP and a negative inotropic effect mediated by a PKG-dependent reduction in myofilament responsiveness to Ca
2+
. Low levels of NO increase cAMP, at least in part, by a novel cGMP-independent activation of AC and induce a positive contractile response.
Mouse Model of a Familial Hypertrophic Cardiomyopathy Mutation in α-Tropomyosin Manifests Cardiac Dysfunction
Authors: Muthuchamy Mariappan , Pieples Kathy , Rethinasamy Prabhakar , Hoit Brian , Grupp Ingrid L. , Boivin Greg P. , Wolska Beata , Evans Christian , Solaro R. John , Wieczorek David F.
Published: June 2012

Abstract
—To investigate the functional consequences of a tropomyosin (TM) mutation associated with familial hypertrophic cardiomyopathy (FHC), we generated transgenic mice that express mutant α-TM in the adult heart. The missense mutation, which results in the substitution of asparagine for aspartic acid at amino acid position 175, occurs in a troponin T binding region of TM. S1 nuclease mapping and Western blot analyses demonstrate that increased expression of the α-TM 175 transgene in different lines causes a concomitant decrease in levels of endogenous α-TM mRNA and protein expression. In vivo physiological analyses show a severe impairment of both contractility and relaxation in hearts of the FHC mice, with a significant change in left ventricular fractional shortening. Myofilaments that contain α-TM 175 demonstrate an increased activation of the thin filament through enhanced Ca
2+
sensitivity of steady-state force. Histological analyses show patchy areas of mild ventricular myocyte disorganization and hypertrophy, with occasional thrombi formation in the left atria. Thus, the FHC α-TM transgenic mouse can serve as a model system for the examination of pathological and physiological alterations imparted through aberrant TM isoforms.
Activation of Epithelial Growth Factor Receptor Pathway by Unsaturated Fatty Acids
Authors: Vacaresse Nathalie , Lajoie-Mazenc Isabelle , Augé Nathalie , Suc Isabelle , Frisach Marie-Françoise , Salvayre Robert , Nègre-Salvayre Anne
Published: June 2012

Abstract
—Nonesterified fatty acids (NEFAs) are acutely liberated during lipolysis and are chronically elevated in pathological conditions, such as insulin resistance, hypertension, and obesity, which are known risk factors for atherosclerosis. The purpose of this study was to investigate the effect and mechanism of action of NEFAs on the epithelial growth factor (EGF) receptor (EGFR). In the ECV-304 endothelial cell line, unsaturated fatty acids triggered a time- and dose-dependent tyrosine phosphorylation of EGFR (polyunsaturated fatty acids [PUFAs] were the most active), whereas saturated FAs were inactive. Although less potent than PUFAs, oleic acid (OA) was used because it is prominent in the South European diet and is only slightly oxidizable (thus excluding oxidation derivatives). EGFR is activated by OA independent of any autocrine secretion of EGF or other related mediators. OA-induced EGFR autophosphorylation triggered EGFR signaling pathway activation (as assessed through coimmunoprecipitation of SH2 proteins such as SHC, GRB2, and SHP-2) and subsequent p42/p44 mitogen-activated protein kinase (as shown by the use of EGFR- deficient B82L and EGFR- transduced B82LK
+
cell lines). OA induced in vitro both autophosphorylation and activation of intrinsic tyrosine kinase of immunopurified EGFR, thus suggesting that EGFR is a primary target of OA. EGFR was also activated by mild surfactants, Tween-20 and Triton X-100, both in vitro (on immunopurified EGFR) and in intact living cells, thus indicating that EGFR is sensitive to amphiphilic molecules. These data suggest that EGFR is activated by OA and PUFAs, acts as a sensor for unsaturated fatty acids (and amphiphilic molecules), and is a potential transducer by which diet composition may influence vascular wall biology.
Vascular Matrix Metalloproteinase-2 Cleaves Big Endothelin-1 Yielding a Novel Vasoconstrictor
Authors: Fernandez-Patron Carlos , Radomski Marek W. , Davidge Sandra T.
Published: June 2012

Abstract
—Matrix metalloproteinase-2 (MMP-2, gelatinase A) and its tissue inhibitor (TIMP-2) are mainly known for their roles in the (patho)physiological remodeling of the vasculature, angiogenesis, tissue repair, tumor invasion, inflammation, and atherosclerotic plaque rupture. A mechanism of action of MMP-2 is the proteolytic breakdown of specific extracellular matrix proteins. The amino acid sequences in interstitial collagen (Gly-Leu/Ile) and laminin-5 (Ala-Leu) that are cleaved by MMP-2 are homologous to a region (Gly
32
-Leu
33
) within human big endothelin-1[1 to 38] (big ET-1). Big ET-1 requires cleavage to an active form to produce vasoconstriction. We tested the hypothesis that vascular MMP-2 can cleave big ET-1, thus generating a vasoconstrictor peptide. In perfused rat mesenteric arteries with an intact endothelium, inhibition of vascular MMP-2 with TIMP-2 reduced (by 16.2±4.2%) the vasoconstrictor effects of big ET-1 (50 pmol). However, when the endothelium was mechanically removed, TIMP-2 abolished (>90%) the vasoconstriction of big ET-1, and this effect was mimicked by an anti–MMP-2 antibody. Incubation of big ET-1 with recombinant human MMP-2 resulted in the specific cleavage of the Gly
32
-Leu
33
bond of big ET-1. Moreover, the resultant peptide ET-1[1 to 32] exerted greater vasoconstrictor effects than big ET-1. We conclude that vascular MMP-2 contributes to the vasoconstrictor effects of big ET-1 by cleaving big ET-1 to yield a novel and potent vasoconstrictor, ET-1[1 to 32]. These data implicate, for the first time, the endogenous MMP-2/TIMP-2 system in the regulation of vascular reactivity.
Regulation of Cardiac Myocyte Protein Turnover and Myofibrillar Structure In Vitro by Specific Directions of Stretch
Authors: Simpson D. G. , Majeski M. , Borg T. K. , Terracio L.
Published: June 2012

Abstract
—We have examined how different degrees (0.5%, 1.0%, 2.5%, 5.0%, and 10.0%) and directions of stretch regulate the turnover and accumulation of contractile proteins in cultured neonatal cardiac myocytes (NCMs). In pulse-chase experiments, stellate-shaped NCMs with random arrays of myofibrils (MFs) exhibited a threshold response to stretch. With respect to unstretched controls, the turnover of the contractile protein pool was suppressed 50% to 100% in stellate NCMs stretched 1.0% to 5.0% and was unaltered in stellate NCMs stretched 0.5% or 10.0%. The posttranslational metabolism of myosin heavy chain (MHC) and actin was regulated in parallel with the total contractile protein pool. The turnover of the cytoplasmic protein pool remained unchanged in response to stretch. NCMs plated onto an aligned matrix of type I collagen expressed an elongated, rod-like cell shape. The MFs of these cells were distributed in parallel with one another along a single unique axis. The tissue-like pattern of organization of these cultures made it possible to assay how specific directions of stretch affected cardiac protein turnover and MF organization. In pulse-chase experiments, stretch in parallel with the MFs did not alter the turnover of the total contractile protein pool, the cytoplasmic protein pool, MHC, or actin. The total cellular concentration of MHC and actin remained constant, and MF alignment was not overtly affected. In contrast, even modest degrees of stretch across the short axis of the MFs suppressed total contractile protein turnover, the turnover of MHC and actin, and promoted the accumulation of these MF subunits. The parallel alignment of MFs deteriorated in myocytes stretched greater than 5%. The characteristic response of aligned myocytes to stretch was not affected by the contractile state of the cells. Isoproterenol (ISO) treatment in concert with stretch in parallel with the MFs modestly accelerated contractile protein turnover. Conversely, contractile protein turnover was suppressed in cells treated with ISO and stretched across the short axis of the MFs. Contractile arrest with nifedipine (NIFED) accelerated total myofibrillar protein turnover. Stretch across the short axis, but not in parallel with the MFs, suppressed protein turnover in cells treated with NIFED. The turnover of the cytosolic proteins remained constant under all conditions assayed. These data suggest that specific directions of stretch may play a crucial role in regulating MF organization and the metabolism of contractile proteins in the cardiac myocyte. The full text of this article is availabale at http://www.circresaha.org.
Overexpression of the Cardiac β
2
-Adrenergic Receptor and Expression of a β-Adrenergic Receptor Kinase-1 (βARK1) Inhibitor Both Increase Myocardial Contractility but Have Differential Effects on Susceptibility to Ischemic Injury
Authors: Cross Heather R. , Steenbergen Charles , Lefkowitz Robert J. , Koch Walter J. , Murphy Elizabeth
Published: June 2012

Abstract
—Cardiac β
2
-adrenergic receptor (β
2
AR) overexpression is a potential contractile therapy for heart failure. Cardiac contractility was elevated in mice overexpressing β
2
ARs (TG4s) with no adverse effects under normal conditions. To assess the consequences of β
2
AR overexpression during ischemia, perfused hearts from TG4 and wild-type mice were subjected to 20-minute ischemia and 40-minute reperfusion. During ischemia, ATP and pH fell lower in TG4 hearts than wild type. Ischemic injury was greater in TG4 hearts, as indicated by lower postischemic recoveries of contractile function, ATP, and phosphocreatine. Because β
2
ARs, unlike β
1
ARs, couple to G
i
as well as G
s
, we pretreated mice with the G
i
inhibitor pertussis toxin (PTX). PTX treatment increased basal contractility in TG4 hearts and abolished the contractile resistance to isoproterenol. During ischemia, ATP fell lower in TG4+PTX than in TG4 hearts. Recoveries of contractile function and ATP were lower in TG4+PTX than in TG4 hearts. We also studied mice that overexpressed either βARK1 (TGβARK1) or a βARK1 inhibitor (TGβARKct). Recoveries of function, ATP, and phosphocreatine were higher in TGβARK1 hearts than in wild-type hearts. Despite basal contractility being elevated in TGβARKct hearts to the same level as that of TG4s, ischemic injury was not increased. In summary, β
2
AR overexpression increased ischemic injury, whereas βARK1 overexpression was protective. Ischemic injury in the β
2
AR overexpressors was exacerbated by PTX treatment, implying that it was G
s
not G
i
activity that enhanced injury. Unlike β
2
AR overexpression, basal contractility was increased by βARK1 inhibitor expression without increasing ischemic injury, thus implicating a safer potential therapy for heart failure.
Signal Transduction in Atria and Ventricles of Mice With Transient Cardiac Expression of Activated G Protein α
q
Authors: Mende U. , Kagen A. , Meister M. , Neer E. J.
Published: June 2012

Abstract
—We recently showed that the transient expression of a hemagglutinin (HA) epitope–tagged, constitutively active mutant of the G protein α
q
subunit (HAα
q
*) in the hearts of transgenic mice is sufficient to induce cardiac hypertrophy and dilatation that continue to progress after HAα
q
* protein becomes undetectable. We demonstrated that the activity of phospholipase Cβ, the immediate downstream target of activated Gα
q
, is increased at 2 weeks, when HAα
q
* is expressed, but also at 10 weeks, when HAα
q
* is no longer detectable. This observation suggested that the transient HAα
q
* expression causes multiple, persistent changes in cellular signaling pathways. We now demonstrate changes in the level, activity, or both of several signaling components, including changes in the amount and hormone responsiveness of phospholipase Cβ enzymes, in the basal level of diacylglycerol (which predominantly reflects activation of phospholipase D), in the amount or distribution of protein kinase C (PKC) isoforms (PKCα, PKCδ, and PKCε), and in the amount of several endogenous G proteins. These changes vary depending on the isoform of the signaling molecule, the chamber in which it is expressed, and the presence or absence of HAα
q
*. Our results suggest that a network of linked signaling functions determines the development of hypertrophy. They also suggest that atria and ventricles represent different signaling domains. It is likely that such changes occur in other model systems in which the activity of a single signaling component is increased, either due to an activating mutation or due to overexpression of the wild type.
Matrix Metalloproteinase-9 Overexpression Enhances Vascular Smooth Muscle Cell Migration and Alters Remodeling in the Injured Rat Carotid Artery
Authors: Mason David P. , Kenagy Richard D. , Hasenstab David , Bowen-Pope Daniel F. , Seifert Ronald A. , Coats Scott , Hawkins Suzanne M. , Clowes Alexander W.
Published: June 2012

Abstract
—Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of atherosclerosis as well as intimal hyperplasia after vascular injury. We used Fischer rat smooth muscle cells (SMCs) overexpressing MMP-9 to determine the role of MMP-9 in migration and proliferation as well as in vessel remodeling after balloon denudation. Fischer rat SMCs were stably transfected with a cDNA for rat MMP-9 under the control of a tetracycline-regulatable promoter. In this system, MMP-9 was overexpressed in the absence, but not in the presence, of tetracycline. In vitro SMC migration was determined using a collagen invasion assay as well as a Boyden chamber assay. In vivo migration was determined by measuring the invasion into the medial and intimal layers of transduced SMCs seeded on the outside of the artery. Transduced SMCs were also seeded on the luminal surface, and the effect of local MMP-9 overexpression on vascular structure was measured morphometrically at intervals up to 28 days. MMP-9 overexpression enhanced SMC migration in both the collagen invasion assay and Boyden chamber in vitro, increased SMC migration into an arterial matrix in vivo, and altered vessel remodeling by increasing the vessel circumference, thinning the vessel wall and decreasing intimal matrix content. These results demonstrate that MMP-9 enhances vascular SMC migration in vitro and in vivo and alters postinjury vascular remodeling.
A Single Na
+
Channel Mutation Causing Both Long-QT and Brugada Syndromes
Authors: Bezzina Connie , Veldkamp Marieke W. , van den Berg Maarten P. , Postma Alex V. , Rook Martin B. , Viersma Jan-Willem , van Langen Irene M. , Tan-Sindhunata Ghita , Bink-Boelkens Margreet Th. E. , van der Hout Annemarie H. , Mannens Marcel M. A. M. , Wilde Arthur A. M.
Published: June 2012

Abstract
—Mutations in
SCN5A
, the gene encoding the cardiac Na
+
channel, have been identified in 2 distinct diseases associated with sudden death: one form of the long-QT syndrome (LQT
3
) and the Brugada syndrome. We have screened
SCN5A
in a large 8-generation kindred characterized by a high incidence of nocturnal sudden death, and QT-interval prolongation and the “Brugada ECG” occurring in the same subjects. An insertion of 3 nucleotides (TGA) at position 5537, predicted to cause an insertion of aspartic acid (1795insD) in the C-terminal domain of the protein, was linked to the phenotype and was identified in all electrocardiographically affected family members. ECGs were obtained from 79 adults with a defined genetic status (carriers, n=43; noncarriers, n=36). In affected individuals, PR and QRS durations and QT intervals are prolonged (
P
<0.0001 for all parameters). ST segment elevation in the right precordial leads is present as well (
P
<0.0001). Twenty-five family members died suddenly, 16 of them during the night. Expression of wild-type and mutant Na
+
channels in
Xenopus
oocytes revealed that the 1795insD mutation gives rise to a 7.3-mV negative shift of the steady-state inactivation curve and an 8.1-mV positive shift of the steady-state activation curve. The functional consequence of both shifts is likely to be a reduced Na
+
current during the upstroke of the action potential. LQT
3
and Brugada syndrome are allelic disorders but may also share a common genotype.
Mechanical Stretch and Angiotensin II Differentially Upregulate the Renin-Angiotensin System in Cardiac Myocytes In Vitro
Authors: Malhotra Ricky , Sadoshima Junichi , Brosius Frank C. , Izumo Seigo
Published: June 2012

Abstract
—Pressure overload in vivo results in left ventricular hypertrophy and activation of the renin-angiotensin system in the heart. Mechanical stretch of neonatal rat cardiac myocytes in vitro causes secretion of angiotensin II (Ang II), which in turn plays a pivotal role in mechanical stretch–induced hypertrophy. Although in vivo data suggest that the stimulus of hemodynamic overload serves as an important modulator of cardiac renin-angiotensin system (RAS) activity, it is not clear whether observed upregulation of RAS genes is a direct effect of hemodynamic stress or is secondary to neurohumoral effects in response to hemodynamic overload. Moreover, it is unclear whether activation of the local RAS in response to hemodynamic overload predominantly occurs in cardiac myocytes or fibroblasts or both. In the present study, we examined the effect of mechanical stretch on expression of angiotensinogen, renin, angiotensin-converting enzyme (ACE), and Ang II receptor (AT
1A
, AT
1B
, and AT
2
) genes in neonatal rat cardiac myocytes and cardiac fibroblasts in vitro. The level of expression of angiotensinogen, renin, ACE, and AT
1A
genes was low in unstretched cardiac myocytes, but stretch upregulated expression of these genes at 8 to 24 hours. Stimulation of cardiac myocytes with Ang II also upregulated expression of angiotensinogen, renin, and ACE genes, whereas it downregulated AT
1A
and did not affect AT
1B
gene expression. Although losartan, a specific AT
1
antagonist, completely inhibited Ang II–induced upregulation of angiotensinogen, renin, and ACE genes, as well as stretch-induced upregulation of AT
1A
expression, it did not block upregulation of angiotensinogen, renin, and ACE genes by stretch. Western blot analyses showed increased expression of angiotensinogen and renin protein at 16 to 24 hours of stretch. The ACE-like activity was also significantly elevated at 24 hours after stretch. Radioligand binding assays revealed that stretch significantly upregulated the AT
1
density on cardiac myocytes. Interestingly, stretch of cardiac fibroblasts did not result in any discernible increases in the expression of RAS genes. Our results indicate that mechanical stretch in vitro upregulates both mRNA and protein expression of RAS components specifically in cardiac myocytes. Furthermore, components of the cardiac RAS are independently and differentially regulated by mechanical stretch and Ang II in neonatal rat cardiac myocytes.
Reentry and Fibrillation in the Mouse Heart
Authors: Vaidya Dhananjay , Morley Gregory E. , Samie Faramarz H. , Jalife José
Published: June 2012

Abstract
—The idea that fibrillation is only possible in hearts exceeding a critical size was introduced by W. Garrey >80 years ago and has since been generally accepted. In ventricular tissue, this critical size was originally estimated to be 400 mm
2
. Recent estimates suggest that the critical size required for sustained reentry is ≈100 to 200 mm
2
, whereas 6 times this area is required for ventricular fibrillation. According to these estimates, fibrillation is not possible in the mouse heart, where the ventricular surface area is ≈100 mm
2
. To test whether sustained ventricular fibrillation could be induced in such an area, we used a high-speed video imaging system and a voltage-sensitive dye to quantify electrical activity on the epicardial surface of the Langendorff-perfused adult mouse heart. In 6 hearts, measurements during ventricular pacing at a basic cycle length (BCL) of 120 ms yielded maximum and minimum conduction velocities (CV
max
and CV
min
) of 0.63±0.04 and 0.38±0.02 mm/ms, respectively. At a BCL of 80 ms, CV
max
and CV
min
changed to 0.55±0.03 and 0.34±0.02 mm/ms. Action potential durations (APDs), measured at 70% repolarization at those pacing frequencies were found to be 44.5±2.9 and 40.4±2.6 ms, respectively. The wavelengths (CV×APD) were calculated to be 28.6±3.4 mm in the CV
max
direction and 16.8±1.5 mm in the CV
min
direction at BCL 120 ms. Wavelengths were significantly reduced (
P
<0.05) at BCL 80 ms (CV
max
, 22.2±1.8 mm; CV
min
, 13.7±0.9 mm). In 5 hearts, stationary vortex-like reentry organized by single rotors (4 of 5 hearts) or by pairs of rotors (1 of 5 hearts) was induced by burst pacing. In the ECG, the activity manifested as sustained monomorphic tachycardia. Detailed analysis showed that the local CVs were reduced in the vicinity of the rotor center, which allowed the reentry to take place within a smaller area than was calculated from wavelength measurements during pacing. In 4 of 7 hearts, burst pacing resulted in a polymorphic ECG pattern indistinguishable from ventricular fibrillation. These data challenge the critical mass hypothesis by demonstrating that ventricular tissue with an area as small as 100 mm
2
is capable of undergoing sustained fibrillatory activity.
Tumor Necrosis Factor-α Upregulates Angiotensin II Type 1 Receptors on Cardiac Fibroblasts
Authors: Gurantz Devorah , Cowling Randy T. , Villarreal Francisco J. , Greenberg Barry H.
Published: June 2012

Abstract
—Angiotensin II (Ang II) plays an important role in post–myocardial infarction (MI) remodeling. Most Ang II effects related to remodeling involve activation of the type 1 receptor (AT
1
). Although the AT
1
receptor is upregulated on cardiac fibroblasts post-MI, little is known about the mechanisms involved in the process. Consequently, we tested whether growth factors known to be present in the remodeling heart increased AT
1
mRNA levels. Using quantitative competitive reverse transcription–polymerase chain reaction, we found that norepinephrine, endothelin, atrial natriuretic peptide, and bradykinin had no significant effect on AT
1
mRNA levels. Ang II, transforming growth factor-β
1
, and basic fibroblast growth factor reduced AT
1
mRNA levels (
P
<0.02). Tumor necrosis factor-α (TNF-α), however, produced a marked increase in AT
1
mRNA. After 24 hours of TNF-α incubation, AT
1
mRNA increased by 5-fold above control levels (
P
<0.01). The EC
50
for the TNF-α effect was 4.6 ng/mL (0.2 nmol/L). Interleukin (IL)-1β caused a 2.4-fold increase, whereas IL-2 and IL-6 had no significant effect. Studies of TNF-α enhancement of AT
1
mRNA levels demonstrate that the increase was not due to a change in transcript stability. TNF-α treatment for 48 hours also resulted in a 3-fold increase in AT
1
surface receptor and a 2-fold increase in Ang II–induced production of inositol phosphates. The present findings provide evidence for TNF-α regulation of AT
1
receptor density on cardiac fibroblasts. Because TNF-α concentration and AT
1
receptor density increase in the myocardium after MI, these results raise the possibility that TNF-α modulates post-MI remodeling by enhancing Ang II effects on cardiac fibroblasts.
Propagation of Cardiomyocyte Hypercontracture by Passage of Na
+
Through Gap Junctions
Authors: Ruiz-Meana Marisol , Garcia-Dorado David , Hofstaetter Bettina , Piper H. Michael , Soler-Soler Jordi
Published: June 2012

Abstract
—Prolonged ischemia increases cytosolic Ca
2+
concentration in cardiomyocytes. Cells with severely elevated cytosolic Ca
2+
may respond to reperfusion, developing hypercontracture, sarcolemmal disruption, and death. Cardiomyocytes are efficiently connected through gap junctions (GJs) to form a functional syncytium, and it has been shown that hypercontracture can be propagated to adjacent myocytes through a GJ-mediated mechanism. This study investigated the mechanism of propagation of cell injury associated with sarcolemmal rupture in end-to-end connected pairs of isolated rat cardiomyocytes. Microinjection of extracellular medium into one of the cells to simulate sarcolemmal disruption induced a marked increase in cytosolic Ca
2+
(fura-2) and Na
+
(SBFI) in the adjacent cell and its hypercontracture in <30 seconds (22 of 22 cell pairs). This process was not modified when Ca
2+
release from the sarcoplasmic reticulum was blocked with 10 μmol/L ryanodine (5 of 5 cell pairs), but it was fully dependent on the presence of Ca
2+
in the extracellular buffer. Blockade of L-type Ca
2+
channels with 10 μmol/L nifedipine did not alter propagation of hypercontracture. However, the presence of 15 to 20 μmol/L KB-R7943, a highly selective blocker of reverse Na
+
/Ca
2+
exchange, prevented propagation of hypercontracture in 16 of 20 cell pairs (
P
<0.01) without interfering with GJ permeability, as assessed by the Lucifer Yellow transfer method. Addition of the Ca
2+
chelator EGTA (2 mmol/L) to the injection solution prevented hypercontracture in the injected cell but not in the adjacent one (n=5). These results indicate that passage of Na
+
through GJ from hypercontracting myocytes with ruptured sarcolemma to adjacent cells, and secondary entry of [Ca
2+
]
o
via reverse Na
+
/Ca
2+
exchange, can contribute to cell-to-cell propagation of hypercontracture. This previously unrecognized mechanism could increase myocardial necrosis during ischemia-reperfusion in vivo and be the target of new treatments aimed to limit it.
Impaired Long-Chain Fatty Acid Utilization by Cardiac Myocytes Isolated From Mice Lacking the Heart-Type Fatty Acid Binding Protein Gene
Authors: Schaap Frank G. , Binas Bert , Danneberg Heike , van der Vusse Ger J. , Glatz Jan F. C.
Published: June 2012

Abstract
—Heart-type fatty acid binding protein (H-FABP), abundantly expressed in cardiac myocytes, has been postulated to facilitate the cardiac uptake of long-chain fatty acids (LCFAs) and to promote their intracellular trafficking to sites of metabolic conversion. Mice with a disrupted H-FABP gene were recently shown to have elevated plasma LCFA levels, decreased cardiac deposition of a LCFA analogue, and increased cardiac deoxyglucose uptake, which qualitatively establishes a requirement for H-FABP in cardiac LCFA utilization. To study the underlying defect, we developed a method to isolate intact, electrically stimulatable cardiac myocytes from adult mice and then studied substrate utilization under defined conditions in quiescent and in contracting cells from wild-type and H-FABP
−/−
mice. Our results demonstrate that in resting and in contracting myocytes from H-FABP
−/−
mice, both uptake and oxidation of palmitate are markedly reduced (between –45% and –65%), whereas cellular octanoate uptake, and the capacities of heart homogenates for palmitate oxidation and for octanoate oxidation, and the cardiac levels of mRNAs encoding sarcolemmal FA transporters remain unaltered. In contrast, in resting H-FABP
−/−
cardiac myocytes, glucose oxidation is increased (+80%) to a level that would require electrical stimulation in wild-type cells. These findings provide a physiological demonstration of a crucial role of H-FABP in uptake and oxidation of LCFAs in cardiac muscle cells and indicate that in H-FABP
−/−
mice the diminished contribution of LCFAs to cardiac energy production is, at least in part, compensated for by an increase in glucose oxidation.
Evidence From a Novel Human Cell Clone That Adult Vascular Smooth Muscle Cells Can Convert Reversibly Between Noncontractile and Contractile Phenotypes
Authors: Li Shaohua , Sims Stephen , Jiao Yang , Chow Lawrence H. , Pickering J. Geoffrey
Published: June 2012

Abstract
—Smooth muscle cells (SMCs) perform diverse functions that can be categorized as contractile and synthetic. A traditional model holds that these distinct functions are performed by the same cell, by virtue of its capacity for bidirectional modulation of phenotype. However, this model has been challenged, in part because there is no physiological evidence that an adult synthetic SMC can acquire the ability to contract. We sought evidence for this by cloning adult SMCs from human internal thoracic artery. One clone, HITB5, expressed smooth muscle α-actin, smooth myosin heavy chains, heavy caldesmon, and calponin and showed robust calcium transients in response to histamine and angiotensin II, which confirmed intact transmembrane signaling cascades. On serum withdrawal, these cells adopted an elongated and spindle-shaped morphology, random migration slowed, extracellular matrix protein production fell, and cell proliferation and [
3
H]thymidine incorporation fell to near 0. Cell viability was not compromised, however; in fact, apoptosis rate fell significantly. In this state, agonist-induced elevation of cytoplasmic calcium was even more pronounced and was accompanied by SMC contraction. Readdition of 10% serum completely returned HITB5 cells to a noncontractile, proliferative phenotype. Contractile protein expression increased after serum withdrawal, although modestly, which suggested that the switch to contractile function involved reorganization or sensitization of existing contractile structures. To our knowledge, the physiological properties of HITB5 SMCs provide the first direct demonstration that cultured human adult SMCs can convert between a synthetic, noncontracting state and a contracting state. HITB5 cells should be valuable for characterizing the basis of this critical transition.
11,12-Epoxyeicosatrienoic Acid Stimulates Endogenous Mono-ADP-Ribosylation in Bovine Coronary Arterial Smooth Muscle
Authors: Li Pin-Lan , Chen Cai-Lian , Bortell Rita , Campbell William B.
Published: June 2012

Abstract
—The role of endogenous ADP-ribosylation in mediating the activation of the Ca
2+
-activated K
+
channels was determined in bovine coronary arteries. Endogenous ADP-ribosylation was examined by incubating coronary arterial homogenates or lysates of cultured coronary arterial smooth muscle cells with [adenylate-
32
P]NAD. Four
32
P-labeled proteins were observed at 51, 52, 80, and 124 kDa in the homogenates and lysates. This reaction was enhanced by the addition of 11,12-epoxyeicosatrienoic acid (11,12-EET), a cytochrome P450–derived eicosanoid, and GTP to the incubation. By Western blot analysis, 42- and 70-kDa proteins were recognized by specific antibodies against ADP-ribosyltransferase in the coronary arterial homogenates and smooth muscle cell lysate but not in the lysate of endothelial cells. The 52-kDa acceptor protein of endogenous ADP-ribosylation comigrated with a protein ADP-ribosylated by cholera toxin and was recognized and immunoprecipitated by an anti–G
S
α antibody. These results suggest that G
S
α is one of several acceptors of the ADP-ribose moiety. As shown by the patch-clamp technique, 11,12-EET stimulated the activation of the K
+
channels in the smooth muscle cells, and this activation was completely blocked by novobiocin, vitamin K
1
, 3-aminobenzamide, and
m
-iodobenzylguanidine, inhibitors of endogenous mono-ADP-ribosyltransferases. We conclude that endogenous mono-ADP-ribosyltransferases are present in smooth muscle from bovine coronary arteries. These enzymes transfer ADP-ribose to the cellular proteins such as G
S
α and may mediate intracellular signal transduction in coronary vascular smooth muscle. In the coronary circulation, the ADP-ribosylation signaling pathway may play an important role in mediating the activation of the K
+
channels induced by 11,12-EET.
Matrix Metalloproteinase Inhibition During the Development of Congestive Heart Failure
Authors: Spinale Francis G. , Coker Mytsi L. , Krombach Stephen R. , Mukherjee Rupak , Hallak Hussein , Houck Ward V. , Clair Mark J. , Kribbs Scott B. , Johnson Linda L. , Peterson J. Thomas , Zile Michael R.
Published: June 2012

Abstract
—The development of congestive heart failure (CHF) is associated with left ventricle (LV) dilation and myocardial remodeling. The matrix metalloproteinases (MMPs) play a significant role in extracellular remodeling, and recent studies have demonstrated increased MMP expression and activity with CHF. Whether increased MMP activity directly contributes to the LV remodeling with CHF remains unknown. Accordingly, this study examined the effects of chronic MMP inhibition (MMPi) on LV size and function during the progression of CHF. Pigs were assigned to the following groups: (1) CHF, rapid pacing for 3 weeks at 240 bpm (n=12); (2) CHF/MMPi, rapid pacing and concomitant MMPi (PD166793, 20 mg/kg per day [n=10]), and (3) control (n=11). With pacing CHF, LV fractional shortening was reduced (19±1 versus 45±1%), and end-diastolic dimension increased (5.67±0.11 versus 3.55±0.05 cm), compared with baseline values (
P
<0.05). In the CHF/MMPi group, LV endocardial shortening increased (25±2%) and the end-diastolic dimension was reduced (4.92±0.17 cm) compared with CHF-only values (
P
<0.05). LV midwall shortening was reduced to a comparable degree in the CHF-only and CHF/MMPi groups. LV peak wall stress increased 3-fold with pacing CHF compared with controls and was significantly reduced in the CHF/MMPi group. LV myocardial stiffness was unchanged with CHF but was increased in the CHF/MMPi group. LV myocyte length was increased with pacing CHF compared with controls (180±3 versus 125±4 μm,
P
<0.05) and was reduced in the CHF/MMPi group (169±4 μm,
P
<0.05). Basal-state myocyte shortening velocity was reduced with pacing CHF compared with controls (33±2 versus 66±1 μm/s,
P
<0.05) and was unchanged in the CHF/MMPi group (31±2 μm/s). Using an ex vivo assay system, myocardial MMP activity was increased with pacing CHF and was reduced with chronic MMPi. In summary, concomitant MMPi with developing CHF limited LV dilation and reduced wall stress. These results suggest that increased myocardial MMP activity contributes to LV myocardial remodeling in developing CHF.
The Mitochondrial Apoptotic Pathway Is Activated by Serum and Glucose Deprivation in Cardiac Myocytes
Authors: Bialik Shani , Cryns Vincent L. , Drincic Andjela , Miyata Setsuya , Wollowick Adam L. , Srinivasan Anu , Kitsis Richard N.
Published: June 2012

Abstract
—Many cell types undergo apoptosis under conditions of ischemia. Little is known, however, about the molecular pathways that mediate this response. A cellular and biochemical approach to elucidate such signaling pathways was undertaken in primary cultures of cardiac myocytes, a cell type that is especially sensitive to ischemia-induced apoptosis. Deprivation of serum and glucose, components of ischemia in vivo, resulted in myocyte apoptosis, as determined by nuclear fragmentation, internucleosomal cleavage of DNA, and processing of caspase substrates. These manifestations of apoptosis were blocked by zVAD-fmk, a peptide caspase inhibitor, indicating that caspase activity is necessary for the progression of apoptosis in this model. In contrast to control cells, apoptotic myocytes exhibited cytoplasmic accumulation of cytochrome
c
, indicating release from the mitochondria. Furthermore, both caspase-9 and caspase-3 were processed to their active forms in serum-/glucose-deprived myocytes. Caspase processing, but not cytochrome
c
release, was inhibited by zVAD-fmk, placing the latter event upstream of caspase activation. This evidence demonstrates that components of ischemia activate the mitochondrial death pathway in cardiac myocytes.
Role of Respiratory Motor Output in Within-Breath Modulation of Muscle Sympathetic Nerve Activity in Humans
Authors: St. Croix Claudette M. , Satoh Makoto , Morgan Barbara J. , Skatrud James B. , Dempsey Jerome A.
Published: June 2012

Abstract
—We measured muscle sympathetic nerve activity (MSNA, peroneal microneurography) in 5 healthy humans under conditions of matched tidal volume, breathing frequency, and end-tidal CO
2
, but varying respiratory motor output as follows: (1) passive positive pressure mechanical ventilation, (2) voluntary hyperventilation, (3) assisted mechanical ventilation that required the subject to generate –2.5 cm H
2
O to trigger each positive pressure breath, and (4) added inspiratory resistance. Spectral analyses showed marked respiratory periodicities in MSNA; however, the amplitude of the peak power was not changed with changing inspiratory effort. Time domain analyses showed that maximum MSNA always occurred at end expiration (25% to 30% of total activity) and minimum activity at end inspiration (2% to 3% of total activity), and the amplitude of the variation was not different among conditions despite marked changes in respiratory motor output. Furthermore, qualitative changes in intrathoracic pressure were without influence on the respiratory modulation of MSNA. In all conditions, within-breath changes in MSNA were inversely related to small changes in diastolic pressure (1 to 3 mm Hg), suggesting that respiratory rhythmicity in MSNA was secondary to loading/unloading of carotid sinus baroreceptors. Furthermore, at any given diastolic pressure, within-breath MSNA varied inversely with lung volume, demonstrating an additional influence of lung inflation feedback on sympathetic discharge. Our data provide evidence against a significant effect of respiratory motor output on the within-breath modulation of MSNA and suggest that feedback from baroreceptors and pulmonary stretch receptors are the dominant determinants of the respiratory modulation of MSNA in the intact human.
Susceptibility to Myocarditis Is Dependent on the Response of αβ T Lymphocytes to Coxsackieviral Infection
Authors: Opavsky Mary Anne , Penninger Josef , Aitken Karen , Wen Wen-Hu , Dawood Fayez , Mak Tak , Liu Peter
Published: June 2012

Abstract
—Viral myocarditis is an important cause of heart failure and dilated cardiomyopathy. T lymphocytes are implicated in myocardial damage in murine models of coxsackievirus B3 (CVB3) myocarditis. We used knockout mice lacking CD4 (CD4
−/−
), CD8 (CD8
−/−
), both coreceptors (CD4
−/−
CD8
−/−
), or the T-cell receptor β chain (TCRβ
−/−
) to address the contribution of T-cell subpopulations to host susceptibility to CVB3 myocarditis. Severity of disease was magnified in CD8
−/−
mice but attenuated in CD4
−/−
mice, consistent with a pathogenic role for CD4
+
lymphocytes. Elimination of both CD4 and CD8 molecules from T lymphocytes by genetic knockout better protected mice from myocarditis, demonstrating that both CD4
+
and CD8
+
T cells contribute to host susceptibility. The same benefit occurred in TCRβ
−/−
mice, with prolonged survival and minimal myocardial disease observed after CVB3 infection. Elevated interferon-γ and decreased tumor necrosis factor-α expression are associated with attenuated myocardial damage in CD4
−/−
CD8
−/−
mice. These results show that the presence of TCRαβ
+
T cells enhances host susceptibility to myocarditis. The severity of myocardial damage and associated mortality are dependent on the predominant T-cell type available to respond to CVB3 infection. One mechanism by which CD4
+
and CD8
+
T-cell subsets influence the pathogenesis of myocarditis may involve specific cytokine expression patterns.
cAMP-Independent Dilation of Coronary Arterioles to Adenosine
Authors: Hein Travis W. , Kuo Lih
Published: June 2012

Abstract
—Adenosine is known to play an important role in the regulation of coronary blood flow during metabolic stress. However, there is sparse information on the mechanism of adenosine-induced dilation at the microcirculatory levels. In the present study, we examined the role of endothelial nitric oxide (NO), G proteins, cyclic nucleotides, and potassium channels in coronary arteriolar dilation to adenosine. Pig subepicardial coronary arterioles (50 to 100 μm in diameter) were isolated, cannulated, and pressurized to 60 cm H
2
O without flow for in vitro study. The arterioles developed basal tone and dilated dose dependently to adenosine. Disruption of endothelium, blocking of endothelial ATP-sensitive potassium (K
ATP
) channels by glibenclamide, and inhibition of NO synthase by
N
G
-nitro-
l
-arginine methyl ester and of soluble guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one produced identical attenuation of vasodilation to adenosine. Combined administration of these inhibitors did not further attenuate the vasodilatory response. Production of NO from coronary arterioles was significantly increased by adenosine. Pertussis toxin, but not cholera toxin, significantly inhibited vasodilation to adenosine, and this inhibitory effect was only evident in vessels with an intact endothelium. Tetraethylammonium, glibenclamide, and a high concentration of extraluminal KCl abolished vasodilation of denuded vessels to adenosine; however, inhibition of calcium-activated potassium channels by iberiotoxin had no effect on this dilation. Rp-8-Br-cAMPS, a cAMP antagonist, inhibited vasodilation to cAMP analog 8-Br-cAMP but failed to block adenosine-induced dilation. Furthermore, vasodilations to 8-Br-cAMP and sodium nitroprusside were not inhibited by glibenclamide, indicating that cAMP- and cGMP-induced dilations are not mediated by the activation of K
ATP
channels. These results suggest that adenosine activates both endothelial and smooth muscle pathways to exert its vasodilatory function. On one hand, adenosine opens endothelial K
ATP
channels through activation of pertussis toxin–sensitive G proteins. This signaling leads to the production and release of NO, which subsequently activates smooth muscle soluble guanylyl cyclase for vasodilation. On the other hand, adenosine activates smooth muscle K
ATP
channels and leads to vasodilation through hyperpolarization. It appears that the latter vasodilatory process is independent of G proteins and of cAMP/cGMP pathways.
Induction of Heme Oxygenase-1 Suppresses Venular Leukocyte Adhesion Elicited by Oxidative Stress
Authors: Hayashi Shinobu , Takamiya Rina , Yamaguchi Tokio , Matsumoto Kenji , Tojo Shinichiro J. , Tamatani Takuya , Kitajima Masaki , Makino Nobuya , Ishimura Yuzuru , Suematsu Makoto
Published: June 2012

Abstract
—This study aimed to examine whether an elevated activity of heme oxygenase (HO)-1 in the tissue attenuates endothelial cell–leukocyte interactions microvessels in vivo. When rats were pretreated with an intraperitoneal injection of hemin, an HO-1 inducer, mesenteric tissues, including their microvessels, displayed a marked induction of HO-1 concurrent with an increase in plasma concentrations of bilirubin-IXα, the product of HO-catalyzed degradation of protoheme IX. In these rats, oxidative stress such as superfusion with H
2
O
2
and ischemia-reperfusion of the tissues neither induced rolling nor exhibited adherent responses of leukocytes in venules. In contrast, the oxidative stresses evoked marked rolling and adhesion of leukocytes in the control rats without HO-1 induction. The HO-1 induction also downregulated leukocyte adhesion elicited by other pro-oxidant stimuli such as
N
ω
-nitro-
l
-arginine methyl ester. The decreases in the oxidant-elicited leukocyte adhesive responses under HO-1–inducing conditions were restored by perfusion with zinc protoporphyrin-IX, an HO inhibitor, but not with copper protoporphyrin-IX, which did not inhibit the enzyme. Furthermore, the effects of zinc protoporphyrin-IX were repressed by superfusion with bilirubin or biliverdin at the micromolar level, but not by the same concentration of carbon monoxide, another product of HO. These results indicate that induction of the HO-1 activity serves as a potential stratagem to prevent oxidant-induced microvascular leukocyte adhesion through the action of bilirubin, a product of HO reaction.