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Abstract: Objective: Insulin protects cardiomyocytes from apoptosis. Insulin resistance usually refers to a defect in the ability of insulin to stimulate glucose uptake. It is unknown, however, whether or not insulin resistance compromises the cell-protective effect of the hormone. Caspases are a family of cysteine proteases that regulate apoptosis. We explored the effects of insulin resistance on hypoxia-induced caspase-3 activation in cardiomyocytes. Methods: Experiments were performed in cultured neonatal rat cardiomyocytes. Insulin resistance was induced by treating cardiac myocytes with isoproterenol, a β-adrenergic receptor agonist. Results: Twelve hours of hypoxia-induced caspase-3 cleavage, which was inhibited by treatment with insulin, while pre-treatment with isoproterenol abolished the insulin effect. Hypoxia-induced cleavage of caspase-3 was mediated by p38 mitogen-activated protein kinase (MAPK). Insulin inhibited hypoxia-induced phosphorylation of p38 through MAPK phosphatase-1 (MKP-1). Insulin-induced MKP-1 expression was mediated by extracellular signal-regulated protein kinases (ERK) 1/2, c-Jun NH2-terminal kinases (JNK) MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. Isoproterenol stimulation failed to induce expression of MKP-1; moreover, insulin resistance induced by long-term β-adrenergic stimulation inhibited insulin-evoked expression of MKP-1 by impairing insulin-induced phosphorylation of both ERK1/2 and JNK without affecting Akt kinase activity. Furthermore, concomitant activation of Akt, ERK 1/2, and JNK was required for insulin to exert its protective effect against the hypoxia-induced cleavage of caspase-3. Conclusions: The results of this study lead to the conclusions that, in cardiac myocytes, antiapoptotic signals induced by insulin are mediated by more than one signaling pathway, and that long-term β-adrenergic receptor stimulation impairing some of these pathways affects the cytoprotective action of insulin. [Copyright &y& Elsevier]

Abstract: Objective : The outward current flowing through the two-pore domain acid-sensitive potassium channel TASK-1 (I TASK) and its inhibition via α1-adrenergic receptors was studied in rat ventricular cardiomyocytes. Methods : Quantitative RT-PCR experiments were carried out with mRNA from rat heart. Patch-clamp recordings were performed in isolated rat cardiomyocytes. TASK-1 and other K+ channels were expressed in Xenopus oocytes to study the pharmacological properties of a new TASK-1 channel blocker, A293. Results : TASK-1 channels were found to be strongly expressed in rat heart. Analysis of the sensitivity of various K+ channels to A293 in Xenopus oocytes showed that at low concentrations A293 was a selective blocker of TASK-1 channels. I TASK in rat cardiomyocytes was dissected by application of A293 and by extracellular acidification to pH 6.0; it had an amplitude of ∼0.30 pA/pF at +30 mV. Application of 200 nM A293 increased action potential duration (APD50) by 31±3% at a stimulation rate of 4 Hz. The plausibility of the effects of A293 on APD50 was checked with a mathematical action potential model. Application of the α1-adrenergic agonist methoxamine inhibited I TASK in Xenopus oocytes co-injected with cRNA for TASK-1 and α1A-receptors. In cardiomyocytes, methoxamine inhibited an outward current with characteristics similar to I TASK. This effect was abolished in the presence of the α1A-antagonist 5-methyl-urapidil. Conclusions : Our results suggest that in rat cardiomyocytes I TASK makes a substantial contribution to the outward current flowing in the plateau range of potentials and that this current component can be inhibited via α1A-adrenergic receptors. [Copyright &y& Elsevier]

Abstract: Objective: Mice deficient for the AP-1 transcription factor JunD, the only Jun protein constitutively expressed and clearly detectable in the mammalian heart, develop enhanced cardiac hypertrophy in response to chronic pressure overload. Catecholamines inducing α-adrenergic receptor-mediated signaling have been implicated in the neurohumoral response to pressure overload and the development of left ventricular hypertrophy. In the present study we analyzed the mechanistic role of JunD in cardiomyocyte hypertrophy in vitro in response to α-adrenergic agonist phenylephrine (PE). Methods: Cardiomyocytes were isolated from 1- to 3-day-old rats and transfected with adenoviruses expressing LacZ or wild-type JunD, or with expression vectors encoding LacZ, wild-type JunD, mutated JunD forming only JunD homodimers (JunDeb1), mutated JunD lacking the JNK site (JunD-Δ162), or c-Jun. After stimulation with PE (10−5 mol/L), hypertrophic growth of cardiomyocytes (cross-sectional area and [3H]-leucine incorporation) and mRNA expression of JunD, c-Jun, c-Fos, and atrial natriuretic peptide (ANP) were analyzed. Transcriptional activation was determined by luciferase activity in cardiomyocytes transfected with AP-1 or ANP luciferase reporter plasmids. Gel shift assays with an AP-1 consensus oligonucleotide were performed to analyze AP-1 DNA binding activities. Results: PE augmented mRNA levels of c-Jun and c-Fos, but decreased JunD transcript levels. Adenoviral over-expression of wild-type JunD blunted PE-induced hypertrophic growth and expression of ANP mRNA. Over-expression of JunD in cardiomyocytes caused enhanced AP-1 protein–DNA binding, without increasing the transcriptional response from AP-1 or ANP luciferase reporter plasmids at baseline or upon PE stimulation. Moreover, over-expression of JunDeb1 attenuated transcription from AP-1 or ANP luciferase reporter plasmids and blunted c-Jun-mediated acceleration of AP-1 transcriptional activity at baseline and in response to PE. Conclusions: Our observations establish a novel role for JunD as a negative regulator of cardiomyocyte hypertrophy in response to hypertrophic stimuli by inhibiting AP-1 transcriptional activity. [Copyright &y& Elsevier]

Abstract: Objective: Caveolae, flask shaped invaginations of the cell membrane, influence signalling cascades in many cell types. We have tested the hypothesis that caveolae modulate excitation–contraction coupling (ECC) and β-adrenergic stimulation in the adult cardiac myocyte. Methods: Shortening, [Ca2+]i and L-type Ca2+ current (I Ca,L) were recorded in rat ventricular myocytes. Caveolae were disrupted with methyl-β-cyclodextrin (MβC). Results: Shortening and [Ca2+]i transient amplitude were reduced in myocytes treated with MβC. MβC did not alter the density or characteristics of I Ca,L or the sarcoplasmic reticulum (SR) Ca2+ load, but significantly reduced fractional SR Ca2+ release. The inotropic response of myocytes to β1-adrenoceptor stimulation was insensitive to caveolae disruption. By contrast, the increase in shortening, [Ca2+]i transient and I Ca,L seen following β2 stimulation was markedly enhanced (3–5 fold) following MβC treatment, and the effect on I Ca,L could be mimicked by dialyzing cells with an antibody to caveolin 3. When the Gαi pathway was disabled with pertussis toxin (PTX), control cells showed a similar response to β2 stimulation as seen in MβC-treated myocytes, whereas MβC-treated cells were insensitive to PTX. Conclusions: Caveolae modulate ECC via the efficiency of the Ca2+-induced Ca2+ release process, rather than Ca2+ influx. Our data are also consistent with the hypothesis that interaction of Gi protein cascade components with caveolin in the caveolae is necessary for effective signalling by this pathway. This suggests that changes in caveolin expression in the adult heart seen during aging and in disease will have consequences for baseline cardiac function and β-adrenergic responsiveness. [Copyright &y& Elsevier]

Abstract: Objective: Sympathetic nervous system activity in the myocardium is increased in patients with heart failure. However, the in vivo mechanisms responsible for β-adrenoceptor-mediated cardiac hypertrophy or remodeling remain unclear. This study aimed to clarify the role of reactive oxygen species (ROS) in mitogen-activated protein (MAP) kinase activation and tissue remodeling of the heart of isoproterenol (ISO)-infused rats. Methods and results: Different doses of ISO (up to1000 ng/kg/min) were given intravenously to conscious rats for 30 min. Phosphorylated MAP kinase levels (ERK1/2, JNK, p38) and lipid peroxidation were measured in the cardiac left ventricle, revealing the dose-dependent augmentation of MAP kinase phosphorylation and increased lipid peroxidation levels. Simultaneous treatment with 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (Tempol), a membrane-permeable radical scavenger, completely eliminated the increases of phosphorylated MAP kinases and their upstream elements (Raf-1, Rac-1, ASK-1) as well as the increases of cardiac lipid peroxidation induced by the highest dose of ISO infusion. In chronically ISO-infused rats (3 mg/kg/day, s.c. for 10 days), cardiac hypertrophy developed with accompanying increases of collagen content, whereas cardiac phosphorylated MAP kinases returned to normal. Tempol treatment prevented increases of collagen accumulation and type I collagen mRNA without any significant reduction of cardiac mass enlargement induced by chronic ISO infusion. Conclusion: β-Adrenoceptor stimulation provokes cardiac oxidative stress. In the acute phase of ISO infusion, ROS are important activators of cardiac MAP kinase cascades; while, in the chronic phase, ROS may participate in cardiac remodeling, especially in respect to wall stiffness, based on fibrogenesis. [Copyright &y& Elsevier]

Objective: Catechol O-methyltransferase (COMT) is believed to exert degradative action at high norepinephrine (NE) levels. Although COMT exists in cardiac tissues, the contribution of cardiac COMT activity to regional NE kinetics, particularly in ischemia-induced NE accumulation, remains unclear. We investigated the role of cardiac COMT in NE kinetics in the ischemic region. Methods: We implanted a microdialysis probe into the left ventricular myocardium of anesthetized rabbits and induced myocardial ischemia by 60-min coronary artery occlusion. We monitored myocardial interstitial levels of NE and its metabolites in the presence and absence of a COMT inhibitor. We intraperitoneally administered entacapone (10 mg/kg) 120 min before control sampling. Results: In control, entacapone increased interstitial dihydroxyphenylglycol (DHPG, intraneuronal NE metabolite by monoamine oxidase (MAO)) levels and decreased interstitial normetanephrine (NMN, extraneuronal NE metabolite by COMT) and 3-methoxy-4-hydroxyphenylglycol (MHPG, extraneuronal DHPG metabolite by COMT) levels, but did not change interstitial NE levels. Coronary occlusion increased NE levels to 165±48 nM at 45–60 min of occlusion. This increase was accompanied by increases in DHPG and NMN levels (11.3±1.1 and 9.3±1.3 nM at 45–60 min of occlusion). Entacapone augmented the ischemia-induced NE and DHPG responses (333±51 and 22.9±2.4 nM at 45–60 min of occlusion). In contrast, the ischemia-induced NMN response was suppressed by entacapone (2.0±0.4 nM at 45–60 min of occlusion). Reperfusion decreased interstitial NE levels and increased interstitial DHPG and NMN levels. Entacapone suppressed changes in NE and NMN levels, but augmented the increase in dialysate DHPG. Conclusion: Myocardial ischemia evoked increases in myocardial interstitial NE and NMN levels. COMT inhibition augmented the increase in NE (substrate of COMT) levels and suppressed the increase in NMN (metabolite by COMT) levels. In the ischemic heart, COMT contributes to the removal of accumulated NE in the myocardium. [Copyright &y& Elsevier]

Objective: Overexpression of erythropoietin (Epo) in mice (Epo-tg6) leads to an increase in hematocrit and blood volume, and strongly reduces endurance upon exercise. It was the aim of this study to characterize the mechanisms underlying the reduced cardiac performance. Methods: Left (LV) and right (RV) ventricular function was measured with and without norepinephrine (NE) stimulation in 12 anaesthetized Epo-tg6 and in 13 wild-type (WT) control mice. Results: There were no differences in heart function under baseline resting conditions. Stimulation with NE (10 μl bolus injections of 1–100 ng per mouse) in WT mice led to a dose-dependent increase in heart rate (HR), LV developed pressure (LVDP) and rate of rise in LV pressure (LV dP/dtmax), while LV end-diastolic pressure (LVEDP) was unchanged. Except for HR, these parameters increased to a lesser extent in EPO-tg6 mice. Strikingly, LVEDP strongly increased in Epo-tg6 mice after NE (up to >20 mmHg). Eleven out of 13 Epo-tg6, but none of the WT mice died or required resuscitation after high-doses of NE. In these cases severe diastolic dysfunction became overt since the relative myocardial relaxation time was significantly prolonged and the duration of diastole was shortened. Moreover, the ECG showed a marked ST segment depression as well as deep negative T-waves. The NE-induced reduction in myocardial adenosin-triphosphate (ATP) content was more pronounced in Epo-tg6 mice after 10 min of continuous NE infusion (50 ng/min per mouse). Conclusion: NE-induced stress in Epo-tg6 mice led to acute heart failure associated with diastolic dysfunction and myocardial ischemia. [Copyright &y& Elsevier]

Objectives: Plasminogen activator inhibitor-1 (PAI-1) is a major anti-fibrinolytic glycoprotein thought to promote vascular diseases. Recently we have shown that systemically injecting mice with kainate, an analog of the principal brain excitatory neurotransmitter glutamate, immediately induced PAI-1 mRNA in brain vascular cells which are not known to contain glutamate receptors. Here we further investigated whether: (a) kainate also increases PAI-1 gene expression in the cardiac vascular bed; (b) subunits of kainate/AMPA receptors could be expressed in cardiac and brain vascular cells; and (c) PAI-1 mRNA could be similarly induced by agonists of adrenergic receptors that are candidates to act downstream in kainate-activated pathways. Methods: We analyzed cardiac and brain cryosections for PAI-1 mRNA, as well as mRNAs encoding three receptor subunits, by in situ hybridization using 35S-labeled specific riboprobes. PAI-1 activity was tested in cardiac homogenates using one-phase reverse zymography. Results: Prominent PAI-1 mRNA hybridization signals were induced in the vascular cells of the heart, and unexpectedly, also in cardiocytes, within 1–2 h after injection of kainate (i.p., 11–25 mg/kg body weight); the signals persisted for at least 8 h and disappeared after 24 h. In addition, PAI-1 activity increased (∼5 fold) 2–10 h after the treatment. In contrast, mRNAs encoding the kainate/AMPA receptor subunits could not be detected. The adrenergic agents adrenaline (3.5 mg/kg) and isoproterenol (200 mg/kg) exerted kainate-like effects in cardiovascular cells. Conclusions: These results revealed, for the first time, that PAI-1 gene expression can be enhanced locally in the cardiovascular system by a fast-acting neurological mechanism triggered by glutamate receptors, whose pathway and relation to catecholamines, which exerted similar effects, have yet to be resolved. These findings raised the possibility that excessive glutamate, or stress-related catecholamines, may increase the risk of stroke and myocardial infarction. [ABSTRACT FROM PUBLISHER]

Objective: The present study was designed to examine regional differences in the response of α1 adrenoceptor stimulation in the canine ventricle. Methods: Standard microelectrode techniques were used to record transmembrane action potentials from epicardial, M cell, and endocardial as well as Purkinje fiber preparations isolated from the canine left ventricle. Results: Phenylephrine (0.1–10 μM+propranolol 0.2 μM) and methoxamine (1–10 μM) produced dose- and rate-dependent prolongation of action potential duration (APD90) in Purkinje fibers (P<0.05, at 0.1–10 μM, BCL=0.5–2 s), but an abbreviation of APD90 in tissues from the M region (P<0.05, at 10 μM, BCL=0.5–2 s). At slow pacing rates (≥2 s), phenylephrine (1 μM) exerted a small, significant (P<0.05) prolongation of APD90 in epicardium and endocardium which returned to control values when the concentration was increased to 10 μM. The amplitude of phase 1 of the action potential in M and epicardial cells was significantly increased by phenylephrine at concentrations of 10 μM (P<0.05). Prazosin (1 μM), a nonspecific α1 antagonist, reversed these effects of phenylephrine (10 μM) and methoxamine (10 μM) on APD90 and the action potential notch. The α1b-antagonist, chloroethylclonidine (0.1–1.0 μM), but not the α1a-antagonist, WB-4101 (0.1–1.0 μM), reversed the APD-abbreviating effect of methoxamine in the M cell. Conclusion: Our results demonstrate striking regional differences in the electrophysiological response of the four canine ventricular cell types to α1 adrenergic agonists. Our data provide support for the hypothesis that different adrenoceptor subtypes underlie the opposite response of M cells (α1b-APD abbreviation) and Purkinje fibers (α1a-APD prolongation) to α1-adrenoceptor activation. [ABSTRACT FROM PUBLISHER]

Objective: Prior studies of the modulation of the Na+ current by sympathetic stimulation have yielded controversial results. Separation of the Na+ and Ca2+ currents poses a problem in myocyte preparations. The gating of cloned Na+ channels is different in oocytes compared with mammalian expression systems. We have examined the sympathetic modulation of the α-subunit of the wild-type human cardiac Na+ channel (hH1) and the long QT-associated mutant, ΔKPQ, expressed in human embryonic kidney cells. Methods: Stable cell lines of hH1 and ΔKPQ were established in human embryonic kidney cells. Whole-cell and single-channel currents were measured with the patch–clamp technique. Sympathetic stimulation was effected by exposure to isoproterenol or 8-bromo-cAMP. Na+ channel activation and inactivation were determined using standard voltage clamp protocols. Ca2+: Na+ permeability ratio was determined under bi-ionic conditions. Results: We observed a qualitatively different effect of sympathetic stimulation on the cardiac Na+ current from that reported in frog oocytes: activation and inactivation kinetics were shifted to more negative potentials. This shift was similar for both hH1 and ΔKPQ. [ΔV0.5 for inactivation: 8.3±1.7 mV, p<0.001 (hH1); 6.8±0.9 mV, p<0.001 (ΔKPQ)]. Increased rate of closed-state inactivation contributed to the shifting of the inactivation–voltage relationship. Open-state inactivation was not affected as mean open times were unchanged. Reversal potential measurement in hH1 suggested a low Ca2+: Na+ permeability ratio of 0.017, uninfluenced by sympathetic stimulation. In ΔKPQ, the size of the persistent relative to the peak current was increased with 8-bromo-cAMP from 3.0±0.7% to 4.3±0.6% (p=0.056). Conclusions: Sympathetic stimulation exerts multiple effects on the gating of hH1. Similar effects are also seen in ΔKPQ which may increase arrhythmia susceptibility in long QT syndrome by modifying the Na+ channel contribution to the action potential. [ABSTRACT FROM PUBLISHER]

Objective: The aims of the study were to establish the roles of angiotensin II and of the cardiopulmonary baroreceptor reflex in the regulation of peripheral vascular tone in patients with cirrhosis. Methods: Forearm blood flow responses to subsystemic, locally active intrabrachial infusions were measured in patients with Child's Grade C cirrhosis and matched controls using bilateral venous occlusion plethysmography. Responses were determined to the angiotensin II type I receptor antagonist, losartan, noradrenaline, angiotensin II and the nitric oxide synthase inhibitor, l-NG-monomethyl arginine. Results: Losartan at 30 and 90 μg/min caused no significant change in blood flow in controls, but caused 23±6% and 27±5% increases in patients respectively (p<0.001). Lower body negative pressure caused a mean bilateral reduction in forearm blood flow of 20±4% in controls (p<0.001) but only tended to reduce flow (9±5%; p=0.06) in patients (p<0.001; controls vs. patients). Noradrenaline, angiotensin II and l-NG-monomethyl arginine caused significant vasoconstriction (p<0.001) in both patients and controls although angiotensin II caused significantly less vasoconstriction in patients (p=0.01). Conclusions: We conclude that angiotensin II makes an important contribution to basal peripheral vascular tone in patients with cirrhosis in the face of reduced vascular responses to its local administration. In addition, the vasoconstrictor response to cardiopulmonary baroreceptor unloading is attenuated despite normal vascular responses to noradrenaline. These responses are consistent with chronic activation of the renin–angiotensin and sympathetic nervous systems in patients with advanced cirrhosis. [ABSTRACT FROM PUBLISHER]