Loss of spontaneous fluctuations in resting microcirculatory flow has been described in diabetes mellitus, but its mechanism remains unexplained.
The autonomic control of forearm skin microcirculation was investigated in 23 insulin-dependent diabetic human subjects (median age 39 years, range 27-50) and in 23 age-matched controls (median age 38 years, range 20-57), by laser-Doppler flowmetry. Using spectral analysis of spontaneous microvascular fluctuations, we measured the power of 0.1 Hz ('10-second rhythm') fluctuations, dependent on sympathetic control, and of respiration-related, high-frequency fluctuations, due to the transmission of mechanical chest activity. Autonomic function abnormalities were assessed by 5 tests of cardiovascular reflexes.
Abnormalities in cardiovascular autonomic tests were present in 7/23 patients: deep breathing was abnormal 4 in patients, standing in 2, handgrip in 3, cross-correlation in 4, and Valsalva ratio in 0. The power of 0.1 Hz microcirculatory fluctuations was significantly lower in diabetic than in control subjects (2.57 +/- 0.16 vs 3.48 +/- 0.09 In-mV2, mean +/- s.e.m., P < 0.001), whereas that of respiratory fluctuations was similar (2.60 +/- 0.24 vs 2.56 +/- 0.19 In-mV2, P = n.s.). The 0.1 Hz power was 2 standard deviations below the mean of controls (P < 0.05) in 13/23 diabetic patients; this abnormality was significantly more frequent than abnormalities in any other autonomic test (P < 0.001).
Since the observed reduction was confined to those microvascular fluctuations under autonomic control, but not to those dependent on passive mechanical transmission, the reduction in spontaneous microcirculatory vasomotion appears to be determined mainly by sympathetic dysfunction. Sympathetic impairment of skin microvascular control seems to be a common finding, and is probably an early index of autonomic dysfunction in insulin-dependent diabetes.
Mesenchymal stem cells (MSCs) are widely used for cell therapy, particularly for the treatment of ischemic heart disease. Mechanisms underlying control of their metabolism and proliferation capacity, critical elements for their survival and differentiation, have not been fully characterized. AMP-activated protein kinase (AMPK) is a key regulator known to metabolically protect cardiomyocytes against ischemic injuries and, more generally, to inhibit cell proliferation. We hypothesized that AMPK plays a role in control of MSC metabolism and proliferation.Methods and ResultsMSCs isolated from murine bone marrow exclusively expressed the AMPKα1 catalytic subunit. In contrast to cardiomyocytes, a chronic exposure of MSCs to hypoxia failed to induce cell death despite the absence of AMPK activation. This hypoxic tolerance was the consequence of a preference of MSC towards glycolytic metabolism independently of oxygen availability and AMPK signalling. On the other hand, A-769662, a well-characterized AMPK activator, was able to induce a robust and sustained AMPK activation. We showed that A-769662-induced AMPK activation inhibited MSC proliferation. Proliferation was not arrested in MSCs derived from AMPKα1-knockout mice, providing genetic evidence that AMPK is essential for this process. Among AMPK downstream targets proposed to regulate cell proliferation, we showed that neither the p70 ribosomal S6 protein kinase/eukaryotic elongation factor 2-dependent protein synthesis pathway nor p21 was involved, whereas p27 expression was increased by A-769662. Silencing p27 expression partially prevented the A-769662-dependent inhibition of MSC proliferation.
MSCs resist hypoxia independently of AMPK whereas chronic AMPK activation inhibits MSC proliferation, p27 being involved in this regulation.
Proliferation of pulmonary arterial smooth muscle cells (PASMCs) is one histological sign of pulmonary arterial hypertension (PAH). We hypothesized that a signalling cascade from fibroblast growth factor 2 (FGF₂) to plasminogen activator inhibitor 1 (PAI-1) and monocyte chemotactic protein-1 (MCP-1) via nuclear transcription factor nuclear factor kappaB (NF-kB) play a critical role in progression of PAH, and tested this hypothesis both in vivo and in vitro using a synthetic selective NF-kB inhibitor, N-(3,5-Bis-trifluoromethyl-phenyl)-5-chloro-2-hydroxy-benzamide (IMD-0354).
Methods and results:
Monocrotaline (MCT) was injected into 75 Sprague-Dawley rats. Starting at day 14 after MCT injection, we administered IMD-0354 (MCT + IMD group) or vehicle (MCT group) daily. At day 32, 65% of the MCT + IMD group were alive compared with 0% of the MCT group. IMD-0354 prevented increase of right ventricular pressure, and suppressed proliferation and induced apoptosis of PASMCs. mRNA transcript levels of FGF₂, PAI-1, and tissue plasminogen activator (t-PA) were lower in MCT + IMD compared with MCT. In in vitro experiments, IMD-0354 inhibited p65 translocation to the nucleus promoted by FGF₂ in PASMCs. Furthermore, the time courses of extracellular signal-regulated kinase (Erk) 1/2, MCP-1, and PAI-1 stimulated with FGF₂ were each markedly shortened by IMD-0354.
We speculate that the positive-feedback loop (Erk1/2-NF-kB-MCP-1-Erk1/2) is associated with progression of PAH by causing FGF₂-induced inflammation in MCT rats. IMD-0354 has potential as a new therapeutic tool for PAH.
This paper describes a catheter-tip micromanometer for chronic left ventricular pressure measurements which can be calibrated in the animal after implantation. The calibration curves are linear, both in vitro and in vivo. Good zero-stability is reached after 1 week of implantation. The duration of function of the device is 3 to 6 months.
Hemostasis interrupts bleeding from disrupted blood vessels by activating platelet aggregation and coagulation. A similar mechanism termed thrombosis generates obstructive thrombi inside diseased arteries. As a consequence of this similarity, current antithrombotic agents increase the risk of bleeding. Atherosclerotic plaques produce significant amounts of prostaglandin E2 (PGE2) which activates its receptor EP3 on platelets and aggravates atherothrombosis. We investigated whether blocking EP3 could dissociate atherothrombosis from hemostasis.Methods and ResultsInhibiting in vivo the receptor EP3 for PGE2 with the blocking agent DG-041 reduced murine thrombosis triggered by local delivery of arachidonic acid or ferric chloride on healthy arteries. Importantly, it also reduced thrombosis triggered by scratching murine atherosclerotic plaques. PGE2 was not produced at the bleeding site after tail clipping. Consistently, blocking EP3 did not alter murine tail, liver or cerebral hemostasis. Furthermore, blocking EP3 reduced murine pulmonary embolism and intensified platelet inhibition by clopidogrel leaving tail bleeding times unchanged. Human atherosclerotic plaques produced PGE2, which facilitated platelet aggregation in human blood and rescued function of P2Y12-blocked platelets. Finally, in healthy patients DG-041 reduced platelet aggregation, but did not significantly alter the cutaneous bleeding time at doses up to 8-times the dose that inhibited the facilitating effect of PGE2 on platelets.
In mice, blocking EP3 inhibited atherothrombosis without affecting hemostasis and intensified efficiency of conventional antiplatelet treatment without aggravating the bleeding risk. In patients, blocking EP3 should improve the prevention of cardiovascular diseases which is currently limited by the risk of bleeding.
The aim of our study was to investigate the effect of IRFI 042, a novel dual vitamin E-like antioxidant, on nuclear factor-kappaB (NF-kappaB) activation, TNF-alpha gene priming and on the release of the mature protein during endotoxin shock.
Endotoxin shock was produced in male rats by a single intravenous (i.v.) injection of 20 mg kg(-1) of Salmonella enteritidis lipopolysaccharide (LPS). Survival rate, mean arterial blood pressure, serum TNF-alpha and plasma malondialdehyde (MAL) levels were investigated. We then evaluated in the liver TNF-alpha mRNA levels, NF-kappaB binding activity and the inhibitory protein IkappaBalpha. Moreover we studied in LPS stimulated (50 microg ml(-1)) peritoneal macrophages (Mphi), NF-kappaB activation, cytoplasmic IkappaB-alpha degradation, the message for TNF-alpha, and TNF-alpha and MAL levels.
LPS administration reduced survival rate (0%, 72 h after LPS administration), decreased mean arterial blood pressure, augmented serum TNF-alpha (60+/-11 ng ml(-1)) and enhanced plasma malondialdehyde (MAL) levels (55+/-7.1 nmol l(-1)). LPS shocked rats also had increased TNF-alpha mRNA levels, augmented liver NF-kappaB binding activity in the nucleus and decreased levels of the inhibitory protein IkappaBalpha. In addition, in vitro LPS stimulation (50 microg ml(-1)) significantly induced NF-kappaB activation and cytoplasmic IkappaBalpha degradation in Mphi, enhanced TNF-alpha mRNA levels and increased Mphi TNF-alpha and MAL. Treatment with IRFI 042 (20 mg kg(-1), i.v., 5 min after endotoxin challenge) protected against LPS-induced lethality (90% survival rate 24 h and 80% survival rate 72 h after LPS injection, respectively), reduced hypotension, blunted plasma MAL (9.0+/-0.9 nmol l(-1)) and decreased serum TNF-alpha (15+/-3 ng ml(-1)). The antioxidant also inhibited the loss of IkappaBalpha protein from the hepatic cytoplasm, blunted the increased NF-kappaB binding activity in the liver and decreased hepatic liver mRNA for TNF-alpha. Furthermore 'in vitro' IRFI 042 (50 microM) significantly inhibited activation of NF-kappaB through inhibition of IkappaBalpha degradation, reduced the amount of TNF-alpha mRNA, decreased LPS-induced TNF-alpha release and blunted lipid peroxidation (MAL) in LPS stimulated Mphi.
These data suggest that IRFI 042 blocks the activation of NF-kappaB, reduces TNF-alpha mRNA levels, and finally reverses endotoxic shock.
Nuclear factor-kappaB (NF-kappaB) is a ubiquitous rapid response transcription factor involved in inflammatory reactions which exerts its effect by expressing cytokines, chemokines, and cell adhesion molecules. Oxidative stress causes NF-kappaB activation. IRFI 042 is a novel dual vitamin E-like antioxidant and we, therefore, investigated its ability to protect the heart from oxidative stress and to halt the inflammatory response in a model of myocardial ischaemia-reperfusion injury.
Anaesthetized rats were subjected to total occlusion (45 min) of the left main coronary artery followed by 5-h reperfusion (MI/R). Sham myocardial ischaemia rats (sham-operated rats) were used as controls. Myocardial necrosis, cardiac output, cardiac and plasma vitamin E levels, myocardial malondialdehyde (MAL), cardiac SOD activity and elastase content (an index of leukocyte infiltration), hydroxyl radical (OH&z.ccirf;) formation, cardiac amount of mRNA codifying for ICAM-1 (evaluated by the means of reverse transcriptase polymerase chain reaction) and ICAM-1 immunostaining in the at-risk myocardium were investigated. NF-kappaB activation and the inhibitory protein of NF-kappaB, I-kappaBalpha, were also studied in at-risk myocardium by using electrophoretic mobility shift assay (EMSA) and Western blot analysis, respectively.
The ischaemia-reperfusion model produced wide heart necrosis (area at risk-necrotic area=52+/-5%; necrotic area-left ventricle=28+/-3%), increased cardiac MAL, an index of lipid peroxidation (area at risk=62.5+/-3.9 nmol/g tissue; necrotic area=80.3+/-5.1 nmol/g tissue), induced tissue neutrophil infiltration, and caused a marked decrease in endogenous antioxidants. Furthermore, myocardial ischaemia plus reperfusion caused in the area at risk peak message for ICAM-1 at 3 h of reperfusion and increased cardiac ICAM-1 immunostaining at 5 h of reperfusion. NF-kappaB activation was also evident at 0.5 h of reperfusion and reached its maximum at 2 h of reperfusion. I-kappaBalpha was markedly decreased at 45 min of occlusion; peak reduction was observed at 1 h of reperfusion and thereafter it was gradually restored. Intraperitoneal administration of IRFI 042 (5, 10, 20 mg/kg, 5 min after reperfusion) reduced myocardial necrosis expressed as a percentage either of the area at risk (18+/-4%) or the total left ventricle (11+/-2%), and improved cardiac output. This treatment also limited membrane lipid peroxidation in the area at risk (MAL=14.3+/-2.5 nmol/g tissue) and in the necrotic area (MAL=26.5+/-3.7 nmol/g tissue), restored the endogenous antioxidants vitamin E and superoxide dismutase, and inhibited detrimental hydroxyl radical formation. Finally, IRFI 042 blocked the activation of NF-kappaB, reduced cardiac ICAM-1 expression, and blunted tissue elastase content, an index of leukocytes accumulation at the site of injury.
Our data suggest that IRFI 042 is cardioprotective during myocardial infarction by limiting reperfusion-induced oxidative stress and by halting the inflammatory response.
The effects of chagasic sera containing an antibody (EVI antibody), which reacts with the plasma membrane of striated muscle fibres and endothelial cells, on the inotropic and chronotropic influences of noradrenaline upon isolated rat atria suspended in different media, were explored. Also explored were the action of cocaine, normetanephrine and U-0521 (3′-4′-dihydroxy-2- methyl propiophenone) on the atrial effects of the agonist in preparations immersed in Krebs-Ringer bicarbonate, alone and with the addition of normal human sera or EVI positive human chagasic sera (EVI(+)S). The positive inotropic action of a single dose of noradrenaline (3.0 x 10-7 mol·litre-1) was significantly smaller in spontaneously beating auricles exposed to the EVI(+)S. Cumulative dose-response curves for the agonist showed depression and reduced slope suggesting that EVI(+)S may nihilate the effect of the amine by means of a "noncompetitive-like" mechanism.A similar diminished reactivity towards noradrenaline was found regarding atrial contractile rate. Incubation in a solution with U-0521 enhanced the maximal augmentation of atrial isometric tension evoked by the amine in the three media whereas cocaine failed to elicit significant changes. However, normal human sera not only restored but actually potentiated the inotropic influence of noradrenaline although only in auricles bathed with a solution containing EVI(+)S. None of these three agents altered the reduced chronotropic effect of the agonist exposed to the EVI antibody. Contractile influences on atria paced at single fixed rates were similar to those observed in spontaneously beating preparations. The experimental findings suggest that: a) at the contractile frequencies in the present studies neuronal uptake is neither an important mechanism for terminating the contractile effect of exogenously added noradrenaline, nor a factor involved in the altered action of the agonist in the presence of EVI(+)S; and b) the importance of extraneuronal uptake of noradrenaline became clear after the incubation in a media containing EVI(+)S. It is, therefore plausible to suggest that the EVI antibody might influence the contractile effect of the agonist through a reversible enhancement of extraneuronal uptake. This would in turn modify the interaction between the amine and adrenoceptive sites needed for the full positive inotropic effectiveness of noradrenaline. The possible significance of these findings in the alterations observed in chronic Chagas heart disease is discussed.
The aim was to compare the electrophysiological and inotropic effects of the novel class III agents H 234/09, UK-68,798, and E-4031 in vitro.
The electrophysiological effects were investigated by recording transmembrane action potentials in the isolated ventricular muscle and Purkinje fibres of the rabbit; effects on force (adjusted to the maximum isoprenaline response) and refractoriness were investigated in the isolated cat papillary muscle.
It was shown that all the drugs induced a concentration dependent prolongation of the action potential duration, which was much more pronounced in the Purkinje fibres than in the ventricular muscle. However, when compared at concentrations giving a 15% increase of the action potential duration in ventricular muscle, H 234/09 was significantly less effective in the Purkinje fibres than the other two drugs. In the cat papillary muscle all drugs induced an increase in force development. This increase tended to parallel the increase in effective refractory period. However, at prolongations of effective refractory period of more than approximately 50% the increase in developed force levelled off.
All the class III agents investigated showed a positive inotropic effect, which may be of advantage when compared to conventional class I antiarrhythmic agents, which have cardiodepressant actions. Compared to UK-68,798 and E-4031, H 234/09 showed a less unfavourable profile in terms of dispersion of repolarisation, which theoretically may reduce the risk of arrhythmias associated with delayed repolarisation. However, this less unfavourable profile must, like the positive inotropic effect, ultimately be investigated in clinical trials.
1,2-Diacylglycerol (1,2-DAG) and phosphatidic acid (PA) are produced by phospholipase C and D activity and play a key role as second messengers in receptor-mediated signal transduction. So far, little is known about alterations of endogenous 1,2-DAG and PA production during myocardial ischemia.
Rat isolated perfused hearts were subjected to global ischemia, total lipids were extracted, and separated by thin-layer chromatography. The mass of PA and 1,2-DAG were quantified using laserdensitometric analysis of visualized lipids.
Compared to normoxic control values (1,2-DAG 713 +/- 45 ng/mg protein, PA 171 +/- 11 ng/mg protein), the myocardial content of 1,2-DAG and PA was unaltered after 10 min of ischemia. Prolonged myocardial ischemia (20 min), however, which was accompanied by marked overflow of endogenous norepinephrine, significantly increased the mass of both second messengers (1,2-DAG 1062 +/- 100 ng/mg protein, PA 340 +/- 29 ng/mg protein). The increase in PA and 1,2-DAG in response to ischemia was abolished by inhibition of ischemia-induced norepinephrine release as well as by alpha1-adrenergic blockade but unaffected by beta-adrenergic blockade. While inhibition of diacylglycerol kinase did not affect ischemia-induced increase in PA and 1,2-DAG, inhibition of phosphatidylinositol-specific phospholipase C activity significantly suppressed ischemia-induced increase in 1,2-DAG but did not affect endogenous production of PA indicating phospholipase C-independent formation of PA and activation of both, phospholipase C and D, in the ischemic heart.
Ischemia elicits an alpha1-adrenergic receptor-mediated increase in the mass of myocardial PA and 1,2-DAG. The increase in endogenous PA is suggested to be due to the activation of myocardial phospholipase D, whereas 1,2-DAG is formed predominantly by activation of phospholipase C in the ischemic heart.
The aim was to investigate the consequences of simultaneous stimulation of phospholipase C and D by agonists for the molecular species composition of 1,2-diacylglycerol and phospholipids in cardiomyocytes.
Serum-free cultured neonatal rat cardiomyocytes were stimulated by endothelin-1, phenylephrine or phorbolester. The molecular species of 1,2-diacylglycerol (in mol%) and those derived from phosphatidylcholine and phosphatidylinositol were analyzed by high-performance liquid chromatography and their absolute total concentration (nmol per dish) by gas-liquid chromatography. Phospholipids were labelled with [14C]glycerol or double-labelled with [14C]16:0 and [3H]20:4n6 for measurements of respectively, the amount of or relative rate of label incorporation into 1,2-diacylglycerol.
The major molecular species of 1,2-diacylglycerol in unstimulated cells was found to be 18:0/20:4 (57 mol%). The same species was observed predominantly in phosphatidylinositol (73 mol% compared to 11 mol% in phosphatidylcholine). A significant decrease (about 10 mol%) was found for the 18:0/20:4 species of 1,2-diacylglycerol during stimulation (10-40 min) with endothelin-1 or phorbolester, but not phenylephrine. The results of the double-labelling experiments were consistent with the latter finding: the ratio [3H]20:4 over [14C]16:0 in 1,2-diacylglycerol decreased from 1.70 in the control to 1.40 during 10-min endothelin-1 or phorbolester stimulation, but not during phenylephrine stimulation. The [14C]glycerol incorporation into 1,2-diacylglycerol remained relatively constant under agonist-stimulated conditions as did the total concentration of 1,2-diacylglycerol.
1,2-Diacylglycerol present in unstimulated cardiomyocytes is likely derived from phosphatidylinositol. During stimulation with endothelin-1 and phorbolester, but not phenylephrine, phosphatidylcholine becomes an increasingly important source for 1,2-diacylglycerol due to sustained activation of phospholipase D. The 1,2-diacylglycerol level remains relatively constant during agonist stimulation which strongly indicates that particular molecular species of 1,2-diacylglycerol more than its total concentration determine the activation of protein kinase C isoenzymes.
1,2-Diacylglycerol may initiate cardiac hypertrophy, probably by activating protein kinase C. To test this hypothesis we determined the 1,2-diacylglycerol content of hypertrophied tissue.
Rats were treated with monocrotaline and developed severe right ventricular hypertrophy followed by congestive heart failure. 1,2-Diacylglycerol content and fatty acid composition, DNA concentrations, and RNA concentrations in the right ventricle from monocrotaline treated rats were compared with values obtained from the left side or from control rats.
During the first week, the right ventricle showed no significant change in 1,2-diacylglycerol content and a small increase in RNA concentration. However, the 1,2-diacylglycerol content was significantly increased by 55% at two weeks after monocrotaline injection, when DNA and RNA synthesis was also enhanced to its highest level when compared with control rats (37% and 18%, respectively). At four weeks after monocrotaline injection, conversely, the 1,2-diacylglycerol content was decreased by 25% in the right ventricle from monocrotaline treated rats, most of which had pleural and peritoneal effusions indicating congestive heart failure, although RNA synthesis was sustained at a high level. The fatty acid composition of 1,2-diacylglycerol did not differ significantly between the right and left ventricles or control rat ventricles.
These results suggest that 1,2-diacylglycerol accumulation is associated with development of hypertrophy in monocrotaline treated rats. In contrast, at a stage of congestive heart failure 1,2-diacylglycerol production decreased, suggesting that intracellular transduction mechanisms may be attenuated.
The juvenile visceral steatosis (JVS) mouse, a genetic model of systemic carnitine deficiency resulting from carnitine transport mutation, develops cardiac hypertrophy. We determined two putative lipid messengers, 1,2-diacylglycerol (DAG) and ceramide, in JVS and carnitine palmitoyltransferase-I (CPT-I) inhibitor etomoxir-treated mice because these lipids function as co-messengers in the myocardium via modification of protein kinase C activity.
JVS mice were evaluated at 4 and 8 weeks of age. The effect of long-term etomoxir treatment (45 mg/day) (ET) on mice was investigated in control mice from 4 to 8 weeks of age. As a model of inhibited cardiac hypertrophy, carnitine-treated JVS (CT) mice were produced. Myocardial DAG and ceramide levels and their fatty acid composition were measured.
The heart/body weight ratio increased by 100% in JVS mice compared with that in controls, while that of CT mice was normalized in comparison with controls at 8 weeks of age. DAG markedly increased in both JVS and ET mice compared with that in controls (1,677+/-84, 1,258+/-49, and 585+/-58 ng/dry wt, respectively; P<0.01 for controls versus JVS or ET mice), whereas it was decreased significantly in CT mice compared with that in JVS mice (1,066+/-54 ng/dry wt, P<0.01). Furthermore, the fatty acid composition of DAG was similar in JVS and ET mice; in particular, 18:1 and 18:2 were significantly elevated in the myocardium (P<0.01 versus controls). On the other hand, that of DAG in CT mice was similar to that of the control group. In contrast, no difference was observed in myocardial ceramide levels among the groups.
Pharmacological intervention with etomoxir mimics changes in the lipid second messenger characteristic of genetic JVS mice. The results suggest that the increases in distinct DAG species might be involved in the pathogenesis of cardiac hypertrophy as a result of disorder of fatty acid transport.
The juvenile visceral steatosis (JVS) mouse, a murine model of systemic carnitine deficiency, shows a disorder of fatty acid oxidation and develops cardiac hypertrophy associated with lipid accumulation. Recently, alpha-tocopherol was shown to decrease 1,2-diacylglycerol (DAG) levels. We investigated the involvement of DAG in cardiac hypertrophy due to energy metabolism disorder by evaluating the effects of alpha-tocopherol administration on the hearts of JVS mice.
Both JVS and control mice were fed a high alpha-tocopherol diet or a standard diet from 4 to 8 weeks of age. Myocardial DAG levels and fatty acid composition were assessed at 8 weeks of age.
The ventricular to body weight ratio in the JVS mice was significantly higher than that in the control mice [11.2+/-0.1 (mean+/-S.E.M.) versus 3.8+/-0.1 mg/g, P<0.01], and was reduced by alpha-tocopherol treatment (9.7+/-0.2 mg/g, P<0.01 versus JVS mice). However, echocardiographic analysis showed the exaggeration of left ventricular dilatation in the alpha-tocopherol treated JVS mice (P<0.01 versus JVS mice). The myocardial thiobarbituric-acid-reactive substance level was not affected by alpha-tocopherol treatment. The myocardial DAG level was 2.5-fold higher in the JVS mice compared with that in the control mice (2004+/-136 versus 806+/-36 ng/mg dry weight, P<0.01) with a significant increase in 18:1 and 18:2 fatty acids. alpha-Tocopherol treatment reduced myocardial DAG levels in the JVS mice (1443+/-49 ng/mg dry weight, P<0.01 versus JVS mice) without any alteration of the fatty acid composition.
alpha-Tocopherol treatment may partially reduce cardiac hypertrophy but it may also depress cardiac function in the JVS mice by decreasing the myocardial DAG level. An increase in DAG might be involved in the development of cardiac hypertrophy and in the maintenance of cardiac function in energy metabolism disorder of the heart.
STUDY OBJECTIVE - To examine the effect of 9-amino-1,2,3,4-tetrahydroacridine (THA), a compound similar to the K+ blocker 4-aminopyridine, on potassium channels in the sinoatrial node. DESIGN - The pacemaking portion of rabbit sinoatrial nodes was studied using the double microelectrode voltage clamp method in the presence of THA at various concentrations. MEASUREMENTS AND RESULTS - Above 1 mumol.litre-1, THA prolonged the spontaneous cycle length and the transmembrane action potential duration at 50% repolarisation. Above 10 mumol.litre-1, the compound also decreased the maximum rate of rise, the action potential amplitude, and the rate of diastolic depolarisation. Under voltage clamp conditions, THA reduced the time dependent K+ current (IK) in a dose dependent manner. Neither the decay process of IK nor its activation process were altered by THA. CONCLUSIONS - THA depresses sinoatrial node IK without changing its kinetics. Thus it may inhibit the open state of the potassium channels.
There is now substantial evidence that Ins(1,4,5)P3, by virtue of its role in Ca2+ release, can initiate electro-physiological disturbances which develop into cardiac arryhtmias, and in addition appears to be involved in the progression of the cardiomyocytes to apoptosis. Therapeutic regimens targeting the generation or the actions of Ins(1,4,5)P3 may thus prove beneficial in treating or preventing a number of different cardiac pathologies.
Cardiac hypertrophy is a compensatory response to increased mechanical load. Since Fas receptor activation is an important component in hypertrophy induced by pressure- and volume-overload, deciphering the underlying signaling pathways is of prime importance. Based on our previous work showing that in mice and rats ventricular myocytes the electrophysiological disturbances and diastolic [Ca2+]i-rise caused by 3 h of Fas activation are dependent on the Fas-->phospholipase C (PLC)-->1,4,5-inositol trisphosphate (1,4,5-IP3)-->sarcoplasmic reticulum (SR) [Ca2+]i release pathway, we tested the hypothesis that this pathway is also critical for Fas-mediated hypertrophy.
The effects of 24 h Fas activation in cultured neonatal rat ventricular myocytes (NRVM) were analyzed by means of RT-PCR, Western blot, immunofluorescence and fura-2 fluorescence.
Fas activation increased nuclei surface area, atrial natriuretic peptide and connexin43 (Cx43) mRNA, the protein levels of total Cx43 and non-phosphorylated Cx43, and sarcomeric actin, all indicating hypertrophy. Concomitantly, Fas activation decreased mRNA of SERCA2a, the ryanodine receptor (RyR) and nuclear IP3R3. Further, Fas activation caused NFAT nuclear translocation. The hypertrophy was abolished by U73122, xestospongin C (blockers of the 1,4,5-IP3 pathway), genistein and by the PI3K blocker LY294002.
Fas-mediated hypertrophy is dependent on the 1,4,5-IP3 pathway, which is functionally inter-connected to the PI3K/AKT/GSK3beta pathway. Both pathways act in concert to cause NFAT nuclear translocation and subsequent hypertrophy.
We studied the effects of intracellularly applied inositol-1,4,5-trisphosphate (InsP3) to test the hypothesis that InsP3 is a messenger for stimulation of L-type calcium current (ICa(L)) and contractions by muscarinic agonists.
Voltage clamp pulses elicited ICa(L) that evoked contractions recorded with an edge detector in single guinea pig ventricular myocytes superfused with Tyrode's solution (36 degrees C). InsP3 or cyclic AMP (cAMP) was dialyzed into the cell at selected times via the patch electrode.
InsP3 (1-10 microM) transiently increased isotonic contractions when applied for 4-5 min; higher concentrations (50-300 microM) caused a sustained decrease in contractions. InsP3 had no effect on ICa(L) at any concentration tested. Caffeine (10 mM)-induced contractures were increased and decreased, respectively, at 3 and 100 microM InsP3. Pentosan polysulfate (50 micrograms/ml), an InsP3 receptor antagonist, opposed the increased contractions by InsP3. Intrapipette cyclic AMP (10-300 microM) caused sustained increases of ICa(L) and contractions. Cyclic AMP, but not InsP3, also increased ICa(L) when intrapipette Cs+ suppressed K+ currents.
Increased myocyte shortening at low InsP3 concentrations accords with receptor-initiated sarcoplasmic reticulum Ca2+ release. The transient stimulation of contractions at low concentrations and the sustained reduction of contractions at high concentrations are not consistent with a role for InsP in the persistent increase of contractions by muscarinic agonist in ventricular muscle and myocytes. The failure of InsP3 to change ICa(L) when contractions were increased or decreased militates against the L-type calcium channel being an effector of InsP3.
The effect of intravenous fructose-1,6-diphosphate (FDP) infusion on haemodynamic and biochemical variables was studied in dogs after ligation of the anterior descending branch of the left coronary artery. In the control series cardiogenic shock was present in every case 4 h after ligation. In FDP treated animals 4 h after ligation there was no fall in cardiac output and the systolic blood pressure was restored to pre-ligation values. Levels of serum creatine kinase isoenzyme (CK-MB), a highly specific indicator of myocardial cell damage, rose in the shocked (no FDP given) group, but remained low in the FDP treated group, equalling the levels measured in sham operated (no ligation) dogs. Samples of myocardium were taken from infarcted and adjacent normal regions 4 h after ligation for biochemical analysis. CK-MB concentrations in the infarcted region did not change from normal levels with FDP infusion; in the infarcted region lactate concentration (mumol.g-1 wet weight) fell from 18.48 in the control group to 7.90 in the FDP treated group. ATP levels in the infarcted region remained the same as those in the adjacent normal region with FDP treatment. It is concluded that infusion of FDP improves myocardial performance and metabolism following acute myocardial ischaemia.
Isoflurane has been hailed as the anaesthetic of the eighties. We examined the effects of isoflurane anaesthesia on regional distribution of brain and myocardial blood flow in 11 healthy isocapnic pigs using 15 μm diameter radionuclide labelled microspheres that were injected into the left atrium. Each animal was studied during five of the following six conditions: (i) unanaesthetised (control; n=8); (ii) 1.45% end-tidal (ET; 1.0 MAC) isoflurane anaesthesia (n=10); (iii) 2.18% ET (1.5 MAC) isoflurane anaesthesia (n=9); (iv) 0.95% ET isoflurane + 50% N20 anaesthesia (equivalent to 1 MAC; n=8); (v) 1.68% ET isoflurane + 50% N20 anaesthesia (equivalent to 1.5 MAC; n=8); and (vi) 50% N20 alone (n=8). The order of anaesthetised steps was randomised for each pig. At every step 50 to 55 min were allowed for equilibration with isoflurane, and for N20 35 to 40 min were allowed for equilibration. Recovery periods of 60 min each were interposed between anaesthetised steps to allow pigs to recover towards control values.
Control values of blood flow in the cerebrum, cerebellum, and brain-stem were 81±5, 87±8, and 64±6 ml·min⁻¹ 100 g⁻¹, respectively. During 1.45% isoflurane anaesthesia, cerebral, cerebellar and brainstem blood flows were 120%, 152%, and 145% of respective control values. With 2.18% isoflurane, perfusion in these regions of the brain was 140%, 200%, and 226% of respective control values. Substitution of 50% N20 to maintain equipotent anaesthesia markedly exaggerated the increment in cerebral blood flow, while changes in cerebellar and brain-stem blood flow were similar. Cerebral blood flow during 0.95% isoflurane + 50% N20 and 1.68% isoflurane + 50% N20 anaesthesia was 137% and 210% of the control value, respectively. Regional brain blood flow was only insignificantly altered by 50% N20 alone. It is concluded that isoflurane caused dose-dependent vasodilatation in all regions of the brain, the magnitude being greater in the cerebellum and the brain-stem. The administration of N20 with isoflurane to maintain equipotent anaesthesia exaggerated cerebral vasodilatation, especially at deeper level of anaesthesia.
Myocardial blood flow in isoflurane anaesthetised pigs decreased, especially in the inner layers, in a dose-related manner. The use of 50% N20 with isoflurane permitted higher heart rate, perfusion pressure, rate-pressure product, and transmural myocardial blood flow.
Nitric oxide (NO) elicits relaxation in vascular smooth muscle cells (VSMC) that is associated with guanylate cyclase (GC) and K(+) channel activation. In this study we determined the mechanisms that lead to ERK1/2 MAP kinase dephosphorylation in response to NO.
VSMC were treated with the NO donor SNAP or sodium nitroprusside (SNP), and ERK1/2, Src homology (SH) 1 domain-containing protein tyrosine phosphatase (SHP-1), and Kv.1.2 phosphorylation were assessed by immunoprecipitation and Western blot analysis.
NO decreased basal ERK1/2 phosphorylation in a dose- and time-dependent manner. NO-induced ERK1/2 dephosphorylation was detected at 1 min and sustained for 30 min. Pre-treatment with the GC inhibitor ODQ or the protein tyrosine phosphatase inhibitor I prevented ERK1/2 dephosphorylation induced by SNAP. The inhibition of protein phosphatase 1A/2A had no effect on ERK1/2 dephosphorylation induced by SNAP. Treatment with cromakalim A, a nonspecific K(+) channel activator, also induced ERK1/2 dephosphorylation, while blockade of Kv.1.2 K(+) channels (AM92016 hydrochloride) prevented NO-induced ERK1/2 dephosphorylation. In addition, SNAP induced SHP-1 phosphorylation, and the Kv.1.2 dephosphorylation increase and SHP-1 phosphorylation was blocked by ODQ or AM92016. The basal interaction between ERK1/2 and SHP-1 was decreased in response to SNAP stimulation. SHP-1 also interacted with Kv.1.2 under basal conditions and participates in Kv.1.2 activation. Using the mouse mesenteric resistance artery, we found that ERK1/2 MAP kinase is involved in regulation of myogenic tone.
Thus, our study provides the first evidence that NO controls basal ERK1/2 phosphorylation by a signaling cascade that involves a dynamic signaling complex between cGMP, Kv.1.2 and SHP-1.
This editorial refers to ‘Super-resolution imaging reveals that loss of the C-terminus of connexin43 limits microtubule plus-end capture and NaV1.5 localization at the intercalated disc’ by E. Agullo-Pascual et al. , pp. 371–381, this issue.
The cardiac voltage-gated sodium channel, NaV1.5, is responsible for conducting the inward sodium current ( I Na), which leads to the fast depolarization of the cardiac cell membrane. Mutations in SCN5A, the gene encoding NaV1.5, that lead to alterations in I Na are linked to many cardiac phenotypes including congenital long QT syndrome type 3, Brugada syndrome, atrial fibrillation, conduction slowing, and dilated cardiomyopathy. Several partner proteins have been described to associate with NaV1.5, and the genes encoding some of these regulatory proteins have also been found to be mutated in patients with inherited forms of cardiac arrhythmias.1
Recent investigations have revealed that the expression level, cellular localization, and activity of NaV1.5 are finely regulated by complex molecular and cellular mechanisms. Multiple pools of NaV1.5 in cardiac cells have been identified,2 depending on where they are targeted and with which partner proteins they interact ( Figure 1A ). Thus, proteins such as SAP97,3 ankyrin-G, plakophilin 2 (PKP2), and connexin43 (Cx43)4,5 have been described to interact with NaV1.5. The importance of these interactions in targeting and stabilizing NaV1.5 at the intercalated disc (ID), where cells are electrically and mechanically coupled, is only partially understood. NaV1.5 is also expressed at the lateral membrane of cardiomyocytes, and its targeting to this compartment is regulated by …
We have previously reported that interleukin (IL)-6 is induced by hypoxic stimulation in cardiac myocytes. In the present study, we examined the induction of potent transcription factors of IL-6, nuclear factor (NF)-kappa B and NF-IL6, in cardiac myocytes subjected to hypoxia.
Five different lengths of IL-6 promoter-luciferase reporter plasmids and three mutant plasmids, in which the binding sites of NF-kappa B and/or NF-IL6 were disrupted, were transfected into neonatal rat cardiac myocytes. Luciferase activities after hypoxic stimulation were measured. Electrophoretic mobility shift assays were performed using oligonucleotides containing the binding site for NF-kappa B or NF-IL6 as a probe.
Hypoxic stimulation for 4 h increased luciferase activity by 5.7 fold in -179/+12-luciferase reporter plasmid, whereas no significant increase was observed in -60/+12-luciferase plasmid. Decrease in luciferase activity was more prominent when the NF-kappa B binding site was disrupted rather than when the NF-IL6 binding site was disrupted. Moreover, when both sites were disrupted, luciferase activity increased only by 1.5 fold. Electrophoretic mobility shift assays demonstrated enhanced binding activity to oligonucleotides containing the NF-kappa B binding site in hypoxic cardiac myocytes, which displayed a supershift with antibody to its subunit, p50 or p65. The binding activity to the NF-IL6 probe also enhanced and displayed a supershift with antibody to NF-IL6.
Although hypoxic stimulation induced NF-kappa B and NF-IL6 in cardiac myocytes, NF-kappa B may be the primary positive regulator of transcriptional activation of the IL-6 gene in the context of hypoxia.
<$O_ST_ABS>AUTHORS' SYNOPSIS<$C_ST_ABS>The effects were investigated of acute selective coronary hypotension with or without nitroglycerin or Bay a 1040 on regional distribution of Rb86 clearance. When the left circumflex coronary artery was partially obstructed and its mean pressure was reduced below 50-60 mm Hg, Rb86 clearance of the left ventricle, especially of the subendocardium, decreased. In these conditions, nitroglycerin recovered partially to normal distribution of Rb86 clearance without altering total coronary flow and Bay a 1040 did not recover the disproportionate distribution but increased Rb86 clearance of non-ischaemic myocardium, especially of the subepicardium of the left ventricle. The effects of selective coronary hypotension, with or without nitroglycerin or Bay a 1040, on regional distribution of myocardial blood flow of the right ventricle were different from those of the left ventricle.
Agonists selective for the A1 adenosine receptor mimic the protective effect of ischaemic preconditioning against infarction in the rabbit heart. Unselective adenosine antagonists block this protection but, paradoxically, the A1 adenosine receptor selective antagonist 8-cyclopentyl- 1,3-dipropylxanthine (DPCPX) does not. The aim of this study was to test the hypothesis that the newly described A3 adenosine receptor, which has an agonist profile similar to the A1 receptor but is insensitive to DPCPX, might mediate preconditioning.
Isolated rabbit hearts perfused with Krebs buffer experienced 30 min of regional ischaemia followed by 120 min of reperfusion. Infarct size was measured by tetrazolium staining.
In control hearts infarction was 32.2(SEM 1.5)% of the risk zone. Preconditioning by 5 min ischaemia and 10 min reperfusion reduced infarct size to 8.8(2.3)%. Replacing the regional ischaemia with 5 min perfusion with 10 microM adenosine or 65 nM N6-[2-(4-aminophenyl)ethyl]adenosine (APNEA), an adenosine A3 receptor agonist, was equally protective. The unselective antagonist 8-p-sulphophenyl theophylline at 100 microM abolished protection by preconditioning, adenosine, and APNEA, but 200 nM DPCPX did not block protection by any of the interventions. Likewise the potent but unselective A3 receptor antagonist 8-(4-carboxyethenylphenyl)-1,3-dipropylxanthine (BW A1433) completely blocked protection from ischaemic preconditioning.
Because protection against infarction afforded by ischaemic preconditioning, adenosine, or the A3 receptor agonist APNEA could not be blocked by DPCPX and because the potent A3 receptor antagonist BW A1433 blocked protection from ischaemic preconditioning, these data indicate that the protection of preconditioning is not exclusively mediated by the adenosine A1 receptor in rabbit heart and could involve the A3 receptor.