Contractile effects of adenosine A1 and A2A receptors in isolated murine hearts

The adenosine A1 receptor (A1R) inhibits beta-adrenergic-induced contractile effects (antiadrenergic action), and the adenosine A2A receptor (A2AR) both opposes the A1R action and enhances contractility in the heart. This study investigated the A1R and A2AR function in beta-adrenergic-stimulated, isolated wild-type and A2AR knockout murine hearts. Constant flow and pressure perfused preparations were employed, and the maximal rate of left ventricular pressure (LVP) development (+dp/dt(max)) was used as an index of cardiac function. A1R activation with 2-chloro-N6-cyclopentyladenosine (CCPA) resulted in a 27% reduction in contractile response to the beta-adrenergic agonist isoproterenol (ISO). Stimulation of A2AR with 2-P(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxyamidoadenosine (CGS-21680) attenuated this antiadrenergic effect, resulting in a partial (constant flow preparation) or complete (constant pressure preparation) restoration of the ISO contractile response. These effects of A2AR were absent in knockout hearts. Up to 63% of the A2AR influence was estimated to be mediated through its inhibition of the A1R antiadrenergic effect, with the remainder being the direct contractile effect. Further experiments examined the effects of A2AR activation and associated vasodilation with low-flow ischemia in the absence of beta-adrenergic stimulation. A2AR activation reduced by 5% the depression of contractile function caused by the flow reduction and also increased contractile performance over a wide range of perfusion flows. This effect was prevented by the A2AR antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385). It is concluded that in the murine heart, A1R and A2AR modulate the response to beta-adrenergic stimulation with A2AR, attenuating the effects of A1R and also increasing contractility directly. In addition, A2AR supports myocardial contractility in a setting of low-flow ischemia.     

Functional alterations after cardiac sodium-calcium exchanger overexpression in heart failure

The sodium-calcium exchanger (NCX) is discussed as one of the key proteins involved in heart failure. However, the causal role and the extent to which NCX contributes to contractile dysfunction during heart failure are poorly understood. NCX overexpression was induced by infection with an adenovirus coding for NCX, which coexpressed green fluorescence protein (GFP) (AdNCX) by ex vivo gene transfer to nonfailing and failing rabbit cardiomyocytes. Myocardial gene transfer in rabbits in vivo was achieved by adenoviral delivery via aortic cross-clamping. Peak cell shortening of cardiomyocytes was determined photo-optically. Hemodynamic parameters in vivo were determined by echocardiography (fractional shortening) and tip catheter [maximal first derivative of left ventricular (LV) pressure (dP/dt(max)); maximal negative derivative of LV pressure (-dP/dt(max))]. Peak cell shortening was depressed after NCX gene delivery in isolated nonfailing and in failing cardiomyocytes. In nonfailing rabbits in vivo, basal systolic contractility (fractional shortening and dP/dt(max)) and maximum rate of LV relaxation (-dP/dt(max)) in vivo were largely unaffected after NCX overexpression. However, during heart failure, long-term NCX overexpression over 2 wk significantly improved fractional shortening and dP/dt(max) compared with AdGFP-infected rabbits, both without inotropic stimulation and after beta-adrenergic stimulation with isoproterenol. -dP/dt(max) was also improved after NCX overexpression in the failing rabbits group. These results indicate that short-term effects of NCX overexpression impair contractility of isolated failing and nonfailing rabbit cardiomyocytes. NCX overexpression over 2 wk in vivo does not seem to affect myocardial contractility in nonfailing rabbits. Interestingly, in vivo overexpression of NCX decreased the progression of systolic and diastolic contractile dysfunction and improved beta-adrenoceptor-mediated contractile reserve in heart failure in rabbits in vivo.     

Peroxynitrite and myocardial contractility: in vivo versus in vitro effects

Generation of peroxynitrite (ONOO-) as a result of altered redox balance has been shown to affect cardiac function; however, inconsistencies in the data exist, particularly for myocardial contractility. The hypothesis that the cardiac impact of ONOO- formation depends on its site of generation, intravascular or intramyocardial, was examined. Cardiac contractility was assessed by pressure-volume analysis to delineate vascular versus cardiac changes on direct infusion of ONOO- into the right atria of conscious dogs both with normal cardiac function and in heart failure. Additionally, ONOO- was administered to isolated murine cardiomyocytes to mimic in situ cardiac generation. When infused in vivo, ONOO- had little impact on inotropy but led to systemic arterial dilation, likely as a result of rapid decomposition to NO2- and NO3-. In contrast, infused ONOO- was long lived enough to abolish beta-adrenergic (dobutamine)-stimulated contractility/relaxation, most likely through catecholamine oxidation to aminochrome. When administered to isolated murine cardiomyocytes, ONOO- induced a rapid reduction in sarcomere shortening and whole cell calcium transients, although neither decomposed ONOO- or NaNO2 had any effect. Thus, systemic generation of ONOO- is unlikely to have primary cardiac effects, but may modulate cardiac contractile reserve, via blunted beta-adrenergic stimulation, and vascular tone, as a result of generation of NO2- and NO3-. However, myocyte generation of ONOO- may impair contractile function by directly altering Ca2+ handling. These data demonstrate that the site of generation within the cardiovascular system largely dictates the ability of ONOO- to directly or indirectly modulate cardiac pump function.     

模拟失重降低大鼠心肌细胞对异丙肾上腺素的反应性

本文旨在研究模拟失重对大鼠单个心肌细胞无负荷收缩功能的影响以及对异丙肾上腺素(isoproterenol,ISO)反应性的变化.采用人鼠尾部悬吊法在地面模拟失重状态,4周后以胶原酶I消化分离心肌细胞,分别对左、右两心室心肌细胞进行收缩功能测量.结果显示,悬吊4周大鼠(悬吊组)左,右心室心肌细胞的长度和宽度与正常大鼠(对照组)相比均无显著差异.随刺激频率增加,对照组与悬吊组大鼠心肌细胞缩短幅值均逐步增加.在1.0、2.0与4.0 Hz刺激下,对照组大鼠左心室心肌细胞缩短幅值分别为(8.50±1.26)%、(9.00±1.38)%与(9.23±1.83)%,右心室心肌细胞缩短幅值分别为(9.80±2.48)%、(10.03±2.48)%与(10.28±2.27)%;与对照组大鼠相比,在1.0与2.0Hz刺激下,悬吊组大鼠左心室心肌细胞无负荷缩短幅值分别降低12.2%、10.9%(P《0.05),右心室则分别降低16.5%、16.3%(P《0.05);但是在4.0 Hz刺激下却无显著性改变.与同一频率刺激下的对照组大鼠相比,悬吊组大鼠左、右心室心肌细胞达到缩短峰值的时程(time to peak shortening,TPS)明显缩短(P《0.05);而从缩短峰值至75%舒张的时程(TR75)则明显延长(P《0.05).在各刺激频率下,悬吊组大鼠左、右心室心肌细胞缩短(+dL/dtmax)与舒张(-dL/dtmax)速度均未发生明显改变.用1、5、10 nmol/L ISO灌流达稳态水平后,对照组大鼠心肌细胞缩短幅值分别增加了(10.63±0.83)%、(35.06±5.22)%和(71.64±6.83)%;而悬吊组大鼠心肌细胞缩短幅值仅增加(5.75±0.76)%、(23.97±4.50)%和(26.38±8.13)%,均有显著性差异(P《0.05,P《0.01).用10、50、100 nmol/L forskolin 灌流达稳定水平后,对照组大鼠心肌细胞缩短幅值分别增加了(3.04±0.27)%、(9.81±2.66)%、(20.20±3.47)%;而悬吊组大鼠心肌细胞缩短幅值仅增加了(1.42±0.53)%、(3.83±1.71)%、(5.49±4.08)%,均有显著性差异(P《0.05).以上结果表明,模拟失重4周降低人鼠心肌细胞无负荷缩短幅值以及对ISO的反应性.     

Effect of SIN-1 in rat ventricular myocytes: interference with beta-adrenergic stimulation

We have examined the effects of the nitric oxide (NO) donor, 3-morpholino-sydnonimine (SIN-1), on Ca(2+) transients, L-type Ca(2+) current (I(Ca,L)), and cGMP/cAMP content in electrically-stimulated rat ventricular myocytes in the absence and presence of the beta-adrenergic stimulation with isoproterenol. SIN-1 had no effect at low concentrations, but decreased the amplitude of electrically-induced Ca(2+) transients at higher concentrations. SIN-1 attenuated the increase in Ca(2+) transients induced by isoproterenol in a concentration-dependent manner. SIN-1 Also reduced the amplitude of caffeine-induced Ca(2+) transients, and the increase in I(Ca,L) induced by isoproterenol. These effects of SIN-1 were associated with an increased cGMP and a decreased cAMP content in ventricular myocytes in either the absence or presence of isoproterenol. These data suggest that the inhibitory effect of SIN-1 on basal and beta-adrenergic stimulated Ca2+ signal in ventricular myocytes could be due to the depression in the SR function and I(Ca,L), possibly mediated by a cGMP/cAMP-dependent mechanism. Taken together, the present study supports the idea that NO acts as an inhibitory modulator of the cardiac function during pathological conditions associated with an abnormal production of NO such as septic shock.     

The cardiotonic effects of levosimendan in guinea pig hearts are modulated by beta-adrenergic stimulation

The effects of the Ca2+-sensitiser levosimendan alone or in combination with beta-adrenergic stimulation on the contractile function were studied in various guinea pig cardiac preparations. Echocardiography in narcotised animals indicated that a maximal dose of levosimendan (50 microg x kg(-1)) increased the left ventricular posterior wall movement velocity during systoles and diastoles by 25 +/- 3% (mean +/- S.E.M.) and 17 +/- 2%, respectively. In Langendorff hearts, a saturating concentration of levosimendan (0.3 micromol x l(-1) for 5 min) increased +dP/dt(max) and dP/dt(max) by 28 +/- 3% and 14 +/- 2%, respectively. Further, the Ca2+-sensitising potential of levosimendan in Triton-skinned cardiomyocytes (EC50: 5 +/- 3 nmol x l(-1)) was illustrated by a maximal increase in the isometric force production by 51 +/- 5% (at pCa 6.2). However, following stimulation by isoproterenol, when the level of troponin I phosphorylation was elevated, no significant additional increase in the contractile parameters could be demonstrated upon levosimendan administration. Moreover, the levosimendan-induced increase in force production in isolated skinned myocytes could be prevented by incubation with the catalytic subunit of protein kinase A (100 U x ml(-1) for 40 min). These data indicate that thin filament-targeted Ca2+-sensitisation by levosimendan is modulated by phosphorylation of the contractile filaments, an effect that should be considered during combination therapy with levosimendan.     

cGMP-independent inotropic effects of nitric oxide and peroxynitrite donors: potential role for nitrosylation

Nitric oxide (NO) has concentration-dependent biphasic myocardial contractile effects. We tested the hypothesis, in isolated rat hearts, that NO cardiostimulation is primarily non-cGMP dependent. Infusion of 3-morpholinosydnonimine (SIN-1, 10(-5) M), which may participate in S-nitrosylation (S-NO) via peroxynitrite formation, increased the rate of left ventricular pressure rise (+dP/dt; 19 +/- 4%, P < 0.001, n = 11) without increasing effluent cGMP or cAMP. Superoxide dismutase (SOD; 150 U/ml) blocked SIN-1 cardiostimulation and led to cGMP elaboration. Sodium nitroprusside (10(-10)-10(-7) M), an iron nitrosyl compound, did not augment +dP/dt but increased cGMP approximately eightfold (P < 0.001), whereas diethylamine/NO (DEA/NO; 10(-7) M), a spontaneous NO. donor, increased +dP/dt (5 +/- 2%, P < 0.05, n = 6) without augmenting cGMP. SIN-1 and DEA/NO +dP/dt increase persisted despite guanylyl cyclase inhibition with 1H-(1,2,4)oxadiazolo-(4,3,-a)quinoxalin-1-one (10(-5) M, P < 0.05 for both donors), suggesting a cGMP-independent mechanism. Glutathione (5 x 10(-4) M, n = 15) prevented SIN-1 cardiostimulation, suggesting S-NO formation. SIN-1 also produced SOD-inhibitable cardiostimulation in vivo in mice. Thus peroxynitrite and NO donors can stimulate myocardial contractility independently of guanylyl cyclase activation, suggesting a role for S-NO reactions in NO/peroxynitrite-positive inotropic effects in intact hearts.     

Activity of amylin at CGRP1-preferring receptors coupled to positive contractile response in rat ventricular cardiomyocytes

Calcitonin gene-related peptide (CGRP) exerts a positive contractile response directly in rat ventricular cardiomyocytes. This response is mediated by receptors of the CGRP₁-subtype. Amylin is 46% homologous with CGRP and binds to receptors selective for CGRP in a range of tissues. The ability of amylin to influence ventricular contractility has been assessed using cardiomyocytes isolated from the ventricles of adult rats. Cardiomyocytes were subjected to biphasic electrical stimulation at 0.5 Hz. CGRP produced a concentration-dependent positive contractile response which became maximal 4 min after initial stimulation. CGRP increased the contractile amplitude maximally at 1 nM and to a value which was 23.3% greater than in the absence of peptide (EC₅₀ VALUE = 21 pM). Amylin increased the contractile amplitude maximally at 20 nM and to a value which was 17.3% greater than in the absence of peptide (EC₅₀ VALUE = 216 pM). In the presence of amylin (20 nM), the concentration-dependence of the contractile response to CGRP was shifted to the left, so that the response became maximal when CGRP was present at 50 pM. In the presence of CGRP_8–37 (100 nM), a selective antagonist at CGRP₁-preferring receptors, the concentration-dependence of the contractile response to CGRP was shifted to the right (dose RATIO = 54). Similarly, in the presence of CGRP_8–37 (100 nM), the contractile response to amylin was inhibited significantly (P ≤ 0.01). Amylin_8–37 (100 nM) did not inhibit the concentration-dependence of the contractile responses to CGRP and amylin significantly (dose RATIOS = 4.2 and 2.4, respectively). In conclusion, these data indicate that amylin exerts a contractile response directly in rat ventricular cardiomyocytes via CGRP₁-preferring receptors. This effect could assume greater significance in non-insulin-dependent diabetes mellitus and in hypertensive states, in which the concentration of amylin is elevated in plasma.     

Negative feedback exerted by cAMP-dependent protein kinase and cAMP phosphodiesterase on subsarcolemmal cAMP signals in intact cardiac myocytes: an in vivo study using adenovirus-mediated expression of CNG channels

Intracardiac cAMP levels are modulated by hormones and neuromediators with specific effects on contractility and metabolism. To understand how the same second messenger conveys different information, mutants of the rat olfactory cyclic nucleotide-gated (CNG) channel alpha-subunit CNGA2, encoded into adenoviruses, were used to monitor cAMP in adult rat ventricular myocytes. CNGA2 was not found in native myocytes but was strongly expressed in infected cells. In whole cell patch-clamp experiments, the forskolin analogue L-858051 (L-85) elicited a non-selective, Mg2+ -sensitive current observed only in infected cells, which was thus identified as the CNG current (ICNG). The beta-adrenergic agonist isoprenaline (ISO) also activated ICNG, although the maximal efficiency was approximately 5 times lower than with L-85. However, ISO and L-85 exerted a similar maximal increase of the L-type Ca2+ current. The use of a CNGA2 mutant with a higher sensitivity for cAMP indicated that this difference is caused by the activation of a localized fraction of CNG channels by ISO. cAMP-dependent protein kinase (PKA) blockade with H89 or PKI, or phosphodiesterase (PDE) inhibition with IBMX, dramatically potentiated ISO- and L-85-stimulated ICNG. A similar potentiation of beta-adrenergic stimulation occurred when PDE4 was blocked, whereas PDE3 inhibition had a smaller effect (by 2-fold). ISO and L-85 increased total PDE3 and PDE4 activities in cardiomyocytes, although this effect was insensitive to H89. However, in the presence of IBMX, H89 had no effect on ISO stimulation of ICNG. This study demonstrates that subsarcolemmal cAMP levels are dynamically regulated by a negative feedback involving PKA stimulation of subsarcolemmal cAMP-PDE.     

Phosphoinositide 3-kinasegamma (PI3Kgamma) controls L-type calcium current (ICa,L) through its positive modulation of type-3 phosphodiesterase (PDE3)

The modulation of L-type calcium current (ICa,L) is mainly due to mediators acting through activation of G protein-coupled receptors (GPCR) and different protein kinases; among them, phosphoinositide 3-kinasegamma (PI3Kgamma) has been recently discovered to play an important role in the regulation of cardiac contractility and beta-adrenergic signal transduction. Recent reports have demonstrated that, in the heart, different subtypes of beta-adrenergic receptors are coupled to both Gi and/or Gs proteins. While beta1-adrenergic receptors (beta1-AR) couple only to Gs and evoke a strong ICa,L, beta2-adrenergic receptors (beta2-AR) can activate both Gs and Gi proteins and trigger only a limited ICa,L. Here we demonstrate that (i) PI3Kgamma-/- ventricular myocytes are characterized by an higher basal ICa,L density, even if the responsiveness of adenylyl cyclase to Forskolin is comparable to that observed in PI3Kgamma+/+ cardiomyocytes; (ii) both in basal conditions and after beta-AR stimulation, the activity of phosphodiesterase (PDE) type 3 depends on PI3Kgamma; (iii) in PI3Kgamma-/- cardiac myocytes, specific stimulation of beta2-AR is followed by a increase in ICa,L stronger than in wild-type controls. Taken together, our results suggest that the higher values of ICa,L observed both in basal conditions and after beta-AR stimulation in PI3Kgamma-/- ventricular myocytes are mainly due to a positive modulation of PDE3 activity exerted by PI3Kgamma. As observed in PI3Kgamma-/- neonatal cardiomyocytes, cells lacking PI3Kgamma are more sensitive to stimulation of beta2-adrenergic receptors.     

Bidirectional regulation of insulin receptor autophosphorylation and kinase activity by peroxynitrite

Accumulating evidence suggests that enhanced peroxynitrite formation occurs during diabetes. This report describes the effect of peroxynitrite on insulin receptor (IR) function. Addition of peroxynitrite to purified IR resulted in concentration-dependent tyrosine nitration and thiol oxidation. Interestingly, the basal and insulin-stimulated IR autophosphorylation and tyrosine kinase activity were upregulated at low peroxynitrite concentrations, but downregulated at high peroxynitrite concentrations. Concomitantly, peroxynitrite dramatically reduced ¹²⁵I-insulin binding capacity and phosphotyrosine phosphatase activity of IR preparations. Moreover, SIN-1 administration decreased blood glucose levels in normal mice via upregulation of IR/IRS-1 tyrosine phosphorylation. In contrast, SIN-1 markedly increased blood glucose levels in diabetic mice concomitant with downregulation of IR/IRS-1 tyrosine phosphorylation. Taken together, these data provide new insights regarding how peroxynitrite influences IR function in vitro and in vivo, suggesting that peroxynitrite plays a dual role in regulation of IR autophosphorylation and tyrosine kinase activity, and SIN-1 has hyperglycemic effect in diabetic mice.     

Subthreshold nitric oxide synthase inhibition improves synergistic effects of subthreshold MMP‐2/MLCK‐mediated cardiomyocyte protection from hypoxic injury

Injury of myocardium during ischaemia/reperfusion (I/R) is a complex and multifactorial process involving uncontrolled protein phosphorylation, nitration/nitrosylation by increased production of nitric oxide and accelerated contractile protein degradation by matrix metalloproteinase‐2 (MMP‐2). It has been shown that simultaneous inhibition of MMP‐2 with doxycycline (Doxy) and myosin light chain kinase (MLCK) with ML‐7 at subthreshold concentrations protects the heart from contractile dysfunction triggered by I/R in a synergistic manner. In this study, we showed that additional co‐administration of nitric oxide synthase (NOS) inhibitor (1400W or L‐NAME) in subthreshold concentrations improves this synergistic protection in the model of hypoxia–reoxygenation (H‐R)‐induced contractile dysfunction of cardiomyocytes. Isolated cardiomyocytes were subjected to 3 min. of hypoxia and 20 min. of reoxygenation in the presence or absence of the inhibitor cocktails. Contractility of cardiomyocytes was expressed as myocyte peak shortening. Inhibition of MMP‐2 by Doxy (25–100 μM), MLCK by ML‐7 (0.5–5 μM) and NOS by L‐NAME (25–100 μM) or 1400W (25–100 μM) protected myocyte contractility after H‐R in a concentration‐dependent manner. Inhibition of these activities resulted in full recovery of cardiomyocyte contractility after H‐R at the level of highest single‐drug concentration. The combination of subthreshold concentrations of NOS, MMP‐2 and MLCK inhibitors fully protected cardiomyocyte contractility and MLC1 from degradation by MMP‐2. The observed protection with addition of L‐NAME or 1400W was better than previously reported combination of ML‐7 and Doxy. The results of this study suggest that addition of NOS inhibitor to the mixture of inhibitors is better strategy for protecting cardiomyocyte contractility.     

Alteration of cytosolic free calcium homeostasis by SIN-1: high sensitivity of L-type Ca2+ channels to extracellular oxidative/nitrosative stress in cerebellar granule cells

Exposure of cerebellar granule neurones in 25 mm KCl HEPES-containing Locke's buffer (pH 7.4) to 50-100 microm SIN-1 during 2 h decreased the steady-state free cytosolic Ca2+ concentration ([Ca2+]i) from 168 +/- 33 nm to 60 +/- 10 nm, whereas exposure to > or = 0.3 mm SIN-1 produced biphasic kinetics: (i) decrease of [Ca2+]i during the first 30 min, reaching a limiting value of 75 +/- 10 nm (due to inactivation of L-type Ca2+ channels) and (ii) a delayed increase of [Ca2+]i at longer exposures, which correlated with SIN-1-induced necrotic cell death. Both effects of SIN-1 on [Ca2+]i are blocked by superoxide dismutase plus catalase and by Mn(III)tetrakis(4-benzoic acid)porphyrin chloride. Supplementation of Locke's buffer with catalase before addition of 0.5-1 mm SIN-1 had no effect on the decrease of [Ca2+]i but further delayed and attenuated the increase of [Ca2+]i observed after 60-120 min exposure to SIN-1 and also protected against SIN-1-induced necrotic cell death. alpha-Tocopherol, the potent NMDA receptor antagonist (+)-MK-801 and the N- and P-type Ca2+ channels blocker omega-conotoxin MVIIC had no effect on the alterations of [Ca2+]i upon exposure to SIN-1. However, inhibition of the plasma membrane Ca2+ ATPase can account for the increase of [Ca2+]i observed after 60-120 min exposure to 0.5-1 mm SIN-1. It is concluded that L-type Ca2+ channels are a primary target of SIN-1-induced extracellular nitrosative/oxidative stress, being inactivated by chronic exposure to fluxes of peroxynitrite of 0.5-1 microm/min, while higher concentrations of peroxynitrite and hydrogen peroxide are required for the inhibition of the plasma membrane Ca2+ ATPase and induction of necrotic cell death, respectively.     

Nitric oxide reduces energy supply by direct action on the respiratory chain in isolated cardiomyocytes

To investigate the effect of nitric oxide (NO) on cardiac energy metabolism, isolated cardiomyocytes of Wistar rats were incubated in an Oxystat system at a constant ambient PO2 (25 mmHg) and oxygen consumption (VO2); free intracellular Ca(2+) (fura 2), free cytosolic adenosine [S-adenosylhomocysteine (SAH) method], and mitochondrial NADH (autofluorescence) were measured after application of the NO donor morpholinosydnonimine (SIN-1). In Na(+)-free medium (contracting cardiomyocytes), VO2 increased from 7.9 +/- 1.2 to 26.4 +/- 3.1 nmol x min(-1) x mg protein(-1). SIN-1 (100 micromol/l) decreased VO2 in contracting (-21 +/- 3%) and in quiescent cells (-24 +/- 7%) by the same extent. Inhibition of VO2 was dose dependent (EC(50): 10(-7) mol/l). S-nitroso-N-acetyl-penicillamine, another NO donor, also inhibited VO2, whereas SIN-1C (100 micromol/l), the degradation product of SIN-1, displayed no inhibitory effect. Intracellular Ca(2+) remained unchanged, and inhibition of protein kinases G, A, or C did not antagonize the effect of NO. Mitochondrial NADH increased with NO, indicating a reduced flux through the respiratory chain. In quiescent but not in contracting cardiomyocytes, NO significantly increased adenosine, indicating a reduced energy status. These data suggest the following. 1) NO decreases cardiac respiration, most likely via direct inhibition of the respiratory chain. 2) Whereas in quiescent cardiomyocytes the inhibition of aerobic ATP formation by NO causes reduction in energy status, contracting cells are able to compensate for the NO-induced inhibition of oxidative phosphorylation, maintaining energy status constant.     

Overexpression of β(1)-adrenoceptor can not protect rat cardiomyocytes from injury induced by isoprenaline

The purpose of this study was to investigate the effect of the overexpression of β(1)-adrenoceptor (β(1)-AR) on the contractile function and cell survival of rat cardiomyocytes injured by isoprenaline (ISO). The rat cardiomyocytes were isolated using the collagenase perfusion method and then transfected with β(1)-AR gene using adenoviruses vector. Four hours after the infection, the rat cardiomyocytes were treated with ISO for 24 h to imitate the high catecholamine levels of chronic heart failure. Western blot was performed to measure the protein expression of β(1)-AR. The percentages of rod cells were measured to test cell survival. Video-based edge-detection system was used to measure the contractile function of the cardiomyocytes. The results indicated that the expression of β(1)-AR in β(1)-AR-transfected cardiomyocytes was significantly increased compared with that in control group (P<0.01). Meanwhile, β(1)-AR transfection also increased β(1)-AR protein levels in ISO-injured cardiomyocytes. The cardiomyocyte survival was significantly decreased in ISO group compared with that in control group. β(1)-AR-transfection alone had no effect on cardiomyocyte survival in β(1)-AR group, but it further decreased cardiomyocyte survival in β(1)-AR+ISO group. Contractile amplitudes of ISO-injured cardiomyocytes were significantly decreased regardless of whether they were transfected with β(1)-AR or not, although β(1)-AR-transfected cardiomyocytes showed significantly increased contractile function compared with control group (P<0.05). These results suggest that the overexpression of β(1)-AR has no significant protective effect on rat cardiomyocytes injured by ISO.     

Inhibition of SIN-1–Induced Change in Mitochondrial Membrane Permeability in PC12 Cells by Dopamine

Dopamine (50 or 100 microM) attenuated the nuclear damage and cell death due to 500 microM SIN-1, a donor of superoxide and nitric oxide, in differentiated PC12 cells whereas 200 microM dopamine did not depress cell death. Dopamine at 50-100 microM for a 4-h treatment did not show a significant cytotoxic effect on PC12 cells. Dopamine (100 microM) inhibited the decrease in mitochondrial transmembrane potential, cytochrome c release, activation of caspase-3, formation of reactive oxygen species, and depletion of glutathione (GSH) due to 500 microM SIN-1 in PC12 cells. The reaction of dopamine with peroxynitrite reduced an amount of peroxynitrite. The results suggest that dopamine exhibits a biphasic effect against the cytotoxicity of SIN-1 depending on concentrations. Dopamine at 50-100 microM may attenuate the reactive nitrogen species-induced viability loss in PC12 cells by suppressing the mitochondrial membrane permeability change through inhibition of the formation of reactive species, including peroxynitrite.     

Subsensitivity to beta-adrenergic stimulation in atria from rats infested with Syphacia sp

Syphacia sp. is a common intestinal parasite in conventionally-housed laboratory rodents. Although gross lesions are rare in oxyuriasis, it is possible that more subtle changes may develop, which may affect research results. In this study, we analysed the responsiveness to beta-adrenergic stimulation by isoproterenol (ISO) of left atria isolated from Syphacia-infested (SYPH) and control, non-infested adult male Wistar rats (CONT). In the non-infested animals, ISO pD(2) was not significantly changed by ivermectin treatment. Whereas the maximal inotropic response to ISO was not significantly affected, the pD(2) value was decreased in SYPH (7.61 +/- 0.09, n = 7, vs 8.21 +/- 0.25 in CONT, n = 5, P < 0.05), indicating lower sensitivity to beta-adrenergic stimulation. This change was similar to that caused by a classic stressor, namely repeated immobilization, in non-infested rats (IMMO). In this group, ISO pD(2) was 7.62 +/- 0.14, n = 6 (P < 0.05 with relation to CONT). The results indicate that infestation with Syphacia sp. is as effective as immobilization at diminishing cardiac reactivity to beta-adrenergic stimulation. It is thus possible that oxyuriasis may affect the response of other tissues to physiological modulators.     

白细胞介素-2对大鼠心肌收缩功能的影响涉及一氧化氮机制

The aim of the present study was to investigate the effect of interleukin-2 (IL-2) on the contractility in cardiomyocytes and the underlying mechanisms. Ventricular myocytes were isolated from adult male Sprague-Dawley rats. Contractile responses were evaluated by use of the video tracking system. Contractile parameters in cardiomyocytes electrically stimulated at 0.2 Hz included peak velocity of cell shortening (+dL/dtmax), peak velocity of cell relengthening (-dL/dtmax), contractile amplitude (dL), and end-diastolic cell length. Calcium transients of ventricular myocytes were determined by the spectrofluorometric techniques. Dose-dependent inhibition in + dL/dtmax, -dL/dtmax, dL and end-diastolic cell length were induced by IL-2 at 2-1000 U/ml. Pretreatment with the nitric oxide synthase inhibitor N(w)-nitro-L-arginine methyl ester (L-NAME, 100 micromol/L) and soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo [4,3a]quinoxalin-1-one (ODQ, 10 micromol/L) attenuated IL-2-induced inhibition of contractility. Aminoguanidine, an inhibitor of inducible nitric oxide synthase, had no effect on the inhibition by IL-2. IL-2 at 200 U/ml decreased the amplitude of electrically induced [Ca2+]i transients of ventricular myocytes. Pretreatment with ODQ diminished IL-2-induced inhibition of amplitude of the calcium transient. In conclusion, the present study indicates a direct action of IL-2 on cardiomyocyte contraction, possibly through an increased NO production, activation of soluble guanylyl cyclase and inhibition in intracellular Ca2+ level.     

Anti-adrenergic effects of nitric oxide donor SIN-1 in rat cardiac myocytes

We studied how the nitric oxide (NO*) donor 3-morpholinosydnonimine (SIN-1) alters the response to beta-adrenergic stimulation in cardiac rat myocytes. We found that SIN-1 decreases the positive inotropic effect of isoproterenol (Iso) and decreases the extent of both cell shortening and Ca2+ transient. These effects of SIN-1 were associated with an increased intracellular concentration of cGMP, a decreased intracellular concentration of cAMP, and a reduction in the levels of phosphorylation of phospholamban (PLB) and troponin I (TnI). The guanylyl cyclase inhibitor 1H-8-bromo-1,2,4-oxadiazolo (3,4-d)benz(b)(1,4)oxazin-1-one (ODQ) was not able to prevent the SIN-1-induced reduction of phosphorylation levels of PLB and TnI. However, the effects of SIN-1 were abolished in the presence of superoxide dismutase (SOD) or SOD and catalase. These data suggest that, in the presence of Iso, NO-related congeners, rather than NO*, are responsible for SIN-1 effects. Our results provide new insights into the mechanism by which SIN-1 alters the positive inotropic effects of beta-adrenergic stimulation.     

Endothelium-derived nitric oxide mediates the antiadrenergic effect of human vasostatin-1 in rat ventricular myocardium

Vasostatins (VSs) are vasoactive peptides derived from chromogranin A (CgA), a protein contained in secretory granules of chromaffin and other cells. The negative inotropic effect and the reduction of isoproterenol (Iso)-dependent inotropism induced by VSs in the heart suggest that they have an antiadrenergic function. However, further investigation of the mechanisms of action of VSs is needed. The aim of the present study was to define the signaling pathways activated by VS-1 in mammalian ventricular myocardium and cultured endothelial cells that lead to the modulation of cardiac contractility. Ca(2+) and nitric oxide (NO) fluorometric confocal imaging was used to study the effects induced by recombinant human VS-1 [STA-CgA-(1-76)] on contractile force, L-type Ca(2+) current, and Ca(2+) transients under basal conditions and after beta-adrenergic stimulation in rat papillary muscles and ventricular cells and the effects on intracellular Ca(2+) concentration and NO production in cultured bovine aortic endothelial (BAE-1) cells. VS-1 had no effect on basal contractility of papillary muscle, but the effect of Iso stimulation was reduced by 27%. Removal of endocardial endothelium and inhibition of NO synthesis and phosphatidylinositol 3-kinase (PI3K) activity abolished the antiadrenergic effect of VS-1 on papillary muscle. In cardiomyocytes, 10 nM VS-1 was ineffective on basal and Iso (1 microM)-stimulated L-type Ca(2+) current and Ca(2+) transients. In BAE-1 cells, VS-1 induced a Ca(2+)-independent increase in NO production that was blocked by the PI3K inhibitor wortmannin. Our results suggest that the antiadrenergic effect of VS-1 is mainly due to a PI3K-dependent NO release by endothelial cells, rather than a direct action on cardiomyocytes.